Microbiological Food Hygiene

 

MICROBIOLOGICAL HYGIENE 

MICROBIOLOGICAL FOOD HYGIENE 

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MICROBIOLOGICAL HYGIENE   E   INO E   LIAS H   AKALEHTO  AKALEHT O , P   P   H   D –    –  S   S   ERIES E   DITOR -

University of Eastern Finland, Kuopio, Finland Microbiological Clinical Hygiene Microbiological  Eino Elias Hakalehto (Editor)

2015 - 1st Quarter. ISBN: 978-1-63463-428-1 (Hardcover) Microbiological Food Hygiene Microbiological  Eino Elias Hakalehto (Editor)

2015 - 4th Quarter. ISBN: 978-1-63483-646-3 (Hardcover)

 

MICROBIOLOGICAL HYGIENE 

MICROBIOLOGICAL FOOD HYGIENE 

EINO ELIAS HAKALEHTO  EDITOR  

 New York

 

Copyright © 2015 by Nova Science Publishers, Inc. All rights reserved.   No part of this book may be reproduced, stored in a retrieval system or

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CONTENTS  Preface

vii 

Introduction

 Elias Hakalehto Hakalehto  Chapter 1

Microbial Presence in Foods and in Their Digestion   Elias Hakalehto Hakalehto 

Chapter 2

Trends toward Clean and Healthy Nutrition 

ix  1  19 

 Mikko Immonen, Immonen, Jukka-Pekk Jukka-Pekka a Hakaleht Hakalehto o and Elias Hakalehto  Chapter 3

Hazards and Prevention of Food Spoilage   Elias Hakalehto Hakalehto 

33 

Chapter 4

Foodborne Foodbor ne Viruses   Robert Armon 

75 

Chapter 5

Prevalence, Detection and Prevention of Foodborne Outbreaks Related to Large Hospital Kitchens   Markus Hell, Hell, Christa Bernhofer, Jouni Pesola, Pesola,   Ilkka Pesola and and Elias Hakalehto Hakalehto 

Chapter 6

Biofilm Formation in Food 

91 

111 

 Robert Armon  Chapter 7

First Detection of Salmonella Contaminations 

131 

 Elias Hakalehto, Hakalehto, Joun Jounii Pesola, An Anneli neli Heitto, Heitto, Henri H Hänninen, änninen,  Panu Hendolin, Hendolin, Osmo Osmo Hänninen, Hänninen, Robert Armo Armon, n,  Tarmo Humppi and Heikki Paakkanen  Chapter 8

Hygienic Lessons from the Dairy Microbiology Cases   Elias Hakalehto Hakalehto 

155 

Chapter 9

Detection of Bacillus Cereus Cereus 

175 

 Elias Hakalehto Hakalehto and Anneli Heitto   

Chapter 10

Method Development for Clostridium Botulinum Toxin Detection   

 Jouni Pesola, Pesola, Tarmo H Humppi umppi a and nd Elias Ha Hakalehto kalehto

 

197 

vi

Contents

Chapter 11

Infant Milk Formulas   Jouni Pesola Pesola 

211 

Chapter 12

Foodborne Zoonosis   Robert Armon 

227 

Chapter 13

Antibiotic Resistance in Foods   Elias Hakalehto Hakalehto 

233 

Chapter 14

Monitoring Food and Water Sources with the PMEU 

253 

 Elias Hakalehto, Hakalehto, Annel Annelii Heitto, Heitto, Juha Jokela Jokelainen, inen, Aki Immonen Immonen and Lauri Heitto  Chapter 15

Food, Polyphenols and Health   Osmo Hänninen, Tuomo Tompuri and Riitta Törrönen  

Chapter 16

Microbial Effects on Selenium Nutritional Values in Biofortification   Zhi-Qing Lin, Solo Solomon mon Miller Miller and Jie Hong  

Chapter 17

Catering Services and Hygienic Food Deliveries   Jukka Sauramäki Sauramäki and Eli Elias as Hakalehto Hakalehto 

Appendix I:

Recommended Recommend ed Hygiene Measures in Food Preparation

Appendix II:

Appendix III:

271 

283  297 

and Storage –  A  A Global View  Methodologies for Bacillus cereus cereus Laboratory Investigations under the Supervision of Elias Hakalehto in 1993-1994 in the Rapid Microbial Detection Project 

315 

Biosafe Packaging of Samples for Transport 

325 

Index

311 

327 

 

 

PREFACE  This volume is the second in the  Microbiolo  Microbiological gical Hygiene  series. As a discipline,  Microbiological  Microbiol ogical Food Hygiene  is a closely related and integrated discipline with clinical hygiene, or health related microbiology, and discusses many interrelated topics. Food microbiological studies help in safeguarding one of the main entrances into the human body system and the digestive tract. Because food is produced on the basis of the circulation of substances in nature, there is a link between food and environmental microbiology. Most of our food is nowadays being produced by industrial processes. This underlines the importance of industrial hygiene maintenance and its link to the quality of our daily diets. The microbes in various foods, besides constituting potential health hazards, also take  part in the absorption absorption of food substances in the alimentary alimentary tract. Consequently, Consequently, the varieties varieties of micro-organisms, micro-organi sms, as well as their interactions in food materials, influence our health and well being. Different foods contain a variable population of microbes. Understanding their  behavior and distribution are the core functions of Microbiological Food Hygien Hygiene. e. This helps us in preventing disease and food poisonings. poisonings. This book presents practically related projects which focus on different sources of food and some of their microbes. As already initiated in “Microbial Clinical Hygiene,”  the molecular basis of different activities is highlighted, as well as the use of novel methods and tools in the overall control of the hygienic situation. This wide approach to microbiological food quality control is extended to include air, water, facilities, raw materials and personnel of food producing units. Also, the hygienic quality of packaging materials should be continuously monitored. Last but not least, ways of influencing microbiological and nutritional food quality should be jointly researched. Some ideas regarding this novel field are introduced. For this  purpose, chapters on food polyphenols polyphenols as well as the microbial contribution to food Selenium content have been included. In all cultures, food is a central role of everyday life. Different traditions have developed in various climates and geographical areas. In today´s world, increased international trade and tourism as well as immigration dissolve the borderlines between ethnic groups also with respect to food acquisition, preservation and preparation. Following the first two volumes, one on clinical hygiene and the other on food hygiene, this series of books on microbial hygiene will continue with another two books focusing on issues of industrial and environmental microbiology as well as hygiene. Many of the

 

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important microbial species and strains in industries or in the environment are the same as in the ones in our food or inside our bodies. These four disciplines interact with each other. The current occurrence of emerging bacterial and viral epidemics emphasizes the need for integrated microbiological control in all aspects life. Our actions as a species have to challenge many threats; these include viral ones such as Ebola, or bacterial hazards caused by antibiotic resistant strains of many bacteria, such as tuberculosis, salmonella and others. Also, less hazardous agents compromise our health and cause huge economic losses. Then, the most vulnerable individuals are at risk. Sincefood foodmicrobiology and health arewith interlinked, current trendsofincrease the need for developing modern a broadtheunderstanding basic microbiological factors related to our nutrition. These principles can be applied to both individual diets at home and to large institutional kitchens, catering operations and other related aspects. Microbes could  provide us with keys for future clean and healthy food supplies on this planet. Different geographical areas have their typical issues, besides the global ones. My sincere thanks are due to all the contributors of this volume as well as to Nova Science Publishers Inc. —   — the the publisher of the  Microbio  Microbiological logical Hygiene series — for for smooth and constructive cooperation. In Kuopio, Finland, 11th of March, 2015.  Elias Hakalehto, Hakalehto, Edit Editor  or   Department of Environmental Sciences

University of Eastern Finland P.O.B. 1627, FI-70211 Kuopio, Finland

 

 

INTRODUCTION  E lilia as H Ha akaleht lehto o Department of Environmen Environmental tal Sciences University of Eastern Finland, Kuopio, Finland

Food is our source of nutrition. Our body system acquires chemical energy for its billions of living cells via its principle material uptake route, the digestive system. Moreover, food  provides us with with building blocks blocks for anabolic metabolism. Microbes, in turn, are omnipresent in nature. They occupy most food substances, and numerous fermented foods are prepared by using them. The micro-organisms have many metabolic activities which may also be used for food preservation purposes. However, occurrence of unwanted microbes in foods can cause spoilage, toxication or dissemination of contagious diseases. The intestinal microflora takes part in food processing and nutrient uptake (Hakalehto, 2012; 2015a). Its composition and functions resemble that of human organs. Therefore, it has been designated as an “alimentary microbiome” by Joshua Lederberg (2000). This illustrates the close interaction of microbiota and its human host, which could be considered as a symbiotic relationship. This has been proven by the stabile nature of the infantile flora which forms the basis for a lifelong metabolic and immunogenic cooperation from the viewpoint of man (Pesola & Hakalehto, 2011). After food uptake in the upper GI tract, the colonic microflora takes part in the waste outlet of the body system. Usually, this microbial community is considered non-hygienic, disgusting and most destructive from the health point of view. Consequently, great care is taken to keep colonic microflora away from all food and raw water sources. The environmental strain on food sources also consists of many other contaminants: climate oriented factors, water circulation, man-made emission and other origins of microbes that may cause a challenge or threat for human bodily defenses. Thus, food microbiological hygiene should — and and has been developed —  to   to safeguard our health and nutrition against the risks that evolve from our environment and that pose a threat to us in the form of our food materials. Besides actual hazards or dangers, poor microbiological quality of foods may cause intestinal imbalances, allergies, lack of trace elements or minerals, and several other malfunctions. It has to be taken into account that the most advantageous food microbe content is not always the most deprived. For example, probiotic microbes in foods or supplements can

 

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 preserve food quality, may prevent diseases, complete the diet and ease intestinal complications (Hakalehto et al., 2015). Many human diseases are so-called “zoonosis,” which spread between man and animals (Armon and Cheruti, 2012). Therefore, the monitoring of food cleanliness has to be based on the careful consideration of the risks of various zoonotic illnesses. In food materials, microbes use all available opportunities to spread and distribute. This takes place under metabolic control and is facilitated also by interspecies interaction. Microorganisms form biofilms which have an elaborated structure and make the diffusion of substances possible. These structures form channels or hollow constitutions that originate from the fibrous formations of bacterial or other microbial cells and are available for liquid movement (Hakalehto, 2015b). Full insight of food hygienic principles requires knowledge and information about the many raw materials, their combinations, storage, distribution, preparation of food and meals, and catering, and overall microbiological context of the consumer, consumer, his body system, digestion, normal flora and environment. Due to the complexity of the situation in manufacturing and trading of foods, certain practices, such as HACCP (Hazard Analysis and Critical Control Points), have been established for keeping the hygienic standards. Additionally, continuous development of methods and equipment is a necessity for eliminating risks related to food  production. Besides the actual pathogens, several toxigenic functions are associated with food spoilage. Many microbes are classified as food spoilers and contaminators which compromises food safety and quality. These include salmonellae, campylobacteria, aerobic  bacilli, staphylococci, many strict anaerobes and numerous other bacterial groups, as well as molds, yeasts and viruses. A clear distinction has to be made between malicious and  beneficial food microbes, keeping in mind that many organisms can act in both roles or  belong to the commensal flora (Hakalehto, 2013). Also, many opportunistic food pathogens or spoilage organisms do occur. The monitoring of food microbial content and communities as well as raw material and  product hygiene is under continuous development. development. Historical steps after the Petri dish have  been. hygienic ATP (Adenosine (Adenosine triphosphate) determination, fluorescent dyes, chromogenic media, immunological and genetic methods, gas chromatography and mass spectrometry, nucleic magnetic resonance and many other techniques. Whatever final detection and identification tool is selected, it can be boosted with automation, careful sample preparation, and the design of sampling strategy. The issues of food hygiene can be crystallized into three situational questions: 1.  How can we guarantee that sixteen tons of nuts in a port are free from salmonellas? 2.  When will the liter of packed pasteurized milk in my refrigerator go bad, and which microbes cause its spoiling? 3.  What impact does the eating of this or that food item have on gut microflora? microflora? Searching for answers to these questions will eventually lead to the key points of food microbiology. In the practical investigations, source-tracking of contaminations can give valuable information and help in avoiding future risks or mistakes.

 

Introduction

xi

From the consumer’s point of view, it is crucial to monitor the entire food chain from raw

material production to the table. Maintaining the cold chain has a key position in food  protection. In our modern societies, the demand for healthy nutrition is increasing. This prerequisite for decent living should be provided for all. This does not mean only that, quantitatively, enough food should be produced, but it casts a focus on the nutritional qualities and cleanliness of food, also. Microbes are involved in all food chains. Therefore, investigations on their very existence, interactions and metabolic capabilities could provide us with tools for increasing our understanding on the linkages between mankind and the planet we live on. After all, we belong to our “global village,”  as Marshall McLuhan stated in the early sixties (1964). During the era of the intensive electronic transfer and storage of ideas, the transformation of goods and food products has also intensified. Movements of microbial strains have become global not only by the climatological climat ological or ecological causes, but also by the man himself.

FOOD FOR HEALING  Corresponding to any cream or liniment which calms our skin, food is an ointment for our epithelia. It protects the body system, flushes away metabolic wastes, and nourishes the cells and our inner organs. These influences of healthy food are achieved by the contribution of microbes which inhabit the alimentary tract. It is with them that our body has learned to cometabolize, interact and cooperate. In fact, prior to nutrient uptake, the food is processed into chyme where the intestinal microbes actively process it. To remain healthy, we need a functioning relationship with our gut flora. This also keeps out the potentially harmful organisms. In food hygienic measures, the microbiological and  biochemical stress from from outside sources should be kept kept below bearable bearable limits. During recovery from various diseases, the body system needs replacement for lost reserves, as well as additional vitamins, antioxidants and trace elements for returning back to the normal state. It is of essential importance that an adequate supply of the full scale of nutritional factors is made available. Otherwise the health of the individual could be  permanently harmed or compromised. compromised. Our forefathers were hunter-gatherers. They had to catch their proteins from the lakes and the forests. They also had to learn the clean preparation of their food serves, as well as the  preservation of them in their commonplace, everyday setting. Products from the plant kingdom, such as fruits and berries, contain useful vitamins and antioxidants (Figure 1). It is often taught that refrigeration below a temperature of +8 Celsius degrees keeps food reasonably well preserved. However, there are psychrophilic organisms capable of producing growth also in colder environments. One particular organism of this kind is aerobic sporeforming  Bacillus cereus. Besides being a known spoiling agent of refrigerated milk, it is also causing so called fried rice syndrome (Gilbert et al., 1974). Thus, this bacterium is able to withstand the thermal extremes of food preservation and processing rather well, and consequently propagates in them. It is characteristically an environmental microbe, e.g., the rice fields (Figure 2).

 

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Figure 1. Lingonberries contain relatively high concentrations of benzoic acid, a known preservative. It can also give the foods health promoting qualities, together with flavonoid substances. Photo: Vicente Serra.

Figure 2. Universal distribution and robust cell structure with rapid growth capabilities have made B. cereus a widely spread, food spoiling organism. It can originate from rice fields, which produce food from the borderlines of land, water and air. This cropland is from Japan. These fields contain nitrogen fixing bacteria which recycle atmospheric nitrogen into plant proteins and tissues. Circulation of elements has thus become an important part of human nutrition. Native farmers in the rice-growing areas have learnt to cultivate their crops in close understanding of their nature, on the basis of traditional methods and knowledge of local conditions (Gladwell, 2008). Photo: Jukka-Pekka Hakalehto.

Since microbes naturally belong to the food chain — as as well as to the digestion and food uptake as member strains of alimentary microbiome — their their contribution to our health and well-being should not be overlooked. In our body system, where the food particles are

 

Introduction

xiii

exploited, the intestinal microbiota takes part in metabolizing the various substances of the  biochemical pathways. pathways. This metabolism related to host food uptak uptakee takes place particularly in

the duodenum and the subsequent part of the small intestine (Hakalehto et al., 2008, 2010). It takes about 6-7 hours for the degrading food mass to pass through the small bowel, during which 80% of the nutrients are digested by hydrolysis, subsequent microbial processing and nutrient uptake by the host. However, most stages of the surprisingly fast digestion process happen with the help of gastric acid and enzymes, and pancreatic and intestinal enzymes of human origin. These host activities are under hormonal control. Many microbial strains also have hormone-like regulators that also influence the host metabolism. This integration and unity of metabolic activities in various cells and tissues has led scientific thinking into the direction which implies that nervous control is actually under a regulatory system partially independent and distinct from the CNS (central nervous system) and the spine-mediated neuronal control (Brookes & Costa, 2012). On the level of different cells and groups of cells, it is fascinating to see the unity of all life in the metabolic sense (Kluyver & van Niel, 1956). Therefore, it can very well be said that food energizes our very being in the form of an energy flow and uploading energy storages. It is also the source of various molecular building blocks. Microbes are, in a way, “hitchhikers.”   On their way through the alimentary tract, they exploit their share of the nutrients. At the same time, they contribute to the host health by  processing the food. It is one of our duties as microbiologists to remain on guard in order to guide this influence into a positive direction. Clean food and water are prerequisites for healthy living. Once we have learned more about the ways the microbes take part in the converting of food sources into our diet, we can  better master the processes processes of hygienic hygienic protection and healing healing phenomena. phenomena.

EFERENCES  R EFERENCES

Armon,  R.; Cheruti, U. Environmental Aspects of Zoonotic Diseases.  IWA Press, pp. 492; Armon,  2012. Brookes, S. J.H.; Costa, M. Cellular organisation of the mammalian enteric nervous system. In: Brookes, S. J. H.; Costa, M (eds).  Innervation of the gastrointest gastrointestinal inal tract. Taylor and Frances, London & New York, pp 393-467; 2002. Gilbert, R.J.; Stringer, M.F.; Peace, T.C. The survival and growth of Bacillus cereus in boiled and fried rice in relation to outbreaks of food poisoning.  J Hyg (Lond); 1974, 73, 433 –  444. Gladwell, M. Outliers: The story of success. Little, Brown and Company, Boston, USA; 2008. Hakalehto, E. (Ed.):  Alimentary Microbiome - a PMEU approach. Nova Science Publishers, Inc., New York, NY, USA,; 2012. Hakalehto, E. Interactions of  Klebsiell  Klebsiella a sp. with other intestinal flora. In Pereira, L.A. & Santos, A. (eds.)  Klebsiella infectio infections: ns: Epidemiology, Epidemiology, pathogenesi pathogenesiss and clinical outcomes. Nova Science Publishers, Inc. New York, USA; 2013. Hakalehto, E. Microbes and human digestive system.. In: Hakalehto, E. (Ed.):  Microbiological  Microbiol ogical Clinical Hygiene. Nova Science Publishers, Inc., New York, NY, USA,  pp. 219-258; 219-258; 2015a.

 

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Hakalehto, E. Structure and function of bacteria in contact with host. In: Hakalehto, E. (Ed.):  Microbiological  Microbiol ogical Clinical Hygiene. Nova Science Publishers, Inc., New York, NY, USA,  pp. 15-34; 2015b.

Hakalehto, E.; Humppi, T.; Paakkanen, H. Dualistic acidic and neutral glucose fermentation  balance in small intestine: intestine: Simulation in vitro. Pathophys  Pathophysiology; iology; 2008, 15, 211-220. Hakalehto, E.; Hell, M.; Bernhofer, C.; Heitto, A.; Pesola, J.; Humppi, T.; Paakkanen, H. Growth and gaseous emissions of pure and mixed small intestinal bacterial cultures: Effects of bile and vancomycin. Pathophysi  Pathophysiology ology; 2010, 17, 45-53. Hakalehto, E.; Jaakkola, K.; Pesola, J.; Heitto, A; Hell, M.; Hänninen, O. Tendencies in  Microbiological ogical Clinical Hygiene.  Nova  probiotic treatments.. In: Hakalehto, E. (Ed.):  Microbiol Science Publishers, Inc., New York, NY, USA, pp. 301-322; 2015. Kluyver, A.J.; van Niel, C.B. (eds.). The Microbes’s Contribution to Biology.  Harvard University Press, Cambridge, Massachusetts, USA ; 1956. Lederberg, J. Infectious history. Science, 2000; 288, 287-293. Mcluhan, M. Understanding media: The extensions of man. McGraw-Hill, New York, USA; 1964. Pesola, J; Hakalehto, E. Enterobacterial microflora in infancy - a case study with enhanced enrichment. Indian J Pediatr, Pediatr, 2011; 78, 562-568.

 

In: Microbiological Microbiolo gical Food Hygiene Editor: Eino Elias Hakalehto

ISBN: 978-1-63483-646-3 © 2015 Nova Science Publishers, Inc.

Chapter 1

MICROBIAL PRESENCE IN FOODS AND IN THEIR DIGESTION  E lilia as H Ha akaleht lehto o Department of Environmen Environmental tal Sciences University of Eastern Finland, Kuopio, Finland

ABSTRACT  Mankind has always utilized the raw materials from animal and plant kingdoms for establishing and maintaining healthy nutrition. Besides meat, animals provide us with e.g., milk and eggs. Many vegetables, meats and milk can be used for manufacturing various fermented foods with beneficial microbes. Several other means for preservation or food preparation can be used for controlling and adjusting the environmental (or incoming) microbes of the food sources. Our intestinal microflora is the ultimate barrier, which functions in cooperation with our defenses and other body functions. Interfaces  between different physiochemical phases, or in between various organisms, or in the  border zone of two ecosystems, these tend to form areas of elevated biochemical activity.

1. INTRODUCTION  The filamentous bacteria belonging to e.g.,  Actinomycete  Actinomycetess as well as fungi carry out vast numbers of enzymatic reactions in the soil and in the proximity of the plant roots. It could be  postulated that zones of enhanced hydrolytic hydrolytic activity are formed around the hairy roots of many plants. It is in these zones, where the fiercest competition between various microbial species occurs. In the soil this is a natural phenomenon. It is most likely that in these areas also high numbers of antimicrobial substances are being produced as a consequence of interspecies battle for space and nutrients (the latter of which are to some extent exhausted near the plant roots). On the other hand, the plants secrete there various substances. The plant root surfaces are also borderlines between the so called scattered and enhanced microbial ecosystems (Hakalehto, 2012a). Without much direct scientific evidence, it could then be anticipated that the soil matrix in poorly washed vegetables could constitute some kind of risk

 

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for spreading of the antibiotic resistance markers into the foods. The corresponding strains are enriched in the food production, our body system, or in the wastes, provided that there the selection is prevalent in the form of the antimicrobial substances e.g. , ,  in medicines. Soil ecosystems form a reservoir of the antibiotic resistant strains.

Table 1. Different categories of microbial ecosystem according to Hakalehto (2012a)

Type Scattered ecosystem

Nutrients Scarce and dispersed

Diffusion limitation Present

Concentrated ecosystem Enhanced ecosystem

Highly abundant

Present

Occasionally highly abundant Variable

Removed

Resting state ecosystem

Insignificant

Example Common soil habitat Manure Duodenal epithelia Hot or salt spring

In the human body, the micro-organisms of the digestive tract take part in the rapid degradation of food substances into more exploitable biochemical form for the body system. The very same degradation process takes place both in the slaughterhouse waste and even in the unfrozen lakeside mud sediment under a snow belt in Finland (Hakalehto, 2015a). In these cases local microflora produces organic acids and alcohols, which are further reduced to aliphatic compounds, whichand potentially can be used regarding to combustible gas (Schwedevolatile et al., 2015). Further ideas hygienic considerations chickenliquid abattoirs and other slaughterhouses are illustrated in the project reports of the six nation European Union Baltic Sea Region ABOWE Project (see www.abowe.eu) (Figure 1). The analogies as well as the distinctive features of soil or biorefinery microbial ecosystems with the human digestion give ideas about the microbial degradation in general.

Figure 1. Uses of chicken (Hakalehto et al., 2015a).

 

Microbial Presence in Foods and in Their Digestion

3

2. ASPECTS OF HYGIENIC INDICATIONS  Some bacterial species and strains have been selected to provide the so called hygienic indications (Heitto et al., 2012; Hakalehto, 2015a). The most important strains belong to the

enterobacterial group, or  Enterobacteri  Enterobacteriaceae aceae, especially to the fecal coliforms. Besides these small rod-shaped gram negative organisms, fecal enterococci are also often used as basis for hygienic evaluations. The rapid detection of the former ones could be facilitated by specific cultivation media, such as Colilert™  (Idexx Corporation, Maine, USA), automated sample collection system (ASCS) (Finnoflag Oy and Samplion Oy, Finland), and optimal growth conditions (Wirtanen and Salo, 2010; Hakalehto et al., 2013). Also a viable method for the detection of enterococci and other additional indicator groups could be facilitated by the PMEU (Portable Microbe Enrichment Unit). Even though the microbial hygiene indicators could potentially give a sign of fecal contamination as described above, they may originate from various other sources as well. For example, paper and pulp industries enrich considerably the presence of hygienic indicators and many other frequent contaminants in their water circulation systems (Mentu et al., 2009). In nature, the human impact on microbial communities is, however, not restricted to the elevated levels of intestinal bacteria only (Hakalehto, 2015a). Different authropogenic activities related to housing, industries, agriculture and forestry, fisheries, different land use  purposes etc. produce produce significant changes in the natural natural microbial ecosystems. ecosystems. The hygiene indicator species are most often used as markers of lowered water or food quality. Their methods use hasinherently: to be considered in the light of many additional aspects. The corresponding 1.  are giving a limited view on the microbiological/bacteriological reality 2.  are not always a sure sign of fecal contamination 3.  seldom give a direct proof of the hazardous pathogens themselves However, since the presence of fecal indicators is a warning sign, which indicates a risk of true contamination by fecal strains and/or serious pathogens, it should be taken seriously. However, in some cases also direct monitoring of microbial pathogens is recommendable (Hakalehto and Heitto, 2012). It should be remembered that a food contamination is not usually limited to a hygienic problem of food only, but relates to some other of the following fields:                 

hygiene of animal husbandry and feed agricultural crop hygiene food processing hygiene  biology of of the buildings water hygiene air hygiene clinical hygiene environmental environm ental microbiology

 

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All these disciplines have to be taken into account, when food microbiological cases are under investigation. For instance, in Burkina Faso in West Africa, a hygiene problem of irrigation waters was bringing up an issue of chicken meat contaminated by Campylobacter   sp. in local markets (Hakalehto et al., 2014a). The same strains or subspecies were then identified as disease-causing organisms in a nearby pediatric hospital.

3. CAMPYLOBACTERIAL, CLOSTRIDIAL, ENTEROBACTERIAL AND MYCOBACTERIAL GROWTH BOOSTED WITH CO2  Since bacterial pathogens belonging of the genus Campylobacter  are  are common c ommon occasional contaminants in various foods manufactured from the corresponding meat or other parts of the chicken, their presence should be carefully monitored. In Tokyo, Japan, there is a restaurant called “Matsumoto,” where all fragments of slaughtered chicken are exploited in  preparing tasty food varieties. See also the internet blog: blog.livedoor.jp/ blog.livedoor.jp/ iessi/archives/ 22191912.html. In such situations related to food preparation, contaminants like the campylobacteria or salmonellae need to be prevented from causing intestinal or other diseases. The required high-standard cooking is a part of cultural tradition in many countries. In case of accelerated growth and detection of Campylobacter coli  and C. jejuni in the PMEU (Portable Microbe Enrichment Unit), this method worked out in a more sensitive, fast and effective manner when compared to the international standard method (Pitkänen et al., 2009). During these experiences of natural and spiked samples by gas the flow Finnish  National Institute of Health and Welfare, the potential of using the conducted microaerobic for growth promotion was fully exploited. The final cell concentration of campylobacteria was 10-100 fold in comparison with the standard culture method. Consequently, the bacterial detection took place in about 10 hours shorter time. In case of treating the chicken waste, various hygienic problems could be supposed to  play a significant role. However, However, during our experimentation experimentation in the EU Baltic Sea region ABOWE project (Advanced concepts for the biological utilization of waste), the hygienization of chicken slaughterhouse waste and manure was successfully carried out in the Pilot A experimental station in Enköping, Sweden (Anderson et al., 2015; Schwede et al., 2015). The ABOWE Pilot A had been designed by the author on the basis of preceding Finnoflag Oy project experiences, and the concept exploited the natural microbes fortified by specific production organisms, such as  sp. In practice, the mixed cultures were treated with gas flows containing 50 Clostridium - 100% CO 2. The various clostridial strains can withstand high CO2 concentrations (Hell et al., 2010). They can even get significantly boosted  by carbon dioxide, as was the case with Clostridium butyricum  (Hakalehto and Hänninen, 2012). In these experiments the onset of bacterial growth was speeded up by many hours, whereas lack of CO2  in the carrier gas resulted in growth inactivation (Hakalehto, 2015c). This is an interesting phenomenon, since CO2 is usually considered as a disinfection agent, too (Garcia-Conzalez et al., 2007). Food derived contaminations by  E. coli  and Staphylococcus aureus were inactivated in liquid culture with strong bactericidal effect by synergistic action of high hydrostatic pressure and dissolved CO2  (Wang et al., 2010). Also fruit juices were pasteurized with this same

 

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approach, combining the pressure and CO 2  effect for the inactivation of  E. coli,  Listeria innocua and Lactobacil  Lactobacillus lus pla plantarum ntarum (Li et al., 2012). In case of removing contaminating  E. coli from water, more than 5.0-log reduction was obtained by the CO2 (Vo et al., 2013). For comparison, disinfection by bubbling N2O or N2  under optimal conditions (0,7 MPa with exposure time of 23 min) produced 3.3-log and 2.4-

log reductions, respectively. However, in the PMEU less intense CO2  flow was shown to  promote bacterial onset of growth (Hakalehto, 2011; Hakalehto and Hänninen, 2012; Hakalehto, 2013a; Hakalehto et al., 2013). Also the N 2  can be used for the acceleration of  bacterial growth in the PMEU. P MEU. Then the gas flow is mixing up nutrients and facilitates their  proper diffusion diffusion in the process process broth (Hakalehto, (Hakalehto, 2015c). In natural microbial communities, it seems to be evident that the volatile substances of some microbial cells can boost the growth of some others. Human digestive tract is one of the most obvious set of niches, where these phenomena do occur, and where they have a significant effect on the microbial ecosystems (Hakalehto. 2012a; 2015b) This was documented in a case of  Lactobacill  Lactobacillus us brevis boosting C. butyricum in an experiment where PMEU (Portable Microbe Enrichment Unit) enrichment and cultivation syringes were connected in series (Hakalehto and Hänninen, 2012). The CO2 liberating from the preceding syringe could activate the growth in the following one. This illustrates the course of events in silage feed, or in the cecum, or even during food spoilage. Once any type of microbial activity is producing CO2, this can accelerate the spoilage (or activate other strains).

Figure 2. Common routes of contamination of e.g. , , campylobacteria, salmonellae and mycobacteria from chicken farms into human consumption. Source-tracking Source-tracking of infections and contaminations is essential for the eradication of any hygienic problems.

 

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During silage production, the propionibacteria have been used for improving the quality and stability of the animal feed (Dawson et al., 1998). In the distal large intestines, these  bacteria contribute to the the final digestion of food residues residues (Hakalehto, 2015b). 2015b). In pure cultures of e.g.,  Escherichia coli and  Klebsiell  Klebsiella a sp., the boosting effect of carbon dioxide bubbling has been measured (Hakalehto, 2011, 2013a; Hakalehto et al., 2013). These hygienic indicators are not necessarily pathogens but merely belong to the normal microbiota

since our early childhood (Pesola et al., 2009). Similar methods have turned out valuable in the detection of mycobacterial contamination where the pathogens or environmental contaminants in enrichment cultures could be drastically speeded up by the PMEU technology (Hakalehto, 2013b; Hakalehto et al., 2014b). This offers potential benefits in the detection of e.g. , , M. tuberculosis (Hakalehto, 2013b) or M. avium-paratubercu avium-paratuberculosis losis, the latter of which is a potential spoilage organism and  pathogen in milk and meat meat products (Eltholt et al., 2009). See Figure 2 for the po potential tential routes for the spreading of infections.

4. HYGIENIC MAINTENANCE WITH WORK PRACTICES  In studies on many of the common routes or vehicles of pathogenic or antibiotic resistant strain distribution the hazardous organisms themselves should be daily monitored. Their  presence is as such a natural phenomenon, but is often scaled up by the modern methods of industrial food production. Consequently, the HACCP (Hazard Analysis and Critical Control Points) approach (ICMSF, 1980) has been developed in the meat industries in the USA where vast amounts of hygienically risky substances are transported and processed as a flow of refined animal-based new materials (Tompkin, 1990). The animal or meat production in huge industrial production units brings many of the consequences of the hygienic protection on the table (Clayton, 1967). For example, from the industrial point of view antibiotic medications and vaccinations can sometimes even be seen as elements of the hygienic maintenance. However, the former are highly controversial due to the risks of disseminating resistance (van den Bogaard et al., 2001). In fact, the prevalence of antibiotic resistant Salmonell Salmonella a enterica  and  E. coli  were 18% and 53%, respectively, at the supermarkets in Bangkok, Thailand already in 2001 (Chaisatit et al., 2012). In the ABOWE tests referred earlier in this chapter, the elevated temperatures during the  biomass hydrolysis eliminated most of the tedious contaminants. According to the microbiological analysis carried out in the Finnofl ag Oy’s laboratory in Siilinjärvi, Finland , the risk of any serious contamination could be avoided by this procedure. The elements of  process hygiene hygiene maintenance in the biorefinery biorefinery runs are consequently consequently the follow following: ing: 1.  elevated temperature and low pH during the biomass hydrolysis hydrolysis 2.  effective occasional distribution of gas flow containing ith high CO2 concentration into the process fluid or suspension 3.  application of nutrient-bed type of inoculation where the production bioreactor is heavily inoculated with desired production strain as a mixture with optimal nutrients

 

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During the experimentation with chicken waste it turned out that addition of trace elements or minerals could boost the desired bacterial (clostridial) growth. In practice, the anaerobic fermentation was boosted with blueberry juice rich in the micronutrients (Figure 3). The organic products from chicken waste indicate the potential of the conversion of food wastes into useful products. In this case, the natural microflora is kept under control by the dominance of the production strains and by adjusting the conditions accordingly. Food ecosystems consist of several basic elements:

         



original microbiota degradation final products down regulation ecological succession

Basics of healthy nutrition are (according to Hakalehto, 2012b): 1.  2.  3.  4. 

Adequate supply of food, vitamins and antioxidants Proper digestive hormone function Functionable human and microbial enzymes in food degradation Balanced GI microbiome (BIB, Bacteriological Intestinal Balance)

Figure 3. Effect of blueberry juice addition (vertical line on o n the right in both b oth figures) to the bacterial  production of ethanol (a.) and 2,3 -butanediol -butanediol (b.) during ABO ABOWE WE Pilot A experimentation experimentation with chicken waste (Anderson et al., 2015).

5. MILK IS THE PREMIUM NUTRIENT  The most important nutritional component of the human early development is usually  breast milk, which is the first source of of::        



energy carbon skeletons for anabolic metabolism minerals trace elements

 

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Elias Hakalehto   immunoglobulins   other defensive biomolecules



The composition of human breast milk as well that of cow, goat, sheep or any other animal milk used for human nutrition is an important factor contributing to the general health. The issues of dairy hygiene are further discussed in Chapter 8. Natural milks should remain free from toxic or otherwise harmful substances. Some parts of the adult population are

lactose intolerant, and several milk allergies can also occur. On the other hand, unprocessed milk contains numerous health promoting molecules. Many antimicrobials and immunoglobulins, immunoglobu lins, as well as anti-carcinogenic substructures can be found in unprocessed raw milk. Any medications in the early childhood, especially the antibiotics, may interfere with the formation of intestinal microflora and nutritional patterns (Pesola and Hakalehto, 2011). Fortunately, in most cases this interference is of a temporary nature. The concept “probiotic” refers to a microbe, which could positively contribut e to human health and well-being as a member of intestinal or other microflora (Hakalehto et al., 2012, 2015b). As food substituents probiotics are often combined with so called “prebiotics,” food substances that positively influence on the intestinal milieu (Hakalehto and Jaakkola, 2013). Most probiotic products contain lactic acid bacteria. This is a bacterial group generally considered as safe for human consumption. However, some other potential probiotics are already in the market. Since the human normal flora is developing to its full composition during the early childhood, the importance of milk and its derivatives on the formation of the alimentary microbiota various and balances in the digestive system in general as well as in food uptake are widely recognized (Hakalehto, 2012b). The orally administered probiotic lactobacillic strains have a tendency to disappear from the feces if they are not given to the test persons (Jacobsen et al., 1999). This illustrates the need for more or less incessant usage of the probiotic strains in the treatment of prevention of digestive disorders. It also implies to the fact that newcomer strains are not easily finding a niche in the gastrointestinal tract. On the contrary, with mouse model systems it was indicated that probiotic addition had multiple effects on the host-microbe metabolic functions (Martin et al., 2008). From this data it could  be deduced that even though the additions of probiotic bacteria into the gut ecosystems clearly has immediate effects on the host and the microbiome, it is difficult to trigger any  profound or long-lasting changes in the microbial content of the alimentary tract. Correspondingly, to repair the damages in the intestinal balances could be time consuming and challenging from the point of view of the treatment strategies. However in the case of a newborn baby, the effects of antibiotic medications on the enterobacterial flora took only several months to recover (Pesola and Hakalehto, 2011). For more grown up patients it could  be more difficult to re-establish the balanced intestinal microbiota. The metabolic profiling could offer means for screening up the effects of dietary supplementation of probiotics on the health status of the patients (Martin et al., 2008). For the long term surveillance of the components of the microflora, some immunological techniques could be developed (Hakalehto et al., 2007).

 

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6. MICROBIAL CONTRIBUTION TO HUMAN AND ANIMAL DIGESTION DURING THE INDIVIDUAL LIFE SPAN  Recently, human milk oligosaccharides (HMOs) have been suggested as prebiotics in order to modulate the growth of beneficial gut microbiota (Krausova et al., 2015), The in vitro  growth of six strains of lactobacilli in media containing purified HMOs obtained from breast milk. Based on the evaluation of bacterial densities in the growth media, pH values and

 bacterial metabolite detection, the authors concluded concluded that the lactobacilli tested did not appear to be active HMO consumers. In the case of four strains ( L.  fermentum,  L.  animalis and two strains of  L.  delbrueckii  subsp. bulgaricus), no increase in bacterial density was detected.  L. acidophilus and  L. casei  subsp.  paracasei) showed a slight, but insignificant Two strains ( L. increase in bacterial densities during 24 h of incubation. However, it has been shown earlier that the presence of the enzyme lacto-N-biose I phosphorylase was responsible for the cleavage of lacto-N-biose I which is an important component of the HMOs (Satoh et al.,   2013). This enzyme is possessed by the  Bifidoba  Bifidobacterium cterium bifidum and  B. langu langum m, two of the main bacterial strains that are important in the developing flora of the new-born infants already during their very first weeks and months. These strains were also the main components of the probiotic products which effectively attenuated the enterobacterial strains of human intestines when combined with flax seed prebiotic specifically manufactured and containing berry antioxidants (Hakalehto and Jaakkola, 2013). HMOs are the key selective factors in establishing the bifidobacterial microflora during the first periods of our lives (Kitaoke, 2012). The lactobacilli have been shown to boost the butyric acid bacteria by carbon dioxide (Hakalehto and Hänninen, 2012). Butyrate, in turn, is supposed to protect us from colonic cancer (Sengupta et al.,  2006). In Finland, where the consumption of fermented milk products is traditionally much higher than in most other European countries, the  propotional incidence of colorectal cancers is approximately approximately 25% less than in the rest of Europe (on the basis of Ferlay et al., 2013) Similar observations were in the background when the author of this chapter outlining in 1986 the strategies for Valio corporation to establish the  probiotic commercialization. This led to the purchasement of the GG (Goldin and Gorbach) strain to Finland from the USA a couple of years later. The statistical data behind the  probiotic usage enables enables hypoth hypothetical etical consideration on the microflora microflora development: 1.  human breast milk oligosaccharides select the bifidos ( Bifidobact  Bifidobacterium erium sp. strains) capable of utilizing these HMOs 2.  together with lactose fermenting lactobacilli they bind to the oligosaccharides, thus  preventing too early outgrowth of e.g., the enterics (members of the family  Enterobactericeae)  Enterobacterice ae)  3.  later on the enteries and other prevalent flora in the adult intestines is attenuated by the lactobasilli and bifidobacteria attached on plant fibers derived from the food 4.  the small intestinal microflora influence positively on the cecal and colonic flora, e.g., by favouring the butyric acid bacteria Since the small intestines is the major site for nutrient absorption, documented probiotic health effect relate most essentially to food uptake and to the Bacterial Intestinal Balance (BIB) are introduced by Hakalehto (2012b). This multitude of irreplaceable tasks lead to

 

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vulnerability if the small and large bowels cease to function in a healthy manner: the intestinal link could explain the dissemination of some zoonotic diseases, on one hand, and the onset of septic infection of vulnerable patients on the other. Also the Irritable Bowel Sydrome (IBS) and Small Intestinal Bacterial Overgrowth can be associated with microbiological imbalances inside the small bowel. The duodenal tract is a crucial area both in the protection against infections and in healthy nutrition (Hakalehto et al., 2008, 2010). The retention of food-derived chyme in different parts of the intestines, with some digestive microbiological and biochemical events is illustrated in Figure 4.

Figure 4. Some basics of the BIB (Bacteriological Intestinal Balance) according to PMEU studies (drawing by Ronja Hakalehto). This is a reproduction from Hakalehto 2015b. The elapsed periods of time from the food intake are rough estimates.

The adult burbot fishes in the Finnish lakes feed mainly on smaller fishes (Figure 5). They lay their eggs in winter time. The main method for their fishing is under ice nets (Figure 6). In Finland burbot roe is sold as caviar as a seasonal delicacy. This fish is known also as an ingredient for winter fish soup. As juveniles the fishes are active at night, but take shelter under rocks and among debris or sediment during the daytime, feeding on plankton and amphipods. The burbots tolerate an array of substrate types, including mud, sand, rubble,

 

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 boulder, silt, and gravel, for feeding. For example, in Michigan they can be found in deep cold lakes and large cool rivers (http://www.michigandnr.com/PUBLICATIONS/PDFS/ ifr/ifrlibra/special/ reports/sr37/SR37_app02_p reports/sr37/SR37_app02_pp108_thru p108_thru_119.pdf _119.pdf). ). The digestive organs and immune system of the burbots is compatible with the feed type these fishes consume. Interestingly, this is seen in some physiological and anatomical features. For example, in the proximal part of the intestines, the hydrolysis of fats occurs in the pyloric caeca (Izvekova et al.,   2013). Therefore the fat gradient is the highest in this  proximal section, whereas the concentrations of degraded proteins and carbohydrates carbohydrates grow into the opposite direction. The parts of the burbot inner organs responsible for the fat

digestion seem to consist of numerous appendixes, possibly for the production and secretion of the lipolytic enzymes (Figure 7).

Figure 5. One edible cold water fish species is burbot. It is caught mostly in the wintertime, during the spawning season between December and March. Its benthic ecological niche locates at the lowest level of a body of water in a lake, including the sediment surface and some sub-surface layers. The burbots tolerate many substrate types, including mud, sand, rubble, boulder, silt, and gravel, for feeding  platforms. Photo: Maretarium, Maretarium, Kotka, Finland / Petri Päiväri Päivärinta. nta.

Figure 6. Burbots and other lake fishes are often caught by nets which have been laid under ice in the wintertime in Finland. The microbes which accommodate any food raw material originate from the natural surroundings of a plant or an animal used as this food source, such as the burbots.

 

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Figure 7. The intestines of the burbot contain ramified branches probably related to the feeding habits and nutrient hygiene of this benthic fish (see text). Intestinal protrusions in the pyloric caeca are indicated by an arrow. This anatomic structure reflects the feeding habits of this particular fish.

The example of the burbot digestion is corresponding to the high importance of the upper intestines in the human digestion (Hakalehto et al.,  2008). It is this part of the gut system that also receives the microbiological and digestible food load from the stomach. Therefore, the human duodenal tract could be well called as the "heart of our digestive system.” There the nutrient uptake and stabilization of the incoming microbes in the food actually starts, following the gastric acidification and initial enzymatic hydrolysis. In the duodenum the pH of the food mass, or chyme, is adjusted to 6 by pancreatic bicarbonates. Many cycles of elements are influential in natural ecosystems. They are kept circulating  by the microbiological microbiological activities (Hakalehto, 2015a). The same cycles take part in regulating industrial bioprocesses, and have to be taken into account in waste management (Hakalehto, 2015c). In fish GI tract many cellulolytic bacteria have been found in the 16 S rRNA survey of the microbiota of such species as catfish ( Panaque  Panaque nigrolin nigrolineatus eatus), for instance (Watts et al., 2013). The bacterial genera include Clostridium , Cellulomonas,  Bacteroides,  Eubacterium  and  Aeromonas. It seems evident that the wood – eating eating catfish can accommodate a cellulosedegrading microbial community in its digestive tract. Consequently, there is a direct link  between the preferred nutrition of an organism to the intestinal flora, and further to the microbes emitted by it to the environment. However, in these different surroundings, the microbes live in a variable ecosystemic setting. In the GI tract nutrients are relatively well available, whereas in the environment they are less uniformly achievable. Therefore, the basic nature of the microbial ecosystem in the former is enhanced, and in the latter scattered one (Hakalehto, 2012b). See also Table 1. The interdependence of food microbiota on the natural microflora is schematized in Figure 8. This over-simplification is illustrating the food chains with transfer of microbiological load (expressed by arrows).

 

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Figure 8. Beyond the big picture on ecosystem level interactions, each type of organisms have their typical microbiological diseases, which depend on the virulence factors in case of plant pathogens (Salmond, 1994). On the contrary, animals and man often face or share the same pathogens between them. The diseases caused by them are called zoonoses, zoonos es, and they are introduced elsewhere in this book (see Chapters 3 and 12).

CONCLUSION  Any food material carries the inherent microbes in it. During the preparation of meals, or in the manufacturing of food products, this microbiological load is under control and adjustment. If the hygienic maintenance of the chain of events starting from the acquisition and selection of raw materials is failing at some point, this could cause food spoilage,  poisoning, or disease. Many malfunctions of the intestinal microbial communities can lead to rapidly occurring health consequences. On the other hand, also long term effects of the unfavourable microflora microflora composition and function may also lead to disastrous effects, such as carcinogenesis. Our digestive system is at forefront and is contacting with the outside microbes, and in our intestines a rich microflora is interacting with our bodily defenses in order to maintain the balance (BIB, Bacteriological Intestinal Balance). The stability of this system is a basic element of our general health. Food preservation, processing and preparation  practices have developed in various cultural and historical backgrounds into a reservoir of hygienic knowledge. This information is worth using together with scientific research and reasoning for healthy and clean nutrition.

R EFERENCES EFERENCES Anderson, H.; Thorin, E.; Lindmark, J.; Schwede, S.; Jansson, J.; Suhonen, A.; Jääskeläinen, A.; Reijonen, T.; Laatikainen, R.; Heitto, A.; Hakalehto, E. Technical Output Report  –   Pilot A in Sweden. www.abowe.eu; 2015.

 

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van den Bogaard, A.E.; London, N.; Driasson, C.; Stobbaringh, E.E. Antibiotic resistance of faecal  Escherichia coli in poultry, poultry farmers and poultry slaughters.  J. Antimicrob. Chemother.; 2001, 47, 763-771. Carcia-Conzalez, L.; Geeraerd, A.H.; Spiliunbergo, S.; Elst, K.; Van Ginneken, L.; Debevere, J.; Van Impe, J.F.; Devlieghere, F. High pressure carbon dioxide inactivation of microorganisms in foods: The past, the present and the future.  Int. J. Food. Microbiol.; Microbiol.;  2007, 117, 1-28. Chaisatit, C.; Tribuddharat, C.; Pulsrikarn, C.; Dejsirilert, S. Molecular characterization of antibiotic-resistant bacteria in contaminated chicken meat sold at supermarkets in Bangkok, Thailand. Jpn J. Infect Infect Dis.; 2012, 65, 527-534.

Clayton, E.S. The economics of the poultry industry. Longmans, Green, London, UK; 1967. Dawson, T.E.; Rust, S.R.; Yokoyama, M.T. Improved fermentation and aerobic stability of ensiled high moisture corn with the use of  Propionibac  Propionibacterium terium acidopropionici.  J. Dairy Sci.; 1998, 81, 1015-1021. Eltholt, M.M.; Marsh, V.R.; Van Winden, S.; Guitian, S.J. Contamination of food products with  Mycobacteriu  Mycobacterium m avium paratubercul paratuberculosis osis: a systematic review.  J. Appl. Microbi Microbiol. ol.; 2009, 107, 1061-1071. Ferlay, J.; Steliarova-Foucher, E.; Lortet-Tieulent, J.; Rosso, S.; Coebergh, J.W.W.; Comber, H.; Forman, D.; Bray, F. Cancer incidence and mortality patterns in Europe: Estimates for 40 countries in 2012. European Journal Journal of Cancer; 2013, 49, 1374-1403. Hakalehto, E. Simulation of enhanced growth and metabolism of intestinal Escherichia coli coli in the Portable Microbe Enrichment Unit (PMEU). In: Rogers MC, Peterson ND (eds.)  E. coli infections: causes, treatment and prevention.   New York, USA: Nova Science Publishers, pp.159-175; 2011. Hakalehto, E. Development of microbial ecosystems. In: Hakalehto, E. (Ed.):  Alimentary  Microbiome - a PMEU approach. approach. Nova Science Publishers, Inc., New York, NY, USA,  pp. 95-126; 95-126; 2012a. Hakalehto, E. (Ed.):  Alimentary Microbiome - a PMEU approach. Nova Science Publishers, Inc., New York, NY, USA,; 2012b. Hakalehto, E. Interactions of  Klebsiella sp. with other intestinal flora. In Pereira, L.A. and Santos, A. (eds.)  Klebsiella infectio infections: ns: Epidemiology, Epidemiology, pathogenesi pathogenesiss and clinical outcomes. Nova Science Publishers, Inc. New York, USA; 2013a. Hakalehto, E. Enhanced mycobacterial diagnostics in liquid medium by microaerobic bubble flow in Portable Microbe Enrichment Unit. Pathophysiol  Pathophysiology ogy; 2013b, 20: 177 – 180. 180. Hakalehto, E. Bacteriological indications of human activities in the ecosystems. In: Armon,  Environmental al indicato indicators rs. Springer, Dortdrecht, Germany; R. and Hänninen, O. (eds.)  Environment 2015a. Hakalehto, E. Microbes and human digestive system. In: Hakalehto, E. (Ed.): Microbiol  Microbiological ogical Clinical Hygiene.  Nova Science Publishers, Inc., New York, NY, USA, pp. 219-258; 2015b. Hakalehto, E., 2015. Enhanced microbial process in the sustainable fuel production. In: Boehm, R.F., Yang, H., Yan, J. (Eds.), (Eds.), 2015. Handbook Handbook of clean energy systems. John Wiley & Sons, Inc, Chichester, West Sussex, UK; 2015c. Hakalehto, E.; Heitto, L. Minute microbial levels detection in water samples by Portable Microbe Enrichment Unit Technology.  Environment and Natural Resources Research, Vol. 2 (4). Canadian Center of Science and Education; 2012.

 

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Hakalehto, E.; Hänninen, O. Gaseous CO 2  signal initiate growth of butyric acid producing Clostridium butyricum  both in pure culture and in mixed cultures with  Lactobacill  Lactobacillus us brevis. Can. J. Microbiol.; 2012, 58, 928-931. Hakalehto, E; Jaakkola, K. Synergistic effect of probiotics and prebiotic flax product on intestinal bacterial balance , Clinical Nutritio Nutrition; n; 2013, 32, S200. Hakalehto, E., Pesola, J., Heitto, L., Narvanen, A., Heitto, A. Aerobic and anaerobic growth modes and expression of type 1 fimbriae in Salmonella .  Pathophysiol  Pathophysiology, ogy, 2007; 14, 6169. Hakalehto, E.; Humppi, T.; Paakkanen, H. Dualistic acidic and neutral glucose fermentation  balance in small intestine: intestine: Simulation in vitro. Pathophys  Pathophysiology; iology; 2008, 15, 211-220. Hakalehto, E.; Hell, M.; Bernhofer, C.; Heitto, A.; Pesola, J.; Humppi, T.; Paakkanen, H.

Growth and gaseous emissions of pure and mixed small intestinal bacterial cultures: Effects of bile and vancomycin. Pathophysi  Pathophysiology ology; 2010, 17, 45-53. Hakalehto, E.; Hell, M.; Hänninen, O. What should a future probiotic be like? In: Hakalehto, E. (Ed.):  Alimentary microbiome - a PMEU approach.  Nova Science Publishers, Inc.,  New York, NY, USA, pp. pp. 233-245; 233-245; 2012. Hakalehto E.; Heitto A.; Heitto L. Fast coliform detection in portable microbe enrichment unit (PMEU) with Colilert(®) medium and bubbling.  Pathophysi  Pathophysiology ology; 2013, 20,257262. Hakalehto, E.; Nyholm, O.; Bonkoungou, I.J.O.; Kagambega, A.; Rissanen, K.; Heitto, A.; Barro, N.; Haukka, K. Development of microbiological field methodology for water and food-chain hygiene analysis of Campylobacter   spp. and Yersinia  spp. in Burkina Faso, West Africa. Pathophysio  Pathophysiology logy; 2014a, 21, 219-229. Hakalehto, E.; Heitto, A.; Heitto, L.; Rissanen, K.; Pesola, I.; Pesola, J. Enhanced recovery, enrichment and detection of  Mycobacteri  Mycobacterium um marinum  with the Portable Microbe Enrichment Unit (PMEU). Pathophysi  Pathophysiology ology; 2014b, 21, 231-235. Hakalehto, E.; Heitto, A.; Jääskeläinen, A. The Proof of Technology.  EU ABOWE Project  Report ; 2015a. www.abowe.eu Hakalehto, E.; Jaakkola, K.; Pesola, J.; Heitto, A; Hell, M.; Hänninen, O. Tendencies in  probiotic treatments.. In: Hakalehto, E. (Ed.):  Microbiol  Microbiological ogical Clinical Hygiene.  Nova Science Publishers, Inc., New York, NY, USA, pp. 301-322; 2015b. Izvekova, G.; Solovgev, M.M; Kashinskaya, E.N.; Izvekov, E.I. Variations in the activity of digestive enzymes along the intestine of the burbot ( Lota  Lota lota) expressed by different methods. Fish Physiol. Biochem.; 2013, 39, 1181-1193.  Heitto, L.; Heitto, A.; Hakalehto, E. Environmental monitoring using the enrichment of  Alimentary ary Microbiome - a PMEU hygienic indicators. In: Hakalehto, E. (Ed.):  Aliment approach. Nova Science Publishers, Inc., New York, NY, USA, pp. 215-231; 2012.

Hell, M.; Bernhofer, C.; Huhulescu, S.; Indra, A.; Allerberger, F.; Maass, M.; Hakalehto, E. How safe is colonoscope-reprocessing regarding Clostridium difficile spores? The th  Journal of Hospital Hospital IInfection nfection;2010, 76, 21-22, Abstracts, 8  International Congress of the Hospital Infection Society, 10-13 October 2010, Liverpool, UK. ICMSF (International Commission of Microbiological Specifications for Foods).  Microbial  Ecology of Foods.  Vol. 2. Food Commodities. Academic Press, New York, USA. 997 p; 1980. Jacobsen, C.N.; Rosenfeldt Nielsen, V.; Hayford, A.E.; Møller, P.L.; Michaelsen, K.F.; Paerregaard, A.; Sandström, B.; Tvede, M.; Jakobsen, M. Screening of probiotic

 

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activities of forty-seven strains of  Lactobacil  Lactobacillus lus sp. by in vitro techniques and evaluation of the colonization ability of five selected strains in humans.   Appl. Environ. Microbiol ..,, 1999; 65, 4949-4956. Kitaoka, M. Bifidobacterial enzymes involved in the metabolism of human milk oligosaccharides. Adv. Nutr.; 2012, 3, 422-429. Krausova, G.; Rada, V.; Marsik, P.; Musilova, S.; Svejstil, R.; Dreb, V.; Hyrslov Hyrslova, a, I.; Vlkova, E. Impact of purified human milk oligosaccharides as a sole carbon source on the growth of lactobacilli in in vitro model. African Journal Journal of Micr Microbiology obiology Resear Research ch; 2015, 9, 565571. Li, W.; Pan, J.; Xie, H.; Yang, Y.; Zhou, D.; Zhu, Z. Pasteurization of fruit juices of different  pH values by combined high hydrostatic pressure and carbon dioxide.  J. Food Prot.;

2012, 75, 1873-1877. Martin, F.P.; Wang, Y.; Sprenger, N.; Yap, I.K.; Lundstedt, T.; Lek, P.; Rezzi, S.; Ramadan, Z.; van Bladeren, P.; Fay, L.B.; Kochhar, S.; Lindon, J.C.; Holmes, E.; Nicholson, J.K. Probiotic modulation of symbiotic gut microbial-host metabolic interactions in a humanized microbiome mouse model.  Mol. Syst. Biol., 2008; 4: 157. doi: 10.1038/msb4100190. Mentu, J.V.; Heitto, L.; Keitel, H.V.; Hakalehto, E. Rapid Microbiological Control of Paper Machines with PMEU Method. Paperi ja Puu / Paper and Timber ; 2009, 91, 7-8 (90th  Anniversary). Pesola, J., Hakalehto, E. Enterobacterial microflora in infancy - a case study with enhanced enrichment. Indian J. Pediatr; Pediatr; 2011, 78, 562-568. Pesola, J.; Vaarala, O.; Heitto, A.; Hakalehto, E. Use of portable enrichment unit in rapid characterization of infantile intestinal enterobacterial microbiota.  Microb. Ecol. Health

 Dis.; 2009, 21, 203-210.

Pitkänen, T.; Bräcker, J.; Miettinen, I.; Heitto, A.; Pesola, J.; Hakalehto, E. Enhanced enrichment and detection of thermotolerant Campylobacter  species   species from water using the Portable Microbe Enrichment Unit (PMEU) and realtime PCR. Can. J. Microbiol.; 2009,  55, 849-858. Salmond, G.P.C. Secretion of extracellular virulence factors by plant pathogenic bacteria. In: Cook, R.J.; Zentmyer, G.A.; Shanes, G. (Eds.). Annual review of phytopathol phytopathology ogy. Annual Reviews Inc., Palo Alto, CA, USA, Vol 32. pp. 181-200; 1994. Satoh, T.; Odamaki, T; Namura, M.; Shimizu, T.; Iwatsuki, K.; Kitaoka, M.; Nishimoto, M.; Xiao, J. In vitro comparative evaluation of the impact of lacto-N-biose I, a major building  block of human milk oligosaccharides, on the fecal microbiota of formula-fed formula-fed infants.  Anaerobe

2013, 19,E.;50-57. Schwede,S.; ,Thorin, Lindmark, J.; Klintenberg,P.; Jääskeläinen, A.; Reijonen,T.; Laatikainen, R.; Heitto, A.; Hakalehto, E. Using slaughter house waste in a biochemical  based biorefinery -results from pilot scale tests. Manuscript. Manuscript. Sengupta, S.; Muir, J.G.; Gibson, B.R. Does butyrate protect from colorectal cancer?  J. Gastroenterol.. Hepatol.; 2006, 1, 209-218. Gastroenterol Tompkin, R.B. The use of HACCP in the production of meat and poultry products.  J. Food  Prot.; 1990, 9, 734-817. Wang, L.; Pan, J.; Xie, H.; Yang, Y.; Lin, C. Inactivation of Staphylococcus aureus  and  Escherichia coli by the synergistic action of high hydrostatic pressure and dissolved CO2.  Int. J. Food Microbiol Microbiol..; 2010, 144, 118-125.

 

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Watts, J.E.M; McDonald, R.; Daniel, R.; Schreier, H.J. Examonation of a culturable microbial  population from the gastrointestinal tract of the woal.eating locariid catfish ( Panaque  Panaque nigrolineatus). Diversity; 2013, 5, 641-656. Vo, H.T.; Imci, T.; Teeke, J.; Sekine, M.; Kanno, A.; Le, T.V.; Higwehi, T.; Phummala, K.; Yamamoto, K. Comparison of disinfectant effect of pressurized gases of CO 2, N2O and  N2 on Escherichia coli coli. Water Res. ; 2013, 47, 4286-4293. Wirtanen G,; Salo S. PMEU-laitteen validointi koliformeilla (Validation of the PMEU equipment with coliforms, in Finnish). 2010; Report  Report VTT-S-01705-10 VTT-S-01705-10, Statement VTT-S02231-10.

 

In: Microbiological Microbiolo gical Food Hygiene Editor: Eino Elias Hakalehto

ISBN: 978-1-63483-646-3 © 2015 Nova Science Publishers, Inc.

Chapter 2

TRENDS TOW TOWARD ARD CLEAN AND HEALTHY NUTRITION 

 Mii kko  M kk o I mmone nen n1 , J ukka-P ukka-Pe ekk kka a H akale kaleht hto o 2 and E lia li as H akale lehto hto 3  1

Department of Food and Environmental Sciences, University of Helsinki, Finland 2 Finnoflag Oy, Kuopio, Finland 3 Department of Environmental Sciences, University of Eastern Finland, Kuopio, Finland

ABSTRACT  Food safety is a factor that is complicated and desired worldwide as it determines our health andofwellbeing significantly. Health regarding safetydifferent can be assessed in terms microbiological, chemical and hazards nutritional quality.food Several kinds of  properties define whether food material is a suitable ground for microbes to propagate. One of the most important properties is water activity which is related to a product’s humidity as well as mobility of water in the material. Reduction in water activity leads to higher osmotic pressure and reduced microbial activity. Extensive use of preservatives and highly developed packaging methods has enabled longer shelf lives and further a greater variety in products. Maintaining a healthy balanced diet decreases risks of several common diseases, partly through the compounds formed in the metabolism of gut microbes.

1. INTRODUCTION  In the modern industrialized world, few people produce food for themselves. In fact, most of us are hardly at all connected to the origins of the food we are eating. Cultivating,  processing and distributing food relies increasingly on centralized food marketing chains  beginning with large monocultures in primary production and ending up into supermarket supermarket shelves. This is the prerequisite of producing enough food for big urban populations and also the reason, why the responsibility for food safety is not only the issue of consumer. Food safety enforcement can be roughly divided into three sections, microbiological, chemical and nutritional safety. They are all interlinked to each other. The consumer market is very sensitive to taking into account scientific information and also the current trends toward

 

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healthy diets and supplementations. These mainstream developments in our eating habits often pose specific needs to the hygiene surveillance and nutritional considerations.

2. TRADITIONAL FOOD PROCESSING  Over the history, several kinds of processing methods have been used to modify food materials for longer preservation. It is necessary for food to have adequate water content or humidity for microbes to maintain their metabolism, but an even more significant factor is water activity (aw), which describes the amount of free water not attached to water-binding molecules. These water related parameters of foods reflect on the hygienic, nutritional and culinary aspectsarriving of foodinto usage, well assystem. on the health contributions which the food varieties carry on when our as digestive “Microorganisms that are capable of growth   at reduced water activities all rely on a

common strategy for survival, i.e., the intracellular accumulation of a solute or solutes to  balance the external water activity (a w), thus preventing the mass movement of water out of the cell” (Hocking 1988). This reveals the relationship between water activity and osmotic  pressure. Because of their small size, swarming bacterial cells can protect themselves by the above-mentioned means. In biofilms, in turn, the outermost cells may form a borderline, which compromises the unfavorable environmental conditions. Brownian motion of water molecules affects the active movement of bacteria near surfaces (Li et al., 2008). The  purpose-oriented  purpose-or iented structure and function of the flagellar molecular arrangements reflects the complexity found in these microscopic “motors” (Hakalehto et al., 1997). The microbial cells   navigate in the fluidic environment environment existing in the foods.

Figure 1. Key factors affecting microbial activity in the product.

 

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Figure 2. Half-split “Kalakukko” (“Chicken rooster” in English). A traditional food  from East Finland. Fish and other ingredients are baked inside the rye bread which makes them sterile and gives

 preservation time as long as the cover is intact. Photo by Vicente Serra, City of Kuopio.

Some of the common processing methods disturb these basic mechanisms. Properties affecting microbial growth in foods depend on them. Water activity (aw), relates to mobility of water molecules inside the food product in terms of, for example, sugar, salt and other water binding substance concentrations concentrations (Roos 2000). Macromolecules like proteins, starches and fibers are extremely effective in binding water into themselves. Therefore, their quality and  proportions in foods is highly influential with respect to both microflora of the food product and the digestive tract. Consequently, nutrient uptake, host-microbe interaction and gastrointestinal symptoms could thus arise indirectly from the water activity. High osmotic  pressure kills microbial cells by forcing water molecules to move outside the cell. This  phenomenon is directly dependent dependent on water activity. activity. Besides water movement characteristics, also pH, temperature, preserving agents, quality and quantity of gases available can be used to prevent the microbial survival, and their  propagation. See also Figure 1. In preservation these methods mostly change circumstances  beyond microbial tolerance. Common traditional processing methods include for example drying, fermentation, smoking, salting, baking into crust, heating and mixing with sour  berries, all of them in their way decreasing the possibilities possibilities of microbes to survive. Before Before the development of fine packaging methods, smoking and baking into crust have been useful ways to handle certain foods in northern Europe. Smoke, which in fact consists of small liquid drops, forms a mechanical and chemical barrier around the product containing hundreds of anti-microbial compounds. Foods in overall, mainly from plant source, themselves contain a lot of antimicrobial compounds. Plants produce for example phenolic compounds and organic acids that are repellants to microbes or insects as well. One interesting example of traditional food processing is a fish pie called kalakukko (Figure 2) common in the area of Savo in Eastern Finland. Kalakukko contains small white fishes like perch or whitefish that are mixed with pork meat and baked into a hard rye crust. This way the easily spoilable parts of the product are secured inside a handy edible cover. The hard rye crust offers a fairly poor living environment for microbes. Very low water activity occurs and rye’s polymers, fibers, starches and proteins form a tight barrier between the fish and the exterior.

 

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3. MODERN FOOD PRESERVATION AND CONTROL  The modern western diet contains various practically new food ingredients and substances. Many of them ar e effective in improving a product’s preservation , texture, color and taste, but simultaneously they modify the living environment of microbes in food and  possibly in the GI-tract as well. Consumers are used to very little seasonal variation in the retail supply, which forces producers and the industry to manufacture products with longer shelf lives. Another thing to favor longer shelf lives is the variety of food products available. In concentrated food manufacturing, product volumes are high and making products with shorter shelf lives would result in significant increase in delivery input as well as product losses at retailers. A chain of hygiene related events in the food production and consumption is presented in Figure 3.

Figure 3. Besides raw material quality and storage, also the composition and preparation of foods contribute to the health effects. In this context microbiological, chemical and nutritional safety qualities are interlinked.

The large scale concentrated and controlled food industry relies on efficient selfmonitoring, which includes trained personnel and approved HACCP (Hazard Analysis and Critical Control Points) procedure. Scientific research on food microbiology and chemistry gives officials tools for elaborating regulations that are a re made at country level but increasingly also by international or nationwide stakeholders such as World Health Organization (WHO), Food and Agriculture Organization (FAO), Food and Drug Administration (FDA) and European Commission (EC). Foods with microbiological or chemical risk entering an EU country are reported in RASFF (the Rapid Alert System for Food and Feed) which is a public system available for anyone to follow (RASFF EC). Some technological aspects might still battle against microbiological safety, for example, heating processes of milk, such as pasteurizing or UHT shock heating (Ultra High Temperature). In some cases the heating of raw milk is avoided due to several different points of view. Heating, depending on temperature and duration, inactivates most of the enzymes in raw milk. The making of several traditional cheeses require a portion of those enzymes for

 

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distinct flavor compound’s formation  during maturation. Heating may also slightly change structures of some proteins existing in raw milk (Fox 1993). The use of food additives today is common while still regulated. Preservatives are a group of additives such as sorbic acid, benzoic acid, citric acid and nitrites, targeted to disturb microbial function. Due to its 2,4-diene-structure, sorbic acid is especially useful for  preventing the growth of molds that would otherwise be possible producers of toxins like aflatoxin and okratoxin (Alagöz et al., 2015). For significant growth molds usually need the  presence of oxygen, oxygen, but relatively low water activity. activity.  Nitrites, in forms of sodium nitrite or potassium nitrite, are added to most most processed meat  products, such as sausages, cutlery and bacon. Nitrite addition has two distinct purposes. It efficiently prevents the growth of Clostridium botulinum, bacteria found in soil and capable of producing one of the most powerful known toxin, botulin. The other purpose for nitrite is the preservation of meat’s red color  during  during and after cooking to maintain an appealing look of the product. In meat myoglobin molecules provide the red color when they bind oxygen molecules into themselves. However, myoglobin has an even higher affinity for binding nitrite or nitrogen monoxide, which keeps them attached also during a heat-treatment process.

The same reaction occurs in the human body too, when nitrites are consumed and absorbed into circulation. They can replace oxygen in hemoglobin and myoglobin resulting in lower oxygen-binding capacity (Otsuka et al., 2010, Lu et al., 2014). Another health concern  prevails due to nitrite nitrite consuming. R Reactions eactions in the acidic stomach stomach with the presence of of nitrites may result in formation of N-nitroso compounds, performing also carcinogenic features (Ohsawa et al., 2003, Catsburg et al., 2014). However, simultaneous presence of ascorbic acid (vitamin C) can reduce the formation of these end products (Pourazrang et al., 2002). To meet these concerns the European Commission has established an ADI-value (Allowed Daily Intake) of 0,07mg/kg body weight/day for nitrites. This safe daily intake may be exceeded even with a quite moderate usage of processed meat products, especially with small children. When considering the use of preservatives in food products the question should also be raised whether they are affecting the human alimentary microbiome in the small intestine, or if not absorbed there, in the large intestine. So far this is not included in the safety assessment ass essment  procedure for food additives additives. The intestines form a “column of microbes” which significantly influences our health, and any preservative has its own spectrum of effects on the microbial gut population from the duodenum onwards onwards (Hakalehto et al., 2008).Within the EU, additives used have gone through safety assessments including toxicological animal trials. The target of reducing the use of possibly detrimental preservatives requires stricter hygiene control in production and packaging and preparedness to shorten the shelf lives of  products. Preservatives, unlike other additives, are allowed to be used in some traditional foods, but their use in organic foods has specific regulations. Locally produced and consumed food naturally requires fewer preservatives due to shorter transportation and storage. In addition to microbiological risks, modern food control requires the assessment of chemical risk factors. Arable soil and raw materials for fertilizers naturally contain some harmful heavy metals, such as cadmium and arsenic, varying greatly on the area. Also some of the cultivars used are more efficient than others, in absorbing these substances from the soil (Evira 2014). For example rice, due to its high consumption and arsenic levels, is one of the important staple foods that need to be monitored (Meharg et al., 2009). The use of hazardous chemicals and radioactive compounds in industry or various consumer goods has led to contamination of some of the environments that are producing our food. Especially

 

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 pregnant women and small children are vulnerable to adverse effects of heavy metals and environmental environmen tal toxins.

4. TRENDS IN DIETARY HABITS  When considering the safety of food, we have to pay attention to its nutritional quality as well. Diet is a major factor in our welfare. Nutritional habits strongly reflect to our risks of common diseases such as diabetes, cardiovascular diseases and even cancer. While being fairly difficult to recognize links between certain foods and cancer, an estimate of the role of diet in total cancer prevention is around one third (Anand et al., 2008). Especially some types of cancer are strongly related to dietary habits. Understandably the quality of food consumed is a key factor in colorectal cancer for instance.

4.1. Sugars Are Important but Risky Parts of the Diet

One of the main concerns about modern diet consumed in the industrial countries, but increasingly in the developing countries as well, is the consumption of sugar. All mono- and dimeric carbohydrate molecules (saccharides) are defined as sugars, but the actual concern is regarding added sugar in forms of sucrose (disaccharide of glucose and fructose), glucose syrup or high fructose corn syrup. Sugars provide us a quick source of energy and sweet taste is what humans naturally aspire. Glucose is the form of sugar that all human cells are able to utilize as energy. The breakdown of glucose includes an anaerobic step, the glycolysis, followed by step by step cleavage of carboxyl groups releasing carbon dioxide, and finally oxidative phosphorylation producing high energy compounds, ATPs. According to Nordic Nutrition recommendation (NNR 2012), the consumption of added sugar should in all age groups be restricted to less than 10% of the total daily energy. Overall, added refined sugar increases the energy amount and glycemic load of food, without  providing any micronutrients for the body. Therefore, a diet rich in added sugar is more than likely energy dense and has to contain huge amounts of energy to meet all micronutrient requirements. This is probably one of the key factors linking sugar consumption to high body mass index. Sugar-sweetened beverages such as soft drinks or juices might be particularly effective in boosting weight gain, due to their energy content not reflecting simultaneous satiety effect (Millar et al., 2014). High blood sugar levels are also linked to higher inflammation marker, such as interleukin-6 and tumor necrosis factor- α, levels in the blood (Esposito et al., 2002). Dietary sucrose provides fast elevation in plasma glucose, but also in  plasma fructose, which is not used as energy before transforming into glucose or endogenous fat in the liver. This is why consumed fructose generates significantly lower blood sugar levels two hours after the meal compared to glucose or sucrose (Wiebe et al., 2011). Fructose is present in honey and most fruits and berries, usually attached to glucose or sometimes as free form. Free fructose is slightly sweeter than sucrose and therefore is favored in some food  products. In fact, sweetness of sugars can be somewhat modified by using their mutarotation in aqueous solution. First heated and then cooled fructose solution increases its relative sweetness (Damodaran et al., 2008). Despite these appealing possibilities, wide use of

 

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fructose for sweetening purposes may not be advisable, especially because of the burden it is  putting on liver functions. High High sugar intake promotes, promotes, in only a couple of weeks, lipogenesis in the liver, the main suspect being fructose (Sevastianova et al., 2012).In children, high intake of fructose could be related to non-alcoholic fatty liver and metabolic syndrome (Mager et al., 2010).

4.2. Additional or Alternative Sweeteners

Since taste of sweet is sensed by chemoreceptors probing the shape of the molecules, numerous non-sugar sweet tasting substances occur. Some of these molecules are already exploited by the food industry. Sweeteners include molecules found in the nature as well as artificial ones. Sugar alcohols are a group of sweet tasting molecules chemically close to sugars, but while sugars contain one carbonyl group per monosaccharide, sugar alcohols only contain variable amounts of hydroxyl groups attached to carbon backbone. The relative sweetness of sugar alcohols is close to sucrose or slightly less, but they don’t raise blood sugar levels until

 processed in the liver. They are not absorbed as efficiently in the small intestine as sugars, so  part of them is carried into colon, where they are used by microbes. A large quantity of consumed sugar alcohols most probably causes laxative effect in the combination of water shift from tissues to intestinal lumen and gases produced by microbes in the colon (Sakurai et al., 2007). Sorbitol is a widely used six-carbon sugar alcohol often derived from glucose. When sorbitol is absorbed from intestinal lumen, it is transported into liver and first transformed to fructose (Caudill and Stipanuk 2013). Often the amounts of sorbitol used for sweetening purposes are marginal compared to those of fructose, but same health concerns should be taken into consideration. Mannitol, another type of sugar alcohol is found small amounts in many edible plant species. However, various wild and cultivated mushroom species are significant sources of mannitol, which can be used for various purposes from sweetener to carrier substance matrix uses (Barros et al., 2008). Erythritol is a small four-carbon sugar alcohol. Compared to other sugar alcohols, erythritol has milder laxative effects. For example the diarrhea-causing amount for sorbitol is shown to be 0,15 to 0,25g/kg body weight, whereas the amount for erythritol is around 0,65 to 0,80g/kg body weight (Oku and Okazaki 1996). Other sweetening options besides the sugars and sugar alcohols occur as well. Stevia glycosides are 200-300 times sweeter molecules than sucrose and they are extracted from stevia plant common especially in South America. Also artificial sweeteners are currently used widely. Some of the most popular are aspartame, asesulfame K, sucralose and saccharine. 4.3. Our Lifestyle and the Glycemic Load

One of the dietary trends with the objective of a healthier lifestyle is reducing carbohydrates, especially those with a high glycemic load. A concern when reducing carbohydrates might be the simultaneous reduction in dietary fiber intake. Fibers perform  prebiotic activity and other health benefits, for example the production of butyrate, acetate and propionate. Fermentation of indigestible carbohydrates may increase energy intake up to

 

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10%. Formed butyrate is absorbed into the body and used as energy, while it also performs an anti-inflammatory effect (Singh et al., 2014). General benign activity of intestinal bacteria speeds up the onset of butyric acid formation by colonic anaerobes (Hakalehto and Hänninen 2012). Especially pectins, oligosaccharides and resistant starch are fermented efficiently in the colon. Water insoluble fibers such as cellulose are partially fermented (FAO 1998). Small amounts of protein end up into the colon as residues of food protein mixed with excreted old enterocytes and enzymes (Caudill and Stipanuk 2013). Reducing carbohydrates in the diet may result in an increase of protein and fat intake. This leads to a larger amount of protein residue in the colon although the absorption efficiency of amino acids would maintain. Fermentation of protein in the colon produces short chain fatty acids similarly to fermenting fibers, but also nitrogen containing compounds such as ammonia. These nitrogen containing  protein degradation end products may perform activity increasing colon cancer risk (Geboes et 2006). Long-term high (Linn proteinetconsumption 1,5g/kg body weight/day may contribute to theal., risk of insulin resistance al., 2000). However, relative protein intake should rise for people older than 65 years to maintain muscle tissue (Levine et al., 2014).

4.4. Dietary Fats

The debate over dietary fatty acid composition has lasted for decades. Fat is an important source of energy, but since only alfalinolenic acid (ALA, n-3) and linoleic acid (LA, n-6) are considered as essential fatty acids, they need to be consumed regularly to meet physiological demand. Linoleic acid itself has a role of being a specific part of sphingolipids forming the hydrophobic hydrophob ic layer in the skin. Both ALA and LA are precursors of long chain fatty acids. For instance arachidonic (AA) in the elongation and acid desaturation of LA,in and eicosapentaenoic acid acid (EPA) andresults eventually dokosahexaenoic (DHA) results the elongation and desaturation of ALA (Dubois et al., 2007). Those long chain fatty acids can be used as eicosanoid family tissue hormones (AA and EPA) or as structural components of membranes and other lipid structures. DHA is especially important for the development of the central nervous system and eyes. In addition to ALA and LA, AA, EPA and DHA are present in animal based and marine foods. As vital as they are, these polyunsaturated fatty acids (PUFA) are extremely sensitive to oxidation caused by light and heating during preservation and processing. While ignoring the necessity of essential fatty acids, the dietary fatty acid composition should be favored towards unsaturated fatty acids to maintain healthy serum lipid profile, although the evidence for direct adverse effects of saturated fatty acids is more or less contradictory (Mozaffarian et al., 2010, Hooper et al., 2012). In the human body system, the pancreatic lipases are secreted for breaking up the fats of the foods (Lowet 2002). In the intestines, in addition to several strictly anaerobic bacteria such as  Bacteroides sp., also some lactobacilli carry out bile acid deconjugation, which is related to the lipid uptake by the body (Gilliland and Speck 1977). Since the balance between various microbes essentially influences the bile acid secretion, bile circulation, nutrient uptake, and many other essential functions of the body system, it is of high importance to continue studies on “micro bial hygiene” with reference to the fate of food substances inside our GI tract and beyond (Hakalehto 2012).

 

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4.5. Vitamins and the Microflora

Overall trends to improve nutritional quality of foods have recently included vitamin (especially vitamin D and folate) and mineral fortification while reducing fat, sugar and salt contents. Vitamin D, for instance, is an important contributor to human innate immunity (Bischoff-Ferrari et al., 2012). Therefore, many pathogens tend to block the vitamin D receptors, which increase its demand during infections and inflammations (Haussler et al., 2011, Marchal et al., 2011). This vitamin affects also the fat uptake. Therefore, the intestinal fate of various fractions in foods is an outcome of many factors:   nutritional    body functions functions (immunology, (immunology, humoral, humoral, hormones, neurological) 



      

food hygienic quality and constitution intestinal microbiota stress, activity

Water content usually increases when reducing fat concentrations. On the other hand, water activity increases when reducing sugar and salt. The balance between microbiological

and nutritional quality has their conflicts, but is currently solved in many cases by the excess use of preservatives. Unfortunately, these can have adverse effects on health by misregulating the intestinal flora. Therefore more “natural” means for keeping up the microbiological standards in foods could be recommended. These include the use of fermentation, mild organic acids, and other such means. Correspondingly to many other preservative treatments, heating is effective in destroying pathogens but meanwhile it may deprive the food from some of itsIntestinal beneficialmicrobes micronutrients. affect the functions of an organism both on biochemical and immunological levels (Mitra et al., 1998). Therefore, adequate supplies of probiotic bacteria need to be available regardless of the nutritional or health conditions of an individual (Giorgi 2009, Hakalehto et al., 2015).

CONCLUSION  Cultivating, processing and distributing food relies increasingly on centralized food marketing chains which is the prerequisite of producing enough food for big urban  populations and also the reason why the responsibility for food safety is not only the issue of a consumer. One of the most important hygienic factors is water activity (aw) which relates to the mobility of water molecules inside the food product in terms of, for example, sugar, salt and other water-binding substance concentrations. Consumers are used to very little seasonal variation in the retail supply, which forces producers and the industry to manufacture  products with longer shelf lives. Efficient self-monitoring equipped with organized heat treatments and the wide use of preservatives offers tools to meet hygienic requirements with some problematic issues following by side.  Nutritional habits strongly reflect to our risks of common diseases and therefore are a valid part of safety related to eating. The common trend of high sugar consumption causes

 

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 problems, especially combined withkind a low fibers an unnecessarily high intake of protein. Effects of this of intake a diet of candietary be seen for and example in fermentation  products formed in the colon. All aspects of food safety are interlinked and sometimes competing with each other. Methods preventing acute risks are often favored, partly at the expense of nutritional quality, effect to gut microbes, or even technological desire.

EFERENCES  R EFERENCES

Alagöz, S., Türkyilmaz, M., Tai, Ş & Özkan, M. 2015. Effects of different sorbic acid  and moisture levels on chemical and microbial qualities of sun-dried apricots during storage.  Food Chemistry Chemistry 174. Elsevier Ltd. 356-364 pp. Anand, P., B. Kunnumakara, Sundaram , C., Harikumar, K. B.,disease Tharakan, T., Lai, major O. S., Sung, & Aggarwal,A.B.B.,B.Sundaram, 2008. Cancer is a preventable thatS.requires lifestyle changes. Pharmaceut  Pharmaceutical ical Research 25: 2097-2116. Barros, L., Cruz, T., Baptista, P., Estevinho, L. M. & Ferreira, I. C. F. R. 2008. Wild and commercial mushrooms as source of nutrients and nutraceuticals.  Food and Chemical Toxicology 46. 2742-2747 pp.

Bischoff-Ferrari, H. A., Dawson-Hughes, B., Stöcklin, E., Sidelnikov, E., Willett, W. C., Edel, J. O., Stähelin, H. B., Wolfram, S., Jetter, A., Schwager, J., Henschkowski, J., Von Eckardstein, A. & Egli, A. 2012. Oral supplementation with 25(OH)D3 versus vitamin D3: Effects on 25(OH)D levels, lower extremity function, blood pressure, and markers of innate immunity. Journal of Bone Bone and Mineral Mineral Research 27: 160-169. Catsburg, C. E., Gago-Dominguez, M., Yuan, J., Castelao, J. E., Cortessis, V. K., Pike, M. C. & Stern, M. C. 2014. Dietary sources of N-nitroso compounds and bladder cancer risk: Findings from the Los Angeles bladder cancer study.  Internatio  International nal Journal of Cancer 134: 125-135. Caudill, M. A. & Stipanuk, M. H. 2013.,  Biochemical  Biochemical,, physiological, and   molecular aspects of human nutrition. 3rd ed. ed. St. Louis, Mo: Elsevier/Saunders.ISBN 1437709591 (pbk.). xix, 948 p. Damodaran, S., Fennema, O. R. & Parkin, K. L. 2008. Fennema's food chemistry. 4 th  ed. Boca Raton: CRC Press, Food science and technology technology, ISBN 0-8493-9272-1. 1144 s p. Dubois, V., Breton, S., Linder, M., Fanni, J. & Parmentier, M. 2007. Fatty acid profiles of 80 vegetable oils with regard to their nutritional potential.  European Journal of Lipid Science and Technology Technology 109: 710-732. Esposito, K., Nappo, F., Marfella, R., Giugliano, G., Giugliano, F., Ciotola, M., Quagliaro, L., Ceriello, A. & Giugliano, D. 2002. Inflammatory cytokine concentrations are acutely increased by hyperglycemia in humans: Role of oxidative stress. Circulation 106: 20672072. Evira 2014.  Elintarvikkei  Elintarvikkeiden den ja talousveden kemialliset vaarat . Elintarviketurvallisuusvirasto Elintarviketurvallisuusvirasto Eviran julkaisuja. Uudistettu painos. (In Finnish). FAO 1998.  Physiological Effects of Dietary Fibre . Cited 2/15/2015. 1998. Available on the Internet:http://www.fao.org/docrep/w Internet:http://www .fao.org/docrep/w8079E/w807 8079E/w8079e0l. 9e0l. htm. Fox, P. J. 1993. Cheese: Chemistry, Physics and Microbiology: Volume 1: General Aspects Volume 2: Major Cheese Groups. Springer.ISBN 0834213389.

 

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Geboes, K. P., De Hertogh, G., De Preter, V., Luypaerts, A., Bammens, B., Evenepoel, P., Ghoos, Y., Geboes, K., Rutgeerts, P. & Verbeke, K. 2006. The influence of inulin on the absorption of nitrogen and the production of metabolites of protein fermentation in the colon. British Journal Journal of Nutrition Nutrition 96: 1078-1086. Gilliland, S. E. & Speck, M. L., 1977. Deconjugation of bile acids by intestinal lactobacilli.  Applied and and Environmental Environmental Mi Microbiology crobiology 33: 15-18. Giorgi, P. L., 2009. Probiotics. A review. Recenti Progressi Progressi in Medicina Medicina 100: 40-47. Hakalehto, E., Jaakkola, K., Pesola, J., Heitto, A., Hell, M. & Hänninen, O. 2015. Tendencies in probiotic treatments. In: Hakalehto, E. (ed.).  Microbi  Microbiological ological Clini Clinical cal Hygiene. N. Y.:  Nova Sciences Publishers Publishers Inc. pp. pp. 301-321. 301-321. Hakalehto, E. 2012. Alimentary microbiome: A PMEU approach.  Alimentary Microbiome: Microbiome: A  PMEU Approach Approach Nova Science Publishers, Publishers, Inc. 1-263 1-263 pp. Hakalehto, E. & Hänninen, O. 2012. Gaseous CO2 signal initiates growth of butyricacid producing Clostridium butyricum in both pure culture and mixed cultures with Lactobacillus brevis. Canadian Journal of Microbiology 58: 928-931. Hakalehto, E., Humppi, T. & Paakkanen, H. 2008. Dualistic acidic and neutral glucose fermentation balance in small intestine: Simulation in vitro.  Pathophysi  Pathophysiology ology 15: 211220. Hakalehto, E., Santa, H., Vepsäläinen, J., Laatikainen, R. & Finne, J. 1997. Identification of a

common structural motif in the disordered N-terminal region of bacterial flagellins. Evidence of a new class of fibril-forming peptides.  European Journal of Biochemistry 250: 19-29. Haussler, M. R., Jurutka, P. W., Mizwicki, M. & Norman, A. W. 2011., Vitamin D receptor (VDR)-mediated actions of 1α,25(OH) 2vitamin D 3: Genomic and non-genomic mechanisms.  Best Practice and Research: Clinical Endocrin Endocrinology ology and Metaboli Metabolism sm 25. 543-559 pp. Hocking, A. D., 1988, Strategies for microbial growth at reduced water activities.  Microbiological  Microbiol ogical Sci Sciences ences 5: 280-284. Hooper, L., Summerbell, C. D., Thompson, R., Sills, D., Roberts, F. G., Moore, H. J. & Davey Smith, G. 2012. Reduced or modified dietary fat for preventing cardiovascular disease. Cochrane Database of Systematic Reviews (Online) 5. Levine, M. E., Suarez, J. A., Brandhorst, S., Balasubramanian, P., Cheng, C., Madia, F., Fontana, L., Mirisola, M. G., Guevara-Aguirre, J., Wan, J., Passarino, G., Kennedy, B. K., Wei, M., Cohen, P., Crimmins, E. M. & Longo, V. D. 2014. Low protein intake is associated with a major reduction in IGF-1, cancer, and overall mortality in the 65 and younger but not older population. Cell Metabolism 19: 407-417. Li, G., Tam, L. & Tang, J. X. 2008. Amplified effect of Brownian motion in bacterial nearthe Nationa Nationall Academy of Sci Sciences ences of the United States States surface swimming. Proceedings of the of America 105: 18355-18359. Linn, T., Santosa, B., Grönemeyer, D., Aygen, S., Scholz, N., Busch, M. & Bretzel, R.G. 2000. Effect of long-term dietary protein intake on glucose metabolism in humans.  Diabetologia  Diabetolog ia 43: 1257-1265. Lowet, M. E. 2002. The triglyceride lipases of the pancreas.  Journal of Lipid Research 43: 2007-2016. Lu, N., Chen, C., He, Y., Tian, R., Xiao, Q. & Peng, Y., 2014, The dual effects of nitrite on hemoglobin-dependent hemoglobindependent redox reactions. Nitric Oxide: Oxide: Biolo Biology gy and Chemistry Chemistry 40: 1-9.

 

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Mager, D. R., Patterson, C., So, S., Rogenstein, C. D., Wykes, L. J. & Roberts, E. A. 2010. Dietary and physical activity patterns in children with fatty liver.  European Journal of Clinical Nutrition 64: 628-635. Marchal, C., Schramm, F., Kern, A., Luft, B. J., Yang, X., Schuijt, T. J., Hovius, J. W., Jaulhac, B. & Boulanger, N. 2011. Antialarmin effect of tick saliva during the transmission of lyme disease. Infection and and Immuni Immunity ty 79: 5039. Meharg, A. A., Williams, P. N., Adomako, E., Lawgali, Y. Y., Deacon, C., Villada, A., Cambell, R. C. J., Sun, G., Zhu, Y., Feldmann, J., Raab, A., Zhao, F., Islam, R., Hossain, S. & Yanai, J. 2009. Geographical variation in total and inorganic arsenic content of  polished (white) rice. Environmenta  Environmentall Science a and nd Technol Technology ogy 43: 1612-1617. Millar, L., Rowland, B., Nichols, M., Swinburn, B., Bennett, C., Skouteris, H. & Allender, S. 2014. Relationship between raised BMI and sugar sweetened beverage and high fat food consumption among children. Obesity 22: E96-E103. Mitra, R., Saha, P. K., Basu, I., Venkataraman, A., Ramakrishna, B. S., Albert, M. J., Takeda, Y. & Nair, G. B. 1998. Characterization of non-membrane-damaging cytotoxin of nontoxigenic Vibrio cholerae O1 and its relevance to disease.  FEMS Microbiol Microbiology ogy Letters 169: 331-339. Mozaffarian, D., Micha, R. & Wallace, S. 2010. Effects on coronary heart disease of increasing polyunsaturated fat in place of saturated fat: a systematic review and metaanalysis of randomized controlled trials. PLoS Medicine Medicine 7.

 NNR 2012.  Nordic Nutrition Nutrition Recommendations Recommendations 2012: FULLTEXT01.pdf. Cited 2/21/2015 2015. 2012. Available on the Internet: http://norden.diva-portal.org/smash/get/diva2: 704251/FULLTEXT01.pdf. Ohsawa, K., Nakagawa, S., Kimura, M., Shimada, C., Tsuda, S., Kabasawa, K., Kawaguchi, S. & Sasaki, Y. F. 2003. Detection of in vivo genotoxicity of endogenously formed N Mutation ion nitroso compounds and suppression by ascorbic acid, teas and fruit juices.  Mutat  Research: Genetic Genetic Toxicolog Toxicologyy and Environ Environmental mental M Mutagenesi utagenesiss 539: 65-76. Oku, T. & Okazaki, M. 1996. Laxative threshold of sugar alcohol erythritol in human subjects. Nutriti  Nutrition on Research 16: 577-589. Otsuka, M., Marks, S. A., Winnica, D. E., Amoscato, A. A., Pearce, L. L. & Peterson, J. 2010, Covalent modifications of hemoglobin by nitrite anion: Formation kinetics and  properties of nitrihemoglobin. nitrihemoglobin. Chemical Research in Toxicology 23: 1786-1795. Pourazrang, H., Moazzami, A. A. & Fazly Bazzaz, B.S. 2002. Inhibition of mutagenic Nnitroso compound formation in sausage samples by using L-ascorbic acid and atocopherol. Meat Science Science 62: 479-483. RASFF EC. RASFF:  Food and Feed Safety Alerts: European Commissi Commission on. Cited 3/6/2015 2015. EC. Available on the Internet: http://ec.europa.eu/ food/safety/rasff food/safety/rasff/index_en.htm /index_en.htm..

Roos, Y. H. 2000. Water activity and plasticization, In: Eskin, N. A. M. & Robinson, D. S. (eds.) Food Shelf Life Stability: Chemical, Biochemical and Microbiological Changes. Boca Raton, Florida, USA: CRC Press. P ress. Sakurai, H., Takahashi, H., Takemura, K. & Fukahori, M. 2007. Augmented laxative action of sugar alcohol by organic acids and inorganic salts,  Japanese Pharmacolo Pharmacology gy and Therapeutics 35: 143-149. Sevastianova, K., Santos, A., Kotronen, A., Hakkarainen, A., Makkonen, J., Silander, K., Peltonen, M., Romeo, S., Lundbom, J., Lundbom, N., Olkkonen, V. M., Gylling, H., Fielding, B. A., Rissanen, A. & Yki-Jar¨vinen, H. 2012. Effect of short-term

 

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carbohydrate overfeeding and long-term weight loss on liver fat in overweight humans.  American Journal Journal of Cl Clinical inical Nu Nutrition trition 96: 727-734. Singh, N., Gurav, A., Sivaprakasam, S., Brady, E., Padia, R., Shi, H., Thangaraju, M., Prasad, P. D., Manicassamy, S., Munn, D. H., Lee, J. R., Offermanns, S. & Ganapathy, V. 2014. Activation of Gpr109a, receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and carcinogenesis. Immunit  Immunityy 40: 128-139. Wiebe, N., Padwal, R., Field, C., Marks, S., Jacobs, R. & Tonelli, M. 2011. A systematic review on the effect of sweeteners on glycemic response and clinically relevant outcomes. BMC Medicine Medicine 9.

 

In: Microbiological Microbiolo gical Food Hygiene Editor: Eino Elias Hakalehto

ISBN: 978-1-63483-646-3 © 2015 Nova Science Publishers, Inc.

Chapter 3

HAZARDS AND PREVENTION  OF FOOD SPOILAGE  E lilia as H Ha akaleht lehto o Department of Environmental Sciences, University of Eastern Finland, Kuopio, Finland

ABSTRACT  Food microbiology is an extremely wide research topic. It should not be restricted to  partial solutions only. Norms Nor ms and directions are important guidelines, guidelines , but they should not impede development of technology or improvement of safety practices. Whenever human food is acquired or prepared from various raw materials, this includes aspects of hygienic safety. We have inherited the everyday instructions from the older generations, and we try to teach them to our children. Whether the food is acquired by hunting or by gathering it from the Nature, or grown in local small farms, or using urban greenhouses, it is important that our nutrition is on healthy, scientifically sound basis. This casts a great responsibility to the quality management, hygiene control staff, biochemists and microbiologists. Also the legislative innovations and good information management and editing is warranted during our modern times. The history of mankind is full of results from ignorance of the basic microbiological facts, and their negligence. Microbial impacts on our food raw materials are numerous. Some simplification is required for dealing with the issues of potential causative agents of bacterial food  poisoning. The most common microbiological surveillance methods base on the screening of hygiene indicators, but for deeper understanding of the problem, the actual  pathogens need to be revealed and investigated. Besides these disease-causing agents, food may become spoiled by any degradative actions of miscellaneous microflora.

1. INTRODUCTION  Sometimes intentional ageing or “spoilage” of the food   is used for preparing special culinary effects or experiences, or it is exploited as an actual means of preserving food. Then

 

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some desirable microbial strains and their fermentative reactions are favored during the maturing of the food. This kind of deliberate use of microbes always requires experience and skills. In a way it resembles a biotechnical process. Such topics are discussed elsewhere in this book. The epidemiological triangle is illustrated in Figure 1. This simple model describes the basic elements of food hygiene considerations. In all food manufacturing the utmost goal is to prevent any hazardous hygienic event from causing threat or damage to the consumers. Four bacterial causative agents of food poisoning are some of the most common reasons for food-mediated food-mediated pathological conditions of this kind: 1.   Bacillus cereus 2.  Clostridium perfringens 3.  staphylococcal enterotoxins 4.  Salmonella/Campylobacter  Naturally this list of some of the most common bacteriological agents is highly variable depending on the geographical location, climate, food sorts (various dishes), general health and other environment or host related functions. In any case, it has been stated that the annual growth of biomass could be enough for both the energy production and to feed the global  population (Dahlqvist (Dahlqvist et al. 2013). 2013). Besides the organisms listed above, many other bacteria can potentially cause equally hazardous or even more serious situations, but the list above is a general check-list of some of

the most common kinds of causative agents of food spoilage. These pathogens are now considered on the basis of their microbiological characteristics as well as the potential symptoms and consequences they cause for the human host. Also some other species are being introduced. The viral food poisoning should not be mixed with these bacteriological agents, and it will be discussed elsewhere in this book (in chapter 4).

2. CONTAMINATION AND DEGRADATION PATTERNS  Food spoilage is a common phenomenon occurring when any food substance is in a  physicochemical condition which allows allows the deterioration deterioration process to start and and proceed.

Modified from Bryan (1979). Figure 1. The epidemiological triangle.

 

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This condition can be divided into different stages of the food matter consisting of: A.  food constituents; their biochemical nature (protein, fat, carbohydrates, minerals etc.) B.  fundamental chemical conditions in food (pH, redox, osmolarity etc.) C.  natural microflora in the food D.   prevailing storage storage or environmental environmental conditions (temperature, humidity humidity etc.) E.  environm environmental ental microbial load It is inevitable that any food will get spoiled, if not preserved effectively by drying, freezing, salting, or by any other means known by man. The severity of the spoilage is usually related to the time elapsed. However, the level of hazardousness is dependent on many other factors besides the abundance of microbes. For example, when studying the barley barrels contaminated with  Fusarium  molds, it was resembling detected thattheroughly 1 out 100 rounds harmful levels cearulenon, a substance structure andofinfluences of contained female steroid-hormone. steroid-hormon e. of

These experiments were carried out in the Biotechnology laboratory of the VTT (State Research Center of Finland) in 1982-83. It was also found out that of all months the weather conditions in March (particularly with respect to rainfall) were the most important determinants of mold is barley crops in the next autumn (Hakalehto, 2015a). It seems evident that a thick, slowly melting snow layer on the agricultural land produces environmental conditions that are most beneficial for the reproduction of microfungi and their spreading into the emerging sprouts later.

From the above mentioned example of barley grains, it could be deduced that the severity  of contamination was largely caused by weather-related humidity, but the hazardousness  was dependent on an overwhelming number of factors which influenced not only the growth of a  particular microbe, microbe, but its toxic or infective infective properties, too. Moreover, when the spread of mold spores in the atmosphere were studied at the altitude of 300-1000 m, equally high numbers of mold spores were detected in the samples taken high in the air than on the ground (Hakalehto, 2015a). The collection method was done by aircraft sampling (Kantolahti et al., 1986; Hakalehto et al., 1994). Sometimes it seemed to be the case that the mold spores could concentrate high up in the air masses, whose movements and turbulences determine the transportation distance of these infective entities. Gravitation is less influential, having practically no effect on particles with a diameter less than 10 microns. Consequently, mold spores and other contaminating agents could, in theory, travel with the air to other side of the globe, providing that they can resist drying up, surviving UV radiation, variable temperatures and a nd other environmental challenges. It was found that in urban air and in its upper layers, Penicillium was the most abundant mold genus, whereas in the countryside such genera as  Aspergill  Aspergillus us  and  Alternaria  dominated.  Penicillium  Penicilliu m was disseminated to the atmosphere from ventilation systems, but the latter ones mainly from the vegetation. If a mold spore lands on some sugary and acidic substance, e.g., a berry dessert, it slowly  begins to change the condition of this food, starting from the surface (Hakalehto, 2012a). Eventually the growing mycelium will change the physicochemical characteristics of the food  by producing enzymes and metabolic products that alter the inner milieu of the dessert as well.

 

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Elias Hakalehto Then the fungal hyphae grow deeper into the originally hostile space, starting to

increasinglythan exploit the available nutrients it. The fungi often withstand pH “submersion” of can the fungal growth, thelower conditions bacterial strains. Prior to the in mycelia on the surface forms a tightening network and produces more spores. After this “pioneering” spoilage activity of the mold culture, started from a single spore, numerous other microbes can facilitate their growth and metabolism in the food product. The deteriorating microbial growth in a food product, besides starting from the surfaces, often progresses through specific channels, routes or pores. For example, the spoilage of an avocado fruit initiates at a specific point where the strains penetrate into the flesh of the fruit (Figure 2). This type of disqualified part or area of the product is often removed prior to consumption or during the preparation of the food in a kitchen. However, in the food industries it is generally not considered acceptable to use any  partially spoiled raw materials, even though the potentially malicious parts are excluded from the product. This is justified also because the microbial growth, before becoming visually detectable, can cause alterations in the product or its raw materials. For example, bacteria or molds may secrete toxins into their surroundings. Anaerobic putrefaction also causes food products to  become inedible because they often often become poisonous. poisonous. The spread of pathogenic influences should be prevented during the collection of raw materials, and in the actual preparation p reparation of food. Zoonoses, for exam example, ple, may infect consumers via animal products (Table 1). In a more profound sense, microbiological deterioration begins right after the collection of the food substance, for example after a berry has been picked.

Any plant material usually contains antioxidants to prevent damages to its structure and function. These protective substances include vitamins or flavonoids. They are considered as healthy foods for humans. Therefore, the antioxidant therapies can treat many diseases or conditions successfully (Sharma et al., 2015). Antioxidants can also be used for maintaining digestive health (Jaakkola, 2010, Jaakkola and Hakalehto, 2012).

Figure 2. Food spoilage usually has a starting point. The spoilage of an avocado fruit by  Penicillium molds (right). The removal of the contaminated part (in the middle) does not necessarily remove the contaminating microbes or their hyphae or spores. For example, the mold mycelium which is not visible to the eye may have grown into the entire fruit.

 

Hazards and Prevention of Food Spoilage Table 1. Bacterial zoonoses in Finland 2000-2010

1. Lyme disease ( Borrelia sp.)  –  a  a few thousand cases annually 2. Botulinum (Clostridium botulinum-toxin)  –  proportion  proportion of seropositive dogs about 10% 3. Brucellosis ( Brucella  Brucella sp.)  –  Finland  Finland is brucellosis free, but Zoonosis Centre of EVIRA is surveying the cattle, sheep and goat. 4. EHEC (Enterohaemorrhagie E. coli)  –  some  some tens of cases reported annuall 5. Tularemia ( Francisella  Francisella tularensis)  –  reservoir  reservoir in hares and small rodents  –   epidemics epidemics in some years with a few hundred human cases  spreads mostly with insect bites, also in aerosols from dead animals  –  spreads  –  highly  highly infective (small infective doses) 6. Campylobacterial disease ( Campylobacter  sp.)  sp.)  –  some  some 4000-5000 thousand cases reported annually  –  derived  derived mostly from infections abroad (tourism)  –  in  in 2004 17,6% of slaughtered broilers and 29,7% of turkeys turned out carriers, in 2003 only 3% of the cows 7. Listeriosis ( Listeria  Listeria monocytogenes)  –  potentially  potentially hazardous infection for immunocompromised, elderly and pregnant women  –  below  below 100 reported human cases annually

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 –  in  in cold smoked fish, for example, some 20-30% of the packages contain Listeria sp. (in small amounts, only 3-4% with more than 100 cfu/g)  Mycobacterium bovis) 8. Cow tuberculosis ( Mycobacterium  –  below  below 5 infected animals per 100.000 slaughtered ones 9. Anthrax ( Bacillus  Bacillus anthracis)  –  spore-forming  spore-forming agent, no reported human case after 1995 in Finland  –  in  in animals once in 5-10 years 10. Psittacosis (Chlamydia psittaei)  –  derived  derived from birds, ”undulate fever”   –  a  a few animal (bird) cases annually 11. Q fever  –   0,2% 0,2% of cattle possess antibodies against this intracellular pathogen 12. Salmonellosis  around 2500 patient cases in Finland annually  –  around  Erysipelothrix rhusiopathea) 13. Erysipeles ( Erysipelothrix  –   0-3  0-3 human cases reported annually  –  less  less than 50 cases in pig farms 14. Yersiniosis  –  normal  normal annual prevalence less than 100 patients, in year 2006 nearly 500 cases

The list is an overview of the EVIRA Zoonotic Centre Report. For further information on the zoonotic diseases in foods, see Chapter 12.

 

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Microbes in the foods, for their part, can cause spoilage by breaking up the structures by oxidation and by metabolic products which undermine the nutritional and other qualities of the food. On the contrary, microbial metabolism can be exploited in the preparation or  preservation of foods, the fermented food products being a good example of this. For example, each spoonful of yoghurt contains approximately as many bacterial cells by number as there are human beings on this planet. In their outstanding book on the biological effects of the free radicals, Halliwell and Gutteridge (1989) define oxidative stress as a result from the free radical action. Oxygen is toxic to plant, animal and human cells. The damaging effect of oxygen on the obligate anaerobes is based on the oxidation of essential cellular components. These bacteria thrive in reduced environments. By oxygen such substances as NAD(P)H, thiols or iron-sulphur proteins get oxidized which harms the substances or prevents the  biosynthesis. In foods, oxidation compromises compromises the chemical qualities of the product by  boosting aerobic bacterial growth. Therefore, many food materials are preserved by packing them into specific protective gas atmosphere. Within the body system we have ten times more  bacterial cells than there are cells of our own. own. These bacterial cells concentrate concentrate mostly into the alimentary tract. Therefore their contribution to our digestive processes is of essential kind which can lead to a disease causing sequence of events, in the worst case, or to the promotion of health and protection, at its best (Hakalehto 2012b, Hakalehto, 2015b).

3. SOME TYPES OF BACTERIAL CONTAMINATIONS   This section focuses on the above-mentioned four common types of food-poisoning (see the Introduction section of this chapter) and discusses some related bacterial varieties. One

main group Salmonella/ Campylobacter , for instance, is not formed on the basis of the combination of close taxonomical relatedness of the two genera, but more on the similarities in the disease-causing patterns. This group of major food-poisoning bacteria represents the Gram-negative eubacteria. Therefore, alongside with the characterization of the salmonellae and cambylobacteria, numerous pathogenic or toxicogenic forms of e.g., enteric species (or coliforms) will be discussed. As a matter of fact, the Gram-negative outer cell structure makes the pathogenesis or the  pathological conditions caused by these organisms significantly more variable than the ones  provoked by the Gram-positives. The latter ones can be grouped into three generalized categories: 1.  the intestinal growth or toxin formation of aerobic spore-forming bacilli (e.g.,  Bacillus) 2.  the intestinal growth or toxin formation of anaerobic spore-forming bacilli (e.g., Clostridium ) 3.  the intestinal growth or toxin formation of facultative anaerobic Gram-positive cocci (e.g., Staphylococcus ) The specific characteristics of each of these groups are somewhat distinct from the Gramnegative mode of pathogenesis or food poisoning.

 

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In the case of Gram-negatives, one major difference is the presence of lipopolysaccharides (endotoxins) in them (Helander et al., 1983). These molecules vary from strain to strain causing “camouflage” protection   for the bacterial cells against the immune system. They also produce inflammation, and toxigenic or allergenic reactions in the host. The biological activities of the endotoxins and other bacterial structures are discussed in the  previous volume volume of this book book series (Hakalehto (Hakalehto,, 2015c). The above mentioned division of the major food poisoning causes is and could well turn out a somewhat controversial classification. However, the three Gram-positive agents or groups of agents together with their versatile toxins may cause numerous incidents of minor ill-health and digestive disorders. The Gram-negative genera of Salmonella  and Campylobacter   cause gastrointestinal diseases of many kinds, and the milder forms are usually not even reported to the healthcare system, while the severe ones truly incapacitate the patients and can be even life-threating. However, if the above-categorized four classes of causative agents are prevented from contaminating food materials, most other agents will simultaneously be confined with them. Hence this approach of focusing the attention to these “indicators of food poisoning”could be considered as a pragmatic one. The mode of agricultural practices can influence on the  bacterial presence. For example, in organic farms most antibiotic resistant variants of Campylobacter sp. were less frequent than in conventional farms (Halbert et al., 2006). More considerations on the antibiotic resistance distribution in food and agriculture are presented in Chapter 13. These traits may change the picture of pathogenesis of any particular bacterium from the healthcare point-of-view (Hakalehto, 2006). Since our division into four food poisoning categories does not follow the statistics regarding the severe or life-threatening foodborne infections, we wish to include some more malicious examples into our survey.

4. STATISTICS OF FOOD EPIDEMICS  According to the list of 10 worst foodborne illness outbreaks 2014 in the US, the most deadly cases were caused by  Listeria  (www.foodsafetynews.com). Nine fatal cases were reported, related to caramelized apples, cheese and bean sprouts. In these incidences the total number of sickened was 47. On the list, there were three Salmonella  outbreaks with 828 sickened by spoiled chicken, bean sprouts and chia seeds. Clostridium perfringens caused the food-poisoning food-poison ing in two of the most difficult cases, with more than 500 diseased people. In both of the cases, it was a hot dish that caused the poisoning. The remaining two cases on the list of the worst outbreaks in the US during 2014 were a Cyclospora  in salad outbreak, and an incident with raw milk contaminated with Campylobacter. Food poisoning statistics in the UK (published on 26.6.2014) give an idea of the most common causative agents of the food  borne illnesses (www.food.gov.uk). (www.food.gov.uk). The number of total registered cases of known human foodborne pathogens causing severe illnesses exceeded 500,000 per year. The approximate numbers of diseased persons by different bacteria were   Campylobacter , about 280,000 cases   Clostridium perfringens, about 80,000 cases  

 

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Elias Hakalehto   norovirus, about 74,000 cases   Salmonella, about 33,000 cases



In this study, Salmonella caused the highest number of hospital admissions, about 2500 cases. With respect to food commodity, commodity, poultry was reported to cause about 244,000 cases. Salad or cooked vegetables, various nuts, seeds and fruits contributed about 43,000 cases. The numbers of beef and lamb, seafood or eggs as a vector for foodborne cases were estimated to be 43,000, 32,000 and 26,000, respectively.

5. ECOLOGICAL SUCCESSIONS IN  INTESTINAL AND “MANMADE” ECOSYSTEMS  There are specific continuums which illustrate the microbial presence in men, in manmade systems or in the environment. Microbiological Microbiological effects of our actions in the latter can be of surmounting nature (Hakalehto and Heitto, 2012; Hakalehto, 2015a). In Figures 3a and 3b two successions of microbiological ecosystems ecosystems are outlined.

a

 b Figure 3. Typical process flow situations with respect to food consumption and human digestion (A) and to the bioprocess design including recycling (B). In a sense, these are analogous situations with each other, with coherent types of ecological successions of microbial strains and effects.

 

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Figure 4. The antigen dots on the upper row from the left: human Candida yeast isolates nro 1 and 2, S. aureus strain Hambi 178, S. epidermis Hambi 184, S. haemolyticus Hambi 43, and in the lower row from the left E. coli Hambi 93, Klebsiella mobilis ATCC 13048, , Bacillus cereus strain, Salmonella enterica Serovar Typhimurium HIS 59929 and Clostridium butyricum ATCC 97426. The primary serum was a patient sera of a severely s everely ill patient suffering from staphy staphylococcal lococcal chronic infection, which  probably had entered his inner organs organs from the intestines. The pale and poorly visible dot of the B. cereus may reflect the presence of some capsid on the surface. We have noticed that such layers la yers of this  bacterium may bind surrounding surrounding macromolecules from the solu solutions tions around (results not shown here). Is this a “camouflage” means to hide from f rom the immune system?

The “ecosystem  thinking” has been introduced earlier, especially with   respect to the intestinal microbial communities (Hakalehto, 2012b). The same line of thinking could be applied in food microbiology. In fact, there is no clear-cut distinction between the microbes in foods and the microbes of human (or animal) digestion.

In the case of studies with a severely immunodeficient patient, some common alimentary microbes were tested against his serum in a dot blot test (Figure 4). It was then obvious that the highest relative immunoreaction were obtained by having invasive type of bacteria as antigens. Actually the antigen solution was prepared with a 0.05 M HCl extraction from the microbial cell surface antigens (Hakalehto et al., 1984). Such microbes as Candida  yeasts or  Klebsiell  Klebsiella a mobilis  bacterial strain, did not provoked much reactivity which is likely to correlate with the lack of penetration (or even attachment) into the patient epithelia or tissues. On the contrary, there are microbes that invade the intestines in high numbers, being at the same time adsorbed on some parts of the digestive system, such as  E. coli or Clostridium butyricum. These types of colonizing bacteria could also initiate remarkable antigen formation. Staphylococci Staphylococci have been documented as influential members of our intestinal flora  but they can also intrude the human tissues via skin ruptures or through the epithelial cell monolayer in the intestines (Edwards et al., 2012). This type of process may explain the health problems of many young individuals which seemingly are related to the food consumption. It could also be anticipated that skin and gut colonization could often be interlinked. In the case of the pediatric leaky gut syndrome, the staphylococcal influences were divided into two causes of intestinal inflammation:

 

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Elias Hakalehto 1.  Direct epithelial cytopathy 2.  Superantigen-m Superantigen-mediated ediated T-cell activation

With respect to potential immunodeficiencies caused by e.g., chemical toxication, a working hypothesis can be presented about one potential sequence of events for the  penetration of intruding intruding staphylococcal staphylococcal strains via via the intestines: 1.  A toxic substance causes a leaky gut and temporarily weakens the defense system. 2.  A bacterial strain such as Staphylococcus sp. invades the body and forms capsules into the adipose tissue/intracellular space. 3.  The nutrient drain caused by inflammation etc. is replaced by storage lipids and staphylococci are liberated into the lymphoid tissues, and stay there. 4.  The bacterium is distributed to the entire body system where it spreads and breaks various tissues enzymatically. 5.  The capsules around the bacterial cellstoprovoke thebile cholecystokinin secretion and lipid the intestines are acidified e.g., due increased secretion [pH 1 was measured from the duodenal patient samples]. 6.  Osmotic pressure in the lymphoid tissue increases, causing swelling and thirstiness. 7.  As the bacterium attacks basically defenseless (with respect to innate immunity) tissues, B cells increase. When the situation calms down the cells die. However, B memory cells increase in number all the time causing superantigen formation. 8.  IgG production of B cells is based on T helper cells and their growth factors. T cells require a contact with bacterial cells, which do not form interaction through the lipophilic capsule (perhaps consisting partially of host lipids). 9.  Antibiotic treatments and high hygienic standards limit the t he direct contacts of T helper cells with bacterial cells. This can lower the IgG level slightly, but IgM acts

independently. The above-mentioned immunodeficient patient turned out to be a carrier of an internal total invasion of staphylococci, some S. haemolyticus  were isolated from his peritoneum (Hakalehto, 2015c). He also had a strong reaction with S. aureus  isolates from his stools. Another patient with corresponding symptomalogy also hosted bacterial strains exhibiting cross-reactions with the antibodies of the first patient. The latter patient was a 30-year old mother who had just delivered a baby and suffered more depressive traits. In fact, this kind of superantigen-mediated superantigen-m ediated profile has been characterized in literature (Rossi-George (Rossi-George et al., 2005). In individuals with decreased body defenses, the adverse effects of food spoilage or  poisoning can be extremely serious even with minor concentrations of the harmful strains or toxins. This kind of successions of pathogenic events may well describe an ever-increasing number of cases of bacterial infections with novel modes of pathogenesis. This malicious action could be a joint effect of toxicity, epigenetic influences, adverse or weakened immunodefenses, as well as chronic infections and inflammations in the gut and in other tissues, including the adipose tissue. Staphylococci are typical members of our normal flora, but the overstressing, chemicalized environment of modern urban societies may  provoke staphylococcal staphylococcal strains increasingl increasinglyy often to convert convert into devastating devastating pathogens.

 

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6. FOOD DEGRADATION, SPOILAGE AND ITS EARLY DETECTION  The human digestive system is subordinated to hormonal, humoral and neural regulation (Lyte, 2010). Similarly, the microbial metabolic system is directed by multiple control systems. This makes up pathways by which reaction sequences the bacterial or other microbial cells deal with their raw materials and make up the end products. The types of bacterial metabolism can be divided into: 1.  catabolism 2.  anabolism 3.  overflow metabolism These different modes function in degrading food, regardless of its composition or deposition. In the digestion the microbes take part in the enzymatic and other food disruption and utilization. They in a more or lessfor manner also in mmunities. spoiling foods or  beverages. Multip Multiple le or function mixed cultures are typical fosimilar r such situations and communities. co Often the joint processing along networks of pathways is called cascade fermentation (Schoen et al., 2009). Those substances which are difficult to get degraded are sometimes subjected to co-metabolic action by several microbes (Dalton et al., 1982). The antioxidants can also influence the shelf life of foods which can have an effect on their marketing (Eskin and Przybylski, 2001). These antioxidants can be divided into several categories (simplified from Eskin and Przybylski P rzybylski,, 2001): 1.  Primary A.  Phenols (gellate, hydroquinone, hydroquinone, etc.) B.  “Hindered” phenols (BHA, BHT, TBHQ, Tocophenols, Tocotrienols,

Plastochromano etc.) C.  Plastochromanols, Miscellaneous ls, (Flavonoids, Herb and spices extracts, carotenoids, lignans ascorbate, processing antioxidants etc.) 2.  Secondary (Thiopropionic (Thiopropionic acid (TDPA), Dilanryl and distearyl, Esters of TDPA etc.) 3.  Synergistic A.  Oxygen Scavengers (sulfites, ascorbic acid, ascorbyl palmitate, erythorbic acid etc.) B.  Chelating Agents (polyphosphates, EDTA, Citric acid, tartaric acid, phytic acid, lecithin etc.) C.  Miscellaneous (Nitrates, amino acids, flavonoids, carotenoids, tea extracts, zinc, selenium, lignans, ascorbate, processing antioxidants etc.)

7. DIAUXIC GROWTH  Since many common microbes express catabolite repression, they also produce growth with at least two acceleration periods, which mode of microbial growth is designated as the diauxic growth when glucose is the primary carbon and energy source (Narang and Pilyugin, 2007).

 

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In practice, the organism prioritizes glucose as a source of energy or carbon as long as it is available. Consequently, food degradation is often initiated by the degradation of the sugars available in the substrate. Corresponding to the organisms situation ininthe human intestinestheorsmall in a fermentation broth of a bioreactor, the bacterial food first exhaust carbohydrates, carbohydr ates, sugar moieties, and especially the glucose. In fact, in the human alimentary tract the host body is deprived from some of the monosaccharides due to their fast consumption by rapidly-growing microbes. Then, our digestion collects the energy-rich outcomes of the anaerobic microbial metabolism, such as organic acids and alcohols. Besides diauxic growth, also simultaneous consumption of different substrates or bistable growth are possible metabolic modes. If the food material is rich in more slowly degrading substances, such as plant macromolecules like cellulose, or alternatively, proteins and fats mainly from animal sources, different metabolic activities and microflora take part in the utilization. In case of high initial sugar content, the microbes often convert these carbohydrates into organic acids and the pH sinks. This sequence of events is often, but not necessarily  preventing the formation formation of toxic compounds from e.g., the the above-mentioned above-mentioned sources. As a matter of fact, these more complex metabolites are usually a result of secondary metabolism. This, in turn, takes place only after the prior exhaustion of sugar moieties. Food hygiene should not focus on food degradation only, but the understanding of it can help in developing preventive preservation means for commonplace practices. Various microbial metabolites come up at different time points of the degradation processes and they may explain different events during the spoilage. Consequently, these degradation events have effects on the health of the human host. Fermentation processes in food manufacturing come close to “controlled spoilage.” Moreover, the utilization of food wastes in biorefineries is often directed toward the formation of microbial metabolites (Hakalehto, 2015d).

8. SITES OR SPOTS OF FOOD CONTAMINATION ALERTS  If we consider the transfer of food substances to the consumer table, there are specific  points of interest with with respect to ruling ruling out a contamination contamination before it gets pathogenic. Here is a short list of proposed control sites in the food chain: 1.  animal or plant production facilities 2.  reception of raw material into food industry 3.   processing units and and unit oper operations ations 4.   packaging 5.  transport to the wholesale storage 6.  reception at the retailer shop 7.  setting the product onto the selves 8.   purchasing by a client 9.  storage in the refrigerator 10.  meal preparation 11.  dining

 

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As conscious professionals and/or consumers we tend to think about food quality almost “automatically” when coming across these situations. Often the visual inspection and our nose give a quick estimate of the food microbiological quality. In Finland, the official registers for 2013 recorded the total number for water or food borne outbreaks to be 47 (www.zonoosikeskus.fi). (www.zonoosikeskus.fi). In 43 cases (91%), the disease was transmitted by food, the total number of diseased persons being 793 in the epidemics. In water-borne outbreaks about 200 individuals fell ill. Out of bacterial agents, EHEC (Enterohemorragic  Escherichia coli) caused two less widely spread, but serious minor epidemics. The total number of patient cases in them was 20, with 15 individuals being hospitalized. According to the numbers of the foodborne patient cases, the most common etiological agents were Salmonella sp. and Bacillus cereus.

9. ENTEROBACTERIAL FOOD  OL I  O157:H7 POISONING AND E . C OLI  A wide variety of intestinal bacteria belong to the family  Enterobacteria  Enterobacteriaceae ceae. Many important indicator species, commensal strains as well as pathogens belong to this family. The close relatedness of the species in this family can be seen e.g., in the homologies of their  N-terminal sequences (Hakalehto et al., 1997). Also their type 1 fimbrial sequences bear many similarities (Clegg et al., 1987; Madison et al., 1994). Some members of the family, such as  Klebsiell  Klebsiella a pmeumoniae, can be both an important member of the gut microbiota of a developing infant and a serious respirative pathogen (Hakalehto, 2013a). Differences in  pathogenecities can thus depend on the ecological position and niche of the organisms, but also on the potential toxin formation. This is the case with respect to the  E. coli  O157:H7 variant, which is a foodborne pathogen capable of inducing hemorrhagic colitis or hemolytic uremic syndrome (Normanno, (Normanno, 2012). This organism is highly acid resistant, which ca cann help it in

1.  surviving in acidic foods 2.   passing the acid barrier barrier of the stomach stomach and reaching the the intestinal areas However, this acid tolerance is more than 1000 times more effective in the cells of the stationary phase than during the exponential phase. Consequently, metabolic, structural and toxin qualities of the contaminating strains can explain their infective behavior.

10. NATURE OF CONTAMINATIONS AND  A SHORT HISTORY OF THEIR DETECTION 

It has become evident from studies on the medical relevance of specific microbial contaminations that some taxonomical entities or species, such as  Klebsiella pneumoniae pneumoniae can  be either a harmless member of the normal flora or it can constitute a principal health hazard (Hakalehto, 2013a).

 

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Correspondingly, any organism can often be considered as a potential threat, but it’s manifestation as a contaminant should be estimated according to the highest risk level. Also the precautions, preventive measures, and surveillance should be planned accordingly. Especially, in the case of monitoring pathogens or potentially food-poisoning microbes, because of the multitudes of potential hazardous species, only the hygienic indicators are routinely screened. However, it is recommendable that the techniques used for this purpose could also offer a broader scope for monitoring true pathogens as well (Hakalehto and Heitto, 2012). Several examples on using the PMEU (Portable Microbe Enrichment Unit) technologies for rapid screening of pathogens include: 1.  Indication of septic contaminations which often initiate from intestinal leakages (Hakalehto et al., 2009; Hakalehto et al., 2010; Laitiomäki et al., 2015, Pesola and Hakalehto, 2015). 2.  Infections and contaminations of members of the family  Enterobacteri  Enterobacteriaceae aceae (Pesola et al., 2009; Pesola and Hakalehto, 2011; Pesola et al., 2012; Hakalehto et al., 2013b). 3.  Rapid diagnostics of the salmonellae (Hakalehto et al., 2007; Hakalehto et al., 2011) and the campylobacteria (Pitkänen et al., 2009; Hakalehto et al., 2014a). 4.  Detection of Gram-positive spore-forming bacteria, such as  Bacillus  sp. (Mentu et al., 2009) and Clostridium   sp. (Hell et al., 2010; Hakalehto and Hänninen, 2012; Hakalehto, 2015d). 5.  Shortened enrichment of clinical and environmental mycobacteria (Hakalehto 2013b; Hakalehto et al., 2014b). In order to rapidly identify e.g., septic contaminations, modern genetic techniques offer a selection for verifying specific infections amongst a big number of potential causative agents. Several commercial  products have been developed for that purpose (e.g., Bioquelle™). Also in the food sector such methods have been introduced during the recent years. One of the most widely used molecular biology methods is gene sequencing which can be

used for besides identifying e.g., microbes but also for characterizing specific genes (Sanger and Coulson, 1975). Nowadays, combining microdroplet PCR with flow-cell technologies  provides a fast approach approach for sequencing thousands thousands of genomic genomic targets (Kirkness, (Kirkness, 2009). 2009). However, if the genetic techniques such as PCR (Polymerase Chain Reaction) are used for the detection and identification, their limitations have to be kept in mind regardless of the many lucrative options offered by them. The limitations include the following:   disturbances of the enzyme kits by chemicals or physical conditions in the samples



may lead to false negative results   “picking up” of the desired  strains by the probes could be difficult due to many



dominating miscellaneous, commensal strains sample matrix of organic materials may also interfere with the determination   false positives may be caused by contaminated equipment or laboratory premises;   also by crossreacting caused by other strains sophisticated genetic methods require special skills from the user  

 

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  it is not always easy to isolate the causative agent for confirmation or further



characterization   complicated protocols and fine chemicals increase the cost of analysis. In a nutshell, the history of the development of food hygiene mainstream technologies could be roughly presented as follows:    before 1970: 1970: traditional cultivation methods methods   1970-85: ELISA-methods (this was boosted by the development of monoclonal 

 



      

antibodies) 1985-95: automated cultivation and screening machines (such as Bioscreen™ by Labsystems Oy in Finland) 1995-2005: ATP methods 2005-2015:: Genetic methods 2005-2015 2010-2015:: Mass spectroscopy and volatile sensing techniques 2010-2015

 Naturally, these periods overlap: the genetic techniques, for example, have been developed during many decades. The consideration above is an attempt to characterize the arsenal of methods in food hygiene determinations. The next period could well be the era of sensing the volatile emissions. This has in fact already started many years ago, e.g., in the form of MALTI-TOF MS (Jadhav et al., 2015). In the future, the diagnostics of food  poisoning and contamination control is to be developed also into the direction of increased automation and intelligent technology te chnology packages.

11. PRETREATMENTS AND DATA ACQUISITION  The microbiological detection protocol is a sequence of events, steps or phases

(Hakalehto, 2010; Hakalehto and Heitto, 2012). Each of them is equally important for the end result (Figure 5). A process surveillance of the type was uused sed for evaluating mixed microflora in the degradation of organic wastes from car washing, for instance (Hakalehto et al., 2013b). The effects of gaseous emissions from some cells onto the adjacent ones could be simulated in the PMEU (Hakalehto, 2011; Hakalehto and Hänninen, 2012; Hakalehto, 2013a). In the first and last references,  Escherichia coli  and  Klebsiella mobilis  were monitored. If these species are cultivated in the same vessel (together in one PMEU enrichment syringe) it seems evident that the  E. coli  exploited substances derived from the metabolism of  Klebsiell  Klebsiella a  (Hakalehto et al., 2010). Part of the substances were in the gaseous form. In the Canadian Journal of Microbiology it was described that the bacterial emission of CO2  boosted the onset of growth (Hakalehto and Hänninen, 2012). This fast onset of the  propagation of cells was proven to be due to the activation of both intraspecies and interspecies by the emitted CO2. The same effect has been used in a biorefinery process process (Hakalehto, 2015d). In fact, all a ll different areas of microbiological microbiological research stem from the adjustment and simulation of both the effects of circumstances on the microbes and the interactions between the strains:

 

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Elias Hakalehto 1.  Pharmaceutical microbiology 2.  Medical plant microbiology 3.  Clinical nutrition microbiology 4.  Food supplement microbiology 5.  Industrial food raw material hygiene 6.  Agricultural food microbiology 7.  Farm animal feeding microbiology 8.   Nutritional compounds compounds produced produced by microbes microbes of animal products products (e.g., milk) milk) 9.  Medical production in animals or cell lines 10.  Farm animal hygiene and health

Environmental microbiology is related to all fields since microbes are mostly derived from the “environment.”  The kaleidoscope model assists in the linking of any specific hygienic issue into a corresponding scientific discipline (Figure 6). That “mapping” or “orienteering” activity, in turn, is helping in finding out appropriate categories and correct terms for identifying the problems and addressing them to the public.

Figure 5. Phases (steps) of a diagnostic process in food microbiology. microbiology. For example, if a sample is collected from the wrong place, or preserved or stored with inadequate care, the entire chain of events in the diagnostic process is carried out more or less in vain, and may lead to falsified results. In order to avoid mistakes caused by the careless performance of personnel, unsuitable growth medium, inadequate capacity of the chosen detection method, poor sample quality, too long preservation of the specimens, or any other drawback or negligence of the diagnostic operation, the planning of the most sensible  procedure has to be exercised. In the PMEU PMEU technology, the results can be followed up when they develop. And the growth curves can be followed up in real-time. Data of the previous, and corresponding, diagnostic tasks and determinations can be compared with the current information. If artificial intelligence is used, the software could give predictions while the result is developing provided that such an early warning is required. This opens up new possibilities for diagnostic uses. For example, emerging epidemics could be spotted out from large numbers of results. This is based on, as indicated above, the exploitation of accumulating data from the diagnostic process.

 

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Figure 6. “Kaleidoscope model” of the microbiological  disciplines.

ISK LEVELS ASSOCIATED WITH SPECIFIC FOODS  12. HIGH R ISK

Besides on the aspects of the epidemiological triangle (Figure 1), food contaminations depend on and reflect around the local conditions, practices and traditions regarding food  production, as well as the food type. It is of high relevance, whether the meals consist mainly of meat and fish, or of fruits and vegetables, or of cereals. In fact, three global major types of intestinal microflora have been identified (Wu et al., 2011). The microflora types correlate with the diet, and they can be classified as follows:

1.  Protein and animal fat diet ( Bacteroides  Bacteroides) type of flora 2.  Carbohydrates ( Provotella  Provotella) type of flora 3.   Ruminococu  Ruminococuss type of flora  Some traditional food varieties may cause specific hygienic risks. For example unripened natural cheeses from different milk sources (cow, goat, sheep, camel etc.) could in some geographical areas contain endemic  Brucella  sources (Atluri et al., 2011). The topic of  Brucella sp as the hazardous industrial raw material, milk sources, will be dealt with in the

next part.” of the “microbiological  Hygiene” series, namely the “microbiological Industrial Hygiene Different  Brucella  species are related to various host animals. Brucellosis is usually acquired by consumption of unpasteurized milk products or by contact with infected animals. Half a million new cases are globally reported each year. It is thus advisable to avoid food  products made of of unpasteurized milk especially in the the endemic areas of brucellosis.

 

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Scromboid food poisoning is a condition caused by the consumption of spoiled or decayed fish food (Clark et al., 1999). One of the toxic agents in this disease is histidine, which is converted into histamine as a result of the action of the histidine decarboxylase enzyme which is produced by enteric bacteria. Since this compound is resistant to boiling or cooking, it is contaminating fish meals if the fish is preserved e.g., in too high temperatures  before the preparation preparation of the food food sorts. This food food poisoning poisoning resembles an allergic reaction. Another marine food poisoning that is common in the warm tropical and subtropical waters is Ciquatera (Friedman et al., 2008). It is caused by dinoflagellate toxins, such as ciguatoxin in the fish. This type of toxin is not destroyed by cooking, and therefore this dangerous form of food poisoning can be very treacherous. Although certain marine areas are endemic for this disease, it has been recently causing epidemics also e.g., in the New York City (Center for Disease Control and Prevention, CDC, 2013). In order to avoid this type of food poisoning it is important to educate the public health authorities, seafood suppliers and general public about this severe disease causing long lasting gastrointestinal, cardiovascular and neurological symptoms. For this illness, no specific treatment known, the use of intravenous mannitol been successfully used in some bothisacute and although chronic cases (Schnort et al., 2002; Mitchellhas 2005). Many cyanobacterial toxins may also contaminate seafood (Carmichael, 1994). Particularly risky meals could derive from some algal foods. The toxic cyanobacteria are found in fresh, marine and brackish waters (Sivonen, 1996; Dittman and Wiegand, 2006). Their neurotoxins can cause detrimental poisoning also in case of using contaminated water sources (Boyer, 2008). Moreover, Moreover, many waterborne illnesses and toxications relate to specific environmental environmen tal conditions, such as algal blooms (Armon and Starosvetsky, 2015). Similarly, dissemination of many bacterial contaminants especially in rural conditions could relate to such natural catastrophes as floods (Hakalehto, 2015a), or oceanic distribution of protozoan giruses (gigantic viruses) (van Etten, 2011) or other vehemently influential environmental, biological or physicochemical, or climatological conditions. In these circumstances, it is ofandcrucial importanceortofood-borne re-examine the procedures for their monitoring environmental health, the water-borne microbial diseases and routes of contamination into the food (Hakalehto and Heitto, 2012). One example of the postulated spread of Campylobacter   infection infection via contaminated food production chain was recorded in

trials in Burkina Faso where field capable PMEU method was applied for bacterial monitoring (Hakalehto et al., 2014a). See also Figure 7. Another microbiological toxigenic risk in the consumption of raw or insufficiently heat treated fish is the toxin formation by anaerobic Clostridium botulinum (Aberoumand, 2010). For example, hot smoking in too mild conditions (less than 65°C) does not inactivate or inhibit all bacteria. Even at higher temperatures bacterial spores may remain intact, and can cause bacterial growth or toxin formation after germination. The detection method development for C. botulinum is presented in chapter 10 of this book. Some wild animals can carry less familiar zoonotic agents, some of which are potentially deadly ones. For example, bats on the British Isles and in Finland have shown to carry  Bartonella bacteria (Veikkolainen (Veikkolainen et al., 2014).  No human cases have been reported so far due to small amount of physical contacts  between bats and humans. Researchers handle the bats with gloves. In the US,  Bartonell  Bartonella a mayotimonensis has caused lethal epidemics in man. It could transmit from the bat feces, and  by the fleas or flies or tick bites.  Bartonella sp. pathogens may be transmitted also from the

 

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domestic animals, such as cats, to other animals or man by vectors, such as fleas (http://www. cdc.gov/bartonella/veterinarians/).

According to Hakalehto et al., (2014a). Figure 7. Hypothetical routes of contamination of Campylobacter  sp.  sp. during the outbreaks in Burkina Faso.

The bats constitute about 20% of known the mammalian species with some 1100 specie globally. During recent times, the horrifying Ebola and Massa viral hemorrhagic fevers have received a lot of publicity (see the Economist magazine, October 18th-24th, 2014) (Leroy et al.,  2005; Towner et al.,  2009). The bats are also potential vectors of some milder viral infections, but could transmit rabies, too (Drexler et al., 2012). However, the role of bats as intermediate hosts of bacterial diseases is less studied.

13. HISTORICAL ASPECTS OF EPIDEMICS 

The order Rodentia is the only mammalian group which outnumbers the amount of the  bat species. Among Among the rodents, numerous bacterial bacterial zoonotic infections infections are known. One of the most famous one is the plague which has devastated mankind over centuries influencing strongly to the course of history and civilization as it spread also via dirty water and contaminated food sources. A citation from Wilson (2004) (p. 25): "In 1500 there were about 75,000 Londoners. By 1600 there were around 200,000 and by 1650 perhaps double that. Indeed, the persistent recurrence of the disease makes it all the more remarkable that the late was the timeand when English actually began. Every time thereit was sixteenth a plaque,century the theatres closed thethe actors and drama managers lost their income." In fact, was 1592 when William Shakespeare´s (1564-1616) plays were on stage in London for the first time (Chambers, 1930). Further citations, now from Scott (2013): "A mood of pessimism descended over western Europe during the 1590s. In the face of unabating religious wars and inflation, of massive

 

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 plague outbreaks and bad harvests, it seemed that humans could merely hope to endure the flux of time..." (p. 103) and .”..The courtiers themselves were no cleaner. When the court decamped to Oxford in 1665 to escape the plague, they scandalized one academic by leaving `theirThis excrements in every e veryofcorner..´..." (p. 212). "Great Plague" the 1660s was the last episode in Britain of this disease caused by Yersinia pestis, and originally distributed by fleas from the black rats ( Rattus rattus). These rodents arrived to Europe from the Orient in ships to e.g., Venice in the fourteenth century causing the epidemic "Black Death" killing roughly one third of the population starting from 1347. This bubonic plague is a lymphatic infection, usually resulting from the bite of an infected flea,  Xenopsylla cheopsis (the rat flea). In this sense, the poor food hygiene which favoured the rats, was the major indirect cause of the epidemics.

14. ASPECTS OF MICROBIAL DISSEMINATION  ELATED TO SOME FIELD HYGIENIC ISSUES  R ELATED In addition to the Ebola, Marburg, plague, several other hazardous diseases are transmitted by the insect bites. These include malaria (mosquitos as vectors),  Leishmania  (sandflies) and Trypanosoma (tsetse flies) protozoan illnesses (Hurd, 2003), and dengue fever (mosquitos) (Lee et al.,  2010). In the prevention of these diseases, cleanliness of water and food sources, and the eradication of breeding areas of the insects is one of the key targets. Mosquito larvae that get infected with  Bacillus thuringiensis var. israelensis die off, which has been used as an elegant way for biological control of malaria in many waterways and around them (Goldberg and Margalit, 1977). In poor hygienic conditions, flies can transmit also many ordinary causes of food  poisonings and gut infections. Also for their prevention, natural bacteriological means are available. For example,  B. thuringiensis strains can be used for biological control in outdoor lavatories in tropical countries, in cow B.houses etc. in severywhere 1986). This  biological control method with or different thuringiensis thuringiensi   toxins could(Carlberg, remarkably decrease the infection frequencies, because of the prevention of the pathogen distribution from the feces or manure to the tables via the flies. Consequently, also the warehouses of various foods remain more hygienic.

Leptospirosis is transmitted by the urine of infected animals, mostly rodents, and it is contagious as long as the urine is still moist (Guerra, 2009). It is caused by a spirochaetal  bacterium (spiral cell form). form). Its vehicle of transmission (the urine) emph emphasizes asizes the importance of high hygiene, and protected shelters and food stores are highly recommendable. Also water sources need to be protected. The spread of cholera is also related to the effects of similar hygienic measures (Hakalehto, 2015a). Campylobacteria were distributed via irrigation water into the vegetables and chicken feed, and further to human diet from the local markets in Burkina Faso, East Africa (Hakalehto al.,  2014a). also Figure 7. In a project work supervised by theetauthor at theSee University of Eastern Finland in Kuopio, commercialization potential of PMEU (Portable Microbe Enrichment Unit, designed by Finnoflag Oy, Kuopio, Finland) approach for water control in rural India was investigated (Project Report "The Story of Ice and Water," University of Eastern Finland, Faculty of Business, Social Innovation and Strategy Course Report 25.4.2012).

 

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A citation from the Kuopio report (“Finnoflag” in this context refers mainly to the PMEU technology): "Water is political issue in India as the government has promised free water to everyone. Also the government has woken up to humanitarian issues and the viewpoint has slowly changing from ´hard´ issues e.g., economical factors to ´softer´ issues. One of the main problems in political arena is quite high level of corruption. In environmental point of view, the main problem seems to be toxic water which on the other hand encourages Finnoflag’s vision  and mission in India. Also social factors e.g., tight housing, poor hygiene etc., encourages that there is need for Finnoflag in India." In a continuation of this student group work report it is stated: "It seems that there is a clear need for water testing in India. The technology used in India is far behind the one that Finnoflag offers. On the other hand, there is a high number of technically skilled people which provides demand for Finnoflag’s products and co -operative projects. The legal issues in India are causing problems to standard Western point of view. Different states have their own “legislative” systems: Various states have adopted legislation  that make panchayats can control over water supply at the local level. Old and new laws have no linkages. The old expertise and experience is not taken into account when defining new laws. In conclusion, there are both possibilities and threats in Indian environment and markets. Still, we saw the situation which encourages Finnoflag’s market entry." 

15. FIELD MICROBIOLOGICAL  HYGIENE –  FOODS FROM THE WILDERNESS  When hunting with rifles or shotguns, it is of crucial importance to estimate what is the impact of the bullet or shots on the killed animal. Well targeted bullets or shots will not unnecessarily tear the animal apart, or damage the digestive tract. The intestinal microbes can easily cause spoilage if the intestines are damaged. Therefore, the removal of the guts and other alimentary tract organs is field. On thetoother hand, it sometimes be more advisable to often leave carried this partout of in thethe game handling be carried outcan at home. For example, in wintertime the fishes or animals get frozen rather soon, and their skinning or other preparation could cause more damage hygienically if performed outdoors. For the quality of meat, it is important to shed the blood out as soon as possible, and to

remove badly damaged parts of the game animal. Washing of the internal cavities with water may initiate spoilage, and it should be avoided in most cases. Sometimes small branches of spruce trees are put inside the animal to prevent anaerobiosis. The wood material is also thought to give some aroma to the meat, and possibly the wood polyphenols could have a role in the preservation of the meat (see chapter 15). During the preliminary storage the meat is usually hanged to get ripened by the own enzymes of the animal (Figure 8). This improves the quality of meat prior to meal preparation, or the final storage. However, since this preliminary maturing is carried out during several days, the critical inspection of the meat is warranted. In Finland the annual moose hunt produces 10 Million kg of meat. The total consumption of meat is 419 Million kg (78 kg per citizen). About 100 Million kg of this amount is cow meat. The moose meat production is thus reaching important  proportions economically, not only in culinary sense. The reindeer meat production is an inherited right of the native Lappish people. It is aamounting mounting about 2 Million kg annually. The

 

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nutritional importance of the game meat should not be underestimated. The moose hunting in Finland is arranged in groups (clubs) which have their designated hunting territories with acquired permissions for the kills (Figure 9). The catch is transported from the forests often quite long ways to the game kitchens or equivalent. Some preliminary treatment has to take  place on site.

Figure 8. Hunted hare is ripened after the evisceration. In wintertime this does not cause microbiological problems provided the animal is healthy and clean. For hygienic purposes it is much  better if the bullets or shots have not permeated the digestive tract. Traditionally the hares are hunted by tripwires in Northern Eurasia and North America, also rifles and shotguns are used. Hare can carry some hazardous zoonotic pathogens, such as Francisella tularensis. This risk has to be taken into account in the endemic areas of that disease.

Figure 9. The result of moose hunt by a group of hunters belonging to a local club in Central Finland. Finland . After the successful result, the success has to be guaranteed by correct hygienic hygienic measures.

 

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Besides the actual pathogens, the meat or the entire wild animal can be contaminated. During one recent episode, a gorgeous Finnish deer ( Rangifer tarandus fennicus fennicus) (Figure 10) was caught and processed in a specific game kitchen unit, when it was detected to have a septic infection. The meat had to be discarded, and the facilities profoundly disinfected.

Drawing by Ronja Hakalehto.  Rangifer tarandus fennicus) is a large relative of reindeer. It is believed that Figure 10. Finnish deer ( Rangifer the first Finns arrived to their country in order to hunt this animal, which was an important source for meat, hides and bone utensils.

It is important to pay attention to the hunted or caught haul. If it looks out sick or otherwise the quality seems to be susceptible, immediate rejection and possibly destruction of the animal is recommended. Sometimes the evisceration can cause damage to the meat. Therefore, extra caution is needed when removing the intestines or other internal organs. Some of them, such as liver, can be collected and prepared as specialties of the game kitchen. In winter during under ice fishing with nets (Figure 6 in Chapter 1), the caught fishes usually stay alive which maintains their freshness for a longer time. Fishermen often keep crushed ice in their boats or sleighs for keeping the quality of the

catch high. Also fish chests are used for transporting the fishes alive. In case of pretreated catch (either on site or in the boat), salt is sometimes used for safeguarding the quality, if the keeping time is expected to get prolonged before the further processing of the fish. From ancient times folks prepared special food sorts out of their catches. One traditional fish course in the Eastern Finland is the "kalakukko" ("fish rooster" in English) in which the local lake fishes (usually perches or vendaces) are baked into a complete rye bread cover with some fat (Figure 2 in Chapter 2). Since the “kalakukko” is prepared in a hot oven, the fillings remain unspoiled for long periods of time even without refrigeration. This is of course the case only if the bread cover is kept intact. Also some vegetables, such as cooked turnips, have  been preserved with this method. In In old times, this approach was a handy way to hy hygienically gienically  produce and pack pack food for for long hunting hunting and fishing fishing excursions, excursions, for instance.

 

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16. CHALLENGES OF THE CONTROL  Integrated food sources and raw materials cause a challenging situation, as all potential routes of contaminations have to be checked. In industries this means strict control of:                   



           



raw materials water supplements air in the production units facilities and machinery transport chain  personnel and work clothing  packaging material material storages sterilization UHT  pasteurization, UHT etc. disinfection  product shell-life shell-life cold chain equipment for food preparation

One example of complicated hygiene control tasks is in chocolate manufacturing (http:// www.gmaonline.org/down www.gmao nline.org/downloads/technical-guid loads/technical-guidance-and-tools/Salm ance-and-tools/SalmonellaControlGuid onellaControlGuidance. ance.  pdf). In this production line, multiple raw materials are mixed with each other and each one has to be controlled. Many of the original stocks contain hygienically vulnerable substances, which offer a good growth medium for microbes. However, the microbiological control of the actual product and the production line may  be tedious as the spoilage microbes or pathogens may occur in small quantities or in resting forms. Another example of such hygienically complicated food commodity is ice-cream. It contains e.g., milk and eggs which are potentially high risk materials. In hot climates their hygienic control is a particularly demanding task. Not only is the hazardous microbe load

multiplying rapidly, but also the less meaningful flora may propagate fiercely. The latter is often overgrowing or shadowing the strains which are important from the viewpoint of hygiene control. In Burkina Faso, East Africa, the tracking of Campylobacter  sp.  sp. was successfully performed from water, food-chain and clinical samples regardless of the other flora (Hakalehto et al., 2014a; Hakalehto, 2015c). In Finland, the surveillance of the thermotolerant Campylobacter   spp. and adenoviruses from andwaters sewage has been considered as an important issue of public healthbathing becausewaters surface areeffluents often used for drinking water or for industrial production (Hokajärvi et al., 2013). In Malian hospitals, during the years 2000-2010, food borne illnesses accounted for 58.5% of all accidental ac cidental poisonings (Hami et al., 2013). The average age of victims was 19 and

 

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more than ¼ of these microbial illnesses occurred with children under the age of 6 years. Main sources of contamination were dengue, milk and smoked fish. It has been postulated that the human intestinal flora is built up into the community and configuration which will more or less remain persistent throughout the entire life span of an individual by the age of six (Hakalehto, 2012b). Consequences of antibiotic medications can be relatively quickly repaired (Pesola and Hakalehto, 2011). Consequently, the treatment of severe diarrhea or other episodic infective diseases of the digestive tract with effective antibiotics should be carefully considered. In  patient cases whose prophylaxis could involve involve a risk of initiating a chronic disease otherwise, it could be justified to attack the foodborne infections with appropriate antibiotics without delay. Naturally, it is also important to avoid provoking the formation of antibiotic-resistant  bacterial variants (Hakalehto, (Hakalehto, 2006). The PMEU method offers a tool to rapidly evaluate the situation in case of alimentary or other rapidly progressing infection (Hakalehto et al., 2007, 2009, 2010; Pesola et al., 2012; Hakalehto and Heitto, 2012; Laitiomäki et al., 2015). One potential method for avoiding tedious and persistent intestinal infections during early years of life could be the use of probiotics which dominate the nutrient utilization in the food digestate and chyme. They take part in these important nutrient adsorption reactions in the course of the entire digestive system (Hatakka et al., 2001; Hakalehto 2015c). Continuous supplementation of probiotics could then serve as a preventive measure for the abovementioned intestinal infections. In the environment, many indicator bacteria as well as pathogens are disseminated as a result of anthropogenic activities (Hakalehto, 2015a). The root causes of water biological  pollution, and consequently food contaminations and human or animal illnesses lie in inadequate waste management, purification and environmental monitoring.  Natural phenomena, such as floods, may cause the liberation of pathogens from manmade installations (Baig et al., 2012). These microbiological hazards need to be confined and contained in order to prevent water or food contaminations (Best et al., 1990). the bacterial causes Bacteriophages of foodborne infections, many viruses originatecoli from raw In or addition drinking to water (Armon, 2015). of such species as  Escherichia   or  Bacteroides fragilis fragilis have often been used as potential indicators of enteric viruses. In the case of food testing, it could be recommendable to use indicator viruses for the surveillance of fecal viral contaminations. This kind of approach could prevent many epidemics originating from contaminated foods. One example could be from Southern Europe where strawberries are irrigated with waste water. These strawberries were used as raw

material for jam that later disseminated a viral infection to hundreds of school-children and their teachers and families in Kuopio, Eastern Finland. Rapid screening of the potentially health-compromising health-comprom ising microbes, whatever their nature, is an essential prerequisite for effective food hygiene. Besides the heat treatment also al so irradiation is used for improving food product shell-life. shell-life. However, in this case, the effects of the preservation method on the nutritional food quality have toalso be taken into account. In general, at least the following changes in parameters follow from food processing (also as losses of nutritional quality) (modified from Birch and Parker, 1984).

 

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Elias Hakalehto Major factors: 1.  Dry matter (milling, peeling etc. leeching) 2.  Dietary fiber (milling, peeling etc.) 3.  Protein quality (excessive heat treatment) 4.  Sugar (leeching) 5.  Lipid (oxidation -> rancidity and loss of acceptability) Minor factors: 1.  Water-soluble vitamins (milling, peeling, etc., leeching, instability) 2.  Fat-soluble vitamins 3.  Minerals (milling, peeling etc.)

It was highlighted that milling of cereals leads to big losses of nutrients, which has a large effect on the overall nutrient content of the diet. Cereals represent approximately 1/3 of the total energy of the diet.proteins Nowadays, various trends have influenced the  proportions of carbohydrates, carbohy drates, and fatshowever, in our diets. diets.

17. METHODS FOR IMPROVEMENT –  LEARNING FROM THE PAST  The principle type of the diet is essentially influencing the gut microbiota (Hakalehto, 2012b). In fact, only three principal groups or patterns of digestive tract microflora composition have been identified globally (Wu et al., 2011). Therefore, a chain of influences can be identified: Fertilization/animal feed -> Raw material quality -> Processing -> Food type -> Microflora composition -> Health effects This kind of chain of events is behind our general health, aand nd bacteria and other microbes  position themselves into this chain. For example, some hidden infection in the teeth could have detrimental effects on health if remaining untreated (Pesola et al., 2015). Food quality and production influence these infections, in turn the overwhelming microbial presence and  physiological strain to the body then easily accelerate the health compromising effects.

Consequently, determined action on improving all or any phases or steps in the chain above could lead to improved 1.  hygienic level 2.  nutrition/food quality 3. 4.   health environment For example, it has been recorded that increasing conversion of forest land into agricultural use with alarming ecological and climatological consequences has become one of

 

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the major drivers of deforestation (Sonza, 2006). This development, in turn, leads to changing modes of microbiological communities in the ecosystems (Hakalehto, 2015a). In the forest ecosystems, the microbes of soil, trees, epiphytes, other vegetation, water and air phases are in constant circulation. In the Nature, this “microbiological “microbiological biosphere” contributes to the food  production environm entand with hygienic and health with effects. In the global ecosystems,and our environment life, health realdemonstrable standard of living are interlinked the well-being of natural communities of plants, animals and microbes. Local, ancient, rural or otherwise traditional cultures have learnt to live in harmony with Nature. One example of this is seen in the agricultural tradition of the Far East based on rice cultivation (Gladwell, 2008). Miniature land use on rice fields are based on inherited knowledge and wisdom regarding soil quality (and consequently about the hidden, invisible microbes!), microclimate, water circulation and many other factors. According to Gladwell this generation-to-generation generation-to-generation know-how on the food production at the origins of the food chains has essentially influenced the cultures and ways of learning and thinking in the corresponding Asian societies. It may have contributed also to the aboveaverage learning results in these countries. Similar wisdom can be found and recognized in all countries with respect food production andfor hygienic One example of atoconsiderable threat entire maintenance. global agriculture is the decrease of honey bees (Cox-Foster et al., 2007). These insects have sophisticated communities housing their hives into which they collect nectar from the nearby blossoming flowers (Figure 11). At the same time they pollinate the plants thus ensuring the crops. If the pollinating bees and other insects disappear, the results could be catastrophic also for the entire ecosystems. The root causes for the colony collapse disorder (CCD) hav havee been suggested to be:        



 pesticides diseases  parasites flowerless landscapes



   plant monoculture monoculture

Also other hypothetical causes, such as increase of electromagnetic radiation, have been suggested. Anyhow, Anyhow, it is of crucial importance not for the apiculture only, but for the survival of mankind to stop this kind of negative development. Besides the invaluable pollinating effect, bees are producing honey. As a sweetening agent, honey is used for preservation. Its crystallization during storage is dependent on the ratio between glucose and fructose. The higher the concentration of the latter monosaccharide, the longer the honey remains

in liquid form. In a Finnish village shop after the Second World War no last utility utilit y dates were marked on the unpacked sausages. Before the weekend all the sausages were washed and put into a large  barrel filled with salty salty water.

 

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Photo: Lauri Heitto. Figure 11. A beehive in the Finnish countryside is a biological indicator on human manifold impacts in food production chain.

Consequently, no contamination could spread into the products (since the microbial growth on meat, for example, takes place on the surfaces and slowly inward into the  products). Hence, the contamination control was not needed in this local small shop.  –   However, this approach to food hygiene is absolutely not recommendable in the modern societies, for mass consumption, or industrial food production, transport, storage and deliveries. In the modern context, we need many kinds of precaution and monitoring. In a way the today’s challenges  in the food hygiene are often encountered with inherited and learnt way of thinking, a “traditional mindset.” It has been stated that we often adopt short term goals that we strive to achieve but not necessarily to exceed (Kahnemann, 2011). One example of this “limited vision” in food production  and distribution is the overwhelming wasting of food in the urban societies, where the hygienically packed food products are manufactured with high energy demand and consumption of natural resources. This kind of wasting of the resources would have been inconceivable for the past generations. As a result, the sausages, for example, are produced from selected and controlled raw materials, packed in standardized conditions by highly educated work force to be transported in cold-chains from factory store rooms into dealers’ warehouses, to retail   shops, using refrigerated lorries. However, high percentages of these safeguarded food products ultimately  pass the last utility dates. Then the packages are removed in a waste treatment unit and the unwrapped food is discarded. In the best case it is used for bioenergy production, for instance. In any case, the spoilage

of food products is an immense economic problem, and also a moral issue in our global village where many of us live li ve their lives undernourished. Apart from being a question of justice or implementing high ethical standards, this matter has also technological and microbiological dimensions. The monitoring of food quality needs to be rationalized in order toIndeed, compensate the drawbacks of the over-rationalized oversimplified mass production. we need new fast tracking methods for foodand quality,  preferably based on real-time monitoring. monitoring.

 

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As crystallized by Tversky and Kahnemannn (1974): “These heuristics are highly economical and usually effective, but they lead to systematic and predictable errors. A better understanding of these heuristics and of their biases to which they lead would improve  judgements and decisions decisions in situations situations of uncertainty uncertainty.” .”  Since food microbiological hygiene is a tightly regulated field, it is clear that the methodology can develop only relatively slowly. This is true particularly with respect to the gradually accepted protocols for specific foods or micro-organisms. These methods are not the best ones, or most effective or most modern ones, but their long-term development has  been orchestrated by by big diagnostics diagnostics manufactures. Therefore, the current market is an outcome of the historical progress. Thus, many validated, accredited and accepted methods have emerged from the shadows of history, and many others have also disappeared. These shadows often reflect the borderlines between the above-mentioned big companies and relatively small research and development companies. The latter do not usually have the resources to widely distribute their innovations, not mentioning the costly efforts for acquiring the approvals needed for the marketing.

18. SOPHISTICATION OF CULTURE METHODS  Petri dish (or plate) was named after the German bacteriologist Julius Petri (Voswinckel, 2001). His idea of solidifying warm agar liquid onto plates, where this nutrients, growth factors and regulators containing liquid is forming solid growth medium after the agar is cooling. Normally the liquid is poured onto the plates at around 50°C, and it solidifies at about 45°C. Then it can be dissolved again by boiling. The microbial samples are inoculated and spread onto the agar surface, and the colonies arise from individual cells or clumps or aggregates of cells, after incubation period. By spreading the colonies onto new plates, colonies originating from single cells are obtained (Figure 12). This produces the pure cultures, which are the foundation of microbiology. Such famous pioneers as French Louis Pasteur, German Robert Koch and Ferdinand Cohn and Hungarian Ignaz Semmelweis can be considered as the fathers of microbiological hygiene (Hakalehto, 2006). Their findings often related to contagious disease, but since many of them are zoonoses, the link to food spoilage is most evident. Significant improvements have been to the Petri culture method. For example, the chromogenic dyes were implemented into the agar medium by Dr. Alain Rambach of Pasteur Institute in Paris (see www.chromagar.com). As in many other techniques of microbiological diagnostics, the cultivation of samples on selective media leads to the growth of desired

microbial cells as colonies. The chromogenic reagents are then processed by the microbial strains, and produce confirmative colour reaction onto the plates (Rambach, 1994). For example, CHROMagar™ ECC can be used for the simultaneous detection  and enumeration of E. coli and other coliforms in food or water samples (Figure 12).

 

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19. MICROBIOLOGY ON THE BORDERLINES  OF SURFACES, AIR AND LIQUID  John Tyndall’s experiments in England  in 1875 with “optically pure air,” which unintentionally provedpetri the dishes existence of hygiene microbes in the air, was giving leads for the development of fallout for the studies.

Figure 12. CHROMagar™ plates with  E. coli (pink) and Klebsiella mobilis (blue) colonies.

Later on Tyndall developed so called tyndallization, which facilitated the sterilization of  bacterial endospores containing solutions by sequential boiling and cooling for at least three times. Tyndall was in correspondence with Louis Pasteur, and this contact boosted the development of bacteriology remarkably (Conant, 1957). The experiments of William Firth Wells on the airborne mechanisms of pathogen distribution and air hygiene (Wells, 1955) is widely discussed in “Microbial Clinical Hygiene” (Hakalehto, 2015c).

20. ENZYMATIC R EACTIONS EACTIONS IN  LIQUID CULTURES FOR VERIFICATION  In order to facilitate faster growth, or a quicker onset of growth, as well as more complete recovery of various strains, liquid culture in selective broths could be used. Also, some generalized media, such as TYG (Tryptone, Yeast Extract, Glucose) can be applied for initial screening of micro-organisms. Urinary tract pathogenic  E. coli  has been screened and

identified by media containing beta-D-glucuronidase enzyme in forming the indicator color onto agar media (Menafi, 2000). This enzyme has been tested with automated sample collection system (ASCS) of the Coliline™ PMEU (Berner Oy, Helsinki, Finland) (Hakalehto et al., 2013a). The method has been developed for the automated control of the water departments (Hakalehto, 2011; Heitto et al., 2012; Hakalehto and Heitto, 2012). The coliform tests with PMEU units have been validated by the Finnish State Technical Research Centre (VTT) (Wirtanen and Salo, 2010).

 

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Further enhancement of the E.coli diagnostics and coliform analytics in the UTI (urinary tract infections) diagnostics have also been developed using sensors for volatile emissions (Pesola et al., 2012). All these methods and corresponding PMEU devices could be used for monitoring E. coli and other coliforms (Hakalehto, 2013a).

21. AUTOMATED MICROBIOLOGICAL METHODS  For large number s of hygiene control samples, many devices have been introduced, such as Bioscreen™ (Venus  et al., 1992; Cherotre-Vialette and Lebert, 2000). These systems normally can harbor only relatively small sample volumes, but offer a standardized testing  platform. In the PMEU technology, technology, also the sampling protocols have been automatized (Hakalehto and Heitto, 2012; Hakalehto et al., 2013a). Different gas condition can be tested, for example for the salmonellas (Hakalehto et al., 2007). After the enrichment, detection and identification steps, the microbial strains need to be further characterized. Real-time PCR has been used together with the PMEU system for validating the detection of Campylobacter  sp.  sp. (Pitkänen et al., 2009). This type of combined detection of food and water contaminants has been carried out in Burkina Faso, East Africa (Hakalehto et al., 2014a). Further characterization of bacterial isolates could be carried out using e.g., molecular characterization as carried out in the food isolates of E. coli in Slovenia (Trkov et al., 2014). In the processes for heat sterilization or treatment, other parameters besides the actual temperature determine the outcomes (Stumbo, 1965). As the term sterilization can be defined as “freeing o f the material of all microorganisms,” no such concept as “commercial  sterilization” or “partial sterilization” or “pasteurization” fit into this definition. However, they are commonly used   in the food industries. Even the canned foods are not sterilized with perfection. To avoid confusion, “acceptable level of contamination” means production rounds which have become contaminated, not the concentration of contaminants. Under moist heat bacterial cell death in population follows the logarithmic order (Rahn, 1945). This was explained by the death of a single cell to be due to the denaturation of a single key molecule. Naturally, in larger volumes it also becomes increasingly challenging to  produce identical thermal conditions in all parts of the product or process fluid. One solution could be using several simultaneous factors for eliminating or eradicating the microbes, but this also increases the unit costs for operation. Microbiological hazard analysis (HACCP) can  be used for determining the risks of a single hazardous microbe in specific foods. VTEC (verotoxigenic  Escherichia coli) is a threat in many products including the fermented sausages manufactured and processed from raw meat (Arinder and Borch, 2007; Arinder et

al., 2009). This bacterium can be present in raw meat and has a low infection dose, which increases the risk of hazardous infections. If different measurement points along the  production chain are used for determining the bacterial numbers, it is possible to predict mathematically the percentage of the contaminated final products (sausages). This underlines the importance of good process hygiene control in the quality assessment for safer food  products.

 

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22. SLAUGHTERHOUSE WASTE DEGRADATION  AND ITS IMPLICATIONS TO NUTRITION  During the ABOWE project of the European Union Baltic Sea Region, the Pilot A unit was tested in three different countries, namely Finland, Poland and Sweden (Hakalehto, 2015d). In the Swedish tests, the chicken manure from the Hagby farm in Enköping (owned  by Jenny and Sven-Åke Lundgren) and the wastes from the nearby Västerås ecological  poultry abattoir were tested with respect to their potential as raw materials for biorefinery  purposes (Anderson et al., 2015; Schwede et al., 2015). Utilization of these types of wastes had been studied earlier in the Finnoflag Oy’s laboratory in Siilinjärvi, Finland.  In these experiments rapid overnight step by step conversion of fats and other tissues was documented into: 1.  organic acids (1. day) 2.  alcohols (2. day) 3.  aliphatic compounds (3. day)  Naturally this timetable is an oversimplification, oversimplification, but it illustrates the potential of the methods developed by Finnoflag Oy for microbial cultivation and bioprocesses for the waste treatment and reuse purposes. In case of the chicken abattoir and farm, useful production ideas could be further developed (Hakalehto et al., 2015). Interestingly, this kind of multiple use of agricultural raw material sources is parallel to the activities of a microflora on food when it arrives into the digestion. The degradation takes place also during the spoilage. This digestive process is enhanced  by the host enzymes, enzymes, but the final outcome is highly highly dependent on 1.  food hygienic quality 2.  gut microflora Therefore, it must be taken into account that food hygiene is not just avoiding infections or toxic reactions, but it comprises also of the contribution of microbes on the food nutritional value. Our body system and its alimentary microbiome are maintaining a Bacteriological Intestinal Balance (BIB) which helps our system to optimally utilize the available nutrients (Hakalehto, 2011; Hakalehto, 2012b; Hakalehto, 2013a; Hakalehto, 2015c). During food spoilage, the prevailing microbes of the food materials, and sometimes from outside cause the takeover of the space starting to exhaust the nutritional factors available. Particularly in cases where the food substance is effectively pasteurized or sterilized or  preserved, the outside contamination may turn out more important factor than the indigenous

microflora.

23. HAZARD ANALYSIS  The spoilage of canned food is usually caused by anaerobic thermophiles, such as thermophilic species of the genus Clostridium  (Tortora et al., 2010). Radiation is often used

 

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for canned foods in order to achieve sterilization. For vegetative bacterial cells, the required dose for that purpose is 0,5-10 kGy (kiloGrays), for bacterial spores 10-50 kGy, and for viruses 10-200 kGy. In practice, low doses below 1 kGy are used to killing insects, or inhibit sprouting of stored potatoes. So called pasteurizing doses (1-10 kGy) are used on meats to critically reduce the numbers of pathogens. Doses above 10 kGy are often used to sterilize, or greatly lower the bacterial contamination in spices. Radiation is considered as effective and safe a method from the consumer point of view as the thermal treatments, but paradoxically it is often bringing along prejudices regarding the safety. In case of food microbiology, microbiology, we may apply the same ecosystem principle, as in case of e.g., intestinal microbiome, to comprehend the interactions of different microbes (Hakalehto, 2012b). The composition of the flora in any food substance is qualitatively dependent on the physicochemical parameters in it. For example, protein-rich foods maintain different microbial communities from carbohydrates. After being subjected to the modern industrial manufacturing processes many foods contain unexploited nutrient storages or can maintain the microbial metabolic activities. By  preservation techniques the relatively low microbial cell numbers are kept latent, or inactive. In fact, this cleanliness of newly prepared food product may lead to rapid spoilage when any microbial contaminant commences growth and active metabolism in a less competitive environment.

24. BIOLOGICAL TERRORISM  Some potential bioweapons have been listed, including highly pathogenic bacteria and viruses (Table 2). However, biological weapons or toxic agents can include also less lethal or directly harmful organisms. For example, crops or domestic animals can be damaged causing decreased food production or quality. It is also possible to use microbes that corrode fuel tanks or machines and instruments, for incapacitating the societies. Moreover, less pathogenic or poisonous micro-organisms could be reused for increasing short-term or long-term illnesses, or for spoiling water or food sources. The Portable Microbe Enrichment Unit (PMEU) could be used for rapidly screening and verifying these contaminations (Hakalehto and Heitto, 2012).

CONCLUSION  However horrendous the microbiological threat is, it cannot be hidden away if it is a real one. Throughout the centuries, in various geographical zones and in different cultures, the human race has survived by finding the most appropriate sources for the nutrition that are available. The avoidance of risks consists of careful planning of hygienic operations on the

 basis of acquired and accumulated information. This is the optimal way to feed the growing  populations, and for safeguarding safeguarding the diet qualities on the basis of m modern odern research. Microb Microbes es can be utilized for biotechnological waste management and in the production of fuels, chemicals and fertilizers. Sustainability and nutrient recycling promote health in a long run. More sophisticated but yet simple enough methods are needed for the microbiological hygiene monitoring.

 

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Table 2. List of some potential bioweapons according to Tortora et al., (2010). Milder pathogenic or food poisoning agents are lacking from this list. Also, no such agents as prions are included in these categories of most hazardous bacteria and viruses. On the other hand, M  Myyco cob bacter ium tub ube er culo ulosis is not ranked, although this bacterium has been approximated approxim ated to be responsible for more than 50% of the deaths caused by the contagious diseases in human history. Even today, the mycobacterial diagnostics is much needed for increased clinical, environmental and food safety (Hakaleht (Hakalehto, o, 2013a; Hakalehto et al., 2014b; Hakalehto, 2015a; Hakalehto 2105c). Even though this bacterium is growing slowly, it could be spread internationally to cause terror. Especially the antibiotic resistant strains (MDR, XDR) are extremely potent agents of natural or man-made devastation

Bacteria  Bacillus anthracis  Brucella sp. Chlamydophila psittaci Clostridium botulinum toxin Coxiella burnetii  Francisella tularensis  Rickettsia prowazekii Shigella sp. Vibrio cholera Yersinia pestis

Viruses  polio measles encephalitis viruses influenza A monkey pox nipah virus small pox yellow fever Ebola, Marburg, Lassa and other hemorrhagic fever viruses  plague

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Rossi-George, A.; Urbach, D.; Colas, D.; Goldfarb, Y.; Kusnecov, A. W. Neuronal, endocrine, and anorexic responses to the T-cell superantigen staphylococcal enterotoxin A: dependence on tumor necrosis factor-alpha. J. Neuroscl .;.; 2005, 25, 5314-5322. Sanger, F.; Coulson, A. R. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J. Mol. Biol  Biol .; .; 1975, 94, 441-448. Schnort, H.; Taurani, M.; Candy, H. Ciquatera fish poisoning: a double-blind randomized trial of mannitol therapy. Neurology; 2002, 58, 873-880. Schoen, C.; C.;  Schulz, A.; A.; Schweikart, J.; J.;  Schütt, S.; S.; von Baehr, V.  V.  Regulatory effects of a fermented food concentrate on immune function parameters in healthy volunteers.  Nutrition  Nutritio n; 2009, 25, 499-505. Scott, D. Leviathan. The rise of Britain as a world power . William Collins, London, UK; 2013. Sharma, S.;  S.;  Raghuvanshi, S.; S.;  Jaswal, A.;, A.;,   Shrivastava, S.; S.;  Shukla, S. Lead acetate-induced hepatoxicity in wistar rats: possible protective role.  role.   Sivonen, K. Cyanobacterial toxins and toxin production. Phycologia; 1996, 35, 12-24. Sonza, C. M. Mapping land use of tropical regions from space.  PNAS ; 2006, 39, 1426114262. Stumbo, C. R. Thermobacteriology in food processing . Academic Press; 1965, London and  New York, 236 p. Schwede, S.; Thorin, E.; Lindmark, J.; Klintenberg, P.; Jääskeläinen, A.; Reijonen, T.; Suhonen, A.; Laatikainen, R.; Heitto, A.; Hakalehto, E. Using slaughterhouse waste in biochemical based biorefinery biorefinery - results from pilot scale tests, 2015; Manuscript submitted for publication. Tortora, G. J.; Funke, B. R.; Case, C. C.  Microbiol  Microbiology ogy –   –  an   an Introduction. Pearson Benjamin Cumminys, San Francisco, US; 2010. Towner, J. S.; Amman, B. R.; Sealy, T. K.; Carrol, S. A.; Corner, J. A.; Kemp, A.; Swanepoel, R.; Paddock, C. D.; Balinandi, S.; Khristova, M. L.; Formenty, P. B.; Albarino, C. G.; Miller, D. M.; Reed, Z. D.; Kayiwa, J. T.; Mills, J. N.; Cannon, D. L.; Greer, P. W.; Byaruhanga, E.; Farnon, E. C.; Atimnedi, P.; Okware, S.; KatongoleMbidde, E.; Downing, R.; Tappero, J. W.; Zaki, S. R.; Ksiazek, T. G.; Nichol, S. T.; Rollin, P. E. Isolation of genetically diverse Marburg viruses from Egyptian fruit bats.

 PLoS Pathog  Pathog .; .; 2009, 5 (7), DOI:10.1371/journal.ppat.1000536.

Trkov, M.; Rupl, T.; Žgur -Bertok, D.; Trontelj, S.; Avguštin,  G.; Avguštin, J. A. Molecular characterization of  Escherichia coli  strains isolated from different food sources.  Food Technology and Biotechnology; 2014, 52, 255-262. Tversky, A.; Kahnemann, D. Judgement under uncertainty: heuristics and biases. Science; 1974, 185. Van Etten, J. L. Giant viruses. American Scientist  Scientist ; 2011, 99, 304-311. Veikkolainen, V.; Vesterinen, E. J.; Lilley, T. M.; Pulliainen, A. T. Bats as reservoir host of human bacterial pathogen,  Bartonella mayotimonen mayotimonensis. sis.  Emerging infectious diseases; 2014, 20, DOI: 10.3201/eid2006.130956. Wells, W. F.  Airborne  contagion and air hygiene. Harvard University Press, Cambridge, Massachusetts, US; 1955. 423 p.

Venus, J.; Idler, F.; Albrecht, C. New ways of selecting lactic acid bacteria for  biotechnological processes. processes. Applied Microbiolog Microbiologyy and Biotechnology Biotechnology; 1992, 37, 240-243. Wilson, A. N. London  London –   –  A  A short history. Orion Books, London, UK, 166 p.; 2004.

 

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Wirtanen, G.; Salo, S. PMEU-laitteen validointi koliformeilla (Validation of the PMEU equipment with coliforms, in Finnish). 2010; Report VTT-S-01705-10 VTT-S-01705-10, Statement VTT-S02231-10. Vosvinckel, P.; Petri, J. R.  Neue Deutche Biographi Biographiee (NDB)  (In German). Duncker and Humblot, Berlin; 2001, 263-264. Wu, G. D.; Chen, J.; Hoffmann, C.; Bittinger, K.; Chen, Y.-Y.; Keilbaugh, S. A.; Bewtra, M.; Knights, D.; Walters, W. A.; Knight, R.; Sinha, R.; Gilroy, E.; Gupta, K.; Baldassano, R.;  Nessel, L.; Li, H.; Bushman, F. D.; Lewis, J. D. Linking Long-Term Dietary Patterns with Gut Microbial Enterotypes. Science; 2011, 334 (6052), 105-108.

 

In: Microbiological Microbiolo gical Food Hygiene Editor: Eino Elias Hakalehto

ISBN: 978-1-63483-646-3 © 2015 Nova Science Publishers, Inc.

Chapter 4

FOODBORNE VIRUSES  R ober t A r mon Faculty of Civil and Environmental Engineering, Technion Israel Institute of Technology, Haifa, Israel

ABSTRACT  Similar to water, food may also be contaminated with human viruses, originating from human faecal material. Most of foodborne viruses belong to enteric viruses group that clinically manifest as acute gastroenteritis in infants and adults. In spite of viruses inability to multiply in food contrasting bacterial pathogens that can reach high numbers following contamination, they have a low infectivity dose facilitating infections at low numbers. Since food is preserved under refrigeration, to maintain reduced bacterial growth, low temperatures favour viruses thus surviving for extended periods. Among the global foodborne viruses, noroviruses, Hepatitis E virus and rotavirus are the most widespread. This chapter describes the various foodborne viruses, their potential outbreak in large population and ways to prevent foodborne viral diseases.

1. INTRODUCTION  Water and food are the two main components that in their absence humans will perish, therefore they are defined as the two most important life supporting elements. In absence of water, humans can survive only several days (2-3) but in absence of food a person can survive for weeks (3-5). Our body intake requirements also dictate the quality of water and food from the hygienic point of view. For example, water that is essential for survival should be free of any contaminants at any point in time. This is also correct for food, but in this case we have a much higher choice to select from in the event of contamination and still provide protection in those cases. Another major difference between water and food lays on production sources and treatment chain. Water is basically produced from several main sources: i.e., groundwater, rivers, and lakes or following desalination and further up scaled via a certain treatment sequence in order to provide safe water. Food is produced from two principal sources: plants

and animals that have a much larger variability. Food production processes are much more

 

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versatile and diverse along the continents in comparison to water, consequently potential viral contamination has a higher risk, particularly that many foods are handmade or in direct contact with human hands. Maunula and von Bonsdorff (2014) pointed out the following statement: “ongoing changes in food commerce and production from national to internationally-distributed foodstuffs are leading to widespread infections and multinational outbreaks. Recent large viral outbreaks have been linked to oysters, frozen berries and semidried tomatoes.” Among foodborne microorganisms that infect humans, viruses are the smallest organisms (on the nanoscale). In contrast to bacteria, viruses as obligatory parasites unable to multiply when present in food and the degree of infection is related to the degree of food contamination (meaning the absolute viral particles number). The term infectious dose (ID) can differ from virus to virus and can range from few PFU (Plaque forming units) to > 100 PFU (Flint et al. 2009; Martin, 1978). In this aspect, bacteria in small numbers can  proliferate and reach high high numbers numbers based on their capability to utilize food food components, components, while viruses don’t. The origin of all foodborne viruses are humans and animals intestines and as such viruses are often shed in faeces or other body fluids. The transmission through water and food is difficult to distinct hence to obtain sound epidemic evidences, therefore some data is reported as water/foodborne transmission (Table 1). Foodbornee transmission of viruses occurs through three main routes: 1) contamination of Foodborn food by infected food handlers due to poor hygienic practices; 2) contact of food with animal waste, human sewage or sewage-polluted water: 3) consumption of products of animal origin contaminated with viruses (e.g., meat, fish etc.).  Foodborne viruses are of public concern due to their low infectious dose, e.g., the probability of infection from ingestion of one rotavirus virion is 31% and ~ 1PFU can cause infection in 1% of healthy adults never exposed to this virus (Schiff et al. 1984). Haas et al. (1993) calculated the risk of infection with enteric viruses when consuming drinking water is 10- to 10,000-fold greater than that for pathogenic bacteria at similar exposures (Haas et al. 1993). When dealing with food, the infective dose number should be lower due to food nature and interaction with viruses, though it should not be ignored that certain food components can provide an improved and affable environment for viruses compared to water. A good example related to foodborne diseases are the large variety of fruits and vegetable consumed mostly raw and shellfish that are consumed mostly steamed (Appleton and Pereira, 1977). In this regard, shellfish are among the most efficient biofilters, able to pump large volumes of water, thus retaining small particles like bacteria and viruses in their digestive system. Viral outbreaks from shellfish consumption have been well documented (Diez-Valcarce et al. 2012; Gormley et al. 2011). 2 011).

2. FOOD PRODUCTION TRAIN  There are two major sources of human food: sea and land. Fishing industry is still one of the major source for food in maritime countries harvesting different products such as: fish (a large geographical variety), shellfish (e.g., crustacean and molluscs), roe, Echinoderms (e.g., sea cucumber). In comparison with land production, beside hatcheries and sea farming most of the food products grow without human involvement, but it is very much impacted by two factors: pollution and overfishing.

 

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Food land production is much different and requires human involvement that developed for hundreds of years to an advanced step where we can control quantities and qualities. For example, cereal production is based on natural and genetic selection in order to produce the utmost and sturdiest crop able to face tough weather and vermin! Along with this tremendous scientific development able to support practically all global population without starvation, an increase in pollution has been also associated due to rapid industrial development. Beside chemical and physical pollution, the biological one is the one referred to in the present chapter. Figure 1 briefly presents the food production chain from raw material to consumer table. Along this trail there are many points where viruses may come in contact with food causing different diseases. As mentioned before, human domestic sewage is the main source of viruses, excreted from sick people. Viruses are faecally excreted in large numbers, therefore very extensive sewage treatment should be applied in order to reduce viruses’ number through inactivation.

Figure 1. Food production chain (From CDC, http://www.cdc.gov/foodsafety/outbreak http://www.cdc.gov/foodsafety/outbreaks/investigatings/investigatingoutbreaks/production-chain.html, outbreaks/production-chain .html, retrieved 2015.02.02).

Effluents, the last product of sewage treatment process should be free of human enteric viruses in order to be allowed for reuse in irrigation and other practices. In malfunction cases, crop contamination with poorly treated effluents is one of the route to cause outbreaks involving fruits and vegetables consumption (Brassard et al. 2012; Maunula et al. 2013). The same effluents if disposed in sea/ocean, may also reach the sea fauna (e.g., seafood) that in turn may concentrate viruses internally to reach high numbers.

 

 

Table 1. Global Foodborne transmission and supporting evidences [Modified from Anonymous (2003); Anonymous (2008); Koopmans et al, 2003] Country USA Australia  Netherlands

Population size (approx.) 300 million 20 million 16 million

Viral Infections (x 103) 9200 470 90

Burden of viral illness 1 in 33 1 in 45 1 in 178

UK  New Zealand Japan

60 million 4 million 126 million

77 17 13.5

1 in 780 Not estimated 1 in 9333

Denmark 5.5 million England and wales 56 million Finland 5.47 million France 66.6 million Germany 80.7 million Italy 60.7 million Slovenia 2 million Spain 46.4 million Sweden 9.7 million  Netherlands 16.9 million 1  Crude estimates and extrapolations 2  Range (not shown) calculated based on 2 investigated years only. 3 The time range of reports covers the period of 1995-2000.

Total outbreaks

17 290 58 28 227 2 14 14 190 41

Food/waterborne outbreaks (%)

16(94) 20(7) 14(24) 28(100)

2(14) 1(7) 7(17)

Source Mead et al. (1999) Hall et al. (2005) de Wit et al. (2003) (2003) Kreijl et al. (2006) Adak et al. (2002) Lake et al, 2000 Report from Ministry of Health, Welfare and Labour Lopman et al. (2003) Lopman et al. (2003) Lopman et al. (2003) Lopman et al. (2003) Lopman et al. (2003) Lopman et al. (2003) Lopman et al. (2003) Lopman et al. (2003) Lopman et al. (2003) Lopman et al al.. (2003)

 

 

Figure 2. Viruses classification and their biochemical and physical properties (from http://www.nlv.ch/Virologytutorials/Classification.htm).

 

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Here we face another bottleneck as shellfish are consumed almost raw, therefore enteric viruses are practically not inactivated. On the land production, animals for meat have the same problem and continuous veterinary surveillance surveillance is eessential. ssential. Perhaps the best example is the 1989 global episode of Bovine spongiform spongiform encephalopathy (BSE), the notorious mad cow disease, a disease that causes a fatal neurodegenerative neurodegenerative illness (encephalopathy) in cattle. The disease is easily transmitted to humans by means of meat consumption originating  – Jakob from infected animals. In humans the pathogen express itself as Creutzfeldt –  Jakob disease (vCJD or nvCJD) with similar symptoms (Anonymous, 2009). In 2014, 177 people were killed in UK and 52 elsewhere by an organism recognized today as prion (Anonymous, 2009; Armon and Cheruti, 2012). Beside human life loss, the economic burden was huge: 4.4 million cows had been slaughtered along the eradication program (Brown 2001). Another problem at that time was that between 460,000 to 482,000 BSE  – infected infected animals had entered the human food chain before governmental control measures had been introduced (Valleron et al. 2001). Along the food production chain, there are other vulnerable location prone to viral contamination such as: processing, distribution, retail, restaurant and home. At present, these locations are guarded by good personal hygiene, continuous cleaning and disinfection. Reviewing the vast literature on foodborne viruses outbreaks it is clear that the human factor is the most important one revealing that one infected person can spread viruses to a large number of people simultaneously, causing severe mass outbreaks (Stevenson et al. 1994; Franck et al. 2015; Lee and Greig, 2010).

3. FOODBORNE VIRUSES  Most of foodborne viruses belong to enteric viruses that infect both humans and animals, where some human strains are closely related to animal strains (Mead et al. 1999). These viruses are of particular interest as they are transmitted and acquired by the faecal-oral route and are human- specific (though animal strains of the same virus may also exist and evolutionary it is reasonable that they originate from animals) (Cliver 1997a,b). As such, these viruses cross the digestive system owing stability at low pH-s and in the presence of lipid solvents, and resist freezing and drying. It should be mentioned that most of known viruses involved in food and waterborne are naked viruses. The lack of envelope seems to impart these viruses with higher resistance to inactivation (Yates et al. 1987). Figure 2 represents the classical classification of viruses according to their biochemical  properties. Foodborne Foodborne viruses are relatively small viruses with an icosahedron structure containing a small RNA/DNA (from 5-8 kb, except adenov a denovirus irus with 36-38 kb).

4. NORWALK VIRUS (AKA NOROVIRUS)

 Norwalk virus owes its name after the geographical site where it caused an acute gastroenteritis in November 1968 among children of elementary school in Norwalk, Ohio, USA. Norwalk like virus known by this name till 2002 is now classified as Norovirus which

 

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81

 belongs to Caliciviridae   family. As being the only species of Norovirus genus, the name  Norwalk virus virus is basically a synonym, synonym, less frequently frequently used in the the media.

4.1. Norovirus (Formerly Norwalk Virus)

 Norovirus has been previously known as Norwalk virus and famous for its contagiousness. The particle size is between 27 to 38 nm and contains a positive-sense RNA genome. Norovirus has a high estimated mutation rate compared with other RNA viruses. Transmission occurs by contact with an infected person, contaminated food and water as a s well surfaces. The clinical symptoms of an infection manifest as acute gastroenteritis with stomach  pain, nausea, vomiting and diarrhoea. Illness in most people pe ople infected with norovirus will last from 1 to 3 days. Norovirus infectivity is high with no age difference and may occur several times along a life time (see above the high mutation rate). In USA alone, it has been rated as the highest acute gastroenteritis with ~20 million cases, ~60,000 hospitalization and ~685 deaths. Most of these outbreaks occur in the food service settings like restaurants (at least in US). Infected food workers are frequently the source of the outbreaks, often by handling bare hands ready-to-eat foods, such as raw fruits and vegetables (Maunula et al. 2013). Norovirus outbreaks may also occur from foods, such as oysters, fruits, and vegetables possibly contaminated at their source (Linco and Grohmann,1980). Another frequent infection source are cruise vessels, mostly attributed to person to person transmission and spread.

4.2. Hepatitis A Virus (HAV)

Hepatitis A virus has been classified as a new genus  Hepatovirus  of the family Picornaviridae. In contrast to other picornaviruses that causes gastroenteritis, HAV owes hepatotropism characteristic as infection site (causing mild to severe illness) and an exceptional stability to acid and heat. WHO estimates 1.4 million global cases of HAV infections each year. Ingestion of contaminated water and food are the main routes of infection but also direct contact with an infectious person can also transmit the virus especially in areas with poor sanitation (Dentinger et al. 2001). Virus reservoirs are humans,  both symptomatic symptomatic and non-symptomatic. non-symptomatic. A well-known route of infection is via sewage contaminated shellfish beds that are not  previously depurated but marketed without health inspection (Melnick 1995). In contrast to other picornaviruses, HAV may cause to serious health problem (even leaver cancer) and vast economic damages due to long convalescence periods. Perhaps the best example of HAV contagiousness is the Shanghai 1988 outbreak that had an overall attack rate of 4083/100,000  population (with 292,301 cases) cases) (Halliday et al. 1991). A case-control study of 1208 matched  pairs revealed that clams were the vehicle of transmission (OR=9.47, p≤ 0.001). Additional evidences were obtained from virus presence in clams sold at Shanghai markets and the three  peaks of consumption consumption curves co correlating rrelating remarkably remarkably with epidemic epidemic data!

 

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4.3. Rotaviruses

Worldwide infant gastroenteritis is caused by Rotaviruses. In developing countries fatalities are common among infants < 5 years old, though in general adults can also be infected with Rotavirus though the disease manifests itself from mild diarrhoea to unapparent. Food and water outbreaks have been reported in a number of countries. In New York (between 1985-1990) 11 food outbreaks were reported with 460 cases of rotaviral gastroenteritis. These cases were associated with food service premises implicating salad, cold foods, shepherd’s pie and water/ice. In Japan, a large – scale scale rotaviral outbreak has been reported in primary school (>3000) (Matsumoto et al. 1989). The immediate suspect was a central facility that prepared school lunches, but direct evidence from food and water was not obtained in this area. Other countries reporting in the past on waterborne rotaviral outbreaks are: Germany, Israel, Sweden China and Russia. It should be emphasized here that one of the main difficulty from the epidemiological point of view is to separate the two main sources of infection: water and food when coming to trace the viral source. Beside contaminated water and care-givers, food handlers are also a source of infection. Currently we are able to grow this virus group in tissue cultures to confirm their viability  beside immunological methods methods that can detect past infections and presen presence ce of viruses in food. The infectious dose is < 100 virions and keeping in mind that an infected person can excrete 10 up to 10  PFU/g of faecal material, this is a very low infective dose! Human rotaviruses can survive for several weeks in river water at 4 and 20 oC. Heat inactivation at 50oC for 30 minutes will reduce infectivity by 99% (two orders of magnitude) thus normal cooking temperatures should be sufficient for inactivation. Another mean of inactivation is pH alteration to 10.0 that will cause a rapid loss in infectivity.

4.4. Astroviruses

Astroviruses are still unclassified viruses that consistent with their structure they should  be located beside Picornaviridae (Group IV, (+) ssRNA, see Figure 1). Astrovirus contains a single positive strand of RNA of about 7.5 kb surrounded by a protein capsid of 28-30 nm in diameter. It has a typical five or six pointed star shape as observed by electron microscope. In UK, at least five human serotypes have been already identified, and some strains are culturable in tissue cultures. Similar to rotaviruses, astroviruses can also infect animals and humans and cause gastroenteritis. In adults, astroviruses can cause mild infection but are mostly associated with infants ( 50 days (i.e., Hepatitis A virus-HAV). Pathogens presence in food is mainly due to contamination, defective food treatment and disinfection (sterilization or pasteurization).

 

 

Table 2. Domestically acquired foodborne bacteria, viruses, protozoan parasites and cyanobacteria linked to human diseases in USA and France and estimated annual number of hospitalizations and deaths (where available) (Scallan et al. 2011a,b; Nelson and Williams, 2007; Carmichael and Falconer, 1993; Rocourt et al. 2003; Saker et al. 2005; Vaillant et al. 2005) Bacteria 

Country/case/   (death) 

Viruses 

Country/cases/   (death) 

Protozoan parasites 

Country/cases/   (death) 

Cyanobacteria  

 Listeria monocytogenes monocytogenes

 Norovirus

USA/14,663/( USA/14,663/(149) 149)

Cryptosporidium sp

USA/210/(4)

Spirulina platensis 

Staphylococcus Staphylococ cus aureus4 

USA/1,455/(255)a  France/300/(80) USA/1,064/(6) USA/1,064/ (6)

Hepatitis A

USA/99/(7) France/60/(2)

Giardia intestinalis

USA/225/(2)

 Aphanizomenon  Aphanizomenon  flos-aquae 

 Bacillus cereus cereus 4 

USA/20

Hepatitis E

Cyclospora

USA/11

cayetanensis

Adenovirus (Enteric)

 Bacillus anthracis anthracis

Clostridium botulinum 4 

USA/42/(9)

Rotaviruses

Clostridium perfringens 

USA/ 438/(26)

Nipah virus

Clostridium difficile 

 Entamoeba histolytica

USA/348

 Balantadium coli coli Toxoplasma gondii 

USA/4,428 /( 327) France/~400/(40) 95% due to toxoplasma

Protozoan parasites

Country/cases/ Country/cases/ (death)

SARS-causing Coronavirus 2 

Salmonella sp.

USA/ 19,336/(378) France/ ~8,000/(~300)

Shigella sp. Campylobacter  spp  Escherichia coli coli 0157:H7 Yersinia enterocolitica   Brucella spp Vibrio cholerae 

USA/ 1,456/(10) USA/ 8,463/(76) France ~3,000/(~13) USA/12

Highly Pathogenic Avian Influenza virus (HPAI) 2  Enterovirus Astrovirus

USA/87

Sapovirus

USA/87

USA/533/(29) USA/55/(1) USA/2

 

 

Bacteria Vibrio parahaemoly parahaemolyticus ticus  Vibrio vulnificus Corynebacterium Corynebacter ium ulcerans1  Y. pseudotuber pseudotuberculosis culosis  Coxiella burnetii or Q fever 1   Plesiomonas shigelloides 1   Aeromonas hydrophila,  Aeromonas caviae,  Aeromonas  sobria3   Pseudoalteromons  Pseudoalterom ons tetraodonis  Pseudomonass sp.4   Pseudomona

Country/case/ (death) USA/100/(4) USA/93/(36)

Viruses

Country/cases/ (death)

Cyanobacteria

a

 –  Country/  Country/ Hospitalizations, mean (90% credible interval)/ Deaths, mean (90% credible interval). Less common infection agents. 2  Emerging viruses. 3 Emerging bacteria. 4 Enterotoxin producers. 1

 

 

Table 3. Traditional and emerging pathogens in meat, poultry and shellfish (Mor-Mur and Yuste, 2010; Behravesh et al. 2012) "Traditional pathogens" 

Source  

Illness

"Emerging Pathogen Pathogens" s" 

Illness 

Contaminated eggs, poultry, meat Beef, poultry

Gastroenteritis

Gastroenteritis

Staphylococcus Staphylococ cus aureus

Raw and undercooked poultry  Fresh and frozen meat and meat  products

 E. coli

Contaminated food, especially undercooked ground beef

Salmonella spp. Clostridium perfringens Campylobacter spp.

Campylobacter jejuni (O:19, O:4, O:1), Campylobacter Campylobacter Campylobact er lanienae

Bacterial enteritis Salmonella typhimurium (DT104, DTU302), Salmonella enteridis (PT4, PT8,

Gastroenteritis

PT13, PT14b) Enterohemorrhagic  E. coli  O157:O7(EHEC)

Gastroenteritis

 Listeria monocytogenes monocytogenes

Gastroenteritis

 paratuberculosis   paratuberculosis  Aeromonas hydrophila hydrophila

 Bacillus cereus cereus

Vibrio vulnificus and Vibrio  parahaemolyticus   parahaemolyticus

meats and poultry Sauces, meat leftovers Raw or undercooke undercookedd shellfish,  particularly raw raw oysters

Gastroenteritis Gastroenteritis Gastroente ritis and emetic toxin syndrome

 Enterobacter sakazakii sakazakii

Chronic reactive arthritis

Hemorrhagic colitis, hemolytic uremic syndrome, thrombotic

 Mycobacterium  Mycobacteri um avium subsp.

Raw or undercooked pork, other Yersinia enterocolitic enterocolitica a Clostridium botulinum

Reactive arthritis, pancreatitis, meningitis, endocarditis, Guillain –  Barré and MillerFisher MillerFisher syndromes

thrombocytopenic thrombocytope nic purpura Meningitis or meningoencephalitis, septicemia, abortion

 Helicobacter pylori, H. pullorum  Arcobacter butzleri butzleri, other Arcobacter  spp. 

Crohn’s disease  Peritonitis, endocarditis,  pneumonia, conjunctivitis, urinary tract infections  Neonatal meningitis, meningitis, bacteremia, bacteremia, necrotizing enterocolitis enterocolitis,, appendicitis, conjunctiviti conjunctivitiss Gastric ulcer and cancer, liver disease Septicemia, bacteremia

 

 

Table 4. Cheese contamination with different pathogens in outbreaks with fatalities (modified from Kousta et al. 2010) Product  

Common Pathogens found in cheese products 

Cheese type (pathogen) 

Cheese

Staphylococcus aureusa, Listeria monocytogenes b, Escherichia coli coli  O157:H7 b and Salmonella  b spp. and  Mycobacterium avium subsp. paratubercul  paratuberculosis osis 

Soft cheese made of unpasteurized milk ( L.  L. monocytogenes monocytogenes)

Cases number (deaths)  

Country (year) 

122 (33)

Switzerland (1983-87)

142 (48)

USA (1985)

26 (6) 37 (11) 12 (3) 321 (2) 273 (1) 25 (5) 14 (1) 4 (1) >1700 (0)*

Denmark (1989-90) France (1995) Switzerland (2005) USA (1981) France (1993) France (1995) France (1993) France (1992) Canada (1984)

(MAP)c  Mexican-style Mexican-sty le soft cheese made of pasteurized milk ( L. monocytogenes) Blue-mold cheese/hard cheese ( L. monocytogenes) Soft cheese ( L. monocytogenes monocytogenes) Soft cheese ( L. monocytogenes monocytogenes) Mozzarellaa made of pasteurized milk (Salmonella) Mozzarell Goat’s milk cheese made of raw milk ( Salmonella) Mont d’Or cheese made of raw milk ( Salmonella) Mont d’Or cheese made of raw milk ( Salmonella) Farm fromage frais made of raw milk ( E. coli) Cheddar made of unpasteurized milk (Salmonella)

*One of the largest published outbreak (D’Aoust et al,1985).  a S. aureus contamination of raw milk originates primarily from staphylococcus mastitis of mammary glands.  b Salmonella, E. coli O157:H7 and L. monocytogenes f eces). monocytogenes contaminate raw milk from farm environment (e.g., feces). c  Mycobacterium Mycobacterium avium subsp. paratubercul  paratuberculosis osis (Map) is the cause of Johne's disease (a chronic infection of ruminant animals gut, animals that provide milk and/or meat for human consumption).

 

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A decent example of cheeseborne pathogens is shown in Table 4. Cheese is an excellent example of a product that if not treated according to regulations and contaminated, can harm human health (including death) (Figure 3)(Somers et al, 2001). The first step in cheese  preparation is pasteurization pasteurization of raw milk milk to prevent pathogens pathogens such as Staphylococcus aureus,  Listeria monocytogenes monocytogenes,  Escherichia coli  O157:H7,  Salmonella  spp. and  Mycobact  Mycobacterium erium   avium subsp.  paratubercu  paratuberculosis losis  (MAP) to be present along fermentation process. Table 4 depicts exactly those pathogens detected in cheese products, in countries known for high standards of production, which had also a fatal outcome. The main question is: what went wrong that favored such a bacterial contamination that caused human death? It is clear, that if  present in the final product, product, those pathogens pathogens survived pasteurization. There are are several pattern of survival for bacteria (spore, thermotolerance, capsule, biofilm) among which biofilm umbrella can protect bacteria from the denaturing process of pasteurization (Burgess et al., 2010). It is theoretically conceivable that if one bacterial cell is left alive after different disinfection process, the reach milk milieu will encourage future growth up to high numbers. Definitely, the various fermentation processes are expected to lower the pH, increasing the stress on the contaminant bacterial survivors, but then again a biofilm can annul this effect easily and conceivably this is what happened in most cases listed in Table 4.

Figure 3. Sources of milk contamination (from Anonymous, 2010).

Food Contact Surfaces

When dealing with surfaces, the critical surface tension is one of the major factors in  bacterial attachment. As higher the high free surface energy or its wettability the higher the attachment (Boulange-Peterman, 1993) .For example stainless steel and glass that have high free surface energy, therefore more hydrophilic, are better colonized by bacteria in comparison with nylon, Teflon, fluorinated polymers and buna-N rubber that are more hydrophobic (Sinde and Carballo 2000). In general, attachment rate is faster on hydrophilic surfaces however the attachment strength is higher on hydrophobic surfaces; while already attached, bacteria will form a monolayer on hydrophilic surface and a patchy pattern on hydrophobic surface (Figure 4). Interestingly, when a hydrophilic surface is treated with strong alkali or acid it will become more hydrophilic while treatment with weak acid will  produce effect (Sindethis andway Carballo, 2000). An interesting with stainless steel wasa hydrophobic observed when treated and further exposed to aireffect or water it goes

 

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 passivation by formation of a chromium oxide layer to which organic soil particles adhere creating a conditioning film favoring bacterial adherence (Li and Logan, 2004;Verran and Jones, 2000). In relation to surfaces, several attempts have been made to avoid biofilm formation. Such attempts have been described where antimicrobial products were incorporated into surface materials (Park et al. 2004; Weng et al. 1999), antimicrobial coatings (e.g., silver) (Gottenbos et al. 2001; Thouvenin et al. 2003; Hashimoto, 2001) or by  physicochemical properties modification of surfaces (e.g., surfactants) (Rosmaninho (Rosmaninho et al. 2007; Cloete and Jacobs, 2001). Most of these applications were tested on biomedical devices, but it is not excluded to be further implemented in food industry, pending safety. Here it should be pointed out a very basic principle of biofouling: the conditioning film; any of the above proposed solutions to prevent microorganisms attachment will be overwhelmed  by residual organics that will screen any antibacterial material present on the modified surface, making it obsolete.

Figure 4. Hydrophilic/hydrophobic surfaces and bacteria and their characteristics in attachment process.

Food Disinfection

In order to achieve efficient control over undesirable biofilms in food industry, it is recommended to understand the nature of contaminating residue materials present on contact surfaces. These materials can be carbohydrates, fat, proteins, minerals or various combinations. In addition it is very much required to identify the microorganism attached to those surfaces in order to choose the right disinfectant agent. There are many disinfectants and detergents and they have to be selected based on their efficacy, safety (especially in food  production) and ease of removal (Table 5). The basic requirements from these chemicals are to be non-corrosive and not to impact organoleptic quality of the product (Mosteller and Bishop, 1993; Wirtanen et al. 2000).

 

 

Table 5. Biofilm control by known chemical and biological means in food industry and some other potential application (adapted from Simões et al, 2010) Antimicrobial Products

Biofilm type

Other treatments

Biofilm type

Ozone, commercial chlorinated sanitizer

 P. fluorescens/Alcaligenes fluorescens/Alcaligenes faecalis faecalis

Enzymatic cleaning products

Benzalkonium chloride, hexadecyl trimethylammonium  bromide, sodium hypochlorite, hypochlorite, pera peracetic cetic acid, hydrogen peroxide, o-cresol, phenol Chlorine, peracetic acid, peroctanoic acid

 E. coli

Applying ultrasonic waves and  proteolytic and glycolytic enzymes enzymes

 Lactobacillus bulgaricus, bulgaricus, Lacto Lactobacillus bacillus lactis, Streptococcus t hermophilus  E. coli

 L. monocytogenes monocytogenes and Pseudomon  Pseudomonas as 

Proteolytic enzymes with surfactants

 Bacillus sp.

sp. mixed biofilms Chlorine dioxide containing sanitizer

 B. cereus/P. fluorescens fluorescens single and

Bacteriophages

 Enterobacter agglomerans agglomerans 

Chlorine Chlorinated-alkaline Chlorinated-al kaline solution; low-phospha low-phosphate te  buffer detergent; detergent; dual peracid solution; alkaline solution; hypochlorite Sodium hydroxide; commercial alkaline cleaner Chorine; ozone

 E. coli  L. monocytogenes monocytogenes

Bacteriophages A bacteriophage ( L.  L. monocytogenes monocytogenes  phage ATCC 23074-B1) 23074-B1)

 P. fluorescens  L. monocytogenes monocytogenes

 P. putida

Bacteriophage T4

 E. coli

 P. fluorescens, P. fragi fragi and P. putida 

Alkaline cleaner and a bacteriophage bacteriophage

Chlorine, hydrogen peroxide, ozone

 L. monocytogenes monocytogenes

Glutaraldehyde, ortho-phtalalde Glutaraldehyde, ortho-phtalaldehyde, hyde, hexadecyl trimethylammonium bromide, sodium dodecyl sulfate, chlorine solution sodium hydroxide Sodium hydroxide; nitric acid

 P. fluorescens

Engineered enzymatic bacteriophage bacteriophage (T7)that hydrolizes β-1,6- N  -acetyl-d N -acetyl-dglucosamine, a crucial adhesin Biosurfactants produced by  Lactococcus lactis lactis 53

 E. coli O157:H7  E. coli

mixed biofilms

Mixed species

Surfactin from Bacillus subtilis subtilis 

Staphylococcus epidermidis, Streptococcu Streptococcuss  salivarius, Staphylococcus Staphylococcus aure aureus us

Salmonella enterica, E. coli, and Proteus mirabilis mirabilis 

 

 

Antimicrobial Products

Biofilm type

Other treatments

Biofilm type

Chlorine; chlorine dioxide; commercial detergent

 B. cereus and Pseudomonas spp.

 Pseudomonas aeruginosa produces

Sodium hypochlorite

S. typhimurium

 B. subtilis, E. coli, Staphylococcus Staphylococcus a aureus, ureus,  Klebsiella pneumoniae, pneumoniae,  P. aeruginosa, P. mirabilis mirabilis, Streptococcus  pyogenes and the yeast Candida albicans  E. coli, S. typhimurium , Shewanella  putrefacians, L. monocytogenes monocytogenes 

Peroxydes; quaternarium ammonium compounds; chlorine Hydrogen peroxide; sodium dichloroisocyanurate; dichloroisocy anurate; peracetic acid

 L. monocytogenes monocytogenes

cis-2decenoic acid disperser

Staph. aureus

Iron sequestration by chemical or  biological chelators chelators (siderophores, (siderophores, Quorum sensing disturbers D-amino acids that trigger biofilm disassembly (from B. subtilis)

Different sp. Staphylococcus aureus and Pseudomonas aeruginosa (Kolodnik-Gal et al. 2010)

 

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Another important aspect linked to disinfection is construction material of equipment in which food is processed. Perhaps stainless steel is the most practical and most used in food industry. It can be easily cleaned by brushing, lapping, mechanical grinding and polish (electrolytic or mechanically) (Maukonen et al. 2003). However, even though construction material meets industry top quality requirements, there are some bottle necks in processing line where biofilms can accumulate, such as: joints, gaskets, crevices, cracks, dead ends and valves. The most efficient disinfection program is based on the following steps: removal of foreign bodies, microorganisms, nutrients and residual disinfectants/detergents (Dosti et al. 2005). For example, in milk industry the cleaning-in-place (CIP) procedures are commonly used in this order: 1. prerinse with cold water to remove bulk residues; 2. circulation of detergent for left minor residues removal (i.e., 1% sodium hydroxide, 70 oC for 10 minutes, water rinse than 0.8% nitric acid at 70oC for additional 10 minutes); 3. flash of the detergent with cold water; 4. circulation of a disinfectant to inactivate/kill residual microorganisms (i.e., chlorine or combination of nisdin, lauricidin and lactoperoxidase system for different exposure intervals) and 5. final flash to remove the disinfectant with cold water (Forsythe and Hayes, 1998; Dufour et al. 2004). This CIP procedure has been shown to be less effective in  biofilm eradication and the decrease in viable cell numbers was between 0 to 2 log reduction, while additional antimicrobial treatment reached only 2.8 log reduction after 2 hours of chlorine exposure (Bremer et al. 2006). In diary industry, the subsequent disinfectants/agents are in use: acidic compounds, aldehyde-based biocides, caustic products; chlorine, hydrogen peroxide, iodine, isothiazolinones, ozone, peracetic acid, phenolics, biguanidines, and surfactants. Some of them highly oxidative therefore caution should be implemented related to processing equipment material, in order to prevent corrosion, which may intensify biofouling (Simões et al. 2010). Beside aggressive chemicals some milder approaches have been applied such as: enzyme, ultrasonic waves, bacteriophages, etc., however they are limited by surface and microorganism variability (Table 5).

SUMMARY AND R ECOMMENDATIONS ECOMMENDATIONS  The two main food industries: meat and dairy are the most vulnerable to contamination and biofilm formation. For meat, several recommendations are valid in order to prevent  biofilm formation (e.g., by  L. monocytogenes). First to have a clean water source for all cleaning procedures; ensure hygienic pre-slicing; introduction of high-risk zone; personnel hygiene training; define critical and observation sites and expose them to frequent rotation sanitation (frequent sanitation means that certain parts of the processing equipment are thoroughly sanitized at e.g., 7-days or 14-days intervals and rotation sanitation means that critical sites are given special attention in daily turns and if necessary, special treatment like scrubbing); regular bacteriological tests and selection of appropriate detergents and disinfectants. Indicator bacteria such as Enterobacteriaceae can be used to follow hygienic status (Jessen and Lammert, 2003). For dairy industries the following recommendations are valid: First to have a clean water source for all cleaning procedures; production animals should be registered and subjected to  periodical or when necessary to veterinary check-up; raw milk and colostrum must come

 

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from healthy animals (to which no unauthorized substances or products have been administered and that have not undergone illegal treatment and if positive to follow the withdrawal periods prescribed); Milking equipment and premises in contact with milk and colostrum must be located and constructed so as to limit the risk of contamination and contact with vermin/birds; operator’s hands and forearms must be thoroughly washed before milking and these parts as well as gloves, if worn, kept clean during milking and milk handling; dairy equipment and premises should be separated from animal housing, storage tanks must be sited, constructed and enclosed so as to limit contamination risk and prevent physical contamination; valid refrigeration equipment; strict temperature application in all thermal and refrigeration processes (see Figure 5 for optimal temperatures); the milk vat, including mobile vats, must be emptied and cleaned after each use; milk collection tankers and reload tankers must be cleaned and disinfected at least once a day; material used for wrapping and  packaging are not to be a source of contamination. Similar to meat, dairy products should be tested for bacteriological parameters such as  Listeria monocytogenes  in ready to eat foods, coagulase positive-Staphylocci in dairy products and Enterobacteriaceae, Salmonella  and  E.  sakazakii (Cronobacter (Cronobacter sa sakazakii kazakii) in dried infant formula and foods (Anonymous, 2010).

Figure 5. Temperature requirements in dairy industry in order to inhibit bacteria spoilage and also to extend the shelf life of the milk. Note: milk cooled below 6 oC is an excellent method to prevent microbial growth (including biofilms) and also slow down chemical processes (i.e., enzymes). enz ymes).

 

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In summary, prevention of bacterial biofilms in food industry is not an easy task in light of products nature, therefore high hygienic measures and standards are required to prevent foodborne diseases.

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Anonymous (2010) Industry Guide to Good Hygiene Practice. Milk and Dairy Products. Dairy UK, pp. 97. http://www.dairytransport.co.uk/resources/000/634/680/Final_Draft_ August_2010_-_Industry_guide_to_good_hygiene_practice.pdf   Bagge-Ravn D, Ng Y, Hjelm M et al. (2003) The microbial ecology of processing equipment in different fish industries e analysis of the microflora during processing and following cleaning and disinfection. Internatio  International  nal  Journal  Journal of Food Microbiology Microbiology, 87(3):239-250. Behravesh CB, Williams IT, Tauxe RV (2012) Emerging food foodborne borne pathogens and problems: expanding prevention efforts before slaughter or harvest. In: Institute of Medicine (US). Improving Food Safety Through a One Health Approach: Workshop Summary. Washington (DC): National Academies Press (US); 2012. A14. Available from:http://www.ncbi.nlm.nih.gov from:http://www .ncbi.nlm.nih.gov/books/NBK /books/NBK114501/ 114501/ Boulange-Peterman, L, Barroux B, Bellon-Fontaine M-N (1993) The influence of metallic Technology 7(3):221wettability on bacterial adhesion.  Journal of Adhesion Science and Technology 230. Bremer PJ, Fillery S, McQuillan AJ (2006) Laboratory scale clean-in-place (CIP) studies on the effectiveness of different caustic and acid wash steps on the removal of dairy  biofilms. Internati  International onal Journ Journal al of Food Microbio Microbiology logy, 106:254-262. Burgess SA, Lindsay D, Flint SH (2010) Thermophilic bacilli and their importance in dairy  processing. Internati  International onal Jou Journal rnal of Fo Food od Microbi Microbiology ology 144:215-225. Carmichael WW, Falconer IR (1993) Diseases related to freshwater blue-green algal toxins, and control measures. In  Algal toxins in  seafood seafood and drinking water Edited by: Falconer IR. London: Academic Press; pp.187-209. Carpentier B, Cerf O (1993) Biofilms and their consequences, with particular reference to hygiene in the food industry. Journal of Applied Bacteriology Bacteriology, 75: 499 – 511. 511.

Chmielewsky RAN, Frank JF (2003)inBiofilm formation and control in food processing facilities. Comprehensive Reviews Food Science and Food Safety  2:22-32. Cloete TE, Jacobs L (2001) Surfactants and the attachment of Pseudomonas aeruginosa to 3CR12 stainless steel and glass. Water SA, 27:21-26. Costerton JW, Stewart PS, Greenberg EP (1999) Bacterial biofilms: A common cause of  persistent infections. Science 284:1318-1322. D’Aoust JY, Warburton DW, Sewell AM (1985). Salmonella   typhimurium phage-type 10 from cheddar cheese implicated in a major Canadian foodborne outbreak.  Journal of  Food Protection Protection, 48:1062 – 1066. 1066. Donlan RM, Costerton JW (2002) Biofilms: survival mechanisms of clinically relevant microorganisms. Clinical Microbiological Review 15(2):167-193. Dosti B, Guzel-Seydim Z, Greene AK (2005) Effectiveness of ozone, heat and chlorine for  Internationall destroying common food ogy spoilage  Journal of Dairy Technol Technology , 58:19bacteria  – 24. 24. in synthetic media and biofilms.  Internationa

 

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Dufour M, Simmonds RS, Bremer PJ (2004) Development of a laboratory scale clean-in place system to test the effectiveness of ‘‘natural’’ antimicrobials against dairy biofilms.  Journal of Food Protect Protection ion, 67:1438-1443. Elhariry HM (2008) Biofilm formation by endospore-forming Bacilli on plastic surface under some food-related and environmental stress conditions. Gobal Journal of Biotechnology and Biochemistry Biochemistry, 3(2):69-78. 3(2):69-78. Flemming HC, Wingender J (2010) The biofilm matrix. Nature Reviews Microbiology Microbiology 8:623633. SJ, Hayes PR (1998)  Food hygiene, microbiol Forsythe microbiology ogy and HACCP   (3rd ed.). Aspen Publishers. Gibson H, Taylor JH, Hall KE et al. (1999) Effectiveness of cleaning techniques used in the food industry in terms of the removal of bacterial biofilms.  Journal of Applied  Microbiology,  Microbiol ogy, 87:41-48. Gottenbos B, van der Mei HC, Klatter F et al. (2001) In vitro and in vivo antimicrobial activity of covalently coupled quaternary ammonium silane coatings on silicone rubber.  Biomaterials  Biomateria ls, 23:1417 – 1423. 1423. Gunduz GT, Tuncel G (2006) Biofilm formation in an ice cream plant.  Antonie Van  Leeuwenhoek , 89(3-4):329-336. Guobjornsdottir B, Einarsson H, Thorkelsson G (2005) Microbial adhesion to processing lines for fish fillets and cooked shrimp: influence of stainless steel surface finish and  presence of gram negative bacteria on the attachment of Listeria monocytogenes. monocytogenes.  Food Technology and Biotechnology, 43(1):55-61. Hall-Stoodly L, Costerton JW, Stoodly P (2004) Bacterial biofilms: from the natural environmentt to infectious diseases. Nature Reviwes environmen Reviwes Microb Microbiology iology 2:95-108. Hashimoto H (2001) Evaluation of the anti-biofilm effect of a new antibacterial silver citrate/lecithin coating in an in-vitro experimental system using a modified Robbins device. Journal of the Japanese Japanese Association Association for Infect Infectious ious Diseas Diseases es, 75:678-685. Jessen B, Lammert L (2003) Biofilm and disinfection in meat  processing processing  plants. plants.  International  International   –  269.  Biodeterioration  Biodeteriorat ion & Biodegradation  Biodegradation 51:265  –  Kim KY, Frank JF (1995) Effect of nutrients on biofilm formation by Listeria monocytogenes on stainless steel. Journal of Food Protection Protection, 58(1): 24-28. Kousta M, Mataragas M, Skandamis P et al. (2010) Prevalence and sources of cheese contamination with pathogens at farm and processing levels. Food Control  Control  21:805  21:805 – 815. 815. Kumar C, Anand SK (1998) Significance of microbial biofilms in food industry: a review.    International  Internatio nal Journal of Food Microbiology Microbiology, 42:9-27. Li B, Logan BE (2004) Bacterial adhesion to glass and metal-oxide surfaces. Colloids and Surfaces B: Biointerfa Biointerfaces ces 36(2):81 – 90. 90. Maukonen J, Mättö J, Wirtanen G et al. (2003) Methodologies for the characterization of microbes in industrial environments: a review.  Journal of Industria Industriall Microbiology Microbiology and  Biotechnologyy, 30: 327-356.  Biotechnolog Monroe D (2007) Looking for Chinks in the Armor of Bacterial Biofilms.  PLoS Biology  5(11): e307. Mor-Mur M, Yuste J (2010) Emerging Bacterial Pathogens in Meat and Poultry: An Bioprocessing Technol Technology ogy 3:24 – 35. Overview.  Food Bioprocessing 35. Mosteller TM, Bishop JR (1993) Sanitizer efficacy against attached bacteria in milk biofilm.  Journal of Food Protect Protection ion, 56:34 – 41. 41.

 

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 Nelson K, Williams C (Ed.) (2007) Secon Secondd Edition, Infectious Infectious Disease Epidemiology, Epidemiology, Theory and Practice. New York: Aspen Publishers. O'Toole GA, Stweart PS (2005) Biofilms strike back.  Nature Biotechnol Biotechnology ogy  23(11):13781379. Park A-I, Daeschel MA, Zhao Y (2004) Functional properties of antimicrobial lysozyme –  chitosan composite films. Journal of Food Safety Safety, 69:215-221. Rocourt J, Moy G, Vierk K, Schlundt J (2003) The present state of foodborne disease in OECD countries. World Health Organization, pp.43, ISBN 9241591099. Rosmaninho R, Santos O, Nylander T, Paulsson M, et al. (2007) Modified stainless steel surfaces targeted to reduce fouling -evaluation of fouling by milk components. Journal of  Food Engineering  Engineering , 80:1176-1187. Saker ML, Jungblut AD, Neilan BA et al. (2005) Detection of microcystin synthetase genes in health food supplements containing the freshwater cyanobacterium  Aphanizomen  Aphanizomenon on  flos-aquaee. Toxicon 46:555-562.  flos-aqua Scallan E, Griffin PM, Angulo FJ, Tauxe RV, Hoekstra RM (2011a) Foodborne illness acquired in the United States-unspecified agents.  Emerging Infectious Diseases. 17(1):16-22. Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV et al. (2011b) Foodborne illness acquired in the United States-major pathogens. Emerging Infectious Infectious Diseases Diseases 17(1):7 – 15. 15. Sharma M, Anand SK (2002) Biofilms evaluation as an essential component of HACCP for food/dairy processing industry industry e a case. Food Control  Control , 13(6-7), 469-477.   Simões M, Simões LC, Vieira MJ (2010) A review of current and emergent biofilm control strategies. LWT - Food Science and and Technology Technology 43:573 – 583. 583. Sinde E, Carballo J (2000) Attachment of Salmonella spp. and Listeria monocytogenes to stainless steel, rubber and polytetrafluorethylene: the influence of free energy and the effect of commercial sanitizers. Food Microbiology 17:439-47. Somers EB, Johnson ME, Wong ACL (2001) Biofilm Formation and Contamination of Cheese by Nonstarter Lactic Acid Bacteria in the Dairy Environment.  Journal of Dairy Science 84:1926 – 1936. 1936. Thouvenin M, Langlois V, Briandet R et al. (2003) Study of erodable paint properties involved in antifouling activity. Biofoulin  Biofouling  g , 19:177-186. Vaillant V,  de Valk H,  Baron E  et  al.  (2005)  Foodborne  infections  in  France.  Foodborne  Pathogens Diseases 2(3):221-232. van der Kooij D (1992) Assimilable organic carbon as an indicator of bacterial regrowth.  Journal American Water  Works Association, 84(2):57 – 65. 65. van der Kooij D, Vrouwenvelder JS, Veenendaal HR (2003) Elucidation and control of  biofilm formation processes in water treatment and distribution using the unified biofilm approach. Water Science and Technology 47(5):83 – 90. 90. Verran J, Jones M (2000) Problems of biofilm in the food and beverage industry. In: Walker J, Suramn S, Jass J, editors. Industrial Biofouling. Cichester, N.Y.: John Wile, pp. 14573. Weng Y-M, Chen M-J, Chen W (1999) Antimicrobial food packing materials from  poly(ethylene-co-methacrylic  poly(ethyleneco-methacrylic acid). LWT  LWT –   –  Food  Food Science and Technology, 32:191-195. Microbiological Wimpenny J, Manz W, Szewzyk U (2000) Heterogeneity in biofilms.  FEMS Microbiological  Reviews 24:661-671.

 

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Wimpenny JWT, Kinniment SL, Scourfield MA (1993) The physiology and biochemistry of  biofilm. In: Denyer SP, Gorman SP, Sussman M, editors.  Microbial biofilms: formation formation and control. London: Blackwell Scientific. pp 51-94. Wirtanen G, Husmark U, Matilla-Sandholm T (1996) Microbial evaluation of the biotransfer  potential from surfaces with Bacillus biofilms after rinsing and cleaning procedures in closed food-processing food-processing system. Journal of Food Food Protection Protection, 59(7):727-733. Wirtanen G, Saarela M, Mattila-Sandholm T (2000) Biofilms  –  impact   impact on hygiene in food industries. In J. D. Bryers (Ed.),  Biofilms II: Process analysis analysis and applications (pp. 327 –  372).

 

In: Microbiological Microbiolo gical Food Hygiene Editor: Eino Elias Hakalehto

ISBN: 978-1-63483-646-3 © 2015 Nova Science Publishers, Inc.

Chapter 7

FIRST DETECTION OF S A  AL L M ONE L L A   CONTAMINATIONS  E lilia as H Ha akale kaleht hto o1,3 , J ouni uni Peso Pesola la 2 , A nne nneli H He ei tto 3 , H enri H änn nnii ne nen n4 , P anu H endo ndolilin n 5 , Os Osm mo H änn nnii ne nen n6 , R ober t A r mon7   , T ar mo Hum H ump ppi 8 a  and nd H ei kki Paakkane nen n9  1

Department of Environmental Sciences, University of Eastern Finland, Kuopio, Finland 2Department of Pediatrics, Kuopio University Hospital, Kuopio, Finland 3 Finnoflag Oy, Kuopio, Finland 4  Department of Physics, University of Jyväskylä, Jyväskylä, Finland 5  AIV Institute, University of Eastern Finland, Kuopio, Finland 6 Department of Physiology, University of Eastern Finland, Kuopio, Finland 7 Faculty of Civil and Environmental Engineering, Technion, Haifa, Israel 8 Finnish Defence Forces Technical Research Centre, Lakiala, Finland 9 Environics Oy, Mikkeli, Finland

ABSTRACT  Salmonella sp. is a wide and versatile group of human pathogens. The members of

this genus are able to cause somewhat milder but also fatal intestinal infections. Salmonella enterica Serovar Typhi is an example of the latter, and it has changed the course of history by several epidemics throughout centuries. The various Salmonella  strains can act as agents of salmonellosis. They can also cause latent infections, intracellular infiltration, and diseases elsewhere in the body system beyond the gut areas which are their primary distribution. Because of the relative big infective doses required for contagion, various salmonellae are typically agents of food and waterborne diseases. They can quickly captivate the surfaces of the intestinal epithelia, and attempt penetration into deeper tissues. In order to face the challenges, we present here some research of ours from a  period of 22 years to give an idea of the efforts in made to develop the fast monitoring of these widely distributed food contaminating agents.

 

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Elias Hakalehto, Jouni Pesola, Anneli Heitto et al. In case of Salmonella sp., the detection, identification and characterization of strains and the screening of the contamination problems need to progress stepwise. It is important to achieve the first detection as promptly as possible, and then continue further with the confirmation and typing efforts.

1. INTRODUCTION  the prevalence numbers ofAccording cases of different wellare as theirFigures generalon threat picture varyand considerably. to some salmonellosis, statistical data,asthere annually 1.3 billion cases of human gastroenteritis due to non-typhoid Salmonella , leading to 3 million deaths worldwide (Thong et al., 1995). Typhoid fever, in turn, has been estimated to cause 21 million cases causing more than 700.000 deaths (Thong et al ., 1994). These estimates are about 20 years old, and particularly the somewhat less hazardous non-typhoid salmonellosis can often remain undetected and unidentified as a cause of intestinal infection or food poisoning. Opposite to the more hiding or milder cases of salmonellosis, Salmonella enterica  Serovar Typhi (or Salmonella Typhi, or   S. Typhi  )) can become the cause of devastating epidemics. This risk is higher in poor hygiene conditions. In the US Civil War (1861-65) it was killing twice as many humans as the weapons altogether. In a later estimate, typhoid fever approximated to have2012). causedIn21.7 illnesses and 217.000 deaths in the year 2000 was (Crump and Sugarman, thatmillion survey, regardless of the somewhat smaller number of incidences than in the earlier estimate, the emerging resistance to traditional firstline drugs, fluoroquinolones, and third-generation cephalosporins was strongly highlighted. Azithromycin was reported to be an effective alternative for treatment of common type of typhoid fever (Trivedi and Shah, 2012). It has been documented that azithromycin inhibits the formation of flagellin filaments by suppressing their synthesis in Salmonella enterica Serovar Typhimurium (Matsui et al., 2005). However, increasing resistance toward azithromycin has  been reported among cases of both typhoid and non-typhoid salmonellosis (Sjölund-K (Sjölund-Karlsson arlsson et al., 2011). Fast detection and characterization of the contaminating salmonellae is often a matter of urgency in clinical or industrial setting. In this chapter we wish to document some steps of the method development which started in 1992 in the Rapid Microbial Detection project in Kuopio, Finland, and continued in the laboratory of Finnoflag Oy until this date.

1.1. S  Sa almone lmonell lla a As a Pathogen Salmonella  Typhi  is a rod-shaped, motile, non-sporulating, Gram-negative bacterium.

Typhoid fever is a systemic infection characterized by continued fever, lymphoid tissue involvement, ulceration of the intestines, enlargement of the t he spleen, rose-colored spots on the skin, diarrhea and constitutional disturbances. The incubation time is from 3 to 38 days, usually 7 to 14 days. The mortality in untreated cases ranges from 0-10% (Jane's NBC Protection Equipment, listing 1990-1991). Other agents of the Salmonella   infection are usually milder ones, but it is of great importance to monitor them in foods, and in healthcare, for establishing better hygienic conditions.

 

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With respect to the pattern of pathogenesis, salmonellae are attacking relatively fiercely on the intestinal epithelia. They have several surface structures which are able to penetrate and intrude the host membranes (Figure 1). This is partially based on the effector functions in which this pathogen begins to modulate the host activities. For example, these complex events are characterized in case of Salmonella enterica Serovar Typhimurium with studies using tissue culture models and the neonatal calf model (Zhang et al., 2003). The host pathogen interactions leading leading to diarrhea diarrhea are listed accordingly: accordingly: 1.  Salmonella type III effector proteins translocated into host cells 2.  elicit infiltration of neutrophils by inducing the chemoattractant chemokines in ideal tissues 3.  resulting in acute inflammatory response is associated with an increase in vascular  permeability causing causing mucosal edema 4.  with ideal surface mucosal necrosis 5.  initiating the leakage of extravascular fluids and transmigration of neutrophils into the intestinal lumen In the light of this “prognosis” of Salmonella infection, it seems essential to prevent this  pathogen from getting a hold to the epithelial surface and from breaking its permeability  barrier.

Figure 1. Salmonella sp. cell with invasive surface structures and protrusions. Electron micrograph by Elias Hakalehto.

Although more than 10 different adhesins have been identified on the gene cluster level of Salmonella enterica, their participation in the infection and the invasion of various human or animal hosts is only partially known (Wagner and Hensel, 2011). In a recent Chinese study, 323 poultry Salmonella  isolates were subdivided into 41 fimbrial genotypes (Gong et al., 2014). Of these strains, 285 (88.2%) had 12 to 14 fimbrial genes. Quite uniformly, uniformly, both in enteric bacteria and in the salmonellae, Type 1 fimbriae have been associated with intestinal infections (Finley and Falkow, 1989, Muller et al., 1991). We could demonstrate a stronger expression of Salmonella type 1 fimbriae in aerobiosis than in anaerobisis (Hakalehto et al.,

 

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2007). Ernst et al. (1990) reported higher tendencies for this expression in anaerobiosis. In  both studies, the fimbrial fimbrial expression peaked during exponential exponential growth growth period. Since the genus Salmonella is a wide group of human pathogens and cause diseases of many warm-blooded animals, its engulfment into the GI tract from food or water deserves some exploration. This bacterium is also an integral part of the commensal flora of many  birds, rodents, amphibians and reptiles, reptiles, which can transfer salmonellae salmonellae into food commodities and thus act as vectors for human infections and epidemics. The Salmonella sp. strains can usually attach on the gut epithelia, with various mechanisms, one of the most important ones  being the type fimbriae mediated This is often one of the first steps in the colonization of 1human intestines by adsorption. Salmonella . The type 1 fimbriae are expressed at 3740°C but not at 18-20°C which could explain the less potent pathogenic nature of the salmonellae in cold-blooded animals (Hakalehto, 2000). The colonies developed by the attached cells onto the gut epithelium liberate effectively the so called swarmer cells. This  phenomenon is behind the fast spreading  of the Salmonella  infection along the intestinal “column.”  It is probably based on the fast detachment of the fimbrial extrusions and their almost equally fast regrowth in a few seconds (Hakalehto et al., 2000). This makes the switching between colonized cell in a biofilm and the swarmer cells relatively easy.

1.2. Some Lessons from the History

The principal habitats of Salmonella   sp. arethe intestinal tracts of man and animals. Salmonellosis is transmitted from man to man through fecal contamination of food and water. An oral infective dose of at least 10 5 S. Typhi cells is needed to cause typhoid fever in 50% of human volunteers; and at least 10 9 cells are needed to cause symptoms of a toxic infection. The organism remains viable for 2 to 3 weeks in water, up to 3 months in ice and snow and several years in soil if the conditions are favorable (LeMinor, 1981). The effects of the sample matrices on the functions of the detection method are discussed later in this Chapter. In the series of books of “Hornblower” by C. S. Forester, the fictive main character is a naval officer, a son of a physician. He refers to typhoid fever as “the scourge of armies and fleet.”  In the books the main character contracted this incapacitating disease in northern Europe during epidemics in connection with an armed conflict some 200 years ago (Forester, 1945). The slow by recovery took one year   For theofcontagion, intestinal  –  For infection caused S . Typhi requires on (Forester, average an1946). infective dose about one this million cells. Therefore, the contaminated food and water sources have always been the main route of contamination due to the relatively high infective dose of the disease. Therefore individual  bacterial cells hardly cause this disease. One example of a typhoid fever outbreak was reported by Duncan et al . (1946). In this case the disease was distributed by one worker in a resident hotel in Cleveland, Ohio, USA, for less than one month. She was a carrier of the bacterium “ Eberthella typhosa” (later on designated as Salmonella Typhi), which was the causative agent. Her only task was to prepare orange juice for breakfast every morning for about 360 young women. After she had been identified as the source of contagion, her blood test showed a titer of 1:80 for the flagellar H antigen, and 1:160 for the O-specific polysaccharide side chain of the specific bacterial lipopolysaccharidee (endotoxin). lipopolysaccharid (endotoxin).

 

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This above-mentioned outbreak caused 18 illnesses and one death of the diseased individuals, 14-15 had consumed the orange juice every morning. Seven had suffered from a  previous attack of typhoid fever. Out of the 368 subjected individuals 211 were immunized  by vaccination. Among this group of pre-immunized pre-immunized persons only one individual got the disease. Her last vaccine shot had been received on November 30th, 1943. The onset of this case of illness was given as January, less than two months later. In theory, her immunoprotection should have been at some level awake, but obviously it was not enough to  prevent the infection. infection. –  Out  Out of the 140 non-immunized individuals 17 were developed typhoid fever, giving anthe attack  Nowadays numrate number ber of of 12,1%. Salmonella serotypes or serovars has been calculated to be over 2600 serovars that can be divided into typhoidal and non-typhoidal non-typhoidal Salmonella  serovars (Zou et al., 2009, Gal-Mor et al., 2014). Serovars Typhimurium and Enteritidis belong to nontyphoidal serovars, while serovars Typhi, Sendai, Paratyphi A and Paratyphi B are referred to as typhoidal Salmonella serovars. Enteric fever caused by these serovars is called also typhoid fever or paratyphoid fever, when caused by serovars Typhi or Paratyphi, respectively. Regarding enteric fever Salmonella serovars that cause human infection can change over time and location. In some areas in Asia, for example, multidrug-resistant S. Typhi has been the main cause of enteric fever, but now S. Typhi is being displaced by the drug-resistant S. enterica Serovar Paratyphi A (Wain et al., 2015). Salmonella  strains occur globally in different animals, in sewage and sewagecontaminated surface waters. Due to increasing global trade and transport, growing amounts fresh foodstuffs are contaminated with the salmonellae (Krauss et al., 2003). There was also another case of recorded outbreak where orange juice was the cause of an epidemic. This took place in a New York hotel in year 1989 (Birkhead et al., 1993). This outbreak started from a breakfast of a single day and was initiated by a hotel employee who was not known to be a carrier. In the subsequent investigation of stool samples, there were 43 culture-confirmed and 24 probable cases among the quests, one culture-positive case among the hotel workers and one culture-confirmed secondary case. In a statistical study on food related epidemics, where employees have been the source of contamination, 816 outbreaks were studied in a wider study. Eleven of the outbreaks involved involved more than 1,000 persons, and in 4 epidemics more than 3,000 fell ill (Todd et al., 2007). The  pathogens most likely to be transmitted by the food workers are norovirus, norovirus, hepatitis A virus, Salmonella, Shigella and Staphylococcus aureus (Todd et al., 2008). According to Wareing and Davenport (2015) microbial problems with soft drinks and fruit juices can be divided into two groups: 1.  growth in, and deterioration of, the product by general organisms to cause spoilage 2.  growth in, or contamination of, the product by pathogens to cause food poisoning There have been relatively few instances of food poisonings caused by contaminated fruit  juices, despite the typhoid fever examples as outlined earlier. However, general spoilage is very common. This situation is somewhat analogous to the water microbiology practices, among which the fecal indicators are generally searched for in order to avoid distribution of  pathogens (Hakalehto and Heitto, 2012). However, in water science the “heterotrophic” microbial load is often neglected, but in practical water monitoring at the Finnish water departments this type of analysis is often carried out. The reason for this “unorthodox”

 

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 practice is that a better picture of the overall quality of products and production lines is obtained on the basis of total microbial counts. At the Kuopion Vesi Oy (the water company of the city of Kuopio, Finland) a survey of the 350 km water distribution network was carried out with the PMEU technology for one month (Hakalehto et al., 2011). During this period of time, about 30 cases of compromised microbiological water quality were revealed on the  basis of increased heterotrophic counts, while only a couple of them were recorded using hygienic indications with the “official” methodologies. Moreover, when the water was artificially contaminated with Salmonella enterica serovars, a single bacterial cell was detected in approximately 10 hours using the PMEU enrichment and detection principles. For more information on the practical methodology regarding water and food microbiological analysis, the reader is recommended to see chapter 14 of this book as well.

1.3. Endangered by Antibiotic Resistance

Mapping the exchange of genetic material between pathogens and non-pathogens opens up new perspectives for understanding and managing the spread of drug-resistant strains (Dantas and Sommer, 2014). The treatment of antibiotic-resistant infections costs annually USD 35 billion in the USA. Some of the worst kinds of infections are the drug-resisting salmonellae and the MRSA. Although the intestinal infections caused by non-typhoid salmonellae are quite seldom treated with antibiotics, each patient represents an individual case and the need for antibiotic treatment has to be carefully evaluated case by case. One reason for the avoidance of antibiotics in the treatment of the intestinal diseases is the rapid  progress of the infection after the onset of diarrhea and other tedious symptoms, and its usually relatively speedy recovery with more conventional means of cure. It is also noteworthy that many long-lasting Salmonella   cases are not necessarily intestinal ones. In case of the staphylococci, the toxic effect of food-poisoning is often formed already in the contaminated food substances. In many countries, including Sub-Saharan Africa there is an urgent need of a sustainable surveillance system for various nosocomial infections caused by e.g. , , Salmonella, Salmonella, Klebsi Klebsiella, ella, E. col colii and V. cholerae (Foley et al., 2007, Mshana et al., 2013). Typhoid fever caused by S. Typhi can cause significant morbidity and mortality, hence requiring antibiotic therapy. Empiric first-line treatment has been a combination of ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole. The use of this kind of treatment approach has resulted in the development of transmissible multidrug-resistant strains. The disease outbreaks caused by these strains have been successfully treated with ciprofloxacin. However, decreased ciprofloxacin susceptibility has caused treatment failures. These characteristics are due to point mutations in the S. Typhi genes gyrA and/or parC. Tatavarthy et al. (2014) stated that selective pressure resulting from empirical use of first-line empiric drugs and fluoroquinolones has led to the global emergence of S. Typhi haplotype H-58. Although azithromycin has been useful in many multidrug-resistant cases (Trivedi and Shah, 2012), careful use of antimicrobials in treatment of typhoid fever is warranted in order to decrease the selective pressure leading to new mutations increasing the prevalence of antibiotic resistance.

 

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The PMEU method was successfully used for verifying antibiotic resistances by

Hakalehto (2011). With the PMEU it could be possible to screen simultaneously particular strains e.g., against 10 different antibiotics. More considerations on the aspects related to  bacterial antibiotic markers markers in foods are discussed in chapter chapter 13 of this book. book.

1.4. Protection from Vaccines

One approach to fight increasing antibiotic resistance is the vaccine development (Crump (Crump et al., 2010, Cogan et al., 2004). Efforts are underway to develop vaccines that are immunogenic in infants after a single dose. They could ideally be produced in countries of endemic infectivity areas,  e.g., against Salmonella enterica Serovar Paratyphi A in Asia. For rapid response against emerging infections, passive immunization with e.g., egg yolk antibodies could be advisable (Hakalehto and Kuronen, 1998). With such procedure we managed to produce effective epithelial surface lgY-type antibodies against such severe  pathogens as  Francisella tularensi tularensiss  and  Bacillus anthracis. One daily dosage for epithelial  protection against the pathogen pathogen is produced produced into one one egg.

1.5. Patient Case of Latent Salmonellosis

One specific patient case was the presence of Salmonella sp. in the stool samples of an 80 years old senior male citizen travelling from Finland to a Mediterranean country. He was showing no symptoms but was tested because of several cases of salmonellosis in his tourist group. On the basis of the above-mentioned cultivation results doctors prescribed him ciprofloxacin medication with high doses. However, he exhibited no signs of the disease, and actually could have been a carrier of Salmonella  from his younger days (no classification or typing of the strains was carried out). Consequently, he was advised to delay the onset of the medication. The heavy antibiotic load, considering his old age, could have ruined his health. After all he was not suffering from his parasitic strain (3% of the population in Finland are carriers of Salmonella   without knowing about it). The senior patient was instructed to maintain a high level of personal hygiene which would prevent him from spreading the disease to others. During one month, the above-mentioned patient used daily the combination of probiotics and prebiotics together with some herbal treatments (Hakalehto and Jaakkola, 2013; Hakalehto et al., 2015). After that, he did not have any traces of the pathogen in his stool samples. And the ciprofloxacin turned out to be unnecessary in his case. However, this incidence cannot be taken as an example of a recommendable treatment policy of salmonellosis in a general sense, but this patient history reflects the variety of individual cases that may be encountered during an outbreak of a food poisoning epidemic. Many other members of his touristic group got salmonellosis with more powerful symptoms, and they all received antibiotic therapies.

 

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2. LUMINESCENCE DETECTION METHOD

FOR S AL M ONE L L A

E NTERICA SEROVAR TYPHI 

A project was aimed at developing a luminescence detection method for S. enterica Serovar Typhi (Kuosmanen et al., 1995). The work was started with producing antibodies against the target organism. The antibodies were screened with cross-reaction and sensitivity tests, and the most suitable ones were selected for further testing. By combining the produced antibody with a luminescence detection and sample handling method, the target organism was 6

detected specifically the level ofthe10 period  cells perofmltime or less. Luminescence the sensitivity and atshortened required for the detection detectionincreased in the immunological assays. In the future, the sensitivity of the method can be increased by producing an improved secondary antibody needed in the sandwich-assay. Some difficulties could be connected with the natural immunological responses against enterobacteria in mice or in other experimental animals. This has probably caused the cross-reactivity problems that occur when using the method. An example of the potential of combined enrichment with detection using antibodies is  presented in Figure 2. By this approach of elevated cultivation temperature, several crossreactivities were avoided.

Figure 2. Growth temperature +42°C in the Rappaport Vassiliadis soya peptone (RVS) broth selected the other cross-reactive enteric species. In the lower temperature of 37°C cross-reactions occurred (results not shown here). The strains used for the experiment were Salmonella enterica serovar Typhimurium  (59929), S. enterica serovar Enteritidis (59813), S. enterica serovar Infantis (Vsa 162), Citrobacter freundii (1282),  Klebsiella mobilis  (1276),  Escherichia coli (99) and  Enterobacter aerogenes (101/3). In ELISA testing the measurement time was 1 h. The primary antibody N:o 14 was  purified with protein A (antibody dilution 1:100). Growth time (h) is shown in X axis and absorbance (A) in Y axis. Graph by Sanna Airaksinen.

 

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Alternative solutions to overcome the cross-reactivity problem could also be the use of monoclonal antibodies, producing the secondary antibodies in an alternative species or

the labeling of the specific antibodies with enzymatic labels. In this surveillance method, specific recognition of antigenic proteins from the surface of the cells with antibodies and a following luminescence detection is performed. The luminescence detection enables a better signal-to-noise ratio and shortens the time needed for the detection compared to the colorimetric reactions (e.g., ELISA) method. The antigenic proteins are collected with a method based on acidic extraction of the cell surface proteins, which reduces considerably the possibility of hazards during the handling of the samples (Hakalehto et al., 1984). Sensitivity of the method is an important matter, because of the remarkable hazards often caused by the target organisms or their toxins even with small doses. Immunological methods methods are faster and less laborious compared to the traditional methods based on biochemical tests or selective cultivation. In addition to this, the immunological methods can detect weakened or dead microbes, which is not possible with the traditional methods. In most cases the traditional methods require a pre-incubation or an enrichment step in order to increase the concentration of the bioagents, which increases the time ti me needed for their successful detection. This leads to detection times of 24 - 72 hours minimum ranging to several days. Immunological methods also have their disadvantages. For example, they are extremely dependent on the reagents used which is the case also with also in the genetic techniques.  Nonspecific binding of the antibodies or their cross-reactivity with other microbes lead into false positive results. During the projected development work we tried to minimize these sideeffects by screening the antibodies and by optimizing the parameters included in the method (time, temperature, antibody and antigen concentrations etc.).

2.1. Details of Luminometric Detection Method for enteric Serovar  Salm  Sa lmo onell nella a Typhi

This research work was originally presented in the symposium "Protection against Chemical and Biological Warfare Agents,” Stockholm, 1995 (Kuosmanen et al., 1995). Salmonella Typhi strains RHS 671, RHS 672, RHS 673 and RHS 674 were received as a gift from the Public Health Institute, Helsinki, Finland (nowadays the National Institute of Health and Welfare, THL). The antigenic proteins of individual strains were extracted with mild acid according to the method developed by Hakalehto et al. (1984). T The he antigen extracts were treated with SDS-PAGE gel electrophoresis, and a 65 kD protein fraction isolated from the gel was used as an antigen for the immunizations. The immunizations and the collection of the sera were carried out according to the standard procedures (Harlow and Lane, 1988). In addition to this, we also tested some sera which were produced earlier against synthetic  peptides. The antisera were tested with the acid extracts of the different S.  Typhi strains with Western blotting and luminescence analyses. Western blots were made as described by Harlow and Lane (1988). The principle of the luminescence analysis is described in Figure 3. The immunological analyses were performed according to the standard procedures (Harlow and Lane, 1988) using 1.5% BSA in TBS for blocking and dilution buffer for antibodies. Acid extracts were immobilized on microtiter plates and cuvettes with 0.5%

 

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glutaraldehyde activation as described by Hermanson et al. (1992). In the analyses we used Zymed AFOS-labelled anti-mouse or anti-rabbit antibodies. The luminescence analyses were

carried out with Clinicon or Sarsted cuvettes, Bio Orbit 1253 luminometer and Boehringer Mannheim's Lumi-Phos™  530-substrate. The colorimetric analyses were performed with Labsystems Microstrip® microtiter plates, Labsystems Multiskan® Plus reader and Sigma's  pNPP substrate. The detection method was either luminescence detection with LumiPhos or colorimetric detection (Catty et al., 1988) with pNPP as a substrate. In the luminescence detection we used in each cuvette 200 µl LumiPhos TM 530 substrate diluted with an equal volume of 0.1 M diethanolamine (pH 9.5) containing 1 MMMmol MgC1 2. The colorimetric detection was carried out as described earlier (Harlow and Lane, 1988). The incubation time was 20 min in  both analyses.

Figure 3. Principle of luminescence detection (Kuosmanen et al., 1995).

2.2. Results from the Luminescence Analysis

Figure 4 demonstrates the affinities of the anti-synthetic peptide and anti-surface protein antibodies for the S. Typhi acid extracts (Hakalehto et al., 1984). The lesser reactivity against strain RHS 673 can be explained with the smaller protein concentration of the extract in question. This was seen in the SDS-PAGE gels (data not shown here). Because cross-reactivity with other microbes were noticed when using the anti-synthetic  peptide antibodies, we tested also the anti-surface protein antibodies against some microbes. These microbes are known to cross-react with many commercial Salmonella tests (Citrobacter freundii) or showed cross-reactivity with anti-synthetic peptide antibodies ( Klebsiella  Klebsiella mobilis, E. coli).

 

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Figure 4. The responses of different antisera against acid extracts (Hakalehto et al., 1984) of Salmonella Typhi strains RHS 671, 672, 673 and 674. The intensities of luminescence signals are shown on the yaxis (Kuosmanen et al., 1995).

Figure 5. Results of luminometric analyses when crossreactions were examined by using rabbit antisera S404 and S405 and mouse antiserum (99 =  Escherichia coli, 1282 = Citrobacter freundii, 1276 =  Klebsiella mobilis, 672 = Salmonella Typhi). The intensities of luminometric signals are shown on the y-axis (Kuosmanen et al., 1995). 

The antibodies produced in rabbits detected S . Typhi more specifically and with higher affinities than the antibodies produced in mice (Figure 5). The latter antibodies were pooled from the sera of several animals. They could have contained natural activities and crossreactivities with various enteric bacteria. The next step was to test the cross-reactions against different microbes with a sandwichassay. A rabbit antibody S405 was the capturing antibody and pooled mouse antibodies from three individual mice were used in a mixture as the secondary antibody. Based on the cross-

 

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reaction tests, the rabbit polyclonal antibody S404 was chosen for further examinations (Figure 6). For the determination of the optimal dilution, the chosen antibody was titrated against a standard amount of antigen and unspecific background. On the basis of the signal-to-

 background ratio, the optimal titer for the antibody was wa s found fou nd to be 1:400 - 1:800. After the determination of the working titer for the antibody the detection limit of the method was tested. Cell surface antigens were diluted and detected according to the method. The results are shown in Figure 7.

Figure 6. Results of luminescence analyses when cross-reactions were examined by using rabbit antisera S404 after dilution of the samples. (The abbreviations of the species: 99 =  Escherichia coli, 1282 = Citrobacter freundii, 1276 =  Klebsiella mobilis, 672 = Salmonella Typhi.) The intensities of luminescence signals are shown on the y-axis (Kuosmanen et al., 1995).  

Figure 7. The detection limits of different dilutions of acid extract (Hakalehto et al., 1984) of strain RHS 672 by the rabbit antiserum S404. The intensities of luminescence signals are shown on the y-axis (Kuosmanen et al., 1995).

 

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The limiting antigen dilution   was 1:1600, which is equal to the detection limit of 10 6  cells per ml. This could be further improved by using the monoclonal antibodies in the sandwich-assay. We compared the luminescence detection method with the traditional colorimetric

detection (Figure 8). Remarkable advantages were achieved with the luminescence protocol. It was faster and more sensitive. The detection limit was almost 20 times lower during the 20 minutes detection time applied using this method. If the incubation time is extended considerably, the colorimetric method could become almost as sensitive as the luminescence detection. For instance, to reach the sensitivity of the luminescence detection in the example presented indetection. Figure 8, the incubation time of over two hours would have been needed for the colorimetric

Figure 8. Comparison between luminescence and colorimetric ELISA detection of acid extracts (Hakalehto et al., 1984) of S. Typhi strain RHS 672 by rabbit polyclonal antiserum S404. Incubation time was 20 minutes. - The graph indicates the methodological improvement achieved by the luminescence signals measured from labels bound to specific antibody sandwiches (Kuosmanen et al., 1995).

During the Rapid Microbial Detection project (1992-95) we also started the development of an immuno-PCR method and protocol for Salmonella detection using this combination of genetic techniques together with an immunoassay.

 

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3. PCR  DETECTION OF S A  AL L M ONE L L A E NT NTE E R I CA   SEROVAR TYPHIMURIUM  The selected primers on the basis of computer assisted evaluation (melting temperature in

 brackets): - upper primer: - lower primer 1: - lower primer 2:

ATG TCC AAG ATG CCT ACA CC (51,7°C) TAG TGG TTT TAG CAG TAA TTG C (48,8°C) CTT GCT AAG TAA GTC TTA CCA T (48,8°C)

For the genetic map, see Figure 9.

Figure 9. Results of the PCR primer analysis  by using Amplify™ computer  software.   software. The darker the arrowhead, the stronger is the annealing capacity of the primer. The thickness of the line in the lower  part of the figure is related to the the intensity of the polym polymerization. erization.

PCR running program was the following: 1.  2.  3.  4.  5. 

Denatura tion Denaturation Enzyme addition Denaturation Annealing of primers Extension of the strands

6.  DNA assembly

94°C 80°C 94°C 55°C 72°C

5 min  5 min  30 s  ┐  30 s ├   1 min  ┘ 

72°C

5 min 

34 x 

Table 1. The components of the PCR reaction when the reaction volume is 50 µl

Reagent dNTP, 1mM of each  primer 1 approx. 20 ρM   primer 2 approx. 20 ρM  10 x reaction buffer (DZB ᵇ)    Aqua Sterilisata (Orion Oy) 

Volume 10 μl  1 μl  1 μl  5 μl  29 μl 

Final concentration 200 μl  0,4 pM 0,4 pM 1x -

 

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Reagent Volume Final concentration DynaZyme™ -polymerase 2,5 μl  1,5 U DNA -template 1 μl  0,5-5 x 10⁴ copies of gene ᵃ  ᵃ assuming that each bacterium  has only one copy of the gene. ᵇ DynaZyme buffer (ingredients  of 1 x buffer: 10mM Tris-HCl, pH 8,8 in 25°C, 1,5mM MgCl ₂, 50mM

KCl, 0,1% Triton X-100).

The products were separated by electrophoresis with 1% agarose gel. The products were visualized by ethidiumbromide dye in the UV light (Figure 10). primers are specificity shown in Table 2. No clearly significant crossreaction was Crossreactions detected at 60oof C. the As PCR one can see the of primers depended on the attachment temperature.

Figure 10. Effect of biological background material matrices on the PCR detection of Salmonella enterica Serovar Typhimurium strain Ty 21a. The amplified products derive from two distinct PCR runs: (A)  557 basepair amplicon (up-lo1) and 666 basepair amplicon (up-lo2). The different sample wells: 1.  –  3.   3. dilutions to saliva: 1,84 x 10Exp4 bacterial cells (1); 1,84 x 10Exp3 bacterial cells (2); 1,84 x 10Exp2 bacterial cells (3); reagent control with H 2O in well (4); molecule weight markers in well (5); 18 bacterial cells (6); 2 bacterial cells (7); 0,2 bacterial cells (8); positive control (9); molecular weight markers (10); (B)  dilution series to blood: 7,45 x 10Exp3 (1); 7,45 x 10Exp2 (2);  blood control (3), water (4); molecular weight markers (5); 70 bacterial cells (6); 7 bacterial cells (7); 0,7 bacterial cells (8); positive posi tive control (9); molecular weight markers (10). Table 2. Results of cross-reactions when selected PCR primers were used Annealing temperature

Sample

Salmonella Typhi 

55°C a

60°C b

60°C a 

+++++ c 

+++++

++++++

(+) d 

-

-

 Aeromonas salmonicida salmonicida

 

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Sample

Citrobacter freundii

Annealing temperature 55°C a

60°C b

60°C a 

 ND

-

-

+++

-

-

-

-

-

+++++

-

-

 K. pneumoniae

-

-

-

 Proteus vulgaris vulgaris

-

-

-

 Pseudomonas aeruginosa aeruginosa

 ND

-

-

 Pseudomonas pseudomallei pseudomallei

++++

-

-

S. Enteritidis 

-

-

-

S. Typhimurium 

-

-

-

Serratia marcescens

-

-

-

(++) d

-

-

Earth sample

ND

-

-

Dental plaque

ND

-

-

 Enterobacter aerogenes aerogenes  Escheria coli  Klebsiella mobilis mobilis

Yersinia ruckeri

ᵃ primers up-lo2; PCR product 666 base pairs product intensity 557 baseof pairs primers up-lo1; ᶜᵇ concentration ofPCR the product; signal: +……..++++++. No signal: ᵈ when brackets used, length of PCR  product  product different than in S. Typhi.  ND = Not done.

4. ENRICHMENT AND DETECTION OF S A  AL L MON MONE E L L A STRAINS IN PROTEIN R ICH ICH FOODS  Salmonella enterica Serovars Typhimurium and Enteritidis were used as well-known

 pathogens contaminating protein rich food at low concentrations. Our aim was to monitor the growth of Salmonella  bacteria with the PMEU (Portable Microbe Enrichment Unit) device (Hakalehto al.,production 2007). The specific live bacterial growth was2009a). monitored on was the detection ofetgas from the cultures (Hakalehto et al., Thisbased method  previously tested with household water samples to selectively detect single Salmonella cells in ~10 hours (Hakalehto et al., 2011). These results were further confirmed by immunoassay immunoassayss or PCR analysis (Kumar et al., 2005). PMEU was used to detect the bacteria at variable concentrations ranging from 1 to 10,000 cfu per ml. With some exceptions (cocoa with one cfu per ml) all contaminations were confirmed in seven hours or less, and confirmed by PCR or immunological analysis (results not shown here). PMEU Scentrion® equipped with sensors for Volatile Organic Compounds (VOC's), revealed a detection time as low as 2 hours after the initial contamination of minced meat with Salmonella  sp. This detection method can be effectively applied for ultra-fast recognition of Salmonella sp. in contaminated foods.

 

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4.1. Materials and Methods for the Detection of Meat Salmonellae

The bacterial strains used in the study are presented in Table 3. S. Typhimur Typhimurium ium and S. Enteritidis strains were obtained from EVIRA Salmonella Center, Kuopio, Finland. The acidcoli E 17 was isolated from a fecal sample of a male infant (Pesola and  producing Escherichia coli Hakalehto, 2011). The neutral end-products producing  Klebsiell  Klebsiella a mobilis ATCC 1276 was

obtained from the American Type Culture Collection (ATCC). Enterobacterial probiotic strain was obtained from an in-house i n-house culture repository of Finnoflag Oy. All the cultures were grown on nutrient agar (2%) slants at 30 ± 1°C for 24 hrs and further stored at 4°C. A 2- to 3-day-old culture was used to inoculate 10 ml of TYG broth consisting (% w/v) 1.0 tryptone, 0.5 Yeast extract and 0.5 glucose, in 20-ml test tubes. Inoculation was carried out by scraping bacterial culture from slants into sterile medium (1 ml), which was subsequently transferred to test tubes. Incubation was carried out in a 37 ± 1 oC incubator for 24 hrs. For PMEU enrichment of meat samples, serial dilutions of bacterial strains grown overnight were prepared. 1 ml of the dilutions was used to inoculate 12 ml of RVS medium containing 2.7 g of minced meat in PMEU syringes (20 ml capacity). An initial concentration of 100, 1000 cfu per ml was used for the enrichment. The mixture was manually agitated and directly transferred to the PMEU. Table 3. Bacterial strains and samples used in the experiment. Brain Heart Infusion broth was used for f or maintenance and enrichment

Syringe syringe 1

Sample S. Enteritidis B 678/95/1 (Evira Salmonella Center, Kuopio, Finland) + minced meat + BHI syringe 2 S. Typhimurium Sa111/01 (Evira)* + minced meat + BHI syringe 3  E. coli E17 (VTT, Espoo, Finland) + minced meat + BHI syringe 4  Klebsiella mobilis ATCC 1276+ minced meat + BHI syringe 5 Probiotic strain + minced meat + BHI syringe 6 minced meat + BHI syringe 7 S. Enteritidis B 678/95/1 (Evira)** + BHI (minced meat) syringe 8 S. Typhimurium Sa111/01 (Evira)** + BHI (minced meat) syringe 9 only BHI (control) *after several times of rejuvenation in the laboratory **directed from the culture collection

The PMEU enrichment of bacterial strains was carried out as described earlier (Hakalehto et al. 2007). Briefly, strains were cultivated under aerobic conditions. In order to mix broth and bacterial cells, gas flow was impelled into 20 ml syringes (ONCE, CODAN Medical ApS, DK-4970 Rødby, Denmark) through a sterile filter (0.2 µm, SY13TF-S, Advanced Microdevices Pvt. Ltd., Ambala Cantt, India) and a needle (0.80mm×120 mm, Sterican, B. Braun, Melsungen, Germany). S. Enteritidis, S. Typhimurium ,  E. coli,  K. mobilis and probiotic cultures were used separately for external contamination of minced meat. Approximately 100 cfu per ml of the above strains were mixed aseptically with 5 g of minced meat with a glass rod and then transferred into 12 ml of BHI (Brain Heart Infusion) medium. PMEU conditions were similar

 

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as described above except that 100% nitrogen was made to flow into the syringes to produce an anaerobic environment. An initial temperature of 37 ± 1°C was maintained for 2 hrs and then raised to 40 ± 1°C for the rest of fermentation. Gas measurements by the PMEU Scentrion® prototype prototype were carried out at intervals of one hour. All strains were grown overnight at 37°C in the TYG broth (Tryptone, Yeast Extract, Glucose). Then they were diluted to sterile water aiming for final concentration of 10 000 cfu  per ml.

About 5 g of minced meat was taken into decanters and 100 µl of the bacterial dilutions (6) were added to it and mixed carefully with sterile glass rods. The mixtures were transferred into 60 ml enrichment syringes. Then the cultivations took place in the BHI medium using 100% nitrogen flow at 37°C for 2 hours and after that at 40°C. The gas measurements in the PMEU Scentrion® prototype were performed in intervals of one hour. 

4.2. Results from Anaerobic Culturing in the PMEU and the Gas Measurements

Anaerobic culturing of the S. Enteritidis, S. Typhimurium ,  E. E. coli,  Klebsiell  Klebsiella a mobilis and  probiotic strain with with minced meat resulted in detectable colony counts after an incubation of of 5 hrs. That growth was detected both with real-time monitoring of volatile compounds and by the inoculation of the plate cultures. The time dependent Volatile Organic Compound (VOC)  production from each bacterial culture was detectable after 7 hour or less time of cultivation, regardless of the initial concentration (results not shown here). This provisional data could facilitate fast-track detection of salmonellae during one working day, even if started from a single cell. Real-time PCR proved out a useful approach for quick verification (results not shown here). Minced meat had very little or no effect on the growth of the different cultures and the counts were comparable in its presence and absence. Bacterial colonies in the syringes containing the minced meat additions are presented in Table 4. Table 4. The growth of various bacteria on t he CHROMagar Orientation™ plates. Colony forming units in syringes 1 –  9  9 after 5 hours of cultivation. The following inocula and samples were taken to the syringes as described in Table 3 5h

5h

5 h Probiotic strain or

contamination

 Klebsiella 

 E. coli 

equivalent colonies

 Bacillus?) ( Bacillus

80000

115000

3

4000

3690000

4

4995000

5

70000

Syringe

5 h Salmonella 

1

100000000

2

200000000

6

17000

7

107000

8

86500

100000

1000

1695000 20000

49000

1000

9

 

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The laboratory strains seemed to have started their growth more rapidly than the fewer times reinoculated isolates (in the Syringes 7 and 8). However, in all circumstances the  Salmonella strains reached the level of detectability in 5 hours only. In the detection of food contaminants it is the ultimate goal to rule out any sources of bacterial contaminations. On that occasion even one living cell is too much, regardless of the possibility that it may not have sufficient potential to initiate the microbial growth alone. Therefore, its presence should  be verified as reliably as possible. In recent experiments where samples were collected from

tap water, we were able to recognize one single cell of Salmonella enterica Serovar Typhimurium and Salmonella enterica Serovar Enteritidis after about ten hours of selective enrichment in the PMEU cultivator (Hakalehto et al., 2011). The method of detection was ultrasensitive gas recording originally developed for monitoring hospital infections (Hakalehto et al., 2009a). In the case of salmonellae in the household water samples the onset of the growth was probably somewhat delayed by the selective conditions prevailing in the RVS broth. From the food samples studied here we were able to detect the contaminating salmonellae from low concentrations by a procedure of seven hours of enrichment and consequent PCR analysis. In this procedure we used two phased enrichment starting with peptone water for  better recovery of the cells switched to the RVS culture in the PMEU after three hours of initial PMEU cultivation in the peptone water. Gas monitoring with PMEU Scentrion® was used for the verification of meat contamination. The required levels for the PCR analysis were reached in most of the PMEU cultivations in 7 hours only.

THE CHICKEN SPOILAGE EXPERIMENT  In this experiment the applicability of PMEU Scentrion®  for the detection of food spoilage was investigated. This device has been previously used for the recording of the minor emissions caused by the metabolizing bacteria in the patient blood samples (Hakalehto et al. 2009b; Pesola and Hakalehto 2012, Hakalehto and Heitto 2012, Laitiomäki et al. 2014). In the experiment, food-grade chicken slices bought from a supermarket were cut into  pieces of less than one gram each. These pieces were added in equal total weights into five syringes, sample sizes varying then from 1.03 g to 1.07 g. Four of these syringes were kept refrigerated for two and half days and the fifth one was taken out to room temperature after a day in order to provoke spoilage. On the third day, fresh growth medium was added to the syringes.

Salmonella and E. coli were inoculated into three out of the four refrigerated syringes, in

addition to the growth media. Both bacteria were rejuvenated three days prior to the start of the experiment. The syringes were sealed with filters appropriate for PMEU Scentrion ® and the measurement was started at 37°C. Gas flow through the syringes was initially around 50 ml/min. Due to poor filter performance and the adverse reaction to the humidity and foam, the filters exposed to the flow declined, and some of the syringes became fully obstructed in the course of the experiment. For these syringes further measurements were impossible to be carried out. For the functionable ones, the measurement was stopped after 24 hours. In future experiments, pre-filtering the vapors and emissions could prevent the obstruction of the air or

 

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gas flow. Results from the growth of the natural bacterial flora in the refrigerated chicken are  presented in Figure 11. The artificially contaminated chicken meet produced different volatile  profiles from the natural background flora (Figure 12). The latter caused peak formation into the curves (6-7 hour time point from the onset of cultivation). This peak caused by the salmonellae is clearly recognizable from the emissions of the other flora, aand nd it was produced in 6-7 hours after the onset of the PMEU enrichment.

Figure 11. The growth of natural microflora in spoiled chicken kept in room temperature for three days, as measured at 37°C in the PMEU Scentrion ® (Hakalehto et al., 2009a; Pesola et al., 2012; Laitiomäki et al., 2014). TYG broth was used as the culture media. MOS 1 - 3 and ScCell 1  –   2 are sensors detecting the vapors resulting from the microbial metabolism. The cultivation time (h) is shown in the x-axis. Arbitrary Chempro units illustrate the amounts of vaporized compounds detected by the sensors in the y-axis.

Figure 12. The growth of Salmonella in artificially contaminated chicken meat (by the strain ® Salmonella Typhimurium) as measured at 37°C in the PMEU Scentrion   (Hakalehto et al., 2009a; Pesola et al., 2012; Laitiomäki et al., 2014). The sample and Salmonella inocula were added to TYG  broth. MOS 1 - 3 and ScCell 1  –   2 are sensors detecting the volatiles resulting from the microbial metabolism. The cultivation time (h) is shown in the x-axis and arbitrary Chempro units illustrating the amounts of vaporized compounds detected by the sensors in the y-axis.

 

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CONCLUSION  In a survey on the methods for fast primary detection of Salmonella contaminations of various foods, it turned out that any amount of cells, regardless of the serovar that they belong to, could be detected within one working day. Immunological, genetic and volatile emission measurements represented the fastest and most accurate means for the practical verification of the Salmonella   sp. The immunoassay and PCR techniques could be used for the specific detection of individual serovars, such as S . Typhi. Volatile sensing with the PMEU

Scentrion® could give a fast track detection of overall Salmonella  contamination in food. Combined with real-time PCR the detection of individual Salmonella  cells, this approach could be accomplished with the PMEU cultivation also in 7 hours or less.

R EFERENCES EFERENCES  Birkhead, G. S., Morse, D. L., Levine, W. C., Fudala, J. K., Kondracki, S. F., Chang, H. G., Shayegani, M., Novick, L., Blake, B. A. Typhoid fever at a resort hotel in New York; a large outbreak with an unusual vehicle. J. Infect. Dis., 1993; 167, 1228-1232. Catty, D., Raykundalia, C. ELISA and related enzyme immunoassays.   In:  Catty, D., editor.   Antibodies a Practical Approach. Approach. Oxford IRL Press; 1988, pp. 98-155. Cogan, T. A., Jørgensen, F., Lappin-Scott, H. M., Benson, C. E., Woodward, M. J., Humphrey, T. J. Flagella and curli fimbriae are important for the growth of Salmonella enterica serovars in hen eggs. Microbiol  Microbiology ogy, 2004; 150, 1063-1071.

Crump, J.A., Sugarman, J. Examining the scale and outcomes of global health fellowship  programs in the United States. J Grad Med Educ., 2012; 4, 261-262. Dantas, G., Sommer, M. O. A. How to fight back against antibiotic resistance.  American Scientist , 2014; 102, 42-51. Duncan, T. G., Doull, J. A., Miller, E. R.; Bancroft, H. Outbreak of typhoid fever with orange  juice as a vehicle, illustrating the value of immunization.  Am. J. Public Health Nations  Health, 1946; 36, 34-36. Ernst, R. K., Dombroski, D. M., Merrick, J. M. Anaerobiosis, type 1 fimbriae, and growth  phase are factors that affect invasion of HEp-2 cells by Salmonella typhimurium. Infect.  Immun., 1990; 38, 2014-2016. Finlay, B. B., Falkow, S. Common themes in microbial pathogenicity.  Microbiol  Microbiol.. Rev., 1989; 53, 210-230. Foley, S. L., Lynne, A. M. Food animal-associated Salmonella  challenges: Pathogenicity and antimicrobial resistance. American Society Society of Anim Animal al Science, 2007; 86, E173-E187. Forester, C. S. The Commodore (Hornblower). Boston, Ma, USA: Brown, Little and Co.; 1945. Forester, C. S. Lord Hornblower  Hornblower . Boston, Ma, USA: Brown, Little and Co.; 1946. Gal-Mor, O., Boyle, E. C., Grassl, G. A. Same species, different diseases: how and why typhoidal and non-typhoidal Salmonella enterica serovars differ. Front Microbiol .,., 2014; 5, 391.

 

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Gong, J., Zhang, J., Xu, M., Zhu, C., Yu, Y., Liu, X., Kelly, P., Xu, B., Wang, C. Prevalence and fimbrial genotype distribution of poultry Salmonella   isolates in China (2006 to 2012). Applied and and Environmental Environmental Mi Microbiolog crobiology, y, 2014; 80, 687-693. Hakalehto, E. Characterization of  Pectinatus cerevisiiphilus cerevisiiphilus  and  P.frisingi  P.frisingiensis ensis  surface components. Use of synthetic peptides in the detection of some gram-negative bacteria. PhD Thesis. Kuopio, Finland: Kuopio University Publications C, Natural and Environmental Environmen tal Sciences 112; 2000. Hakalehto, E. Antibiotic resistance traits of facultative  Enterobacter cloacae  strain studied with the PMEU (Portable Microbe Enrichment Unit). In: Méndez-Vilas, A., editor.  

Science Against Microbial Pathogens: Communicating Current Research and Technological Advances. Badajoz, Spain: Formatex Research Center: Microbiology

Series N:o 3, Vol. 2; 2011, pp.786-796. Hakalehto, E., Heitto, L. Minute microbial levels detection in water samples by Portable Microbe Enrichment Unit Technology.  Environment and Natural Resources Research, Vol. 2 (4). Canadian Center of Science and Education; 2012 Hakalehto, E., Jaakkola, K. Synergistic effect of probiotics and prebiotic flax product on intestinal bacterial balance , Clinical Nutritio Nutrition n 2013 vol. 32, Supplement 1, S200. Hakalehto, E., Kuronen, I. A Method For Producing Jelly Sweets Which Contain Antibodies. Document Type and Number: 1998 WIPO Patent Application WO/1998/043610. WO/1998/043610. Hakalehto, E., Haikara, A., Enari, T. M., Lounatmaa, K. Hydrocloric acid extractable protein  patterns of Pectinatus cerevisiophil cerevisiophilus us strains. Food Microbiology Microbiology, 1984; 1, 209-216. Hakalehto, E., Hujakka, H., Airaksinen, S., Ratilainen, J., Närvänen, A. Growth-phase limited expression and rapid detection of Salmonella type 1 fimbriae. In: Hakalehto, E., editor. Characterization of Pectinatus cerevisiiphilus and P. frisingiensis surface components. Use of synthetic peptides in the detection of some Gram-negative bacteria.  Kuopio,

Finland: Kuopio University Publications C. Natural and Environmental Sciences 112; 2000. Hakalehto, E., Pesola, J., Heitto, L., Närvänen, A., Heitto, A. Aerobic and anaerobic growth modes and expression of type 1 fimbriae in Salmonella .  Pathophysiol  Pathophysiology ogy, 2007; 14, 6169. Hakalehto, E., Pesola, J., Heitto, A., Bhanj, Deo, B., Rissanen, K., Sankilampi, U., Humppi, T., Paakkanen, H. Fast detection of bacterial growth by using portable microbe enrichment unit (PMEU) and ChemPro100i® gas sensor.  Pathophysi  Pathophysiology, ology, 2009a; 16, 57-62. Hakalehto, E., Paakkanen, H., Hänninen, O. Fast detection of environmental microbes by combination of PMEU enrichment and ChemPro100i® ion mobility spectrometer detection. 17 th International Conference on Bioindicators. Moscow; 2009b. Hakalehto, E., Heitto, A., Heitto, L., Humppi, T., Rissanen, K., Jääskeläinen, A., Hänninen, O. Fast monitoring of water distribution system with portable enrichment unit  –   Measurement of volatile compounds of coliforms and Salmonella sp. in tap water.  Journal of Toxicology Toxicology a and nd Environmental Environmental Hea Health lth Sciences, Sciences, 2011; 3, 223-233. Hakalehto, E., Jaakkola, K., Pesola, J., Heitto, A., Hell, M., Hänninen, O. Tendencies in  probiotic treatments. In: Hakalehto, E., editor.  Microbi  Microbiological ological Clinical Hygiene. New York, NY, USA: Nova Science Publishers, Inc.; 2015; pp. 301-321. Harlow, E., Lane, D. Antibodi  Antibodies. es. Cold Spring Harbor Laboratory; 1988, pp. 553-612.

 

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Hermanson, G. T., Mallia, A. K., Smith, P. K.  Immobil  Immobilized ized Affinity Affinity Ligand Techniques. San Diego, CA, USA: Academic Press.; 1992, 454 s. Jane’s NBC Protection equipmen equipmentt listing, 1990-1991. 1990-1991. Krauss, H., Weber, A., Appel, M., Euders, B., Isenberg, H.D., Schiefer, H.G., Slenezka, W., von Graevenity, A., Zahner, H.  Zoonoses  –   Infectious diseases transmissible from animals to humans. AMS Press, Washington D.C., USA; 2003.  Kumar, S., Balakrishna, K., Singh, G. P., Batra, H. V. Rapid detection of Salmonella Typhi in foods by combination of immunomagnetic separation and polymerase chain reaction. World Journal of Microbiology and Biotechnology, 2005; 21, 625-628. Kuosmanen, T., Torvinen, A., Humppi, T., Hakalehto, E. A luminometric detection method

for Salmonella typhi. Poster. In:  Protection Against Chemical and Biological Warfare  Agents. Symposium. Stockholm, 11.-16.6.1995. Laitiomäki, E., Pesola, J., Hakalehto, E. Projected improvement in the fast microbiological analysis of neonatal blood samples.  Journal of Neonatal Nursing , 2014; http://dx.doi.org/10.1016/j.jnn http://dx.doi.org /10.1016/j.jnn.2014.10.0 .2014.10.005. 05. LeMinor, L. The genus Salmonella.  In: Starr, M. P., Stolp, H., Trüper, H. G., Balows, A., Schlegel, H. G., editors. The Prokaryotes  - A Handbook on Habitats, Isolation and Identification of Bacteria. Berlin, Germany: Germany: Springer Springe r Verlag; 1981, pp. 1149-1159. 1149-1159. Matsui, H., Eguchi, M., Ohsumi, K., Nakamura, A., Isshiki, Y.,Sekiya, K., Kikuchi, Y.,  Nagamitsu, T. Azithromycin Azithromycin inhibits the formation of flagellar filaments without suppressing flagellin synthesis in Salmonella enterica  serovar Typhimurium.  Antimicrobial  Antimicrobi al agents and chemotherapy chemotherapy, 2005; 49, 3396 – 3403. 3403. Mshana, S. E., Matee, M., Rweyemamu, M. Antimicrobial resistance in human and animal  pathogens in Zambia, Democratic Republic of Congo, Mozambique and Tanzania: an urgent need of a sustainable surveillance system.  Annals of Clinical Microbiology Microbiology and  Antimicrobials  Antimicrobi als, 2013; 12:28. Müller, K. H., Collinson, S. K., Trust, T. J., Kay, W. W. Type 1 fimbriae of Salmonella Enteritidis, J. Bacteriol .,., 1991; 173, 4765-4772. Pesola, J., Hakalehto, E. Enterobacterial microflora in infancy - a case study with enhanced enrichment. Indian J. Pediatr. Pediatr., 2011; 78, 562-568. Pesola, J., Hakalehto, E. Importance of intestinal microbiota on immunocompromised  pediatric patients. In: Hakalehto, E, editor.  Alimentary microbiome microbiome - a PMEU approach.  New York, NY, USA: Nova Nova Science Publishers, Inc.; Inc.; 2012; pp. pp. 161-178. 161-178. Pesola, J., Paakkanen, H., Hakalehto, E. Enhanced diagnostics of pyelonephritis  –   a case study. Int. J. Med. Med. Sci., 2012; 2, 273-277. Sjölund-Karlsson, Sjölund-Karl sson, M., Joyce, K., Blickenstaff, K., Ball, T., Haro, J., Medalla, F. M., FedorkaCray, P., Zhao, S., Crump, J. A., Wichard, J. M. Antimicrobial susceptibility to azithromycin among Salmonella enterica  isolates from the United States.  Antimicrob.  Agents Chemother., Chemother., 2011; 55, 3985-3989. Tatavarthy, A., Luna, V. A., Amuso, P. T. How multidrug resistance in typhoid fever affects treatment options. Ann. N. Y. Acad. Sci., 2014; 1323, 76-90. Thong, K.-L., Cheong Y.-M., Puthucheary, S., Koh, C.-L., Pang, T. Epidemiologic analysis of sporadic Salmonella  Typhi isolates and those from outbreaks by pulsed-field gel electrophoresis. J. Clin. Microbiol .,., 1994; 32, 1135-1141.

 

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Thong, K.-L., Ngeow, Y.-F., Altwegg, M., Navaratnam, P., Pang, T. Molecular analysis of Salmonella  Enteritidis by pulsed-field gel electrophoresis and ribotyping.  J. Clin.  Microbiol.,  Microbiol ., 1995; 33, 1070-1074. Todd, E. C., Creig, J. D., Bartleson, C. A., Michaelis, B. S. Outbreaks where food workers have been implicated in the spread of foodborne disease. Part 2. Description of outbreaks  by size, severity, severity, and setting. J. Food Prot. Prot., 2007; 70, 1975-1993. Todd, E. C., Creig, J. D., Bartleson, C. A., Michaelis, B. S. Outbreaks where food workers have been implicated in the spread of foodborne disease. Part 4. Infective doses and  pathogen carriage. J. Food Prot., Prot., 2008; 71, 2339-2373. Trivedi, N.A., Shah, P.C. A meta-analysis comparing the safety and efficacy of azithromycin

over the alternate drugs used for treatment of uncomplicated enteric fever.  J Postgrad  Med ., ., 2012; 58, 112-118. Wagner, C., Hensel, M. Adhesive mechanisms of Salmonella enterica.  Adv. Exp. Med. Biol ..,, 2011; 715, 17-34. Wain, J., Hendriksen, R. S., Mikoleit, M. L., Keddy, K. H., Ochiai, R. L. Typhoid fever.  Lancet, 2015; 385, 1136-1145. Wareing, P. W., Davenport, R. R. Microbiology of soft drinks and fruit juices. In: Chemistry and Technology of Soft Drinks and Fruit Juices , Second Edition. Ashurst and Associates Consulting Chemists for the Food Industry Hereford, UK; 2015, pp. 279  –  299.  299. Zhang, S., Kingsley, R. A., Santos, R. L., Andrews-Polymenis, Andrews-Polymenis, H., Raffatellu, M., Figueiredo, J., Nunes, J., Tsolis, R. M., Adams, R. M., Bäumler, A. J. Molecular pathogenesis of Salmonella enterica serotype Typhimurium - induced diarrhea.  Infect. Immun., 2003; 71, 1-12. Zou, X., Huang, X., Xu, S., Zhou, L., Sheng, X., Zhang, H., Xu, H., Ezaki, T. Identification of a fljA gene on a linear plasmid as the repressor gene of fliC in Salmonella enterica   Microbiol.. Immunol Immunol., ., 2009; 53, 191-197. serovar Typhi. Microbiol

 

In: Microbiological Microbiolo gical Food Hygiene Editor: Eino Elias Hakalehto

Chapter 8

ISBN: 978-1-63483-646-3 © 2015 Nova Science Publishers, Inc.

HYGIENIC LESSONS FROM THE DAIRY MICROBIOLOGY CASES  E lilia as H Ha akaleht lehto o Department of Environmen Environmental tal Sciences University of Eastern Finland, Kuopio, Finland

ABSTRACT  Various play after a central role in diets inmany manyindividuals countries. However, it ismilk alsoproducts evident that childhood or the laterhuman in adulthood develop lactose intolerance or allergic reactions toward milk. These are often based on inherited genetic traits. Among some Southeast Asian nations, nearly 100% of the adult  population is lactose intolerant (in Thailand 97%, for example). In Finland, specific  products such as the lactose-free, or low-lactose, items that have been developed for different groups of milk consumers. In any case, industrial dairy manufacturing is causing several risks about which the awareness and caution need to be developed and exercised. The purpose of this chapter is to highlight some means for milk safety. Also some nutritional aspects are discussed. In the hygienic planning in the dairies, modern variants in the milk products need sometimes extra caution. The production processes have to be  planned according to the specific requirements caused by the product types and the  production techniques.

1. INTRODUCTION  Demand for the uniformity of microbiological methods started from water hygiene analysis. In the year 1895, the American Public Health Association recognized the need to diminish or abolish large laboratory-to-laboratory variation in the diagnostic methods, and appointed a committee to draft uniform procedures for some chemical determinations (Doyle et al., 2001). In 1905, also microbiological methods were included in a committee meeting regarding water analysis, producing a manual, Standard Methods of Water Analysis. Interestingly, this manual was published only 13 years after the description of one remarkable

 

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fecal bacterium  Bacillus coli (nowadays  Escherichia coli) by T. Escherich. The concept of “fecal coliforms” was introduced in 1904 by C. Eijkman. The species  E. E. coli and the group of thermotolerant fecal coliformic bacteria (bacteria resembling  E. coli) are the basic hygienic indicators of water quality, especially with respect to the contamination control. However, it is essential to recognize that the water and food qualities are reflected by the presence or absence of many other bacterial groups, besides the coliforms or the fecal strains (Hakalehto, 2015a). Following the water microbiology specifications, standardization of the dairy microbiology field was also started in 1905 leading to the “Standard Methods of Bacterial Milk Analysis” by S.C. Prescott (Doyle et al., 2001). Later on, dairy bacteriology has expanded into the microbiological analysis of various milk products, such as butter,

fermented milks, cheeses and starter cultures (Marth and Steale, 2001). It became also evident that the quality of milk is dependent on the health status of lactating animals. Therefore, we have proposed the use of mobile PMEU (Portable Microbe Enrichment Unit) units in a milk lorry collecting the raw milk from the tanks in different farms into the three collection tanks of the car. Early warnings on the contaminated products are then obtained before the contents of the three tanks in the lorry will be mixed with the milk in the larger tanks of the dairy factory. In the industrial production it is of crucial importance to take into account: 1.  the food type (raw material risks) 2.  risks of contamination inside the production unit 3.  routes of the contamination 4.  “hygienic history” of the unit  5.  distribution chain hygienic surveillance

2. STRATEGIES AND CASES OF MILK HYGIENE CONTROL  Any standardized method or liquid growth medium could be used in the enhanced enrichment by the PMEU (Hakalehto and Heitto, 2012). After pasteurization and homogenization the milk is filled into bottles or cardboard containers of e.g., one liter in volume. During the packaging and storage of the retail milk, many often unforeseeable dangers or hazards or risks could occur, compromising the product’s microbiological  qualities. One of them is introduced in Figure 1. More industrial contamination events are presented in the next volume of the “Microbiological Hygiene” series, namely “Microbiological Industrial Hygiene.”  This example related to the packing line clearly demonstrates, how unacceptable microbial contaminations can sneak into food product regardless of high-class monitoring. These contributing risk factors or elements may overlap as like in the above-mentioned case of one liter containers of pasteurized milk filled in a production unit. The milk for retail shops was filled into containers on a conveyor belt line (Figure 1). Although the packaging line was cleaned according to high hygienic standards, surprisingly high bacterial contamination rates occurred in some of the product packages. The risks which were

 

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identified on the basis of the “hygienic history” of the unit, turned out to have been caused by neglecting one possible source of contamination. See figure text for further explanation.

a

b

c

d

Figure 1 a. The rubber belt line is moving in the milk packaging unit. Severe contamination problems were detected as “spot-like” ín some of the one liter containers . b.  A proteinaceous lubricating substance is pumped from a barrel behind the wall, and sprayed onto the rubber conveyor belt. c. With closer inspection, there is a considerable accumulation of mixed microbial growth found in the barrel attached to the rubber tube. d. Within the lubricating liquid, a steady flow of bacterial inoculation is sprayed onto the belt line. Then the microbial cells adhere to the bottom of the one liter containers. After packing and the closure of the containers, they are moved into basket holders with 4x5 containers in each of them. These baskets are loaded on top of each other and washed with water. Then the microbial cells from the bottoms of the container are rinsed onto the upper parts of the containers below them. When opening these containers, e.g., at homes, the contaminating strains can spread into the milk and start growing in it. Drawings by Ronja Hakalehto.

In Finland, 50% of the of the revenues from one liter of milk are collected by dairy industry and trade organizations (2/3 of this goes to wholesale and retail trade compensations, 1/3 to the dairy industries), only two thirds of the remaining half goes to the producer, and the rest is required for the taxes. The major expenses for hygienic monitoring are covered by the industries and the animal husbandry, which collect only 1/6 and 1/3 of the retail sales income, respectively.

 

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Especially the food industries are strained by these hygiene monitoring expenses. Revised cost structure in hygiene control could make sense also from the consumer point of view and it could encourage better hygiene control if the distributing organization had to take a remarkable share of these costs. Improved and cost-efficient technologies for the microbial monitoring add value to the hygiene screening. On the shelves of the shops, intellectual  packages could give give alarms to the clients abo about ut the contaminated contaminated products.

Figure 2. Graphs of the measurements on the volatile emissions out of the mixed cultures in the PMEU Scentrion® demonstrate dairy contaminants. The Semiconductor Sensor Cell N:o 1 recognized and  primarily verified the contamination contamination by mixed flora in a freshly opened one lliter iter container. The package where the variable gas emitting microflora was detected from, had identical last utility date than another  package (till seven days ahead), the latter of which was clean from any contamination (Hakalehto (Hakalehto et al., 2013a). Both containers were packed at the same hour and originated from the same dairy factory according to the packaging codes. They were also purchased from the same shop at the same time. Such dramatic differences in the purity of the pasteurized milk quality could be explained by a spot-like contamination in the manufacturing industrial unit of the kind that was described above. See also the Figure 1, for considerations.

 

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3. R ESULTS ESULTS SEEN IN THE SUPERMARKET SELVES  The above-mentioned contamination pattern according to which individual product  packages could get randomly contaminated may explain such spoilage incidents as described  by Hakalehto et al. (2013a). In this case, two similar retail milk packages of one liter each were purchased from same shop with identical last utility dates one week ahead from the time of purchasing. One of them was completely spoiled by a mixed culture, whereas the other one was almost sterile. The verification of the contamination was performed by 1.  standard culture method in 2-4 days 2.  PMEU Scentrion® detection of volatiles in 2-4 hours The graphs of the latter method are presented in Figure 2. Regarding the specificity of the

PMEU method in milk analysis, we screened the detection of mastitis contaminants from the milk of individual cows with a combination of real-time PCR and pre-enrichment with PMEU. This project was performed in cooperation with Finnzymes Oy (Mikko Koskinen, Laura Salmikivi). In this case, 1-2 cases out of 50 samples (from different animals) with mastitis (2-4% of the samples) would have passed the tests unnoticed if the PMEU was not in use (Hakalehto et al., unpublished unpublished results).

4. EFFECTS OF CARBON DIOXIDE  Several methods have been suggested for controlling microbial growth in milk. For example, addition of 10-30 mm/liter of carbon dioxide to milk inhibits the growth of a common spoilage organism  Pseudomonas fluorescens  (Muir, 1996). This technique was  believed to extend the shelf life of refrigerated milk by several days. On the other hand, CO₂  can also boost the onset of bacterial growth (see below). In any case, if the concentration of viable cells can get lowered, this will gradually delay the spoilage. It has been demonstrated that in the PMEU cultivation and detection units the onset of  Pseudomona  Pseudomonass growth was delayed by short periods only when the original inoculum was diluted in series of 10 (Figure 3). In case of  Escherichia coli  each 10-fold dilution corresponded to a delay of one hour in the onset of bacterial growth (Hakalehto, 2011). 2011). Both E. coli and klebsiellas were shown to benefit from the volatile metabolites produced  by the adjacent cells (Hakalehto, 2013). However, it was documented more recently that many Clostridium  species, such as C. difficile (Hell et al., 2010), or C. butyricum (Hakalehto and Hänninen, 2012), or C. acetobutylicum  (Hakalehto, 2015b) were able to withstand a  permanent gas flow flow of even 1100% 00% CO2 in the PMEU. In Figure 4. Dr. Markus Hell and the author carry out the tests indicating the clostridial resistance to medicinal carbon dioxide at SALK (Salzburgers Landesklinikum) hospital in Austria in 2008. During these experiments it was demonstrated that not only the spores but also actively growing clostridial cells could survive complete 100% CO2 gas atmosphere. In the PMEU with such gas flow at +37°C, all C. difficile contaminants started their growth in max 19 hours whatever was their initial concentration. Exhaust bubbles from bacterial

 

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cultures containing the CO2 also boosted the onset of the microbial growth (Hakalehto, 2011, 2013).

Figure 3. The growth of  Pseudomonas fluorescens in the PMEU Spectrion® is dependent on the initial size of the inoculum. In comparison with larger inoculum, a ten-fold dilution delays the growth by one hour. With less dense cultures, the time window is still a narrower one. See also Hakalehto (2012).

Figure 4. Dr. Markus Hell (in white coat) and the author (right) carrying out experiments with hospital isolates of Clostridium difficile  at SALK hospital laboratory, Salzburg, Austria. The complete anaerobiosis was achieved in the PMEU using bubbling with 100% CO 2 which seemed to have growth  promoting effects on the clostridial strains. In our later experiments with mixed cultures of Clostridium butyricum and  Lactobacillus brevis  we were able to demonstrate that the carbon dioxide produced by the lactobacilli speeded up remarkably the onset of the growth of the clostridia (Hakalehto and Hänninen, 2012). This finding could explain the sometimes quicker spoilage of some food products by mixed cultures. In other words, some microbial strains interact for mutual benefit or one can boost the other. This leads to enhanced growth resulting in both situations. This is the case also with symbiotic mixed cultures of  E. coli and klebsiellas (Hakalehto et al., 2008). Photo by Kari Rissanen.

 

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5. HEAT TREATMENTS  In the pasteurization of milk and milk products, the dependence of the treatment time on the temperature is illustrated in Table 1. If the milk fat content is more than 10%, each temperature setting should be increased by 3°C (5°F). In the Introduction section of this chapter, usage of a PMEU unit in milk lorries was suggested to be started as a common practice. Then the growth result could be transformed via internet to the dairy unit (Hakalehto, 2011). This could give an idea about the microbe levels, and even quantitative information regarding the milk quality in various partitions of the tanks already before the lorries arrive to the plant. Heat treatments and other procedures could then be adjusted accordingly, or in the worst case over-contaminated milk of a tank section could be discarded. Table 1. Pasteurization time in relation to the treatment temperature temperature according to the

U.S. Public Health Service instructions, 1995

Temperature o C 63 72 89 90 94 96 100

o

F 145 161 191 194 201 204 212

Time s 1800 15 1 0.5 0.1 0.05 0.01

In the industrial processes, it has to be taken into account that required sterilization or  pasteurization temperature has to be achieved and prevailed in all parts of product in an equal way. In other words, it has to be maintained in all droplets of milk simultaneously and unanimously.. On the other hand, heat treatment destroys many beneficial health effects of the unanimously raw milk (Hakalehto, 1998). Nevertheless, it seems to be the method of choice for eliminating many risks during industrial mass production of various milk products. In ultrapasteurization, milk is held at a minimum temperature of 138°C for at least 2 s, and in UHT processing at 140-150°C for a few seconds (Bylund, 1995). During autoclavation with hot steam, the treatment for isabsolute sterility is considered to requirebacteria the temperature of 121°C for 20 minutes. This supposed to eliminate all spore-forming from the sterilized objects. In HACCP analysis (Hazard Analysis and Critical Control Point), the hazard is assessed  by two factors, risks and severity (ICMSF, 1980). In milk products, the serious outbreaks of salmonellosis and listeriosis are associated with high severity and risk level. Therefore, any improved step toward speedy and sensitive detection of milk contamination is justified . Modern intelligent packages and the gas analysis could offer means for fast development in this field. An example of highly severe hazards, but very low risks, is the potential contamination of canned meat by Clostridium botulinum. Even though the amounts of shelf-stable canned

 

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meats are vast, the occurrence of this type of contamination is almost non-existing. The extremely toxigenic C. botulinum would rather occasionally spoil fish products from which they have been isolated more frequently. Therefore, the cold-chain and other precautions of the raw fish have to be properly taken care of from the first stages of the preparation of the catch. Sophisticated techniques developed for molecular biology applications for C. botulinum toxin detection are presented in the Chapter 10 of this book. Some issues on fish spoilage are discussed also in the Chapter 3. Besides pasteurization, UHT (Ultra high treatment or Ultra-high temperature) is often used for the preservation of raw milk. Temperature for UHT treatment of milk is elevated up to some 140-150°C for a few seconds (in pasteurization for 15 seconds at 80°C treatment is enough to remove hazardous vegetative cells). Effect of hygienization method on the  preservation of milk vitamins was summarized summarized by Renner (1974). (1974). See also Table 2. 2. Table 2. Dependence of vitamin loss upon heat treatments of milk (modified from Renner 1974). The numbers indicate the percentage of various vitamin molecules losing

their functionality after the treatment Preservation method

Thiamin

Pyridoxine

Cobalamin

Folic acid Vitamin C

Pasteurization