Cocoa and Coffee Fermentation [MyeBookShelf]

Cocoa and Coffee Fermentations Edited by

Rosane F. Schwan • Graham H. Fleet

Cocoa and Coffee Fermentations

FERMENTED FOODS AND BEVERAGES SERIES Series Editors

M.J.R. Nout and Prabir K. Sarkar Cocoa and Coffee Fermentations (2014) Editors: Rosane F. Schwan and Graham H. Fleet

Handbook of Indigenous Foods Involving Alkaline Fermentation (2014) Editors: Prabir K. Sarkar and M.J.R. Nout

Solid State Fermentation for Foods and Beverages (2013) Editors: Jian Chen and Yang Zhu

Valorization of Food Processing By-Products (2013) Editor: M. Chandrasekaran

Fermented Foods and Beverages Series

Cocoa and Coffee Fermentations

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2015 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Version Date: 20140624 International Standard Book Number-13: 978-1-4398-4793-0 (eBook - PDF) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright. com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

Contents S e r i e s P r e fa c e vii

P r e fa c e ix E d i t o r s xiii

C o n t r i b u t o r s xv C h a p t e r 1 M i x e d M i c r o b i a l F e r m e n tat i o n s a n d M e t h o d o l o g i e s f o r Th e i r I n v e s t i g at i o n 1 D E N N I S S . N I E L S E N , N I L S A R N E B O RG , A N D L E N E J E S P E R S E N

C h a p t e r 2 B o ta n y

and

Production

of

C o c o a 43

U I L S O N V. L O P E S A N D J O S E L U I S P I R E S

C h a p t e r 3 M e t h o d s o f C o c oa F e r m e n tat i o n a n d D ry i n g 71 W I S D O M KO F I A M OA-AW UA

C h a p t e r 4 M i c r o b i a l A c t i v i t i e s d u r i n g C o c o a F e r m e n tat i o n 129 RO S A N E F. S C H WA N , G I L BE R T O V. D E M E L O PER EIR A, A N D GR A H A M H. F LEE T

C h a p t e r 5 B i o c h e m i s t r y

of

C o c o a F e r m e n tat i o n 193

J Ü RGE N VO I G T A N D R E I N H A R D L I E BE R E I

v

vi

C o n t en t s

C h a p t e r 6 Q ua l i t y

and

Safety

of

C o c o a B e a n s 227

L Í D I A J . R . L I M A A N D M . J . RO B N O U T

C h a p t e r 7 C o c o a P r o c e ss i n g a n d C h o c o l at e Te c h n o l o gy 271 E M M A N U E L O H E N E A F OA K WA

C h a p t e r 8 A g r o - I n d u s t r i a l U s e s o f C o c o a B y - P r o d u c t s 309 D I S N E Y R I BE I RO D I A S

C h a p t e r 9 B o ta n y

and

Production

of

C o f f e e 341

N E Y S U S S U M U S A K I YA M A A N D M A R I A A M É L I A G AVA F E R R ÃO

C h a p t e r 10 M e t h o d s o f C o f f e e F e r m e n tat i o n a n d  D r y i n g 367 C A R L O S H . J . BR A N D O A N D M A R I A F E R N A N DA P. BR A N D O

C h a p t e r 11 M i c r o b i a l A c t i v i t y d u r i n g C o f f e e F e r m e n tat i o n 397 C R I S T I N A F E R R E I R A S I LVA

C h a p t e r 12 M e ta b o l i c R e sp o n s e s o f C o f f e e B e a n s d u r i n g P r o c e ss i n g a n d Th e i r I mpa c t o n  C o f f e e F l av o r 431 D I R K S E L M A R , M A I K K L E I N WÄC H T E R , A N D GE R H A R D B Y T O F

C h a p t e r 13 Q ua l i t y

of

C o f f e e B e a n s 477

L U Í S RO BE R T O B AT I S TA A N D S A R A M A R I A CH A LFOU N

C h a p t e r 14 Tox i g e n i c F u n g i

and

M yc o t ox i n s

in

C o f f e e 509

M A R TA H . TA N I WA K I , BE AT R I Z T. I A M A N A K A , A N D M A R I A H E L E N A P. F U N G A RO

C h a p t e r 15 M a n a g e m e n t a n d U t i l i z at i o n o f W a s t e s f r o m C o f f e e P r o c e ss i n g 545 D I S N E Y R I BE I RO D I A S , N E L S O N RO D R Í G U E Z VA L E N C I A , D I E G O A . Z A M BR A N O F R A N C O, A N D J UA N C A R L O S L Ó P E Z-N Ú Ñ E Z

Series Preface Natural fermentation precedes human history, and since ancient times humans have been controlling the fermentation process. Fermentation, the anaerobic way of life, has attained a wider meaning in the biotransformations resulting in a wide variety of fermented foods and beverages. Fermented products, made with uncontrolled natural fermentations or with defined starter cultures, achieve their characteristic flavor, taste, consistency, and nutritional properties through the combined effects of microbial assimilation and metabolite production, as well as from enzyme activities derived from food ingredients. Fermented foods and beverages span a wide diversity range of starchy root crops, cereals, pulses, vegetables, nuts and fruits, as well as animal products such as meats, fish, seafood, and dairy. The science of chemical, microbiological, and technological factors and changes associated with manufacture, quality, and safety is progressing and is aimed at achieving higher levels of control of quality, safety, and profitability of food manufacture. Both producers and consumers benefit from scientific, technological, and consumer-oriented research. Small-scale production needs to be better controlled and safeguarded. Traditional products need to be characterized and described to establish, maintain, and protect their authenticity. Medium- and large-scale food fermentation vii

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Serie s P refac e

require selected, tailor-made, or improved processes that provide sustainable solutions for the future conservation of energy and water, and responsible utilization of resources and disposal of by-products in the environment. The scope of the CRC book series on Fermented Foods and Beverages will include (1) globally known foods and beverages of plant and animal origin (such as dairy, meat, fish, vegetables, cereals, root crops, soybeans, legumes, pickles, cocoa and coffee, wines, beers, spirits, starter cultures, and probiotic cultures), their manufacture, chemical and microbiological composition, processing, compositional, and functional modifications taking place as a result of microbial and enzymic effects, their safety, legislation, development of novel products, and opportunities for industrialization; (2) indigenous commodities from Africa, Asia (South, East, and South-East), Europe, Latin America, and the Middle East, their traditional and industrialized processes and their contribution to livelihood; and (3) several aspects of general interest such as valorization of food-processing by-products, biotechnology, engineering of solid-state processes, modern chemical and biological analytical approaches (genomics, transcriptomics, metabolomics, and other -omics), safety, health, and consumer perception. The third book born in the series is Cocoa and Coffee Fermentation. This treatise, edited by Dr. Rosane Schwan and Professor Graham Fleet, deals with the fermentation of cocoa beans and coffee berries. Needless to say, cocoa and coffee as stimulating beverages are known and appreciated worldwide. Their fermentation forms an essential step in the process of generating flavor, and most of the fermentation is carried out under uncontrolled conditions. In view of ever-increasing requirements for quality and safety by a sophisticated world market, it is essential to upgrade and optimize cocoa and coffee processing based on scientific evidence. We are convinced that this authoritative book provides a balanced dataset contributed by experts in their respective domains.

Preface Cocoa and coffee beans are two of the most traded agricultural commodities on international markets. Currently, about 5 million metric tons of cocoa beans, valued at about US$10 billion, are produced and traded annually. These beans provide the raw material to a global chocolate confectionery industry valued at around US$100 billion annually. For coffee beans, about 8 million metric tons, valued about US$15 billion, are produced, and supply a global retail industry valued at about US$150 billion annually. (Data from the International Cocoa Organization (www.­icco.­org) and the International Coffee Organization (www.­ico.­org).­) Despite the vast economic value of these two raw materials, very few people, including many scientists, know that microorganisms and microbial fermentation play key roles in their production and have major impacts on the quality, safety, and value of the chocolate and coffee products derived from them. Cocoa beans occur in the fruit pods of the tree, Theobroma cacao. After harvesting, the beans are removed from the pods and are embedded in a viscous, sugary pulp. Microbial fermentation of this pulp occurs naturally. This fermentation degrades the pulp and facilitates subsequent drying of the beans, after which they are traded and used in chocolate manufacture. Pulp fermentation also generates many microbial metabolites, such as ethanol and organic acids, ix

x

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which, along with increasing temperature, kill the cocoa bean (seed) and trigger an array of endogenous biochemical reactions that produce the precursors of chocolate flavor and color. This characteristic flavor and color are then further developed during bean drying, and the subsequent roasting and conching processes used for chocolate manufacture. Fermentation is essential to developing these chocolate properties along with other flavors impacted by microbial metabolites. Coffee beans occur in the fruit berries of the tree Coffea arabica or C. canephora. The beans, two per fruit, are surrounded by layers of mucilage, pulp, and fruit skin. A combination of mechanical and microbial fermentation processes is used to degrade and remove these external layers, after which the beans are dried and then sold for coffee production. Two different processes may be used to liberate the beans from the berry; the dry process, or the wet process. In the dry process, the harvested fruits are spread as a thin layer on platforms and allowed to dry in the sun for 10–20 days. During this time, natural microbial fermentation occurs within the berry, leading to degradation of the pulp and mucilage layers. The residual material surrounding the beans is then mechanically removed and the beans are then dried. In the wet process, the skin is mechanically removed from the berry, after which the berries are transferred to a tank of water where a natural fermentation occurs over a period of 24–48 hours to degrade residual pulp and mucilage. The beans are then dried and sold. In both cases, the main function of fermentation is to solubilize and remove pulp and mucilage materials, but microbial metabolites produced during this process could also affect bean aroma and flavor. Although the contributions of microorganisms to these fermentations have been realized for over 100 years in the case of cocoa, and 50 years in the case of coffee, these processes are still conducted as traditional, uncontrolled operations. Such traditional methods give rise to process inefficiencies and inconsistencies in product quality that negatively impact on production economy and product value. During the last 25 years, significant progress has been made in understanding the microbial ecology and biochemistry of cocoa and coffee bean fermentations, leading to suggestions that they be developed into wellcontrolled industrialized processes using defined microbial starter cultures, as has occurred for fermentations now used to produce bread, wine, beer, cheese, yogurts, and so on. Despite these calls and the

P refac e

xi

advantages such technological development would bring, both cocoa and coffee fermentations still remain as traditional processes. Recent research has revealed the microbial and biochemical complexity of these fermentations and the need for further information that links the impacts of particular microbial species and strains to functional outcomes of product aroma and flavor, product safety, and process economy. To achieve such goals, we believe it is important to have a good understanding of the total production chain, where microorganisms fit into this chain, what factors affect the presence, growth, and activity of microorganisms throughout this chain, how such microbial action determines product quality, safety, and acceptability, and how this microbial activity might be best managed to optimize process and operation efficiency, including waste utilization. We have selected and designed the contents of this book to help answer these questions. Both cocoa and coffee fermentations have a complex microbial ecology, involving interactions between species of yeasts, bacteria, and filamentous fungi. Chapter 1 examines the scientific bases of these interactions in microbial fermentations, along with methods for their investigation. Subsequently, there are chapters that systematically cover each of the following topics for cocoa and then coffee: botanical and production background; methods of bean fermentation and drying; microbial ecology and activities of fermentation; biochemistry of fermentation; product quality and safety; and waste utilization. Two additional chapters cover more specific topics; the linkage between cocoa bean quality and chocolate production; and mycotoxin production and safety in coffee products. We hope that this book will inspire further research that links the microbiology and biochemistry of cocoa and coffee bean fermentations with the sensory and safety attributes of the final product; the development of better controlled fermentations using defined microbial starter cultures and improved fermentor design; and the implementation of good manufacturing, quality assurance, and certification programs across the total chain of production. Rosane F. Schwan Graham H. Fleet

Editors Dr. Rosane F. Schwan is an associate professor in the Microbiology Group, Department of Biology at the Federal University of Lavras, Minas Gerais, Brazil, where she is involved in research on the microbiology of fermented foods and beverages. She has specialized interests in cocoa and coffee fermentations and has authored numerous publications on the microbiology and biotechnology of these processes over the past almost 30 years. Graham H. Fleet is an emeritus professor in the Food Science Group, School of Chemical Engineering, the University of New South Wales, Sydney, Australia. He has been active as a researcher on the microbiology and biotechnology of fermented foods and beverages for more than 35 years, including studies on cocoa bean fermentations in Indonesia and north Australia.

x iii

Contributors Emmanuel Ohene Afoakwa Department of Nutrition and Food Science University of Ghana Legon-Accra, Ghana Wisdom Kofi Amoa-Awua Food Research Institute Council for Scientific and Industrial Research Accra, Ghana

Carlos H. J. Brando P&A International Marketing Praça Rio Branco E.S. Pinhal-SP, Brazil Maria Fernanda P. Brando P&A International Marketing Praça Rio Branco E.S. Pinhal-SP, Brazil

Nils Arneborg Department of Food Science University of Copenhagen Copenhagen, Denmark

Gerhard Bytof Tchibo GmbH Coffee Research and Development Hamburg, Germany

Luís Roberto Batista Food Science Department Federal University of Lavras Lavras-MG, Brazil

Sara Maria Chalfoun Agricultural Institute of Minas Gerais-EPAMIG Lavras-MG, Brazil xv

xvi

C o n t ribu t o rs

Disney Ribeiro Dias Food Science Department Federal University of Lavras Lavras-MG, Brazil Maria Amélia Gava Ferrão Incaper Rua Afonso Sarlo Vitória-ES, Brazil Graham H. Fleet School of Chemical Engineering University of New South Wales New South Wales, Australia Diego A. Zambrano Franco Cenicafé FNC Chinchina, Colombia Maria Helena P. Fungaro Universidade Estadual de Londrina (UEL) Londrina-PR, Brazil

Maik Kleinwächter Institute for Plant Biology Technische Universität Braunschweig Braunschweig, Germany Reinhard Lieberei Biozentrum Klein-Flottbek und Botanischer Garten Universität Hamburg Hamburg, Germany Lídia J. R. Lima Laboratory of Food Microbiology Wageningen University Wageningen, the Netherlands Uilson V. Lopes Cocoa Research Centre (CEPEC/CEPLAC) Rod. Ilhéus-Itabuna, Itabuna-BA, Brazil Juan Carlos López-Núñez Cenicafé FNC Chinchina, Colombia

Beatriz T. Iamanaka Instituto de Tecnologia de Alimentos (ITAL) Campinas-SP, Brazil

Dennis S. Nielsen Department of Food Science University of Copenhagen Copenhagen, Denmark

Lene Jespersen Department of Food Science University of Copenhagen Copenhagen, Denmark

M. J. Rob Nout Laboratory of Food Microbiology Wageningen University Wageningen, the Netherlands

C o n t ribu t o rs

Gilberto V. de Melo Pereira Biology Department Federal University of Lavras Lavras-MG, Brazil Jose Luis Pires Cocoa Research Centre (CEPEC/CEPLAC) Rod. Ilhéus-Itabuna, Itabuna-BA, Brazil

x vii

Cristina Ferreira Silva Department of Biology Federal University of Lavras Lavras-MG, Brazil Marta H. Taniwaki Instituto de Tecnologia de Alimentos (ITAL) Campinas-SP, Brazil

Ney Sussumu Sakiyama Plant Breeding Department Federal University of Viçosa Vicosa-MG, Brazil

Nelson Rodríguez Valencia Cenicafé FNC Chinchina, Colombia

Rosane F. Schwan Biology Department Federal University of Lavras Lavras-MG, Brazil

Jürgen Voigt Institute of Microbiology Friedrich-Schiller-University Jena, Germany

Dirk Selmar Institute for Plant Biology Technische Universität Braunschweig Braunschweig, Germany

1 M ixed M i crobial

Fermentati ons and M e thod olo g ies for Their I n v esti g ati on DEN N IS S. N I ELSEN, N I LS A R N EBORG, A ND LENE JESPER SEN Contents

1.1 Introduction 2 1.2 Mixed Microbial Fermentations: Ecological, Biochemical, and Physiological Background 3 1.2.1 Environmental Stress Factors Influencing Microbial Growth and Interactions 3 1.2.1.1 Competition for Nutrients 3 1.2.1.2 The Substrate as a Stress Factor 5 1.2.2 Microbial Interactions 6 1.2.2.1 Inhibitory and Stimulatory Effects of Metabolites 6 1.2.2.2 Production of Antimicrobial Compounds 7 1.2.2.3 Quorum Sensing 7 1.2.2.4 Competition for Space 9 1.3 Solid and Semi-Solid Fermentations 10 1.3.1 Characteristics of Solid-State Fermentations 10 1.3.2 Solid-State Fermented Foods 10 1.3.2.1 Determination of Microbial Physiology and Biochemistry in SSF 10 1.3.2.2 Control of Growth Conditions in SSF 11 1.3.2.3 Cultivation Technique and Bioreactor Design 11 1.4 Methods to Study the Microbial Ecology and Chemistry of Mixed Fermentations 12 1.4.1 Culture-Dependent Approach 13 1.4.1.1 Lactic Acid Bacteria 13 1

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1.4.1.2 Acetic Acid Bacteria 14 1.4.1.3 Aerobic Spore Formers 15 1.4.1.4 Yeasts 16 1.4.1.5 Molds 17 1.4.2 Culture-Independent Approach 17 1.4.2.1 Denaturing Gradient Gel Electrophoresis and Temperature Gradient Gel Electrophoresis 17 1.4.2.2 Terminal Restriction Fragment-Length Polymorphism 20 1.4.2.3 Clone Library Analysis 21 1.4.2.4 Quantitative Real-Time PCR 23 1.4.3 Microscopy-Based Methods 24 1.4.3.1 Direct Monitoring of Microbial Growth 24 1.4.3.2 Intracellular pH 24 1.4.3.3 Fluorescence In Situ Hybridization 25 1.4.4 Technological Properties Approach 26 1.5 Future Prospects and Conclusions 27 References 28 1.1 Introduction

The fermentations of cocoa and coffee are rather complex microbiological processes involving the activity of a wide range of microorganisms such as yeasts, lactic acid bacteria (LAB), acetic acid bacteria (AAB), spore-forming bacteria, and molds (Masoud et  al. 2004; Schwan and Wheals 2004; Nielsen et al. 2007, 2013). The fermentations are driven by a complex interplay between raw materials, different microorganisms, and their metabolites. To control the processes and consequently the quality of the final product, a thorough understanding of this interplay is essential. Not only cocoa and coffee, but numerous other food products are produced by mixed microbial fermentations. Even though the substrates and the end products of these fermentations have very different properties, the underlying mechanisms share many similarities. Consequently, fermentations of cocoa and coffee should not be seen as microbiologically unique processes but, instead, in the broader context of mixed microbial fermentations. In this chapter, the mechanisms underlying food-relevant mixed fermentations

Mi x ed Mi c r o bia l F erm en tati o ns

3

and the available methods to investigate these fermentations are thus reviewed, with the aim of placing the fermentation of cocoa and coffee in a broader scientific context and to provide an overview of the methods that can be used to investigate these fermentations. 1.2 Mixed Microbial Fermentations: Ecological, Biochemical, and Physiological Background

Numerous microbiological interactions take place during mixed fermentations, such as (i) interactions between microorganisms and substrate; (ii) interactions between microorganisms and metabolites; and (iii) interactions between different microbiological groups at both species and strain level. In addition, the microbiological processes during fermentation induce biochemical and/or physical changes in the substrate which also influence the fermentation. 1.2.1 Environmental Stress Factors Influencing Microbial Growth and Interactions

1.2.1.1  Competition for Nutrients  The majority of food-relevant fer-

mentations are carried out under batch conditions, even though some nutrients may drain away, as exemplified by the carbohydrate-rich sweatings that drain away during cocoa fermentations (Schwan and Rose 1994). Under such conditions, the amount of nutrients available is, to a large extent, fixed and those microorganisms that exploit the limited amount of nutrients most efficiently have a competitive advantage (Hibbing et al. 2010) as illustrated in the following discussion. In carbohydrate-rich environments, nitrogen is often a limiting nutrient that directly impacts the fermentation. As an example, the limited nitrogen content during wine or beer fermentations may result in stuck fermentations that are characterized by an unacceptably slow fermentation rate or a fermentation that completely stops, prematurely (Alexandre and Charpentier 1998; Malherbe et al. 2007). Consequently, the rate and degree of fermentation are affected and, for wine fermentation, may even result in a change in the microbiota leading to flavor changes (Alexandre and Charpentier 1998; Malherbe et al. 2007). Similarly, competition for nitrogen between Yarrowia lipolytica and Staphylococcus xylosus, both involved in sausage fermentation,

4

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can give a 100-fold reduction in the Y. lipolytica numbers in mixed fermentations compared to fermentations where Y. lipolytica did not encounter the competition from S. xylosus (Mansour et al. 2009). Lactobacillus plantarum is able to ferment citrate, but has a higher affinity for glucose as a carbon source. When grown as a mono-culture in a substrate containing both carbon sources, the glucose will be metabolized into lactate before the citric acid is metabolized to acetic acid and CO2 (Kennes et  al. 1991). However, when co-cultured with Saccharomyces cerevisiae, the yeast outcompetes L. plantarum for glucose, causing L. plantarum to grow to lower cell numbers and produce less lactic acid compared to when grown as a mono-culture. The impaired growth of L. plantarum makes citric acid metabolism more efficient. Consequently, the pH of the fermenting substrate is higher because the L. plantarum produces less lactic acid and metabolizes the citric acid (Kennes et al. 1991). In other words, due to competition with S. cerevisiae for glucose, L. plantarum reaches smaller numbers compared to mono-culture growth, but the cells remaining are able to metabolize citric acid faster and more efficiently. Similar mechanisms are likely to be of importance during fermentation of cocoa, where assimilation of citrate is an important aspect in driving the microbial succession during the process (Schwan and Wheals 2004). Oxygen availability has a strong impact on microbial growth and competition for oxygen will affect the composition of the microbiota during a given fermentation. As an example, Hansen et  al. (2001) found that Torulaspora delbrueckii and Kluyveromyces marxianus are less tolerant to oxygen-limited conditions than S. cerevisiae which, in mixed culture fermentations, gives S. cerevisiae a competitive advantage, thereby enabling it to become dominant. Even small changes in oxygen tension can significantly influence microbial growth as exemplified by AAB, which require oxygen for growth. During storage/ maturation of wine in barrels, tanks, or bottles, access to even small amounts of oxygen will potentially lead to growth of AAB, thereby spoiling the product (Drysdale and Fleet 1988). In other cases, such as vinegar and cocoa fermentations, growth of AAB is desirable and essential for the quality of the final product. Consequently, oxygen access/aeration strongly influences the progress of such fermentation processes and influences the quality of the final product (Allison and Rohan 1958; Camu et al. 2008; Vegas et al. 2010b).

Mi x ed Mi c r o bia l F erm en tati o ns

5

1.2.1.2  The Substrate as a Stress Factor  The environmental conditions

during mixed fermentations will strongly influence the growth and fermentation capacity of the microorganisms. During mixed fermentations, the stress factors such as low water activity, high sugar concentration, high salt concentration, low/high pH, low content of O2, or high CO2 tension, together with limited nutrient availability, will have a strong impact on the microbial succession at both the species and strain level. In high sugar containing environments such as fermentations of grape and other fruit juices, beer wort, cocoa pulp, and marzipan, the substrate itself exerts a strong stress on the microorganisms present, and is a decisive factor in shaping the microbial community that grows. Only a limited number of organisms, mainly yeasts and LAB, are able to grow under conditions where the sugar concentration exceeds 200 g/L. Furthermore, the often acidic conditions in such environments exert additional stress on the organisms present (Fleet 2001; Hohmann 2002). For most microorganisms, osmotic stress leads to efflux of water from the cell, reduction in cellular volume and accumulation of osmolytes in the cytosol (Hohmann 2002). As a response, the cell seeks to counteract osmotic stress by producing the so-called compatible solutes (mainly glycerol and trehalose) that balance the loss of water and enable the cell to continue metabolism and growth. Even for organisms capable of growing under the high osmotic stress present in high sugar-containing environments, the lag phase is increased significantly when grown at low water activity (Hohmann 2002). Many mixed fermentations such as sauerkraut and traditional vinegar are acid fermentations that give an end-pH as low as 3.0 (Mäki 2004; Vegas et al. 2010a). However, alkaline fermentations that give an end-pH of about 8.5 are common in Asia and Africa, and examples include soya and seed- (African locus bean, roselle (Hibiscus sabdariffa) and baobab) based fermentations (Ouoba et al. 2004, 2008; Parkouda et al. 2009, 2010). Whether pH decreases or increases during fermentation, it will have a strong impact on the microbial succession taking place during the fermentation as seen during the fermentation of kenkey, a spontaneous fermented African maize dough (Jespersen et al. 1994; Halm et al. 2004). The fermentation is dominated by LAB and the two yeast species S. cerevisiae and Candida krusei. As the fermentation progresses, pH decreases due to increasing concentrations of lactic

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C o c oa a n d C o f f ee F erm en tati o ns

acid produced by the LAB. This condition impairs the ability of S. cerevisiae to maintain a proton gradient over its membrane and causes C. krusei to become the dominant yeast during the later stages of fermentation due to its better ability to cope with low pH and high concentrations of organic acids (Jespersen et al. 1994; Halm et al. 2004). 1.2.2  Microbial Interactions

Microbial interactions are classified into indirect (competition, commensalism, mutalism, ammensalism, and neutralism) and direct (predation and parasitism) interactions (Viljoen 2001). In mixed microbial fermentations, a number of different interactions are often taking place simultaneously and/or in succession, influencing not only the microbial composition, but also organoleptic properties of the final product (Jakobsen and Narvhus 1996; Viljoen 2001, 2006). 1.2.2.1 Inhibitory and Stimulatory Effects of Metabolites  Many end

products of microbiological metabolism such as organic acids and ethanol have bacteriostatic/fungistatic effects and, at higher concentrations, they may be bacteriocidal/fungicidal. Well-known examples are ethanol produced by yeasts in wine and lactic acid produced by LAB in dairy products (Fleet 2001, 2007; Carr et al. 2002). However, in many products, these metabolites may also serve as substrates for the growth of microorganisms other than those that produced them. In cocoa fermentation, ethanol produced by yeasts will be converted into acetic acid by AAB, which is essential for the process and quality of the final product (Biehl 1969; Schwan and Wheals 2004; Aculey et al. 2010) and, likewise, in vinegar production, ethanol produced by yeasts is metabolized to acetic acid by Acetobacter and Gluconacetobacter spp. (Vegas et al. 2010b). In Swiss-type cheeses such as Emmental, lactose is metabolized to galactose and l-lactate by Streptococcus thermophilus and, subsequently, the galactose is fermented to l-lactate and/or d-lactate by various lactobacilli (Turner and Martley 1983; Turner et al. 1983; Daly et al. 2010). During ripening of the cheese, some of the lactates are metabolized to propionate, acetate, and CO2 by propionibacteria. This reaction is essential for the characteristic eye formation and flavor development in Swiss-type cheeses but it can also lead to spoilage defects if propionibacteria continue their activity

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during the cold room ripening (Turner et al. 1983; Daly et al. 2010). In other types of cheeses, lactic acid produced by LAB can be utilized by yeasts and molds during maturation, causing an increase in cheese pH to values above 5.0–6.0, thereby allowing the growth of other bacteria such as staphylococci that may contribute to further cheese maturation or spoilage (Addis et al. 2001). 1.2.2.2  Production of Antimicrobial Compounds  In addition to the inhib-

itory effect of many microbial metabolites, a wide range of organisms produce compounds with inhibitory effect against other microorganisms. Among bacteria, for example, various species of LAB, Bacillus, and Enterobacteriaceae produce bacteriocins (peptides and smaller proteins) that inhibit or kill other microorganisms (Riley and Wertz 2002; Franz et al. 2007; Abriouel et al. 2011). Some yeast species have been found to produce killer toxins, also peptides or small proteins that destroy other yeasts (Schmitt and Breinig 2006). Bacteriocins and killer toxins are, in general, mainly active against closely related organisms (Riley and Wertz 2002; Schmitt and Breinig 2006). However, cross-genus and cross-kingdom effects by bacteriocins and yeast killer toxins have been described (Fleet 1999) such as the secretion by S. cerevisiae of peptides that kill non-Saccharomyces winerelated yeasts (Perez-Nevado et al. 2006; Albergaria et al. 2010) and inhibit the bacterium, Oenococcus oeni (Osborne and Edwards 2007). The inhibitory effects exerted by volatile compounds produced by the yeasts Pichia anomala, Pichia kluyveri, and Hanseniaspora uvarum have been reported against the ochratoxin producing mould Aspergillus ochraceus (Masoud et al. 2005). Some broad-spectrum-effective compounds such as reuterin (3-hydroxypropionaldehyde) produced by Lactobacillus reuteri are known as well. Reuterin is active against a range of Gram-positive and Gram-negative bacteria, yeasts, and molds, probably by inducing oxidative stress in the cells by modifying thiol groups in proteins (Talarico et  al. 1988; Axelsson et  al. 1989; Chung et al. 1989; El-Ziney et al. 1999; Cleusix et al. 2007; Rasch et al. 2007; Schaefer et al. 2010). 1.2.2.3  Quorum Sensing  Quorum sensing (QS) is a process by which

microorganisms communicate by signaling molecules in a cell densitydependent manner. Both prokaryotic and eukaryotic microorganisms

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have been shown to communicate in a QS manner, even though the sensing molecules and their mode of action vary to a great extent. QS has been shown to regulate a number of species-dependent physiological functions such as bacteriocin production, biofilm formation, stress response, virulence, and bioluminescence (Gobbetti et al. 2007; Antunes and Ferreira 2009; Moslehi-Jenabian et al. 2009). The collectively controlled gene expression is accomplished through the production, secretion, and detection of small signaling molecules (Xavier and Bassler 2005). In bacteria, QS has been reported both at the intra- and interspecies level. For intra-species communication, Gram-negative bacteria communicate via LuxI/LuxR-type QS systems involving acyl homoserine lactones as signaling molecules, whereas Gram-positive bacteria communicate via two-component-type QS systems involving small autoinduced peptides as signaling molecules. Additionally, the LuxS-mediated QS system producing a universal signaling molecule called autoinducer-2 (AI-2) appears in both Gram-negative and Gram-positive bacteria and is used for interspecies communication (Federle and Bassler 2003). LuxS homologues have been found in the genomes of different lactobacilli and production of AI-2 by some Lactobacillus species has been reported (Gobbetti et al. 2007; Lebeer et al. 2007; Moslehi-Jenabian et al. 2009; Gobbetti et al. 2011a). Also, yeasts are capable of QS even though very little is known about its possible role in mixed fermentations. In yeasts, QS has been reported to coordinate metabolism, to stimulate meiosis and sporulation, to stimulate mycelium formation, and to be involved in the development and survival of neighboring colonies (Richard et  al. 1996; Palkova et  al. 1997; Hayashi et  al. 1998; Chen et  al. 2004; Chen and Fink 2006; Gori et al. 2011b). In Candida albicans, the aromatic alcohol tyrosol has been found to be a QS molecule simulating filamentous growth and thus to shorten lag phase time (Chen et al. 2004), whereas the sesquiterpene farnesol has been found to be a QS molecule that inhibits filamentous growth (Hornby et al. 2001). More recently, the aromatic alcohols phenylethanol and tryptophol were identified as QS molecules stimulating pseudohyphal growth in S. cerevisiae and phenylethanol was additionally found to stimulate invasive growth (Chen and Fink 2006). Ammonia-mediated QS-like

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mechanisms that coordinate colony growth have been observed for yeasts of the genera Candida, Cryptococcus, Endomyces, Debaryomyces, Hansenula, Kluyveromyces, Rhodosporidium, Rhodotorula, Saccharomyces, and Schwanniomyces. Similar mechanisms are likely to be of importance in solid and semi-solid mixed fermentation systems (Palkova et al. 1997; Gori et al. 2007). 1.2.2.4  Competition for Space  During the initial stages of wine fer-

mentation, a range of different yeast species including S. cerevisiae and various non-Saccharomyces yeasts such as Candida, Kloeckera, Torulaspora, Kluyveromyces, and Hanseniaspora spp. have been found to proliferate, but after 2–4 days the non-Saccharomyces yeasts, in general, stop growing and die off (Fleet 2001, 2007). This growth pattern has mainly been attributed to the inability of the non-Saccharomyces yeasts to tolerate the increasing concentration of alcohol in the environment produced by S. cerevisiae (Fleet 2001, 2007). However, some non-Saccharomyces yeasts have been found to tolerate much higher concentrations of alcohol when grown as monoculture compared to growth in co-culture with S. cerevisiae (Nissen et al. 2003). In a series of experiments, it was found that only viable S. cerevisiae cells in physical contact (i.e., not separated by a dialysis membrane) were able to kill the non-Saccharomyces cells, and it was suggested that S. cerevisiae is more efficient in competing for space compared to non-Saccharomyces yeasts which then gives S. cerevisiae a competitive advantage (Nissen et  al. 2003, 2004). In a later study using optical tweezers to trap individual cells, it was shown that confinement of a non-Saccharomyces yeast (Hanseniaspora uvarum) by S. cerevisiae cells indeed slowed down the growth of H. uvarum, thereby demonstrating the importance of cell–cell contactmediated mechanisms in yeast interactions (Arneborg et al. 2005). Apparently, this phenomenon is not restricted to yeasts, as a similar mechanism has been observed in Escherichia coli (Aoki et  al. 2005; Slechta and Mulvey 2006). Due to the often high cell densities reached in mixed microbial fermentations, it is indeed likely that cell–cell contact-mediated interactions play an important role for the microbial succession often observed in mixed fermentations such as cocoa fermentation.

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1.3  Solid and Semi-Solid Fermentations 1.3.1  Characteristics of Solid-State Fermentations

Solid-state fermentation (SSF) processes are defined as any fermentation process taking place on or within solid substrates or supports in the absence or near absence of free water (Pandey et al. 2000). However, during fermentation of cocoa beans and during wet processing of coffee beans, small quantities of free water are present during fermentation. Thus, these fermentations may be characterized as a semi-solid-state fermentation process (Couto et  al. 2001). This kind of fermentation process is very poorly described in the literature. However, it shares more features with SSF than with submerged liquid fermentations, which may be defined as any fermentation process occurring “on” solids dissolved (or submerged) in plenty of free water (Murthy et al. 1993; Ray and Sivakumar 2009). One of the main characteristics is that growth and metabolism of the microorganisms during semi-solidstate fermentations occur on the solid substrate and not in the liquid. Thus, the focus of this chapter will be on the SSF process. 1.3.2  Solid-State Fermented Foods

Some of the first known uses of SSF were, in fact, within the manufacture of foodstuffs, dating back to 2000 bc (bread making in Egypt) and 3000 bc (soy sauce koji making in China) (Pandey et al. 2008). 1.3.2.1  Determination of Microbial Physiology and Biochemistry in SSF

In SSF involving filamentous fungi, the determination of fermentation kinetics (i.e., biomass production), substrate uptake, and metabolite production is difficult, as cells, substrate, and metabolites are maintained within the solid matrix. Thus, in these types of SSF, growth kinetic data are generally provided by the use of indirect methods, comprising measurements of cell components, such as DNA, glucosamine, ergosterol and protein, or measurements of metabolic activity, such as respirometry and microcalorimetry (Bellon-Maurel et al. 2003; Rodriguez-Leon et  al. 2008). However, in SSF involving bacteria and yeasts, the growth of cells in single cultures can be quantified by standard microbial isolation and detection methods; that is, sampling, pretreatment, dilution, inoculation on plates, incubation, and plate

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counting or by quantitative real-time PCR (qPCR). For mixed culture fermentations, the same approach can be used but aided by species or group specific isolation media and subsequent identification, or the use of species-specific qPCR (Rantsiou et al. 2008; Falentin et al. 2010). For the estimation of substrate uptake and metabolite production, not only high pressure liquid chromatography (HPLC) analysis of extracts can be applied (Raimbault 1998), but also new innovative techniques, such as aroma sensing and infrared spectrometry, may be used (Bellon-Maurel et al. 2003). Moreover, as will be described later in this chapter, microbial growth and physiology on solid surfaces may be determined at a single cell level using fluorescence microscopy and image analysis techniques. 1.3.2.2  Control of Growth Conditions in SSF  SSF is a complex and

heterogeneous process, due to the fact that cells, nutrients, and products occur in three different physicochemical phases; for example, the carbon and energy source is in the solid phase, oxygen is in the gas phase, and microbial cells and their metabolic products are in the liquid phase surrounding the solids. In contrast, a submerged fermentation is a more homogeneous process, due to the homogeneity of the suspension of cells, and the solution of nutrients and products, in the liquid phase. Thus in SSF, the fermentation problems may arise due to lack of mixing, resulting in gradients of temperature, oxygen, water, pH, nutrients, and products. These problems render measurement and control of crucial growth conditions, such as temperature, pH, water activity, and oxygen levels, difficult (Raimbault 1998). 1.3.2.3  Cultivation Technique and Bioreactor Design  Although it is possi-

ble to operate SSF in fed-batch and continuous modes, batch processes are most commonly used. Mainly two bioreactor types are nowadays available for SSF batch processes; that is, the packed bed (with forced aeration) and the tray (without forced aeration) bioreactor (Mitchell et al. 2000). To the best of our knowledge, the packed-bed bioreactor, as defined above, is not used for industrial food fermentations. As will be seen in later chapters of this book, a tray-like bioreactor, that is, the tray fermentation system (Allison and Rohan 1958; Allison and Kenten 1963), has been developed for controlled fermentation of cocoa in Ghana (Amoa-Awua 2014, Chapter 3 of this book). The tray bioreactor

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consists of a chamber; the size of which can vary from as small as an incubator to as large as a room, containing a number of trays. The individual trays, often of a depth of 10 cm in the case of cocoa fermentation (Allison and Kenten 1963; Nielsen et al. 2007) and the bottoms of which are usually perforated, may be made of plastic, metal, or wood as it is the case with fermentation of cocoa (Allison and Kenten 1963; Nielsen et al. 2007). The substrate bed in a tray may be turned by hand, due to the difficulty in automating the handling of trays, or it may simply be left static. The growth conditions are controlled by regulating the temperature and humidity of the air which is blown through the chamber (Mitchell et al. 2000). Another system, which is widely used for controlled fermentation of cocoa in Brazil, Indonesia, and Malaysia, is the box bioreactor (Wood and Lass 1985). This system may also fall into the category of a tray bioreactor as a less-controlled heap fermentation system, but it may be considered to contain only one tray, the bottom of which is not perforated, and with a much larger depth of the substrate bed than the ordinary tray bioreactor, thus rendering control of growth conditions more difficult. Alternatively, it may be regarded as a packed-bed bioreactor without forced aeration. In small-scale production sites, coffee is fermented in wooden drums or boxes, whereas in large coffee production facilities the wet fermentation takes place in concrete tanks (Wrigley 1988). As for the tanks, the substrate bed may be turned by hand, or it may be left static. The growth conditions during the wet processing of coffee beans may be controlled by regulating the temperature of the water. As mentioned previously, however, the coffee fermentation process is virtually not described in the scientific literature from a technological point of view, leaving plenty of opportunities for further investigation. 1.4 Methods to Study the Microbial Ecology and Chemistry of Mixed Fermentations

In principle, four different but complementary approaches can be used to study the microbiology and chemistry of complex fermentation systems: 1. The culture-dependent approach, where the organisms involved in the fermentation are isolated by culture on plates of agar media. Identification of organisms isolated from the

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plates is then done by a combination of cultural and molecular methods. 2. The culture-independent approach, where the organisms involved in the fermentation are detected without cultivating them. Here, the molecular methods based on the extraction and analyses of DNA, RNA, or protein from the fermenting substrate are used. 3. The microscopy-based methods, where the organisms are observed directly using light microscopy or by various specific probes using epifluorescence or confocal laser scanning microscopy techniques. 4. The technological properties approach, where changes in substrate composition, metabolite profiles, texture, color, and so on are determined using various chemical and physical analytical methods. In the ideal case, several of the approaches are combined to give a thorough knowledge of the microbiology and biochemistry of the process being investigated. 1.4.1  Culture-Dependent Approach

Numerous microbiological media have been developed for purposes ranging from cultivating broad groups of microorganisms such as aerobic mesophilic organisms on plate count agar (PCA) and yeasts and molds on dichloran Rose Bengal chloramphenicol agar (DRBC) to particular species and sub-species on specially developed selectivedifferential media, such as Escherichia coli O157:H7 on Fluorocult E. coli O157:H7 agar (Szabo et al. 1986). No universal microbiological medium suitable for all food-relevant microorganisms exists. Consequently, it is necessary to use a range of different isolation and detection media and incubation conditions to be able to cultivate all or at least the majority of microorganisms involved in a spontaneous mixed fermentation. 1.4.1.1  Lactic Acid Bacteria  LAB are involved in a wide range of fer-

mentations. LAB are, in general, fastidious and rather difficult to cultivate unless optimal substrates and optimal incubation conditions are used. Various media have been described for the isolation of LAB

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and these have been reviewed in Carr et al. (2002). In brief, de Man, Rogosa and Sharpe (MRS) agar is well suited for the cultivation of lactobacilli and, depending on the pH of the medium, other closely related LAB such as some species of Weisella, some Leuconostoc and Pediococcus (de Man et al. 1960; Carr et al. 2002). Medium 17 (M17) agar supports the growth of most coccal LAB (lactococci, streptococci, leuconostoc), whereas the growth of lactobacilli is suppressed (Terzaghi and Sandine 1975). It is recommended to use MRS and M17 in combination to favor isolation of all readily culturable LAB. Plates of media should be incubated under micro-aerobic conditions, although some members of the lactobacilli will grow even if incubated aerobically. To enhance the growth of oxygen-sensitive species, 0.1% cystein-HCl can be added to the medium (Nielsen et al. 2007). LAB often occupy the same ecological niche as yeasts that, in many cases, readily grow on MRS and M17. To avoid this possibility, cycloheximide (0.04–0.1%) and sorbic acid (0.2%) can be added to the medium (Camu et al. 2007; Nielsen et al. 2007). The addition of cycloheximide and sorbic acid, furthermore, has the advantage that it slows down or completely inhibits mold growth that may be a problem when examining spontaneous fermentations. More selective media targeting specific groups of LAB have been developed such as bile esculin agar and kanamycin esculin azide (KAA) agar (Mossel et al. 1978) for the selective cultivation of enterococci (Swan 1954; Facklam and Moody 1970; Carr et al. 2002). Substrates designed specifically for the cultivation of LAB from stressful environments such as beer take advantage of the antimicrobial properties of the product itself, and beer forms an important ingredient in, for example, universal beer agar (UBA) and Nachweismedium für Bierschädliche Bacterien (NBB) (Jespersen and Jakobsen 1996). The addition of tomato juice or apple juice to MRS facilitates the isolation of LAB from grapes, wines, and other substrates (Fleet 2007). Some LAB are very sensitive to stresses such as oxygen and might die during prolonged storage and transport but can be stored at –80°C for years using 20% glycerol as a cryoprotectant (Camu et al. 2007; Nielsen et al. 2007). 1.4.1.2  Acetic Acid Bacteria  AAB are widely distributed in the envi-

ronment and occur on flowers, fruits, grapes, roots and stems of

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plants, and in the digestive system of insects (Kounatidis et al. 2009; Sengun and Karabiyikli 2011; Loganathan and Nair 2004; Raspor and Goranovic 2008). They also occur as spoilage organisms in products such as beer and wine (Drysdale and Fleet 1988; Sengun and Karabiyikli 2011; Jespersen and Jakobsen 1996). In other systems, AAB play a positive role and are obviously essential for the production of vinegar (Fernández-Pérez et al. 2010; Vegas et al. 2010b) but are also essential in other fermentation processes such as the fermentation of cocoa (Camu et al. 2007; Nielsen et al. 2007) and add important flavor to beers of the lambic type (Martens et al. 1997). Cultivating and maintaining cultures of AAB are not without challenges, especially for strains isolated from high acidity environments (Entani et al. 1985; Sengun and Karabiyikli 2011). From high acidity environments, it is recommended to include acetic acid in the substrate such as in the acetic acid–ethanol medium developed by Entani et al. (1985). The other substrates for isolation and enumeration of AAB include glucose yeast extract carbonate (GYC) agar, deoxycholatemannitol-sorbitol (DMS) agar, and yeast extract-peptone-mannitol (YPM) agar (Drysdale and Fleet 1988; Nielsen et al. 2007; Raspor and Goranovic 2008). AAB often coexist with yeasts and LAB that in many cases also grow well on the mentioned media. Consequently, antibiotics/bacteriocins such as cycloheximide or pimiracin to inhibit yeasts and penicillin or nisin to inhibit LAB have to be added to the medium (Drysdale and Fleet 1988; du Toit and Lambrechts 2002; Nielsen et al. 2007; Raspor and Goranovic 2008). AAB can be stored in liquid media such as yeast extract glucose (YG) or DMS containing 20–40% glycerol at –80°C (du Toit and Lambrechts 2002; Nielsen et al. 2007). 1.4.1.3 Aerobic Spore Formers  Bacillus spp. and other aerobic spore

formers are involved in the production of a wide range of alkalinefermented African and East Asian products as recently reviewed by Parkouda et al. (2009). Owing to their ability to form spores, Bacillus spp. and other aerobic spore formers will survive processing steps that kill vegetative cells. This ability gives these organisms a competitive advantage in products where processing involves a boiling step as, for example, in maari, an African-fermented condiment based on baobab seeds (Parkouda et  al. 2010) and tempe produced from soy

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bean fermentation (Mulyowidarso et  al. 1990). In other processes, such as the fermentation of cocoa beans, the microbial activity causes the temperature to increase to levels inhibitory to other microorganisms, thereby giving Bacillus spp. a competitive advantage to survive and grow during the later stages of this process (Nielsen et al. 2007; Schwan and Wheals 2004). Aerobic spore formers grow on a wide range of media. Nutrient agar, brain–heart infusion agar, and PCA all support the growth of these organisms. To inhibit mold and yeasts, cycloheximide can be added to the substrate (Nielsen et al. 2007; Padonou et al. 2009). In order to isolate aerobic spore formers from an environment with a heavy load of competing microbiota, spore formation can be induced and vegetative cells are killed by heating the product. The recommended time/temperature regime differs widely but 70°C for 15–30 min has been found to give a good agreement between the number of colonyforming units of Bacillus before and after heat treatment (Turnbull et al. 2007). 1.4.1.4 Yeasts  Yeasts are involved in the fermentation of many

products, ranging from beer, wine, bread, and cheese to indigenous African-fermented foods such as lafun, a fermented cassava-based product (Jakobsen and Narvhus 1996; Petersen et al. 2002; Jespersen 2003; Fleet 2007; Padonou et al. 2009; Tamang and Fleet 2009). Yeasts in general grow readily on a range of media such as yeast extract peptone glucose agar, malt extract yeast extract glucose peptone agar, and malt extract agar. LAB often occupy the same ecological niche as yeasts, and a number of LAB also grow well on these media, but LAB and other bacteria can be inhibited by addition of antibiotics such as chlortetracycline and chloramphenicol (Jespersen et al. 2005; Padonou et al. 2009). Deak (2003) offers a comprehensive list and discussion of media for isolation of yeasts from food. In wine and beer, S. cerevisiae and Saccharomyces pastorianus often outnumber other yeast species, which are thus difficult to detect. However, S. cerevisiae does not utilize lysine whereas a wide range of other food relevant yeasts do (Kurtzmann et al. 2011). Lysine is the sole nitrogen source in lysine agar, thereby allowing the growth of a range of non-Saccharomyces yeast while growth of S. cerevisiae is inhibited. This medium has been used for detecting wild yeasts in beer and

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non-Saccharomyces in wine fermentations (van der Kühle and Jespersen 1998; Nissen et al. 2003). 1.4.1.5 Molds  Molds are common spoilage organisms but also play

a positive role in a range of fermented products, such as white and blue-veined cheese, botrytized wine, tempe, and soysauce. Potato dextrose agar and Sabouraud’s agar are commonly used as “general” media for mold isolation and cultivation of pure isolates for identification (Samson et al. 1995; Pitt and Hocking 2009). A number of other substrates have been developed for cultivation of more defined groups. Some examples are Czapeks agar, especially suitable for Penicillium and Aspergillus species (Malloch 1982; Abildgren et al. 1987), DG18 for xerophilic fungi, and DRYES (dichloran Rose Bengal yeast extract agar sucrose agar) for toxigenic Penicillium and Aspergillus spp. (Samson et  al. 1995; Pitt and Hocking 2009). If molds are to be isolated from environments containing high bacterial counts, it is advisable to add broad-spectrum antibiotics to the medium to inhibit bacterial growth. 1.4.2  Culture-Independent Approach

Owing to the microbiological complexity of mixed microbial fermentations, culture-based investigations using a portfolio of different media are tedious and time consuming. Molecular methods based on denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), terminal restriction fragment-length polymorphism (T-RFLP), qPCR and high-throughput sequencing (HTS), and metagenome characterization offer alternative strategies for investigating the microbial ecology of complex fermentations. As culture-independent techniques are based on the detection and subsequent analysis of DNA and/or RNA extracted directly from the samples, they enable the detection of organisms difficult to cultivate by culture-based methods (Muyzer and Smalla 1998; Juste et al. 2008; Metzker 2010). 1.4.2.1 Denaturing Gradient Gel Electrophoresis and Temperature Gradient Gel Electrophoresis  DGGE and TGGE are techniques

based on sequence-specific separation of PCR-derived rRNA gene

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amplicons in polyacrylamide gels. The PCR fragments have similar size but differ in sequence and, consequently, also have different melting properties (Muyzer and Smalla 1998). In DGGE, the gels contain a linearly increasing concentration of denaturant (urea and formamide) and, as the PCR fragments migrate through the gel, they encounter increasing concentrations of denaturants causing them to denature which lowers their mobility and allows them to be separated as discrete bands based on their melting properties (Muyzer and Smalla 1998). In TGGE, the same principle is applied, but the PCR-amplicons migrate through a temperature gradient instead of a chemical gradient (Muyzer and Smalla 1998). If needed, the separated bands of interest can be excised from the gels, re-amplified, and sequenced identifying the microorganisms present to at least genus, often species level (Muyzer and Smalla 1998; Nielsen et al. 2005, 2007). During recent years, DGGE and TGGE have been used to investigate the microbiological populations in numerous fermented products, including wine, fermented cassava, maize dough, fermented sausages, cheese, cocoa, and coffee beans (Ampe et  al. 1999, 2001; Cocolin et al. 2000, 2001; Ercolini et al. 2003; Masoud et al. 2004; Prakitchaiwattana et al. 2004; Nielsen et al. 2005, 2007). The two techniques are efficient tools for rapid, low-cost “fingerprinting” of the microbial ecology of fermented products. For investigating bacterial communities, the 16S rRNA gene is normally targeted for PCR amplification, whereas the 26S rRNA gene (D1-region) is usually targeted when investigating yeast and mold (eukaryotic) communities, but the 18S rRNA gene has also been used. For bacteria, several universal prokaryotic primer sets targeting different variable regions of the 16S rRNA gene have been developed, including the V1, V1–V3, V3, and V6–V8 regions. In general, primers targeting the V3-region have been found to give the highest resolution (number of bands) (Nielsen et al. 2007). However, some genera such as Lactococcus and some Leuconostoc spp. are difficult to differentiate based on the V3-region but are highly variable in the V1-region. Consequently, if the fermentation is expected to contain complex Lactococcus and Leuconostoc communities, the V1-region is a more optimal target (Vogensen, F.K. 2010, unpublished results). Using universal primers, only the most abundant species (