Probiotics From Human Breast Milk and Their Application - Copy (2)

CONTENT S. NO.

CONTENT

PAGE NO.

1

Introduction

2

1.1

Application of probiotics

2

1.2

Selection criteria for probiotics

9

2

Human Breast Milk- A Source of probiotics

10

2.1

Importance of human milk probiotics

11

2.2

Application of probiotics isolated from human milk

12

2.3

Probiotics isolated from human milk and their isolation

13

2.4

Lactic Acid Bacteria (LAB)

14

2.5

Isolation of Lactobacilli

15

2.6

Isolation of Bifidobacteria

16

3

Reference

18

1

1. INTRODUCTION The word ‘Probiotic’ comes from Greek language ‘Pro bios’ which means ‘for life’ opposed to ‘antibiotics’ which means ‘against life’. The history of Probiotics began with the history of man by consuming fermented foods that is well known Greek and Romans consume very much and as a result they consume ‘probiotics’ in large quantity and hence live long. In 1908 a Russian researcher Ellie Metchnikoff, firstly proposed the beneficial effects of probiotic microorganisms on human health (Cakir,et al. 2003) . Metchnikoff hypothesized that Bulgarians are healthy and long lived people because of the consumption of fermented milk products which consists of rod shaped bacteria (Lactobacillus spp.). Therefore, these bacteria affect the gut microflora positively and decrease the microbial toxic activity (Gismondo, et al.1999). The term ‘Probiotic’ firstly used in 1965 by Lilly and Stillwell to describe substances which stimulate the growth of other microorganisms. After this year the word ‘Probiotic’ was used in different meaning according to its mechanism and the affects on human health. The meaning was improved to closest by Parker in 1974. Parker defined ‘Probiotic’ as ‘substances and organisms which contribute to intestinal microbial balance’. In 1989, the meaning was improved by Fuller. Thus, Probiotic is a live microbial supplement which affects host’s health positively by improving its intestinal microbial balance. Then this definition was broadened by Havenaar and Huis in’t Veld in 1992 including mono or mixed culture of live microorganisms which applied for animal and man (Guarner, et al. 2005 ). ‘Live microorganisms which when administered in adequate amounts confer a health benefit on the host’ is called Probiotics is accepted by FAO/WHO (Sanders, et al. 2003). For example- Lactic acid Bacteria (LAB) and Bifidobacteria are the most common types of microbes used as Probiotics. 1.1 Applications of probiotics There are a number of applications of Probiotics some of them are listed below, and Table 1 shows some of the strains of bacteria, their applications and their source of isolation

2

Table 1. Few probiotic microorganism and their application Sr.

Name of

Source of

No.

microorganism

isolation

1

S. cervisiae

Skin of grapes

Probiotic property

Reference

Antitoxic

Jenabian, et al. 2010

2

S. cervisiae

Lychee fruit

Anti –inflammatory

boulardi 3

Lactobacilli

Jenabian, et al. 2010

Fermented

Production of

Hoover, et al.

vegetable and

vitamin- K

1991

Glycerol in aerobic

Increasing levels of

Yavuzdurmaz, et

condition

immunoglobulins

al. 2010

Human oral

Repair of intestinal

Villoslada, et al.

cavities and

tract by providing

2007

intestine

needed enzymes

cultured milk 4

5

L.rhamnosus

L. salivarious

and essential nutrients 6

B.subtilis

Soil and human

Immune system

Turroni, et al.

feces and malt

stimulator

2011

Production of

Turroni, et al.

antibiotics and

2011

waste 7

Strepttococcus

Dairy product

thermophilis

Supress tumor development and growth in laboratory studies 8

9

L.sporogens or

Soil receiving

Modify serum

Yavuzdurmaz, et

Bacillus

electroplating

lipoprotein or blood

al. 2010

coagulans

waste water

fat levels

B. longum

Fermented dairy

Against formation

Hoover, et al.

products and teas

of liver tumors

1991

related to pathogenic microbe in intestine 3

1.1.1 Application of Probiotics on Human health Probiotics have been applied on human to Confer a health benefit on the host. There are a number of effect of Probiotics on Human health. Lots of studies have been carried on the benefits of fermented foods and Probiotics. However in most of these studies researchers dis not use sufficient test subject or they use microorganisms were not identified definitely (Cakir 2003). So, while a number of reported effects have been only partially established, Some can be regarded as well established and well documented. The most common health effect seen on the host is given below (Cakir 2003) -Managing lactose intolerance. -Preventing of colon cancer. -Reduction of allergic systems. -Supression of pathogenic microorganisms (antimicrobial effect). -Reduction of cholesterol. 1.1.2 Lactose Intolerance Most of human become lactose intolerant after weaning. These intolerant people cannot metabolize lactose due to the lack of essential enzyme Beta galactosidose. When they consume milk or lactose-Containing products, Symptoms including abdominal pain, bloating, flatulence, crampiny and diarrhea ensue. As lactose passes through large intestine, it is converted to gas and acid by the colonic microflora. The studies provide that the addition of certain starter cultures to milk products, allows the lactose intolerant people to consume those products without the actual rise of breath hydrogen or associated symptoms (Fooks, et al. 1999). The beneficial effects of probiotics on lactose intolerance are explained by two methods. One of them is lower lactose concentration in the fermented food due to the high lactase activity of bacterial preparations used in the production. The second by increasing lactase enzyme enters the small intestine with the fermented product or with the viable probiotic bacteria (Salminen, et al . 2004) Furthermore, the (AB which is used to produce yogurt, Cactobacillus bulgaricus and Streptococcus thermophilus, are not resistant to gastric acidity. Hence, the products with probiotic bacteria are more efficient for lactose intolerant human. It is thaught that the major fector improves the digestibility by the hydrolyses of lactose is the bacterial enzyme B- galactosidase. Another fector is the 4

slower gastric emptying of Semi- Solid milk products such yogurt. So, the Bgalactosidase activity of probiotic strain and other lactic acid bacteria used in dairy products is really important. B- galactosidase activity within probiotics varies in a huge range. It has to be considered both the enzyme activity of probiotic strain and the activity left in the final product for their used in lactose intolerant Sabjects. (Salminen, et al. 2004). 1.1.3 Immune system Improvement The effect of immune system are promising. However, the mechanism is not well understood. Human, studies have shown that probiotic bacteria can have positive effects on the immune system of their hosts. (Mombelli and Gismondo 2000). Several researches have studied on the effects of in vitro and in Vivo Searchs have been carried out in mice some with human. Data indicate that oral bacteriotherapy and living bacteria feeding against some pathogen (Scheinbach 1998). Probiotics affect the immune system in different ways such as; Producing Cytokines, Stimulating macrophages, increasing Secretory 1g A Concentrations (Cakir 2003, Scheinbach 1998). Some of these effects are related to adhesion while some of them are not (Quwenhand, et al. 1999) Eating fermented milk containing Lactobacillus acidophilus and bifidobacteria could modulate the immune response in human. They give volunteers the test fermented milk over a period of three weeks during which attenuated Salmonella typhi was administered to mimic an enteropathogenic infection. After three weeks, the Specific serum I g A increased. These results Showed that LAB which can Survive in the gastrointestinal tract can act as adjuvants to the humoral immune response (Lime Amster, et al. 1994, Quwehand, et al. 1999). Mechanism- 1 Production of inhibitory substances Production of some organic acids, hydrogen peroxide and bacteriocins which are inhibitory to both gram-positive and gram-negative bacteria. 2- Stimulating of immunity Stimulating of specific and nonspecific immunity may be one possible mechanism of probiotics to protect the host from intestinal disease. This mechanism is not well documented, but it is thought that specific cell wall components or cell layers may act as adjuvants and increase humoral immune response.(Rolfe,et al.2000).

1.1.4 Prevention And Cure of Diarrhea 5

Diarrhea has many causes and many types so it is difficult to evaluate the effects of Probiotics on diarrhea. But there are lots of searches and evidence that Probiotics have beneficial effects on some types of diarrhea. Diarrhea is a severe reason of children death in the worldwide and rotavirus is its Common Cause (Scheinbach 1998). In the treatment of rotavirus diarrhea, Lactobacillus is reported really effective. The best documented Probiotic effect is shortened duration of rotavirus diarrhea using Lactobacillus. It has been given proof in several studies around the world by some like Guandalini, Pant, etc. Also Lactobacillus acidophilus LBI, Bifidobacterium lactis and Lactobacillus reuterii are reported to have beneficial effects on shortening the diarrhea (Salminen, et al.2004) Once of the types of diarrhea is traveller’s not only in developing countries but also in Europe. Probiotics have beneficial effects in preventing some forms of TD. Oksanen in 1990 evaluated the efficacy of Lactobacillus in preventing diaeehea in 820 people travelling from Finland to Turkey. In a double-blind study by Black et al. (1989) lyophylised bacteria (L. acidophilus, B. bifidum, L. bulgaricus, S. thermophilus) were given to 56 Danish tourists on a 2- week trip to Egypt. The occurrence of diarrhea in the group receiving the lactic acid bacteria was 43% while it was 71% in the Placebo group (Gismondo, et al. 1999). Antibiotic therapy causes mild and severe outbreaks of diarrhea. The normal microflora may be Suppressed during the microbial therapy and resulting with filling with pathogenic Strains. The resistant Strains at least Colostridium difficile which is the reason of antibiotic associated diarrhea (ADD). Several Clinical trails have used

Saccharomyces boulardii,

Lactobacillus SPP. And Bifidobacterium SPP. In ADD. Probiotics which are able to restore and replace the normal flora should be used. Also they Should be used in high risk Patients such as old, hospitallised or immune Compromised. Studies with Saccharomyces houlardii proved that Colostridium difficile Concentration is decreased in the presence Saccharomyces boulardii (Ceismondo, et al. 1999). Mechanism- Degradation of toxin receptor: Because of the degredation of toxin receptor on the intestinal mucosa, it was shown that S. boulardii protects the host against C. difficile intestinal disease. (Rolfe,et al. 2000). 1.1.5 Prevention of Cancer Epidemiological Studies point out that if the Consumption of Saturated fats increases in the diet, the occurrence of colon Cancer increases in Western World. Bacterial enzymes

(Beta

glucornidase,

nitroreductase

and

azoreductase)

Convert

precarcinogens to active Carcinogens in the colon. It is thought that Probiotics Could 6

reduce the risk of cancer by decreasing the bacterial enzyme activity. Although the exact mechanisms for the anti tumor action is not Known, some suggestions have been proposed by MeIntosh as follows (Fooks, et al. 1999, Scheinbach 1998) 1)Carcinogen/ Procarcinogen are Suppressed by binding, blocking or removal. 2)Suppressing the growth of bacteria with enzyme activities that may convert the procarcinogens to Carcinogens. 3)Changing the intestinal PH thus altering microflora activity and bile Solubility. 4)Altering Colonic transit time to remove fecal mutagens more efficiently. 5)Stimulating the immune system. (Brady, et al. 2000). 1.1.6 Cholesterol Reduction Lots of researchers proposed that probiotics have cholesterol reduction effects. However, the mechanism of this effect could not be explained definitely. There are two hypotheses trying to explain the mechanism. One of them is that bacteria may bind or incorporated cholesterol directly into the cell membrane. The other one is, bile salt hydrolysis enzymes deconjujate the bile salts which are more likely to be extracted resulting in increased cholesterol breakdown (Cakir 2003). A study on the reduction of cholesterol showed Lactobacillus ruteri decreased total cholesterol by 38% when it is given to mice for seven days in the rate of 10 4 cells/day. This dose of Lactobacillus reuteri caused a 40% reduction in triglyceride and 20% increase in the ratio of High Density Lipoprotein (HDL) toLow Density Lipoprotein (LDL) without bacterial translocation of the native microflora into the spleen and liver ( Kaur, et al. 2002). Some more probiotic strain have been shown in Table -1 with their Application including their source of isolation. 1.1.7 Application of probiotics in Industries The probiotic concept is open to lots of different applications on a large variety of fields relevant for human and animal health. Probiotic products consist of different enzymes, vitamins capsules or tablet and some fermented foods contain microorganisms which have beneficial effects on the health of host. They can contain one or several species of probiotic bacteria; Most of products which destine human consumption are produced in 7

fermented milk or given in powders or tablets. These capsules and tablets are not used for medical applications. They are just used as health supporting products. The oral consumption of probiotic microorganisms produces a protective effect on the gut flora. Lots of studies suggest that probiotics have beneficial effects on microbial disorders of the gut, but it is really difficult to show the clinical effects of such products. The probiotic preperations used for traveller’s diarrhea, antibiotic associated diarrhea and acute diarrhea which is showed that they have positive therapeutic effect. In addition to this the probiotics perceived health benefits, probiotics have been incorporated into a semihard and hard cheese, ice cream, milk powders and frozen dairy desserts. However, there are still several problems with respect to the low viability of probiotic bacteria in dairy foods. Microencapsulation of probiotic bacteria can be used to enhance the viability during processing. 1.1.8 Application of probiotic in Poultry A. Pathogen control (particularly salmonella). It was reported by Francis, et al. that the addition of Lactobacillus product at 75 mg/kg of feed significantly decrease the coliform counts in the ceca and small intestine. Kizerwetter-swida and Binek demonstrated that L.Salivarius 3d strain reduces the number of Salmonella in the group of chickens treated with Lactobacillus. (S.M.Lutful Kabir, 2009). B .Improved health and associated production. C. Pressure to reduce antibiotic use in animal agriculture. D. Improved health and performance. -Improved weight gain and feed conversion. -Help prevent and overcome stress during production. -Improved birds ability to Immunologically respond to challenges. (J.Stan Bailey). Mechanism- suppression of toxin production, reduction of gut pH, attenuation of virulence (Fooks, et al. 1999).

8

1.1.9 Application of probiotics in Aquaculture -The research of Probiotics for aquatic animals is increasing with the demand for environment friendly aquaculture. -Most attempts to propose probiotics have been undertaken by isolating and selecting strains from aquatic environment. -These microbes were vibrionaceae, pseudomonas, Lactic acid bacteria, Bacillus spp. And yeast. -Some strains could increase the resistance to disease of their host. -Many other beneficial effects may be expected from Probiotics, e.g. Competition with pathogens for nutrients or for adhesion sites, and stimulation of the immune system. Mechanism- Blocking of adhesion sites: Probiotics and pathogenic bacteria are in a competition. Probiotics inhibit the pathogens by adhering to the intestinal epithelial surfaces by blocking the adhesion sites.(Rolfe, et al. 2000). 1.2 Selection Criteria for Probiotics

In order to be able to exert its beneficial effect a successful potential probiotic strain is expected to have a number of desirable properties. The selection criteria are listed are in Table -2. A potential probiotic strain may not fulfill all such selection criteria ( Quwehand, et al. 1999).

The selection criteria can be categorized in four basic groups. Appropriateness, technological suitability, competitiveness, performance and functionality (Klaenhammer and Kullen 1999). Strains which have these criteria should be used in order to get effective on health and functional probiotic strains. The properties of probiotics are divided into three basic groups (i) safety aspects, (ii) aspects of functionality and (iii) technological aspects.

Table-2 Selection criteria for probiotics Properties

Remarks

9

Human origin for

Although the human probiotic Saccharomyces boulardii

human usage

is not human origin, this criteria is important for species dependent health effects.

Acid and bile

Important for oral consumption even if it may not be for

tolerance

other applications for survival through the intestine, maintaining adhesiveness and metabolic activity.

Adhesion to

Imortant to improve immune system, competition with

mucosal

pathogens, maintain metabolic activity, prevent pathogens

Surface

to adhesion and colonization.

Safe for food and

Identification and characterization of strains accuratly,

clinical use

documented safety. No invasion and no degradation of intestinal mucus.

Clinically validated

Minumum effective dosage has to be known for each

and

particular strain and in different products. Placebocontrolled,

documented

double-blinded and randomized studies have

health effects

to be run.

Good technological

Survival in products if viable organisms are required,

Properties

phage resistance, strain stability, culturable in large scales, oxygen resistance, have no negative effects on product flavour.

2. Human Breast Milk – A Source of Probiotics After birth, breast milk is the best food for infants because it fulfills all nutritional requirements. Also breast milk protects the newborn against infectious diseases. This effect seems a result of the action of some breast milk components, like different antimicrobial compounds, immunoglobulins and immunocomponent cells. Moreover breast milk also contains prebiotic substances which stimulate the growth of the benefical bacteria in the neonate gut (Martin, etal. 2004, Martin, et al. 2003). It is estimated that an infant ingests 1x105 – 1x107 commensal bacteria while suckling if the infant comsumes approximately 800 ml breast milk per day. From the studies the fact is that, the bacterial composition of the infant fecal flora seems to reflect the bacterial 10

composition of breast milk (Heikilla and Saris 2003). The composition of the gut microflora is thoroughly influenced by the diet of the infant. Thus, the presence of a few predominant Gram-positive species in breast milk may be a reason explaining why microbiota of breast-fed infants is composed of a narrow spectrum of species, and a more diverse microbiota develops after weaning (Martin, et al. 2004, Favier, et al. 2002). Recently there is a growing interest in the probiotic microorganisms isolated from human milk. Studies have been reported the isolation of Lactobacillus gasseri, Lactobacillus fermentum and Enterococcus faecium. These species are considered among the probiotic bacteria, and contain strains that are used in commercial probiotic products. They concluded as their work indicates that breast milk contains lactic acid bacteria is a natural source of LAB for the newborns and may be considered a synbiotic food. Martin et al.(2003).

2.1 Importance of human milk probiotics Besides, a wide variety of source, human milk is considered as the best source for probiotic bacteria due to many reasons such as (i) Breast milk probiotics are more potential than any other Probiotics, (ii) breast milk probiotics are the only probiotics responsible for the initiation and development and of course composition of the neonatal gut microflora, (iii) probiotics from the breast milk are the only Probiotics which protect the baby from many infectious diseases and also responsible for the development of immune system, (iv) the probiotics in human milk have a vital role in protecting the infant against the pathogen such as Staphylococcus Aureus (iv) Breast milk Probiotics are the only Probiotics helpful to the neonates as other Probiotics can not be supplemented to the neonate. (Hatice Yavuzdurmaz, Sebnem Harsha 2008, Francesca Turroni,et al. 2011, Federico Lara-Villoslada,et al.2007).

There are lots of studies on the effect of human milk on the health of infants and the infant diseases but surprisingly lack of studies on the microbiology of breast milk. From the few studies, it is found that human milk is an attractive source for potential probiotic strains. As, the bacteria implement some of the main criteria for being probiotic strains such as, human origin, survival in the gastrointestinal conditions and particularly low pH and

bile,

production

antimicrobial

compounds,

adhesion

to

the

intestinal

mucosa(Olivares 2005).

11

From the bacteria isolated from breast milk, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus fermentum, or Enterococcus feacium are found and they can be regarded as potential probiotic bacteria. Hence, breast milk, a natural source of potentially probiotic or biotherapeutic LAB, protects mother and infants against infectious diseases (Martin, et al. 2004).

2.2 Application of probiotics isolated from human milk A study was focused on the antimicrobial activity against Staphylococcus aureus of bacteria isolated from human milk. They identified the bacteria by different molecular characterization techniques and named the bacteria as staphylococci, streptococci, and LAB as Lactobacillus crispatus, Lactobacillus rhamnosus, Lactococcus lactis and Leuonostoc mesenteroides and also Enterococcus feacalis. Then they examined the antimicrobial activity of these bacteria against Staphylococcus aureus. They concluded that the commensal bacteria in human milk may have a role in protecting the infant and mother against Staphylococcus aureus. Also the results supported that the commensal staphylococci and streptococci are predominant bacterial species in breast milk. The other isolated bacteria Lactobacillus rhamnosus had RAPD profile identical to the commercial strain Lactobacillus rhamnosus GG, which is a commonly used probiotic strain in milk products in Finland (Heikila and Saris 2003). Human milk is an important food for neonates during some months to grow them up and protect the infants against some infectious. The high concentration of LAB in milk from healthy mother may play an important biological role during the first months of life. Studies on this biological fluid indicate that human milk is a challenging source for potential probiotic bacteria. Table-3 shows some of the probiotics isolated from breast milk with their application.

Table-3 Probiotics isolated from breast milk and their application.

12

S.No

Micro-

Application

Source

Reference

organism 1

B. longum

Development and maintenance Human

Zivkovic et al. 2011

of a balanced immune system Breast Milk as well as to aid in the nutrition activities of intestinal cells. 2

B. breeve

Development and maintenance Human

Turroni, et al. 2011

of a balanced immune system Breast Milk as well as to aid in the nutrition activities of intestinal cells. 3

L.fermentum

Beneficial effects in intestinal Human

Villoslada,

inflammation

2007

reducing

the Breast Milk

et

al.

inflammatory response and the intestinal damage. 4

Pediococcus

Antimicrobial

activity

pentasaceus

gram positive bacteria species, Breast Milk

biotechnology

ozf

including

volume -10

the

against Human

food

borne

African

journal

of

pathogen. 5

L. salivarius

An alternative or a complement Human for

treating

Jimenez, et al. 2008

staphoylococcal Breast Milk

lactational mastitis. 6

L. oris

Used as potential probiotics and Human

Yavuzdurmaz,

have

Sebnem Harsa

a

considerable

anti Breast Milk

microbial effect.

2.3 Probiotics isolated from human milk and their isolation Altough, there are limited knowledge about the commensal or probiotic bacteria in breast milk, bacteria commonly isolated from this biological fluid include Staphylococci, Streptococci, Micrococci, Lactobacilli, Enterococci

and Bifidobacteria . (Hatice

Yavuzdurmaz, Sebnera Harsa).

2.4 Lactic Acid Bacteria (LAB) Lactic acid bacteria is one of the vital probiotics isolated from human breast milk. Although, it constitute for  1 % of the total microbiota in infants, but plays a very important role in forming the neonatal gut microflora. The strains of LAB isolated from 13

human breast milk with their application is shown in Table 4. (Martin, et al. 2006, Villoslada, et al. 2007).

S. NO.

1

Table-4 Beneficial effect of LAB isolated from breast milk Strain

Beneficial Effect

L. salivarius

Intestinal colonisation Production of antimicrobial compounds No D-lactic production Anti-microbial effect Immunomodulatory effect Anti-inflammatory effect

2

L. gasseri

Intestinal colonisation Improved gastrointestinal function Production of antimicrobial compounds Anti-microbial effect Immunomodulatory effect

3

L. fermentum

Anti-allergic effect L. fermentum CECT5716 Intestinal colonisation Production of antimicrobial compounds Anti-microbial effect Immunomodulatory effect Enhanced effects of vaccination Anti-inflammatory effect

14

2.5 Isolation of Lactobacilli

2.5.1 Materials and Methods The isolation source was human milk obtained from 7 healthy mother volunteers in Jayna Hospital and Krishna hospital from the Anand and Karamsad town of Gujrat state, India. The milk sample were collected in a sterile tube by manual expression using sterile gloves. Before sample collection, nipples and mammary areola were cleaned with soap and sterile water and soaked in chlorhexidine. The first drop (~500 micolitre) was discarded. Skin sampling was performed briefly, a 4cm 2 area of the upper outer quarter of each breast was gently rubbed gently using sterile cotton swabs soaked in ST solution (0.15 M NaCl with 0.1% Tween 20). The head of each swab was aseptically cut from the handle, Placed into a falcon tube. The samples were collected in sterile carriers and stored on ice until delivery to the laboratory.

Pour plate technique was used to isolate the organism. 1 ml aliquots of the sample were plated into MRS (Man, Rogosa and Sharpe) agar (pH 6.2). The plates were incubated at 370C for 2-3 days under anaerobic conditions (in anaerobe jar using Oxoid anaerogen compact). After incubation, individual colonies were selected and transferred into sterile broth mediums. The isolates were purified by selecting colonies with streak plate tecgnique. (Bhatt, et al.2012).

2.5.2 Colony/Culture characterization

All the isolates were spreaded on nonselective media (N-Agar) and incubated for 24 hours at 370C. Isolated colonies were examined for striking differences in size, shape, margin, elevation, consistency, texture opacity, pigmentation which assist in identification of different group of micro organism.

2.5.3 Morphological characterization Size, shape, arrangement and gram’s nature of the isolates were studied using gram’s staning.

15

2.5.4 Growth on selective media

Man, Rgosa and Sharpe agar (MRS medium). All the isolates were streaked on MRS agar plates and incubated at 370C for 48 hours. Isolates expected to belong in the genus lactobacilli were grown only in this medium.

2.5.5 Long term Preservation of Isolates

Gram positive and catalase negative isolates were preserved in MRS broth medium containing 20% (v/v) glycerol as frozen stocks at -800C. The glycerol stocks of sample were prepared by mixing 0.5 ml of active cultures and 0.5 ml MRS medium including 40% sterile glycerol. (Bhatt, et al. 2012).

2.6 Isolation of Bifidobacteria

Development of intestinal microbiota is an important factor affecting the health of the newborn. Recent studies suggest that specific bacterial components, especially the bifidobacteria, have a key impact on development of a healthy balanced infant microbiota. The composition of infant and child intestinal microbiota may become aberrant and thus influence the development of diarrheal, inflammatory, and allergic diseases. Based on this understanding, positive health effects of probiotics have been reported in the management of diarrheal, inflammatory, and allergic diseases in infants. bifidobacteria make up 60 to 90% of the total fecal microbiota in breast-fed infants. The composition of bifidobacteria microbiota in infants was first clarified by Benno and Mitsuoka. Usually, bifidobacteria appear after birth, and, within a week, they have been reported

as

the

dominant

bacterial

group,

with

Bifidobacterium breve

and

Bifidobacterium bifidum being the most common species in healthy infants. More recent molecular studies have identified Bifidobacterium infantis, Bifidobacterium longum and B. breve as the species most often found in infants. In addition to this, some of the application of strains of Bifidobacterium has been shown above in Table-3. (Salminen, et.al.2005).

2.6.1 Materials and Methods

16

A total of 23 women and their respective infants, which were fed by exclusive breastfeeding, participated in the study, and they were enrolled according to the following criteria: (i) healthy women without present or past underlying conditions; (ii) normal, full-term pregnancy; and (iii) absence of infant and/or maternal perinatal problems, including mastitis. None of the subjects enrolled in this study had received a probiotic treatment during pregnancy or after birth. All volunteers gave written informed consent to the protocol, which was approved by the Ethical Committee of Hospital Clinico (Madrid, Spain). The participants provided samples of breast milk, breast skin swabs, and infant feces between days 4 and 7 after birth. The milk samples were collected in a sterile tube by manual expression using sterile gloves. Previously, nipples and mammary areola had been cleaned with soap and sterile water and soaked in chlorhexidine (Cristalmina; Salvat, Barcelona, Spain). The first drops (~500 microlitre) were discarded. Skin sampling was performed as described previously; briefly, a 4-cm2 area of the upper outer quarter of each breast was gently rubbed using sterile cotton swabs soaked in ST solution (0.15 M NaCl with 0.1% Tween 20). The head of each swab was aseptically cut from the handle, placed into a microcentrifuge tube containing 100 microlitre of ST solution, centrifuged for 5 min, and then removed. Recently, it was shown that this procedure provides a representative profile of the microbial community of human skin. To detect possible contamination, negative controls were prepared using cotton swabs in ST solution without any contact with skin and then subjected to the abovementioned procedures.

All the samples were kept at 4°C until delivery to the laboratory, which occurred within 1 h after collection.

2.6.2 Isolation

Proper peptone water dilutions of the milk, skin, and fecal samples were plated in triplicate onto Man-Rogosa-Sharpe (MRS; Oxoid, Basingstoke, United Kingdom) medium supplemented with L-cysteine (0.5 g/liter) (MRS-Cys) agar plates, which were incubated anaerobically (85% nitrogen, 10% hydrogen, 5% carbon dioxide) in an anaerobic workstation (MINI-MACS; DW Scientific, Shipley, United Kingdom) at 37°C for 48 h. Between 50 and 75 isolates from each sample were randomly selected, grown in MRS-Cys broth, and stored at -80°C in the presence of glycerol (30%, vol/vol).(Martin, et al. 2009). 17

2.6.3 Characterization of the bacterial isolates

The selected isolates were observed by optical microscopy to determine their morphology and Gram staining results. Additionally, they were tested for catalase, oxidase, nitrate reductase, gelatinase activities, production of indol, and production of gas from glucose. All the gram-positive and catalase-negative isolates with typical bifidobacterial shapes were identified to the genus level by demonstration of fructose-6phosphate phosphoketolase (F6PPK) activity in cellular extracts and to the species level by PCR sequencing of a 470-bp fragment of the 16S rRNA gene, using primers plb16

(5’-AGAGTTTGATCCTGGCTCAG-3’)

and

mlb16

(5’-GGCTG

CTGGCACGTAGTTAG-3’) (positions 8 to 27 and 507 to 526 in the 16S rRNA gene sequence of Escherichia coli, respectively). The PCR conditions were as follows: 96°C for 30 s, 48°C for 30 s, and 72°C for 45 s (40 cycles) and a final extension at 72°C for 4 min. The amplicons were purified using a Nucleospin Extract II kit (Macherey-Nagel, Du’ren, Germany) and sequenced at the Genomics Unit of the Universidad Complutense de Madrid, Spain. The resulting sequences were used to search sequences deposited in the EMBL database by using the BLAST algorithm, and the identities of the isolates were determined on the basis of the highest scores (>98%).(Martin, et al. 2009).

Other Bacteria isolated from human breast milk except the above discussed in detail have found to have some adverse effect on human health and thus can not be termed as Probiotics. (Cermak, et al. 2009, Delgado, et al. 2009).

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31. LAN-SZU CHOU and BART WEIMER, Department of Nutrition and Food Sciences, Center for Microbe Detection and Physiology, Utah State University, Logan, Isolation and Characterization of Acid- and Bile-Tolerant Isolates from Strains of Lactobacillus acidophilus.82:23-31 32. Valerie Coeuret, Micheline Gueguen, Jean Paul Vernoux, Laboratoire de Microbiologie Alimentaire USC INRA, Universite de Caen Basse-Normandie, Esplanade de la Paix, 14032 Caen Cedex, FranceLaboratoire de Microbiologie Alimentaire USC INRA, Universite´ de Caen Basse-Normandie, Esplanade de la Paix, 14032 Caen Cedex, France, Numbers and strains of lactobacilli in some probiotic products.97:147-156 33. Martin N. Matu, George O. Orinda, Eliud N.M. Njagi, Craig R. Cohen, Elizabeth A. Bukusi, 2008 In vitro inhibitory activity of human vaginal lactobacilli against pathogenic bacteria associated with bacterial vaginosis in Kenyan women.75:965-969 34. Francesca Turroni, Clelia Pean, Daniel A. Pass, Elena Foroni, Marco Severgnini, Marcus J. Claesson, Colm Kerr, Jonathan Hourihane, Deirdre Murray, Fabio Fuligni, Miguel Gueimonde, Abelardo Margolles, Gianluca De Bellis, Paul W. O’Toole, Douwe van Sinderen, Julian R. Marchesi,Marco Ventura, 2011, Diversity of Bifidobacteria within the Infant Gut Microbiota.77:7408-7417 35. Federica Guaraldi and Guglielmo Salvatori, 2012, Effect of breast and formula feeding on gut microbiota shaping in newborns.2:1-4 36. Susanna Cunningham-Rundles, Siv Ahrne, Rosemary Johann-Liang, Rachel Abuav Ann-Margaret Dunn-Navarra, Claudia Grassey, Stig Bengmark and Joseph S. Cervia, 2011, Effect of Probiotic Bacteria on Microbial Host Defense, Growth, and Immune Function in Human Immunodeficiency Virus Type-1 Infection.3:1042-1070 37. Klaenhammer TR, Kullen MJ. 1999. Selection and design of probiotics. International Journal of Food Microbiology.50(1):45-57 38. Linda J. Brady, Daniel D. Gallaher and Frank F. Busta, The Role of Probiotic Cultures in the Prevention of Colon Cancer.130:410s-414s 39. M.P. Heikkila and P.E.J. Saris Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, Finland, 2003, Inhibition of Staphylococcus aureus by the commensal bacteria of human milk. 95:471-478 40. Kara M. Levri, MD, MPH, Kari Ketvertis, MD, Mark Deramo, MD, Joel H. Merenstein, MD, and Frank D’Amico, PhD, Do probiotics reduce adult lactose intolerance?54:613-620 41. Gregor Reid, Jana Jass, Tom Sebulsky, and John K. McCormick Canadian Research and Development Centre for Probiotics, The Lawson Health Research Institute, London, Ontario N6A 4V2 and Departments of Microbiology and Immunology2 and Surgery,3 University of Western Ontario, London, Ontario N6A 5C1, Canada, 2003, Potential Uses of Probiotics in Clinical Practice.4:658-672 42. Sunita Grover, Hogarehalli Mallapa Rashmi, Anil Kumar Srivastava and Virender Kumar Batish, Probiotics for human health –new innovations and emerging trends.4:15 21

43. Geoffrey A. Preidis, Colin Hill, Richard L. Guerrant, B.S. Ramakrishna, Gerald W. Tannock, and James Versalovic, 2012, Probiotics, Enteric and Diarrheal Diseases, and Global Health.1:8-14 44. M.R. Gismondo, L. Drago, A. Lombardi Clinical Microbiology Laboratory, L. Sacco Teaching Hospital, Uni6ersity of Milan, Via G.B. Grassi 74, 20157 Milan, Italy, 1999, Review of probiotics available to modify gastrointestinal flora.12:287-292 45. Cisem BULUT, İzmir Institute of Technology Izmir, Turkey, 2003, Isolation and Molecular Characterization of Lactic Acid Bacteria From Cheese. 46. Flavia Indrio, M.D. and University of Bari, Bari, Italy Josef Neu, M.D. University of Florida, Gainesville, Florida, USA, 2012, The Intestinal Microbiome Of Infants And The Use Of Probiotics.23:145-150 47. S. M. Lutful Kabir, 2009, The Role of Probiotics in the Poultry Industry.10:35313546 48. K. M. Hunt,a,b J. Preuss, C. Nissan, C. A. Davlin, J. E. Williams, B. Shafii, A. D. Richardson,f M. K. McGuire, L. Bode, and M. A. McGuire, Human Milk Oligosaccharides Promote the Growth of Staphylococci. 49. Seppo J. Salminen, Miguel Gueimonde, and Erika Isolauri, Functional Foods Forum and Department of Biochemistry and Food Chemistry, Department of Pediatrics, University of Turku, Finland, 2005, Probiotics That Modify Disease Risk.135:1294-1298 50. Vaibhav D. Bhatt, Yati H. Vaidya, Prashant D. Junjadia and Anju P. Kunjalia, 2012, Isolation And Characterisation of probiotic Bacteria From Human Milk.3:62-70 51. Federico Lara-Villoslada, Monica Olivares, Saleta Sierra, Juan Miguel Rodrı guez, Julio Boza and Jordi Xaus, 2007, Beneficial effects of probiotic bacteria isolated from breast milk.98:96-100

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