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

8/12/2019 Probiotics From Human Breast Milk and Their Application - Copy (2)

1/22

1

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


2/22

2

1. INTRODUCTION

The word Probiotic comes from Greek language Pro bios which means for lifeopposed 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 quantityand 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 te rm Probiotic firstly used in 1965 by Lilly and Stillwell to describe substanceswhich 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 microbialbalance. In 1989, the meaning was improved by Fuller. Thus, Probiotic is a livemicrobial supplement which affects hosts health po sitively by improving its intestinal

microbial balance. Then this definition was broadened by Havenaar and Huis int Veldin 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 healthbenefit on the host is called Prob iotics 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


3/22

3

Table 1. Few probiotic microorganism and their application

Sr.

No.

Name of

microorganism

Source of

isolation

Probiotic property Reference

1 S. cervisiae Skin of grapes Antitoxic Jenabian, et al.

2010

2 S. cervisiae

boulardi

Lychee fruit Anti inflammatory Jenabian, et al.2010

3 Lactobacilli Fermented

vegetable and

cultured milk

Production of

vitamin- K

Hoover, et al.

1991

4 L.rhamnosus Glycerol in aerobic

condition

Increasing levels of

immunoglobulins

Yavuzdurmaz, et

al. 2010

5 L. salivarious Human oral

cavities and

intestine

Repair of intestinal

tract by providing

needed enzymes

and essential

nutrients

Villoslada, et al.

2007

6 B.subtilis Soil and human

feces and malt

waste

Immune system

stimulator

Turroni, et al.

2011

7 Strepttococcus

thermophilis

Dairy product Production of

antibiotics and

Supress tumor

development and

growth in laboratory

studies

Turroni, et al.

2011

8 L.sporogens or

Bacillus

coagulans

Soil receiving

electroplating

waste water

Modify serum

lipoprotein or blood

fat levels

Yavuzdurmaz, et

al. 2010

9 B. longum Fermented dairy

products and teas

Against formation

of liver tumors

related to

pathogenic microbe

in intestine

Hoover, et al.

1991


4/22

4

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 notidentified 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 lowerlactose 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 thehydrolyses of lactose is the bacterial enzyme B- galactosidase. Another fector is the


5/22

5

slower gastric emptying of Semi- Solid milk products such yogurt. So, the B-

galactosidase 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


6/22

6

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 travellers 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


7/22

7

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


8/22

8

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 travellers 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 semi-

hard 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).


9/22

9

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


10/22

10

Human origin for

human usage

Although the human probiotic Saccharomyces boulardii

is not human origin, this criteria is important for species

dependent health effects.

Acid and bile

tolerance

Important for oral consumption even if it may not be for

other applications for survival through the intestine,

maintaining adhesiveness and metabolic activity.

Adhesion to

mucosal

Surface

Imortant to improve immune system, competition with

pathogens, maintain metabolic activity, prevent pathogens

to adhesion and colonization.

Safe for food and

clinical use

Identification and characterization of strains accuratly,

documented safety. No invasion and no degradation ofintestinal mucus.

Clinically validated

and

documented

health effects

Minumum effective dosage has to be known for each

particular strain and in different products. Placebocontrolled,

double-blinded and randomized studies have

to be run.

Good technological

Properties

Survival in products if viable organisms are required,

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 isthat, the bacterial composition of the infant fecal flora seems to reflect the bacterial


11/22

11

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).


12/22

12

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.


13/22

13

S.No Micro-

organism

Application Source Reference

1 B. longum Development and maintenance

of a balanced immune system

as well as to aid in the nutrition

activities of intestinal cells.

Human

Breast Milk

Zivkovic et al. 2011

2 B. breeve Development and maintenance

of a balanced immune system

as well as to aid in the nutrition

activities of intestinal cells.

Human

Breast Milk

Turroni, et al. 2011

3 L.fermentum Beneficial effects in intestinal

inflammation reducing the

inflammatory response and the

intestinal damage.

Human

Breast Milk

Villoslada, et al.

2007

4 Pediococcus

pentasaceus

ozf

Antimicrobial activity against

gram positive bacteria species,

including the food borne

pathogen.

Human

Breast Milk

African journal of

biotechnology

volume -10

5 L. salivarius An alternative or a complement

for treating staphoylococcal

lactational mastitis.

Human

Breast Milk

Jimenez, et al. 2008

6 L. oris Used as potential probiotics and

have a considerable anti

microbial effect.

Human

Breast Milk

Yavuzdurmaz,

Sebnem Harsa

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


14/22

14

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

Villoslada, et al. 2007).

S. NO. Table-4 Beneficial effect of LAB isolated from breast milk

Strain Beneficial Effect

1 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




3 L. fermentum Anti-allergic effect

L. fermentum CECT5716 Intestinal

colonisation




Enhanced effects of vaccination

Anti-inflammatory effect


15/22

15

2.5 Isolation of L a c t o b a c i l l i

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 37 0C 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 37 0C. 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 grams nature of the isolates were studied using gramsstaning.


16/22

16

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 37 0C 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 -80 0C. 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 recentmolecular 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


17/22

17

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 4C 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 37C

for 48 h. Between 50 and 75 isolates from each sample were randomly selected, grown

in MRS-Cys broth, and stored at -80C in the presence of glycerol (30%,vol/vol).(Martin, et al. 2009).


18/22

18

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-6-

phosphate 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-GGCTGCTGGCACGTAGTTAG- 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: 96C

for 30 s, 48C for 30 s, and 72C for 45 s (40 cycles) and a final extension at 72C for 4

min. The amplicons were purified using a Nucleospin Extract II kit (Macherey-Nagel,

Duren, Germany) and sequenced at the Genomics Unit of the UniversidadComplutense 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).


19/22

19

3. References

1. Cakr, 2003. Determination of some probiotic properties on Lactobacilli andBifidobacteria. Ankara University Thesis of Ph.D.

2. Gismondo, M.R., Drago, L., Lombardi, A. 1999. review of probiotics available tomodify gastrointestinal flora. International Journal of Antimicrobial Agents. 12:287-292

3. Guarner, F., Perdigon, G., Corthier, G., Salminen, S., Koletzko, B. and Morelli, L.2005. Should yoghurt cultures be considered probiotic?. British Journal ofNutrition .93:783-786

4. Fooks, L.J., Fuller, R., Gibson, G.R. 1999. Prebiotics, probiotics and human gutmicrobiology. International Dairy Journal.9:53-61

5. Salminen, S. 1999. Probiotics: scientific support for use. Food Technology.

6. Salminen, S., Gorbach, S., Lee, Y.K., Benno, Y. 2004. Human studies on probiotics:What is scientifically proven today?. Lactic Acid Bacteria Microbiological and Functional

Aspects, New York: Marcel Dekker Inc.134:1190-1194

7. Salminen, S., Ouwehand, A., Benno, Y., and Lee, Y.K. 1999. Probiotics: How shouldthey be defined?. Trends in Food Science&Technology 135:1294-1298.

8. Mombelli, B., Gismondo, M.R. 2000. The use of probiotics in medical practice.International Journal of Antimicrobial Agents .16:531-536

9. Scheinbach, S. 1998. Probiotics: Functionality and commercial status. Biotechnology Advances.16(3):581-608

10. Martin, R., Langa, S., Reviriego, C., Jimenez, E., Marin, L.M., Olivares, M., Boza,J., Jimenez, J., Fernandez, L., Xaus, J. and Rodriguez, J.M. 2004. the commensalmicroflora of human milk: New perspectives for food bacteriotherapy and probiotics.Trends in Food Science&Technology.15:121-127

11. Martin, R., Langa, S., Reviriego, C., Jimenez, E., Marin, L.M., Xaus, J., Fernandez,L. and Rodriguez, J.M. 2003. Human milk is a source of lactic acid bacteria for infantgut. The Journal of Pediatrics.

12. Martin, R., Olivares, M., Marin, M.L., Fernandez, L., Xaus, J., Rodriguez, J.M. 2005.Probiotic potential of 3 Lactobacilli strains isolated from breast milk. J Hum Lact . 21(1):8-17

13 . Martin, R., Olivares, M., Marin, M.L., Fernandez, L., Xaus, J., Rodriguez, J.M. 2004.Probiotic potential of 3 Lactobacilli strains isolated from breast milk. J Hum Lact .15:121-127

14. Olivares, M., Diaz-Ropero, M.P., Martin, R., Rodriguez, J.M. and Xaus, J. 2005. Antimicrobial potential of four Lactobacillus strains isolated from breast milk. Journal of Applied Microbiology. 101:72-79

15. Quwehand, A.C., Kirjavainen, P.V., Shortt, C., Salminen, S. 1999. Probiotics:Mechanisms and established effects. International Dairy Journal.9:43-52


20/22

20

16. Rolfe, R.D. 2000. The Role of probiotic cultures in the control of gastrointestinalhealth. J. Nutr. 130:396s-420s

17. Karaoglu, . A., Aydn, F., Klc, S. S., Klc, A.O. 2003. antimicrobial activity and characteristics of bacteriocins produced by vaginal Lactobacilli. Turk J Med. Sci. 33:7-13

18. Holzapfel, W.H., Haberer, P., Snell, J., Schillinger, U., Huis int Veld, J. 1998. Overview of gut flora and probiotics. International Journal of Food Microbiology.

19. Heikkila, M.P. and Saris, P.E.J. 2003. Inhibition of Staphylococcus aureus by thecommensal bacteria of human milk. Journal of Applied Microbiology.95:471-478

20. Favier, C.F., Vaughan, E.E., De Vos, W.M., Akkermans, A.D.L. 2002. Molecularmonitoring of succession of bacterial communities in human neonates. Appl. EnvironMicrobiol ogy.68:219-226

21. Dunne, C., OMahony, L., Murphy, L., Thornton, G., Morrissey, D., OHalloran, S.,Feeney, M., Flynn, S., Fitzgerald, G., Daly, C., Kiely, B., C OSullivan, G., Shanahan,F., and Collins J.K., 2001. In vitro selection criteria for probiotic bacteria of humanorigin: Correlation with in vivo findings.73:386s-392s

22. Chuayana, Jr. E.L., V. Ponce, C., Rivera, M.R.B., Cabrera, E.C. 2003. Antimicrobialactivity of probiotics from milk products. Phil J. Microbiol. Infect. Dis .32(2):71-74

23. Brady, L.J., Gallaher, D.D., Busta, F.F. 2000. The role of probiotic cultures in theprevention of colon cancer. The Journal of Nutrition. 130:410s-414s

24. Kaur, I.P., Chopra, K., Saini, A. 2002. Probiotics: Potential pharmaceuticalapplications. European Journal of Pharmaceutical Sciences. 15:1-9

25. Klaenhammer, T.R. and Kullen, M.J. 1999. Selection and design probiotics.International Journal of Food Microbiology. 50(1):45-57

26. Federico Lara- Villoslada, Monica Olivares, Saleta Sierra, Juan Miguel Rodrguez,2007 Beneficial effects of probiotic bacteria isolated from breast milk.98:96-100

27. Hatice Yavuzdurmaz, Sebnem Harsa, Izmir Institute of Technology, Izmir, Turkey, Selection of potential probiotic Lactobacillus strains from human milk.

28. Pavel Cermak, Ales Landfill, Pavel Mericka and Milan Houska, 2009 Enterococcusfaecim growth model.5:361-371

29. Susana Delgado, Rebeca Arroyo, Esther Jimenez, Maria L Martin, Rosa delCampo, 2009 Staphylococcus epidermidis strains isolated from breast milk of womensuffering infectious mastitis; potential virulence traits and resistance to antibiotics.9:82

30. Francesca Turroni, Elena Foroni, Fausta Serafini, Alice Viappiani, BarbaraMontanini, Francesca Bottacini, Alberto Ferrarini, Pier Luigi Bacchini, Claudio Rota,Massimo Delledonne, Simone Ottonello, Douwe van Sinderen,3 and Marco Ventura,2011 Ability of Bifidobacterium breve To Grow on Different Types of Milk: Exploring theMetabolism of Milk through Genome Analysis.77:7408-7417


21/22

21

31. LAN-SZU CHOU and BART WEIMER , Department of Nutrition and Food Sciences, Center for Microbe Detection and Physiology, Utah State University, Logan, Isolationand Characterization of Acid- and Bile-Tolerant Isolates from Strains of Lactobacillusacidophilus.82:23-3132. Valerie Coeuret, Micheline Gueguen, Jean Paul Vernoux, Laboratoire deMicrobiologie 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 probioticproducts.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 pathogenicbacteria 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, Gian luca De Bellis, Paul W. OToole, Douwevan Sinderen, Julian R. Marchesi,Marco Ventura, 2011, Diversity of Bifidobacteriawithin the Infant Gut Microbiota.77:7408-7417

35. Federica Guaraldi and Guglielmo Salvatori, 2012, Effect of breast and formulafeeding 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. InternationalJournal of Food Microbiology.50(1):45-57

38. Linda J. Brady, Daniel D. Gallaher and Frank F. Busta, The Role of ProbioticCultures 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 bythe 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 DAmico, PhD, Do probiotics reduce adult lactoseintolerance?54:613-620

41. Gregor Reid, Jana Jass, Tom Sebulsky, and John K. McCormick CanadianResearch and Development Centre for Probiotics, The Lawson Health ResearchInstitute, London, Ontario N6A 4V2 and Departments of Microbiology and Immunology2and 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 VirenderKumar Batish, Probiotics for human health new innovations and emerging trends.4:15


22/22

22

43. Geoffrey A. Preidis, Colin Hill, Richard L. Guerrant, B.S. Ramakrishna, Gerald W.Tannock, and James Versalovic, 2012, Probiotics, Enteric and Diarrheal Diseases, andGlobal Health.1:8-14

44. M.R. Gismondo, L. Drago, A. Lombardi Clinical Microbiology Laboratory, L. SaccoTeaching 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, zmir Institute ofTechnology Izmir, Turkey, 2003, Isolation andMolecular Characterization of Lactic Acid Bacteria From Cheese.

46. Flavia Indrio, M.D. and University of Bari, Bari, Italy Josef Neu, M.D. University ofFlorida, Gainesville, Florida, USA, 2012, The Intestinal Microbiome Of Infants And TheUse Of Probiotics.23:145-150

47. S. M. Lutful Kabir, 2009, The Role of Probiotics in the Poultry Industry.10:3531-

354648. 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 MilkOligosaccharides Promote the Growth of Staphylococci .

49. Seppo J. Salminen, Miguel Gueimonde, and Erika Isolauri, Functional Foods Forumand 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 Rodr guez,Julio Boza and Jordi Xaus, 2007, Beneficial effects of probiotic bacteria isolated frombreast milk.98:96-100

Documents

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