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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
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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
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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
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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
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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
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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
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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
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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).
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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
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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
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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).
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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.
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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
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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
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
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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.
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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
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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).
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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).
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