The document discusses developing a breast milk substitute called MIMIC-BM made from buffalo milk. It aims to resemble human breast milk by making buffalo milk more compatible and functional. The need arises when mothers cannot breastfeed for medical reasons, poor lactation, or lack of availability. The document provides nutritional analyses of human milk, animal milks like cow and buffalo milk, and reviews the establishment of infant gut microbiota from breastfeeding versus formula feeding. It also discusses digestion in neonates and their nutritional requirements.

1. MIMIC-BM
Dairy Processed
Buffalo Milk
Made
more compatible
and total
in it’s functional
parameters
resembling
Healthy
Mother’s
Breast Milk
NEED OF MIMIC-BM ARISES IN THE CASES OF
• Mothers those who cannot breast feed owing to medical reasons
Without any intervention, 30-35% of mother-to-child transmission (MTCT) cases are
due to breastfeeding. The remaining portion of MTCT cases occur in utero and at
childbirth. This is called perinatal transmission. As the total percentage of MTCT
ranges from between 15-45% by region, it's expected that a child born to an HIV-
positive mother has a 5-10% chance of acquiring the virus via breastfeeding
• Mothers those who cannot breast feed owing to poor lactation
• Non availability of Mothers.
• Other Reasons
Despite all the public-health campaigns encouraging new mothers to breastfeed,
tens of millions of babies worldwide are raised on infant formula rather than breast
milk.
PREAMBLE
Given that so many babies are being bottlefed, we are bound to do something and
everything that is possible on this Earth to make it better and worthwhile not in the
interest of Economics but due to our accountability to give our best to our next
generations. However It is becoming feasible to manufacture human proteins like
those in milk, thanks to the advancement in Science and Technology, which could be
added to such breast milk replacements.
The buffalo milk is normally hesitated to be given to infants in India for unknown
reasons. Some of such reasons might be that
Buffalo milk contains more energy, protein, fat or total solid as compared to the cow’s or
other milk types.
Buffalo Milk will not be easily digested
Buffalo Milk may cause flatulence recurrent vomiting abdominal distension and difficulty
in stool evacuation.

2. NUTRITIVE ANALYSIS OF HUMAN MILK
Amino Acids (mg)
Constituent Human Milk
Alanine 56.00
Arginine 51.00
Aspartic Acid 120.00
Cystine 24.00
Glutamic Acid 220.00
Glycine 36.00
Histidine 31.00
Isoleucine 77.00
Leucine 130.00
Lysine 86.00
Methionine 24.00
Phenylalanine 54.00
Proline 120.00
Serine 59.00
Threonine 63.00
Tryptophan 22.00
Tyrosine 56.00
Valine 81.00
Carbohydrates (select) (gm)
Constituent Human Milk
Lactose 7.00
Glucose --
Fructose --
Sucrose --
Starch --
Fatty Acids (mg)
Constituent Human Milk

3. Capric Acid 61.00
Lauric Acid 213
Myristic Acid 342
Palmitic Acid 963
Stearic Acid 293
Arachidic Acid 46
Palmitoleic Acid 129
Oleic Acid 1,340.00
Linoleic Acid 380
Linolenic Acid 22
Arachidonic Acid 4.2
Other (varies) --
Sterols (mg)
Constituent Human Milk
Cholesterol 25.00
Miscellaneous (mg)
Casein 360
Whey Protein 500
COMPOSITION OF ANIMAL MILK
MILK COMPOSITION ANALYSIS
Constituents Cow Goat Sheep Buffalo
Protein grm 3.2 3.1 5.4 4.5
Fat grm 3.9 3.5 6 8
Carbohydrate grm 4.8 4.4 5.1 4.9
Energy K cal 66 60 95 110
K J 275 253 396 463
Sugars (Lactose) grm 4.8 4.4 5.1 4.9
Fatty Acids- grm 2.4 2.3 3.8 4.2
Saturated grm 1.1 0.8 1.5 1.7
Monounsaturated grm 0.1 0.1 0.3 0.2

4. Polyunsaturated
Cholesterol mg 14 10 11 8
Calcium iu 120 100 170 195
COMPOSITOIONS OF ANIMAL VS HUMAN MILK
Various types of milk composition per 100 ml of milk
Milk Type
Human’s Milk* Cow’s Milk* Buffalo’s Milk* Lactogen-1**
Moisture (gms) 88 88 81 90.4
Protein (gms) 1.1 3.2 4.3 1.7
Fat (gms) 3.4 4.1 8.8 3.4
Carbohydrates (gms) 7.4 4.4 5.0 7.41
Energy (kcal) 65 67 117 67
Minerals (gms) 0.1 0.8 0.8 0.4
Calcium (mgs) 28 120 219 73
Phosphorus (mgs) 11 90 198 53
Iron (mgs) 0.5 0.2 0.2 0.8
Vitamin A (IU) 137 174 160 230
Thiamin (mgs) 0.02 0.05 0.04 0.05
Riboflavin (mgs) 0.02 0.19 0.10 0.1
Niacin (mgs) - 0.1 0.1 0.67
Vitamin C (mgs) 3 2 1 7
*Goplan et al. (1981). Nutritive value of Indian Foods. National Institute of Nutrition, Hyderabad-7 (A.P.),
Indian Council of Medical Research. P.O. Box No. 4508. Assari Nagar, New Delhi-16. **Nestle Milkpack
Limited 308 Upper Mall, Lahore 54000
INITIAL COLONIZATION OF THE NEONATE
There is no publication or work describing the presence of lactic acid bacterial strains in
normal human amniotic fluid. It has been so far nowhere reported that such non-
pathogenic microbial strains could be beneficial for the gestating baby. Just during the
gestation, the initial microbial populations are able to colonize the fetal gut.
INTESTINAL MICROFLORA IN THE NEWBORN INFANT
Fetuses are sterile in the womb, but beginning with the birth process, infants are
exposed to microbes that originate from the mother and the surrounding environment.
The infant tends to acquire the flora swallowed from the vaginal fluid at the time
of delivery. Because vaginal flora and intestinal flora are similar, an infant's flora
may closely mimic the intestinal flora of the mother.
Another factor affecting the intestinal flora of the newborn is delivery mode.
A normal vaginal delivery commonly permits transfer of bacteria from the mother to the
infant.
During cesarean deliveries, this transfer is completely absent. These infants commonly
acquire and are colonized with flora from the hospital's environment and, therefore, their
flora may differ from maternal flora. Infants delivered by cesarean section are colonized
with more anaerobic bacteria, especially Bacteroides, than vaginally delivered infants.
Clostridium perfringens is the anaerobic bacterium most frequently isolated after

5. cesarean deliveries. When colonized, cesarean delivered infants less frequently harbor
E. coli, and more often klebsiella and enterobacteria.
The initial colonizing bacteria vary with the food source of the infant including breast
milk or formulated dairy products fed by bottle.
Breast milk provides along with the basic fats, proteins, carbohydrates, minerals and
vitamins that babies need to survive; hormones, immune signalling molecules,
antibodies and even living immune cells. It also contains live bacteria that help colonise
a baby's gut, along with substances that promote the growth of beneficial gut bacteria.
In breast-fed infants, Bifidobacteria account for more than 90% of the total intestinal
bacteria. The low concentration of protein in human milk, the presence of specific anti-
infective proteins such as immunoglobulin A, lactoferrin, lysozyme, and oligosacharides
(prebiotics), as well as production of lactic acid, cause an acid milieu and are the main
reasons for its bifidogenic charachtersitics.
It has been recently found that breast milk contains endocannabinoids that
stimulate suckling and appetite.
It is well known that Breast Milk contains sugars that stop bacteria sticking to gut cells,
and fats that disrupt certain kinds of viruses, and an array of signalling molecules that
stimulate immune development.
In bottle-fed infants, Bifidobacteria are not predominant. Instead enterobacteria and
gram-negative organisms dominate because of a more alkaline milieu and the
absence of the prebiotic modulatory factors present in breast milk.
The establishment of an intestinal microbial ecology is very variable at the
beginning but will become a more stable system similar to the adult microflora by
the end of the breast feeding period.
Other factors affecting the intestinal microflora of the infant include geographical
differences (industrialized vs. developing countries) and administration of
antibiotics in neonatal intensive care.
(http://www.customprobiotics.com/about_probiotics_a.htm)
DIGESTION PROCESS IN NEONATALS
The triglyceride digestive process of the neonate is complex. It is initiated by a gastric
phase catalyzed by gastric or lingual lipase [Hamosh M. (1990) Nutrition ; 6: 421-8]. This
initial lipolysis allows maximal activity of pancreatic colipase-dependent lipase during the
intestinal phase of digestion. The pancreatic lipase system attacks the triglyceride with a
high degree of positional specificity. Lipolysis occurs predominantly at the son-l and sn-3
positions, yielding two free fatty acids and a 2-monoglyceride
[Mattson FH. & Beck LH. (1956) J. Biol. Chem. ; 219: 735-740].
Monoglycerides are well absorbed independent of their constituent fatty acid.
In contrast, the absorption of free fatty acids varies greatly, depending on their chemical
structure.
Mono and polyunsaturated fatty acids are well absorbed, as are saturated fatty acids of 12
carbons or less in chain length. The coefficient of absorption of free long chain saturated
fatty acids i. e. palmitic acid is relatively low due in part to a melting point above body
temperature (~63°) and the tendency of these fatty acids to form hydrated fatty acid soaps
with minerals such as calcium or magnesium at the pH of the intestine
[Jensen C, et al. (1988) Am. J. Clin. Nutr. ; 43: 745-51].
The greater absorption of fat and calcium in breast-fed infants compared with those fed
animal milk has been ascribed to two factors: the presence in breast milk of a lipolytic

6. enzyme (the bile salt- stimulated lipase) and the relatively high proportion of palmitic acid
at the sua-2 position of the triglyceride
(Hernell O. et al. (1988) Perinatal Nutrition.)
NUTRITIONAL REQUIREMENTS OF INFANTS
During the first year of life an infant's birth weight triples and the length is increased by
50%. To meet the requirements of their rapidly expanding skeletal mass, growing infants
require bio available sources of Micro Nutrients like Calcium, Phosphorus, Magnesium,
Manganese, Zinc and Vitamin D3 apart from the regular Macro Nutrients like Amino Acids,
Carbohydrates and Lipids.
PROTEIN:
Recommended level is 2.46 g/kg bodyweight/day
(FAO/WHO/UOU Expert Consultation, 1985)
FATTY ACID PROFILE
Studies have shown that fatty acid composition of the diet influences the fatty acid
composition of developing infant tissue
(Widdowson E. M. (1975) Br. Med. J.; 1: 633-5; Carlson SE. et al. (1986) Am. J. Clin. Nutr.
; 44: 798-804 ; Innis SM. et al.)
CACIUM AND FATTY ACIDS
The higher the amount of unsaturated fatty acids, such as oleic acid, the better, since this
indicates that most of the sn-1, 3 positions are occupied by fatty acids that will not create
harmful complexes with calcium.
Consequently, the infant will not lose either energy (in the form of fatty acids) or calcium.
LINSEED OIL
Fatty Acid Composition of Linseed Oil
Fatty Acid Formula %ge
Palmitic CH3 (CH2)14 COOH 4 to 7
Stearic CH3 (CH2)16 COOH 2 to 4
Oleic CH3 (CH2)7 CH-CH (CH2)7 COOH 14 to 38
Linoleic CH3 (CH2)4 CH-CH (CH2) CH-CH (CH2)7 COOH 7 to 18
Linolenic CH3 CH2 CH-CH CH2 CH-CHCH2CH-CHCH(CH2 )7 COOH 35 to 66
Content of 3 Omega Fatty Acids in Flax / Linseed oil : 58 g / 100g
linolenic acid
ALA (alpha-linolenic acid) is an omega-3 essential fatty acid. GLA (gamma-linolenic acid)
is an essential omega-6 fatty acid manufactured by the body from the essential fatty acid
linoleic acid. An essential fatty acid is one that must be supplied by the diet, since the body
cannot manufacture it or cannot manufacture enough of it. Both ALA and GLA are
polyunsaturated fats ("good" fats, as opposed to saturated fats which increase the risk of
heart disease). ALA and GLA are found in the seed oils of certain plants.

7. ALA is an omega-3 fatty acid that, to a limited extent, can be converted in the body into
two other important omega-3's — EPA (eicosapentaenoic acid) and DHA
(docosahexaenoic acid) — (see The body has a limited ability to manufacture both EPA
and DHA from ALA (only about 10% is converted), and even this is lessened if the diet is
too high in omega-6 fatty acids, because they compete with omega 3's for certain
enzymes as they are metabolized.
GLA is an omega-6 fatty acid. The body ordinarily is able to produce sufficient GLA from
the essential fatty acid linoleic acid (LA), However, GLA production may be reduced in
several conditions (advanced age, diabetes, high alcohol intake, eczema, cyclic mastitis,
viral infections, excessive saturated fat intake, elevated cholesterol, and some
vitamin/mineral deficiencies). In such cases, supplements may be beneficial.
ALA:
The Mediterranean diet, which is associated with a lower risk of coronary artery disease
and certain types of cancer, is high in ALA. However, the beneficial effects of EPA and
DHA (which include cardiovascular benefits and reduced pain associated with rheumatoid
arthritis and menstrual cramps) have not been seen with ALA alone. There is preliminary
evidence that ALA might be useful in bipolar disorder.
GLA:
GLA may be useful in diseases that involve inflammation. GLA is useful in treating breast
pain associated with the menstrual cycle (cyclic mastalgia). It appears to be helpful in
about 50% of women studied, vs. less than half that for placebo. However, it may not be
effective in more severe cases involving actual breast cysts or lumps. GLA may also be
helpful in reducing other PMS symptoms. GLA is used in Europe to treat diabetic
neuropathy and eczema — although the evidence that it works for these conditions is
mixed. It may also have some benefit in treating rheumatoid arthritis (especially as purified
GLA and when combined with traditional treatments) and Raynaud's phenomenon. Many
other potential uses, including some in conjunction with fish oils, have been explored, but
evidence is either weak or very preliminary. Linoleic acid, which the body can convert into
GLA, may also have a role in treating symptoms of multiple sclerosis.
Most clinical studies of GLA, such as those on breast pain and diabetic neuropathy,
Experts now believe that the CURRENT HUMAN diet contains too little omega-3 fatty
acids and too much omega-6 fatty acids. They compete with each other for certain
enzymes needed in their metabolism. Consequently, too much omega-6 fatty acids in the
diet will interfere with the body's conversion of ALA into DHA and EPA and may contribute
to an increased risk of heart disease and cancer. While the ratio of omega-6's to omega-
3's in the CURRENT HUMAN diet is believed to be as high as 14:1 (14 grams of omega
6's for every gram of omega-3's), a ratio of no more than 3:1 (3 grams of omega-6's for
every 1 gram of omega-3) is recommended.
Galacto-Oligosaccharide
A low level of galacto-oligosaccharide (0.24 g/100 mL) in infant formula can improve
stool frequency, decrease fecal pH, and stimulate intestinal Bifidobacteria and
Lactobacilli as in those fed with human milk.
(Xiao-Ming Ben, Juan Li, Zong-Tai Feng, Sheng-Yun Shi, Ya-Dong Lu, Rui Chen, Xiao-Yu Zhou;
R World J Gastroenterol 2008 November 14; 14(42): 6564-6568)
PRETERM BABIES AND B COMPLEX REQUIREMENTS

8. Samples of milk were taken at intervals during lactation from 35 mothers of term and 26
mothers of preterm infants and assayed for 8 B complex vitamins. Both term and
preterm milks varied widely in vitamin content between mothers. Mean concentrations of
thiamin, vitamin B6, nicotinic acid, pantothenic acid, biotin, and folic acid increased
progressively over several weeks after parturition but vitamin B12 concentrations
declined generally and riboflavin values showed little change. Preterm milk was not
richer in vitamins than term milk of the corresponding stage of lactation and it appeared
that intake of B vitamins differed widely among preterm infants given their own mothers'
milk. These infants may have meager body reserves and an increased need for
vitamins, and breast milk whether from their own mother or from the milk bank may not
meet their needs. There is a strong case for supplementing breast milk given to preterm
babies with the B complex vitamins.
(J E Ford, A Zechalko, J Murphy, and O G Brooke; Arch Dis Child. 1983 May; 58(5):
367–372.)
L. gasseri
Breast milk of healthy women is a major source of lactic acid bacteria to the infant gut,
and that Lactobacillus gasseri is among the predominant species.
L. gasseri(CECT 5714) and L. coryniformis (CECT 5711), enhanced immunity in healthy
people to a greater extent than the standard yogurt consumption.
(Mónica Olivares, M. Paz Díaz-Ropero, Nuria Gómez, Federico Lara-Villoslada, Saleta
Sierra, Juan A. Maldonado, Rocío Martín, Juan M. Rodríguez, Jordi Xaus; Int Microbiol
2006; 9(1):47-52)
CONSTIPATION PROBLEM
Another important issue which is associated with animal milk feeding is constipation in
both term and preterm infants which, in the latter, can lead to life threatening
complications. By contrast, constipation is rare in breast fed term infants. A study
comparing breast fed and formula fed infant stool hardness and composition showed
that calcium fatty acid soaps are positively correlated to stool hardness. Stools from
formula-fed infants were significantly harder than those of the breast-fed infants
suggesting different handling of saturated fatty acids
[Quinlan PT. et al. (1995) J. Pediatr. Gastr. and Nutr. ; 20: 81-90].
TARGETS OF MIMIC-BM
Manufacture of a new milk formulation for feeding Neonatal based on Buffalo Milk can
be done by doing the following modifications.
• Addition of Lipase and suitable DFM which can secrete suitable Lipase enzymes
fit for the lipolysis in neonatal.
• Calcium in the milk is to be reduced by electro dialysis which decreases the curd
tension and improves the heat stability; or Fatty acid profiles are to be modified
which will not interfere with Calcium.
• Fortification with lactose and vitamin mixture.
• Milk proteins particularly αs casein is to be degraded by regulated proteolysis of
milk with trypsin or Direct Fed Microbes which secrete these enzymes are to be
incorporated.
• Suitable viable specific strains of bifidobacterium and lactobacillus are to be
incorporated.

9. • Vegetable oil and milk fat in distinct proportions are to be incorporated resulting
in improved polyunsaturated fatty acid profiles;
SELECTION OF THE CONSTITUENTS OF MIMIC-BM
Probiotics used in MIMIC-BM have been obtained from different sources apart from
feces, such as goat cheese and from human breast milk and amniotic fluid, and have
been chosen by a method consisting in the ability of these strains to survive in breast
milk and/or amniotic fluid, and by their ability to be transferred to breast milk and/or
amniotic fluid after oral intake and by their ability to perform the tasks required as by
Breast Milk.
This selection method ensures that these bacterial strains present in MIMIC-BM have
implicitly most of the characteristics attributed to a potential probiotic strain, namely
• Absolute safety aspects,
• Adhesion capabilities
• Amicable origin,
• Good resistance to digestion process
• Other beneficial characteristics.
• Regulating some human niches other than the gut.
• The ability of gut colonization,
• Secretion of quality immunomodulators
• Secretion of quality enzymes necessary for improvising the food conversion
• Secretion of quality Vitamins etc
Improving the Characteristics of these Selective Probiotic
Strains
Is done during the harvesting of these microbes by replacing
• the nitrogen sources to lactic casein
• the lipids to Linseed Oil
to make them target specific.
CONTENTS OF MIMIC –BM
Probiotics like
Bifidobacterium bifidum,
Bifidobacterium breve,
Bifidobacterium longum
Enterococcus faecalis,
Lactobacillus acidophilus,
Lactobacillus bulgaricus,
Lactobacillus casei,
Lactobacillus delbrueckii,
Lactobacillus fermentum,
Lactobacillus gasseri,
Lactobacillus lactis,
Lactobacillus plantrum,
Lactobacillus reuteri,
Lactobacillus rhamnosus,

10. Lactobacillus salivarius,
Saccharomyces boulardii
Streptococcus thermophilus
1,3/1,6 Beta Glucons
Oligo Mannon Saccharides
Amino Acids
Taurine
Enzymes
Alpha Amylase
Protease
Lipase
Prebiotics like
Inulin
Choline
Cassein
Ferrous Salts
Galacto Oligo Saccharides
All in well balanced proportions.
SALIENT FEATURES OF MIMIC-BM
Found useful in
• controling serum cholesterol levels
• cow's milk protein allergy presenting with Hirschsprung's disease-mimicking
symptoms.
• decreasing the diaper rash among babies drinking the milk containing MIMIC-
BM.
• enhancing immunity by regulating lymphocytes as well as antibodies.
• establishing a normal healthy micro flora.
• improving the nutrient bioavailability, including B vitamins, calcium, iron, zinc,
copper, magnesium and phosphorus.
• improving the nutritional value of foods ~ via nutrient synthesis and bioavailability
• Improving the shelf life of the milk from dairy industry to the bottle of the infant.
• producing vitamins (namely, vitamin B & K)
• producing lactic acid and natural antibiotics, necessary for the healthy functioning
and inhibition of pathogens in the intestines
• promoting the growth of healthy bacteria in the colon and reducing the
conversion of bile into carcinogens (cancer causing substances).
• providing Essential nutrients.

11. • significantly decreasing the rate of acute diarrhea and rotavirus shedding.
• the break down of lactose (milk sugar) making dairy products more easily
digestible, particularly for those with lactose intolerance
TRADITIONAL METHOD OF MANUFACTURING MODIFIED
BUFFALO MILK FOR USE IN INFANTS
Buffalo milk was diluted with four parts of whey that was produced by addition of citric
acid or lemon juice to milk and neutralization with sodium bicarbonate.
SUGGESTED METHOD OF PREPARATION OF ARTIFICIAL
BREAST MILK IN THE DAIRY PROCESSING PLANT
Preparation of a fermented liquid milk formula using Buffalo Milk for use in the
cases of bottle fed infants.
A normal fermented liquid milk composition with MIMIC-BM was prepared using the
following Process:
Dilute Buffalo Milk with sterile water free from Chlorine so as the
Resultant Milk possess 1.5% Fat; 3% protein 997g/kg
MIMIC- BM (10^10 CfU/g) 3 g/kg
The fat and dry solids contents of the milk were standardized before use, according to
the requirements of the formulation as described above.
After that, the milk was homogenized at 20-25 Mpa and 65-70°C to obtain optimum
physical properties in the product.
The preparation was heated at 90-95°C and a holding time of about 5 minutes.
This period of time causes the de naturation of about 70-80% of whey proteins.
Cooled milk (40-45°C) was inoculated with the MIMIC-BM in absence of any other
starter culture and fermented in the incubation tank at 40-45°C for 10 hours without
agitation until reaching a final pH (pH 4.5-5).
Then Add Vit. A, B Complex and Vitamin C.
After clot formation, this mixture was homogenized by mechanical methods.
Once the homogenization was carried out, the preparation was cooled down to a
temperature below 10°C in 60 minutes.
After that, the composition was packaged.
Final cooling, normally down to 5°C, took place in a cold room till dispatches are made in
a cold chain to the end consumer point.
SPRAY DRIED INFANT FOODS
Specifications of the spray-dried powder obtained from the modified buffalo milk 3.l-3.2g
protein, 3.5g fat, and 628 mg of linoleic acid per 100 calories with a Ca/P ratio of 2.2.
(As per WHO)
METHOD OF ANALYSIS OF MIMIC-BM
Adhesion Properties
The adhesion of the probiotic strains present in MIMIC-BM were assessed using Caco-2
(grey bars) or HT-29 (black bars) intestinal cell lines and compared to commercial
probiotic strains. Twenty randomized fields were counted and the results expressed as
the mean of the number of bacteria attached to the cells per field SD. The adhesion
capability of a probiotic strain to each intestinal cell line was considered high if the

12. number of attached bacteria was >250, moderate between 100 and 250, and slight
>100.
Survival to digestion conditions.
The resistance of the probiotic strains present in MIMIC-BM, to acidic (grey bars) and
high bile salt content (black bars) was assessed in vitro by culture of bacteria in MRS pH
3.0 or 0.15% bile salts for 90 minutes. The results are represented as the mean SD of
three independent experiments. The resistance of a probiotic strain was considered high
if the survival was >80%, moderate between 60% and 80%, and slight >60%.
Generation time of probiotic strains present in MIMIC-BM.
The time of generation of the probiotic strains present in MIMIC-BM was assessed in
vitro by cultivating bacteria in MRS 0.2% glucose for 120 minutes. The results are
represented in minutes and as the mean SD of three independent experiments. The
generation time of a probiotic strain was considered rapid if the time was less than 60,
moderate between 60 and 120, and slow >120 minutes.
Fermentation capabilities of probiotic strains present in MIMIC-BM
The fermentation capabilities of the probiotic strains present in MIMIC-BM, to use
complex carbohydrates as an unique source of carbohydrates was assessed in vitro by
cultivating bacteria in MRS without glucose and supplemented with 2% of indicated
carbohydrates for 24 and 48 hours. Reduction of the pH was assessed using brom
cresol purple. The results are represented as the fold-induction in absorbance after 24
hours compared with a control culture without carbohydrate source (A) and the sum of
all independent fold- induction values (B). The fermentation capability of a probiotic
strain was considered high if the total value was >30, moderate between 25 and 30, and
slight <25.
Resistance to antibiotic of probiotic strains present in MIMIC-BM.
The resistance of antibiotic treatment of the probiotic strains present in MIMIC-BM was
assessed in vitro by an agar well diffusion assay in Mueller-Hinton plates for 24-48
hours. The diameter of the hallo of inhibition determines the antibiotic effect. The results
are represented as R (resistant) if the hallo has a diameter < 12 mm, I (intermediate)
from 12 to 15 mm, and S (sensible) if >15 mm. After that, a numerical value was
assigned to each condition: R=3, I=2, and S=1. The resistance capability of a probiotic
strain was considered high if the total value was >17, moderate between 15 and 17, and
slight <15.
Acid production by the probiotic strains present in MIMIC-BM.
The production of acid (lactic, propionic, acetic and butyric acid) by the probiotic strains
present in MIMIC-BM was assessed in vitro by the measurement of the pH in milk
cultures for 24 (grey bars) and 48 (black bars) hours. The production of acid by a
probiotic strain was considered high if the milk pH value after 48 hours was <4,
moderate between 4 and 4.5, and slight >4.5.
Production of bactericide metabolites by the probiotic strains present in MIMIC-
BM.
The production of antimicrobial metabolites by the probiotic strains present in MIMIC-BM
was assessed in vitro by an agar well diffusion assay in TSA plates cultured with S.
typhimuriumi (black bars) or Escherichia coli (grey bars). The diameter of the hallo (in
millimeters) of inhibition induced by the bacterial supernatants determines the

13. bactericide effect. The antimicrobial capability of a probiotic strain was considered high if
the hallo was &gt;12, moderate between 8 and 12, and slight <8 for both pathogenic
strains.
Inhibition of the adhesion of pathogenic bacteria.
The adhesion of the pathogenic strains E. coli (grey bars) and S. typhimurium (black
bars) to Caco-2 cells was assessed in the presence of the probiotic strains present in
MIMIC-BM and compared to other such commercial products available in the global
markets. Ten randomized fields were counted and the results expressed as the mean of
the % of adhered gram-negative bacteria attached to the cells compared to the number
of pathogenic bacteria adhered in absence of MIMIC-BM. The capability of a probiotic
strain to inhibit pathogenic bacteria adherence was considered high if the % of both
attached pathogenic bacteria was <25, moderate between 25 and 75, and slight &gt;75.
Gut colonization by L. Salivarius
The number of fecal lactobacillus, bifidobacteria and coliform bacteria in mice
supplemented daily for 14 days with 108 cfu of L. salivarius was analyzed by bacterial
platting. Fecal samples (200 mg aprox) were collected at day 0, 7 and 14 of probiotic
supplementation and also one and two weeks (day 21 and 28) after supplementation
was terminated. (* p< 0.05 ; ** p<0. 01).
Effect of L. Fermentum on Salmonella infection.
L. fermentum CECT5716 inhibits Salmonella translocation to the spleen. The number of
Salmonella colonies was measured in the spleens of mice treated with L. fermentum
with or without vaccination with 108 inactivated cfu of Salmonella after 24 hour of an oral
challenge with 10'° cfu Salmonella. B) The same mice were used to measure the IgA
content in feces.
Effect of probiotic strains present in MIMIC-BM on cytokine expression.
The TNF-(x (A) ot IL-10 (B) cytokine production was analyzed in bone marrow derived
macrophages stimulated with LPS and the indicated probiotic strain for 12 hours.
Cytokine production was detected by an ELISA technique.
Effect of probiotic strains present in MIMIC-BM on Ig G expression.
The IgG production was analyzed in lymphocytes obtained from the spleen of Balb/c
mice (6-8 weeks old) stimulated with LPS and the indicated probiotic strain for 6 days.
Immunoglobulin production was detected by an ELISA technique from Bethyl.
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