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MIMIC-BMDairy ProcessedBuffalo MilkMademore compatibleand totalin it’s functionalparametersresemblingHealthyMother’sBreast...
NUTRITIVE ANALYSIS OF HUMAN MILKAmino Acids (mg)Constituent Human MilkAlanine 56.00Arginine 51.00Aspartic Acid 120.00Cysti...
Capric Acid 61.00Lauric Acid 213Myristic Acid 342Palmitic Acid 963Stearic Acid 293Arachidic Acid 46Palmitoleic Acid 129Ole...
PolyunsaturatedCholesterol mg 14 10 11 8Calcium iu 120 100 170 195COMPOSITOIONS OF ANIMAL VS HUMAN MILKVarious types of mi...
cesarean deliveries. When colonized, cesarean delivered infants less frequently harborE. coli, and more often klebsiella a...
enzyme (the bile salt- stimulated lipase) and the relatively high proportion of palmitic acidat the sua-2 position of the ...
ALA is an omega-3 fatty acid that, to a limited extent, can be converted in the body intotwo other important omega-3s — EP...
Samples of milk were taken at intervals during lactation from 35 mothers of term and 26mothers of preterm infants and assa...
• Vegetable oil and milk fat in distinct proportions are to be incorporated resultingin improved polyunsaturated fatty aci...
Lactobacillus salivarius,Saccharomyces boulardiiStreptococcus thermophilus1,3/1,6 Beta GluconsOligo Mannon SaccharidesAmin...
• significantly decreasing the rate of acute diarrhea and rotavirus shedding.• the break down of lactose (milk sugar) maki...
number of attached bacteria was >250, moderate between 100 and 250, and slight>100.Survival to digestion conditions.The re...
bactericide effect. The antimicrobial capability of a probiotic strain was considered high ifthe hallo was >12, moderat...
5. Miller JB, McVeagh P. Human milk oligosaccharides: 130 reasons to breast-feed. Br J Nutr 1999;82: 333-335 PubMed6. Fana...
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IMPROVING BUFFALO MILK FOR FEEDING NEONATALS AS A REPLACEMENT OF MOTHER'S MILK

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MIMIC is a process technology employed to improve Buffalo Milk FOR FEEDING NEONATALS AS A REPLACEMENT OF MOTHER'S MILK

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IMPROVING BUFFALO MILK FOR FEEDING NEONATALS AS A REPLACEMENT OF MOTHER'S MILK

  1. 1. MIMIC-BMDairy ProcessedBuffalo MilkMademore compatibleand totalin it’s functionalparametersresemblingHealthyMother’sBreast MilkNEED OF MIMIC-BM ARISES IN THE CASES OF• Mothers those who cannot breast feed owing to medical reasonsWithout any intervention, 30-35% of mother-to-child transmission (MTCT) cases aredue to breastfeeding. The remaining portion of MTCT cases occur in utero and atchildbirth. This is called perinatal transmission. As the total percentage of MTCTranges from between 15-45% by region, its 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 ReasonsDespite all the public-health campaigns encouraging new mothers to breastfeed,tens of millions of babies worldwide are raised on infant formula rather than breastmilk.PREAMBLEGiven that so many babies are being bottlefed, we are bound to do something andeverything that is possible on this Earth to make it better and worthwhile not in theinterest of Economics but due to our accountability to give our best to our nextgenerations. However It is becoming feasible to manufacture human proteins likethose in milk, thanks to the advancement in Science and Technology, which could beadded to such breast milk replacements.The buffalo milk is normally hesitated to be given to infants in India for unknownreasons. Some of such reasons might be thatBuffalo milk contains more energy, protein, fat or total solid as compared to the cow’s orother milk types.Buffalo Milk will not be easily digestedBuffalo Milk may cause flatulence recurrent vomiting abdominal distension and difficultyin stool evacuation.
  2. 2. NUTRITIVE ANALYSIS OF HUMAN MILKAmino Acids (mg)Constituent Human MilkAlanine 56.00Arginine 51.00Aspartic Acid 120.00Cystine 24.00Glutamic Acid 220.00Glycine 36.00Histidine 31.00Isoleucine 77.00Leucine 130.00Lysine 86.00Methionine 24.00Phenylalanine 54.00Proline 120.00Serine 59.00Threonine 63.00Tryptophan 22.00Tyrosine 56.00Valine 81.00Carbohydrates (select) (gm)Constituent Human MilkLactose 7.00Glucose --Fructose --Sucrose --Starch --Fatty Acids (mg)Constituent Human Milk
  3. 3. Capric Acid 61.00Lauric Acid 213Myristic Acid 342Palmitic Acid 963Stearic Acid 293Arachidic Acid 46Palmitoleic Acid 129Oleic Acid 1,340.00Linoleic Acid 380Linolenic Acid 22Arachidonic Acid 4.2Other (varies) --Sterols (mg)Constituent Human MilkCholesterol 25.00Miscellaneous (mg)Casein 360Whey Protein 500COMPOSITION OF ANIMAL MILKMILK COMPOSITION ANALYSISConstituents Cow Goat Sheep BuffaloProtein grm 3.2 3.1 5.4 4.5Fat grm 3.9 3.5 6 8Carbohydrate grm 4.8 4.4 5.1 4.9Energy K cal 66 60 95 110K J 275 253 396 463Sugars (Lactose) grm 4.8 4.4 5.1 4.9Fatty Acids- grm 2.4 2.3 3.8 4.2Saturated grm 1.1 0.8 1.5 1.7Monounsaturated grm 0.1 0.1 0.3 0.2
  4. 4. PolyunsaturatedCholesterol mg 14 10 11 8Calcium iu 120 100 170 195COMPOSITOIONS OF ANIMAL VS HUMAN MILKVarious types of milk composition per 100 ml of milkMilk TypeHuman’s Milk* Cow’s Milk* Buffalo’s Milk* Lactogen-1**Moisture (gms) 88 88 81 90.4Protein (gms) 1.1 3.2 4.3 1.7Fat (gms) 3.4 4.1 8.8 3.4Carbohydrates (gms) 7.4 4.4 5.0 7.41Energy (kcal) 65 67 117 67Minerals (gms) 0.1 0.8 0.8 0.4Calcium (mgs) 28 120 219 73Phosphorus (mgs) 11 90 198 53Iron (mgs) 0.5 0.2 0.2 0.8Vitamin A (IU) 137 174 160 230Thiamin (mgs) 0.02 0.05 0.04 0.05Riboflavin (mgs) 0.02 0.19 0.10 0.1Niacin (mgs) - 0.1 0.1 0.67Vitamin 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 MilkpackLimited 308 Upper Mall, Lahore 54000INITIAL COLONIZATION OF THE NEONATEThere is no publication or work describing the presence of lactic acid bacterial strains innormal 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 thegestation, the initial microbial populations are able to colonize the fetal gut.INTESTINAL MICROFLORA IN THE NEWBORN INFANTFetuses are sterile in the womb, but beginning with the birth process, infants areexposed 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 timeof delivery. Because vaginal flora and intestinal flora are similar, an infants floramay 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 theinfant.During cesarean deliveries, this transfer is completely absent. These infants commonlyacquire and are colonized with flora from the hospitals environment and, therefore, theirflora may differ from maternal flora. Infants delivered by cesarean section are colonizedwith more anaerobic bacteria, especially Bacteroides, than vaginally delivered infants.Clostridium perfringens is the anaerobic bacterium most frequently isolated after
  5. 5. cesarean deliveries. When colonized, cesarean delivered infants less frequently harborE. coli, and more often klebsiella and enterobacteria.The initial colonizing bacteria vary with the food source of the infant including breastmilk or formulated dairy products fed by bottle.Breast milk provides along with the basic fats, proteins, carbohydrates, minerals andvitamins that babies need to survive; hormones, immune signalling molecules,antibodies and even living immune cells. It also contains live bacteria that help colonisea babys 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 intestinalbacteria. 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 mainreasons for its bifidogenic charachtersitics.It has been recently found that breast milk contains endocannabinoids thatstimulate 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 thatstimulate immune development.In bottle-fed infants, Bifidobacteria are not predominant. Instead enterobacteria andgram-negative organisms dominate because of a more alkaline milieu and theabsence of the prebiotic modulatory factors present in breast milk.The establishment of an intestinal microbial ecology is very variable at thebeginning but will become a more stable system similar to the adult microflora bythe end of the breast feeding period.Other factors affecting the intestinal microflora of the infant include geographicaldifferences (industrialized vs. developing countries) and administration ofantibiotics in neonatal intensive care.(http://www.customprobiotics.com/about_probiotics_a.htm)DIGESTION PROCESS IN NEONATALSThe triglyceride digestive process of the neonate is complex. It is initiated by a gastricphase catalyzed by gastric or lingual lipase [Hamosh M. (1990) Nutrition ; 6: 421-8]. Thisinitial lipolysis allows maximal activity of pancreatic colipase-dependent lipase during theintestinal phase of digestion. The pancreatic lipase system attacks the triglyceride with ahigh degree of positional specificity. Lipolysis occurs predominantly at the son-l and sn-3positions, 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 chemicalstructure.Mono and polyunsaturated fatty acids are well absorbed, as are saturated fatty acids of 12carbons or less in chain length. The coefficient of absorption of free long chain saturatedfatty acids i. e. palmitic acid is relatively low due in part to a melting point above bodytemperature (~63°) and the tendency of these fatty acids to form hydrated fatty acid soapswith 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 fedanimal milk has been ascribed to two factors: the presence in breast milk of a lipolytic
  6. 6. enzyme (the bile salt- stimulated lipase) and the relatively high proportion of palmitic acidat the sua-2 position of the triglyceride(Hernell O. et al. (1988) Perinatal Nutrition.)NUTRITIONAL REQUIREMENTS OF INFANTSDuring the first year of life an infants birth weight triples and the length is increased by50%. To meet the requirements of their rapidly expanding skeletal mass, growing infantsrequire 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 PROFILEStudies have shown that fatty acid composition of the diet influences the fatty acidcomposition 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 ACIDSThe higher the amount of unsaturated fatty acids, such as oleic acid, the better, since thisindicates that most of the sn-1, 3 positions are occupied by fatty acids that will not createharmful complexes with calcium.Consequently, the infant will not lose either energy (in the form of fatty acids) or calcium.LINSEED OILFatty Acid Composition of Linseed OilFatty Acid Formula %gePalmitic CH3 (CH2)14 COOH 4 to 7Stearic CH3 (CH2)16 COOH 2 to 4Oleic CH3 (CH2)7 CH-CH (CH2)7 COOH 14 to 38Linoleic CH3 (CH2)4 CH-CH (CH2) CH-CH (CH2)7 COOH 7 to 18Linolenic CH3 CH2 CH-CH CH2 CH-CHCH2CH-CHCH(CH2 )7 COOH 35 to 66Content of 3 Omega Fatty Acids in Flax / Linseed oil : 58 g / 100glinolenic acidALA (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 acidlinoleic acid. An essential fatty acid is one that must be supplied by the diet, since the bodycannot manufacture it or cannot manufacture enough of it. Both ALA and GLA arepolyunsaturated fats ("good" fats, as opposed to saturated fats which increase the risk ofheart disease). ALA and GLA are found in the seed oils of certain plants.
  7. 7. ALA is an omega-3 fatty acid that, to a limited extent, can be converted in the body intotwo other important omega-3s — EPA (eicosapentaenoic acid) and DHA(docosahexaenoic acid) — (see The body has a limited ability to manufacture both EPAand DHA from ALA (only about 10% is converted), and even this is lessened if the diet istoo high in omega-6 fatty acids, because they compete with omega 3s for certainenzymes as they are metabolized.GLA is an omega-6 fatty acid. The body ordinarily is able to produce sufficient GLA fromthe essential fatty acid linoleic acid (LA), However, GLA production may be reduced inseveral conditions (advanced age, diabetes, high alcohol intake, eczema, cyclic mastitis,viral infections, excessive saturated fat intake, elevated cholesterol, and somevitamin/mineral deficiencies). In such cases, supplements may be beneficial.ALA:The Mediterranean diet, which is associated with a lower risk of coronary artery diseaseand certain types of cancer, is high in ALA. However, the beneficial effects of EPA andDHA (which include cardiovascular benefits and reduced pain associated with rheumatoidarthritis and menstrual cramps) have not been seen with ALA alone. There is preliminaryevidence that ALA might be useful in bipolar disorder.GLA:GLA may be useful in diseases that involve inflammation. GLA is useful in treating breastpain associated with the menstrual cycle (cyclic mastalgia). It appears to be helpful inabout 50% of women studied, vs. less than half that for placebo. However, it may not beeffective in more severe cases involving actual breast cysts or lumps. GLA may also behelpful in reducing other PMS symptoms. GLA is used in Europe to treat diabeticneuropathy and eczema — although the evidence that it works for these conditions ismixed. It may also have some benefit in treating rheumatoid arthritis (especially as purifiedGLA and when combined with traditional treatments) and Raynauds phenomenon. Manyother potential uses, including some in conjunction with fish oils, have been explored, butevidence is either weak or very preliminary. Linoleic acid, which the body can convert intoGLA, 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 fattyacids and too much omega-6 fatty acids. They compete with each other for certainenzymes needed in their metabolism. Consequently, too much omega-6 fatty acids in thediet will interfere with the bodys conversion of ALA into DHA and EPA and may contributeto an increased risk of heart disease and cancer. While the ratio of omega-6s to omega-3s in the CURRENT HUMAN diet is believed to be as high as 14:1 (14 grams of omega6s for every gram of omega-3s), a ratio of no more than 3:1 (3 grams of omega-6s forevery 1 gram of omega-3) is recommended.Galacto-OligosaccharideA low level of galacto-oligosaccharide (0.24 g/100 mL) in infant formula can improvestool frequency, decrease fecal pH, and stimulate intestinal Bifidobacteria andLactobacilli 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. 8. Samples of milk were taken at intervals during lactation from 35 mothers of term and 26mothers of preterm infants and assayed for 8 B complex vitamins. Both term andpreterm milks varied widely in vitamin content between mothers. Mean concentrations ofthiamin, vitamin B6, nicotinic acid, pantothenic acid, biotin, and folic acid increasedprogressively over several weeks after parturition but vitamin B12 concentrationsdeclined generally and riboflavin values showed little change. Preterm milk was notricher in vitamins than term milk of the corresponding stage of lactation and it appearedthat intake of B vitamins differed widely among preterm infants given their own mothersmilk. These infants may have meager body reserves and an increased need forvitamins, and breast milk whether from their own mother or from the milk bank may notmeet their needs. There is a strong case for supplementing breast milk given to pretermbabies 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. gasseriBreast 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 healthypeople to a greater extent than the standard yogurt consumption.(Mónica Olivares, M. Paz Díaz-Ropero, Nuria Gómez, Federico Lara-Villoslada, SaletaSierra, Juan A. Maldonado, Rocío Martín, Juan M. Rodríguez, Jordi Xaus; Int Microbiol2006; 9(1):47-52)CONSTIPATION PROBLEMAnother important issue which is associated with animal milk feeding is constipation inboth term and preterm infants which, in the latter, can lead to life threateningcomplications. By contrast, constipation is rare in breast fed term infants. A studycomparing breast fed and formula fed infant stool hardness and composition showedthat calcium fatty acid soaps are positively correlated to stool hardness. Stools fromformula-fed infants were significantly harder than those of the breast-fed infantssuggesting different handling of saturated fatty acids[Quinlan PT. et al. (1995) J. Pediatr. Gastr. and Nutr. ; 20: 81-90].TARGETS OF MIMIC-BMManufacture of a new milk formulation for feeding Neonatal based on Buffalo Milk canbe done by doing the following modifications.• Addition of Lipase and suitable DFM which can secrete suitable Lipase enzymesfit for the lipolysis in neonatal.• Calcium in the milk is to be reduced by electro dialysis which decreases the curdtension and improves the heat stability; or Fatty acid profiles are to be modifiedwhich will not interfere with Calcium.• Fortification with lactose and vitamin mixture.• Milk proteins particularly αs casein is to be degraded by regulated proteolysis ofmilk with trypsin or Direct Fed Microbes which secrete these enzymes are to beincorporated.• Suitable viable specific strains of bifidobacterium and lactobacillus are to beincorporated.
  9. 9. • Vegetable oil and milk fat in distinct proportions are to be incorporated resultingin improved polyunsaturated fatty acid profiles;SELECTION OF THE CONSTITUENTS OF MIMIC-BMProbiotics used in MIMIC-BM have been obtained from different sources apart fromfeces, such as goat cheese and from human breast milk and amniotic fluid, and havebeen chosen by a method consisting in the ability of these strains to survive in breastmilk and/or amniotic fluid, and by their ability to be transferred to breast milk and/oramniotic fluid after oral intake and by their ability to perform the tasks required as byBreast Milk.This selection method ensures that these bacterial strains present in MIMIC-BM haveimplicitly 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 etcImproving the Characteristics of these Selective ProbioticStrainsIs done during the harvesting of these microbes by replacing• the nitrogen sources to lactic casein• the lipids to Linseed Oilto make them target specific.CONTENTS OF MIMIC –BMProbiotics likeBifidobacterium bifidum,Bifidobacterium breve,Bifidobacterium longumEnterococcus faecalis,Lactobacillus acidophilus,Lactobacillus bulgaricus,Lactobacillus casei,Lactobacillus delbrueckii,Lactobacillus fermentum,Lactobacillus gasseri,Lactobacillus lactis,Lactobacillus plantrum,Lactobacillus reuteri,Lactobacillus rhamnosus,
  10. 10. Lactobacillus salivarius,Saccharomyces boulardiiStreptococcus thermophilus1,3/1,6 Beta GluconsOligo Mannon SaccharidesAmino AcidsTaurineEnzymesAlpha AmylaseProteaseLipasePrebiotics likeInulinCholineCasseinFerrous SaltsGalacto Oligo SaccharidesAll in well balanced proportions.SALIENT FEATURES OF MIMIC-BMFound useful in• controling serum cholesterol levels• cows milk protein allergy presenting with Hirschsprungs disease-mimickingsymptoms.• 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 functioningand inhibition of pathogens in the intestines• promoting the growth of healthy bacteria in the colon and reducing theconversion of bile into carcinogens (cancer causing substances).• providing Essential nutrients.
  11. 11. • significantly decreasing the rate of acute diarrhea and rotavirus shedding.• the break down of lactose (milk sugar) making dairy products more easilydigestible, particularly for those with lactose intoleranceTRADITIONAL METHOD OF MANUFACTURING MODIFIEDBUFFALO MILK FOR USE IN INFANTSBuffalo milk was diluted with four parts of whey that was produced by addition of citricacid or lemon juice to milk and neutralization with sodium bicarbonate.SUGGESTED METHOD OF PREPARATION OF ARTIFICIALBREAST MILK IN THE DAIRY PROCESSING PLANTPreparation of a fermented liquid milk formula using Buffalo Milk for use in thecases of bottle fed infants.A normal fermented liquid milk composition with MIMIC-BM was prepared using thefollowing Process:Dilute Buffalo Milk with sterile water free from Chlorine so as theResultant Milk possess 1.5% Fat; 3% protein 997g/kgMIMIC- BM (10^10 CfU/g) 3 g/kgThe fat and dry solids contents of the milk were standardized before use, according tothe requirements of the formulation as described above.After that, the milk was homogenized at 20-25 Mpa and 65-70°C to obtain optimumphysical 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 otherstarter culture and fermented in the incubation tank at 40-45°C for 10 hours withoutagitation 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 atemperature 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 ina cold chain to the end consumer point.SPRAY DRIED INFANT FOODSSpecifications of the spray-dried powder obtained from the modified buffalo milk 3.l-3.2gprotein, 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-BMAdhesion PropertiesThe 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 commercialprobiotic strains. Twenty randomized fields were counted and the results expressed asthe mean of the number of bacteria attached to the cells per field SD. The adhesioncapability of a probiotic strain to each intestinal cell line was considered high if the
  12. 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) andhigh bile salt content (black bars) was assessed in vitro by culture of bacteria in MRS pH3.0 or 0.15% bile salts for 90 minutes. The results are represented as the mean SD ofthree independent experiments. The resistance of a probiotic strain was considered highif 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 invitro by cultivating bacteria in MRS 0.2% glucose for 120 minutes. The results arerepresented in minutes and as the mean SD of three independent experiments. Thegeneration 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-BMThe fermentation capabilities of the probiotic strains present in MIMIC-BM, to usecomplex carbohydrates as an unique source of carbohydrates was assessed in vitro bycultivating bacteria in MRS without glucose and supplemented with 2% of indicatedcarbohydrates for 24 and 48 hours. Reduction of the pH was assessed using bromcresol purple. The results are represented as the fold-induction in absorbance after 24hours compared with a control culture without carbohydrate source (A) and the sum ofall independent fold- induction values (B). The fermentation capability of a probioticstrain was considered high if the total value was >30, moderate between 25 and 30, andslight <25.Resistance to antibiotic of probiotic strains present in MIMIC-BM.The resistance of antibiotic treatment of the probiotic strains present in MIMIC-BM wasassessed in vitro by an agar well diffusion assay in Mueller-Hinton plates for 24-48hours. The diameter of the hallo of inhibition determines the antibiotic effect. The resultsare 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 wasassigned to each condition: R=3, I=2, and S=1. The resistance capability of a probioticstrain was considered high if the total value was >17, moderate between 15 and 17, andslight <15.Acid production by the probiotic strains present in MIMIC-BM.The production of acid (lactic, propionic, acetic and butyric acid) by the probiotic strainspresent in MIMIC-BM was assessed in vitro by the measurement of the pH in milkcultures for 24 (grey bars) and 48 (black bars) hours. The production of acid by aprobiotic 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-BMwas 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 (inmillimeters) of inhibition induced by the bacterial supernatants determines the
  13. 13. bactericide effect. The antimicrobial capability of a probiotic strain was considered high ifthe hallo was &gt;12, moderate between 8 and 12, and slight <8 for both pathogenicstrains.Inhibition of the adhesion of pathogenic bacteria.The adhesion of the pathogenic strains E. coli (grey bars) and S. typhimurium (blackbars) to Caco-2 cells was assessed in the presence of the probiotic strains present inMIMIC-BM and compared to other such commercial products available in the globalmarkets. Ten randomized fields were counted and the results expressed as the mean ofthe % of adhered gram-negative bacteria attached to the cells compared to the numberof pathogenic bacteria adhered in absence of MIMIC-BM. The capability of a probioticstrain to inhibit pathogenic bacteria adherence was considered high if the % of bothattached pathogenic bacteria was <25, moderate between 25 and 75, and slight &gt;75.Gut colonization by L. SalivariusThe number of fecal lactobacillus, bifidobacteria and coliform bacteria in micesupplemented daily for 14 days with 108 cfu of L. salivarius was analyzed by bacterialplatting. Fecal samples (200 mg aprox) were collected at day 0, 7 and 14 of probioticsupplementation and also one and two weeks (day 21 and 28) after supplementationwas 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 ofSalmonella colonies was measured in the spleens of mice treated with L. fermentumwith or without vaccination with 108 inactivated cfu of Salmonella after 24 hour of an oralchallenge with 10° cfu Salmonella. B) The same mice were used to measure the IgAcontent 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 derivedmacrophages 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/cmice (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.REFERENCES1. Harmsen HJ, Wildeboer-Veloo AC, Raangs GC, Wagendorp AA, Klijn N, Bindels JG, Welling GW.Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecularidentification and detection methods. J Pediatr Gastroenterol Nutr 2000; 30: 61-67 PubMed DOI2. Gronlund MM, Arvilommi H, Kero P, Lehtonen OP, Isolauri E. Importance of intestinal colonisationin the maturation of humoral immunity in early infancy: a prospective follow up study of healthyinfants aged 0-6 months. Arch Dis Child Fetal Neonatal Ed 2000; 83: F186-F192 PubMed DOI3. Newburg DS. Oligosaccharides in human milk and bacterial colonization. J Pediatr GastroenterolNutr 2000; 30 Suppl 2: S8-S17 PubMed DOI4. Picciano MF. Nutrient composition of human milk. Pediatr Clin North Am 2001; 48: 53-67PubMed DOI
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