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Nutrition Research, Vol. 19, No. 10, pp. 1559-1597, 1999Copyright 0 199 9 Elsevier Science Inc.Printed in the USA. All rights reserved

0271.5317/99/ x front matterP I I SO271-5317(9 9)00112-8

ROLE OF BIFIDOBACTERIA IN NUTRITION, MEDICINE AND TECHNO LOGYKantha D. Arunachalam, Ph.D.

Immunology Department, Faculty of Medicine, Health Sciences Centre,Memorial University of Newfoundland, St.Johns, NF, Canada AlB 3V6

ABSTRACTThe purpose of this review is to discuss the relatively recent revival ofinterest in bacterial interference, particularly the use of B i f i d obuc f e r i umspp. in nutrition, for treatment and prevention of disease in medicine andcomm ercial food produ cts by using recently develop ed techn ology. In1910, Metchnikoff (1) first put forward the idea that the regularconsumption of fermen ted milks might offer health benefits, the possibleprophylactic and/or therapeutic properties of yogurt and related productshave been the subject of much speculation. New fermented dairy productscontaining Lac tobac i l l u s spec ies and B i f i d obac t e n um spp . have beendeveloped and marketed in Europe, North America and the Far East.The potential health-promoting proper ties of Lac t obac i l l u s a c i d oph i l u s iswell docum ented, but possible roles of ingested bifidobacteria have notbeen reviewed much. B i j i d o b u c t e r i a are normal inhabitants of the humanand animal gut, and newborns are colonized within days after birth. Thepopulation seem s to be relatively stable until advanced age when it hasbeen repor ted to decline. Although the population of bifidobacteria in theintestine is stable , it is influenced by diet, antibiotics, stress etc.Bifidobacteria were first described by Tissier in 1899 (2) as predominantflora in breast-fed infants. This review will consider the character istics,ecology and role in human system s, the therapeutic and prophylac ticactivities of BQ idobac t e r i a , and the potential pharm aceutical and fermen tedproduc ts manufactured using bifidobacteria and advanced technology. Thefacts and the results of the some of the experiments done by differentauthors and the current areas of research interest and future developmenthave also been reviewed.0 1999lscvlacience nc.KEY WORDS: Bifidobacteria, Nutrition, Medicine, Technology, Health.

Corresponding Author: K. D. Arunachalam, Ph.D. Immunology Department, Faculty ofMedicine, Health Sciences Centre, M emorial university of Newfoundland, St.Johns, NF, CanadaAlB 3V6. e-mail: [email protected].

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1560 K.D. ARUNACHALAM

INTRODUCTIONThe present review discusses the recent revival of interest in bacterial

interference, particularly the use of Bifidobacterium spp. in nutrition, for treatment andprevention of disease in medicine and comm ercial food produc ts using recent advancetechnology. Ever since Metchnikoff first put forward the idea that the regularconsumption of ferme nted milks might offer health benefits, the possible pr ophylacticand/or therapeutic properties of yogurt and related products have been the subject ofmuch speculation( 1). Russians have long advoc ated the administration of kefir andkoumiss to patients with a wide range of illnesses (3), but because of the varyingmicrobiology of these products it is difficult to confirm any theoretical basis for theclaims. In US also the fermented products are promoted for good health but the poorquality control with respe ct to microbiological and organoleptic proper ties of the retailitems m ade its claim a dispute.

In recent years new fermented dairy products have been developed and marketedin Europe, North America and the Far East. The products contain Lac tobac i l l u s spec iesand B i f i d obac t e r i um spp . at lo6 viable cells/ml of produ ct at the time o f consumption.The potential health-prom oting properties of Lac t obac i l l u s a c i d oph i l u s is welldocum ented (4,5,6,7) , but possible roles of ingested bifidobacteria have less reviewe d.The importance of the bifidobacteria with respect to the healthy operation of thehuman digestive system encourage the consumption of bifidus products. This reviewwill consider taxonomy and the charac teristics , ecology and role in human system sand the potential health produ cts m anufactured using bifidobacterium. The facts and theresults of the some of the experiments done by different authors and the current areasof resea rch interest and future developme nt have also been reviewe d.

WHAT ARE BIFIDOBACTERIAB i f i d obac t e r i a spp . are normal inhabitants of the human and animal gut, and

newborns are colonized within days after birth. Th e population seem s to be relativelystable until a dvanced age when it has been reporte d to decline (8). Although thepopulation of bifidobacteria in the intestine is stable, it is influenced by diet,antibiotics, stress etc. B i f i d obac t e r i a wer e first describe d by Tissier in 1899 aspredominant flora in breast-fed infants (2). Their name comes from the observationthat these gram positive rods often exist in a Y - shaped or bifid form (Figure 1).B i f i d obac t e r i a are anaerobes with special nutritional requiremen ts, thus often difficultto isolate and grow in the laboratory. Tissier was the first one to prom ote thetherapeutic use of bifidobacteria for infant diarrhoe a by giving large doses ofbifidobacteria orally. Since then much research has been undertaken in an attemp t toelucidate the mechanism of action of these bacteria in the intestine.

CLASSIFICATIONThe taxonomy of bifidobacteria has change d continuously since they wer e first

isolated. They have been assigned to the genera Bac i l l u s , Bac te ro ides , Noca rd ia ,


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ROLE OF BIFIDOBACTERIA 1561

Lac t obac i l l u s and Co rynebac t e r i um among others, before being recognized as a separategenera in 1974 (9). Currently there are 29 species been described. These organismswere isolated from different sources such as i) the faeces of humans (infants andadults, animals, and sewage ii) bees, iii) the human vagina and iv) dental c aries(pathoge n). Many species ar e present in both animals and humans and while humanflora have been studied extensively, studies on animal flora are less abundant. Arecent study, using DNA -DN A hybridization to classify recent isolates, su ggests thatstrains isolated f rom calves may represen t a continuum between B . l o ngum and B .i n f a n t i s , two human strains (10). At present only five species of Bifidobacterium (i.e.B. ado lescen t i s , B . b i f i dum , B. b reve, B. i n fan t i s and B. lon gum) have interested thedairy manufacturers for manufacturing the therapeutic fermented milk products.

Figure 1: Photomicrograph of B i f i d obac t e r i a Grown on Glucose-Blood Agarwith China Blue. (Incubated Anaerobically at 37C for 72 h)

Structure:The structure of B i f i d obac t r i um spp . grown anaerobically in tripticase-phytone

yeast extrac t stabs show ed distinctive cellular shap es and arrangem ents. These traits arei) grouping of amp hora-like cells are Bz j i d . b i@dum, ii) specific ep ithet, thinnest and


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shortest cells are Bi j i d . b reve and iii) very elongated relatively thin cells with slightlyirregular contours and rare branching are B i f i d . l o ngum. ,. B i j i d ado l e scen t i s and i n f a n t i s .The principal compone nt of the cell wall is peptidoglycan (murein). Table 1 .summ arises the cell wall constituents of different species of bifidobacteria.

Table 1. Principal Cell Wall Constituents of Different Spec ies of Bifidobactria

Bacteria PeptidoglycanPolysaccharides

Galactose Glucose BhamnoseBi f i d . ado l escen t i s Lys-or Orn-D-Asp + +B i d . b i f i d um Orn or Lys-D-Ser-D-Asp + + +B@d. breve Lys-Gly + + +B i j i d . i n f an t i s Orn or Lys-Ser-Ala-Thr-Ala + + +B@d. longum Orn or Lys-Ser-Ala-Thr-Ala + + +

The polysacc haride compone nt like glucose, galactos e and rham nose will varyquantitatively and qualitatively among species, strain and grow th medium . Myristic,palmitic, palmitoleic and oleic are the major fatty acids in the cell wall structure butit will vary depending on the growth medium, growth temperature and the presenceor absence of human milk. Lipoteicho ic acids help in cell adhesion to the intestinewall. Phosphatidylglycerol, diphosphatidylglycer ol and alanylphosphatidylglycerol arespecific to bifidobacteria (11).Fermentation of Sugars :

All Bifid. species can fermen t lactose and grow well in milk. B. ado lescent is ,B . b reve, B . in f an t i s and B. l ongum can utilize many carbohydr ates while Bifld.bifidum can utilize fru ctose, galactos e and lactose. Bifidobacteria ferment glucose viathe fructose 6 -phosphate shunt, which has been reported and the key enzyme involvedin the hexose metabolism, fructose-6-phosphate phosphoketolase (F6PPK; EC 4.1.2.22)is present in the cellular extrac ts and the path way is called a s bifid shunt(12,1 3).

PHYSIOL OGY OF BIFIDOBACTERIABifidobacterium can grow between 20C to 46C (14), and dies at 60C (11).

Scardovi (15) reported the optimum pH for growth as 6.5 -7 and no growth at pH< 5.1 or > 8.0. These organisms do not grow in the synthetic medium (16).Bifidobactria are able to survive under different oxygen conditions and ferment widerange of substrates. Bifidobacteria are anaerobic organisms,but som e species can tolerateoxygen. The enzymes like superoxide dismutase (EC 1.15. 1.1) and catalase (EC1.11.1.6), help the organism to defence against the toxic effects of superoxide and


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hydrogen peroxid e. Thes e enzymes are present as a very low activity in B i j i d . i n f a n t i s ,b reve , lo ngum and ado l escen t i s and the oxygen sensitivity in these strains variesindependently of the superox ide dismutase activity (17). Shimamura et al have repor tedthat in the absence of exogenous carbohydrate, some Bifid strains utilize oxygen formetabolizing the intercellular substrates for its survival after fermentation and ingestion(18). NADH oxidase (EC 1.6.99.3) and NAD H peroxidase (EC 1. 11.1.1) are thesignificant enzymes involved in oxygen utilization. These enzymes catalyse the followingreactions

0, -I- NADH + H+ + H,O, + NAD+and

H,Oz + NA DH + H+ --, 2 H,O + NAD+without a ccumulation of hydrogen peroxid e. The bacterial grow th will be impairedwhen the hydrogen perox ide gets accum ulated due to disruption in the reaction.Metabolism of Carbohvdrates:

Bifidobacteria are differentiated from Lac t obac i l l u s spp . by their metabolic activityto utilize carbohydrates. Bifids use fructose 6-phosphate pathway for hexose fermentationwhile lactobacillus uses glucose 6-phosphate shunt (19). Madhi have reported theincrease in uric and formic acids (2 pg/g) when cow and ewes milk were fermentedby B i f i d . b i j i d um (20 ) . Recently Biavati e t al have detected low levels of glucoseB-phosphate dehydrogenase and aldolase in some species of Bifidobacteria (21).Metabolism of Nitrogen:

Amm onium sulphate is used as the nitrogen source by B i f i d obac t e r i a spp . whileB@d . cun i cu l i and B i f i d . su i s requires exogenous organic source (14). Matteuzzi et alhave reported the production of alanine, valine, aspar tate and threonine by Bifid.bifidum at in vitro conditions (22). Since milk contains limited number of freeaminoacids and peptides; it has to be suppleme nted with a nutrient rich broth (23),casein hydrolysate (24), or yeast extract (25).Metabolism of Linids:

There is no repor t available on the lipid metabolism of Bifidobacterium . Mah diin 1990 have repor ted the increase in total fatty acids when fermenting cow s andewesmilk with B i j i d . b i f i d um (20 ) .Growth Factors:

Presence of many grow th promoting factors have been reported in Bifidobacteriain vitro (26, 27). A bifidogenic grow th stimulator for Bi f id . ado lescen t i s 6003 havebeen identified by Kaneko et al in 1994 in the cell free filtrate of P r o p i o n i b a c t e r i umf reudenre ich i i 7025 and in the methanol extrac t fraction of the cells (28); how ever,


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short-chain fatty acids (form ate, acetate, propionate and butyrate also stimulated thegrow th of bifidobacteria. Bifidus factor wa s first described by Gyijrgy et al as a partof milk and colostrum and mad e up of glycoproteins (29). It can be sepa rated in totwo factors by isocratic HPL C (30) and the glyco moiety contains N-acetylglucosamine. Bezkorovainy et al have described the promoters as human caseinor its trypsin d erivative (31). Glycoma cropeptide which is the resulting p roduct oftrypsin hydrolysis of human k-casein contains active sugars like galatosamine andglucosamine. Azum a et al. found that the chymotrypsin hydrolysis produc t of humank-casein is more effective in promoting the growth of B i f i d . b i f i d um than the trypsinderivative (32). The sugar and polypeptide component are responsible for the promotingactivity of the bifidus factor. Allen have reported the presence of bifidus factor inmucus secretion of salivary glands, small intestine and colon (33). The majorcompon ents of this factor is also glycoproteins with N-acetylgalactosam ine, N-acytylglucosamine and sialic acid. Gyorgy et al have identified the Bifidus f actor 2as a non-glycosylated peptides of casein ( 34). It is a byproduc t of proteinasehydrolysis, found to be effective in milk and whey .Synthesis of Vitamins:

Bifidobacterium strains isolated from humans need vitamins like thiamin (B l),pyridoxine (Be), folic acid (B,) and cyanocobalamin (B,J for its growth. But the sameorganisms can synthesize B vitamins. Degu chi et al. have listed (Table 2) thefollowing B vitamins synthesized by different B ifidobacteria (35).

Table 2 . Synthesis of Vitamin by Different B i f i d obac t e r i a spp .

Vitaminpg ml-

ThiaminFolic acidPyridoxineNicotineCyanocobalaminAscorbic acid BiotinRiboflavin (35) 2 (14)

MicroorganismB i f i d . B i f i d . B i j i d . b r eve B i j i d . B i f i d .

ado lescent is b i f i d um i n f a n t i s l o n g um0.02 0.23 0.09 0.2 0.090.01 0.058 0.008 0.040 0.02

0.043 0.046 0.02 0.059 0.420.17 1.04 0.39 1.23 0.610.35 0.65 0.49 0.39 0.46

I.C. n.s. l.c. 1.C l.c.l.c. n.s. l.c.. 1.c l.c.n.s ns. n.s. n.s. n.s.

l.c. low concentration, n.s. not synthesized.


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ENUMER ATION OF BIFIDOFACTERIATwo facto rs are important in the cultivation of bifidobacteria: an adequ ate

culture m edium and anaerobic conditions. The traditional technique is easy to use andit contains 0.1 yeast extrac t or corn steap liquor yields viable cell counts ofBifidobacteria equivalent to ferme nted milk (36). The following Table 3 describes som eselective m edia for the enumeration of B i f i d obac t e r i um spp . from fermented dairyprodu cts. The synthesis of B vitamins by bifidobacteria enhances the nutritional qualityof the ferme nted milk product. But whe ther this improv es the bioavailability of thesevitamins in the human sto ma ch needs mo re evidence and resear ch. Acidified mediasuch as MR S, Rogosa are extensively been used to enumerate bifidobacteria frommixed cultures (37). B ut it is evident that more resea rch is needed in formulating theselective media which is easy to prepare, inexpensive and be able to recove r mor ethan 95 of BQ idobac t e r i a i p p . _ _

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Diarrhoea. gastroceteritisinfections. meningitis,cndocarditis. septicaemia.urinary tract infection,brain abscesses, liverabscesses, lung abscesses)

Figure 2. Hos t Relationships of the Intestinal Bacte riaBIFIDOBACTEIA AS PROBIOTICS

Fuller defined probiotics as a live microbial feed supplement which beneficially


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affects the host animal by improving its intestinal microbial balance (38).Bifidobacteria have been called resistance factors by Homm a (39). A resistance factoris described as a substance which carries ou t certain physiological functions but which ,when lacking in a healthy pe rson in optimum condition has no advers e effect.However, once a person is affected by outside forces, which is essentially always, thelack of this resistance factor causes negative changes in the body, due to the bodysdecre ased ability to respond to the outside challenge. The host relationships of theintestinal bacteria are shown in Figure 2.

The way the probiotics work is not known, but our knowledge of gutmicrobiology suggests four ways in which they may be operating. They are i)production of antimicrobial substances (50, 51) ii) competition for adhesion recep tors(52, 53, 54) iii) competition for nutrients with pathogen s (55), iv) stimulating immunity(56). There are many claims for beneficial effec ts of probiotics in constipation, cancer,heart disease and ulcerative colitis but the evidences are from animals. Table 4 and5 summ arize som e of the stated beneficial effec ts of probiotics use in both animalsand human.

Table 4. Beneficial Effe cts of Probiotics (57).

Animals HumansEffe ct on indigenous microbes:

Noninfectious disbacteriosisafter high dosag es of antibiotics

Stress (eg. feed change, transport)Deficiency in the developm ent ofmicrofloraGrowth promotion

Higher feed conversionAntinutritional factors(ANFs)destructionVitamin synthesis

Protein predigestion

Noninfectious disbacteriosis afterhigh dosa ges of antibiotics andafter radiation therapy.In Neona te intensive care unitHealth promotionInhibition of carcinogenesisAnticholesteremic effectHigh calcium resorptionDestruction of ANFsVitamin synthesisProtein predigestion

The two notable evidences from humans are the effect on lactase deficiency (68) andon Pseudom em branous co l i t i s (69) . As seen from Table 5, bifidobacteria have beenreported to be responsible for many different effects. T hese range from increasedproduction of secretor y IgA, to altering the compo sition of the intestinal flora. A lthoughseveral studies have been performed in humans, most of these were carried out ona limited number of subjects and som e were without c ontrols.


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SELECTING BIFIDOBACTERIA AS STARTER CULTURESStarter cultures refer to the specific Bifidobacteria (BIB)which are used to

inoculate milk and whose metabolism leads to the fermented products. There are twotypes of starter cultures. The first one is the traditional me thod which containscom plex, poorly defined mixtures of bacterial strains. Th e second one is selected ordefinedculture which contains one or mor e identified pure cultures with knownspecific properties. Specific properties means Bifidobacterias rate of acid production,type of polysaccharide fermentation, proteolysis characteristics (which sometimes leadsto bitter compounds) and their capacity to produce flavour compounds. Anothercriterion is based on the Bifidobacterias ability to synthesis vitamins and increase thenutritional value and health prope rties of the fermen ted produc ts. For exam ple incertain countries the Bifidobacteria is adde d as the starter culture because of itspossible beneficial effects in the gastrointestinal tract or to improve the vitamin profile(70, 71). Traditional bulk starter milk is prepa red by reconstituting antibiotic-free,skimmed milk pow der (total solid lOO-12Og/l) and heating to 90 -95C for 1 5 to 30min which w ould destro y the native microbes in the milk, inactivate the naturalinhibitors, modify the proteins, and reduce the dissolved oxygen. All these changewould help to provide the right growth conditions for B i j i d obac t e r i um spp . Howevera growth stimulatory factor(s) will be added to the bulk starter milk. Comm erciallyproduced Bifidobacteria starter cultures are more consistent than the home producedliquid cultures. Com mercial cultures can be bought in concentrated freez e - dried formwhich can be stored at < 5C or at -20C or as concentrated cultures deep frozenat -190C; which can be stored at -40 to -80C. The average count is 1 lOi cfu/gand the bacterial cells are concentrated to achieve these numbers and the activity ofthese cultures depend s on the survival rate of the organisms .

BIFIDOBACTERIA AND HUMA N GITStom ach and Intestinal Transit:

At least 400 types of bacteria have been detected in the faeces of humans(72,73) among which 25 species of bacteria were identified as B@d. ado lescent is ,i n f a n t i s , and l o ngum. As seen from Figure 3 the digestive tract of newly-born infantsrapidly colonized bacteria from the vagina during birth (74) and within 48 hours ofbirth, the stools of infants may contain up to 4.0 x 10 cfu/g (75). Yuh ara et al.have reporte d that in breast fed infants bifidobacterium species as the mos t frequentlyisolated spec ies (76). The human small intestine contains a pproxim ately lo5 bacterialcells per gram w hile the colon contains 10 bacterial cells per gram o f contents. Ithas been sugge sted that for bacteria to act as probiotics they must arrive in theintestines alive and in sufficient number lo6 cells/g, (37) or lo7 cells/g (64) to havean effec t per se or they should adhe re or implant and multiply (Figure 4) (77).

Thus f or the bifidobacteria in a fermen ted milk to have an effect, they mustresist the acidic conditions of the stom ach for 90 minutes (an averag e time for gastricemptying of dairy pro ducts), and be resistant to bile in orde r to transit the small


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intestine. Berr ada obser ved that the strain of bifidobacteria in one comm ercial fermen tedmilk pro duct had a survival rate after in vitro incubation at pH 3 for 3 hourswhe reas the strain in another comm ercial produ ct dit not. This sam e survival patternwas subsequently confirmed in humans ( 78). Bifidobacteria do survive intestinal transitin high numbers, although not all strains are equally resistant (79, 80, 81). Onestudy, using a marked strain, has shown that a bifidobacterial strain present infermented milk could be recovered at a rate of 30 from the faeces of humanvolunteers. This agrees with the 23.5 to 37.5 recovery rate of bifidobacteriafrom the illeum of other volunteers (79, 81).

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Effects on Gut Flora:It has been observed that feeding bifidobacteria fermen ted milk alters the

intestinal population, as determined by faecal enumerations (61, 66, 81). In one studythe faecal bifidobacterial recovery was temporarily increased by a factor of 20 (81),


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ROLE OF BIFIDOBACTERIA

while in another the increase was by a factor of 8000 (66).

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BacteriodesEubacteriumAnaerobic streptococcus

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Birth Weaning Maturity Old ageFigure 4: Chan ges in the Intestinal Micro flora with Age.

Another recent study with humans suggests that consumption of 5. lo9bifidobacteria 3 times daily for 5 wee ks in the form of lyophilized pow der alters theprofile of the faecal flora (61 ). In this study the relative proportion of bifidobacteriaincreased while the proportion of bacteroide s and clostridia de creas ed. In addition, asignificant reduction in faecal pH value was observe d at the end of the study. Themechanism of action by which bifidobacteria affect the gut microflor a remains to beelucidated. In a study with labelled bifidobacteria, it was determined that the increasewas due to the ingested bacteria, whereas the authors of another study attribute thisincrease to implantation. One recent study has shown that bifidobacteria, given orallyto humans, are unable to implant in the gut even under conditions which favourcolonization (65). Othe r studies have shown that the increased population ofbifidobacteria in the faece s observe d while feeding bifidobacteria-ferm ented milk returnedto pre-intervention levels within 1 to 2 wee ks when the produc t was no longer fed.


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Although the mechanism by which bifidobacteria may alter the intestinal microflora hasnot been clearly determined, it is possibly related to the production of acetic acid andlactic acid which may restrict the grow th of potential pathogenic and putrefactivebacteria (82). That acetic acid is more bacteriostatic than lactic acid at the same p Hmay explain a greater effect of bifidobacteria compared to certain other bacteria, suchas those found in yogurt, which pro duce only lactic acid.Phvsiological Effe cts in GIT:

The microflor a effec ts have been mor e clearly demonstrated , both in animalsand humans. These fall into two types of categories, effects on the gut microflora,as mea sured indirectly by gut transit (6 0, 63, 67, 83) or mea sured directly by faecalenumerations and enzyme activities such as P-glucuronidase and nitroreductase (62, 84).Although numerous articles cite the in vivo effects in both animals or humansfollowing ingestion of large numbers of live bifidobacteria, few double-blind studieshave been sufficiently large enough and with appro priate controls to dem onstratestatistical differences . In one recent study, using 60 healthy v olunteers, it was observe dthat feeding 125 g of ferme nted milk containing bifidobacteria, 3 times per day,reduc ed intestinal transit times, but still within normal p hysiological limits (67). Thiseffec t in the sigmoid colon wa s statistically significant for wom en on a normal diet.Transit time for the colon in women tend to be slower than in men. It is possiblethat this physiological effect was media ted through a change in the intestinal floraeffec ted by feeding large numbers of bifidobacteria.Ecolog v and Identification of Svecies in GIT:

The colonization of the colon and the species com position depends on thehygiene in the delivery area and/or me thod of birth that is natural or caesare an. Thedifferences in morp hological and biochemical variations exists among Bifidobacteria mak eidentification to species level difficult. The walls of the colon provide a specialecological niche in which bifidobacteria can proliferate at the expense of other group s.The strains of B@d . b i f d um and i n f a n t i s can metabolize the oligosacch arides found inthe mucilage secre ted by the cell walls of the colon, and hence the mucin tends tohave a selective influence on the microflora (85). W ith respect to the actual specie sinvolved, som e strain like B i j i d . i n f a n t i s secre tes the polysacc harides which initiatesadhesion to the epithelial cells of the intestine. The binding of lipoteichoic acids ofBifidobacteria to human e pithelial cells depend upon the cell concentration and thecontact time. The number of cells detec ted in the stools may not reflect thepopulation in the intestine because the cells may be sent out from the body bef oreadhesion. Guerina and Neutra have reported that some times the host cell may growaround the bacterial cell as a mem brane and host bacterium relationship is create dwhich depends on the age of the individual cells as well as the age of the person.This interaction between the epithelial cells and the bacteria also depends on the fattyacid composition of the lipoteichoic acid, higher the fatty acid the better the adhesion(86).


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II

10

2Points

LengthTime taken for

food to arrive

Anaerobtc strepococcusBibidobacterium

StreptococcusE c o l iLactobacillus

---+----iIVeillonellaC. p e r f r i n g e n s

15 Stomach Jejunum Caecum2 Duodenum Ileum RectumY- 65 cm--w : 5-6 m -1.5 m -

__c I-60 --- 3-5 * - 24-72hr 4h-r6 hr

Figure 5: Variations of Bacterial Populations and Spec ies along the GastrointestinalTract in Human Adult under Non-fasting Conditions.

(Cu = Colony forming unit, s= second, h=hour, cm= centimeter, m = meter)

Age and Feeding RegimenThe microflora in the colon of a bottle-fed, one month old child will be

occupied with about 80 of bifidobacteria, but the number will gradually decre asewith age. In adults bacteroides are the major group with Bifld. adolescentis, longumand bijidum. The abundance of bifidobacterial species depends on the healthy functionof the colon. Eventhou gh numerous repor ts are existing about the general trend thatthe colons o f breast-fed infants have higher levels of bifidobacteria than the bottle-fedinfantswhether or not the species frequ ency changes with the type of feed in notclear. Ballongue suggested that Bifid. adolescentis was the dominant organism in bottlefed neonates (14) and Beerens found Bifid. longum as the frequently isolated s peciesfrom th e stools of form ulated infants ( 87) and on the contrary Biavati et al. isolatedmixed populations, namely Bijid. bifidum , infantis, longu m and breve, (8 8) irrespectiveof the type of milk. There were some suggestions that the difference between the


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resultant microflor as may be due to the absence of specific stim ulatory factor s incommercial feeds. Even the variations in the human milk from mother to mother alsoaffec t the intestinal microbial flora of the infants. Figure 5 show s the dram aticvariations in the total population and species along the GIT, with the highestconcentrations in the colon (89).

Ther e is a wide variation of bacteria among individuals, but globally thenumber of species and the population of bacteria are relatively stable in healthy adults.In addition to the normal m icroflora (resident microflo ra) already established in theGIT, bacteria are introduced into the body as a normal part of food (transientmicroflora), or as contaminants (accidental microflor a). Transient bacteria have agreater effect on the upper GIT than on the colon because of their lower numbersof resident microorg anisms. Ingestion of the LAB found in ferme nted milks poses nohealth problem s for the general population (90). In spite of its stability, the intestinalmicroflora can be affected by a variety of factors. Host factors, such as gastric acid,bile salts, and mucus in the intestinal wall can affect the compo sition of themicroflo ra. In addition, diet, medication, infections, stress, aging, and climate can alterthe microflor a. Bacterial interactions such as antagonism or symbiosis can also influencethe contents of the microflora . The intestinal microflora is capable of adapting to andmetabolizing mos t of the substances that enter the intestines from the oral tract, orthroug h the biliary tract, o r compo unds which are secre ted directly into the intestines.Adaptation can occur within several days.

BIB in Prevention of Intestinal Disorde r:Imnroved Protein Metabolism:

Bifidobacteria have phosph oprotein phos phata se activity which helps in increaseabsorption of human milk protein by breaking down the casein in human milk. Thisis though t to contribute to the satisfactory absorption of human milk (91). Nitrogenretention is good in infants with a bifidus microflora; bifidobacteria prom otes theaminoacids metabolism, but the mechanism is not clear. O ne of the roles whichbifidobacteria fulfil in the intestinal tract of the infants is to suppr ess the multiplicationof putrefactive bacteria thereby stopping losses of nutrients.

Imoroved Vitamin MetabolismBifidobacteria are predominant in the intestinal m icroflora of healthy people

irrespective of age and the vitamins produ ced by them needs warran t attention. Thevalues repor ted for vitamins prod uced by bifidobacteria are : vitamin B, 7.5 pg andB, 25 pg per g dry weight for intracellular bacterial vitamins, and B, 25 - 25O pg,B, lOO pg, B ,, O.O6pg, nicotinic acid 400 pg, and folic acid 25 pg per litre ofmedium for vitamins produ ced outside the bacterial cells. It is known th at BuciZZusthiamonolyticus inhabiting the intestinal tract break down the vitamin B, and cause B,


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deficiency. In such cases oral administration of vitamin B, is not effective (92). S othe oral administration of bifidobacterial preparations are sugge sted to increase theintestinal microflor a and to suppre ss the thiaminolytic bacteria. With bifidus microflorait would also be enable the beneficial utilization of the extracelluar vitamin B,produ ced by the bifidobacteria.BIB in Prevention of Constination:

Constipation can be defined as the failure to defecate for 3 or 4 days andwhen combined with pain and treatment it becom es a illness. Constipation is causedby the poor quality of food, re gularity in taking fo od, and the mortality of thedigestive tract microflor a. The organic acids produ ced by bifidobacteria are thought tostimulate the intestinal peristalsis and help in normal bow el movement. Bifiduspreparations for constipation sufferers and people with irregular bowel movements havebeen on sale for years. Doerbeck et al. reported an improvement in bowel m ovementsand a simultaneous increase in bifidobacterial count in the faece s when they give milkcontaining bifidobacteria to constipation patients (93). Administration of bifidobacteriaalso leads to a higher moisture content in faeces and Tanaka and Shimosaka reportedthis as a factor in the beneficial effect ( 63).BIB and Antibiotic Action:

In vitro, bifidobacteria have been noted to have antibacterial activity againstpathogenic E. Co l i , S taphy lo coccus au reus, Sh igel l a dy sen t er i ae, Sa l m onel l a t y ph i , Pr o t eusspp . and Cand ida a lb icans . The antibacterial action shown by bifidobacteria is fromthe organic acids they produce. Bifidobacteria make 1 mol lactic acid, 1.5 mol aceticacid and small amount of formic acid from 1 mol of glucose. Rasic and Kurmannrepo rted that the intensity of the antibiotic action varies with acids: for exam ple theminimum pH at which Sa lmone l la spp . can grow is 5.4 for acetic acid, 4.4 for lacticacid and 4.05 f or citric/ hyd rochloric acid (11). Anand et al. have isolated anantibiotic called Bifidin from B i j i d . b i f i d um 1452 . Bifidin is stable to heating 100Cfor 30 min; it gives a positive ninhydrin reaction, and its main compo nents arephenylalanine and glutamic acid. It show s antibacterial activity against M ic ro co ccu sj lavus and Staphy loccoccus au reus , by being active at pH 4.8 to 5.5 (9 4). Ferrari etal. have shown that the bifidobacterial cells breakdow n the conjugated bile acids tofree bile acids which intern inhibit the grow th of pathogen s (95).

BIB in the Treatment of Liver D amape:Disturbances to liver function caus e metabolic abnormalities, with ill effects from

amm onia and amines, the produ cts o f putrefactive bacteria. Muting et al. repor ted thatwhen they gave bifidus milk fo r a long period to patients with hepatitis or cirrhosisof the liver, blood ammonia, phenol and urinary indican dec rease d significantly asbifidobacteria in faece s increased, and the patients show ed increased appetite and gainedweigh t (96). The dietary administration of B i j i d . b i d um togethe r with lactulose assistin re-establishing the normal gut flora in advanced cirrhosis. The re-establishment of


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the normal microbial balance is accom panied by a reduction of amm onia and freephenols in the blood (F igure 6).

Blood Ammon ia3x70 3*too 3s70 3 70 3

/ng/~roog

100 ml250,

wmwA bi f idus-mi lk preparat ion

mg/KXImlFree Serum Phenols

8. bif idu m in the faeces: ; j

21.12 26.1 7.3 11.5 21.7 10.3 23.513.12 5.1 10.2 23.3 2.6 30.8 14.2 18.4 20.7

1965 1966 1967

Figure 6: Long Term Treatment of Decom posed Liver Cirrhosis with Bifidus M ilkPreparation Eugalan Forte in 12 Yea r Old Patient


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A normalization in protein metabolism has occure d and it allows the patient toincrease protein intake ( to 70 g per day). The ability of bifidobacteria to utilizeammonia as a source of nitrogen may also lead to decrease of ammonia in the colon(15). Lactulose is administered as a 50 (w/w) syrup after meals. The dose isadjusted so as to induce two soft stools a day, under which conditions the faecal pHreaches 5-6 or less. It is important to start with small doses eg. 3 x 10 g per day,followed by a gradual increase to 3 x 100 g or better 5 x 60 g per day (97). Mutinget al. have proven the beneficial effect of bifidus m ilk for protein metabolism in livercirrhosis (96). It is important to use non -urease producing strains of B . bi f i d um inthe manufa cture of bifidus milk preparation s. How ever, if lactulose and /or bifidus milkfails to reduce b lood ammonia in portal encephalopathy then neomycin should betemporarily administered to the patients.BIB in the Reduction of the Risk of Colon Cancer:

Research in the past fifteen years has focused on the potential role of BIB inthe prevention of cancer initiation. Studies first showed a slower grow th of certainexperimental cancers in animals (98) . B. b i f i dum (99) , have been shown in humanclinical studies to reduce the levels of som e colonic enzymes (/I-glucuronidase,nitroreductase, azore ductase , and glycoholic acid hydrolas e), which are implicated in theconversion of procarcinoge ns to carcinogens (100, 10 1) such as nitrosamines orsecondary bile salts (102). Most studies report a decrease in these enzymes during thestudy period when live BIB wer e consumed with a return to baseline levels duringfollow-up when no BIB were consumed. The mechanisms and long-term effects ofthese changes are not clear. Recent epidemiological studies (103, 104) have found thatcolon cance r risk was inversely related to the consumption of diets which includedfermented milks. Other results (105) show no relation to colorectal cancer risk.

Conclusions for the reduction of risk of colon cance r can not yet beextrapolated from these preliminary results. Other dietary factors have been consideredin the prevention of colon cancer, including fibre and calcium ferme nted milks maybe one factor of many that affect risk of colon cancer.BIB and Antitumour Activitv:

Studies with laboratory animals hav e indicated antitumour effec t of bifidobacteria.No significant difference in tumour size was observe d when meth-a ascites tumour cell(25 x 103) were transplanted and a suspension of B i f i d . i n f a n t i s (10 cells)wasintraregionally injected in to BA LB /c mice. The antitumour effect decreased when thefirst bacterial injection started 3 or 7 days after tumour inoculation, or when the micewer e inoculated with a large number (500~ 10~ ) of tumour cells. The stimulation ofthe host immune response has been sugges ted to be involved in the antitumour effec tof Bifidobacteria (106). L iver tumorigenesis has been reported to be reduced by thepresence of B i f i d . l o n g um in the gut of gnotobiotics C3H/H e male m ice (107) dueto the stimulation of immune respons e of the host, and decreas e in the faecal bacterialenzymes by bifidobacteria; but mor e evidence is needed. Studies have shown that


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chemically induced colon tumorigenesis may be inhibited by feeding w ith sour milk.The results of DM H- induced tumours in rats fed with different diet is given in theTable 6 (108)

Table 6. Incidence of DM H-induce d Tumo urs in Rats Fed with Different Diets.

Group No. of rats Rats withtumours

No. of colon tumours per ratTotal Adenoma Carcinoma

ControlSour milkAcidifiedmilkStarter cells

9 9 2.6 1.3 1.39 6 1.0 0.4 0.69 9 3.4 2.0 1.4

8 7 2.3 1.8 0.5The anti tumour effec t may be explained by the beneficial activities of

bifidobacteria present in increasing numbers in the intestine of rats fed with sourmilk.Feeding studies have shown that the life span of mice fed with 14 sour milkwas , on avera ge 8 longer than control diet and the bifidobacterial count was 10times higher than the control diet. But the potential mechanism for antitumour activityneeds further research.BIB and the Host Immune Svstem

Current evidence sugges ts that microorg anisms indigenous to the gastrointestinaltract are less immunogenic in their hosts than allochthonous microorga nisms of theirsimilar typ es (109). S tarter bacteria provide a significant source of antigens, whichinduce som e immunological activity in their hosts. Administration of viable and nonviable mixture s of various stains of intestinal bacteria to germ free mice has shownthat intestinal production of immunoglobulin A (IgA) plasma cytes was greate r than thatfollowing the implantation of single strain or species. Lactic acid bacteria capable o festablishing in the intestinal tract wer e less immunogenic than the transitory strains.Perdigon and Alavarez have recently s umm erised the very active and interestingresea rch d eveloping in the field immu nomodulatory effect of lactic acid bacteria (110 ).The effects of ingested lactic acid bacteria with the immune system first start in thegut associated lymphoid tissue (GALT).

Few studies have been reported on the effect of Bifidobacterium on GALT andnone of the on human. In mice peridigon et al. (111) sh owe d th at oar1 administrationof L. casei, L.acidophilus, L. bulgaricus and S. thermophilus increased the levels ofimmunoglobulins in the intestinal fluid. How ever, only L. casei pretreated mice hadincreased secretion of specific antibodies aga inst Salmonellae when challenged withSalmonellae (111). A recent study of the comparative effects of diets, enriched


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respectively with live and heat treated yogurt on the response of the murineimmunosystem has shown that large inocula stimulate the immunological activity of thespleen and thymus gland. Live bacteria were more effective than dead ones inincreasing the serum imrnunoglobulin G (IgG& (112) . Supplem entary feeding withyogurt stimulated the enlargement of lymph nodes in the spleen and the number ofB lymphocytes associated w ith humoral immunity and T lymphocytes associated withcell-mediated immunity wer e increased significantly ( 113). Experim ents with germ -freemice monoassociated with Bid. longum shown antilethal effec t and cell-mediatedimmunity against invading E. Coli (114). Subsequent studies with germ free micemonoassociated with B@d. longum have indicated th e production of anti-bifid. longumIgA and cell-mediated immunity. These data suppo rt the speculation that ingestion offerme nted milks containing large numbers of viable bifidobacteria and lactic acidbacteria m ay induce an immunological respons e in humans. How ever, the effect ofstarter bacteria on the immunosy stem of their hosts require further investigation. Asshown in Table 5, recently published findings with Bifidobacteria fall in two generalcategor ies, immunological and microflor a effects (115 ) and the immunological effec tshave been clearly d emon strated invitro (58) and a significant increase in humo ralimmunity was observe d in a study involving 15 human volunteers (59)BIB Do They L ower B lood Cholesterol Levels?

Since Mann and Spoerrys observation in 1974 that East African Maasai warriorswho consume four liters per day of fermented milk had low blood levels of serumcholestero l, and that, when intake increased to eight liters per day, they had additionaldecre ase in levels (116), m any studies have been conducted to find ahypoch olesterolem ic factor in ferme nted milks. Hydroxymethyl glutarate has beensugge sted as active fa ctor ( 117). The results of this resea rch is given in Table 7.

Table 7. Influence of Yoghurt in Human Serum Cholesterol Level (117).

ProductWhole MilkYogurt from Whole milkYogurt from Skim Milk

Before196193211

Serum Cholesterol (mg/dl)After Significant reduction177 No175 Yes150 Yes

This factor inhibits cholestero l synthesis in the body resulting in reduce d serumcholestero l levels. Res ults are difficult to interpret because of confounding variables,such as exercise and dietary patterns and varying levels of initial s erum cholestero l.Thakur and Jha showed that rabbits fed a high cholesterol diet supplemented withyogurt exhibited lower serum cholestero l levels than did rabbits on the nonsupplemented control diet (118). The effects of BIB on cholesterol levels are therefore


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inconsistent, and range from a significant reduction to no reduction, with intermediateresults o f transient effec ts and positive ou tcom es only in wom en (119) . Th e exactmechanism is unknown. Most of the research have used L. acidophilus; otherpossibilities have included L . casei , L . p l an t a rum , B i f i dobac te r ium spp . (120) andEnterococcus fuecium (121,122). Ther e is concern that consuming yogurt on a regularbasis may increase serum cholesterol because of the milk fat. Halpem et al. andMcN amara et al. both showed that in young normolipidemic adults consuming 450 g(around 16 ounces) per day of lowfat yogurt (7 g fat/d) for up to four months, therewere no deleterious effects on serum cholesterol, triglycerides, HDL or LDL levels.Yogu rt can be consum ed regularly without concern for raising cholesterol levels (123,124) . Consum ption of yogurt itself may not help in controlling cholestero l but som efactor produ ced by the yogurt ba cteria during fermentation of the milk is responsible.So, there may be some ways to concentrate the active factor(s) into usable volumefor practical use. Additional resea rch is certainly needed in order to clarify thepossible hypocholesterplaemic effect of cultured yogurt.BIB in the Treatm ent of Urogenital Infections:

The predominant microorgan ism in the normal m icroflora of the fema le urinarytract is lactobacilli, in particular, L . a c i d oph i l u s . Hormonal changes, age, andmenop ause can alter the flora. During urinary tract infections, pathogen s such asCund ida a l b i cun s (a yeast responsible for candidiasis) and E. co l i , travel from thecolon to the urethra and the bladder. A recent study by Hilton et al. suggests thatdaily oral ingestion of 227 gm (8 ounces) of yogurt containing L . a c i d oph i l u s maybe useful in reducing the recurrence of candidal vaginal infections (125) . Lactic acidbacteria c omp ete w ith uropatho gens for nutrition, space and habitat and reduce itspathogenicity. The mechanisms been suggested was coaggregation of the pathogen withlactobacilli BiJ id . f rag i l i s . In the small prospe ctive cross-o ver study, wom en consumingyogurt exp erienced few er recurrence s of vaginal candidiasis than during the controlperiod (1 26). Further research is necessary.BIB and Possible Adverse Effects:

Som e strains of Bifidobacteria have been implicated in possible advers e eff ectsfor the host, when found in the soft tissues. They usually occur in mixed populationsalong with facultative anaerobic bacteria ( 81). Subsequently these strains have beenidentified as B i d . den t i um in dental caries and other clinical materials includingpleural fluid (127 ), suggesting its possible harm ful potentiality.

THERAPEUTIC AND PROPHYL ACTIC ACTIVITIES OF BIFIDOBACTERIACertain antibiotic, irradiation of the abdomen with gamm a or X-rays , and stress

conditions may also disturb the the balance of intestinal microflor a. The consumptionof ferm ented milk containing bifidobacterial species like Bifid. longum, and bifidumhelped in the restoration of the microflor a after oral antibiotic therapy . Korshunov etal. have rapidly restored the decreased population of bifidobacteria due to the oral


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administration of kanamycin 40 mg /kg to adult humans by three oral administrationof autostrains of bifidobacteria and lactobacilli (128) . A disturbed balance of intestinalmicroflora in leukaemia patients been improv ed by oral administration of bifidobacteriaand acidophilus bacteria (129) . The results of the treatment are given in Table 8.

Table 8 . Indicates the Chan ges in Minor M emb ers of Intestinal Bacte riaduring Antileukaemic Therap y

Organism

K lebsie l l a sp .C i t robac te r sp .P ro t eu s vu l g a r - i s

Treatm ent with antileukaemic drugControl (56 cases)

(locases) Without Bifidobacteria With Bifidobacteria(28 cases) in (28 cases) in

0 3 (10.7) 2 (7.1)0 6 (21.4) 5 (17.9)0 8 (28.6) 2 (7.1)

Cases in which more than lo6 colonies were observed

The antagonistic effect of bifidobacteria against many undesirable microbe s maybe due to the acetic and lactic a cids they produc e and possibly, to their effe ct on theimmunosystem of the host. Schneegan et al have shown that the oral dietaryadministration of a freez e dried cultures of B. b i j i d um to children with entericinfections, could eradicate enteropathogenic E. C o l i strains in about 60 of thecases, and more than 80 when lactulose was included (130). B i f i d . b@dum, togetherwith lactulose, has also shown to assist in reestablishing the balance of intestinalmicroflora which is disturbed in liver cirrhosis, and this is accom panied by low faecalpH and low ammonia and phenol levels in the blood ( 97). To consider bifidobacteriato be the major group in the colon then the following possible routes should beencoura ged i) feeding the subject with a diet that will stimulate his or her nativemicrof lora, ii) consumption of dairy produ cts manu factured with bifidobacteria (102) ,iii) the use of suppository containing viable cells of bifidobacteria.

How ever, the position with foods containing bifidobacteria may be different andin assessing the potential advantages of consuming bioactive milks or yoghurts, it hasto be assumed that i) consumption will be on a regular basis, at a level of som e400-500g product/week, ii) the dairy product should contain minimum of 1.0~10~ cfu/gof produ ct at the time of consumption (131) iii) the origin of Bifidobacterium shouldbe of human origin so that it can with stand the intestinal tract transit. If theseconditions are met then there is a clinical evidence that suppo rt that the ingestedbacteria may i) stimulate the existing population of bifidobacteria ii) act in conjunctionwith the native flora and iii) replace the disturbed microflor a (132) . The potential


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benefits for healthy subjects is mor e difficult to predict, how ever regular intake ofB i f i d . b@dum by healthy infants did provide a degre e of protection against entericinfections (133 ). The increased sales of biomilks and bio-yoghurts in Euro pe and Nor thAm erica indicates that the consum ers have acquired the interest in these productsloyalty w ithout any genuine belief or understanding in its therapeu tic and orprophylactic properties.

BIFIDOBACTERIAL PRODUCTSLar ge number of bifidobacterial produc ts are available in the mark et. Reuter

conducted a survey of fermen ted milk produ cts containing Bifidobacteria in Germany,Japan, and France and found out that B i f i d . l o n g um is widely use d in Germany (134) .The most suitable combination is B@d . l o ngum and St r . the rmoph i lus . The selectionof comm ercial strain is important that it has a higher survival rates in acidic produ cts(135, 136, 137). B i j i d . b@dum an d B i j i d . Z o n gum are employed as mixed cultures incombination with St rep tococcus sa l i va r ious and/or Lb . ac idoph i lus . Decription of someof those products are as follows:Acidonhilus Bifidus Yogur t:

This is similar to Bifidus yo gurt. It is manu factured in many countries byfermenting cows m ilk. The homogenized milk is heated to 85C for 30 min or 90Cfor 5 min and co oled to 40 - 42 C inoculated with sep arate cultures of L .a ci d o p h i l u s a n d B i f i d . b i f i d um o r l o n gum packaged, incubated at 40 - 44C for 3-5h, followed by cooling and storage . T he therapeu tic organism is of human origin andthe viable cell count averages l-3 x 10 &u/ml for each of Lb . a c i d oph i l u s andBi f idobac te r ia sp .Bifidus Milk:

Cow s milk is fortified with 150 - 200 g total solids per kg. T his milk ishomogenized and heated to 80-120 C for 15 min, cooled to 37 C, inoculated with100 g/l of B i f i d . b i f i d um o r Zo n g um as starter culture at pH -4.5. The coagulumis cooled, packaged and moved to the cold store (138). The characteristic features ofBifidus milk are i) It has a mild acidic and slightly spicy taste, ii) the molar ratioof lactic acid to acetic acid is 2:3 iii) set or stirred types are produced with orwithout fruit flavours and iv) the viable count of B i f i d . b i f i d um is lo*-lo9 cfu perml. Ferm ented milk containing Bifidobacteria are becoming popular in UK because itis milder in taste when compared to yogurt. Misra and Kuila have reported acomm ercial me thod for the production of bifidus milk in India (139) . Skim med milkwas heated to 90C for 30 min, cooled to 37 C, inoculated with Bifid. bifidum NDR I(National Dairy Res earch Institute of India) at the concentration of 1OO g per 1, andincubated for 19h. The products has good storage life of minimum 3 weeks, goodtaste and microbial count of 10 cfu per g. The antibacterial activity of the fermen tedmilk against four pathogens showed that it was dependent on i) the type of milk used(skimmed, cow, buffalo or reconstituted baby formula) ii) heating tem perature iii)


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concentration of the inoculum and iv) concentration of sugar. Bifidus or acidophilusdrinks are becoming popular. The viable cells of >2x1 06 cfu per ml is added to coldpasteurize d milk followed by packaging . These products complies with statutoryregulations in California and Orego n. A similar product containing Lb. ac i d oph i l u s andB i f z d . b i j i d um is also marketed in USA called Nu-trish A/B which is supplied by Chr.Hansens laboratory (140) These products are sweet in taste and not fermented butoffer a good way to provide an abundance of bifidobacteria to consumers.Bifidus Yoghurt:

It is a mixed starter culture of B i j i d . b i f i d um o r l o n gum and yogurt culturewith or with out Lb . ac idoph i lus . The concentration used is at a rate of 50 - 100g/l to ferment milk at 40 - 42C for 3-5 h which is followed by mixing beforecooling and packaging.Bifighurt:

This is similar to Bifidus milk or yogurt but the starter cu lture is exclusivelyof B i f i d . l o n g um CKL 1969 or DSM 2054 (a slime- producer) and St r . the rmoph i lus .The bifidobacterial count in the produc t is 10 cfu/ml, and produ ces only lactic acid(95 ).

PHARMACEUTICAL PRODUCTSSpecial freeze-dried pharm aceutical dietary preparations containing viable cells of

B i f i d obac t e r i um spp . alone or in combination with other organisms are available astablets. They are prepa red by the pharm aceutical and food industries, and containautochthono us intestinal microorg anisms. The primary objective of these produc ts is toachieve implantation of the bacteria in the gut during the treatment of various ailmentsand diseases. They are utilized fo r the therapy o f gastro-intestinal disturbances likediarrho ea, side effects of antibiotic and radiation therap y, chro nic constipation, chronicduodenitis, peptic ulcers in children, after irradiation therapy and as for specialpreparations for certain liver diseases. Some of the pharmaceutical products made fromferme nted milks containing viable cells of B i j i d . b i f i d um or other B i f i d obac t e r i a spp .are Bifider (Japanese) , Bifidogene (French) and Liobif (Yugoslavian). Eugalan TopferForte is a German produ ct containing 94g milk protein, 7g plant protein, 6268 lactose,59g lactulose and 30g minerals per kg; with high number of bifidobacteria. It is afat free and gluten -free preparation. Another G erman product Euga-Lein Topfercontains 18g fat, 1 51g protein, 71 lg carboh ydrate, 31g minerals, 48 g dietary fibre,and 2g vitamin C. Kurmann et al. have described Omniflora as a germanpharmaceutical preparation containing Lb . a c i d oph i l u s , B i f i d . l o ngum and a saprophyticE. co l i (138) . Th e utilization of soya milk to prepa re ferme nted soya produc ts usingB i f i d obac t e r i um spp . been described by Maslov et al. (141). This product is preparedby incubating the fermented produ ct at 37C for 16h till the lactic acid concentrationreach -50g per 1 and the viable count ranged from lo* and lo9 cfu per ml. Murtiet al. has used culture combination of yogurt strains and Bifidobacteria for the


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fermentation of cows m ilk and soya milk (142). From his results he came to theconclusions that the soya milk could be used successfully for the manufacture of avegetarian yogurt for European and North American markets and the mixed cultureof Bifidobacteria with other species could be an ideal combination.

ROLE OF TECHNOLOGYThe International Dairy Federation publishes mon ographs updating the

technological and scientific aspec ts of dairy manufacturing and International DairyFederation (143, 144) review the recent technological developments for fermented milks,hygienic design o f dairy processing equipment and hygiene m anagement in dairy plants.Over the years, equipme nt manu facturers have specifically develop ed and designedprocesses to meet the technological requirements of fermented milk products; theprocessing plants are universal. The highly sophisticate d techniques have develop ed inthis line; for exam ple the use of de-aeration equipment in a yogurt production but itis highly desired to de-aera te milk if it is fermen ted by Bifidobacteria. The presenceof oxygen will prolong the incubation period. To avoid the oxygen problem, aninteresting appr oach was applied by mixing Bifido with S. themophilus having highoxygen utilization ability. In order to keep the oxygen perme ation low glass bottlesor aluminium-laminated packa ges are used. Since polyethylene or polystyrene are notoxygen barriers they are not suitable for bifidus products. PVD C or EVO H maintaina very good oxygen barrier these materials were used for coating or laminating thepackaging materials. Strains of Bifidobacreia should be pH and heat resistant becauseof the high drying temperature used in processing for the preparation of driedfermented products. Hinterwaldner have recommended the addition of 0.1 to 0.5yeast extra ct to the production of fermen ted milk if it is proc essed by drying (145) .For spray drying the numbers of the bacterial should be higher than 10.

CURRENT AREAS OF RESEARCH AND FUTURE DEVELOPMENTA number of other possible health benefits of BIB have been postulated, with

varying deg rees of validity. T he following exam ples require rigorous resea rch befor econclusions can be drawn . The possible benefits o f the calcium compon ent of yogurtin reducing hypertension. Yam amo to et al. recently reported antihypertensive activityin spontaneously hypertensive rats given milks ferme nted by L. helveticus (146). Notall LAB tested exerted this effect. LAB may help regulate blood pressure withinnormal range. A few studies have noted an inverse relationship between theconsumption of fermented milks and the risk of breast cancer (147, 148). To ourknow ledge, no experime ntal studies have been perfo rme d. The current scientific rese archand the manufa cture of dairy produ cts as adjuncts is centered in Japan and in Euro pe.In US the poor consumer awareness of the beneficial health aspects of Bifidobacteriain dairy products has to be overcome. The possible future development has to bedone in the following areas i) selection of suitable strains for fermentation and startercultures ii) wider ap plication of bifidobacteria in the dairy industry ii) geneticmanipulation to enhance the grow th in milk and produ ce less acetic acid iv)improvem ents in designing the experim ents involving human volunteers v) increasing


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the research efforts to study the therapeutic aspects of Bifidobacterium, vi)improvedfunding for bifidobacteria resea rch and greate r collaboration between sc ientists,nutritionists and technologists in many countries. Som e preliminary studies provideexciting possibilities for future researc h, particularly in the reduction of the risk ofcolon cancer. Othe r areas of resea rch urogenital tract infections, hypertension are mo respeculative, and may provide positive p aths in the future. The mechan isms of certainactions hav e yet to be uncovered. Developm ent of validated mod el system s will helpachieve this goal more quickly. Todays consumers demand food products that not onlytaste good, but are also good for their health. As resea rch continues, strain selectionwill becom e m ore significant, and the subsequent fermen ted milks will answer m oreand more of consumers needs.

CONCLUSIONIt has been established that certain strains of bifidobacteria, which hav e been

selected for their ability to resist acid digestion and the action of bile salts, surviveintestinal transit and reach the colon in significant numbers. Thes e live bacteria havethe potential to influence the endogenous intestinal m icroflora. Altering the endogenousmicroflora may result in such physiological effects as altering enzyme activities of themicroflora , and affecting gut transit. For exam ple, it has been observe d that certaintypes of clostridia constitute a high proportion of the faecal microflora in patients withcolorectal cancer (1 49). T hus altering the profile of the intestinal microflora could havelong-term health benefits, as propo sed by Metch nikoff. While studies on the effectsof consuming live bifidobacteria are numerous, the lack of rigorous, well-structured,double-blind trials with appro priate controls ham pers the interpretation of the results.In addition, more studies a re needed to establish the mechan ism(s) by which thesebacteria exert their probiotic effects . Lactic acid bacteria in the form of ferme ntedmilks have been im plicated in the maintenance of good health and in the preventionof many disorde rs. It is clear that som e strains of LAB posse ss the ability to alterthe activity of the intestinal microflor a and thus to effect changes related to the gutYogu rt is an exellant alternative for the treatment of lactose intolerance. Billions ofpeople around the world are lactose maldigesters, and can benefit from this every daysimple traditional food.

ACKNOWLEDGMENTThe author would like to thank D r. T. R. Pate1 for reviewing this manuscript

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