Arunachalam Et Al.

Nutrition Research, Vol. 19, No. 10, pp. 1559-1597, 1999 Copyright 0 1999 Elsevier Science Inc.
Printed in the USA. All rights reserved 0271.5317/99/%x front matter
ELSEVIER
PII SO271-5317(9!9)00112-8
ROLE
OF
BIFIDOBACTERIA
IN NUTRITION,
MEDICINE
AND
TECHNOLOGY
Kantha D. Arunachalam, Ph.D. Immunology Department, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St.John s, NF, Canada AlB 3V6
ABSTRACT The purpose of this review is to discuss the relatively recent revival of interest in bacterial interference, particularly the use of Bifidobucferium spp. in nutrition, for treatment and prevention of disease in medicine and commercial food products by using recently developed technology. In 1910, Metchnikoff (1) first put forward the idea that the regular consumption of fermented milks might offer health benefits, the possible prophylactic and/or therapeutic properties of yogurt and related products have been the subject of much speculation. New fermented dairy products containing Lactobacillus species and Bifidobacten um spp. have been developed and marketed in Europe, North America and the Far East. The potential health-promoting properties of Lactobacillus acidophilus is well documented, but possible roles of ingested bifidobacteria have not been reviewed much. Bijidobucteria are normal inhabitants of the human and animal gut, and newborns are colonized within days after birth. The population seems to be relatively stable until advanced age when it has been reported to decline. Although the population of bifidobacteria in the it is influenced by diet, antibiotics, stress etc. intestine is stable, Bifidobacteria were first described by Tissier in 1899 (2) as predominant flora in breast-fed infants. This review will consider the characteristics, ecology and role in human systems, the therapeutic and prophylactic activities of BQidobacteria, and the potential pharmaceutical and fermented products manufactured using bifidobacteria and advanced technology. The facts and the results of the some of the experiments done by different authors and the current areas of research interest and future development have also been reviewed. 0 1999 Elscvla ScienceInc. KEY WORDS: Bifidobacteria, Nutrition, Medicine, Technology, Health.
Corresponding Author: K. D. Arunachalam, Ph.D. Immunology Department, Medicine, Health Sciences Centre, Memorial university of Newfoundland, St.John s, AlB 3V6. e-mail: karunach@plato.mun.ca.
Faculty of NF, Canada
1559
1560
K.D. ARUNACHALAM
INTRODUCTION The present review discusses the recent revival of interest in bacterial interference, particularly the use of Bifidobacterium spp. in nutrition, for treatment and prevention of disease in medicine and commercial food products using recent advance technology. Ever since Metchnikoff first put forward the idea that the regular consumption of fermented milks might offer health benefits, the possible prophylactic and/or therapeutic properties of yogurt and related products have been the subject of much speculation( 1). Russians have long advocated the administration of kefir and koumiss to patients with a wide range of illnesses (3), but because of the varying microbiology of these products it is difficult to confirm any theoretical basis for the claims. In US also the fermented products are promoted for good health but the poor quality control with respect to microbiological and organoleptic properties of the retail items made its claim a dispute. In recent years new fermented dairy products have been developed and marketed in Europe, North America and the Far East. The products contain Lactobacillus species and Bifidobacterium spp. at lo6 viable cells/ml of product at the time of consumption. The potential health-promoting properties of Lactobacillus acidophilus is well documented (4,5,6,7), but possible roles of ingested bifidobacteria have less reviewed. The importance of the bifidobacteria with respect to the healthy operation of the human digestive system encourage the consumption of bifidus products. This review will consider taxonomy and the characteristics , ecology and role in human systems and the potential health products manufactured using bifidobacterium. The facts and the results of the some of the experiments done by different authors and the current areas of research interest and future development have also been reviewed. WHAT ARE BIFIDOBACTERIA
Bifidobacteria spp. are normal inhabitants of the human and animal gut, and newborns are colonized within days after birth. The population seems to be relatively stable until advanced age when it has been reported to decline (8). Although the population of bifidobacteria in the intestine is stable, it is influenced by diet, antibiotics, stress etc. Bifidobacteria were first described by Tissier in 1899 as predominant flora in breast-fed infants (2). Their name comes from the observation that these gram positive rods often exist in a Y - shaped or bifid form (Figure 1). Bifidobacteria are anaerobes with special nutritional requirements, thus often difficult to isolate and grow in the laboratory. Tissier was the first one to promote the therapeutic use of bifidobacteria for infant diarrhoea by giving large doses of bifidobacteria orally. Since then much research has been undertaken in an attempt to elucidate the mechanism of action of these bacteria in the intestine. CLASSIFICATION The taxonomy They have of bifidobacteria has changed continuously since they been assigned to the genera Bacillus, Bacteroides, were first Nocardia,
isolated.
ROLE OF BIFIDOBACTERIA
1561
Lactobacillus and Corynebacterium among others, before being recognized as a separate genera in 1974 (9). Currently there are 29 species been described. These organisms were isolated from different sources such as i) the faeces of humans (infants and adults, animals, and sewage ii) bees, iii) the human vagina and iv) dental caries (pathogen). Many species are present in both animals and humans and while human studies on animal flora are less abundant. A flora have been studied extensively, recent study, using DNA-DNA hybridization to classify recent isolates, suggests that strains isolated from calves may represent a continuum between B. longum and B. infantis, two human strains (10). At present only five species of Bifidobacterium (i.e. B. adolescentis, B. bifidum, B. breve, B. infantis and B. longum) have interested the dairy manufacturers for manufacturing the therapeutic fermented milk products.
Figure
1:
Photomicrograph of Bifidobacteria Grown with China Blue. (Incubated Anaerobically
on Glucose-Blood Agar at 37C for 72 h)
Structure: The structure of Bifidobactrium spp. grown anaerobically in tripticase-phytone yeast extract stabs showed distinctive cellular shapes and arrangements. These traits are i) grouping of amphora-like cells are Bzjid. bi@dum, ii) specific epithet, thinnest and
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K.D. ARUNACHALAM
shortest cells are Bijid. breve and iii) very elongated relatively thin cells with slightly irregular contours and rare branching are Bifid. longum.,. Bijid adolescentis and infantis. The principal component of the cell wall is peptidoglycan (murein). Table 1. summarises the cell wall constituents of different species of bifidobacteria. Table 1. Principal Cell Wall Constituents of Different Species of Bifidobactria
Polysaccharides Bacteria Bifid. adolescentis Bi d. bifidum B@d. breve Bijid. infantis B@d. longum Peptidoglycan Lys-or Orn-D-Asp Orn or Lys-D-Ser-D-Asp Lys-Gly Orn or Lys-Ser-Ala-Thr-Ala Orn or Lys-Ser-Ala-Thr-Ala Galactose + + + + + Glucose + + + + + + + + + Bhamnose
The polysaccharide component like glucose, galactose and rhamnose will vary quantitatively and qualitatively among species, strain and growth medium. Myristic, palmitic, palmitoleic and oleic are the major fatty acids in the cell wall structure but it will vary depending on the growth medium, growth temperature and the presence or absence of human milk. Lipoteichoic acids help in cell adhesion to the intestine diphosphatidylglycerol and alanylphosphatidylglycerol are wall. Phosphatidylglycerol, specific to bifidobacteria (11). Fermentation of Sugars:
All Bifid. species can ferment lactose and grow well in milk. B. adolescentis, while Bifld. B. breve, B. infantis and B. longum can utilize many carbohydrates ferment glucose via bifidum can utilize fructose, galactose and lactose. Bifidobacteria the fructose 6 -phosphate shunt, which has been reported and the key enzyme involved in the hexose metabolism, fructose-6-phosphate phosphoketolase (F6PPK; EC 4.1.2.22) is present in the cellular extracts and the path way is called as bifid shunt (12,13). PHYSIOLOGY OF BIFIDOBACTERIA
Bifidobacterium 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 wide range of substrates. Bifidobacteria are anaerobic organisms,but some species can tolerate The enzymes like superoxide dismutase (EC 1.15. 1.1) and catalase (EC oxygen. 1.11.1.6), help the organism to defence against the toxic effects of superoxide and
1563
hydrogen peroxide. These enzymes are present as a very low activity in Bijid. infantis, in these strains varies breve, longum and adolescentis and the oxygen sensitivity independently of the superoxide dismutase activity (17). Shimamura et al have reported some Bifid strains utilize oxygen for that in the absence of exogenous carbohydrate, metabolizing the intercellular substrates for its survival after fermentation and ingestion (18). NADH oxidase (EC 1.6.99.3) and NADH peroxidase (EC 1. 11.1.1) are the significant enzymes involved in oxygen utilization. These enzymes catalyse the following reactions 0, -I- NADH + H+ + H,O, + NAD+ and H,Oz + NADH + H+ --, 2 H,O + NAD+ without accumulation of hydrogen peroxide. The bacterial growth will be impaired when the hydrogen peroxide gets accumulated due to disruption in the reaction. Metabolism of Carbohvdrates:
Bifidobacteria are differentiated from Lactobacillus spp. by their metabolic activity to utilize carbohydrates. Bifids use fructose 6-phosphate pathway for hexose fermentation while lactobacillus uses glucose 6-phosphate shunt (19). Madhi have reported the increase in uric and formic acids (2 pg/g) when cow and ewe s milk were fermented by Bifid. bijidum (20). Recently Biavati et al have detected low levels of glucoseBphosphate dehydrogenase and aldolase in some species of Bifidobacteria (21). Metabolism of Nitrogen:
Ammonium sulphate is used as the nitrogen source by Bifidobacteria spp. while B@d. cuniculi and Bifid. suis requires exogenous organic source (14). Matteuzzi et al have reported the production of alanine, valine, aspartate and threonine by Bifid. bifidum at in vitro conditions (22). Since milk contains limited number of free aminoacids and peptides; it has to be supplemented with a nutrient rich broth (23), casein hydrolysate (24), or yeast extract (25). Metabolism of Linids:
There is no report available on the lipid metabolism of Bifidobacterium. Mahdi in 1990 have reported the increase in total fatty acids when fermenting cow s and ewe smilk with Bijid. bifidum (20). Growth Factors:
Presence of many growth promoting factors have been reported in Bifidobacteria in vitro (26, 27). A bifidogenic growth stimulator for Bifid. adolescentis 6003 have been identified by Kaneko et al in 1994 in the cell free filtrate of Propionibacterium freudenreichii 7025 and in the methanol extract fraction of the cells (28); however,
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K.D. ARUNACHALAM
short-chain fatty acids (formate, acetate, propionate and butyrate also stimulated the growth of bifidobacteria. Bifidus factor was first described by Gyijrgy et al as a part of milk and colostrum and made up of glycoproteins (29). It can be separated in to two factors by isocratic HPLC (30) and the glyco moiety contains Nacetylglucosamine. Bezkorovainy et al have described the promoters as human casein or its trypsin derivative (31). Glycomacropeptide which is the resulting product of trypsin hydrolysis of human k-casein contains active sugars like galatosamine and glucosamine. Azuma et al. found that the chymotrypsin hydrolysis product of human k-casein is more effective in promoting the growth of Bifid. bifidum than the trypsin derivative (32). The sugar and polypeptide component are responsible for the promoting activity of the bifidus factor. Allen have reported the presence of bifidus factor in mucus secretion of salivary glands, small intestine and colon (33). The major components of this factor is also glycoproteins with N-acetylgalactosamine, Nacytylglucosamine and sialic acid. Gyorgy et al have identified the Bifidus factor 2 as a non-glycosylated peptides of casein (34). It is a byproduct of proteinase hydrolysis, found to be effective in milk and whey. Synthesis of Vitamins:
Bifidobacterium strains isolated from humans need vitamins like thiamin (Bl), pyridoxine (Be), folic acid (B,) and cyanocobalamin (B,J for its growth. But the same organisms can synthesize B vitamins. Deguchi et al. have listed (Table 2) the following B vitamins synthesized by different Bifidobacteria (35).
Table
2. Synthesis
of Vitamin
by Different
Bifidobacteria spp.
Microorganism Vitamin pg ml- Thiamin Folic acid Pyridoxine Nicotine Cyanocobalamin Ascorbic acid Biotin Riboflavin (35) 2 (14)
Bifid. adolescentis 0.02 0.01 0.043 0.17 0.35

I.C.
Bifid. bifidum 0.23 0.058 0.046 1.04 0.65 n.s. n.s. ns.
Bijid. breve 0.09 0.008 0.02 0.39 0.49 l.c. l.c.. n.s.
Bijid. infantis 0.2 0.040 0.059 1.23 0.39

1.C
Bifid. longum 0.09 0.02 0.42 0.61 0.46 l.c. l.c. n.s.
l.c. n.s
1.c n.s.
l.c. low concentration,
n.s. not synthesized.
Table Source with different 40 It is a selective medium for Bifidobacteria from human application needs further investigation because of the inhibitors. origin. Its Suggestion
3: Media
Used
for Enumeration
of BiJidobacterium
spp.
Media
Reinforced clostridial agar(RCA) inhibitory components. chloride 41
fortified
Galactose
agar with 0.4 g/l of lithium
The viable bifidobacterial count ranged from lo4 to 10 &/ml. In Europe five strains were isolated from eight commercial products. but only one was identified as Bitid.longum and others were classified as animalis. This is a selective medium for the enumeration of Bifidobacteria from dairy products. Arabinose is used only for Bitid.longum and adolescentis.
Transgalactosylated oligosaccharide (TOS) agar medium with NPNL. Milieu Selectif Bitidobacterium spp. (MSB) agar or arabinose agar (instead of TOS) 43
42
1OOmg/l neomycin MRS agar supplemented with sulphate, 15 mg/l nalidixic acid, and 3g/l lithium chloride (NNL) solution choridate 44 Selective medium milk products. for the enumeration
This is a selective medium using the oxygen reducing membrane fraction (ORMF). The plates will be incubated aerobically and there was no difference in the bacterial counts. of Bifidobacterium from fermented
MRS agar with added dicloxacillin
and cystein
Trypticase-phytone-yeast medium dicloxacillin. Better medium than agar for Bifids. with (LP 46
with 2pg/ml of Mann-Rogosa-Sharpe
45
Human strains of Bifidobacteria grow well. Pure strains were isolated from the commercial nroducts like Otilus, Bifidus yogurt, and Fromage _ bitidus. The growth of Lactobacilli and Streptococci will be inhibited. _ Selective medium for commercial yogurt samples. All the strains of Bitidobacteria of dairy products grew in this medium. Growth of other yogurt bacteria like Lactobacillus and Streptococcus were inhibited. It is non cultures. selective media for enumerating Bitidobacteria from yogurt
Modified liver-cystine-lactose agar supplemented 2gil lithium chloride and 3gil sodium propionate
agar) 47
Rogosa medium with 40pg/ml 5-bromo-4-chloro-3-indolB-D-glucopyranoside known as X-Glu agar. lithium sodium 48
Modified chloride, propionate agar. 49
VF Bouillion agar with 0.5mg/l 20pglml lam-y1 sulphate, 5 mgil and lOpg/ml neomycin sulphate.
This medium is used for enumerating viable cells from dried probiotic products. It is used in combination with triple layer diffusion technique to selectively enumerate Bitid.bifidum. Yogurt distinct cultures and BiIid. colony morphology. bifidum could be enumerated by their
Reinforce
clostridial
Prussian
blue (RCPB)
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K.D. ARUNACHALAM
ENUMERATION
OF BIFIDOFACTERIA
Two factors are important in the cultivation of bifidobacteria: an adequate culture medium and anaerobic conditions. The traditional technique is easy to use and it contains 0.1% yeast extract or corn steap liquor yields viable cell counts of Bifidobacteria equivalent to fermented milk (36). The following Table 3 describes some dairy selective media for the enumeration of Bifidobacterium spp. from fermented products. The synthesis of B vitamins by bifidobacteria enhances the nutritional quality of the fermented milk product. But whether this improves the bioavailability of these vitamins in the human stomach needs more evidence and research. Acidified media such as MRS, Rogosa are extensively been used to enumerate bifidobacteria from mixed cultures (37). But it is evident that more research is needed in formulating the selective media which is easy to prepare, inexpensive and be able to recover more than 95% of BQidobacteria ipp. _ _
Structure
of the mtestinal mlcroflora 7
Functions I
of the intestinal mw9lora , Useful
Effects
on the host \
CONItS
groups Backnodes Eubactcrium Anaerobic Synthesize vitamins/protems Aid digestion/absorption Suppress external bacteria Stimulate unmune function
(per g
.y
.j
5
faeces i
log10
Streptococcus
-1
Maintam
health
Bafidobactcnum
Produce
toxins
Cl. I? 3 s B , I
perfringens lHypertenrion
Sraphylococcus O-IO Proleus Ps. aeruglnosa
Diarrhoea. gastroceteritis infections. (meningitis, cndocarditis. septicaemia. urinary tract infection, brain abscesses, liver abscesses, lung abscesses)
Figure
2. Host Relationships BIFIDOBACTEIA
of the Intestinal AS PROBIOTICS
Bacteria
Fuller
defined
probiotics
as a live microbial
feed supplement
which beneficially
1567
affects the host animal by improving its intestinal microbial balance (38). Bifidobacteria have been called resistance factors by Homma (39). A resistance factor is described as a substance which carries out certain physiological functions but which, when lacking in a healthy person in optimum condition has no adverse effect. However, once a person is affected by outside forces, which is essentially always, the lack of this resistance factor causes negative changes in the body, due to the body s decreased ability to respond to the outside challenge. The host relationships of the intestinal bacteria are shown in Figure 2. The way the probiotics work is not known, but our knowledge of gut microbiology suggests four ways in which they may be operating. They are i) production of antimicrobial substances (50, 51) ii) competition for adhesion receptors (52, 53, 54) iii) competition for nutrients with pathogens (55), iv) stimulating immunity (56). There are many claims for beneficial effects of probiotics in constipation, cancer, heart disease and ulcerative colitis but the evidences are from animals. Table 4 and 5 summarize some of the stated beneficial effects of probiotics use in both animals and human. Table 4. Beneficial Effects of Probiotics (57).
Animals Effect on indigenous microbes: Noninfectious disbacteriosis after high dosages of antibiotics
Humans Noninfectious disbacteriosis after high dosages of antibiotics and after radiation therapy. In Neonate intensive care unit Health promotion Inhibition of carcinogenesis Anticholesteremic effect High calcium resorption Destruction of ANFs Vitamin synthesis Protein predigestion
Stress (eg. feed change, transport) Deficiency in the development of microflora Growth promotion Higher feed conversion Antinutritional factors(ANFs) destruction Vitamin synthesis Protein predigestion
The two notable evidences from humans are the effect on lactase deficiency (68) and on Pseudomembranous colitis (69). As seen from Table 5, bifidobacteria have been reported to be responsible for many different effects. These range from increased production of secretory IgA, to altering the composition of the intestinal flora. Although several studies have been performed in humans, most of these were carried out on a limited number of subjects and some were without controls.
Table 5. Experimental
Results Showing the Effect of Bifidobacteria.
Organism in vitro (phagocyte) in vitro in vivo (mice) in vivo ( 15 humans) in vivo (15 humans) in vivo (5 humans) in vivo (mice) in vivo (humans) in vivo (10 humans) in vivo (6 humans) in vivo (humans) in vivo (48 humans) in vivo (60 humans) fermented milk fermented milk fermented milk fermented milk fermented milk fermented milk fermented milk lyophilized bacteria -stool frequency in intractable diarrhea :W ,, i;k,,,_ ~~~~ronidase in feLes* -pH in faeces* + life span* -creatinine* + stool frequency elderly) + (in bedridden -erythromycin -induced gastrointestinal effects -nitroreductase -glucuronidase survival during intestinal transit -population clostridia, bacteroides, coliforms -colonic transit time
??
Control Reference 58 58 antibody* 58 59 60 61 lyophilized heatkilled cells + secretory IgA water suspension (stomach tube) lyophilized -chronic inflamation of sigmoid colon + humoral immunity* + antigen-specific (cholera toxm) + IL- 1 production + cytotoxicity
Test
Form
Effect
B. longum BL 2928
latex
B. breve 4064
B. breve Ka-6
B. breve 4064
B. breve Ka-6
B. bijidum
placebo
Bijidobacterium SPP.
B. longum
same ersons before and a Rer bifidus
B. longum
Skim milk and yogurt
62 63 60 64 61, 65 66 67
Bifidobacterium same ersons before and a Rer bifidus fPP.
B. longum
Yogurt without bifidobacteria
Bi$dobacterium SPP.
same ersons before and a Ker bifidus
Bijidobacterium SPP.
;sym
B.longum ATCC 15707
Bijidobacterium asteurized bifidus Permented milk spp.
1569
SELECTING
BIFIDOBACTERIA
AS STARTER
CULTURES
Starter cultures refer to the specific Bifidobacteria (BIB)which are used to inoculate milk and whose metabolism leads to the fermented products. There are two types of starter cultures. The first one is the traditional method which contains complex, poorly defined mixtures of bacterial strains. The second one is selected or definedculture which contains one or more identified pure cultures with known specific properties. Specific properties means Bifidobacteria s rate of acid production, type of polysaccharide fermentation, proteolysis characteristics (which sometimes leads to bitter compounds) and their capacity to produce flavour compounds. Another criterion is based on the Bifidobacteria s ability to synthesis vitamins and increase the nutritional value and health properties of the fermented products. For example in certain countries the Bifidobacteria is added as the starter culture because of its possible beneficial effects in the gastrointestinal tract or to improve the vitamin profile (70, 71). Traditional bulk starter milk is prepared by reconstituting antibiotic-free, skimmed milk powder (total solid lOO-12Og/l) and heating to 90 -95C for 15 to 30 min which would destroy the native microbes in the milk, inactivate the natural inhibitors, modify the proteins, and reduce the dissolved oxygen. All these change would help to provide the right growth conditions for Bijidobacterium spp. However a growth stimulatory factor(s) will be added to the bulk starter milk. Commercially produced Bifidobacteria starter cultures are more consistent than the home produced liquid cultures. Commercial cultures can be bought in concentrated freeze - dried form which can be stored at < 5C or at -20C or as concentrated cultures deep frozen at -190C; which can be stored at -40 to -80C. The average count is 1 lOi cfu/g and the bacterial cells are concentrated to achieve these numbers and the activity of these cultures depends on the survival rate of the organisms. BIFIDOBACTERIA Stomach and Intestinal Transit: AND HUMAN GIT
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. adolescentis, infants infantis, and longum. As seen from Figure 3 the digestive tract of newly-born rapidly colonized bacteria from the vagina during birth (74) and within 48 hours of birth, the stools of infants may contain up to 4.0 x 10 cfu/g (75). Yuhara et al. have reported that in breast fed infants bifidobacterium species as the most frequently isolated species (76). The human small intestine contains approximately lo5 bacterial cells per gram while the colon contains 10 bacterial cells per gram of contents. It has been suggested that for bacteria to act as probiotics they must arrive in the intestines alive and in sufficient number lo6 cells/g, (37) or lo7 cells/g (64) to have an effect per se or they should adhere or implant and multiply (Figure 4) (77). Thus for the bifidobacteria in a fermented milk to have an effect, they must resist the acidic conditions of the stomach for 90 minutes (an average time for gastric emptying of dairy products), and be resistant to bile in order to transit the small
1570
K.D. ARUNACHALAM
intestine. Berrada observed that the strain of bifidobacteria in one commercial fermented milk product had a survival rate after in vitro incubation at pH 3 for 3 hours whereas the strain in another commercial product dit not. This same survival pattern was subsequently confirmed in humans (78). Bifidobacteria do survive intestinal transit in high numbers, although not all strains are equally resistant (79, 80, 81). One study, using a marked strain, has shown that a bifidobacterial strain present in fermented milk could be recovered at a rate of 30 % from the faeces of human volunteers. This agrees with the 23.5 % to 37.5 % recovery rate of bifidobacteria from the illeum of other volunteers (79, 81).
11 10
-1 qa ;
,
,
: / t
I
x-K:>x : f-- , -a\

I ,
Bifidobacteria
t-y
\\
x
0.
/I /
x-x-x
e__
__ _e----.
Escherichia coli Enterococci Lactococci
,&__-,-
-a-
__--
&-.
- g ,_a/. &
I
i;
I ; I
-=-& _*
.X,
./
o_.-.
-Ip\
N.\.-.--o
Staphylococci Clostridia
Days of age
Figure 3. Changes in the Intestinal Microflora in Babies from Birth to 7 Days.
Effects
on Gut Flora:
It has been observed that feeding bifidobacteria fermented milk alters the intestinal population, as determined by faecal enumerations (61, 66, 81). In one study the faecal bifidobacterial recovery was temporarily increased by a factor of 20 (81),
1571
while
in another
the increase
was by a factor
of 8000 (66).
Bacteriodes Eubacterium Anaerobic streptococcus
Bifidobacteria
C. perfringens
-+ \ / --
Birth
Figure
Weaning
4: Changes in the Intestinal
Maturity
Microflora with
Old age
Age.
suggests that consumption of 5. lo9 Another recent study with humans bifidobacteria 3 times daily for 5 weeks in the form of lyophilized powder alters the profile of the faecal flora (61). In this study the relative proportion of bifidobacteria increased while the proportion of bacteroides and clostridia decreased. In addition, a significant reduction in faecal pH value was observed at the end of the study. The mechanism of action by which bifidobacteria affect the gut microflora remains to be elucidated. In a study with labelled bifidobacteria, it was determined that the increase was due to the ingested bacteria, whereas the authors of another study attribute this increase to implantation. One recent study has shown that bifidobacteria, given orally to humans, are unable to implant in the gut even under conditions which favour that the increased population of colonization (65). Other studies have shown bifidobacteria in the faeces observed while feeding bifidobacteria-fermented milk returned to pre-intervention levels within 1 to 2 weeks when the product was no longer fed.
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K.D. ARUNACHALAM
Although the mechanism by which bifidobacteria may alter the intestinal microflora has not been clearly determined, it is possibly related to the production of acetic acid and lactic acid which may restrict the growth of potential pathogenic and putrefactive bacteria (82). That acetic acid is more bacteriostatic than lactic acid at the same pH may explain a greater effect of bifidobacteria compared to certain other bacteria, such as those found in yogurt, which produce only lactic acid. Phvsiological Effects in GIT:
The microflora effects have been more clearly demonstrated, both in animals and humans. These fall into two types of categories, effects on the gut microflora, as measured indirectly by gut transit (60, 63, 67, 83) or measured directly by faecal enumerations and enzyme activities such as P-glucuronidase and nitroreductase (62, 84). Although numerous articles cite the in vivo effects in both animals or humans following ingestion of large numbers of live bifidobacteria, few double-blind studies have been sufficiently large enough and with appropriate controls to demonstrate statistical differences. In one recent study, using 60 healthy volunteers, it was observed that feeding 125 g of fermented milk containing bifidobacteria, 3 times per day, reduced intestinal transit times, but still within normal physiological limits (67). This effect in the sigmoid colon was statistically significant for women on a normal diet. Transit time for the colon in women tend to be slower than in men. It is possible that this physiological effect was mediated through a change in the intestinal flora effected by feeding large numbers of bifidobacteria. Ecologv and Identification of Svecies in GIT:
The colonization of the colon and the species composition depends on the hygiene in the delivery area and/or method of birth that is natural or caesarean. The differences in morphological and biochemical variations exists among Bifidobacteria make identification to species level difficult. The walls of the colon provide a special ecological niche in which bifidobacteria can proliferate at the expense of other groups. The strains of B@d. bifdum and infantis can metabolize the oligosaccharides found in the mucilage secreted by the cell walls of the colon, and hence the mucin tends to have a selective influence on the microflora (85). With respect to the actual species involved, some strain like Bijid. infantis secretes the polysaccharides which initiates adhesion to the epithelial cells of the intestine. The binding of lipoteichoic acids of Bifidobacteria to human epithelial cells depend upon the cell concentration and the The number of cells detected in the stools may not reflect the contact time. population in the intestine because the cells may be sent out from the body before adhesion. Guerina and Neutra have reported that some times the host cell may grow around the bacterial cell as a membrane and host bacterium relationship is created which 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 fatty acid composition of the lipoteichoic acid, higher the fatty acid the better the adhesion (86).
1573
II 10 Anaerobtc strepococcus Bibidobacterium
Streptococcus E. coli Lactobacillus
---+----iIVeillonella C. perfringens
2 Points Length Time taken for food to arrive -
1 5
2 Y 65 cm--w __c Stomach Duodenum : 3-5 hr 5-6 m Jejunum Ileum -1.5 * 4h-r6 Caecum Rectum m 24-72 hr
I-60 ---) s
Figure (Cu
5: Variations of Bacterial Populations and Species along the Gastrointestinal Tract in Human Adult under Non-fasting Conditions. = Colony forming unit, s= second, h=hour, cm= centimeter, m = meter)
Age
and
Feeding
Regimen
The 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 decrease with age. In adults bacteroides are the major group with Bifld. adolescentis, longum and bijidum. The abundance of bifidobacterial species depends on the healthy function of the colon. Eventhough numerous reports are existing about the general trend that the colons of breast-fed infants have higher levels of bifidobacteria than the bottle-fed infantswhether or not the species frequency changes with the type of feed in not clear. Ballongue suggested that Bifid. adolescentis was the dominant organism in bottle fed neonates (14) and Beerens found Bifid. longum as the frequently isolated species from the stools of formulated infants (87) and on the contrary Biavati et al. isolated mixed populations, namely Bijid. bifidum, infantis, longum and breve, (88) irrespective of the type of milk. There were some suggestions that the difference between the
1574
K.D. ARUNACHALAM
resultant microfloras may be due to the absence of specific stimulatory factors in commercial feeds. Even the variations in the human milk from mother to mother also affect the intestinal microbial flora of the infants. Figure 5 shows the dramatic variations in the total population and species along the GIT, with the highest concentrations in the colon (89). There is a wide variation of bacteria among individuals, but globally the number of species and the population of bacteria are relatively stable in healthy adults. In addition to the normal microflora (resident microflora) already established in the GIT, bacteria are introduced into the body as a normal part of food (transient or as contaminants (accidental microflora). Transient bacteria have a microflora), greater effect on the upper GIT than on the colon because of their lower numbers of resident microorganisms. Ingestion of the LAB found in fermented milks poses no health problems for the general population (90). In spite of its stability, the intestinal microflora can be affected by a variety of factors. Host factors, such as gastric acid, wall can affect the composition of the bile salts, and mucus in the intestinal microflora. In addition, diet, medication, infections, stress, aging, and climate can alter the microflora. Bacterial interactions such as antagonism or symbiosis can also influence the contents of the microflora. The intestinal microflora is capable of adapting to and metabolizing most of the substances that enter the intestines from the oral tract, or through the biliary tract, or compounds which are secreted directly into the intestines. Adaptation can occur within several days.
BIB in Prevention Imnroved Protein
of Intestinal Metabolism:
Disorder:
Bifidobacteria have phosphoprotein phosphatase activity which helps in increase absorption of human milk protein by breaking down the casein in human milk. This is thought to contribute to the satisfactory absorption of human milk (91). Nitrogen retention is good in infants with a bifidus microflora; bifidobacteria promotes the aminoacids metabolism, but the mechanism is not clear. One of the roles which bifidobacteria fulfil in the intestinal tract of the infants is to suppress the multiplication of putrefactive bacteria thereby stopping losses of nutrients. Imoroved Vitamin Metabolism
Bifidobacteria are predominant in the intestinal microflora of healthy people irrespective of age and the vitamins produced by them needs warrant attention. The values reported for vitamins produced by bifidobacteria are : vitamin B, 7.5 pg and B, 25 pg per g dry weight for intracellular bacterial vitamins, and B, 25 - 25Opg, B, lOOpg, B,, O.O6pg, nicotinic acid 400 pg, and folic acid 25 pg per litre of medium for vitamins produced outside the bacterial cells. It is known that BuciZZus thiamonolyticus inhabiting the intestinal tract break down the vitamin B, and cause B,
1575
deficiency. In such cases oral administration of vitamin B, is not effective (92). So the oral administration of bifidobacterial preparations are suggested to increase the intestinal microflora and to suppress the thiaminolytic bacteria. With bifidus microflora it would also be enable the beneficial utilization of the extracelluar vitamin B, produced by the bifidobacteria. BIB in Prevention of Constination:
Constipation can be defined as the failure to defecate for 3 or 4 days and when combined with pain and treatment it becomes a illness. Constipation is caused by the poor quality of food, regularity in taking food, and the mortality of the digestive tract microflora. The organic acids produced by bifidobacteria are thought to stimulate the intestinal peristalsis and help in normal bowel movement. Bifidus preparations for constipation sufferers and people with irregular bowel movements have been on sale for years. Doerbeck et al. reported an improvement in bowel movements and a simultaneous increase in bifidobacterial count in the faeces when they give milk containing bifidobacteria to constipation patients (93). Administration of bifidobacteria also leads to a higher moisture content in faeces and Tanaka and Shimosaka reported this as a factor in the beneficial effect (63). BIB and Antibiotic Action:
In vitro, bifidobacteria have been noted to have antibacterial activity against pathogenic E. Coli, Staphylococcus aureus, Shigella dysenteriae, Salmonella typhi, Proteus spp. and Candida albicans. The antibacterial action shown by bifidobacteria is from the organic acids they produce. Bifidobacteria make 1 mol lactic acid, 1.5 mol acetic acid and small amount of formic acid from 1 mol of glucose. Rasic and Kurmann reported that the intensity of the antibiotic action varies with acids: for example the minimum pH at which Salmonella spp. can grow is 5.4 for acetic acid, 4.4 for lactic acid and 4.05 for citric/ hydrochloric acid (11). Anand et al. have isolated an antibiotic called Bifidin from Bijid. bifidum 1452. Bifidin is stable to heating 100C for 30 min; it gives a positive ninhydrin reaction, and its main components are phenylalanine and glutamic acid. It shows antibacterial activity against Micrococcus jlavus and Staphyloccoccus aureus, by being active at pH 4.8 to 5.5 (94). Ferrari et al. have shown that the bifidobacterial cells breakdown the conjugated bile acids to free bile acids which intern inhibit the growth of pathogens (95). BIB in the Treatment of Liver Damape:
Disturbances to liver function cause metabolic abnormalities, with ill effects from ammonia and amines, the products of putrefactive bacteria. Muting et al. reported that when they gave bifidus milk for a long period to patients with hepatitis or cirrhosis of the liver, blood ammonia, phenol and urinary indican decreased significantly as bifidobacteria in faeces increased, and the patients showed increased appetite and gained weight (96). The dietary administration of Bijid. bi$dum together with lactulose assist in re-establishing the normal gut flora in advanced cirrhosis. The re-establishment of
1576 the normal microbial phenols in the blood balance (Figure
K.D. ARUNACHALAM
is accompanied 6).
by
a reduction
of ammonia
and
free
Blood 3x70 3*too /ng/ 100 ml wmw 250,
Ammonia 3s70 3#70 3 ~roog
A bifidus-milk
preparation
Free mg/KXIml
Serum
Phenols
8. bifidum :;j
in the
faeces
21.12 26.1 7.3 11.5 21.7 13.12 5.1 10.2 23.3 2.6 30.8 1965 1966
10.3 23.5 14.2 18.4 20.7 1967
Figure
6:
Long Term Treatment of Decomposed Liver Cirrhosis Preparation Eugalan Forte in 12 Year Old Patient
with Bifidus
Milk
1577
A normalization in protein metabolism has occured and it allows the patient to increase protein intake (to 70 g per day). The ability of bifidobacteria to utilize ammonia 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 is adjusted so as to induce two soft stools a day, under which conditions the faecal pH reaches 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). Muting et al. have proven the beneficial effect of bifidus milk for protein metabolism in liver cirrhosis (96). It is important to use non -urease producing strains of B. bifidum in the manufacture of bifidus milk preparations. However, if lactulose and/or bifidus milk fails to reduce blood ammonia in portal encephalopathy then neomycin should be temporarily 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 in the prevention of cancer initiation. Studies first showed a slower growth of certain experimental cancers in animals (98). B. bifidum (99), have been shown in human clinical studies to reduce the levels of some colonic enzymes (/I-glucuronidase, nitroreductase, azoreductase, and glycoholic acid hydrolase), which are implicated in the conversion of procarcinogens to carcinogens (100, 101) such as nitrosamines or secondary bile salts (102). Most studies report a decrease in these enzymes during the study period when live BIB were consumed with a return to baseline levels during follow-up when no BIB were consumed. The mechanisms and long-term effects of these changes are not clear. Recent epidemiological studies (103, 104) have found that colon cancer risk was inversely related to the consumption of diets which included fermented milks. Other results (105) show no relation to colorectal cancer risk. Conclusions for the reduction of risk of colon cancer can not yet be extrapolated from these preliminary results. Other dietary factors have been considered in the prevention of colon cancer, including fibre and calcium fermented milks may be one factor of many that affect risk of colon cancer. BIB and Antitumour Activitv:
Studies with laboratory animals have indicated antitumour effect of bifidobacteria. No significant difference in tumour size was observed when meth-a ascites tumour cell (25 x 103) were transplanted and a suspension of Bifid. infantis (10 cells)was intraregionally injected in to BALB/c mice. The antitumour effect decreased when the first bacterial injection started 3 or 7 days after tumour inoculation, or when the mice were inoculated with a large number (500~10~) of tumour cells. The stimulation of the host immune response has been suggested to be involved in the antitumour effect of Bifidobacteria (106). Liver tumorigenesis has been reported to be reduced by the presence of Bifid. longum in the gut of gnotobiotics C3H/He male mice (107) due to the stimulation of immune response of the host, and decrease in the faecal bacterial enzymes by bifidobacteria; but more evidence is needed. Studies have shown that
1578
K.D. ARUNACHALAM
chemically induced colon tumorigenesis The results of DMH- induced tumours Table 6 (108) Table Group 6. Incidence of DMH-induced Rats with tumours 9 6 9 7
may be inhibited by feeding with sour milk. in rats fed with different diet is given in the
Tumours
in Rats Fed
with
Different
Diets.
No. of rats
No. of colon tumours per rat Total 2.6 1.0 3.4 2.3 Adenoma 1.3 0.4 2.0 1.8 Carcinoma 1.3 0.6 1.4 0.5
Control Sour milk Acidified milk Starter cells
9 9 9 8
The anti tumour effect may be explained by the beneficial activities of bifidobacteria present in increasing numbers in the intestine of rats fed with sour milk.Feeding studies have shown that the life span of mice fed with 14 % sour milk was, on average 8% longer than control diet and the bifidobacterial count was 10 times higher than the control diet. But the potential mechanism for antitumour activity needs further research. BIB and the Host Immune Svstem
Current evidence suggests that microorganisms indigenous to the gastrointestinal tract are less immunogenic in their hosts than allochthonous microorganisms of their similar types (109). Starter bacteria provide a significant source of antigens, which of viable and non induce some immunological activity in their hosts. Administration viable mixtures of various stains of intestinal bacteria to germ free mice has shown that intestinal production of immunoglobulin A (IgA) plasmacytes was greater than that following the implantation of single strain or species. Lactic acid bacteria capable of establishing in the intestinal tract were less immunogenic than the transitory strains. Perdigon and Alavarez have recently summerised the very active and interesting research developing in the field immunomodulatory effect of lactic acid bacteria (110). The effects of ingested lactic acid bacteria with the immune system first start in the gut associated lymphoid tissue (GALT). Few studies have been reported on the effect of Bifidobacterium on GALT and none of the on human. In mice peridigon et al. (111) showed that oar1 administration of L. casei, L.acidophilus, L. bulgaricus and S. thermophilus increased the levels of immunoglobulins in the intestinal fluid. However, only L. casei pretreated mice had increased secretion of specific antibodies against Salmonellae when challenged with Salmonellae (111). A recent study of the comparative effects of diets, enriched
1579
respectively with live and heat treated yogurt on the response of the murine immunosystem has shown that large inocula stimulate the immunological activity of the spleen and thymus gland. Live bacteria were more effective than dead ones in increasing the serum imrnunoglobulin G (IgG& (112). Supplementary feeding with yogurt stimulated the enlargement of lymph nodes in the spleen and the number of B lymphocytes associated with humoral immunity and T lymphocytes associated with cell-mediated immunity were increased significantly ( 113). Experiments with germ-free mice monoassociated with Bi d. longum shown antilethal effect and cell-mediated immunity against invading E. Coli (114). Subsequent studies with germ free mice monoassociated with B@d. longum have indicated the production of anti-bifid. longum IgA and cell-mediated immunity. These data support the speculation that ingestion of fermented milks containing large numbers of viable bifidobacteria and lactic acid bacteria may induce an immunological response in humans. However, the effect of starter bacteria on the immunosystem of their hosts require further investigation. As shown in Table 5, recently published findings with Bifidobacteria fall in two general categories, immunological and microflora effects (115) and the immunological effects have been clearly demonstrated invitro (58) and a significant increase in humoral immunity was observed in a study involving 15 human volunteers (59) BIB Do They Lower Blood Cholesterol Levels?
Since Mann and Spoerry s observation in 1974 that East African Maasai warriors who consume four liters per day of fermented milk had low blood levels of serum cholesterol, and that, when intake increased to eight liters per day, they had additional decrease in levels (116), many studies have been conducted to find a hypocholesterolemic factor in fermented milks. Hydroxymethyl glutarate has been suggested as active factor (117). The results of this research is given in Table 7. Table 7. Influence of Yoghurt in Human Serum Cholesterol (mg/dl) Significant No Yes Yes reduction Level (117).
Serum Cholesterol Product Whole Milk Yogurt from Whole milk Yogurt from Skim Milk Before 196 193 211 After 177 175 150
This factor inhibits cholesterol synthesis in the body resulting in reduced serum cholesterol levels. Results are difficult to interpret because of confounding variables, such as exercise and dietary patterns and varying levels of initial serum cholesterol. Thakur and Jha showed that rabbits fed a high cholesterol diet supplemented with yogurt exhibited lower serum cholesterol levels than did rabbits on the non supplemented control diet (118). The effects of BIB on cholesterol levels are therefore
1580
K.D. ARUNACHALAM
inconsistent, and range from a significant reduction to no reduction, with intermediate results of transient effects and positive outcomes only in women (119). The exact mechanism is unknown. Most of the research have used L. acidophilus; other possibilities have included L. casei, L. plantarum, Bifidobacterium spp. (120) and Enterococcus fuecium (121,122). There is concern that consuming yogurt on a regular basis may increase serum cholesterol because of the milk fat. Halpem et al. and McNamara 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, there were no deleterious effects on serum cholesterol, triglycerides, HDL or LDL levels. Yogurt can be consumed regularly without concern for raising cholesterol levels (123, 124). Consumption of yogurt itself may not help in controlling cholesterol but some factor produced by the yogurt bacteria during fermentation of the milk is responsible. So, there may be some ways to concentrate the active factor(s) into usable volume for practical use. Additional research is certainly needed in order to clarify the possible hypocholesterplaemic effect of cultured yogurt. BIB in the Treatment of Urogenital Infections:
The predominant microorganism in the normal microflora of the female urinary tract is lactobacilli, in particular, L. acidophilus. Hormonal changes, age, and menopause can alter the flora. During urinary tract infections, pathogens such as Cundida albicuns (a yeast responsible for candidiasis) and E. coli, travel from the colon to the urethra and the bladder. A recent study by Hilton et al. suggests that daily oral ingestion of 227 gm (8 ounces) of yogurt containing L. acidophilus may be useful in reducing the recurrence of candidal vaginal infections (125). Lactic acid space and habitat and reduce its bacteria compete with uropathogens for nutrition, pathogenicity. The mechanisms been suggested was coaggregation of the pathogen with lactobacilli BiJid. fragilis. In the small prospective cross-over study, women consuming yogurt experienced fewer recurrences of vaginal candidiasis than during the control period (126). Further research is necessary. BIB and Possible Adverse Effects:
Some strains of Bifidobacteria have been implicated in possible adverse effects for the host, when found in the soft tissues. They usually occur in mixed populations along with facultative anaerobic bacteria (81). Subsequently these strains have been identified as Bi d. dentium in dental caries and other clinical materials including pleural fluid (127), suggesting its possible harmful potentiality. THERAPEUTIC AND PROPHYLACTIC ACTIVITIES OF BIFIDOBACTERIA
Certain antibiotic, irradiation of the abdomen with gamma or X-rays, and stress conditions may also disturb the the balance of intestinal microflora. The consumption of fermented milk containing bifidobacterial species like Bifid. longum, and bifidum helped in the restoration of the microflora after oral antibiotic therapy. Korshunov et al. have rapidly restored the decreased population of bifidobacteria due to the oral
1581
administration of kanamycin 40 mg/kg to adult humans by three oral administration of autostrains of bifidobacteria and lactobacilli (128). A disturbed balance of intestinal microflora in leukaemia patients been improved by oral administration of bifidobacteria and acidophilus bacteria (129). The results of the treatment are given in Table 8.
Table
8. Indicates
the Changes in Minor during Antileukaemic
Members of Intestinal Therapy with antileukaemic (56 cases)
Bacteria
Treatment Organism Control (locases)
drug
Without Bifidobacteria (28 cases) in % 3 (10.7) 6 (21.4) 8 (28.6)
With Bifidobacteria (28 cases) in % 2 (7.1) 5 (17.9) 2 (7.1)
Klebsiella sp. Citrobacter sp. Proteus vulgar-is
0 0 0
Cases in which more than lo6 colonies were observed
The antagonistic effect of bifidobacteria against many undesirable microbes may be due to the acetic and lactic acids they produce and possibly, to their effect on the et al have shown that the oral dietary immunosystem of the host. Schneegan with enteric administration of a freeze dried cultures of B. bijidum to children infections, could eradicate enteropathogenic E. Coli strains in about 60 % of the cases, and more than 80% when lactulose was included (130). Bifid. b@dum, together with lactulose, has also shown to assist in reestablishing the balance of intestinal microflora which is disturbed in liver cirrhosis, and this is accompanied by low faecal pH and low ammonia and phenol levels in the blood (97). To consider bifidobacteria to be the major group in the colon then the following possible routes should be encouraged i) feeding the subject with a diet that will stimulate his or her native microflora, ii) consumption of dairy products manufactured with bifidobacteria (102), iii) the use of suppository containing viable cells of bifidobacteria. However, the position with foods containing bifidobacteria may be different and in assessing the potential advantages of consuming bioactive milks or yoghurts, it has to be assumed that i) consumption will be on a regular basis, at a level of some 400-500g product/week, ii) the dairy product should contain minimum of 1.0~10~ cfu/g of product at the time of consumption (131) iii) the origin of Bifidobacterium should be of human origin so that it can with stand the intestinal tract transit. If these conditions are met then there is a clinical evidence that support that the ingested bacteria may i) stimulate the existing population of bifidobacteria ii) act in conjunction with the native flora and iii) replace the disturbed microflora (132). The potential
1582
K.D. ARUNACHALAM
benefits for healthy subjects is more Bifid. b@dum by healthy infants did infections (133). The increased sales of America indicates that the consumers loyalty without any genuine belief prophylactic properties.
difficult to predict, however regular intake of provide a degree of protection against enteric biomilks and bio-yoghurts in Europe and North have acquired the interest in these products or understanding in its therapeutic and or
BIFIDOBACTERIAL
PRODUCTS
Large number of bifidobacterial products are available in the market. Reuter conducted a survey of fermented milk products containing Bifidobacteria in Germany, Japan, and France and found out that Bifid. longum is widely used in Germany (134). The most suitable combination is B@d. longum and Str. thermophilus. The selection of commercial strain is important that it has a higher survival rates in acidic products (135, 136, 137). Bijid. b@dum and Bijid. Zongum are employed as mixed cultures in combination with Streptococcus salivarious and/or Lb. acidophilus. Decription of some of those products are as follows: Acidonhilus Bifidus Yogurt:
This is similar to Bifidus yogurt. It is manufactured in many countries by fermenting cow s milk. The homogenized milk is heated to 85C for 30 min or 90C for 5 min and cooled to 40 - 42 C inoculated with separate cultures of L. acidophilus and Bifid. bifidum or longum packaged, incubated at 40 - 44C for 3-5 h, followed by cooling and storage. The therapeutic organism is of human origin and the viable cell count averages l-3 x 10 &u/ml for each of Lb. acidophilus and Bifidobacteria sp. Bifidus Milk:
Cow s milk is fortified with 150 - 200 g total solids per kg. This milk is homogenized and heated to 80-120 C for 15 min, cooled to 37 C, inoculated with 100 g/l of Bifid. bifidum or Zongum as starter culture at pH -4.5. The coagulum is cooled, packaged and moved to the cold store (138). The characteristic features of Bifidus milk are i) It has a mild acidic and slightly spicy taste, ii) the molar ratio of lactic acid to acetic acid is 2:3 iii) set or stirred types are produced with or without fruit flavours and iv) the viable count of Bifid. bifidum is lo*-lo9 cfu per ml. Fermented milk containing Bifidobacteria are becoming popular in UK because it Misra and Kuila have reported a is milder in taste when compared to yogurt. commercial method for the production of bifidus milk in India (139). Skimmed milk was heated to 90C for 30 min, cooled to 37C, inoculated with Bifid. bifidum NDRI (National Dairy Research Institute of India) at the concentration of 1OOg per 1, and incubated for 19h. The products has good storage life of minimum 3 weeks, good taste and microbial count of 10 cfu per g. The antibacterial activity of the fermented milk against four pathogens showed that it was dependent on i) the type of milk used baby formula) ii) heating temperature iii) (skimmed, cow, buffalo or reconstituted
1583
concentration of the inoculum and iv) concentration of sugar. Bifidus or acidophilus drinks are becoming popular. The viable cells of >2x106 cfu per ml is added to cold These products complies with statutory pasteurized milk followed by packaging. regulations in California and Oregon. A similar product containing Lb. acidophilus and Bifzd. bijidum is also marketed in USA called Nu-trish A/B which is supplied by Chr. Hansen s laboratory (140) These products are sweet in taste and not fermented but offer a good way to provide an abundance of bifidobacteria to consumers. Bifidus Yoghurt:
It is a mixed starter culture of Bijid. bifidum or longum and yogurt culture used is at a rate of 50 - 100 with or with out Lb. acidophilus. The concentration g/l to ferment milk at 40 - 42C for 3-5 h which is followed by mixing before cooling and packaging. Bifighurt: This is similar to Bifidus milk or yogurt but the starter culture is exclusively of Bifid. longum CKL 1969 or DSM 2054 (a slime- producer) and Str. thermophilus. The bifidobacterial count in the product is 10 cfu/ml, and produces only lactic acid (95%). PHARMACEUTICAL PRODUCTS
Special freeze-dried pharmaceutical dietary preparations containing viable cells of with other organisms are available as Bifidobacterium spp. alone or in combination tablets. They are prepared by the pharmaceutical and food industries, and contain autochthonous intestinal microorganisms. The primary objective of these products is to achieve implantation of the bacteria in the gut during the treatment of various ailments disturbances like and diseases. They are utilized for the therapy of gastro-intestinal diarrhoea, side effects of antibiotic and radiation therapy, chronic constipation, chronic peptic ulcers in children, after irradiation therapy and as for special duodenitis, preparations for certain liver diseases. Some of the pharmaceutical products made from fermented milks containing viable cells of Bijid. bifidum or other Bifidobacteria spp. are Bifider (Japanese), Bifidogene (French) and Liobif (Yugoslavian). Eugalan Topfer Forte is a German product containing 94g milk protein, 7g plant protein, 6268 lactose, 59g lactulose and 30g minerals per kg; with high number of bifidobacteria. It is a fat free and gluten -free preparation. Another German product Euga-Lein Topfer contains 18g fat, 151g protein, 71 lg carbohydrate, 31g minerals, 48 g dietary fibre, Omniflora as a german and 2g vitamin C. Kurmann et al. have described pharmaceutical preparation containing Lb. acidophilus, Bifid. longum and a saprophytic E. coli (138). The utilization of soya milk to prepare fermented soya products using Bifidobacterium spp. been described by Maslov et al. (141). This product is prepared by incubating the fermented product at 37C for 16h till the lactic acid concentration per 1 and the viable count ranged from lo* and lo9 cfu per ml. Murti reach -50g et al. has used culture combination of yogurt strains and Bifidobacteria for the
1584
K.D. ARUNACHALAM
fermentation of cow s milk and soya milk (142). From his results he came to the conclusions that the soya milk could be used successfully for the manufacture of a vegetarian yogurt for European and North American markets and the mixed culture of Bifidobacteria with other species could be an ideal combination. ROLE OF TECHNOLOGY
The International Dairy Federation publishes monographs updating the technological and scientific aspects of dairy manufacturing and International Dairy Federation (143, 144) review the recent technological developments for fermented milks, hygienic design of dairy processing equipment and hygiene management in dairy plants. Over the years, equipment manufacturers have specifically developed and designed processes to meet the technological requirements of fermented milk products; the processing plants are universal. The highly sophisticated techniques have developed in this line; for example the use of de-aeration equipment in a yogurt production but it is highly desired to de-aerate milk if it is fermented by Bifidobacteria. The presence of oxygen will prolong the incubation period. To avoid the oxygen problem, an interesting approach was applied by mixing Bifido with S. themophilus having high oxygen utilization ability. In order to keep the oxygen permeation low glass bottles or aluminium-laminated packages are used. Since polyethylene or polystyrene are not oxygen barriers they are not suitable for bifidus products. PVDC or EVOH maintain a very good oxygen barrier these materials were used for coating or laminating the packaging materials. Strains of Bifidobacreia should be pH and heat resistant because of the high drying temperature used in processing for the preparation of dried fermented products. Hinterwaldner have recommended the addition of 0.1% to 0.5% yeast extract to the production of fermented milk if it is processed by drying (145). For spray drying the numbers of the bacterial should be higher than 10 . CURRENT AREAS OF RESEARCH AND FUTURE DEVELOPMENT
A number of other possible health benefits of BIB have been postulated, with varying degrees of validity. The following examples require rigorous research before conclusions can be drawn. The possible benefits of the calcium component of yogurt in reducing hypertension. Yamamoto et al. recently reported antihypertensive activity in spontaneously hypertensive rats given milks fermented by L. helveticus (146). Not all LAB tested exerted this effect. LAB may help regulate blood pressure within normal range. A few studies have noted an inverse relationship between the consumption of fermented milks and the risk of breast cancer (147, 148). To our knowledge, no experimental studies have been performed. The current scientific research and the manufacture of dairy products as adjuncts is centered in Japan and in Europe. In US the poor consumer awareness of the beneficial health aspects of Bifidobacteria in dairy products has to be overcome. The possible future development has to be done in the following areas i) selection of suitable strains for fermentation and starter of bifidobacteria in the dairy industry ii) genetic cultures ii) wider application manipulation to enhance the growth in milk and produce less acetic acid iv) improvements in designing the experiments involving human volunteers v) increasing
1585
the research efforts to study the therapeutic aspects of Bifidobacterium, vi)improved funding for bifidobacteria research and greater collaboration between scientists, Some preliminary studies provide nutritionists and technologists in many countries. exciting possibilities for future research, particularly in the reduction of the risk of colon cancer. Other areas of research urogenital tract infections, hypertension are more speculative, and may provide positive paths in the future. The mechanisms of certain actions have yet to be uncovered. Development of validated model systems will help achieve this goal more quickly. Today s consumers demand food products that not only taste good, but are also good for their health. As research continues, strain selection will become more significant, and the subsequent fermented milks will answer more and more of consumer s needs. CONCLUSION It has been established that certain strains of bifidobacteria, which have been selected for their ability to resist acid digestion and the action of bile salts, survive intestinal transit and reach the colon in significant numbers. These live bacteria have the potential to influence the endogenous intestinal microflora. Altering the endogenous microflora may result in such physiological effects as altering enzyme activities of the microflora, and affecting gut transit. For example, it has been observed that certain types of clostridia constitute a high proportion of the faecal microflora in patients with colorectal cancer (149). Thus altering the profile of the intestinal microflora could have long-term health benefits, as proposed by Metchnikoff. While studies on the effects of consuming live bifidobacteria are numerous, the lack of rigorous, well-structured, double-blind trials with appropriate controls hampers the interpretation of the results. In addition, more studies are needed to establish the mechanism(s) by which these bacteria exert their probiotic effects. Lactic acid bacteria in the form of fermented milks have been implicated in the maintenance of good health and in the prevention of many disorders. It is clear that some strains of LAB possess the ability to alter the activity of the intestinal microflora and thus to effect changes related to the gut Yogurt is an exellant alternative for the treatment of lactose intolerance. Billions of people around the world are lactose maldigesters, and can benefit from this every day simple traditional food. ACKNOWLEDGMENT The author would like to thank Dr. T. R. Pate1 for reviewing this manuscript and to Dr. R. K. Chandra for his suggestions and encouragements in the preparation and publication. REFERENCES 1. Metchnikoff E. The prolongation of life. 1907, Heineman, London. 1910. Tissier MH. La reaction chromophile Optimistic Studies. Revised edition of
2.
d Escherich
et Bacterium
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C R Sot
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1899;51:943-945. NS. In: Therapeutic properties of Fermented Milks, Elsevier Applied Sciences Publishers 1991: 159-180. (Ed, RK Robinson)
Koroleva London:
4.
Gilliland SF and Walker DK. Factors to consider when selecting a culture of Lactobacillus acidophilus for a dietary adjunct to produce hypocholesterolemic effect in humans. J of Diary Science 1990;73:905-911. Sellars RL. In: Robinson London, Elsevier Applied RK, ed. Therapeutic Properties Science Publishers 1991;81-116. of Fermented Milks.
Salji J. Acidophilus milk products: Food with a special dimension. and Technology Today 1992;6(3): 142-147. Salji J. In: Macrae R, Robinson RK and Sadler Science, Food Technology and Nutrition, London: 1, pp 3-5. 8.
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M edEncyclopedia of Food Academic Press 1993; vol.
Mitsuoka T, and Hayakawa K. Die Faekalflora bei Menschen: I. Mitteilung: Die Zusammensetzung der Faekalflora der verscheidenen Altersgruppen. Zentralbl. Bakteriolk. Hyg I Abt Orig 1972;A223:333-342. Modler factors. HW, McKellar Can Inst Food RC, and Yaguchi M. Bifidobacteria Sci Technol J 1990:23:29-41. and Bifidogenic
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Bahaka D, Neut C, Khattabi A, Monget D and Gavini F. Phenotypic and genomic analysis of human strains belonging or related to Bifidobacterium longum, Sinfantis and B.breve. Int J Syst Bacterial 1993;43:565-573. Rasic JL, Experientia Scardovi peculiar Microbil and Kurmann JA. Bifidobacteria 1983;39 Supplement: 3-34 V, and Trovatelli LD. (1965), pattern of hexose degradation Enzymol 1965;15: 19-29. and their Role, Basel: Birkhauser,
11.
12.
The fructose -6- phosphate shunt in the genus Bifidobacterium,
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Nutrition Research, Vol. 19, No. 10, pp. 1559-1597, 1999 Copyright 0 1999 Elsevier Science Inc.

Printed in the USA. All rights reserved 0271.5317/99/%x front matter

Faculty of NF, Canada

Photomicrograph of Bifidobacteria Grown with China Blue. (Incubated Anaerobically

on Glucose-Blood Agar at 37C for 72 h)

Bifid. adolescentis 0.02 0.01 0.043 0.17 0.35

Bijid. infantis 0.2 0.040 0.059 1.23 0.39

l.c. low concentration,

n.s. not synthesized.

Reinforced clostridial agar(RCA) inhibitory components. chloride 41

agar with 0.4 g/l of lithium

MRS agar with added dicloxacillin

with 2pg/ml of Mann-Rogosa-Sharpe

Modified chloride, propionate agar. 49

of the mtestinal mlcroflora 7

of the intestinal mw9lora , Useful

Sraphylococcus O-IO Proleus Ps. aeruglnosa

2. Host Relationships BIFIDOBACTEIA

of the Intestinal AS PROBIOTICS

Results Showing the Effect of Bifidobacteria.

same ersons before and a Rer bifidus

Skim milk and yogurt

Bifidobacterium same ersons before and a Rer bifidus fPP.

Yogurt without bifidobacteria

same ersons before and a Ker bifidus

B.longum ATCC 15707

Bijidobacterium asteurized bifidus Permented milk spp.

x-K:>x : f-- , -a\

Escherichia coli Enterococci Lactococci

Bacteriodes Eubacterium Anaerobic streptococcus

II 10 Anaerobtc strepococcus Bibidobacterium

Streptococcus E. coli Lactobacillus

2 Points Length Time taken for food to arrive -

BIB in Prevention Imnroved Protein

1576 the normal microbial phenols in the blood balance (Figure

Blood 3x70 3*too /ng/ 100 ml wmw 250,

Ammonia 3s70 3#70 3 ~roog

10.3 23.5 14.2 18.4 20.7 1967

Control Sour milk Acidified milk Starter cells

the Changes in Minor during Antileukaemic

Members of Intestinal Therapy with antileukaemic (56 cases)

Treatment Organism Control (locases)

Without Bifidobacteria (28 cases) in % 3 (10.7) 6 (21.4) 8 (28.6)

With Bifidobacteria (28 cases) in % 2 (7.1) 5 (17.9) 2 (7.1)

Klebsiella sp. Citrobacter sp. Proteus vulgar-is

Cases in which more than lo6 colonies were observed

M edEncyclopedia of Food Academic Press 1993; vol.

The fructose -6- phosphate shunt in the genus Bifidobacterium,

of glucose, lactose, 1967;96(2):472-478. A, eds. Lactic Acid

Mair NS, Sharpe ME Bacteriology. Baltimore,

and Holt JG, MD: Williams

of growth and acid 1990;73(6): 1478-1484. of

Growth and survival Journal 1993;47:151-164. of

P. Growth requirements 1990;45:500-502.

factor II. Its occurence products. Archives of

1991;32:439-442. beings. Bifidobacteria

Homma N. Bifidobacteria Microflora 1988;7:35-43.

for isolation Microbilogy. H, and

and enumeration of 1988;54: 1715-1718. T. Bifidobacteria

PM and De Groote FH. dairy products. Netherlands

Selective enumeration of Milk and Dairy Journal.

for the 1990;70:

P. Role of adhering young chicks. Journal