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come from investigations of the antidiarrheal effect of

bifidobacteria. Indeed, the biotherapeutic potential of

foods containing bifidobacteria (and other LAB) has

been well established for antibiotic-associated diar-

rhea outbreaks and rotavirus infections. In a double-

blind, placebo-controlled trial in 55 hospitalized

infants, oral administration of Bifidobacterium bifi-

dum and Streptococcus thermophilus reduced the

incidence and severity of rotavirus diarrhea. There is

also some evidence that fermented milk products

containing bifidobacteria may protect against travel-

er’s diarrhea. However, work to date suggests that the

tbl0002 Table 2 Compilation of experimental results showing the effect of feeding bifidobacteria in humans

Organism Control Test Effect

Bifidobacterium bifidum,

Lactobacillus

acidophilus

15 humans receiving no

fermented milk

15 humans Increased total IgA levels

Fermented milk

B. bifidum, L. acidophilus Baseline

14 humans Increased phagocytic activity

B. bifidum, Streptococcus

thermophilus

Placebo 55 hospitalized infants Reduced incidence of diarrhea (70% of

observed diarrhea cases caused by

rotatvirus)

Bifidobacterium spp. Baseline Humans Lowered b-glucuronidase levels

Fermented milk

B. bifidum, L. acidophilus,

S.lactis,S.cremoris

Baseline Humans Lowered nitroreductase levels

Fermented milk

B. longum Baseline 10 humans Lower fecal pH

Fermented milk

B. longum, L. acidophilus Placebo 30 humans Lowered fecal volatile fatty acids levels and

reduced GI discomfortFermented milk

B. longum Baseline 6 humans Increased bifidobacterial dominance

Fermented milk

B. longum Placebo 12 humans Increased fecal bifidobacteria levels

Fermented milk

B. bifidum, L. acidophilus Placebo Humans Delayed reduction of bifidobacteria due to

antibiotic treatment and induced earlier

re-establishment of bifidobacteria,

postantibiotic therapy

Lyophilized bacteria

B. bifidum Baseline 12 elderly dyspepsia patients Increased fecal bifidobacteria levels and

reduced symptomsLyophilized bacteria

B. bifidum, L. acidophilus Placebo 20 humans No significant differences during amplicillin

administrationLyophilized bacteria

B. bifidum Placebo 15 humans Decreased chronic inflammation of sigmoid

colon and increased humoral immunityLyophilized bacteria

B. longum BB536 B. longum ATCC15707 48 Humans Reduced clostridia, bacteroides, coliforms

population levelsFermented milk

Bifidobacterium spp. Pasteurized bifidus-

fermented milk

60 Humans Reduced colonic transit time

Fermented milk

Bifidobacterium spp. Baseline Patients with hepatitis or liver

cirrhosis

Lowered blood ammonia and phenol levels,

reduced urinary indican levels, increased

fecal bifidobacteria levels and improved

appetite and weight gain

B. lactis 13 control subjects

given placebo milk

12 healthy elderly humans Increased interferon-a levels and

polymorphonuclear cell phagocytic capacitySupplemented milk

B. longum Baseline 5 humans Reduced lecithinase negative clostridia

numbers, lowered NH

and b-glucuronidase

levels in feces and reduced fecal pH

Lyophilized bacteria

Bifidobacterium spp. Baseline Bedridden elderly humans Increased stool frequency

Fermented milk

B. bifidum, L. acidophilus 10 elderly humans 15 elderly humans Reduced inflammation of sigmoid and

descending colon

Bifidobacterium spp. Baseline 15 humans Reduced stool frequency in intractable diarrhea

B. longum Yogurt without

bifidobacteria

10 Humans Reduced erythromycin induced gastrointestinal

effectsFermented milk

Bifidobacterium spp. Baseline Humans Lowered nitroreductase and glucuronidase

levelsFermented milk

Same subjects prior to feeding.

BIFIDOBACTERIA

IN FOODS 465

probiotic strain/mixture as well as the destination are

paramount.

Physiological Effects

0008 The production of organic acids (acetic and lactic

acid) by bifidobacteria inhibits the growth of patho-

genic organisms (directly and indirectly) and stimu-

lates intestinal peristalsis. Acetic acid is a stronger

antagonist against Gram-negative bacteria than lactic

acid. As such, the potential applications of bifido-

bacteria against microbial perturbation may surpass

those of lactobacilli. Additionally, the organic acids

produced by bifidobacteria have been shown to in-

hibit the growth of many nitrate-reducing bacteria.

Human studies using bifidobacteria as a dietary ad-

junct have shown the relief of symptoms in consti-

pated elderly persons (Table 2). Several investigations

have also reported a reduction of fecal pH in individ-

uals consuming fermented milk products containing

bifidobacteria. Lowering the pH of the intestinal tract

produces an environment less favorable to some

pathogenic bacteria and thus helps prevent their over-

growth.

Anticarcinogenic Activity

0009 Increasing evidence, from in vitro experiments and

animal studies, indicates the potential protective

influence of probiotic bacteria (including bifido-

bacteria) against cancer. To date, three plausible

mechanisms have been identified: (1) inhibition of

putrefactive organisms that produce carcinogens

(such as N-nitroso compounds, phenolic products of

tyrosine and tryptophan, and metabolites of biliary

steroids); (2) binding and/or inactivation of carcino-

gens; and (3) inhibition of tumor cell formation.

Bacterial enzymes (including b-glucuronidase, b-

glucosidase, nitroreductase, and azoreductase) are

responsible for converting some procarcinogens into

carcinogens. As such, the levels/activities of these

enzymes are considered a useful biomarker for cancer

risk in humans, enabling noninvasive estimation

of carcinogen levels. Several human studies have

employed this method to determine the effects of

different diets on the risk of colon cancer. A number

of investigations have shown a reduction in fecal

enzyme activity during ingestion of bifidobacteria-

containing milk products, compared with initial

levels before bifidobacterial consumption (i.e., base-

line levels). Although these results are encouraging,

they are inconclusive regarding the effects on cancer

risk. Much more research is required to establish clear

links between enzyme activity and cancer risk.

Animal studies have enabled more definitive investi-

gations of the anticancer effects of bifidobacterial

feeding. Several such reports from murine models

have identified an antitumor effect of bifidobacterial

feeding (with or without prebiotics). Cases affording

protection against both aberrant crypts (precancerous

lesions) and tumors have been published. However,

animal studies are speculative at best, regarding the

protective nature of these strains against the develop-

ment of cancer in humans.

Stimulation of Immune Functions

0010Several probiotics, including some bifidobacterial

strains, are claimed to enhance the immune system

in a nonspecific manner, thereby stimulating immun-

ity to a number of antigens. A number of studies have

also shown the ability of certain LAB strains to alter

cytokine production and/or increase secretory IgA

levels. However, most work in this area has either

involved inappropriate animal models or concen-

trated on intermediary endpoints (such as cytokine

production) rather than disease symptoms. Poor

study designs have also led to difficulty in confirming

the immunomodulatory agent. Preliminary data dem-

onstrate the potential of probiotics in modulating

certain immune responses and indicate their potential

role in allergy, autoimmunity, and gastrointestinal

disease.

Cholesterol-lowering Ability

0011Mann and Spoerry identified correlations between

daily consumption of fermented milk and low serum

cholesterol levels in East African Masai warriors.

Subsequent in vitro work has demonstrated the abil-

ity of bifidobacteria to both assimilate cholesterol

and coprecipitate it with deconjugated bile acids.

Such observations have led to great interest in the

cholesterol-lowering capacity of a diet containing fer-

mented milks. However, much contradictory data

exist regarding the effects of consuming foods con-

taining bifidobacteria on serum cholesterol levels.

Confounding the issue has been the use of different

strains, dosages, and/or food vehicles in the various

studies carried out so far. Additional criticisms of the

current data have included lack of stabilization of

baseline cholesterol levels, inadequate size and/or

duration of studies, and difficulty in controlling the

diet and physical activity of subjects.

Lactose Intolerance

0012Lactose malabsorption affects large portions of the

population (estimated by some to affect over half the

world’s population), with a higher prevalence in those

of Oriental or African ancestry. Symptoms normally

include abdominal discomfort, flatulence, and/or

diarrhea. However, lactose-intolerant individuals

466

BIFIDOBACTERIA

IN FOODS

can consume cultured milk products (containing

bifidobacteria and/or lactobacilli) without any dele-

terious effects. Two mechanisms have been proposed

for the improved digestibility of lactose in such

products: (1) the b-galactosidase activity of the pro-

biotic strains; and (2) stimulation of host mucosal

b-galactosidase activity by the ingested strains.

Nutritional Value

0013 Bifidobacteria are known to produce thiamine, ribo-

flavin, vitamin B

, and vitamin K. There have also

been reports of their ability to synthesize folic acid,

niacin, and pyridoxine. These vitamin B complexes

are slowly absorbed in the human body. However, the

impact on human nutrition of such vitamin synthesis

by bifidobacteria in the colon is unknown. Available

information on the nutritional properties of fer-

mented milks containing bifidobacteria indicates

that they have lower residual lactose and higher levels

of free amino acids and vitamins than nonfermented

milks. Additionally, they preferentially contain l(þ)-

lactic acid [produced by bifidobacteria in addition

to acetic acid, whereas lactobacilli produce d/l()-

lactic acid], which is more easily metabolized by

humans. This is particularly important for infants

less than 1 year old, in whom metabolic acidosis can

be a problem. Consuming bifidobacterial food prod-

ucts may also improve the bioavailability of certain

minerals, including calcium, zinc, and iron, by

lowering the gastric pH (facilitating ionization of

minerals, which is necessary for their uptake).

Product Development

0014 Technically, bifidobacteria may be incorporated into

foods either to produce certain product characteris-

tics, such as organoleptic and/or nutritional proper-

ties, or as a probiotic. In the latter case, the viability

of the strain is essential during both manufacture and

storage of the food product. Indeed, the generally

accepted recommendation is that probiotic strains

must be present at levels of > 10

viable cells per

milliliter at the time of consumption. This is based

on the minimum therapeutic daily dose of 10

–10

cells.

0015 Bifidobacterial cultures incorporated into foods for

therapeutic purposes may be added either during

fermentation (along with or as part of the starter

culture) or to the finished fermented or fresh product

prior to shipment. Storage of food products under the

appropriate refrigeration conditions after manufac-

turing restricts subsequent fermentation. As such,

products to which bifidobacteria are added immedi-

ately prior to distribution are organoleptically indis-

tinguishable from the unfortified version. Products

fermented with bifidobacteria, however, often have

a mild acidic flavor. The selection of culture strains

and ratios is as critical in determining the final prod-

uct characteristics as the fermentation parameters

(temperature, duration, chemical composition and

level of inoculum).

Selection Criterion

0016The selection of bacterial strains is vital since differ-

ent strains (even within the same species) have dis-

tinctive metabolic and probiotic characteristics, as

well as different growth rates. The main factors to

consider include rate of acid production, type of poly-

saccharide fermentation, ability to synthesize vita-

mins, proteolytic characteristics (which may result

in bitter compounds) and capacity to produce flavor

compounds. An additional consideration is the growth

characteristics of the bacterial strains. This is particu-

larly important regarding mixed inocula and deter-

mining both the optimal mixture and the ratio of

strains, as well as the appropriate time in processing

to add the organisms. Recent studies have shown that

bifidobacteria inoculated into sour cream and butter-

milk either at the time of setting or at the time of

breaking allowed appropriate numbers of bacteria to

survive during normal storage of the products. Add-

itional work suggests that the best procedure for in-

corporating bifidobacteria in cottage cheese is in the

creaming mixture, whereas addition immediately

after cooking (prior to hooping) is suitable for

Edam cheeses. In the production of yogurts contain-

ing bifdiobacteria, it is common practice to use

premixed cultures of Streptococcus thermophilus,

Lactobacillus delbrueckii ssp. bulgaricus, L. acidoph-

ilus, and Bifidobacterium species. However, bacterial

‘loading’ is also practiced, where the lactobacilli

and bifidobacteria are grown separately prior to

addition to the product. A number of studies have

also demonstrated successful manufature of probiotic

cheeses, especially hard cheeses (e.g. cheddar,

Canestrato Pug liese and Gouda-type), by either in-

corporating bifidobacterial strains in the starter cul-

ture or using bifidobacterial fermented cream.

0017As bifidobacteria are anaerobes, it is useful to select

species or strains that are less oxygen-sensitive (such

as B. infantis and B. longum), perform gas flushing

with nitrogen (a process often used for preserving

yogurts), or include an oxygen scavenger in the

fermentation inocula (such as S. thermophilus). The

growth and survival characteristics depend on the pH

(and its buffering capacity), the fermentation and

storage conditions (especially temperature and dur-

ation), and the presence of competing micro-

organisms or microbial inhibitors (such as H

and NaCl).

BIFIDOBACTERIA

IN FOODS 467

0018 Regarding biotherapeutic considerations, the

strain(s) must be selected, based on their ability to

elicit the desired beneficial effect at the target. This

necessitates their survivability within the product and

during transit through the upper GI tract. As such, it

is pertinent to use bifidobacteria of human intestinal

origin as they are better suited to the physiological

needs of the host than strains from other sources.

Finally, it is essential to demonstrate the safety of

probiotic strains. They must be confirmed as non-

pathogenic, have a well-established safety record, or

have ‘generally regarded as safe’ (GRAS) status.

Many publications have listed recommendations of

selection criteria for probiotic strains. These fall into

the four general categories of (1) appropriateness, (2)

technological suitability, (3) competitiveness, and (4)

performance and functionality.

Technical Considerations

0019 Besides strain selection, careful monitoring through-

out the manufacturing process is required to insure

efficient control of the final product (pH, metabolic

products, cell density, and flavor). Incorporation of

LAB above the necessary starter organism to produce

specific product characteristics demands examination

of the final product to confirm the desired property.

For example, in vitro demonstration of a strain’s abil-

ity to synthesize vitamins is inadequate on its own, as

the fermentation characteristics and final product

composition (including the levels of the desired vita-

mins) need to be determined.

0020 A number of methods have been reported to

increase the survival of LAB whilst maintaining

the desirable attributes of yogurt. These include

enrichment with whey protein concentrate; lowering

incubation temperature (to 37



C); terminating fer-

mentation at a higher pH (e.g., > 5); hydrostatic pres-

sure treatment or heat-shock of yogurt (to prevent

postmanufacture acidification); and storing below

3–4



Market for Foods Containing

Bifidobacteria

0021 To date, food products containing bifidobacteria

have largely been of dairy origin and include yogurts,

fermented milks, fresh milks, cheeses, buttermilk,

sour cream, cream cheeses, cottage cheeses, frozen

dairy desserts (including icecream and frozen yogurts),

and dips. Research has further demonstrated the

feasibility to incorporate bifidobacteria in vegetable

spreads, fermented vegetable products (such as kim-

chi, a traditional Korean food), mayonnaise, cookies,

and some beverages. A list of some of the commer-

cially available fermented milk products containing

bifidobacteria and some of their claims is provided

in Table 3.

0022The increased awareness of consumers towards

general health and well-being, together with the

accepted impact of diet on health, has led to a greater

demand for healthy and/or functional food products.

As such, the upward trend currently seen in bifido-

bacterial foods is projected to continue. As much as

70% of milk products on the market in some Euro-

pean countries, such as Sweden, contain bifidobac-

teria. The Japanese and European food industry and

markets are well ahead of the USA and Canada,

regarding functional foods. However, the influx of

foods containing bifidobacteria on these markets has

begun. A recent article estimated double-digit annual

growth rates of the world-wide market for such

products.

Regulations and Legislation

0023The generally accepted minimum level of probiotics

in foods at the time of consumption is > 10

cells per

milliliter. However, the standard developed by the

Fermented Milks and Lactic Acid Bacteria Beverages

Association of Japan is > 10

cells per gram at the

time of manufacture. In the USA, 10

bacteria per

gram at the time of manufacture is the criterion of

the National Yogurt Association (NYA). As well as

the concern of viability of probiotics within the con-

sumed product, there is increasing interest (from the

scientific community, food industry, and national au-

thorities) in establishing codes of practice regarding

the legislation and regulations of health claims for

food products. To this end, definitive evidence

demonstrating the efficacy of products is essential.

As such, statically significant, well-designed (double-

blind, placebo-controlled) clinical trials are demanded

for health-promoting food products. The need to

prove safety is equally important to substantiating

efficacy. Key areas for demonstrating nutritional

safety include intake, extent of use, implications/

impact on gut microflora and metabolic pathways,

compositional analysis (including the presence of

antinutritional or toxic factors), and potential effects

in compromised individuals or specific target popula-

tions.

0024Currently, there is no world-wide regulatory or

legislative control on the labeling and health claims

of food products. However, most legislative bodies

prohibit the use of ‘medicinal’ claims for food prod-

ucts (including functional foods). That is, claims sug-

gesting the prevention of, treatment, and/or cure for

human disease are not permissible for food products.

However, health claims are deemed acceptable and

usually involve the promotion of health benefits,

468

BIFIDOBACTERIA

IN FOODS

reduction of disease risk, or general well-being of the

host. Health claims may not include specific claims

such as ‘improves resistance against pathogenic infec-

tion’ but could state ‘improves normal intestinal func-

tion.’ As one can see, the line between medical and

health claims is very gray, and the presentation or

wording of the claims is usually important in defining

under which heading they fall. Indeed, one published

explanation of medical foods (medicine) and func-

tional foods gave the following definitions. Medicines

include ‘a food product which has a direct preventa-

tive effect on a disease,’ whereas health claims may

tbl0003 Table 3 Commercially available fermented milk products containing bifidobacteria

Product Bacterial strains Claims Country

A-38 L. acidophilus, B. bifidum, Leuconostoc

mesenteroides ssp. cremoris, mesophilic

lactococci

Denmark

A-B Yogurt B. bifidum, L. acidophilus France

Aktifit S. thermophilus, L. casei, L. acidophilus,

B. bifidum

Defense system for bowel Switzerland

Akult B. bifidum, L. acidophilus, B. breve,

L. casei ssp. casei

Normalize the balance of human intestinal

flora

Japan

B-Active L. acidophilus, B. bifidum, S. thermophilus,

L. delbrueckii ssp. bulgaricus

France

Bifidus milk B. bifidum, B. longum Several

Bifiel B. breve strain Yakult, S. thermophilus,

Lactococcus lactis

Normalize the balance of human intestinal

flora

Japan

Bifighurt B. bifidum, S. thermophilus Germany

Bifilus B. longum, L. bulgaricus, S. thermophilus Sweden

Biokys B. bifidum, L. acidophilus, Pediococcus

acidilactici

Czech Republic

Biomilk Bifidobacterium spp., L. acidophilus Several

Bio Pot L. acidophilus, B. bifidum Improve intestinal flora Germany

Bio-Garde L. acidophilus, B. bifidum, S. thermophilus Australia

Biola B. lactis, L. acidophilus, L. GG Balance microflora Norway

Bulla AB Live L. acidophilus, Bifidus Singapore, Australia,

Indonesia

GEFILUS S. thermophilus, L. bulgaricus, L. acidophilus,

B. bifidum, L. casei GG

Finland

LA7 L. acidophilus LA7, Bifidus Improve intestinal flora Germany

Leisure Live L. acidophilus, B. bifidum UK

Little Swallow L. acidophilus, B. bifidum UK, Germany

Mil-Mil B. breve strain

Yakult, B. bifidum strain Yakult, L.

acidophilus

Normalize the balance of human intestinal

flora

Japan

Mil-Mil E B. breve strain Yakult, L. acidophilus,

S. thermophilus

Normalize the balance of human intestinal

flora

Japan

Morinaga Bifidus B. longum, L. acidophilus Normalize the balance of human intestinal

flora

Japan

Morinaga Caldus B. longum, L. acidophilus Normalize the balance of human intestinal

flora

Japan

Nu-trish A/B B. bifidum, L. acidophilus USA

Procult 3 B. lo ngum Germany

Progurt B. bifidum, L. acidophilus, mesophilic

lactococci

Several

Ski L. acidophilus, Bifidus Promote health Australia, Singapore

Sym-Balance L. reuteri, L. acidophilus, L. casei,

bifidobacteria

Defense system for bowel Switzerland

VAALIA L. acidophilus, B. lactis, L. GG Promote health, balance intestinal

microflora

Australia

Vifit S. thermophilus, L. bulgaricus, L. acidophilus,

B. bifidum, L. casei GG

Keep the intestinal flora in good shape,

enhance immune response

Netherlands, Belgium,

Vifit S. thermophilus, L. bulgaricus, L. acidophilus,

B. bifidum, L. casei GG

Keep the intestinal flora in good shape,

enhance the immune response

Germany

Yoplait Yoplus L. acidophilus, Bifidus Singapore, Australia

Yukijirushi Nachure L. acidophilus, B. longum Promote the maintenance of a good

intestinal environment

Japan

BIFIDOBACTERIA

IN FOODS 469

include maintenance of ‘good health as part of a life

style’ and/or potential reduction of ‘the risk of disease

occurring.’

0025 In Japan, a voluntary system is in place where

industry may apply for ‘Foods for Specified Health

Use’ (FOSHU) approval. Information on the safety

and efficacy in humans (including definitive scientific

evidence) and nutritional analyses are required for

such approval. FOSHU labeling, therefore, effectively

demonstrates that the product is approved as a func-

tional food, according to the advertised claim.

0026 In the USA, Federal Drug Agency (FDA) approval

is necessary regarding any labeling of health claims on

food products. Furthermore, the claims must be

founded on an ‘authoritative statement’ from an

appropriate Government scientific agency respon-

sible for ‘public health protection or research.’ How-

ever, ‘structure/function’ statements are permitted on

dietary supplements relating the food ingredient to

‘normal, healthy functioning of the human body.’

Such claims are not normally used on conventional

foods, but rather for foods intended to supplement

the diet. That said, foods containing bifidobacteria

are generally marketed as dietary supplements in the

USA, and therefore fall within a loophole. Currently,

however, most manufacturers of such products

simply name the added bacteria (at least to genus

level) for labeling purposes and do not state any

functions or claims.

0027 In Europe, there is no collective regulation or legis-

lation regarding functional foods. In general, the

primary focus is on the safety of the products. Food

products or food ingredients that were not available

on the European market for human consumption

prior to April 1997, and can be classified as novel

(owing to raw materials, composition, or manufac-

turing process), must undergo regulatory approval.

The second regulatory focus in Europe is the labeling

and health claims of the food product. The general

rule of thumb is that health claims may not attribute

the property of preventing, treating, or curing a

human disease, or any such implications. Currently,

seven European Union member states are developing

their own Codes of Practice regarding legislation and

regulation of health claims and labeling of functional

foods. The level of scientific substantiation needed for

such health claims differs between some codes. How-

ever, the Codes of Practice for the UK, Belgium, and

The Netherlands all include principles for human

(clinical) trials.

Summary

0028 Results to date clearly indicate the potential benefits

of consuming a diet incorporating foods containing

bifidobacteria. Several food vehicles have been

identified within the dairy industry, and appropriate

manufacturing techniques are available to insure that

the desired product characteristics and viability of

probiotic strains are attained. Essential to the future

of functional foods are adequate studies confirming

the safety, efficacy, and viability of such products

(especially well-designed human clinical trials). Cur-

rent developments within the scientific community,

food industry, and regulatory bodies are all pursuing

this end.

See also: Dairy Products – Nutritional Contribution;

Fermented Milks: Types of Fermented Milks; Products

from Northern Europe; Other Relevant Products; Dietary

Importance; Lactic Acid Bacteria; Microbiology:

Detection of Foodborne Pathogens and their Toxins;

Microflora of the Intestine: Role and Effects; Probiotics;

Prebiotics; Probiotics; Yogurt: The Product and its

Manufacture; Yogurt-based Products; Dietary Importance

Further Reading

American Society for Nutritional Sciences (various authors)

(2000) Symposium: Probiotic bacteria: Implications

for human health. Journal of Nutrition 130: 382S–

416S.

Arunachalam KD (1999) Role of bifidobacteria in nutri-

tion, medicine and technology. Nutrition Research 19:

1559–1597.

de Roos NM and Katan MB (2000) Effects of probiotic

bacteria on diarrhea, lipid metabolism, and carcino-

genesis: a review of papers published between 1988

and 1998. American Journal of Clinical Nutrition 71:

405–411.

Gibson GR and Williams CA (2000) Functional Foods:

Concept to Product. Cambridge: Woodhead Publishing.

Heller KJ (2001) Probiotic bacteria in fermented foods:

product characteristics and starter organisms. Amer-

ican Journal of Clinical Nutrition 73(Suppl): 374S–

379S.

Kailasapathy K and Rybka S (1997) L. acidophilus and

Bifidobacterium spp. – their therapeutic potential and

survival in yogurt. The Australian Journal of Dairy

Technology 52: 28–34.

Lee Yuan-Kun Nomoto K, Salminen S and Gorbach SL

(1999) Handbook of Probiotics. Wiley-interscience

Publication. New York: Wiley.

Rasic JL and Kurmann JA (1983) Bifidobacteria and Their

Role. Experientia Supplementum, vol. 39. Basel: Birk-

hauser.

Sanders ME and in’t Veld JH (1999) Bringing a probiotic-

containing functional food to the market: microbio-

logical, product, regulatory and labeling issues. Antonie

van Leeuwehoek 76: 293–315.

Tannock GW (1999) Probiotics: A Critical Review.

Wymondham, Norfolk, UK: Horizon Scientific Press.

470

BIFIDOBACTERIA

IN FOODS

BILE

A Lanzini, University of Brescia, Brescia, Italy

Background

0001 Bile is a complex aqueous solution of organic and

inorganic compounds secreted by the liver. It contains

bile acids, a class of detergent-like molecules that

exert their biological functions as lipid solubilizers

within the biliary tree and the gut lumen. The bile

acids are restricted to the enterohepatic circulation,

so as to reutilize many times these biologically

valuable molecules. This chapter reviews the com-

position and physicochemical properties of bile, the

mechanism involved in bile formation, and the func-

tion of bile acids as solubilizers of biliary and dietary

lipids.

Composition of Bile

0002 Inorganic compounds in human hepatic bile comprise

electrolytes in concentrations similar to those in

plasma, with the noticeable exception of HCO



con-

centration which is higher in bile. The osmolarity of

human hepatic bile is also similar to plasma osmolar-

ity, at about 300 mOsm l

1

0003 Organic compounds in bile comprise protein

and bile pigments in addition to the three biliary

lipids, bile acid, cholesterol, and phospholipid. Pro-

teins account for 4.5% of organic compounds in bile,

and their concentration ranges between 0.3 and 3.0 g

1

in typical human bile. The most abundant bil-

iary protein is albumin derived from the plasma pool.

In general, the biliary concentration of other plasma

proteins is inversely related to their molecular weight.

Bile also contains immunoglobulin A, lysosomal

enzymes, and plasma membrane enzymes. The secre-

tion of these latter proteins is influenced by the bile

acid secretion rate.

0004 Bile pigments constitute less than 0.3% of organic

compounds in bile, and their concentrations range

between 0.8 and 3.2 mmol l

1

in human hepatic and

gallbladder bile, respectively. Conjugation with glu-

curonic acid is essential for biliary bilirubin excretion,

and bilirubin diglucuronide is the major pigment in

human bile. The maximal bilirubin excretion capacity

depends on bile flow and is increased during enhanced

bile acid secretion rates. About 30% of bilirubin ex-

cretion is thought to be bile acid-independent, sug-

gesting that incorporation of bilirubin into mixed

micelles is not essential for its biliary excretion.

0005Many other endogenous substances are present in

human bile in addition to those listed above. These

include vitamins (mainly D

, folic acid), steroid

(estrogens), and thyroid hormones. Exogenous com-

pounds such as contrast media, some antibiotics

(ampicillin, metronidazole), cardiac glycosides, and

opiates may also be excreted in bile and undergo

some degree of enterohepatic circulation.

Biliary Lipid Chemistry and

Physicochemical Properties

Bile Acids

0006Bile acids comprise a class of molecules derived from

the hepatic catabolism of cholesterol. More than 200

bile acids have been isolated in human bile, and 92–

99% of total bile acids are constituted by mono-, di-,

and tri-hydroxy derivatives of a 24-carbon atom ster-

oid, cholanoic acid (Figure 1). The brief and system-

atic names and the relative proportions of individual

bile acids in human bile are shown in Table 1.

0007Virtually all biliary bile acid are amidated in man

almost exclusively with glycine or taurine at a ratio

of about 2:1. Sulfation on the steroid nucleus of

bile acids occurs to a significant extent in man only

for lithocholic acid, and glucuronidation is a trace

metabolic pathway in man.

0008From a physicochemical point of view, bile acids

are planar amphiphiles in that they exhibit both

hydrophilicity with one part of the molecule and

hydrophobicity with the remainder. In commonly oc-

curring bile acid molecules, the hydrophobic face

is constituted by the b side of the steroid nucleus,

and the hydrophilic face by the a side of the nucleus

and by the side chain. The relative potency of the

hydrophilic and hydrophobic functional groups in

affecting the physicochemical properties of bile

acids is referred to as the hydrophilic–hydrophobic

balance. The order of increasing hydrophilicity,

as assessed by high-pressure liquid chromatogra-

phy, follows the order taurine conjugates > glycine

conjugates > unconjugated bile acid; and trihydroxy-

lated > dihydroxylated>monohydroxylated bile acids.

0009Bile acid solubility is strongly dependent on amida-

tion. Unconjugated bile acids (pK

5.0) are insoluble

at a pH below 6–7, and glycine-(pK

3.8) and taurine-

conjugates (pK

< 1.0) are soluble at pH 4–5and

below 2, respectively. Fully ionized common bile

acids are present in physiological solutions as their

sodium salts. They are extremely water-soluble, with

a monomeric solubility of 1–3 mmol l

1

(Figure 2).

BILE 471

They are present in diluted water solutions as mono-

mers in the bulk water phase, and as an unstable film

on the water surface. Upon concentration, a critical

monomeric concentration is reached, called the crit-

ical micellar concentration (CMC), at which bile acid

monomers self associate to form multimolecular

aggregates called micelles. Micelle formation involves

back-to-back agglomeration of the hydrophobic side

of the bile acid molecules, with the hydrophilic

side facing water. At concentrations above the

CMC, bile-salt micelles have the capacity of incorpor-

ating and solubilizing otherwise insoluble com-

pounds, thus acting as detergents. In general terms,

hydrophobic bile acids form micelles at lower concen-

tration than hydrophilic bile acids, and the CMC

ranges between 0.5 and 11 mmol l

1

for common

bile acid (Table 1).

Phospholipids

0010The most abundant biliary phospholipid species are

phosphatidylcholines (lecithins), which account for

80–95% of phospholipids in human bile. Phosphati-

dylcholines are insoluble swelling amphiphiles, with a

monomeric solubility in water of about 1 pmol l

1

(Figure 2). Upon hydration (> 45% water), phospho-

lipids swell to form liquid crystalline phases consist-

ing of choline bilayers with interposed water layers.

These liquid crystalline phases may fold and aggre-

gate to form vesicular structures.

COOH

Cholesterol

Liver

Primary

Secondary

Intestinal

bacteria

Intestinal

bacteria

COOH

Cholic acid

Tertiary

Deoxycholic acid Lithocholic acid

Sulpholithocholic acid Ursodeoxycholic acid

COOH

Chenodeoxycholic acid

COOH

−

COOH

7-Oxolithocholic acid

COOH

Liver

fig0001 Figure 1 Structural formulae and sites of synthesis and metabolism of common bile acids in man. From Carey MC and Duane WC

(1994) Enterohepatic circulation. In: Arias IM, Boyer JL, Fausto N et al. (eds.) The Liver: Biology and Pathobiology, 3rd edn, pp. 719–767.

New York: Raven Press, with permission.

472 BILE

Cholesterol

0011 From a physicochemical point of view, cholesterol

is an insoluble nonswelling amphiphile, with a mono-

meric solubility in water of about 1 nmol l

1

at 37



(Figure 2). Upon hydration, crystals of cholesterol

monohydrate are in equilibrium with water-contain-

ing monomers of cholesterol monohydrate.

Bile Formation

0012Bile formation involves a number of secretory

processes in the hepatocyte and in the ductular cells

generating an osmotic gradient between the cells and

the biliary canaliculus and ductulus, with passive

water movement along this gradient until osmotic

equilibration is achieved. Canalicular and ductular

bile flow contribute 470 and 150 ml, respectively to

the typical total daily bile flow of 620 ml in man. Bile

flow varies from 1.5 to 15.4 ml min

1

,andis

linearly related to bile acid secretion rate (Figure 3).

The contribution of canalicular to total bile flow is

defined by the linear relationship between bile acid

secretion and biliary clearance of sugars (erythritol or

mannitol) acting as bile-flow markers. This relation-

ship identifies the so-called bile acid-dependent cana-

licular bile flow (about 60% of total canalicular bile

flow) generated by active bile acid transport into the

canaliculus. Extrapolation of the linear regression

function relating bile acid secretion rate and erythri-

tol clearance to a value of zero bile acid secretion rate

yields a positive zero intercept. This linear extrapo-

lation identifies the so called bile acid-independent

bile flow (about 40% of total canalicular bile flow).

0013Bile acid-independent bile flow is generated by

active transport of glutathione and organic anions

tbl0001 Table 1 Name and composition of biliary bile acids of typical

human bile

Brief name

of bile acid

Systematicname CMC

(mmoll

1

)

Percentage

in bile

Cholic 3a,7a,12a-

Trihydroxy-

5-cholanoic acid

11 35

Chenodeoxycholic 3a,7a-Dihydroxy-

5-cholanoic acid

435

Deoxycolic 3a,12a-Dihydroxy-

5-cholanoic acid

324

Ursodeoxycholic 3a,7b-Dihydroxy-

5-cholanoic acid

7 tr.–4

Lithocolic 3a-Monohydroxy-

5-cholanoic acid

0.5 tr.–3

Individual bile acids as a percentage of total bile acids. CMC, critical

micellar concentration; tr, trace.

(A)

(B)

BS−Ch micelle

(Rh, 1 nm)

'Mixed disc'

BS−L−Ch micelle

(Rh, 2−3 nm)

Unilamellar

BS−L−Ch vesicles

(Rh, 30−40 nm)

1−3 mmoll

−1

~1 pmol l

−1

~1 nmol l

−1

fig0002 Figure 2 Molecular models of (A) monomeric and (B) aggregated biliary lipids. Monomeric solubility in water and mean hydro-

dynamic radius (Rh) of aggregated lipids are also shown. BS, bile acids; L, lecithin; Ch, cholesterol). From Carey MC and Duane WC

(1994) Enterohepatic circulation. In: Arias IM, Boyer JL, Fausto N et al. (eds.) The Liver: Biology and Pathobiology, 3rd edn, pp. 719–767.

New York: Raven Press, with permission.

BILE 473

(bilirubin glucuronide, thyroid and steroid hormones,

drugs) into the canaliculus. Glutathione is synthesized

in the hepatocyte, secreted into bile, where it is hydro-

lyzed into the constituent amino acids. These amino

acids exert the osmotic gradient responsible for bile

acid-independent paracellular entry of water into

the biliary canaliculus. Na

and HCO



also contrib-

ute to generating the osmotic gradient responsible for

the bile acid-independent bile flow.

0014 Bile acid-dependent bile flow is a complex phe-

nomenon involving captation, intracellular transport

and biliary secretion of bile acids (Figure 4). Bile acid

captation occurs on the basolateral membrane of the

hepatocyte and involves two transport systems.

The first is a Na

-taurocholate cotransporter poly-

peptide dependent on a sodium gradient energized by

the Na

–K

–ATPase system. The second transport

mechanism is independent of sodium gradient and

consists of an organic anion transporting polypep-

tide. This system is not specific for bile acids, and

transports other organic anions such as bilirubin,

bromosulfophthalein, and estrogens.

0015 The process of intracellular bile acid transport is

poorly understood, and it is thought to occur by

diffusion of bile acid bound to cytosolic protein

from the sinusoidal to the canalicular pole of the

hepatocyte. Vesicular transport may also play a role

in regulating intracellular bile acid traffic, as sug-

gested by the finding that vesicle poisoning results in

cholestasis.

0016 Bile acid secretion into the biliary canaliculus in-

volves two ATP-dependent transport systems and

an electrogenic system driven by membrane poten-

tial. Monovalent bile acids are transported by an

ATP-dependent canalicular bile salt export pump,

and sulfated or glucuronated divalent bile acids are

transported by a canalicular multispecific organic

anion transporter. This latter transport system is

also involved in biliary excretion of bilirubin diglu-

curonide, glutathione, bromosulfophthalein, and

other anionic dyes.

0017Phospholipid secretion into bile is also an active

ATP-dependent process mediated by p-glycoprotein

encoded by two genes in humans (multidrug-

resistant; MDR1 and MDR3) that make cells resist-

ant to a variety of amphipatic drugs (colchicine,

vincristine, doxorubicin, and others) by stimulating

their extrusion from the cell. MDR3 protein is ex-

pressed exclusively on the bile canalicular membrane

and is specialized in biliary export of phospholipid,

as indicated by the finding that homozygous

disruption of the murine mdr2 gene (the equivalent

of human MDR3) leads to complete absence of phos-

pholipid in bile.

0018Cholesterol secretion into bile is tightly coupled

with that of phospholipid, but it is not clear whether

cholesterol secretion is carrier-mediated, as demon-

strated for phospholipids. It is likely that cholesterol

translocation to the outer leaflet of the bile canalicu-

lar membrane is mediated by a spontaneous flip-flop

mechanism.

0019The current hypothesis to explain the phenomenon

of lipid secretion into bile is summarized in Figure 5.

Bile acids are continuously pumped out of the bile

canalicular membrane into bile. When the outer leaf-

let of the bile canalicular membrane is sufficiently

enriched with phosphatidylcholine, it ‘buds out,’

forming a phospholipid–cholesterol bilayer that ad-

sorbs to bile acids and dissolves into mixed bile acid–

cholesterol–phospholipid micelles (see below).

0020Bile flow and composition changes along the bil-

iary tree, due to water and inorganic electrolyte

movements across the duct system. The net result of

these movements is an increase in bile flow due to

secretion of bicarbonate and water. This process is

under control of the enteric hormone secretin that

stimulates secretion of Cl



by a ‘cystic fibrosis

GSH

HCO

−

HCO

−

,OA

−

,OA

−

NTCP

OATP

MOAT

AE2

MDR3BSEP

−

fig0004Figure 4 Major membrane transporters involved in bile forma-

tion. NTCP, Na

taurocholate cotransporter polypetide; OATP,

organic anion transporting polypetide; BSEP, bile salt export

pump; MOAT, multispecific organic anion transporter; MDR3,

multi drug resistance 3; AE2, anion exchanger 2.

Bile acid secretion

Bile acid independent flow

Bile flow

Ductal flow

Canalicular flow

fig0003 Figure 3 Relationship between bile acid secretion into bile and

bile flow.

474 BILE