Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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MICROFLORA OF THE INTESTINE

Contents

Role and Effects

Probiotics

Role and Effects

B R Goldin, Tufts University School of Medicine,

Boston, USA

Description of the Microflora of the

Normal Adult Gastrointestinal Tract

0001 The gastrointestinal microflora of humans is a com-

plex ecosystem. More than 400 bacterial species have

been identified in feces from a single person. Ninety

per cent of the total microflora is represented by 30–

40 different species. Anaerobic bacteria are the pre-

dominant microorganisms in the gastrointestinal

tract, outnumbering aerobes by a factor of 10

. The

distribution and composition by genera of the most

prevalent microflora in the gastrointestinal tract are

outlined in Table 1.

0002 The saliva washes the bacteria in the oral cavity

deposited on teeth, soft tissue surfaces, and food into

the stomach where most of the bacteria are killed by

the low pH of the gastric juice. Consequently, only

10–100 organisms are isolated per milliliter of gastric

juice. The most commonly isolated bacteria in the

stomach are Gram-positive facultative forms such as

Streptococcus and Lactobacillus.

0003The bacterial population in the small intestine in-

creases from the duodenum to the distal ileum. The

bacterial composition of the proximal small bowel is

similar to the stomach and the number of organisms

per milliliter of contents increases from 10

to 10

the distal jejunum. The bacterial counts and complex-

ity of the microflora increase dramatically in the dis-

tal ileum. The total concentration of bacteria is

between 10

and 10

per milliliter of contents, and

anaerobic bacteria such as Bacteroides, Bifidobacter-

ium, and Clostridium are found in substantial

numbers. Distal to the ileocecal sphincter, the number

of bacterias increase dramatically. In the colon, the

total bacterial count approaches 10

per milliliter of

fecal material, and one-third of the fecal dry weight

consists of viable bacteria. The complexity of the

microflora also increases in the colon, and a number

of different strict anaerobes (see Table 1) become

predominant components of the flora.

Development of the Human Intestinal

Microflora

0004The human fetus exists in a sterile environment

until birth. Upon passage through the vaginal canal

and exposure to the outside environment, the new-

born gastrointestinal tract is rapidly colonized. In

breastfed and bottlefed infants, by the third day

after birth, Bacteroides, Bifidobacterium, Clostri-

dium, and Enterobacter are isolated from the feces.

For breastfed infants, by day 7, Bifidobacterium is the

predominant organism accounting for more than

90% of the bacterial population. For bottlefed

infants, by day 7, Enterobacterium accounts for the

majority of the fecal population. At 1 month of age,

Bifidobacterium is predominant in both breast and

bottlefed infants, but breastfed infants have approxi-

mately 10-fold higher Bifidobacterium counts. With

the introduction of a varied diet for bottlefed infants

and weaning for breastfed infants, the microflora

tbl0001 Table 1 Distribution of the bacterial flora in human

gastrointestinal tract

Region of the GI tract Bacteria Number per milliliter

of contents

Stomach Streptococcus 10

–10

Lactobacillus 10

–10

Small intestine

Duodenum and jejunum Streptococcus 10

–10

Lactobacillus 10

–10

Veillonella 10

–10

Ileum–cecum Bacteroides 10

–10

Clostridium

Streptococcus

Lactobacillus

Enterobacteria

Bifidobacterium

Colon and feces Bacteroides 10

Bifidobacterium 10

–10

Enterobacter 10

Peptococcus 10

Clostridium 10

Streptococcus 10

–10

Fusobacterium 10

–10

Lactobacillus 10

–10

Veillonella 10

–10

3904 MICROFLORA OF THE INTESTINE/Role and Effects

becomes more complex, and the numbers of Bacter-

oides and anaerobic Gram-positive cocci increase

over a period of a few months until their numbers

equal those of Bifidobacterium. By the second year of

life, the child’s intestinal microflora come to resemble

those of an adult.

Intestinal Microflora of the Elderly

0005 Studies in the elderly have indicated that there are

some changes that occur in the flora of humans in

the seventh through the ninth decade of life. Some

of the more significant changes are shown in Table 2.

The numbers of Lactobacillus, Streptococcus,

Enterobacter, and Clostridium perfringes increase,

and Bifidobacterium decreases.

Interaction of the Microflora with the

Intestinal Mucosa

0006 Owing to the difficulty of sampling human colonic

mucosa, there is limited information on the type (and

number) of bacteria that are firmly attached to the

colonic epithelial mucosa. The current information

available indicates that Gram-positive rods and

cocci and spirochetes are located on the mucosal

layer. There is a maximum of 10

bacteria adhering

per gram of colonic mucosal epithelial tissue, and

Bacteroides is the most common isolated organism.

Antibiotics can reduce by 5 logs the number of

bacteria on the colonic surface.

Alteration of Normal Gastrointestinal

Microflora

0007 The composition of the gastrointestinal microflora is

relatively stable. New bacteria introduced into the

gastrointestinal tract have difficulty permanently im-

planting into an established flora. Diet can affect

alterations in the microflora, but comparative studies

on human populations eating radically different diets

have revealed only selective changes (see below) and

not a general shift in the flora. In animals, it has been

demonstrated that limiting nutrient intake can cause

major changes in the composition of the microflora.

In humans, it is not known if self-imposed severe

dieting or anorexia will result in substantial changes

in the composition or distribution of the microflora.

0008Two known perturbants of human gastrointestinal

ecosystem: antibiotics and chemotherapeutic agents

can drastically alter the microflora, and this is often

reflected in diarrhea and, in some cases, an enteritis.

A specific example is the development of Clostridium

difficile-induced pseudomembraneous colitis after

antibiotic treatment. The antibiotic alteration of the

intestinal flora interferes with the normal suppression

of sporulation of Clostridium difficile with subse-

quent pathogenic toxin production.

Diet and the Gastrointestinal Microflora

0009There have been a number of studies in humans inves-

tigating the ability of diet to alter specific components

of the adult flora. One study reported no change in

the predominant organisms in the fecal flora of indi-

viduals changing from an omnivorous to a vegetarian

diet. The most detailed study involved a comparison

of subjects eating a Western diet with Japanese eating a

traditional diet and vegetarian and nonvegetarian

Seventh-Day Adventists. The subjects eating a Japan-

ese diet had higher fecal counts of Streptococcus

faecalis, Eubacterium lentum, and Eubacterium con-

tortium, and lower counts of Bacteroides.

0010In summation, dietary studies indicate that differ-

ent eating patterns influence specific bacteria in the

intestine, but overall, changes in the composition of

the flora are hardly dramatic.

Effect and Role of the Gastrointestinal

Microflora

Metabolic Activities of Microflora

0011In order to discuss the effects and role the microflora

has on the host, a discussion of the metabolic activity

of the flora should be considered. The more than 10

organisms that occupy the intestinal tract at any given

time can perform a large number of different reac-

tions, which can impact on the host. Any compound

taken orally or any substance entering the intestine

through the biliary tract or the blood or by secretion

directly into the lumen can potentially be a substrate

for bacterial conversion. Table 3 lists some of the

major reactions performed by the intestinal bacteria.

Most bacterial reactions can be classified as reductive,

hydrolytic, or removal of various functional groups. A

brief description of these reactions is presented below.

The potential impact on the health and physiology of

the host is discussed later in this article.

tbl0002 Table 2 Comparison of counts per gram of feces for selected

bacteria of the fecal flora

Bacterial genera Log of counts

Adults Elderly

Lactobacillus 5.8 7.5

Enterobacter 7.8 8.2

Streptococcus 7.9 7.4

Clostridium perfringens 4.4 6.6

Bifidobacterium 10.0 9.4

MICROFLORA OF THE INTESTINE/Role and Effects 3905

Role and Effect of the Intestinal Flora

Metabolic Role

0012 The hydrolysis of the glycoside linkage is one of the

most important reactions performed by the intestinal

microflora. Among the reactions performed by the

bacteria are a- and b-glucosidase and galactosidase,

b-glucuronidase, xylosidase, and cellobiase, and

a-mannosidase and fucosidase. These reactions are

responsible for the hydrolysis of oligo- and polysac-

charides, as well as a wide range of exogenous and

endogenous compounds conjugated by a glyosidic

bond with glucuronic acid or other sugar moities.

The bacterial glycosidases can remove terminal

sugars as well as hydrolyzed bonds in the middle of

a polysaccharide. The hydrolysis of amides, glucuro-

nides, esters, and nitrites, and a number of other

reactions listed in Table 3 relate to conversion of

drugs, pesticides, and various endogenous com-

pounds, such as cholesterol, estrogens, bile acids,

and their conjugates produced in the liver. Decarbox-

ylation and deamination are reactions related to

the bacterial fermentation of amino acid, and the

bacterial reactions of the Embden–Meyerhof–Parnas

glycolytic pathway result in fermentation of hexoses

to short-chain fatty acids.

Glycosides

0013Glycosides enter the intestine from two major

sources: from the diet or via the biliary tract. The

diet contains fiber, resistant starch, and phytochem-

icals, such as the glycosides of flavonoids and iso-

flavonoids. These compounds are not normally

absorbed in the upper gastrointestinal tract and,

therefore, serve as substrates for the vast majority of

microflora that reside in the distal ileum, colon,

and rectum. Glycosides coming from the liver

include endogenous compounds, an example being

estrogens and other biological molecules, and drugs

or environmental agents that are conjugated by the

liver via glycoside formation primarily with glucuro-

nic acid subsequently secreted into the bile, which is

deposited in the duodenum. The intestinal flora then

hydrolyze the glycoside bond, leading to the release of

potentially biologically active agycones (nonsugar

moiety).

0014There are a number of proven examples of the

importance of the action of bacterial glycosidases on

the host. Cycasin is a compound that is found in the

cycad plant, also referred to as tropical fern. The

compound is a glycoside of methylazoxymethanol,

which can cause intestinal tumors in conventional

and germ-free mice and rats. Germ-free animals lack

an intestinal microflora and do not develop tumors

when given cycasin; in contrast, conventional animals

when fed cycasin develop intestinal tumors. These

data suggest that the bacterial glycosidase in the in-

testine is necessary for the carcinogenic activity of

cycasin. This has been confirmed when germ-free

animals are monocontaminated with bacteria having

different levels of glycosidase. The carcinogenicity

after feeding cycasin has been found to be positively

correlated with the specific implanted bacterial

strain.

0015The action of the intestinal microflora on the plant

glycoside rutin is another example of the potential for

generating carcinogens. Rutin is not mutagenic prior

to the hydrolysis of the glycoside linkage, but the

hydrolysate is mutagenic.

0016There are a number of drugs whose action and/or

pharmacokinetics are influenced by bacterial glycosi-

dases. The metabolism of the cardiac glycoside

digoxin is an example of the effect of bacterial action.

To produce the drug’s pharmacological action, the

tbl0003 Table 3 Reactions performed by the gastrointestinal

microflora

Reaction Example of substrate orproducts

Hydrolysis Estrogen glucuronide

Glycosides Lactose

Sulfamates Cyclamate

Amides Methotrexate

Esters Acetyldtigoxin

Nitrates Pentaerythritol trinitrate

Dehydroxylation Bile acids

C-hydroxy groups N-hydroxyfluorenyl-acetamide

N-hydroxy groups

Decarboxylation Amino acids

Demethylation Biochanin A

Deamination Amino acids

Dehydrogenation Cholesterol, bile acids

Dehalogenation DDT

Reductive p-nitrobenzoic acid

Nitro groups Polysaturated unsaturated

Double bonds Fatty acids

Azo bonds Azo dyes

Aldehyeles Benzaldehdes

Alcohols Benzyl alcohols

N-Oxides 4-Nitroquinoline-1-oxide

Nitrosamines Dimethylnitrosamine

Gas production Products CO

Fermentation of sugars Products lactate, butyrate,

propionate, acetate, formate

Fiber, inulin,

oligosacchrides, starch

Disaccharides, monosacchides,

short-chain fatty acids

Fermentation of amino

acids

Products H

,CO

,CH

, amines,

phenols, indoles, NH

, organic

acids

Aromatization Quinic acid

Aceylation Histamine

Esterification Galic acid

3906 MICROFLORA OF THE INTESTINE/Role and Effects

bacterial flora must remove a trisaccharide from the

parent compound, releasing diagoxigenin. The fecal

flora of 36% of the residents of New York given

digoxin has been found to reduce the double bond

in the lactone ring, resulting in the formation of

dihydrodigoxigenin. This metabolite is not pharma-

cologically active. The extent of this bacterial modifi-

cation is reflected in the observation that 14% of

New Yorkers had high levels of digoxin metabolites in

their feces. This would result in lower-than-predicted

serum levels in these individuals. In this example, the

intestinal microflora inhibit the action of an orally

administered drug. The opposite is true for the cath-

artic agent, Cascara Sagrada. This agent is a mixture

of glycosides that are not pharmacologically active

until bacterial glycosidases in the intestine release

the active aglylone.

0017 An important physiological implication of the bac-

terial hydrolytic glycosidic reaction is the cleavage of

disaccharides. Lactose, sucrose, and maltose are dis-

acchrides that are normally absorbed in the upper

small intestine after hydrolysis by tissue enzymes

located in the intestinal mucosal brush border. There-

fore, contact with the majority of intestinal micro-

flora does not occur. However, because of either a

genetic defect or an acquired disorder, individuals

who have few or no disaccharidases do not absorb

the disaccharides in their small intestine. The sugars

are transported to the ileum and large bowel, where

bacterial sucrase, lactase, and maltase hydrolyze the

disaccharidases. The final bacterial end products are

short-chain fatty acids. An osmotic imbalance results

from the increased concentration of short-chain fatty

acids, and water flows into the lumen of the bowel,

resulting in diarrhea.

0018 The most common disorder is lactose intolerance,

which is common in adults residing in temperate

and warm climates. Less frequently encountered is

intolerance towards sucrose or maltose.

Amino Acids and Other Amines

0019 To some extent, food-derived proteins can remain

intact until they reach the large bowel. In addi-

tion, proteins from the epithelial cells, and digestive

secretions and mucins all contain protein. Bacteria

deaminate amino acids by five different mechanisms.

Amino acids also undergo bacterially derived decar-

boxylation. These reactions result in the formation of

a wide range of different amines and short-chain

organic acids that can be absorbed from the colon.

This process helps the host reclaim energy. The over-

production of, or lack of, absorption of hydroxy

short-chain fatty acids derived from amino acid

fermentation can cause diarrhea.

Fermentation of Carbohydrates

0020In this section, a more general description will be

given of the fermentation of carbohydrates by the

intestinal microflora. The major source of ferment-

able carbohydrates in the human colon is plant cell-

wall polysaccharides, such as pectins, cellulose, and

hemicellulose. Other sources of carbohydrate in the

colon are resistant starch and intestinal mucus. It is

estimated that between 20 and 70% of carbohydrates

are fermented per day. The end products of carbohy-

drate fermentation in the feces are acetate, propion-

ate, and butrarate. This fermentation also leads to the

production of hydrogen, carbon dioxide, methane,

and water.

Bacterial Lipid Metabolism

0021In humans, most of the fatty acids derived from dietary

lipids are absorbed in the small intestine. The human

colonic anaerobic microflora can hydrogenate and hy-

drate unsaturated fatty acids. Evidence for these reac-

tions comes from the isolation of 10-hydroxystearic

acid in the feces. The metabolism of lipids by the intes-

tinal flora has only a minor role in humans.

Bacterial Conversion of Cholesterol, Bile

Acids, and Bile Pigments

0022Cholesterol is a precursor of bile acids, and both

classes of compounds are structurally similar. These

compounds are endogeously synthesized in the liver

initially from two carbon units.

0023Cholesterol is also a component of the diet. Be-

tween 75 and 200 mg of cholesterol and its metabol-

ites are excreted in the feces daily. There are two

principal fecal metabolites of cholesterol, coprosta-

none and coprostanol, and one minor product, cho-

lestenone. The formation of coprostanol requires a

steroid nuclear hydrogenation of the 5,6 double

bond. The conversion of cholesterol to coprostanone

results from the reduction of the 4,5 bond and a C3

oxido-reductase converting the hydroxyl to a keto

group. Coprostanol accounts for approximately

50% of the total fecal neutral sterols, coprostanone

10–15%, and unmetabolized cholesterol the remain-

der. In germ-free animals, only cholesterol is re-

covered in the feces, providing evidence that the

intestinal microflora is solely responsible for the me-

tabolism of cholesterol in the intestine. The precen-

tage of intestinal cholesterol conversion varies from

70 to 85% in Americans and Western Europeans to

55 to 65% in Africans and Asians.

0024Bile acids synthesized in the liver are conjugated

viaan amide bondtoglycine or taurine.Theconjugated

MICROFLORA OF THE INTESTINE/Role and Effects 3907

bile acids are secreted in bile and are deposited in the

upper small intestine. The principal intestinal bacter-

ial reactions involve bile acids, primarily occuring in

the distal ileum and colon. The bacterial conversion

of bile acids includes: the hydrolysis of the amide

bond to release free bile acid from the glycine and

taurine conjugates; an oxidoreduction of the hy-

droxyl groups at C3, C7, and C12 to form either

oxo bile acids (hydroxyl to keto groups) or a-hy-

droxyl groups after the reduction of the keto groups

(inversion products); and dehydroxylation at C7, and

to smaller extent at the C3 and C12 positions. The

result of these reactions is the conversion of primary

bile acids to secondary bile acids and the reabsorption

of free bile acids from the terminal ileum and, to a

lesser extent, from the large bowel.

0025 Bile pigments derive from the breakdown of hemo-

globin. Between 200 and 300 mg of bilirubin, the

product derived from heme, are excreted daily in the

bile. Approximately 90% of bilirubin is conjugated to

glucuronic acid. The bilirubin conjugate is hydro-

lyzed by the intestinal microflora releasing the free

compound, which can be reabsorbed in the intestine.

Alternatively, bilirubin can be reduced by the

microflora to urobilonogen. Approximately 20% of

urobilinogen is reabsorbed from the intestine of

humans. The major components of bile, namely, chol-

esterol, bile acids, and bile pigments, are subject to

bacterial action in the intestine. Additional com-

pounds secreted in the bile such as, androgens and

estrogens (discussed below), are also subject to the

action of the intestinal microflora.

Additional Intestinal Bacterial Reactions

Vitamins

0026 The bacteria residing in the human intestinal tract can

synthesize homologs of vitamin K

(menoquinone 7).

The bacterial conversion, in part, occurs in the ileum

where menoquinone can be absorbed. Human adults

given low vitamin K diets for several weeks did not

exhibit a deficiency. Treatment of subjects with anti-

biotics at the same time as they were fed a low vita-

min K diet had a significant decrease in plasma

prothrombin levels. The synthesis of the peptides in

the prothrombin blood-clotting complex requires

menoquinone. These human studies demonstrate the

importance of intestinal bacterial metabolism with

respect to vitamin K synthesis.

0027 Humans depend solely on this action of intestinal

microflora for their vitamin B

(cyanocobalamin)

requirement indirectly via the consumption of meat

from ruminants that derive B

from synthesis by

their microflora. In addition, the human intestinal

microflora contributes significant amounts of vitamin

, and approximately 5 mg is excreted in feces daily.

At least two organisms, Pseudomonas and Klebsiella,

have been shown to synthesize vitamin B

in the

small intestine.

0028Biotin is synthesized to a major extent by the intes-

tinal flora. The administration of high doses of

antibiotics can cause biotin deficiency in laboratory

animals. The administration of antibiotics can lower

biotin urinary levels.

0029Several other B complex vitamins, namely folic

acid and thiamin, are synthesized by the human intes-

tinal microflora. The amount synthesized by bacteria

is not sufficient to eliminate the requirement for

dietary sources of folate and thiamin.

Estrogens and Androgens

0030Estrogens and androgens are modified by the intes-

tinal microflora. Estrone, estradiol, and estriol are

three major circulating estrogens excreted in the

bile, conjugated to glucuronic acid and/or sulfate.

Bacteria found in the feces can hydrolyze these conju-

gates, releasing the free estrogens. The nonconjugated

estrogens are then subject to further bacterial action.

A major reaction involves oxidoreduction of the

C17 position. Bacteria can convert estrone to

estradiol, and the fecal flora can also convert 16 a-

hydroxyestrone to estriol.

0031Several intestinal bacterial conversions of andro-

gens are also catalyzed by the human fecal flora.

The intestinal microflora can reversibly oxidize and

reduce the 3-hydroxy group, reducing steroid nuclear

double bonds at the 1- and 4-positions and resulting

in a complex interconversion of androgens. Evidence

for an introduction of a double bond into the andro-

gen nucleus has also been described. However, this

reaction only occurs under aerobic conditions and

therefore would be unlikely to occur in the highly

anaerobic environment of the colon.

Role of the Intestinal Flora in Health

Colon Cancer

0032Colon cancer is a common disease in Western Societies.

Epidemiological studies have shown that the incidence

of colon cancer is higher in North America and Western

Europe than in Africa, Asia, and South America. A

number of studies have shown a positive correlation

between consumption of beef, fat, and protein, and a

negative correlation with fiber, and the incidence of

colon cancer. It has been proposed that the dietary

influence on the etiology of colon cancer results, in

part, from alteration of the metabolic activity of the

3908 MICROFLORA OF THE INTESTINE/Role and Effects

intestinal microflora, more specifically, the ability of

intestinal bacteria to convert procarcinogens to prox-

imate carcinogens. This conversion would be greatest

in the large bowel where the bacterial population is

highest, and the transit time for the fecal stream is the

slowest. Several bacterial enzymes have been impli-

cated in the formation of mutagens, carcinogens,

and tumor promoters. Among the enzymes that have

the potential to increase the incidence of colon cancer

are: b-glucosidase, b-glucuronidase, b-galactosidase,

azoreductase, nitroreductase, 7-a-steroid dehydrogen-

ase, and 7-a-hydroxy-steroid dehydroxylase.

0033 There have been at least five different classes of

mutagens isolated in the feces that have the potential

to cause colorectal cancer. The final step in the syn-

thesis of fecapentenes (dodecapentaenyloxy-1,2 pro-

panediol), one class of mutagen, involves the action of

the intestinal flora.

0034 Bile acids, particularly the secondary bile acids, can

act as colon tumor promoters, and, as discussed pre-

viously, the fecal microflora are responsible for the

generation of secondary bile acids in the colon.

0035 Heterocyclic aromatic amines are also procarcino-

gens, which can be acted on by bacterial and

intestinal tissue enzymes to generate proximal car-

cinogens. Nitrosamines, which are also present in

the colon, are often direct-acting and spontaneously

breakdown to electrophiles, which react with DNA

and do not require activation by bacteria or tissue

enzymes. The final broad class of known potential

colon carcinogens are polycyclic aromatic hydrocar-

bons, which are generally activated by tissue micro-

somal enzymes. From this brief discussion, it is

apparent that the intestinal flora can be an important

factor in the etiology of colon cancer.

Bacterial Overgrowth

0036 Bacterial overgrowth occurs when there is a signifi-

cant increase in the bacterial population in the small

intestine and stomach. The proliferation of bacteria

in the upper gastrointestinal tract can be caused by

anatomic or physiologic derangements. One common

cause of bacterial overgrowth is the underproduction

of gastric acid. Gastric acid limits bacterial growth in

the stomach and upper small intestine. There are a

number of different causes for decreased gastric acid

production, such as atrophic gastritis, pernicious

anemia, surgical resections, and drug therapy. The

consequences of bacterial overgrowth are numerous,

including steatorrhea, vitamin deficiencies, and

carbohydrate malabsorption.

0037 More than 20 different species of bacteria have

been identified in the upper small intestine of patients

with bacterial overgrowth. The most common

clinical manifestation of bacterial overgrowth is

malabsorption of fat and fat-soluble vitamins. Small

bowel bacterial overgrowth can also cause megalo-

blastic anemia, which results from vitamin B

defi-

ciency. Binding of vitamin B

to bacteria in the upper

small intestine can prevent absorption of the vitamin

in the distal ileum. In-vitro studies have demonstrated

that Bacteroides, among the most common of intes-

tinal bacteria, bind the intrinsic factor–cobalamin

complex. Patients with bacterial overgrowth can

have high levels of B

in the lumen of the small

intestine, from nonabsorbed dietary sources and

from local bacterial synthesis. Despite this, patients

can still suffer from vitamin B

deficiency because of

the lack of absorption from the intestine.

0038Other consequences of bacterial overgrowth in-

clude impaired amino acid and carbohydrate absorp-

tion. Fecal nitrogen content is increased, serum

proteins are low, and this could result in a clinical

protein-calorie malnutrition.

Diarrheal Diseases

0039Disruption of the intestinal flora by antibiotics or

other drugs can lead to diarrhea. A disruption in the

balance of the intestinal microflora can lead to the

overgrowth of pathogens in the large bowel. An

example is antibiotic-induced Clostridium difficile

colitis. This pathogen resides as a spore in the colon

of a significant number of healthy individuals. Pa-

tients receiving antibiotics for various infections

have, as a result of their treatment, a decrease and

shift in the population of the normal flora. In some

instances, this leads to situations in which sporulation

of Clostridium difficile takes places with a concomi-

tant production of toxin, resulting in severe diarrhea

and, in some cases, a pseudomembraneous colitis.

The use of antibiotics in general, in a hospital setting,

results in diarrheal disease in 30–50% of the antibi-

otic-treated patients.

0040The etiological agents in most of the cases are

unknown. Another common cause of diarrhea is the

introduction of pathogens from food, water, or other

orally ingested material. The enterobacteriaceae

groups of Gram-negative rods include a number of

diarrheal pathogens, including specific strains of

Escherichia, Shigella, Salmonella, and Yersinia.

Other pathogens are Vibrio cholerae and Mycobac-

terium. Most of these pathogens are not normal resi-

dents of the intestinal tract; however, these agents can

disrupt the normal intestinal flora.

Health Implications of Probiotics

0041Just as bacterial pathogens introduced into the lumen

of the intestine can cause disease, the feeding of

MICROFLORA OF THE INTESTINE/Role and Effects 3909

specific types of bacteria can be beneficial to the host.

The term probiotic has been given to bacteria that,

when fed, confer health benefits to the host.

0042 Table 4 lists some of the microorganisms currently

being fed to humans and animals. A large number of

health benefits have been attributed to probiotics. A

partial list of these therapeutic claims for probiotics is

given in Table 5. A brief discussion of some of these

claims is included below.

Diarrheal Diseases

0043 An initial report with five patients treated with Lacto-

bacillus rhamnosum strain GG (LGG) for relapsing

diarrhea caused by Clostridium difficile toxin showed

that this probiotic could stop the diarrheal symptoms.

An additional 32 subjects were subsequently studied.

Of these 32 patients, 84% were cured by initial treat-

ment with LGG daily for 2 weeks. Three additional

patients were cured upon retreatment with LGG,

resulting in an overall cure rate of 94%.

0044 LGG was also shown to lower the rate of diarrhea

in Finnish travelers to Turkey. A study has also been

performed with American travelers to developing

countries fed either LGG or a placebo in a double-

blind study design. The subjects taking the placebo

had a 7.4% per day risk of developing diarrhea, and

travelers who ingested LGG had a 3.9% per day risk.

These data indicate that LGG provides a 47% protec-

tion rate against traveler’s diarrhea.

0045LGG has also been shown to be effective for

treating children hospitalized for severe diarrhea.

Children given LGG had a decrease in the duration

of their diarrhea from 2.5 to 1.1 days.

0046These studies indicate that a probiotic can be help-

ful in preventing antibiotic-induced, travelers’, and

young childhood diarrhea.

Lactose Intolerance

0047Ingestion of yogurt when compared with feeding

milk containing equal amounts of lactose resulted in

one-third the amount of hydrogen in the breath.

Breath hydrogen is an indicator of the passage of

intact lactose to the lower intestine. The reason

for this observation rests on the fact that the Lacto-

bacillus and Streptococcus in yogurt hydrolyze the

lactose in the yogurt, as well as in the upper small

intestine.

Food Allergy

0048Casein in milk can cause the first allergic reaction in

infants. Lactobacillus can degrade milk proteins to

smaller peptides and amino acids. One of the symp-

toms of milk allergy is atopic eczema. Several studies

have been performed showing that the feeding

of LGG to infants significantly decreased clinical

allergic symptoms when compared with a placebo

group. These results suggest that probiotics down-

regulate intestinal inflammation and hypersensitivity

tbl0004 Table 4 Probiotics in use for humans and animals

Genera Lactobacillus Bifidobacterium Streptococcus Additionalbacteria Yeast andmolds

Species acidophilus infantis cremoris Leuconostoc Aspergillus niger

casei longum lactis Pediococcus A. cerevisiae

rhamnosus adolescents salivarius ssp. thermophilus Bacillus A. oryzae

reuterii animalis intermedius Propionibacterium Candida pintolopesii

delbrueckii ssp. bulgaricus bifidum Enterococcus

brevis thermophilus E. faecium

fermentum

lactis

cellobiosus

plantarum

tbl0005 Table 5 Some of the health claims made for probiotics

Intestinalproblems Other disorders and diseases Other uses

Constipation Cystic fibrosis Adjuvant for vaccines

Colitis Vaginitis Recolonization of bowel after antibiotic use

Lactose intolerance Alcohol-induced liver disease

Salmonella and Shigella infections Cancer

Flatulence Hypercholesterolemia

Crohn’s disease Food allergies

Diarrhea

Antibiotic-induced

Infantile

Travelers

3910 MICROFLORA OF THE INTESTINE/Role and Effects

reactions in infants with food allergies and resulting

atopic eczema.

Liver Disease

0049 Studies in rats chronically fed alcohol have shown

that feeding LGG reduced liver pathology and plasma

endotoxin levels. These findings suggest that probio-

tics may be useful in preventing alcohol-induced liver

damage, a common human medical problem.

Colon Cancer

0050 There have been a number of studies showing that

feeding Lactobacillus acidophilus or LGG lowers

colon tumor incidence in rats administered the car-

cinogen dimethylhydrazine. There are no human clin-

ical intervention trials that have been performed to

determine if probiotics can prevent colon cancer.

See also: Amino Acids: Properties and Occurrence;

Bifidobacteria in Foods; Bile; Biotin: Properties and

Determination; Physiology; Cholesterol: Absorption,

Function, and Metabolism; Colon: Structure and Function;

Diseases and Disorders; Cancer of the Colon; Fats:

Digestion, Absorption, and Transport; Food Intolerance:

Food Allergies; Lactose Intolerance; Hormones: Steroid

Hormones; Lactic Acid Bacteria; Probiotics; Vitamin K:

Properties and Determination

Further Reading

De Kok TMCM and Van Maanen JMS (2000) Evaluation

of fecal mutagenicity and colorectal cancer risk. Muta-

tion Research 463: 53–101.

Gibson GR and Macfarlane GT (1995) Human Colonic

Bacteria. Boca Raton, FL: CRC Press.

Goldin BR (1986) In situ bacterial metabolism and colon

mutagens. Annual Review of Microbiology 40: 367–393.

Goldin BR (1998) Health benefits of probiotics. British

Journal of Nutrition 80 (supplement 2): S203–S207.

Goldin BR and Gorbach SL (1989) Impact of Anaerobic

Bowel Flora on Metabolism of Endogenous and

Exogenous Compounds. In: Anaerobic Infections in

Humans. New York: Academic Press.

Goldin BR, Lichtenstein AH and Gorbach SL (1994) Nutri-

tional and metabolic roles of intestinal flora. In: Modern

Nutrition in Health and Disease, 8th edn. Philadelphia,

PA: Lea & Febiger.

Hill MJ (1986) Microbial Metabolism in the Digestive

Tract. Boca Raton, FL: CRC Press.

Mitsuoka T (1992) The Human Gastrointestinal Tract. The

Lactic Acid Bacteria in Health and Disease. London:

Elsevier Applied Science.

Salminen S (1996) Lactobacillus GG. Nutrition Today

31(No. 6 supplement 1): 1S–52S.

Schrezenmeir J, de Vrese M and Heller K (2001) Inter-

national Symposium on Probiotics and Prebiotics.

American Journal of Clinical Nutrition 73(2S):

361S–498S.

Probiotics

J L Ras

, Jasenova 11/II (Lerak), Belgrade,

Yugoslavia

Introduction

0001The word ‘probiotic’ derives from the Greek words

meaning ‘for life.’ It refers to live microbial dietary

supplements which contribute to the intestinal micro-

bial balance, thus benefiting host health; they can also

beneficially affect the properties of the indigenous

flora. The use of probiotics in the diet stems from

Metchnikoff’s theory (1907) that the consumption

of large amounts of yogurt containing lactobacilli

prolongs life. Many reports since then have shown

that gut organisms, such as bifidobacteria, and other

lactobacilli also beneficially affect the host, including

human hosts.

0002The indigenous gut microorganisms have their own

protective function against invading pathogens. Vari-

ous factors, including diet, can affect the protective

gut flora. Special ingredients selectively stimulate the

growth and/or activity of a small number of beneficial

organisms in the colon. The potential benefits of

ingested bifidobacteria and lactobacilli include vari-

ous healthful aspects. Selection of microbial strains is

important in the development of improved probio-

tics. There is evidence that probiotics ingested can

influence the gut flora.

Overall Concept of the Importance of the

Gut Flora

0003The gut microorganisms colonize available habitats

in the gastrointestinal tract and, although not essen-

tial for life, they live in a stable relationship with their

host. Their presence is manifested by their effects on

the development of resistance factors, including de-

fense systems of the host, the protective effect of the

flora, and the production of microbial products.

Defense Systems and the Protective Flora

0004Experiments with gnotobiotes (animals which are

either germfree or have been inoculated with one or

more known species of microorganisms) have shown

that the lymph nodes are larger and more numerous,

and the number of lymphocytes within the nodes is

greater in conventional animals (those which retain

their normal gut flora) than in germfree animals.

Lymphocytes are the major constituents of the lymph-

atic tissues, and are associated with immunity. Spe-

cific lymphocytes participate, via plasma cells, in the

production of immunoglobulins.

MICROFLORA OF THE INTESTINE/Probiotics 3911

0005 The levels of plasma cells and immunoglobulins

are much lower in germfree than in conventional

animals, but when germfree animals are exposed to

microorganisms, the lymphatic tissues rapidly enlarge

and there is a gradual increase of plasma cells and

immunoglobulins. Consequently, microorganisms are

the major source of antigens in normal animals, in-

cluding humans, with dietary constituents making

a secondary contribution. The macrophages from

germfree animals digest bacteria, but more slowly

than those from conventional animals.

0006 The gut microorganisms have their own protective

function. They prevent colonization of an area in the

gut by invading pathogens by competition for essen-

tial nutrients and for attachment sites on the epithe-

lium. They inhibit the growth of invading pathogens

by the production of organic acids, particularly vola-

tile fatty acids, by the deconjugation of bile salts, and

by the production of antibacterial substances other

than acids. The protective effect of the indigenous gut

flora against pathogens has been proved experimen-

tally. For example, Shigella spp. are lethal to germfree

guinea-pigs, whereas they are unable to establish

in conventional guinea-pigs. Similarly, mice treated

with streptomycin show increased sensitivity to Sal-

monella. The indigenous gut flora normally acts syn-

ergistically with its host’s immunological system in

protecting against infections by intestinal pathogens.

Microbial Metabolic Products

0007 The gut microorganisms break down undigested or

unabsorbed dietary constituents, body secretions, and

ingested foreign compounds; they also produce cer-

tain vitamins. Consequently, numerous metabolic

products occur in the gut. These include a variety of

organic acids, protein degradation products, dehy-

droxylated bile acids, cholesterol metabolites, and

others. Many such products are potentially harmful

to humans, but most of them are detoxified in the

liver before excretion. High levels of some products

(e.g., dehydroxylated bile acids, cholesterol metabol-

ites) found in the gut, due to the effect of long-term

changes in diet on the metabolic activities of the indi-

genous flora, may be implicated in the etiology of

some degenerative diseases and cancerous conditions.

The type of diet influences the levels of harmful mi-

crobial products. (See Bile; Protein: Synthesis and

Turnover.)

Influence of Diet on the Gut Flora

Effects of Composition of the Flora

0008 It is difficult to induce major changes in the indigen-

ous gut flora by alterations in the diet of healthy

adults. Dietary effects on the composition of the

fecal flora are demonstrably slow and their long-

term effects may be variable. Subsequent studies sug-

gest that the type of diet may be the major factor

causing the variation in fecal flora and enzyme activ-

ity among various populations.

0009One study examined the fecal flora of nine rural

healthy Japanese people and eight urban healthy Can-

adians, who were consuming typical Japanese and

western diets, respectively. The number of eubacteria,

bifidobacteria, lactobacilli, and veillonelae, and the

frequency of occurrence of bifidobacteria were higher

in the Japanese than in the Canadians, but larger

numbers of Bacteroides and lecithinase-negative

Clostridium were found in the Canadians. In another

study, the fecal flora of 15 healthy elderly persons in a

rural area in Japan were compared with the flora of

persons living in urban areas. The diet of elderly

persons was characterized by a high intake of dietary

fiber. Significantly larger numbers of bifidobacteria,

but not of clostridia, were found in the group of

elderly persons. Dietary fiber composed of cellulose,

noncellulosic polysaccharides (hemicellulose, pectin,

inulin, guar, and plant gums) and lignin is resistant to

digestion by the human intestinal tract. Some of the

fiber is degraded in the colon by the flora, and the

main products of digestion are volatile fatty acids,

gases, and energy. (See Dietary Fiber: Properties and

Sources.)

Effects on Microbial Metabolism

0010The metabolic activities of the gut flora may be

affected by alterations in the diet, and extreme diets

may induce great changes in the bacterial activities.

For example:

0011Bacteria isolated from persons living on a high-fat

diet were more active in the dehydroxylation of

bile acids than were similar bacteria isolated from

persons living on a low-fat diet.

0012Bacteria isolated from persons living on a diet rich

in dietary fiber may be more active in digesting

cellulose and noncellulosic polysaccharides than

are similar bacteria isolated from persons living

on a low-fiber diet, because the enzymes catalyz-

ing polysaccharide degradation are inducible.

0013A diet rich in meat products but low in fiber

indirectly provides the flora with urea, amino

acids, bile acids, and neutral steroids. As a result,

increasing concentrations of ammonia, amines,

indole, dehydroxylated bile acids, and cholesterol

metabolites occur in the gut, along with increasing

activities of beta-glucoronidase, azoreductase, and

nitroreductase enzymes. (See Dietary Fiber:

Physiological Effects.)

3912 MICROFLORA OF THE INTESTINE/Probiotics

Influence of Special Ingredients (Prebiotics)

0014 Special ingredients refer mainly to nondigestible

oligosaccharides, that benefit the host by selectively

stimulating the growth and/or activity of one or a

limited number of beneficial bacteria (e.g., bifidobac-

teria and lactobacilli) in the colon. These ingredients,

called prebiotics, are not hydrolyzed in the small

intestine; they are not absorbed and reach the colon

where they are selectively utilized by the bacterial

flora.

0015 The best-known prebiotics are: fructooligosac-

charides such as oligofructose and inulin, lactulose,

maltooligosaccharides, galacto-, xylo-, gentio-, pala-

tinose-oligosaccharides, etc. Among many nondiges-

tible ingredients, lactulose has been most extensively

studied for its growth-promoting effect in vivo for

bifidobacteria and lactobacilli. It is not present in a

free state in milk but, upon heating milk, a part of the

lactose is converted to lactulose, and in sterilized

liquid formula feeds it amounts to 2–8% of the total

carbohydrate content. High levels of lactulose have

laxative effects, leading to diarrhea. (See Carbohy-

drates: Metabolism of Sugars.)

0016 Lactulose is used for babies in some formula feeds,

and for adults to promote the growth of bifidobac-

teria and lactobacilli. It is also used in the manage-

ment of liver disease, and in the treatment of chronic

constipation.

0017 Other nondigestible ingredients occur naturally in

many foods (e.g., Jerusalem artichoke, onion, wheat),

and some are isolated or synthesized. They are com-

mercially available under various trade names.

Potential of Ingested Bifidobacteria and

Lactobacilli

0018 Selected strains of bifidobacteria and lactobacilli are

commonly used in probiotics to suppress harmful

microbial populations in the gut and encourage bene-

ficial organisms.

0019 The major potential benefits of ingested bifidobac-

teria and lactobacilli are claimed to be:

0020 modification of the gut flora in infants (bifidobac-

teria)

0021 prophylactic and therapeutic functions

0022 improved lactose utilization (lactobacilli)

0023 possible anticarcinogenic activity

0024 control of serum cholesterol

0025 stimulation of the immune system

Modification of the Gut Flora in Infants

0026 It may be possible to modify the gut flora of formula-

fed infants if large numbers of Bifidobacterium bifi-

dum (10

–10

cells day

1

) are fed, if they survive

gastric transit, and if a fermentable carbohydrate

is available to the cells in the large intestine. Since

lactose of formula feeds does not reach the large

intestine in sufficient amounts to promote the

growth of bifidobacteria, the addition of bifidogenic

substances could provide a fermentable carbohy-

drate. The improved formulation of milk is also

an important factor. (See Infants: Breast- and Bottle-

feeding.)

Prophylactic and Therapeutic Functions

0027There is a clear evidence that bifidobacteria and

lactobacilli are antagonistic to various harmful bac-

teria in the gut. Bifidobacteria (B. bifidum, B. longum,

B. infantis, etc.) produce acetic and lactic acids, with

small amounts of formic acid; and some strains pro-

duce antimicrobial substances other than organic

acids. The antagonistic effect of acetic acid against

Gram-negative bacteria is stronger than that of lactic

acid, and the former is produced in greater amounts

by bifidobacteria. Lactobacilli, such as Lactobacillus

acidophilus and L. casei, create lactic acid and small

amounts of hydrogen peroxide to suppress harmful

bacteria; some strains of L. acidophilus produce anti-

biotic-like substances, including acidophilin, lactoci-

din, acidolin, and possibly others. Deconjugation of

bile salts to free bile acids by both bifidobacteria and

lactobacilli may also function in the control of the

bacterial flora. Large numbers of bifidobacteria and

lactobacilli in the gut may involve competitive antag-

onism against invading pathogens.

0028The protective effect of bifidobacteria and lactoba-

cilli against enteric infections and the side-effects of

oral antibiotic therapy was shown in human studies.

Comparative feeding studies of two groups of infants,

one receiving formula feeds containing viable

B. bifidum and lactulose, and the other receiving a

buttermilk preparation, showed that enteric infec-

tions were eight times more frequent in the butter-

milk-fed group than in the formula-fed group. The

beneficial effect of L. acidophilus or L. casei in the

prevention of side-effects of oral antibiotic therapy

(e.g., moniliasis, staphylococcal enteritis) in children

was also shown. It has been suggested that daily

ingestion of large numbers of the lactobacilli (e.g.,

L. acidophilus) may protect consumers from diarrhea

caused by intestinal pathogens (e.g., traveler’s diar-

rhea). Dietary supplements of bifidobacteria and

lactobacilli have beneficial effects in children with

enteric infections. For example, the ingestion of a

freeze-dried culture of B. bifidum in conjunction

with lactulose was shown to eradicate enteropatho-

genic Escherichia coli strains in more than 80% of

cases. Many reports indicated the beneficial roles of

bifidobacteria in the management of chronic liver

MICROFLORA OF THE INTESTINE/Probiotics 3913