PROBIOTICS

ELSEVIER

Biotechnology Advances, Vol. 16, No. 3, pp. 581-608, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All fights reserved

0734-9750/98 $19.00 + .00

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PROBIOTICS: FUNCTIONALITY AND COMMERCIAL STATUS

SAUL SCHEINBACH

Nabisco Research, Schaeberle Technology Center, 200 DeForest Ave., E. Hanover, New Jersey 07936 email:[email protected]

ABSTRACT

Probiotics in the form of fermented milk products have been consumed for centuries. In this

century various health benefits have been purported to result from consumption of foods

containing live microorganisms, particularly lactic acid bacteria (LAB). Probiotics can

provide relief for lactose intolerant individuals and reduce bouts of diarrhea. Evidence for

other claims such as lowering serum cholesterol, suppressing cancer and stimulating the

immune system remains to be clearly established by conducting well-controlled, statistically-

valid clinical trials. Although the benefits to healthy individuals are uncertain, many

consumers especially in Japan and Europe, perceive probiotic products to be healthful, and

sales are robust. © 1998 Elsevier Science Inc.

KEY WORDS

Probiotics, functional food, enteric microflora, lactic acid bacteria, lactose intolerance,

diarrhea, cancer, yogurt

I N T R O D U C T I O N

In recent years the concept o f providing functional foods containing healthful

components rather than removing potentially harmful ones (e.g., saturated fat) is gaining

ground in the United States. Functional foods, designer foods, pharmafoods and

nutraceuticals are synonyms for foods with ingredients that can prevent and treat diseases

581

582 s. SCHEINBACH

[39]. A probiotic may also be a functional food, but more specifically it is a live microbial

feed supplement that beneficially affects the host beyond correcting for traditional nutrient

deficiencies by improving its intestinal balance [31 ]. Hence, it may be considered a functional

food with the special property of containing live, beneficial microorganisms [84]. Regulation

of the intestinal microbial balance results from the competition among the many bacterial

species that survive passage through the upper gastrointestinal tract and colonize the human

colon. A health benefit can also arise from the ability of an ingested microorganism to

contribute an enzyme to the small intestine e.g., I]-galactosidase (lactase) that many adults

lack.

Considering that 75% of the wet weight of our fecal output is composed of bacteria and

each gram contains at least 1 x 1011 organisms from at least 50 genera, belonging to over 400

species, we may be thought of as the outer covering of the most complex microbial ecosystem

that we know [67]. Through fermentation the colonic bacteria are able to produce

compounds that have positive and negative effects on both gut and systemic physiology. For

example, they produce short chain fatty acids (SCFA) from the metabolism of complex

carbohydrates that reach the colon [21]. The SCFA become an energy source for the host's

colonocytes and the microbes themselves respond to fluctuations in substrate availability, redox potential, pH and 02 tension in the colon [22]. With few exceptions, ingested bacteria

do not survive the high acid conditions in the stomach or the bile acids secreted by the liver

into the duodenum. In addition, the high numbers of well-adapted indigenous

(autochthonous) microfiora present severe competition for any transient (allochthonous)

organism attempting to compete for nutrients and mucosal attachment. These considerations

greatly restrict the scope of organisms that may be considered for probiotics.

Although outside the scope of this review, an alternate approach to that of consuming

probiotics is to consume substances (prebiotics) that get to the colon undigested and

stimulate the growth of beneficial autochthonous microflora.

Most studies on probiotics have focused on the LAB, particularly the genera that are of

human intestinal origin, Lactobacillus, Bifidobacterium and Streptococcus, or Enterococcus, either singly or in mixed culture. For a culture to be considered a good probiotic candidate it

should have several characteristics [20]. For example, it should normally be found in the

intestinal tract, maintain viability in the carrier (food), survive passage from stomach to colon

where it can adhere to the mucosa and, of course, be beneficial.

EFFICACY OF PROBIOTICS

At the beginning of the century it was proposed that by displacing toxin-producing

intestinal microflora, the live cultures in yogurt (later shown to be Lactobacillus bulgaricus

PROBIOTICS 583

and Streptococcus thermophilus) were responsible for the longevity of Bulgarians who

regularly consumed yogurt [85]. These organisms were later shown not to survive in the

intestinal tract, but a variety of claims have since been made for the health benefits derived

from ingesting these and other live LAB. According to reviews by various authors, probiotics

can alleviate lactose intolerance, lower serum cholesterol, reduce diarrheal incidence, stimulate

the immune system, control infections, act as antibiotics, suppress tumors and protect

against colon/bladder cancer by maintaining a healthy intestinal microflora balance [20, 26, 32,

68, 103]. Others have cautioned that in many instances, well-documented evidence is lacking

for the healthful effects claimed for probiotics and that more careful work needs to be done on

characterizing the bacterial strains and their effects [54, 108, 124]. Justification for health

claims comes mainly from animal studies. These have shown that germ-free (gnotobiotic)

animals or animals whose gut microflora have been perturbed by antibiotics are more

susceptible to disease, and resistance can be restored by oral administration of fecal

suspensions from healthy individuals, a route taken by the USDA to reduce salmonellosis

infection in commercially reared chicks [52]. The organisms used in probiotics are known to

produce antimicrobial substances that might affect the colonic microflora balance [87].

Mechanisms by which probiotics could improve health include:

1. production of acids, peroxides or bacteriocins bactericidal to groups that negatively

impact health;

2. competition with pathogens for mucosal binding sites;

3. competition for substrates;

4. stimulation of the immune system.

While there is some evidence to indicate these processes occur in vitro and in animal

models, the efficacy of probiotics for humans remains speculative because well-designed and

controlled studies that yield more conclusive evidence with proper statistical analyses

allowing claims to be incorporated into established nutritional pathways are often lacking [56,

95]. Notwithstanding a lack of clear-cut mechanisms, probiotics may provide health benefits

under certain circumstances.

Lactose intolerance

Most of the world's population (60-90% of non-Caucasians and 6-12% of Caucasians)

become lactose intolerant after weaning [6, 66]. This stems from a 90-95% decline in the

production of lactase [34]. Any lactose reaching the large intestine is metabolized by the colonic microflora to yield CO2, methane and hydrogen and the latter is typically noted as an

increase in breath hydrogen. The presence of lactose also alters the osmotic balance in the

colonic lumen. Thus, symptoms including abdominal bloating, cramping, flatulence and

584 S. SCHEINBACH

diarrhea ensue [121]. Alleviation of lactose intolerance is probably the best established health

claim for probiotics. A summary of recent clinical trials on controlling symptoms in lactose

intolerant subjects by LAB is presented in Table 1. A number of these human studies

provide convincing evidence that the addition of certain starter cultures to milk products,

even with high levels of lactose, allows them to be consumed by lactose intolerant individuals

without experiencing the usual rise in breath hydrogen or associated symptoms [65, 69, 78,

79, 113]. Fermented milk products like yogurt are less likely to cause gastric upsets in two

ways. First, they usually contain less lactose (4% vs 6%) due to microbial digestion during

fermentation. Second, indigenous lactase, present in the ingested cells can make up for the

host's deficiency.

Adding Lactobacillus acidophilus concentrate to cold milk results in unfermented

acidophilus milk. Lactose intolerant individuals who want to include a dairy product in their

diet, but dislike the taste or texture of yogurt can consume acidophilus milk, however, it has

not been consistently effective in preventing malabsorption in adults [63, 69, 83, 113]. In

children, acidophilus milk reduced symptoms but not breath hydrogen [89]. Whether or not

malabsorption is prevented appears to depend, in part, upon the extent to which intracellular

bacterial enzyme is released following ingestion. Sonication of the L. acidophilus cells prior

to their addition significantly alleviated lactose malabsorption [82, 83], indicating that cell

lysis promotes lactose digestion. This premise is supported by the fact that milk containing

or fermented by L. acidophilus, which survives intestinal transit intact, is not as effective as

milk containing or fermented by S. thermophilus, which is killed upon ingestion [63, 69].

Strain differences may also play a role. In a recent double-blind study comparing four L.

acidophilus strains, it was shown that effectiveness is most dependent upon the tolerance of

a strain to bile and acid rather than its lactase level or lactose transport [91]. In addition, cells

from a variety of S. thermophilus strains were shown to contain more lactase than strains of

Lactobacilli or Bifidobacteria [110]. As a consequence, amelioration of lactose intolerance

effects is best accomplished by consumption of ordinary yogurt or thermophilus milk [65],

which are both fermented by S. thermophilus.

The psychological state of subjects also appears to be important [122]. In a double-

blind trial, 15-30% of self-described lactose intolerant individuals were actually lactase

persistent, possibly causing unwarranted conclusions in some earlier trials. There are few

double-blind studies demonstrating efficacy in reducing symptoms because it is difficult to

blind a study involving yogurt consumption; nevertheless, yogurt is still recommended for

symptomatic people wishing to consume lactose-containing products [121].

PROBIOTICS

Table 1. Summary of Clinical Trials on Controlling Symptoms of Lactose Intolerance by

LAB (adapted from 109)

585

N u m b e r of Lactose Product/Culture Tested aSignif icant Breath Intolerant Subjects Hydrogen Reduction [ref.] vs Milk or Lactose

Unfermented L. acidophilus milk + +

12163] 10165] 91113]

14182]

¥o~t Yogurt Buttermilk, Unfermented L. acidophilus milk Heated ),o~:urt Yogurt Heated yogurt Unfermented L. acidophilus milk As above but sonicated

+

+ +/-

+

8 [ 129] Three yogurts + Dannon® +Royal Maid® -Borden®

10169] +/- @107 cfu/ml + @108 cfu/ml

7[79]

12[79]

22[78]

Unfermented S. thermophilus, L. bulgaricus milk Unfermented L. acidophilus milk with: LA1 LA2 N C F M 7 yogurts with different S. thermophilus/L, bulg, aricus strains S. thermophilus/L, bulgaricus yogurt Fermented milk with:S, thermophilus

:L. bulgaricus :L. acidophilus :B. bifidus

Yogurt(n=22) Yogurt and a meal(n = 12) Yogurt and lactose(n = 10) Milk with L. acidophilus NCFM Milk with S. thermophilus and L. lactis Unfermented acidophilus milk with:

strain ATCC4356 strain B strain N 1 strain E

8 children[89]

11191]

- @ 107 cfu/ml +/- @108 cfu/ml - @107 or 108 cfu/ml +

+ + +

+ +

_b +b

+/- +/- +

a+ = positive result; - = negative result; b other symptoms significantly reduced compared to control.

586 S. SCHEINBACH

Cholesterol reduction

Reports that LAB consumption is hypocholesterolemic are controversial, often lacking

controls or using too few subjects to be significant. Two possible mechanisms for

microbially-induced lowering of serum cholesterol have been suggested, but neither have been

shown conclusively. The first proposes that, since cholesterol is used in the production of

bile acids, enhanced catabolism and excretion of bile acids might reduce serum cholesterol

[15]. The major primary bile acids made by the liver are cholic and chenodeoxycholic acids.

These are often conjugated with glycine or taurine to form glycocholic or taurocholic acid,

respectively, but may be deconjugated by a microbial enzyme, bile salt hydrolase (BSH).

Deconjugated bile salts are more likely to be excreted, resulting in increased cholesterol

breakdown.

Under anaerobic conditions human isolates ofL. acidophilus can deconjugate taurocholic

and glycocholic acid [36]. Chikai et al. [15] observed that gnotobiotic rats exhibited an

increase in total fecal bile acids when inoculated with intestinal microflora of human origin

able to deconjugate bile acids. They suggested that free bile acids, formed in the colon, are

more readily excreted than their conjugates and that the adherence of free bile acids to

bacterial fiber enhanced bile excretion. Fletcher [29] found that serum cholesterol levels

dropped in pigs, but not in hamsters or rats that were fed LAB having high BSH activity.

The report also considered the potential safety of releasing more bile to the colon where it

could be metabolized to secondary bile acids by microbial enzymes (see Cancer below).

Currently, 300 strains of Lactobacilli are being tested for BSH activity as a possible measure

of cholesterol-reducing ability [30].

It has also been proposed that microbes directly assimilate cholesterol [35]. Significant

cholesterol assimilation in the presence of bile was reported in vitro for one strain of L.

acidophilus isolated from a pig [35], while others from humans assimilated cholesterol to

lesser extents [38]. The pig strain also inhibited increases in serum cholesterol levels in pigs

fed a high-cholesterol diet [35]; however, these claims are controversial and have not been

confirmed. For example, another group tested the ability of the pig strain, at a dose of lxl 0 ll

cfu/day, to lower serum cholesterol in pigs and hamsters and found no effect [29].

Various LAB have been tested for a hypocholesterolemic effect in humans. Lin et al.

[70] set up the largest human study to date involving 354 subjects in a double-blind, placebo-

controlled trial. They tested tablets (Lactinex TM) containing about 2 x 106cfu/tablet of L.

acidophilus and L. bulgaricus. The cultures were shown to assimilate cholesterol under

certain in vitro conditions. However, after 157 people consumed four tablets a day for six

weeks, they showed no significant differences in serum cholesterol levels as compared to the

placebo group.

PROBIOTICS 587

More recently, two double-blind, placebo-controlled studies looked at the

hypocholesterolemic effects of a Danish milk product (Gaio) fermented by Enterococcus

faecium (about 2 x 108cfu/ml) and two strains ofS. thermophilus (about 7xl08cfu/ml). Both

studies were sponsored by the manufacturer. Neither could show an unambiguous effect.

The first study [1] tested 29 normocholesterolemic men, all aged 44 years. The test group

exhibited a 10% drop in serum LDL-cholesterol following six weeks of diet supplemented

daily with 200 ml of the product as compared to a placebo group of 28 subjects who drank an

unfermented low-fat milk that was chemically acidified. Unfortunately, the two groups were

not randomized beforehand, resulting in a significantly higher LDL-cholesterol mean for the

test group at the start of the trial. Thus, at least a portion of the observed decrease could

have resulted from regression to the "true" mean. In the second study 44 healthy men and

women 50-70 years old consumed 200 ml daily of fermented product for six months [104].

The placebo group, comprising 43 subjects, drank chemically acidified milk. Subjects

receiving bacteria exhibited a rapid fall in LDL-cholesterol within the first month. However,

by the end of the study the placebo group showed a similar decrease such that no significant

difference existed between the two groups. A major complication was the fact that the cell

titer of E.faecium in the fermented product dropped three to four logs during the trial period.

Whether or not a greater response would have been seen if the higher dose had been

maintained is unknown. Moreover, it is unclear from these studies what dose must be

reached before an in vivo effect can be observed.

Conflicting and variable results among studies may occur from the use of different

doses, strains and even subjects. For example, Richelsen, et al. [104] found that three months

into their study they could divide subjects into responders, who exhibited a decrease in LDL-

cholesterol, and non-responders who did not. The biological basis for this is unknown. Since

there are no convincing double-blind trials in humans, a hypocholesterolemic effect of feeding

LAB to humans has yet to be scientifically proven.

Diarrhea

Diarrhea has many causes so it is difficult to evaluate the effects of probiotics on

limiting its severity. Worldwide, diarrhea is the greatest killer of children and rotavirus is its

most common cause [16]. The gastrointestinal tract of the newborn is inoculated by the

mother's vaginal and fecal flora [86]. Following a brief bloom of facultative anaerobes,

bifidobacteria are the first anaerobic group to establish themselves in high numbers

(1 x 1011cfu/gm feces), comprising over 99% of the total population. This proportion is lower

in formula-fed infants, but still constitutes about 95% [60]. Their growth creates a low pH

environment in the colon by producing lactic and acetic acids which may exert a protective

588 S. SCHEINBACH

effect against diarrhea by inhibiting the growth of Gram-negative facultative anaerobes that

could be pathogenic. Breast vs. formula feeding of infants helps to maintain high

bifidobacteria levels [5, 9] resulting in a colonic pH of 4.5-5.0 for breast-fed vs. pH 5.7-6.7

for bottle-fed infants. A decreased pH for the former probably occurs, in part, because

human milk has a lower protein content and less buffering capacity than cow's milk [14].

Breast milk also contains a glycoprotein bifidus growth promoting factor composed of

glucose, galactose, fucose and N-acetylglucosamine [12].

Microbial succession proceeds with either diet, but occurs more rapidly in babies fed

formula, so that by two years of age Bacteroides spp., clostridia, streptococci and other

anaerobes establish themselves, and the bacterial colonic community approaches that of

adults, with the bifidobacteria population under 25% [60]. Once the gut ecosystem has

matured it acts as a physical and physiological barrier to the entry of pathogenic bacteria and

antigens from the gut lumen. At the genus and species level the gut microflora appears to

resist changes in diet [27], being perturbed only by major stressors such as antibiotic therapy

or pathogen colonization [67, 112]. However, the recent use of pulsed-field gel

electrophoresis and ribotyping to differentiate between the bifidobacterial and lactobacillus

populations of two individuals revealed that stability is not common to all individuals.

Humans can harbor nonoverlapping strains and, at least in some people, these strains can

exhibit shifts over time [81]. It is likely that as more studies employ the increased

discriminatory power of molecular genotypic techniques the intestinal microflora will be seen

to be less immutable than originally thought.

Four well-designed, double-blind, placebo-controlled human clinical trials on the use of

probiotics to alleviate diarrhea have yielded positive results. These studies, done in children

suffering from rotaviral diarrhea, have shown that accelerated recoveries and/or less severe

symptoms can occur following administration of certain probiotics [8, 61, 106, 116].

Saavedra et al. [106] performed a trial with 55 children, 5-24 months old, admitted to a

hospital over a 17-month period. They evaluated the efficacy of a formula containing

Bifdobacterium bifidum and S. thermophilus for the prevention of acute diarrheal episodes and

rotaviral shedding. They found that significantly fewer children (6.9% vs 31%) receiving the

microbially-supplemented formula (n=29) contracted diarrhea as compared to the

unsupplemented control group (n=26). In addition, those in the test group were less likely to

shed rotavirus (10% vs 38%). A similar study [116] was conducted with children (1-36

mos.) suffering from rotaviral, bacterial or diarrhea of unknown cause. Children in the test

group received a Lactobacillus casei GG dose of 1 x 101° cfu over a five day period. The

duration of diarrhea was shortened from 3.8 days in the placebo group (n=64) to 2.7 days in

patients receiving the probiotic (n=59). The rotaviral-positive subgroup especially benefited

from L. case i GG therapy (n = 13), whereas patients with confirmed bacterial diarrhea (n=l 1)

PROBIOTICS 589

Table 2. S u m m a r y o f Clinical Trials to Control Diar rhea by L A B (adapted f rom 109)

Culture (ref.) L. acid.[7]

S. therm. L. acid. L. bulg.[97]

Subjects a 59 adults

94 children < 3 years

old

C a u s e Abdominal symptoms Acute onset

> 10 months

Lactobacilli[100] 26 healthy Travelers' diarrhea adults

53 children Various causes 23 healthy

adults 23 healthy

adults 36 adults

with ETEC 61 adults

E. faecium[8] Lactinex TM

Lactobacilli[ 171 Lactinex TM

Lactobacilli[ 18] Lactinex TM

Lactobacilli[ 18] Sweet acid. milk

[931 B. longum[19] 10 healthy

adults

Enterotoxigenic E. coli (ETEC)

ETEC

Neomycin

Irritable bowel

Erythromycin

L. casei GG[46] 5 adults C. difficle colitis with colitis 15 children Intractable B. brevi, B.bif.

L. casei[58] L. acid.[107] 11 adult

females Undergoing

radiation therapy

L. casei GG [117] 8 healthy Erythromycin adults

L. casei GG[94] 349 healthy Traveler's diarrhea adults

Amoxicillin 15 children (5-76 mos.) 47 children (4-45 mos.) 29 children (5-24 mos.)

Lactinex TM

Lactobacilli[ 123 ] L. casei GG[61]

B. bifidum S. therm.[106]

L. caseiGG, L. caseirh.[73]

L. caseiGG[101]

L. casei GG[96]

L. casei GG[62]

L. casei GG [116]

30 children (4-35 mos.) 21 children

20 children (1-24 mos) 21 children (5-28 mos.) 59 children (1-36 mos.)

Acute viral (mainly rotaviral)

Acute gastroenteritis

(mainly rotaviral) Acute rotaviral

Acute (some rotaviral)

Acute (some rotaviral)

Acute rotaviral

Acute rotaviral, bacter., unknown

Resu l t s $ symptoms

$ duration

C o m m e n t s

$ symptoms

No controls

Lower incidence

+

,[, symptoms

$ symptoms

$ duration

$ incidence

$ duration, symptoms $ incidence

No prevention

$ duration

$incidence androtavkal

shedding $ duration,

IgA $ duration

$ vomiting, watery stool $ duration

$ duration

Lot to lot cell variability

Few subjects

Few subjects, no controls

No controls ?

? No controls, subjective reporting

Few subjects, no statistics

Only in 1 of 2 destinations

+

+

Neg. for 14 L. + casei rh. cases

+ Pos. for acute non-bloody cases

Pos. for acute watery diarrhea No placebo for control group No effect on

bacterial cases

Efficacy b ?

aDoes not include controls; b? = insufficient information to determine if results were truly

oositive.

590 S. SCHEINBACH

did not. The third study [61] done with 71 children (4-45 months), 47 of whom received L

casei GG, either as a fermented milk (Gefilac) or freeze-dried powder, indicated that the mean

duration of rotaviral diarrhea could be shortened from 2.4 to 1.4 days. The positive effect

was abolished by pasteurization. In the fourth study E. faecium (SF68) was tested for its

effect on children (1 month-9 years) with diarrhea of various causes [8]. A significantly

greater proportion of the children receiving 2-4 x 107 cfu of SF68 daily (n=53) recovered as

compared to the control group (n=51) who were fed daily a mix o fL . acidophilus (5 x 108

cfu), L. bulgaricus (5 x 108 cfu) and Lactococcus lactis (4 x 109 cfu). For example, after two

days o f treatment, recovery was 62% vs 35% for test and controls groups, respectively. If a

control group not fed live bacteria had been used instead, greater differences might have been

seen.

Other trials with children have suffered from one or another experimental flaw [62, 73,

96, 101]. In a double blind study [73] 49 children (4-35 months) admitted to a hospital

during a six-month rotavirus epidemic were divided into three groups receiving L. casei GG,

L. acidophilus or a mix orS. thermophilus and L. bulgaricus. Each group received one of the

three probiotics twice daily for five days. The duration of diarrheal episodes decreased

signficantly and the immune response to infection was enhanced in children fed L. casei GG,

but not L. acidophilus or those fed the mix. The L. acidophilus thought to be present in the

commercial preparation (Lactophilus) fed to the second group was actually a Lactobacillus

casei rhamnosus strain at a titer considerably lower than expected. A shortened duration for

rotaviral diarrhea was also found for 21 children (5-28 mos.) fed 1 x 101° cfu L. casei GG

twice daily for five days as compared to an equal number in the control group [62].

Unfortunately, the control group did not receive a placebo. Two studies where the etiology

was not apparent [96, 101 ] observed a significant decrease in symptoms for children (1-24

mos.) suffering from watery, non-bloody diarrhea who were fed about 1 x 1011 cfu L. casei

GG twice daily over two days as compared with children fed a placebo.

In the case of adults, the effect of probiotics on diarrheal symptoms is less evident.

Oksanen et al. [94] gave packets containing either L. casei GG or a placebo to 756 travelers

going to two different Turkish cities. Subjects were asked to drink the contents of one

package in water twice daily, yielding a dose of 2 x 109 cells, and fill out a questionnaire upon

their return. The results showed that the GG group (349 subjects) exhibited a small but

statistically significant drop in diarrheal incidences (43.8% vs 46.5%), but only for those

traveling to one of the destinations. Clements et al. [17] administered a preparation of L.

acidophilus and L. bulgaricus (Lactinex TM) in milk four times daily to prevent neomycin-

induced diarrhea in patients receiving therapy against enterotoxigenic Escherichia coli

(ETEC). One lot of Lactinex TM produced a 50% reduction in diarrheal incidence among 20

patients as compared to 19 controls, but a second lot produced no effect.

PROBIOTICS 591

Lactinex TM also proved ineffective in preventing amoxicillin-induced diarrhea among 15

children who received one-gram doses four times daily during ten days of antibiotic therapy

[123]. The preparation was claimed by the manufacturer to contain 5.1 x 108 cfu/g, but no

independent count was performed during the study. Other reports alleging positive results

from administering LAB were either not controlled, used too few subjects to be significant or

suffered from some other experimental flaw.

Table 2 describes the results of studies to control diarrhea in humans with probiotics.

In summary, some beneficial effects of certain probiotic strains on the course of rotaviral-

induced diarrhea in children have been found.

Cancer

Epidemiological studies indicate that the rise in the incidence of colon cancer in the

Western world is associated with a high-fat "Westernized" diet. Dietary fat stimulates bile

acid turnover leading to increased bile acids in the colon. Secondary bile acids, produced in

the colon as a result of bacterial metabolism, have been implicated as factors promoting colon

cancer [102]. Fecal bacterial enzymes, such as (-glucuronidase, nitroreductase and

azoreductase produced by the autochthonous microflora, are thought to convert

procarcinogens in the colon to carcinogens. This has prompted research into the intriguing

possibility that probiotics could reduce the risk of colon cancer.

A number of mechanisms for the anti-tumor action of probiotics have been proposed as

follows [84]:

1. suppressing the carcinogen/procarcinogen by binding, blocking or removing it;

2. curbing the growth of bacteria with enzyme activities that directly or indirectly convert

procarcinogens to carcinogens;

3. reducing the intestinal pH, thereby altering microflora activity and bile acid solubility;

4. altering colonic transit time to remove fecal mutagens more rapidly;

5. stimulating the immune system.

While dietary fiber appears to reduce cancer rates by acting through 1, 3 and 4 above,

the mechanistic role played by probiotics is uncertain, but may involve decreasing certain

fecal bacterial enzyme levels. For example, ([3-glucuronidase is made by several genera

including Bacteroides spp. [51], a major component (20%) of the microflora [55, 90]. This

enzyme deconjugates compounds with a (13-glucosidic bond, releasing aglycones that can act

as mutagens. For instance, deconjugation of N-hydroxy-N-2-fluorenylacetamide-[3-

glucuronide formed by the liver results in the carcinogenic N-hydroxy-N-2-

fluorenylacetamide [128]. Nitroreductase enhances the conversion of aromatic nitro

compounds into potentially harmful amines such as reactive N-nitroso and N-hydroxy

592 S. SCHEINBACH

intermediates [25]. Azoreductase performs a similar conversion on azo compounds. Another

class of bacterial enzymes, dehydroxylases, act in the colon to convert primary bile acids to

secondary bile acids like deoxycholic and lithocholic which are also thought to promote colon

cancer by acting as co-carcinogens. For example, secondary bile acids promote binding of

benzopyrene to DNA in human coloncytes in vi tro [3] and act as promoters of

nitrosoguanidine-induced colon tumors in rats [102]. In man, over 80% of fecal bile acids are

dehydroxylated [105].

It has been suggested that LAB have lower fecal enzyme activities than coliforms,

clostridia and bacteroides [105]. Since LAB produce acids, peroxides and bacteriocins, they

could influence cancer rates by acting through mechanism 2 above, lowering fecal enzyme

activity by outcompeting other groups. Alternatively, a decrease in fecal enzymes could

occur without an appreciable change in cell numbers by altering the metabolism of the

producer colonic microflora as might result from a drop in pH. While these possibilities

appear attractive, a demonstration that consuming LAB reduces either the numbers or

metabolic activities of other colonic microflora remains elusive. In contrast, reductions in

fecal enzyme activities have been observed and these findings provide indirect evidence that

LAB lower cancer rates. Exactly how fecal enzyme activities are lowered remains unclear.

Studies examining the effect on fecal enzyme levels of feeding probiotics have

commonly used L. acidophilus strains or L. casei GG as a fermented product or in powdered

form. Both species are of human origin, can be recovered in high numbers from the feces

following consumption [37, 43] and have been shown to adhere to human colonic mucosa in

vitro [11, 111]. A bifidobacteria culture has also been investigated for its effect. The human

trials summarized in Table 3 show that activity levels of (I]-glucuronidase and nitroreductase

were generally reduced by oral consumption of certain LAB strains, but an effect on

azoreductase levels was observed less often.

Two reports appear most convincing. In one [41], the ([~-glucuronidase, azoreductase

and nitroreductase activities of 21 subjects were followed during a 30-day period of milk

supplementation (500 ml daily) succeeded by a normal diet for 30 days (baseline). The

ingestion of milk had no effect on enzyme activities. Following the baseline diet, subjects

again consumed milk but it was supplemented with either L. acidophilus strain NCFM or

N-2 (2 x 106 cfu/ml). The results demonstrated a decrease in all three enzyme activities

during culture consumption which returned to baseline following its cessation. The other

study [71 ] found that activities of (I]-glucuronidase and nitroreductase dropped in 14 woman

receiving L. casei GG-supplemented (3 x 10 l° cfu/day) yogurt during a four-week period. L.

casei GG was recovered from the feces of these subjects and no drop was found for 12

woman fed pasteurized yogurt. The decrease in enzyme activities did not persist when

feeding stopped, indicating that the probiotic organisms could not successfully colonize the

PROBIOTICS

Table 3. Effects of Oral Consumption of LAB on Fecal Enzymes (adapted from 109)

593

Number of Organism(s) and Daily Dose Reduction of Fecal Enzyme Activity b Subjects a (cfu)

1214] L. acidophilus DDS1 in milk; 4x109

71451

7[42]

21141]

91801

81431

L. acidophilus; 4x101°

L. acidophilus NCFM; l x l 0 I°

L. acidophilus NCFM; lxl09 (n=l 0) or L. acidophilus N-2; lx l 09 (n=l 1) L. acidophilus; 3x 10 9

B. bifidum; 3x101° S. lactis; 3x101° S. cremoris; 3x101° L. casei GG; lxl01° (frozen concentrate) L. casei GG yogurt; 3x101°

? ~-glucuronidase (no statistics) + ([~-glucuronidase + nitroreductase - azoreductase - 7-~-deh~cdrox~clase + nitroreductase - azoreductase + nitroreductase + azoreductase + p-glucuronidase + nitroreductase - azoreductase - ~-glucuronidase

+ 13-glucuronidase

14 [71] + 13-glucuronidase + nitroreductase

6113] Bifidobacteria; 4x1011 (fermented + 13 glucuronidase milk) - nitroreductase

- azoreductase

aNumber of subjects does not include controls, bstatistically significant positive = +; negative = - ; not definitive = ?.

gut in the high numbers caused by feeding. A less convincing study, using only six subjects,

also found a decrease in (-glucuronidase activity, but not for nitroreductase or azoreductase

during an eight-day period in which milk fermented by a strain of bifidobacteria was

consumed [13]. Another less convincing report, using only seven subjects, found that the

activities of 13-glucuronidase and nitroreductase, but not azoreductase, were significantly

reduced during one month of feeding an L. acidophilus concentrate at 1 x 1010 cfu/day [45].

It appears that LAB supplementation can play a role in reducing the activities of certain

fecal enzymes thought to play a role in tumor formation. The evidence is encouraging, but it

is another thing to infer that they can suppress cancer. These studies lasted no more than

several weeks so they suffer from the fact that they are looking for a short-term connection to

a long-term disease. A longitudinal study focusing on enzyme activities and cancer incidence

in long-term consumers of fermented dairy products could go a long way in establishing these

connections. At this time the ability of probiotics to lower cancer risk is unclear.

594 S. SCHEINBACH

Immune system stimulation

Research on immune system stimulation by probiotics has focused on:

1. response of mammalian cell cultures to exposure to LAB or their cellular products;

2. response of mice to intraperitoneal injection of LAB or their cellular products;

3. response of mice to oral administration of LAB or fermented milks;

4. human feeding studies.

Studies involving 1 or 2 have shown effects, but these do not involve oral consumption

and their significance is unclear. In vivo studies have been carried out in mice and some with

humans. The work of Perdigon and her colleagues is most often cited. Feeding lactobacilli or

yogurt to mice stimulated macrophages and increased secretory IgA concentrations [98].

Mice were also somewhat protected against infection with Salmonella thyphimurium by

prefeeding L. casei or S. thermophilus, but not L. acidophilus or L. bulgaricus [99]. In one

human trial [53], 24 subjects consumed 450 g of yogurt per day for four months. Blood

chemistry in this group showed no T-interferon in the sera, but when T-lymphocytes were

tested for T-interferon production, they exhibited a marked increase. It has been suggested

[54] that findings such as these result from the known ability of the muramyl dipeptide,

present in the cell wall of live as well as dead lactobacilli, to stimulate the immune system

[50, 125]. The limited number of studies in humans (see next section), make it difficult to

link probiotic consumption to disease prevention in normally healthy humans.

C O M M E R C I A L D E V E L O P M E N T OF PROBIOTICS

Probiotic product innovation

The lack of sound scientific evidence to support some claims for the efficacy of

probiotics in healthy consumers has not prevented food companies from investing time and

money to enter the business arena. In 1989 one company launched a research project to

screen certain LAB for health benefits in order to identify a probiotic culture for use in a

product [103]. The researchers focused on strains of human origin that had already been used

industrially for many years. First, they checked for the ability to survive transit to the colon

in human volunteers. Next, they examined the ability of various cultures to adhere to human

intestinal cell lines in vitro as an indication that they could persist in the gut and perhaps act

as a barrier against mucosal adhesion by enteropathogens.

A strain ofL. acidophilus, named Lal , could be recovered in the feces of subjects who

daily consumed Lal in a fermented milk product, indicating it was resistant to stomach acid

and intestinal bile. The organism strongly adhered to human intestinal Caco-2 cells in vitro

PROBIOTICS 595

and could block adhesion of Salmonella spp., Yersinia spp. and Escherichia coli, but not if the

pathogens were added first. The adhesion was calcium-dependent and required a

proteinaceous substance secreted into the culture supernatant [10]. In addition, a non-

proteinaceous substance, active against intestinal pathogens but not normal gut LAB, was

found to be secreted by strain Lal [11]. The unidentified substance exhibited limited

protection against S. typhimurium infection in gnotobiotic mice by delaying mortality, and in

vitro it blocked invasion of human enterocytes by the pathogen. Lastly, two human studies

provided evidence that ingestion of a fermented milk product containing Lal stimulated the

immune system. One study with 28 subjects consuming 7 x 101° Lal cfu/day for three

weeks found an increase in the number of activated macrophages [115]. In the other, 30

volunteers consumed 5 x 109 Lal cfu/day and after three days were challenged with an oral

Salmonella typhi vaccine to which they exhibited an increase in secretory IgA specific for S.

typhi over that shown for the control group [72].

With these findings in hand, patent applications were filed in Europe and the United

States claiming to possess a probiotic organism that protects against enteropathogenic

infections and stimulates the immune response. The company now produces a milk product

fermented with Lal called LC1, "a new concept in healthy eating which, when eaten daily and

as part of a healthy, balanced diet, helps your body to protect itself" [103]. Currently, this

product accounts for 20% of its European yogurt business [130].

Two other lactobacillus probiotics have been studied extensively. L. acidophilus

NCFM was first isolated in the 1970's. It can survive transit to the colon [37], adhere to

Caco-2 cells in vitro via a protein-mediated mechanism [49] and reduce fecal enzyme activity

levels (see Table 3). The culture, is sold mainly to the dairy industry as a yogurt

supplement. Another well studied lactobacillus probiotic is L. casei GG. First isolated in

1983 [24], it is acid and bile resistant and adheres to epithelial cells in vitro. Like Lal, it

produces a non-proteinaceous substance with antimicrobial activity [ 118] that acts against S.

typhimurium to block adhesion to Caco-2 cells in vitro and protect against infection in mice

[57] In human feeding trials it can readily be recovered from feces [43] and it alleviates

diarrheal symptoms and reduces fecal enzyme activity levels (see Tables 2 and 3). The

patented culture [47, 48] is sold in Finland.

Enterococcusfaecium hs also been examined for probiotic properties. A novel strain of

E.faecium, PR88, was patented but is not currently in use[2]. It is purported to be effective

in alleviating irritable bowel syndrome (IBS) in humans The claim is based on a study using

17 IBS patients, treated with PR88 either by intracecal intubation or oral consumption of

yogurt. Either treatment resulted in fecal recovery of PR88 at a level of 105-109cfu/gm with a

concomitant reduction in IBS symptoms.

596 S. SCHEINBACH

Probiot ic commerc ia l i za t ion

In terms of consumer awareness of probiotics, Japan has taken the lead with Europe

coming in second and the United States a distant third. Recognizing the benefits of diet and

health to an aging population, the Japanese government in 1991 set up a licensing system for

"foods for specified health use", i.e. a functional food "containing ingredients that aid specific

body functions in addition to being nutritious" [59, 126 ]. In 1930 L. casei Shirota was

isolated from the human colon [76] and it became the basis for the Yakult Honsha Co. which

produces several probiotic dairy products. For example, Mil-Mil E, introduced in 1982 is a

bifidobacteria-containing yogurt supplemented with vitamins A, C, D, and E. The company

also holds a number of United States patents from the late 1970's and early 1980's

concerning methods for producing bifidobacteria-fermented milk products. In Japan full-scale

bifidus milk and yogurt production began in 1977, with home delivery of products containing

Bifdobacterium longum and S. thermophilus. By 1984 the number of fermented products had

reached 20 [59].

In both Japan and Europe probiotic-supplemented dairy foods and drinks continue to

be a fast-growing industry, numbering over 70 products [ 119]. Over 70% of the functional

foods developed in Japan are liquids and one citrus-flavored probiotic drink (Yakult),

containing L. casei, sells 1 million 50-ml bottles a day in Japan [59] and 23 million word-wide

[76]. Estimates indicate that functional food may capture 5% of the total Japanese market

[59]. In 1995 Yakult Honsha extended the distribution of Yakult into several European

countries [130] where new probiotic dairy products appear regularly. Examples of recent

introductions are: Actimel Orange, a milk drink containing L. casei, sold by Danone; ProCult3

with B. longum BB536, sold by Muller; Symbalance, a yogurt sold in Switzerland by Tonilait

and containing Lactobacillus reuteri, L. acidophilus and L. casei; Probiotic plu Oligofructose,

made in Germany and containing L. acidophilus and Lactobalillus bifidus LA7. These now

join the list of older products such as Ofilus and Cultura. The former is a fermented milk

product made by Yoplait and the latter is made by Dairy Denmark using Chr Hansen's

cultures [23]. Gaio, containing S. thermophilus and E.faecium, and made in Denmark by MD

Foods, captured 15% of the Danish yogurt market following its introduction in 1993. But

when an advertising campaign claiming serum cholesterol reductions accompanied its recent

introduction in the United Kingdom, negative government response resulted in it being

withdrawn [130]. A list of some probiotic fermented milk products currently produced in

Japan and Europe is presented in Table 4.

In order to obtain good organoleptic properties the bifidus-supplemented products are

fermented with S. thermophilus to provide proteolytic activity, omitting L. bulgaricus

because it can cause overacidification [74]. The Cultura product uses selected strains that

PROBIOTICS 597

grow symbiotically in milk to yield the desired pH without the bifidibacteria producing too

much acetic acid. Similarly, Biogarde from Germany contains L. acidophilus, S. thermophilus

and B. bifidum, but not L. bulgaricus in order to reduce the level of lactic acid in the product.

Table 4. Commercial Probiotic Dairy Products in Japan and Europe (adapted from 23)

Product ACO-yogurt Cultura-AB AB-yogurt Biogarde Bifighurt Gefilac Yakult

Miru Miru Mil Mil E

Biokys

Ofilus

Gaio LC1

Symbalance* Probiotic plus Oligofructose*

ProCult3 Actimel Orange

Fysiq* *contains FOS

Countrf of Origin Switzerland Denmark Denmark GeNTlany Germany Finland Japan Japan Japan

Slovakia

France

Denmark Europe

Switzerland Germany

Germany Germany

Netherlands

Organism(s) S. thermol~hilus; L. bul~,aricus; L. acidophilus

L. acidophilus; B. bifidum L. acidophilus; B. bifidum; yogurt culture

L. acidophilus; B. bifidum; S. thermophilus B. lon~um; S. thermophilus L. casei GG (rhamnosus)

L. casei L. acidophilus; L. casei

B. bifidum; yogurt culture B. bifidum; L. acidophilus; Pediococcus

acidilactici B. bifidum/B, longum; L. acidophilus;

S. lactis; S. cremoris E. faecium; S. thermophilus

L. acidophilus Lal L. reuteri; L. casei; L. acidophilus

L. acidophilus; L. bifidus; LA7

B. lon~um BB536 L. acidophilus

L. acidophilus Gilliland

Cultures of L. acidophilus, L. casei GG and Bifdobacteria spp. make good probiotics

because they are of human origin, surviving stomach acid, intestinal bile and capable of

colonizing the colon. But milk products fermented with these organisms could not readily

expand into the market place because human strains grow slowly in milk, lose viability upon

storage and do not yield products with favorable organoleptic qualities [74]. Better growth

could be achieved by supplementing milk with yeast extract or whey protein, and by adding

threonine to promote acetaldehyde production and enhance flavor [75]. Most

Bifidobacterium strains cannot ferment milk by themselves because they require low redox

potentials and peptides generated from the breakdown of casein [64]. Moreover, when co-

cultured with lactobacilli, they quickly become inhibited as the pH drops. In order to

overcome these manufacturing problems, most fermented milk products containing

598 S. SCHEINBACH

bifidobacteria are inoculated with the final number of cells needed in the product [64]. It has

been reported that the enzyme oxyrase stimulates growth of bifidobacteria in skim milk [77].

Substances that pass through the gut undigested have been investigated for their

intestinal bifidogenic ability. The fructooligosaccharides (FOS), especially inulin, were

shown to stimulate bifidobacteria growth in vitro [127] and in healthy human subjects [33].

Natural FOS are found in plants such as Jerusalem artichokes, bananas, barely, garlic and

onion [28] or can be commercially produced by the action of fructofuranosidase on sucrose

[120]. Several probiotic dairy products containing FOS are now being marketed in Europe

[130]. Other bifidogenic factors are lactulose (4-O-B-D-galactopyranosyl-D-fructose),

derived from isomerization of the lactose in whey [88], and transgalactosyl oligosaccharides.

The latter have been patented as bifidus-growth factors by Yakult Honsha in Japan [92].

CONCLUSIONS

While human studies show that feeding probiotics can be efficacious under certain

circumstances, most of the claims have yet to be proven. For healthy individuals, the

benefits of probiotics are unproven. Where disease is present, those who are lactose

intolerant probably benefit the most. In children limited protection from diarrhea, especially

rotaviral, also occurs with certain strains of lactobacilli. However, more work is needed using

adults and children with known diarrheal causes to test specific cultures in well-controlled

experiments and with enough subjects to yield significant results. Oral consumption of LAB

may reduce the levels of certain fecal enzymes thought to promote cancer, but claims of

cancer suppression remain to be proven. Efficacy with regard to cholesterol reduction and

immune system stimulation is also unproven. A major problem in assessing the true benefit

of probiotics is the inability to perform certain types of trials in humans as they are done

with animals. Obviously, it would be unethical to try to show that LAB can protect against

experimentally induced tumors in humans as has been done for rats [40, 42, 44] and

translating animal results to humans must be done with care because there are significant

differences between them. For example, changing the diet of rats from chow to ground beef

causes dramatic changes in the colonic microflora [114]. In contrast, the human intestinal

flora are refractory to dietary changes [27].

Another failing of many probiotic studies that makes comparisons problematic is the

variety of strains used and the lack of standard preparation. For example, molecular

diagnostic techniques have shown that several commercial LAB cultures are different species

than previously believed [110]. Even two apparently identical probiotics with the same cell

titer may, in fact, be different due to differing growth conditions, timing of harvest,

preparation of concentrate and storage conditions. More studies need to be published in

PROBIOTICS 599

peer-reviewed journals and confirmed in other laboratories. The work should be performed

with well defined strains in well designed, properly controlled, randomized trials.

Without doubt there are some beneficial effects associated with the consumption of

probiotics and sales are rising worldwide as consumers become more aware of their benefits.

In Japan, where the government has established a licensing procedure for foods used for

health, consumer demand for probiotic products is high. This fits in well with the traditional

Japanese concept of gaining good health through the consumption of natural products.

European consumers also perceive health benefits from probiotics and have greatly increased

their consumption of fermented dairy products in recent years. If consumer trust and interest

in the health benefits of probiotics is to continue and even expand, it is important that

product claims are based upon sound scientific evidence.

ACKNOWLEDGEMENT

I would like to thank Dr. M. E. Sanders for critically reading the manuscript and making

helpful suggestions. The editing performed by Dr. Martin Cole is also greatly appreciated.

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