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BiotechnologyAdvances,Vol.16, No. 3, pp. 581-608, 1998 Copyright© 1998ElsevierScienceInc. Printed in the USA.

All fightsreserved 0734-9750/98 $19.00 + .00 ELSEVIER PII S0734-9750(98)00002-0

Nabisco Research, Schaeberle Technology Center, 200 DeForest Ave., E. Hanover, New Jersey 07936 email:ScheinbachS@Nabisco.com

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, statisticallyvalid 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 W O R D S Probiotics, functional food, enteric microflora, lactic acid bacteria, lactose intolerance, diarrhea, cancer, yogurt INTRODUCTION 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




[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.

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 can alleviate lactose intolerance. This stems from a 90-95% decline in the production of lactase [34]. act as antibiotics. 103]. 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. Mechanisms by which probiotics could improve health include: 1. Any lactose reaching the large intestine is metabolized by the colonic microflora to yield CO2. 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. 2. Notwithstanding a lack of clear-cut mechanisms. Lactose intolerance Most of the world's population (60-90% of non-Caucasians and 6-12% of Caucasians) become lactose intolerant after weaning [6. These have shown that germ-free (gnotobiotic) animals or animals whose gut microflora have been perturbed by antibiotics are more susceptible to disease. These organisms were later shown not to survive in the intestinal tract.PROBIOTICS 583 and Streptococcus thermophilus) were responsible for the longevity of Bulgarians who regularly consumed yogurt [85]. control infections. competition for substrates. 32. While there is some evidence to indicate these processes occur in vitro and in animal models. symptoms including abdominal bloating. Thus. probiotics may provide health benefits under certain circumstances. 108. suppress tumors and protect against colon/bladder cancer by maintaining a healthy intestinal microflora balance [20. Others have cautioned that in many instances. stimulation of the immune system. reduce diarrheal incidence. 95]. The presence of lactose also alters the osmotic balance in the colonic lumen. and resistance can be restored by oral administration of fecal suspensions from healthy individuals. flatulence and . peroxides or bacteriocins bactericidal to groups that negatively impact health. competition with pathogens for mucosal binding sites. production of acids. methane and hydrogen and the latter is typically noted as an increase in breath hydrogen. 124]. stimulate the immune system. 4. 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. Justification for health claims comes mainly from animal studies. a route taken by the USDA to reduce salmonellosis infection in commercially reared chicks [52]. 3. lower serum cholesterol. The organisms used in probiotics are known to produce antimicrobial substances that might affect the colonic microflora balance [87]. 68. cramping. 26. 66].

Alleviation of lactose intolerance is probably the best established health claim for probiotics. . thermophilus. yogurt is still recommended for symptomatic people wishing to consume lactose-containing products [121]. thermophilus strains were shown to contain more lactase than strains of Lactobacilli or Bifidobacteria [110]. 83]. which survives intestinal transit intact. 69. even with high levels of lactose. they usually contain less lactose (4% vs 6%) due to microbial digestion during fermentation. 79. acidophilus cells prior to their addition significantly alleviated lactose malabsorption [82. 69]. amelioration of lactose intolerance effects is best accomplished by consumption of ordinary yogurt or thermophilus milk [65]. Second. possibly causing unwarranted conclusions in some earlier trials. which are both fermented by S. indicating that cell lysis promotes lactose digestion. 113]. Fermented milk products like yogurt are less likely to cause gastric upsets in two ways. First. 113]. but dislike the taste or texture of yogurt can consume acidophilus milk. In a recent double-blind study comparing four L. Sonication of the L. 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]. Lactose intolerant individuals who want to include a dairy product in their diet. In children. acidophilus milk reduced symptoms but not breath hydrogen [89]. Adding Lactobacillus acidophilus concentrate to cold milk results in unfermented acidophilus milk. allows them to be consumed by lactose intolerant individuals without experiencing the usual rise in breath hydrogen or associated symptoms [65. however. it has not been consistently effective in preventing malabsorption in adults [63. in part. indigenous lactase. This premise is supported by the fact that milk containing or fermented by L. Strain differences may also play a role. 15-30% of self-described lactose intolerant individuals were actually lactase persistent. upon the extent to which intracellular bacterial enzyme is released following ingestion. present in the ingested cells can make up for the host's deficiency. 83. nevertheless. acidophilus strains. SCHEINBACH diarrhea ensue [121]. acidophilus. cells from a variety of S. In addition. As a consequence. The psychological state of subjects also appears to be important [122]. A summary of recent clinical trials on controlling symptoms in lactose intolerant subjects by LAB is presented in Table 1. which is killed upon ingestion [63. Whether or not malabsorption is prevented appears to depend. A number of these human studies provide convincing evidence that the addition of certain starter cultures to milk products. is not as effective as milk containing or fermented by S. thermophilus. In a doubleblind trial.584 S. 69. 78. There are few double-blind studies demonstrating efficacy in reducing symptoms because it is difficult to blind a study involving yogurt consumption.

acidophilus milk Heated ).= negative result. bulgaricus yogurt Fermented milk with:S. L. bifidus 22[78] Yogurt(n=22) Yogurt and a meal(n= 12) Yogurt and lactose(n= 10) Milk with L. b other symptoms significantlyreduced compared to control. bulgaricus milk Unfermented L. + + _b +b 8 children[89] lactis Unfermented acidophilus milk with: strain ATCC4356 +/strain B +/strain N 1 + strain E a+ = positive result. thermophilus and L.] 12163] 10165] 91113] Product/Culture Tested aSignificant Breath Hydrogen Reduction vs Milk or Lactose + + + Unfermented L.@107 or 108 cfu/ml + + + + 8 [ 129] 10169] 7[79] 12[79] Unfermented S. Summary of Clinical Trials on Controlling Symptoms of Lactose Intolerance by LAB (adapted from 109) N u m b e r of Lactose Intolerant Subjects [ref. bulgaricus :L. acidophilus milk ¥o~t Yogurt Buttermilk.o~:urt Yogurt Heated yogurt Unfermented L. acidophilus milk As above but sonicated Three yogurts 14182] + +/- + + Dannon® +Royal Maid® -Borden® +/. acidophilus milk with: LA1 LA2 NCFM 7 yogurts with different S.@107 cfu/ml + @108 cfu/ml . acidophilus :B. thermophilus/L. thermophilus :L. bulg. .@ 107 cfu/ml +/. 11191] . thermophilus.aricus strains S. acidophilus NCFM Milk with S.@108 cfu/ml .PROBIOTICS 585 Table 1. thermophilus/L. Unfermented L.

placebocontrolled trial. another group tested the ability of the pig strain. Deconjugated bile salts are more likely to be excreted. after 157 people consumed four tablets a day for six weeks. to lower serum cholesterol in pigs and hamsters and found no effect [29]. formed in the colon. but not in hamsters or rats that were fed LAB having high BSH activity. resulting in increased cholesterol breakdown. Lin et al. since cholesterol is used in the production of bile acids. are more readily excreted than their conjugates and that the adherence of free bile acids to bacterial fiber enhanced bile excretion. 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). bile salt hydrolase (BSH). The cultures were shown to assimilate cholesterol under certain in vitro conditions. they showed no significant differences in serum cholesterol levels as compared to the placebo group. They suggested that free bile acids. enhanced catabolism and excretion of bile acids might reduce serum cholesterol [15]. acidophilus can deconjugate taurocholic and glycocholic acid [36]. respectively. Chikai et al. . For example. while others from humans assimilated cholesterol to lesser extents [38]. Currently. acidophilus isolated from a pig [35]. but neither have been shown conclusively. acidophilus and L. It has also been proposed that microbes directly assimilate cholesterol [35]. These are often conjugated with glycine or taurine to form glycocholic or taurocholic acid. However. The pig strain also inhibited increases in serum cholesterol levels in pigs fed a high-cholesterol diet [35]. Under anaerobic conditions human isolates ofL. Significant cholesterol assimilation in the presence of bile was reported in vitro for one strain of L. Fletcher [29] found that serum cholesterol levels dropped in pigs. SCHEINBACH Reports that LAB consumption is hypocholesterolemic are controversial. Two possible mechanisms for microbially-inducedlowering of serum cholesterol have been suggested. bulgaricus. at a dose of lxl 0 ll cfu/day. 300 strains of Lactobacilli are being tested for BSH activity as a possible measure of cholesterol-reducing ability [30]. but may be deconjugated by a microbial enzyme. They tested tablets (LactinexTM) containing about 2 x 106cfu/tablet of L. [70] set up the largest human study to date involving 354 subjects in a double-blind.586 Cholesterol reduction S. however. [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. often lacking controls or using too few subjects to be significant. The first proposes that. Various LAB have been tested for a hypocholesterolemic effect in humans. these claims are controversial and have not been confirmed. The major primary bile acids made by the liver are cholic and chenodeoxycholic acids.

diarrhea is the greatest killer of children and rotavirus is its most common cause [16]. Worldwide.faecium in the fermented product dropped three to four logs during the trial period. Unfortunately.PROBIOTICS 587 More recently. comprising 43 subjects. all aged 44 years. it is unclear from these studies what dose must be reached before an in vivo effect can be observed. a hypocholesterolemic effect of feeding LAB to humans has yet to be scientifically proven. thermophilus (about 7xl08cfu/ml). Both studies were sponsored by the manufacturer. et al. Diarrhea Diarrhea has many causes so it is difficult to evaluate the effects of probiotics on limiting its severity. two double-blind. drank chemically acidified milk. Following a brief bloom of facultative anaerobes. [104] found that three months into their study they could divide subjects into responders. strains and even subjects. Neither could show an unambiguous effect. but still constitutes about 95% [60]. at least a portion of the observed decrease could have resulted from regression to the "true" mean. 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. Since there are no convincing double-blind trials in humans. the two groups were not randomized beforehand. 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. The biological basis for this is unknown. who exhibited a decrease in LDLcholesterol. Subjects receiving bacteria exhibited a rapid fall in LDL-cholesterol within the first month. A major complication was the fact that the cell titer of E. For example. resulting in a significantly higher LDL-cholesterol mean for the test group at the start of the trial. comprising over 99% of the total population. Their growth creates a low pH environment in the colon by producing lactic and acetic acids which may exert a protective . Moreover. Richelsen. Thus. by the end of the study the placebo group showed a similar decrease such that no significant difference existed between the two groups. The gastrointestinal tract of the newborn is inoculated by the mother's vaginal and fecal flora [86]. In the second study 44 healthy men and women 50-70 years old consumed 200 ml daily of fermented product for six months [104]. bifidobacteria are the first anaerobic group to establish themselves in high numbers (1 x 1011cfu/gm feces). Conflicting and variable results among studies may occur from the use of different doses. and non-responders who did not. The placebo group. This proportion is lower in formula-fed infants. However. The first study [1] tested 29 normocholesterolemic men. Whether or not a greater response would have been seen if the higher dose had been maintained is unknown.

116].9% vs 31%) receiving the microbially-supplemented formula (n=29) contracted diarrhea as compared to the unsupplemented control group (n=26). 106. Saavedra et al.5-5.8 days in the placebo group (n=64) to 2. those in the test group were less likely to shed rotavirus (10% vs 38%). being perturbed only by major stressors such as antibiotic therapy or pathogen colonization [67. because human milk has a lower protein content and less buffering capacity than cow's milk [14]. 112].7 for bottle-fed infants. bacterial or diarrhea of unknown cause. done in children suffering from rotaviral diarrhea. galactose. at least in some people. admitted to a hospital over a 17-month period. 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. streptococci and other anaerobes establish themselves. formula feeding of infants helps to maintain high bifidobacteria levels [5. A similar study [116] was conducted with children (1-36 mos. thermophilus for the prevention of acute diarrheal episodes and rotaviral shedding. these strains can exhibit shifts over time [81]. They found that significantly fewer children (6. The rotaviral-positive subgroup especially benefited from L. placebo-controlled human clinical trials on the use of probiotics to alleviate diarrhea have yielded positive results. so that by two years of age Bacteroides spp. in part. clostridia. whereas patients with confirmed bacterial diarrhea (n=l 1) . Four well-designed. pH 5. but occurs more rapidly in babies fed formula. 5-24 months old. Breast milk also contains a glycoprotein bifidus growth promoting factor composed of glucose. Humans can harbor nonoverlapping strains and. The duration of diarrhea was shortened from 3.7-6. 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. A decreased pH for the former probably occurs. 9] resulting in a colonic pH of 4. case i GG therapy (n= 13). Breast vs. with the bifidobacteria population under 25% [60]. At the genus and species level the gut microflora appears to resist changes in diet [27]. However. [106] performed a trial with 55 children. 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. 61. These studies. double-blind.0 for breast-fed vs. Children in the test group received a Lactobacillus casei GG dose of 1 x 101° cfu over a five day period.) suffering from rotaviral.7 days in patients receiving the probiotic (n=59). Microbial succession proceeds with either diet. They evaluated the efficacy of a formula containing Bifdobacterium bifidum and S. SCHEINBACH effect against diarrhea by inhibiting the growth of Gram-negative facultative anaerobes that could be pathogenic.. and the bacterial colonic community approaches that of adults. have shown that accelerated recoveries and/or less severe symptoms can occur following administration of certain probiotics [8.588 S. fucose and N-acetylglucosamine [12]. In addition.

for 14 L. casei GG[61] B. brevi. L. symptoms $ symptoms $ duration $ incidence $ duration. caseiGG.. bifidum S.[73] L. casei GG [117] L. difficle colitis Intractable Undergoing radiation therapy Erythromycin Traveler's diarrhea Amoxicillin Acute viral (mainly rotaviral) Acute gastroenteritis (mainly rotaviral) Acute rotaviral Acute (some rotaviral) Acute (some rotaviral) Acute rotaviral Acute rotaviral. S u m m a r y o f Clinical Trials to Control D i a r r h e a by L A B (adapted f r o m 109) Culture (ref.) 21 children 20 children (1-24 mos) 21 children (5-28 mos. bacter. for acute watery diarrhea No placebo for control group No effect on bacterial cases + + aDoes not include controls. caseirh. no statistics Only in 1 of 2 destinations ? ? + Neg.[.[97] Lactobacilli[100] E. acid. unknown . for acute non-bloody cases Pos. therm.) L. B.) 30 children (4-35 mos.[107] L. longum[19] L. casei GG[46] B. casei[58] L. b? = insufficient information to determine if results were truly oositive. caseiGG[101] L. L. watery stool $ duration $ duration + $ symptoms Lot to lot cell variability Few subjects Few subjects. therm.) 47 children (4-45 mos.) 29 children (5-24 mos. L. acid. casei GG [116] Subjects a 59 adults 94 children < 3 years old 26 healthy adults 53 children 23 healthy adults 23 healthy adults 36 adults with ETEC 61 adults 10 healthy adults 5 adults with colitis 15 children 11 adult females 8 healthy adults 349 healthy adults 15 children (5-76 mos.[7] S. acid. symptoms $ incidence No prevention $ duration $incidence androtavkal shedding $ duration. casei GG[62] L.PROBIOTICS 589 T a b l e 2.) 59 children (1-36 mos. L.) Cause Abdominal symptoms Acute onset > 10 months Travelers' diarrhea Various causes Results $ symptoms Comments No controls Efficacy b ? Lower incidence $ duration + Enterotoxigenic E. casei GG[96] L.[106] L. milk [931 B. no controls No controls No controls. faecium[8] LactinexTM Lactobacilli[ 171 LactinexTM Lactobacilli[ 18] LactinexTM Lactobacilli[ 18] Sweet acid. subjective reporting Few subjects. cases Pos.bif. casei GG[94] LactinexTM Lactobacilli[ 123] L. coli (ETEC) ETEC Neomycin Irritable bowel Erythromycin C. bulg. . IgA $ duration $ vomiting. casei rh.

casei GG. 96. Two studies where the etiology was not apparent [96. One lot of Lactinex TM produced a 50% reduction in diarrheal incidence among 20 patients as compared to 19 controls. [17] administered a preparation of L. The duration of diarrheal episodes decreased signficantly and the immune response to infection was enhanced in children fed 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. casei GG or a placebo to 756 travelers going to two different Turkish cities. acidophilus and L. but only for those traveling to one of the destinations. acidophilus (5 x 108 cfu). bulgaricus (Lactinex TM) in milk four times daily to prevent neomycininduced diarrhea in patients receiving therapy against enterotoxigenic Escherichia coli (ETEC). respectively. but a second lot produced no effect. Clements et al. their return. 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. In the fourth study E. yielding a dose of 2 x 109 cells. bulgaricus (5 x 108 cfu) and Lactococcus lactis (4 x 109 cfu). the control group did not receive a placebo. In the case of adults. Other trials with children have suffered from one or another experimental flaw [62. L. and fill out a questionnaire upon The results showed that the GG group (349 subjects) exhibited a small but statistically significant drop in diarrheal incidences (43. casei GG.) suffering from watery. 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 f L . The L. SCHEINBACH did not.) fed 1 x 101° cfu L. Each group received one of the three probiotics twice daily for five days. Unfortunately. after two days o f treatment.5%). thermophilus and L. 73. non-bloody diarrhea who were fed about 1 x 1011 cfu L. A shortened duration for rotaviral diarrhea was also found for 21 children (5-28 mos. bulgaricus. acidophilus or those fed the mix. acidophilus or a mix orS. but not L. 47 of whom received L casei GG.4 to 1. L.8% vs 46. The third study [61] done with 71 children (4-45 months).4 days. 101]. recovery was 62% vs 35% for test and controls groups. The positive effect was abolished by pasteurization. indicated that the mean duration of rotaviral diarrhea could be shortened from 2. For example. faecium (SF68) was tested for its effect on children (1 month-9 years) with diarrhea of various causes [8]. If a control group not fed live bacteria had been used instead. 101 ] observed a significant decrease in symptoms for children (1-24 mos. [94] gave packets containing either L. the effect of probiotics on diarrheal symptoms is less evident. casei GG twice daily for five days as compared to an equal number in the control group [62]. either as a fermented milk (Gefilac) or freeze-dried powder. Oksanen et al. . casei GG twice daily over two days as compared with children fed a placebo. greater differences might have been seen.590 S. Subjects were asked to drink the contents of one package in water twice daily.

PROBIOTICS 591 LactinexT M 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]. thereby altering microflora activity and bile acid solubility. ([3-glucuronidase is made by several genera including Bacteroides spp. a major component (20%) of the microflora [55.1 x 108 cfu/g. Secondary bile acids. For example. curbing the growth of bacteria with enzyme activities that directly or indirectly convert procarcinogens to carcinogens. altering colonic transit time to remove fecal mutagens more rapidly. 2. reducing the intestinal pH. Nitroreductase enhances the conversion of aromatic nitro compounds into potentially harmful amines such as reactive N-nitroso and N-hydroxy . used too few subjects to be significant or suffered from some other experimental flaw. the mechanistic role played by probiotics is uncertain. 3. A number of mechanisms for the anti-tumor action of probiotics have been proposed as follows [84]: 1. such as (-glucuronidase. This has prompted research into the intriguing possibility that probiotics could reduce the risk of colon cancer. Dietary fat stimulates bile acid turnover leading to increased bile acids in the colon. produced in the colon as a result of bacterial metabolism. Other reports alleging positive results from administering LAB were either not controlled. Fecal bacterial enzymes. some beneficial effects of certain probiotic strains on the course of rotaviralinduced diarrhea in children have been found. 5. 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. 3 and 4 above. but may involve decreasing certain fecal bacterial enzyme levels. deconjugation of N-hydroxy-N-2-fluorenylacetamide-[3glucuronide formed by the liver results in the carcinogenic N-hydroxy-N-2fluorenylacetamide [128]. While dietary fiber appears to reduce cancer rates by acting through 1. stimulating the immune system. 4. are thought to convert procarcinogens in the colon to carcinogens. releasing aglycones that can act as mutagens. Table 2 describes the results of studies to control diarrhea in humans with probiotics. The preparation was claimed by the manufacturer to contain 5. blocking or removing it. but no independent count was performed during the study. This enzyme deconjugates compounds with a (13-glucosidic bond. For instance. nitroreductase and azoreductase produced by the autochthonous microflora. In summary. [51]. have been implicated as factors promoting colon cancer [102]. suppressing the carcinogen/procarcinogen by binding. 90].

subjects again consumed milk but it was supplemented with either L. the ([~-glucuronidase. acidophilus strains or L. Both species are of human origin. but an effect on azoreductase levels was observed less often. Alternatively. peroxides and bacteriocins. 43] and have been shown to adhere to human colonic mucosa in vitro [11. secondary bile acids promote binding of benzopyrene to DNA in human coloncytes in vitro [3] and act as promoters of nitrosoguanidine-inducedcolon tumors in rats [102]. The other study [71 ] found that activities of (I]-glucuronidase and nitroreductase dropped in 14 woman receiving L. Two reports appear most convincing. acidophilus strain NCFM or N-2 (2 x 106 cfu/ml). a demonstration that consuming LAB reduces either the numbers or metabolic activities of other colonic microflora remains elusive. In man. 111]. reductions in fecal enzyme activities have been observed and these findings provide indirect evidence that LAB lower cancer rates. SCHEINBACH intermediates [25]. For example. In one [41]. The results demonstrated a decrease in all three enzyme activities during culture consumption which returned to baseline following its cessation. they could influence cancer rates by acting through mechanism 2 above. Another class of bacterial enzymes.592 S. indicating that the probiotic organisms could not successfully colonize the . Following the baseline diet. Exactly how fecal enzyme activities are lowered remains unclear. In contrast. casei GG as a fermented product or in powdered form. clostridia and bacteroides [105]. While these possibilities appear attractive. Since LAB produce acids. The ingestion of milk had no effect on enzyme activities. casei GG-supplemented (3 x 10 l° cfu/day) yogurt during a four-week period. 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. It has been suggested that LAB have lower fecal enzyme activities than coliforms. 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. over 80% of fecal bile acids are dehydroxylated [105]. Azoreductase performs a similar conversion on azo compounds. 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. The decrease in enzyme activities did not persist when feeding stopped. lowering fecal enzyme activity by outcompeting other groups. L. 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). A bifidobacteria culture has also been investigated for its effect. casei GG was recovered from the feces of these subjects and no drop was found for 12 woman fed pasteurized yogurt. Studies examining the effect on fecal enzyme levels of feeding probiotics have commonly used L. can be recovered in high numbers from the feces following consumption [37.

3x101° . 3x101° + 13-glucuronidase + nitroreductase Bifidobacteria. negative = . using only six subjects. casei GG yogurt. but not for nitroreductase or azoreductase during an eight-day period in which milk fermented by a strain of bifidobacteria was consumed [13]. but not azoreductase. At this time the ability of probiotics to lower cancer risk is unclear. acidophilus.azoreductase L. l x l 09 + azoreductase + p-glucuronidase (n=l 1) L. casei GG. but it is another thing to infer that they can suppress cancer. acidophilus concentrate at 1 x 1010 cfu/day [45]. . using only seven subjects.azoreductase aNumber of subjects does not include controls.~-glucuronidase S. 4x109 7[42] 21141] 91801 81431 14 [71] 6113] ? ~-glucuronidase (no statistics) L. 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. acidophilus NCFM. l x l 0 I° + nitroreductase . found that the activities of 13-glucuronidase and nitroreductase.. cremoris. lxl01° (frozen + 13-glucuronidase concentrate) L. 3x101° L. lactis. acidophilus NCFM. 3x101° S. acidophilus DDS1 in milk. not definitive = ?. 4x1011 (fermented + 13glucuronidase milk) . Effects of Oral Consumption of LAB on Fecal Enzymes (adapted from 109) N u m b e r of Subjects a O r g a n i s m ( s ) and Daily Dose (cfu) Reduction of Fecal E n z y m e Activity b 593 1214] 71451 L.azoreductase .7-~-deh~cdrox~clase L. lxl09 + nitroreductase (n=l 0) or L. A less convincing study. acidophilus N-2.azoreductase B. Another less convincing report. 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.PROBIOTICS Table 3. The evidence is encouraging. 3x 109 + nitroreductase .nitroreductase . were significantly reduced during one month of feeding an L. 4x101° + ([~-glucuronidase + nitroreductase . gut in the high numbers caused by feeding. acidophilus. bifidum. also found a decrease in (-glucuronidase activity. bstatistically significant positive = +. 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.

response of mice to intraperitoneal injection of LAB or their cellular products. Feeding lactobacilli or yogurt to mice stimulated macrophages and increased secretory IgA concentrations [98]. The organism strongly adhered to human intestinal Caco-2 cells in vitro . casei or S. 3. named Lal. make it difficult to link probiotic consumption to disease prevention in normally healthy humans. First. In one human trial [53]. indicating it was resistant to stomach acid and intestinal bile. they exhibited a marked increase. bulgaricus [99]. 24 subjects consumed 450 g of yogurt per day for four months. It has been suggested [54] that findings such as these result from the known ability of the muramyl dipeptide. 125]. human feeding studies. The researchers focused on strains of human origin that had already been used industrially for many years. Blood chemistry in this group showed no T-interferon in the sera. could be recovered in the feces of subjects who daily consumed Lal in a fermented milk product. acidophilus. A strain ofL. but these do not involve oral consumption and their significance is unclear. 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. COMMERCIAL DEVELOPMENT 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.594 S. but not L. 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]. 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. but when T-lymphocytes were tested for T-interferon production. acidophilus or L. 2. response of mice to oral administration of LAB or fermented milks. 4. thermophilus. In vivo studies have been carried out in mice and some with humans. The work of Perdigon and her colleagues is most often cited. they checked for the ability to survive transit to the colon in human volunteers. Mice were also somewhat protected against infection with Salmonella thyphimurium by prefeeding L. Studies involving 1 or 2 have shown effects. to stimulate the immune system [50. Next. present in the cell wall of live as well as dead lactobacilli. The limited number of studies in humans (see next section).

Two other lactobacillus probiotics have been studied extensively.PROBIOTICS and could block adhesion of Salmonella spp. Yersinia spp.faecium. With these findings in hand. The company now produces a milk product fermented with Lal called LC1. this product accounts for 20% of its European yogurt business [130]. 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. 595 proteinaceous substance. The patented culture [47. was The unidentified substance exhibited limited protection against S. Lastly. L. Enterococcusfaecium hs also been examined for probiotic properties. Currently. treated with PR88 either by intracecal intubation or oral consumption of yogurt. Like Lal. when eaten daily and as part of a healthy. In the other. 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. found to be secreted by strain Lal [11]. 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). 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]. First isolated in 1983 [24]. is sold mainly to the dairy industry as a yogurt supplement. casei GG. but not if the pathogens were added first. adhere to Caco-2 cells in vitro via a protein-mediated mechanism [49] and reduce fecal enzyme activity levels (see Table 3). and Escherichia coli. typhi over that shown for the control group [72]. typhimurium infection in gnotobiotic mice by delaying mortality. A novel strain of E. helps your body to protect itself" [103]. 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. PR88. Either treatment resulted in fecal recovery of PR88 at a level of 105-109cfu/gm with a concomitant reduction in IBS symptoms. It can survive transit to the colon [37]. two human studies provided evidence that ingestion of a fermented milk product containing Lal stimulated the immune system. The culture. it is acid and bile resistant and adheres to epithelial cells in vitro. active against intestinal pathogens but not normal gut LAB. "a new concept in healthy eating which. and in vitro it blocked invasion of human enterocytes by the pathogen. balanced diet. acidophilus NCFM was first isolated in the 1970's. it produces a non-proteinaceous substance with antimicrobial activity [ 118] that acts against S. . was patented but is not currently in use[2].. Another well studied lactobacillus probiotic is L. 48] is sold in Finland. The adhesion was calcium-dependent and required a In addition. a nonproteinaceous substance secreted into the culture supernatant [10].

bulgaricus because it can cause overacidification [74]. For example. the Japanese government in 1991 set up a licensing system for "foods for specified health use". 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. casei. longum BB536. thermophilus to provide proteolytic activity. 126 ]. captured 15% of the Danish yogurt market following its introduction in 1993. In 1930 L. a yogurt sold in Switzerland by Tonilait and containing Lactobacillus reuteri. and E. D. Over 70% of the functional foods developed in Japan are liquids and one citrus-flavored probiotic drink (Yakult). Probiotic plu Oligofructose. a functional food "containing ingredients that aid specific body functions in addition to being nutritious" [59. omitting L. casei Shirota was isolated from the human colon [76] and it became the basis for the Yakult Honsha Co. A list of some probiotic fermented milk products currently produced in Japan and Europe is presented in Table 4. thermophilus. Symbalance. Mil-Mil E. i. C. In order to obtain good organoleptic properties the bifidus-supplemented products are fermented with S. which produces several probiotic dairy products. containing L. sold by Danone.596 Probiotic c o m m e r c i a l i z a t i o n S. SCHEINBACH In terms of consumer awareness of probiotics. sells 1 million 50-ml bottles a day in Japan [59] and 23 million word-wide [76]. made in Germany and containing L. But when an advertising campaign claiming serum cholesterol reductions accompanied its recent introduction in the United Kingdom.e. and made in Denmark by MD Foods. a milk drink containing L. ProCult3 with B. The former is a fermented milk product made by Yoplait and the latter is made by Dairy Denmark using Chr Hansen's cultures [23]. numbering over 70 products [ 119]. In Japan full-scale bifidus milk and yogurt production began in 1977. casei. The Cultura product uses selected strains that . Gaio. Examples of recent introductions are: Actimel Orange. containing S. sold by Muller. negative government response resulted in it being withdrawn [130]. Japan has taken the lead with Europe coming in second and the United States a distant third. acidophilus and Lactobalillus bifidus LA7. acidophilus and L. introduced in 1982 is a bifidobacteria-containingyogurt supplemented with vitamins A. with home delivery of products containing Bifdobacterium longum and S. By 1984 the number of fermented products had reached 20 [59]. casei. L. In both Japan and Europe probiotic-supplemented dairy foods and drinks continue to be a fast-growing industry. Estimates indicate that functional food may capture 5% of the total Japanese market [59]. These now join the list of older products such as Ofilus and Cultura. thermophilus and E.faecium. In 1995 Yakult Honsha extended the distribution of Yakult into several European countries [130] where new probiotic dairy products appear regularly. Recognizing the benefits of diet and health to an aging population.

PROBIOTICS grow symbiotically in milk to yield the desired pH without the bifidibacteria producing too much acetic acid. L. casei. lon~um. most fermented milk products containing . acidophilus. Biogarde from Germany contains L. bifidum. Better growth could be achieved by supplementing milk with yeast extract or whey protein. In order to overcome these manufacturing problems. bifidum. bulgaricus in order to reduce the level of lactic acid in the product. S. and by adding threonine to promote acetaldehyde production and enhance flavor [75]. bifidum. lactis. acidophilus. S. acidophilus. casei B. casei GG (rhamnosus) L. acidophilus. cremoris E. longum. thermophilus L. acidophilus. S. acidophilus. Pediococcus acidilactici B. but not L. Similarly. L. acidophilus. thermophilus and B. thermol~hilus. acidophilus. bifidus.aricus. yogurt culture L. S. L. L. B. S. bifidum. acidophilus L. LA7 B. bifidum. acidophilus Gilliland GeNTlany Germany Finland Japan Japan Japan Slovakia France Denmark Europe Switzerland Germany Germany Germany Netherlands Cultures of L. bul~. thermophilus B. when cocultured with lactobacilli. L. acidophilus L. yogurt culture B. B. acidophilus Lal L. S. L. they quickly become inhibited as the pH drops. Commercial Probiotic Dairy Products in Japan and Europe (adapted from 23) 597 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 Organism(s) S. bifidum/B. faecium. thermophilus L. L. lon~um BB536 L. acidophilus. lose viability upon storage and do not yield products with favorable organoleptic qualities [74]. L. casei GG and Bifdobacteria spp. intestinal bile and capable of colonizing the colon. bifidum L. reuteri. Table 4. surviving stomach acid. Moreover. L. casei L. make good probiotics because they are of human origin. acidophilus L. Most Bifidobacterium strains cannot ferment milk by themselves because they require low redox potentials and peptides generated from the breakdown of casein [64]. B. But milk products fermented with these organisms could not readily expand into the market place because human strains grow slowly in milk.

Natural FOS are found in plants such as Jerusalem artichokes. timing of harvest. the benefits of probiotics are unproven. most of the claims have yet to be proven. also occurs with certain strains of lactobacilli. 44] and translating animal results to humans must be done with care because there are significant differences between them. were shown to stimulate bifidobacteria growth in vitro [127] and in healthy human subjects [33]. the human intestinal flora are refractory to dietary changes [27]. It has been reported that the enzyme oxyrase stimulates growth of bifidobacteria in skim milk [77]. More studies need to be published in . 42. The latter have been patented as bifidus-growth factors by Yakult Honsha in Japan [92]. 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. Substances that pass through the gut undigested have been investigated for their intestinal bifidogenic ability. Efficacy with regard to cholesterol reduction and immune system stimulation is also unproven. preparation of concentrate and storage conditions. For example. 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. Where disease is present. but claims of cancer suppression remain to be proven. and transgalactosyl oligosaccharides. CONCLUSIONS While human studies show that feeding probiotics can be efficacious under certain circumstances. barely. Even two apparently identical probiotics with the same cell titer may. those who are lactose intolerant probably benefit the most. in fact. Obviously. Other bifidogenic factors are lactulose (4-O-B-D-galactopyranosyl-D-fructose). In children limited protection from diarrhea. be different due to differing growth conditions. In contrast. For example. Another failing of many probiotic studies that makes comparisons problematic is the variety of strains used and the lack of standard preparation. molecular diagnostic techniques have shown that several commercial LAB cultures are different species than previously believed [110]. garlic and onion [28] or can be commercially produced by the action of fructofuranosidase on sucrose [120]. However. For healthy individuals. changing the diet of rats from chow to ground beef causes dramatic changes in the colonic microflora [114]. Several probiotic dairy products containing FOS are now being marketed in Europe [130].598 S. Oral consumption of LAB may reduce the levels of certain fecal enzymes thought to promote cancer. especially inulin. The fructooligosaccharides (FOS). bananas. especially rotaviral. 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. SCHEINBACH bifidobacteria are inoculated with the final number of cells needed in the product [64]. derived from isomerization of the lactose in whey [88].

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