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

PROBIOTICS: FUNCTIONALITY AND COMMERCIAL STATUS

SAUL SCHEINBACH

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, 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 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

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 585

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

LAB (adapted from 109)

N u m b e r of Lactose Product/Culture Tested aSignificant Breath

Intolerant Subjects Hydrogen Reduction
[ref.] vs Milk or Lactose
12163] Unfermented L. acidophilus milk +
10165] ¥o~t +
91113] Yogurt +
Buttermilk,
Unfermented L. acidophilus milk
Heated ),o~:urt
14182] Yogurt +
Heated yogurt +/-
Unfermented L. acidophilus milk
As above but sonicated +
8 [ 129] Three yogurts + Dannon®
+Royal Maid®
-Borden®
10169] Unfermented S. thermophilus, L. +/- @107 cfu/ml
bulgaricus milk + @108 cfu/ml
Unfermented L. acidophilus milk with:
LA1 - @ 107 cfu/ml
LA2 +/- @108 cfu/ml
NCFM - @107 or 108 cfu/ml
7[79] 7 yogurts with different S. +
thermophilus/L, bulg,aricus strains
12[79] S. thermophilus/L, bulgaricus yogurt +
Fermented milk with:S, thermophilus +
:L. bulgaricus +
:L. acidophilus
:B. bifidus
22[78] Yogurt(n=22) +
Yogurt and a meal(n= 12) +
Yogurt and lactose(n= 10)
8 children[89] Milk with L. acidophilus NCFM _b
Milk with S. thermophilus and L. +b
lactis
11191] Unfermented acidophilus milk with:
strain ATCC4356
strain B +/-
strain N 1 +/-
strain E +
a+ = positive result; - = negative result; b other symptoms significantlyreduced 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-inducedlowering 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 (LactinexTM) 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

T a b l e 2. 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.) Subjects a Cause Results Comments Efficacy b

L. acid.[7] 59 adults Abdominal $ symptoms No controls ?
symptoms
S. therm. L. acid. 94 children Acute onset
L. bulg.[97] < 3 years > 10 months
old
Lactobacilli[100] 26 healthy Travelers' diarrhea Lower incidence
adults
E. faecium[8] 53 children Various causes $ duration +
LactinexTM 23 healthy Enterotoxigenic E.
Lactobacilli[ 171 adults coli (ETEC)
LactinexTM 23 healthy ETEC
Lactobacilli[ 18] adults
LactinexTM 36 adults Neomycin $ symptoms Lot to lot cell
Lactobacilli[ 18] with ETEC variability
Sweet acid. milk 61 adults Irritable bowel
[931
B. longum[19] 10 healthy Erythromycin ,[, symptoms Few subjects
adults
L. casei GG[46] 5 adults C. difficle colitis $ symptoms Few subjects, no
with colitis controls
B. brevi, B.bif. 15 children Intractable $ duration No controls ?
L. casei[58]
L. acid.[107] 11 adult Undergoing $ incidence No controls, ?
females radiation therapy subjective
reporting
L. casei GG [117] 8 healthy Erythromycin $ duration, Few subjects, no
adults symptoms statistics
L. casei GG[94] 349 healthy Traveler's diarrhea $ incidence Only in 1 of 2
adults destinations
LactinexTM 15 children Amoxicillin No prevention
Lactobacilli[ 123] (5-76 mos.)
L. casei GG[61] 47 children Acute viral (mainly $ duration +

(4-45 mos.) rotaviral)

B. bifidum 29 children Acute $incidence +
S. therm.[106] (5-24 mos.) gastroenteritis androtavkal
(mainly rotaviral) shedding
L. caseiGG, L. 30 children Acute rotaviral $ duration, Neg. for 14 L. +
caseirh.[73] (4-35 mos.) IgA casei rh. cases
L. caseiGG[101] 21 children Acute (some $ duration Pos. for acute +
rotaviral) non-bloody cases
L. casei GG[96] 20 children Acute (some $ vomiting, Pos. for acute
(1-24 mos) rotaviral) watery stool watery diarrhea
L. casei GG[62] 21 children Acute rotaviral $ duration No placebo for
(5-28 mos.) control group
L. casei GG [116] 59 children Acute rotaviral, $ duration No effect on
(1-36 mos.) bacter., unknown bacterial cases
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 f L . 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

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]. 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 vitro [3] and act as promoters of
nitrosoguanidine-inducedcolon 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 593

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

N u m b e r of O r g a n i s m ( s ) and Daily Dose Reduction of Fecal E n z y m e Activity b

Subjects a (cfu)
1214] L. acidophilus DDS1 in milk; 4x109 ? ~-glucuronidase
(no statistics)
71451 L. acidophilus; 4x101° + ([~-glucuronidase
+ nitroreductase
- azoreductase
- 7-~-deh~cdrox~clase
7[42] L. acidophilus NCFM; l x l 0 I° + nitroreductase
- azoreductase
21141] L. acidophilus NCFM; lxl09 + nitroreductase
(n=l 0) or L. acidophilus N-2; l x l 09 + azoreductase
(n=l 1) + p-glucuronidase
91801 L. acidophilus; 3x 109 + nitroreductase
B. bifidum; 3x101° - azoreductase
S. lactis; 3x101° - ~-glucuronidase
S. cremoris; 3x101°
81431 L. casei GG; lxl01° (frozen + 13-glucuronidase
concentrate)
14 [71] L. casei GG yogurt; 3x101° + 13-glucuronidase
+ nitroreductase
6113] Bifidobacteria; 4x1011 (fermented + 13glucuronidase
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.

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. 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

Probiotic c o m m e r c i a l i z a t i o n

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-containingyogurt 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 Countrf of Origin Organism(s)

ACO-yogurt Switzerland S. thermol~hilus; L. bul~,aricus; L. acidophilus
Cultura-AB Denmark L. acidophilus; B. bifidum
AB-yogurt Denmark L. acidophilus; B. bifidum; yogurt culture
Biogarde GeNTlany L. acidophilus; B. bifidum; S. thermophilus
Bifighurt Germany B. lon~um; S. thermophilus
Gefilac Finland L. casei GG (rhamnosus)
Yakult Japan L. casei
Miru Miru Japan L. acidophilus; L. casei
Mil Mil E Japan B. bifidum; yogurt culture
Biokys Slovakia B. bifidum; L. acidophilus; Pediococcus
acidilactici
Ofilus France B. bifidum/B, longum; L. acidophilus;
S. lactis; S. cremoris
Gaio Denmark E. faecium; S. thermophilus
LC1 Europe L. acidophilus Lal
Symbalance* Switzerland L. reuteri; L. casei; L. acidophilus
Probiotic plus Germany L. acidophilus; L. bifidus; LA7
Oligofructose*
ProCult3 Germany B. lon~um BB536
Actimel Orange Germany L. acidophilus
Fysiq* Netherlands L. acidophilus Gilliland
*contains FOS

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.

REFERENCES

1. Agerbaek, M., Gerdes, L.U. and Richelsen, B. (1995), Hypocholesterolaemic effect of a

new fermented milk product in healthy middle-aged men. Eur. J. Clin. Nutr. 49, 346-
352.
2. Allen, W.D., Linggood, M. and Porter, P. (1996), Probiotic. U.S. Patent 5,589,168
Unilever Patent Holdings B.V.
3. Autrup, H., Harris, C.C. and Jeffrey, A.M. (1978), Metabolism of benzo(a)pyrene and
identification of the major benzo(a)pyrene-DNA adducts in cultured human
colonocytes. Cancer Res. 38, 3689.
4. Ayebo, A.D., Angelo, I.A. and Shahani, K.M. (1980), Effect of ingesting Lactobacillus
acidophilus milk upon fecal flora and enzyme activities in humans. Milchwissenschaft
35, 730-733.
5, Ballongue, J. (1993), In Lactic Acid Bacteria., (Salimen, S. and von Wright, A., editors),
Marcel Dekker Publishers (New York). Bifidobacteria and probiotic action
6. Bayless, T.M., Paige, M. and Ferry, G.D. (1971), Lactose intolerance and milk drinking
habit. Gastroenterology 60, 605.
7. Beck, C. and Necheles, H. (1961), Beneficial effects of administering Lactobacillus
acidophilus in diarrheal and other intestinal disorders. Amer. J. Gastroenterol. 35, 522-
530.
600 S. SCHEINBACH

8. Bellomo, G., Mangiagle, A., Nicastro, L. and G. Frigerio. (1980), A controlled double-
blind study of SF68 strain as a new biological preparation for the treatment of diarrhea
in pediatrics. Curr. Therap. Res. 28, 927-936.
9. Benno, Y., Sawada, K. and Mitsuoka, T. (1984), The intestinal microflora of infants:
Composition of fecal flora in breast-fed and bottle-fed infants. Microbiol. Immunol. 28,
975-986.
10. Bernet, M.F., Brassart, D., Neeser, J.R and Servin, A.L. (1994), Lactobacillus
acidophilus Lal binds to cultured human intestinal cell lines and inhibits cell attachment
and cell invasion by enterovirulent bacteria. Gut 35, 483-489.
11. Bernet-Camard, M.F., Li6vin, V., Brassert, D., Neeser, J-R., Servin, A.L. and Hudault,
S. (1997), The human Lactobacillus acidophilus strain Lal secretes a nonbacteriocin
antibacterial substance(s) active in vitro and in vivo. Appl. Environ. Microbiol. 63,
2747-2753.
12. Bezkorovainy, A. (1989), Ecology of bifidobacteria. In Biochemistry and Physiology oJ
Bifidobacteria, (Bezkorovainy, A. and Miller-Catchpole, R. editors), CRC Press (Boca
Raton).
13. Bouhnik, Y., Flourie, B., Andrieux, C., Bisetti, N., Briet, F. and Rambaud, J-C. (1996),
Effects of Bifidobacterium sp fermented milk ingested with or without inulin on colonic
bifdobacteria and enzymatic activities in healthy humans. Eur. J. Clin. Nutr. 50, 269-
273.
14. Bullen, C.L., Teale, P.V. and Stewart, M.G. (1977), The effect of "humanized" milks
and supplemented breast feeding on the fecal flora of infants. J. Med. Microbiol. 10,
403-413.
15. Chikai, T., Nakao, H. and Uchida, K. (1987), Deconjugation of bile acids by human
intestinal bacteria implanted in germ-free rats. Lipids 22, 669-671.
16. Claeson, M. and Merson, M.H. (1990), Global progress in the control of diarrheal
disease. Pediatr. Infect. Diseases. 9, 345-355.
17. Clements, M.L., Levine, M.M. and Black, R.E. (1981), Lactobacillus prophylaxis for
diarrhea due to enterotoxigenic Escherichia coli. Antimicrob. Agents Chemother. 20,
104-108.
18. Clements, M.L., Levine, M.M, Ristaino, P.A., Daya, V.E. and Hughes, T.P. (1983),
Exogenous lactobacilli fed to man(their fate and ability to prevent diarrheal disease.
Prog. Fd. Nutr. Sci. 7, 29-37.
19. Columbel, J.F., Cortot, A., Neut, C. and Romond, C. (1978), Yogurt with
Bifidobacterium longum reduces erythromycin-induced gastrointestinal effects. Lancet
ii~ 43.
 PROBIOTICS 601

20. Conway, P.L. (1996), Selection criteria for probiotic microorganisms. Asia Pac. J. Clin.
Nutr. 5, 10-14.
21. Cummings, J.H. (1981), Short chain fatty acids in the human colon. Gut 22, 763-779.
22. Cummings, J.H. and Macfarlane, G.T. (1991), A review: the control and consequences
of bacterial fermentation in the human colon. J. Appl. Bacteriol. 70, 443-459.
23. Daly, C. (1991), Lactic acid bacteria and milk fermentation. J. Chem. Technol.
Biotechnol. 51,544-548.
24. Deneke, C.F., McGowan, K., Thorne, G.M. and Gorbach, S.L. (1983), Attachment of
enterotoxigenic Escherichia coli to human intestinal cells. Infect. Immun. 39, 1102-1106.
25. Drasar, B.S., Renwick, A.G. and Williams, R.T. (1972), The role of the gut flora in the
metabolism of cyclamate. Biochem. J. 129, 881-890.
26. Fernandes, C.F. and Shahani, K.M. (1990), Anticarcinogenic and immunological
properties of dietary lactobacilli. J. Food Prot. 53, 704-710.
27. Finegold, S.M., Attebery, H.R. and Sutter, V.L. (1974), Effect of diet on human fecal
flora: Comparison of Japanese and American diets. Amer. J. Clin. Nutr. 27, 1456-1469.
28. Fishbein, L., Kaplan, M. and Gough, M. (1988), Fructooligosaccharides: A review. Vet.
Human Toxicol. 30(2), 104-107.
29. Fletcher, J.M. 1995. Second generation functional foods. Flair-Flow reports. F-FE
190/95.
30. Fletcher, J.M. and van Noortlaan, O. (1997), Emerging Food R&D Report. June 1997.
31. Fuller, R. (1992), History and development of probiotics. In Probiotics. The Scientific
Basis, (Fuller, R., editor), Chapman & Hall Publishers (London).
32. Fuller, R. (1996), Probiotics-panacea or nostrum? BNF Nutr. Bull. 21,204-208.
33. Gibson, G.R., Beatty, E.R., Wang, X. and Cummings, J.H. (1995), Selective stimulation
of bifldobacteria in the human colon by oligofructose and inulin. Gastroenterology 108,
975-982.
34. Gilat, T., Russo, S., Gelman-Malachi, E. and Aldor, T.A. (1972), Lactose in man: A
non-adaptable enzyme. Gastroenterology 102, 1269-1277.
35. Gilliland, S.E., Nelson, C.R. and Maxwell, C. (1985), Assimilation of cholesterol by
Lactobacillus acidophilus. Appl. Environ. Microbiol. 49, 377-381.
36. Gilliland, S.E. and Speck, M.L. (1977), Deconjugation of bile acids by intestinal
lactobacilli. Appl. Environ. Microbiol. 33, 15-18.
37. Gilliland, S.E., Speck, M.L., Nauyok, Jr., G.F. and Giesbrecht, F. G. (1978), Influence
of consuming nonfermented milk containing Lactobacillus acidophilus on fecal flora of
healthy males. J. Dairy Sci. 61, 1-10.
602 S. SCI-IEINBACH

38. Gilliland, S.E. and Walker, D.K. (1990), Factors to consider when selecting a culture of
Lactobacillus acidophilus as a dietary adjunct to produce a hypocholesterolemic effect
in humans. J. Dairy Sci. 73, 905-911.
39, Goldberg, I. (1994), Introduction. In Functional Foods, Designer Foods, Pharmafoods,
Neutraceuticals, (Goldberg, I., editor), Chapman & Hall Publishers(New York).
40. Goldin, B.R. and Gorbach, S.L. (1980), Effect of Lactobacillus acidophilus dietary
supplements on 1,2-dimethylhydrazine dihydrochloride-induced intestinal cancer in
rats. J. Natl. Cancer Inst. 64, 263-265.
41. Goldin, B.R. and Gorbach, S.L. (1984), The effect of milk and lactobacillus feeding on
human intestinal bacterial enzyme activity. Am. J. Clin. Nutr. 39, 756-761.
42. Goldin, B.R. and Gorbach, S.L. (1984), The effect of oral administration on
Lactobacillus and antibiotics on intestinal bacterial activity and chemical induction of
large bowel tumors. Dev. Indust. Microbiol. 25, 139-150.
43. Goldin, B.R., Gorbach, S.L., Saxelin, M., Barakat, S., Gualitieri, L. and Salminen, S.
(1992), Survival of Lactobacillus species (strain GG) in human gastrointestinal tract.
Digestive Diseases and Sci. 37,121-128.
44. Goldin, B.R. and Gualtieri, L.J. (1996), The effect of Lactobacillus GG on the initiation
and promotion of DMH-induced intestinal tumors in the rat. Nutr. Cancer. 25, 197-204.
45. Goldin, B.R., Swenson, L., Dwyer, J., Sexton, M. and Gorbach, S.L. (1980), Effect of
diet and Lactobacillus acidophilus supplements on human fecal bacterial enzymes. J.
Natl. Cane. Instit. 64, 255-261.
46. Gorbach, S.L., Chang, T-W. and Goldin, B. (1987), Successful treatment of relapsing
Clostridium difficle colitis with Lactobacillus GG. Lancet ii, 1519.
47. Gorbach, S.L. and Goldin, B. (1989), Lactobacillus strains and method of selection. U.S.
Patent 4,839,281.
48. Gorbach, S.L. and Goldin, B. (1991), L. acidophilus strains. U.S. Patent 5,032399.
49. Greene, J.D. and Klaenhammer, T.R. (1994), Factors involved in adherence of
lactobacilli to human Caco-2 cells. Appl. Environ. Micrbiol. 60, 4487-4494.
50. Hashimoto, S., Nomoto, K., Matsuzaki, T., Yokokura, T. and Mutai, M. (1984),
Oxygen radical production by peritoneal macrophages and kupffer cells elicited with
Lactobacillus casei. Infect. Immun. 44, 61-67.
51. Hawksworth, G., Drasar, B.S. and Hill, M.J. (1971), Intestinal bacteria and the
hydrolysis of glycosidic bonds. J. Med. Microbiol. 4, 451-459.
52. Hays, S.M. (1994), Natural microbes curb salmonella. Agile. Res. 42, 22-26.
53. Halpern, G.M., Vruwink, K.G., Van de Water, J, Keen, C.L. and Gershwin, M.E.
(1991), Influence of long-term yoghurt consumption in young adults. Internat. J.
Immunotherapy 7, 205-210.
 PROBIOTICS 603

54. Hamilton-Miller, J.M.T., (1996), Probiotics-panacea or nostrum? BNF Nutr. Bull. 21,
199-208.
55. Holdeman, L.V., Good, I.J. and Moore, W.E.C. (1976), Human fecal flora: Variation in
bacterial composition within individuals and a possible effect of emotional stress. Appl.
Environ. Microbiol. 31,359-375.
56. Hose, H. and Sozzi, T. (1991), Probiotics, fact or fiction. J. Chem. Technol. Biotechnol.
51,540-544.
57. Hudault, S., Li6vin, S.,Bernet-Camard, M.F. and Servin, A.L. (1997), Antagonistic
activity exerted in vitro and in vivo by Lactobacillus casei (strain GG) against
Salmonella typhimurium C5 infection. Appl. Environ. Microbiol. 63, 513-518.
58. Hotta, M., Sato, Y., Iwata, S., Yamashita, N., Sunakawa, K., Oikawa, T., Tanaka, R.,
Watanabe, K., Takayarna, H., Yajima, M., Sekiguchi, S., Arai, S., Sakurai, T. and Mutai,
M. (1987), Clinical effects of Bifidobacterium preparations on pediatric intractable
diarrhea. Keio. J. Med. 36, 298-314.
59. Ichikawa, T. (1994), Functional foods in Japan. In Functional Foods, Designer Foods,
Pharmafoods, Neutraceuticals, (Goldberg, I., editor), Chapman & Hall Publishers (New
York).
60. Ishibashi, N. and Shimamura, S. (1993), Bifidobacteria: Research and development in
Japan. Food Tech. 47(6), 126-136.
61. Isolauri, E., Juntunen, M., Rautenen, T., Sillanaukee, P. and Koivula, T. (1991), A
human Lactobacillus strain (Lactobacillus casei sp. strain GG) promotes recovery from
acute diarrhea in children. Pediatrics 88, 90-97.
62. Isolauri, E., Kaila, M., Mykk~nen, H., Ling, W.H. and Salminen, S. (1994), Oral
bacteriotherapy for viral gastroenteritis. Dig. Dis. Sci. 39, 2595-2600.
63. Kim, H.S. and Gilliland, S.E. (1983), Lactobacillus acidophilus as a dietary adjunct for
milk to aid lactose digestion in humans. J. Dairy Sci. 66, 959-966.
64. Klaver, F.A.M., Kingma, F. and Weerkamp, A.H. (1993), Growth and survival of
bifidobacteria in milk. Netherlands Milk Dairy J. 47, 151-164.
65. Kolars, J.C., Levitt, M.D., Aouji, M. and Savaiano, D.A. (1984), Yogurt-an
autodigesting source of lactose. N. Engl. J. Med. 310, 1-3.
66. Kretchmer, N. (1972), Lactose and lactase. Sci. Amer. 227:71.
67. Lee, A. (1985), In Advances in Microbial Ecology, (Marshall, K.C. editor), Plenum
Press (New York). Neglected niches: The microbial ecology of the gastrointestinal tract.
68. Lee, Y-K and Salminen, S. (1995), The coming age ofprobiotics. Trends Fd. Sci. Tech.
6, 241-245.
604 S. SCHEINBACH

69. Lin, M-Y., Savaiano, D. and Harlander, S. (1991), Influence of nonfermented dairy
products containing bacterial starter cultures on lactose maldigestion in humans. J.
Dairy Sci. 74, 87-95.
70. Lin. S.Y., Ayres, J.W., Winkler Jr., W. and Sandine, W. (1989), Lactobacillus effects on
cholesterol: In vitro and in vivo results. J. Dairy Sci. 72, 2885-2899.
71. Ling. W. H., Korpela, R., Mykk~inen, H., Salminen, S. and H~inninen, O. (1994),
Lactobacillus strain GG supplementation decreases colonic hydrolytic and reductive
enzyme activities in healthy female adults. J. Nutr. 124, 18-23.
72. Link-Amster, H., Rochat, F., Saudan, K.Y., Mignot, O. and Aeschlimann, J.M. (1994),
Modulation of a specific humoral immune response and changes in intestinal flora
mediated through fermented milk intake. FEMS Immunol. Med. Microbiol. 10, 55-64.
73. Majamaa, H., Isolauri,E., Saxelin, M. and Vesikari, T. (1995), Lactic acid bacteria in the
treatment of acute rotavirus gastroenteritis. J. Pediatr.Gastroenterol. & Nutr. 20, 333-
338.
74. Marshall, V.M. (1991), Gut-derived organisms for milk fermentations. J. Chem.
Technol. Biotechnol. 51,548-553.
75. Marshall, V. M., Cole, W.M. and Mabbitt, L.A. (1982), Fermentation of specially
formulated milk with single strains of bifidobacteria. J. Dairy Technol. 35, 143-144.
76. Martin, C. (1996), The elixir of life? Chemistry in Britain. Aug. 1996.34-36.
77. Martin, J.H. (1996), Technical considerations for incorporating bifidobacteria and
bifidogenic factors into dairy products. Bulletin Internat. Dairy Fed. 313, 49-51.
78. Martini, M., Kukiella, D. and Savaiani, D.A. (1991), Lactose digestion from yogurt:
Influence of a meal and additional lactose. Am. J. Clin. Nutr. 53, 1253-1258.
79. Martini, M., Lerebours, E.C., Lin, W-J., Harlander, S., Berrada, N.M., Antoine, J.M.
and Savaiano, D.A. (1991), Strains and species of lactic acid bacteria in fermented milks
(yogurts): Effect on in vivo lactose digestion. Am. J. Clin. Nutr. 54, 1041-1046.
80. Marteau, P., Pochart, P., Flourir, B.,Pellier, P.,Santos, L., Desjeux, J-F and Rambaud, J-
C. (1990), Effect of chronic ingestion of a fermented dairy product containing
Lactobacillus acidophilus and Bifidobacterium bifidum on metabolic activities of the
colonic microflora in humans. Am. J. Clin. Nutr. 52, 685-688.
81. McCartney, A.L., Wang,W. and Tannock,G.W. (1997), Molecular analysis of the
composition of the Bifidobacterial and Lactobacillus microflora of humans. Appl.
Environ. Microbiol. 62, 4608-4613.
82. McDonough, F.E., Wong, N.P., Hitchins, A.D. and Bodwell, C.E. (1987), Alleviation of
lactose malabsorption from sweet acidophilus milk. Am. J. Clin. Nutr. 42, 345-346.
 PROBIOTICS 605

83. McDonough, F.E., Hitchins, A.D., Wong, N.P., Wells P. and Bodwell, C.E. (1987),
Modification of sweet acidophilus milk to improve utilization by lactose-intolerant
persons. Am. J. Clin. Nutr. 45, 570-574.
84. Mclntosh, G.H. (1996), Probiotics and colon cancer prevention. Asia Pac. J. Clin. Nutr,
5, 48-52.
85. Metchnikoff, E. (1907), In The prolongation of life, Heinemarm Publishers (London).
86. Mevissen-Verhage, E.A.E., Marcelis, J.H., De Vos, M.N., Harmsen-Van Amerongen,
W.C.M. and Verhoef, J. (1987), Bifdobacterium, Bacteroides and Clostridium spp. in
fecal samples from breast-fed and bottle-fed infants with and without iron supplement.
J. Clin. Microbiol. 25, 285-289.
87. Mishra, C. and Lambert, J. (1996), Production of anti-microbial substances by
probiotics. Asia Pac. J. Clin. Nutr. 5, 20-24.
88. Mizota, T., Tamura, Y., Tomita, M. and Okonogi, S. (1987), Lactulose as a sugar with
physiological significance. Bull. Internat. Dairy Fed. 212, 69-76.
89. Montes, R.G., Bayless, T.M., Saavedra, J.M. and Perman, J.A. (1995). Effect of milks
inoculated with Lactobacillus acidophilus or a yogurt starter culture in lactose-
maldigesting children. J. Dairy Sci. 78, 1657-1664.
90. Moore, W.E.C. and Holdeman, L.V. (1974), Human fecal flora: The normal flora of 20
Japanese-Hawaiians. Appl. Microbiol. 27, 961-979.
91. Mustapha, A., Jiang, A. and Savaiano, D.A. (1997), Improvement of lactose digestion
by humans following ingestion of unfermented acidophilus milk: Influence of bile
sensitivity, lactose transport, and acid tolerance of Lactobacillus acidophilus. J. Dairy
Sci. 80,1537-1545.
92. Mutai, M., Terashima, T,, Takashi, T., Tanaka, R., Kuroda, A., Ueyama, S. and
Matsumoto, K. (1980), Composition for promoting growth of bifidobacteria and the
method for manufacture thereof. U.K. Patent App. 2,080,330. Kabushiki Kaisha Yakult
Honsha.
93. Newcomer, A.D., Park, H.S., O'Brien, P.C. and McGill, D.B. (1983), Response of
patients with irritable bowel syndrome and lactase deficiency using unfermented
acidophilus milk. Amer. J. Clin. Nutr. 38, 257-263.
94. Oksanen, P.J., Salminen, S., Saxelin, M., Htimtiltiinen, P., Ihantola-Vormisto, A.,
Muurasniemi-Isoviita, L., Nikkari, S., Oksanen, T., Ptirsti, I., Salminen, E., Siitonen, S.,
Stuckey, H., Toppila, A. and Vapaatalo, H. (1990), Prevention of travellers' diarrhea by
Lactobacillus GG. Annals of Medicine. 22, 53-56.
95. O'Sullivan, M.G., Thornton, G., O'Sullivan, G.C. and Collins, J.K. (1992). Probiotic
bacteria: myth or reality? Trends Fd. Sci. Tech. 3, 309-314.
606 S. SCHEINBACH

96. Pant, A.R., Graham, S.M., Allen, S.J., Harikul, S., Sabchareon, A., Cuevas, L. and Hart,
C. A. (1996), Lactobacillus GG and acute diarrhea in young children in the tropics. J.
Trop. Pediatr. 42, 162-165.
97. Pearce, J.L., and Hamilton, J.R. (1974), Controlled study of of orally administered
lactobacilli in acute infantile diarrhea. J. Pediat. 84, 261-262.
98. Perdigon, G., Nader de Macias, M. E., Alvarez, S., Medici, M., Oliver, G. and Pesce de
Ruiz Holgado, A. (1986), Effect of a mixture of Lactobacillus casei and Lactobacillus
acidophilus administered orally on the immune system in mice. J. Food. Prot. 49, 986-
989.
99. Perdigon, G., Alvarez, S., Nader de Macias, M. E., Roux, M.E., and Pesce de Ruiz
Holgado, A. (1990), The oral administration of lactic acid bacteria increase the mucosal
intestinal immunity in response to enteropathogens. J. Food Prot. 53, 404-410.
100. Pozo-Olano, J.D., Warram, J.G., Jr., Gomez, R.G. and Cavazos, M.G. (1978), Effect of
a lactobacilli preparation on traveler's diarrhea. A randomized, double blind clinical trial.
Gastroenterology 74, 829-830.
101. Raza, S., Graham, S.M., Allen, S.J., Sultana, S. and Cuevas, L. (1995), Lactobacillus
GG promotes recovery from acute nonbloody diarrhea in Pakistan. Pediatr. Infect. Dis.
J. 14, 107-111.
102. Reddy, B.S., Watanabe, K., Weisburger, J.H. and Wynder, E.L. (1977), Promoting
effect of bile acids in colon carcinogenensis in germ-flee and conventional F344 rats.
Cancer Res. 37, 3238-3242.
103. Richardson, D. (1996), Probiotics and product innovation. Nutr. Food Sci. 6, 27-33.
104. Richelsen, B., Kristensen, K. and Pederson, S.B. (1996), Long-term (6 months) effect of
a new fermented milk product on the level of plasma lipoproteins-a placebo-controlled
and double blind study. Eur. J. Clin. Nutr. 50, 811-815.
105. Rowland, I.R. (1992), In Probiotics. The Scientific Basis, (Fuller, R., editor), Chapman
& Hall Publishers (London). Metabolic interactions in the gut.
106. Saavedra, J.M., Bauman, I.O., Perman, J.A. and Yolken, R.H. (1994), Feeding of
Bifidobacterium bifidum and Streptococcus thermophilus to infants in hospitals for the
prevention of diarrhea and shedding of rotavirus. Lancet 344, 1046-1049.
107. Salminen, E., Elmoaa, I., Minkkinen, J., Vapaatalo, H. and Salminen, S. (1988),
Preservation of intestinal integrity during radiotherapy using live Lactobacillus
acidophilus cultures. Clin. Radiol. 39, 435-437.
108. Sanders, M.E. (1993), Effect of consumption of lactic cultures on human health. Adv. in
Food Nutr. Res. 37, 67-130.
 PROBIOTICS 607

109. Sanders, M.E. (1994), In Functional Foods, Designer Foods, Pharmafoods,

Neutraceuticals, (Goldberg, I., editor), Chapman & Hall Publishers (New York). Lactic
acid bacteria as promoters of human health.
110. Sanders, M.E., Walker, D.C., Walker, K.M., Aoyama, K. and Klaenhammer, T.R.
(1996), Performance of commercial cultures in fluid milk applications. J. Dairy. Sci. 79,
943-955.
111. Sarem-Damerdji, L., Sarem, F., Marchal, L., and Nicolas, J-P. (1995), In vitro
colonization ability of human colon mucosa by exogenous Lactobacillus strains. FEMS
Microbiol. Lett. 131, 133-137.
112. Savage, D.C. (1977), Microbial ecology of the gastrointestinal tract. Ann. Rev.
Microbiol. 31, 107-133.
113. Savaiano, D.A., AbouELAnouar, A., Smith, D.E. and Levitt, M.D. (1984), Lactose
malabsorption from yogurt, pasteurized yogurt, sweet acidophilus milk and cultured
milk in lactase-deficient individuals. Am. J. Clin. Nutr. 40, 1219-1223.
114. Scheinbach, S., Hayes, J.R., Carman, R.J., Zhou, D., Van Tassel. R.L. and Wilkins, T.D.
(1994), Effects of structured triacylglycerols containing stearic, acetic, and propionic
acids on the intestinal microflora of rats. J. Agric. Fd. Chem. 42, 572-580.
115. Schiffren, E.J., Rochat, F., Link-Amster, H., Aeschlimann, J.M. and Donner-Hughes.
(1995), Immunomodulation of human blood cells following the ingestion of lactic acid
bacteria. J. Dairy Sci. 78,491-497.
116. Shomikova, A-V, Isolauri, E., Burkanova, L., Lukovnikova, S. and Vesikari, T. (1997),
A trial in the Karelian republic of oral rehydration (sic) and Lactobacillus GG for
treatment of acute diarrhea. Acta Pediatr. 86, 460-465.
117. Siitonen, S., Vapaatalo, H., Salminen, S., Gordin, A., Saxelin, M., Wikberg, R. and
Kirkkola, A-L. (1990), Effect of Lactobacillus GG yogurt in prevention of antibiotic
associated diarrhea. Ann. Medicine (Helsinki) 22, 57-59.
118. Silva, M., Jacobus, N.V., Deneke, C. and Gorbach, S.L. (1987), Antimicrobial substance
from a human Lactobacillus strain. Antimicrob. Agents Chemother. 31, 1231-1233.
119. Sloan, A.E. (1994), Top 10 trends to watch and work on. Food Tech. 48(7), 89-100.
120. Spiegel, J.E., Rose, R., Karabell, P., Frankos, V.H. and Schmitt, D.F. (1994), Safety and
benefits of fructooligosaccharides as food ingredients. Food Tech. 48(1), 85-89.
121. Suarez, F.L. and Savaiano, D.A. (1997), Diet, genetics, and lactose intolerance. Food
Tech. 51(3), 74-76.
122. Suarez, F.L., Savaiano, D.L. and Levitt, M.D. (1995), A comparison of symptoms after
the consumption of milk or lactose-hydrolyzed milk by people with self-reported
severe lactose intolerance. New Eng. J. Med. 333, 1-4.
608 S. SCHEINBACH

123. Tankanow, R.M., Ross, M.B., Ertel, I.J., Dickinson, D.G., McCormick, L.S. and
Garfinkel, J.F. (1990), A double-blind, placebo-controlled study of the efficacy of
Lactinex in the prophylaxis of amoxicillin-induced diarrhea. DCIP Ann. Pharmacother.
24, 382-384
124. Tannock, G.W. (1997), Probiotic properties of lactic-acid bacteria: Plenty of scope for
fundamental R & D. Trends Biotech. 15, 270-274.
125. Tomioka, H., Sato,K. and Saito, H. (1990), Effect of oxofloxacin combined with
Lactobacillus casei against Mycobacterium fortuitum infection induced in mice.
Antimicrob. Ag. Chemother. 34, 632-636.
126. Van den Broek, A. (1993), Functional foods. The Japanese approach. Internat. Food
Ingred. Feb/Mar., 4-9.
127. Wang, X. and Gibson, G.R. (1993), Effects of the in vitro fermentation of oligofructose
and inulin by bacteria growing in the human large intestine. J. Appl. Micro. 75, 373-
380.
128. Weisburger, J.H., Grontham, R.E. and Weisburger, E.K. (1970), Metabolism of the
carcinogenic N-hydroxy-N-2-fluorenylacetamide in germ-free rats. Biochem. Pharrnacol.
19, 151-162,
129. Wytock, D.H. and DiPalma, J.A. (1988), All yogurts are not created equal. Am. J. Clin.
Nutr. 47,454-457.
130. Young, J. (1997), Developments in probiotics and prebiotics and symbiotics. A
European perspective. Presented at the Annual Meeting of the International Food
Technologists 1997.