Current Pharmaceutical Design, 2005, 11, 11-16 11

The Importance of Guidelines in the Development and Application of

Probiotics

Gregor Reid*

Canadian Research and Development Centre for Probiotics, Lawson Health Research Institute, and Departments of
Microbiology & Immunology, and Surgery, University of Western Ontario, London, Canada

Abstract: Probiotics, defined as ‘‘Live microorganisms which when administered in adequate amounts confer a health
benefit on the host’’ have many attributes including the lack of adverse side effects associated with their use. While
probiotics have proven benefits, the optimism associated with their use is counterbalanced by the fact that many so-called
‘probiotic’ products are unreliable in content and unproven clinically. Therefore much remains to be done to gain the
acceptance of the broader medical community.

Recognition of the obvious product inequality and the lack of any regulatory guidelines lead to the development of
Operating Standards in 2002 (FAO/WHO), that would ensure product safety, reliability and a level playing field for all
companies producing probiotic products.

The guidelines constitute a set of parameters required for a product/strain to be termed ‘probiotic’ and also the clinically
relevant steps to be followed to move probiotics closer to being embraced by the medical community. These include i)
implementation of Guidelines for use of probiotics; ii) phase I, II and III clinical trials to prove health benefits that are as
good as or better than standard prevention or treatments for a particular condition or disease; iii) Good Manufacturing
Practice and production of high quality products; iv) studies to identify mechanism of action in vivo; v) informative/
precise labelling; vi) development of probiotic organisms that can carry vaccines to hosts and/or anti- viral probiotics; vii)
expansion of proven strains to benefit the oral cavity, nasopharynx, respiratory tract, stomach, vagina, bladder and skin as
well as for cancer, allergies and recovery from surgery/ injury.

Key Words: Probiotics, prebiotics, regulatory guidelines, FAO, WHO.

INTRODUCTION OPTIMISM VERSUS SKEPTICISM

The Food and Agriculture Organization of the United
Nations (FAO) and the World Health Organization (WHO)
define probiotics as “Live microorganisms which when
administered in adequate amounts confer a health benefit on
the host” [1]. This definition reflects the fact that probiotic
remedies have been, or are being, targeted not only at the
intestine but also the oral cavity, nasopharynx, stomach,
vagina, bladder and skin. However, while this topic is
gathering momentum amongst the scientific and “alternative
medicine” communities, much remains to be done to gain the
acceptance of the broader medical community.
Historically, the levels of evidence required to compel
clinicians have been higher than those needed by other
professions. Despite this, many drugs reach the marketplace
before their clinical efficacy has been thoroughly elucidated;
and, as a result, numerous drugs are withdrawn from the
market, often because of unacceptable side effects. One
major attribute of probiotics is the lack of adverse events
associated with their use.
*Address correspondence to this author at the Canadian Research and
Development Centre for Probiotics, Lawson Health Research Institute, 268
Grosvenor Street, London, Ontario N6A 4V2, Canada; Tel: 519-646-6100
ext. 65256; Fax: 519-646-6031; E-mail: gregor@uwo.ca
The optimism associated with probiotics is counter-
balanced by skepticism, often from physicians and pharma-
ceutical representatives trained in, and still motivated by, the
use of pharmaceutical or chemical agents. Unfortunately,
some of this skepticism is warranted. Many so-called
“probiotic” products are unreliable in content and unproven
clinically. Three studies and one web-based report have
highlighted the extent to which even well known companies
have not yet reached an acceptable level of product quality
[2-5]. In the study published on the web, ConsumerLab.com
purchased twenty-five probiotic products; nineteen for use
by the general population, three specifically marketed for use
by children, and three yogurts. Of these, the manufacturers
of eight products claimed that daily servings contained
between 1 billion to 6 billion live organisms; the producers
of a further thirteen products claimed specific numbers of
live organisms (some as high as 60 billion) but only at the
time of manufacture, not indicating the number at the time of
use. On testing, eight of these specific products contained
less than 1% of the claimed number of culturable bacteria or
of the expected minimum of 1 billion. In fact, six products
contained only a few thousand viable bacteria. Another
recent study of this type, again analysed some well-known
brands and indicated that “a rather high percentage of
probiotic products suffered from incorrect labeling and

1381-6128/05 $50.00+.00 © 2005 Bentham Science Publishers Ltd.

12 Current Pharmaceutical Design, 2005, Vol. 11, No. 1
yielded low bacterial counts” [6]. Similar results were found
in Italian and Canadian studies [7]. Particularly troublesome
was the fact that products Neolactoflorene, Lacto5 and
Bifilact, bearing labels stating that they contained a non-
existent organism, "L. sporogenes" [8] (it should be named
B. coagulans), did not even contain the bacilli in question.
Recognizing the inequality of products and the lack of
any regulatory guidelines, the Argentinian government
lobbied the FAO/WHO to host an Expert Consultation to
examine whether or not probiotics had proven benefits and if
so, to establish a set of guidelines that would ensure product
safety, reliability and a level playing field. The resultant
report [1] not only established that there were indeed
excellent data on probiotics, but that a set of Guidelines or
Operating Standards was required. These emerged in 2002
[1]. The guidelines have since been received favourably by
Codex Alimentarius, the agency which oversees food safety
and health benefits of food. The guidelines make a number
of critical points which will now be presented and illustrated
in terms of relevance to clinical practice.
GUIDELINES FOR THE EVALUATION OF PRO-
BIOTICS
The guidelines constitute a set of parameters required in
order for a product or strain to be termed ‘probiotic’. At first
glance, these standards may seem overly stringent, especially
for food and small dietary supplement companies who
currently produce most of the available probiotic products
and whose margins are said to be relatively low. But, the
onus is on those who make a profit from such sales, to
provide evidence supporting their claims of health benefits.
Without this data, the manufacturers/distributors may claim
that their products are “bacterial-containing” not probiotic.
By raising their standards, such companies will, in time, be
able to access the enormous physician-based market for
probiotic sales, thereby expanding sales and profits.
STEP ONE: IDENTIFY THE EXACT STRAIN THAT
IS BEING USED
The first step in an organism(s) or product being referred
to as a probiotic is to identify and characterize the organism
to genus and species level using internationally accepted
methodologies, such as DNA-DNA hybridization, sequen-
cing of DNA encoding 16S rRNA [9], Pulsed Field Gel
Electrophoresis (PFGE) or Randomly Amplified Polymor-
phic DNA (RAPD)[10]. Newer systems are being developed
such as terminal restriction fragment-length polymorphism
(T-RFLP) [11, 12], digoxigenin-labelled peptide nucleic acid
to detect lactobacilli PCR amplicons immobilized on
membranes from denaturing gradient gel electrophoresis [13]
and enterobacterial repetitive intergenic consensus PCR
(ERIC-PCR) and repetitive extragenic palindromic PCR
(REP-PCR) [14].
This is important in order to differentiate reliable and
proven strains and products from those of dubious quality as
referenced above. It will, therefore, ensure that consumers
know exactly what strains are in the products.
The next stage is to ensure that the particular strains used
are properly designated (e.g., Lactobacillus acidophilus
Gregor Reid
NCFMTM). Too often, product labels simply state “Lactoba-
cillus acidophilus” or “acidophilus” and reference generic
studies showing benefits of Lactobacillus acidophilus strains
such as probiotic NCFMTM. This is unacceptable. As with
pathogens, each “probiotic” strain is different. For example,
uropathogenic p fimbriated E. coli causing pyelonephritis is
totally distinct clinically from E. coli 0157:H7 causing
gastroenteritis and hemolytic uremic syndrome. In order for
a strain (or several strains in a combination product) to be
probiotic, each organism must be designated and their
importance in conferring the health benefit documented
(more on this later).
Few studies have been undertaken to compare probiotic
strains. Several performed in our laboratory have suggested
that L. acidophilus NCFMTM [15] and L. rhamnosus GG [10,
16] do not appear to be ideally suited for urogenital applica-
tions, and anti-infective proteins are not universally present
[17]. With the possibe exceptions of L. delbrueckii subsp.
bulgaricus and S. thermophilus (where all known strains
appear capable of enhancing lactose digestion in lactose
intolerant individuals), strain selection is important if bene-
ficial physiological effects are to be conferred [1]. The
selection process and the commercial marketing of products
has long relied upon the presumption that strains such as L.
acidophilus and L. reuteri are the most commonly found
bacterial populations in the gut. A study of the feces of
healthy young adults and elderly showed that this perception
is indeed incorrect, and L. delbrueckii subsp. lactis or L.
paracasei subsp. paracasei are more common [18]. Origin of
strains is also a factor for consideration. Studies from 1982
suggest that strains for human use should come from humans
[19]. The extent to which this remains true is unknown, as
dairy and perhaps livestock strains may be the source of
some human strains. The importance may be one of con-
sumer perception, in that humans may not wish to be
consuming strains that originated from pigs, rats or mice (or
worse, if from the fecal matter of these animals).
STEP TWO: IN VITRO EVIDENCE IS INSUFFICIENT
TO CALL A STRAIN A PROBIOTIC
The selection of suitable strains may be further refined by
undertaking a series of in vitro experiments, invariably to
examine ability to adhere to surfaces, and inhibit growth and
attachment of pathogens [20, 21]. While there are benefits to
these types of studies, there has been little direct correlation
between in vitro properties and prediction of functionality of
probiotic microorganisms in the human body. Exceptions
could be the documentation of resistance to bile salts, which
can correlate with gastric survival in vivo [22], and resistance
to spermicide which could aid survival in the vagina of
women using these products [23]. However, in general in
vitro data alone is not sufficient to describe strains as probio-
tic [1]. This may change in the future, through research
which proves, for example, that a specific clinical outcome is
due to a specific bacterial physiological or genetic trait (e.g.,
finding that expression of a bactericidal agent correlates with
depletion of pathogen numbers). One recent study comes
close to providing this level of correlation. Two Lactoba-
cillus strains displayed similar growth, survival, and adhe-
rence properties in vitro, yet L. johnsonii NCC 533 colonized
the intestinal lumen and translocated into mucosal lymphoid
The Importance of Guidelines in the Development
tissue more effectively than L. paracasei NCC 2461, due to
an induction of different types of immune responses [24].
Still, in vitro assays remain valuable and can provide
scientific insight into characteristics of probiotic organisms,
such as signaling mucus production on intestinal cells [25],
or the identification of proteins that can prevent
Staphylococcus aureus surgical implant infections in animals
[26].
A major advantage of adherence to these guidelines is
that scientists, physicians and consumers can track the scien-
tific and clinical data accumulated for these strains/products.
In an era when consumer self-learning is influencing many
health care and marketing decisions, it is important that the
end users recognize the practical benefits and limitations of
their therapy. Trends show that consumers, mainly women,
are using more foods and natural supplements to diagnose
and treat disease as well as maintain health [27, 28], and
therefore, one should not underestimate the lengths to which
people will go to find a proven probiotic strain/product. One
analysis of the North American market showed that sales of
specialty supplements, fortified/functional foods, natural
personal care products and cosmeceuticals are soaring
because of the “do-it-yourself” approach, resulting in a $42
billion retail value [29]. Furthermore, 79% of primary food
shoppers used vitamins/minerals to help maintain health,
while 79% used OTC drugs, 73% fortified foods, 72% Rx
medications, 52% foods without artificial additives/preserva-
tives, 39% herbal remedies, 37% organic foods, 24% aroma-
therapy, 19% homeopathic remedies and 14% in-home
diagnostic kits. This desire to self-treat or manage a specific
health condition or illness using foods was evident in 63-
72% of shoppers surveyed. While the N. American market is
not necessarily reflective of other parts of the world, it can
be assumed that consumer education has a major role to play
in the future of healthcare, including the use of probiotics.
Thus, consumers will soon become aware of proven products
versus ones that are poorly labeled and unproven clinically.
STEP THREE: SAFETY OF PROBIOTICS
There is historical data to indicate that lactobacilli and
bifidobacteria are safe for human use [30, 31]. If a probiotic
strain is of human origin, a strong case can be made that it is
a normal commensal and, therefore, safe for use. Minor side
effects can occur when using probiotics, but infections rarely
occur and invariably only in immunocompromised patients
or those with intestinal bleeding [32-34]. Given that an
estimated 20 billion doses of probiotics are consumed each
year, with only 4 cases of bacteremia associated with their
use over the past ten years, probiotic therapy is significantly
safer than pharmaceutical agents [35]. In human tests,
Lactobacillus rhamnosus 19070-2, L. reuteri DSM 12246, L.
rhamnosus LGG, L. delbrueckii subsp. lactis CHCC 2329,
and L. casei subsp. alactus CHCC 3137 were found to be
safe and well tolerated at two daily doses of 10 10 freeze-dried
bacteria for 18 days [36]. Even in HIV patients, where some
concern has been raised about administration of viable
bacteria, a study showed that a L. reuteri strain could be fed
at 1010 cfu/day without any clinically significant safety or
tolerance problems [37].
Current Pharmaceutical Design, 2005, Vol. 11, No. 1 13
Elements of safety of a probiotic strain/product should be
tested in humans, and using laboratory studies. The FAO/
WHO Guidelines recommend that, at a minimum, probiotic
strains be characterized using a series of specific tests: (a)
Determination of antibiotic resistance patterns; (b) Assess-
ment of certain metabolic activities (e.g., D-lactate produc-
tion, bile salt deconjugation); (c) Assessment of side-effects
during human studies; (d) Epidemiological surveillance of
adverse incidents in consumers (post-market); (e) If the
strain under evaluation belongs to a species that is a known
mammalian toxin producer, it must be tested for toxin
production (one possible scheme for testing toxin production
has been recommended by the European Union Scientific
Committee on Animal Nutrition [38]); (f) If the strain under
evaluation belongs to a species with known hemolytic
potential, determination of hemolytic activity is required;
and (g) Assessment of lack of infectivity by a probiotic strain
in immunocompromized animals (adding a measure of
confidence in the safety of a probiotic).
The issue of safety becomes more complicated and per-
haps controversial for organisms such as enterococci. These
bacteria are present in relatively high numbers in the intes-
tine and are sometimes used in so-called probiotic products.
Their emergence as a nosocomial pathogen and their carriage
of vancomycin resistance raises the question of whether or
not they should be recommended as a probiotic. Organisms
such as Saccharomyces boullardii, have been associated with
cases of fungemia [39], so users of probiotic products con-
taining these strains need to be cautioned by the distributor
through labeling or enclosed information directed at the
consumer. Producers should take into consideration the
health condition of the end user (for example those about to
undergo bowel surgery compared to a healthy person), and
should provide a means for this person to contact the
company if a perceived side effect occurs.
STEP FOUR: EFFICACY - PROVING THAT THE
PROBIOTIC CONFERS A SIGNIFICANT IMPROVE-
MENT IN HEALTH
While phase II clinical trials are part of the drug approval
process, they have not been widely used to assess probiotic
products. The FAO/WHO Guidelines indicate that this must
change in the future, otherwise health benefits cannot be
measured. The advantage of a phase II study is that evidence
is obtained and directly compared to a placebo. If a
statistically and biologically significant improvement occurs
in a patient’s condition, symptoms, signs, well-being or
quality of life, risk of disease or speedier recovery from
illness, then the producers of the probiotic will have impor-
tant supportive material to convince physicians to consider
their proposed remedy. In preventing diseases such as
urinary tract infection, kidney stones, and flair-ups of
Crohn’s disease, as some probiotics appear capable of doing
[40-42], the outcome is relatively easy to identify. However,
for many conditions such as asymptomatic bacterial vagino-
sis (BV), hypercholesterolemia, irritable bowel disease and
general wellness, measuring the impact of probiotics is more
difficult. Thus, the primary and secondary outcomes must be
clear and measurable, and the protocol preferably should
involve a randomized, double blind, placebo-controlled
14 Current Pharmaceutical Design, 2005, Vol. 11, No. 1
(DBPC) design. Study sites different to those where the
inventors are located will enhance credibility.
By generating high quality, independent clinical data,
more members of the medical community are likely to
prescribe and recommend specific probiotics. Such evidence
has been accrued for several probiotic strains. The bulk of
the data shows that strains L. rhamnosus GG and L. reuteri
SD2222 can reduce the duration of bacterial and rotavirus
associated diarrhea [43-46] when combined with oral
rehydration therapy. The requirement for oral rehydration is
best exemplified in Kenya, where failure of a child to access
a District hospital for this treatment can be the difference
between life and death [47]. Prevention of rotavirus infection
is not easy to assess because it is difficult to ensure that
active and placebo groups are equally exposed to the virus.
Nevertheless, one study in Peru did indicate a significant
reduction in incidence of infection due to prophylaxis with L.
rhamnosus GG [48]. Arguably, prevention of infection is
easier to assess for urogenital infections (urinary tract
infection [UTI], BV and yeast vaginitis) where recurrences
are common within a 3-6 month timeframe. Patients with a
history of UTI who were given once-weekly vaginal suppo-
sitories containing 10 9 L. rhamnosus GR-1 and L. fermentum
B-54 cells had 79% fewer episodes during treatment
compared to the previous year [40]. Despite the fact that this
patient population is highly prone to yeast vaginitis no
episodes were observed.
STEP 5: EFFECTIVENESS
Phase III studies, usually as randomized, blinded designs,
are necessary to determine whether a probiotic therapy is as
good as, or better than a standard treatment for a particular
disease. For example, this might compare lactobacilli against
metronidazole to eradicate BV. As most probiotic products
are in the form of food, it is rare for a phase III study to be
completed. Foods and dietary supplements are used for
nutritional purposes with some added functional value. Once
reference is made to curing or treating a disease, most
government regulatory agencies would regard this as drug
therapy, and require extensive testing - including a phase III
study. At the time of preparing this article, no phase III
studies on probiotics could be identified using a PubMed
search.
Some probiotics are legislated as drugs, for example
VSL#3 for Crohn’s disease. The L. rhamnosus GG strain is
mostly distributed in dairy product formulation, but as a
freeze dried capsule it can be registered as a drug. Such
products must be manufactured under FDA, European
Pharmacopia or equivalent quality assurance regulations.
Very few probiotic manufacturers have reached this Good
Manufacturing Practices (GMP) standard, with Chr Hansen
Laboratories, Denmark being the most notable exception. An
advantage for clinicians is that products prepared in this way
are likely to be more reliable in terms of content, shelf-life
and quality.
STEP 6: HEALTH CLAIMS AND LABELING
An important component of the FAO/WHO Guidelines
involves labeling and use of specific health claims. For foods
and dietary supplements, only general health claims are
Gregor Reid
currently allowed in most countries. The Guidelines recom-
mend that this be changed in order to provide the consumer
with more useful and precise information. Statements such
as ‘helps lactose intolerant people better digest milk’ are
much more instructive than ‘helps intestinal well-being’. It
may be several years before such standards are implemented
in United Nations member countries, and even longer for
them to filter through to industry. Whether or not this will
take place and be enforced is another matter. However, for
those companies that invest in proving efficacy of strains and
creating high quality end goods, the reward could well be a
leadership position in an expanding market.
The need for legislation is certainly evident by a brief
examination of web sites claiming to sell probiotics. For
example, one site states that “Lactobacillus acidophilus is
one of the most important microorganisms found in the small
intestine. It is known to implant itself on the intestinal wall,
and in the lining of the wall of the vagina, cervix and
urethra.” Of course there are no peer-reviewed data to
support these claims for this bacterium and no scientific
evidence either that L. acidophilus is one of the most
important intestinal organisms, or that probiotics implant
themselves anywhere. Another site suggests that their
proprietary probiotic organisms “multiply in the intestines
and actually compete with "harmful" bacteria for receptor
sites, crowding out the pathogens and taking their place”.
Again, there is no proof that receptor site competition occurs
in vivo in the gut, little evidence of depletion of pathogen
numbers and as for ‘taking their place’, this seems unlikely
given that probiotic organisms are washed out within, at
most, a few weeks. If companies want to inform consumers,
then accurate, science-based information is required.
References to reviews and somebody else’s strains are of no
use unless in the proper context.
Much is made of enhancing immunity and detoxifying
the intestine. Some facts need to be first understood. Our
knowledge of the microflora of the gut and other body sites
is still evolving. The use of PCR-DGGE methods without
culture, is allowing identification of organisms that were
previously undetected. Studies have shown that an organism
that is very difficult to culture, L. iners, is the most common
Lactobacillus in the vagina of one population [49], while a
relatively unknown organism, Atopobium, may in fact be a
major cause of BV [50]. In short, legends of the great
‘acidophilus’ need to be put in perspective and more
attention must be placed on evolving science.
THE IMPORTANCE OF THE FIRST SIX MONTHS
OF LIFE
Humans contain ten fold more microbes than the human
cells from which they are themselves comprised. The
organisms which colonize us, through vaginal and fecal
contact from the birthing mother, handling and feeding, play
a major role in life-long health. Exposure of young children
to broad spectrum antibiotics can perturb the intestinal
microflora, potentially resulting in Th2 dominance over Th1
immune responses to environmental antigens and an incre-
ased incidence of atopic disorders [51]. This negative effect
need not necessarily be caused by depletion of lactobacilli or
bifidobacteria, but rather organisms such as Bacteroides
The Importance of Guidelines in the Development
thetaiotaomicron, a predominant member of the gut micro-
flora. Substances produced by such bacteria have now been
shown to shape innate immunity [52]. An imbalance of the
Bacteroides flora may have other consequences. A recent
study showed a correlation between sulfate reducing bacteria
and ankylosing spondylitis as well as an elevated T-cell
proliferation response to Bacteroides [53]. In short, the
microbial content of the body plays an important role in
health and disease, yet little attention is given to the
organisms that each of us inherits.
In low birth weight premature babies, the gut microflora
is dominated by potentially pathogenic Enterococcus faecalis,
Escherichia coli, Staphylococcus epidermidis, Enterobacter
cloacae, Klebsiella pneumoniae and Staphylococcus
haemolyticus [54]. One attempt to alter this flora and reduce
the risk of necrotizing enterocolitis proved successful using
Lactobacillus acidophilus and Bifidobacterium infantis [55],
while another using Lactobacillus GG failed [56]. The extent
to which the infant microflora can be permanently
manipulated will likely be the subject of much research in
the future, especially if it leads to improved health outcomes.
The high rates of morbidity and mortality associated with
diarrhea in children, especially in the developing world,
make it essential that use of probiotics be considered as an
interventional therapy.
THE FUTURE
In summary, the following is a list of clinically relevant
steps that will move probiotics closer to being embraced by
the medical community: i) implementation of Guidelines for
the use of probiotics; ii) phase I and II clinical trial data on
strains and end products to prove health benefits; iii) use of
Good Manufacturing Practices and production of high
quality products; iv) Studies which identify mechanisms of
action of probiotic strains in vivo; v) appropriate information
dissemination about products to physicians, health
professionals and lay people; vi) development of probiotic
organisms that carry vaccines or other beneficial substances
to the host; vii) development of anti-viral probiotics; viii)
expansion of proven strains to benefit the oral cavity,
nasopharynx, respiratory tract, stomach, vagina, bladder, and
skin as well as for cancer, allergies and recovery from
surgery and injury.
ACKNOWLEDGEMENTS
The author is supported in part by by the Natural
Sciences and Engineering Research Council of Canada
(NSERC) and The Ontario Research and Development
Challenge Fund (ORDCF).
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