1000 Asia Pacific J Clin Nutr (1996) 5: 10-14

Asia Pacific J Clin Nutr (1996) 5: 10-14

Selection criteria for probiotic microorganisms

Patricia L Conway, MSc, PhD

School of Microbiology and Immunology, University of New South Wales, Sydney, Australia

Probiotics are preparations of live microorganisms which beneficially affect the host by improving the properties of the indigenous microbes. Since the human intestinal flora plays an important role in health and disease of man, probiotics are used to improve intestinal health and to stimulate the immune system. The microbes commonly used as probiotics for humans are the lactic acid bacteria (LAB). In early studies the strains used for fermenting milk products for human consumption were frequently used as probiotics. Subsequently, it was realised that it would be more appropriate if the strains originated from the human intestinal tract and that in addition to LABs, other microorganisms could be used either singly or in combination. Today, strict selection criteria are employed to obtain functional probiotic strains. It is generally agreed that the strain should be of host origin, well characterised, able to survive the rigours of the digestive tract and possibly colonise, biologically active against the target as well as to be stable and amenable to commercial production and distribution. In addition, information on dosages and evidence of efficacy needs to be obtained. In vitro and in vivo studies are frequently combined to allow investigation of the various parameters, and ultimately clinical trials are required. Although lactic acid bacteria have been generally recognised as safe, the question of safety is discussed for LAB and non-LAB probiotic strains in terms of potential pathogenicity of the strains and risk to the individual and the community. Finally, even though the techniques for genetic manipulation of many probiotic strains are available, it is not envisaged that this issue will be addressed in the near future because of regulatory implications. It is proposed that when this type of selection criteria is employed, probiotics strains with demonstrable efficacy can be obtained.

Background

The concept of probiotics was in use in the early 1900s, however, the term was only coined in 1965 by Lilly and Stillwell and has subsequently evolved. Numerous definitions have been proposed. Initially it was used by Lilly and Stillwell to refer to the stimulation of the growth of one microbe by another, in other words, the opposite of antibiotic. Today it is generally agreed that a probiotic is a preparation "of live microorganisms which, applied to man or animal 1000 , beneficially affects the host by improving the properties of the indigenous microbiota" 17.

The indigenous microbiota of the gastrointestinal tract plays an important role in the health and well being of the host27. Some of the beneficial and harmful effects of the gastrointestinal microbiota are summarised in Table 1. It is envisaged that these parameters may be influenced by probiotic administration. Initially lactic acid bacteria (LAB), particularly lactobacilli, were orally administered to man with promising but often non-conclusive effects. In fact, there has been considerable controversy over the validity of statements about the beneficial effects of lactobacilli and probiotic preparations15. This is best exemplified by the titles of some reviews around 1990, namely, "Lactobacillus: fact and fiction"4, "Probiotics-- fact or fiction?"24 and "Probiotic bacteria: myth or reality?"28 The most striking common feature for all reviews of the time is the comment that not all lactobacilli strains behave the same, and a stringent criterium for strain selection is required in order to consistently achieve positive results using probiotics. The careful analysis in the early 1990s of probiotics lead to recommendations for the future and heralded the way for effective probiotic preparations of tomorrow by strict attention to strain selection. This is summarised in a recent review entitled "The coming of age of probiotics" 22. Table 1. Influences of the human intestinal microbiota on the host

Beneficial effects

Harmful effects

Inhibition of pathogens

Constipation

Stimulation of immune system

Diarrhoea

Synthesis of vitamins

Infections

Aid in digestion

Liver damage

Produce metabol 1000 ic fuel for enterocytes

Cancer

Maintain stability of ecosystem

Flatulence

Metabolise drugs

  

In this paper, the range of microorganisms used as probiotics is presented and the mechanisms by which probiotics are beneficial to the host are discussed. The parameters for selecting and evaluating strains of microorganisms for use as probiotics are then discussed in terms of the beneficial effects that can be achieved.

Microorganisms used as probiotics

Although many of the early studies primarily used lactobacilli and in particular those used for production of fermented milks, preparations of microorganisms have not been limited to fermented milk products. In the 1920s, it was shown that the bacillus in bulgarian fermented milk did not survive in the human gut and consequently intestinal isolates of Lactobacillus acidophilus were used as dietary supplements and clinical trials gave encouraging results. Over the years, a number of microbes have been utilised as probiotics for humans and these are presented in Table 3.

The rational for using the LAB is historically based since Metchnikoff26 originally proposed use of this type. In addition, this group of bacteria is very rarely pathogenic.

 Table 2. Areas of application of probiotics for humansReduction in cholesterol
Enhancement of resistance against pathogens
Stimulation of the immune system
Lactose intolerance
Prevention or reduction of constipation
Prevention of diarrhoea
Reduction in the risk of colon cancer
Reduction in levels of faecal enzymes associated with cancer
Reduction of irritable bowel syndrome
Improved health
Today, there is increasing interest in the use of strains other than the traditional LAB (lactobacilli and bifidobacteria). For example, Saccharomyces boulardii, non-pathogenic Escherichia coli,6 Salmonella typhimurium1 and Clostridium difficile3 and complex mixtures of intestinal microbes35 have been used.

Beneficial effects of probiotics

The beneficial and harmful effects that the indigenous microbiota exert on the host have been summarised in Table 1. These are consistent with the reported or proposed beneficial effects of probiotics as presented in Table 2. While evidence is accumulating that probiotics may be beneficial to man, there is still a sparsity in the literature of well conducted clinical trials proving efficacy. The subject has been extensively reviewed9,10,20,29,30,34.

There is a positive trend that probiotics can function in a diverse range of applications. Unfortunately, studies are usually carried out on a limited number of subjects. Inherent with the use of human subjects, the researchers are plagued with variations (between individuals themselves and with their diets) which can only be reduced by using a large number of subjects. A very promising article presenting evidence of immuno-modulation in man following the ingestion of LAB has recently been published31.

Table 3. Microorganisms used as probiotics for humans 1000
Lactobacillus acidophilus

Bifidobacterium bifidum
L. plantarum

Bif. infantis
L. casei

Bif. adolescentis
L. casei ssp. rhamnosus

Bif. longum
L. delbrueckii ssp. bulgaricus

Bif. breve
L. fermentum

Strep. salivarius ssp. thermophilus
L. reuteri

Enterococcus faecalis

Saccharomyces boulardii

Enterococcus faecium
Lactococcus lactis ssp lactis

Lactococcus lactis ssp cremoris

In some instances, it is the probiotic microbes themselves which function for the benefit of the host, such as antimicrobial mechanisms, while in other cases, the probiotic microbe may trigger the indigenous microbes or the host physiology to induce the action. It can be envisaged that any one probiotic preparation may be, but need not be, multi-functional. The beneficial effects presented in Table 2 can be grouped as follows:

Antimicrobial mechanisms refer to the actions of the probiotic preparation on another microbe or group of microbes. These are directly applicable to the use of probiotics for enhanced resistance against intestinal pathogens and prevention of diarrhoea7. The types of interactions include competitive colonisation as well as adhesion and growth inhibition.

Competitive colonisation refers to the fact that the probiotic strain can successfully outcompete the pathogen for either nutrients or the site of colonisation. Since many gastrointestinal pathogens attach to the intestinal mucosa as the first step in infection, it would be beneficial to the host if this adhesion could be inhibited. There are reports that lactobacilli produce components which inhibit attachment of enterotoxigenic Escherichia coli to intestinal mucosa,2 however, there is no evidence as yet that this occurs in the digestive tract. In addition, various compounds produced during growth of the probiotic have been shown to inhibit pathogen growth7,21. These include organic acids such as lactic and acetic acid, reuterin and bacteriocins33. The organic acids lower the pH and thereby can indirectly affect growth of the pathogen. In addition, the lactic and acetic acids can be toxic to microbes. Reuterin which inhibits the growth of a very broad range of cells23, is produced by Lactobacillus reuteri when grown in the presence of glycerol. Numerous bacteriocins have been reported to be produced by lactobacilli, for example, Acidophilin, Acidolin, Lactocidin, Bacteriocin, Bulgar-ican, Lactolin, Lactobacillin and Lactobrevin7,21. They can either have a very broad range of activity or alternatively specifically inhibit the growth of a very limited range of closely related microbes. For example, Lactobacillus sp exhibited specific antagonistic effects towards Clostridium ramosum.25

Biochemical effects of the probiotic include:

The ingestion of lactobacilli has resulted in a reduction in faecal enzymes such as beta-glucuronidase, azoreductase and nitro-reductase in humans14. These enzymes can be produced by the bacteroides group of bacteria and it is probable that the presence of the probiotic strains influences either the production of the enzyme or the levels of the specific microbes which produce the enzyme. Lactose intolerance occurs in subjects who lack the enzyme, lactase (a beta-galactosidase). Symptoms include abdominal pain and osmotic diarrhoea after eating foods high in lactose since the lactose is not degraded and absorbed in the upper regions of the small intestine and hence can be used by the indigenous microbiota. This results in production of gases and organic acids which give rise to the symptoms in lactose intolerant patients. Ingestion of probiotic microbes which contain and produce beta-galactosidase results in degradation of the lactose before it reaches the indigenous microbes in the lower part of the small intestine.

It is reported that probiotics such as lactobacilli can assimilate cholesterol12 and deconjugate bile acids11 and that this will lead to a reduction in serum cholesterol levels. At present the evidence for this is based on the laboratory evidence of assimilation of cholesterol and in vivo action in one study, however, the findings have not been confirmed by other workers and it is proposed that the assimilation may in fact be co-precipitation of the cholesterol with bile acids at low pH. Since propionic acid can reduce de novo synthesis of cholesterol in the liver, it has been suggested that probiotics that produce propionic acid could reduce cholesterol synthesis. Unfortunately, it is unlikely that sufficient levels of propionic acid in the liver can be achieved for this mechanism to influence cholesterol levels.

Physiological mechanisms of probiotics refer to the influences of these microbes on the host responses and include the following:

There is accumulating evidence that lactobacillus cell components directly stimulate the immune response18. This has ramifications for both protecting the host from infection and for conditions which involve the immune response, such as irritable bowel syndrome and colon cancer. In some cases an adjuvant effect has been noted and this represents a general enhancement of the immune status of the host as a result of probiotic dosage. Such a general enhancement may also assist the host is suppressing tumours and there is evidence available from animal model studies that this can occur13.

Parameters for evaluating probiotic strains

Non-conclusive and even contradictory studies on probiotic usage have been reported over the years. It is now generally agreed that more rigorous attention to strain selection would yield more conclusive results4. For example, it is now acknowledged that not all Lactobacillus acidophilus are the same, and that bacterial strains which produce desirable food products may not necessarily have a beneficial effect on the host. There is an increasing demand that strains used in probiotic preparations are stringently selected5,8,16.

1000
The parameters recommended to be included for selecting functional probiotic strains are presented in Table 4. This list of parameters for screening microorganisms for potentially valuable probiotic strains is based on the fact that we need strains which can be viable and metabolically active within the gastrointestinal tract and are biologically active against the identified target. In addition, it is imperative that viability of the strain and stability of the desirable characteristics of the strain can be maintained during commercial production and in the final product. Finally, it is crucial that the strain is safe. The parameters included in a strain selection criterium will be influenced by the intended target for use. Table 4. Parameters used for selecting a functional probiotic strain.
Specified target

Host origin
Strain identified

Biological activity against target
Colonisation potential

Survival in situ
Stability of numbers

Stability of characteristics
Safe

Demonstrable efficacy
Dosage required  

Since it cannot be assumed that a probiotic strain will be effective for a broad range of applications, the target for the use of the probiotic needs to be identified. This would allow one to select strains with biological activity against the target. For example, the antibiotic associated diarrhoea has been one condition which could be treated by using probiotic microbes. The causative agent of antibiotic associated diarrhoea is often the bacterium, Clostridium difficile. Consequently, in order to select a probiotic strain for use in antibiotic associated diarrhoea conditions, one would select a strain which has an antagonistic effect against the C. difficile cells32. It is also necessary to ensure that antagonistic effects demonstrable in vitro may also be effective in vivo.

In order for the strain to be viable and metabolically active in the digestive tract, it is recommended that the strain be of host origin, have the potential to colonise the tract and 1000 be able to survive the rigours of the tract, such as low pH and bile acids. The rational for selecting strains of host origin is that there have been several studies reporting that strains isolated from the digestive tract of one animal, can not survive or colonise another animal. Consequently, strains originating from the human gastointestinal tract are screened for use as human probiotic microbes19. If the probiotic can colonise, it will ensure that the strain is maintained in the tract for a longer period of time.

Since it is technically difficult to test which of a number of strains can colonise the digestive tract of man, the colonisation potential is tested in vitro by studying the capacity of the strains to adhere to gastrointestinal mucosa and their capacity to grow in intestinal extracts. While this procedure is relatively straight forward, close attention to the controls in the assay is required. This aspect has been discussed in detail in a previous paper5. Briefly, although bacteria can adhere in an in vitro assay, non-specific adhesion may be involved. This can be examined by using control proteins as well as intestinal mucosa in the in vitro assay. Survival in conditions within the intestine can be relatively easily studied in the laboratory by using buffers of defined pH values and by the addition to buffers and growth media of bile acids or other components such as digestive enzymes, antibiotics and food additives to which the probiotic may be exposed.

Stability of viability during preparation and storage is easily monitored, and is a very important parameter. It has been shown that microbes can lose some characteristics when maintained in laboratory conditions. For example, some strains originating from the gastrointestinal tract rapidly loose the capacity to adhere to epithelial mucosa while others retain this capacity during extensive sub-culturing in the laboratory4. It is therefore important to ensure that the potential probiotic strain can retain desirable characteristics both in the laboratory and during commercial production and storage. Similarly, standard tests must be established to confirm that the strain is safe and identified taxonomically.

Clinical studies

Clinical studies are a pre-requisite for proving efficacy of a particular probiotic because of limitations with extrapolating from data obtained from in vitro and animals studies. Ideally, clinical studies should be conducted double blind in a cross-over fashion. The term double blind describes how the probiotic preparation must be evaluated against a placebo control and the identity of the preparations should not be know to either the medical staff or the subjects involved in the study until completion of the study. By cross-over, we refer to the fact that each subject is its own control and that all subjects are treated with both placebo and test preparations, separated by a washout period. This approach reduces the limitations of low numbers of subjects and large individual variations since each subject functions as its own control.

Strain improvement possibilities

Frequently today, microorganisms used for commercial purposes are improved by genetic manipulation. Techniques are now available to perform such manipulations on many of the strains used as probiotics as discussed by Tannock34. It is therefore reasonable to propose that desirable characteristics can be combined in a single strain by gene technology. While this is most probably the future for probiotics, it is not envisaged that such preparations will be introduced in the near future.

Summary

Evidence is accumulating that confirms that probiotics can benefit the host by improving intestinal well being. In order to have functional probiotic strains with predictable and measurable beneficial effects, strict attention to strain selection is required. A combination of in vitro and in vivo studies culminating in clinical trials are therefore required. It is envisaged that probiotics can be targeted for specific uses or be used to generally maintain stability of the indigenous microbes in the digestive tract.

Chinese abstract

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