Probiotics: A Critical Review Chapter Abstracts

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G. W. Tannock.

A "probiotic", by the generally accepted definition, is a "live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance". Although referring to the supplementation of animal feeds for farm animals, the definition is easily applied to the human situation. The major consumption of probiotics by humans is in the form of dairy-based foods containing intestinal species of lactobacilli and bifidobacteria. It is implicit in the definition that consumption of the probiotic affects the composition of the intestinal microflora. This effect of the probiotic on the intestinal ecosystem, it is proposed, impacts in some beneficial way on the consumer. A number of potential benefits arising from changes to the intestinal milieu through the agency of probiotics have been proposed, including:

· increased resistance to infectious diseases, particularly of the intestine · decreased duration of diarrhoea · reduction in blood pressure · reduction in serum cholesterol concentration · reduction in allergy · stimulation of phagocytosis by peripheral blood leucocytes · modulation of cytokine gene expression · adjuvant effect · regression of tumours · reduction in carcinogen or co-carcinogen production

Perhaps surprisingly, despite this impressive list of therapeutic and prophylactic attributes, probiotics are not commonly part of the medical practitioner's armamentarium of prescription drugs. Instead, probiotics are available from retail outlets, usually supermarkets, grocery and health food stores. The probiotics are available to the consumer as powders or tablets, but most commonly as milk-based products. The growth in the production of probiotics by the dairy industry means that it is now increasingly difficult to purchase yogurts that do not contain "probiotic" bacteria such as Lactobacillus acidophilus. They sell well, but it is very doubtful that probiotic yogurts are purchased entirely for health reasons. Consumers may be purchasing probiotics with the vague idea that it is "good for them": that consumption of the product will contribute to their well-being. Since even some scientists treat the "probiotics" concept with scepticism, how can the average consumer hope to comprehend the significance of "acidophilus" or "bifidus" in any but the simplest of contexts? Many others purchase probiotic dairy products because they prefer the organoleptic and rheologic characteristics of these products in comparison to "regular" yogurts. Whatever the reason, there are consumers who hold a firm belief that their health is improved by regular consumption of a probiotic. It becomes almost an article of faith. Others are suspicious of the probiotic industry, and question the validity of the claims made in relation to health benefits. And, it must be admitted, they have some basis for these doubts: the marketing strategy for probiotics relates to health benefits and therefore to medical science. Yet even the most ardent advocates of the probiotic concept must admit that the "science" associated with probiotics over many decades has been remarkably weak (16). Why else has the medical profession, and much of the scientific community, remained aloof from the results of probiotic research?

Despite doubts in scientific circles as to its validity, the probiotics industry is flourishing, and interest in establishing scientific credibility has attained importance for many companies and scientists. European Union programmes are to the fore in this work. It is an unusual and difficult situation for scientists, however, because probiotic products have been in existence for decades, yet they must now search for experimental evidence to support long-held beliefs associated with products already in the retail market. The probiotics industry is burdened with myth (anecdotal studies) and a reliance on in vitro experimentation. Much effort has been devoted to screening bacterial isolates for properties deemed appropriate for a "probiotic" strain, mostly characteristics that might enable the microbes to at least survive passage through the digestive tract. There must be millions of such strains to choose from, because the intestinal milieu of humans is already the home to bacteria with these properties. Probably the biggest obstacle to allaying scepticism is that the probiotic concept is based on a very poor understanding of the intestinal microflora. The concept concentrates essentially on two groups of bacteria, lactobacilli and bifidobacteria, while practically ignoring the vast array of other species that inhabit the intestinal tract of humans. It is the impact of probiotics on the composition of the intestinal microflora, nevertheless, that forms the basis for the probiotic concept. What can currently be said about the impact of probiotic bacteria on microbial balance at the moment? "They go in at one end of the digestive tract and come out the other, and hopefully something good happens along the way" is probably not too harsh a statement. Can anyone define the "microbial balance" to which one aspires in the definition of a probiotic?

While there is a current trend to conduct "clinical trials" to prove the efficacy of exisiting probiotics, it may be better to return to a consideration of fundamental principles of microbial ecology, human physiology and immunology before embarking on these very costly exercises. What are the "beneficial effects" that one wants to produce by using probiotics? Do we know how to recognise the beneficial effects mentioned in the definition of a probiotic? Better, perhaps, to allocate funds to the utilisation of molecular biological tools in the analysis of the complicated systems which must be investigated. With these tools, a better understanding of the interactions between members of the intestinal microflora and between the microflora and the human host could be established. The intestinal bacteria could then be used as mediators to modulate phenomena that are of significance to human health. The development of modern pharmaceutical drugs is based on fundmental knowledge of processes occurring within the human body. Mechanisms of drug action are known, and explanations of efficacy can be publicised. To attain scientific validity, probiotics must be derived by the application of logic.

All of the authors that have contributed chapters to this book share the same conviction: fundamental knowledge of intestinal bacteria and their interactions with each other and with the human or other animal host are a prerequisite for successful probiotic research and development. The topics discussed by the authors of this book include:

· the development of a modern concept of the intestinal microflora and microflora-host
relationships to use as a working basis for probiotic research (Chapters 1 and 7). Molecular technologies will aid greatly in this work (Chapter 3).
· the scientific rigour of trials involving farm animals or humans (Chapters 2 and 9).
· the accurate identification of intestinal bacteria (Chapters 4 ), safety, and maintainenance
of bacterial viabilty under industrial conditions (Chapter 5).
· the development of methods for the derivation of genetically modified intestinal
bacteria, their application in studies of microbial ecology, and in the generation of beneficial host-microbe relationships (Chapters 6 and 8).
· the potential for modulation of the intestinal microflora using fermentable substrates as
dietary additives (Chapter 10).

The chapters in this book contain state-of-the-art commentaries on these aspects of the intestinal microflora and probiotics. Lactobacilli and bifidobacteria are, of necessity, the main points of discussion in these chapters because they are the two bacterial groups most commonly incorporated in probiotic products. I am much obliged to the contributing authors for their timely and informative chapters which together provide an authoritative review of important aspects of probiotic research. Thank you very much.

Chapter 1

A FRESH LOOK AT THE INTESTINAL MICROFLORA

Gerald W. Tannock

The study of the intestinal microflora is a crucial aspect of probiotic research and development. Comparative studies with germfree and conventional animals, strictly anaerobic culture methods and microscopy have provided the knowledge on which current concepts of the intestinal microflora are based. The application of molecular methodologies will enhance knowledge of the complex microbial ecology of the intestinal tract and enable a modern concept of the intestinal microflora and microflora-host relationships to be developed.

Chapter 2

PROBIOTICS FOR FARM ANIMALS

Roy Fuller

The development of probiotics for farm animals is based on the knowledge that the gut microflora is involved in resistance to disease. The stressful conditions experienced by the young animal causes changes in the composition and/or activity of the gut microflora. Probiotic supplementation seeks to repair these deficiencies and provide the type of microflora which exists in feral animals uninfluenced by modern farm rearing methods. The products available are of varying composition and efficacy but the concept is scientifically-based and intellectually sound. Under the right conditions the claims made for probiotic preparations can be realised.

Chapter 3

METHODS FOR ANALYSIS OF THE INTESTINAL MICROFLORA

Daniel J. O'Sullivan

The concept of probiotics has been around for nearly 100 years. Yet its impact on human nutrition is still an emerging concept. Lack of convincing scientific validation for the efficacy of any ingested probiotic bacterium on intestinal health, has been a major reason for the low impact of probiotics on human nutrition. Obtaining positive scientific validation requires the use of suitable probiotic strains and also the necessary tools to monitor the performance of these bacteria in the intestines of individuals. To date, selection of strains for probiotic purposes has not been based on a scientific directed approach, primarily because it is not yet fully known what specific traits a desirable probiotic strain should possess. Filling this knowledge void will depend largely on furthering our understanding of the human intestinal ecosystem and the functional role of specific bacteria for intestinal health. Traditional approaches for studying this ecosystem have provided a good foundation in this knowledge base. Complementation of the traditional approaches with the emergence of sophisticated molecular tools shows enormous promise for obtaining the necessary insight into the intestinal microflora. This review will cover the traditional methodologies which have been used to analyze the human intestinal microflora. It will also reveal the development of modern molecular approaches for studying the diversity and phylogeny of its flora, and the rapid molecular tools for monitoring the presence of specific strains in the intestine. Finally, it will address the advent of in situ analysis of individual microbial cells, which promises to provide tremendous advances in our understanding of the microflora and their metabolic activities in the human intestine.

Chapter 4

IDENTIFICATION OF LACTOBACILLI AND BIFIDOBACTERIA

Gerald W. Tannock

Selective culture media and phenotypic tests enable lactobacilli to be differentiated from morphologically similar bacteria. The accurate identification of Lactobacillus species can be accomplished by reference to 16S rRNA gene sequences. Species-specific, PCR primers that target the 16S-23S rRNA spacer region are available for a limited number of Lactobacillus species. Molecular methods for the comprehensive identification of Bifidobacterium species are not yet available. Only DNA-DNA reassociation provides a reliable means of species identification for this genus at present. Bifidobacteria can be differentiated from morphologically similar bacteria by the use of genus-specific, PCR primers or oligonucleotide probes.

Chapter 5

INDUSTRIAL PERSPECTIVES

Ulla Svensson

General approaches to the preparation of probiotic milk products with good rheological and organoleptic properties, texture and adequate survival of probiotic bacteria are described. Choice of strains of recognised safety and probiotic efficacy, size of inoculum, incubation conditions, microbial interactions, and survival of bacterial strains during storage of the product, and the regulation of health-associated claims for probiotic products, are of prime importance from a production perspective.

Chapter 6

GENETIC MODIFICATION OF INTESTINAL LACTOBACILLI AND BIFIDOBACTERIA

Martin J. Kullen and Todd R. Klaenhammer

Lactobacilli and bifidobacteria are important members of the gastrointestinal microflora of man and animals. There is a substantial and growing body of evidence that these microbes provide benefits to the host in which they reside. Understanding the roles of these two groups of bacteria in the intestine continues to be a significant challenge. To this end, genetic characterisation and manipulation of intestinal lactobacilli and bifidobacteria is essential to define their contributions to the intestinal microflora, and to potentially exploit any beneficial or unique properties. This chapter will describe the tools and strategies currently available for the genetic manipulation of lactobacilli and bifidobacteria. Additionally, the ramifications and opportunities that may arise as a result of the genetic manipulation of probiotic lactobacilli and bifidobacteria will be addressed.

Chapter 7

PROBIOTICS AND THE IMMUNE SYSTEM

Vance J. McCracken and H. Rex Gaskins

The intestinal microflora is an important component of host defence. A critical review of the literature indicates that probiotic supplementation of the intestinal microflora may enhance defence, primarily by preventing colonisation by pathogens and by an indirect, adjuvant-like stimulation of innate and acquired immune functions. However, evidence for probiotic-mediated enhancement of immunity remains inconclusive. To fully explore mechanisms by which probiotic microorganisms might modulate intestinal immune function, we have reviewed the role of nonpathogenic bacteria in the development of the intestinal immune system and in protecting the host from pathogenic challenges. Specifically, in addition to reviewing the potential of probiotic therapy to modulate human or animal immune function, we discuss the relative immunogenicity of various members of the intestinal microflora and consequences of "broken" immune tolerance to the members of the microflora based on data from studies using germfree (GF) animals, animals with a defined microflora, and rodent models of inflammatory disorders, including inflammatory bowel disease (IBD).

Chapter 8

LACTIC ACID BACTERIA AS LIVE VACCINES

Annick Mercenier

Mucosal routes for vaccine delivery offer several advantages over systemic inoculation from both immunological and practical points of view. The development of efficient mucosal vaccines therefore represents a top prority in modern vaccinology. One way to deliver protective antigens at the mucosal surfaces is to use live bacterial vectors. Until recently most of these were derived from attenuated pathogenic microorganisms. As an alternative to this strategy, non-pathogenic food grade bacteria such as lactic acid bacteria (LAB) are being tested for their efficacy as live antigen carriers. The LABVAC european research network is presently comparing the vaccine potential of Lactococcus lactis, Streptococcus gordonii and Lactobacillus spp. To date, it has been shown that systemic and mucosal antigen-specific immune responses can be elicited in mice through the nasal route using the three LAB systems under study. Data on successful oral and vaginal immunizations are also accumulating for L. lactis and S. gordonii, respectively. Moreover, the immune responses can be potentiated by co-expression of interleukins. Future areas of research include improvement of local immunization efficiency, analysis of in vivo antigen production, unravelling of the Lactobacillus colonization mechanisms and construction of biologically contained strains.

Chapter 9

TESTING THE EFFICACY OF PROBIOTICS

Gregor Reid

There is a relatively large volume of literature which supports the use of probiotics to prevent and treat intestinal and urogenital infections and other ailments. However, the basis for the claims is often weakened by a lack of proven reliability of the preparations, and an inability to prove conclusively that the contents are safe and efficacious. In addition, not enough emphasis has been placed upon selecting strains which have specific properties shown to be important in colonising the host and acting antagonistically against pathogens. For probiotic therapy to truly be accepted in general medical practice, it must undergo rigorous clinical trials, and as these are expensive, government agencies and industry partners should, and indeed must, begin to invest in such studies.

Chapter 10

PREBIOTICS

Ross G. Crittenden

A range of non-digestible dietary supplements have now been identified that modify the balance of the intestinal microflora, stimulating the growth and/or activity of beneficial organisms and suppressing potentially deleterious bacteria. Termed "prebiotics" these supplements include lactulose, lactitol, a variety of oligosaccharides, and inulin. In particular, prebiotics promote the proliferation of bifidobacteria in the colon. The science of prebiotics is still in its infancy and as yet there is a dearth of reported clinical trials demonstrating clear efficacy in the prophylaxis or treatment of human disease. However, research to date indicates that prebiotics have potential to positively influence human health. Prebiotics have shown promise in the prevention and control of exogenous and endogenous intestinal infections; control of serum triglycerides and cholesterol; improvement of mineral uptake; and reduction in putative risk factors for colon cancer. This review summarises recent research into the impact of prebiotics on the microecology in the human colon, and proposed mechanisms and effects of prebiotics on human health.

G. W. Tannock.

I conclude that probiotic research and development is poised to make great advances during the next five years. A detailed understanding of the intestinal microflora will quickly emerge as molecular technologies are applied increasingly to analysis of intestinal communities, in conjunction with the use of biochemical and bacteriological methodologies. Similarly, the availability and application of molecular tools, particularly in studies utilising experimental animal models, will provide critical knowledge of the role of specific bacterial groups in the modulation of the immune system. The development of probiotic-type products for administration to other body sites, such as the vagina, will doubtless be of increasing interest. Taken together, the fruits of this fundamental research will form the basis for logical research and development programmes that will produce efficacious probiotic products. These programmes will involve controlled trials that have clearly defined and quantifiable goals with regard to expected health benefits. While national and international collaboration is already common in probiotic research, in order to make the greatest gains, it would be optimal for a team of scientists with appropriate skills to be assembled and funded to carry out the fundamental research in a cohesive manner. The results of this research would then be available to all who wished, under their own auspices, to bring novel probiotics to the market. Just as the hydrolysis of complex molecules is achieved by consortia of bacteria in the intestinal tract, so too could knowledge of the intestinal microflora and probiotics be advanced by an appropriately constituted consortium of scientists. It is an exciting time to be involved in intestinal microflora research.

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