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Probiotics

Probiotics are living bacteria or other microorganisms which may be beneficial to the health of humans or animals when consumed. Beneficial bacteria include Lactobacillus and Bifidobacterium spp. and other lactic acid bacteria. Much has been written in both the medical literature and lay press about their potential benefits, some of it well validated by randomized controlled trials but other claims remain unsubstantiated. There is, however, a growing body of evidence for the role of probiotics in gastrointestinal infections, irritable bowel syndrome and inflammatory bowel disease. Provided here is information on the science of probiotics, recent scientific research on probiotics and current research topics of interest to scientists and clinicians.

Contents

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What are Probiotics?

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. 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.

The Intestinal Microflora

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.

Prebiotics

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.

Probiotics for Farm Animals

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.

Methods for Analysis of the Intestinal Microflora

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.

The Hygiene Hypothesis: The Role of Microbes in the Prevention of Atopy and Atopic Disease

From: The Microbe-Host Interface in Respiratory Tract Infections. Eds. Jan L.L. Kimpen and Octavio Ramilo. Microbiology Books.
Probiotics are microbes present in the human gut that have a beneficial effect on the human immune system, opposed to pathogens that can have harmful effects (Schrezenmeier, 2001). Several mechanisms of action have been proposed by in-vitro studies, but the precise mechanism remains to be elucidated (Isolauri et al., 2001). In the only primary prevention study performed so far, 1010 colony forming units (cfu) of Lactobacillus GG was administered daily for 2-4 weeks before expected delivery to pregnant women with a positive family history of atopy, and to their offspring during the first 6 months of life in a randomised, placebo-controlled design. At the age of 2 years, the frequency of eczema in the intervention group was half that of the placebo group. This suggests that LGG given prior to birth, and for a prolonged period early in life may decrease the incidence of eczema (Kalliomaki et al., 2001). At the age of four years, follow-up was performed in 107 of the children who participated in the study at the age of two (Kalliomaki et al., 2003). The incidence of eczema was significantly lower in the children who received LGG (14 out of 53) compared to the children who received the placebo (25 out of 54), implicating a relative risk of 0.57 with a 95% confidence interval of 0.33-0.97. No significant difference in the incidence of asthma and rhinitis was found between the two groups. The authors conclude that these results suggest that the preventive effect of LGG on AD extends beyond infancy. A shortcoming of this study is the low number of children examined at the last follow up, implicating that a selection bias cannot be excluded. Furthermore, no difference in serum IgE concentration and in the number of positive skin-prick tests was found between the intervention and the control-group. This suggests that treatment with LGG results in a reduction of the incidence of non-atopic eczema rather than atopic dermatitis (Niers et al., 2003). Other studies addressing this issue are definitely required before definite conclusions can be drawn on the preventive effect of LGG in high-risk infants with respect to the development of atopic disease. In a tertiary prevention study, LGG (2x1010 cfu twice daily) was administered for 1 month to 14 infants (2.5-15.7 months of age) with cow's milk allergy and AD in a randomised double-blind, placebo-controlled study design. All children were fed an extensively hydrolysed whey formula. In the intervention-group, a reduction in the SCORAD-score, in faecal TNF-a, and in a-1-antitrypsin was found compared with the placebo-group. This suggests that administration of LGG results in a decrease in intestinal and systemic inflammation and in a decrease in mucosal permeability (Majamaa et al., 1997). Comparable results were found with Bifidobacterium Bb-12 and LGG administration in even younger infants (mean age 4.6 months) with AD. Despite several shortcomings in both studies, the authors of these studies suggested that these probiotics may be used as treatment of infant AD (Isolauri, 2000). However, these results could not be confirmed in a recently performed study, in which a combination of 2 strains of Lactobacilli (rhamnosus and reuteri) was administered in 1-13 years old children with AD. The only significant difference between the probiotic- and the placebo-group was found in a subjective dermatitis severity parameter (the children or parents evaluated the eczema as "better, unchanged or worse" after either treatment). During administration of probiotics, the eczema was significant more frequently evaluated as better (Rosenfeldt et al., 2003). The reason for the discrepancy between the latter study and the former studies is not entirely clear, despite the different age-groups studied, the different probiotics administered, and the different dose of probiotics used. More studies are needed on this issue before definite conclusions can be drawn.

Probiotics and Prebiotics: Where are We Going?

Perspectives concerning the composition of the human gut microflora have changed drastically since the concept of probiotics, and even prebiotics, was introduced. Culture-independent, nucleic acid-based, methods of analysis have provided results that demonstrate the complexity of the gut microflora and the quantitative and qualitative dominance of previously little known bacterial species. The need for invasive sampling techniques has also become apparent because reliance on the analysis of the faecal microflora may fail to accurately reflect the true state of affairs in the proximal colon. Probiotic administration transiently alters the gut microflora by donating bacterial cells to the ecosystem. It may be possible to use carefully formulated products in the alleviation of inflammatory bowel diseases or allergic diseases if the safety of such probiotics can be guaranteed. Prebiotics may be useful in the study of the regulation of the gut ecosystem as well as in attempts to rectify "abnormal" microfloras. The molecular foundations of the gut microflora-host relationship should be pursued using functional genomics, and clarification of the impact of the gut microflora on host nutrition and host physiology is necessary. To recognise the potential advantages of probiotic and prebiotic use, we need to understand how bacterial cells function in the gut ecosystem, and how bacterial functions impact on the host.

Fluorescence In Situ Hybridization as a Tool in Intestinal Bacteriology

The human gut microflora has an important function in health and disease. During the last six years fluorescence in situ hybridization (FISH) has been increasingly used to analyze bacterial communities in the gastrointestinal tract. This method is easy and inexpensive, and it can be performed in every laboratory equipped with a fluorescence microscope. However, for large studies involving many samples the enumeration of the fluorescent bacteria can be laborious. Automation of this process made this method into a reliable tool to perform comparative studies of larger sets of samples in an objective way. In this chapter the developments that are going on in the field of (automated) FISH analysis of colonic and fecal samples are discussed (i.e. developments in automation, standardization and validation of the protocol) and the design of a useful set of probes covering the total microflora. Experiments with fecal samples of volunteers show that 90% of the total hybridizable bacteria are covered by the current probe set. Furthermore, it is shown that stability of the microflora is difficult to measure due to population dynamics and variations in the protocol that was used. However, the results do indicate that the microflora is characteristic for an individual even over a long period of time. FISH has become a valuable tool to study the dynamics of the gut microflora.

From Composition to Functionality of the Intestinal Microbial Communities

The mammalian gastrointestinal (GI) tract harbours a large bacterial community that has an essential role in creating optimum health conditions for the host. This chapter focuses on the use of molecular fingerprinting tools to describe the taxonomic and functional diversity of the microbial community in the GI tract. Special attention is given to the composition analysis of microbial communities based on 16S rDNA sequence diversity. Basic principles and new developments of several PCR-based methods, such as denaturing gradient gel electrophoresis (DGGE) and related fingerprint methods as well as methods to analyse these finger prints are described. Advantages and drawbacks of DGGE are described and compared with the terminal restriction fragment length polymorphism (T-RFLP) method. In addition to methods investigating the taxonomic diversity of microbial communities in the GI tract, we also address the recent progress to describe the functional diversity of bacterial communities in the GI tract. Although relatively little information is available yet, we anticipate that our insight in the occurrence and activity of functional bacterial genes in the GI tract will rapidly expand in the next decade due to the enormous increase in sequence information and developments in microarrays technology.

Genus- and Species-Specific PCR Primers for the Detection and Identification of Bifidobacteria

16SrDNA-targeted genus- and species-specific PCR primers have been developed and used for the identification and detection of bifidobacteria. These primers cover all of the described species that inhabit the human gut, or occur in dairy products. Identification of cultured bifidobacteria using PCR primer pairs is rapid and accurate, being based on nucleic acid sequences. Detection of bifidobacteria can be achieved using DNA extracted from human faeces as template in PCR reactions. We have found that, in adult faeces, the Bifidobacterium catenulatum group was the most commonly detected species, followed by Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium bifidum. In breast-fed infants, Bifidobacterium breve was the most frequently detected species, followed by Bifidobacterium infantis, B. longum and B. bifidum. It was notable that the B. catenulatum group was detected with the highest frequency in adults, although it has often been reported that B. adolescentis is the most common species. Real time, quantitative PCR using primers targeting 16S rDNA shows promise in the enumeration of bifidobacteria in faecal samples. The approach to detect the target bacteria with quantitative PCR described in this chapter will contribute to future studies of the composition and dynamics of the intestinal microflora.

Prebiotic Oligosaccharides: Evaluation Of Biological Activities and Potential Future Developments

Prebiotics are recognised for their ability to increase levels of 'health promoting' bacteria in the intestinal tract of humans or animals. This normally involves targeting the activities of bifidobacteria and/or lactobacilli. Non digestible oligosaccharides such as fructo-oligosaccharides, lactulose and trans-galacto-oligosaccharides seem to be efficacious prebiotics in that they confer the degree of selective fermentation required. Other oligomers are used as prebiotics in Japan e.g. xylo-oligosaccharides, soybean oligosaccharides, isomalto-oligosaccharides. To determine prebiotic functionality, various in vitro systems may be used. These range from simple batch culture fermenters to complex models of the gastrointestinal tract. The definitive test however is an in vivo study. The advent of molecular based procedures in gut microbiology has alleviated many concerns over the reliability of microbial characterisation, in response to prebiotic intake. Techniques such as DNA probing and molecular fingerprinting are now being applied to both laboratory and human studies. These will help to further identify prebiotics that can be added to the diet and thereby fortify 'beneficial' bacteria. Such robust technologies can also be used in structure-function assays to identify the mechanisms behind prebiotic effects. Considerable research effort is currently being expended in developing so called 'second generation' prebiotics. These are forms that have multiple biological activity that attempts health enhancement properties beyond the genus level stimulation of bifidobacteria or lactobacilli within the gut microbiota. Examples include higher molecular weight oligomers than is conventional for prebiotics, such that targeted activities in the distal colon are feasible (the left side of the human large gut being the frequent area for colonic disorder). Glycobiology is also developing anti-adhesive prebiotics that incorporate receptor sites for common gut pathogens and/or their activities. Through the use of reverse enzyme technology, as applied to b-galactosidase activity in probiotics, oligosaccharides that enhance a lactic flora at the species, rather than genus, level are possible. This review gives an account of how second generation prebiotics may be manufactured, through a variety of biotechnological techniques, and tested for their biological activity. The health attributes of such molecules as well as existing prebiotics is also discussed, with reference to specific target populations. The prebiotic concept is a much more recent development in dietary intervention for enhanced gut function than is probiotics. Not surprisingly therefore, research developments are proceeding quickly. Because oligosaccharides can be added to a wide variety of foodstuffs, new functional food developments are continuing. It is important that these are tested using reliable methodologies and that any health effects are underpinned by realistic mechanisms of effect.

Prebiotics and Calcium Bioavailability

A prebiotic substance has been defined as a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon. Therefore, compared to probiotics, which introduce exogenous bacteria into the colonic microflora, a prebiotic aims at stimulating the growth of one or a limited number of the potentially health-promoting indigenous micro-organisms, thus modulating the composition of the natural ecosystem. In recent years, increasing attention has been focussed on the possible beneficial effects of prebiotics, such as enhanced resistance to invading pathogens, improved bowel function, anti-colon cancer properties, lipid lowering action, improved calcium bioavailability, amongst others. The objective of this chapter is to critically assess the available data on the effects of prebiotics on calcium bioavailability, and place it in the context of human physiology and, when possible, explain the underlying cellular and molecular mechanisms. The chapter will also try to highlight future areas of research that may help in the evaluation of prebiotics as potential ingredients for functional foods aimed at enhancing calcium bioavailability and protecting against osteoporosis.

The Possible Role of Probiotic Therapy in Inflammatory Bowel Disease

Despite many years of extensive research, the role of the luminal bacterial flora in the pathogenesis of chronic inflammatory bowel diseases has not been fully clarified. There is mounting evidence that a genetically determined immune response is reacting overly aggressive towards components of the intestinal microflora. Recent work suggested, that the course of the disease might be altered by manipulating the intestinal microflora with the use of antibiotics or probiotics. However, few clinical trials have been conducted and the results of in vitro experiments are still contradictoy regarding the effects on the human immune system. This chapter will summarize recent in vitro and in vivo data regarding possible disease initiating and perpetuating microorganisms and suggested therapeutic mechanisms of probiotic bacteria relevant to inflammatory bowel disease. Furthermore, we will review clinical trials examining the efficacy of probiotic microorganisms in IBD.

Gut Microflora and Atopic Disease

In recent years many countries have experienced a rise in allergic disease, which cannot be genetic in origin. Over the same period many aspects of modern life have changed and theories have been put forward to explain this trend. In 1989, Strachan introduced his "Hygiene Hypothesis", in which he proposed that allergic diseases could be prevented by infection in early childhood. However, despite numerous studies a specific "infective protective factor" has not been identified. Recently, attention has turned towards the intestinal microflora and the possibility that colonisation with specific microbes may be more important than sporadic infections. The immune system is Th-2 skewed in newborn babies, and the intestinal microflora may act as a counter-regulator, driving towards Th-1 differentiation. Colonisation with microbes begins immediately after birth and soon outnumber the human host cells. Thus, the microflora is the earliest and by far the largest stimulus to the immune system, and outweighs that of any occasional infection. There is evidence suggestive of an association between intestinal microflora and allergic disease and also suggestive that probiotics may improve or even prevent disease. However, these studies are on small numbers of children and long-term follow up is awaited. Longitudinal studies are necessary to establish whether the intestinal microflora plays an active role in the aetiology of allergic disease and whether manipulation can lead to a decrease in prevalence.

Genomic Perspectives on Probiotics and the Gastrointestinal Microflora

The explosion of genomic technologies in recent years has revolutionized every aspect of biology in an unprecedented manner. From a long and successful history of "reductionist" science, it has now become possible to understand how component parts interact collectively to create an organism. The growing collection of genomic sequence information, the high-throughput analysis of expression profiles using DNA microarrays, and the ability to deal with this information using advanced bioinformatics offer many possibilites to advance our knowledge of the microbial world. Developments such as these will enable a better understanding of the gastrointestinal (GI) tract as a complex and delicately balanced ecosystem. Genetic characterization of probiotic cultures is essential to unequivocally define their contributions to human health. Functional genomic approaches may help improve the functionality of these strains from an industrial and health-promoting perspective, and help to scientifically substantiate some of the health claims made for probiotic strains. This paper describes some of the recent developments in the rapidly growing area of genomics, and how these advances may be exploited to identify the molecular foundations of the relationships between probiotic organisms and their hosts, and how they contribute to our health and well-being.

Intestinal Microflora and Homeostasis of the Mucosal Immune Response: Implications for Probiotic Bacteria?

The intestinal microflora can be considered a post-natally acquired organ that is composed of a large diversity of bacteria that perform important functions for the host and can be modulated by environmental factors, such as nutrition. Specific components of the intestinal microflora, including lactobacilli and bifidobacteria, have been associated with beneficial effects on the host, such as promotion of gut maturation and integrity, antagonisms against pathogens and immune modulation. Beyond this, the microflora seems to play a significant role in the maintenance of intestinal immune homeostasis and prevention of inflammation. The contribution of the intestinal epithelial cell in the first line of defense against pathogenic bacteria and microbial antigens has been recognized. However, the interactions of intestinal epithelial cells with indigenous bacteria are less well understood. This chapter will summarize the increasing scientific attention to mechanisms of the innate immune response of the host towards different components of the microflora, and suggest a potential role for selected probiotic bacteria in the regulation of intestinal inflammation.