Abstract 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.
Abstract 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.
Abstract 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.
Abstract 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.
Abstract 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.
Abstract
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.
Abstract 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.
Abstract 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.
Abstract 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.
Abstract 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.
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