Über die Autorin bzw. den Autor

YUAN KUN LEE, PHD, is an Associate Professor in the Department of Microbiology at the National University of Singapore, where he researches fermentation and microbe-host interactions and probiotics. Dr. Lee has written ninety journal papers, sixty conference proceedings, twenty-eight book chapters, and three books. He is also the holder of three patents.

SEPPO SALMINEN, PHD, is a Professor in the Department of Biochemistry and Food Chemistry at the University of Turku in Finland. He is also a Visiting Professor of Food Toxicology at RMIT University in Australia. Prior to his academic career, Dr. Salminen worked as a nutrition specialist, project manager, and toxicologist for private industry and government agencies. Dr. Salminen has written more than 240 refereed publications, several book chapters, and five books.

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The Second Edition of the Handbook of Probiotics and Prebiotics brings together all the latest information and technology needed to develop successful probiotic and prebiotic products. From the laboratory to the marketplace, every phase of development is covered, including evaluating, testing, analyzing, manufacturing, and processing.

In addition to probiotics, this Second Edition now covers prebiotics, examining the results from the latest scientific research and clinical interventions. Thoroughly revised and updated, the Second Edition features much more new material, including:

• A new chapter dedicated to the mechanisms of probiotics
• A new chapter on commercially available human probiotic microorganisms
• The latest methods for the analysis, enumeration, and identification of gastrointestinal microbiota
• The most recent developments in cell line and animal models
• New information on the safety of novel probiotic bacteria, reflecting new regulatory requirements for novel food products in Asia, the EU, and North America
• The latest information on the role probiotics and prebiotics play in health and nutrition

This handbook features a team of expert authors whose experience encompasses every stage of probiotic and prebiotic analysis and development. As a result, the book addresses many of the practical issues that manufacturers and researchers face when dealing with probiotic organisms and prebiotic compounds.

With more and more interest in probiotics and prebiotics as well as a growing number of commercially available products, the Handbook of Probiotics and Prebiotics will help you understand how these products work and help you create the next generation of probiotic and prebiotic products.

Aus dem Klappentext

The Second Edition of the Handbook of Probiotics and Prebiotics brings together all the latest information and technology needed to develop successful probiotic and prebiotic products. From the laboratory to the marketplace, every phase of development is covered, including evaluating, testing, analyzing, manufacturing, and processing.

In addition to probiotics, this Second Edition now covers prebiotics, examining the results from the latest scientific research and clinical interventions. Thoroughly revised and updated, the Second Edition features much more new material, including:

• A new chapter dedicated to the mechanisms of probiotics
• A new chapter on commercially available human probiotic microorganisms
• The latest methods for the analysis, enumeration, and identification of gastrointestinal microbiota
• The most recent developments in cell line and animal models
• New information on the safety of novel probiotic bacteria, reflecting new regulatory requirements for novel food products in Asia, the EU, and North America
• The latest information on the role probiotics and prebiotics play in health and nutrition

This handbook features a team of expert authors whose experience encompasses every stage of probiotic and prebiotic analysis and development. As a result, the book addresses many of the practical issues that manufacturers and researchers face when dealing with probiotic organisms and prebiotic compounds.

With more and more interest in probiotics and prebiotics as well as a growing number of commercially available products, the Handbook of Probiotics and Prebiotics will help you understand how these products work and help you create the next generation of probiotic and prebiotic products.

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Handbook of Probiotics and Prebiotics

John Wiley & Sons

Chapter One

1.1 DEFINITIONS

Yuan Kun Lee

Department of Microbiology, National University of Singapore, Singapore

"Probiotics" is derived from Greek and means "prolife." It has been redefined throughout the years as more scientific knowledge and better understanding on its relationship between intestinal health and general well-being has been gained. The following are definitions of "probiotics" derived through times.

Lilly and Stillwell in 1965 defined probiotics as "Growth promoting factors produced by microorganisms."

Parker in 1974 suggested an interaction between microorganisms with the host: "Organisms and substances with beneficial effects for animals by influencing the intestinal microflora."

Fuller in 1989 defined it as "A live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance."

Havenaar and Huis Int Veld in 1992 said probiotics are "A mono- or mixed culture of live microorganisms which, applied to animal or man, affect beneficially the host by improving the properties of the indigenous microflora."

ILSI (International Life Sciences Institute) Europe Working Group (1998): "A viable microbial food supplement which beneficially influences the health of the host."

Diplock et al. in 1999 puts it as

"Probiotic food is functional if they have been satisfactorily demonstrated to beneficially affect one or more target functions in the body beyond adequate nutritional effects, in a way that is relevant to either an improved state of health and well-being and/or reduction in the risk of diseases."

Naidu et al. in 1999 said "A microbial dietary adjuvant that beneficially affects the host physiology by modulating mucosal and systemic immunity, as well as improving nutritional and microbial balance in the intestinal tract."

Tannock in 2000 observed that long-term consumption of probiotics was not associated with any drastic change in the intestinal microbiota composition, and thus proposed an alternative definition: "Microbial cells which transit the GI tract and which, in doing so, benefit the health of consumer."

Schrezenmeir and de Vrese in 2001 defined probiotics as "A preparation of a product containing viable, defined microorganisms in sufficient numbers, which alter the microflora (by implantation or colonization) in a compartment of the host and by that exert beneficial health effects in this host."

FAO/WHO (Food and Agriculture Organization and World Health Organization) (2001) and Reid et al. (2003) concentrated exclusively on its health purpose: "Live microorganisms which when administered in adequate amounts confer a health benefit on the host."

1.2 SCREENING, IDENTIFICATION, AND CHARACTERIZATION OF Lactobacillus AND Bifidobacterium STRAINS

Abelardo Margolles, Baltasar Mayo, and Patricia Ruas-Madiedo

Instituto de Productos Lcteos de Asturias (CSIC), Villaviciosa, Asturias, Spain

Several genera of bacteria (and yeast) have been proposed as probiotic cultures, the most commonly used are Lactobacillus and Bifidobacterium species. However, the selection of a strain to be used as an effective probiotic is a complex process (Fig. 1.1). The work begins with the source of screening of strains, the most suitable approach being the natural intestinal environment.

According to FAO/WHO guidelines it is necessary to identify the microorganism to species/strain level given that the evidence suggests that the probiotic effects are strain specific. It is recommended to employ a combination of phenotypic and genetic techniques to accomplish the identification, classification, and typing. For the nomenclature of bacteria, scientifically recognized names must be employed and it is recommended to deposit the strains in an internationally recognized culture collection. Further characterization of strains must be undertaken taking into account the "functional" or probiotic aspects and safety assessment. In vitro tests, some of them summarized in Fig.1.1, are useful to gain knowledge of both strains and mechanisms of the probiotic effect. In addition, even if these genera have a long history of safe consumption in traditionally fermented products and several species have been awarded a "General Recognised As Safe" (GRAS) status by the American Food and Drug Association or a qualified presumption of safety (QPS) consideration by the European Food Safety Authority (EFSA), some characteristics (Fig. 1.1) must be studied to ensure the safety of the novel lactobacilli and bifidobacteria strains. Several of the in vitro tests can be correlated with in vivo studies with animal models, but probiotics for human use must be validated with human studies covering both safety (phase 1 trials) and efficacy (phase 2 trials) aspects. Phase 2 studies should be designed as double-blind, randomized, and placebo-controlled to measure the efficacy of the probiotic strain compared with a placebo and also to determine possible adverse effects.

This chapter focuses on the current techniques for bacterial identification, taxonomic classification, and typing of Lactobacillus and Bifidobacterium strains, and also reviews the in vitro probiotic characterization of strains based on their functional aspects.

1.2.1 Sources of Screening for Probiotic Strains

Even though essentially all animals contain strains of both Lactobacillus and Bifidobacterium genera, it is well accepted that an effective human probiotic should be of human origin. The underlying reason for this is that human intestines are sufficiently different from those of animals, such that the isolates suited to those environments would not necessarily be suited to the human intestine. The human gastrointestinal tract (GIT) is a very complex ecological niche and its bacteria inhabitants can achieve the highest cell densities recorded for any ecosystem. Nonetheless, diversity at a division level is among the lowest and the lactobacilli and bifidobacteria comprise less than 5% of the total microbiota. A number of articles have been published in the last few years studying the diversity of the GIT ecosystem employing several culture-independent genetic tools. But, for the isolation of novel strains, classical cultivation techniques must be employed. Enrichment, selective media, and specific culture conditions are employed for the isolation of strains from human samples that are initially identified by morphological characterization under the microscope. Molecular tools, mainly based on the sequencing of the 16S rRNA gene, allow identification down to the species level. Using this basic scheme several collections of strains have been isolated from human (and other animal) samples. Commonly, fecal samples are donated by healthy adult or infant volunteers. But other GIT sections obtained from healthy individuals and patients submitted to biopsies such as the terminal ileum or colonic mucosa can be screened. Also the oral cavity seems to be the origin of some allochthonous lactobacilli of the intestine. Recently, it has been indicated that the infant fecal microbiota reflects the bacterial composition of the breast milk. Therefore, the natural microbiota of human milk could be proposed as a source for the isolation of novel probiotic bacteria.

Another approach to search for improved probiotic strains (Fig....