Applications for biotechnology: present and future improvements in lactic acid bacteria

The lactic acid bacteria are involved in the manufacture of fermented foods from raw agricultural materials such as milk, meat, vegetables, and cereals. These fermented foods are a significant part of the food processing industry and are often prepared using selected strains that have the ability to produce desired products or changes efficiently. The application of genetic engineering technology to improve existing strains or develop novel strains for these fermentations is an active research area world-wide. As knowledge about the genetics and physiology of lactic acid bacteria accumulates, it becomes possible to genetically construct strains with characteristics shaped for specific purposes. Examples of present and future applications of biotechnology to lactic acid bacteria to improve product quality are described. Studies of the basic biology of these bacteria are being actively conducted and must be continued, in order for the food fermentation industry to reap the benefits of biotechnology.     

Applications for biotechnology: present and future improvements in lactic acid bacteria

Abstract The lactic acid bacteria are involved in the manufacture of fermented foods from raw agricultural materials such as milk, meat, vegetables, and cereals. These fermented foods are a significant part of the food processing industry and are often prepared using selected strains that have the ability to produce desired products or changes efficiently. The application of genetic engineering technology to improve existing strains or develop novel strains for these fermentations is an active research area world-wide. As knowledge about the genetics and physiology of lactic acid bacteria accumulates, it becomes possible to genetically construct strains with characteristics shaped for specific purposes. Examples of present and future applications of biotechnology to lactic acid bacteria to improve product quality are described. Studies of the basic biology of these bacteria are being actively conducted and must be continued, in order for the food fermentation industry to reap the benefits of biotechnology.     

Biotechnology of health-promoting bacteria

Over the last decade, there has been an increasing scientific and public interest in bacteria that may positively contribute to human gut health and well-being. This interest is reflected by the ever-increasing number of developed functional food products containing health-promoting bacteria and reaching the market place as well as by the growing revenue and profits of notably bacterial supplements worldwide. Traditionally, the origin of probiotic-marketed bacteria was limited to a rather small number of bacterial species that mostly belong to lactic acid bacteria and bifidobacteria. Intensifying research efforts on the human gut microbiome offered novel insights into the role of human gut microbiota in health and disease, while also providing a deep and increasingly comprehensive understanding of the bacterial communities present in this complex ecosystem and their interactions with the gut-liver-brain axis. This resulted in rational and systematic approaches to select novel health-promoting bacteria or to engineer existing bacteria with enhanced probiotic properties. In parallel, the field of gut microbiomics developed into a fertile framework for the identification, isolation and characterization of a phylogenetically diverse array of health-promoting bacterial species, also called next-generation therapeutic bacteria. The present review will address these developments with specific attention for the selection and improvement of a selected number of health-promoting bacterial species and strains that are extensively studied or hold promise for future food or pharma product development.     

Lactic acid bacteria in a changing legislative environment

The benefits of using lactic acid bacteria in the food chain, both through direct consumption and production of ingredients, are increasingly recognised by the food industry and consumers alike. The regulatory environment surrounding these products is diverse, covering foods and food ingredients, processing aids, feed additives and dietary supplements. On a global basis, there are different approaches taken by the various regulatory authorities. While in Europe, the national legislation is gradually being harmonised, predominantly through the Novel Foods Regulation, there is still a wide disparity between the stringency of regulation of microbial products fed to animals and the comparatively relaxed approach to non-novel microbial products intended for human consumption. In the United States, the onus is on self-regulation of the manufacturer, with the Generally Recognised As Safe (GRAS) and Dietary Supplement Health Education Act (DSHEA) notification schemes encouraging industry to be more open about the ingredients they market. In Japan, the Foods for Special Health Use system continues to gain recognition as more products are approved, and is a potential model for other countries in regulating functional foods. Despite the different approaches to regulating these products, safety of microorganisms such as lactic acid bacteria in the food chain is paramount in all countries. This paper discusses the regulatory requirements of microbial products, predominantly lactic acid bacteria within the global markets, focusing mainly on the developments in Europe.     

Histamine-producing pathway encoded on an unstable plasmid in Lactobacillus hilgardii 0006

Histamine production from histidine in fermented food products by lactic acid bacteria results in food spoilage and is harmful to consumers. We have isolated a histamine-producing lactic acid bacterium, Lactobacillus hilgardii strain IOEB 0006, which could retain or lose the ability to produce histamine depending on culture conditions. The hdcA gene, coding for the histidine decarboxylase of L. hilgardii IOEB 0006, was located on an 80-kb plasmid that proved to be unstable. Sequencing of the hdcA locus disclosed a four-gene cluster encoding the histidine decarboxylase, a protein of unknown function, a histidyl-tRNA synthetase, and a protein, which we named HdcP, showing similarities to integral membrane transporters driving substrate/product exchange. The gene coding for HdcP was cloned downstream of a sequence specifying a histidine tag and expressed in Lactococcus lactis. The recombinant HdcP could drive the uptake of histidine into the cell and the exchange of histidine and histamine. The combination of HdcP and the histidine decarboxylase forms a typical bacterial decarboxylation pathway that may generate metabolic energy or be involved in the acid stress response. Analyses of sequences present in databases suggest that the other two proteins have dispensable functions. These results describe for the first time the genes encoding a histamine-producing pathway and provide clues to the parsimonious distribution and the instability of histamine-producing lactic acid bacteria.     

乳酸菌对真菌毒素的脱毒作用研究进展

真菌毒素广泛存在于农业产品中,对人和动物的健康构成巨大威胁。乳酸菌作为一种公认安全的微生物,在食品生物减毒方面具有巨大的应用潜力,成本低廉且不会对食品品质及生态环境造成不良影响。文章主要根据近年来国内外研究进展,阐述乳酸菌对食品和饲料中几种常见真菌毒素的脱毒作用(抑制真菌生长、毒素的吸附和降解),关注乳酸菌在生物脱毒方面的实际应用,为乳酸菌在食品保鲜领域的应用提供理论指导。     

Ecological studies on the occurrence of bacteria utilizing lactic acid at pH values below 4.5

Sites within two cheese factories, a silage tower and silage pit and two pet food factories were investigated for lactic acid utilizing bacteria. The bacteria isolated, together with known lactic acid utilizing and aciduric bacteria, were tested for their ability to 'alkalinize' a fermented meat product containing more than 20 g/1 lactic acid at pH > 4.3 a ten-fold dilution of this product and synthetic media, both at pH 4.5. Strains able to alkalinize the diluted product were 13 aerobic isolates (all Acetobacter spp.), nine anaerobic cultures, one strain of Megasphaera elsdenii and two strains of Clostridium tyrobutyricum. Eight of the aerobes could alkalinize undiluted product containing < 1 g/1 residual potassium sorbate, but had no effect on product containing > 3 g/1 sorbate; none of the anaerobic cultures was able to alkalinize undiluted product. Acetobacter spp., therefore, threatened the microbial stability of a fermented product containing < 3 g/1 potassium sorbate. The cultures able to alkalinize diluted product were all from sites contaminated with whey, silage or fermented product for at least 2 d, suggesting substantial accumulation of lactic acid utilizing bacteria at these sites.     

Health and nutritional benefits from lactic acid bacteria

There are several potential health or nutritional benefits possible from some species of lactic acid bacteria. Among these are: improved nutritional value of food, control of intestinal infections, improved digestion of lactose, control of some types of cancer, and control of serum cholesterol levels. Some potential benefits may result from growth and action of the bacteria during the manufacture of cultured foods. Some may result from growth and action of certain species of the lactic acid bacteria in the intestinal tract following ingestion of foods containing them. In selecting a culture to produce a specific benefit it is necessary to consider not only the wide variation among species of the lactic acid bacteria but also that among strains within a given species. With the possible exception of improving lactose utilization by persons who are lactose maldigestors, no specific health or nutritional claims can yet be made for the lactic acid bacteria.     

Citrate metabolism in lactic acid bacteria

Abstract: Citrate metabolism plays an important role in many food fermentations involving lactic acid bacteria. Since citrate is a highly oxidized substrate, no reducing equivalents are produced during its degradation, resulting in the formation of metabolic end products other than lactic acid. Some of these end products, such as diacetyl and acetaldehyde, have very distinct aroma properties and contribute significantly to the quality of the fermented foods. In this review the metabolic pathways involved in product formation from citrate are described, the bioenergetic consequences of this metabolism for the lactic acid bacteria are discussed and detailed information on some key enzymes in the citrate metabolism is presented. The combined knowledge is used for devising strategies to avoid, control or improve product formation from citrate.     

Health and nutritional benefits from lactic acid bacteria

Abstract There are several potential health or nutritional benefits possible from some species of lactic acid bacteria. Among these are: improved nutritional value of food, control of intestinal infections, improved digestion of lactose, control of some types of cancer, and control of serum cholesterol levels. Some potential benefits may result from growth and action of the bacteria during the manufacture of cultured foods. Some may result from growth and action of certain species of the lactic acid bacteria in the intestinal tract following ingestion of foods containing them. In selecting a culture to produce a specific benefit it is necessary to consider not only the wide variation among species of the lactic acid bacteria but also that among strains within a given species. With the possible exception of improving lactose utilization by persons who are lactose maldigestors, no specific health or nutritional claims can yet be made for the lactic acid bacteria.     

Post-genomics of lactic acid bacteria and other food-grade bacteria to discover gut functionality

Recent years have seen an explosion in the number of complete or almost complete genomic sequences of lactic acid bacteria and other food-grade bacteria that are used in functional foods to increase the health of the consumer. These have been instrumental in the development of functional, comparative and other post-genomics approaches that provide the possibility to detect, unravel and understand their functionality in the human intestinal tract. In conjunction with other high-throughput approaches, these advances can be exploited in the functional food innovation cycle for developing new or designed probiotic and other bacterial products that impact gut health.     

A review of food-grade vectors in lactic acid bacteria: from the laboratory to their application

Lactic acid bacteria (LAB) have a long history of use in fermented foods and as probiotics. Genetic manipulation of these microorganisms has great potential for new applications in food safety, as well as in the development of improved food products and in health. While genetic engineering of LAB could have a major positive impact on the food and pharmaceutical industries, progress could be prevented by legal issues related to the controversy surrounding this technology. The safe use of genetically modified LAB requires the development of food-grade cloning systems containing only the DNA from homologous hosts or generally considered as safe organisms, and not dependent antibiotic markers. The rationale for the development of cloning vectors derived from cryptic LAB plasmids is the need for new genetic engineering tools, therefore a vision from cryptic plasmids to applications in food-grade vectors for LAB plasmids is shown in this review. Replicative and integrative vectors for the construction of food-grade vectors, and the relationship between resistance mechanism and expression systems, will be treated in depth in this paper. Finally, we will discuss the limited use of these vectors, and the problems arising from their use.     

基因工程乳酸菌的应用研究进展

随着分子生物学技术的不断发展,重组基因工程乳酸菌取得了较快的进展.本文主要从基因工程乳酸菌在食品、药品、保健品、饲料养殖领域中的应用进行综述,以便正确认识、了解和使用基因工程乳酸菌,对其进一步的研究以及开发应用具有深远的意义.     

Review: Biosafety of E. coli β-glucuronidase (GUS) in plants

The -glucuronidase (GUS) gene is to date the most frequently used reporter gene in plants. Marketing of crops containing this gene requires prior evaluation of their biosafety. To aid such evaluations of the GUS gene, irrespective of the plant into which the gene has been introduced, the ecological and toxicological aspects of the gene and gene product have been examined. GUS activity is found in many bacterial species, is common in all tissues of vertebrates and is also present in organisms of various invertebrate taxa. The transgenic GUS originates from the enterobacterial species Escherichia coli that is widespread in the vertebrate intestine, and in soil and water ecosystems. Any GUS activity added to the ecosystem through genetically modified plants will be of no or minor influence. Selective advantages to genetically modified plants that posses and express the E. coli GUS transgene are unlikely. No increase of weediness of E. coli GUS expressing crop plants, or wild relatives that might have received the transgene through outcrossing, is expected. Since E. coli GUS naturally occurs ubiquitously in the digestive tract of consumers, its presence in food and feed from genetically modified plants is unlikely to cause any harm. E. coli GUS in genetically modified plants and their products can be regarded as safe for the environment and consumers     

Histamine-Producing Pathway Encoded on an Unstable Plasmid in Lactobacillus hilgardii 0006

Histamine production from histidine in fermented food products by lactic acid bacteria results in food spoilage and is harmful to consumers. We have isolated a histamine-producing lactic acid bacterium, Lactobacillus hilgardii strain IOEB 0006, which could retain or lose the ability to produce histamine depending on culture conditions. The hdcA gene, coding for the histidine decarboxylase of L. hilgardii IOEB 0006, was located on an 80-kb plasmid that proved to be unstable. Sequencing of the hdcA locus disclosed a four-gene cluster encoding the histidine decarboxylase, a protein of unknown function, a histidyl-tRNA synthetase, and a protein, which we named HdcP, showing similarities to integral membrane transporters driving substrate/product exchange. The gene coding for HdcP was cloned downstream of a sequence specifying a histidine tag and expressed in Lactococcus lactis. The recombinant HdcP could drive the uptake of histidine into the cell and the exchange of histidine and histamine. The combination of HdcP and the histidine decarboxylase forms a typical bacterial decarboxylation pathway that may generate metabolic energy or be involved in the acid stress response. Analyses of sequences present in databases suggest that the other two proteins have dispensable functions. These results describe for the first time the genes encoding a histamine-producing pathway and provide clues to the parsimonious distribution and the instability of histamine-producing lactic acid bacteria.     

Traditional healthful fermented products of Japan

A variety of fermentation products, such as foods containing probiotic bacteria, black rice vinegar (kurosu), soy sauce (shoyu), soybean-barley paste (miso), natto and tempeh, are sold in food stores in Japan. These fermented food products are produced by traditional methods that exploit mixed cultures of various non-toxic microorganisms. These microorganisms include lactic acid bacteria, acetic acid bacteria, sake yeast, koji molds and natto bacteria. Many traditional fermented foods have been studied and their effects on metabolism and/or immune system have been demonstrated in animal and/or human cells. This review summarizes the scientific basis for the effects of these traditional food products, which are currently produced commercially in Japan.     

Fermentation of plant-based dairy alternatives by lactic acid bacteria

Ethical, environmental and health concerns around dairy products are driving a fast-growing industry for plant-based dairy alternatives, but undesirable flavours and textures in available products are limiting their uptake into the mainstream. The molecular processes initiated during fermentation by lactic acid bacteria in dairy products is well understood, such as proteolysis of caseins into peptides and amino acids, and the utilisation of carbohydrates to form lactic acid and exopolysaccharides. These processes are fundamental to developing the flavour and texture of fermented dairy products like cheese and yoghurt, yet how these processes work in plant-based alternatives is poorly understood. With this knowledge, bespoke fermentative processes could be engineered for specific food qualities in plant-based foods. This review will provide an overview of recent research that reveals how fermentation occurs in plant-based milk, with a focus on how differences in plant proteins and carbohydrate structure affect how they undergo the fermentation process. The practical aspects of how this knowledge has been used to develop plant-based cheeses and yoghurts is also discussed.     

Probiotics: functionality and commercial status

Probiotics in the form of fermented milk products have been consumed for centuries. In this century various health benefits have been purported to result from consumption of foods containing live microorganisms, particularly lactic acid bacteria (LAB). Probiotics can provide relief for lactose intolerant individuals and reduce bouts of diarrhea. Evidence for other claims such as lowering serum cholesterol, suppressing cancer and stimulating the immune system remains to be clearly established by conducting well-controlled, statistically-valid clinical trials. Although the benefits to healthy individuals are uncertain, many consumers especially in Japan and Europe, perceive probiotic products to be healthful, and sales are robust.     

Genetically modified lactic acid bacteria: applications to food or health and risk assessment

Lactic acid bacteria have a long history of use in fermented food products. Progress in gene technology allows their modification by introducing new genes or by modifying their metabolic functions. These modifications may lead to improvements in food technology (bacteria better fitted to technological processes, leading to improved organoleptic properties em leader ), or to new applications including bacteria producing therapeutic molecules that could be delivered by mouth. Examples in these two fields will be discussed, at the same time evaluating their potential benefit to society and the possible risks associated with their use. Risk assessment and expected benefits will determine the future use of modified bacteria in the domains of food technology and health.     

Functional genomics of lactic acid bacteria: from food to health

Genome analysis using next generation sequencing technologies has revolutionized the characterization of lactic acid bacteria and complete genomes of all major groups are now available. Comparative genomics has provided new insights into the natural and laboratory evolution of lactic acid bacteria and their environmental interactions. Moreover, functional genomics approaches have been used to understand the response of lactic acid bacteria to their environment. The results have been instrumental in understanding the adaptation of lactic acid bacteria in artisanal and industrial food fermentations as well as their interactions with the human host. Collectively, this has led to a detailed analysis of genes involved in colonization, persistence, interaction and signaling towards to the human host and its health. Finally, massive parallel genome re-sequencing has provided new opportunities in applied genomics, specifically in the characterization of novel non-GMO strains that have potential to be used in the food industry. Here, we provide an overview of the state of the art of these functional genomics approaches and their impact in understanding, applying and designing lactic acid bacteria for food and health.