Production of gamma-aminobutyric acid by <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> NCL912 using fed-batch fermentation

Background  

Gamma-aminobutyric acid is a major inhibitory neurotransmitter in mammalian brains, and has several well-known physiological functions. Lactic acid bacteria possess special physiological activities and are generally regarded as safe. Therefore, using lactic acid bacteria as cell factories for gamma-aminobutyric acid production is a fascinating project and opens up a vast range of prospects for making use of GABA and LAB. We previously screened a high GABA-producer Lactobacillus brevis NCL912 and optimized its fermentation medium composition. The results indicated that the strain showed potential in large-scale fermentation for the production of gamma-aminobutyric acid. To increase the yielding of GABA, further study on the fermentation process is needed before the industrial application in the future. In this article we investigated the impacts of pyridoxal-5'-phosphate, pH, temperature and initial glutamate concentration on gamma-aminobutyric acid production by Lactobacillus brevis NCL912 in flask cultures. According to the data obtained in the above, a simple and effective fed-batch fermentation method was developed to highly efficiently convert glutamate to gamma-aminobutyric acid.     

Selecting for lactic acid producing and utilising bacteria in anaerobic enrichment cultures

Lactic acid-producing bacteria are important in many fermentations, such as the production of biobased plastics. Insight in the competitive advantage of lactic acid bacteria over other fermentative bacteria in a mixed culture enables ecology-based process design and can aid the development of sustainable and energy-efficient bioprocesses. Here we demonstrate the enrichment of lactic acid bacteria in a controlled sequencing batch bioreactor environment using a glucose-based medium supplemented with peptides and B vitamins. A mineral medium enrichment operated in parallel was dominated by Ethanoligenens species and fermented glucose to acetate, butyrate and hydrogen. The complex medium enrichment was populated by Lactococcus, Lactobacillus and Megasphaera species and showed a product spectrum of acetate, ethanol, propionate, butyrate and valerate. An intermediate peak of lactate was observed, showing the simultaneous production and consumption of lactate, which is of concern for lactic acid production purposes. This study underlines that the competitive advantage for lactic acid-producing bacteria primarily lies in their ability to attain a high biomass specific uptake rate of glucose, which was two times higher for the complex medium enrichment when compared to the mineral medium enrichment. The competitive advantage of lactic acid production in rich media can be explained using a resource allocation theory for microbial growth processes.     

耐乙酸乳杆菌的筛选及其产乳酸特性

【背景】耐受乙酸的乳酸菌是传统谷物醋醋酸发酵过程中产生乳酸及其风味衍生物的重要功能微生物。【目的】从镇江香醋醋醅中分离鉴定具有耐乙酸特性的乳酸菌,并评价不同条件下该菌株的产乳酸能力。【方法】利用4%(体积比)乙酸含量的MRS培养基分离耐乙酸乳酸菌;对其进行16S rRNA基因鉴定、基因组测序、形态观察以及生理生化特性研究;考察不同乙酸浓度、葡萄糖浓度、发酵温度和时间对菌株产乳酸能力的影响。【结果】分离得到一株可耐受6%乙酸的乳杆菌Lactobacillus sp. JN500903;在厌氧静置、接种量5%、乙酸浓度5%、葡萄糖浓度40 g/L、发酵温度37°C、发酵时间10 d条件下,该菌株乳酸产量为16.1 g/L。【结论】乳杆菌JN500903能够耐受6%乙酸浓度,具有在酸性环境下合成乳酸的能力,有一定的应用潜力。     

Peptidases and amino acid catabolism in lactic acid bacteria

The conversion of peptides to free amino acids and their subsequent utilization is a central metabolic activity in prokaryotes. At least 16 peptidases from lactic acid bacteria (LAB) have been characterized biochemically and/or genetically. Among LAB, the peptidase systems of Lactobacillus helveticus and Lactococcus lactis have been examined in greatest detail. While there are homologous enzymes common to both systems, significant differences exist in the peptidase complement of these organisms. The characterization of single and multiple peptidase mutants indicate that these strains generally exhibit reduced specific growth rates in milk compared to the parental strains. LAB can also catabolize amino acids produced by peptide hydrolysis. While the catabolism of amino acids such as Arg, Thr, and His is well understood, few other amino acid catabolic pathways from lactic acid bacteria have been characterized in significant detail. Increasing research attention is being directed toward elucidating these pathways as well as characterizing their physiological and industrial significance.     

Heteropolysaccharides from lactic acid bacteria

Microbial exopolysaccharides are biothickeners that can be added to a wide variety of food products, where they serve as viscosifying, stabilizing, emulsifying or gelling agents. Numerous exopolysaccharides with different composition, size and structure are synthesized by lactic acid bacteria. The heteropolysaccharides from both mesophilic and thermophilic lactic acid bacteria have received renewed interest recently. Structural analysis combined with rheological studies revealed that there is considerable variation among the different exopolysaccharides; some of them exhibit remarkable thickening and shear-thinning properties and display high intrinsic viscosities. Hence, several slime-producing lactic acid bacterium strains and their biopolymers have interesting functional and technological properties, which may be exploited towards different products, in particular, natural fermented milks. However, information on the biosynthesis, molecular organization and fermentation conditions is rather scarce, and the kinetics of exopolysaccharide formation are poorly described. Moreover, the production of exopolysaccharides is low and often unstable, and their downstream processing is difficult. This review particularly deals with microbiological, biochemical and technological aspects of heteropolysaccharides from, and their production by, lactic acid bacteria. The chemical composition and structure, the biosynthesis, genetics and molecular organization, the nutritional and physiological aspects, the process technology, and both food additive and in situ applications (in particular in yogurt) of heterotype exopolysaccharides from lactic acid bacteria are described. Where appropriate, suggestions are made for strain improvement, enhanced productivities and advanced modification and production processes (involving enzyme and/or fermentation technology) that may contribute to the economic soundness of applications with this promising group of biomolecules.     

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。     

乳酸菌食品级表达载体的研究与应用

乳酸菌是能够发酵糖类产生大量有机酸的革兰氏阳性菌的通称,在发酵食品中有着悠久的应用历史。乳酸菌通常被认为是安全菌株,这些微生物的基因工程操作在食品、医学等方面具有广阔的应用前景。表达载体是基因工程中常用的工具之一,大多数乳酸菌的表达载体通常以抗生素抗性基因作为选择标记,然而抗性基因具有潜在的转移性,因此需要开发食品级表达载体。食品级表达载体不含有抗生素的抗性基因,仅包含来自同源宿主或通常被认为是安全生物的DNA。本文介绍了乳酸菌食品级表达载体的构成及其常用宿主,同时对乳酸菌食品级表达载体的应用进行了归纳总结。     

低温环境中乳酸菌的开发利用

乳酸菌作为一种益生资源,越来越为人们所重视.在自然界存在的乳酸菌中,有一类虽然人们一直在利用,但是还没有充分的研究和开发,这类乳酸菌就是低温环境中生长的乳酸菌.国外目前所见有关低温乳酸菌的报道多集中于低温冷藏肉、鱼制品及泡菜中的乳酸菌,集中研究和应用的菌属主要包括Leuconostoc和Lactobacillus,而国内这方面的研究并不多见.笔者根据目前国内外低温乳酸菌的研究现状,阐述了其存在环境、种类及相应作用,并对其研究涉及的领域、趋势及应用前景进行了探讨和展望,以期为国内这类乳酸菌资源的研究开发提供参考和依据.     

乳酸菌生理功能的系统解析与代谢调控

作为工业化的细胞工厂,乳酸菌广泛应用于食品、农业和医药等行业。然而在乳酸菌的工业生产中以及作为益生菌在人体胃肠道系统中都会面临多种环境胁迫,这些胁迫环境严重影响乳酸菌的生理功能,从而影响食品微生物制造的效率。近年来,随着代谢工程和系统生物学的发展,为乳酸菌生理功能的改造带来了前所未有的机遇。本文综述了系统生物学和代谢工程在乳酸菌生理功能的优化和调控中的具体应用。     

Amylolytic bacterial lactic acid fermentation - a review

Lactic acid, an enigmatic chemical has wide applications in food, pharmaceutical, leather, textile industries and as chemical feed stock. Novel applications in synthesis of biodegradable plastics have increased the demand for lactic acid. Microbial fermentations are preferred over chemical synthesis of lactic acid due to various factors. Refined sugars, though costly, are the choice substrates for lactic acid production using Lactobacillus sps. Complex natural starchy raw materials used for production of lactic acid involve pretreatment by gelatinization and liquefaction followed by enzymatic saccharification to glucose and subsequent conversion of glucose to lactic acid by Lactobacillus fermentation. Direct conversion of starchy biomass to lactic acid by bacteria possessing both amylolytic and lactic acid producing character will eliminate the two step process to make it economical. Very few amylolytic lactic acid bacteria with high potential to produce lactic acid at high substrate concentrations are reported till date. In this view, a search has been made for various amylolytic LAB involved in production of lactic acid and utilization of cheaply available renewable agricultural starchy biomass. Lactobacillus amylophilus GV6 is an efficient and widely studied amylolytic lactic acid producing bacteria capable of utilizing inexpensive carbon and nitrogen substrates with high lactic acid production efficiency. This is the first review on amylolytic bacterial lactic acid fermentations till date.     

Genetics of the proteolytic system of lactic acid bacteria

Abstract The proteolytic system of lactic acid bacteria is of eminent importance for the rapid growth of these organisms in protein-rich media. The combined action of proteinases and peptidases provides the cell with small peptides and essential amino acids. The amino acids and peptides thus liberated have to be translocated across the cytoplasmic membrane. To that purpose, the cell contains specific transport proteins. The internalized peptides are further degraded to amino acids by intracellular peptidases. The world-wide economic importance of the lactic acid bacteria and their proteolytic system has led to an intensive research effort in this area and a considerable amount of biochemical data has been collected during the last two decades. Since the development of systems to genetically manipulate lactic acid bacteria, data on the genetics of enzymes and processes involved in proteolysis are rapidly being generated. In this review an overview of the latest genetic data on the proteolytic system of lactic acid bacteria will be presented. As most of the work in this field has been done with lactococi, the emphasis will, inevitably, be on this group of organisms. Where possible, links will be made with other species of lactic acid bacteria.     

具有优良抑菌特性乳酸菌的筛选鉴定及活性物质检测

【背景】有益性乳酸菌在人体和动物体内分布极为广泛,是维持胃肠道菌群平衡、提高机体免疫力的主力军。近年来,为了解决禁用抗生素而导致动物发病率不断增高的问题,分析和研究乳酸菌及其所产活性物质的益生特性并开发新型饲料添加剂成为一个重要手段。【目的】本实验旨在从土壤中分离筛选出具有优良抑菌特性的乳酸菌,并对其所产活性物质的特性进行分析评价。【方法】采用溴甲酚紫平板法筛选并结合抑菌能力检测,得到2株具有优良抑菌特性的产酸菌株,分别命名为H-3和H-4。经形态学鉴定及16S rRNA基因序列测定后,对2株菌分别进行生长曲线和产酸量检测;通过排除酸处理、蛋白酶处理和热处理的方法分析2株菌所产抑菌物质的有效成分。【结果】H-3和H-4菌株经初步鉴定为乳酸片球菌(Pediococcus acidilactici),2株菌均具有良好的生长性能及产酸性能。菌株发酵上清液对大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、猪霍乱沙门氏菌(Salmonella choleraesuis)、福氏志贺氏菌(Shigella flexneri)均表现出明显的抑...     

Genetics of the proteolytic system of lactic acid bacteria

The proteolytic system of lactic acid bacteria is of eminent importance for the rapid growth of these organisms in protein-rich media. The combined action of proteinases and peptidases provides the cell with small peptides and essential amino acids. The amino acids and peptides thus liberated have to be translocated across the cytoplasmic membrane. To that purpose, the cell contains specific transport proteins. The internalized peptides are further degraded to amino acids by intracellular peptidases. The world-wide economic importance of the lactic acid bacteria and their proteolytic system has led to an intensive research effort in this area and a considerable amount of biochemical data has been collected during the last two decades. Since the development of systems to genetically manipulate lactic acid bacteria, data on the genetics of enzymes and processes involved in proteolysis are rapidly being generated. In this review an overview of the latest genetic data on the proteolytic system of lactic acid bacteria will be presented. As most of the work in this field has been done with lactococci, the emphasis will, inevitably, be on this group of organisms. Where possible, links will be made with other species of lactic acid bacteria.     

Lactic acid bacteria of wines: stimulation of growth and malolactic fermentation

After the appearance of “Etudes sur le vin” by Pasteur, in enology lactic acid bacteria have been considered as deteriorating agents for more than 50 years. About 1920, Ferré in Burgundy and Ribéreau-Gayon in Bordeaux demonstrated the enological importance of the transformation of malic to lactic acid. This notion is now generally accepted in most vinicultural areas. Malolactic fermentation is encouraged, especially for red wines, for two reasons: a) it eliminates the taste of malic acid and lowers the acidity of the wine, b) it assures the biological stability of wines conserved with a minimum of sulphurous anhydride. In traditional vinification, malolactic fermentation is the result of bacterial growth. It is spontaneous, that means induced by the endogenous lactic acid bacteria of grapes and winery equipment. In the must, yeasts and bacteria develop simultaneously; in the antagonism between yeasts and bacteria the bacterial population is more often becoming dominant than being suppressed. The grapes are sulphited so that bacterial growth occurs only after complete exhaustion of sugars by the yeasts. Consequently, alteration of the wine, as a result of sugar fermentation by the bacteria, is prevented. In a well-controlled vinification lactic acid bacteria can complete their growth cycle in the wine. Wine, however, is a poor culture medium and the bacteria multiply under restricted nutritional, physical and chemical conditions. As a consequence, malolactic fermentation is difficult to control in practice, in spite of all the research done for more than 30 years. For a long time, one has tried to stimulate malolactic fermentation by inoculating wine with bacteria. Until now, the problem has been to determine the biomass of bacteria, sufficient for fermentation to take place as well as the quality required. The desired physiological state of the bacteria in the inoculum is also not known.     

Recombinant lactic acid bacteria as mucosal biotherapeutic agents

The safety status of lactic acid bacteria (LAB) and their capacity to survive the passage through the gastrointestinal tract (GI tract) have rendered them excellent candidates for the production of therapeutic proteins and their delivery in situ to the GI tract. During the past two decades, major health benefits of mucosally administered recombinant LAB have been successfully demonstrated, predominantly using animal models. However, the field has recently moved into the era of human clinical trials. In this review, we provide a timely update on the recent important advances made in this field, and outline the potential of recombinant LAB as therapeutic tools for their safe and efficient use in human health.     

Probiotics and their fermented food products are beneficial for health

Probiotics are usually defined as microbial food supplements with beneficial effects on the consumers. Most probiotics fall into the group of organisms' known as lactic acid-producing bacteria and are normally consumed in the form of yogurt, fermented milks or other fermented foods. Some of the beneficial effect of lactic acid bacteria consumption include: (i) improving intestinal tract health; (ii) enhancing the immune system, synthesizing and enhancing the bioavailability of nutrients; (iii) reducing symptoms of lactose intolerance, decreasing the prevalence of allergy in susceptible individuals; and (iv) reducing risk of certain cancers. The mechanisms by which probiotics exert their effects are largely unknown, but may involve modifying gut pH, antagonizing pathogens through production of antimicrobial compounds, competing for pathogen binding and receptor sites as well as for available nutrients and growth factors, stimulating immunomodulatory cells, and producing lactase. Selection criteria, efficacy, food and supplement sources and safety issues around probiotics are reviewed. Recent scientific investigation has supported the important role of probiotics as a part of a healthy diet for human as well as for animals and may be an avenue to provide a safe, cost effective, and 'natural' approach that adds a barrier against microbial infection. This paper presents a review of probiotics in health maintenance and disease prevention.     

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.     

Antifungal-activity-producing lactic acid bacteria as biocontrol agents in plants

Fungal infection represents a severe problem that decreases the yield and market value of fruit crops. The use of fungicides is a conventional method to control infections but it is associated with disadvantages, such as hazardous impact on public health, environmental contamination, resistance development among pathogens and high cost of agrochemicals. Biological control is an alternative approach for the treatment of fungal infections. The species of Bacillus, Pseudomonas, Enterobacter, Pantoea, Burkholderia, Lysobacter and Serratia have been successfully used in the control of fungal infections. The mechanisms involved in biocontrol are hyperparasitism or predation, production of antibiotics, lytic enzymes and induction of host resistance. Lactic acid bacteria have been used as biopreservative organisms in food and feed systems. They are a cluster of Gram-positive bacteria and include species of the genera Enterococcus, Lactobacillus, Leuconostoc, Lactococcus and Pediococcus. The ability to produce several antibacterial and antifungal substances confers biopreservation potential to lactic acid bacteria. Many have ‘generally regarded as safe’ status and are considered as safe from both human and environmental points of view. Their isolation is reported from vegetables, aerial plant surfaces, pickled cabbage, grass silage, malted cereals and also from soil. They produce antifungal substances, such as cyclic dipeptides, proteinaceous compounds, organic acids, fatty acids and reuterin. The biocontrol potential of lactic acid bacteria is demonstrated in the prevention of fungal infections of fruits, such as apples and grapes. Thus, living cells or product formulations of antifungal lactic acid bacteria may be prepared and used as an alternative biocontrol technology.     

Metabolic engineering of sugar catabolism in lactic acid bacteria

Lactic acid bacteria are characterized by a relatively simple sugar fermentation pathway that, by definition, results in the formation of lactic acid. The extensive knowledge of traditional pathways and the accumulating genetic information on these and novel ones, allows for the rerouting of metabolic processes in lactic acid bacteria by physiological approaches, genetic methods, or a combination of these two. This review will discuss past and present examples and future possibilities of metabolic engineering of lactic acid bacteria for the production of important compounds, including lactic and other acids, flavor compounds, and exopolysaccharides.     

碳酸盐对谷氨酸产生菌在缺氧条件下产有机酸的影响

考察谷氛酸产生菌在缺氧条件下积累L-乳酸和琥珀酸的情况.结果表明:在缺氧条件下,嗜乙酰乙酸棒杆菌(Corynebacterium acetoacidophilum)ATCC 13870积累有机酸的浓度随菌体密度的增大而增加,其中琥珀酸和乳酸积累的最适pH分别为7.5和8.0,最高质量浓度分别为22.5和60g/L.碳酸盐是影响产酸与有机酸分布的主要因素.比较ATCC 13870在NaHC03浓度为40和400mmol/L时的代谢通量,发现后者合成琥珀酸的代谢通量比前者提高了214.1%,合成乳酸的代谢通量降低了61.8%,说明PEP节点处的代谢通量分配明显受NaHCO3浓度的影响,而PYR节点受环境因素的影响不明显.