From physiology to systems metabolic engineering for the production of biochemicals by lactic acid bacteria

The lactic acid bacteria (LAB) are a functionally related group of low-GC Gram-positive bacteria known essentially for their roles in bioprocessing of foods and animal feeds. Due to extensive industrial use and enormous economical value, LAB have been intensively studied and a large body of comprehensive data on their metabolism and genetics was generated throughout the years. This knowledge has been instrumental in the implementation of successful applications in the food industry, such as the selection of robust starter cultures with desired phenotypic traits. The advent of genomics, functional genomics and high-throughput experimentation combined with powerful computational tools currently allows for a systems level understanding of these food industry workhorses. The technological developments in the last decade have provided the foundation for the use of LAB in applications beyond the classic food fermentations. Here we discuss recent metabolic engineering strategies to improve particular cellular traits of LAB and to design LAB cell factories for the bioproduction of added value chemicals.     

应用于乳酸菌的非抗生素抗性选择标记系统

 乳酸菌是一类重要的安全型微生物,在免疫载体疫苗开发及食品菌株改良等医疗、食品领域均有广泛的应用.非抗生素抗性选择标记是乳酸菌基因工程菌株构建中必不可少的关键组成部分,也是目前乳酸菌研究的前沿和热点.根据筛选时质粒和受体菌之间的表型关系及特征,主要分为显性选择标记、互补型选择标记、显性/互补型选择标记、双质粒选择标记4大类.其中显性选择标记中的细菌素抗性/免疫性选择标记及互补型选择标记中的糖类选择标记均有较大的发展潜力及应用空间;双质粒选择标记系统构建的筛选过程新颖独特,为整个选择标记系统的发展开辟了新的途径及思路.     

Influence of temperature and backslopping time on the microbiota of a type I propagated laboratory wheat sourdough fermentation

Sourdough fermentation is a cereal fermentation that is characterized by the formation of stable yeast/lactic acid bacteria (LAB) associations. It is a unique process among food fermentations in that the LAB that mostly dominate these fermentations are heterofermentative. In the present study, four wheat sourdough fermentations were carried out under different conditions of temperature and backslopping time to determine their effect on the composition of the microbiota of the final sourdoughs. A substantial effect of temperature was observed. A fermentation with 10 backsloppings (once every 24 h) at 23°C resulted in a microbiota composed of Leuconostoc citreum as the dominant species, whereas fermentations at 30 and 37°C with backslopping every 24 h resulted in ecosystems dominated by Lactobacillus fermentum. Longer backslopping times (every 48 h at 30°C) resulted in a combination of Lactobacillus fermentum and Lactobacillus plantarum. Residual maltose remained present in all fermentations, except those with longer backslopping times, and ornithine was found in almost all fermentations, indicating enhanced sourdough-typical LAB activity. The sourdough-typical species Lactobacillus sanfranciscensis was not found. Finally, a nonflour origin for this species was hypothesized.     

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.     

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.     

Engineering Tools for the Development of Recombinant Lactic Acid Bacteria

Lactic acid bacteria (LAB) is mainly used in food fermentation. In addition, LAB fermentation technology has been studied in the development of industrial food additives, nutrients, or enzymes used in food processing. In the field of red biotechnology, LAB is approved and is generally recognized as a safe organism and is considered safe for biotherapeutic treatments. Recent clinical trials have demonstrated the medicinal value of therapeutic recombinant LAB and the suitability of innate mechanisms of secretion and anchoring for therapeutic applications such as antibody or vaccine production. However, the gram‐positive phenotypic trait of LAB creates challenges for genetic modifications when compared to other conventional workhorse bacteria, resulting in exclusive developments of genetic tools for engineering LAB. In this review, several distinct approaches in gene expression for engineering LAB are discussed.     

Lactic acid bacteria producing B-group vitamins: a great potential for functional cereals products

Wheat contains various essential nutrients including the B group of vitamins. However, B group vitamins, normally present in cereals-derived products, are easily removed or destroyed during milling, food processing or cooking. Lactic acid bacteria (LAB) are widely used as starter cultures for the fermentation of a large variety of foods and can improve the safety, shelf life, nutritional value, flavor and overall quality of the fermented products. In this regard, the identification and application of strains delivering health-promoting compounds is a fascinating field. Besides their key role in food fermentations, several LAB found in the gastrointestinal tract of humans and animals are commercially used as probiotics and possess generally recognized as safe status. LAB are usually auxotrophic for several vitamins although certain strains of LAB have the capability to synthesize water-soluble vitamins such as those included in the B group. In recent years, a number of biotechnological processes have been explored to perform a more economical and sustainable vitamin production than that obtained via chemical synthesis. This review article will briefly report the current knowledge on lactic acid bacteria synthesis of vitamins B2, B11 and B12 and the potential strategies to increase B-group vitamin content in cereals-based products, where vitamins-producing LAB have been leading to the elaboration of novel fermented functional foods. In addition, the use of genetic strategies to increase vitamin production or to create novel vitamin-producing strains will be also discussed.     

Ecology of lactic acid bacteria in Italian fermented sausages: isolation, identification and molecular characterization

In this study, the ecology of the lactic acid bacteria (LAB) of three naturally fermented sausages produced in the Friuli-Venezia-Giulia region, in the North East of Italy, was investigated. A total of 465 strains isolated from three fermentations were identified by molecular methods and 12 different species of LAB were detected. Lactobacillus curvatus and Lactobacillus sakei were the most numerous (67 and 353 strains isolated, respectively) and they were subjected to RAPD-PCR. Clusters containing strains isolated from different plants were observed, underlining a coherent population distribution in three different fermentations. However, we also observed clusters formed by strains isolated from a specific fermentation, only. This could be explained considering the different technologies and recipes used for the production in three plants. Ingredient composition, fermentation and maturation parameters could play an important role in the selection of specific populations adapted in a specific environment.     

Antagonistic activities of lactic acid bacteria in food and feed fermentations

Abstract Many factors contribute to a successful natural fermentation of carbohydrate-rich food and feed products. Metabolic activities of lactic acid bacteria (LAB) play a leading role. Their ability to rapidly produce copious amounts of acidic end products with a concomitant pH reduction is the major factor in these fermentations. Although their specific effects are difficult to quantitate, other LAB metabolic products such as hydrogen peroxide and diacetyl can also contribute to the overall antibiosis and preservative potential of these products. The contribution of bacteriocins is also difficult to evaluate. It is suggested that they may play a role in selecting the microflora which initiates the fermentation. Bacteriocins are believed to be important in the ability of LAB to compete in non-fermentative ecosystems such as the gastro-intestinal tract. During the past few decades interest has arisen in the use of the varied antagonistic activities of LAB to extent the shelf-life of protein-rich products such as meats and fish. Recent findings indicate that the newly discovered Lactobacillus reuteri reuterin system may be used for this purpose.     

Antagonistic activities of lactic acid bacteria in food and feed fermentations

Many factors contribute to a successful natural fermentation of carbohydrate-rich food and feed products. Metabolic activities of lactic acid bacteria (LAB) play a leading role. Their ability to rapidly produce copious amounts of acidic end products with a concomitant pH reduction is the major factor in these fermentations. Although their specific effects are difficult to quantitate, other LAB metabolic products such as hydrogen peroxide and diacetyl can also contribute to the overall antibiosis and preservative potential of these products. The contribution of bacteriocins is also difficult to evaluate. It is suggested that they may play a role in selecting the microflora which initiates the fermentation. Bacteriocins are believed to be important in the ability of LAB to compete in non-fermentative ecosystems such as the gastrointestinal tract. During the past few decades interest has arisen in the use of the varied antagonistic activities of LAB to extend the shelf-life of protein-rich products such as meats and fish. Recent findings indicate that the newly discovered Lactobacillus reuteri reuterin system may be used for this purpose.     

Cloning vectors based on cryptic plasmids isolated from lactic acid bacteria: their characteristics and potential applications in biotechnology

Lactic acid bacteria (LAB) are Gram positive bacteria, widely distributed in nature, and industrially important as they are used in a variety of industrial food fermentations. The use of genetic engineering techniques is an effective means of enhancing the industrial applicability of LAB. However, when using genetic engineering technology, safety becomes an essential factor for the application of improved LAB to the food industry. Cloning and expression systems should be derived preferably from LAB cryptic plasmids that generally encode genes for which functions can be proposed, but no phenotypes can be observed. However, some plasmid-encoded functions have been discovered in cryptic plasmids originating from Lactobacillus, Streptococcus thermophilus, and Pediococcus spp. and can be used as selective marker systems in vector construction. This article presents information concerning LAB cryptic plasmids, and their structures, functions, and applications. A total of 134 cryptic plasmids collated are discussed.     

乳酸菌食品级基因表达系统

酸菌是一类重要工业菌株。最近,乳酸菌遗传学和分子生物学的研究取得长足进步,导致发展了乳酸菌食品级基因表达系统。通过介绍乳酸菌食品级基因表达系统的基本要求、食品级选择性标记、食品级诱导物及该系统的研究进展,展示了乳酸菌食品级基因表达系统的建立对研究乳酸菌的基因表达调控和它的深层次的开发利用所具有的重要意义。     

Recent update on lactic acid bacteria producing riboflavin and folates: application for food fortification and treatment of intestinal inflammation

Lactic acid bacteria (LAB), widely used as starter cultures for the fermentation of a large variety of food, can improve the safety, shelf life, nutritional value and overall quality of the fermented products. In this regard, the selection of strains delivering health-promoting compounds is now the main objective of many researchers. Although most LAB are auxotrophic for several vitamins, it is known that certain strains have the capability to synthesize B-group vitamins. This is an important property since humans cannot synthesize most vitamins, and these could be obtained by consuming LAB fermented foods. This review discusses the use of LAB as an alternative to fortification by the chemical synthesis to increase riboflavin and folate concentrations in food. Moreover, it provides an overview of the recent applications of vitamin-producing LAB with anti-inflammatory/antioxidant activities against gastrointestinal tract inflammation. This review shows the potential uses of riboflavin and folates producing LAB for the biofortification of food, as therapeutics against intestinal pathologies and to complement anti-inflammatory/anti-neoplastic treatments.     

Bacteriocinogenic LAB Strains for Fermented Meat Preservation: Perspectives,Challenges, and Limitations

Over the last decades, much research has focused on lactic acid bacteria (LAB) bacteriocins because of their potential as biopreservatives and their action against the growth of spoilage microbes. Meat and fermented meat products are prone to microbial contamination, causing health risks, as well as economic losses in the meat industry. The use of bacteriocin-producing LAB starter or protective cultures is suitable for fermented meats. However, although bacteriocins can be produced during meat processing, their levels are usually much lower than those achieved during in vitro fermentations under optimal environmental conditions. Thus, the direct addition of a bacteriocin food additive would be desirable. Moreover, safety and technological characteristics of the bacteriocinogenic LAB must be considered before their widespread applications. This review describes the perspectives and challenges toward the complete disclosure of new bacteriocins as effective preservatives in the production of safe and “healthy” fermented meat products.     

Engineering metabolic highways in Lactococci and other lactic acid bacteria

Lactic acid bacteria (LAB) are widely used in industrial food fermentations and are receiving increased attention for use as cell factories for the production of food and pharmaceutical products. Glycolytic conversion of sugars into lactic acid is the main metabolic highway in these Gram-positive bacteria and Lactococcus lactis has become the model organism because of its small genome, genetic accessibility and simple metabolism. Here we discuss the metabolic engineering of L. lactis and the value of metabolic models compared with other LAB, with a particular focus on the food-grade production of metabolites involved in flavour, texture and health.     

Multi-Loci Sequence Typing (MLST) for Two Lacto-Acid Bacteria (LAB) Species: <Emphasis Type="Italic">Pediococcus parvulus</Emphasis> and <Emphasis Type="Italic">P. damnosus</Emphasis>

The control of wine microbial population during and beyond fermentation is of huge importance for wine quality. Lactic acid bacteria (LAB) in wine are responsible for malolactic fermentation (MLF) which can be desired in some cases and undesirable in others. Some LAB do not perform MLF and their uncontrolled growth could contribute to severe wine spoilage such as undesired flavours. Their identification and detection is considered crucial for numerous biotechnological applications in food fermentations, where, through acidification and secretion of bacteriocins, they contribute to reduce food spoilage and growth of pathogenic microorganisms. LAB have traditionally been classified using morphological or biochemical features. Primary isolation, biochemical identification and phenotypic analysis are laborious, time consuming and inaccurate and often lead to misidentification within some genera such as Pediococcus. Molecular identification based on suitable marker genes could be an attractive alternative to conventional morphological and biochemical methods. We assessed here the applicability of four housekeeping genes recA, rplB, pyrG and leuS in combination with the mle gene in multi-loci sequence typing (MLST) of Pediococcus parvulus and Pediococcus damnosus. Sequencing and comparative analysis of sequence data were performed on 19 strains collected during wine fermentation. A combination of these five marker genes allowed for a clear differentiation of the strains analysed, indicating their applicability in molecular typing. Analysis of the observed nucleotide polymorphisms allowed designing highly discriminative primers for a multi-loci sequence typing (MLST) method that proved successful in detecting a particular isolate or sequence type of P. parvulus when using either conventional PCR or Real Time PCR.     

Bacteriophage ecology in commercial sauerkraut fermentations

Knowledge of bacteriophage ecology in vegetable fermentations is essential for developing phage control strategies for consistent and high quality of fermented vegetable products. The ecology of phages infecting lactic acid bacteria (LAB) in commercial sauerkraut fermentations was investigated. Brine samples were taken from four commercial sauerkraut fermentation tanks over a 60- or 100-day period in 2000 and 2001. A total of 171 phage isolates, including at least 26 distinct phages, were obtained. In addition, 28 distinct host strains were isolated and identified as LAB by restriction analysis of the intergenic transcribed spacer region and 16S rRNA sequence analysis. These host strains included Leuconostoc, Weissella, and Lactobacillus species. It was found that there were two phage-host systems in the fermentations corresponding to the population shift from heterofermentative to homofermentative LAB between 3 and 7 days after the start of the fermentations. The data suggested that phages may play an important role in the microbial ecology and succession of LAB species in vegetable fermentations. Eight phage isolates, which were independently obtained two or more times, were further characterized. They belonged to the family Myoviridae or Siphoviridae and showed distinct host ranges and DNA fingerprints. Two of the phage isolates were found to be capable of infecting two Lactobacillus species. The results from this study demonstrated for the first time the complex phage ecology present in commercial sauerkraut fermentations, providing new insights into the bioprocess of vegetable fermentations.     

Growth of yeast contaminants in an immobilized lactic acid bacteria system

Calcium alginate-immobilized lactic acid bacteria (LAB) were used to acidify milk and re-utilized for five successive fermentations. The yeast Kluyveromyces marxianus was added to the milk in order to simulate contamination of the bioreactor. Growth of the yeast was studied over the successive re-utilizations of the immobilized LAB. When the system was contaminated only in the first fermentation, the yeast population decreased in the successive re-utilizations; rinsing of the system following each fermentation was effective in further reducing yeast contamination. Yeast population increased only when contaminants were added at the beginning of each fermentation. Presence of yeast in the system did not influence acidification rate of the immobilized LAB or cell release from the alginate beads.     

Next-generation sequencing reveals significant bacterial diversity of botrytized wine

While wine fermentation has long been known to involve complex microbial communities, the composition and role of bacteria other than a select set of lactic acid bacteria (LAB) has often been assumed either negligible or detrimental. This study served as a pilot study for using barcoded amplicon next-generation sequencing to profile bacterial community structure in wines and grape musts, comparing the taxonomic depth achieved by sequencing two different domains of prokaryotic 16S rDNA (V4 and V5). This study was designed to serve two goals: 1) to empirically determine the most taxonomically informative 16S rDNA target region for barcoded amplicon sequencing of wine, comparing V4 and V5 domains of bacterial 16S rDNA to terminal restriction fragment length polymorphism (TRFLP) of LAB communities; and 2) to explore the bacterial communities of wine fermentation to better understand the biodiversity of wine at a depth previously unattainable using other techniques. Analysis of amplicons from the V4 and V5 provided similar views of the bacterial communities of botrytized wine fermentations, revealing a broad diversity of low-abundance taxa not traditionally associated with wine, as well as atypical LAB communities initially detected by TRFLP. The V4 domain was determined as the more suitable read for wine ecology studies, as it provided greater taxonomic depth for profiling LAB communities. In addition, targeted enrichment was used to isolate two species of Alphaproteobacteria from a finished fermentation. Significant differences in diversity between inoculated and uninoculated samples suggest that Saccharomyces inoculation exerts selective pressure on bacterial diversity in these fermentations, most notably suppressing abundance of acetic acid bacteria. These results determine the bacterial diversity of botrytized wines to be far higher than previously realized, providing further insight into the fermentation dynamics of these wines, and demonstrate the utility of next-generation sequencing for wine ecology studies.     

Cloning Vectors Based on Cryptic Plasmids Isolated from Lactic Acid Bacteria:Their Characteristics and Potential Applications in Biotechnology

Lactic acid bacteria (LAB) are Gram positive bacteria, widely distributed in nature, and industrially important as they are used in a variety of industrial food fermentations. The use of genetic engineering techniques is an effective means of enhancing the industrial applicability of LAB. However, when using genetic engineering technology, safety becomes an essential factor for the application of improved LAB to the food industry. Cloning and expression systems should be derived preferably from LAB cryptic plasmids that generally encode genes for which functions can be proposed, but no phenotypes can be observed. However, some plasmid-encoded functions have been discovered in cryptic plasmids originating from Lactobacillus, Streptococcus thermophilus, and Pediococcus spp. and can be used as selective marker systems in vector construction. This article presents information concerning LAB cryptic plasmids, and their structures, functions, and applications. A total of 134 cryptic plasmids collated are discussed.