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.     

Progress and potential in the biotechnology of lactic acid bacteria

Abstract: Current activities and future prospects for the biotechnology of lactic acid bacteria are reviewed. Genetic engineering technology, including advances and limitations of plasmid vectors and chromosomal integration strategies are discussed together with the status of gene expression and the importance of in vivo gene transfer systems and transposition. Areas of biotechnological application considered include proteolysis and flavour generation, bacteriophage resistance, antimicrobials, metabolic engineering and the possible uses of lactic acid bacteria in relation to health.     

Food-grade gene expression in lactic acid bacteria

In the 1990s, significant efforts were invested in the research and development of food-grade expression systems in lactic acid bacteria (LAB). At this time, Lactococcus lactis in particular was demonstrated to be an ideal cell factory for the food-grade production of recombinant proteins. Steady progress has since been made in research on LAB, including Lactococcus, Lactobacillus and Streptococcus, in the areas of recombinant enzyme production, industrial food fermentation, and gene and metabolic pathway regulation. Over the past decade, this work has also led to new approaches on chromosomal integration vectors and host/vector systems. These newly constructed food-grade gene expression systems were designed with specific attention to self-cloning strategies, food-grade selection markers, plasmid replication and chromosomal gene replacements. In this review, we discuss some well-characterized chromosomal integration and food-grade host/vector systems used in LAB, with a special focus on sustainability, stability and overall safety, and give some attractive examples of protein expression that are based on these systems.     

Potential of bacteriocin-producing lactic acid bacteria for improvements in food safety and quality

Lactic acid bacteria (LAB) have been used for centuries in the fermentation of a variety of dairy products. The preservative ability of LAB in foods is attributed to the production of anti-microbial metabolites including organic acids and bacteriocins. Bacteriocins generally exert their anti-microbial action by interfering with the cell wall or the membrane of target organisms, either by inhibiting cell wall biosynthesis or causing pore formation, subsequently resulting in death. The incorporation of bacteriocins as a biopreservative ingredient into model food systems has been studied extensively and has been shown to be effective in the control of pathogenic and spoilage microorganisms. However, a more practical and economic option of incorporating bacteriocins into foods can be the direct addition of bacteriocin-producing cultures into food. This paper presents an overview of the potential for using bacteriocin-producing LAB in foods for the improvement of the safety and quality of the final product. It describes the different genera of LAB with potential as biopreservatives, and presents an up-to-date classification system for the bacteriocins they produce. While the problems associated with the use of some bacteriocin-producing cultures in certain foods are elucidated, so also are the situations in which incorporation of the bacteriocin-producer into model food systems have been shown to be very effective.     

Drug delivery in biotechnology: present and future

Drug delivery is becoming a whole interdisciplinary and independent field of research and is gaining the attention of pharmaceutical makers, medical doctors and industry. A targeted and safe drug delivery could improve the performance of some classical medicines already on the market and, moreover, will have implications for the development and success of new therapeutic strategies, such as peptide and protein delivery, glycoprotein administration, gene therapy and RNA interference. Many innovative technologies for effective drug delivery have been developed, including implants, nanotechnology, cell and peptide encapsulation, microfabrication, chemical modification and others. On the long way from the clinic to market, however, several issues will have to be addressed, including suitable scientific development, specific financial support as a result of altered scientific policy, government regulations and market forces.     

乳酸菌对微囊藻毒素的清除

【目的】研究唾液乳杆菌(Lactobacillus salivarius BBE09-18)、发酵乳杆菌(L.fermentum BBE09-29)以及干酪乳杆菌(L.casei Zhang)对藻毒素的清除能力以及影响乳酸菌清除藻毒素的主要因素,以期为进一步解析乳酸菌清除藻毒素的作用机制提供理论基础,并为去除食品体系中的藻毒素提供新的思路。【方法】研究乳酸菌细胞的不同生理状态(活细胞与死细胞)对藻毒素清除能力的影响,考察菌浓差异、起始藻毒素浓度差异、葡萄糖的供给等对乳酸菌清除藻毒素效能的影响。【结果】3株实验乳酸菌均具有清除藻毒素的能力,其中,L.casei Zhang的藻毒素清除能力最强,当藻毒素初始浓度为150μg/L时,24h后残留藻毒素浓度为85.5μg/L,清除率可达43%。此外,研究还发现乳酸菌活细胞的清除能力显著高于热失活后的死细胞。添加外源物质葡萄糖能显著提高实验菌株清除藻毒素的效率,在初始浓度为1800μg/L的藻毒素溶液中,当添加5%(w/v)葡萄糖后,L.casei Zhang经24h可清除92%的藻毒素。【结论】3株乳酸菌均具有藻毒素清除能力;同时,菌体浓度以及菌体自身的生理状态对藻毒素的清除效率具有重要影响,乳酸菌对藻毒素的清除可能与菌体的代谢活性相关。     

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.     

Genotypic diversity of lactic acid bacteria isolated from African traditional alkaline‐fermented foods

Aim: To identify and compare lactic acid bacteria (LAB) isolated from alkaline fermentations of cassava (Manihot esculenta Crantz) leaves, roselle (Hibiscus sabdariffa) and African locust bean (Parkia biglobosa) seeds for production of, respectively, Ntoba Mbodi, Bikalga and Soumbala. Methods and Results: A total of 121 LAB were isolated, identified and compared by phenotyping and genotyping using PCR amplification of 16S–23S rDNA intergenic transcribed spacer (ITS‐PCR), repetitive sequence‐based PCR (rep‐PCR) and DNA sequencing. The results revealed a diversity of genera, species and subspecies of LAB in African alkaline fermentations. The isolates were characterized as nonmotile (in most cases) Gram‐positive rods, cocci or coccobacilli, catalase and oxidase negative. ITS‐PCR allowed typing mainly at species level, with differentiation of a few bacteria at subspecies level. Rep‐PCR permitted typing at subspecies level and revealed significant genotypic differences between the same species of bacteria from different raw materials. DNA sequencing combined with use of API 50CHL and API 20Strep systems allowed identification of bacteria as Weissella confusa, Weissella cibaria, Lactobacillus plantarum, Pediococcus pentosaceus, Enterococcus casseliflavus, Enterococcus faecium, Enterococcus faecalis, Enterococcus avium and Enterococcus hirae from Ntoba Mbodi; Ent. faecium, Ent. hirae and Pediococcus acidilactici from Bikalga and Soumbala. Conclusion: LAB found in African alkaline‐fermented foods belong to a range of genera, species and subspecies of bacteria and vary considerably according to raw material. Significance and Impact of the Study: Our study confirms that LAB survive in alkaline fermentations, a first crucial stage in determining their significance and possible value as probiotic bacteria.     

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.     

Plastid biotechnology for crop production: present status and future perspectives

The world population is expected to reach an estimated 9.2 billion by 2050. Therefore, food production globally has to increase by 70% in order to feed the world, while total arable land, which has reached its maximal utilization, may even decrease. Moreover, climate change adds yet another challenge to global food security. In order to feed the world in 2050, biotechnological advances in modern agriculture are essential. Plant genetic engineering, which has created a new wave of global crop production after the first green revolution, will continue to play an important role in modern agriculture to meet these challenges. Plastid genetic engineering, with several unique advantages including transgene containment, has made significant progress in the last two decades in various biotechnology applications including development of crops with high levels of resistance to insects, bacterial, fungal and viral diseases, different types of herbicides, drought, salt and cold tolerance, cytoplasmic male sterility, metabolic engineering, phytoremediation of toxic metals and production of many vaccine antigens, biopharmaceuticals and biofuels. However, useful traits should be engineered via chloroplast genomes of several major crops. This review provides insight into the current state of the art of plastid engineering in relation to agricultural production, especially for engineering agronomic traits. Understanding the bottleneck of this technology and challenges for improvement of major crops in a changing climate are discussed.     

Looking backward and forward at the practical applications of genetic researches on lactic acid bacteria

Abstract Background research findings which have led to current knowledge on the genetics of lactic acid bacteria are reviewed. Important early contributions by researchers with other microorganisms are highlighted to acknowledge milestone discoveries, especially those which have made genetic engineering of industrially important genera a reality. Current genetics in the dairy Streptococcus , Lactobacillus , Pediococcus , Leuconostoc , Bifidobacterium and Propionibacterium genera are discussed. Regulatory, environmental, and research funding concerns also are discussed.     

Beneficial properties of lactic acid bacteria naturally present in dairy production

Background

Consumers are increasingly demanding for natural and beneficial foods, in order to improve their health and well-being. Probiotics play an important role in such demand, and dairy foods are commonly used as vehicles for such bacteria, represented predominantly by lactic acid bacteria. Due to consumers demand, food industry is constantly looking for novel bacterial strains, leading to studies that aims the isolation and characterization of their beneficial features. This study aimed to characterize the naturally occurring lactic acid bacteria obtained from a dairy environment, in order to assess their potential use as probiotics.

Results

Preliminary screening and PCR analysis, based on 16S rRNA sequencing, were applied to select and identify 15 LAB strains from the genera Lactobacillus (n?=?11), Pediococcus (n?=?2) and Weissella (n?=?2). All strains showed resistance to low pH and the evaluated bile salt concentrations in vitro. The API ZYM test characterized the enzymatic activity of the strains, and a high β-galactosidase activity was observed in 13 strains. All strains presented resistance to simulated gastric (3?h) and intestinal (4?h) conditions in vitro, the ability to auto- and co-aggregate with indicator microorganisms and a high cell surface hydrophobicity. Most of the strains were positive for map and EFTu beneficial genes. All strains exhibited strong deconjugation of bile salts in vitro and all assimilated lactose.

Conclusions

The phenotypes exhibited in vitro and the presence of beneficial genes revealed the beneficial potential of the studied strains, demanding further analyses in a food matrix and in vivo to allow the development of a functional product, with health-related properties.

     

Evaluation of lactic acid bacteria autolysate for the supplementation of lactic acid bacteria fermentation

At the end of culture in a carbon-limited medium, i.e. the best conditions for subsequent autolysis, lactic acid bacteria were harvested and autolysed at 50 °C for 24 h. The resulting supernatant was then successfully tested as a substitute for industrial yeast extract for the supplementation of whey permeate and its conversion into lactic acid: for almost equivalent total nitrogen amounts of both supplements, the same growth and production rates were recorded.     

Bacteriocins from lactic acid bacteria and their potential in the preservation of fruit products

Bacteriocins produced by lactic acid bacteria (LAB) are well-recognized for their potential as natural food preservatives. These antimicrobial peptides usually do not change the sensorial properties of food products and can be used in combination with traditional preservation methods to ensure microbial stability. In recent years, fruit products are increasingly being associated with food-borne pathogens and spoilage microorganisms, and bacteriocins are important candidates to preserve these products. Bacteriocins have been extensively studied to preserve foods of animal origin. However, little information is available for their use in vegetable products, especially in minimally processed ready-to-eat fruits. Although, many bacteriocins possess useful characteristics that can be used to preserve fruit products, to date, only nisin, enterocin AS-48, bovicin HC5, enterocin 416K1, pediocin and bificin C6165 have been tested for their activity against spoilage and pathogenic microorganisms in these products. Among these, only nisin and pediocin are approved to be commercially used as food additives, and their use in fruit products is still limited to certain countries. Considering the increasing demand for fresh-tasting fruit products and concern for public safety, the study of other bacteriocins with biochemical characteristics that make them candidates for the preservation of these products are of great interest. Efforts for their approval as food additives are also important. In this review, we discuss why the study of bacteriocins as an alternative method to preserve fruit products is important; we detail the biotechnological approaches for the use of bacteriocins in fruit products; and describe some bacteriocins that have been tested and have potential to be tested for the preservation of fruit products.     

Comparative studies of lactic acid dehydrogenases in lactic acid bacteria

The stability, pH-dependence and kinetic properties of the Mn²⁺ and FDP-activated NAD-dependent lactic acid dehydrogenases from Lactobacillus casei ssp. casei (ATCC 393) and L. curvatus (DSM) 20010) were studied after the enzymes were purified to homogeneity by affinity chromatography. Both enzymes are virtually unidirectional, catalysing efficiently only the reduction of pyruvate. They are similar with respect to the effector requirement and pH-optimum. They differ, however, in their electrophoretic mobility, heat stability, pH-dependence of the Mn²⁺ requirement and several kinetic properties. It is suggested that most of these differences are caused by differences of the negative charges in the vicinity of the FDP-binding site or the site responsible for the interaction of the subunits of the enzymatically active oligomeres.Abbreviations l-LDH l-Lactic acid dehydrogenase - FDP Fructose-1,6-bisphosphate - DTE Dithioerythrol AddendumIn the case of the L. casei-LDH the shape of the NADH saturation curve is not changed by omitting the effectors FDP and Mn 2⁺. The K _M under these conditions is 3 fold higher (10.10 ^–5 M).     

乳酸菌的基因组编辑技术研究进展

乳球菌(Lactococcussp.)和乳杆菌(Lactobacillussp.)是工业上常用的乳酸菌(lacticacid bacteria,LAB),长期应用于食品和饮料的发酵。近年来,随着分子操作及遗传改造技术的不断完善,推动了乳球菌和乳杆菌的基础和应用研究,其作为功能菌株和工业微生物细胞工厂的重要潜能也不断突显出来。本文综述了工业常用乳酸菌的基因组编辑技术研究进展,着重介绍了基于整合质粒的敲除、敲入,基于基因组重组工程的精细修饰和敲除、敲入,以及基于成簇的规律性间隔的短回文重复序列及其相关蛋白9[(clusteredregularlyinterspacedshortpalindromicrepeats(CRISPR)/CRISPR-associated nuclease9 (Cas9), CRISPR/Cas9]系统的基因组编辑技术。     

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.     

Searching for bacteriocin-producing lactic acid bacteria in soil

A survey was conducted on the isolation and characterization of bacteriocin-producing lactic acid bacteria in soil. Forty-two acid-producing bacterial strains were isolated from 55 soil samples collected in Yamanashi prefecture, Japan. Investigation of antibacterial activities of isolates revealed that three isolates, Lactobacillus animalis C060203, Enterococcus durans C102901 and Leuconostoc mesenteroides subsp. mesenteroides C060204, showed antibacterial activities against the indicator strain of Lactobacillus sakei JCM 1157T. Bacteriocin from Enterococcus durans C102901 showed different characteristics from the known durancin L28-1A, produced by Enterococcus durans L28-1. Furthermore, this is the first report of a bacteriocin being produced by Lactobacillus animalis. Viewing from the species, bacteriocins from strains C102901 and C060203 showed high possibilities for the novel substances. These significant findings suggest that soil may be a common source for the isolation of novel bacteriocin-producing lactic acid bacteria.