New trends and opportunities in the development and use of inoculants for silage

Abstract: Inoculants are used as silage additives to improve preservation efficiency and to enhance animal performance. In most commercially available inoculants, homofermentative lactic acid bacteria (LAB) have been used because they are fast and efficient producers of lactic acid, improving natural silage fermentation. Specific LAB inuculants may also have beneficial effects on animal performance even if there is no effect on fermentation. However, these types of inoculants are not always advantageous. They do not necessarily prevent sermentation by clostridia in moist silages, and they sometimes impair the aerobic stability of grass and small grain silages. Therefore, new criteria for silage inoculants should be established which consider the specific needs of the crop being ensiled. New approaches which are being taken to develop improved inoculants for silage include the following: (1) using LAB isolates which are more specific to the target crops; (2) inclusion of heterofermentative LAB to produce volatile fatty acids to inhibit yeasts and moulds upon aerobic exposure; (3) inclusion of organisms other than LAB in inoculants to inhibit detrimental microorganisms; (4) selection or engineering of LAB strains to inhibit specific microorganisms; and (5) cloning and expression of genes which would enable selected LAB strains to utilize polysaccharides in crops which are low in soluble carbohydrates. Many of these new strategies for formulating inoculants are being tested, but further research is needed to determine the most successful approaches.     

Proteolytic systems of lactic acid bacteria

Lactic acid bacteria (LAB) have a very long history of use in the manufacturing processes of fermented foods and a great deal of effort was made to investigate and manipulate the role of LAB in these processes. Today, the diverse group of LAB includes species that are among the best-studied microorganisms and proteolysis is one of the particular physiological traits of LAB of which detailed knowledge was obtained. The proteolytic system involved in casein utilization provides cells with essential amino acids during growth in milk and is also of industrial importance due to its contribution to the development of the organoleptic properties of fermented milk products. For the most extensively studied LAB, Lactococcus lactis, a model for casein proteolysis, transport, peptidolysis, and regulation thereof is now established. In addition to nutrient processing, cellular proteolysis plays a critical role in polypeptide quality control and in many regulatory circuits by keeping basal levels of regulatory proteins low and removing them when they are no longer needed. As part of the industrial processes, LAB are challenged by various stress conditions that are likely to affect metabolic activities, including proteolysis. While environmental stress responses of LAB have received increasing interest in recent years, our current knowledge on stress-related proteolysis in LAB is almost exclusively based on studies on L. lactis. This review provides the current status in the research of proteolytic systems of LAB with industrial relevance.     

细菌素的合成与作用机制

细菌素是由细菌产生的抗菌蛋白,可以杀死与产生菌相近的细菌。很多乳酸菌产生不同多样性的细菌素,虽然这些细菌素都是由发酵或非发酵食品中发现的乳酸菌产生的,但是迄今只有乳酸链球菌素(Nisin)作为食品防腐剂被广泛应用。和抗生素不同的是,细菌素由核糖体合成,需经翻译后修饰活化并且通过特定转运系统输到胞外才能发挥其功能,它一般通过作用于靶细胞膜来抑制靶细胞的生长,同时本身合成细菌素的细胞对其产物具有免疫性。细菌素能安全有效地抑制病原体生长,在食品行业中具有广阔的应用前景。     

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.     

Perspectives of engineering lactic acid bacteria for biotechnological polyol production

Polyols are sugar alcohols largely used as sweeteners and they are claimed to have several health-promoting effects (low-caloric, low-glycemic, low-insulinemic, anticariogenic, and prebiotic). While at present chemical synthesis is the only strategy able to assure the polyol market demand, the biotechnological production of polyols has been implemented in yeasts, fungi, and bacteria. Lactic acid bacteria (LAB) are a group of microorganisms particularly suited for polyol production as they display a fermentative metabolism associated with an important redox modulation and a limited biosynthetic capacity. In addition, LAB participate in food fermentation processes, where in situ production of polyols during fermentation may be useful in the development of novel functional foods. Here, we review the polyol production by LAB, focusing on metabolic engineering strategies aimed to redirect sugar fermentation pathways towards the synthesis of biotechnologically important sugar alcohols such as sorbitol, mannitol, and xylitol. Furthermore, possible approaches are presented for engineering new fermentation routes in LAB for production of arabitol, ribitol, and erythritol.     

Immunobiosis and probiosis: antimicrobial activity of lactic acid bacteria with a focus on their antiviral and antifungal properties

Lactic acid bacteria (LAB), a heterogeneous group of bacteria that produce lactic acid as the main product of carbohydrate degradation, play an important role in the production and protection of fermented foods. Moreover, beside the technological use of these microorganisms added to control and steer food fermentations, their beneficial healthy properties are largely overt. Thus, numerous LAB strains have obtained the probiotic status, which entails the ability to maintain and promote a good health of consumers. In particular, increasing consideration is being focused on probiotic microorganisms that can improve the human immune response against dangerous viral and fungal enemies. For such beneficial microbes, the term “immunobiotics” has been coined. Together with an indirect host-mediated adverse effect against undesirable microorganisms, also a direct antagonistic activity of several LAB strains has been largely demonstrated. The purpose of this review is to provide a fullest possible overview of the antiviral and antifungal activities ascribed to probiotic LAB. The interest in this research field is substantiated by a large number of studies exploring the potential application of these beneficial microorganisms both as biopreservatives and immune-enhancers, aiming to reduce and/or eliminate the use of chemical agents to prevent the development of pathogenic, infectious, and/or degrading causes.

     

Safety of industrial lactic acid bacteria.

Lactic acid bacteria (LAB) are ubiquitous in fermented and non-fermented foods and are common components of the human commensal microflora. This long history of human exposure and consumption has led to the reasonable conclusion that they are generally safe. Recent attention has also focused on their possible role as probiotic bacteria, promoting beneficial health effects. There have, however, been a number of reports of human infections caused by LAB and these are reviewed. In most cases, the source of the infection was the commensal LAB flora rather than ingested bacteria and the patient had some underlying disease or predisposing condition. Even as opportunistic pathogens, the LAB, with the notable exception of the enterococci, are much less successful than a number of other members of the commensal microflora. The use of new strains for probiotic use is likely to require more detailed evidence for their safety, particularly if the strains have been genetically modified or have been derived from animals. Procedures that have been proposed for assessing the safety of new strains are described.     

Lactic acid bacteria biofilms and their ability to mitigate Escherichia coli O157:H7 surface colonization

Lactic acid bacteria (LAB) exert antagonistic activities against diverse microorganisms, including pathogens. In this work, we aimed to investigate the ability of LAB strains isolated from food to produce biofilms and to inhibit growth and surface colonization of Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 at 10°C. The ability of 100 isolated LAB to inhibit EHEC O157:H7 NCTC12900 growth was evaluated in agar diffusion assays. Thirty-seven LAB strains showed strong growth inhibitory effect on EHEC. The highest inhibitory activities corresponded to LAB strains belonging to Lactiplantibacillus plantarum, Pediococcus acidilactici and Pediococcus pentosaceus species. Eighteen out of the 37 strains that showed growth inhibitory effects on EHEC also had the ability to form biofilms on polystyrene surfaces at 10°C and 30°C. Pre-established biofilms on polystyrene of four of these LAB strains were able to reduce significantly surface colonization by EHEC at low temperature (10°C). Among these four strains, Lact. plantarum CRL 1075 not only inhibited EHEC but also was able to grow in the presence of the enteric pathogen. Therefore, this strain proved to be a good candidate for further technological studies oriented to its application in food-processing environments to mitigate undesirable surface contaminations of E. coli.     

Evaluation of Lactic Acid Bacteria Isolated from Fermented Plant Products for Antagonistic Activity Against Urinary Tract Pathogen <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis>

Urinary tract infections (UTIs) are the most common infectious diseases in infants and the elderly; they are also the most common among nosocomial infections. The treatment of UTIs usually involves a short-term course of antibiotics. The purpose of this study was to identify the strains of lactic acid bacteria (LAB) that can inhibit the urinary tract pathogen Staphylococcus saprophyticus, as alternatives to antibiotics. In this study, we collected 370 LAB strains from fermented plant products and reference strains from the Bioresources Collection and Research Center (BCRC). Using spent culture supernatants (SCS), we then screened these LAB strains with for antimicrobial effects on urinary tract pathogens by the well-diffusion assay. Seven LAB strains—PM2, PM68, PM78, PM201, PM206, PM229, and RY2—exhibited inhibitory activity and were evaluated for anti-growth activity against urinary tract pathogens by the co-culture inhibition assay. Anti-adhesion and anti-invasion activities against urinary tract pathogens were evaluated using the SV-HUC-1 urothelial cell cultures. The results revealed that the survival rate of S. saprophyticus ranged from 0.9–2.96%, with the pH continuously decreasing after co-culture with LAB strains for 4 h. In the competitive adhesion assay, the exclusion and competition groups performed better than the displacement group. In the SV-HUC-1 cell invasion assay, PM201, PM206, PM229, and RY2 were found to inhibit the invasion of SV-HUC-1 cells by S. saprophyticus BCRC 10786. To conclude, RY2, PM229, and PM68 strains exhibited inhibitory activity against the urinary tract pathogen S. saprophyticus.     

Recent investigations and updated criteria for the assessment of antibiotic resistance in food lactic acid bacteria

The worldwide use, and misuse, of antibiotics for about sixty years in the so-called antibiotic era, has been estimated in some one to ten million tons, a relevant part of which destined for non-therapeutic purposes such as growth promoting treatments for livestock or crop protection. As highly adaptable organisms, bacteria have reacted to this dramatic change in their environment by developing several well-known mechanisms of antibiotic resistance and are becoming increasingly resistant to conventional antibiotics. In recent years, commensal bacteria have become a cause of concern since they may act as reservoirs for the antibiotic resistance genes found in human pathogens. In particular, the food chain has been considered the main route for the introduction of animal and environment associated antibiotic resistant bacteria into the human gastrointestinal tract (GIT) where these genes may be transferred to pathogenic and opportunistic bacteria. As fundamental microbial communities in a large variety of fermented foods and feed, the anaerobe facultative, aerotolerant lactic acid bacteria (LAB) are likely to play a pivotal role in the resistance gene exchange occurring in the environment, food, feed and animal and human GIT. Therefore their antibiotic resistance features and their genetic basis have recently received increasing attention. The present article summarises the results of the latest studies on the most typical genera belonging to the low G + C branch of LAB. The evolution of the criteria established by European regulatory bodies to ensure a safe use of microorganisms in food and feed, including the assessment of their antibiotic resistance is also reviewed.     

Importance of lactic acid bacteria in Asian fermented foods

Lactic acid bacteria play important roles in various fermented foods in Asia. Besides being the main component in kimchi and other fermented foods, they are used to preserve edible food materials through fermentation of other raw-materials such as rice wine/beer, rice cakes, and fish by producing organic acids to control putrefactive microorganisms and pathogens. These bacteria also provide a selective environment favoring fermentative microorganisms and produce desirable flavors in various fermented foods. This paper discusses the role of lactic acid bacteria in various non-dairy fermented food products in Asia and their nutritional and physiological functions in the Asian diet.     

应用乳酸菌生物保护剂控制肉制品中单增李斯特菌的研究进展

肉制品营养丰富,极易被微生物污染,单增李斯特菌是污染肉制品主要病原菌之一。乳酸菌做为生物保护剂已经被广泛应用于食品中控制单增李斯特菌。本文首先分析了我国肉制品中单增李斯特菌的污染状况,总结了乳酸菌应用于肉制品安全控制的概况;然后进一步详细介绍了乳酸菌对单增李斯特菌的抑菌机理,着重探讨了乳酸菌对单增李斯特菌致病能力(生长、抗性和毒性)的影响;文章最后指出了乳酸菌在食品应用中存在的问题,并对未来的研究方向提供了建议,以期为乳酸菌在食品安全控制中的应用提供参考。     

Interaction of lactic acid bacteria with metal ions: opportunities for improving food safety and quality

Certain species of lactic acid bacteria (LAB), as well as other microorganisms, can bind metal ions to their cells surface or transport and store them inside the cell. Due to this fact, over the past few years interactions of metal ions with LAB have been intensively investigated in order to develop the usage of these bacteria in new biotechnology processes in addition to their health and probiotic aspects. Preliminary studies in model aqueous solutions yielded LAB with high absorption potential for toxic and essential metal ions, which can be used for improving food safety and quality. This paper provides an overview of results obtained by LAB application in toxic metal ions removing from drinking water, food and human body, as well as production of functional foods and nutraceutics. The biosorption abilities of LAB towards metal ions are emphasized. The binding mechanisms, as well as the parameters influencing the passive and active uptake are analyzed.     

Lactococci and lactobacilli as mucosal delivery vectors for therapeutic proteins and DNA vaccines

Food-grade Lactic Acid Bacteria (LAB) have been safely consumed for centuries by humans in fermented foods. Thus, they are good candidates to develop novel oral vectors, constituting attractive alternatives to attenuated pathogens, for mucosal delivery strategies. Herein, this review summarizes our research, up until now, on the use of LAB as mucosal delivery vectors for therapeutic proteins and DNA vaccines. Most of our work has been based on the model LAB Lactococcus lactis, for which we have developed efficient genetic tools, including expression signals and host strains, for the heterologous expression of therapeutic proteins such as antigens, cytokines and enzymes. Resulting recombinant lactococci strains have been tested successfully for their prophylactic and therapeutic effects in different animal models: i) against human papillomavirus type 16 (HPV-16)-induced tumors in mice, ii) to partially prevent a bovine β-lactoglobulin (BLG)-allergic reaction in mice and iii) to regulate body weight and food consumption in obese mice. Strikingly, all of these tools have been successfully transposed to the Lactobacillus genus, in recent years, within our laboratory. Notably, anti-oxidative Lactobacillus casei strains were constructed and tested in two chemically-induced colitis models. In parallel, we also developed a strategy based on the use of L. lactis to deliver DNA at the mucosal level, and were able to show that L. lactis is able to modulate the host response through DNA delivery. Today, we consider that all of our consistent data, together with those obtained by other groups, demonstrate and reinforce the interest of using LAB, particularly lactococci and lactobacilli strains, to develop novel therapeutic protein mucosal delivery vectors which should be tested now in human clinical trials.     

乳酸菌基因组学研究进展

伴随着高通量测序技术的快速发展和测序成本的降低,越来越多的微生物基因组全序列测定得以实现,从基因组学的层面了解乳酸菌的遗传结构和组成,进而分析和掌握其生物学功能已经逐渐成为可能.迄今为止已经有22株乳酸菌的基因组完成测序并公开发表,还有至少12株测序工作仍在进行中.本文在分析相关文献和生物信息数据基础上,从乳酸菌基因组特点、代谢多样性、进化及共线性四个方面对乳酸菌基因组学研究进展进行了总结,旨在为乳酸菌研究和应用提供参考.     

乳酸菌食品级nisin控制的基因表达系统NICE

乳酸菌安全应用于人们的生产和生活已有上千年的历史,是一种食品级的微生物。在过去二十年里,其生理及遗传学特性已被彻底研究。由于其遗传可行且操作简单,乳酸菌除了其传统应用外已被广泛用于表达异源基因,在食品、农业及医药工程领域具有重要的应用前景。人们已开发了一系列乳酸菌食品级基因表达系统。本文主要介绍了乳酸菌,重点是其模式菌Lactococcus lactis最常见的食品级诱导表达系统--nisin控制的基因表达系统NIC E及其食品级诱导物nisin、食品级的宿主及表达载体系统,以及NICE系统在表达异源基因方面的应用。     

Use of mixed cultures for the fermentation of cereal-based substrates with potential probiotic properties

The production of a new cereal-based probiotic foods with suitable aroma, flavor and pH using mixed culture fermentation has been investigated. This required the selection of suitable types of cereal grains and probiotic microorganisms. In a medium of 5% (w/v) malt suspension the effects of yeast presence on the fermentation of a lactic acid bacterium (LAB), Lactobacillus reuteri, was studied. With different inoculum ratios between the yeast and the LAB, the characteristics of the fermentation broth including pH and the contents of free amino nitrogen (FAN), reducing sugar, lactic acid and ethanol were investigated. It was found that LAB growth was enhanced by the introduction of the yeast. In mixed culture broth pH was lowered and the production of lactic acid and ethanol were increased in comparison against pure LAB culture.     

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.     

Use of green fluorescent protein to tag lactic acid bacterium strains under development as live vaccine vectors

The lactic acid bacteria (LAB) are safe microorganisms which are mainly used for the preparation of fermented foods and for probiotic applications. The potential of LAB as live vehicles for the production and delivery of therapeutic molecules such as antigens is also being actively investigated today. However, very little is known about the fate of live LAB when administered in vivo and about the interaction of these microorganisms with the nasal or gastrointestinal ecosystem. For future applications, it is essential to be able to discriminate the biotherapeutic strain from the endogenous microflora and to unravel the mechanisms underlying the postulated health-beneficial effect. We therefore started to investigate both aspects in a mouse model with two LAB species presently under development as live vaccine vectors, i.e., Lactococcus lactis and Lactobacillus plantarum. We have constructed different expression vectors carrying the gfp (green fluorescent protein [GFP]) gene from the jellyfish Aequoria victoria, and we found that this visible marker was best expressed when placed under the control of the inducible strong nisA promoter from L. lactis. Notably, a threshold amount of GFP was necessary to obtain a bright fluorescent phenotype. We further demonstrated that fluorescent L. plantarum NCIMB8826 can be enumerated and sorted by flow cytometry. Moreover, tagging of this strain with GFP allowed us to visualize its phagocytosis by macrophages in vitro and ex vivo and to trace it in the gastrointestinal tract of mice upon oral administration.