提高乳酸菌细菌素合成量方法的研究进展

细菌素是某些细菌通过核糖体合成机制产生的蛋白质或多肽,能够抑制与其亲源关系相同或相近的微生物,某些细菌素在食品加工和发酵过程中能抑制致病菌和腐败菌。乳酸菌被认为是一般公认安全,其细菌素具有安全性高、稳定性好、抑菌谱广等优点,作为一种新型食品防腐剂备受关注,但商品化的乳酸菌细菌素十分有限,仅限于Nisin和Pediocin PA-1等少数几种,合成量低是细菌素在食品中应用受限的主要原因之一。从不同原料中筛选高产菌株、发酵培养基和发酵条件优化、诱变育种、原生质体融合、基因工程方法、群体感应系统调控六个方面,论述了增加乳酸菌细菌素合成量的方法,以期为实现乳酸菌细菌素的工业化生产提供一定的借鉴。     

Bacteriocins and their Food Applications

Over the last 2 decades, a variety of bacteriocins, produced by bacteria that kill or inhibit the growth of other bacteria, have been identified and characterized biochemically and genetically. This review article focuses on the ecology of bacteriocins, determination of bacteriocin activity, biosynthesis of bacteriocins, and mode of action. Bacteriocin production and modeling are discussed in the article. Nisin is discussed in some detail in this article since it is currently the only purified bacteriocin approved for food use in the U.S. and has been successfully used for several decades as a food preservative in more than 50 countries. For activity spectra and food applications, the review article focuses primarily on class I and class IIa bacteriocins produced by lactic acid bacteria (LAB) given their development as food preservatives.     

Charakterisierung einiger Bacteriocine im Hinblick auf mögliche Anwendungen im Lebensmittelbereich. 1. Mitt. Stammauswahl

Characterization of some bacteriocins with regard to possible applications in food industry. Part 1. Selection of strains. The ability to produce bacteriocins is common in all genera of lactic acid bacteria. Thirty-four out of 223 strains of lactic acid bacteria from the culture collection of the University Potsdam produced bacteriocins active against one or more indicator strains. Seventeen Gram-positive and Gram-negative strains, frequent occur as spoilage bacteria in food industry, were chosen as indicator strains, and 11 of them were inhibited by one or several bacteriocin producers. Most of the bacteriocin producers belong to the genera Enterococcus or Lactobacillus. Bacteriocins derived from Enterococcus faecium and Pediococcus acidilactici exhibited the widest inhibitory spectrum. The properties of Enterococcus faecium 10.051 and Enterococcus faecium 10.211 bacteriocins were further investigated to evaluate their potential use as natural food preservatives. The bacteriocin produced by Enterococcus faecium 10.211 exhibited a very wide inhibitory spectrum against food-spoiling strains. It was heat stable (100 °C for 60 min) and stable in a wide range of pH (1.2–10.0). The bacteriocin produced by Enterococcus faecium 10.051 showed a smaller inhibitory spectrum, and it was less stable against temperature and pH as the bacteriocin from strain 10.211. Both bacteriocins were inactivated by proteinase K. pronase E, and α-chymotrypsin. Bacteriocin from strain 10.211 will be the object of further investigations for application of bacteriocins in food industry.     

Physical and nutritional conditions for optimized production of bacteriocins by lactic acid bacteria – A review

ABSTRACT

There has been an increasing debate about the use of synthetic chemical compounds and the consequences of their use in food preservation. In this context, the utilization of some natural compounds produced by bacteria, showing an inhibitory effect against microorganisms associated with food contamination, have gained attention as preservation technology. In order to improve the production and yield costs of bacteriocins, detailed studies are necessary to determine the conditions that allow an optimized production and extraction of bacteriocins from lactic acid bacteria (LAB). In this context, this article aims to discuss the information regarding the main factors that influence bacteriocin production by LAB. The biosynthesis of bacteriocins can be influenced by various culture conditions, such as the composition of the medium, pH, temperature and growth kinetics of the microorganisms. One of the limiting factors for the use of bacteriocins on a large scale in food preservation is the economic factor. In order for the production costs of bacteriocins to be reduced, making them attractive, it is necessary to know the optimum parameters of production, thus maximizing productivity and making costs more attractive.     

Development of Wide-Spectrum Hybrid Bacteriocins for Food Biopreservation

The food industry demands new procedures and methods to produce minimally processed, ready to eat food with intact nutritional, taste, and flavor properties. The biopreservation and the use of both bacteriocins produced by lactic acid bacteria (LAB) and bacteriocinogenic strains as an alternative to substitute chemical antimicrobial for food preservation became increasingly important in the last two decades. When the new proposed natural preservatives techniques are applied, probiotics food can be obtained and, simultaneously, foodborne pathogens and spoilage contaminants can diminish. However, bacteriocins produced by LAB have a narrow antibacterial spectrum and are inactive against Gram-negative bacteria like Salmonella and the emergent enterohemorrhagic Escherichia coli. Knowing the mechanism of action and the structural features of microcins synthesized by Gram-negative bacteria and with potent antimicrobial activity against the mentioned microorganism, the proposal is to obtain hybrid peptides (microcin–bacteriocin) with broad antimicrobial spectrum. This review explains how the inability of bacteriocins to cross the outer membrane of Gram-negative bacteria unable them to act on the bacteria. It will also be discussed how a hybrid bacteriocin can be obtained.     

Bacteriocins: safe, natural antimicrobials for food preservation.

Bacteriocins are antibacterial proteins produced by bacteria that kill or inhibit the growth of other bacteria. Many lactic acid bacteria (LAB) produce a high diversity of different bacteriocins. Though these bacteriocins are produced by LAB found in numerous fermented and non-fermented foods, nisin is currently the only bacteriocin widely used as a food preservative. Many bacteriocins have been characterized biochemically and genetically, and though there is a basic understanding of their structure-function, biosynthesis, and mode of action, many aspects of these compounds are still unknown. This article gives an overview of bacteriocin applications, and differentiates bacteriocins from antibiotics. A comparison of the synthesis. mode of action, resistance and safety of the two types of molecules is covered. Toxicity data exist for only a few bacteriocins, but research and their long-time intentional use strongly suggest that bacteriocins can be safely used.     

Identification and characterization of two bacteriocin-producing strains of Lactococcus lactis isolated from vegetables.

Isolated from mixed salad and fermented carrots, 123 strains of lactic acid bacteria were screened for bacteriocin production. Two strains, D53 and 23, identified as Lactococcus lactis by DNA-DNA hybridizations, produced heat stable bacteriocins which were resistant to trypsin and pepsin, but were inactivated by alpha-chymotrypsin and proteinase K. The bacteriocins were active from pH 2 to 9 and inhibited species of Listeria, Lactobacillus, Lactococcus, Pediococcus, Leuconostoc, Carnobacterium, Bacillus and Staphylococcus. Strain D53 produced bacteriocin at pH values of 4.5-8.0 and from 10 to 37 degrees C.     

Heterologous production of bacteriocins by lactic acid bacteria

Over the last two decades, bacteriocins produced by lactic acid bacteria (LAB) have been the subject of considerable research and industrial interest due to their potential as food biopreservatives. The development of heterologous expression systems for such antimicrobial compounds may offer a number of advantages over native systems, such as facilitating the control of bacteriocin gene expression or achieving higher production levels. In addition, the heterologous production by food-grade LAB offers an attractive method for overcoming some of the adverse situations that may affect the effectiveness of some bacteriocins in food systems. Construction of multibacteriocinogenic strains or acquisition of antimicrobial properties by industrial strains are further objectives that can be achieved through the use of heterologous gene expression systems. The development of new biotechnological tools and recent advances in LAB genetics account for the escalating number of studies dealing with heterologous production of bacteriocins by such hosts. This paper reviews the literature published on the subject and compares the different experimental strategies that have been used up to the present for this purpose.     

Induction of bacteriocin production by coculture is widespread among plantaricin-producing Lactobacillus plantarum strains with different regulatory operons

We describe the bacteriocin-production phenotype in a group of eight singular bacteriocinogenic Lactobacillus plantarum strains with three distinct genotypes regarding the plantaricin locus. Genotyping of these strains revealed the existence of two different plantaricin-production regulatory operons, plNC8-plNC8HK-plnD or plnABCD, involving three-component systems controlled each of them by a specific autoinducer peptide (AIP), i.e. PLNC8IF or PlnA. While all of the strains produced antimicrobial activity when growing on solid medium, most of them halted this production when cultured in broth, thus reflecting the functionality of regulatory mechanisms. Antimicrobial activity in broth cultures was re-established or enhanced when the specific AIP was added to the culture or by coculturing with specific bacterial strains. The latter trait appeared to be widespread in bacteriocinogenic L. plantarum strains independently of the regulatory system used to regulate bacteriocin production or the specific bacteriocins produced. The induction spectrum through coculture, i.e. the pattern of bacterial strains able to induce bacteriocin production, was characteristic of each individual L. plantarum strain. Also, the ability of some bacteria to induce bacteriocin production in L. plantarum by coculture appeared to be strain specific. The fact that induction of bacteriocin production by coculturing appeared to be a common feature in L. plantarum can be exploited accordingly to enhance the viability of this species in food and feed fermentations, as well as to contribute to probiotic functionality when colonising the gastrointestinal tract.     

Influence of pH and temperature on growth and bacteriocin production by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442

The aim of this study was to investigate the relationship between the microbial growth, the bacteriocin production and the effect of pH and temperature on the occurrence and the concentration of the maximum activity, to optimize the bacteriocin synthesis during the growth cycle. Two bacteriocins produced by lactic acid strains Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442 were studied. A slight increase of the final biomass resulted in the improvement of the bacteriocin activity in the growth medium under controlled pH (5.0 and 5.5). Temperature and pH had a significant effect on the production of the two bacteriocins and was enhanced by the relatively low growth rates. The optimum production conditions of the bacteriocins did not coincide with those for growth. The optimum pH and temperature values for growth were 6.0-6.5 and 30?°C and for bacteriocin production were 5.5 and 25?°C.     

安全的天然食品防腐剂细菌素

细菌素是细菌产生的可以杀死或抑制别的细菌生长的抗菌蛋白。许多乳酸菌(LAB)产生各种各样的细菌素，其中的尼生素是唯一的在食品中作为防腐剂广泛使用的细菌素。文章综述了以尼生素为重点对细菌素与抗生素的区别、活性细菌素分子的形成以及其在食品中的应用，并比较了这两种类型的分子的合成、作用模式、抵抗力和安全性。     

Characterisation of anti-Listeria monocytogenes bacteriocins from Enterococcus faecium strains isolated from dairy products

Bacteriocin-producing lactic acid bacteria were isolated from 34 samples of dairy products. Nine bacteriocin producers were phenotypically and genotypically identified as Enterococcus faecium . By means of PCR-techniques, enterocin A was characterised in all of the nine bacteriocin-producing Enterococcus isolates. Enterocin-producing lactic acid bacteria were the most abundant in dairy products collected from different areas in Iran. Maximum bacteriocin production by Enterococcus faecium strains was detected in the stationary phase of growth. Bacteriocins produced by all isolates were found to have anti-listerial activity in sterile milk. The purified bacteriocins were identified as <6.5 kDa peptide by sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE). The molecular weights of bacteriocins were found to be the same in all strains. This bacteriocin might be useful as a natural preservative.     

Class IIa bacteriocins from lactic acid bacteria: antibacterial activity and food preservation

In the last decade, a variety of ribosomally synthesized antimicrobial peptides, or bacteriocins, produced by lactic acid bacteria have been identified and characterized. As a result of these studies, insight has been gained into various fundamental aspects of biology and biochemistry such as bacteriocin processing and secretion, mechanisms of cell immunity, and structure-function relationships. In parallel, there has been a growing awareness that bacteriocins may be developed into useful antimicrobial food additives. Class IIa bacteriocins can be considered as the major subgroup of bacteriocins from lactic acid bacteria, not only because of their large number, but also because of their significant biological activities and potential applications. The present review provides an overview of the knowledge available for class IIa bacteriocins and discusses common features and recent findings concerning these substances. The activity and potential food applications of class IIa bacteriocins are a major focus of this review.     

Enterococcus faecium RZS C5, an interesting bacteriocin producer to be used as a co-culture in food fermentation

Enterocins, bacteriocins produced by enterococci, are gaining interest because of their industrial potential. Due to its bacteriocin production, Enterococcus faecium RZS C5, a natural cheese isolate, has a strong activity towards Listeria monocytogenes. For this reason, the strain may be applicable as a bacteriocin-producing co-culture in food fermentation in order to reduce the risk on Listeria outgrowth. The strain displays remarkable bacteriocin production kinetics. Whereas most lactic acid bacteria produce bacteriocin in a growth-associated way until the beginning of the stationary phase, bacteriocin production by E. faecium RZS C5 in MRS broth at controlled pH values below 7.5 is characterised by a boost of bacteriocin activity levels in the very early growth phase. In addition, bacteriocin production kinetics are closely linked to the environmental and cultural conditions. However, no straightforward statement about the effect of environmental stress on bacteriocin production can be made since the effect is dependent on the type of stress applied. Kinetic experiments in milk and on pilot scale, applying Cheddar cheese-making conditions, have indicated that the strain may be effective as a bacteriocin-producing co-culture. Further research is needed to evaluate the use of E. faecium RZS C5 as a co-culture for the production of fermented sausage.     

The locus responsible for production of plantaricin S,a class IIb bacteriocin produced by Lactobacillus plantarum LPCO10, is widely distributed among wild-type Lact. plantarum strains isolated from olive fermentations

The genes plsA and plsB encoding for production of plantaricin S (Pls), a two-peptide bacteriocin produced by Lactobacillus plantarum LPCO10, are commonly distributed among wild-type Lact. plantarum strains isolated from olive fermentations. Among 68 independent isolates from different olive processing plants in South Spain, 15 of them were shown to produce bacteriocins that were active against other lactic acid bacteria, as well as spoilage and pathogenic bacteria. On the basis of PCR amplification and hybridization with specific probes, the Pls operon was detected in all the bacteriocin producer strains but not in the non-producer ones. Purification and subsequent amino acid sequencing of the bacteriocin produced by some of the 15 isolates yielded both the alpha and beta peptides from Pls. These results suggest that bacteriocin production contributes an ecological advantage for the wild-type Lact. plantanum strains in the colonization of the spontaneous, traditional olive fermentation process.     

A novel area of predictive modelling: describing the functionality of beneficial microorganisms in foods

Predictive microbiology generally focuses on the potential outgrowth of spoilage bacteria and foodborne pathogens in foods. Little attention has been paid to the biokinetics of beneficial foodgrade microorganisms, such as lactic acid bacteria. The latter is commonly used in the food fermentation industry, mainly for the in situ production of the antimicrobial lactic acid to extend the shelf life of the food. Furthermore, many strains show additional industrial potential as novel starter cultures since they produce functional metabolites, such as bacteriocins and exopolysaccharides. The production of these functional metabolites has been demonstrated during in vitro experiments, but in many cases these novel starter cultures seem to be less efficient when applied in a food system. A modelling approach may contribute to a better understanding of the tight relation between the food environment and bacterial functionality. Primary modelling can be applied to fit the experimental data concerning cell growth, sugar metabolism, and the production of functional metabolites for a given set of environmental conditions. This led to conclusions concerning the growth-associated production of bacteriocin and exopolysaccharides, the inactivation of these molecules when cell growth levels off, and a minimum cell concentration to trigger on bacteriocin production. Examples deal with the production of the bacteriocin sakacin K by the natural fermented sausage isolate Lactobacillus sakei CTC 494, and the production of heteropolysaccharides by the yoghurt starter culture Streptococcus thermophilus LY03. Secondary modelling of biokinetic parameters quantifies the production of bacteriocin and exopolysaccharides in function of environmental factors. As an example, the specific bacteriocin production by Lb. sakei CTC 494 decreases with increasing sodium chloride concentrations. Furthermore, since the assessment of functionality is frequently hampered by the nature of the food system, mathematical modelling techniques may help to predict the functional behaviour of novel lactic acid bacteria starter cultures in a food matrix, and hence quantify in situ production. For example, a model may simulate cell growth and exopolysaccharide production of S. thermophilus LY03 in a milk environment, where direct measurements are difficult to perform.     

Incidence and antibiotic susceptibility of bacteriocin‐producing lactic acid bacteria from dairy products

Screening for bacteriocin production by strains of lactic acid bacteria (LAB) from local dairy products in Iran resulted in the detection of 10 bacteriocin‐producing strains. Among 105 isolated, 10 bacteriocin producers were phenotypically and genotypically identified as Enterococcus spp. The antimicrobial compounds produced by these novel strains were inactivated by trypsin, proteinase k. These bacteriocins also were active in a wide range of pH and temperature values, and inhibited not only the closely related LAB, but also Listeria monocytogenes.     

Bacteriocins of lactic acid bacteria: Their potentials as food biopreservative

Abstract

Numerous strains of lactic acid bacteria used in the fermentation of foods are known to produce bacteriocins. In general, bacteriocins are a group of proteinaceous antimicrobial substances that inhibit the growth of closely related bacteria. However, some bacteriocins produced by lactic acid bacteria (LAB) exhibit a relatively broad antimicrobial spectrum and are active against several food‐spoilage and health‐threatening microorganisms. Many investigators have reported on the use of bacteriocins as food preservative to extend the shelflife of various foods. This review decribes the research that has been conducted on bacteriocinogenic lactic acid bacteria— isolated from a wide variety of foods and in some instances of animal origin—and the characteristics of bacteriocins. Special emphasis is placed on their potentials for use as food preservative and on their physicochemical nature, antibacterial spectrum, and genetic behavior.     

Detection and preliminary characterization of a bacteriocin (plantaricin 35d) produced by a Lactobacillus plantarum strain

Lactic acid bacteria (134) from Italian sausages were tested for the production of antimicrobial substances (bacteriocins). Six percent of these showed antibacterial activity against one or several closely related microorganisms used as indicators. Lactobacillus plantarum 35d in particular produced a bacteriocin of high activity (320 AU ml(-1)) and a wide range of antimicrobial activity including S. aureus, L. monocytogenes, and A. hydrophila. The bacteriocin withstood heating at 80 degrees C for 120 min and storage at 4 degrees C for 6 months. The mode of action was identified as bactericidal. The apparent molecular weight of the bacteriocin extracted with n-butanol was estimated to be 4.5 kDa.     

Preservation and fermentation: past,present and future

Preservation of food and beverages resulting from fermentation has been an effective form of extending the shelf-life of foods for millennia. Traditionally, foods were preserved through naturally occurring fermentations, however, modern large scale production generally now exploits the use of defined strain starter systems to ensure consistency and quality in the final product. This review will mainly focus on the use of lactic acid bacteria (LAB) for food improvement, given their extensive application in a wide range of fermented foods. These microorganisms can produce a wide variety of antagonistic primary and secondary metabolites including organic acids, diacetyl, CO2 and even antibiotics such as reuterocyclin produced by Lactobacillus reuteri. In addition, members of the group can also produce a wide range of bacteriocins, some of which have activity against food pathogens such as Listeria monocytogenes and Clostridium botulinum. Indeed, the bacteriocin nisin has been used as an effective biopreservative in some dairy products for decades, while a number of more recently discovered bacteriocins, such as lacticin 3147, demonstrate increasing potential in a number of food applications. Both of these lactococcal bacteriocins belong to the lantibiotic family of posttranslationally modified bacteriocins that contain lanthionine, beta-methyllanthionine and dehydrated amino acids. The exploitation of such naturally produced antagonists holds tremendous potential for extension of shelf-life and improvement of safety of a variety of foods.