Potential applications of lactic acid bacteria and bacteriocins in antimycobacterial therapy

Tuberculosis(TB) is a communicable disease caused by Mycobacterium tuberculosis(M. tuberculosis). WHO estimated that 10.4 million new(incident) TB cases worldwide in year 2016. The increased prevalence of drug resistant strains and side effects associated with the current anti-tubercular drugs make the treatment options more complicated. Hence, there are necessities to identify new drug candidates to fight against various sub-populations of M. tuberculosis with less or no toxicity/side effects and shorter treatment duration. Bacteriocins produced by lactic acid bacteria(LAB) attract attention of researchers because of its "Generally recognized as safe" status. LAB and its bacteriocins possess an effective antimicrobial activity against various bacteria and fungi. Interestingly bacteriocins such as nisin and lacticin 3147 have shown antimycobacterial activity in vitro. As probiotics, LAB plays a vital role in promoting various health benefits including ability to modulate immune response against various infectious diseases. LAB and its metabolic products activate immune system and thereby limiting the M. tuberculosis pathogenesis. The protein and peptide engineering techniques paved the ways to obtain hybrid bacteriocin derivatives from the known peptide sequence of existing bacteriocin. In this review, we focus on the antimycobacterial property and immunomodulatory role of LAB and its metabolic products. Techniques for large scale synthesis of potential bacteriocin with multifunctional activity and enhanced stability are also discussed.

Isolation and Characterization of Lactic Acid Bacteria Producing Bacteriocin like Inhibitory Substance (BLIS) from “Gappal”, a Dairy Product from Burkina Faso

Indigenous fermented foods are known for their nutritional and functional properties but they are often spoiled by pathogenic bacteria that can constitute a food safety problem. “Gappal” is a no-thermal treat food based on millet dough and milk and its production conditions can constitute a food safety problem. The aim of this study was to screen and identify LAB producing Bacteriocin-like inhibitory substances using a matrix similar to “Gappal”. The detection of potential BLIS was first performed using overlaid method after enrichment of samples in whey and millet dough. The isolates demonstrating inhibiting area were preselected, purified and tested for the presence of antibacterial properties using their neutralized cell-free culture supernatant and subsequently treated with catalase in combination with protease, pepsin or trypsin. The antimicrobial effect of two isolates (Gbf48 and Gbf50) after growth on MRS broth over 12 h at 30?C were active against E. faecalis ATCC 19433, M. luteus ATCC 49732, S. aureus ATCC 2523, L. monocytogenes, B. megaterium, B. sphaericus and B. cereus with an activity of 2560 AU/mL. The 16S RNA gene sequencing identification indicated that these isolates are Pediococcus acidilactici. Gbf 48 and Gbf 50 could be used to improve preservative factors for a controlled fermentation of non thermal treatment fermented food for their potential of acidification adds to BLIS production.

Antimicrobial Profile of Lactic Acid Bacteria Isolated from Vegetables and Indigenous Fermented Foods of India against Clinical Pathogens Using Microdilution Method

In dairy and food industries lactic acid bacteria （LAB） have been used in form of starter culture that plays vital role in fermentation; as flavouring and texturizing or as preservative agents. There is increasing evidence that lactobacilli which inhabit the gastrointestinal tract develop antimicrobial activities and participate in the host＇s defence system[1]. During fermentation, most of the LAB produces a number of different compounds like organic acids, hydrogen peroxide, diacetyl, acetaldehyde, carbon dioxide, polysaccharides, and proteinaceous compounds called bacteriocins or bacteriocinogenic peptides.

Evaluation of the Method Based on Restriction Fragment Length Polymorphism Analysis as Simple Analysis Method of Lactic Acid Bacteria in Foods

Lactic acid bacteria have not only been used to produce various kinds of fermented food, but also used as probiotic products. As lactic acid bacterial group was consisted from diverse genera, a simple inspection method by which numbers and contained microorganisms could be automatically analyzed without any preliminary information was required to use them more effectively. In this manuscript, lactic acid bacterial groups in commercial products of kimuchi, komekouji-miso, and yoghurt were identified and enumerated by our newly developed method [1]-[3], to evaluate whether the method could be used as an inspection method of various food samples. In kimuchi, numerically dominant bacteria were Lactobacillus sakei, and L. casei (1.4 × 104 MPN g-1) and Leuconostoc spp. (l.4 × 104 MPN). In kouji-miso, numerically dominant bacteria was Bacillus spp. (3 × 103 MPN), which mainly included B. subtilis group and B. cereus group. Lactic acid bacteria such as Lactobacillus spp., or Lactococcus spp., included in the komekouji-miso, could be enumerated after 3 days incubation (1.24 × 104 MPN), but not detected after 7 days incubation. In yoghurt A and C, Lactococcus lactis was detected as numerically dominant lactic acid bacteria (3.0 × 105 MPN). In yoghurt B, Lactobacillus spp., or Lactococcus spp., was detected not only by a culturebased method but also by an unculture-based method, although there was a difference between the both estimated numbers. The present results suggested that the method might become useful as a simple inspection method of food microorganisms, because time and labor of the analysis could be reduced by using an unculture-based method and MCE-202 MultiNA. In this study, Bifidobacteriium spp. was not detected in B and C yoghurt, in spite of indicating their existence, and numbers of lactic acid bacteria were lower than the level of the daily product regulation, because 16S rDNA of Bifidobacteriium spp. might not be amplified by the used PCR condition. The PCR condition must be changed so as to amplify Bifidobacterium spp., before the method will be used as an inspection method for lactic acid bacteria.

Partial Characterization of Bacteriocins from Two <i>Pediococcus acidilactici</i>Strains Isolated during Traditional Sorghum Beer Processing in Côte d’Ivoire

Two lactic acid bacteria strains (At1BEAE22 and At344E21) isolated during tchapalo production were identified on the basis of phenotypic analyses. Bacteriocins produced by these strains were tested for their antimicrobial activities using well diffusion agar method. Heat resistance, pH sensitivity and enzyme treatments were also analyzed. Results showed that both lactic acid bacteria strains were identified as Pediococcus acidilactici. Their bacteriocins inhibited growth of Lactobacillus delbrueckii F/31, Listeria innocua ATCC 33090, Enterococcus faecalis, Enterococcus faecalis ATCC 29212, Streptococcus sp, Enterococcus faecalis CIP 105042 and Enterococcus faecium ATCC 51558. These bacteriocins were heat stable at 60&degC for 30 min for all indicator bacteria. However, they remained active only against Lactobacillus delbrueckii and Listeria innocua at 121&degC for 60 min. Moreover, they were active in a wide range of pH (3 to 9) with a maximum activity observed at pH 5 and 6 on all indicator bacteria. But, bacteriocin from Pediococcus acidilactici At34E21 was more stable at acidic pH than basic one. The fact that the bacteriocin was inactivated by proteinase K and α-chymotrypsin indicated its proteinaceous nature, a general characteristics of bacteriocins.

Screening beneficial bacteriostatic lactic acid bacteria in the intestine and studies of bacteriostatic substances

Lactic acid bacteria(LAB)are a representative probiotic.As the dominant flora in the human intestinal tract,LAB can regulate the balance of human intestinal flora and improve host health.The purpose of this study was to isolate and screen LAB that are well suited to the intestinal characteristics of the Chinese population,with excellent probiotics and high antibacterial activity.After 16S ribosomal RNA(rRNA)homology and phylogenetic tree analysis,potential probiotics were tested for their antibacterial activity,resistance to artificial gastrointestinal fluid and drugs,surface hydrophobicity,and safety.Three strains of LAB with acid resistance,bile salt resistance,epithelial cell adhesion,and no multidrug resistance were selected:Lactobacillus salivarius,Leuconostoc lactis,and Lactobacillus paracasei.Analysis of the antibacterial active substances in the three strains and their fermentation broths revealed that the main antibacterial substances of L.lactis were organic acids,whereas those of L.salivarius and L.paracasei were organic acids and bacteriocins with broad-spectrum antibacterial activity.These three strains of probiotic LAB with high antibacterial activity were identified as bacterial resources that could potentially be used to develop probiotic preparations for the prevention and treatment of intestinal diseases caused by intestinal pathogens.

Study Bacteriocin Production and Optimization Using New Isolates of <i>Lactobacillus</i>spp. Isolated from Some Dairy Products under Different Culture Conditions

Lactobacilli belong to the group of lactic acid bacteria (LAB), that have several distinguished abilities such as production of lactic acid, enzymes such as β-Galactosidase and natural antimicrobial substances called bacteriocins. Bacteriocin is a biopreservative agent potential of suppressing growth of some contaminant bacteria in food industry but its commercial availability is limited and costly. The study aimed to select isolates of Lactobacillus spp. potential for producing bacteriocins to suppress the growth of Escherichia coli ATCC 25922 and Bacillus subtilis NCIB3610, and to optimize the process of bacteriocin production. Results obtained in this study showed that L. acidophilus isolate CH1 was selected as the best candidate for bacteriocin among the four isolates that tested. The largest amounts of the bacteriocins were synthesized only in MRS medium was supplemented with K2HPO4 (1.0%), Tween 80 (1%), Beef extract (1%), glucose, cyctein and peptone extract (1%). The optimization of culture conditions for bacteriocin production areas showed that corn steep liquor medium was the best medium for all isolates against Bacillus subtilis while no effect was observed on Escherichia coli ATCC 25922 except when used MRS medium. The optimum conditions for bacteriocin production were pH 6.0, temperature 34?C with 4% Phenyl acetamide showing the greatest growth inhibition areas.

Use of Bacteriocin Producing <i>Lactococcus lactis</i>subsp. <i>lactis</i>LABW4 to Prevent <i>Listeria monocytogenes</i>Induced Spoilage of Meat

A bacteriocin producing strain of Lactococcus lactis subsp. lactis LABW4 was isolated from naturally fermented milk product which exhibited strong antibacterial activity against Listeria monocytogenes MTCC657, a food spoilage psychrophilic organism. Both cell free and heat killed supernatants of LABW4 were effective to produce zones of inhibition against L. monocytogenes in vitro. The antibacterial metabolite(s) of LABW4 showed strong cidal effect on the growth of L. monocytogenes. Meat samples, mixed with heat killed supernatant of LABW4 when inoculated with Listeria, remain fresh up to 25 days in refrigerated condition whereas spoilage started immediately after 24 hours of inoculation for control sets. Enhancement of Lactate dehydrogenase of L. monocytogenes upon treatment with LABW4 cell free supernatant suggested its lytic mode of action. Cell lysis or degradations were also supported by scanning electron micrograph of treated cells.

Recent biotechnological trends in lactic acid bacterial fermentation for food processing industries

Lactic acid bacteria(LAB)are non-mobile,gram-positive,non-spore-forming,micro-aerophilic microorganisms widely explored as starter cultures food industry to enhance the gustatory,nutritional value,imparts appetizing favour,texture to milk,vegetative,meat foods and prolongs their shelf life.This vast review emphasis various LABs widely explored in the food industry.Herein,we have summarized the classifcation of LAB strains,their metabolic pathways for biosynthesis of lactic acid,ethanol,acetic acid and demonstrated their application in various food industries for making fermented milk(yoghurt),cheese,beverages,bread,and animal foods.The wide spectrum of LAB-based probiotics,bacteriocins,exopolysaccharides,bio preservative and their relevant benefts towards human health has also been discussed.Moreover,LAB bacteriocins and probiotics in food application may limit the growth of pathogenic,while boosting health immunity.Microbial exopolysaccharides have interesting characteristics for the fermented food industry as new functional foods.Later on,we have discussed the various advancement in metabolic engineering,synthetic biology tools,which have gained considerable interest to elucidate the biosynthetic pathway for tailoring cellular metabolism for high activity.

Natural populations of lactic acid bacteria in douchi from Yunnan Province,China

This research was aimed at isolating and identifying the predominant lactic acid bacteria(LAB) in the traditional Chinese salt-fermented soybean food,douchi,from Yunnan,China.The predominant LAB present were isolated and identified by conventional culture-dependent methods combined with molecular biological methods.Two hundred and sixty isolates were obtained from thirty kinds of traditional fermented douchi from six cities and counties in Yunnan,and those strains were divided into twelve groups by their morphological and biochemical characteristics.Based on 16S ribosomal DNA(rDNA) sequencing and analysis,56 representative strains were identified as belonging to 6 genera and 14 species:Lactobacillus(4 spp.),Weissella(3 spp.),Pediococcus(2 spp.),Staphylococcus(2 spp.),Enterococcus(1 sp.),and Bacillus(2 spp.).The results show that douchi contains a large natural population of LAB of diverse composition from which some strains could be selected as starters for functional fermented foods.This is the first study on the original douchi from Yunnan,and the results suggest that it may be a useful source for the isolation of LAB.This study has also laid a foundation for further research on developing functional douchi products.

Biosurfactants from lactic acid bacteria:A critical review on production,extraction,structural characterization and food application

Biosurfactants are structurally diverse classes of amphiphilic molecules derived from microorganisms.They have various properties such as surface,emulsification,antibacterial,anti-adhesive,anti-biofilm,antioxidant activity that can be used in many industrial fields.The interest in these molecules compared to their synthetic counterparts is due to their biodegradability,low toxicity,structural diversity,and stability over a wide range of pH,temperature,and salinity.Among biosurfactants,those derived from lactic acid bacteria(LAB)attracted attention nowadays due to the GRAS(Generally recognized as safe)status of these microorganisms.Biosurfactants production by LAB strains has been documented in several investigations,and they deal with the functionality and chemical nature of biosurfactants.LAB biosurfactants were mainly a complex mixture of proteinaceous compounds,glycolipids,glycoproteins,or glycolipopeptides.There are only a few studies reporting on the elucidation of the structure of LAB biosurfactants.Generally,biosurfactants’production process includes several unit operations starting from screening methods to the characterization of the biosurfactant compound.This review highlights the different techniques used in the screening of biosurfactants production by LAB,their extraction and purification processes,and their structural characterization and application in the food industry.

Bacteriocins as antimicrobial and preservative agents in food:Biosynthesis,separation and application

Emerging evidence shows the trend of using safe and natural preservatives like bacteriocins in food processing.The increasing demand by the food industry to extend keeping quality and prevent spoilage of various food products has appealed for new preservatives and new methods of conservation.Further,to discover the novel spectrum of antimicrobial compounds that could effectively battle food-borne pathogens,bacteriocins have been intensively studied in the last few years.Recently,great attention has been paid to the application of lactic acid-bacterial bacteriocins targeting food spoilage-causing or pathogenic microorganisms with no significant side effects.Different mechanisms of action like pore-formation,retarding cell-wall/nucleic acid/protein synthesis have been proposed and described for diverse bacteriocins.Therefore,the syntheses,purification of bacteriocins in addition to their applications as antimicrobial and preservative agents in food processing are reviewed in detail.The review will also discuss the preservative effect of bacteriocins and their combinations having multiple modes of action(hurdle approach)to reduce the microbial load as well as antimicrobial resistance.

Retrospecting the concept and industrial significance of LAB bacteriocins

Ever increasing demand for natural products with minimum chemical additives has necessitated the search for innovative green,potent,environment friendly,natural substitutes for prolonging the shelf life of food products.The lactic acid bacteria(LAB)are a group of Gram-positive microorganisms with great biotechnological potential in the food industry.To reduce pathogen invasion,they can produce inhibitory compounds(bacteriocins,organic acids,hydrogen peroxide,diacetyl,and carbon dioxide).LAB-bacteriocin are proteinaceous antimicrobial molecules with a diverse genetic origin,post-translationally modified or not,that can help the producer organism to outcompete other bacterial species.In addition,they are emerging as a novel wave of potential antibiotics with potent in vitro and in vivo activities.Moreover,LAB-bacteriocin serves as an effective method of natural bio preservation in the food industry and enables the reduction of other costly or user-unfriendly treatments while increasing the product self-life.Therefore,this article provides a recapitulation of classification of bacteriocins,potential of LAB to produce different bacteriocins,their biology,and purification methods.Additionally,it will give a critical snapshot of industrial applications of LAB-bacteriocin as a starter culture,preservative,antibiotic,and probiotics,etc.

Development of Efficient Fermentation Process at Bioreactor Level by Taguchi's Orthogonal Array Methodology for Enhanced Dextransucrase Production from <i>Weissella confusa</i>Cab3

The influence of medium ingredients on extracellular dextransucrase production by a new bacterial strain Weissella confusa Cab3 (Genbank Accession Number JX649223) was evaluated using fractional factorial design of Taguchi's orthogonal array. Four metabolism influencing factors viz. sucrose, yeast extract, K2HPO4 and Tween80 were selected to optimize dextransucrase production by W. confusa Cab3 using fractional factorial design of Taguchi methodology. Based on the influence of interaction components of fermentation, least significant factors of individual level have higher interaction severity index and vice versa for enzyme production from Weissella confusa Cab3. Sucrose and yeast extract were found to be the most significant factors which positively influenced the dextransucrase production. The optimized medium composition consisted of sucrose—5%;yeast extract—2%;K2HPO4—1.0%;Tween80—0.5%, based on Taguchi orthogonal array method. The optimized composition gave an experimental value of dextransucrase activity of 17.9 U/ml at shake flask level which corresponded well with the predicted value of 17.54 U/ml by the model. The optimized medium by Taguchi method gave significant (3 fold) enhancement of dextransucrase activity as compared to unoptimised enzyme activity of 6.0 U/ml. The dextransucrase production was scaled up in lab scale bioreactor resulting in further enhancement of enzyme activity (22.0 U/ml).

传统发酵乳品中产广谱细菌素乳酸菌选育

为选育产广谱细菌素乳酸菌,采用琼脂扩散法对甘肃牧区传统发酵乳品中分离纯化的96株乳酸菌进行了筛选,并确定产细菌素菌株,具有最佳抑菌效果的菌株经重离子束12C6+辐照诱变,再采用培养皿分区法进行6种指示菌广谱抑菌性能比较。结果表明,琼脂扩散法筛选出抑菌效果最佳的产细菌素菌株Lp1,经辐照诱变得到98株突变菌,选育出对大肠埃希氏菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、单核细胞增生李斯特氏菌(Listeria monocytogenes)、沙门氏菌(Salmonella)、志贺氏菌(Shigella)和克罗诺杆菌(Cronobacter)抑菌性能均明显优于出发菌株Lp1的5株突变株L088、L087、L063、L049、L010进行广谱抑菌试验,其中菌株L088和L063为广谱抑菌性能较好菌株,突变株L088对上述6种指示菌的抑菌扇环半径比出发菌株Lp1抑菌扇环半径分别提高47.81%、33.28%、22.97%、45.25%、21.39%、40.21%,突变株L063对上述6种指示菌的抑菌扇环半径比出发菌株Lp1抑菌扇环半径分别提高54.27%、29.18%、25.37%、58.03%、17.85%、42.13%。采用培养皿分区法可同时在1个培养皿中完成检测6种指示菌的抑菌试验,可高效筛选产广谱细菌素乳酸菌。

益生菌基因组学在乳酸菌筛选和功能评价中的应用

乳杆菌和双歧杆菌是益生菌筛选的主要微生物类群,在促进人类、动物甚至植物健康方面都有重要的应用价值,被广泛应用于多种功能性食品、动物饲料和微生物菌肥。然而,这些细菌对宿主健康产生积极影响的分子机制还远未完全了解。另外,传统的益生菌筛选技术主要依靠表型和生理生化特性的测定以及随机对照试验,这些方法不仅耗时、费力,而且数据难以重复。不同研究小组之间数据难以共享,不能实现菌株有益特性的均一性和标准化验证,严重制约了益生菌科学研究的发展和菌株的产业化开发利用。基于新的测序技术和强大的计算机分析方法,被称为益生菌基因组学新学科的出现,允许研究者在短时间内通过筛选大量的生物学和基因组数据,显著提高益生菌筛选的效率和准确性。本文综述了通过基因组数据的分析,帮助人们高效、快速地确定益生菌,开辟益生菌新功能的评价途径。总结益生菌基因组学在阐明益生特性功效评价的分子基础等方面的最新研究成果,以期为扩展研究人员对这些有益微生物生物学的认知,揭示益生菌促进健康特性的分子机制提供新视角。

新疆传统发酵乳制品调研及其发酵微生物种质资源分析

为挖掘新疆传统发酵乳制品的微生物菌种资源,对不同地区传统发酵乳的制作方式及其发酵微生物进行了调研。采集了全疆12个地州及乌鲁木齐周边共45个县165个乡村483份家庭作坊生产的传统发酵乳制品,并对其进行了发酵微生物筛选。从采集的不涉及任何商品发酵剂的新疆传统发酵乳中,共筛选出497株乳酸菌和364株酵母菌,并首次发现了假肠膜明串珠菌、汉逊德巴利酵母和巴氏醋杆菌等菌。此研究成果可为开发新疆传统发酵乳制品的优质菌种资源提供基础。

原驼乳中乳酸菌的分离筛选及特性分析

【目的】挖掘和获得原驼乳中的乳酸菌资源。【方法】采集新疆乌鲁木齐县近效南山周边原驼乳样品,采用平板稀释法分离菌株,观察比对16S rRNA基因序列测序和菌株形态学特性,分析菌株分类学地位,并检测菌株溶血性和有害代谢物质等食用安全性。【结果】获得29株乳酸菌,经鉴定其归属于Leuconostoc、Lentilactobacillus两个属的4个种;菌株均可耐受0.5%胆盐,5%NaCl,且具有较好耐高温、牛奶胨化、抗氧化和抑制细菌等特性;所有菌株均无溶血现象,不产生硝基还原酶;除T12外,均利用氨基酸不产生物胺。【结论】从原驼乳中获得的乳酸菌具有良好的发酵特性和食品安全性。

LuxS/AI-2群体感应系统在乳酸菌细菌素合成中的作用

乳酸菌细菌素具有抑菌谱广、稳定性高、安全性高等优势,作为新型生物防腐剂备受青睐,但目前工业化应用的细菌素数量有限,合成量低是其应用受限的重要原因。国内外研究显示与特定的微生物共培养是提高乳酸菌细菌素合成量的有效方法,该过程受到LuxS/AI-2群体感应系统的调控。文章从乳酸菌LuxS/AI-2群体感应系统的组成及LuxS/AI-2群体感应系统在乳酸菌细菌素合成中的作用两个方面进行论述,为乳酸菌细菌素合成量的提高和工业化应用的实现提供了一定的理论依据。

泡菜中乳酸菌的抑菌性能及生长特性探究

从四川传统泡菜母水中分离出具有抑菌功能的乳酸菌分别为XZ5和XZ11,并对其进行鉴定和生长特性研究。利用形态、生理生化和16S rRNA测序等方法对菌种进行鉴定,并研究这两株菌的生长特性。结果表明,菌株XZ5对沙门氏菌和大肠杆菌的抑菌直径分别为11.3±0.3 mm和10.9±0.5 mm,而菌株XZ11的抑菌圈直径为9.3±0.7 mm和6.8±0.3 mm。菌株XZ5和XZ11通过革兰氏染色鉴定为阳性的杆菌,其测序结果分别为德氏乳杆菌(Lactobacillus delbrueckii)和短乳杆菌(Lactobacillus brevis)。菌株XZ5和XZ11都在8 h进入对数期,并且在生长繁殖时期产生大量酸性物质使菌液pH值降低,而较低的pH值有利于泡菜的保存。综上所述德氏乳杆菌XZ5具有发酵泡菜的益生潜能,可作为泡菜等发酵食品的微生物资源。