酸菜中具有抑菌活性乳酸菌的分离鉴定及其产细菌素特性

Isolation and Identification of Lactic Acid Bacteria with Antibacterial Activity in Pickles and Their Bacteriocin Production Characteristics

目前已有研究陆续从不同的乳酸菌中分离到细菌素,但是这些细菌素存在着抑菌谱窄的问题,寻找广谱抗菌性能的新一代细菌素具有重要的理论价值和应用前景。以市售保质期较长的8种不同品牌的酸菜为材料,将青霉菌作为指示菌,通过抑菌实验筛选出一株能产生抑菌物质的菌株L_(3),经过生理生化鉴定、16S rDNA测序分析确定该菌株为植物乳杆菌(Lactobacillus plantarum),故命名为植物乳杆菌L_(3),NCBI序列号为MT781360。该菌株在MRS培养基中于37℃培养18~24 h时产细菌素L_(3)的抑菌活性最强。将该培养上清液采用乙酸乙酯萃取,葡聚糖凝胶柱Sephadex G-50过滤分离纯化,再经Trinice-SDS-PAGE电泳结果显示,细菌素L_(3)的分子质量约为4~5 kDa。细菌素L_(3)对蛋白酶敏感,但其活性不受过氧化氢酶的影响,初步结果显示细菌素L_(3)为蛋白质类物质。细菌素L_(3)在60~100℃处理20 min或121℃处理15 min仍具有较高的抑菌活性,pH值2~10范围内有良好的稳定性;对革兰氏阳性菌和革兰氏阴性菌以及部分真菌具有抑菌活性。植物乳杆菌L_(3)分泌的细菌素L_(3)除了具备现有细菌素的稳定性和对革兰氏阳性细菌的杀菌能力以外,还能对革兰氏阴性细菌和真菌起到抑菌效果,这对于易被真菌污染导致腐败的果蔬类产品及乳制品的保藏具有重要意义,希望研究可为具有广谱抗菌性能的天然防腐剂的生产提供新的菌种。

Bacteriocins from different lactic acid bacteria have been successively isolated currently,however,these bacteriocins often suffer from the problem of a narrow antibacterial spectrum.Exploring new bacteriocins with broad-spectrum antibacterial properties has important theoretical value and application prospects.Commercially available pickled vegetables(sauerkraut)of 8 different brands with a long shelf life were selected as materials and bacteriostasis tests were done using Penicillium as indicator bacteria,the strain L_(3) which could produce bacteriostatic substances was screened out.The strain was identified as Lactobacillus plantarum and named Lactobacillus plantarum L_(3) after physiological and biochemical identification and 16S rDNA sequencing analysis,and the NCBI serial number was assigned MT781360.The bacteriocin L_(3) showed the strongest bacteriostatic activity when the strain L_(3) was cultured in MRS medium at 37℃for 18-24 h.The cell-free supernatant was extracted with ethyl acetate and purified by Sephadex G-50 gel filtration on a dextran gel column,and then subjected to Trinice-SDS-PAGE electrophoresis.The results showed that the molecular weight of the bacteriocin L_(3) was around 4-5 kDa.The bacteriocin L_(3) was sensitive to proteases,but its activity was not affected by catalase,indicating that the bacteriocin L_(3) was proteinaceous substance.The bacteriocin L_(3) still kept high antibacterial activity after subjected to 60-100℃treatment for 20 min or 121℃treatment for 15 min and was stable over the range of pH 2-10.In addition,the bacteriocin L_(3) had good antibacterial activity against Gram-positive bacteria,Gram-negative bacteria and some fungi.In summary,the bacteriocin L_(3) produced by Lactobacillus plantarum L_(3) not only had good stability comparable to existing bacteriocins and the ability to kill Gram-positive bacteria,but also exerted an antibacterial effect on Gram-negative bacteria and some fungi.It is of great significance for the preservation of fruits,vegetables and dairy products,all of which are easily contaminated by fungi and spoilage.Therefore,this study was expected to provide new strain for the production of natural preservatives with broad-spectrum antibacterial properties.