二氧化碳联合益生菌对猪肉中单增李斯特菌抗性的影响及其作用机制

Effect of CO_(2)combined with probiotic on resistance of Listeria monocytogenes and its action mechanism

人类患李斯特菌病的概率和单增李斯特菌进入宿主感染周期的剂量紧密相关,对不利环境的抗性影响了单增李斯特菌进入宿主感染周期的剂量。前期研究结果表明60%CO_(2)+20%O_(2)+20%N_(2)气调包装结合植物乳杆菌(Lactobacillus plantarum)处理(以下简称CO_(2)-LP)可以抑制单增李斯特菌的生长,但是对其抗性的影响尚待探究。该研究旨在探究CO_(2)-LP处理对单增李斯特菌抗性的影响及其作用机制。在整个货架期内,研究CO_(2)-LP处理前后猪肉中单增李斯特菌对55℃(温和热处理)和65℃(中低温处理)热处理、唾液、胃液(强酸)和肠液(高渗透压)的耐受性。用实时荧光定量PCR方法检测压力调控基因sigB的相对表达量,研究单增李斯特菌抗性变化的机制。结果表明,13℃贮存3 d时,55℃和65℃热处理后,CO_(2)-LP处理组单增李斯特菌每分钟的减少量分别是对照组的2.47倍和1.55倍;模拟胃液过程后,CO_(2)-LP组单增李斯特菌的减少量是对照组的3.31倍;模拟肠液过程后,对照组单增李斯特菌增加了0.09 lg CFU/g,而60%CO_(2)-LP组减少了0.84 lg CFU/g。在25℃贮存的整个货架期内,CO_(2)-LP处理显著(P<0.05)降低了单增李斯特菌的热耐受性、胃酸耐受性和肠液耐受性,但是对唾液耐受性无显著(P≥0.05)影响。和对照组相比,在13℃贮存3 d,25℃贮存12 h和24 h时,sigB的相对表达量显著下调(P<0.05),说明CO_(2)-LP处理引起压力调控基因sigB下调是单增李斯特菌抗性降低的主要原因。该研究为CO_(2)联合益生菌处理应用到食品中降低单增李斯特菌危害、进而提高食品的微生物安全提供理论支持和科学依据。

The dose of Listeria monocytogenes entering host infection cycle plays an important role in the probability of human suffering listeriosis,and the resistance of L.monocytogenes to adverse environment is the main factor affecting the dose of the bacterium entering host infection cycle.In early stage of the research,it is demonstrated that the combination of 60%CO_(2)+20%O_(2)+20%N_(2) modified atmosphere packaging and Lactobacillus plantarum treatment(CO_(2)-LP)could inhibit the growth of L.monocytogenes.However,the effect of CO_(2)-LP on the resistance of L.monocytogenes has not been investigated.This paper aimed to study the effect of CO_(2)-LP on the resistance of L.monocytogenes and its action mechanism.During the shelf-life,the effect of CO_(2)-LP on the resistance of L.monocytogenes to 55℃(mild thermal treatment)and 65℃(medium thermal treatment)thermal process,to simulated salivary fluid(SSF),to simulated gastric fluid(SGF,strong acid)and simulated intestinal fluid(SIF,high osmolality)was evaluated.To explore the potential resistance mechanism,the relative expression levels of the sigB gene,a gene involved in regulating large numbers of stress-related genes,was analyzed by the reverse transcription quantitative real-time PCR(qPCR).Results indicated that at 13℃,on day 3,the reduction of L.monocytogenes per minute in T_(CO_(2)-LP) after 55℃(65℃)treatment was 2.47(1.55)times as many as T_(C).After SGF phase,the reduction of L.monocytogenes in T_(CO_(2)-LP) was 3.31-fold as many as T_(C).After SIF phase,the number of L.monocytogenes in T_(C) increased by 0.09 lg CFU/g,but reduced 0.84 lg CFU/g in T_(CO_(2)-LP).Similarly,CO_(2)-LP treatment significantly(P<0.05)reduced the resistances of L.monocytogenes to thermal process,to SGF and to SIF at 25℃,while had no significant effect(P≥0.05)on the resistances of L.monocytogenes to SSF.Compared with T_(C),the sigB was down-expressed(P<0.05)on day 3 at 13℃,in 12 h and 24 h at 25℃,which indicated that the down-regulation of sigB was the main reason for the decreased resistance of L.monocytogenes.This study provides theoretical support and scientific basis for the application of CO_(2)-LP in food to reduce the harm of L.monocytogenes and thereby improve the microbial safety of food.