产气荚膜梭菌芽孢萌发剂筛选及其萌发特性研究

Screening of germinants for inducing the germination of Clostridium perfringens spores and study on its germination characteristics

目的 研究产气荚膜梭菌(Clostridium perfringens,C.perfringens)芽孢的萌发特性。方法 本研究以C.perfringens芽孢为研究对象,研究L-丙氨酸、L-谷氨酰胺、L-赖氨酸、L-天冬酰胺(L-asparagine)、氯化钾(KCl)、钙离子与2,6-吡啶二羧酸(pyridine-2,6-dicarboxylic acid,DPA)的螯合物(a chelate of Ca^(2+)with DPA,Ca^(2+)-DPA)、十二烷胺、L-asparagine和KCl的混合物(a mixture of L-asparagine and KCl,AK)、L-asparagine、D-葡萄糖、D-果糖和KCl的混合物(a mixture of L-asparagine,D-glucose,D-fructose and KCl,AGFK)、脑心浸液肉汤(brain heart infusion broth,BHI) 10种萌发剂对芽孢萌发的影响,选取处理后芽孢DPA释放量、热抗损失、芽孢溶液吸光度(optical density at 600 nm,OD_(600))及微观图像的变化来表征芽孢萌发,筛选出诱导C.perfringens芽孢萌发效果最好的萌发剂,并对其浓度进一步优化。结果 AK是诱导C.perfringens芽孢萌发效果最好的萌发剂;100 mmol/L AK与C.perfringens芽孢共孵育120 min时,C.perfringens芽孢溶液OD_(600)下降至49.42%,88.56%的DPA从芽孢核内释放,1.76个log芽孢被随后的湿热处理杀灭,在相差显微镜下由“亮相”变成“暗相”状态的芽孢数量最多;比较不同浓度的AK诱导芽孢萌发的效果,发现150 mmol/L AK诱导C.perfringens芽孢萌发的效果最好,其萌发率达63.17%。结论 AK诱导C.perfringens芽孢萌发的效果较好,为揭示C.perfringens芽孢的萌发机制奠定理论基础,同时为更高效、定向地防控肉制品中的C.perfringens提供科学依据。

Objective To study the germination characteristics of Clostridium perfringens (C.perfringens)spores.Methods This paper took C.perfringens spores as the research object,the effects of 10 kinds of germinants[L-alanine,L-glutamine,L-lysine,L-asparagine,potassium chloride (KCl),a chelate of Ca^(2+)with pyridine-2,6-dicarboxylic acid (DPA)(Ca^(2+)-DPA),dodecylamine,a mixture of L-asparagine and KCl (AK),a mixture of L-asparagine,D-glucose,D-fructose and KCl (AGFK),and brain heart infusion broth (BHI) on germination of C.perfringens spores were studied.The release of DPA,the heat resistance loss,the absorbance of spore solution(optical density at 600 nm,OD_(600)) and microscopic image changes of spores after treatment were selected to characterize spore germination,and the germinant with the best effect on inducing C.perfringens spore germination was screened out and its concentration was further optimized.Results The results showed that AK was the most effective germinant to induce germination of C.perfringens spores;when 100 mmol/L AK was incubated with C.perfringens spores for 120 min,the OD_(600)of C.perfringens spore solution decreased to 49.42%,88.56%DPA was released from spore core,and 1.76 log spores were killed by subsequent wet heat treatment.The number of spores changed from“bright phase”to“dark phase”under the phase contrast microscope was the highest.To compare the effect of different concentrations of AK in inducing spore germination,it was found that 150 mmol/L AK had the best effect on germination of C.prerfringens spores,with a germination rate of 63.17%.Conclusion AK has a good effect in inducing C.prerfringens spore germination,which lays a theoretical foundation for revealing the germination mechanism of C.perfringens spores,and provides scientific basis and support for more efficient and targeted prevention and control of C.perfringens in meat products.