摘要
采用叠氮溴化丙锭(propidium monoazide,PMA)结合实时荧光定量PCR对乳制品中活菌DNA进行定量分析,建立一种快速而准确检测发酵乳品中植物乳杆菌P-8(Lactobacillus plantarum P-8)活菌数的新方法。通过对影响PMA作用的浓度、暗孵育和曝光时间等因素进行试验,确定最佳PMA处理方案。结果表明:L. plantarum P-8经80℃处理60 s,即为膜损伤菌;当PMA质量浓度为40μg/m L,暗孵育时间10 min,曝光时间为20 min时,PMA既不影响活菌DNA的PCR扩增,又能渗透进入细胞膜受损的死菌并抑制其PCR扩增;通过制备L.plantarum P-8质粒标准品并建立标准曲线,其表现出良好的线性关系,相关系数(R2)为0. 992 9,最低检测限为103CFU/m L,特异性良好。该方法为完善发酵乳产品益生菌活菌数的检测奠定了基础。
Propidium monoazide (PMA) was combined with real-time fluorescent quantitative PCR to detect DNA of living bacteria in milk products.A new,fast,and accurate method to detect Lactobacillus plantarum P-8 in fermented dairy products was developed.Influencing factors,such as concentrations,dark incubation,duration of illumination were determined to establish the optimal PMA treatment scheme.The results showed that after treating L.plantarum P-8 at 80℃ for 60s,they became membrane-damaging.When 40ug/mL PMA was used for 10min dark incubation,followed by espousing for 20min,PMA did not influence the living bacteria's DNA amplification.In addition,it inhibited DNA amplification of dead cells.A standard curve with a good linear relationship was set up based on L.plantarum P-8 plasmid standards.The relative coefficient of the curve was 0.9929,and the lowest detection limit was 10^3CFU/mL with good specificity.This method laid a foundation for improving the detection of viable probiotics in fermented milk products.
作者
韩之皓
郭帅
黄天
郑岩
王月娇
白梅
王记成
张和平
HAN Zhihao;GUO Shuai;HUANG Tian;ZHENG Yan;WANG Yuejiao;BAI Mei;WANG Jicheng;ZHANG Heping(Key Laboratory of Dairy Biotechnology and Engineering,Ministry of Education,Key Laboratory of Dairy Products Processing,Ministry of Agriculture,Inner Mongolia Agricultural University,Huhhot 010018,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2019年第1期183-189,共7页
Food and Fermentation Industries
基金
国家奶牛体系建设项目(CARS-36)
内蒙古自治区科技计划重点项目(201702070)
