摘要
为快速检测发酵豆乳中主要乳酸菌植物乳杆菌、副干酪乳杆菌含量,建立实时荧光定量聚合酶链式反应检测方法。根据植物乳杆菌、副干酪乳杆菌保守区域设计特异性引物和探针,验证建立的实时荧光定量PCR法的特异性、灵敏度和重复性,并与国标法进行比较。结果表明:引物特异性强,实时荧光定量PCR法特异性及重复性较好;植物乳杆菌和副干酪乳杆菌的检验灵敏度分别达到1.3×10^-4和1.0×10^-5 ng·μL^-1。分别建立植物乳杆菌和副干酪乳杆菌标准菌株的实时荧光定量PCR检验法的标准曲线,得出R2分别为0.994 3和0.999 6,表明线性关系较好,可进行发酵豆乳中2种菌株含量的检测,测得供试发酵豆乳中植物乳杆菌与副干酪乳杆菌比例为4∶1。实时荧光定量PCR法测得发酵豆乳中乳酸菌总量为(5.5±0.26)×10^9 CFU·mL^-1,国标法检测为(5.3±0.43)×10^9 CFU·mL^-1,2种方法检测结果无差异(P>0.05),表明建立的实时荧光定量PCR方法可快速、准确地检测出发酵豆乳中植物乳杆菌和副干酪乳杆菌的含量。
In order to detect the contents of Lactobacillus plantarum and Lactobacillus paracei in fermented soybean milk rapidly, we established a real-time fluorescence quantitative polymerase chain reaction method. Specific primers and probes were designed according to the conserved regions of Lactobacillus plantarum and Lactobacillus paracei, and the established real-time fluorescence quantitative PCR method was verified for specificity, sensitivity and repeatability, and we compared it with the national standard method. The results showed that the specificity of the primer was strong, and the specificity and repeatability of real-time fluorescence quantitative PCR were stable. The sensitivity of Lactobacillus plantarum and Lactobacillus paracei was 1.3×10^-4 and 1.0×10^-5 ng·μL^-1, respectively. The standard curve of real-time fluorescence quantitative PCR was established for the standard strains of Lactobacillus plantarum and Lactobacillus parmesei, and the R2 was 0.994 3 and 0.999 6, respectively, indicating a good linear relationship, and the content of the two strains in fermented soybean milk could be detected. The ratio of Lactobacillus plantarum and Lactobacillus parmesei in fermented soybean milk was 4∶1. The total amount of lactic acid bacteria in fermented soybean milk was(5.5±0.26) ×10^9 CFU·mL^-1 by real-time fluorescence quantitative PCR, and was(5.3±0.43) ×10^9 CFU·mL-^1 by national standard method. There was no difference between the two methods(P> 0.05), indicating that the established real-time fluorescence quantitative PCR method could quickly and accurately detect the contents of Lactobacillus plantarum and Lactobacillus paracei in fermented soybean milk.
作者
孙畅
安彬
杨柳
王国超
冯时荣
于寒松
王玉华
任大勇
SUN Chang;AN Bin;YANG Liu;WANG Guo-chao;FENG Shi-rong;YU Han-song;WANG Yu-hua;REN Da-yong(School of Food Science and Engineering,Jilin Agricultural University/National Processing Laboratory for Soybean Industry and Technology,Changchun 130118,China)
出处
《大豆科学》
CAS
CSCD
北大核心
2020年第3期464-471,共8页
Soybean Science
基金
现代农业产业技术体系建设专项(CARS-04)
吉林省教育厅“十三五”科学技术项目(JJKH20190926KJ)
国家重点研发项目(2017YFE0105400)
吉林农业大学大学生科技创新基金(2018088)。
关键词
发酵豆乳
植物乳杆菌
副干酪乳杆菌
实时荧光定量PCR
Fermented soybean milk
Lactobacillus plantarum
Lactobacillus paracasei
Quantitative real-time fluorescence PCR
