SUN Chang,AN Bin,YANG Liu,et al.Establishment of Real-time Fluorescent Quantitative PCR Method for the Detection of Lactic Acid Bacteria in Fermented Soybean Milk[J].Soybean Science,2020,39(03):464-471.[doi:10.11861/j.issn.1000-9841.2020.03.0464]
发酵豆乳主要乳酸菌实时荧光定量PCR检测方法的建立
- Title:
- Establishment of Real-time Fluorescent Quantitative PCR Method for the Detection of Lactic Acid Bacteria in Fermented Soybean Milk
- Keywords:
- Fermented soybean milk; Lactobacillus plantarum; Lactobacillus paracasei; Quantitative real-time fluorescence PCR
- 文献标志码:
- A
- 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）×109 CFU?mL-1，国标法检测为（5.3±0.43）×109 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。
- Abstract:
- 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) ×109 CFU?mL-1 by real-time fluorescence quantitative PCR, and was (5.3±0.43) ×109 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.
参考文献/References:
［1］李学莉,胡海娥，张金桃，等.乳酸菌发酵豆乳研究进展［J］. 食品工业科技, 2016, 37(12): 385-390.（Li X L, Hu H E, Zhang J T, et al. Progress in researches on lactic acid bacteria-fermented soymilk［J］. Science and Technology of Food Industry, 2016, 37(12): 385-390.）［2］Zhang X L, Wu Y F, Wang Y S, et al．The protective effects of probiotic-fermented soymilk on high-fat diet-induced hyperlipidemia and liver injury［J］. Journal of Functional Foods, 2017, 30: 220-227.［3］叶春苗.发酵酸豆乳生产工艺及问题研究［J］.农业科技与装备, 2016(4): 33-34. (Ye C M. Research on manufacturing technique and problems of fermentation sour soybean milk［J］. Agricultural Science and Technology and Equipment, 2016(4): 33-34.)［4］沈建华. 基于植物乳杆菌的发酵豆乳制品研究［J］.食品工业, 2017, 38(7): 216-218. (Shen J H. Processing research of fermented soybean milk products based on Lactobacillus plantarum［J］.The Food Industry, 2017, 38(7): 216-218.)［5］Alan Y, Topalcengiz Z, D〖AKgˇD〗rak M. Biogenic amine and fermentation metabolite production assessments of Lactobacillus plantarum isolates for naturally fermented pickles［J］. LWT-Food Science and Technology, 2018, 98: 322-328.［6］舒梨, 刘苑皓, 何义国, 等. 副干酪乳杆菌发酵冷吃兔［J］. 食品工业, 2019, 40(4): 203-208. (Shu L, Liu Y H, He Y G, et al. Fermentation condition of piquancy rabbit meat with Lactobacillus paracasei［J］.The Food Industry, 2019,40(4): 203-208.）［7］张晨曦，贺稚非. 乳酸菌细菌素研究进展及其在肉制品防腐保鲜领域的应用［J］. 食品与发酵工业, 2017, 43(7): 271-277.（Zhang C X, He Z F. Research of bacteriocins from lactic acid bacteria and their applications in preservation of meat products［J］. Food and Fermentation Industries, 2017, 43(7): 271-277.）［8］唐雅茹, 于上富, 国立东, 等. 一株降胆固醇乳杆菌的筛选及其益生作用的研究［J］.食品工业科技, 2016, 37(1): 142-144, 152.（Tang Y R, Yu S F, Guo L D, et al. Screening and study on probiotic characteristics of a cholesterol- lowering Lactobacillu［J］. Science and Technology of Food Industry, 2016, 37(1):142-144, 152.）［9］Chiang S S, Pan T M. Beneficial effects of Lactobacillus paracasei sub sp. paracasei NTU 101 and its fermented products［J］. Applied Microbiology and Biotechnology, 2012, 93(3): 903-916.〖ZK)〗［10］Zhang H，Sun J，Liu X T，et al. Lactobacillus paracasei subsp. paracasei LC-01 positively modulates intestinal microflora in healthy young adultes［J］. Journal of Microbiology, 2013, 51(6):777-782.［11］Arasu M V, Al-Dhabi N A, Ilavenil S, et al. In vitro importance of probiotic Lactobacillus plantarum related to medical field［J］.Saudi Journal of Biological Sciences, 2016, 23(1): 6-10.［12］张莉丽, 崔宪, 马微, 等. 6株乳酸菌在豆乳中的发酵特性［J］. 食品科学, 2014, 35(15): 141-144.（Zhang L L, Cui X, Ma W, et al. Fermentation characteristics of different lactic acid bacteria in soymilk［J］. Food Science, 2014,35(15):141-144.）［13］陈晓华, 李建周, 董艺凝, 等. 豆乳发酵饮料的研制［J］. 食品研究与开发, 2014, 35(15): 57-60. (Chen X H, Li J Z, Dong Y N, et al. The development of fermented soybean-yoghurt［J］.Food Research and Development, 2014,35(15): 57-60.）［14］陈红霞. 发酵豆乳的研制［J］. 当代畜禽养殖业, 2012(11): 23-31.（Chen H X. Development of fermented soybean milk ［J］. Modern Animal Husbandry, 2012(11): 23-31.）［15］中华人民共和国国家卫生和计划生育委员会. 食品安全国家标准—饮料［S］.GB 7101-2015，北京: 中国标准出版社, 2015.（National Health and Family Planning Commission of the people′s Republic of China. National food safety standard: Beverage［S］. GB 7101-2015，Beijing: China Standard Press, 2015.）［16］张娜娜, 刘洋, 俞漪, 等.乳酸菌饮料中嗜酸乳杆菌的实时荧光定量PCR检测方法［J］. 食品科学, 2019, 40(8): 27-32.（Zhang N N, Liu Y, Yu Y, et al. Real-time fluorescent quantitative PCR method for detection of Lactobacillus acidophilus in probiotic beverages［J］. Food Science, 2019, 40(8): 27-32.）［17］姜鸿瑞, 韩紫音, 夏海磊, 等. 荧光定量PCR检测原料乳中粘质沙雷氏菌［J］. 中国奶牛, 2019(5): 48-51. (Jiang H R, Han Z Y, Xia H L, et al. Fluorescence quantitative PCR detection of Serratia marcescens in raw milk［J］. China Dairy Cattle, 2019(5): 48-51.)［18］李文学, 肖瑞刚, 吕苗苗, 等. 葡萄霜霉病菌实时荧光定量PCR检测体系的建立和应用［J］.中国农业科学, 2019, 52(9): 1529-1540. (Li W X, Xiao R G, Lyu M M, et al. Establishment and application of real-time PCR for quantitatively detecting Plasmopara viticola in Vitis vinifera［J］. Scientia Agricultura Sinica, 2019,52(9):1529-1540.）［19］Tyug T S, Prasad K N, Ismail A. Antioxidant capacity, phenolics and isoflavones in soybean by-products［J］. Food Chemistry, 2010, 123(3): 583-589.［20］Guo Z H, Fang H, Xia Z S, et al. Detection of Lactobacillus acidophilus in fermented material by real-time fluorescent quantitative PCR［J］. Animal Husbandry and Feed Science, 2016, 8(1):54-57.［21］包秋华, 李梅花, 徐洁, 等. 一种检测发酵乳中嗜酸乳杆菌的定量定性方法［J］. 食品研究与开发, 2012, 33(11): 153-155.（Bao Q H, Li M H, Xu J, et al. Study on the methods of qualitative and quantitative detection L. acidophilus in the fermented milk［J］. Food Research and Development, 2012, 33(11): 153-155.）［22］牛犇, 洪斌, 穆丽丽, 等. 荧光定量PCR在预测微生物学中的应用［J］. 食品工业科技, 2019, 40(5): 275-282. (Niu B, Hong B, Mu L L, et al. Application of fluorescent quantitative PCR in predictive microbiology［J］. Science and Technology of Food Industry, 2019, 40(5): 275-282.)［23］韦兰芳. 关于荧光定量关于荧光定量PCR在预测微生物学中的运用分析［J］. 现代农业研究, 2019(1): 111-112. (Wei L F. Application and analysis of fluorescent quantitative PCR in predicting microbiology［J］. Modern Agriculture Research, 2019(1): 111-112.）
备注/Memo