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40 μg/mL,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103 CFU/mL,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 60 s, they became membrane-damaging. When 40 ug/mL PMA was used for 10 min dark incubation, followed by espousing for 20 min, 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 0.992 9, and the lowest detection limit was 103 CFU/mL with good specificity. This method laid a foundation for improving the detection of viable probiotics in fermented milk products.
[1] PEREZCANO F J,DONG H,YAQOOB P. <i>In vitro</i> immunomodulatory activity of <i>Lactobacillus fermentum</i> CECT5716 and <i>Lactobacillus salivarius</i> CECT5713: two probiotic strains isolated from human breast milk[J].Immunobiology,2010,215(12):996-1 004.<br />
[2] WANG Z,BAO Y,ZHANG Y,et al.Effect of soymilk fermented with <i>Lactobacillus plantarum</i> P-8 on lipid metabolism and fecal microbiota in experimental hyperlipidemic rats[J].Food Biophysics,2013,8(1):43-49.<br />
[3] BAO Y,WANG Z,ZHANG Y,et al.Effect of <i>Lactobacillus plantarum</i>, P-8 on lipid metabolism in hyperlipidemic rat model[J]. European Journal of Lipid Science & Technology,2012,114(11):1 230-1 236.<br />
[4] 王记成.基于转录组学和蛋白质组学对益生菌<i>Lactobacillus casei</i> Zhang在牛乳和豆乳中生长机理的研究[D].呼和浩特:内蒙古农业大学, 2012.<br />
[5] S C I,SHALLCROSS J A,MACKEY B M.Effect of stress treatments on the detection of <i>Listeria monocytogenes</i> and enterotoxigenic <i>Escherichia coli</i> by the polymerase chain reaction[J].Journal of Applied Microbiology,1994,77(1):73-79.<br />
[6] NOCKER A,CAMPER A K.Novel approaches toward preferential detection of viable cells using nucleic acid amplification techniques[J].Fems Microbiology Letters,2009,291(2):137-142.<br />
[7] CHANG B,TAGURI T,SUGIYAMA K,et al.Comparison of ethidium monoazide and propidium monoazide for the selective detection of viable <i>Legionella</i> cells[J].Japanese Journal of Infectious Diseases,2010,63(2):119.<br />
[8] 张佳超,王丽凤,高鹏飞.一株可以在人肠道中定植并繁殖的益生菌及其检测方法:中国,102747012 A[P]. 2012-05-15.<br />
[9] 王力均,谭强来,朱江.等.应用PMA-qPCR方法快速准确检测发酵乳制品中副干酪乳杆菌活菌的研究[J].中国微生态学杂志, 2013,25(1):1-4.<br />
[10] NOCKER A,SOSSA K E,CAMPER A K.Molecular monitoring of disinfection efficacy using propidium monoazide in combination with quantitative PCR[J].Journal of Microbiological Methods,2007,70(2):252-260.<br />
[11] SHAO Y,WANG Z,BAO Q,et al.Application of propidium monoazide quantitative real-time PCR to quantify the viability of <i>Lactobacillus delbrueckii</i> ssp. <i>bulgaricus</i>[J].Journal of Dairy Science,2016,99(12):9 570.<br />
[12] NOCKER A,CAMPER AK.Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide[J].Applied and Environmental Microbiology, 2006,72(3):1 997-2 004.<br />
[13] TAKASHI S,KEN-ICHIRO I,TIAN Q,et al.Method to detect only live bacteria during PCR amplification[J].Journal of Clinical Microbiology,2008,7:2 305-2 313.<br />
[14] SALMA M,ROUSSEAUX S,SEQUEIRA-LE G A,et al.Characterization of the viable but nonculturable (VBNC) state in <i>Saccharomyces cerevisiae</i>[J].Plos One,2013,8(10):e77 600.<br />
[15] FURET J P,QUéNéE P,TAILLIEZ P.Molecular quantification of lactic acid bacteria in fermented milk products using real-time quantitative PCR.[J].International Journal of Food Microbiology,2004,97(2):197-207.<br />
[16] MASCO L,VANHOUTTE T,TEMMERMAN R,et al.Evaluation of real-time PCR targeting the 16S rRNA and recA, genes for the enumeration of bifidobacteria in probiotic products[J].International Journal of Food Microbiology, 2007,113(3):351-357.<br />
[17] SHEU S J,HWANG W Z,CHIANG Y C,et al.Use of tuf gene-based primers for the PCR detection of probiotic Bifidobacterium species and enumeration of bifidobacteria in fermented milk by cultural and quantitative real-time PCR methods[J].Journal of Food Science,2010, 75(8):521-527.<br />
[18] SHEU S J,HWANG W Z,CHEN H C,et al.Development and use of tuf gene-based primers for the multiplex PCR detection of <i>Lactobacillus acidophilus, Lactobacillus casei</i> group, <i>Lactobacillus delbrueckii</i>, and <i>Bifidobacterium longum</i> in commercial dairy products[J].Journal of Food Protection,2009,72(1):93-100.<br />
[19] GARC A-CAYUELA T,TABASCO R,PEL EZ C,et al.Simultaneous detection and enumeration of viable lactic acid bacteria and bifidobacteria in fermented milk by using propidium monoazide and real-time PCR[J].International Dairy Journal,2009,19(6):405-409.<br />
[20] FALENTIN H,POSTOLLEC F,PARAYRE S,et al.Specific metabolic activity of ripening bacteria quantified by real-time reverse transcription PCR throughout emmental cheese manufacture [J].International Journal of Food Microbiology,2010,144:10-19.