Scariot Mirella Crhistine, Venturelli Gustavo Luiz, Prudêncio Elane Schwinden, Arisi Ana Carolina Maisonnave
CAL CCA UFSC, Food Science and Technology Department, Federal University of Santa Catarina, Rod. Admar Gonzaga, 1346, 88034-001 Florianópolis, SC, Brazil.
CAL CCA UFSC, Food Science and Technology Department, Federal University of Santa Catarina, Rod. Admar Gonzaga, 1346, 88034-001 Florianópolis, SC, Brazil.
Int J Food Microbiol. 2018 Jan 2;264:1-7. doi: 10.1016/j.ijfoodmicro.2017.10.021. Epub 2017 Oct 18.
Propidium monoazide (PMA) coupled with qPCR has been successfully used for specific quantification of viable bacteria cells in diverse matrices food. The present study aimed to develop PMA-qPCR assay for quantification of Lactobacillus paracasei viable cells in probiotic yoghurt. L. paracasei grown in culture medium was submitted to heat treatment at 60°C for different periods of time and probiotic yoghurt containing L. paracasei were prepared and stored at 4°C for 30days. The viable cells were quantified using qPCR and PMA-qPCR assays targeting tuf gene and also by plate counting. Standard curves were prepared and mean efficiency obtained was 94% and 96% (R>0.98) to L. paracasei in culture medium and probiotic yoghurt stored one day, respectively. The limit of detection (LOD) for both samples was 10 genome copies, corresponding to 32.1pg of DNA. For viable cells quantification, standard curves Cq versus log CFU were plotted using mean CFU by plate counting of L. paracasei grown in culture medium and probiotic yoghurt. Results obtained for L. paracasei heat-treated cells were concordant by PMA-qPCR and plate count, CFU decreased as the heat treatment time increased, while qPCR count remained constant. L. paracasei enumerations obtained by qPCR for probiotic yoghurt stored one day and 30days were higher than enumerations by PMA-qPCR for the same samples. The plate count values were similar to CFU values obtained by PMA-qPCR. These results showed that PMA-qPCR is a powerful approach compared with culture-dependent methods for quantification of L. paracasei viable cells in yoghurt. PMA-qPCR allowed reliable obtained results much faster than plate counting.
单叠氮碘化丙啶（PMA）与定量聚合酶链反应（qPCR）联用已成功用于多种食品基质中活菌细胞的特异性定量。本研究旨在开发用于定量益生菌酸奶中副干酪乳杆菌活菌细胞的PMA-qPCR检测方法。将在培养基中生长的副干酪乳杆菌在60°C下进行不同时间的热处理，制备含有副干酪乳杆菌的益生菌酸奶，并在4°C下储存30天。使用针对tuf基因的qPCR和PMA-qPCR检测方法以及平板计数法对活菌细胞进行定量。制备标准曲线，培养基中副干酪乳杆菌和储存一天的益生菌酸奶中获得的平均效率分别为94%和96%（R>0.98）。两个样品的检测限（LOD）均为10个基因组拷贝，相当于32.1 pg的DNA。为了进行活菌细胞定量，使用培养基中生长的副干酪乳杆菌和益生菌酸奶平板计数的平均菌落形成单位（CFU）绘制Cq与log CFU的标准曲线。通过PMA-qPCR和平板计数获得的副干酪乳杆菌热处理细胞的结果一致，随着热处理时间的增加CFU减少，而qPCR计数保持不变。qPCR对储存一天和30天的益生菌酸奶中副干酪乳杆菌的计数高于相同样品的PMA-qPCR计数。平板计数值与PMA-qPCR获得的CFU值相似。这些结果表明，与依赖培养的方法相比，PMA-qPCR是定量酸奶中副干酪乳杆菌活菌细胞的有力方法。PMA-qPCR比平板计数能更快地获得可靠结果。
