Comparison of real-time PCR assay and plate count for Lactobacillus paracasei enumeration in yoghurt

Abstract

Lactobacillus paracasei is a mesophilic lactic acid bacterium technologically active in food fermentation. Culture-independent methods have rapidly been recognized as a valuable alternative to culture-dependent methods for lactic acid bacteria enumeration. In the present work, the efficacy of different protocols to extract DNA from yoghurt were compared, real-time PCR (qPCR) targeting tuf gene for L. paracasei enumeration was evaluated, and qPCR and plate counts of L. paracasei in yoghurt samples were compared. Total DNA concentrations from commercial yoghurts were higher using DNAzol method 2 than using the other tested methods. Standard curves presented suitable mean efficiency values of 91 % (pure L. paracasei strain CTT 7501), 95 % (pure L. paracasei strain FNU), and 103 % (yoghurt with L. paracasei strain FNU). Limit of detection is 3 log DNA copy number, corresponding to 2.78 log CFU, a suitable range of CFU enumeration for probiotic bacteria in yoghurt samples, considering that they should be present in large amounts. The L. paracasei (CFU) enumerated by qPCR were compared to culturable L. paracasei enumerated by plate counts at 7, 14, 21, and 28 days of yoghurt manufacture. Differences between qPCR and plate counts were observed only 28 days after yoghurt preparation, counts were similar at 7, 14, and 21 days. In conclusion, this qPCR assay is a useful and rapid tool to enumerate L. paracasei in yoghurt, although it does not distinguish dead and viable cells.

Dominant Enterobacteriaceae in tempeh were primarily originated from soybean

“…The amplification reaction was done by using CFX96 Real-Time Detection System (Bio-Rad Laboratories Pte. Ltd., Singapore) in the following conditions: pre-denaturation at 95 °C for 5 min, 40 cycles of denaturation at 95 °C for 15 s, annealing at 59 °C for 30 s, and elongation at 72 °C for 60 s. Bacterial population of Enterobacteriaceae (CFU/g) from the sample was then quantified by using a formula based on Ilha et al (2015).…”

Tempeh flour as an excellent source of paraprobiotics

“…Next, the Melt curve analysis was conducted at 65°C to 95°C. Finally, LAB and Enterobacteriaceae (CFU/g) were calculated by the following equation (Ilha et al 2016);…”

“…The reports of previous research results with a detection limit value of 4 log CFU were used to analyze LAB and Enterobacteriaceae populations in tempeh (Erdiansyah et al 2021;Ilham et al 2021). In several reports, the LAB detection limit value obtained was 2.78 log CFU of L. paracasei from yogurt (Ilha et al 2016), 2-3 log CFU of L. plantarum and L. fermentum in cocoa fruit (Schwendimann et al 2015). Then a detection limit of 2-4 log CFU was reported by Takahashi et al (2017) to count Enterobacteriaceae such as Citrobacter freundii, Enterobacter aerogenes, E. coli, Proteus mirabilis, and K. pneumoniae.…”

Microfluidic One-Pot Digital Droplet FISH Using LNA/DNA Molecular Beacons for Bacteria Detection and Absolute Quantification

“…In contrast to culture-based methods, rapid molecular methods can detect both culturable and non-culturable bacteria without an extended incubation time. Among the molecular methods, quantitative real-time polymerase chain reaction (real-time PCR or qPCR) has been demonstrated to be much less time-consuming for identifying and enumerating microbes from complex backgrounds [10,11]. However, DNA extraction is a crucial step in qPCR [12,13].…”