Real-time monitoring of microbial dynamics during fermentation is essential for wine quality control. A method could distinguish between dead and live microbes for Saccharomyces cerevisiae was developed, which combines the fluorescent dye propidium monoazide (PMA) with CELL-qPCR. It also could detect the quantity of microbes efficiently and rapidly without DNA extraction during wine fermentation. The method was optimized for cell wall breakage, PMA treatment concentration, PMA detection sensitivity and multiple conditions of sample pretreatment in wine environment. The results showed that the optimum method can accurately quantify 103-107 CFU/mL of the target strain in multiple matrices; when the concentration of dead microorganisms in the system was 104 times higher than that of live microorganisms, there was an error of 0.5 lg CFU/mL; the optimized PMA-CELL-qPCR method was used to monitor the dynamic changes of S. cerevisiae during the fermentation of Cabernet Sauvignon wine were consistent with the plate count method. In conclusion, the live bacteria quantification method developed for PMA-CELL-qPCR in S. cerevisiae wines is accurate in quantification and simple in operation, and can be used as a mean to accurately monitor microbial dynamics in wine and other fruit wines.
WEI Bo
,
WANG Jie
,
CHEN Yixin
,
WANG Chunguang
,
CHEN Zhuojun
,
ZHANG Bolin
,
ZHU Baoqing
. Development and application of Saccharomyces cerevisiae viable cell quantitative method in wine fermentation based on PMA-CELL-qPCR[J]. Food and Fermentation Industries, 2022
, 48(17)
: 79
-86
.
DOI: 10.13995/j.cnki.11-1802/ts.030337
[1] ALBERGARIA H, ARNEBORG N.Dominance of Saccharomyces cerevisiae in alcoholic fermentation processes:Role of physiological fitness and microbial interactions[J].Applied Microbiology and Biotechnology, 2016, 100(5):2 035-2 046.
[2] BARTLE L, MITCHELL J G, PATERSON J S.Evaluating the cytometric detection and enumeration of the wine bacterium, Oenococcus oeni[J].Cytometry Part A, 2021, 99(4):399-406.
[3] NUNES DE LIMA A, MAGALHÃES R, CAMPOS F M, et al.Survival and metabolism of hydroxycinnamic acids by Dekkera bruxellensis in monovarietal wines[J].Food Microbiology, 2021, 93:103617.
[4] ROUSSEL C, GALIA W, LERICHE F, et al.Comparison of conventional plating, PMA-qPCR, and flow cytometry for the determination of viable enterotoxigenic Escherichia coli along a gastrointestinal in vitro model[J].Applied Microbiology and Biotechnology, 2018, 102(22):9 793-9 802.
[5] QUIRÓS C, HERRERO M, GARCÍA L A, et al.Quantitative approach to determining the contribution of viable-but-nonculturable subpopulations to malolactic fermentation processes[J].Applied and Environmental Microbiology, 2009, 75(9):2 977-2 981.
[6] FITTIPALDI M, NOCKER A, CODONY F.Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification[J].Journal of Microbiological Methods, 2012, 91(2):276-289.
[7] NOCKER A, CHEUNG C-Y, CAMPER A K.Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs.dead bacteria by selective removal of DNA from dead cells[J].Journal of Microbiological Methods, 2006, 67(2):310-320.
[8] LI T T, WANG J S, WANG Z Y, et al.Quantitative determination of mutton adulteration with single-copy nuclear genes by real-time PCR[J].Food Chemistry, 2021, 344:128622.
[9] COCOLIN L, MILLS D.Wine yeast inhibition by sulfur dioxide:A comparison of culture-dependent and independent methods[J].American Journal of Enology and Viticulture, 2003, 54:125-130.
[10] ZHANG S J, WANG L L, LU S Y, et al.A novel, rapid, and simple PMA-qPCR method for detection and counting of viable Brucella organisms[J].Journal of Veterinary Research, 2020, 64(2):253-261.
[11] TANTIKACHORNKIAT M, SAKAKIBARA S, NEUNER M, et al.The use of propidium monoazide in conjunction with qPCR and Illumina sequencing to identify and quantify live yeasts and bacteria[J].International Journal of Food Microbiology, 2016, 234:53-59.
[12] VAN FRANKENHUYZEN J K, TREVORS J T, LEE H, et al.Molecular pathogen detection in biosolids with a focus on quantitative PCR using propidium monoazide for viable cell enumeration[J].Journal of Microbiological Methods, 2011, 87(3):263-272.
[13] SOARES-SANTOS V, PARDO I, FERRER S.Cells-qPCR as a direct quantitative PCR method to avoid microbial DNA extractions in grape musts and wines[J].International Journal of Food Microbiology, 2017, 261:25-34.
[14] SOARES-SANTOS V, PARDO I, FERRER S.Improved detection and enumeration of yeasts in wine by Cells-qPCR[J].LWT, 2018, 90:90-97.
[15] LYU X C, LI Y, QIU W W, et al.Development of propidium monoazide combined with real-time quantitative PCR (PMA-qPCR) assays to quantify viable dominant microorganisms responsible for the traditional brewing of Hong Qu glutinous rice wine[J].Food Control, 2016, 66:69-78.
[16] XIONG T, CHEN J K, HUANG T, et al.Fast evaluation by quantitative PCR of microbial diversity and safety of Chinese Paocai inoculated with Lactobacillus plantarum NCU116 as the culture starter[J].LWT, 2019, 101:201-206.
[17] PAN Y, BREIDT F.Enumeration of viable Listeria monocytogenes cells by real-time PCR with propidium monoazide and ethidium monoazide in the presence of dead cells[J].Applied and Environmental Microbiology, 2007, 73(24):8 028-8 031.
[18] SHAO Y Y, WANG Z X, BAO Q H, et al.Application of propidium monoazide quantitative real-time PCR to quantify the viability of Lactobacillus delbrueckii ssp.bulgaricus[J].Journal of Dairy Science, 2016, 99(12):9 570-9 580.
[19] ANDORRÀ I, ESTEVE-ZARZOSO B, GUILLAMÓN J M, et al.Determination of viable wine yeast using DNA binding dyes and quantitative PCR[J].International Journal of Food Microbiology, 2010, 144(2):257-262.
[20] ZHAO Y K, CHEN H, LIU H M, et al.Quantitative polymerase chain reaction coupled with sodium dodecyl sulfate and propidium monoazide for detection of viable Streptococcus agalactiae in milk[J].Frontiers in Microbiology, 2019, 10:661.
[21] TAKAHASHI H, KASUGA R, MIYA S, et al.Efficacy of propidium monoazide on quantitative real-time PCR based enumeration of Staphylococcus aureus live cells treated with various sanitizers[J].Journal of Food Protection, 2018, 81(11):1 815-1 820.
[22] CAMPBELL G R, PROSSER J, GLOVER A, et al.Detection of Escherichia coli O157:H7 in soil and water using multiplex PCR[J].Journal of Applied Microbiology, 2001, 91(6):1 004-1 010.
