Artigo Acesso aberto Revisado por pares

Detection of culturable and viable but non-culturable cells of beer spoilage lactic acid bacteria by combined use of propidium monoazide and horA -specific polymerase chain reaction

2016; Wiley; Volume: 122; Issue: 1 Linguagem: Inglês

10.1002/jib.289

ISSN

2050-0416

Autores

Yang Deng, Junfeng Zhao, Huiping Li, Zhenbo Xu, Junyan Liu, Jingxia Tu, Tao Xiong,

Tópico(s)

GABA and Rice Research

Resumo

Journal of the Institute of BrewingVolume 122, Issue 1 p. 29-33 Research articleFree Access Detection of culturable and viable but non-culturable cells of beer spoilage lactic acid bacteria by combined use of propidium monoazide and horA-specific polymerase chain reaction Yang Deng, Corresponding Author Yang Deng State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 People's Republic of China College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, 510640 People's Republic of China Technical Centre, Zhujiang brewery Co. Ltd, No. 118, Modiesha Avenue, East Xingang Road, Guangzhou, 510308 People's Republic of ChinaCorrespondence to: Yang Deng and Tao Xiong, State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, People's Republic of China. Email: [email protected] and [email protected]Search for more papers by this authorJunfeng Zhao, Junfeng Zhao College of Food Science and Engineering, Henan University of Science and Technology, Tianjing Road, Luoyang, 471003 People's Republic of ChinaSearch for more papers by this authorHuiping Li, Huiping Li Technical Centre, Zhujiang brewery Co. Ltd, No. 118, Modiesha Avenue, East Xingang Road, Guangzhou, 510308 People's Republic of ChinaSearch for more papers by this authorZhenbo Xu, Zhenbo Xu College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, 510640 People's Republic of ChinaSearch for more papers by this authorJunyan Liu, Junyan Liu College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, 510640 People's Republic of ChinaSearch for more papers by this authorJingxia Tu, Jingxia Tu Technical Centre, Zhujiang brewery Co. Ltd, No. 118, Modiesha Avenue, East Xingang Road, Guangzhou, 510308 People's Republic of ChinaSearch for more papers by this authorTao Xiong, Corresponding Author Tao Xiong State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 People's Republic of ChinaCorrespondence to: Yang Deng and Tao Xiong, State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, People's Republic of China. Email: [email protected] and [email protected]Search for more papers by this author Yang Deng, Corresponding Author Yang Deng State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 People's Republic of China College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, 510640 People's Republic of China Technical Centre, Zhujiang brewery Co. Ltd, No. 118, Modiesha Avenue, East Xingang Road, Guangzhou, 510308 People's Republic of ChinaCorrespondence to: Yang Deng and Tao Xiong, State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, People's Republic of China. Email: [email protected] and [email protected]Search for more papers by this authorJunfeng Zhao, Junfeng Zhao College of Food Science and Engineering, Henan University of Science and Technology, Tianjing Road, Luoyang, 471003 People's Republic of ChinaSearch for more papers by this authorHuiping Li, Huiping Li Technical Centre, Zhujiang brewery Co. Ltd, No. 118, Modiesha Avenue, East Xingang Road, Guangzhou, 510308 People's Republic of ChinaSearch for more papers by this authorZhenbo Xu, Zhenbo Xu College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, 510640 People's Republic of ChinaSearch for more papers by this authorJunyan Liu, Junyan Liu College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, 510640 People's Republic of ChinaSearch for more papers by this authorJingxia Tu, Jingxia Tu Technical Centre, Zhujiang brewery Co. Ltd, No. 118, Modiesha Avenue, East Xingang Road, Guangzhou, 510308 People's Republic of ChinaSearch for more papers by this authorTao Xiong, Corresponding Author Tao Xiong State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 People's Republic of ChinaCorrespondence to: Yang Deng and Tao Xiong, State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, People's Republic of China. Email: [email protected] and [email protected]Search for more papers by this author First published: 04 January 2016 https://doi.org/10.1002/jib.289Citations: 9AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract Current methods of detecting beer spoilage lactic acid bacteria (LAB) are time-consuming and do not differentiate between viable and non-viable bacteria. In this study, a combination of the conventional polymerase chain reaction (PCR) and propidium monoazide (PMA) pretreatment has been described to circumvent the disadvantages. The horA-specific PMA-PCR described here identifies beer spoilage LAB based not on their identity, but on the presence of a gene that is shown to be highly correlated with the ability of LAB to grow in beer. The results suggest that the use of 20 µg/mL or less of PMA did not inhibit the PCR amplification of DNA derived from viable, but putatively non-culturable (VPNC) Lactobacillus acetotolerans. The minimum amount of PMA to completely inhibit the PCR amplification of DNA derived from dead L. acetotolerans cells was 1.5 µg/mL. The detection limit of established PMA-PCR assays was found to be 100 VPNC cells/reaction for the horA gene. Furthermore, the horA-specific PMA-PCR assays were subjected to 18 reference strains, representing 100% specificity with no false positive amplification observed. In conclusion, the use of horA-specific PMA-PCR allows for a substantial reduction in the time required for the detection of potential beer spoilage LAB and efficiently discriminates between live and dead cells. Copyright © 2016 The Institute of Brewing & Distilling Introduction Beer spoilage bacteria have long been a problem for brewers. Among the most problematic beer spoilers are several species of the Gram-positive genera Lactobacilli and Pediococci 1. The most frequently isolated beer spoilage lactic acid bacteria (LAB) include Lactobacillus brevis, Lactobacillus plantarum and Pediococcus damnosus 2. One major problem in the brewing industry is that some specific beer spoilage LAB such as Lactobacillus acetotolerans are difficult to grow in the traditional culture media upon primary isolation from brewery environments 3. In addition, L. acetotolerans can be hidden in beers or brewery environments by entry into a viable, but putatively non-culturable (VPNC) state according to our previous report 4. A VPNC state could be also induced in strong beer spoilage strains Lactobacillus lindneri and L. paracollinoides by beer subculture treatment 5. As a consequence, the brewer requires a rapid, accurate method as a quality control tool for screening samples before release into the marketplace. In order to shorten the detection time, several molecular methods have been developed for the detection of beer spoilage LAB, based on techniques such as the polymerase chain reaction (PCR) 6, 7. However, one of the drawbacks is the inability to differentiate between viable and non-viable cells owing to the persistence of DNA after cell death. To circumvent this problem, intercalating dyes such as ethidium monoazide (EMA) have been applied prior to PCR analysis, allowing a live/dead discrimination of some beer spoilage LAB 8. These dyes enter bacteria with damaged cell membranes and covalently bind to genomic DNA upon exposure to light. The bound DNA cannot be amplified by PCR, thus preventing the detection of dead cells 9, 10. Since EMA is known to penetrate viable cells to some extent, the chemical substance propidium monoazide (PMA) was subsequently tested for its ability to differentiate between viable and dead cells. Results for various bacterial species, for example, Listeria monocytogenes, Serratia marcescens and Staphylococcus aureus, indicated an advantage of PMA over EMA, probably owing to its reduced ability to penetrate live cells 9, 10. Unlike most Gram-positive bacteria, beer spoilage LAB are resistant to hop compounds and thus can spoil beer 1. It is thought that LAB undergo a multifactorial hop adaptation process involving changes in metabolism and morphology, as well as the more energy-dependent multidrug transporter, hop-efflux mechanisms such as horA 11. The wide and exclusive distributions of horA in various beer spoilage LAB species indicate the possibility of comprehensive species-independent detection of beer spoilage LAB with the genetic marker 6, 12, 13. Therefore, the aim of this present paper was to investigate the applicability of PMA-PCR targeting the horA gene to discriminate between viable and non-viable cells of several beer spoilage LAB. Materials and methods Bacterial strains A list of the bacterial species tested is provided in Table 1, with the strains comprising 10 lactobacilli (five species), three pediococci (two species) and five non-lactic acid bacteria (five species). All of the isolates employed in this study were stored in our laboratory previously. Among them, L. acetotolerans 2011–8 (CGMCC accession no. 7.150; China General Microbiological Culture Collection Centre) was originally isolated from finished beers 3 and used to optimize the PMA-PCR assays in subsequent experiments. Other isolates were included to evaluate the specificity of PMA-PCR detection in this study. These lactic acid bacteria were grown anaerobically in de Man Rogosa Sharpe (MRS) broth (Oxoid, UK) at 26 °C for 3–6 days, while the non-lactic acid bacteria were incubated at 37 °C and maintained in Luria–Bertani (LB) broth (Oxoid, UK) for 24 h. The VPNC cells of beer spoilage L. acetotolerans 2011–8 were induced and acquired by beer subculture treatment, as described previously 4. This VPNC state has not been obtained from other common beer spoilage species such as L. brevis, L. plantarum, L. casei and P. damnosus by the same treatment to date (from our unpublished data). Table 1. Bacterial strains, presence of genes and ability to grow in beer Isolate Origin horAa Growth (days)b 1:1c 1:99 1:999 0:1 Beer 1 Beer 2 Lactic acid bacteria Lactobacillus acetotolerans 2011-8 Brewery + + + ̶ + (11) ̶ L. acetotolerans 2011-11 Brewery + + + ̶ + (10) ̶ L. brevis CN003 Brewery + + + ̶ + (2) + (2) L. brevis CN094 Brewery + + + ̶ + (3) + (3) L. brevis CN134 Brewery + + + ̶ + (2) + (3) L. brevis CGMCC 1.2028 Cured meat ̶ ̶ ̶ ̶ ̶ ̶ L. casei CN007 Brewery + + + ̶ + (4) + (6) L. plantarum 11 Brewery + + + ̶ + (3) + (4) L. plantarum CGMCC 1.3919 Pickled cabbage ̶ ̶ ̶ ̶ ̶ ̶ L. rhamnosus CGMCC 1.2568 Milk ̶ ̶ ̶ ̶ ̶ ̶ Pediococcus damnosus SAT Brewery + + + ̶ + (7) + (8) P. damnosus CN004 Brewery + + + ̶ + (9) ̶ P. parvulus CGMCC 1.2696 Wine ̶ ̶ ̶ ̶ ̶ ̶ Non-lactic acid bacteria Bacillus subtilis CGMCC 1.3376 Soil ̶ ̶ ̶ ̶ ̶ ̶ Staphylococcus aureus CGMCC 1.18 Milking machine ̶ ̶ ̶ ̶ ̶ ̶ Enterococcus gallinarum CGMCC 1.9125 Unknown ̶ ̶ ̶ ̶ ̶ ̶ Escherichia coli O157:H7 CGMCC 1.2386 Human feces ̶ ̶ ̶ ̶ ̶ ̶ Salmonella enterica CGMCC 1.10603 Chicken ̶ ̶ ̶ ̶ ̶ ̶ a Determined by horA-specific PMA-PCR. b +, Visible turbidity in beer; ̶ , no visible turbidity in beer; the detection time is shown in parentheses (days). c Different mixtures of live and heat-killed cells in 1:1, 1:99, 1:999 and 0:1 ratios were subjected to horA-specific PMA-PCR analysis. Two lager beers were used in the growth experiments as described earlier 4. Beer 1 was a filter-sterilized 4% vol/vol alcohol beer, pH 5.0, containing an average of 7.8 bitterness units (BU), whereas beer 2 was a pasteurized 5% vol/vol alcohol beer, pH 4.5, containing an average of 10 BU. The 5 day cultures of each strain in MRS or LB broth (~105 cells/mL) were inoculated onto the apical surface of commercial bottled lager beers (330 mL) under sterile conditions at room temperature. Bottle headspaces were flushed with CO2 at a flow rate of 120 mL/min for approximately 3 min to remove the air. These bottles were then tightly recapped with metal lids and incubated at 26 °C and examined regularly for visible growth for up to 1 month. The presence of viable cells in contaminated beers was confirmed by the passive dye exclusion method 4 as mentioned later. Bacteria capable of growing in either beer were considered to be beer-spoilers. The ability of the 18 isolates to grow in beer is recorded in Table 1 for direct comparison with the results on presence or absence of the horA gene. Inactivation of bacterial cells and live/dead cellular viability assays The bacteria were heated at 90 °C in a water bath for 10 min. The resulting heat-treated samples were cooled to room temperature and the absence of viable cells determined by the passive dye exclusion method 4 using a Live/Dead BacLight bacterial viability kit (Molecular Probes, USA). Two fluorescent dyes, SYTO 9 and propidium iodide (PI), in this kit were used following the manufacturer's instructions to evaluate cell membrane integrity. SYTO 9 stain generally labels all bacteria in a population green, while PI penetrates only bacteria with damaged membranes and labels them red, that is, reducing the SYTO 9 stain fluorescence when both dyes are present. Cell samples were stained with the mixture of SYTO 9 (5 µm final concentration) and PI (30 µm) in 0.5 m sodium phosphate buffer at pH 7.0, and incubated in the dark at room temperature for 20 min. The stained cells were analysed under the Guava easyCyte 8HT flow cytometer (Guava Technologies Inc., USA) using blue line excitation at 488 nm. Results are expressed as the number of viable cells per mL of sample. DNA extractions, primer design, and PCR The nucleic acids were extracted from bacterial strains using the TIANamp Bacteria DNA kit (Tiangen Biotech, China) according to the manufacturer's instructions. The primer pairs specific to horA were designed as described by Haakensen et al. 6. The sequences of forward and reverse primers are 5′-AAATCTTAACCCTGCCGG-3′ and 5′-GCGGAACGGCGATAAACATA-3′ respectively, and amplify a 210 bp segment in the conserved region of the horA gene. Taq DNA polymerase and the reaction mixtures were supplied as a kit (TaKaRa Ex Taq, Takara Bio, Japan). PCR reactions were carried out in a PTC-100 Thermocycler (MJ Research, USA), and the particular cycling profile was performed as previously described 6. Amplicons were detected by electrophoresis in 2.0% agarose gels containing ethidium bromide. Digital images were obtained using a Spectroline Model 1000 Electronic Archival System (Spectronics Corp., USA). NIH Image 1.61 software was then used for relative quantitation of DNA bands. The mean values of the fluorescence intensities of bands were derived from triplicate independent assays. Determination of the maximum concentration of PMA that does not inhibit PCR from VPNC L. acetotolerans 2011–8 The intercalating dye PMA (catalogue no. 40013, Biotium Inc., Hayward, CA, USA) was dissolved in 20% dimethyl sulfoxide and stored at −20 °C in the dark until needed. One milliliter suspensions of the VPNC L. acetotolerans 2011–8 containing approximately 105 VPNC cells were transferred to a series of microcentrifuge tubes, respectively. The stock solution of PMA was added to cell suspensions at final concentrations of 1, 5, 10, 20, 30, 50, 75 and 100 µg/mL, respectively. Samples were then incubated for 10 min in the dark at room temperature before being placed in an iced cooling box. Subsequently, the tubes were exposed to a 500 W halogen lamp light source (PROmax-Vega, China) for 10 min unless otherwise indicated, at a distance of 20 cm to activate and photolyse the PMA. After photo-induced cross-linking, cells were pelleted at 10,000 g for 5 min prior to DNA isolation. PMA is potentially carcinogenic and was handled accordingly. Determination of the minimum concentration of PMA inhibiting PCR amplification from dead L. acetotolerans 2011–8 After heat treatment of cell suspensions of L. acetotolerans 2011–8 (1 mL) in microcentrifuge tubes at a density of 105 colony forming units (CFU)/mL, PMA was added to cell suspensions to final concentrations of 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0 and 3.0 µg/mL. The tubes were then placed in dark followed by agitation at room temperature for 10 min to allow the PMA to penetrate the heat-treated dead cells. They were then exposed to the halogen lamp as described above for viable cells. Optimization of light exposure time to active PMA Suspensions of the VPNC L. acetotolerans 2011–8 containing a total of 105 viable cells in microcentrifuge tubes were treated with PMA at a final concentration of 2.0 µg/mL as described above followed by immediate removal to the dark for 10 min. The tubes were then placed in an iced cooling box and subsequently exposed to light for 1, 5, 10, 15 and 20 min at a distance of 20 cm. Sensitivity of horA-specific PCR for detection of VPNC L. acetotolerans 2011–8 The detection limit of PCR assays targeting the horA was ascertained by minimal viable cell number of bacteria. Briefly, the culture of VPNC L. acetotolerans 2011–8 was diluted with sterile water for serial 10-fold, ranging from 102 to 108 viable cells/mL. A negative control was performed using sterile water instead of bacterial culture. DNA extractions and PMA-PCR were conducted as described above. Specificity of PMA-PCR assays targeting the horA The early-stationary-phase and heat-killed cells of each isolate presented in Table 1 were mixed in 1:1, 1:99, 1:999 and 0:1 ratios corresponding to 50, 99, 99.9 and 100% non-viable cells, respectively. The total number of viable plus non-viable cells in 1 mL volumes was kept constant at 105. The live/dead mixtures were treated with PMA at a final concentration of 2.0 µg/mL for 10 min in the dark, and then exposed to the halogen lamp at a distance of 20 cm for 10 min as described above. The resulting bacterial cells were further subjected to DNA extractions and PMA-PCR analysis as aforementioned. Statistical analysis Student's t-test was used to determine statistically significant differences between the mean of the log of genomic targets derived from PCR method and the mean of the log of viable cell number derived from viability assays with the use of the SAS system for Windows 6.12 software (SAS Institute Inc., Cary, NC, USA). A confidence interval at the 99% level (p < 0.01) was considered in all cases. Results Optimization of the conditions of PMA-PCR Firstly, when the VPNC cells of L. acetotolerans 2011–8 were treated with PMA at a concentration of 20 µg/mL or less, no significant inhibition of amplification of the target DNA occurred in the PMA-PCR procedure (Table 2). Secondly, the amplification of target DNA derived from heat-killed (non-viable) cells was completely inhibited when such cells were subjected to treatment with PMA at a concentration of 1.5 µg/mL or higher (Table 2). PMA at a concentration of 2.0 µg/mL was therefore ideally suitable for discrimination of DNA from a mixed population of viable and non-viable beer spoilage LAB by the PMA-PCR procedure. Additionally, the PMA (2.0 µg/mL) in the suspensions of VPNC L. acetotolerans 2011–8 was exposed to the halogen lamp for 1, 5, 10, 15 and 20 min. Inactivation of free PMA was achieved with light exposure from 1 to 20 min (Table 2), which was reflected in the absence of a decrease in DNA amplification with each of these light exposure times compared with the amplification of DNA from the control cells not treated with PMA. Light exposure of dead cells similarly treated with PMA for 1 min or longer completely prevented amplification. A light exposure period of 10 min was chosen for our subsequent standardized assay to ensure inactivation of free PMA that is capable of binding to DNA following cell lysis and thereby preventing amplification of target DNA from the viable cells. Finally, the optimal conditions of PMA-PCR were chosen as 2.0 µg/mL PMA and 10 min light exposure time (Table 2). The detection limit of the PCR assays was found to be 100 viable cells/PCR reaction (namely 105 living cells/mL bacterial culture) for the horA gene. Table 2. The optimization process of the horA-specific propidium monoazide–polymerase chain reaction (PMA-PCR) in this study Optimized conditions Units Values The maximum concentration of PMA that does not inhibit PCR amplification of DNA from viable cellsa µg/mL 20 The minimum concentration of PMA required to inhibit the amplification of DNA from dead cells µg/mL 1.5 The optimal concentration of PMA chosen in this study µg/mL 2 Light exposure time for inactivation of free PMA in suspensions of viable cellsb min 1–20 Light exposure time for inactivation of free PMA in suspensions of dead cells min 1–20 The optimal time of light exposure chosen in this study min 10 a The mixed samples of bacterial cells and PMA were incubated for 10 min in the dark at room temperature before being placed in an iced cooling box. b The incubated samples were then exposed to a 500 W halogen lamp light source for 10 min at a distance of 20 cm to activate and photolyse the PMA. Differentiation between culturable and dead cells in mixed samples by PMA-PCR The established horA-specific PCR assays were applied for differentiation between viable and non-viable cells of some beer spoilage LAB and non-spoilage bacteria listed in Table 1. When the DNA from a constant number of total bacteria was 105/PCR derived from different ratios of culturable and non-viable cells, the amplification of target DNA from the dead cells was effectively inhibited by 2.0 µg/mL of PMA as expected (data not shown). Of these strains, nine strains were detected to be positive for horA by PMA-PCR as shown in Table 1. The fluorescence was not influenced by the presence of the DNA from the dead cells, even when the heat-killed cells constituted 99.9% of the total cell population. This observation is in accord with the results of growth in beer. All the 9 horA-positive isolates were capable of growing in only beer 1 or both beers 1 and 2, showing the horA-specific PMA-PCR had a very high specificity for detecting the beer spoilage LAB (Table 1). Discussion The VPNC beer spoilage LAB are regarded as a serious threat to the production of unpasteurized beers. These earlier findings demonstrated a significant shift in cell size distribution towards shorter rods or coccoids, when L. acetotolerans, L. lindneri or L. paracollinoides entered into the VPNC state 4, 5, 14. Cells entering the VPNC state frequently exhibit dwarfing, and during this period a number of major metabolic changes occur, including reductions in respiration rates, macromolecular synthesis and nutrient transport 15, 16. They can therefore be detected as live with conventional PCR assay but are considered dead on plating. According to Suzuki et al. 5, the sublethal heat treatment (60 °C for 10 min) used as an additional stress factor would facilitate the induction of viable but non-culturable states in beer spoilage LAB strains. Therefore, to ensure the complete inactivation of beer spoilage bacteria tested in this study, the upper range of clean-in-place temperature was chosen rather than the routine pasteurization temperature (60–65 °C). After the heat treatment at 90 °C for 10 min, the absence of viable cells was determined by the passive dye exclusion method (data not shown). In our study, the observation that 100% of horA PCR-positive LAB isolates could grow in beer 1 (67% for beer 2) reinforces the fact that the horA PCR accurately detected LAB capable of rapidly causing beer spoilage. According to the report of Sami et al. 17, the gene horA is considered as a significant and effective predictor of beer spoilage capability. The horA gene has homology to adenosine triphosphate-binding transporter to export trans-isohumulone, preventing its accumulation in the intracellular space 18. The horA hop-resistance gene has been shown to be associated with beer spoilage by isolates from several Lactobacillus spp. and Pediococcus spp. 19. However, there is a situation where a small proportion of horA-negative LAB isolates are capable of growing in beer 19. These isolates can possess horA-independent hop-resistance mechanisms. According to Haakensen et al. 19, Lactobacillus and Pediococcus isolates that tested horA-positive have a 90% chance of spoiling low-hop and low-ethanol beer. Thus, it is similarly possible that the HorA from some LAB isolates is not actually functionally intact. Haakensen et al. 6 found that the multiplex PCR directed to the horA and other hop-resistance genes such as horC and hitA was more accurate in predicting the ability of an isolate to spoil beer. Accordingly, further research is necessary to demonstrate the applicability of PMA-PCR targeting the horC and hitA to detect the beer spoilage LAB. Some inactivated cells are detected with conventional PCR as no differentiation is made between DNA from viable or non-viable cells. This is a considerable weakness of the PCR methodology as it can lead to false-positive results. PMA is able to enter cells with compromised cell walls and intercalate into the DNA of dead cells. On light exposure a covalent DNA-PMA complex is formed; however, this bound DNA cannot be PCR amplified. Here we have found that PMA treatment prior to PCR generally reduces the signal from the dead cells. In the current work, the detection limit was found to be 100 VPNC cells/PCR reaction, which corroborated the earlier findings 8, 11. In summary, the treatment of samples containing beer spoilage LAB with PMA prior to PCR has great potential for reducing the false-positive signal from inactive cells. By specifically targeting organisms capable of beer spoilage through combined use of PMA and horA-specific PCR, brewery quality control laboratories will be able to make rapid, accurate and comprehensive predictions regarding the potential beer spoilage outcome of contamination by LAB in both culturable and VPNC state. Acknowledgements This work was financially supported by the Open Project Program of State Key Laboratory of Food Science and Technology, Nanchang University (no. SKLF-KF-201415) and China Postdoctoral Science Foundation funded project (nos 2014 T70810 and 2015M582063). References 1Sakamoto, K., and Konings, W. N. (2003) Beer spoilage bacteria and hop resistance, Int. J. Food Microbiol. 89, 105– 124. 2Suzuki, K., Iijima, K., Sakamoto, K., Sami, M., and Yamashita, H. (2006) A review of hop resistance in beer spoilage lactic acid bacteria, J. Inst. Brew. 112, 173– 191. 3Deng, Y., Liu, J., Li, H., Li, L., Tu, J., Fang, H., Chen, J., and Qian, F. (2014) An improved plate culture procedure for the rapid detection of beer spoilage lactic acid bacteria, J. Inst. Brew. 120, 127– 132. 4Deng, Y., Liu, J., Li, L., Fang, H., Tu, J., Li, B., Liu, J., Li, H., and Xu, Z. (2015) Reduction and restoration of culturability of beer-stressed and low-temperature-stressed Lactobacillus acetotolerans strain 2011–8, Int. J. Food Microbiol. 206, 96– 101. 5Suzuki, K., Iijima, K., Asano, S., Kuriyama, H., and Kitagawa, Y. (2006) Induction of viable but nonculturable state in beer spoilage lactic acid bacteria, J. Inst. Brew. 112, 295– 301. 6Haakensen, M., Schubert, A., and Ziola, B. (2008) Multiplex PCR for putative Lactobacillus and Pediococcus beer spoilage genes and ability of gene presence to predict growth in beer, J. Am. Soc. Brew. Chem. 66, 63– 70. 7Pfannbecker, J., and Fröhlich, J. (2008) Use of a species-specific multiplex PCR for the identification of pediococci, Int. J. 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