Abstract:This study aimed to investigate the viable but nonculturable (VBNC) state and genomic features of a beer‐spoilage strain, Lactobacillus casei
BM‐LC14617. Induction on the VBNC state of L. casei strain BM‐LC14617 was conducted by both low‐temperature storage and continuous passage in beer, and formation of VBNC state was detected after 196 ± 3.3 days and 32 ± 1.6 subcultures, respectively. Resuscitation of VBNC cells was successfully induced by addition of catalase, and culturable, VBNC, and resuscitated cells … Show more
“…It was also found that the culturability of L. brevis declined significantly during conditions induced by beer subculture or low temperature, indicating the VBNC L. brevis could be potentially neglected in brewery environments due to incapability of detection by traditional culture methods. As Lactobacillus strains were concerned, up to date, a total of five species have been validated for VBNC state formation, including L. lindneri ( Suzuki et al, 2006 ; Liu et al, 2017a ), L. casei ( Liu et al, 2017c ), L. plantarum ( Liu et al, 2017b ), L. paracollinocides , ( Suzuki et al, 2006 ) and L. acetotolerans ( Deng et al, 2015 ), which had been induced to enter into VBNC state by either low-temperature storage or beer subculture treatment. Consequently, this is the first the formation and recovery of VBNC state by the species of L. brevis as the most common beer spoilage and hop resistance strain.…”
Section: Resultsmentioning
confidence: 99%
“…If beer contaminated by VBNC strains has been delivered to commercial markets, even small numbers of hop-resistance bacteria will recover and eventually impart off-flavor and turbidity to beer. Up to date, five species of Lactobacillus strains have been verified for formation of VBNC state, including L. lindneri ( Suzuki et al, 2006 ; Liu et al, 2017a ), L. casei ( Liu et al, 2017c ), L. plantarum ( Liu et al, 2017b ), L. paracollinocides ( Suzuki et al, 2006 ) and L. acetotolerans ( Deng et al, 2015 ), which had been induced to enter into the VBNC state by beer subculture treatment or low-temperature storage.…”
Lactobacillus brevis is a major hop-resistance bacterium which poses significant challenge for the brewing industry, mainly due to the difficulty or incapability in detection by routine culturing methodology and its beer spoilage ability.This study aimed at investigating the VBNC state of a hop-resistance strain, L. brevis BM-LB13908. The culturable, total and viable numbers of L. brevis cells were calculated by MRS agar plate counting, acridine orange direct count (AODC) method and Live/Dead BacLight bacterial viability kit with fluorescence microscope. VBNC formation was induced by 189 ± 5.7 days under low-temperature storage or 27 ± 1.2 subcultures by continuous passage in beer, and VBNC cells induced by both strategies were recovered by adding catalase. In addition, insignificant difference in beer-spoilage ability was found in 3 states of L. brevis, including logarithmic growing, VBNC and recovered cells. This is the first study on the formation of VBNC state for L. brevis and beer-spoilage ability of both VBNC and recovered cells, which indicate L. brevis strain could cause beer spoilage without being detected by routine methodologies. The results derived from this study may support further study on L. brevis and other hop-resistance bacteria, and guidance on beer spoilage prevention and control, such as improvement for brewers on the microbiological quality control by using the improved culture method with catalase supplementation.
“…It was also found that the culturability of L. brevis declined significantly during conditions induced by beer subculture or low temperature, indicating the VBNC L. brevis could be potentially neglected in brewery environments due to incapability of detection by traditional culture methods. As Lactobacillus strains were concerned, up to date, a total of five species have been validated for VBNC state formation, including L. lindneri ( Suzuki et al, 2006 ; Liu et al, 2017a ), L. casei ( Liu et al, 2017c ), L. plantarum ( Liu et al, 2017b ), L. paracollinocides , ( Suzuki et al, 2006 ) and L. acetotolerans ( Deng et al, 2015 ), which had been induced to enter into VBNC state by either low-temperature storage or beer subculture treatment. Consequently, this is the first the formation and recovery of VBNC state by the species of L. brevis as the most common beer spoilage and hop resistance strain.…”
Section: Resultsmentioning
confidence: 99%
“…If beer contaminated by VBNC strains has been delivered to commercial markets, even small numbers of hop-resistance bacteria will recover and eventually impart off-flavor and turbidity to beer. Up to date, five species of Lactobacillus strains have been verified for formation of VBNC state, including L. lindneri ( Suzuki et al, 2006 ; Liu et al, 2017a ), L. casei ( Liu et al, 2017c ), L. plantarum ( Liu et al, 2017b ), L. paracollinocides ( Suzuki et al, 2006 ) and L. acetotolerans ( Deng et al, 2015 ), which had been induced to enter into the VBNC state by beer subculture treatment or low-temperature storage.…”
Lactobacillus brevis is a major hop-resistance bacterium which poses significant challenge for the brewing industry, mainly due to the difficulty or incapability in detection by routine culturing methodology and its beer spoilage ability.This study aimed at investigating the VBNC state of a hop-resistance strain, L. brevis BM-LB13908. The culturable, total and viable numbers of L. brevis cells were calculated by MRS agar plate counting, acridine orange direct count (AODC) method and Live/Dead BacLight bacterial viability kit with fluorescence microscope. VBNC formation was induced by 189 ± 5.7 days under low-temperature storage or 27 ± 1.2 subcultures by continuous passage in beer, and VBNC cells induced by both strategies were recovered by adding catalase. In addition, insignificant difference in beer-spoilage ability was found in 3 states of L. brevis, including logarithmic growing, VBNC and recovered cells. This is the first study on the formation of VBNC state for L. brevis and beer-spoilage ability of both VBNC and recovered cells, which indicate L. brevis strain could cause beer spoilage without being detected by routine methodologies. The results derived from this study may support further study on L. brevis and other hop-resistance bacteria, and guidance on beer spoilage prevention and control, such as improvement for brewers on the microbiological quality control by using the improved culture method with catalase supplementation.
“…Concerning the VBNC formation and identification, rapid detection of the VBNC state of MRSA is of urgent necessity and importance, along with the formation of the VBNC state ( Liu et al, 2016c , 2017a , b , c ). In this study, formation of the VBNC state has been performed on MRSA strains, then development and evaluation of an isothermal nucleic acid amplification-based propidium monoazide (PMA) detection assay have been conducted on MRSA.…”
Formation of viable but non-culturable (VBNC) status in methicillin-resistant Staphylococcus aureus (MRSA) has never been reported, and it poses a significant concern for food safety. Thus, this study aimed to firstly develop a rapid, cost-effective, and efficient testing method to detect and differentiate MRSA strains in the VBNC state and further apply this in real food samples. Two targets were selected for detection of MRSA and toxin, and rapid isothermal amplification detection assays were developed based on cross-priming amplification methodology. VBNC formation was performed for MRSA strain in both pure culture and in artificially contaminated samples, then propidium monoazide (PMA) treatment was further conducted. Development, optimization, and evaluation of PMA-crossing priming amplification (CPA) were further performed on detection of MRSA in the VBNC state. Finally, application of PMA-CPA was further applied for detection on MRSA in the VBNC state in contaminated food samples. As concluded in this study, formation of the VBNC state in MRSA strains has been verified, then two PMA-CPA assays have been developed and applied to detect MRSA in the VBNC state from pure culture and food samples.
“…To validate the existent bacteria, the genomic DNA of bacterial cells from the spoiled beer sample and the isolated L. harbinensis cells were extracted by bacterial genomic DNA extraction kit (Sigma-Aldrich, USA) and subjected to genomic sequencing by Illumina HiSeq. 2500 platform and paired-end libraries, respectively 10 , 47 . Sequences were quality processed using the FastQC pipeline v.0.10.1 before assembly.…”
Occasional beer spoilage incidents caused by false-negative isolation of lactic acid bacteria (LAB) in the viable but non-culturable (VBNC) state, result in significant profit loss and pose a major concern in the brewing industry. In this study, both culturable and VBNC cells of an individual Lactobacillus harbinensis strain BM-LH14723 were identified in one spoiled beer sample by genome sequencing, with the induction and resuscitation of VBNC state for this strain further investigated. Formation of the VBNC state was triggered by low-temperature storage in beer (175 ± 1.4 days) and beer subculturing (25 ± 0.8 subcultures), respectively, and identified by both traditional staining method and PMA-PCR. Resuscitated cells from the VBNC state were obtained by addition of catalase rather than temperature upshift, changing medium concentration, and adding other chemicals, and both VBNC and resuscitated cells retained similar beer-spoilage capability as exponentially growing cells. In addition to the first identification of both culturable and VBNC cells of an individual L. harbinensis strain from spoiled beer, this study also for the first time reported the VBNC induction and resuscitation, as well as verification of beer-spoilage capability of VBNC and resuscitated cells for the L. harbinensis strain. Genes in association with VBNC state were also identified by the first genome sequencing of beer spoilage L. harbinensis. The results derived from this study suggested the contamination and spoilage of beer products by VBNC and resuscitated L. harbinensis strain BM-LH14723.
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