The viable but nonculturable state induction and genomic analyses of Lactobacillus casei BM‐LC14617, a beer‐spoilage bacterium

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 shared similar beer‐spoilage capability. Whole genome sequencing was performed, and out of a total of 3,964 predicted genes, several potential VBNC and beer‐spoilage‐associated genes were identified. L. casei is capable of entering into and resuscitating from the VBNC state and possesses beer‐spoilage capability. The genomic characterization yield insightful elucidation of VBNC state for L. casei. This study represents the first evidence on VBNC state induction of L. casei and beer‐spoilage capability of VBNC and resuscitated cells. Also, this is the first genomic characterization of L. casei as a beer‐spoilage bacterium. The current study may aid in further study on L. casei and other beer‐spoilage bacteria, and guide the prevention and control of beer spoilage.


| INTRODUCTION
As a popular beverage, beer has been recognized as safe due to its high microbiological stability, as a large variety of microorganisms are incapable of survival in beer (Sakamoto & Konings, 2003;Suzuki, Iijima, Asano, Kuriyama, & Kitagawa, 2006). However, despite these unfavorable conditions for microbial growth, a few species of bacteria (primarily Lactobacilli and Pediococci) remain viable in this medium and are designated as beer-spoilage microorganisms. A number of lactic acid bacteria (LAB) have been well documented as a major cause of acidity and turbidity in beer. As an important beer-spoilage bacterium, Lactobacillus casei possesses the ability to produce lactic acid, acetic acid, and diacetyl as end or by-products of carbohydrate fermentation, thus significantly influencing the flavor of beer. This issue is further complicated by the fact that contaminating L. casei strains are able to cause false-negative detection, due to their capability of entering into the viable but noncluturable (VBNC) state under stress conditions (Deng et al., 2015;Suzuki et al., 2006). Bacteria in the VBNC state fail to grow on the routine bacteriological media on which they would normally grow and develop into colonies, but still live and have metabolic activity (Oliver, 2005).
This study aimed to investigate the VBNC state and genomic features through genomic sequencing of L. casei strain BM-LC14617 as a beer-spoilage bacterium.

| Bacterial strains and growth conditions
L. casei strain, designated BM-LC14617, was isolated from finished beer and grown anaerobically at 26°C in MRS broth (Oxoid, UK).

| Induction of entry into VBNC state
Approximately 10 7 cells/ml exponentially growing cells of L. casei strain BM-LC14617 were inoculated and anaerobically subcultured at 26°C in 10 ml aliquots of the degassed commercial beer. Afterward, the bacterial strain was induced to entry into VBNC state by both low-temperature storage and continuous passage in beer. Continuous passage in beer was performed as described by Suzuki et al. (2006) with some modification. The interval of each subculture was 7 days.
Concerning the low-temperature storage, the exponentially growing cells of L. casei BM-LC14617 were harvested by centrifugation at 5000 x g for 15 min at 4°C. The bacterial cells were then resuspended in degassed beer at a final density of 10 7 cells/ml and maintained at 0°C without shaking to induce the VBNC state.

| Culturability and viability assays
In this study, the term "VBNC state" was defined as the inability to grow on MRS agar media until 14 days of incubation. The number of culturable cells were accessed by a conventional plate culture procedure (Suzuki, Asano, Iijima, & Kitamoto, 2008). A portion of subcultures (100 μL) was inoculated on MRS agar and incubated anaerobically at 26°C. The inoculated agar plate was examined each day for the presence of colony formation, and the day on which colonies were first observed was recorded as the detection time. After 14 days of incubation, CFU were counted to determine culturability on agar plates. The number of total cells was determined by AODC method (Hobbie, Daley, & Jasper, 1977). Cell viability was determined using a Live/Dead BacLight bacterial viability kit (Molecular Probes, USA) with fluorescent microscope (Berney, Hammes, Bosshard, Weilenmann, & Egli, 2007). Cellular viability and culturability were evaluated once a week.

| Resuscitation of the VBNC state
After entry into the VBNC state, 100 μL of L. casei cells were inoculated in 10 ml of MRS broth or sterile water (negative control), respectively.
Simultaneously, heat-denatured catalase (60°C for 15 min) served as a control. The culturability of the samples was determined by plating 100 μL of the samples on MRS agar.

| Evaluation of beer-spoilage ability
The beer-spoilage ability was investigated using the traditional "growth in beer test" (Sakamoto & Konings, 2003). Approximately 10 5 cells/ml of the strain in exponentially growing, VBNC, and resuscitated state were inoculated into commercial bottled lager beer under sterile conditions at room temperature, respectively. The inoculated beer were incubated at 26°C and examined daily for visible growth for up to 30 days. Subsequently, lactic acid and acetic acid concentration were analyzed by reversed-phase high performance liquid chromatography. The diacetyl concentration was measured by Head Space Gas Chromatography. Lactic acid, acetic acid, and diacetyl were quantified by the external standard method.

| Statistical analysis
Data are presented as mean ± standard deviation (SD) of three independent biological replicates. Statistical comparisons were made by one-way analysis of variance followed by Tukey's comparison test (XLstat software). A p-value <.05 was considered to be significant.

| Formation of VBNC state
Cellular viability and culturability of L. casei strain were evaluated once a week, and the formation of VBNC state by L. casei was obtained and verified under both processes. According to the low-temperature storage, none of L. casei cells was culturable after 196 ± 3.3 days, while viable cells revealed a low decrease with an approximate of 10 5 cells/ml ( Figure 1A). As for continuous passage in beer, 32 ± 1.6 subcultures were required for the formation of VBNC state of L. casei strains ( Figure 1B).

| Resuscitation of VBNC cells
When the VBNC cells induced by either adaptation protocol were subjected to addition of tween-20, tween-80, vitamin C, or vitamin B2, culturable cells were not obtained (data not shown). However, VBNC L. casei cells regained culturability on media containing catalase. Importantly, heat denaturation of catalase deferred the resuscitation process. The results observed here demonstrate that the addition of catalase is an effective method for the resuscitation of VBNC L. casei cells.

| Beer-spoilage ability
Exponentially growing, VBNC, and resuscitated cells were capable of maintaining in beer within approximately 10 days and further causing turbidity in beer (data not shown). It suggests the maintenance of beer-spoilage capability of L. casei strain BM-LC14617 under both VBNC and resuscitated states. After 30 days of incubation, increased levels of lactic acid and acetic acid were detected, which may eventually lead to beer acidization. Also, no significant difference was identified among the lactic acid, acetic acid, and diacetyl concentrations produced by exponentially growing, VBNC, and resuscitated cells, respectively. In addition, high level of diacetyl was also found under the VBNC states of L. casei strain, suggesting its attribution to imparting "buttery" off flavors in beer ( Table 1). Detection of lactic acid, acetic acid, and diacetyl indicates that the VBNC and resuscitated L. casei cells remained viable and maintained similar beer-spoilage capability as exponentially growing cells.

| Genomic feature
The whole genomic sequence of beer-spoilage L. casei strain BM-  Table S3) annotations were identified, including one each for 23S rRNA and 16S rRNA, and 22 tRNA genes (24 RNA genes).

| DISCUSSION
In this study, similar beer-spoilage capability was found among exponentially growing, VBNC, and resuscitated L. casei cells, which was somewhat in accordance with previous studies (Buck & Oliver, 2010;Du et al., 2007;Imazaki & Nakaho, 2009; Jolivet-Gougeon,   (Suzuki et al., 2006). Also, formation of VBNC state for L. acetotolerans was found after 17 subcultures in beer or incubating in low-temperature condition (Deng et al., 2015). In this study, The VBNC state L. casei strain BM-LC14617 was obtained by continuous beer passages or low-temperature storage, and the beer-spoilage capability under VBNC state was verified. In addition, compared with L. acetotolerans, the shorter growth cycle (L. acetotolerans was hard to culture, requiring 14 days cultivation in MRS medium) (Deng et al., 2015), additional production of diacetyl, and higher frequency in beerspoilage cases for L. casei posed a significant concern for beer brewery industry. Nevertheless, with high identification rate in beer industry and capability of VBNC state formation under low-temperature (common food storage condition), L. casei may be responsible for a large variety of beer-spoilage cases.

F I G U R E 2
The genome circle of L. casei strain BM-LA14617. The circle from outermost to innermost illustrates scaffold sequences, genes in plus strand, genes in minus strand, GC content, low GC content sequences, and high GC content sequences, respectively As a beer-spoilage bacterium, L. casei BM-LC14617 was initially found to show relatively slow growth in MRS agar when isolated from beer sample, but after subculturing, this L. casei strain obtained culturability (data not shown). The addition of catalase is an effective method for the resuscitation of VBNC L. casei cells, and catalase addition has been known to improve flavor stability of beer in the brewing process (Festersen, Elvig, & Bisgaard-Frantzen, 2007). Brewers can improve the microbiological quality control with significantly shorter incubation time by catalase supplementation. Low temperature (Deng et al., 2014;Griffitt, Noriea, Johnson, & Grimes, 2011;Imazaki & Nakaho, 2009;Kong et al., 2014;Zeng et al., 2013) or cold treatment combined with oligotrophic conditions (Chen et al., 2011;Dinu & Bach, 2011;Magajna & Schraft, 2015;Patrone et al., 2013;Sun et al., 2008;Zhong, Chen, Zhang, & Jiang, 2009) (Suzuki et al., 2006), and L. acetotolerans, as induced to enter into the VBNC state by beer subculture treatment or low-temperature storage (Deng et al., 2015). As a consequence, this study represented the first evidence on induction and resuscitation of the VBNC state by the species of L. casei strain.
As part of an initial strategy to investigate the phenotypic behavior  Laub, 2005). Seven transcriptional regulators belong to MarR family involving in degradation of toxic environmental compounds, virulence, export of harmful chemicals, and resistance to oxidative stress (Grove, 2013), and four belongs to LysR family acting as an ortholog of OxyR in Escherichia coli. As a regulatory transcriptional factor sensitive to oxidation, OryR was capable of activating the expression of antioxidant genes in response to hydrogen peroxide in E. coli (Zheng, Aslund, & Storz, 1998). In addition, amino acids have been reported to be important growth substrates in bacteria and regulation on biosynthesis and degradation, and thus are essential for maintaining the carbonnitrogen balance in the VBNC cells (Postnikova, Shao, Mock, Baker, & Nemchinov, 2015). As far as beer spoilage is concerned, presence of genes involved in hop resistance, alcohol-tolerance, and oxidative stress response are considered to be associated with the bacterial growth in beer, with one example being the correlation between carriage of horA, horB, and horC genes and hop resistance (DiMichele & Lewis, 1993;Suzuki et al., 2008). In the draft genome of L. casei BM-LC14617, a homolog of the Lactobacillus brevis plasmid horA gene F I G U R E 5 KEGG pathways enrichment results of genes in L. casei strain BM-LC14617 (GenBank accession no. AB005752.1) was identified, potentially explaining its affinity for growth in this medium. A cluster of genes encoding for alcohol-tolerant enzymes (Brown, Guss, & Karpinets, 2011;Eanes, Merritt, Flowers, Kumagai, & Zhu, 2009;Ram, 2000), such as alcohol dehydrogenase (n = 12), aryl-alcohol dehydrogenase (n = 2), malate dehydrogenase (n = 1), aldehyde dehydrogenase (n = 4), and glycerol-3-phosphate dehydrogenase (n = 3) were harbored by this genome. As a major beer-spoilage compound, diacetyl is produced by dehydrogenation of 2,3-butanediol (Siegel & Eggersdorfer, 2005).
Acquiring (R,R)-butanediol dehydrogenase activity, gene_3949 which might contribute to the diacetyl production, was identified in the genome of L. casei BM-LC14617.

| CONCLUSION
This study focuses on the induction and resuscitation of VBNC state, beer-spoilage ability, as well as the genomic analysis for L. casei. As for phenotype, the VBNC state of L. casei strain BM-LC14617 had been induced by both beer subculture treatment and low-temperature storage, and recovered by addition of catalase.
Also, both VBNC and resuscitated cells remained beer-spoilage capability. According to the phenotype, the genomic characterization of the beer-spoilage L. casei strain BM-LC14617 was also performed for insightful elucidation of VBNC state for L. casei. Information derived from this draft genome sequence may be relevant to study beer-spoilage mechanisms in other lactic acid bacteria in an effort to reduce spoilage and improve food-safety issues in the breweries.
Further transcriptomic analysis and gene knockout studies will aid in additional insight into the genetic mechanisms that govern the VBNC state and beer-spoilage capacity.

NUCLEOTIDE SEQUENCE ACCESSION NUMBERS
The raw data and scaffold sequences of L. casei strain BM-LA14617 were submitted to GenBank under accession number SRX1433289 and LTDP00000000, respectively.