Discovery and control of culturable and viable but non-culturable cells of a distinctive Lactobacillus harbinensis strain from spoiled beer

Junyan Liu,Yang Deng,Brian M Peters,Bing Li,Yanyan Li,Lin Li,Mark E Shirtliff,Zhenbo Xu,Shishui Zhou

doi:10.1038/s41598-018-28949-y

Abstract

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.

Highlights

First reported in 1982, Viable but nonculturable (VBNC) state has been well established and documented to be a survival strategy of nonsporeforming bacteria in response to natural stress, such as starvation, extreme temperature, elevated osmotic pressure, oxygen concentration, or exposure to visible light[1,2]
According to MRS agar growth, acridine orange direct count (AODC) and Live/Dead BacLight bacterial viability kit methodologies with fluorescent microscopy and flow cytometer (Fig. 1), the difference between culturable and viable cell number was approximately 4 × 102 cells/mL, demonstrating the presence of viable but non-culturable (VBNC) cells in the specific spoiled beer sample acquired in Guangzhou of South China in 2014
As occasionally reported, finished beer previously passed random microorganism detection by routine culturing method, are found spoiled after shelf storage. Both culturable and VBNC cells were identified in one spoiled beer sample based on MRS agar growth, AODC and Live/Dead BacLight bacterial viability kit methodologies with fluorescent microscopy and flow cytometer

Summary

Introduction

First reported in 1982, Viable but nonculturable (VBNC) state has been well established and documented to be a survival strategy of nonsporeforming bacteria in response to natural stress, such as starvation, extreme temperature, elevated osmotic pressure, oxygen concentration, or exposure to visible light[1,2]. Occasional beer spoilage incidents due to false-negative detection of causative microorganisms, result in significantly profit loss and have been considered to be a leading problem in the beer brewing industry[6,7,8] Failure to detected these spoilage agents, are partially due to the use of traditional culture-based techniques, which are unable to reliably detect the VBNC microorganisms commonly found under high levels of microbial stress[5,8,9,10,11]. As occasionally reported, finished beer that previously passed random microorganism detection by “golden standard” routine culturing, are found spoiled after shelf storage In this study, both culturable and VBNC cells of an individual L. harbinensis strain BM-LH14723 were identified in one spoiled beer sample, with the induction, resuscitation and characteristics of the VBNC state further defined. The first genome sequence of L. harbinensis is reported

Methods

Results

Conclusion