Study on spoilage capability and VBNC state formation and recovery of Lactobacillus plantarum

Highlights

• L. plantarum strain BM-LP14723 is capable of entering into the VBNC state in beer.

• The VBNC L. plantarum cells acquire similar beer-spoilage capability with culturable cells.

• The addition of catalase is an effective method for the recovery of VBNC L. plantarum cells.

Abstract

Objective

The present study aimed at investigating the capability of L. plantarum strain BM-LP14723 to enter into and recover from the viable but nonculturable (VBNC) state and to cause beer spoilage.

Methods

VBNC state was induced by incubating in beer with subculturing or low temperature treatment. Culturable, total, and viable cells numbers were assessed by MRS agar plate counting, acridine orange direct counting, and Live/Dead BacLight bacterial viability kit, respectively. Organic acids concentrations were measured by reversed-phase high performance liquid chromatography.

Results

VBNC L. plantarum cells were detected after 189 ± 1.9 days low temperature treatment or 29 ± 0.7 subcultures in beer. The VBNC L. plantarum retained spoilage capability. Addition of catalase is an effective method for the recovery of the VBNC L. plantarum cells.

Conclusion

L. plantarum strain BM-LP14723 is capable of entering into and recovery from the VBNC state and maintained spoilage capability. The current study presented that beer-spoilage L. plantarum can hide both in breweries and during transporting and marketing process and thus lead to beer-spoilage incidents.

Introduction

Hop bitter acids which exert antibacterial effects, the presence of alcohol, relatively low pH, and insufficient amounts of oxygen and nutrients, results in high microbiological stability of beer [1], [2]. However, in spite of these unfavorable features, a few microorganisms still capable of growing in beer and cause an increase of turbidity and unpleasant flavor. Furthermore, some beer spoilage microorganisms are also able to produce pathogenic chemicals.

Approximately 70% of microbiological incidents in breweries which result in serious loss of profit and consumers' trust were caused by beer-spoilage lactic acid bacteria (LAB) [3], [4], [5], [6], [7]. One big problem in the brewing industry is that some beer spoilage LAB strains are capable of entering into the viable but nonculturable (VBNC) state which is considered to be a survival mechanism for non-sporulation bacteria to survive under harsh environmental conditions [8], [9], [10], [11], [12], [13]. Bacteria in the VBNC state remain alive, but fail to form a colony on routine bacteriological media that normally support their growth. They have also been reported to remain metabolic activity and continuous gene expression [14], [15], [16], and capable of recovering under certain conditions [17], [18], [19]. Thus, it is urgent to identify specific LAB strains which are able to enter into the VBNC state and propose an available method to solve this problem.

This study focused on the capability of L. plantarum to enter into the VBNC state, its spoilage capability, as well as the recovery of VBNC cells.