Induction and Recovery of the Viable but Nonculturable State of Hop-Resistance Lactobacillus brevis.

Junyan Liu,Yanyan Li,Thanapop Soteyome,Brian M Peters,Yang Deng,Jianyu Su,Lin Li,Zhenbo Xu,Bing Li,Mark E Shirtliff

doi:10.3389/fmicb.2018.02076

Abstract

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.

Highlights

As one of the most common beverage, beer has high microbiological stability and is considered to be safe, as it contains various microorganisms unable to survive in beer
This study aimed to investigate the induction, recovery and beer-spoilage capacity of viable but nonculturable (VBNC) state L. brevis strain BMLB13908 which shows strong resistance to hop compounds
Viable but nonculturable cells of L. brevis strain BM-LB13908 induced by low-temperature storage or beer subculture had been treated by addition of Tween-20 or-80, vitamin C or B2, with

Summary

Introduction

As one of the most common beverage, beer has high microbiological stability and is considered to be safe, as it contains various microorganisms unable to survive in beer. Hop components serve the desirable bitter flavor, characteristic aroma, and apply bacteriostatic effects on most Gram-positive bacteria In spite of such unfavorable conditions for growth of microorganisms, a number of bacterial species (primarily Lactobacilli) are tolerant to hop compounds and capable of growing in hopped beer, designated as hopresistance bacteria. Formation of the viable but nonculturable (VBNC) state under extreme environments enables Lactobacilli strains to cause false-negative detection (Suzuki et al, 2006; Deng et al, 2015). If beer contaminated by VBNC strains has been delivered to commercial markets, even small numbers of hopresistance bacteria will recover and eventually impart off-flavor and turbidity to beer. 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

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Methods

Conclusion