In situ examination of Lactobacillus brevis after exposure to an oxidizing disinfectant.

Yu Zhao,Henrik Siegumfeldt,Susanne Knã¸Chel

doi:10.3389/fmicb.2014.00623

Abstract

Beer is a hostile environment for most microorganisms, but some lactic acid bacteria can grow in this environment. This is primarily because these organisms have developed the ability to grow in the presence of hops. It has been speculated that hop resistance is inversely correlated to resistance against oxidation, and this would have great impact on the use of various disinfectants in the brewing industry. In this study, we cultivated bacteria under aerobic and anaerobic conditions, and then investigated the in situ outgrowth of individual cells into microcolonies on de Man Rogosa Sharpe (MRS) agar after exposure to the oxidizing agent peracetic acid (PAA). An automated microscope stage allowed us to analyse a much larger number of cells over extended periods of incubation. After PAA treatment, the lag time increased markedly, and extensive variation in morphology, μmax as well as stress resistance was observed between and within the tested Lactobacillus brevis strains. The results suggest that aerobic cultivation increased the oxidative stress tolerance in Lactobacillus brevis. The results also show that dead cells are randomly distributed in a microcolony and the majority of non-growing individual cells do not stain with a membrane impermanent dye (Propidium iodide), which indicates that PAA may not destroy the plasma membrane. In conclusion, the developed microscopic analysis of individual cells on MRS agar can provides faster results and more details of cell physiology compared to the traditional CFU method.

Highlights

Lactic acid bacteria (LAB) are fermentative organisms which have been generally regarded as anaerobic bacteria, but most of them can grow under aerobic conditions (Sakamoto and Komagata, 1996)
The results show that dead cells are randomly distributed in a microcolony and the majority of non-growing individual cells do not stain with a membrane impermanent dye (Propidium iodide), which indicates that peracetic acid (PAA) may not destroy the plasma membrane
The two strains HF01 and JK09 were subsequently chosen for further experiments in a microscopic set-up, because they are both beer spoilage bacteria, and exhibited varying levels of sensitivity toward PAA, which was selected as the oxidizing agent for the microscopic analysis

Summary

Introduction

Lactic acid bacteria (LAB) are fermentative organisms which have been generally regarded as anaerobic bacteria, but most of them can grow under aerobic conditions (Sakamoto and Komagata, 1996). The low pH will prevent most Gram-negative bacteria from growing, and the addition of hops will usually prevent LAB from spoiling the beer (Vaughan et al, 2005; Suzuki et al, 2006; Menz et al, 2010; Suzuki, 2011). 60–90% of bacteria isolated from spoiled beer are LAB (Suzuki, 2011). Among those LAB, Lactobacillus brevis is the most common bacteria and frequently detected in breweries (Hollerová and Kubizniaková, 2001; Suzuki et al, 2008a; Menz et al, 2010). It would be interesting to investigate the response of beer spoilage isolates toward oxidative compounds, as some of these are utilized as sanitizers in the food industry (Rossoni and Gaylarde, 2000; Kitis, 2004)

Methods

Results

Conclusion