Effect of bacteriostatic agents on lactic acid bacteria and specific fish spoilage bacteria in a model system

Abstract

Lactic acid bacteria (LAB) are gram positive bacteria, which are the dominant microflora in lightly preserved fish products (LPFP) and many LAB species can spoil LPFP. To increase the shelf-life of LPFP, it is necessary to inhibit the growth of LAB using preservatives. The aim of the study was i) to examine the effect of different concentration of preserving agents (PURAC; Purasal Opti.Form PPA Plus and liquid smoke; Arosmoke P-50) on the growth of Photobacterium phosphoreum, Pseudomonas putida, Vibrio vulnificus, Listeria innocua (as a L. monocytogenes non-pathogenic substitute) and LAB (Carnobacterium inhibens, Carnobacterium maltaromaticum, Lactococcus lactis, Enterococcus faecalis and Lactobacillus curvatus) at 20 ºC by using spectroscopic techniques (Bioscreen C) ii) to compare the effect of different concentrations of natural salt (NaCl), potassium lactate and potassium acetate on the growth of V. vulnificus at 20 ºC. Another objective of this study was to evaluate a panel of candidate reference genes for their potential use for normalization of gene expression in bacteria under food processing relevant conditions. The above mentioned bacteria were inoculated as mono-cultures in tryptic soy broth with yeast extract (TSBYE) supplemented with 8 different combination of preservatives; i) 3% PURAC (Potassium lactate +Potassium acetate), ii) 3% PURAC, iii) 0.07% liquid smoke (LS), iv) 0.14% LS, v) 3% PURAC + 0.07% LS, vi) 3% PURAC + 0.14% LS solution, vii) 6% PURAC + 0.07% LS and viii) 6% PURAC + 0.14% LS. Bacterial growth at 20 °C was measured for up to one week by recording absorbance at 600 nm every 10 minutes using a microplate incubator and reader (Bioscreen C). The treatments had varying effects on growth depending on species. The most detrimental was the effect of PURAC solution on the growth of L. curvatus. Interestingly, growth of this species appeared to be enhanced by the supplement of LS, and it was able to grow in the presence of 6% PURAC + 0.07% LS, but not when 6% PURAC was the sole preservative. However, the apparent lag time in this situation (106 h) was threefold longer compared to 0.07% LS alone. In general, the isolated effect of LS was minor compared to the effect of PURAC, but in most cases, a combinatorial effect of the two preservatives was observed. Another interesting result was the inhibiting effect of PURAC on the growth of V. vulnificus. This bacterium is recognized as a halophilic species by many authors but was not able to grow in any combination with 6% PURAC (all other species where), and was significantly inhibited by the lower PURAC concentration (3%). Eight candidate reference genes and one gene of interest were used in gene expression analysis. Among eight reference genes, six candidate reference genes were stably expressed under the experimental conditions (different temperature). Two genes were sufficient for normalization of gene expression analysis found from geNorm analysis. The expression of the gene of interest (hsp60), was induced significantly when inoculated of high temperature (40 ºC) for 8 hours.