Molecular Approaches for Microbial Diversity, Screening, Characterization of Bacteriocin Producing Lactic Acid Bacteria and Its Application in Fermented Shrimp (Kung-Som) Production

Abstract

Kung-som is a traditionally Thai fermented shrimp which is popularly consumed and distributed in the southern part of Thailand. It is produced by naturally fermentation of small sea shrimp (Fenneropenaeus merguiensis) mixing with sugar, sea salt and water, resulting in the unique characteristic of flavors and tastes. The aim of this study was to evaluate food safety of kung-som that distributed in local markets and to identify bacterial community of kung-som using polymerase chain reaction- denaturing gel gradient electrophoresis (PCR-DGGE) technique. For microbiological safety, all samples of kung-som did not find the pathogenic bacteria including Escherichia coli, Clostridium perfringens, Staphylococcus aureus and Salmonella sp. The bacterial DGGE profile targeting the V3 region of 16S rRNA gene indicated that the predominant lactic acid bacteria (LAB) belonged to Lactobacillus farciminis, Lb. plantarum, Lactococcus garvieae, Tetragenococcus halophilus and Weissella thailandensis. Additionally, DGGE result presented coagulase-negative staphylococci (CNS) as a minor microflora in kung-som. Our finding suggested that understanding of bacterial community presented in kung-som and providing a theoretical basis to develop the starter cultures for production of kung-som. Presently, bacteriocin-producing LAB have been widely used as an alternative food-perseverative agent replacing the antibiotics in food-industry. Lactococcus lactis KTH0-18 strain isolated from Thai traditional fermented shrimp (kung-som) had ability to produce heat-stable bacteriocin and demonstrated the antagonistic activity against food-borne pathogens and food spoilage bacteria. An inhibitory activity of bacteriocin retained active after treatment with different pHs (2- 11) and temperatures (80-121°C) but was sensitive to some proteolytic enzymes. The growth of S. aureus significantly decreased after 9 h co-cultivation with bacteriocin-producing Lc. lactis KTH0-1S. Moreover, the addition of bacteriocin KTH0-1S to S. aureus cultures resulted in viable cell counts sharply decreased by 2.0 log CFU/ml demonstrating a bactericidal mode of action. After four steps of purification (ammonium sulfate precipitation, reverse phase cartridge (C, Sep-Pak), cation- exchange chromatography, RP-HPLC on C8 column), the purified bacteriocin KTHO- 18 had a molecular mass of 3.346 kDa using mass spectrometry (MS/MS) analysis. Bacteriocin KTH0-1S was identified as nisin Z by using PCR amplification and sequencing. Additionally, nisin Z-producing Lc. lactis KTH0-1S was sensible towards most of studied antibiotics, non-biogenic amines production and did not harbor virulence genes. Our finding concluded that Lc. lactis KTH0-1S presented a high potential to be used as a starter culture for improving a microbiological safety in kung-som. In order to reduce the accumulation of biogenic amines in fermented foods, bacteriocin-producing LAB showing positive effect against biogenic amines formation. Tyramine as biogenic amine is most frequently found in various fermented foods that present toxicological effects on human. It occurred from tyraminogenic bacteria by conversion of tyrosine into tyramine through tyrosine decarboxylase activity. Tyramine production of 20 strains of LAB and CNS isolated from Thai traditional fermented shrimp (kung-som) was investigated by using chromatographic and molecular methods. Among 20 of bacterial strains isolated from kung-som, Enterococcus faecalis 9Y, Ent. faecalis DOKS13 and Weissella cibaria DOKS11 were able to produce tyramine and also possess the tyrosine decarboxylase (tdc) gene. The effectiveness of nisin Z-producing Lc. lactis KTH0-18 against tyraminogenic strains was evaluated in co-cultivation. In competition study, Ent. faecalis DOKS13 and W. cibaria DOKS11 decreased significantly the growth rate and the maximal cell concentration, depending on the initial inoculum level of tyraminogenic strain. The reduction of tyramine accumulation decreased strongly when tyraminogenic strain was inoculated at 4.0 log CFU/ml and, to a lesser extent, at 5.0 and 6.0 log CFU/ml. Additionally, kung-som inoculation with nisin Z-producing Lc. lactis KTH0-1S as a starter culture could suppress the growth of undesirable bacteria such as Enterobacteriaceae, S. aureus and total viable count (TVC), comparing with spontaneous fermented kung-som (without starter culture). The addition of Lc. lactis KTHO-18 as a starter culture in kung-som reduced accumulation of tyramine about 100 mg/kg in comparison with naturally fermented kung-som. Our result indicated that Lc. lactis KTH0-1S as a starter culture is advisable for production of safer kung-som with decreasing pathogen contaminants and lowering the tyramine content. Furthermore, the obtained result from DGGE profile showed the coagulase-negative staphylococci (CNS) as minor bacterial flora in kung-som. CNS have been used as starter culture in various fermented foods, they play a major role in the development of sensory properties and color development of fermented foods. The safety evaluation and technological characteristics were primary criteria for selection of CNS to be applied as starter culture in kung-som production. Among 46 CNS strains, only strain Staphylococcus carnosus K21S22 was considered as safe to be used with regard to its sensitiveness to antibiotics tested, non-hemolytic and non-biogenic production. Additionally, its strain showed the lipolytic, proteolytic and nitrate reductase activities, as well as its adaptability under high salt content and acidic conditions. Comparison of kung-som production by using single starter culture (S. carnosus K21S22 or Lc. lactis KTH0-1S), mixed starter cultures and spontaneous kung-som, they were significant differences (p<0.05) in pH, titratable acidity. However, the pathogenic bacteria such as B. cereus, C. perfringens, E. coli, S. aureus and Salmonella sp. did not detect in all kung-som treatments. For sensory evaluation, all kung-som treatments (spontaneous, inoculated with only Lc. lactis KTH0-18 or S. carnosus K21S22 and mixed starter cultures) were significant difference (p<0.05) in appearance, taste, texture, flavor and overall acceptance. The panelists gave the higher score for organoleptic of kung-som inoculated with Lc. lactis KTH0-1S and mixed culture than the spontaneous kung-som and inoculated kung-som with S. carnosus K21S22. Regarding to our results obtained, it indicates that nisin Z-producing Lc. lactis KTHO-IS are good promising starter culture to successfully apply in kung- som fermentation. It will further expand the application area of bacteriocin producing LAB or CNS strain as starter culture to fermented products for shorten time periods of fermentation, increasing food safety, extended shelf-life and improving quality of products.