Screening Of Antagonistic Lactic Acid Bacteria Isolated From Marine Fish Intestinal And Its Application In Turbot Preservation

Lactic acid bacteria(LAB)widely habit in the natural environment,such as traditional fermented foods,animal intestine,feed and sludge,which can produce nisin,reuterin and organic acid to control spoilage organisms and pathogenic bacteria.In this paper,LAB with strong antagonistic activity to Pseudomonas fluorescens and Vibrio harveyi were isolated from marine fish intestine.Its antagonism mechanism and antimicrobial components were evaluated preliminarily.It was applied to control P.fluorescens of turbot using LAB crude extract.The main results were as follows.1.A total of 49 strains LAB were isolated from intestine of ocean perch,coilia ectenes,olive flounder and turbot.Four strains LAB with strong antagonistic activity against P.fluorescens and V.harveyi were screened from 49 strains LAB using the method of Oxford cup agar diffusion.Strains LY1-6 and YP4-2 were identified as Lactobacillus sakei,strains DY4-2 and LP1-4 were identified as Lb.plantarum by physiological and biochemical characteristics and 16S rRNA sequence analysis.The antibacterial components of cell-free supernatants(CFS)from strain LY1-6?YP4-5?DY4-2 and LP1-4 were analyzed.The CFS reached the highest antagonistic activity after 24 h incubation.The inhibitory substance of LAB produced was preliminarily determined as bacteriocin by heat,protease and pH assays.2.The antagonism mechanisms of strain DY4-2 CFS to P.fluorescens and V.harveyi were preliminarily studied.The results showed that cell division of logarithmic phase of P.fluorescens and V.harveyi were inhibited and the stable phase was delayed by CFS of strain DY4-2.The conductivity,protein,alkaline phosphatase(AKP)activity and Na+/K+-ATPase activity of P.fluorescens and V.harveyi analyzed,it was indicated that large amounts of macromolecules have been leaked by CFS of strain DY4-2.Furthermore,the scanning electron microscope results showed that the CFS of strain DY4-2 led to cell integrity damage,cell membrane dissolve and intracellular component leakag.3.The bacteriocin produced by strain DY4-2 was purified and identified by ethyl acetate extraction,ultrafiltration,gel chromatography and high performance liquid chromatography(HPLC).The active peak with a retention time of 5.30 min had inhibitory activity to P.fluorescens and V.harveyi.SDS-PAGE showed that the molecular mass of the bacteriocin was between 1.0?3.3 ku.The mass spectrometry(MS)result showed that the molecular mass of the bacteriocin was 1.46 ku.Amino acid composition analysis showed that a total of 13 amino acid of the bacteriocin was His,Ala,Gln,Ile,Gly,Met,Thr,Met,Asn,Gly,Ser,Phe,Arg.4.The effects of bacteriocin extracted from strain DY4-2 on preservation of turbot slice under 4? were investigated.Results indicated that the sensory quality of the turbot slice was improved by adding bacteriocin extract.The microorganism growth of fish slice was inhibited,and the time of TBC(total number bacterial colonies)exceed bid was extended 2 days at least.The counts of P.fluorescens decreased 3 log units by bacteriocin treatment.The pH,TVB-N and K value of fish slice was decreased slowly.The addition of bacteriocin crude exteact of strain DY4-2 in turbot slice had a good preservation effect and could extend shelf life 2 days at least.