Screening Of Lactic Acid Bacteria With Antagonistic Vibrio Parahaemolyticus And Application In White Shrimp Litopenaeus Vannamei

Lactic acid bacteria(LAB)are distributed in the natural environment widely,such as traditional fermented food and animal intestine,which can produce acidic material and antagonistic material to control Gram-positive bacteria Gram-negative and bacteria.Vibrio parahaemolyticus is one of main halophilic bacterium existed in the marine environment,which can causes foodborne disease by marine products.LAB that have strong antagonistic activity to Vibrio parahaemolyticus were isolated from freshwater fish intestinal in this paper,at the same time,the antagonistic material and mechanism were evaluated preliminarily.The application effects of antagonistic material were verified using shrimp as carriers,which aims to develop biological protective agent.The main results were as follows:1.A total of 46 LAB strains that have strong antagonistic activity to Vibrio parahaemolyticus were isolated from freshwater fish intestinal.The highest inhibition zone diameters of the strain LY-19 from carp intestinal and JY-22 from crucian carp intestinal were 19.87 mm and 20.53 mm,respectively.Strains of LY-19 and JY-22 were identified as Lactobacillus sakei and Lactobacillus plantarum by physiological and biochemical characteristics and 16S rDNA sequence,respectively.2.The antagonistic activity was mainly from cell-free supernatants(CFS)for this two strains.The CFS that LAB fermented for 24h had the highest inhibitory effects,still had antibacterial activity within pH 3.0-4.5.The antagonistic activity lowered by treating with neutral protease,pepsin and papain,but remained by thermal treatment from 40? to 121? for 30 min.The strain LY-19 was not sensitive to a-amylase,however,JY-22 was sensitive to a-amylase.The inhibitory substance of strains LY-19 and JY-22 produced were respectively determined as type ?non-glycosylation bacteriocin and type ? glycosylation bacteriocin preliminarily.3.The MIC of inhibitory substance produced from JY-22 was 6.0mg/mL after extracted with ethyl acetate.Three tubes of moderate purification substance wered collected by Sephadex G-50 chromatography.The molecular mass of purified bacteriocin was estimated about 4.5 ku by SDS-PAGE after freeze-drying.4.The effect of bacteriocin JY-22 to control the V.parahaemolyticus in shrimp were verified.The results showed that the counts of V.parahaemolyticus added bacteriocin JY-22 decreased to 1.13 orders of magnitude compared with the control group,at the same time,the pH,TVB-N and total viable counts of shrimp were almost invariant.5.In order to study the antagonism mechanisms of bacteriocin JY-22,the indexes,such as cell dynamics process,permeability of cell membrance and cell morphology were measured.Compared with the control,the growth curve of V.parahaemolyticus dealed with bacteriocin JY-22 were changed,which always remained at very low level.The electrical conductivity had increased 3.08 times in the experimental group treated by 0.6 MIC for 8h than the control,which indicated that bacteriocin JY-22 can destroyed cell membrance and increased the permeability of cell membrance.The substance such as Na+/K+-ATPase and alkaline phosphatase(AKP)leakage to outside of cell.The result of scanning electron microscopy illustrated that the structure of the bacterial cell was damaged by JY-22 with consequent morphologxical changes,resulted in distorting of cell and killing the cell.