| Vibrio parahaemolyticus is primary causative factor of our country’s reported foodborne diseases outbreak. At the same time, the bacteria can also cause Vibrio disease outbreak in the aquatic animal to lead to great economic losses in aquaculture. The abuse of antibiotics not only caused the emergence of multi-resistant bacteria in aquaculture, but also threatened to human health. Therefore, there is an urgent need to find antibiotic alternatives for preventing V. parahaemolyticus. Among of the alternatives, the biological control method used of probiotics has obvious characteristics of less environmental pollution and source control of pathogen to make it possibly becoming one of the main development directions for Vibrio disease control in aquaculture. At present, the studies on antibacterial mechanisms of the probiotics were still rare from the molecular level. In view of this, this study tried to explore the probiotics antagonistic to V. parahaemolyticus in aquatic environment and to apply the probiotics in the aquatic environment in order to reduce the efficiency drops or disappear caused by probiotics habitat change. The bacteria were separated and identified to select probiotics and confirm its classification status; the fermentation culture medium of probiotic was optimized to obtain the largest amount of antibacterial substances; the antimicrobial substances was separated and purified using chromatographic technique; the purified antibacterial substances were applied to V. parahaemolyticus to study its cellular damage effect; proteomics technology was used to discover the reactive proteins of V. parahaemolyticus treated with the purified antibacterial substances for further study the mechanism of antagonism effect. The main research results are as follows:1. A total of 249 bacteria were isolated from four types of seafood and coastal sediment and all isolates was assayed with spot inoculation. Twenty-four isolates antagonistic to V. parahaemolyticus were obtained. The genetic stability of 16 strains was secondarily screened using the Oxford cup method. Only strains of B16 and J7 showed extracellular antimicrobial activity against V. parahaemolyticus and were identified as Bacillus pumilus and Bacillus mojavensis respectively based on the morphology, the physiological identification and 16S rDNA sequence analysis. Both of the strains B16 and J7 exhibited extracellular hydrolytic enzyme activity and antagonism against more than one indicator bacteria, among them, B. mojavensis J7 was determined as the next step of experimental strain for producing more variety extracellular enzymes and wider antibacterial spectrum.2. The Landy medium which could make B. mojavensis J7 to produce the most antibacterial substances was as a basic medium to screen carbon sources, nitrogen sources and fermentation conditions (fermentation temperature, time, the initial medium pH and inoculum size). The results showed that the sugar and yeast powder were the best carbon source and nitrogen source respectively. The best fermentation conditions were 30 ℃,36 h, initial medium pH7.0,6% inoculum size. The Plackett-Burman experiment design was used to select main factors affecting antimicrobial substances production of B. mojavensis J7 from the above six factors. Sucrose content, yeast powder content and fermentation temperature were main influencing factors. According to the effect of the three main influencing factors the steepest climbing experiment was adopted to determine the center of the response surface experiment. Quadratic polynomial mathematical model was established using Box-Behnken design and the significance of model was tested using the Design Expert 8.0 software. Modified formula (/L) and culture conditions of medium for:sucrose 16.9 g, yeast powder 6.6 g, MgSO40.5 g, KC10.5 g, KH2PO41.0 g, CuSO40.16 mg, FeSO40.15mg, MnSO45.0 mg, fermentation temperature of 29.7 ℃, fermentation time 36 h, initial pH7.0. The bacteriostat titer increased by 38.89% in the same amount of medium than before optimization with the modified medium formula and culture conditions.3. Antibacterial substances existed in fermentation supernatant of B. mojavensis J7 were extracted by using the acidic precipitation method and the methanol extraction method. Four kinds of active stable antibacterial extracts were obtained by the Sephadex LH-20 gel column chromatography and the analytical HPLC chromatography. These active extracts were all peptides and the main amino acid sequences were gained through analysis and identification with MALDI-TOF-TOF/MS. Thermal stability, pH stability, ultraviolet stability and the minimum bacteriostasis concentration of the active component P3 were tested. The results display that the antimicrobial activity of antimicrobial peptides basic remain unchanged when at 100℃ 60min and 121℃ 20min, pH2-12 and the ultraviolet light irradiation,180 min. The MIC of antimicrobial peptides is 1 mg/mL4. When V. parahaemolyticus was treated by more than one MIC concentration of antibacterial peptides, the bacterial multiplication was significantly inhibited, the shape and structure of the cell were damaged, the cell membrane permeability increased to lead to leakage of cell contents, and the cell metabolism reduced. Compared with control, SDS-PAGE electrophoresis bands did not appear obviously missing phenomenon when V. parahaemolyticus was treated by 0.5 MIC antibacterial peptides, while a few bands of color changed which may be due to the expression quantity of the molecular weight of protein has changed.5. By 2-DE technology,56 differentially expressed protein points with abundance ratio greater than 2 or less than 0.5 and p< 0.05 were obtained with the gel map of untreated as control when V. parahaemolyticus was treated by 0.5 MIC antibacterial peptides.36 among 56 differentially expressed protein points were up-regulated,20 protein points were down-regulated. These differentially expressed proteins were identified by mass spectrometry to analysis their physiological functions. The results show that these differentially expressed proteins involved in a variety of metabolic processes:carbohydrate metabolism, energy metabolism, metabolism of cofactors and vitamins, amino acid metabolism, nucleotide metabolism, lipid metabolism, genetic information processing, environmental information processing, redox homeostasis, transport and binding and immune stress. According to identification results of differentially expressed proteins, the expression abundance of periplasmic proteins increased significantly, the expression abundance of outer membrane protein W decreased, the expression abundance of proteins with genetic information processing decreased generally, I speculated that the damage of the outer membrane and the interruption of genetic information processing may be the reasons that the antibacterial peptides could be antagonistic to V. parahaemolyticus. |
