The Application In Oyster Depuration, Large-scale Preparation And Preservation Of Vibrio Parahaemolyticus Bacteriophage VPp1

Vibrio parahaemolyticus is a major food-borne bacterium that is widelydistributed in coastal areas, and is easily accumulated in shellfish such as oysters.Since oysters are typically consumed raw on a half shell or lightly cooked because oftheir great taste and delicate texture. Therefore, oysters can pose a threat to publichealth due to ingestion of this pathogenic marine microorganism.In order to explore an effective method to depurate the V. parahaemolyticus inoysters, a single bacteriophage (VPp1) aimed at V. parahaemolyticus (ATCC17802)was used. Large-scale preparation and preservation conditions of phage VPp1werealso preliminary studied, which laid a foundation for the industrial application ofphage. The main results are as follow:1. This study investigated the potential application of the bacteriophage VPp1during depuration to reduce V. parahaemolyticus in oysters at different multiplicity ofinfection (MOI) and temperature levels. Depuration at16°C with0.1MOI was thebest condition for reducing V. parahaemolyticus in oysters, which decreased by2.35–2.76log CFU/g within36h. Furthermore, we expended the phage application tofactory purification experiment, the V. parahaemolyticus in infected oysters weredecreased by1.11log CFU/g. The proliferation of V. parahaemolyticus and otherkinds of marine vibrio in nature accumulated in oysters was also inhibited. Thisdepuration process required low initial investment and running costs. And phageVPp1lysed the host strain and inhibited the proliferation of other vibrio bacteriawithout affecting the normal flora. Moreover, the organoleptic characteristics ofoysters would not be adversely affected during depuration. Therefore, the applicationof bacteriophage was effectively proven to be useful for shellfish depuration.2. In order to meet the needs of phage industrialized production, the fermentation technology used for large-scale preparation of phage VPp1. The optimum condition ofphage VPp1proliferation was obtained by single factor experiment: the optimumculture medium component obtained were pH7.3(no need of adjustion); theappropriate concentration of Ca2+and Mg2+was20mmol/L; the initial concentrationof phage VPp1was MOI0.0001; the cultivation temperature was37C; theincubation time was10-12h. Under the optimal conditions, the concentration ofphage VPp1could get10.74log PFU/mL in the5L fermenter, and the growth multipleof phage concentration was3.79log PFU/mL more than it did in the flask.3. In order to determine the appropriate media for lyophilization and storage ofphage VPp1, we studied the stability and some physical properties of phage VPp1during lyophilization and storage in different media, the results indicated that theaddition of trehalose concentration (0.3mol/L) stabilized the freeze-dried cakes. Inthis case, the cakes had high thermal stability (the endothermic peak temperature was167.3C) and the moisture content was7.5%. The cakes also showed goodmorphology under transmission electron microscopic (TEM) and high titerpreservation rate (98.8%) after the4months storage.In conclusion, the application of bacteriophage VPp1was effectively proven tobe useful for shellfish depuration. Moreover, studied about the large-scale preparationand lyophilization storage of phage VPp1provided the theory support to industrialapplication of bacteriophage.