| Bioluminescent bacteria have been widely used for rapid biotoxicity monitoring in waterresources or environment including pesticide residue, heavy metal, biotoxin and etc. and alsoare hopeful for food safety detection of veterinary drugs with high sensitivity, rapid responseagainst toxicants and low cost.Recently, in order to develop continuous real time monitoring of toxicants, theimmobilization of V.qinghaiensis-Q67(a kind of bioluminescent strain in fresh water) andP.phosphoreum-T3(a kind of bioluminescent strain in sea) has been commonly studied.In order to make high initial bioluminescence of lyophilized V.qinghaiensis-Q67andP.phosphoreum-T3in a short time of recovery meet the requirements of rapid toxicantmonitoring and industrial production, the present study focuses on the screen of fine,anti-freeze-drying strains and protectants, technological conditions during the freeze-dryingprocess, and subsequent storage. This paper mainly have several findings as follows:(1) the growth curve of V.qinghaiensis-Q67and P.phosphoreum-T3and the effect ofcultural conditions on the cell resistance to freeze drying were investigated. When the culturetemperature were20℃and the culture were harvested at end of the logarithmic phase(18h),the ability of the cell resistance to freeze drying were the highest.(2) The relation of freeze-drying thickness and drying time, water content offreeze-drying culture were studied. The basic technologic parameter of preparing freeze-driedculture were determined. When the freeze-drying thickness was0.75cm, which was1mL inthe freeze-dried vials, drying time was18h, and the ratio and the interaction time of thebacteria and protectants was respectively1:1,30min, the biolumiscence of V.qinghaiensissp.-Q67reached the highest.(3) According to the method of combination of macromolecule and micromolecule, theprotectants of V.qinghaiensis-Q67were optimizedly screened.2kinds of optimal combinedformula were received,(27) the mixture of10%skim milk,6%sucrose,1%sodium glutamateand1%chitosan and (37) the mixture of medium,6%lactose,1%skim milk, and1%sodiumglutamate. The biolumiscence of lyophilized V.qinghaiensis sp.-Q67with2formula after 30min of regeneration were respectively2675%,2567%.(4) The protectants was optimized by Box-Behnken Design and Response SurfaceAnalysis after firstly screened by the single factors. In response to the relative light unit of thelyophilized V.qinghaiensis sp.-Q67after the regeneration for30min, when taken skim milk9.68%, sucrose5.53%, soluble starch0.62%, sodium glutamate1%as the optimal combinedformula, The biolumiscence reached the highest.(5) Protectants with medium were not suitable for the preparation of lyophilizedP.phosphoreum-T3; The biolumiscence of lyophilized P.phosphoreum-T3with skim milkafter30min of regeneration was higher than with medium, and lyophilized bodies hadadvantage in subsequent storage.(6) the effect, stability of storage of the two lyophilized bodies and the suitableconditions for long-term storage were investigated. The results showed that P.phosphoreumsp.-T3was significantly more resistant than V.qinghaiensis sp.-Q67to the freeze-dryingprocess. Lyophilized V.qinghaiensis sp.-Q67can be stored for long term at lower temperaturethan room temperature. In its continuously cultured process during storage, itsbioluminescence was increasing, which means that its vitality was increasing. However, thestorage temperature for P.phosphoreum sp.-T3were in a wide range, from-20℃to roomtemperature. Its initial bioluminescence was higher, when continuously cultured for2h, itsbioluminescence reached the highest.(7) Freeze-dried cells of V.qinghaiensis-Q67and P.phosphoreum-T3were observed byscanning electro-microscope. Protective Mechanism of protectants on on cells waspreliminarily explored. The results demonstrated that the lyophilizaton process made theprotectants have the structure of multi-hole and loose for support. The spatial structure(schistose) provided partial space parclose for bacterial bodies. |
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