| Nile Tilapia （Oreochromis niloticus） is an advantage export of aquatic products in China，and the frozen Nile Tilapia fillet is the main export product. Sodium hypochlorite is used as abacteriostatic agent in Nile Tilapia fillet processing, In order to avoid the problem of sodiumhypochlorite residue, as a novel bacteriostatic agent, ozone has been widely used in cleaningdisinfection and sterilization of food products, in addition, ozone sterilization pretreatmentmethod has been used in aquatic products processing enterprises broadly. The high chemicalactivity free radicals could be produced in the ozone sterilization pretreatment step, freezedenaturation of tilapia protein might be accelerated by the strong oxidizing property of freeradicals as well as lipid oxydrolysis. The degradation products such as carbonyl groups,trimethylamine（TMA） and malonaldehyde（MDA） will cause the flavor and taste of NileTilapia fillet degradation. The safety of ozone sterilization pretreatment in aquatic productsprocessing field has not been evaluated scientifically. For these reasons, the main researchcontents and results are as follows:1. The concentration of O3in ozone-water was determined. The kinds of free radicalsgenerated from ozone-water were detected by spin-trapping electron paramagnetic resonance（EPR） spectroscopy which was used DMPO as the trapping agent, and3510G of center field,10mW of power100kHZ of modulation frequency1G of amplitude and2min of sweeptime. The results show that the concentration of ozone in water is4.5mg/mL. The existenceof superoxide anion free radical （O2-·） and hydroxyl radical （OH） are confirmed inozone-water under room temperature. The kinds of residual free radicals of Nile Tilapia filletsafter ozone sterilization pretreatment were detected by EPR under the low temperature (-196℃) condition, and the existence of O2-· is confirmed.2. Two kinds of novel fluorescence probe:2-SAP and2-APC were synthesized by2-aminopyridine, salicylaldehyde and2-pyridinecarbaldehyde by nucleophilic substitutionreaction and characterized by melting test, elemental analysis, infrared spectrum and1HNMR. The results shows that both of2-SAP and2-APC can produce fluorescence quenchingreaction with O2-·, and the fluorescence intensity of reaction system decreased significantly.Based on this phenomenon, a fluorescence probe method for O2-· detection was established.The optimum conditions of this reaction system are as follows,8.2of pH,40℃oftemperature of reaction system and40min of reaction time. The conditions of fluorimetricdetermination were as follows, λex=295nm, λem=365nm and the slit width was5nm. In therange from0.4×10-6mol/L to8.0×10-6mol/L, relative fluorescence intensity （y） andpyrogallol concentration （x） were shown a good linear relationship, y=37.567x+55.581，R2=0.9834.3. In order to investigate the changes of protein biochemistry during storage at-20℃,myofibrillar protein salt solubility, surface hydrophobicity of actomyosin, sulphur content andATPase activity in Nile Tilapia muscle were determined. The results show that: differentdegrees of protein denaturation of ozone pretreatment group and control group Nile Tilapiafillets were occurred during storage period, thus protein denaturation results in biochemistrychanges. During the extension of storage time， both of the myofibrillar protein salt solubilityand sulphur content are decreased, of which the control group were higher than ozonepretreatment group （P<0.05）. Moreover, surface hydrophobicity of actomyosin is increasedduring storage, due to the hydrophobic amino acid residue exposed on the surface of proteinstructure in Nile Tilapia fillets muscle is destroyed by O2-· generated from ozone-water,surface hydrophobicity of ozone pretreatment group is higher than control group. All of theCa2+-ATPase activity, Mg2+-ATPase activity and Ca2+Mg2+-ATPase activity are decreasedduring storage period, and the ATPase activity of ozone pretreatment group is lower thancontrol group （P<0.05）. In conclusion, the protein denaturation is promoted by O2-· generatedfrom ozone-water during storage period.4. The pH value, MDA content, K-value, color measurement and textural analysis werecarried out to research the quality changes of Nile Tilapia fillets with and without ozone-watersterilization pretreatment during frozen storage at-20℃. The results are as follows: the pHvalue of Nile Tilapia fillets with and without ozone-water sterilization pretreatment is at therange of6.3±0.1to7.1±0.0of which tend is increasing first and then decreasing. MDAcontent of these two groups are gradually increasing during storage, owning to theacceleration of lipid oxidation with free radicals during ozone-water sterilization pretreatment,the MDA content of ozone pretreatment group is higher than control group significantly（P<0.05）. Both K-value of these two groups are increased during storage. Due to the bactericidal effect of ozone, the K-value of ozone pretreatment group is lower than controlgroup （P<0.05）. Because of the bleaching of zone, the L value and HW value of ozonepretreatment group is higher than control group, and the value is lower （P<0.05）. Duringthe extension of storage time, all of the hardness, gumminess and chewiness of Nile Tilapiafillets are decreased significantly （P<0.01）, and the adhesiveness, cohesiveness springiness ofozone pretreatment group and adhesive force are decreased （P<0.05）, but the springiness ofcontrol group shows no significant difference during storage at-20℃. Total plate count of thetwo groups are decreased during the first10days of frozen storage period, and then increasedgradually （P<0.05）, in addition, the control group is higher than ozone pretreatment group（P<0.05）.5. Acute oral toxicity test in SD rats, genetic toxicity test in KM mice and30daysfeeding study in SD rats were used to evaluate the safety of Nile Tilapia fillets treated byozone-water. The results show that the maximum tolerated dose in SD rats is more than15g/kg, the fillets treated by ozone-water belongs to non-toxic class. The result of Ames assayshows that the Colony-Forming Units of bacterial reverse mutation of four strains in the fivedoze groups are less than two times of control group, with or without metabolic activationsystem, as well as the repeated assay. Therefore, the results of Ames assay are negative. Theresults of bone marrow micronucleus test show that there is no significant difference betweenthe three doze groups and control group on micronucleus rate （P>0.05）. The results ofchromosome aberration test show that chromosome aberration rate of mouse testis are0.2%,0.2%and0.1%respectively, there is no significant difference between the three doze groupsand control group （P>0.05）. The results of genetic toxicity test in KM mice are negative. Inthe thirty days feeding study, there are no significant changes on general clinical observation,weight, food intake, food utilization rate, water intake, hematology, blood biochemical index,organ weight, organ indexes and histopathological examination of SD rats. The maximum noneffect level for ozone-treated Nile Tilapia fillets is more than7.5g·kg-1·d-1. |
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