罗非鱼片臭氧减菌化处理中自由基的产生及其对产品品质与安全性的影响
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摘要
罗非鱼片是我国优势出口水产品，出口产品主要以冻罗非鱼片为主。罗非鱼片生产过程中常采用次氯酸钠进行减菌化处理，但易造成次氯酸钠残留，影响产品品质。臭氧作为一种新型的抑菌剂，广泛应用于食品生产过程中的清洗、消毒及灭菌，采用臭氧对水产品进行减菌化处理日益被水产品加工企业广泛采用。但臭氧处理时会产生具有较强的化学活性的自由基，自由基的强氧化性会加速鱼肉蛋白在冻藏过程中的冷冻变性，并可造成鱼肉中脂质的氧化水解，生成各种羰基化合物、三甲胺与丙二醛等，造成鱼肉风味及口感劣化，影响产品品质，且水产品臭氧减菌化处理后产品的安全性尚未有系统科学地评价。鉴于此，本研究以罗非鱼片为研究对象，主要的研究内容与结论如下：
1.测定减菌化处理中臭氧水中O_3浓度，采用自旋捕获-电子顺磁共振（spintrapping-EPR）技术检测罗非鱼片臭氧水减菌化处理过程中臭氧水中产生及处理后罗非鱼片残留自由基的种类。结果表明：试验用臭氧水中臭氧浓度为4.5mg/mL；设置EPR中心磁场强度3510G、微波功率10mW、调制频率100kHz、振幅1G、扫描时间2min，以DMPO作为捕获剂，室温条件下测定臭氧水中自由基的种类，证实臭氧水中存在超氧阴离子自由基（O_2~-·）与羟自由基（· OH）；在低温条件下（~(-1)96℃）检测罗非鱼肉表面残留自由基的种类，臭氧减菌化处理后罗非鱼片表面残留有O_2~-·。
2.以2-氨基吡啶、水杨醛与吡啶-2-甲醛为原料，基于亲核取代反应合成2种荧光探针：2-（2’-亚水杨氨基）吡啶与2-（2’-吡啶亚胺甲基）吡啶，并对合成产物进行表征以确定目标物的生成。结果表明：2种荧光探针均可与O_2~-·发生荧光淬灭反应，导致探针因-C=N-基团的破坏而出现荧光强度显著降低，基于此现象，建立了以2-（2’-吡啶亚胺甲基）吡啶（2-APC）为荧光探针检测邻苯三酚自氧化体系中产生O_2~-·相对含量的方法，该方法反应体系最佳条件为：试剂加入顺序为缓冲溶液、2-APC、邻苯三酚；反应pH值为8.2；反应温度为40℃；反应时间为40min。测定条件为：λ_(ex)=295nm、λ_(em)=365nm，狭缝宽度5nm。邻苯三酚浓度在0.4×10~(-6)~8.0×10~(-6)mol/L范围内与相对荧光强度呈良好线性关系，线性回归方程为y=37.567x+55.581，R~2=0.9834。
3.通过测定罗非鱼肌肉组织中肌原纤维蛋白盐溶性、肌动球蛋白表面疏水性、巯基含量及ATPase活性，研究了经臭氧处理及未经处理罗非鱼片在-20℃冻藏过程中蛋白质生化特性的变化。结果表明：随着冻藏时间的延长，两组鱼肉蛋白质均发生不同程度的变性，从而导致蛋白质各种生化特性的变化；两组鱼肉肌原纤维蛋白盐溶性与巯基含量均呈下降趋势，且对照组鱼肉肌原纤维蛋白盐溶性与总巯基含量高于臭氧处理组（P<0.05），而肌动球蛋白表面疏水性呈上升趋势，同一贮藏时间由于臭氧处理中产生的O_2~-·破坏了罗非鱼肌肉暴露在蛋白质结构表面的疏水性氨基酸残基，导致其疏水性高于对照组；两组鱼肉组织中Ca~(2+)-ATPase活性、Mg~(2+)-ATPase活性及Ca~(2+)Mg~(2+)-ATPase活性在贮藏过程中均显著降低，且臭氧处理组组比对照组低（P<0.05）。总之，臭氧处理中产生的O_2~-·加速了鱼肉蛋白在冻藏过程中的变性作用；
4.分别进行鱼肉pH测定、MDA含量测定、K值测定、色差分析及质构分析系统研究了经臭氧处理及未经臭氧处理罗非鱼片在-20℃冻藏过程中产品品质的变化。结果表明：臭氧处理组与对照组罗非鱼片pH值变化范围为6.3±0.1~7.1±0.0，在贮藏过程中呈先降后升的变化趋势；两组罗非鱼片MDA含量在冻藏过程中均逐渐增加，且由于臭氧处理过程中自由基对鱼肉脂质氧化的促进作用，臭氧处理组MDA含量明显高于对照组（P<0.05）；两组罗非鱼片在冻藏过程中，K值均呈上升趋势，由于臭氧的抑菌作用，相同贮藏时间下臭氧处理组K值较对照组低（P<0.05）；由于臭氧具有漂白作用，臭氧处理组罗非鱼片L值、HW值较对照组大，而值比对照组小（P<0.05）；两组罗非鱼片在-20℃冻藏过程中，随着贮藏时间的延长，鱼肉的硬度、胶着性与咀嚼性有极显著降低（P<0.01），而粘合性、粘聚性、弹性（臭氧处理组）与粘附力随贮藏时间的变化较显著（P<0.05），对照组弹性变化不显著（P>0.05）；菌落总数分析结果表明，两组鱼肉在冻藏过程前10d菌落总数降低，10d后菌落总数逐渐增加（P>0.05），且对照组高于臭氧处理组（P<0.05）。
5.采用SD大鼠急性经口毒性试验、KM小鼠遗传毒性试验及SD大鼠30天喂养试验评价臭氧处理后罗非鱼片产品安全性。结果表明：臭氧处理后罗非鱼片对SD大鼠经口最大耐受剂量MTD＞15g/kg，毒性分级为无毒；Ames试验结果表明，在有无S9活化系统下，罗非鱼片5个剂量组4种菌株的回变菌落数均小于溶剂对照组的2倍，重复试验结果一致，Ames试验结果为阴性；骨髓微核试验结果表明，罗非鱼片高、中、低剂量小鼠的骨髓微核率与溶剂对照组比较无显著性差异（P>0.05）；睾丸染色体畸变试验结果表明，罗非鱼片高、中、低剂量组小鼠睾丸染色体畸变率分别为0.2%、0.2%，0.1%，与溶剂对照组相比无显著差异（P>0.05），KM小鼠遗传毒性试验结果为阴性。30天喂养试验中一般临床观察、体重、摄食量、食物利用率、摄水量、血液学、血液生化、脏器重量及脏器指数、组织病理学检查等各检查指标罗非鱼片各剂量组均未见明显改变，初步认为臭氧处理罗非鱼片对SD大鼠的最大无作用剂量大于7.5g·kg~(-1)·d~(-1)。
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 O_3in 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 (O_2~-·) 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 (~(-1)96℃) condition, and the existence of O_2~-· 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 O_2~-·, and the fluorescence intensity of reaction system decreased significantly.Based on this phenomenon, a fluorescence probe method for O_2~-· 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, λ_(ex)=295nm, λ_(em)=365nm and the slit width was5nm. In therange from0.4×10~(-6)mol/L to8.0×10~(-6)mol/L, relative fluorescence intensity (y) andpyrogallol concentration (x) were shown a good linear relationship, y=37.567x+55.581，R~2=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 O_2~-· generated from ozone-water,surface hydrophobicity of ozone pretreatment group is higher than control group. All of theCa~(2+)-ATPase activity, Mg~(2+)-ATPase activity and Ca~(2+)Mg~(2+)-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 O_2~-· 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).
1.测定减菌化处理中臭氧水中O_3浓度，采用自旋捕获-电子顺磁共振（spintrapping-EPR）技术检测罗非鱼片臭氧水减菌化处理过程中臭氧水中产生及处理后罗非鱼片残留自由基的种类。结果表明：试验用臭氧水中臭氧浓度为4.5mg/mL；设置EPR中心磁场强度3510G、微波功率10mW、调制频率100kHz、振幅1G、扫描时间2min，以DMPO作为捕获剂，室温条件下测定臭氧水中自由基的种类，证实臭氧水中存在超氧阴离子自由基（O_2~-·）与羟自由基（· OH）；在低温条件下（~(-1)96℃）检测罗非鱼肉表面残留自由基的种类，臭氧减菌化处理后罗非鱼片表面残留有O_2~-·。
2.以2-氨基吡啶、水杨醛与吡啶-2-甲醛为原料，基于亲核取代反应合成2种荧光探针：2-（2’-亚水杨氨基）吡啶与2-（2’-吡啶亚胺甲基）吡啶，并对合成产物进行表征以确定目标物的生成。结果表明：2种荧光探针均可与O_2~-·发生荧光淬灭反应，导致探针因-C=N-基团的破坏而出现荧光强度显著降低，基于此现象，建立了以2-（2’-吡啶亚胺甲基）吡啶（2-APC）为荧光探针检测邻苯三酚自氧化体系中产生O_2~-·相对含量的方法，该方法反应体系最佳条件为：试剂加入顺序为缓冲溶液、2-APC、邻苯三酚；反应pH值为8.2；反应温度为40℃；反应时间为40min。测定条件为：λ_(ex)=295nm、λ_(em)=365nm，狭缝宽度5nm。邻苯三酚浓度在0.4×10~(-6)~8.0×10~(-6)mol/L范围内与相对荧光强度呈良好线性关系，线性回归方程为y=37.567x+55.581，R~2=0.9834。
3.通过测定罗非鱼肌肉组织中肌原纤维蛋白盐溶性、肌动球蛋白表面疏水性、巯基含量及ATPase活性，研究了经臭氧处理及未经处理罗非鱼片在-20℃冻藏过程中蛋白质生化特性的变化。结果表明：随着冻藏时间的延长，两组鱼肉蛋白质均发生不同程度的变性，从而导致蛋白质各种生化特性的变化；两组鱼肉肌原纤维蛋白盐溶性与巯基含量均呈下降趋势，且对照组鱼肉肌原纤维蛋白盐溶性与总巯基含量高于臭氧处理组（P<0.05），而肌动球蛋白表面疏水性呈上升趋势，同一贮藏时间由于臭氧处理中产生的O_2~-·破坏了罗非鱼肌肉暴露在蛋白质结构表面的疏水性氨基酸残基，导致其疏水性高于对照组；两组鱼肉组织中Ca~(2+)-ATPase活性、Mg~(2+)-ATPase活性及Ca~(2+)Mg~(2+)-ATPase活性在贮藏过程中均显著降低，且臭氧处理组组比对照组低（P<0.05）。总之，臭氧处理中产生的O_2~-·加速了鱼肉蛋白在冻藏过程中的变性作用；
4.分别进行鱼肉pH测定、MDA含量测定、K值测定、色差分析及质构分析系统研究了经臭氧处理及未经臭氧处理罗非鱼片在-20℃冻藏过程中产品品质的变化。结果表明：臭氧处理组与对照组罗非鱼片pH值变化范围为6.3±0.1~7.1±0.0，在贮藏过程中呈先降后升的变化趋势；两组罗非鱼片MDA含量在冻藏过程中均逐渐增加，且由于臭氧处理过程中自由基对鱼肉脂质氧化的促进作用，臭氧处理组MDA含量明显高于对照组（P<0.05）；两组罗非鱼片在冻藏过程中，K值均呈上升趋势，由于臭氧的抑菌作用，相同贮藏时间下臭氧处理组K值较对照组低（P<0.05）；由于臭氧具有漂白作用，臭氧处理组罗非鱼片L值、HW值较对照组大，而值比对照组小（P<0.05）；两组罗非鱼片在-20℃冻藏过程中，随着贮藏时间的延长，鱼肉的硬度、胶着性与咀嚼性有极显著降低（P<0.01），而粘合性、粘聚性、弹性（臭氧处理组）与粘附力随贮藏时间的变化较显著（P<0.05），对照组弹性变化不显著（P>0.05）；菌落总数分析结果表明，两组鱼肉在冻藏过程前10d菌落总数降低，10d后菌落总数逐渐增加（P>0.05），且对照组高于臭氧处理组（P<0.05）。
5.采用SD大鼠急性经口毒性试验、KM小鼠遗传毒性试验及SD大鼠30天喂养试验评价臭氧处理后罗非鱼片产品安全性。结果表明：臭氧处理后罗非鱼片对SD大鼠经口最大耐受剂量MTD＞15g/kg，毒性分级为无毒；Ames试验结果表明，在有无S9活化系统下，罗非鱼片5个剂量组4种菌株的回变菌落数均小于溶剂对照组的2倍，重复试验结果一致，Ames试验结果为阴性；骨髓微核试验结果表明，罗非鱼片高、中、低剂量小鼠的骨髓微核率与溶剂对照组比较无显著性差异（P>0.05）；睾丸染色体畸变试验结果表明，罗非鱼片高、中、低剂量组小鼠睾丸染色体畸变率分别为0.2%、0.2%，0.1%，与溶剂对照组相比无显著差异（P>0.05），KM小鼠遗传毒性试验结果为阴性。30天喂养试验中一般临床观察、体重、摄食量、食物利用率、摄水量、血液学、血液生化、脏器重量及脏器指数、组织病理学检查等各检查指标罗非鱼片各剂量组均未见明显改变，初步认为臭氧处理罗非鱼片对SD大鼠的最大无作用剂量大于7.5g·kg~(-1)·d~(-1)。
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 O_3in 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 (O_2~-·) 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 (~(-1)96℃) condition, and the existence of O_2~-· 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 O_2~-·, and the fluorescence intensity of reaction system decreased significantly.Based on this phenomenon, a fluorescence probe method for O_2~-· 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, λ_(ex)=295nm, λ_(em)=365nm and the slit width was5nm. In therange from0.4×10~(-6)mol/L to8.0×10~(-6)mol/L, relative fluorescence intensity (y) andpyrogallol concentration (x) were shown a good linear relationship, y=37.567x+55.581，R~2=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 O_2~-· generated from ozone-water,surface hydrophobicity of ozone pretreatment group is higher than control group. All of theCa~(2+)-ATPase activity, Mg~(2+)-ATPase activity and Ca~(2+)Mg~(2+)-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 O_2~-· 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).
引文
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