【作者】 朱军莉；

【导师】 励建荣；

【作者基本信息】 浙江工商大学 ， 食品科学， 2009， 博士

【摘要】 我国已成为水产品生产大国,产量连续18年居世界第一位,国内外市场对水产品的质量与安全提出了更高的要求。但近年来,我国水产品安全事件层出不穷,其中鱿鱼等水产品中甲醛含量超标问题尤为突出,关系到鱿鱼产业乃至全国水产加工产业的发展,亟待解决。因此,探明鱿鱼及制品加工过程中甲醛产生机理,研发鱿鱼及制品中甲醛的控制技术,已成为迫切之需。鉴于此,本课题以资源丰富、开发潜力巨大的秘鲁鱿鱼为研究对象,跟踪鱿鱼加工和储藏过程甲醛的消长规律,研究鱿鱼甲醛生成的酶学途径和高温化学分解途径,探索添加剂控制鱿鱼冷冻和高温过程内源性甲醛的生成,初步闸明了鱿鱼甲醛抑制剂的作用机制。本论文主要围绕以上几个方面开展工作,获得结果如下:1.鱿鱼及其制品储藏加工过程甲醛消长规律的研究显示,鱿鱼原料中FRB-FA含量为5.33-20.13 mg/kg,鱿鱼品种影响甲醛本底含量,鱿鱼制品甲醛含量明显高于鱿鱼原料,FRB-FA含量为17.03-126.23 mg/kg,F-FA含量为1.09-7.09 mg/kg。肝脏甲醛含量明显高于其他组织,胴体靠近内脏和软骨部分较高。鱿鱼肌肉25℃和4℃储存过程随着鱼肉品质的变质,两种甲醛含量逐步下降,高温促进FRB-FA和F-FA的生成,特别是80-100℃。鱿鱼丝加工过程蒸煮和焙烤工序甲醛含量明显上升。秘鲁和北太鱿鱼丝在25℃、4℃和-20℃储存条件下两种甲醛表现相同的变化趋势,25℃储存的鱿鱼丝FRB-FA呈现上升的趋势,低温储藏减少甲醛生成,鱿鱼丝F-FA均呈现下降-再上升的变化趋势,低温储藏,甲醛下降越低,回升越慢。结果表明,鱿鱼加工热处理促进甲醛生成,使鱿鱼制品甲醛含量显著高于原料,鱿鱼和鱿鱼丝储存过程甲醛含量有显著的变化,随着鱼肉变质甲醛含量逐步下降,低温减少鱿鱼丝甲醛的生成。2.秘鲁鱿鱼甲醛生成TMAOase性质及酶活抑制的研究显示,秘鲁鱿鱼TMAOase活性较低,肝脏酶活相对较高。鱿鱼肝脏和肌肉粗酶最佳作用温度为40℃和50℃,表现不耐热性,最佳作用pH分别为4.5和7.0,Na₂SO₃和CA能明显促进酶活,而TP和Na₂S对酶活有较强的抑制作用。肝脏TMAOase经过酸化、DE52和Sephacryl S-300层析,获得较纯化的酶。纯化酶与肝脏粗酶性质相似,分子量为27.0kDa,纯化酶最佳pH为7.0,最佳作用温度为40℃,表现热不稳定性,Km为20.35 mM。EDTA,Na₂SO₃和CA促进酶活,而TP和Na₂S抑制酶活。鱿鱼肝脏.肌肉和TMAOase-肌肉体外重组体甲醛和DMA明显生成,高含量甲醛能促进鱼肉蛋白质交联,使空间结构变得致密。TP、蔗糖和乙酸3种添加剂能显著减少酶-肌肉重组体中甲醛和DMA含量,其中TP抑制甲醛效果最好,蔗糖还有微弱抑制TMA的生成,3种添加剂能延缓TMAO分解和蛋白可溶性的下降,但是对蛋白分布无影响。结果表明,秘鲁鱿鱼存在TMAOase酶催化生成甲醛的途径,该途径对鱿鱼内源性甲醛的贡献不大,TP、蔗糖和乙酸能有效减少冷冻过程鱿鱼的甲醛含量。3.秘鲁鱿鱼高温过程甲醛生成特性和控制的研究显示,鱿鱼和鳕鱼高温具有不同的甲醛生成特点,5种鱿鱼经100℃处理甲醛、DMA和TMA含量显著上升,相应的TMAO含量下降,而4种鳕鱼相同处理后甲醛显著下降,DMA含量无明显变化,TMA含量下升,相应的TMAO含量下降。鱿鱼肌肉和上清高温甲醛生成规律相似,上清pH 7.0时甲醛和DMA生成最高,添加物Na₂SO₃、Fe²⁺+Asc、Fe²⁺+Cys强烈促进鱿鱼体系甲醛的生成,而TP、MF、CA、CaCl₂、MgCl₂显著降低甲醛含量。TMAO标准体系在辅助因子作用下高温分解生成甲醛、DMA和TMA,温度越高,生成量越多;pH 5.0时甲醛和DMA生成最高;添加剂H₂O₂和碘乙酸促进甲醛生成,而CA和Na₂S减少甲醛含量。TMAO-Fe²⁺体系和鱿鱼体系高温均检测到了相似的自由基六重峰,a_N和g相似,a_N分别为6.07G和6.05G,g为2.009G。高温甲醛抑制剂最佳作用浓度CA和CaCl₂为10mM,MgCl₂为50mM、TP为0.1%,MF为0.05%。CA、TP和CaCl₂正交组均能显著降低鱿鱼上清中的甲醛和DMA含量,将0.04%TP和10mM CaCl₂作为最佳的复合甲醛抑制剂。复合甲醛抑制剂能有效降低鱿鱼片甲醛和DMA含量,不影响蛋白含量,但鱿鱼硬度、明度L^*水值和红度a^*略有增加。结果表明,鱿鱼高温具有TMAO热分解生成高含量的甲醛、DMA和TMA的（CH₃）₃N·自由基反应,该途径-高温化学分解是鱿鱼内源性甲醛的主要来源,复合甲醛抑制剂能显著降低高温过程鱿鱼的甲醛含量。4.秘鲁鱿鱼甲醛抑制剂作用机制的初步研究显示,甲醛抑制剂具有两种不同的作用方式,CA、CaCl₂、MgCl₂通过抑制鱿鱼TMAO的热分解,减少甲醛的生成。TP和MF主要捕获鱿鱼中生成的甲醛。5种几茶素类单体中ECg、EGCg结合甲醛能力较高,甲醛捕获率为21-22%,6种简单酚类中间苯三酚结合甲醛能力最高,甲醛捕获率为34%。CA等有机酸明显抑制鱿鱼和TMAO体系高温甲醛和DMA的生成,鱿鱼体系中草酸、CA和柠檬酸三钠效果最好,而TMAO体系酒石酸效果最好。CaCl₂和乳酸钙能显著减少鱿鱼上清甲醛和DMA含量,Ca²⁺能抑制Fe²⁺对甲醛生成的促进作用。CA和Ca²⁺降低鱿鱼高温TMAO分解的自由基信号强度,CA效果更佳。结果表明,TP捕获甲醛主要与几茶素单体A环的间苯三酚型结构有关,CA通过鳌合体系中的Fe²⁺,Ca²⁺通过抑制Fe²⁺参与TMAO脱甲基作用,从而降低自由基的信号,实现抑制鱿鱼高温甲醛生成。更多还原

【Abstract】 China has become the largest producer of aquatic products,and the output has been the first place in the world for 18 years.So,the higher demand for satety and quality of quactic products were proposed.Recently,among the emergences of food safety endless,the great attention has been paid to the problem of formaldehyde（FA） in squid.It had badly affected the development of squid industry,even the whole aquatic product processing industry in China. Therefore,the study has become an urgent task,that found out the formation mechanism of FA in the process of squid and the effective techniques to control the amount of FA in squid products.In view of these situations,Dosidicus gigas having the largest shape and most abundant resource was aimed in the study.The production trend of FA was decteted during the process and storage stage of squid.Furthernmore,the enzyme pathway and chemical decompositon pathway of FA formation was analyzed.The amount of FA in squid was controlled by the inhibitors during the frozen and thermal stage.And the reactivity mechanism of FA inhibitors were evaluated primarily.The main results obtained in this study were as follows.1.The amounts of FA in tour species of squid mantle were from 5.33 to 20.13 mg/kg, different from species.The content of FRB-FA was higher in squid products than mantle,from 17.03 to 126.23 mg/kg,and F-FA was from 1.09 to 7.09 mg/kg.It was found that the liver tissue and the muscle located near the viscera and gristle produced more FA than the other parts of squid.As the quality of squid muscle deteriorated,the amount of F-FA and FBR-FA reduced gradually in muscle stored at 25℃and 4℃.FA formation increased markedly at high temperature,especially from 80 to 100℃.The cooking and baking processes promoted significantly FA formation during the production of dried squid thread.The change of F-FA and FBR-FA showed the similar trend in dried squid thread of Dosidicus gigas and T pacificus stored at 25℃,4℃and -20℃.The content of FRB-FA increased at 25℃,and restrained at low temperature.Then,the content of F-FA showed the trend of droping and rising again,more significantly dropped,more slowly rising at low temperature.The results indicated that the thermal treatment in squid promoted the formation of FA,causing a higher greatly content of FA in squid products than muscle.In the storge,FA content droped gradually with the muscle deterioration for squid muscle,and FA formation was inhibited at the low temperature for squid thread.2.TMAOase activity in Dosidicus gigas was low among the different tissues,found the highest activity in the liver.The crude TMAOase activity in liver and muscle of squid was optimal at temperature 40℃and 50℃,and was optimal at pH 4.5 and 7.0,respectively.Na₂SO₃ and CA promoted the enzyme activity,whereas enzyme activity was inhibited by TP and Na₂S. TMAOase in the liver was purified by deithylaminoethyl-cellulose and sephacryl S-300 chromatography.The molecular mass of the purified TMAOase was defined to be 27.0 kDa by SDS-PAGE.The optimal pH and temperature of the TMAOase was 7.0 and 40℃,respectively. The enzyme was stable to heat treatment up to 50℃.And the TMAOase converted TMAO to DMA and FA soichiometrically with a Km of 20.35 mM.Na₂SO₃ and CA was effective at activating TMAOase,whereas the TMAOase could be significantly inhibited by TP and Na₂S. The FA and DMA were produced significantly in the recombiner with liver or crude TMAOase from liver and muscle stored at -20℃.The more FA caused the protein aggregation and denser spatical structure of protein.The results indicated that the TMAOase catalyzed the conversion of TMAO to FA and DMA in squid,however,the enzymatic pathway was unimportant for the total amount of FA.The additives of TP,saccharose and acetic acid could effectively reduced the FA content in the frozen squid muscle.3.The mode of FA formation was different between squids and cods at high temperature. After five species of squid treated at 100℃,the amount of FA,DMA and TMA increased. Whereas,in the four species of cod treated similarly,the amount of FA reduced,and TMA increased.Corresponding,the amount of TMAO both in squids and cods dropped significantly. The FA formation was silimlar in both muscle and supernatant of squid at high temperature. The most amount of FA and DMA was produced at pH 7.0 in the squid supernatant.Additives of Na₂SO₃,Fe²⁺ in presence of the reductants of Asc and Cys,exhibed stimulating greatly effect, whereas,TP,ME CA,CaCl_2, MgCl₂ decreased markedly the content of FA in squid.The TMAO solutions decomposed FA,DMA and TMA in presence of Cys,Asc and FeCl₂, especially higher than 60℃.The most amount of FA and DMA was produced at pH 5.0. Additives of H₂O₂ and iodoacetic acid promoted the formation of FA,whereas CA and Na₂S had reduced the content of FA.The free radical signal was detected in thermal processing of TMAO-Fe²⁺ system and squid system.The a_N and g value of free radical signal in the two system were resembled,a_N value about 6.07G and 6.05G,respectively,and g value about 2.009G.The optimal inhibiting concentrations were 10mM,10mM,50mM,0.1%and 0.05%for CA,CaCl_2, MgCl_2, TP and MF,respectively.Three-level,three variable orthogonal experiment of CA,CaCl₂ and TP showed that all of groups could reduce the formation of FA and DMA in squid supernatant,0.04%TP and 10mM CaCl₂ as the optimal group of compound inhibitors. The amount of FA and DMA dropped significantly in squid slice immersed in the compound inhibitor,and the content of protein had no effect.However,the hardness,L^* and a^* increased. The results indicated that the（CH₃）₃N·radical existed in the thermal conversion of TMAO to FA,DMA and TMA in squid.This pathway was important for the total amount of FA in squid products.And the compound inhibitors of TP and CaCl₂ could effectively reduced the FA content in the squid treated at high temperature.4.FA inhibitors had two different mode to reduce the content of FA of squid at high temperature,which CA,CaCl_2（?） MgCl₂ restained the thermal decomposition of TMAO in contrast,TP and MF captured the growed FA.The ECG,EGCg and phloroglucinol had a strong ability of capture FA,which reached 21%,22%,24%,respectively.The production of FA and DMA was inhibited greatly by oganic acid on behalf of CA both in the squid and TMAO system. The inhibiting action of oxalic acid,CA and sodium citrate was most effective in the squid system,whereas,tartaric acid were most effective in the TMAO system.CaCl₂ and calcium lactate could reduce significantly the content of FA and DMA.Futhermore,reaction of Fe²⁺ accelerating the formation of FA was inhibited by Ca²⁺ in squid supernatant.CA and Ca²⁺ could reduce the free radical signal strength produced thermal decompostion of TMAO in squid,the better for CA.The results indicated that the capture ability for TP may due to the presence of a phloroglucinolic A-ring structure.It was mechanism of inhibitors that the Fe²⁺ in the squid was chelated by CA,and function group of Fe²⁺ in the TMAO decomposing reaction was combined competively by Ca²⁺,causing the drop of free radical signal to lesser FA formation.更多还原