Study On The Formation Mechanism And Control Of Intrinisic Formaldehyde From Squid (Dosidicus Gigas)

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.