| Oyster is the food with the highest zinc content in nature and protein zinc is abundant.However,the distribution,structural characteristics,digestion and absorption properties,and effects on reproductive system of zinc protein in oyster are still unclear.Therefore,in this thesis,the distributions of protein zinc in different tissue of oyster（Crassostrea hongkongensis）were studied,the representative protein zinc was isolated,purified and structurally characterized,and the effects of heat treatment on the digestion and absorption properties and transport mechanism of protein zinc were discussed,and further studies were carried out.Effects of protein zinc on the reproductive system of zinc-deficient mice.The content and results in this thesis were listed below:（1）Water-soluble protein（WSP）,salt-soluble protein（SSP）,acid-soluble protein（ASP）and alkali-soluble protein（ALSP）were extracted from different tissues of oyster（Crassostrea hongkongensis）,according to the difference of protein solubility,and protein and zinc contents were measured.The results showed that the zinc content in different tissues of oyster was gill,mantle,visceral mass and adductor from high to low,and the proportion trend of zinc was mantle>visceral mass>gill>adductor.The trend of zinc content in protein of oyster is visceral mass>gill>mantle>adductor,and the proportion of zinc in SSP is the highest,followed by ASP and WSP,and ALSP is the lowest,accounting for 36.06%,32.58%,28.01%and 3.35%respectively.The zinc content of gill ASP is the highest（9.85±0.02 mg/g）,and the zinc content and proportion of ALSP are the lowest.The highest zinc content（9.85±0.02 mg/g）was found in the ASP of gills,and the proportion of zinc in ASP was higher in gills than in other protein（67.53%±0.16%）,while the proportion of zinc in WSP and SSP was higher in visceral mass,mantle and adductor.（2）According to the above experimental results,this chapter uses the WSPM and ASPG as raw materials,and the zinc content as the index to track the separation and purification of protein components,and dezincification and zinc chelation treatments were performed to compare the structural characteristics of zinc-binding protein and the effect of zinc on protein structure.The results showed that the zinc-binding protein（W11）-carbonic anhydrase 2 was isolated and purified from WSPM,which had a spherical structure and combined with two zinc ions;the separation and purification of ASPG yielded an unknown zinc-binding protein（G32）with a zinc content of 7.38μg/mg,and a large amount of zinc is adsorbed on its polyhedral surface.Zinc does not affect the secondary structure of liquid zinc-binding protein,but has a significant effect on the stability of solid zinc-binding protein structure.The amino nitrogen,carboxyl oxygen,hydroxyl oxygen and carbonyl oxygen of amino acids in W11 and G32 all participate in the coordination of zinc ions.（3）An in vitro digestion models were used to further study the digestion stability of W11 and G32 before and after heat treatment.The results showed that W11 had good tolerance to pepsin and was sensitive to trypsin before and after heat treatment,and heat treatment promoted the digestion of W11;G32 were sensitive to both pepsin and trypsin before and after heat treatment,and heat treatment inhibited the digestion of G32.In gastric digestion stage,Zn2+of W11 and G32 released before and after heat treatment,and organic zinc content decreased significantly;in intestinal digestion stage,Zn2+and organic zinc content decreased significantly,and digestion time did not affect organic zinc content.The accounted for Zn2+of W11 GI240,RW11GI240,G32 GI240 and RG32GI240 were 16.86±0.58%,11.77±0.29%,16.52±0.18%and 11.47±0.08%,respectively.The proportion of organic zinc were 18.14±2.68%,15.23±0.69%,5.12±0.07%and 7.28±0.33%,respectively.（4）Based on the research in the previous chapter,the absorption properties of W11and G32 before and after heat treatment were studied using the Caco-2 cell absorption model.The results showed that the transport rates of peptides,organic zinc and Zn2+in W11 and G32 digestive products before and after heat treatment increased;the Papp of peptides and organic zinc showed a trend of increasing and then decreasing with the increase of absorption time,and the Papp of Zn2+showed an upward trend;The Papp of peptides,Zn2+and organic zinc in W11 and G32 digestive products before and after heat treatment were all greater than 1.00×10-5 cm/s,indicating a high absorption rate.Heat treatment promoted the transports of peptides and organic zinc in the W11digestion products,inhibited Zn2+transports,and increased the Papp of organic zinc;it also promoted the transports of peptides in the G32 digestive products,inhibited the transports of Zn2+and organic zinc,and decreased the Papp of organic zinc.Both W11and G32 are transported by three pathways:the alternative pathway,endocytosis and carrier transport.（5）The role of zinc-binding protein of oyster in the body after digestion and absorption remains to be studied.In this chapter,a mice models with zinc deficiency were established to study the effects of protein zinc on the reproductive system of zinc-deficient mice.The results showed that protein zinc can improve serum reproductive hormone levels and testicular marker enzyme activities in mice models with zinc deficiency,significantly promote sperm motility（94.09%-97.39%）and improve sperm deformity,and there was a concentration-related effect;It can repair testicular injury and reduce cell apoptosis rate in testicular tissue（2.16±0.48%）. |
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