| Soybean is one of the main source of high quality protein,it contains rich protein.Except for methionine,the composition and proportion of the other essential amino acids in soybean is similar to animal protein.Soybean protein not only can be used in production of bean products,but also can be widely used as the ingredients in many kinds of food.However,soybean is one of eight kinds of food allergens recognized by FAO.Glycinin is one of the major allergen in soybean,accounting to 19.5%?23.1%of the total protein.Survey showed that soybean allergy impairs approximately 0.4%of children worldwide.In addition,the prevalence of soy allergy in adult is on the rise with the increase of soy products.Microbial transglutaminase(MTG)used as a common cross-linking enzyme can improve gel properties,rheological properties,solubility,emulsifying property,water holding and other processing characteristics of protein products,so it is largely used in the soybean protein products and food products containing soybean protein.However,the effects of MTG modification on the allergenicity of soybean allergen are still scarcely understood.The aim of the study is to explore the effects of MTG cross-linking modification on the glycinin structure,digestibility,antigenicity and potential allergenicity,and it provides reliable evidence for the application of MTG control the allergenicity of soybean protein and soybean protein products.Several works are involved in this investigation including the purification of glycinin and isolation of acidic and basic polypeptides,the analysis of amino acid composition,the effect of MTG cross-linking modification on the structure and potential allergenicity of glycinin,the effect of synergistic modification of pH-shifting and MTG cross-linking on the structure and potential allergenicity of glycinin.The main methods,results and conclusions are addressed as follows.1.Glycinin was purified by gel filtration chromatography and affinity chromatography with a purity of about 94%,and the acidic and basic polypeptides of glycinin were successfully separated by isoelectric point precipitation.The amino acid composition of glycinin and its acidic and basic polypeptides were analyzed by acid hydrolysis.The results showed that there were great differences in amino acid composition of acidic and basic polypeptides.The content of glutamic acid and glutamine contained in acidic polypeptides is much higher than that of in basic polypeptides.In addition,basic polypeptides contained higher hydrophobic amino acid comparied with acidic polypeptides.2.The differences in cross-linking reaction between acidic polypeptides and basic polypeptides catalyzed by MTG were discussed by SDS-PAGE identification and solubility analysis.The results showed that both acidic polypeptides and basic polypeptides are good substrates for MTG.However,for natural glycinin,basic polypeptides are buried in the interior of hexamers of glycinin,so MTG cannot be close to the corresponding reactive sites.Acidic polypeptides cross-linked with basic polypeptides can improve the solubility of the basic polypeptides,and it be beneficial for the unfolding of basic polypeptides.3.The structure of the MTG-mediated modified glycinin was analyzed by Far-UV CD spectrum,UV absorption spectra and fluorescence intensity spectra.The results showed that MTG cross-linking modification can improve the thermal stability,maintain its orderly secondary structure and stable spatial structure of natural glycinin.While,for the glycinin processed by thermal denaturation pretreatment,MTG cross-linking modification could promote its structure unfolding and reduce its hydrophobicity.4.The digestibility,antigenicity and potential allergenicity of MTG-mediated modified glycinin were evaluated by in vitro digestion and competitive ELISA.The results showed that the digestibility of MTG cross-linked glycinin was significantly reduced,and the antigenicity of the digests was significantly increased,while the potential allergenicity of digests of MTG cross-linked glycinin is related to the pretreatment conditions.Natural glycinin modified by MTG can increase the potential allergenicity of its digests,but decrease that of digests of thermal denatured glycinin.5.The effect of the comprehensive modification by pH-Shifting and MTG-mediated cross-linking on the structure of glycinin was analyzed by surface free sulfhydryl content detection,Far-UV CD spectrum,UV absorption spectra and fluorescence intensity spectra.The results showed that strong acidic(pH=1)pH-shifting can unfold glycinin spatial structure,change its original oligomer structure,and generate large numbers of polymers linked by disulfide bridged by the rearrangement of disulfide bond,and basic(pH=13)pH-shifting not only destroy the oligomeric structure of glycinin,but also partially hydrolyze glycinin.For glycinin treated by pH1-shifting,its secondary structure was mainly embedded and folded after it was modified by MTG,but the effect of MTG-mediated modification on the spatial structure of glycinin treated by pH13-shifting is negligible.6.The effect of pH-shifting and MTG-mediated modification on the digestibility,antigenicity and potential allergenicity of glycinin was evaluated by in vitro digestion and competitive ELISA.The results showed that the comprehensive modification by pH1-shifting and transglutaminase-mediated cross-linking can reduced the potential allergenicity of glycinin to some certain extent,but significantly increased its antigenicity.While its resistance to gastrointestinal digestion was enhanced.PH13-shifting and transglutaminase-mediated modification significantly reduced the antigenicity and potential allergenicity of glycinin.Simultaneously,gastrointestinal digestibility of comprehensive modification for glycinin was similar to natural glycinin. |
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