| Protein has important influnce on life.Single protein food has not been satisfied peoples' nutritional needs,the production of mixed protein matrix is an area of great potential for future development.In 2017,the state council office issued "National Nutrition Program(2017-2030 years)" to put forward "With high-quality animal source and plant source proteins as the main nutritional basis,developing new nutritious and healthy foods such as double protein food." In the present study,we have developed a novel soymilk and cow milk mixed gel,named as double protein bio-tofu,by means of lactic acid bacteria(LAB)incorporated with microbial transglutaminase(MTGase)as bio-coagulant instead of bittern.Although the nutritional values of soybean and cow milk are very high,both of them are frequently identified as allergen among allergic individuals.It should be explored that whether the antigenicity of soybean proteins and milk proteins in double protein bio-tofu change.In the present study,a proteomic-based approach was used to study the gelling behavior of proteins in soymilk-cow milk mixture gel coagulated by MTGase combined with LAB which lay a good foundation for evaluating the gelling mechanism.The effects of MTGase addition on protein bioaccessibility of bio-tofu were studied by in vitro gastrointestinal digestion model.Also,the antibody(Ab)reactivity of soy proteins and?-lactoglobulin(?-LG)were evaluated present in cow milk or soymilk or soymilk and cow milk mixture polymerized by MTGase combined or not with LAB.Of all samples,the in vitro digestibility studies were also performed,together with Ab reactivity of the digests.Meanwhile,the relationship between antigenicity and protein structure was also discussed.The main contents and results are as follows:1.A proteomic-based approach to study the gelling behavior of soymilk-cow milk proteins coagulated by LAB combined with MTGaseBy monitoring the rheological properties of soymilk-cow milk mixture during coagulation process,it was shown that MTGase alone could not induce gelation of soymilk-cow milk for 4 h,but addition of LAB at the same time result in much higher(P<0.05)storage modulus(G')and denser protein network than treated only with LAB.Moreover,electrophoresis and mass spectrometry results indicated that the use of LAB in conjunction with MTGase promoted the polymerization of soy milk and cow milk proteins and the whole gelling process could be divided into three steps.The first step involves an enzymatic process in which MTGase catalyzed most of the 7S and 11S acidic subunits,during which period,the pH value of the system did not change obviously.The second step involves the cross-linking of all the 7S and 11S acidic subunits and few of the ?-CN and?-CN subunits.During this period,LAB began to produce proton,some negative charges on the surface of protein particles were neutralized.The third step involves the cross-linking of most soy protein subunits(7S and 11S)as well as the caseins and whey proteins.During this period,all the negative charges were neutralized and the bio-tofu gel network was formed.Taken as a whole,all of the 7S(?',?,?)11S acidic(Ala,Alb,A2,A3,and A4)and a portion of 11S basic(B1a)proteins,?s1-CN,?,2-CN,?-CN,?-CN and?-LG were polymerized after 4 h incubation period.And the polymerization of soymilk proteins occurred earlier than that of cow milk proteins,the high glutamine and lysine contents of soy proteins were responsible for this observation.2.Effect of MTGase addition on the protein bioaccesibility of double protein bio-tofuWith addition of MTGase in bio-tofu,the content of soluble proteins,digestibility of proteins,degree of hydrolysis,bioaccessible peptides,free amino acids contents were significantly lower(P<0.05)than without adding MTGase,but the particle size distribution at all digestion phases were much higher(P<0.05).These results confirmed that the addition of MTGase reduced the hydrolysis degree of proteins by digestive enzymes in the gastrointestinal tract.It may have a positive impact on diet pattern with increased satiety and controlled energy intake.3.Study on the antigenicity of soy proteins and ?-LG present in bio-tofuThe antibody(Ab)reactivity of soy proteins and ?-LG were evaluated present in cow milk or soymilk or soymilk and cow milk mixture systems polymerized by MTGase combined or not with LAB by enzyme-linked immunosorbent assay.Of all samples,the in vitro digestibility studies were also performed,together with Ab reactivity of the digests.In comparison with only MTGase or LAB treated samples,the use of LAB in conjunction with MTGase(LAB+MTGase)showed enhanced capacity to reduce the Ab reactivity of soy proteins and ?-LG,expecially in the mixed soymilk-cow milk system.During in vitro digestion,the Ab reactivity of soy proteins in soymilk system or soymilk-cow milk mixture system showed the trend of rising first and then decreased.However,the Ab reactivity of?-LG in cow milk system or soymilk-cow milk mixture system showed the trend of decreasing.In general,the soy protein and ?-LG peptides released from the LAB+MTGase-treated soymilk-cow milk mixture sample showed the lowest Ab-binding before and after the digestion phase among the experimental groups.This provides theoretical support for the development of low-allergenic soymilk and cow milk foods.4.Effects of modification by LAB combined with MTGase on the structure of proteins and prediction and verification of antigenic epitopesIn comparison with only MTGase or LAB treated samples,the use LAB+MTGase could form aggregates with enlarged particle average size(P<0.05),reduced absolute value of Zeta-potential(P<0.05)and free sulfhydryl content in soymilk,cow milk and soymilk-cow milk systems.The secondary structure of soymilk and cow milk proteins could be changed by crosslinking with MTGase which resulted in the increasement of?-helix content and decreasement of random coil content.So that the protein structure tended to be orderly,and the surface hydrophobicity of protein was reduced.In addition,LAB+MTGase treatment could promote the cross-linking degree of proteins,resulted in a more complete entrapment of hydrophobic groups,and further reduction of the surface hydrophobicity of proteins.The epitopes of a chain of ?-conglycinin subunit and?-lactoglobulin were predicted by combined immunoinformatic methods,and it had a good correlation with the antigen epitopes reported by other researchers.It should be noted that the predicted epitopes could be cross-linked by MTGase exactly,which might be the main reason for the decrease of antigenicity of allergic proteins. |
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