Structural Properties Of Soy Protein And Its Relationship To The Nutritional Value

Among oilseeds and legumes, soybeans are the largest source of edible proteins and are important in human and animal nutrition. Soybean meal(SBM), the major byproduct of soybean oil extraction, is the main protein source for animal feed globally. In soybeans, two storage globulins, glycinin(11S globulins) and β-conglycinin(7S globulins), constitute the bulk(-70%) of the total seed proteins. Because of their abundance, 11 S and 7S globulins significantly influence functional properties of soybean seeds and protein meals. Consequently, soybean 11 S and 7S globulins have been extensively investigated to understand relationships between their molecular and functional properties. Although molecular heterogeneity in plant proteins increases the difficulty in establishing clear structure–function relationships, it is important to isolate and investigate individual proteins from their natural sources(“native” proteins) as it is the native proteins. The changs of protein structure were studied on denaturation conditions, thereby permitting refined understanding of the processes involved. The present study targeted purified soybean 11 S and 7S for such investigations.1. Thermal Denaturation on the Solid-State Structure of glycinin and β-conglycinin were investigated by Fourier transform infrared(FTIR) spectroscopy after dry heating and autoclaving thermal treatments. The changes in frequency and signal intensity of IR bands revealed the thermal denaturation on the solid-state structure of glycinin and β-conglycinin. The FTIR spectral changes were subsequently assessed using the second derivative spectroscopy in the amide I region(1700-1600 cm-1). The bands at 1618 cm-1 and 1682 cm-1 were considered to reflect the formation of intermolecular and intramolecular aggregates(A1 and A2), and the contents of β-sheet indicated the degree of denaturation. In autoclaved samples, the contents of the glycinin α-helix, turn, random coil and A2 significantly increased(P<0.05), while the contents of the glycinin β-sheet and A1 significantly decreased(P<0.05); the contents of the β-conglycinin α-helix, random coil and A2 significantly increased(P<0.05), while the contents of the β-conglycinin β-sheet, turn and A1 significantly decreased(P<0.05). The dry heating significantly decreased the contents of the β-conglycinin β-sheet and increased the contents of the β-conglycinin A1 and turn(P<0.05), while slightly affected the secondary structures of glycinin. While both dry thermally treated and autoclaved treated samples showed a high content in β-sheet structures which may adversely affect protein utilization.2. Thermal denaturation on the structure of glycinin and β-conglycinin in solution was analysed by native polyacrylamide gel electrophoresis(Native-PAGE), Fluorescence,c FTIR spectroscopy, and its corresponding immunogenicity was tested with ELISA experiments. Native-PAGE was conducted to analyze the association and dissociation of the glycinin and β-conglycinin subunits. The glycinin contained some oligomeric structures of hexamers, trimers and monomers, and β-conglycinin contained some oligomeric structures of trimers and monomers. The fluorescence probe emission spectra analysis demonstrated that the heat treatment induced an increase in surface hydrophobicity of the glycinin and β-conglycinin. The results of ELISA showed that the immunogenicity of β-conglycinin had a slight increase after being heated at 80℃, while above 60℃ the immunogenicity decreased significantly, and the immunogenicity of glycinin decreased with increasing temperature above 70℃. The frequency and signal intensity of the spectroscopys were assigned, the data suggested that FTIR spectra reflected the large amount of structural information. The contents of β-sheet showed strong positively correlation(r=0.897, r=0.886) with the immunogenicity of glycinin and β-conglycinin, and the contents of α-helix, random coil showed negatively associated with the immunogenicity of glycinin and β-conglycinin. The conformational changes of the protein during thermal denaturation, and discussed the relationship between conformational changes of glycinin and β-conglycinin and immunogenic change, were helpful to the understanding of the mechanism of immunogenic chang of soybean treated by heat.3. On the basis of enzyme mechanism and protein structure, we have extensively studied the reaction behavior of protein during enzymatic hydrolysis. Glycinin and β-conglycinin, the major storage protein of soyabeans was enzymatically modified using pepsin, aspergillus niger acid protease and subtilisin alcalase. The degree of hydrolysis(DH) was monitored by using trinitrobenzene sulphonic acid reaction with liberated α-amino groups. The DH could be varied by varying times of hydrolysis, and its corresponding immunogenicity was tested with ELISA experiments. The structural changes of glycinin and β-conglycinin due to various enzymatic modification were followed by SDS-PAGE, Tricine-SDS-PAGE, SEM, DSC, FTIR spectroscopy. Tricine-SDS-PAGE analysis showed that subunit content of glycinin significantly decreased after hydrolysis, and acidic subunits was more susceptible to hydrolysis than alkaline subunits.SDS-PAGE analysis showed that β-subunit of β-conglycinin was generally less well digested than other subunits. DH of glycinin and β-conglycinin showed strong negatively correlation(P < 0.0001) with its immunogenicity of hydrolysates. The linear regression equations showed that DH of glycinin and β-conglycinin were better predictors of the immunoreactivity of hydrolysates. DSC and FTIR results suggest that enzymatically modified protein immunogenicity are related to changes in the secondary and tertiary packing of the protein. It is important to point out that immunogenicity is dependent on the specificity of antibody-antigen interactions, which may strongly depend on the conformational state of the proteins.4. The objectives of this study were to determine the relationship between the intrinsic molecular structures and the in vitro pepsin-pancreatin digestibility of soy protein products. The protein molecular structures were quantified from spectral data on unique bands such as amid I and amide II, and protein secondary structures such as α-helix and β-sheet and their ratios using Fourier transform infrared spectroscopy. Significant differences were observed in protein amide I and amide II area, model-fitted α-helix and β-sheet height and in the ratio of amide I to II area. The multivariate molecular spectral analyses(PCA, CLA) showed that there were significantly molecular structural differences in the protein amide I and II fingerprint region. The correlation analysis with spearman method showed that there were significantly negative correlation(P<0.01) between the ratio of amide I to amide Ⅱheight and in vitro digestibility of protein, and significantly negative correlation(P<0.01) between the ratio of amide I to amide Ⅱarea and intestinal digestibility of protein in vitro. The contents of the β-sheet showed significantly negative correlation(P<0.05)with intestinal digestibility of protein in vitro, and significantly positively correlation(P<0.01) with the contents of crude protein. The protein digestive behavior was significantly effect on the structural properties of soy protein products. The relationship between mid-IR spectroscopic data and nutritional profiles and digestibility parameters illustrated that intrinsic structures of soy protein are closely related to nutritive quality, nutrient utilization and digestive behavior. Spectral features of different protein products could be used as a potential tool to predict true protein nutritive value.5. Traditional “wet” chemical analyses usually looks for a specific known component(such as protein) through homogenization and separation of the components of interest from the complex tissue matrix. Traditional “wet” chemical analyses rely heavily on the use of harsh chemicals and derivatization, therefore altering the native feed protein structures and possibly generating artifacts. The objective of this study was to introduce a novel and non-destructive method to estimate protein structures in soy proteins within intact tissues using FTIR spectra. The results show that with based FTIR, we are able to localize relatively “pure” protein without destructions of proteins and qualify protein internal structures in terms of the proportions and ratios of α-helix, β-sheet, random coil and β-turns on a relative basis using multi-peak modeling procedures. These protein structure profile(α-helix, β-sheet, etc.) may influence protein quality and availability in animals. Several examples of soy were provided. The implications of this study are that we can use this new method to compare internal protein structures between feeds and between seed verities. We can also use this method to detect the structural changes of protein in feeds.