Optimization Of Preparation Process,Structure Characterization And Emulsification Properties Of Food-grade Casein By Complex Enzymatic Method

In this study,fresh milk was used as raw material,pepsin(pig),flavor protease and neutral protease were prepared into composite enzyme,and the process parameters of casein preparation by composite protease were optimized through mixing experiment,single factor experiment and response surface analysis.The physical and chemical properties and structure of casein prepared were analyzed by physical and chemical analysis,spectral analysis,electrophoresis analysis and other methods.The differences of casein prepared by compound protease and calf rennet were compared,and the possibility of compound protease replacing calf rennet was discussed.After further separation and purification of casein,the structural characteristics of casein were analyzed to explore the influence of its structure on color and emulsifying properties.Through the mixing experiment,the best proportion of each component in the mixed protease was determined as pepsin(pig): flavourzyme: neutral protease 40.8%:28.7%: 30.5%.Under this condition,the casein yield was 2.4318%.Compared with casein prepared by calf rennet,it is found that there is little difference in the four physical and chemical indexes of the two casein(on a dry basis),casein(protein),fat and water.Fourier transform infrared spectroscopy showed that there were only small differences between the two casein.The secondary structure of casein prepared by mixed protease was found to contain 17.63% ?-helix,39.85%?-folding,29.43%?-angle and 13.09% random curl by second derivative analysis of amide ? region of the spectrogram.Casein secondary structure prepared by calf rennet contains 15.97%?-helix,31.81% ?-folding,33.56% ?-angle and 18.68% random curl.The results of SDS-PAGE gel electrophoresis showed that the main components of casein and the molecular weight of each component were the same.The results of colorimetry showed that there was no significant difference between l* value and whiteness value of the two casein,but there was significant difference in a* and b* values(p > 0.05).Through the correlation analysis between chromaticity and casein secondary structure,the results show that only ?-helix and L* value,?-helix and a* value have moderate negative correlation(0.5<?r?< 0.8),?-helix and b* value have moderate positive correlation(0.5<?r?<0.8).The other correlations are low,indicating that the secondary structure of casein is only one of the important factors affecting its color.Response surface methodology was used to optimize the optimal conditions for casein preparation using the mixed protease: the amount of mixed protease was0.014%,the curd temperature was 48?,the pH value of skim milk was 5.98,and the curd time was 11.5min.Under these conditions,the maximum yield of casein was2.9014.The order of influence of the four factors on casein yield is curd temperature > curd time > mixed protease dosage > pH value of skim milk.Fourier transform infrared spectroscopy analysis showed that ?-helix accounted for 17.30%,?-folding accounted for 23.74%,?-corner accounted for 48.56%,random curl accounted for 10.39% of the secondary structure of ?-casein.In the secondary structure of ?-casein,?-helix accounted for 16.82%,?-folding accounted for 30.59%,?-rotation accounted for 41.38%,random curling accounted for 11.21%;In the secondary structure of ?s-casein,?-helix accounted for 17.19%,?-folding accounted for 33.22%,?-corner accounted for 40.32%,and random curling accounted for 9.27%.The analysis of emulsifying properties shows that the order of emulsifying activity from high to low is ?-casein component > casein>? s-casein component >?-casein component.From high to low,the emulsion stability is casein>?-casein component > ?s-casein component > ?-casein component.Correlation analysis between secondary structure of casein and emulsifying properties shows that ?-helix and emulsifying activity,?-rotation angle and emulsifying activity,?-helix and emulsifying stability,?-rotation angle and emulsifying stability are highly positively correlated(r>0.8).?-folding and emulsifying activity,?-folding and emulsifying stability are highly negatively correlated(r>0.8).The correlation between random curling and emulsifying activity and emulsifying stability is very small(r<0.3).