| It would be complicated to figure out the influences of hydrolysis factors, including enzymes types and concentration, substrates, p H, temperature(T) and hydrolysis time, on the enzymatic hydrolysis and the properties of resulting hydrolysates. The goal of this paper was obtaining the hydrolysates with identical properties and similar structure through controlling the hydrolysis process.According to the goal, casein and SPI was chosen as the substrate. Casein was hydrolyzed by Alcalase, trypin, papain and brolemain, and the final degree of hydrolysis(DH) of casein were 13.30%(Alcalase) >6.77%(papain) > 6.76%(trypsin) > 2.79%(brolemain) after 2h. Casein with DH 5% was hydrolyzed by above enzymes, the result of SE-HPLC showed that the molecular weight distribution of hydrolysates were significantly different from each other. Alcalase activity increases as the temperature increase from 40 to 70 oC. However, after incubation at 70 °C for 30 min, there was only 33.64% of proteolytic activity left, while when the Alcalase stock solution were treated at 50 °C and 60°C, the remained activity are 92.56% and 85.09%, respectively. Considering the high efficiency and utilization, Alcalase was chosen as the studied enzyme for the further research.Then θ(h)-method was introduced to analyze the hydrolysis curves. Casein(p H8.5) was hydrolyzed by Alcalase with substrate concentration([S]:5%), temperature(T: 50, 60, and 70°C) and enzyme-substrate ratio(E/S: 1/100, 2/100 and 3/100), The results showed that the kinetics of hydrolysis under 50 and 60 oC could be represented by the first-order kinetics reaction equation and the θ(h) were all constant. However, the hydrolysis curves of 70oC showed different result. Casein hydrolysates at different degree of hydrolysis(DH: 5, 10 and 15%) were prepared by Alcalase, and the properties were determined. The result showed that the properties of hydrolysate which was prepared at 50 and 60oC showed no significant difference between each other. Thus, the θ(h) value were obtained by analyzing hydrolysis curves, and if the θ(h) was constant, then the hydrolysates was obtained with identical structure and properties by controlling the DH.SPI with Alcalase was used to evaluate the correctness of theory above. The hydrolysis curves of SPI with substrate concentration([S]:5%), temperature(T: 50 and 60°C) and enzyme-substrate ratio(E/S: 1/100, 2/100 and 3/100) was obtained, the related θ(h) was constant. However, the TCA-solubility, DPPH scavenging ability and ACE inhibitor activity of the SPI hydrolysates with the same DH were significantly different between each other. Clearly, the properties showed some kind of tendency as the enzyme-substrate ratio changing. The result of SE-HPLC and RP-HPLC also illustrated that the profile of peptide was obviously different when comparing under the same DH. Thus, conside ring the Alcalase-SPI system, hydrolysis temperature, E/S and DH should be controlled strictly to produce the hydrolysates with identical properties.Based on above result, Alcalase was employed to hydrolyzed thermal denatured SPI with substrate concentration, it was found that the θ(h) was constant through calculating the hydrolysis curves. Also, the properties and structure of thermal denatured SPI hydrolysates with the same DH at different hydrolysis conditions showed no significantly difference. The conclusion was different from the situation of SPI with Alcalase, which was confirmed by the result of SE-HPLC and RP-HPLC. It means that if structure of SPI unfolded by heat-denature before enzymatic hydrolysis, hydrolysates with identical properties was easy to produced by controlling the DH. |
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