| Casein is not only rich in nutrition, but also has good functional properties such as water-holding capacity, oil absorption, emulsification, gelation and the important source of bioactive peptides including antibiosis peptide, antioxidant peptide, angiotensin-I-converting enzyme inhibitory peptide etc. , so that it is highly and widespread applied in food and other fields. At present, the researches of the improvement in functional properties and biological properties of casein hydrolysates are popular in food science.In this study, casein hydrolysates were prepared by hydrolysis of casein with three kinds of protease and modified by Fenton reaction and plastein reaction, then, the antioxidant activity and angiotensin-converting enzyme inhibitory activity changes were measured. The results obtained in this study are as follow.(1) Casein was hydrolyzed with Alcalase 2.4L FG protease to prepared Casein hydrolysates. Casein hydrolysates that had the degree of hydrolysis of 10% and the values of IC50 about 2.802±0.072 mg / mL on DPPH radical were hydrolyzed with the substrate concentration 5%(w/v),the addition level of neutral proteinase 11.0 kU/g proteins, the temperature 50℃and the time 4 hours then modified with Fenton reaction. The results showed that, the addition level of Fenton reagent had a significantly influence on the degree of hydroxylation, while little influence on reacetion time. After modification by the Fenton reaction, the values of IC50 about antioxidation activity on DPPH radical had reduced to 0.891±0.017 mg/mL.(2) Casein hydrolysates that had the degree of hydrolysis of 13.0 % and value of IC50 on angiotensin-converting enzyme (ACE) inhibitory activity about 40.35μg/mL were prepared from casein with a protease Neutrase 0.8 L, and then modified by plastein reaction with the same protease. The effects of the addition level of Neutrase, the concentration of casein hydrolysates, temperature and time on the plastein reaction of casein hydrolysates were studied. The results indicated that synthesis reaction was the dominant reaction in the Plastein reaction system as the content of free amino groups in reaction system decreased in all conditions. The addition level of Neutrase, the concentration of casein hydrolysates and reaction time all had significant impact on the plastein reaction of casein hydrolysates, while reaction temperature gave some influence. Three reaction conditions, enzyme addition, concentration of casein hydrolysates and reaction temperature, for Plastein reaction were optimized by a central composite design and response surface methodology analysis with the changes of free amino groups in the reaction mixture as response. quadratic regression equation was obtained therefore, which could reflect the relationship of the changes of free amino groups and enzyme addition, concentration of casein hydrolysates and reaction time. The optimal reaction conditions were that enzyme addition level was 3 KU/g casein hydrolysates, concentration of hydrolysates was 62%(m/m) and reaction time was 6.3 h when the reaction temperature was at 20℃.At this condition the modified casein hydrolysates had a decrease of free amino groups of 210.01μmol/g proteins.Five modified casein hydrolysates with different modification extents were prepared and their ACE inhibitory activity and IC50 values were determined. The analysis results indicated that the ACE inhibitory activity of the modified casein hydrolysates increased as the extent of modification increased. ACE inhibitory activities of the modified casein hydrolysates were all higher than that of the origin casein hydrolysates. When the modified casein hydrolysates had a decrease of free amino groups of 210.01μmol/g proteins, whose IC50 value would decrease to 14.72±0.22μg/mL.(3) Casein hydrolysates that had the degree of hydrolysis of 13.5% and value of IC50 angiotensin-converting enzyme (ACE) inhibitory activity about 45.23μg/mL were prepared from casein with a protease Alcalase, and then modified by plastein reaction with another protease Neutrase. The effects of the addition level of Neutrase, the concentration of casein hydrolysates, temperature and time on the plastein reaction of casein hydrolysates were studied. The results indicated that hydrolysis reaction was the dominant reaction in the Plastein reaction system as the content of free amino groups in reaction system increased in all conditions. The addition level of Neutrase, the concentration of casein hydrolysates and reaction time all had significant impact on the plastein reaction of casein hydrolysates, while reaction temperature gave some influence. Under lower addition level of Neutrase, higher concentration of casein hydrolysates and shorter reaction time, the increase extent of free amino groups in Plastein reaction system showed a trend to decrease which implied that hydrolysis reaction were weakened relatively. Six modified casein hydrolysates with different reaction extents were prepared and their ACE inhibitory activities were determined. The results showed that the values of IC50 of the modified products were in range of 15.56 to 19.98μg/mL, indicating that the ACE inhibitory activity of casein hydrolysates could be improved significantly by plastein reaction catalyzed by Neutrase.
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