Enzymatic Modification Of Casein Hydrolysates In Propanol-water And Ace-Inhibtory Activities Of The Modified Products

Hypertension is the major controllable risk factor associated with cardiovascular disease (CVD) events such as myocardial infarction, stroke, heart failure, and end-stage diabetes. A5mmHg decrease in blood pressure equals about16%decrease in CVD. In the US, annual antihypertensive drug costs are up to approximately$15billion. However, various side effects are associated with the use of the drugs. More attention is thus towards to the natual ACE-inhibitory peptids drived from food proteins. Several studies have demonstrated that casein is one of the major sources of ACE inhibitory peptides and other bioactive peptides. In the present work, casein was applied as raw material. In order to prepare ACE-inhibitiory peptides with higher activity, casein hydrolysate was modified by plastein reaction with Alcalase in propanol-water medium, in the presence of two amino acids. The results obtained from the present study are listed as follows.1. ACE-inhibitiory peptides were prepared by hydrolyzing casein with Alcalase (an alkaline protease2.4L FG). When casein was incubated with Alcalase for6h, casein hydrolysate was observed to exhibit the highest ACE-inhibitory activity. Its DH and ACE-inhibitory activity were12.6and48.2%(at peptide concentration of0.3mg/mL).2. According to Design Expert7.0software, the addition ratio of tyrosine in pure water medium plastein reaction was optimized by a central composite experiment, and the optimum tyrosine addition level was0.54mol/mol when the substrate concentration and reaction time were fixed at45g/100mL and6h respectively.3. The effects of reaction temperature, enzyme to substrate ratio, substrate concentration and propanol concentration in the reaction medium on the plastein reaction of casein hydrolysates were optimized by a central composite design with response surface methodology analysis, by using the decrease of free amino groups in the reaction mixture as a response when the reaction time was fixed at6h. The practical results indicated that the four factors selected had significant impacts on the plastein reaction of the hydrolysate. The reaction conditions optimized by response surface methodology were that reaction temperature was46.8℃, the dosage of enzyme was8.36kU/g proteins, substrate concentration was56.8g/100mL, propanol concentration was58.5%(v/v). The maximum decrease amount of free amino groups in the reaction mixture was327.8μmol/g proteins in theory, and the practical decrease of free amino groups was344.5μmol/g proteins.4. Five casein hydrolysates modified with different reaction extents were prepared by plastein reaction under the optimized conditions in propanol-water medium, and their ACE-inhibitory activities were assayed. The resluts showed that modified product whose reaction time was1h had the highest ACE-inhibitory activity about63.8%. The other two different groups of modified casein hydrolysates were prepared with the same conditions in the presence of tyrosine and phenylalanine with the addition level of0.54mol/mol. The results showed that addition of phenylalanine or tyrosine into the reaction system would induce increase or decrease in the ACE-inhibitory activity of the modified products slightly, compared with the plastein reaction of casein hydrolysates without any amino acid addition. Six modified casein hydrolysates with different reaction extents were prepared under the optimized conditions in pure water medium (without propanol), the results showed that the tendency of the decrease of free amino groups in plastein reaction was different from that in propanol-water medium, but the tendencies of ACE-inhibitory activities in the two mediums shared the similar regularity.5. Six modified products obtained from propanol-water medium with amino acid absence, phenylalanine and tyrosine when the reaction times were fixed at1h and6h. They were conducted for further hydrolysis with Alcalase, Neutrase, trypsin and pepsin respectively. The results show that further hydrolysates with Alcalase and pepsin are more sensitive than those with trypsin and Neutrase.