Materials and reagents
Soy protein isolate, provided by Zhengzhou Tongchuang Yisheng Food Co., Ltd.; alkaline protease (2.4AU/g), flavor complex enzyme (500LAPU/g), Complex enzyme (1.5AU/g), provided by Novozyme; papain (600,000 u/g), provided by Guangxi Nanning Jiewo Biological Products Co., Ltd.; bromelain (2500GDU), provided by Guangzhou Fengtian Chemical Co., Ltd.; Protease (1:10000), trypsin (250.U.F.U/mg), linoleic acid (analytical grade), provided by Sigma; phosphate buffer (pH9.35), FeCl2-EDTA solution (0.05mol/L), FeCl2 Solution (0.1mol/L), KSCN (40), ethanol (75, containing 0.3mol/L HCl), ninhydrin color reagent, glycine solution (nitrogen content 5ug/ml), 1mol/LNaOH solution, 2N HCL solution etc.
1.2 Instruments
UV-visible spectrophotometer, ultrasonic cleaning machine, centrifuge, pH acidometer, Kjeldahl nitrogen analyzer, booster electric stirrer, electric constant temperature water bath , constant temperature drying oven.
1.3 Experimental methods
1.3.1 Determination of nitrogen content
Kjeldahl nitrogen determination (GB/T5009.5-2003).
1.3.2 Preparation of soy peptide solution
Prepare a 4 (W/V) soy protein solution, quickly heat it to 80°C, heat-treat it in an 80°C water bath for 10 minutes, and then cool to enzyme For the optimal hydrolysis temperature, keep the temperature and stirring speed unchanged, and use NaOH (HCl) standard solution to adjust the pH of the solution to the optimal pH for enzymatic hydrolysis. Weigh the protease in proportion and add it to the soy protein solution, start timing, add NaOH (HCl) standard solution in time during the reaction to maintain the pH value, extract a certain volume of solution at the predetermined reaction time, and then quickly heat it to 85°C Inactivate the enzyme for 10 minutes. After cooling, centrifuge at 5000r/min for 10 minutes, and take the supernatant to measure the degree of hydrolysis and antioxidant value.
1.3.3 Determination of degree of hydrolysis [4]
h=(A×10-3×10)/(14×0.1)=A/14(mmol/ml)
ho=4×7.75=0.312 (mmol/ml)
DH ()=h/ho=23A (1)
Note: h: soybean peptide Concentration of -NH2 in the solution (mmol/ml)
ho: Peptide bond equivalent concentration of protein solution (mmol/ml)
A: Nitrogen concentration of soybean peptide solution (ug/ml)
1.3.4 Determination of protease hydrolysis parameters
The parameters that affect the protease hydrolysis speed and protein hydrolysis degree mainly include temperature, pH value, substrate concentration, enzyme concentration, and time. Using the degree of hydrolysis as an indicator, single-factor experiments were first used to determine the optimal values ??of parameters, and then orthogonal experiments were used to optimize factor combinations to determine protease hydrolysis parameters.
1.3.5 Determination of antioxidant value of soybean peptides [5]
Determination of antioxidant value of soybean peptides refers to the reference [5], with appropriate modifications:
Put 10ml of linoleic acid stock solution, 300ml of 0.05mol/L FeCl2-EDTA solution, and 500ul of soy peptide solution into a small Erlenmeyer flask. Mix well and store in the dark at 50°C for 4 hours. Then take out 1000ul and add 200u10.1mol. /L FeCl2 solution, then add 1000ul of 40KSCN solution, mix well, take out 130ul and put it into a dry test tube, then add 6ml of ethanol (75, containing 0.3mol/L HCl), mix and react for 5 minutes, and then measure the mixture Absorbance value at 481nm. Taking the absorbance value of the unhydrolyzed soy protein reaction solution as the blank value, its relative antioxidant value is 1.
Relative antioxidant value = absorbance value of unhydrolyzed protein solution/absorbance value of soy peptide solution
2 Experimental results
2.1 Nitrogen content standard curve
p>
The absorbance values ??of different concentrations of glycine working solutions measured with a spectrophotometer (580nm) are shown in Figure 1.
The regression equation between nitrogen content and absorbance value is:
y=1.3342x 0.0133 (2),
The regression coefficient is R2=0.998.
The inverse function of equation (2) is x=0.425y-0.0095 (3),
Put the x value in equation (3) into the A value in formula (1)
DH()=0.17y(4).
2.2 Determination of protease hydrolysis parameters
Using the degree of hydrolysis as an indicator, first use a single factor test to determine the range of parameters (limited to space, specific data are omitted), and then use orthogonal Test optimization factor combinations, the best parameter combinations of seven proteases are shown in Table 1.
Table 1 The best combination of 7 protease hydrolysis parameters
Temperature (℃) pH Substrate concentration () Enzyme concentration () Time (min)
Alkaline enzyme 60 8.0 3 0.5 60
Flavor enzyme 50 7.0 4 6 30
Complex enzyme 45 7.0 4 6 20
Papaya enzyme 55 7.5 5 3 40
Bromelain 65 8.0 5 6 30
Gastric enzyme 37 2.5 4 2 30
Pancreatin 50 7.0 5 3 302.3 Degree of hydrolysis of soy peptide solution
2.3.1 Absorbance values ??of soy peptide solutions
Use a spectrophotometer (580nm) to measure the absorbance values ??of soy peptide solutions of different concentrations hydrolyzed by different proteases. See Table 2. Unhydrolyzed The absorbance value of the protein solution was 0.178.
2.3.2 Degree of hydrolysis of soy peptide solution
Substitute the absorbance value in Table 2 into formula (4) to obtain the corresponding degree of hydrolysis of soy peptide solution. The results are shown in Figure 2, where The degree of hydrolysis of soy protein solution without protease hydrolysis is 3.03.
As can be seen from Figure 2, the degree of hydrolysis of soybean protein by the seven proteases increases with the extension of the hydrolysis time (within 4 hours), but the hydrolysis rate is larger at first and then gradually becomes smaller and smaller. Among them, alkaline protease has significantly stronger hydrolysis ability than the other six proteases.
2.5 Antioxidant value of soybean peptide solution
After the antioxidant test of the mixed solution of linoleic acid buffer and soybean peptide, use a spectrophotometer to measure the absorbance value at 481nm, and then Calculate the relative peroxide value of the soy peptide solution, and use the relative peroxide value to express the antioxidant capacity of the soy peptide. The results are shown in Figure 3.
Table 2 Absorbance values ??of soy peptide solutions of different protease hydrolysates
Absorbance values
1 (1h) 2 (2h) 3 (3h) 4 (4h) )
Alkaline enzyme 0.605 0.705 0.975 0.985
Flavor enzyme 0.545 0.632 0.877 0.909
Complex enzyme 0.487 0.532 0.587 0.627
Papaya enzyme 0.340 0.349 0.355 0.412
Bromelain 0.422 0.474 0.708 0.808
Gastric enzyme 0.231 0.243 0.293 0.299
Pancreatic enzyme 0.462 0.583 0.599 0.603Fig. 3 It can be seen that protease As the enzymatic hydrolysis time (within 4 hours) increases, the antioxidant capacity of the hydrolyzed soybean peptide solution increases rapidly at first, then slowly, and sometimes even stops. Among these seven enzymes, the soy peptide solution hydrolyzed by alkaline protease has the highest antioxidant value.
3 Conclusion
(1) Soybean peptides have antioxidant properties, and the antioxidant abilities of soybean peptides prepared by different proteases are also different.
(2) Among the seven proteases studied, alkaline protease has the strongest hydrolysis ability and the highest antioxidant value of the soybean peptides prepared by it, so it should be the best enzyme selection. ◇References
［1］Chen H M, Muramoto K.Y, amauchi F, etal. Antioxidative properties of histidine containing peptides desinned from peptide frangments found in the digests of asoybean protein .J. Agric. Food. Chem, 1998, 46: 49-53.
[2] Zhang Xuezhong. Principles and Applications of Soybean Peptide Processing Technology. Beijing: Science and Technology Literature Press, 1999: 181-200.
［ 3] Chen Meizhen. Research on the hydroxyl radical scavenging effect of soybean isolated egg autoenzymatic hydrolyzate. Food Science, 2002, 23 (1); 43-47.
[4] He Zhaofan, Zhang Diqing. Health food chemistry and its detection technology. Beijing: China Light Industry Press.
[5] Adler-Nissen J. Enzymic hydrolysis of food proteins. Elsevier Applied Science Publishers. New York, 1986: 74-78.
*Project funding: Henan Provincial Key Science and Technology Research Plan Project (0523011700).
About the author: Huang Mianming (1971~), male, from Runan, Henan, Ph.D., associate researcher. His research interests include intensive processing of agricultural products and development of functional foods.