The authors have declared that no competing interests exist.
Conceived and designed the experiments: SF. Performed the experiments: SF YH. Analyzed the data: SF CL. Contributed reagents/materials/analysis tools: SF YL. Wrote the paper: SF YH.
Protein isolates of pumpkin (
With the improvement of people’s living standard, many kinds of diseases caused by reactive oxygen species (ROS) and free radicals are bringing serious damage to human health
Considering the nutritional quality and cost, many legumes, oilseeds and their by-products of the oil industry are suitable as protein sources to produce protein hydrolysates for food or non-food application. Some materials have been extensively studied including soybean protein
Pumpkin seed is one of the most important oilseeds in many countries (India, China, etc.), which is also a popular snack. Nearly 90% of the total output in these countries is used for extraction of oil
Most notably, pumpkin seed is a valuable raw material for preparation of antioxidative peptides owing to its high protein content, availability and low cost. In this paper, antioxidative peptides were prepared by hydrolysis from pumpkin seed. The effects of enzyme amount, substrate concentration and hydrolysis time on the DPPH radical scavenging ability of pumpkin seed hydrolysates were investigated. Further more, the optimal hydrolysis conditions were established through RSM.
Pumpkin seeds (
The protein isolate was prepared in alkaline solution followed by isoelectric precipitation, as described by Sathe
In preliminary studies, amount of enzyme, substrate concentration and time were found to have a significant influence on the degree of hydrolysis (DH) and DPPH radical-scavenging activity. The effect of three independent variables on the scavenging activity and their optimum levels were investigated by RSM, using Box-Behnken experimental design. Here, 15 experiments were carried out (with three replicates at the centre of the design). The range and central point values of the independent variables (
Factor | Symbol | Levels | ||
−1 | 0 | 1 | ||
Enzyme amount (U/g) | x_{1} | 2000 | 5000 | 8000 |
Substrate concentration (g/ml) | x_{2} | 0.02 | 0.04 | 0.06 |
Time (h) | x_{3} | 2 | 4 | 6 |
Treat | Variable levels | DPPH free radical scavenging ability (%) | |||
Experimental (Y_{0}) | Predicted (Y_{i}) | ||||
1 | 0 | −1 | −1 | 80.05 | 79.8937 |
2 | 0 | −1 | 1 | 84.47 | 85.0288 |
3 | 0 | 0 | 0 | 91.32 | 90.9767 |
4 | 0 | 0 | 0 | 90.95 | 90.9767 |
5 | 0 | 1 | −1 | 90.84 | 90.2813 |
6 | 0 | 0 | 0 | 90.66 | 90.9767 |
7 | −1 | −1 | 0 | 81.63 | 81.6350 |
8 | 1 | −1 | 0 | 84.72 | 84.3125 |
9 | 1 | 0 | 1 | 91.51 | 91.3588 |
10 | −1 | 1 | 0 | 90.78 | 91.1875 |
11 | 1 | 1 | 0 | 92.56 | 92.5550 |
12 | −1 | 0 | 1 | 90.74 | 90.1763 |
13 | 0 | 1 | 1 | 92.28 | 92.4363 |
14 | 1 | 0 | −1 | 87.99 | 88.5538 |
15 | −1 | 0 | −1 | 85.54 | 85.6913 |
The enzymatic hydrolysis was carried out as stated in
The mixture with inactivated enzyme was centrifuged at 5000 r/min for 10 min and the supernatant was used for further analysis.
The degree of hydrolysis was determined according to the method of Chalamaiah, M. et al.
The scavenging activity of DPPH radical was measured according to the method of Shimada
Data from the Box-Behnken experimental design (
The significance of each coefficient of the resulted model was determined by using the Student t-test and p-value. The proportion of the variance expressed by the models is obtained by determination of multiple coefficient R^{2}. Finally, the fitted polynomial equation was presented as surface plots in order to visualize the relationship between the response and experimental levels of each factor and search out the optimum conditions. The analysis software used for this study was Minitab 15. (Minitab Inc.).
RSM was applied to obtain the regression equation, which represents an empirical relationship between response (scavenging activity of DPPH radical) and the test variables (enzyme amount, substrate concentration and hydrolysis time), as given in the equation (4).
The significance of each coefficient was determined by using the
Variables | Regression coefficient | Standard error | P |
Significance level | |
Constant | 90.9767 | 0.3564 | 255.244 | <0.0001 | ** |
x_{1} | 1.0113 | 0.2183 | 4.633 | 0.006 | ** |
x_{2} | 4.4488 | 0.2183 | 20.382 | <0.0001 | ** |
x_{3} | 1.8225 | 0.2183 | 8.350 | <0.0001 | ** |
x_{1}^{2} | −0.7596 | 0.3213 | −2.364 | 0.064 | |
x_{2}^{2} | −2.7946 | 0.3213 | −8.698 | <0.0001 | ** |
x_{3}^{2} | −1.2721 | 0.3213 | −3.959 | 0.011 | |
x_{1} x_{2} | −0.3275 | 0.3087 | −1.061 | 0.337 | |
x_{1} x_{3} | −0.4200 | 0.3087 | −1.361 | 0.232 | |
x_{2} x_{3} | −0.7450 | 0.3087 | −2.414 | 0.061 |
The calculated values by using the regression model were allowed to do a comparison with experimental values. The coefficient of determination R^{2} of calculated values was 0.9918. This indicates that the fitted model could explain 99.18% of the total variability within the range of values studied. The analysis of variance (
Source | df | Sum of squares | Mean squares | F-value | P-value | Significance level |
Regression | 9 | 230.247 | 25.5830 | 67.12 | <0.0001 | ** |
Linear | 3 | 193.084 | 64.3614 | 168.87 | <0.0001 | ** |
Square | 3 | 33.808 | 11.2694 | 29.57 | 0.001 | ** |
Interaction | 3 | 3.355 | 1.1182 | 22.9 | 0.138 | |
Lack of fit | 3 | 1.687 | 0.5623 | 5.14 | 0.167 | |
Pure error | 2 | 0.219 | 0.1094 | |||
Total | 14 | 232.152 | R^{2} = 0.9918 |
The three-dimensional response surface graphs and contour maps were shown in
Response surface plots (A) and contour plots (B) of the DPPH radical scavenging activity of pumpkin seed hydrolysates affected by enzyme amount and substrate concentration.
Response surface plots (A) and contour plots (B) of the DPPH radical scavenging activity of pumpkin seed hydrolysates affected by enzyme amount and hydrolysis time.
Response surface plots (A) and contour plots (B) of the DPPH radical scavenging activity of pumpkin seed hydrolysates affected by substrate concentration and hydrolysis time.
These results implied that the response surface had a maximum point in the experimental range of the independent variables. The precise coordinates of optimum (i.e the values of three independent variables) were obtained by analytical procedure: enzyme amount 6181.82 U/g, substrate concentration 0.0543 g/ml, time 4.87 h. Thus, we can learn that the maximum predicted scavenging capacity under the optimal condition was 93.17%.
To confirm the validity of the suggested mathematical model, the trial experiments were conducted under the predicted optimal conditions. Take convenience into account, the actual optimum experimental parameters were modified as follows: enzyme amount 6000 U/g, substrate concentration 0.05 g/ml, time 5 h, with the temperature of 50°C and pH 2.5. Three parallel experiments were performed, and the values of DPPH radical scavenging activity were 92.15%, 93.08% and 93.23%, respectively. The mean of three replicate determinations was 92. 82%. The experimental value was in high agreement with the predicted value, which showed the validity of this response model.
To conclude, we carried out hydrolysis experiment for pumpkin seed protein isolate under 15 conditions by different combinations of enzyme amount, substrate concentration and hydrolysis time (independent variables). The second-order model developed for the DPPH radical scavenging activity of pumpkin seed hydrolysates was extremely significant (P<0.01) with a high value of coefficient of determination (0.9918). The surface and contour graphs indicated that maximum scavenging capacity of DPPH radical was 92.82%, which obtained under optimum hydrolysis condition (enzyme amount 6000 U/g, substrate concentration 0.05 g/ml, hydrolysis time 5 h, t temperature 50°C and pH 2.5), which could be validated by experiment. Thus, we believe that the optimum hydrolysis conditions can be obtained by RSM precisely.