Optimization of Processing Conditions by Using Protease to Hydrolyze Bone Powder to Produce Polypeptides and its Antioxidative function

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1 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: 02 8 Optimization of Processing Conditions by Using Protease to Hydrolyze Bone Powder to Produce Polypeptides and its Antioxidative function Wang Yuanliang 1,2, Li Ke 2, Zhang Juan 1, He Jianhua 3, Li Zongjun 1 * 1 College of Food Science and Technology, Hunan Agricultural University, Chanagsha, China 2 Hunan Province Key Laboratory of Food Science and Biotechnology, Chanagsha, China 3 College of Animal Science and Technology, Hunan Agricultural University, Chanagsha, China Abstract: In order to fully utilize pig bones and increase their added value, according to the design principles of Box-Behnken central composite test, with 3 factors and 3 levels of response surface method(rsm), a quadratic polynomial regression mathematics model of Protamax protease hydrolyzing bone powder to produce polypeptides solution was established, its optimal process conditions were at 15% substrate concentration, ph 7.5, 0.25% enzyme substrate ratio (E/S), 55, 13 h enzymatic hydrolysis time. The polypeptides production rate (PPR) was mg/g. Polypeptides powder were obtained by freeze-drying, which contained crude protein 98.91%, amino acid 12.18%, and trichloroacetic acid (TCA) soluble protein more than 95% of total protein. Besides, the total antioxidant capacity (TAC), scavenging action to hydroxyl radical ( OH) and the inhibition superoxide anion radicals capacity of polypeptides were also observed. Compared with glutathione (GSH), when the solution concentration range was from 100 mg/ml to 150 mg/ml, the TAC of bone collagen polypeptides was 71.92% of GSH; from 2.5 mg/ml to 20 mg/ml, its scavenging action to hydroxyl radical was 1.36 times as GSH; from 10 mg/ml to 150 mg/ml, its inhibition superoxide anion radical capacity was lower than GSH. Key words: enzymolysis; bone powder; RSM; bone collagen polypeptides; TAC; scavenging action to hydroxyl radical ( OH); inhibition superoxide anion radical capacity. Polypeptides are bioactivators involving in variety of cell functions in organism. In recent years, to increase food processing performance, tropism and release polypeptides with physical health functions by enzymatic hydrolysis has been widely applied [1]. Edible proteins are hydrolyzed by enzyme to produce small molecular weight polypeptides which are not only improve nutritional values of the original proteins, but also give a variety of bioactivities [2,3]. Inside and outside China, enzyme-hydrolyzing animal bones to get polypeptides were widely reported. Most of them were only limited to analyze the influence factors of enzymatic hydrolysis conditions on the degree hydrolysis of bone proteins, but higher rate of hydrolysis degree blindly made many polypeptides degraded into amino acids, as well as loss of nutritional value and bioactivities of polypeptides [4]. In recent years, Tang Yong degraded pig bones by Lactobacillus to obtain amino acids, free calcium and phosphorus. The maximum hydrolysis rate of bone powder proteins was 63%, in his study, the bone proteins were completely hydrolyzed into amino acids [5]. In relation to the setting of hydrolysis conditions, although the traditional orthogonal test can consider several factors and get the best level combination, this method can t identify the explicit function expression between the factors and the response values, namely, regression models. In consequence, it was unable to find the best combination of all the hydrolysis factors and the optimal response values. This study used Minitab14 software and RSM to optimize the hydrolysis process of bone powder to produce polypeptides, establishing mathematical model between hydrolysis conditions and PPR to obtain the optimal production process parameters [6]. Finally, it increased the yield of active bone polypeptides after using Protamex protease to hydrolyze bone powder, greatly avoided the bone proteins to be hydrolyzed into amino acids and retained the nutritional value and bioactivities of polypeptides. The producing and scavenging of free radicals in normal body keeps a dynamic equilibrium, if the free radicals are too many or the removal rate of them are too slow, they would produce a series of biological damages by accelerating aging and inducing various diseases [7,8]. Many human and animal diseases relate to the metabolic *Corresponding author: Dr Li Zongjun, TEL: , lizongjun@yahoo.com.cn

2 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: 02 9 imbalance of free radicals, such as cancer, Alzheimer's, arthritis, ulcers, arteriosclerosis, etc. [9,10]. External antioxidant supplement can reduce the free radical levels, prevent lipid peroxide, help the body resist disease and delay senility [11]. Antioxidant polypeptides gain more attention, because of their low molecular weight, easy absorption and strong activity, etc., thus they have a broad prospect in medicine, cosmetics, health products and food and feed additives types. Antioxidant polypeptides were abundant in products of bone collagen hydrolyzed by proteases, which possessed many functions, such as a strong inhibition peroxidation of biological macromolecules, removing free radicals, beauty, moisturizing, increasing skin elasticity, promoting calcium deposition,etc. [12-15]. Nowadays, many reports about hydrolyzing soy proteins, lactoproteins, fish proteins, egg ovalbumin by proteases to obtain antioxidant polypeptides at home and abroad were published, while the studies on obtaining antioxidant polypeptides by hydrolyzing animal bones were seldom seen. The TAC, scavenging action to hydroxyl radical and inhibition of superoxide anion radicals capacity of polypeptides were also researched to evaluate the anti-oxidative function of bone polypeptides powder produced in the best enzymolysis conditions. 1 Materials and methods 1.1 Materials Fresh pig hipbones: Hunan Province Zheng Hong Hai Yuan Green Food Co.Ltd Enzyme Preparation: Protamex proteases, Marked activity was 1.5 AU (Anson Unit) per gram, Novozymes company. 1.2 Methods Bone powder preparation Pretreatment of fresh pig hipbones drying coarse grinding superfine comminuting sieving analysis bone powder Operation points: 1)pretreatment: fresh pig hipbones were washed by 1:1 (W/W) water, the pieces of meat on the bones were removed after cooking the bone 20min at 121, the bones were chopped by 3~4cm 2, 1:1(W/W) water, the upper oil were removed after 121 high-pressure cooking 20min, and drained (boil water repeatedly to remove oil). 2) Drying: drying was done at 85 until water content less than 4%. 3) Course grinding: the bone particles were grinded to coarse powder by coarse grinding mill. 4) Superfine comminuting: coarse powder was pulverized to ultrafine powder by Supermicro. 5) Sieving: the ultrafine powder then was sieved by 140 mesh sieve. 6) Analysis: Analytical Center of Hunan Province detects samples Preparation of polypeptides solution by hydrolyzing bone powder process bone powder bone mud sterilizing adjusting ph value adding proteases enzymolysis inactivating proteases centrifugation polypeptides solution Operation points: the bone powder was turned into bone mud of different concentrations and filled into 250 ml flasks, sterilized 15min 85 in water bath, cooled to below 40, ph adjusted. Then different proportions of enzymes were added and hydrolyzed in different times, different temperature and ph, 120 r/min rotational oscillation. After hydrolyzing, proteases were inactivated in water bath 85 15min. The mixtures were finally centrifuged (4000 r/min, 15 min) and the supernatant fluids were collected The enzymatic hydrolysis parameters selection experiment 1) The effect of different temperatures on enzymatic hydrolysis The substrate concentration was 10% (W/V), with E/S 0.2% (W/W), and ph 7.5. The hydrolytic temperatures were 45, 50, 55, 60, 65. The bone mud solutions were hydrolyzed for 5 hours. 2) The effect of different ph value on enzymatic hydrolysis The substrate concentration was 10% (W/V), E/S was 0.2%, hydrolytic temperature was 55 the ph were 5.5, 6.5, 7.5, 8.5, 9.5, the hydrolyzing time for the bone mud solution was 5 hours. 3) The effect of different E/S on enzymatic hydrolysis

3 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: The substrate concentration was 10% (W/V), the ph was 7.5, the hydrolytic temperatures were 55, E/S were 0.05%, 0.10%, 0.15%, 0.20%, 0.25%. The bone mud solutions were hydrolyzed for 5 hours. 4) The effect of substrate concentration and hydrolysis time on hydrolysis and their interaction Minitab14 data statistical analysis software was used. General full factorial design was used to select the best conditions of the single-factor tests, with substrate concentrations (10%, 15%, 20%) and hydrolysis times (6h, 8h, 10h, 12h) as factors. The PPR was the response value factor to determine appropriate combination level of the substrate concentration and hydrolysis time. Experimental factors and levels design can be seen in Table 1. The tests of the above factors were set to three parallel, and their averages were taken as the results. Table 1 The factorial analysis experimental table of the substrate concentration and hydrolysis time serial numbers substrate concentration /% hydrolysis time /h ) RSM test According to the central composite experimental design principles of Box-Behnken RSM, combined with the above results, Minitab14 statistical analysis software was used to select the substrate concentration, E/S, hydrolysis time, respectively. 3 factors and 3 levels of RSM was taken to optimize the hydrolysis conditions [5,6]. Experimental factors and levels are shown in Table 2, a total 15 test points, including 12 factorial points, three for the zero. Zero point tests were carried out 3 times to estimate the error. Table 2 Factors and levels of RSM experimental design factors levels A substrate concentration (%) B E/S( %) C hydrolysis time (h) Preparation process of polypeptides powder The polypeptides hydrolysis solution was concentrated to 1/4 of the original volume by using the rotary evaporation and concentration apparatus, then the concentrated solution was frozen in -30 refrigerator, the polypeptides powder was collected after freeze-drying 24h and was stored in -24 refrigerator Products assay methods 1) Determination of bone powder ingredients The contents of protein, crude fat, ash, total calcium, total phosphorus and water in samples were tested in Analysis and Testing Center of Hunan Province. Test standards: GB/T ~ , GB/T5009.5~ ) Determination the total amount of polypeptides production

4 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: when the molecular weight are less than 10000u and don t be precipitated by trichloroacetic acid (TCA) [16,17,18], this kind of peptides are defined as polypeptides. the total amount of polypeptides production are defined as follows: Where: N=(N (TCA) -N (AA) ) 6.25 (1) N = the total amount of polypeptides production in hydrolysate (mg); N (TCA) = soluble nitrogen content in hydrolysate (mg); N (AA) = the amino acid nitrogen content in hydrolysate (mg); 6.25 = the coefficient of nitrogen in bone proteins converted to protein. 3) N (TCA) Determination: the hydrolysis solution and 10% TCA were mixed by 1:1 (V/V), the mixture was centrifuged (4000 r/min,15 mins), then the amount of total nitrogen in supernatant fluid was determined. 4) Determination of total nitrogen: it was executed according to GB/T (Determination of proteins in food) [19]. 5) N (AA) determination: formaldehyde titration method, referencing GB / T the determination of amino nitrogen in fruit and vegetable juice drinks [20]. 6) PPR: W=N/G (2) Where: W = PPR, (mg/g); N = the total amount of polypeptides production in hydrolysate, (mg); G = the weight of bone powder in hydrolysate, (g) Determination of TAC The definition of TAC, when the absorbance value(od) of the reaction system at 37 per min per ml of sample increased by 0.01, it is a TAC unit, which was determined by the TAC kit produced by Technology Co., Ltd. Nanjing Jiancheng, according to the requirements of instruction Determination of scavenging action to hydroxyl radicals The definition of scavenging action to hydroxyl radical, when the sample per milliliter reacts at 37 for 1min and decreases by 1mmol/L the H 2 O 2 concentration of the reaction system, an inhibition of hydroxyl radicals unit, which was determined by hydroxyl radicals assay kit produced by Technology Co., Ltd. Nanjing jiancheng, according to the requirements of instruction Determination of inhibition superoxide anion radicals capacity The definition of the ability to inhibit the superoxide anion radical, when the sample per liter reacting at 37 for 40min to inhibit the superoxide anion radicals is equivalent to the variation value of 1mg of vitamin C inhibiting the superoxide anion radical, this variation value is a activity unit, which was determined by inhibition and production of superoxide anion radicals ( instruction Data analysis of antioxidant capacity ) assay kit by Technology Co., Ltd. Nanjing jiancheng, according to the requirements of The assay kits of TAC, scavenging action to hydroxyl radicals, inhibition superoxide anion radicals request a preliminary test (determining the optimal concentration required for detection). After the preliminary tests were done, different concentrations of polypeptides solution and GSH solution were prepared, which were in the best test concentration range to detect the above three indicators according to the results of the preliminary tests, each of the test was done seven parallels. Minitab14 Statistical analysis software was used to deal with experimental data. 2 Results and Analysis 2.1 Main ingredients of bone powder The bone powder contained 19.23% protein, 10.42% crude fat, 61.59% ash, 23.90% total calcium, 12.61% total phosphorus, and 3.67% moisture. It was rich in protein, calcium and phosphorus. Fat content was low. It can be used

5 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: as a food additive in various meat products, which not only rich in calcium, had unique flavor, and tasted more delicious, but also can improve the production rate of finished products. 2.2 The affect of different temperature, ph, E/S on the enzymatic hydrolysis The single-factor test results of temperature, ph and E/S on the enzymatic hydrolysis are shown in Fig. 1, Fig.2 and Fig.3 respectively. The results show that the fastest hydrolysis rate was at 55, with optimum ph was 7.5. Therefore, in terms of response surface optimization, 55, 7.5 were selected as the water bath temperature and ph respectively. The result in Fig.3 shows that the increase of the protease amount, protein conversion rate increased firstly, but the increased rate was slow, and when the E/S was 0.2%, the PPR was mg/g. the main reason is that in a certain substrate concentration, when the protease dosage was low, enzyme was saturated, so the higher the enzyme dosage, the higher the hydrolysis rate, PPR increased significantly. With the gradual increase of protease dosage, enzyme concentration in reaction system rose, a competitive inhibition was formed between the enzyme and the enzyme, resulting in that although the enzyme dosage increased, only a small PPR increased. Another important reason is that when the rising of protease dosage increased the polypeptides production, it also increased the speed of formation amino acids, resulting in the PPR didn t raise unceasingly. Fig.1 Effect of different temperature on enzymatic hydrolysis Fig.2 Effect of ph on enzymatic hydrolysis

6 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: Fig.3 Effect of E/S on enzymatic hydrolysis 2.3 The effect of substrate concentration and hydrolysis time on the enzymatic hydrolysis and their interaction As can be seen from Fig.4, when the substrate concentration was 15% and hydrolysis time was 12 h, the highest PPR was obtained, up to mg/g, which was higher than 10%, 20% of the substrate concentration. The main reason is that the rate of polypeptides hydrolyzed into amino acids was accelerated, resulting in the dropping of PPR. According to the center complex theory, when the enzyme catalyzed, the enzyme bound substrate and formed intermediate complex (ES) firstly, and then generated the product (P) and released enzyme. At a higher substrate concentration, the enzyme were completely saturated, resulting in the decreasing of hydrolysis rate, the protein conversion rate in unit time was low finally. However, at a lower substrate concentration, the probability of contact between enzyme and substrate decreased, subsequently leading to the decrease of hydrolysis rate. The highest protein conversion rate obtained when the substrate concentration was 15%, which indicated that the substrate concentration in this level was moderate. 15% substrate concentration was selected for response surface optimization. Fig.4 Interaction results of substrate concentration and reaction time in hydrolysis experiment

7 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: RSM results Experimental design and the results of RSM are shown in Table 3. The encoded unit of Minitab14 statistical analysis software was used to fit multiple linear regression of test data, regression model coefficients and significant test results are shown in Table 4, the quadratic regression model was received which was about substrate concentration (X 1 ), E/S(X 2 ), hydrolysis time (X 3 ). Table 3 items Optimization experimental designs and results of Protamex protease hydrolyzing bone mud test No. substrate concentration(%) E/S(%) Hydrolysis time(h) PPR(mg/g) 1 -(12) -(0.15) 0(10) (18) (0.25) (0.20) -(6) (14) (15) Table 4 Regression model Coefficients and significant test results regression coefficients Standard error of regression coefficients(sb) constant X X X X X X X 1X X 1X X 2X notes:s = Prediction error sum of squares= R-Sq = 98.24% R-Sq(Prediction)= 74.22% R-Sq(adjustment) = 95.08% Significant test result of regression model coefficients was R-Sq = 98.24%, which showed that the model and data had a high degree of fitting. From Table 4 model (1), item X 3 and quadratic item X 3 2 were significant, while other items were insignificant; all of these indicated that the factors for the PPR were not a simple linear relationship. Residual analysis was used to analyze the response data (PPR), the analysis results were shown in Fig.5, and the model can largely explain the variation of the observed data (PPR). T P

8 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: Fig.5 Residual plot of PPR The significance of regression model was tested intensely. The variance analysis results of response data are shown in Table 5, F=31.09>F 0.05 (9,2)=10.38, P=0.001<0.01 in model (2), which indicated that the regression model was significant. Lack-of-fit items F=6.30<F 0.05 (9,3)=8.81, P=0.140>0.05 were insignificant, indicating that the model had a high degree of fitting, and experimental error was small. This model can analyze and predict process results of hydrolyzing bone powder by protease to prepare polypeptides. source degrees of freedom Table 5 Significance test for regression model Sum of Consecutive squares corrected sum of squares corrected mean regression linear square interaction Residual error Lack-of-fit Pure Error total Analyzing and optimizing hydrolysis conditions by RSM The response surfaces and contour lines of model (1) are shown in Fig.6, Fig.7, Fig.8, three figures directly reflects the impact of various factors on the response values. In Fig.6, when reaction time was remained at 14 h, the PPR increased together with the improvement of the E/S. When the substrate concentration was less than 15%, the production rate gradually increased firstly and then began to decreases. As can be seen from Fig.7, the substrate concentration was kept at 18%, the PPR increased with the duration, if the time was less than 12h, the E/S had a small impact on the PPR, after 12 hours with a little impact.in Fig. 8, the E/S was remained at 0.25%, the PPR increased significantly together with increasing duration time. When the substrate concentrations were range from 12% to 18%, they had little effect on the PPR. square F P

9 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: Fig.6 Response surface plot and contour plot of the effect of E/S and substrate concentration and their mutual interactions on the PPR Fig.7 Response surface plot and contour plot of the effect of reaction time and E/S and their mutual interactions on the PPR Fig.8 Response surface plot and contour plot of the effect of hydrolysis time and substrate concentration and their mutual interactions on the PPR The response optimizer of Minitab software optimized the test results, the optimal solution of model (1) was calculated and picture was drawn, the parameters are shown in Table 6, Optimization figure is showed in Fig.9. The optimum substrate concentration was 15.27%, E/S was 0.25%, hydrolysis time was h, predicted polypeptide production was mg/g. Table 6 Parameter setting of response optimizer Parameter target low limit Nominal the Better high limit PPR Large the Better

10 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: Fig.9 Result of response optimization design In order to test the reliability of the results from RSM, the above optimal conditions were adopted for hydrolyzing bone powder repeatedly to prepare polypeptides. The practical operation was taken into account; the extraction process parameters were amended. Substrate concentration was 15%, the enzyme substrate ratio was 0.25%, the hydrolysis time was 13h, ph was 7.5, temperature was 55, and rotation oscillation speed was 120 r/min. In this condition, the actual PPR was mg/g, Compared with the theoretical predicted value, the relative error was about 1.28%. Therefore, optimal process parameters obtained from response surface method was accurate, reliable and practical. 2.6 The test results of polypeptide powder content Crude protein content of polypeptides powder was 98.91%, free amino acid content was 12.18%, TCA soluble protein content was 94.38% which accounted 95.42% of total protein. According to the quality indicators at home and abroad and the actual level of raw materials to produce polypeptide in China, what can be seen is that the levels of polypeptides in bone collagen polypeptides products was very high [21]. 2.7 TAC Preliminary experiments proved that when solution concentration range was from 100 mg/ml to 150 mg/ml, the absorbance value and TAC has a good linearity. The TAC results in optimal detection concentration are shown in Table 7. Table 7 The variance analysis of TAC of polypeptides and GSH at different concentrations Concentration mg/ml polypeptides TAC (U/mL) variance TAC (U/mL) GSH variance ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Note: means not detected, as the incomplete dissolution for 200mg/mL GSH under testing conditions.

11 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: The statistical test shows that the TAC of polypeptides solution and GSH solution at different concentration were significantly different (P<0.01), when polypeptides and GSH concentration range was from 100 mg/ml to 150 mg/ml, the TAC and their concentration were positive correlation, increased with the rise of the concentration. The TAC of two different solutions in the same concentration were significant different (P<0.01), when the solution concentration range was from 100 mg/ml to 150 mg/ml, the TAC of GSH was slightly lower than the polypeptides whose TAC was 71.92% of GSH. 110 mg/ml GSH solution and 130 mg / ml polypeptides solution, the variance analysis results of TAC of two kinds of solution were F = 4.49 <F 0.05 (1,6)=5.99, P=0.056>0.05, which indicated that their TAC were not significant difference (α=0.05), they can be regarded as almost equal. 2.8 Scavenging action to hydroxyl radical Preliminary experiments proved that when two kinds of solution concentration range was from 2.5 mg/ml to 10 mg/ml, the absorbance value and scavenging hydroxyl radical capacity had a good linear relationship. The scavenging hydroxyl radical results in optimal detection concentration are shown in Table 8. Table 8 The variance analysis table of scavenging action to hydroxyl radical of polypeptides and GSH at different concentrations polypeptides GSH concentration Scavenging action to Scavenging action to mg/ml variance hydroxyl radical (U/mL) hydroxyl radical (U/mL) variance ± ± ± ± ± ± ± ± ± ± ± ± The statistical test showed that the scavenging hydroxyl radical capacity of polypeptides solution and GSH solution in different concentration was significantly different (P<0.01), when polypeptides and GSH concentration range was from 2.5 mg/ml to 20 mg/ml, the scavenging hydroxyl radical capacity and their concentration were positively correlated, it was increased with the rise of the concentration. The scavenging hydroxyl radical capacity of two different solutions in the same concentration were significant different (P <0.01), when the solution concentration range was from 2.5 mg/ml to 20 mg/ml, scavenging hydroxyl radical capacity of polypeptides was 1.36 times as GSH. 5 mg/ml polypeptides solution and 7.5 mg/ml GSH solution, the variance analysis results of TAC of two kinds of solution were F=4.49 <F 0.05 (1,6)=5.99, P=0.056>0.05, which indicated that their TAC were not significant difference (α=0.05), can be regarded as almost equal. 2.9 Inhibition the superoxide anion radical capacity Preliminary experiments proved that when polypeptides and GSH solution concentration range was from 10 mg/ml to 50 mg/ml, the absorbance value and the inhibition superoxide anion radical capacity had a good linearity. The inhibition superoxide anion radical results in optimal detection concentration are shown in Table 9. Table 9 The variance analysis table of inhibition superoxide anion radical capacity of polypeptides and GSH at different concentrations concentration mg/ml polypeptides Inhibition the superoxide anion radical capacity (U/L) variance GSH Inhibition the superoxide anion radical capacity (U/L) variance ± ± ± ± ± ± ± ± ± ± ± ± ± ±

12 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: The statistical test shows that the inhibition superoxide anion radical capacity of polypeptides solution and GSH solution in different concentration was significantly different (P<0.01), when polypeptides and GSH concentration range was from100 mg/ml to 150 mg/ml. The inhibition superoxide anion radical capacity and their concentration were positively correlated; it was increased with the rise of the concentration. The inhibition superoxide anion radical capacity of two different solutions in the same concentration were all significant different (P<0.01), when the solution concentration range was from 100 mg/ml to 150 mg/ml, the inhibition superoxide anion radical capacity of GSH was higher than the polypeptides. The average value comparison of polypeptides and GSH inhibiting superoxide anion radical in the same concentration are shown in Table 10, as can be seen from Table 10, with the rise of the concentration, the difference decreased gradually. Table10 Comparison of inhibit ability of superoxide anion free radicalof polypeptides and GSH concentration (mg/ml) Inhibition the superoxide anion radical capacity (U/L) polypeptides GSH polypeptides:gsh(%) Conclutions and discussion 1)Based on the experimental design software Minitab14, by means of quadratic regression design, Protamex proteases were used to hydrolyze pig bone powder to produce polypeptides. Regression model about polypeptides production, substrate concentration, E/S, hydrolysis time was obtained. After testing, the model was reasonable, reliable, and able to predict the PPR. 2)The response surfaces and contour lines of the model were utilized, the key factors affecting PPR and their interactions when ph value was 7.5, temperature was 55, and optimal process parameters (substrate concentration 15.27% (W/V), E/S 0.25% (W/W), hydrolysis time h) were discussed. Considering the convenience of operation, the extraction process parameters were amended: the substrate concentration 15%, hydrolysis time 13h, the PPR was mg/g. Therefore, RSM can be used to optimize the process of Protamex protease hydrolyzing bone powder to produce polypeptides, which obtained optimal process parameters, effectively reduced the blindness of technical operation and lay the foundation for further experimental study. 3)Determination the antioxidant capacity of homemade bone collagen polypeptides and GSH, the results showed that the bone collagen polypeptides had a stronger antioxidant capacity, comparing with GSH. When the solution concentration range was 100 mg/ml to 150 mg/ml, the TAC of GHS was slightly lower than the polypeptides whose antioxidant capacity was 71.92% of GSH. When it was ranged 2.5 mg/ml to 20 mg/ml, scavenging hydroxyl radical capacity of polypeptides was 1.36 times as GSH. The inhibition superoxide anion radical capacity of GSH was stronger than homemade bone collagen polypeptides at the same concentration, the difference of them in inhibiting superoxide anion radical decreased when the concentration increased from10 mg/ml to 150 mg/ml. At present, many reports about enzyme hydrolysis in animal bones are available, but most of them are limited in the effects of enzyme hydrolysis conditions towards the hydrolysis degree of bone protein and how to obtain high degree of hydrolysis. While when the degree of hydrolysis is too high, many polypeptides are degraded into amino acids and lose its original nutritional value and bioactivity. This research took the polypeptides generated rate as the main indicator to ascertain the hydrolysis conditions; gain the optimum production process parameter; improve the

13 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: yield of bone polypeptides; avoid the complete hydrolysis of bone protein into amino acids and keep its nutritive value and bioactivity. When the antioxidant activity of homemade pig bone polypeptides was studied, if the recognized antioxidant peptide glutathione was took as contrast, it made the description of antioxidant properties of big bone polypeptides more accurate and intuitive. The polypeptides in this study were prepared in peptide mixtures form, their components were not separated. Thus further research can separate the different components contained, in order to study the function of big bone polypeptides with different molecular weight ranges. After obtain polypeptides with certain molecular weight, antioxidant and lower blood pressure properties, the enzymatic hydrolysis condition can be optimized in order to expand more functional polypeptide products. In addition, other bioactivities of bone collagen polypeptides were also studied. It was found that bone collagen polypeptides could inhibit the ACE and promote the multiplication of Lactobacillus. Acknowledgment We thank Hunan province key laboratory of food science and biotechnology for providing the experimental equipments. References: [1] LI Chenghui. New period of protein nutrition: Peptide nutrition[j]. Livestock and Poultry Industry, 2005,11:48~50. [2] Kundu B, KHARE SK, SINGH G. Role of polypeptides in the treatment and diagnosis of osteoporosis[j].peptides,1999,20(4): [3] LIU Anjun, SUN Hongchen, LIU Youzhi, et al. Extraction and purification of protein and proteoglycan from bovine bone[j]. Transactions of the Chinese Society of Agricultural Engineering,2007,23(3): [4] SUN Qian, WANG Zhengzheng, LUO Yongkang,et al. Study on enzymatic preparation technology of porcine bone protein bioactive peptides[j].meat Research,2007,5: [5] TANG Yong, LI Hongjun, SUI Daohui. Effect of fermentation by lactobacillus on nutrition and physiochemical property of super-microsmashing hog-bone powder[j]. Transactions of the Chinese Society of Agricultural Engineering, 2002,18(2): [6] Mu Yundong. Response surface methodology and its application in food industry[j]. Journal of Zhengzhou Institute of Technology,2001,22(2): [7] RONG Jianhua, LI Xiaoding, XIE Bijun. Antioxidant effect of soybean peptide research[j]. Food Science, 2003,23(11): [8] LAAKSO S. Inhibiton of lipid peroxidation by casein. Evidence of molecular encapsulation of 1,4-pentadiene fatty acids[j]. Biochim Biophys Acta.1984,792(1): [9] Hu M, Mc Clements D J and Decker E A. Lipid Oxidation in Corn Oil-in-Water Emulsions Stabilized by Casein, Whey Protein Isolate, and Soy Protein Isolate[J]. J Agric Food Chem.2003,51(6): [10] FANG Yunzhong, ZHENG Rongliang. Theory and Application of Free Radical Biology[M]. Beijing: Science Press, 2002: [11] CHENG Shi, DING Haiqin. Glutathione and antioxidant talk today[j]. Progress In Physiological Sciences, 2002,(1): [12] MOSKOWITZ R W. Role of collagen hydrolysate in bone and joint disease[j]. Seminars in arthritis and rheumatism. [Semin Arthritis Rheum].2000, 30 (2): [13] MENDIS E, RAJAPAKSE N, KIM S K. Antioxidant properties of radical-scavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate[j].journal of agricultural and food chemistry[j]. Agric Food Chem, 2005,53(3): [14] GELITA Group. Osteoarthritis; Osteoarthritis patients report improved function in collagen hydrolysate study[j].biotech Business Week.2004,(28):129. [15] TANG Chuanhe, PENG Zhiying. A new functional food ingredient Collagen peptide[j]. Commercial Indusry, 2001,5. [16] Organic Chemistry Department of Tianjin University.Organic Chemistry[J].Tianjin: Tianjin University Press,2004. [17] LI Yong. Current progress and advances of study on bioactive peptides[j].food and Fermentation Industries, 2007,33(1):3-9.

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