Optimization of antioxidant hydrolysate production from flying squid muscle protein using response surface methodology

food and bioproducts processing 9 0 ( 2 0 1 2 ) 676–682

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Optimization of antioxidant hydrolysate production from flying squid muscle protein using response surface methodology Xubo Fang, Ningning Xie, Xiaoe Chen ∗ , Hui Yu, Jing Chen School of Food and Pharmacy, Zhejiang Ocean University, Zhoushan, Zhejiang Province 316004, People’s Republic of China

a b s t r a c t The squid muscle protein, extracted from by-products of flying squid (Ommastrephes bartrami) was hydrolyzed by five proteases (pepsin, trypsin, papain, alcalase and flavourzyme). DPPH radical scavenging power was used to evaluate antioxidative activity of hydrolysates. The hydrolysate obtained by papain exhibited the most excellent potential of antioxidative activity. Furthermore, response surface methodology (RSM) was employed to optimize hydrolysis conditions, including enzyme to substrate (E/S) ratio, reaction temperature, and hydrolysis time. The optimum conditions obtained were as follows: E/S ratio of 1.74%, temperature of 51 ◦ C and time of 46 min, under which, DPPH radical scavenging activity of 74.25% was obtained. Moreover, it was found that the optimum hydrolysate of 8 mg/mL displayed relatively stronger inhibitory effect on lipid peroxidation compared with ␣-Tocopherol of 0.1 mg/mL. © 2012 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Flying squid; Papain; DPPH radical; Lipid peroxidation; Amino acid composition; Response surface methodology

1.

Introduction

In China, the flying squid (Ommastrephes batramii) processing industry, whose annual output exceeds 10 million tons, has become an important part of aquaculture in recent years. Byproducts of flying squid, accounting for approximately 50% of squid weight, include tentacle, fin, funnel, viscera and head of the squid. The flying squid muscle from tentacle, fin and head of the by-products accounts for 15–20% of the residual wastes. Reuse of by-products has attracted considerable attention in aquatic processing industry, due to the increasing economical effectiveness. However, the significance of developing functional property of them has not been realized (Tong and Zhang, 2001). Some kinds of fish proteins, whose hydrolysis mediated by proteolytic enzymes can provide more value-added products (Dong et al., 2008), have been proved to be excellent sources of bioactive food ingredients (Kristinsson and Rasco, 2000). Nevertheless, residual proteins from by-products of the squid have not been fully utilized, and the residual proteins have been commonly processed into fish meal with low economic values. Therefore, further

∗

applications are urgently required to develop value-added products from the squid residual proteins. Bioactive peptides released by enzymatic proteolysis of various proteins that act as potential physiological modulators of metabolism have been investigated in recent reports. Antioxidant activity is thought to be particularly of importance, since oxidation as an unavoidable reaction in all living organisms has been widely accepted (Halliwell, 1994). Generated radicals are very unstable and react rapidly with other groups or substances in human body, which lead to cell or tissue injuries. Lipid oxidation, resulting in undesirable off-flavors and potentially toxic reaction products, becomes a significant concern in food industry (Park et al., 2001). Currently, natural antioxidant like ␣-tocopherol, and many synthetic antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), t-butyl hydroquinone (TBHQ) and propyl gallate, are commonly adopted to attack free radicals in food and biological systems. The synthetic antioxidants are effective and cheap compared with natural ones, while their applications are restricted, due to the potential risks related to human health. Consequently, researches

Corresponding author. Tel.: +86 580 2554038; fax: +86 580 2554781. E-mail address: [email protected] (X. Chen). Received 11 January 2010; Received in revised form 6 February 2012; Accepted 2 April 2012 0960-3085/$ – see front matter © 2012 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fbp.2012.04.001

food and bioproducts processing 9 0 ( 2 0 1 2 ) 676–682

concerning about natural antioxidants as alternatives to artificial ones have become a hot issue. It has been reported that hydrolysate obtained by hydrolysis of squid protein with pepsin, trypsin and ␣-chymotrypsin exhibited antioxidant activities (Rajapakse et al., 2005). Enzymes, such as papain, alcalase and flavourzyme, have also been proved to be capable of hydrolyzing aquatic proteins for antioxidative hydrolysates (Klompong et al., 2007; Yang et al., 2008; Moosman and Behl, 2002). Therefore, hydrolysates from flying squid proteins would also provide potential natural antioxidants. However, there is little research on the antioxidant activity of muscle hydrolysate from by-products of flying squid to date. Response surface methodology (RSM), a useful technique for investigation of complex processes, has been successfully applied to optimize processing conditions, such as E/S ratio, temperature, and time of hydrolysis, in enzymatic reaction (Kim et al., 2001). RSM is also employed in evaluating effective factors and in building models to study interactions of independent variables, and the mathematical model generated can accurately describe the overall process (Shao et al., 2007). In this study, muscle protein was extracted from the by-products of flying squid, and antioxidant hydrolysates were also prepared by various enzymes including pepsin, trypsin, papain, alcalase and flavourzyme. Hydrolysis conditions of papain mediated reactions, by which the resulting hydrolysates displayed the highest antioxidative activity, were optimized using RSM. In addition, the inhibitory efficiency of lipid peroxidation of the hydrolysate bearing highest antioxidative activity was also assayed.

2.

Materials and methods

2.1.

Materials

By-products of flying squid were obtained from Zhejiang Xingye Industrial Group Co., Ltd. Pepsin, trypsin and papain were purchased from Sinopharm Chemical Reagent Co., Ltd. Alcalase and flavourzyme were products of Novozymes (Beijing, China). 2,2-diphenyl-1-picryhydrazyl (DPPH), ␣tocopherol, BHT, and linoleic acid were also purchased from Sigma–Aldrich (Shanghai, China). All other reagents were of analytical grade from standard sources.

2.2.

identified and quantified using an automatic amino acid analyzer (L-8800, HITACHI, Japan).

2.4.

Preparation of flying squid muscle hydrolysates

The squid muscle protein obtained was subjected to hydrolysis using various enzymes (pepsin, trypsin, papain, alcalase and flavourzyme). Muscle protein was mixed with proper deionized water and homogenized before hydrolysis. The obtained mixture were evenly divided into five copies, each of which was adjusted to the required pH with 0.01 mol/L NaOH or 0.05 mol/L HCl, and heated in water bath to the appropriate temperatures before the addition of a proper amount of protease to achieve the same enzyme activity. Each mixture was incubated with stirring (SG-5404, Shuoguang Electron Technology Co., Shanghai, China) and then heated in a boiling water bath for 10 min to inactivate the enzyme. The resulting hydrolysate was centrifuged (CF16RX, HITACHI, Japan) at 8000 × g for 10 min and the lyophilized (EYELA freeze-dryer, Shanghai, China) supernatants were stored under 4 ◦ C for further use.

2.5. Scavenging effect of flying squid muscle hydrolysates on DPPH radical The antioxidant activity was measured by the scavenging effect on DPPH free radical according to the method illustrated in the literature (Shimada et al., 1992) with some modifications. Briefly, a volume of 1.5 mL squid muscle hydrolysate (1.5 mg/mL) in 95% ethanol was added to 1.5 mL of 0.1 mmol/L DPPH in 95% ethanol. The mixture was shaken and left for 30 min at room temperature, and the absorbance of resulting solution was measured at 517 nm with a spectrophotometer (2800UV/VIS, Unico Inc., Shanghai, China). When DPPH encountered a proton-donating substance such as an antioxidant, the radical would be scavenged and the absorbance reduced. A lower absorbance indicated higher DPPH scavenging activity. The scavenging effect can be expressed as shown in the following equation: DPPH scavenging activity (%) = [(blank absorbance − sample absorbance)/blank absorbance] × 100; where the DPPH blank is the value of 1.5 mL of ethanol mixed with the 1.5 mL of ethanol containing 0.1 mmol/L DPPH.

Extraction of muscle protein from flying squid

The muscle protein was obtained according to the method of Sathe and Salunkhe (1981) with a slight modification. After washing manually, the squid meat was minced by a blender (DS-1, Exemplar and Mould Instruments Co., Shanghai, China). The mince was mixed with isopropanol at a ratio of 1:4 (w/v), and then the mixture was homogenized at a speed of 12,000 rpm for 10 min. After supernatant was drained, the residue was degreased at 75 ◦ C for 120 min using isopropanol at a ratio of 1:4 (w/v). The muscle protein was air-dried at room temperature after the removal of the supernatant. The obtained squid muscle protein was stored under −20 ◦ C for further use.

2.3.

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Analysis of amino acid composition

Squid muscle protein was hydrolyzed in 6 mol/L HCl containing 0.1% thioglycolic acid at 110 ◦ C for 24 h under vacuum. Amino acids derived with phenylisothiocyanate were

2.6. Inhibition of lipid peroxidation in linoleic acid model system The antioxidant activity of the hydrolysate was measured based on the method (Nilsang et al., 2005) with a slight modification. One milligram of hydrolysate sample was dissolved in 1.5 mL of 0.1 mol/L phosphate buffer (pH 7.0), and added to 5 mL of linoleic acid (2 mg/mL) dissolved with 95% ethanol in a glass test tube which was sealed tightly with silicon rubber cap and kept at 37 ◦ C in the dark for 7 d. The degree of oxidation was evaluated by measuring the ferric thiocyanate values. The sample solution (100 ␮L) incubated in the linoleic acid model system described above was mixed with 4.7 mL of 75% ethanol, 0.1 mL of 30% (w/v) ammonium thiocyanate, and 0.1 mL of 0.02 mol/L ferrous chloride dissolved in 1 mol/L HCl. After 3 min the degree of color variation that represents linoleic acid oxidation was measured spectrophotometrically at 500 nm. The absorbance of the solution without the hydrolysate samples was considered as the blank.

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0.01 mg/mL BHT and 0.1 mg/mL ␣-tocopherol were used as positive controls.

2.7.

Amino acid

Degree of hydrolysis

Degree of hydrolysis (DH) is defined as the percentage of free amino groups cleaved from proteins, which is calculated from the ratio of ␣-amino nitrogen (AN) to total nitrogen (TPN). According to the method in the literature (Liu et al., 2007), AN was determined by a formaldehyde titration method, while TPN was determined by Kjeldahl method (Ren et al., 2008a,b).

2.8.

Experimental design and optimization

RSM was applied to identify optimum levels of three variables, namely, E/S ratio (X1 ), temperature (X2 ), and time of hydrolysis (X3 ), which were previously proved to be the most effective independent variables in enzymatic reaction (Kim et al., 2001). In addition, antioxidative activity of the hydrolysates, evaluated by DPPH scavenging activity, was taken as the response (Guerard et al., 2007; Peng et al., 2009; Mendis et al., 2004). Box-Behnken Design (BBD) was applied with three variables and three levels, containing three replicates at the center point. Based on the experimental data, multiple regression analysis through the quadratic method was performed, and the data were fitted into an empirical second-order polynomial equation including the effects of the linear, the quadratic and the interaction of the three variables (X1 , X2 and X3 ) on the response (Y). The equation was given as Y = ˇ0 +



ˇi Xi +



ˇii Xi2 +

Table 1 – Amino acid composition of squid muscle protein and giant squid muscle.



ˇij Xi Xj

(1)

Aspartic acid Glutamic acid Serine Histidine Glycine Arginine Alanine Tyrosine Cystine Valine Methionine Phenylalanine Threonine Isoleucine Leucine Lysine Proline a

3.

Results and discussion

3.1.

Amino acid composition

Amino acid composition of flying squid muscle protein was presented in Table 1, compared with that of giant squid muscle protein. The compositions of two kinds of squid muscles were significantly different. Aspartic acid and glutamic acid were the most abundant amino acids in flying squid muscle, while giant squid muscle was rich in glycine. Besides, most of amino acid contents in flying squid muscle, except for glycine, were higher than that of giant squid muscle. Amino acid composition might govern the potential of antioxidative activity (Klompong et al., 2009). Aromatic amino acids including tyrosine, histidine, and phenylanine, hydrophobic amino acids containing valine, alanine, proline and leucine, and also methionine have been reported to be able to scavenge free radicals (Suetsuna et al., 2000; Rajapakse et al., 2005; Kim et al., 2006). As shown in Table 1, contents of aromatic amino acids, the hydrophobic amino acids and methionine were higher in flying squid muscle protein than those in giant squid muscle protein, by 6.98%, 16.31% and 2.81%, respectively. Hence, hydrolysates derived from the

Flying squid muscle

Giant squid musclea

10.17 17.68 4.82 2.00 4.38 7.19 5.38 2.97 1.10 4.15 3.55 4.25 4.46 4.19 8.48 9.78 5.44

3.24 3.90 1.73 0.48 74.47 1.52 2.48 0.77 0.23 1.07 0.74 0.99 1.53 0.90 2.08 2.37 1.51

Rajapakse et al. (2005).

flying squid muscle demonstrated great potential in becoming natural antioxidants. To date, though various fish muscle proteins, such as silver carp, grass carp, hoki, yellow stripe trevally, have been used to prepare hydrolysates with antioxidative activity (Dong et al., 2008; Ren et al., 2008a,b; Kim et al., 2006; Klompong et al., 2007), whether hydrolysis products of flying squid muscle protein remained unexplored.

3.2. where Y is the dependent variables (DPPH scavenging activity). ˇ0 is an offset term. ˇi , ˇii , ˇij are the linear, quadratic, and interaction regression coefficients, respectively and Xi and Xj are levels of the independent variables (Chabeaud et al., 2009). Data were processed for Eq. (1) using analysis of variance (ANOVA) by statistical software (version 7.1.6, Stat-Ease Inc., USA).

Residues/100 residues

Selections of proteolytic enzymes

The types of protease affect the degree of hydrolysis, as well as play an important part on antioxidant activity of protein hydrolysates (Dong et al., 2008). Hence, it is of critical importance for selecting an appropriate protease, which can release antioxidative hydrolysate to a large extent from protein (Klompong et al., 2009). There are many kinds of methods in evaluation antioxidant activity of protein hydrolysate, including assays of FRAP, TEAC, DPPH, ORAC, TRAP, TOSC, F-C, CAP-e, CAA and so on. DPPH radical is independent of any additive-induced complications including metal chelation and enzyme inhibition (Jao and Ko, 2002). DPPH assay is simple, rapid, economical, and convenient, and thus, it has been widely used in evaluation of antioxidant activity (Guerard et al., 2007; Peng et al., 2009; Mendis et al., 2004). Herein, the antioxidative activity of hydrolysates was assessed by DPPH radical scavenging ability. Each of flying squid muscle protein was hydrolyzed by five different proteases, including pepsin, trypsin, papain, alcalase and flavourzyme. The antioxidant activity of hydrolysates was assessed by DPPH radical scavenging ability. As shown in Fig. 1, hydrolysates obtained by these proteases could eliminate DPPH radical in different degrees, with hydrolysate by papain exhibited the highest DPPH scavenging activity (59.51%), followed by alcalase, trypsin, flavourzyme and pepsin, respectively. DH values of flying squid muscle protein were observed to be 16.98%, 15.61% and 13.54% for alcalase, papain and trypsin mediated hydrolysis, respectively. Whereas lower DH value of below 10% was detected for both pepsin and flavourzyme catalyzed hydrolysis. Higher DH resulted in larger quantities of

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Table 2 – The Box–Behnken design for optimizing hydrolysis conditions with DPPH scavenging activity. Run number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 a

Coded levels of variable X1

X2

−1 1 −1 1 −1 1 −1 1 0 0 0 0 0 0 0

−1 −1 1 1 0 0 0 0 −1 1 −1 1 0 0 0

X3 0 0 0 0 −1 −1 1 1 −1 −1 1 1 0 0 0

Experimental values E/S

ratioa

1.0 2.0 1.0 2.0 1.0 2.0 1.0 2.0 1.5 1.5 1.5 1.5 1.5 1.5 1.5

(%)

Temperature 45 45 55 55 50 50 50 50 45 55 45 55 50 50 50

Response value (◦ C)

Time (min)

Y (%)

45 45 45 45 30 30 60 60 30 30 60 60 45 45 45

67.93 72.03 69.18 72.62 64.28 65.80 62.13 69.00 64.62 66.21 66.92 67.04 73.69 73.96 73.95

The ratio of enzyme weight to the weight of substrate (w/w).

low-molecular peptides than that lower DH (Mitsuda et al., 1966). It is generally believed that antioxidative activity of peptides with low molecular is more excellent compared with that of polypeptides. Yang et al. (2008) found that the hydrolysate of cobia skin by papain showed excellent antioxidant activity. Therefore, papain, the hydrolysate by which displayed the highest antioxidant activity and relatively higher DH, was chosen as the best candidate for further studies.

3.3.

Optimization of hydrolysis conditions

RSM has been used to investigate the influence of different hydrolysis variables, including E/S ratio, temperature and time, on antioxidant activity of hydrolysates (Ren et al., 2008a,b; Guerard et al., 2007). Results of 15 runs containing 3 replicates at center point were listed in Table 2. Besides, statistic analysis for linear, quadratic and interaction of the 3 variables (X1 , X2 and X3 ) on the response values (Y) was presented in Table 3. P value of the model was less than 0.0001, implying that the model was significant and could be used to optimize hydrolysis conditions. For the P values of three independent variables, X1 (<0.0001) was lower than X2 and X3 , indicating E/S ratio had the most significant effect on antioxidative activity within a 99%

confidence interval. In addition, effect of hydrolysis temperature (X2 ) and time (X3 ) was significant within a 95% confidence interval. The quadratic terms, X12 (P < 0.01), X22 (P < 0.01), X32 (P < 0.01), as well as the interaction term X1 X3 (0.01 < P < 0.05) were also significant within a 99% confidence interval. The lack of fit test is designed to dertermine whether the selected model is adequate to describe the observed data, or whether a more complicated model should be used. A model is considered adequate (significant) at the 95% confidence level, if the P-value of the lack of fit test is higher than 0.05. Herein, the lack of fit value of 0.1016 indicates the lack of fit is not significant relative to the pure error. The coefficient of variation (R2 = 0.9967) indicates that only 0.33% of the total variations are not explained by the model. The value of the adjusted determination coefficient (Adj R2 = 0.9908) is also high to advocate a high significance of the model. Moreover, “Pred R-Squared” of 0.9506 is in reasonable agreement with “Adj R-Squared” of 0.9908. An adequate precision is a measure of the range of predicted response relative to its associated error or, in other words, a single-to-noise ratio, whose value above 4 is considered to be desirable (Canettieri et al., 2007). For this model, a ratio of 37.759 demonstrated an adequate signal. On the whole, this model proved to be powerful for navigating the design space. Response surface quadratic model was also listed in Table 4. The best explanatory model equation for antioxidant activity was

Y = 73.87 + 1.99X1 + 0.44X2 + 0.52X3 − 0.16X1 X2 + 1.34X1 X3 − 0.37X2 X3 − 2.16X12 − 1.27X22 − 6.40X32

Fig. 1 – The DPPH scavenging activity and DH of flying squid muscle protein hydrolysates prepared by different proteases. Error bars represent mean and SD from triplicate experiments.

(2)

3D response surface and 2D contour plots are the graphical representations of regression equation. All of the response surface plots shown in Fig. 2(A), (C), and (E) are convex in shape, which suggested that there were well-defined optimum conditions (Shao et al., 2007). Shapes of the contour plots, circular or elliptical, indicate whether the mutual interactions between the variables are significant or not. Circular contour plot suggests the interactions between the corresponding variables are negligible, while elliptical contour plot shows

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Table 3 – Analysis of variance for the response of antioxidant activity in the hydrolysates. Source

Sum of squares

DF

Mean square

F Value

P Value

Model X1 X2 X3 X1 X2 X1 X3 X2 X3 X1 2 X2 2 X3 2 Residual Lack of fit Pure error Cor total R-Squared Adj R-Squared Pred R-Squared Adeq Precision

206.61 31.72 1.58 2.18 0.11 7.16 0.54 17.24 5.92 151.39 0.68 0.63 0.047 207.29 0.9967 0.9908 0.9506 37.759

9 1 1 1 1 1 1 1 1 1 5 3 2 14

22.96 31.72 1.58 2.18 0.11 7.16 0.54 17.24 5.92 151.39 0.14 0.21 0.023

168.78 233.21 11.58 16.06 0.80 52.61 3.97 126.75 43.50 1113.04

<0.0001** <0.0001** 0.0192* 0.0103* 0.4119 0.0008** 0.1029 <0.0001** 0.0012** <0.0001**

∗ ∗∗

9.01

0.1016

Significant within a 95% confidence interval. Significant within a 99% confidence interval.

the prominent interactions between corresponding variables (Muralidhar et al., 2001). As shown in Fig. 2(D) and (F), mutual interactions of E/S ratio-time and temperature-time are significant illustrated by the elliptical shape of the contour plots, whereas circular contour plots were observed in Fig. 2(B), demonstrating that the interaction between E/S ratio and temperature could be ignored. Through the three-dimensional plots and their respective contour plots, it is very convenient to locate their optimum ranges (Qiao et al., 2009). By analyzing the contour plots depicted for the interactions of E/S ratio with temperature, of E/S ratio with time, of temperature with time in Fig. 2, DPPH scavenging activity of approximate 74.21% was obtained in the following ranges: E/S ratio 1.59–1.84%, temperature 48.97–52.35 ◦ C and time 43.78–48.47 min. From the above analysis, higher DPPH scavenging ability occurred at a high E/S ratio (Fig. 2B and D), an appropriate hydrolysis temperature (Fig. 2B and F) and time (Fig. 2D and F). The optimum level of the antioxidant activity occurred with 74.40% at the E/S ratio (X1 ) of 1.74%, temperature (X2 ) of 51 ◦ C, and time (X3 ) of 46 min, respectively, calculated by derivazation of Eq. (3) and by solving the inverse matrix. To confirm the validity of the model, three assays were performed under the optimum conditions. The average data showed that the antioxidant activity value was 74.25% with a 99%

confidence interval. The above results indicated that the model was adequate under these conditions and was useful for estimation experimental data. Besides, compared with the original DPPH scavenging activity of papain hydrolysate (59.51%), the optimum one above increased by 24.77% using response surface methodology. The three independent variables, E/S ratio, hydrolysis temperature and time, significantly affected the DPPH radical scavenging activity of the hydrolysates. For E/S ratio, the DPPH scavenging activity increased with the increase of E/S ratio from 1.0% to 1.74%, but further increase from 1.74% to 2.0% led to a decrease of scavenging activity. From 45 ◦ C to 51 ◦ C, the activity increased gradually, while the activity reduced in the range of 51–55 ◦ C. DPPH radical scavenging ability gradually enhanced with increasing the hydrolysis time from 30 to 51 min, while a decline trend was observed from 51 to 60 min.

3.4. Inhibition of lipid peroxidation by the optimum squid muscle hydrolysate in linoleic acid model system Peroxidation of fatty acids might lead to deleterious effects on foods by forming complex mixture of secondary breakdown products of lipid peroxides, which in turn cause a number of adverse effects to mammalian cells (Qian et al., 2008). Therefore, the ability of the optimum squid muscle hydrolysate

Table 4 – Estimates standard errors, 95% confidence boundaries for the response of antioxidant activity in the hydrolysates. Factor

Intercept X1 X2 X3 X1 X2 X1 X3 X2 X3 X1 2 X2 2 X3 2

Coefficient estimate

73.87 1.99 0.44 0.52 −0.16 1.34 −0.37 −2.16 −1.27 −6.40

df

1 1 1 1 1 1 1 1 1 1

Standard error

0.21 0.13 0.13 0.13 0.18 0.18 0.18 0.19 0.19 0.19

95% confidence Low

High

73.32 1.66 0.11 0.19 −0.64 0.86 −0.84 −2.65 −1.76 −6.90

74.41 2.33 0.78 0.86 0.31 1.81 0.11 −1.67 −0.77 −5.91

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Fig. 2 – Response surface plots (A, C and E) and contour plots (B, D and F) for the effect of E/S ratio (X1 ), temperature (X2 ) and time (X3 ) on DPPH scavenging activity. (A and B indicate the effect of E/S ratio and temperature, C and D indicate the effect of E/S ratio and time, E and F indicate the effect of temperature and time.) obtained under the optimal hydrolysis conditions, to protect linoleic acid from oxidation was also evaluated. As shown in Fig. 3, oxidation of linoleic acid was inhibited to different extents at different concentrations, with the addition of flying squid muscle hydrolysate. Inhibitory effect on lipid peroxidation was gradually increased by squid hydrolysate concentrations from 2 mg/mL to 8 mg/mL. Besides, on the first two days, the peptide concentration of 6 mg/mL and 8 mg/mL exhibited noticeable inhibitory effect, which was similar to ␣-Tocopherol. Moreover, hydrolysate with a concentration of

8 mg/mL showed an evident inhibitory effect on lipid peroxidation during the entire reaction period, whose inhibitory effect was stronger than that of ␣-tocopherol, whereas weaker than that of BHT. Inhibitory effect of hydrolysates from by-product of flying squid muscle on lipid peroxidation was comparable with that of flying squid muscle (Rajapakse et al., 2005). Many natural antioxidants were less potent than synthetic antioxidants, but they could be used with higher concentrations, due to lower toxicity of the latter (Li et al., 2008).

4.

Fig. 3 – Inhibitory effect of optimum squid muscle hydrolysate on lipid peroxidation measured in a linoleic acid model system. Lower absorbance at 500 nm represents higher lipid peroxidation inhibition. Experiments were carried out in triplicate, and values were expressed as mean ± SD. ( Blank; 䊉 2 mg/mL;  4 mg/mL;  6 mg/mL;  8 mg/mL;  0.1 mg/mL ␣-Tocopherol; ♦ 0.01 mg/mL BHT).

Conclusions

To utilize the by-products sufficiently, the extraction of flying squid muscle protein was carried out. Meanwhile, the hydrolysis of squid protein using five representative proteases (pepsin, trypsin, papain, alcalase and flavourzyme) was investigated. The hydrolysate derived from papain mediated reaction exhibited excellent DPPH radical scavenging activity, and was therefore chosen for further studies. Statistical analyses based on Box-Behnken designs by response surface methodology showed that the optimum conditions for antioxidant activity were as follows: E/S ratio of 1.74%, reaction temperature of 51 ◦ C and hydrolysis time of 46 min. The maximum DPPH radical scavenging activity of 74.40% was obtained, which was in agreement with the experimental value (74.25%) within a 99% confidence interval, suggesting a good fit between the models and the experimental data. An enhancement of 24.77% of DPPH scavenging activity was compared with the original activity of the hydrolysate (59.51%). Moreover, the optimized flying squid muscle hydrolysate had a notable inhibitory effect on lipid peroxidation. Therefore, the hydrolysate could be exploited to be a promising and potential candidate for natural antioxidant.

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food and bioproducts processing 9 0 ( 2 0 1 2 ) 676–682

Acknowledgment This work is supported by the Project of Science and Technology Department of Zhejiang Province (No. 2008C22052), and Natural science foundations of Zhejiang Province (Nos. Y12C200015 and Y12C200016).

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