Degree of hydrolysis, functional and antioxidant properties of protein hydrolysates from Grass Turtle (Chinemys reevesii) as influenced by enzymatic hydrolysis conditions

Abstract Grass turtle muscle was hydrolyzed with papain enzyme to produce protein hydrolysate (PH) and the degree of hydrolysis (DH) was determined. Under optimal conditions, the highest DH was 19.52% and the yield was recorded as 17.26%. Protein content of the hydrolysates was ranged from 73.35% to 76.63%. Total amino acids were more than 96.77% for each PH. The PH obtained at DH 19.52% achieved excellent solubility and emulsifying activity which were 95.56% and 108.76 m2/g, respectively at pH 6. Foam capacity amounted 100% in PH of DH 19.52% at pH 2, and water‐holding capacity was 4.38 g/g. The antioxidant activity showed the strongest hydroxyl radical scavenging activity (95.25%), ABTS (84.88%), DPPH (75.89%), iron chelating (63.25%), and cupper chelating (66.90%) at DH 11.96%, whereas reducing power (0.88) at DH 19.52%. Thus, the findings indicated that utilization of grass turtle muscle protein hydrolysate is a potential alternative protein resource to improve the nutritional and functional properties in food ingredients and product formulations.


| INTRODUC TI ON
Grass turtle (Chinemys reevesii) is an aquatic animal species of the Geoemydidae family, which is found in many countries, particularly in Hong Kong, China, Taiwan, Japan, and Korea (Dai et al., 2012). At present, research studies are focused on the practical utilization of various aquatic animal species products and their by-products (Zou et al., 2017). Tortoises and turtles have long been used for foods and medicines in the East and Southeast Asia, while China is the largest consumer country in the world. The soft-shelled turtles (Pelodiscus sinensis) is a commercially important and delicious aquatic species due to their higher nutritional value and medicinal benefits, where they can be used for anticancer, antioxidation, and reduces blood pressure. The global production of soft-shelled turtles is estimated to be 355,000 tons in 2014 . In addition, turtles have always been used as a tonic source in Chinese traditional medicine to make a person stronger, nourished, calmed, and also contains necessary elements as narrated in Materia Medica (Rawendra et al., 2014).
Enzymatic hydrolysis of food proteins is an effective way to reveal potent bioactive peptides (Rawendra et al., 2014). The functional properties and bioactivities of protein hydrolysates can be depend on molecular weight, hydrophobicity, and polar groups of the proteins which in turn are strongly affected by enzymatic hydrolysis conditions (Vilailak et al., 2007). Functional properties such as solubility, emulsification, foaming, and other properties of Protein hydrolysates are important in improving functional quality and bioavailability food products (Hall et al., 2018). Protein hydrolysates from natural resources produced through enzymatic hydrolysis possess various bioactivities. Among the bioactivities, antioxidant activity of protein hydrolysates is one of the focuses of current research. Antioxidants from a natural food products can demerit of potential health hazards of artificial antioxidants as food additives.
Additionally, scientific information indicates that the consumption of natural antioxidants leads to reduce the risk of chronic diseases such as heart disease and cancer. PH with antioxidant activity in foods that plays an important role as a health protecting agent. Thus, applications of PH with antioxidant properties are more common in the food industry to improve the functional food (He et al., 2015).
As far as the author's knowledge is concerned, there are no information on the functional and antioxidant properties of protein hydrolysate from grass turtle muscles by using enzymatic hydrolysis in the utilization of food processing as a nutritional and functional value added products. Therefore, the aim of this study was to investigate the effects of enzymatic hydrolysis conditions on DH, functional and antioxidant properties of PH obtained from grass turtle muscles for the potential applications in food and pharmaceutical industries.

| Samples
Grass turtle (Chinemys reevesii) is a kind of usual aquatic food in China. And the grass turtle we use comes from the breeding products of Guangxi zhongtaikang Technology Industry Co., Ltd., Nanning-530029, Guangxi, P.R. China. Each grass turtle was rinsed thoroughly with tap water, and weighed, where their weights ranged between 1,135 and 1874 g and (length 21-25 × wide 12-17 cm) and then immediately slaughtered by using a knife. The muscles were separated from the other parts and by-products were removed.
Samples were put as fresh in ice box, then transported to laboratory. After consultation with relevant Chinese authorities, it is not an experimental animal, and it is unnecessary to issue animal ethics certificate. Finally, muscle(s) were homogenized, packed in vacuum plastic bags and stored at −20°C until further experiments. Before enzymatic hydrolysis process, the sample was transferred to the refrigerator at 4°C for 12 hr.

| Enzyme and Reagents
Papain enzyme (Enzyme activity 800u/mg, pH 5.5 ~ 6.5, 37 ~ 60 ℃ was purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). The enzyme was directly stored at 4°C. All other chemicals and reagents used in the experiments were of high purity and analytical grade.

| Preparation of protein hydrolysates
Papain enzyme was used for enzymatic hydrolysis of grass turtle muscles. Single-factor experiments (E:S, S:L, pH, temperature, and incubation time) were tested to obtain the optimal enzymatic hydrolysis conditions as shown in Table 1. Protein hydrolysates (PH) were prepared according to the method of Noman et al. (2018) with minor modifications. The 25 × 10 − 3 M sodium phosphate buffer was used to keep the pH constant during the incubation time. The layout of the hydrolysis process is presented in Figure 1

| Degree of hydrolysis determination
The degree of hydrolysis (DH) was investigated according to the modified method as described by Noman et al. (2018) with slight modifications. The volume of NaOH was used to calculate the amount of free amino groups. The total nitrogen was analyzed according to standard Kjeldahl method (AOAC, 1995). The percentage of free amino groups and the DH were calculated as follows.
where, V (mL) is the volume of NaOH (0.1N) added, C is the concentration of the solution used for titration (0.1M NaOH), W (g) is the weight of sample.

| Yield determination
The yield of GTPH was determined according to a modified guideline (Dhanabalan et al., 2017), and calculated using the following  Xu et al. (2017) with some modifications. Hundred (100 mg) of PH was taken into 15 ml glass tubes and diluted by 10 ml deionized water, the glass tubes were placed in an ultrasonic bath for 5 min and transferred into centrifuge tubes, centrifuged at 10,000 g for 10 min (SCLOGEX-D3024R, Beijing, China) at 4°C. Then supernatants were filtered and used for MW profile analysis by gel permeation chromatography using a HPLC system (Waters-1525, USA). The TSK-GEL 2000 SWXL (300 x 7.8 mm) column (Tosoh, Japan, Tokyo) was equilibrated with mobile phase composed of acetonitrile/water/trifluoroacetic acid 45/55/0.1(V/V).

| Scanning electron microscopy (SEM)
The images of microstructure of muscles (dry powder) before enzymatic hydrolysis and PH powder were carried out by using a scanning electron microscope (Quanta 200,Fei Company,Netherlands) at an accelerating voltage of 5.0 kV and objective aperture 500 μm.
All samples were coated by using a gold coater (Emitech K550X, Quorum Technologies Inc., UK), and the samples were examined at 160 × magnification.

| Color measurements
The color of the hydrolysate powders was measured using the Hunter Lab colorimeter (D65, UltraScan PRO, Shanghai, China). L*, a*, and b* parameters indicate white 100/ black 0, red positive/ green negative, yellow positive/blue negative, respectively of freeze-dried PH.
The color was evaluated as described by Thiansilakul et al. (2007).

| Water activity
Water activity (a w ) measurements were performed using a Lab Master-a w (Novasina, Switzerland) with an accuracy of 0.001 at 25°C. After the calibration, the PH powders were placed in a sample chamber and kept until equilibrium was reached. Each sample was carried out at list three times.

Protein solubility
The protein solubility of the GTPH was evaluated according to procedure of Jain and Anal (2016) with minor modification. Two hundred milligram GTPH samples were dissolved by 20 ml deionized water, the solution was adjusted to pH 2 to 10 by using 0.1M HCl or 0.1M NaOH. Then, the solutions were incubated at 30°C with stirring (Blue pard Yiheng Technical Co., Ltd, Shanghai, China) at 150 rpm for 30 min, and centrifuged at 10,000 g for 15 min (SCLOGEX-D3024R, Beijing, China). The protein content of supernatant was determined by using the Kjeldahl method (AOAC, 1995). Finally, the percentage of solubility was calculated by following the equation.

Water-and oil-holding capacity
Water-holding capacity (WHC) and oil-holding capacity (OHC) were estimated by using a procedure of Noman et al. (2018) with minor modification. Each PH (0.5 g) was dissolved into 10 ml dd-water or 10 ml soybean oil in a centrifuge tube and dispersed by vortex mixer (XW-80A, Zhejiang, China) for 60 s. The water and oil dispersion were allowed to stand for 7 hr and 20 min, respectively, at 25°C and centrifuged (SCLOGEX-D3024R, Beijing, China) at 5000g for 25 min at 4°C. To get WHC the supernatant was filtered by using a filter paper (Whatman No. 1) and calculated by different weight, while the free oil was taken to obtain OHC from the weight difference. The results were mentioned as g/g PH.

Foaming capacity and foam stability
The foaming capacity (FC) and foam stability ( where, V a is the before whipping Volume (mL) and V b is the after whipping Volume (mL). where, Abs blank = absorbance of control sample and Abs sample = absorbance of sample.

Reducing power capacity
Reducing power of protein hydrolysates was investigated as the procedure (Wu et al., 2003) with slight modification. Two hundred mi- The mixture was allowed to react for 10 min and absorbance was analyzed at 700 nm.
Hydroxyl radical scavenging activity Hydroxyl radical scavenging activity was evaluated according to the guideline of J. Wang et al. (2013) with some modifications. The mixtures were kept in water bath for 90 min at 25°C and the absorbance was analyzed at 522 nm by a UV-1800PC Spectrophotometer (Shanghai Mapada Instruments Co., Ltd., China). The hydroxyl radical scavenging percentage was calculated by the following formula.
where, Ab s , Ab h , and Ab c means the sample absorbance, H 2 O 2 was substituted by distilled water, and sample was replaced by distilled water, respectively.
Sample was prepared at the concentration of 1, 5, 10, 15, and 20 mg/ ml. The absorbance was measured at 562 nm.

Copper (II) ion chelating activity
The Cu 2+ chelating capability was assessed as described by Xu et al. (2014)

| Effect of enzyme-to-substrate ratio
The effect of E:S ratio on the DH was evaluated within the six levels, and the results are revealed in Figure 2a. As it is observed, at 1% enzyme concentration, DH was 4.60% but when the enzyme concentration increased to 2%, the DH was 5.01%, and increased to 6.83% as the concentration of enzyme has increased to 5%. However, DH was decreased to 5.89% as the enzyme concentration is further increased to 6%, this probably due to enzymatic steric effect that prohibits in contact with the protein with catalytic sites enzymes which is promoting the enzymatic hydrolysis process, and the reduction of substrate diffusion, and saturation reaction rate (Noman et al., 2018). Thus, the optimum DH (6.83%) is obtained at 5% of enzyme concentration and the protein hydrolysate at this DH was chosen further experiment.

| Effect of temperature
The DH can be greatly affected by the hydrolysis temperature. The hydrolysis temperature was set at 35, 40, 45, 50, 55, 60, 65, and 70°C to evaluate its effect on the DH, and the results are presented in Figure 2b. When the reaction temperature increased from 35 to 60°C, the DH was significantly increased from 4.99% to 7.49%.
However, by further increasing the reaction temperature to 65 and 70°C, a gradually decline in the DH was observed (Figure 2b). Such a reduction in the DH may be because of thermal denaturation of enzyme, hence leading to a decrease of DH (Noman et al., 2018).
According to this result, temperature of 60°C was selected to further research, where this temperature was close to that was reported X. Wang and Zhang (2012) in their study on Chlorella pyrenoidosa by using papain enzyme.

| Effect of solid-to-liquid ratio
To obtain optimal solid-to-liquid ratio five levels (1:0.5 to 1:4, w/v) were used beside optimal enzyme-to-substrate ratio and temperature that was obtained above in this research, which are 5% and 60°C, respectively. The influence of solid-to-liquid mixing ratio on the DH has demonstrated in Figure 2c. The DH was 11.47% at ratio of 1:0.5, while DH significantly increased to 11.96% when the ratio of 1:1 was used. Further ratio increased to 1:2, 1:3, and 1:4 led to significantly decreased of DH. Therefore, Solid: Liquid ratio 1:1 was selected as optimal ratio to use in the next step. These finding is consistent with those found by Noman et al. (2018) who used the same mixing ratio for the optimization of enzymatic hydrolysis of Chinese sturgeon fish by using papain enzyme.

| Effect of pH
The effect of pH on the DH was investigated in the pH levels of 5, 5.5, 6, 6.5, and 7 as shown in Figure 2d. The DH was increased from 9.28% to 11.06% when the pH increased from 5 to 6. Nevertheless, by further increasing pH to 6.5 and 7, the DH decreased to 10.69% and 10.19%, respectively, such a decline in the DH probably attributed to the denaturation of protein structure of the enzyme (Noman et al., 2018). As a result, pH 6 was selected as optimal pH for further investigation. Similar results were obtained by Wang and Zhang (2012) who revealed that the optimum hydrolysis pH was 6 by using papain enzyme in Chlorella pyrenoidosa.

| Effect of time
The effect of reaction time on the DH was measured within the range of 1-8 hr and these results are shown in Figure 2e. It observed that, when the reaction time increased from 1 to 6 hr, the DH was significantly raised from 11.87% to 19.52%. Nonetheless, upon extending the incubation time 7 and 8 hr, showed no significant increase of DH obtained at 6 hr. According to these results, 6 hr was chosen as the optimal incubation time. Therefore, the optimization profile indicates that an optimum DH (19.52%) can be achieved with including an enzymeto-substrate ratio of 5%, temperature 60°C, substrate to liquid ratio 1:1, pH 6, and incubation time 6 hr. DH under the optimal condition was highly compatible with those reported (Chalamaiah et al., 2010;Wang & Zhang, 2012) which were 17.1% and 14.33% from meriga egg and Chlorella pyrenoidosa, respectively, under optimal conditions.

| Yield
The GTPH yields obtained in this study are demonstrated in

| Chemical composition
The Chemical profiles of raw material and GTPH obtained by using papain enzyme hydrolysis are displayed in

| Molecular weight (MW) profile
The MW distribution of nonhydrolyzed sample and PH obtained from grass turtle muscles under the various DH are demonstrated in Figure 3. The results showed that, the PH obtained at the various DH have small molecular mass peptides compared with untreated sample where higher MW was > 95%, probably due to the effect of enzymatic hydrolysis which led to breakdown of peptides bounds.

TA B L E 2
Yield, Chemical composition, color, a w , and functional properties of raw muscle and GTPH at various DH (n = 3, mean ± SD) between 180 and 500 Da, which were 43.53 to 45.55% from total MW. These result is closely associated with the finding of Wasswa et al. (2007) who reported that proportion of the low MW peptides increased when the DH increased. The dietary proteins rich in low MW peptides could be more available in the food system and may highly contributing to nutritional value (Noman et al., 2018).
Therefore, PH could be used in food products to improve the nutritional value.

| Amino acid composition
Amino acid composition affects the nutritional value of PH particularly essential amino acids beside their effects on the functional properties (Santos et al., 2011). Nineteen different amino acids (total and free) were investigated, which are displayed in Table 3. Inthe fresh muscles, the total amino acids were 21.45 g/100g fresh sample and the major amino acids content such as glutamic acid, aspartic acid, and lysine were 4.03, 2.26, and 1.94 g/100g in fresh sample, respectively. These results were excellently higher than reported by Liang et al. (2018)  Searobin by using enzymes, respectively. The major amino acids in GTPH were glutamic, aspartic, and lysine which ranged 17. 49-17.66, 9.80-10.92, and 8.42-9.25 g/100g protein, respectively. Aspartic acid and glutamic acid are the most important amino acids that contribute to palatability. In addition, alanine, glycine, serine, and threonine deliberate taste sweet (Sriket et al., 2007).
On the other hand, free amino acids increased from 0.46 g/100 g fresh sample to 5.41, 6.87, and 10.95 g/100 g protein in PHs obtained at DH of 11.96%, 13.53%, and 19.52%, respectively. These differences between free amino acids content in the fresh sample and PH possibly due to effect of enzymatic hydrolysis process. Wu et al. (2003) reported that most of the free amino acids increased after enzymatic hydrolysis.

| Scanning electron microscopy
SEM images of fresh sample (dry) and PH for a DH of 11.96%, 13.53%, and 19.52% are presented in Figure 4. From SEMimages, it has TA B L E 3 Amino acid composition of raw muscle (s) and GTPH obtained by using papain enzyme (n = 3, mean ± SD) observed that the protein has degraded into small fragments after enzyme hydrolysis, which led to reduction in particle size of the GTPH (Figure 4b,c, and d) compared with untreated sample of grass turtle muscles (Figure 4a) under the same SEMparameters (Mag = 160×; AV = 5.0 kV). These findings are closely associated of (Agrawal et al., 2019;Elavarasan & Shamasundar, 2016) who mentioned that protein has degraded into small fragments and particle size reduction after treatment by enzyme. Bao et al. (2017) reported that reduced the particle size may attributed to the high solubility.

| Color assessment
The color of freeze-dried PH evaluated by colorimeter is shown in Table 2. As a result, L* (lightness) value was significantly higher (85.82) at DH 11.96% followed by PH obtained 81.59 and 79.34 obtained under the DH 13.53% and DH 19.52%, respectively. Analyzing the values of a* and b*, the PH tended to redness and yellowness. These values were increased when DH increased in the range of a*=1.15-1.76 and b*=16.74-19.97, correspondingly. In our study L*, and a* value was higher than reported by Thiansilakul et al. (2007) who found brownish yellow color (L* = 58.00, a* = 8.38, b* = 28.32) of round scad protein hydrolysate powder. Color differences may be attributed due to the effect of enzymatic hydrolysis on the sample. Several studies have shown that the varying color is mainly depended on the presence of pigments in the muscle, in addition to hydrolysis conditions and nature of raw materials (Rodrigues Freitas et al., 2016;Thiansilakul et al., 2007). Overall, L* value was significantly (p < .05) decreased when the DH was increased, while, a* and b* were gradually increased.

| Water activity
Water activity of a food determines its stability, which is based on availability of water and molecular mobility, and characterizes mainly the physico-chemical and biological degradation in foods (Roudaut et al., 2004). The experimental results of a w are presented in Table 2. The results observed that the best value was 0.19 in PH at DH 19.52% followed by 0.21 at DH 11.96%, while the highest value was 0.24 at DH 13.53%. In these GTPH obtained lower water activity, which could be contributing to excellent stability and increase storage life. Where the water activity is in range of 0.54-0.64 leads to increasing the hardness of food products, and maillard reaction occurs when the a w is in the range of 0.74 to 0.84, as well as encourages the growth of some microorganisms (Rao et al., 2016).

| Protein Solubility
Solubility is an importance functional property of protein hydrolysates, which is required in food industries to controls the utilization of the product in many applications such as gels, emulsions, and foams (Thiansilakul et al., 2007). The protein solubility of GTPH with different DH in the pH ranges of 2-10 are presented in Figure 5a. All the protein hydrolysates showed higher solubility (70.87%-95.56%) depending on the pH used. The highest solubility rate was observed at pH 6 (>95%) in PH at DH 19.52%, while the lowest value was obtained at pH 4 (70.87%) in PH obtained at DH 11.96%. These results may be attributed due to DH and small molecular weights of peptides < 1,000 Da, this findings supported by Naqash and Nazeer (2013) who reported that degradation of proteins to smaller peptides led higher solubility. In this study, the solubility of GTPH was quite low at pH 4, which was in agreement of Vilailak et al. (2007).
Similar results were reported by Foh et al. (2010) who mentioned the isoelectric points (pI) of protein are between pH 4.5 and 5.5, and also near to this range, at which the net charge of the original proteins are minimized, and thus more protein and protein interaction, and less protein-water interaction occur. As a result, the protein solubility is decreased, whenever, the pH moves away from this point, increases protein and water interactions. These results suggest that GTPHs showed excellent protein solubility, which may providing to attractive appearance and smooth mouth feel of the food products (Thiansilakul et al., 2007).

| Emulsifying properties
EAI and ESI of hydrolysates from GTPH with various DH (11.96%, 13.53%, and 19.52%) are revealed in Figure 5b and c. The result shows that EAI and ESI of both are significantly affected by pH level, where EAI was the highest capacity 108.76 m 2 /g in PH of DH 19.52% at pH 6 followed by 83.31 m 2 /g in PH of DH 13.53%. The lowest EAI found 44.53m 2 /g at pH 10 in PH of DH 11.96%. These occurred may be due to protein solubility, DH, and small molecular weight peptides. Santos et al. (2011) reported that emulsifying properties are mostly influenced by protein solubility, DH, and molecular size from Bluewing Searobin by using microbial enzymes.
On the other hand, ESI values were significantly different (p < .05) depending on the DH and pH levels used except pH 8 as shown in Figure 5c. It's observed that the maximum emulsions stability was 89.73% in PH obtained under DH 13.53% at pH 10, while the minimum was 62.23% at pH 4 of DH 19.52%. Generally, ESI was high in pH 10 and lowest at pH 4, which probably be due to the isoelectric point at pH 4 (Naqash & Nazeer, 2013). These results may have the relationship with hydrophobic amino acids such as isoleucine, leucine, valine, alanine, and glycine (Table 3). Vilailak et al. (2007) found that more hydrophobic peptides contribute to the stability of the emulsion.

| Water-and oil-holding capacity
The WHCand OHCare affected by molecular weights. As shown in was achieved the highest OHC(3.66 g oil/g PH), and protein hydrolysate of DH 11.96% and DH 13.53% were found to be 2.98 and 2.33 goil/g PH, respectively. In this study, WHCand OHCvalues were higher than that reported by Noman et al. (2018) but lower than by S. He et al. (2016) who evaluated the OHC of rock lobster hydrolysate. Santos et al. (2011) found that OHCwas ranged from 3.86 to 5.12 and from 2.95 to 3.22 ml oil/g protein of Bluewing Searobin hydrolysates obtained at DH 15% and 10% by using Alcalase and Flavourzyme enzyme, respectively. In the same context, OHCis an important functional property, which influences the taste of products and necessary to application in meat products.

| Foaming capacity and stability
The foaming properties of GTPH with different DH are displayed in Figure 5d and Figure 5e (a, b and c). Figure 5d shows the high- Our all results were higher than reported by Damgaard et al. (2014). Park et al. (2016) reported that amino acids e.g. threonine, isoleucine, and valine, beside to hydrophobic amino acids strongly contribute to positive effects on DPPH scavenging activities.
The IC 50 activity of PH is a concentration used for inhibition of DPPH to 50%. In Figure 6b, the IC 50 value shown that the PH obtained at DH 11.96% was highly active (12.50 mg/ml) followed by PH of DH 13.53% (15.97mg/ml), while PH at DH 19.52% was lowest active (17.58 mg/ml), these finding may be attributed to high amount of hydrophobic amino acids including alanine, valine, leucine, isoleucine, cysteine and phenylalanine in PH of DH 11.96%.

| Reducing power
The reducing power was indicated to increase the absorbance are presented in Figure 6c. Reducing power capacity of GTPH at DH 19.52% was slightly higher (0.88) at concentration of 15 mg/ml than DH 11.96% (0.84) and DH 13.53% (0.81) PHs, however, showing nonsignificant difference (p < .05). These results possibly due to acidic and free amino acids. Park et al. (2016) reported that acidic amino acids such as aspartic acid and glutamic acid have strong positive effects on reducing power. In addition, Binsi et al. (2016) mentioned to protein hydrolysate peptides of aquatics fish having a molecular weight of < 5000Da were mainly responsible to antioxidant activity.
In this study reducing power capabilities were higher than the previous results found by Binsi et al. (2016) of engraved catfish.

| Hydroxyl radical scavenging activity
The hydroxyl radical scavenging activities of GTPHs are presented in Figure 6d. The activity of hydroxyl radical scavenging was the highest (95.25%) in PH with DH 11.96% followed by 81.41% in PH of DH 13.53%, while lowest value (75.72%) in PH of DH 19.52% at the same concentration of 15 mg/ml. In the current study, low DH achieved the highest hydroxyl radical scavenging activity. These is results closely related with the result of He et al. (2015) from Anchovy protein hydrolysate. Our results was higher than by Gao et al. (2019).The lower IC 50 value means the higher free radical scavenging ability. In Figure 6d, IC 50 value obtained at PH of DH 11.96% was the best active (7.89 mg/ml) than the PH of DH 13.53% and DH 19.52% which amounted 9.97% and 9.98%, respectively, with no significant differences (p < .05) between PH of DH 13.53% and DH 19.52%.

| Metal chelating properties
Fe 2+ and Cu 2+ Chelating activity The metal chelating activity of GTPH was evaluated and expressed as a percentage at different concentration (mg/mL). Iron (II) and copper (II) ion chelating capacities of PHs are presented in Figure 6e and f. The Fe 2+ chelating activities observed that PH of DH 11.96% was a highly strong metal (Fe 2+ ) chelating activity (63.25%) at a concentration of 20 mg/ml, while PH of DH 13.53% and DH 19.52% were 56.17 and 52%, respectively. These activities may be related with acidic and basic amino acids. On the other hand, Cu 2+ chelating activity is displayed in Figure 6f. PH of DH 11.96% was significantly high 66.90% at the concentration of 6 mg/ml followed by PH of DH 19.52% (65.01%), without significant difference with PH of DH 13.53% which was 64.78%. These results may be due to essential amino acids specially histidine. Torres-Fuentes et al. (2011) reported that high amount of histidine content, which provided the highly copper chelating activity due to its imidazole ring. can be retard the oxidation reaction of volatile compounds in storage food duration (Saiga et al., 2003).