ANTIOXIDANT ACTIVITY OF PROTEIN HYDROLYSATE PRODUCED FROM TUNA EYE ( Thunnus sp . ) BY ENZYMATIC HYDROLYSIS

Tuna (Thunnus sp.) by-products from frozen loin and canning industry especially the eye is rich in proteins and in lipids consisting of polyunsaturated fatty acids (PUFA). That requires protective agent (antioxidant) to inhibit the oxidation naturally present and predicted to be protein peptides. Enzymatic hydrolysis of protein is an appropriate method to produce bioactive peptide with such nutraceutical/ pharmaceutical function such as an antioxidant peptide. This study aimed to produce protein hydrolysate having a function as anwith an antioxidant activity from eye of tuna through enzymatic hydrolysis and determining the antioxidant activity by DPPH methods. Protein soluble content of tuna’s eye protein hydrolysate (TEPH) ranged from 59.98±0.130 to 94.90±0.002%. The degree of hydrolysis (DH) of TEPH was about 9.10±0.28 to 16.14±0.09%. The highest inhibition of DPPH radical scavenging activity was 93.57±0.05% (at 5 mg/mL) was obtained with a DH of 11.35±0.002% at the concentration 0.1% of papain for 6 hours hydrolysis. The IC50 value of was 1.08±0.008 mg/mL


INTRODUCTION
Fisheries industry is one of the reliable economic sources in some countries.Indonesia's fisheries industry such as tuna industry generate large amount of waste or by-product (head, skin, fin, frame, tail, and viscera).The abundant by-product of tuna, based on the total catch in 2016 reached 132.691 ton (Ruchimat et al. 2017).These byproduct rich in nutrition value has not been optimally utilized yet, discarded without any treatment therefore potentially causing pollution to the environment.Tuna eye, a part of tuna head has high economic value, due to the high lipid and protein content.Vijaykumar (2016) obtained PUFA of tuna eye (EPA and DHA) about 7.07% and 36.72%respectively and protein content according to Sandria (2014) was 19.49%.Long chain omega 3 (EPA and DHA) in lipids are protected by antioxidants from oxidation naturally prior to external treatment.One of the antioxidants that work as a protective PUFA predicted to be peptide.A number of studies on protein hydrolysates showed that peptides derived from fish protein are potential sources of antioxidants.
Some antioxidant peptides had already been identified from several by-product sources such as head (Bougatef et al. 2012), liver (Young-je et al. 2009), backbone (Slizyte et al. 2016), dark meat (Saidi et al. 2014), frame (Kim et al. 2007), skin and viscera (Zhong et al. 2011).Yang et al. (2011) reported, on the antioxidant activity of tuna bigeye protein hydrolysate at 1.34 mg/mL.Food protein have been known for their nutritional and functional properties related to amino acids content that coincides with the physiological utilization of specific amino acids on digestion and absorption.In addition, functional properties of protein associated with the contribution to the physicochemical and sensory properties of the food (Sila and Bougatef 2016).
It plays single role in escalating human health beyond its nutritional value.Several studies in the past two decades has been reported concerning about protein hydrolysate from various food sources, in addition to its nutritional properties, exhibit different biological functions including antioxidant, (Bougatef et al. 2009), antimicrobial (Salampessy et al. 2010;Sila et al. 2014) anticoagulant (Ren et al. 2014), and antihypertensive (Je et al. 2009;Furuta et al. 2016).Derivative bioactive protein, consist of 2 to 20 of amino acids which inactive in sequence of protein core and it can be released by enzymatic hydrolysis during gastrointestinal digestion in the body or food processing.When liberated in the body, derivative bioactive protein can act as a regulatory compound with the activity as hormone.Furthermore, the specific enzyme used for the hydrolysis is important factor for the production of bioactive compound.Enzyme influences the molecular weight, amino acid content and biological activity of the protein hydrolysate ( Van et al. 2002).The objectives of this study were to produce TEPH using different concentration of papain and hydrolysis duration times, and to characterize its antioxidant properties.

Research Methods Separation of tuna eye protein
Tuna eye was cut into small part and blended by using blender, then centrifuged at 10,000 g, 4⁰C for 30 minute to separate the lipid, liquid and precipitant (solid).The precipitant was collected and used in further research stages.

Production of tuna eye protein hydrolysate (TEPH)
Protein hydrolysate from tuna eye was prepared through enzymatic hydrolysis with papain according to the method Imm and Lee (1999) with slight modification.The concentration of papain used were 0.1% (b/v), 0.15% (b/v), 0.2% (b/v), and 0.25% (b/v) with different hydrolysis duration times for 4; 5; 6 and 7 hours.The hydrolysis was carried out in a glass bottle placed in a water bath with stirred in 100-130 g.Distilled water was added in sample by ratio of TEP and distilled water 1:8 (b/v).The hydrolysis started by adding enzyme with the temperature 55⁰C combined with different of hydrolysis times, then heated to 85⁰C for 15 minutes and centrifuged at 6.000 g for 20 minutes, in cold temperature of 4⁰C.Obtained supernatant was then dried using freeze dryer at -55⁰C in order to obtain the hydrolysate powder of tuna eye protein and stored in the freezer until characterization an analysis process.

Measurement of degree of hydrolysis (DH)
Degree of hydrolysis was calculated based on the percentage ratio of trichloroacetic acid (TCA) method (Baharuddin et al. 2016).The supernatant of protein hydrolysate was added with TCA 10% and incubated for a certain time and followed by precipitation, then centrifuged at 7.500 g for 15 minutes.Obtained supernatant was analysed for the nitrogen content by kjeldahl method (AOAC 2005).Degree of hydrolysis was calculated by the following formula:

Determination of soluble protein
The soluble protein content of TEPH Tuna's eye protein hydrolysate was performed Soluble nitrogen TCA 10% Total nitrogen according to Bintang (2010).Two mL of hydrolysate sample was added 5 mL of Cualkali (NaOH 4%, NaCO 3 20%, NaK-tartrate, and 5% CuSO 4 ) and incubated at room temperature for 1 hour.Then added 0.6 mL of folin-ciocalteu, the mixture was kept for 15 minutes at ambient temperature and measured absorbance at a wavelength of 660 nm.

Antioxidant activity 2.2-Diphenil-1picrylhydrazyl (DPPH)
The DPPH Scavenging activity from TEPH was measured according to Shimada et al. (1992) method with slight modifications, 1 mL of sample was mixed with 1 mL of DPPH 0.15 mM solution, vortexed and incubated in a dark room for 30 minutes.The absorbance was measured at wavelength 517 nm using UV-VIS spectrophotometer.Scavenging DPPH activity was calculated by the following equation: Where A is blank absorbance and B is sample absorbance.

Data Analysis
Experimental design was performed referring to Steel and Torrie (1991) method using complete randomized design with factorial pattern: papain concentrations with 4 levels (0.1%; 0.15%; 0.2% and 0.25%) and different hydrolysis times (4; 5; 6 and 7 hours) by twice repetition.Data was processed using Statistical Process for Social Science software (SPSS) version 24.Significant differences (p<0.05) between the means value were evaluated by Least Significant Different (LSD) test.

RESULTS AND DISCUSSION Composition of Tuna Eye
Proximate analysis of tuna eye precipitant was conducted to determine the chemical composition such as moisture, protein, lipid ash of TEP.The results of chemical composition of TEP were shown in Table 1.
Protein content of TEP was 15.06%, indicating that TEP is a good resource of protein for producing protein hydrolysate.Stansby (1982) explained about fish with protein content of 15-20% included into a group of high-protein fish.High protein of fish is one of the requirements in producing protein hydrolysate.

Degree of Hydrolysis
The degree of hydrolysis (DH) is defined as the percent ratio of the number of broken peptide bonds to the total number of peptide bonds on the substrate (hydrolysate) (Bougatef et al. 2012).Sila and Bougatef (2016) stated that DH determination is an important parameter for determining the functional and biological properties of protein hydrolysate.DH value of TEPH can be seen in Figure 1.
Value of DH was changed during hydrolysis.ANOVA data showed that hydrolysis period and enzyme concentration gave significant influence on DH value (p<0.05).DH of TEPH ranged between 9.1%-16.14%.The higher DH value of hydrolysate, the more low-molecular peptides produced.Several factors that influence DH are enzyme concentration, time of hydrolysis and proteases used.Similar DH was obtained by Saidi et al.(2013) about 10.22% hydrolysate from by-products of tuna dark meat and Wasswa et al. (2007) revealed 16.11% in grass carp skin.Differences DH obtained from TEPH due to the various in enzyme concentration used and times of hydrolysis (Jamil et al. 2016).Beveridge (1996) explained that during the hydrolysis process, papain will cleavage the substrate into product by histidine and cysteine groups in active site of catalytic enzyme.Structural changes that occur among them, a highly reactive cysteine (Cys-25) group bonding to the substrate to produce substrate covalent bonds with tetrahedral enzyme.The histidine group (His-159) is protonated so that binds to the  nitrogen present in the substrate.As a result, the amine group on the substrate diffuses and its position is replaced by water molecules which ultimately hydrolyse the intermediate product thus returning the enzyme to original form and function.

Soluble Protein Content
The protein content of TEPH was performed by calculating the absorbance of hydrolysate on standard absorbance using Bovine Serum Albumin serum (BSA).The soluble protein concentration of TEPH illustrated in Figure 2.
The highest soluble protein concentration 94.90±0.002%(p<0.05) was obtained from hydrolysis process for 6 hour with 0.2% papain.Protein is an important component in hydrolysate product.During the Hydrolysis process the insoluble proteins were converted by papain into soluble proteins that will be hydrolysed into smaller component such as peptides and amino acids.Therefore, the total of soluble protein will be increased in optimum condition (Nielsen 1997).

Antioxidant Activity 2.2-Diphenil-1-Picrylhydrazyl (DPPH)
Antioxidants are molecules that can interact with free radicals and form stable compounds that stop oxidation (You et al. 2009).DPPH is a stable radical showing the highest absorbance length at 517 nm.When DPPH discovers a hydrogen donor substance such as an antioxidant, the radical is captured.It is accomplished by the change of colour from purple to yellow and the absorbance will decrease.DPPH scavenger activity shows the ability of antioxidant compounds to donate hydrogen or electrons, thereby altering radical substances to be more stable (Binsan et al. 2008).Antioxidant activity will increase with increasing concentration used.Tuna eye protein inhibits against DPPH radical was 78.44±0.40% at concentration 10 mg/mL.Elias et al. (2008) stated that the ability of proteins and their derivatives to inhibit oxidation of lipids makes it an important component as the antioxidant defence of biological tissue from the food produced.Converting protein structure into short peptide with certain amino acid composition can improve its ability as an antioxidant.The result of antioxidant DPPH radical scavenger of hydrolysate protein from tuna eye presented in Figure 3.
Antioxidant activity changed during hydrolysis process.Hydrolysis period and enzyme concentration gave significant different (p<0.05) in ANOVA data results.Figure 3  Concentration papain 0.1% ( ), 0.15% papain ( ), 2% papain ( ), and 2.5% papain ( ).Soluble protein content (%) Time of hydrolysis (H) ranged from 78-93%.Compared to the raw material, TEPH had high of antioxidant activity.The hydrolysis process enhances the antioxidant activity.Jamil et al. (2016) stated that hydrolysis process had increased the amount hydrophobic amino acids (valine, isoleucine, phenylalanine, methionine) which contributed to the antioxidant activity of protein hydrolysate.The highest activity was obtained from the addition of 0.1% papain with 6 hours hydrolysis about 93.57% with the IC 50 value 1.08±0.008mg/mL.IC 50 value of TEPH is presented in Table 2.
Radical scavenging DPPH can also be expressed with IC 50 value.The lower of IC 50 obtained, the highest the activity of antioxidant.The lowest IC 50 value was obtained in protein hydrolysate with 0.1% enzyme concentration at 6 hours hydrolysis (1.082±0.008mg / mL) (p<0.05) with inhibition percentage of DPPH 93.5% at a concentration of 5 mg/mL.This showed that TEPH with 0.1% concentration for 6 hours probably containing hydrogen donor peptides by quench free radical into stable molecule.High hydrophobic amino acids content has been demonstrated in peptides exhibited high antioxidant activity which is considered in scavenge of free radical.
According to Zou, He, Tang and Xia (2016), hydrophobic amino acids had strong radical scavenging activity in oxidative reaction, especially with enzymatic catalysed reaction due to the present of imidazole ring as important electron donor.Chang et al. (2015)   reported on the hydrolysate protein of bluefin leatherjacket (Navodon septentrionalis) using various enzymes had the highest IC 50 value of 5.22 mg/mL.Hydrolysate of the head protein of bigeye tuna (Thunnus obesus) shows IC 50 value of 1.34 mg/mL (Yang et al. 2011).Zhong et al. (2011) reported on protein hydrolysate byproducts of grass carp has a radical scavenging inhibition percentage 68.4% at a concentration of 5 mg/mL.These showed that tuna eye protein hydrolysate (Thunnus sp.) had better IC 50 value than protein hydrolysate of bigeye tuna, bluefin leatherjacket, and grass carp IC 50 value.There were many studies that reported on the potential of bioactive peptides especially from fish protein as antioxidants.However, it cannot be explained in detail about the mechanism of protein as an antioxidant.Many of the antioxidant mechanisms of proteins depend on their amino acid composition (as free-radical scavenging, metal binder, hydro peroxide reduction, or adduction (giving) aldehyde groups).In addition, the antioxidant activity of amino acid residues is limited to the tertiary structure of the polypeptide, so that many amino acids with antioxidant capabilities are enclosed within the protein core that is inaccessible to the pro-oxidant.

Amino Acid Composition
The quality of a food protein can be evaluated by determining the amino acid composition.The amino acid of hydrolysate protein composition has various roles in its physiological activity, which has also been observed for antioxidant activity.The amino acid composition of HPMT is expressed in percent in Table 3.
Amino acid composition obtained from the protein hydrolysate that had the highest antioxidant activity produced in 0.1% concentration of papain for 6 hours hydrolysis.protein hydrolysate has various roles in its physiological activity, including its activity as an antioxidant.Some amino acids have ability to be antioxidants such as hydrophobic amino acids (alanine, leucine, isoleucine, methionine, proline, tyrosine and valine) and aromatic amino acids (tyrosine, phenylalanine and tryptophan) that have radical scavenging and metal chelating activities (Damodaran 1996).

CONCLUSION
The concentration of the enzyme papain and the duration of hydrolysis significant effect (p<0.05) on the antioxidant activity of tuna eye protein hydrolysate.The highest inhibition of DPPH radical scavenging activity (93.5%) obtained at 0.1% concentration of papain for 6 hours of hydrolysis at the concentration 5 mg/ mL with an IC 50 value of 1.082 ± 0.008 mg/mL.

Table 1
Chemical composition of TEP

Table 2
Antioxidant IC 50 value of TEPH by various condition

Table 3
Chang et al. (2015)cid content was glutamic acid about 19.70/100 ppm.Chang et al. (2015)reported similar results in the hydrolysate protein of skipjack dark meat that had the highest glutamic acid content of 127.6 / 1000.Protein that can provide amino acids that almost match human needs, is a high quality protein.Amino acid composition of food Amino acid composition from tuna eye protein hydrolysate