Antioxidant activity of Sind sardine hydrolysates with pistachio green hull (PGH) extracts

Author’s Accepted Manuscript Antioxidant activity of Sind sardine hydrolysates with pistachio green hull (PGH) extracts Roghayeh Amini Sarteshnizi, Mohammad Ali Sahari, Hassan Ahmadi Gavlighi, Joe M. Regenstein, Mehdi Nikoo www.elsevier.com/locate/sdj

PII: DOI: Reference:

S2212-4292(18)30690-4 https://doi.org/10.1016/j.fbio.2018.11.007 FBIO363

To appear in: Food Bioscience Received date: 16 July 2018 Revised date: 26 September 2018 Accepted date: 12 November 2018 Cite this article as: Roghayeh Amini Sarteshnizi, Mohammad Ali Sahari, Hassan Ahmadi Gavlighi, Joe M. Regenstein and Mehdi Nikoo, Antioxidant activity of Sind sardine hydrolysates with pistachio green hull (PGH) extracts, Food Bioscience, https://doi.org/10.1016/j.fbio.2018.11.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Antioxidant activity of Sind sardine hydrolysates with pistachio green hull (PGH) extracts Roghayeh Amini Sarteshnizi1, Mohammad Ali Sahari1, *, Hassan Ahmadi Gavlighi1, *, Joe M. Regenstein2, Mehdi Nikoo3

1

Department of Food Science and Technology, Faculty of Agriculture, Tarbiat Modares

University, Tehran, Iran 2

Department of Food Science, Cornell University, Ithaca, NY 14853-7201, USA

3

Department of Pathobiology and Quality Control, Artemia and Aquaculture Research Institute,

Urmia University, West Azerbaijan, Urmia, Iran E-mail: sahari@ modares.ac.ir E-mail: [email protected] *Corresponding authors: Tel. +9821 48292328; Fax: +9821 48292328; (Mohammad Ali Sahari), Tel. +9821 48292313; Fax: +9821 48292313; (Hassan Ahmadi Gavlighi)

Abstract High rates of oxidation during hydrolysis are one the main problems with hydrolysate production from fatty fishes such as Sind sardines. The purpose of this study was to control oxidation by different pretreatments with pistachio green hull (PGH) extracts as an antioxidant. Different enzyme levels (1.3, 2.5, and 5%), pretreatments of fish mince (washing, defatting, fish protein isolate (FPI)), and the addition of PGH as well as nitrogen gas treatment were studied on lipid oxidation during hydrolysis of Sind sardines (Sardinella sindensis). The antioxidant properties of the hydrolysates were also studied. Results for thiobarbituric acid reactive substances indicated that the nitrogen gas and 5% (w/w) enzymatic treatment significantly decreased lipid oxidation 1

along with having a higher degree of hydrolysis and higher antioxidant activity for the hydrolysate. FPI was more effective than washing in controlling oxidation while defatting using isopropanol was the most effective. PGH (260 µg/ml) effectively controlled the oxidation of mince and washed mince (P < 0.05) but it was not effective for FPI as seen by the higher oxidation of heme-pigments and weaker metal chelating activity of PGH. Interaction of hydrolysates from all treatments and PGH showed significant combined DPPH radical scavenging and metal chelating activity (P < 0.05). Therefore, defatting using isopropanol and addition of an antioxidant such as PGH can effectively be used for inhibition of lipid oxidation during hydrolysis and improvement of antioxidant activity of hydrolysates. Graphical abstract

Keywords: Sind sardine, pistachio green hulls, fish protein isolate, Sardinella sindensis, Pistachia vera

2

1. Introduction A considerable amount of by-catch and low commercial value fish species such as sardine (Sardina pilchardus), horse mackerel (Trachurus mediterraneus), and axillary seabream (Pagellus acarne) are caught annually (García-Moreno et al., 2014). By-catch, and underutilized fish and shellfish are generally used for the production of fishmeal (Bozzano and Sarda, 2002; García-Moreno et al., 2014; García‐Moreno et al., 2013). However, there is an increasing need for better use of underutilized marine resources (Harnedy and FitzGerald, 2012; Halim et al., 2016; Nikoo and Benjakul, 2015). These marine species contain high quality proteins for the production of bioactive hydrolysates as potential functional food ingredients (Ghaly et al., 2013; Nikoo et al., 2016; Ordóñez-Del Pazo et al., 2014). One of the main challenges during the production of fish protein hydrolysates (FPH) is the high oxidation rate due to the presence of pro-oxidants such as phospholipids in cell membranes, myoglobin and other heme proteins in fish muscle, and undesirable lipid substrates (Khantaphant et al., 2011). These constituents are involved in the undesirable organoleptic properties and oxidative instability of hydrolysates (Benjakul et al., 2014). Primary products of lipid oxidation break down to secondary products with a bad odor and rancid taste. Also, oxidized unsaturated lipids can produce insoluble lipid-protein complexes and cause quality deterioration and loss of nutritional value in the resulting hydrolysates (Chaijan, 2008; Ladikos and Lougovois, 1990). These processes may be more significant when FPH is produced from fatty fishes such as Sardinella species as they contain more mitochondria, myoglobin, fats, glycogen, cytochromes, and dark muscle fibers than white fleshed-fish species (Chaijan et al., 2005).

3

Previously, FPH were produced using different fish substrate such as fish mince (Nazeer et al., 2012; Zhang et al., 2017), defatted fish mince (Chi et al., 2015; Galla et al., 2012; Klompong et al., 2007; Liu et al., 2014; Luo et al., 2013; Quaglia and Orban, 1990), and fish protein isolate (FPI) (Choi et al., 2009; Thuy and Lam, 2015; Zhong et al., 2011). Khantaphant et al. (2011) evaluated different pretreatment effects on lipid oxidation and concluded that membrane separation followed by washing resulted in the lowest myoglobin, phospholipids, and iron content. The effects of Fucus vesiculosus (Fv) extracts on lipid oxidation during cod bone mince hydrolysis showed the protecting effect of natural antioxidants and gave better sensory properties to the FPH (Halldorsdottir et al., 2014). Pistachio green hulls (PGH), the major by-product of the pistachio industry, is a good source of phenolic compounds (Barreca et al., 2016). The antioxidant (Goli et al., 2005; Rajaei et al., 2010, Barreca et al., 2016), antidiabetic (Lalegani et al., 2018), antimutagenic and antimicrobial (Rajaei et al., 2010) properties of PGH extracts have been reported. Because of its strong antioxidant activity, it might be an interesting extract to control oxidative deterioration during production of protein hydrolysates from fatty fishes. There is a need to learn more about producing FPH from fatty fish and the appropriate use of antioxidants during the process. Sind sardine (Sardinella sindensis) is a small pelagic fish species found in the southern coastal waters of Iran. It is mainly used for the production of fishmeal. Production of bioactive peptides could be a better way to use this fish. Previously, production of antioxidant and antihypertensive hydrolysates from canned sardine by-products (Sardina pilchardus) were evaluated using brewer’s spent yeast and commercial proteases (Vieira and Ferreira, 2017; Vieira et al., 2017). It would be interesting if lipid oxidation during hydrolysis of fatty fish such as Sind sardine could be controlled using a local source of antioxidants, e.g., PGH. Therefore, the purpose of this study 4

was to evaluate the effects of different pretreatments of protein substrates and to test PGH extracts as natural antioxidant during hydrolysis and with final products. In addition, the effect of different levels of hydrolysis enzymes on lipid oxidation during production of FPH was measured. 2. Materials and methods 2.1. Materials Sardinella (Sardinella sindensis) with a length of 7-12 cm and weight of 15-30 g was kindly provided by local fishermen (Qeshm Island, Iran). The fish were kept in ice during transportation and stored in sealed plastic bags at -80 ºC until used, a maximum of 6 wk. Pistachio green hulls (Ohadi variety) were obtained from the Rafsanjan Agricultural Research Center of Iran. Protease from Bacillus licheniformis, Alcalase 2.4L (E.C. 3.4.21.62), was obtained from Novozyme® (Bagsvaerd, Denmark). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 3-(2-pyridyl)-5,6-diphenyl1,2,4-triazine-4',4"-disulfonic acid (ferrozine), Trolox (6-hydroxy-2,5,7,8-tetramethylchromane2-carboxylic acid), and 1,10-phenantroline were obtained from Sigma-Aldrich (Vallensbaek Strand, Denmark). 2.2. Preparation of pistachio green hull extract Pistachio green hulls extracts were prepared using the method of Lalegani et al. (2018). Briefly, hulls were oven dried at 45 °C for 12 h. Dried hulls were then ground in a coffee grinder (Tefal Simply Invents, Hong Kong, China), sieved (mesh size 40) and then 10 g of the PGH powder was extracted overnight using deionized water (Millipore, Molsheim, France) at room temperature (~25 to 30 oC). The mixture was then filtered through Whatman No 42 filter paper

5

(Whatman, Maidstone, UK). The concentrated extracts were prepared using a rotary evaporator at 40 °C and then freeze-dried (Labconco Corp., Kansas City, MO, USA). 2.3. Preparation of mince, washed mince, defatted mince, and fish protein isolate Whole fish mince was prepared by washing, removing heads, viscera, and tails, and minced using a Moulinex blender (DR5; Moulinex, Paris, France). The mince was stored at -80 °C until used, a maximum of two wk. Washed mince was prepared using the procedure of Yarnpakdee et al. (2012). Fish mince was homogenized with 5 volumes of cold distilled water (2-4 ºC) (w/v) using an Ultra-Turrax homogenizer (IKA®, T18 D, Staufen, Germany) at 11000 rpm for 2 min. After stirring for 15 min at 4 ºC, the homogenate was centrifuged at 9600 × g using 50 ml test tubes (Sigma D37520, Rotor 19776-H, Osterode am Harz, Germany) for 10 min at 4 ºC. This process was repeated twice. The washed mince was packed and stored at -80 ºC for a maximum of a wk. Defatted mince was prepared using the method of Klompong et al. (2007). Fish mince was mixed with isopropanol (1:4 (w/v), then homogenized at 11000 rpm for 2 min and allowed to stand at 30-35 ºC for 50 min. After draining the liquid, the residue was mixed with isopropanol again (1:4 (w/v)) and defatted at 75 ºC for 90 min. The liquid was removed and the precipitate was air dried at 30-35 ºC. The alkaline process described by Hultin and Kelleher (2000) was used for FPI production. Fish mince was homogenized in 9 volume of cold deionized water at 4 ºC for 1 min. Then, the pH of the homogenate was slowly increased to 11 using NaOH (2 N) for 25 min at 0-4 ºC. Then, the homogenate was centrifuged at 10000 × g for 10 min at 4 ºC and filtered through 4 layers of cheesecloth to remove the insoluble material. The pH of the homogenate was adjusted to 5.5 for myofibrillar proteins precipitation by adding HCl (2 N) and then centrifuged at 10000 × g for 20 min at 4 °C. 2.4. Determination of total pigment, heme iron, and phospholipid content 6

Total pigment and heme iron content were determined using the methods of Merck Index (1989) with slight modification. Freeze-dried samples (200 mg) were mixed with 9 ml acid acetone containing 90% acetone, 8% deionized water and 2% HCl (v/v/w). The mixtures were kept at room temperature for 1 h and then were filtered using Whatman No. 42. The absorbance was measured at 680 nm (Cary 60 UV-Vis spectrophotometer, Aligent Technologies, Santa Clara, CA, USA). The absorbance of samples were multiplied by a factor of 6800 and then divided by the initial sample weight to obtain total pigment content as µg/g dry sample (Hornsey, 1956). Heme iron content was calculated as follow: Heme iron (mg/100 g dry sample) = Total pigment (ppm) × 0.00882 The iron content in haematin is 0.0882 μg Fe/μg haematin (Clark et al., 1997). Phospholipid content was determined using the method of Yarnpakdee et al. (2012) with slight modification. Freeze-dried samples (80 mg) were mixed with 20 ml NaOH (4 N) and then heated in water bath (90-95 ºC) for 30 min. After cooling at room temperature for 1 h the samples were neutralized using HCl (4 N) and centrifuged for 10 min at 10000 × g. Then 2 ml of supernatants were mixed with 2 ml phosphate reagent (4.2% ammonium molybdate solution in 5 M HCl, 0.2% malachite green solution in distilled water, 1:3 v/v). The absorbance was measured at 620 nm after incubation at room temperature for 30 min. A phosphate standard curve (disodium hydrogen phosphate) was used to obtain phosphorus content and a factor of 25 was used to convert to phospholipid content (average molecular weight of phosphatidyl choline/atomic weight of phosphorus) (Sigfusson and Hultin, 2002).Phospholipid content was expressed as mg/100 g dry sample. 2.5. Preparation of hydrolysates

7

Different protein powders were mixed with water to obtain a final protein concentration (N×6.25 as measured using the Kjeldahl test (AOAC 2000)) of 3% (w/v). The pH and temperature of hydrolysis were held at 8 and 50 ºC, respectively (optimum condition for Alcalase) (Guerard et al., 2001). Different conditions of hydrolysis were used and are shown in Table 1. After 3 h of hydrolysis, the enzyme was deactivated at 90-95 ºC for 10 min. Finally, the mixtures were centrifuged at 1000 × g at 4 ºC for 10 min and the supernatants were freeze-dried. 2.6. Determination of degree of hydrolysis (DH) The degree of hydrolysis (DH), was measured using the pH-stat-method of Adler-Nissen (1986). Hydrolysis reactions were done using a stirring system coupled to a thermostatic water bath. The pH of 8 was maintained using NaOH (4 N) and DH calculated using the following equation: DH (%) = B × Nb × 1/α × 1/MP × 1/htot × 100 Where B refers to the amount of base (ml) used to maintain the constant pH during the hydrolysis. Nb is the normality of base (eq/l), α is the average degree of dissociation of the α-NH2 groups expressed as:

Where pK is dependent on pH and temperature. At 50 ºC and pH 8, pK = 7.1 was used for calculation of the degree of amino group dissociation (1/α = 1.13). The pK is the average pK value of α-amino groups liberated during hydrolysis and is calculated to adjust for temperature according to Steinhardt and Beychok (1964): pKa = 7.8 + ((298-T)/298 × T) × 2400

8

MP is the mass of protein of the substrate (g) and htot is the total number of peptide bonds in the protein substrate (meq/g) (estimated from the amino acid composition of the protein by summing up the mmol of individual amino acids/g protein) from an amino acid analysis. For fish proteins, htot is presumed to be 8.6 meq/g of protein (Adler-Nissen 1986). Given the assumptions made, the DH is considered to be an estimate of DH, but a comparison of the different DH of samples should be less influenced by these assumptions. 2.7. Determination of thiobarbituric acid reactive substances (TBARS) TBARS were evaluated during hydrolysis using the method of Lemon (1975) method with some modification. Briefly, 100 µl of homogenate at different times of hydrolysis (0, 30, 60, 90, 120, and 180 min) was Vortexed (FAVS, Bologna, Italy) for 10 min with 600 µl of extraction solution containing

7.5%

trichloroacetic

acid

(TCA),

0.1%

propyl

gallate,

and

0.1%

ethylenediaminetetraacetic acid (EDTA). Then, 400 µl of TCA solution was added to the homogenized samples and centrifuged at 9400 × g for 15 min at 4 ºC using 1.5 ml micro-tubes. After that, 500 µl of supernatant was mixed with an equal volume of thiobarbituric acid (0.02 M) and heated at 95 ºC for 40 min. The samples were cooled immediately and the absorbance was read against a water blank at 532 nm. Malondialdehyde bis (dimethyl acetal) from 0-4.54 µM was used for the standard curve. The TBARS values were expressed as µmol of MDA equivalents/kg of dry sample. 2.8. Determination of antioxidant activity 2.8.1. DPPH• scavenging activity DPPH• scavenging activity was estimated using the method of Shimada et al. (1992). Hydrolysates were dissolved in distilled water and then 500 µl of the samples were mixed with

9

equal volume of 0.1 mM DPPH solution in methanol. The mixtures were Vortexed and allowed to stand for 30 min at room temperature in the dark and then the absorbance was read at 517 nm against a distilled water blank. The DPPH radical scavenging activity was expressed as umoles of Trolox equivalents (μM TE)/g of dry sample. 2.8.2. Ferrous chelating activity The ferrous chelating activity of hydrolysates was estimated using the method of Decker and Welch (1990). Briefly, 1 ml of hydrolysate solution, 3.7 ml distilled water and 100 µl of 2 mM FeCl2 were mixed and allowed to stand for 3 min. Then 200 µl of 5 mM ferrozine was added and left to stand for 20 min in the dark at room temperature. The absorbance was measured at 562 nm. For the control, double distilled water was used instead. EDTA disodium salt dihydrate (EDTA-Na2-2H2O) was used for the standard curve. Ferrous ion chelating activity of the hydrolysates were expressed as umoles of EDTA equivalents/g of dry sample. 2.8.3. Hydroxyl radical scavenging activity Hydroxyl radical scavenging activity (HRSA) was measured using the method of Wang et al. (2014). 1,10-Phenanthroline solution (1 ml of 1.865 mM) and 2 ml of hydrolysates samples (0.5 mg/ml) were mixed. Then, 1 ml of 1.865 mM FeSO4 solution was added. The reaction was started by addition of 1.0 ml H2O2 (0.03%, v/v). The samples were incubated at 37 ºC for 60 min and the absorbance was read against the reagent blank at 536 nm. The hydroxyl radical scavenging activity was calculated as: HRSA (%) = [(As - An)/ (Ab - An)] ×100 Where As, An, Ab refer to the absorbance of sample, negative control (the reaction mixture without any antioxidant) and blank (the reaction mixture without H2O2). 2.9. Analysis of peptide-phenolic complex using fluorescence spectroscopy 10

Complex formation between hydrolysates of mince and polyphenolic compounds of PGH was evaluated using intrinsic fluorescence according to the method of

uimar es

rummond e Silva

et al. (2017) with some modifications. Aqueous samples containing 2500 µg/ml hydrolysates and different concentrations of PGH (250 and 500 µg/ml) were prepared. The fluorescence spectra of samples was determined at λexc = 280 nm and λemi = from 310 to 600 nm using a Cytation 3 multi-mode reader (BioTek Instruments, Winooski, VT, USA). The effect of complex formation between hydrolysates (2500 µg/ml) on the ferrous chelating activity was evaluated. DPPH• scavenging activity of hydrolysates (2500 µg/ml) containing 5 and 10 µg/ml PGH was also evaluated. 2.10. Statistical analysis Data are expressed as the mean ± standard deviation (SD) of three replications. SPSS 19 (SPSS Inc., Chicago, IL, USA) was used for data analysis applying one-way analysis of variance (ANOVA). Significant difference (P < 0.05) between means was done using the least significant differences (LSD) tests.

3. Results and discussion 3.1. Total pigment, heme iron, and phospholipid content of different substrates Different pretreatments showed a significant effect on total pigment and heme iron of mince (P < 0.05) (Table 2). Defatting using solvent was the most effective method and resulted in the lowest pigment and heme iron. FPI production was less effective than washing for removing pigments probably related to the precipitation of heme proteins with decreasing pH to the isoelectric point during production of FPI (Kristinsson and Liang, 2006). Also, FPI production and washing 11

decreased the phospholipid content significantly (P < 0.05), while the effects of defatting using isopropanol were not significant (P ≥ 0.05). This could be related to the low polarity of isopropanol. Gunnlaugsdottir and Ackman (1993) confirmed that hexane/isopropanol were not effective for polar lipid extractions because of the low polarity of isopropanol and poor solubility of polar lipids in hydrocarbon solvents. 3.2. Effect of pretreatments and PGH extracts on DH Different levels of the enzyme (1.3, 2.5, and 5%) showed a significant effect on DH (Table 3). The highest DH (22%) was obtained using 5% Alcalase. Addition of PGH extract as an antioxidant did not influence DH, showing no inhibitory effects of polyphenolic compounds of PGH on the proteolytic activity of Alcalase (Table 3). Washed and FPI production increased DH (19.3 and 18.8%, respectively). This finding was inconsistent with Khantaphant et al. (2011), who indicated that brownstripe red snapper (Lutjanus vitta) washed mince and isolate were more susceptible to protease activity, probably due to the elimination of lipids and other water-soluble proteins, changes of protein configuration, and a looser protein complex. Defatting with isopropanol decreased DH (15.3%), probably due to protein aggregation resulting from water removal from tissues in the presence of solvent (Klompong et al., 2007). 3.3. Effect of PGH extracts and nitrogen gas on lipid oxidation during hydrolysis 3.3.1. Fish mince Fish mince without enzyme addition (mince-No Alcalase) showed the lowest TBARS values compared to any hydrolyzed sample (Fig. 1a). TBARS increased during the first 30 min of reaction and the change was not significant thereafter. Furthermore, TBARS increased 12

significantly when fish mince was hydrolyzed using 2.5% enzyme with no nitrogen. The highest value was observed after 180 min of hydrolysis. This confirmed that the presence of oxygen during hydrolysis affected lipid oxidation. Thus, excluding O2 using an inert gas could decrease the rate of autoxidation (Shahidi et al., 1992), Lipid oxidation decreased by ~19% with N2. Thus, all other reactions were done in the presence of N2. PGH at 260 µg/ml concentration reduced oxidation of mince compared to mince-2.5% Alcalase. Additionally, enzyme concentration significantly influenced lipid oxidation. As the enzyme level went from 1.3 to 2.5%, the TBARS increased significantly, but addition of 5% enzyme showed a protecting effect against oxidation. The different effects could be due to the released lipids and pro-oxidants as well as to the antioxidant activity of hydrolysates produced after 180 min, possibly due to the amount and nature of the smaller peptides obtained with longer hydrolysis times (Halldorsdottir et al., 2014; Senphan and Benjakul, 2015). 3.3.2. Washed mince, defatted mince, and FPI Washed mince, defatted mince and FPI showed significantly lower TBARS than untreated fish mince (Fig. 1, a-b), indicating that pretreatments could remove undesirable lipid substrates or other soluble compounds responsible for oxidation. Among pretreated muscle samples, washed mince showed the highest lipid oxidation, and this value was not significantly different from the values obtained for mince-2.5% Alcalase-PGH and mince-5% Alcalase (P ≥ 0.05). Sarcoplasmic proteins, fat, blood and pigments were removed by washing (Tongnuanchan et al., 2011), contributing to the better oxidative stability of the resulting hydrolysates. The addition of PGH during hydrolysis of washed mince significantly reduced lipid oxidation. TBARS increased significantly during the first 30 min of hydrolysis of washed mince-PGH and 13

the changes were not significant afterward. After 180 min, TBARS were lower for washed mince-PGH compared to washed mince. FPI production was more effective than washing in controlling the rate of oxidation after 180 min of hydrolysis. However, addition of PGH had less benefit with FPI compared to washed mince (P ≥ 0.05). After 180 min, there were no significant differences in TBARS among washed mince-PGH, FPI and FPI-PGH. Defatting using solvent was the most effective method to control lipid oxidation with the lowest TBARS for defatted mince. TBARS did not change significantly after 30 min of hydrolysis and similar results were obtained with defatted and defatted-PGH during the entire time of hydrolysis. It was significantly lower than that of the mince-No Alcalase sample (P < 0.05). Addition of PGH during hydrolysis of mince and washed mince effectively prevented oxidation. The PGH extract contained polyphenolic compounds of phloroglucinol, gallic acid, protocatechuic acid, 4-hydroxybenzoic acid, catechin, vanillic acid, eriodictyol-7-O-glucoside, naringin, and cinnamic acid (Table 4) (Lalegani et al., 2018). A previous study from this laboratory indicated that PGH had strong DPPH• and ABTS• scavenging activity that correlated with polyphenolic compounds, especially with phloroglucinol and gallic acid (Lalegani et al., 2018). PGH showed metal chelating activity of ferrous ions while gallic acid and phloroglucinol showed no chelating activity. A weak metal chelating activity of gallic acid was reported by Yen and Duh (2002), while Shahidi and Ambigaipalan (2015) suggested that flavonoids could act as metal chelators and radical scavengers. PGH extracts contained low amounts of flavonoids and showed low metal chelating activity, thus the decrease in lipid oxidation during hydrolysis of Sind sardine muscle substrates might be due to its termination of the free radical chain reaction. Oxidation of washed mince is more related to 14

the remaining lipid substrates (data not shown) and the effect of heme pigments is less important (Table 2). According to Kristinsson and Liang (2006), a significant removal of heme proteins occurred with washing, while only a small amount of the lipids were removed. Therefore, PGH with high radical scavenging capacity could successfully inhibit oxidation. The pH shift during FPI production decreased the lipids and other pro-oxidants from FPI, but some lipid remained in the product that led to a significant increase in fat oxidation (Shaviklo et al., 2012). Lipid oxidation of hydrolysates from mackerel protein isolate was correlated with the residual phospholipids in the membranes (Yarnpakdee et al., 2012). In addition, co-precipitation of heme proteins (hemoglobin and myoglobin) at pH ~5.5 increased FPI oxidation (Kristinsson and Liang, 2006) as hemoglobin and myoglobin convert to their oxidized form with strong prooxidative activity (Baron et al., 2002). The insignificant effect of PGH on controlling oxidation was probably related to its weak metal chelating activity. Among all substrates, defatted Sind sardine mince showed the lowest oxidation during hydrolysis, which may be related to the greatest removal of lipids from the substrate using isopropanol (data not shown), and the lowest pigment and heme iron content (Table 2). 3.4. Effect of PGH extract on antioxidant activity of hydrolysates from Sind sardine protein substrates 3.4.1. DPPH• scavenging activity There was no significant difference between hydrolysates obtained with addition of 1.3 and 5% enzyme, and hydrolysis with 2.5% enzyme resulted in significantly lower DPPH• scavenging activity (Fig. 2a). This lack of correlation between enzyme dose and DPPH• scavenging activity may be related to the interference of higher fish oil content of mince used for hydrolysate

15

production than other pretreated samples in the DPPH• scavenging activity test. These results were consistent with the findings of Halldorsdottir et al. (2014) who suggested that addition of 5% (v/v) fish oil to washed cod mince and cod protein isolate interfered with DPPH • scavenging activity. Among all substrates that were hydrolyzed without PGH, hydrolysates of mince showed the lowest DPPH• scavenging activity. Defatting, washing, and FPI production increased DPPH• scavenging activity (P < 0.05). PGH addition showed a significant effect on DPPH• scavenging activity of hydrolysate from mince and other pretreated substrates (P < 0.05) (Fig. 2b). Washed mince with PGH in the hydrolysates showed the highest DPPH• scavenging activity. The combined antioxidant activity of phenol and hydrolysate has been attributed to H+ transfer and electron donation ( uimar es rummond e ilva et al., 201 ). 3.4.2. Ferrous chelating activity Ferrous chelating activity is shown in Fig. 3. Antioxidant activity of hydrolysate from defatted mince was significantly higher than mince, washed mince and FPI hydrolysates (P < 0.05) (Fig. 3b). A significant combined effect was observed with PGH and hydrolysates with different substrates except for FPI (P < 0.05). Mince plus PGH showed the highest metal chelating activity while the lowest activity was obtained for washed mince and mince-2.5% Alcalase-O2. Addition of PGH increased metal chelating activity of hydrolysates from mince, defatted mince and washed mince. Similarly, Fv extract improved the metal chelating properties of cod bone mince hydrolysates (Halldorsdottir et al., 2014). Highly oxidized hydrolysates produced from mince-2.5% Alcalase-O2 showed significantly lower metal chelating activity than mince–2.5% Alcalase-N2 hydrolysates. The negative effect of oxidation on metal chelating activity was also reported by Halldorsdottir et al. (2014) who suggested that highly oxidized FPH had significantly lower metal chelating ability than less oxidized hydrolysate. 16

3.4.3. Hydroxyl radical scavenging activity There was no significant difference in antioxidant activity at 1.3 and 2.5% enzyme (P ≥ 0.05), while the addition of 5% enzyme led to a significant increase in hydroxyl radical scavenging activity (Table 3). All hydrolysates of pretreated samples effectively scavenged hydroxyl radicals at 0.5 mg/ml (Table 3). Different pretreatments and addition of PGH did not show any significant effect on hydroxyl radical scavenging activity of hydrolysates except for mince-PGH, where the antioxidant activity was significantly higher (P < 0.05). 3.5. Interaction of hydrolysate-phenolic compounds and antioxidant properties Interactions of mince hydrolysates and PGH polyphenols are shown in Fig. 4. Hydrolysates from mince with no phenolic compounds showed the highest fluorescence intensity, while the lowest intensity was obtained with PGH. Interaction of hydrolysates-PGH showed lower fluorescence intensity than hydrolysates from mince. Fluorescence intensity decreased with increasing PGH concentration. These results suggested that complex formation between hydrolysates and PGH decreased the fluorescence of the aromatic amino acids. Complex formation between hydrolysates and PGH increased the ferrous chelating and DPPH radical scavenging activity significantly (P < 0.05) as shown in Fig. 5 and 6, respectively. By increasing the PGH concentration, the antioxidant activities of hydrolysates were increased. These results were consistent with the results reported by

uimar es

rummond e ilva et al.

(2017) who indicated that the complex formation of protein/peptide-phenolics caused a synergistic antioxidant activity. 4. Conclusions

17

Hydrolysis of mince in the presence of N2 combined with PGH, especially with 5% Alcalase, significantly reduced oxidation. Production of FPI and washed mince improved DH and decreased oxidation. Defatting showed a negative effect on DH while effectively reducing oxidation. Washed mince-PGH, FPI and FPI-PGH were effective methods to reduce oxidation as seen by lower TBARS after 180 min of hydrolysis. A combined effect of substrates and PGH on antioxidant activity was observed. Washed mince-PGH and mince-PGH showed the highest DPPH radical scavenging and metal chelating activity, respectively. Thus, pretreatments, especially when combined with N2 treatment, could successfully control lipid oxidation. Also, the use of antioxidant such as PGH is a new approach for controlling lipid oxidation and improvement of the antioxidant properties of Sind sardine protein hydrolysates. Acknowledgement The authors would like to acknowledge Tarbiat Modares University and the Iran National Science Foundation (Project NO. 94013374) for their financial support.

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Figures captions Fig 1. Changes in TBARS value during 180 min hydrolysis of fish mince (a); washed fish mince, defatted mince, and fish protein isolate (FPI) (b). PGH: pistachio green hull. The data marked with different letters are significantly different (P < 0.05).

Fig 2. Effect of different level of Alcalase on DPPH radical scavenging activity of hydrolysates prepared from mince (a), effect of pretreatments and pistachio green hull (PGH) addition (b). FPI: fish protein isolate. The data marked with different letters are significantly different (P < 0.05). Fig 3. Effect of different level of Alcalase on metal chelating activity of hydrolysates prepared from mince (a), effect of pretreatments and pistachio green hull (PGH) addition (b). FPI: fish protein isolate. The data marked with different letters are significantly different (P < 0.05).

Fig 4. Fluorescence spectra emission of hydrolysate, pistachio green hull (PGH), and hydrolysate-PGH Fig 5. Effect of hydrolysate and pistachio green hull (PGH) polyphenols interaction on metal chelating activity Fig 6. Effect of hydrolysate and pistachio green hull (PGH) polyphenols interaction on DPPH radical scavenging activity

27

Fig. 1

a

300

a µmol MDA/Kg dry sample

250

b 200

c 150

d d e

100

50

0 0

30

60

90

120

150

180

Hydrolysis time (min) mince-No Alcalase

mince-2.5% Alcalase-O₂

mince-2.5% Alcalase

mince-2.5% Alcalase-PGH

mince-1.3% Alcalase

mince-5% Alcalase

28

b

160

f

µmol MDA/Kg dry sample

140 120 100

g g g h h

80 60 40 20 0 0

30

60

90

120

150

180

Hydrolysis time (min)

washed mince

washed mince-PGH

FPI

FPI-PGH

defatted mince

defatted mince-PGH

29

DPPH radical scavenging activity (µmol trolox equivalents/g dry sample)

Fig. 2

a

12

10

8

6

4

2

0 a b

1.3% Alcalase 2.5% Alcalase

30

a

5% Alcalase

Ferrous chelating activity (μmol EDTA/g dry sample)

DPPH radical scavenging activity (µmol trolox equivalents/g dry sample)

b

20 b

12 a

25

15 a

1.3% Alcalase

d

18

20

31

f

14

c

a

2.5% Alcalase

df

16

e g

10

8

6

4

2

0

Fig. 3

c

b

a

10

5

0

5% Alcalase

35

c

Ferrous chelating activity (μg EDTA/g dry sample)

30

e

25

d

d 20 15

b f a

f

a

10 5 0

Fig. 4

Relative Fluorescence Intensity

80000 70000 60000

hydrolysate 50000

hydrolysate-PGH (250 µg/ml)

40000

hydrolysate-PGH (500 µg/ml) PGH (250 µg/ml)

30000 20000 10000 0 310

360

410

460

Wavelength (nm)

32

510

560

610

Ferrous chelating activity (μg EDTA/g dry sample)

Fig. 5

60

e

50 d c

40 30 20

a

10

5.71

0

hydrolysate

hydrolysate-PGHhydrolysate-PGHhydrolysate-PGH (62.5 µg/ml) (250 µg/ml) (500 µg/ml)

33

PGH

DPPH radical scavenging activity (µmol trolox equivalents/ gr dry sample)

Fig. 6

16

c

14

b

12 a

10 8 6 4 2 0 hydrolysate

hydrolysate-PGH (5µg/ml)

34

hydrolysate-PGH (10µg/ml)

Table 1 Hydrolysis conditions of Sind sardine with Alcalase Treatment

Enzyme (%) (w/w

Hydrolysis condition-antioxidant

protein) Mince-No Alcalase

0%

N2

Mince-2.5% Alcalase-O2

2.5%

O2

Mince-2.5% Alcalase

2.5%

N2

Mince-2.5% Alcalase-PGH

2.5%

N2-PGHa

Mince-1.3% Alcalase

1.3%

N2

5%

N2

Washed mince

2.5%

N2

Washed mince-PGH

2.5%

N2-PGHa

Defatted mince

2.5%

N2

Defatted mince-PGH

2.5%

N2-PGHa

FPI

2.5%

N2

FPI-PGH

2.5%

N2-PGHa

Mince-5% Alcalase

PGHa: pistachio green hull (260 µg/ml); FPI: fish protein isolate

35

Table 2 Phospholipid, total pigment and heme iron content of mince and pretreated mince Sample

Phospholipid content

Total pigment (µg/g

Heme iron (mg/100 g

(mg/100 g dry sample)

dry sample)

dry sample)

Mince

700 ± 10 a

2570 ± 100 a

23 ± 1 a

Washed mince

276 ± 3 b

600 ± 80 b

5.3 ± 0.7 b

Defatted mince

690 ± 30 a

140 ± 20 c

1.2 ± 0.2 c

Fish protein isolate

420 ± 10 c

1380 ± 0.00 d

12.1 ± 0.00 d

Values are given as mean ± SD (n = 3). Means within a column with the same letter are not significantly different (P ≥ 0.05)

36

Table 3 Changes in degree of hydrolysis (DH) of different substrates and hydroxyl radical scavenging activity of hydrolysates Treatment

Degree of hydrolysis (%)

Hydroxyl radical scavenging activity (%)

14 ± 1 a

60.7 ± 0.2 a

Mince-2.5% Alcalase-O2

17.7 ± 0.2 b

62.0 ± 0.4 a

Mince-2.5% Alcalase

17.5 ± 0.1 b

60.1 ± 0.3 a

Mince-2.5% Alcalase-PGH

17.6 ± 0.01 b

72.0 ± 0.2 b

22 ± 1 c

72 ± 1 b

Washed mince

19.3 ± 0.04 d

62.8 ± 0.1 a

Washed mince-PGH

19.0 ± 0.01 d

62 ± 1 a

FPI

18.8 ± 0.01 d

62 ± 1 a

FPI-PGH

18.6 ± 0.01 d

62 ± 1 a

Defatted mince-PGH

15.7 ± 0.1 e

60 ± 3 a

Defatted mince

15.3 ± 0.01 e

61 ± 3 a

Mince-1.3% Alcalase

Mince-5% Alcalase

PGH: pistachio green hull; FPI: fish protein isolate Means within a column with the same letter are not significantly different (P ≥ 0.05)

37

Table 4 Phenolic compounds detected in aqueous PGH extract. Data are expressed as mg/g extract powder Compounds

λmax

Polyphenol content

Peak

Rt (min)

Phloroglucinol

1

4.44

268

65± 2

Gallic acid

2

5.33

268

5.2±0.2

Protocatechuic acid

3

10.38

260

0.09±0.08

4-Hydroxybenzoic acid

4

17. 3

280

0.02±0.01

Catechin

5

21.91

280

0.04±0.02

Vanillic acid

6

24.41

260

2.3±0.02

Eriodictyol-7-Oglucoside Naringin

7

43.10

280

0.66±0.03

8

52.40

280

0.18±0.05

Cinnamic acid

9

55.48

280

n.d.

Adapted to Lalegani et al. (2018) Values are means ± SD of three replicates. n.d. = Not detectable

Highlights  Increased dosage of enzyme to 5% can control lipid oxidation during hydrolysis  Pretreated mince with isopropanol was the most effective to control oxidation  Interaction of hydrolysates-PGH showed higher antioxidant activity than hydrolysate

38