Food Research International 54 (2013) 552–561

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ACE inhibitory and antioxidative activities of Goby(Zosterissessor ophiocephalus) fish protein hydrolysates:Effect on meat lipid oxidation

Rim Nasri a,⁎, Islem Younes a, Mourad Jridi a, Mariem Trigui a, Ali Bougatef b, Naïma Nedjar-Arroume c,Pascal Dhulster c, Moncef Nasri a, Maha Karra-Châabouni a

a Laboratoire de Génie Enzymatique et de Microbiologie—Université de Sfax, Ecole Nationale d'Ingénieurs de Sfax, B.P. 1173-3038 Sfax, Tunisiab Institut Supérieur de Biotechnologie de Sfax, Université de Sfax, Tunisiac Laboratoire de Procédés Biologiques, Génie Enzymatique et Microbien, IUT A Lille I, BP 179, 59653 Villeneuve d'Ascq Cedex, France

⁎ Corresponding author. Tel.: +216 74 274 088; fax:E-mail address: ryma47@hotmail.fr (R. Nasri).

0963-9969/$ – see front matter © 2013 Elsevier Ltd. Allhttp://dx.doi.org/10.1016/j.foodres.2013.07.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 28 January 2013Accepted 1 July 2013Available online 8 July 2013

Keywords:Fish protein hydrolysatesDegree of hydrolysisAntioxidant activityACE inhibition activityGastrointestinal model systemLipid oxidationTurkey meat sausage

The incorporation of protein hydrolysates is of increasing commercial interest. The present study investigatedthe angiotensin-I converting enzyme (ACE) inhibitory activities and antioxidant properties of goby proteinhydrolysates (GPHs) obtained by treatment with various gastrointestinal fish proteases.All hydrolysates displayed ACE-inhibitory activity. The hydrolysate generated by the grey triggerfish proteases(GPH-TF) displayed the highest ACE-inhibitory activity (99 ± 1.4%, at 1.5 mg/ml) followed by that obtainedby smooth hound proteases (GPH-SH) (87.5 ± 0.7%).Additionally, results indicated that GPHs showed varying degrees of antioxidant activities, evaluated by usingvarious antioxidant assays. Hydrolysate obtained by treatment with crude protease from golden mullet(GPH-GM) showed the most pronounced DPPH-radical scavenging effect, followed by bovine trypsinhydrolysate (GPH-T), while that obtained by triggerfish proteases (GPH-TF) displayed the highestβ-carotene bleaching inhibitory effects. Moreover, all hydrolysates, except GPH-SH, showed high chelating effectand lipid peroxidation inhibition, even at low concentrations. Further, addition of GPH-TF in turkey meat sausageresulted in products with improved antioxidant activity. The results suggested that GPHs are good source of naturalACE-inhibitory and antioxidant peptides.

© 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Several studies in the past few decades have reported thatpeptides from various food sources, in addition to their nutritionalproperties, exhibited biological activities including antioxidant (Ktariet al., 2012), ACE inhibitory activity (Balti, Nedjar-Arroume, Guillochon,& Nasri, 2010), cholesterol-lowering ability (Ben Khaled et al., 2012),anticoagulant activity (Nasri et al., 2012) etc., and may therefore serveas therapeutic roles in body systems (Erdman, Cheung, & Schröder,2008). These peptides are in the size of 2–20 amino acids (Meisel &FitzGerald, 2003). These peptides can be used as functional food ingre-dients or nutraceuticals to improve human health and prevent disease.Bioactive peptides, are inactive within the sequence of the parentproteins, and can be released by enzymatic hydrolysis either duringgastrointestinal digestion or during food processing (e.g., cheese ripeningandmilk fermentation) or in vitro by treatmentwith proteolytic enzymes.

Oxidation of fats during processing and storage of food products isof great concern to the food industry and consumers because it leads

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to the development of undesirable off-flavors, odors, dark colors andpotentially toxic reaction products (Lin & Liang, 2002). Furthermore,cancer, coronary heart and Alzheimer's diseases are also reported tobe caused in part by oxidation or free radical reactions in the body(Diaz, Frei, Vita, & Keaney, 1997). To prevent oxidative deterioration offoods and to provide protection against serious diseases, it is importantto inhibit the oxidation of lipids and the formation of free radicals occur-ring in the foodstuff and living body. Antioxidants are used to preservefood products by retarding discoloration and deterioration as a result ofoxidation (Halliwell, Murcia, Chirico, & Aruoma, 1995).

Synthetic antioxidants have been widely used in stabilization offoods. The two most commonly used are butylated hydroxyanisole(BHA) and butylated hydroxytoluene (BHT), which are added to fattyand oil foods to prevent oxidative deterioration (Löliger, 1991). However,use of these chemical compounds has begun to be restricted because oftheir induction of DNA damage and their toxicity (Ito et al., 1986). More-over, both BHT and BHA appear to be involved in tumor promotion(Botterweck, Verhagen, Goldbohm, Kleinjans, & Van den Brandt, 2000).Therefore, there is a great interest in finding new and safe antioxidantsfrom natural sources, especially peptides derived from hydrolyzed foodproteins. Bioactive peptides from enzymatic hydrolysis of various food

Table 1Hydrolysis conditions of goby proteins, DH, ACE-inhibitory activities and IC50 values ofGPHs obtained with various proteases treatment.

Enzyme Optimumconditions

Maximum DH(%)

ACE inhibition(%)at 1.5 mg/ml

IC50(mg/ml)

T (°C) pH

Smooth hound 50 8.0 28.4 87.5 ± 0.7 0.833Grey triggerfish 50 10.0 23.3 99 ± 1.4 0.73Golden mullet 55 9.0 11.9 56 ± 1.4 1.33Goby 45 9.0 7.75 83.06 ± 0.6 0.9Bovine trypsin 37 8.0 5.85 71.3 ± 2.8 1.051

E/S ratio: 3 U/mg of protein.

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proteins such as soy protein, casein, whey protein and gelatine have beenshown to possess antioxidant activities (Elias, Kellerby, & Decker, 2008).Recently, a great deal of interest has been expressed regardingmarine-derived bioactive peptides because of their numerous healthbenefits.

On the other hand, protein hydrolysates with angiotensin-Iconverting enzyme (ACE) inhibitory activity have shown great promisein the development of novel therapeutics and functional foods forpreventing hypertension. High blood pressure or hypertension is oneof the major independent risk factor for cardiovascular diseases. ACE(EC 3.4.15.1) plays a central role in the regulation of blood pressurethrough the production of the potent vasoconstrictor, angiotensin-II,and the degradation of the vasodilator, bradykinin. The inhibition ofACE is considered to be a useful therapeutic approach in the treatmentof hypertension. Captopril, lisinopril and other synthetic ACE inhibitors,which have been used in the clinical treatment of hypertension, couldhave some undesirable side effects in humans, such as cough, lost oftaste, renal impairment (Acharya, Sturrock, Riordan, & Ehlers, 2003).Therefore, search for natural safe compounds with high ACE inhibitoryactivity, as alternative one is necessary for the prevention and remedyfor hypertension. A large number of ACE-inhibitory protein hydrolysateshave been produced from various food proteins, such as chickpea(Pedroche et al., 2002) and sunflower (Megias et al., 2009).

The goby (Zosterisessor ophiocephalus), is a common fish inMediterranan Sea, Black Sea, and Sea of Azov. It reaches a maximumlength of 25 cm and is a carnivorous fish; which feed on small crusta-ceans, polychaets and molluscs. It is relatively important in the fish-catches of Tunisia, and is utilised for human consumption. In Tunisia,goby (Z. ophiocephalus) catches were about 130 ton in 2004 (FAO,2004).

In this study, we investigated the antioxidant and ACE-inhibitoryactivities of goby muscle protein hydrolysates (GPHs) obtained bytreatment with various fish crude alkaline protease extracts. GPH-TF,with strong antioxidant activity, was selected for further antioxidantassessment against lipid peroxidation in turkey meat sausage during a25-day storage period.

2. Materials and methods

2.1. Reagents and proteolytic enzymes

Angiotensin-I converting enzyme (ACE) from rabbit lung, ACEsynthetic substrate hippuril-L-histidyl-L-leucine (HHL), 1,1-diphenyl-2-picrylhydrazyl (DPPH), bile salt, butylated hydroxyanisole (BHA),β-carotene, α-tocopherol, ethylene diamine tetra acetic acid (EDTA)and linoleic acid were purchased from Sigma Chemical Co. (St. Louis,MO, USA). Thiobarbituric acid (TBA) was purchased from Suvchem(MH, India). Modified starch (E1422) was provided from Sigma Chem-ical CO., St. Louis, MO.

All other chemicals, namely potassium ferricyanide, trichloroaceticacid (TCA), ferrous chloride, ferrozine, sodium hydroxide, Tween 40,NaCl, NaNO2 and tripolyphosphate (TPP) were of analytical grade.

Trypsin from bovine pancreas was supplied by Fluka Biochcemicals.Crude alkaline protease extracts from the viscera of grey triggerfish(Balistes capriscus) (Jellouli et al., 2009), smooth hound (Mustelusmustelus), golden mullet (Liza aurata) (Ktari et al., in press) and goby(Z. ophiocephalus) (Nasri et al., 2011), used for the preparation of pro-tein hydrolysates, were prepared in our laboratory.

2.2. Fish samples

Goby (Z. ophiocephalus) was purchased from the fish market ofSfax city, Tunisia. The samples were packed in polyethylene bags,placed in ice and then transported to the research laboratory within30 min. Muscle and viscera were separated, rinsed three times withdistilled water to remove salts and other contaminants. Muscle was

stored in sealed plastic bags at −20 °C, until it was used for proteinhydrolysates production; while viscera were used immediately forcrude alkaline enzyme extraction.

2.3. Preparation of endogenous enzyme extracts

Viscera or intestine (150 g) from fish species were thoroughlywashed with distilled water and then homogenized for 60 s with300 ml of extraction buffer (10 mM Tris –HCl, pH 8.0). The homoge-nates were centrifuged at 8.500 ×g for 30 min at 4 °C. The pelletswere discarded and supernatants, referred to as crude alkaline proteaseextracts, were collected. All enzymatic assays were conducted within aweek after extraction. For a long conservation, supernatants werelyophilized.

Alkaline protease activities, in the crude enzyme extracts, weremeasured by the method of Kembhavi, Kulkarni, and Pant (1993)using casein as a substrate. One unit of protease activity was definedas the amount of enzyme required to liberate 1 μg tyrosine per minunder the experimental conditions used. Values are the means ofthree independent experiments.

2.4. Preparation of goby protein hydrolysates (GPHs) using variousdigestive proteases

Goby muscle (500 g), in 500 ml distilled water, were first mincedusing a grinder (Moulinex Charlotte HV3, France) then cooked at90 °C for 20 min to inactivate endogenous enzymes. The cookedmuscle sample was then homogenized in a Moulinex® blender forabout 20 min. The samples were adjusted to optimal pH and temper-ature for each enzyme. Then, the substrate proteins were digestedwith enzymes at a 1:3 (U/mg) enzyme/protein ratio for 360 minunder optimum pH and temperature conditions (Table 1). Enzymeswere used at the same activity levels to compare hydrolytic efficien-cies. During the reaction, the pH of the mixture was maintained atthe desired value by continuous addition of 4 N NaOH solutions.After incubation for 360 min, the reactions were stopped by heatingthe solutions for 20 min at 80 °C to inactivate enzymes. Protein hy-drolysates were then centrifuged at 5000 g for 20 min to separate solu-ble and insoluble fractions. Finally, the soluble fractions, referred to asprotein hydrolysates, were freeze-dried using freeze-dryer at a tempera-ture of −50 ° C and a pressure of about 121 mbar through a lyophilizerlab (Moduloyd-230, ThermoFisher Scientific, USA) and then stored at −20 °C for further use.

The degree of hydrolysis (DH), defined as the percent ratio of thenumber of peptide bonds cleaved to the total number of peptidebonds in the substrate studied, was calculated from the amount ofbase (NaOH) added to keep the pH constant during the hydrolysis(Adler-Nissen, 1986) according to the following equation.

DH %ð Þ ¼ hhtot

� 100 ¼ B� NbMP

� 1α� 1htot

� 100

554 R. Nasri et al. / Food Research International 54 (2013) 552–561

where B is the amount of NaOH consumed (ml) to keep the pH constantduring the proteolysis of the substrate. Nb is the normality of the base,MP is themass (g) of the protein (N × 6.25), andα represents the aver-age degree of dissociation of the α-NH2 groups in the protein substrateexpressed as:

α ¼ 10pH‐pK

1þ 10pH‐pK

where pH and pK are the values at which the proteolysis wasconducted. The total number of peptide bonds (htot) in the proteinsubstrate was assumed to be 8.6 meq/g (Adler-Nissen, 1986).

2.5. Determination of chemical composition of GPHs

Moisture and ash content of GPHs were determined according tothe AOAC methods 930.15 and 942.05 respectively (AOAC, 2000).The protein content was determined by estimating its total nitrogencontent by Kjeldahl method according to the AOAC method number984.13 (AOAC, 2000). A factor of 6.25 was used to convert the nitro-gen value to protein. Lipids were determined gravimetrically afterSoxhlet extraction of dried samples with hexane. All measurementswere performed in triplicate. The protein, ash and fat contents wereexpressed on a dry weight basis.

2.6. Amino acid composition of GPHs

GPH sample was hydrolyzed with 0.5 mL of 6 N HCl at 110 °Cfor 24 h on a heating block, and then filtered through a 0.45 μmmembrane filter prior to analysis. 10 μL of the treated sample wasderivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamateWaters AccQ·Fluor Reagent Kit (according to Waters AccQ·Tag Chemis-try Package Instruction Manual).

The HPLC analyses were performed with a Waters 2996 SeparationModule equipped with a Waters 2475 multi-wavelength fluorescencedetector and amino acids were separated on a Waters AccQ·Tag aminoacid analyzing Column (Nova-Pak C18, 150 × 3.9 mm). The amount ofamino acids was calculated, based on the peak area in comparison withthat of standard. The amino acid content was expressed as a percentageof total amino acids in the sample. All analyses were performed induplicate.

2.7. Characterization of hydrolysates by size exclusion chromatography

The molecular weight analysis of peptides for each protein hy-drolysate was carried out by size-exclusion FPLC (Fast PerformanceLiquid Chromatography) (GE health care-bioscience AB, 751 84,upsale, Sweden) on a superdex peptide 10/300 GL column (7.8 mmI.D × 30 cm L). The eluant used was acetonitrile [ACN] 30% and 0.1%trifluoroacetique acid (TFA) in water filtered through whatman celluloseacetate membrane (0.2 μm). The flow rate was adjusted to 0.5 ml/min.The column was calibrated with standard proteins from SigmaChemicals: albumin (60,000 Da), cytochrome C (12,400 Da), aprotinin(6500 Da), B12 vitamin (1355.5 Da) and gluthation (307.3 Da). Allstandards were loaded separately. Protein hydrolysates were loadedto the column at a concentration of 4 mg/ml.

The liquid chromatographic system consisted of a UPC-900 controllerpump module P-901 and M-925 Mixer w/0.6 ml chamber INV-907Injector UV Detector unit with 254/280 nm filters Frac 950. UNICORNsoftware was used to plot, acquire, and analyze chromatographic data.A calibration curve was obtained by plotting log molecular weight vspeak elution time. The averagemolecular weight of protein hydrolysatewas determined from the standard curve.

Determinations were performed in triplicate and data correspondto mean values. Standard deviations were in all cases lower than 5%.

2.8. Determination of ACE inhibition activity

ACE-inhibitory activity was assayed as reported by Nakamura etal. (1995). A volume of 80 μl containing different concentrations ofGPHs was added to 200 μl of 5 mM HHL, and then preincubated for3 min at 37 °C. GPHs and HHLwere prepared in 100 mMborate buffer(pH 8.3) containing 300 mM NaCl, The reactions were then initiatedby adding 20 μl of 0.1 U/ml ACE from rabbit lung, prepared in thesame buffer. After incubation at 37 °C for 30 min, the enzymatic reac-tions were stopped by adding 250 μl of 0.05 M HCl. The releasedhippuric acid (HA) was extracted with ethyl acetate (1.7 ml) andthen evaporated at 95 °C for 10 min. The residue was dissolved in1 ml of distilled water and the absorbance of the extract at 228 nmwas determined using a UV–visible spectrophotometer (Cecil CE2021, Lab Equip Instruments Ltd). ACE-inhibitory activity was calcu-lated using the equation

ACE inhibition %ð Þ ¼ B‐AB‐C

� �� 100

where A is the absorbance of HA generated in the presence of ACEinhibitor component, B the absorbance of HA generated withoutACE inhibitors and C is the absorbance of HA generated withoutACE (corresponding to HHL autolysis in the course of enzymatic assay).The IC50 value was defined as the concentration of hydrolysate (mg/ml)required to reduce 50% of ACE activity under the above condition.

2.9. Determination of antioxidant activity

2.9.1. DPPH free radical-scavenging activityThe DPPH free radical-scavenging activity of GPHs was deter-

mined as described by Bersuder, Hole, and Smith (1998). A volumeof 500 μl of each sample at different concentrations (1 to 5 mg/ml)was added to 375 μl of 99% ethanol and 125 μl of DPPH solution(0.02% in ethanol) as free radical source. The mixtures were shakenthen incubated for 60 min in a dark at room temperature. Scavengingcapacity was measured spectrophotometrically by monitoring thedecrease in absorbance at 517 nm. In its radical form, DPPH hasan absorption band at 517 nm which disappears upon reductionby an antiradical compound. Lower absorbance of the reaction mixtureindicated higher DPPH free radical-scavenging activity. BHA was usedas positive control. DPPH radical-scavenging activity was calculated asfollows:

DPPH radical‐scavenging activity %ð Þ¼Ablank‐AsampleAblank

� 100

where A blank is the absorbance of the reaction containing all reagentsexcept that distilledwaterwas used instead of the sample, and A sampleis the absorbance in the presence of sample. The experiment wascarried out in triplicate and the results were mean values.

2.9.2. Inhibition of linoleic acid autoxidationThe lipid peroxidation inhibition activity of GPHswasmeasured in a

linoleic acid emulsion system according to the method of Osawa andNamiki (1985). GPHs were dissolved in 2.5 ml of 50 mM phosphatebuffer (pH 7.0) and added to a 0.0325 ml of linoleic acid and 2.5 ml eth-anol (95%). The final volumewas then adjusted to 6.25 mlwith distilledwater. The final assay GPHs concentration usedwas 2 mg/ml. The reac-tion mixture was incubated in a 10 ml tubes with silicon rubber caps at45 °C for 9 days in a dark room. The degree of oxidation of linoleic acidwas evaluated at 0, 4, 6, 8 and 9 days by measuring the ferric thiocya-nate values according to the method of Mitsuta, Yasumoto, and Iwami(1996). Aliquot (0.1 ml) of the reaction mixture was mixed with4.7 ml of 75% ethanol followed by the addition of 0.1 ml of 30%

555R. Nasri et al. / Food Research International 54 (2013) 552–561

ammonium thiocyanate and 0.1 ml of 20 mM ferrous chloride solutionin 3.5% HCl. After stirring for 3 min, the degree of color develop-ment, which represents the linoleic acid oxidation, was measured at500 nm.α-tocopherol, a natural antioxidant agent, was used as a positivecontrol. A tube without sample was used as blank. The test was carriedout in triplicate.

The antioxidative capacity of the inhibition of peroxide formationin linoleic acid system was expressed as follows:

Inhibition %ð Þ ¼ 1‐A500 of sampleA500 of Ablank

� �� 100

2.9.3. Antioxidant assay using the ß-carotene bleaching methodThe ability of GPHs to prevent bleaching of β-carotene was

assessed as described by Koleva, Van Beek, Linssen, de Groot, andEvstatieva (2002). In this test, β-carotene undergoes rapid discolorationin the absence of antioxidant, which results in a reduction in absorbanceof the test solution with increasing reaction time. A stock solution ofβ-carotene/linoleic acid mixture was prepared by dissolving 0.5 mg ofβ-carotene, 25 μl of linoleic acid and 200 μl of Tween 40 in 1 ml of chlo-roform. The chloroform was completely evaporated under vacuum in arotatory evaporator at 40 °C, then 100 ml of bi-distilled water wereadded, and the resulting mixture was vigorously stirred. The emulsionobtained was freshly prepared before each experiment. Aliquots(2.5 ml) of the β-carotene/linoleic acid emulsion were transferredto test tubes containing 0.5 ml of each sample of GPH at differentconcentrations. Following incubation for 2 h at 50 °C, the absorbanceof each sample was measured at 470 nm. A blank consisted of 0.5 mlof distilled water instead of sample. The antioxidant activity of the hy-drolysates was evaluated in terms of bleaching of β-carotene usingthe following formula:

A% ¼ 1– A0–A2hð Þ= A’0–A’2hð Þ½ � � 100

where A0 and A′0 are the absorbance of the sample and the blank, re-spectively, measured at time zero, and A2h and A′2h are the absorbanceof the sample and the blank, respectively, measured after incubation for2 h. The same procedure was repeated with BHA as positive control.Three replicates were done for each test sample.

2.9.4. Metal-chelating activityThe chelating activity of samples towards ferrous ion (Fe2+) was

determined according to the method of Decker and Welch (1990).One milliliter of each sample at different concentrations (1, 2, 5 mg/ml)was mixed with 3.7 ml of distilled water. Thereafter, 0.1 ml of 2 mMFeCl2, 4H2O and 0.2 ml of 5 mM 3-(2-pyridyl)-5,6-bis(4-phenyl-sulfonicacid)-1,2,4-triazine (ferrozine) were added. The mixture was allowed toreact for 20 min at room temperature. The absorbance was thenread at 562 nm. The blank was conducted in the same manner ex-cept that distilled water was used instead of the sample. EDTAwas used as reference. The chelating activity (%) was calculatedas follows:

Chelatingactivity %ð Þ ¼ 1‐A562 of sampleA562 of blank

� �� 100

2.10. pH and thermal stability of GPH-TF

GPH-TF was dissolved in 5 ml of distilled water with final proteinconcentration of 50 mg/ml. The pH of protein hydrolysate solutionswas adjusted to different pHs (from 1.0 to 9.0) using 1 M HCl or1 M NaOH and then the volume of solution was made up to 10 mlwith distilled water previously adjusted to the same pH. The mixtureswere incubated at room temperature for 1 h. Thereafter, the pH of themixtures was adjusted to 7.0 and their volumes were made up to

25 ml with distilled water. The residual antioxidant activities weretested using the β-carotene-linoleate bleaching expressed as the ac-tivity (%) relative to that obtained without pH adjustment.

For the temperature stability, GPH-TF was dissolved in 5 ml of dis-tilled water with final protein concentration of 50 mg/ml, then pH ofprotein hydrolysate solution was adjusted to 7.0 and the volume ofsolution was made up to 25 ml with distilled water. Five millilitresof the GPH-TF solution were transferred to screw-capped test tubeand placed in a boiling water bath (90 °C) for 0, 15, 30, 60, 120, 180and 240 min. Thereafter, the tubes were immediately cooled iniced water. The residual antioxidant activities were tested using theβ-carotene-linoleate bleaching model assays and were expressedas relative activity (%) compared to those without heat treatment.

2.11. In vitro gastrointestinal digestion stability of GPH-TF

The effect of in vitro gastrointestinal digestion on GPH-TF wasevaluated as described by Enari, Takahashi, Kawarasaki, Tada, andTatsuta (2008) with slight modifications. A 100 ml of GPH-TF solution(10 mg/ml) was mixed with 10 ml of 10 mM phosphate buffer(pH 6.8) and incubated for 2 min at 37 °C. Then 1 ml of 1 M HCl wasadded to produce an acidic condition, followed by adding 32 U/ml ofpepsin solution in 1 M HCl–KCl buffer (pH 1.5) (5 ml). The mixturewas incubated for 1 h at 37 °C with continuous shaking (stomach con-dition). Thereafter, the pH of the reaction mixture was adjusted to 6.8with 1 M NaHCO3 (2.5 ml), and 1 ml mixture of bile and pancreaticjuice that contained bile extract (13.5 mg/ml), pancreatin (10 mg/ml),and trypsin (14,600 U/ml) in 10 mM phosphate buffer (pH 8.2), wasadded to the solution, followed by incubation at 37 °C for 3 h to createduodenal condition. To inactivate duodenal enzymes, the test tubeswere kept in boiling water for 10 min. The antioxidant activities weretestedusing theβ-carotene-linoleate bleachingmodel during the diges-tion after 30, 60, 120, 180 and 240 min. The relative activities weremeasured and compared to that without any treatment (at 0 min).

2.12. Effects of GPH-TF on turkey meat sausage lipid oxidation

2.12.1. Turkey meat sausage product preparationGPH-TF with strong antioxidant activity was selected and further

assessed for its antioxidant activity against lipid oxidation in turkeymeat sausage. Turkey sausage products were prepared using mechan-ically separated turkey meat (MSTM) obtained from a local processor(Chahia, Sfax Tunisia). All formulations were prepared with the samecommon ingredients: 60%MSTM, 29% water (ice- and cold-water), 8%modified starch, 0.15% carrageenan, 2% NaCl, 0.5% TPP, 0.8% NaNO2

and 0.045% ascorbic acid or GPH-TF at different concentrations, asdescribed by Ayadi, Kechaou, Makni, and Attia (2009). Dry ingredientswere added slowly to the groundMSTM as powders during processing.Then, cold water was added. The addition of ingredients took less than5 min and the final temperature of the mixture varied between 10 and12 °C. The batters were manually stuffed into collagen reconstitutedcasing and then cooked to an internal temperature of 74 °C in a waterbath. After cooling to room temperature, the turkey meat sausageswere placed in polyethylene bag and then stored at 4 °C. DifferentGPH-TF levels were studied by adding 0.01%, 0.02%, 0.04%, 0.1% and0.2% (w/w) of GPH-TF powder to turkey meat sausages, a samplewhich contain 0.045% vitamin C was used as reference. The processwas replicated twice.

2.12.2. Determination of lipid peroxidation in Turkey meat sausageLipid peroxidation was estimated as evidenced by the formation of

thiobarbituric acid reactive substances (TBARS) such asmalondialdehyde(MDA). TBARS were assayed in tissues by the method described by Yagi(1976). MDA and other TBARS were measured by their reactivity withTBA in an acidic condition to generate pink colored chromospherewhich was read at 530 nm. A sample (375 μl) of turkey meat sausage

Table 2Proximate composition (%) of gobymuscle and freeze-dried GPHs preparedwith differentcrude protease extracts. Values are given as mean ± SD from triplicate determinations.

Protein content (%) Lipids levels (%) Ash (%) Moisture (%)

UGP 74 ± 0.35 7.4 ± 0.7 8.5 ± 0.08 10.2 ± 0.24GPH-SH 69.72 ± 1.01 1.68 ± 0.02 17.6 ± 0.26 10.19 ± 0.11GPH-TF 70.41 ± 1.35 1.6 ± 0.01 16.86 ± 0.05 10.96 ± 0.14GPH-GM 76 ± 2.53 1.08 ± 0.02 12.67 ± 0.05 10.02 ± 0.12GPH-G 77.5 ± 0.53 1.54 ± 0.05 10.28 ± 0.04 10.25 ± 1.06GPH-T 79.3 ± 0.53 1.51 ± 0.05 9.2 ± 0.42 10.21 ± 0.03

UGP: Undigested goby Proteins.GPH-SH, GPH-TF, GPH-GM, GPH-G and GPH-T are goby protein hydrolysates producedusing smooth hound proteases, triggerfish proteases, golden mullet proteases, gobyproteases and bovine trypsin, respectively.

Table 3Amino acids composition (%) of GPHs.

556 R. Nasri et al. / Food Research International 54 (2013) 552–561

was homogenized with 150 μl of TBS (50 mM Tris containing 150 mMNaCl, pH 7.4) and 375 μl of TCA 20% (w/w) in order to precipitateproteins, and then centrifuged (1000 g, 10 min, 4 °C). A 400 μl of thesupernatant was mixed with 80 μl of HCl (0.6 M) and 320 μl of Tris–TBA (Tris 26 mM; TBA 120 mM), and the mixture was heated for10 min at 80 °C. The absorbance of the resulting solution was read at530 nm. TBARS values were calculated from a standard curve of MDAand expressed as mg MDA/kg sample. The blank was the mixturewithout turkey sausage.

2.13. Statistical analysis

Statistical analyses were performed with Stratgraphics ver. 5.1,professional edition (Manugistics Corp., USA) using ANOVA analysis.Differences were considered significant at p b 0.05.

3. Results and discussion

3.1. Preparation of GPHs using different visceral proteases

The most common way to produce bioactive peptides is throughenzymatic hydrolysis of whole protein molecules by appropriateenzymes derived from microorganism, animal or plant. The natureof the protein substrate, the specificity of the enzyme used for theproteolysis, the conditions used during hydrolysis (time and tempera-ture) as well as enzyme/substrate ratio greatly influence the molecularweights and amino acids composition of released peptides and thustheir biological activities (Van der Ven, Gruppen, de Bont, & Voragen,2002). Since proteases have specific cleavage positions on polypeptidechains, treatment of proteins with different proteases produces differenttypes of protein hydrolysates, which contain a mixture of high-medium- and/or low molecular weight peptides, depending on thedegree of hydrolysis of the enzyme. In some cases, more than oneprotease can be used in order to enhance protein hydrolysis andobtain hydrolysate enriched with low molecular weight peptides.

In this study, trypsin and four crude alkaline enzyme extracts fromthe viscera of several fish species, which contained multiple proteases,were used to produce various types of goby protein hydrolysatesenriched with bioactive peptides.

The hydrolysis curves of goby proteins, with the different crudeenzyme extracts, after 360 min of incubation are shown in Fig. 1. Thehydrolysis of proteins with all proteases was characterized by a highrate of hydrolysis during the initial 30 min. The rates of enzymatichydrolysis were subsequently decreased, and then the enzymatic

Fig. 1. Kinetic curves of the proteolysis of goby muscle proteins by various visceralproteases during 360 min.

reaction reached a steady-state phase when no apparent hydrolysistook place. The smooth hound visceral proteases were the most effi-cient, while bovine trypsin was the least efficient. Indeed, after360 min of hydrolysis, the DH values were 28.4%, 23.3%, 11.9%, 7.75%and 5.85% for hydrolysates prepared with visceral proteases of smoothhound, grey triggerfish, goldenmullet, goby and bovine trypsin, respec-tively (Table 1). GPH-SH, which had the highest DH (P b 0.05),should be rich in low molecular weight peptides than the other hy-drolysates. The shape of hydrolysis curves is similar to those previouslypublished for hydrolysates from toothed ponyfish (Klomklao, Kishimura,& Benjakul, 2013), grass goby fish (Nasri et al., 2012), zebra blenny (Ktariet al., 2012) and sardinelle (Ben Khaled et al., 2012).

3.2. Chemical composition of GPHs

The chemical composition of freeze dried GPHs was determinedand compared to that of undigested goby proteins (UGP) (Table 2).The proximate composition of dried UGP showed that it had high pro-tein content (74% of dry matter basis) (P b 0.05). Protein content wasabout 79% in bovine trypsin goby protein hydrolysate (GPH-T) whichwas characterized by the lowest DH (5.8%), while GPH-SH (DH 28.4%)showed a protein content of about 69% (P b 0.05). The liberation ofsmall peptides and amino acids could explain the decrease of theamount of protein through the hydrolysis reaction.

All hydrolysates had approximately the same composition of lipidlevels which ranged from 1.08% to 1.68%, which were lower than UGP

GPH-SH GPH-TF GPH-T GPH-G GPH-GM

Asx 4.91 3.74 3.80 3.87 3.65Ser 6.67 6.54 6.80 7.36 6.75Glx 9.94 7.94 7.59 7.7 8.22Gly 14.21 15.19 14.42 14.03 14.52His 3.05 3.93 4.32 4.21 3.54Arg 5.87 8.68 7.66 7.92 8.12Thr 11.68 12.55 15.25 10.65 13.65Ala 6.54 5.86 5.25 5.42 5.98Pro 3.99 5.28 3.33 3.47 4.12Cys 1.77 1.61 2.59 4.79 2.54Tyr 2.43 3.63 3.46 4.13 3.14Val 4.99 4.71 4.40 4.25 4.32Met 3.00 2.24 3.35 3.01 3.02Lys 5.61 3.99 1.3 3.76 3.76Ile 3.85 3.31 3.32 3.19 3.45Leu 7.51 6.69 6.74 6.58 6.66Phe 3.90 4.01 5.89 5.58 4.56HAA 43.59 41.33 39.9 44.18 41.55

Asx = aspartic acid + asparagine; Glx = glutamic acid + glutamine; Combined ofhydrophobic amino acids (HAA) = alanine, valine, isoleucine, leucine, tyrosine,phenylalanine, tryptophane, proline, methionine, cysteine.GPH-SH, GPH-TF, GPH-GM, GPH-G and GPH-T are goby protein hydrolysates producedusing smooth hound proteases, triggerfish proteases, golden mullet proteases, gobyproteases and bovine trypsin, respectively.

Fig. 2. FPLC profiles of GPHs on a superdex peptide 10/300 GL column. GPH-SH,GPH-TF, GPH-GM, GPH-G and GPH-T are goby protein hydrolysates produced usingsmooth hound proteases, triggerfish proteases, golden mullet proteases, goby proteasesand bovine trypsin, respectively.

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(7.4%) (P b 0.05). Ash content ranged from 9.2% to 17.6%. GPH-SH andGPH-TF, which exhibited the highest DH, had high ash content, 17.6%and 16.84%, respectively (P b 0.05). Similar studies showed that ashcontent in fish hydrolysate increased with the increase of DH whichcould be explained by the increase of the volume of NaOH added tokeep pH constant during enzymatic hydrolysis (Nilsang, Lertsiri,Suphantharika, & Assavanig, 2006).

3.3. Amino acid composition of GPHs

The amino acid composition of proteins hydrolysates is importantbecause of the nutritional value and the influence on the functionalproperties (Santos, Martins, Salas-Mellado, and Prentice (2011). Fishprotein hydrolysates exhibited variation in their amino acids composi-tion. This variation depends on several factors such as enzyme sourceand fish species.

The amino acids compositions of the freeze-dried GPHs, expressedas residues per 100 residues, are presented in Table 3. Hydrolysis withthe different proteolytic enzymes changes slightly the percentages ofseveral amino acid residues of the hydrolysates. The slight differencesin amino acids composition between the five hydrolysates can be at-tributed to the differences in specificity of the five gastrointestinalproteases.

Gly and Thr were the most abundant amino acids which accountedfor 14.03–15% and 10.65–13.65%, respectively. Under the conditions ofthe acid hydrolysis, tryptophan was destroyed. GPHs have a high per-centage of essential amino acids (Val, Met, Lys, Ile, Leu, Phe and Thr).Therefore, GPH could possibly be a dietary protein supplement to poor-ly balanced dietary proteins. Several works have been described theamino acid composition of protein hydrolysates from different fishspecies, including sole (Giménez, Alemán, Montero, & Gomez-Guillén,2009), Cat fish (Yin et al., 2010), Misgurnus anguilliacaudatus (You,Zhao, Regenstein, & Ren, 2011).

3.4. Determination of molecular weight distributions of GPHs

Size-exclusion FPLCwas performed to analyze the overall molecularweight distribution of the different hydrolysates. The profiles reportedin Fig. 2 revealed the differences in the degree of protein hydrolysiswhich depended on the enzyme used. The molecular weight distri-bution of the different hydrolysates divided into seven fractions isshown in Table 4. The molecular ranges of the seven fractions werebelow 100 Da, 100–300 Da, 300–700 Da, 700–1700 Da, 1700–3000,3000–5000 and N5000 Da. The obtained results were in accordancewith DH observed for the different GPHs. Indeed, the results showthat GPH-SH, which had the highest DH contained more small-sized

Table 4Molecular weight distribution of GPHs (results are presented as means +standard de-viations (n = 3)). UNICORN software was used to plat and calculate the area under thecurve based on the molecular weight profile obtained by the superdex peptide 10/300GL column gel filtration.

GPH-SH, GPH-TF, GPH-GM, GPH-G and GPH-T are goby protein hydrolysates producedusing smooth hound proteases, triggerfish proteases, golden mullet proteases, gobyproteases and bovine trypsin, respectively.

Percent of total area under the curve

GPH-T GPH-G GPH-SH GPH-TF GPH-GM

N5000 Da 14.92 3.66 3.51 11.72 4.255000 to 3000 Da 18.1 8.18 6.7 13.48 7.543000 to 1700 Da 23.12 17.3 15.12 16.33 17.541700 to 700 Da 23.65 31.52 24.54 18.81 25.65700 to 300 Da 10.07 17.25 16.84 13.1 18.65300 to 100 Da 7.71 17.55 24.14 20.42 18.96b100 Da 2.41 4.53 9.13 6.12 6.39

peptides with molecular weight below 300 Da, whereas in trypsinhydrolysate, which showed the lowest DH, peptides with molecularweight above 700 Da were predominant (P b 0.05).

3.5. ACE inhibitory activities of GPHs

The hydrolysates obtained by treatment with the five gastrointestinalproteases at 360 min of incubation time were assayed for ACE-inhibitoryactivity. As reported in Table 1, all protein hydrolysates exhibitedACE-inhibitory activities, while no activity was detected with theundigested goby proteins (t = 0) (data not shown). The results soobtained demonstrate that ACE-inhibitory peptides are encryptedwithin goby proteins and could be released by proteolysis. Further,the activity of GPHs was concentration dependent; the values increasedwith increasing hydrolysates concentrations. The highest ACE-inhibitoryactivity (99 ± 1.4%) at 1.5 mg of dryweight/ml (P b 0.05), was observedwith GPH-TF, which exhibited the high DH (23.3%). GPH-SH, whichhad the highest DH (28.4%), also exhibited high ACE-inhibitory activity(87.5% at 1.5 mg/ml). However, GPH-GM showed the weakestACE-inhibitory activity (56% ± 1.4% at 1.5 mg/ml). The differencesin ACE-inhibitory activities of the hydrolysates might be due to the dif-ferent molecular weights and amino acids sequences of ACE-inhibitorypeptides present in protein hydrolysates.

The IC50 values for ACE inhibition of all hydrolysates varied between0.73 and 1.33 mg/mL (Table 1). The IC50 value of GPH-TF (0.730 ±0.02 mg/ml) was lower than those of smooth hound muscle proteinhydrolysates generated by gastrointestinal proteases, whose presentedan IC50 values between 0.85 and 3.55 mg/ml (Bougatef et al., 2010),whereas it was higher (P b 0.05) than that of salmon (IC50 =0.038 mg/ml) (Ono, Hosokawa, Miyashita, & Takahashi, 2005), andsardine hydrolysates (IC50 = 0.082 mg/ml) (Matsufuji et al., 1994).

The obtained results suggest that GPH-TF and GPH-SH possiblycontained more potent ACE-inhibitory peptides than the otherhydrolysates.

3.6. Antioxidant activity of GPHs

Due to the diversity of oxidation processes and antioxidantaction of protein hydrolysates, the use of a single method to evaluatethe antioxidant activity cannot provide a clear idea about its real antiox-idant potential. The overall antioxidant action of protein hydrolysates ismost likely attributed to the cooperative effects of several mechanisms,including, metal ion chelation, free-radical scavenging and singuletoxygen quenching (Chen, Muramoto, Yamauchi, Fujimoto, & Nokihara,1998). Therefore, four chemical in vitro assays based on different antiox-idant mechanisms were used in this study to evaluate the antioxidantactivity of the different GPHs.

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3.6.1. DPPH radical-scavenging activityThe radical scavenging activities of the GPHs, tested at different

concentrations, are shown in Fig. 3A. All GPHs were able to scavengeDPPH radicals. Further, the scavenging activity of all samples increasedwith increasing concentrations of hydrolysates. Our findings are in linewith previous works reported by Ben Khaled et al. (2012) and Ktari etal. (2012) who reported that the DPPH-scavenging activity increasedwith increasing protein hydrolysates concentrations. GPH-GM exhibitedthe highest radical-scavenging activity (57 ± 0.32% at 5 mg/ml) follow-ed by the GPH-T (43 ± 0.47% at the same concentration) (P b 0.05),while, the lowest DPPH radical-scavenging activity was obtained withGPH-TF (17.5 ± 0.36%) (P b 0.05).

The obtained results clearly indicated the high hydrogen donatingability of golden mullet proteases hydrolysate. Hence, GPH-GM proba-bly contained potent peptides which acted as a good proton donorand could react with free radicals to convert them tomore stable prod-ucts and terminate the radical chain reaction (Wu, Chen, & Shiau, 2003).The scavenging activity of GPH-GM was lower than that of smoothhound protein hydrolysate obtained by treatment with low molecularweight alkaline protease from the same species (76.7% at 3 mg/ml)(Bougatef et al., 2009. The differences in the radical scavenging abilityof GPHsmay be attributed to the differences in amino acid compositionof peptides within protein hydrolysates. However, all hydrolysatesshowed lower radical-scavenging activity than BHA which exhibited100% activity at 2 mg/ml.

3.6.2. Lipid peroxidation inhibition assayPeroxidation of lipids is a complex process which refers to the oxida-

tive degradation of lipids that involves formation and propagation of lipid

Fig. 3. A) DPPH free-radical-scavenging activities of GPHs at different concentrations. B) Lipcontrol (2 mM). C) β-carotene bleaching inhibition of GPHs at 5 mg/ml. BHA was used as poEDTA was used as positive control (2 mM). All values are means ± standard deviation (SD)hydrolysates produced using smooth hound proteases, triggerfish proteases, golden mullet

radicals and lipid hydroperoxides formed as primary oxidation productsin the presence of oxygen (Ames, 1983).

The antioxidative activity of the five hydrolysates tested at 2 mg/ml,against theperoxidation of linoleic acid, was investigated and comparedto that of α-tocopherol which has been widely used as a naturalantioxidative agent.

The lipid peroxidation inhibition by the different hydrolysates in-creased with increasing incubation time and reached about 98% forthe most of hydrolysates after incubation for 9 days (Fig. 3B). After6 days of incubation, GPH-GM exhibited high lipid peroxidation inhi-bition (86.5%), which was slightly lower than that obtained withα-tocopherol (91.5%) at 2 mM (P b 0.05). These results are consistentwith those obtained by the DPPH radical-scavenging assay and con-firm the ability of GPHs to donate hydrogen atom to free radicalthus stopping the propagation chain reaction during lipid oxidationprocess.

3.6.3. β-carotene bleaching inhibition activityβ-carotene-linoleic bleaching inhibition assay simulates membrane

lipid oxidation and can be considered a good model for membranebased lipid peroxidation. In oil–water emulsion-based system, linoleicacid acts as a free radical generator that produces peroxyl radicalsunder thermally induced oxidation. The produced free radicals attackthe β-carotene chromophore resulting in bleaching effect, which canbe inhibited by a free-radical scavenger.

The antioxidative activities of GPHs measured by the β-carotenebleaching assay are represented in Fig. 3C. All hydrolysates inhibitedthe oxidation of β-carotene at different degrees. GPH-TF and GPH-Mshowed significantly (p b 0.05) higher antioxidant activities (64.8% and

id peroxidation inhibition assay of GPHs at 2 mg/ml. α-tocopherol was used as positivesitive control (2 mM). D) Metal-chelating activities of GPHs at different concentration.of three determinations. GPH-SH, GPH-TF, GPH-GM, GPH-G and GPH-T are goby proteinproteases, goby proteases and bovine trypsin, respectively.

Fig. 4. Digestive (A) and pH (B) stabilities of GPH-TF as monitored by β-carotenebleaching assay. Values are means ± standard deviation (SD) of three determinations.GPH-TF is goby protein hydrolysate produced using triggerfish proteases.

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55%, respectively; at 5 mg/ml). BHA displayed a better antioxidantactivity than all hydrolysates at the same concentration. This discrepancyin antioxidant activity of GPHs may be related to differences in thesequences of peptides and/or their molecular size. The antioxidantactivity index of peptides or proteins in the free radical-mediatedlipid peroxidation system is influenced by molecular size, chemicalproperties and electron transferring ability of amino acid residuesin the sequence (Qian, Jung, & Kim, 2008).

The results so obtained confirm that the antioxidant activity ofprotein hydrolysates is influenced by the type of proteases investigat-ed, since protease specificity affects the length of peptides as well astheir amino acids sequences.

3.6.4. Metal chelating activityTransition metals such as Fe2+ are well-known stimuli of lipid

peroxidation and their chelation helps to retard the peroxidationand subsequently prevent food rancidity. The reductive capacities ofGPHs were estimated using the method of potassium ferricyanide'sreduction. This method is widely used to assess the ability of an anti-oxidant to reduce an oxidant by donating an electron. It is based onthe principle that substances, which have reduction potential, reactwith potassium ferricyanide (Fe3+) to form potassium ferrocyanide(Fe2+), which then reacts with ferric chloride to form ferric ferrouscomplex that has an absorption maximum at 700 nm (Chung,Chang, Chao, Lin, & Chou, 2002).

The ferrous ion chelating activities of GPHs and EDTA used asreference chelating agent at different concentrations are shown inFig. 3D. The results showed that all hydrolysates, except that treatedwith smooth hound visceral proteases, at a concentration of 5 mg/ml,displayed between 90 and 97% chelating effects on ferrous ion. Further,results reported in Fig. 3D show that metal chelating activity of allhydrolysates increased with increasing concentrations of hydrolysates.Although the chemical EDTA exhibited the highest (P b 0.05) metalchelating ability, natural antioxidants are of growing interest. Severalstudies have shown that the presence of histidine at the N-terminal ofpeptide sequence was effective in metal ion chelation (Chen et al.,1998). The Fe2+ ion chelating activity obtained in this study might bedue to the presence of histidine residues (as evidenced by amino acidanalysis). Indeed, GPH-SH, which exhibited the lowest chelating activi-ty, had the lowest content of histidine. Additionally, Saiga, Tanabe, andNishimura (2003) reported that there is a direct correlation betweenthe presence of acidic and/or basic amino acids and the chelation ofmetal ions by carboxyl and amino groups in their side chains. It hasalso been reported that the scavenging of hydroxyl radicals by antioxi-dant was effective mainly via chelating of metal ions (Pena-Ramos &Xiong, 2002). The results obtained indicate that GPHs can exhibit anti-oxidant activities by capturing ferrous ions or other ions.

3.7. Stability in gastro-intestinal model system

The resistance of bioactive peptides against gastrointestinalproteases (such as pepsin, trypsin, chymotrypsin, etc.) was a pre-requisite for their action in vivo and exploitation for human nutri-tion as functional foods. Indeed, biopeptides which are resistantto digestion in the gastrointestinal tract could be absorbed intheir intact form through the intestine. Therefore, in this study,the stability of GPH-TF (10 mg/ml), with high antioxidant activity,in the presence of digestive enzymes and duodenal juice was stud-ied in order to predict their biological effects in vivo. Interestingly,as shown in Fig. 4A, the antioxidant activity, monitored by theβ-carotene-linoleate bleaching assay, increased slightly after pep-sin and duodenal digestion (P b 0.05). The enhancement of antiox-idant activity could be explained by the liberation of more potentpeptides which might enhance the protection of β-carotene fromdiscoloration. Nalinanon, Benjakul, Kishimura, and Shahidi (2011)also reported an increase in antioxidant activity of protein

hydrolysate from ornate threadfin bream prepared after being ingestedin the simulated model system. Additionally, Kittiphattanabawon,Benjakul, Visessanguan, and Shahidi (2012) reported that oxygen radi-cal absorbance capacity slightly increased after pepsin digestion of gel-atin hydrolysate from blacktip shark skin prepared using papaya latexenzyme. In another study, the antioxidant activity of the protein hy-drolysates from the muscle of brownstripe red sniper prepared byflavorzyme and alcalase increased after the gastro-intestinal digestionin tract model system (Khantaphant, Benjakul, & Kishimura, 2011).

The obtained results ensure that peptides, which exhibited antioxi-dant activities in vitro, are conserved and may display in vivo biologicaleffects.

3.8. Thermal and pH stability of selected GPH-TF

The processing stability of bioactive peptides after thermal treatmentis a prerequisite for their eventual incorporation in food formulations.Therefore, GPH-TF at 50 mg/ml was subjected for 240 min at 90 °C andthen the antioxidant activity was determined. The results showed thatheat treatment did not change the activity of GPG-TF (data not shown).

In addition, the pH of reaction media plays an important role indetermining the antioxidant activity of antioxidant compounds.Therefore, the effect of pH on the stability of peptide's antioxidantactivity was investigated. The results reported in Fig. 4B indicatedclearly that the inhibition of β-carotene discoloration by GHP-TFincreasedwith pH, and the relative activity at pH9.0was approximately143 ± 4%, compared with that of the sample without pH adjustment.Kittiphattanabawon et al. (2012) also reported that oxygen radicalabsorbance capacity (ORAC) and chelating activity of gelatin hydroly-sate with 40% DH from blacktip shark skin increased after pH adjust-ment. At pH 9.0, the metal chelating activity and the ORAC of gelatinhydrolysate increased by approximately 121% and 891%, respectively,compared with that observed in the sample without pH adjustment.The enhancement of antioxidant activity is possibly due to the

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changes in peptides, particularly at N- and C-terminal, mediated by pHadjustment.

3.9. Effect of GPH-TF on lipid peroxidation in turkey meat sausage

The stability of antioxidative peptides in GPH-TF against gastrointes-tinal proteases, heat treatment and in awide range of pH is favorable fortheir use as ingredients in special formulations. Indeed, several worksreported the improvement of some biological activities of foods uponincorporation of protein hydrolysates. (Segura-Campos, Salazar-Vega,Chel-Guerrero, & Betancur-Ancona, 2013). In this study, turkey meatsausagewas used as foodmodel to determinewhether the incorporationof GPH-TF, with high antioxidant activity, can effectively inhibit meatlipid peroxidation during storage.

Indeed, lipid oxidation is a prominent problem in food high in fat,and during storage oxidation secondary products formed may reactwith biomolecules and exert cytotoxic and genotoxic effects. Amongthese products there is malondialdehyde (MDA), which is widelystudied as a marker of oxidative stress and lipid peroxidation index(Fernandez, Pérez-Alvarez, & Fernandez-Lopez, 1997). MDA reactswith TBA to give the TBA reactive substances (TBARS) detectable byspectrophotometry at 532 nm.

In this study, the levels of TBARS were analyzed in turkey meatsausage prepared with or without antioxidant in terms of determina-tion the ability of GHP-TF to inhibit lipid peroxidation during foodstorage. As expected, the results showed that TBARS values of thecontrol increased as a function of storage time (Fig. 5). The valuewas 1.44 mg MDA/kg of cooked meat after 3 days of storage. A de-crease in TBARS values was observed after 14-days of storage. Inter-estingly, all concentrations of GPH-TF assayed inhibited the TBARSformation during storage. On storage day 3, all concentrations re-duced the meat lipid oxidation by more than 50% as compared tothe control. The TBARS values increased in turkey meat sausageadded with GPHs or vitamin C (p b 0.05). After 12 days of storage at4 °C, gradual decreases in TBARS values were observed.

The decrease in TBARS values was probably due to the loss of ox-idation products formed, particularly low molecular weight volatilecompounds. Indeed, MDA and other short-chain products of lipid ox-idation are not stable for a long period of storage. Oxidation of theseproducts yields alcohols and acids, which are not determined by theTBA test (Fernandez et al., 1997).

Similar studies showed that the TBARS of cooked comminuted porkwith and without antioxidants increased as the storage time increasedup to 7 days of storage and thereafter decreased until the end of storage

Fig. 5. Changes in thiobarbituric acid reactive substances (TBARS) values in the absenceand the presence of GPH-TF at different concentrations in turkey sausage model system.GPH-TF: goby protein hydrolysate treated with triggerfish proteases. Values are means ±standard deviation (SD) of three determinations.

(Kittiphattanabawon et al., 2012). In another study, Khantaphant et al.(2011) also reported a sharp increase in TBARS within the first 24 h ofincubationwhen hydrolysate from themuscle of brownstripe red snap-per was added in the lecithin liposome system, and thereafter a gradualdecrease in TBARS were observed.

It was noted that a meat system containing 0.01% of GPH had thesimilar TBARS levels compared with that containing 0.02% vitamin C(p b 0.05). Further, the addition of 0.02 and 0.04% of GPH-TF wasmore effective (p b 0.05) to reduce the mechanisms of peroxide autox-idation during turkey meat sausage storage than vitamin C. The resultsprovide evidence that GPH-TF is a great source of a natural antioxidantthat could replace vitamin C currently used as antioxidant in industryprocess.

4. Conclusion

The results of this study showed that goby proteins can be used toproduce hydrolysates with potent ACE-inhibitory peptides after treat-mentwith gastrointestinal proteases. Further, GPHswere found to exhibitto a variable extent antioxidant activities in different in vitro assaysystems. The differences in biological activities of the five GPHs areprobably due to the fact that peptides in different hydrolysatesmight be different in term of chain length and amino acid sequences.The antioxidant activity of GPH-TF, with high antioxidant activity,was conserved after gastrointestinal digestion in tract model system,and remained constant after heating for 240 min at 90 °C. In addition, itincreased in a wide range of pH (1.0–9.0) after incubation for 1 h.Further, when incorporated in turkey meat sausage GPH-TF efficientlyinhibited lipid peroxidation.

Based on the current results GPHs could be used as a promisingsource of ACE-inhibitory peptides and natural antioxidants in en-hancing biological properties of functional foods and in preventingoxidation reactions in food processing.

Acknowledgment

This work was funded by the Ministry of Higher Education andScientific Research-Tunisia.

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