68 Chiang Mai J. Sci. 2016; 43(1)

Chiang Mai J. Sci. 2016; 43(1) : 68-79http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Optimization of the Preparation of GelatinHydrolysates with Antioxidative Activity fromLizardfish (Saurida spp.) Scales GelatinSutee Wangtueai [a, b]*, Susanne Siebenhandl-Ehn [c] and Dietmar Haltrich [c]

[a] Division of Marine Product Technology, Faculty of Agro-Industry, Chiang Mai University, Mae-Hea,

Muang, Chiang Mai 50100, Thailand.

[b] Burapha University Sakaeo Campus, Watthananakhon, Sakaeo 27160, Thailand.

[c] Department of Food Science and Technology, BOKU University of Natural Resources and Life Sciences

Vienna, Muthgasse 18, 1190 Vienna, Austria.

*Author for correspondence; e-mail: sutee.w@cmu.ac.th

Received: 12 February 2015

Accepted: 23 June 2015

ABSTRACT

The enzymatic hydrolysis of fish-scale gelatin using papain to obtain hydrolysatepreparations with antioxidant properties was optimized using response surface methodology(RSM). The optimized parameters included enzyme-to-substrate ratio, reaction temperatureand hydrolysis time. The optimal conditions for high antioxidant activity were 3% enzyme-to-substrate ratio, reaction temperature of 55°C, and hydrolysis time of 3 h. The RSM modelgave predicted values of 61.7% for the 2, 2’-diphenyl-1-picrylhydrazyl (DPPH) radicalscavenging activity, 485 mM Trolox/g for the 2, 2’-azinobis[3-ethylbenzothiazoline-6-sulfonicacid] (ABTS) radical scavenging activity, and 9.2 mM Fe

2SO

4.7H

2O/g of fish-scale gelatin

hydrolysates (FSGH) for the ferric reducing antioxidant power (FRAP) assay. This wascorroborated by verification experiments under optimal conditions which gave an estimateddegree of hydrolysis (DH) of 73.3±0.5, DPPH radical scavenging activity of 64.1±0.9%, anABTS radical scavenging activity of 480±1 mM Trolox/g, and a FRAP value of 9.1±0.1 mMFe

2SO

4.7H

2O/g of FSGH. The functional properties of FSGH were determined. The solubility

was evaluated over a wide pH range of 2-10, which had a value with the range of 80-94%.The foam expansion and foam stability were evaluated with 0.5% concentration of FSGH.The FSGH had 8.67% foam expansion and 2.0-6.7% foam stability. In addition, the FSGHhad a 3.45% emulsion centrifugation stability (ECS) and a 0.73% emulsion thermal stability(ETS).

Keywords: fish scales, gelatin hydrolysates, papain, antioxidant activity, radical scavenging activity,ferric reducing activity

1. INTRODUCTION

The processing of fishery productstypically leads to large amounts of waste,and this is particularly true for the surimi

industry. Surimi production produces largequantities of by-products rich in collagensuch as fish heads, bones, skins, and scales,

Chiang Mai J. Sci. 2016; 43(1) 69

which can be used as potential raw materialsfor collagen and gelatin extraction [1].Typically, gelatin is produced from mammalianskins and bones. Because of the outbreakof mad cow disease (Bovine SpongiformEncephalopathy; BSE) in the 1980’s as wellas the unacceptability of gelatin producedfrom bovine and/or porcine sources forcertain groups of consumers for religiousreasons (e.g., Muslims, Jews and Hindus),the search for other gelatin raw materials asa source of gelatin been accelerating [2].An alternative to mammalian gelatin, whichis accepted by various religious groups, isfish gelatin. Various sources of fish processingwaste including skins, bones and/or scales,have been evaluated [1].

Fish scales contain various minerals,connective tissue proteins, lipids, andpigments. For example, dried lizardfishscales are high in ash and protein but containvery little lipid [3]. The main proteins infish scales are type I collagen, keratin, andmucin. Scales can be used to extract gelatin[4-6]. Information on gelatin extraction fromfish scales has been limited to fish scales ofAsian carp, lizardfish, and grass carp [3, 5, 7].

Enzymatic fish protein hydrolysatepeptides have antioxidant activity such as thehydrolyzed skin gelatin from Alaska pollack,catfish, sole and squid, with those from Alaskapollack having linoleic acid peroxidationinhibition and catfish, sole, and squid gelatinhydrolysates having antioxidant activity [8-10].Moreover, the skin gelatin hydrolysates fromhoki, cobia, tilapia, and bigeye snaper skinhave also been reported to have free-radicalscavenging activity [11-14].

The purpose of this work was to extractgelatin from fish scales, to study the effectsof key hydrolysis parameters (enzymeconcentration, temperature and time) on thedegree of hydrolysis and antioxidant activityof fish-scale gelatin hydrolysates (FSGH) as

well as to evaluate some functional propertiesof this FSGH.

2. MATERIALS AND METHODS

2.1 Raw Material PreparationLizardfish (Saurida spp.) scales were a

by-product obtained from a surimi processingplants (Kantrang Seafood Co. Ltd.) in Trangprovince, Thailand. Those scales wereremoved from fish by hand and pre-washingby tap water before subjected to blockfreezing. The frozen fish scales weretransported to the laboratory using truckwith freezer containers to control temperature(-18 to -20°C). The frozen fish scales werestored at -18 to -20°C prior to further use(not longer than 2 months). Before use,fish scales were thawed (kept in a refrigeratorat 9-10°C for about 24 h) and washed withby rinsing with tap water to remove anysuperfluous material.

2.2 Gelatin ExtractionGelatin was extracted from fish scales by

the method of Wangtueai and Noomhorm[3]. The thawed scales were treated with2 volumes (v/w) of 0.51% NaOH solutionat room temperature (about 30°C) for 3 h toremove the non-collagen protein and thesubcutaneous tissue after the scales becameswollen. After the alkali treatment, the scaleswere neutralized by washing under runningtap water until the pH became about 7(pH paper, Merck, Darmstadt, Germany).The scales were then given a final washwith distilled water to remove any residualmatter. The extraction was carried out indistilled water at 78.5°C for 3 h in a waterbath without stirring. A ratio of 1 part wetscales to 2 parts distilled water (w/v) was used.The coarse solids were removed by filtrationthrough two layers of cheesecloth, followedby filtration with Whatman No. 1 filter paper(Whatman Interntional Ltd., Maidstone,

70 Chiang Mai J. Sci. 2016; 43(1)

England). The clarified solutions were freeze-dried (Martin Christ GefriertrocknungsanlagenGmbH, Osterode am Harz, Germany) andground to obtain the gelatin powder, whichwas used (not longer than 3 month) withoutany other treatment.

2.3 Preparation of Gelatin HydrolysatesTwo g of fish-scale gelatin was dissolved

in 40 mL of 0.1 M sodium phosphate bufferpH 7.0, and treated with 1-3% (w/w) papain(Sigma-Aldrich, Vienna, Austria) at 45-55°Cfor 1-3 h. The individual conditions of theexperimental design are showed in Table 2.The hydrolysis reaction was done on ashaking water bath at 100 rpm (GLF 1092,Labortechnik, Burgwedel, Germany).Subsequently, papain was inactivated byheating the reaction mixture at 90°C for10 min in a water bath, and the mixture wasthen freeze-dried (Freezone, Labconco,Kansas City, MO, USA). The FSGH powderwas stored at -20°C until further use (notlonger than 3 month).

2.4 Degree of HydrolysisThe degree of hydrolysis (DH) was

estimated by using the trichloroacetic acid(TCA) method. The DH determination wasslightly modified from the method ofHettiarachchy et al. [15] and Chalamaiah et al.[21] which is based on the ratio of solubleprotein in 10% TCA to the total amountof protein in the samples. After hydrolysis,10 mL of hydrolysates were mixed with10 mL of 20% TCA. The mixture was stirredfor 1 min and then centrifuged at 10,000 × gfor 15 min (Eppendorf centrifuge 5430,Hamburg, Germany). The protein content ofthe supernatant was determined by theBiuret method [16]. The estimate of DHwas calculated using the following equation:

Degree of hydrolysis (%) = soluble protein in 10%TCA solution (mg) × 100

total protein in sample (mg)

2.5 DPPH Radical Scavenging ActivityThe determination of the 2, 2’-diphenyl-

1-picrylhydrazyl (DPPH) (Sigma-Aldrich)radical scavenging activity was done accordingto the method of Yang et al. [13], which is amodification of the original work by Shimadaet al. [17]. FSGH solutions (1.0 mL of 25mg/mL in H

2O) were added to 1.0 mL of

0.12 mM DPPH in 95% ethanol. This mixturewas then shaken vigorously using a Vortexmixer (IKA®Labortechnik, Janke & Kunkel,Germany) and kept in the dark at roomtemperature for 30 min. The mixture was thencentrifuged at 10,000 × g for 10 min beforedetermination of the absorbance of thesupernatant at 515 nm (Hitachi U-1100spectrophotometer, Tokyo, Japan). Thereaction blank was prepared in the samemanner, except that distilled water was usedinstead of the sample.

DPPH radical scavenging activity (%) = (A

blank - A

sample) × 100

Ablank

Where: Asample

is the absorbance value for1.0 mL of sample solution mixed with DPPHsolution and A

blank is the absorbance value for

1.0 mL of distilled water mixed with DPPHsolution. All experiments were carried out intriplicates.

2.6 ABTS Radical Scavenging ActivityThe 2, 2’-azinobis[3-ethylbenzothiazoline-

6-sulfonic acid] (ABTS.+) (Sigma-Aldrich)radical scavenging activity of FSGH wasdetermined following the modified methodsof Pellegrini et al. [18] and Moore et al. [19].

Chiang Mai J. Sci. 2016; 43(1) 71

The ABTS activated solution was preparedby mixing 7.0 mM ABTS solution with 140mM potassium persulphate solution (0.1:5)and kept in the dark at room temperaturefor 12-16 h before use. The working solutionwas obtained by mixing 100 mL ABTSactivated solution with 10 mL ethanol toobtain an absorbance of 0.72±0.02 unitsat 734 nm in a 1 cm cuvette. The sample(120 mL of 25 mg/mL in H

2O) was mixed

with 1.5 mL of ABTS working solution,and then shaken vigorously for 30 s beforecentrifugation at 10,000 × g for 10 min.The absorbance of the supernatant wasthen measured at 734 nm. An equivalentvolume of distilled water was used insteadof sample for the reaction blank. Trolox(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; Fluka Chemie, Steinheim,Switzerland) from 10 to 300 μM was usedto prepare the standard curve. The activity isexpressed as mM of Trolox equivalents/g ofFSGH. All testes were done in triplicates.

2.7 Ferric Reducing Antioxidant Power(FRAP)

The FRAP assay is based on the reductionof the ferric tripyridyltriazine (Fe(III)-TPTZ)complex to the ferrous form at low pH,which is monitored at 595 nm [20]. Theworking FRAP solution contained 10volumes of 300 mM acetate buffer (pH 3.6),1 volume of 10 mM TPTZ solution in40 mM HCl, and 1 volume of 20 mMFeCl3.

.6H2O solution. The working solution

was always freshly prepared. This mixturewas then incubated at 37°C for 30 min and isreferred to as the FRAP solution. Sample(0.2 mL of 25 mg/mL in H

2O) was mixed

with 1.3 mL of working FRAP solution in anEppendorf tube (Eppendorf, Hamburg,Germany), mixed by Vortexing and kept for30 min in the dark. The mixture was thencentrifuged at 10,000 × g for 10 min, and the

absorbance of the supernatant was measuredat 595 nm. FeSO

4.7H

2O ranging from 25 to

800 μM was used to prepare the standardcurve. The activity was defined as mMFeSO

4.7H

2O equivalents/g of FSGH. All

experiments were done in triplicate.

2.8 Functional Properties of FSGH2.8.1 Protein solubility

Protein solubility was determined bymethod of Chalamaiah et al. [21]. Briefly,200 mg of FSGH was dispersed in 20 mLof distilled water and the pH of the mixturewas adjusted to 2, 4, 6, 8, and 10 with 0.5 NHCl or 0.5 N NaOH. The mixture was stirredat room temperature (about 25°C) for 30 minand centrifuged at 4,500 × g for 30 min.The protein content of the supernatantwas determined using the Biuret method [16].The protein solubility was calculated asfollows:

Protein solubility index (%) = (A/B) × 100

Where: A is protein content in thesupernatant and B is total protein content inthe sample

2.8.2 Foaming propertiesThe foaming property of FSGH was

obtained according the method of Gim nezet al. [10]. The 50 mL of protein hydrolysatessolution (0.5%, w/v) was homogenized(Ystral, Ystral gmbh, Ballrechten-Dottingen,Germany) in a 100 mL cylinder with a speedof 16,000 rpm to incorporate the air for1 min. The total volume was measured at0, 1, 5 10, 20, and 30 min after whipping.Foam capacity was expressed as foamexpansion at 0 min, which was calculatedaccording the following equation:

Foam expansion (%) = [(A – B)/B] × 100

72 Chiang Mai J. Sci. 2016; 43(1)

Where: A is the volume after whipping(mL) and B is the volume before whipping(mL)

Foam stability (%) = [(At - B)/B] × 100

Where: At is the volume at 1, 5 10, 20,

and 30 min after whipping (mL) and B is thevolume before whipping (mL)

2.8.3 Emulsifying propertiesThe emulsifying properties were

determined according to the method ofZhao et al. [22] with a slight modification.The mixtures included 50 mL pure sunfloweroil (Osana, NT Kft, Kiskunf legyh za,Hungary) and 50 ml of 0.5% (w/v) FSGHsolution. The mixtures were homogenized ata speed of 16,000 rpm for 2 min to form anemulsion. The emulsion was then centrifugedat 1,500 × g for 5 min. The calculation of theemulsion centrifugation stability (ECS) wasobtained as follows:

ECS (%) = (Ve1

/Ve0

) × 100

Where: Ve1

is measuring the volume ofemulsion remaining after centrifugation andV

e0 is measuring the volume of the emulsion

before centrifugationThe emulsion samples were then heated

to 80°C in a water bath (GFL, LabortechnikmbH) for 30 min and cooled down in waterfor 15 min. After cooling, the emulsionswere centrifuged at 1,500 × g for 5 minand recorded the volume of the remainingemulsified fraction (Ve2

). The calculation ofthe emulsion thermal stability (ETS) wasobtained as follows:

ETS (%) = (Ve2

/Ve0

) × 100

2.9 Experimental DesignThe optimization of the hydrolysis

conditions was done using response surfacemethodology (RSM) with a three-level andthree-factor Box-Behnken Design [23].Three factors of enzyme-to-substrate ratio(X

1), hydrolysis time (X

2), and reaction

temperature (X3) were selected for the

optimization, while the responses were theDH (%, Y

1), DPPH radical scavenging activity

(%, Y2), ABTS radical scavenging activity

(mM Trolox/g of FSGH, Y3), and FRAP

assay activity (mM FeSO4.7H

2O/g of FSGH,

Y4). Table 1 shows the coded and real

values of each level of the three factors.The experiments consisted of 15 treatmentsincluding twelve incomplete factorial pointsand three replicates of the central point asgiven in Table 2. Minitab statistical software(Trial version 16, Minitab Inc., State College,PA, USA) was used to design the experiment,and for data analysis and model building.The fitting was done to a second-order modelfor each response. This model can be expressedwith the coded variables (X1

, X2, and X

3) using

the following equation:

Y = B0+B

1X

1+B

2X

2+B

3X

3+B

11X

12 +

B22

X22 +B

33X

32+B

12X

1X

2 +B

13X

1X

3+B

23X

2X

3

where Y represented the estimatedresponse, B

0 represents the equation

parameters for the constant term, B1, B

2 and

B3 represents the linear coefficients, B

11, B

22

and B33

represents the quadratic coefficients,and B

12, B

13 and B

23 represents the interaction

coefficients.After the multifactor analysis of variance

and the second-order model predictiondeterminations, the optimal hydrolysisconditions were obtained by the desirabilityfunction approach for each Y using thestatistical software.

Chiang Mai J. Sci. 2016; 43(1) 73

3. RESULTS AND DISCUSSIONS

3.1 Response Surface ModelFSGH was prepared by enzymatic

hydrolysis using an optimization procedure.Three different parameters, the enzyme-to-substrate ratio (%, X

1), temperature (°C, X

2),

and reaction time (h, X3) were chosen as

independent variables. The optimal conditionfor the production of hydrolysates fromlizardfish-scale gelatin was then determinedby response surface methodology (RSM).Four responses (DH, DPPH, ABTS, andFRAP) were selected as the dependentvariables. Experimental runs were randomized

Table 1. The experimental design and the values of the independent variables in the Box-Behnken design for the production of FSGH.

Table 2. Treatments for optimizing gelatin hydrolysates from fish-scale gelatin together withexperimental value according to the second-order response surface models.

Note: X1= enzyme/substrate ratio (%, w/w); X

2 = reaction temperature (°C); X

3 reaction

time (h); DH = degree of hydrolysis; DPPH = 2,2’-diphenyl-1-picrylhydrazyl radical scavengingactivity; ABTS = 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity;FRAP = ferric reducing antioxidant power assay.

Independent variables

Enzyme/substrate ratio (%, w/w): X1

Temperature (°C): X2

Time (h) : X3

levels-11451

02552

+13653

Treatments

123456789101112131415

FactorsX

1

131313132222222

X2

454565655555555545654565555555

X3

222211331133222

DH(%)

73±664±270±165±273±466±372±364±370±270±267±268±269±168±167±1

DPPH(%)

55.5±1.157.7±0.650.3±1.560.8±0.450.7±5.062.1±0.655.3±0.959.7±0.459.1±0.358.3±0.362.0±0.2

60.2±0.0859.7±0.360.2±0.5

60.1±0.35

VariablesABTS

(mM Trolox/g offish-scale gelatin

hydrolysate)461±2482±2471±3476±1469±3482±1469±3484±1468±3468±1469±3478±2481±3478±1479±2

FRAP(mM FeSO

4.7H

2O/g

of fish-scale gelatinhydrolysate)6.13±0.208.04±0.065.21±0.188.42±0.306.51±0.417.73±0.216.88±0.269.34±0.657.49±0.128.14±0.308.14±0.337.33±0.217.22±0.306.85±0.107.87±0.20

74 Chiang Mai J. Sci. 2016; 43(1)

to minimize the effects of unexpectedvariability in the observed responses. Theregression coefficients for the second-orderresponse surface models and the mathematicalmodels representing the DH, and antioxidantactivity (DPPH, ABTS, and FRAP) of gelatinhydrolysates are showed in Table 3 andTable 4, respectively. The coefficients ofdetermination (R2) for each of the 4 variableswere greater than 0.9, which showed thatthe regression models were suitable [23].

Analysis of variance (ANOVA) showedthat the models for DH, DPPH, ABTS,and FRAP of gelatin hydrolysates weresignificant (P ≤ 0.05) at the 95% probabilitylevel (Table 5). The contribution of all ofthe linear terms to the model was highlysignificant (P ≤ 0.01). Moreover, the lack offits for all of the models was not significant(P > 0.05), which suggesting the models wereappropriate.

Table 3. Regression coefficients of the second-order response surface models for DH, DPPH,ABTS and FRAP.

Note: X1= enzyme/substrate ratio (%, w/w); X

2 = reaction temperature (°C); X

3 = reaction

time (h); DH = degree of hydrolysis; DPPH = 2,2’-diphenyl-1-picrylhydrazyl radical scavengingactivity; ABTS = 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity;FRAP = ferric reducing antioxidant power assay.

Table 4. Response surface model for production of FSGH.

Note: X1= enzyme/substrate ratio (%, w/w); X

2 = reaction temperature (°C); X

3 = reaction

time (h); DH = degree of hydrolysis; DPPH = 2,2’-diphenyl-1-picrylhydrazyl radical scavengingactivity; ABTS = 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity;FRAP = ferric reducing antioxidant power assay.

Parameter

B0

B1

B2

B3

B11

B12

B13

B22

B23

B33

Term

InterceptX

1

X2

X3

X12

X1X

2

X1X

3

X22

X2X

3

X32

DH

Coefficient68.2-3.56

-0.049-0.8790.0381.06

-0.377-0.1300.4050.470

P-value0.0000.0000.8900.0470.9420.0750.4630.8030.4320.384

DPPH

Coefficient58.34.21

-0.6151.05-2.281.51

-0.383-0.780-0.1630.811

P-value0.0000.0000.1700.0410.0100.0390.5120.2260.7770.211

ABTS

Coefficient4796.781.671.70

-0.728-4.220.593-5.9142.37-2.73

P-value0.0000.0000.0650.0610.5160.0080.5800.0020.0640.047

FRAP

Coefficient7.311.10

-0.0890.226-0.2630.3230.307-0.101-0.3660.564

P-value0.0000.0020.6510.2790.3810.2730.2960.7270.2230.095

Response DH

DPPH

ABTS

FRAP

Quadratic polynomial modelY

1 = 68.2 - 3.56X

1- 0.049X

2 - 0.879X

3 + 0.038X

12 - 0.130X

22 +

0.047X32 +1.06X

1X

2 - 0.377X

1X

3 + 0.045X

2X

3

Y2 = 58.3 + 4.21X

1-0.615X

2 + 1.05X

3- 2.28X

12 - 0.780X

22 + 0.811X

32

+ 1.51X1X

2 - 0.383X

1X

3 - 0.163X

2X

3

Y3 = 479 + 6.78X

1+ 1.67X

2 + 1.70X

3- 0.728X

12 - 5.91X

22 - 2.73X

32

- 4.22X1X

2 + 0.593X

1X

3 + 2.37X

2X

3

Y4 = 7.31 + 1.10X

1 - 0.089X

2 + 0.226X

3 - 0.263X

12 - 0.101X

22 +

0.564X32 +0.323X

1X

2 + 0.307X

1X

3 - 0.366X

2X

3

R2

0.962

0.970

0.970

0.904

P-value0.005

0.003

0.003

0.042

Chiang Mai J. Sci. 2016; 43(1) 75

3.2 Optimization of ResponsesThe optimal conditions for the different

responses were calculated using the desirabilityfunction of the statistical software with theresponses to be maximized. The highest DHfor FSGH was calculated to be 1% enzyme-to-substrate ratio at 45°C for 1 h, whichgave a predicted value of 74.1% DH. Theoptimal antioxidant conditions using themultiple response optimizer program.The optimization parameters for highestantioxidant activity included DPPH, ABTS,and FRAB radical scavenging activity were3% (w/w) enzyme-substrate ratio at 55°Cfor 3 h. The predicted values of DPPH,ABTS radical scavenging activity and the

FRAP assay were 61.7%, 485 mM Trolox/gof FSGH, and 9.2 mM FeSO4

.7H2O/g of

FSGH, respectively. The predicted valuesshowed a 0.82 composite desirability. Toconfirm the predicted values, FSGH wereprepared using the optimal condition of themodel and the DPPH, ABTS, and FRAPradical scavenging activity were determined.Those were 64.1 ± 0.9%, 480 ± 1 mMTrolox/g of FSGH, and 9.1 ± 0.1 mMFeSO4

.7H2O/g of FSGH, respectively, which

is very close to the predicted values of thesample.

3.3 Response Surface PlotFigure 1 shows the estimated response

Table 5. Analysis of variance (ANOVA) for the second-order response surface model forDH, DPPH, ABTS and FRAP.

Note: DH = degree of hydrolysis; DPPH = 2,2’-diphenyl-1-picrylhydrazyl radical scavengingactivity; ABTS = 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity;FRAP = ferric reducing antioxidant power assay.

Response

DH

DPPH

ABTS

FRAP

SourceRegressionResidual ErrorLack-of-FitPure ErrorTotalRegressionResidual ErrorLack-of-FitPure ErrorTotalRegressionResidual ErrorLack-of-FitPure ErrorTotalRegressionResidual ErrorLack-of-FitPure ErrorTotal

DF953214953214953214953214

SS1154.493.570.9131191885.905.610.28219465720.114.75.3367713.01.380.8490.53514.4

MS12.70.8971.190.456

20.91.181.870.141

73.04.014.912.66

1.450.280.2830.267

F14.2

2.61

17.7

13.3

18.2

1.84

5.23

1.06

P-value0.005

0.289

0.003

0.071

0.003

0.371

0.042

0.519

76 Chiang Mai J. Sci. 2016; 43(1)

function and the effect of the independentvariables (enzyme to substrate: X

1,

temperature: X2, and reaction time: X

3) on the

responses (DH, DPPH, ABTS, and FRAP).The effects of the dependent variables onthe DH are shown in Figure 1(A), with X

1

and X3 as the main effects on DH. When the

coded values of the X1 and X

3 independent

variables were close to minus one (1 h of X1

and 1% enzyme-to-substrate ratio of X3), the

DH increased indicating greater proteolysis.The enzymatic hydrolysis process dependson the availability of susceptible peptidebonds and the physical structure of theprotein molecules, with the concentration ofhydrolysable bonds mainly controlling thehydrolysis rate in papain-assisted hydrolysis[23, 24].

Figure 1. Response surface plots of the combined effect of the variables on the degree ofhydrolysis (DH), the 2,2’-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH), the2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity (ABTS), andferric reducing antioxidant power assay (FRAP) of fish-scale gelatin hydrolysates: X

1 (enzyme-

to-substrate ratio, %), X2 (temperature, °C), and X

3 (time, h).

Chiang Mai J. Sci. 2016; 43(1) 77

The effects of the dependent variableson the antioxidant activity (DPPH, ABTSradical scavenging activity, and FRAP) areshown in Figure 1(B-D). DPPH radicalscavenging activity was increased enzymesubstrate ratio and time. Temperature hadan impact on the DPPH radical scavengingactivity as well, which is illustrated by theincrease of the activity when the temperaturewas at 55°C. The ABTS radical scavengingactivity was affected by the enzyme-to-substrate ratio as the main effect. When theratio was increased, the ABTS radicalscavenging activity increased as well. Whenthe hydrolysis temperature and time increasedfrom 45 to 55°C and 1 to 2 h, respectively,the ABTS radical scavenging activity increased.Interestingly, the effect of the independentvariables on FRAP differed from the otherdependent variables. As the hydrolysis timeincreased from 1 to 2 h, FRAP decreased.When the time increased from 2 to 3 h, FRAPincreased. The effects of enzyme-to-substrateratio and temperature on FRAP were similarto the ABTS radical scavenging activity.

Mendis et al. [11] reported that theconditions of enzymatic preparation ofgelatin peptides significantly influencedthe molecular structure and molecular massof the bioactive peptides obtained, which inturn relates to their functional properties.Therefore, the most important aspect formodifying the functional properties offood protein and peptides is the enzymatichydrolysis method used.

3.4 Functional Properties of FSGHFSGH was prepared using the optimum

conditions and its functional propertiesdetermined. The DH of the hydrolysate was73.3 ± 0.5. The solubility of FSGH wasevaluated over a wide pH range of 2-10(Figure 2). The solubility was high (80-94%)over the entire range studied. The increasing

of solubility was positively correlated frompH 4. Chalamaiah et al. [21] reported thatprotein hydrolysates from meriga eggwere soluble in the pH range of 2-12 greaterthan 72% protein solubility while sole-skinand squid-skin gelatin hydrolysates hadover 95% protein solubility [10]. Moreover,Ktari et al. [25] reported that proteinshydrolysates from the muscles of zebrablenny was over 80% soluble and over pH 6the solubility rapidly increased up to pH 10.This high solubility of hydrolysates mightbe due to the generation of low molecularweight peptides with an increase in surfacehydrophilic and have proportionally morepolar groups [10, 25, 26]. Protein solubilityis one of the benefits of hydrolysis. Thehigh solubility over a wide pH range suggeststhat it may be appropriate in many foodformulation applications [21, 26].

Figure 2. Protein solubility of FSGHat different pH values. Bars representthe standard deviation from triplicatedeterminations and the different letterindicate significant difference at P ≤ 0.05.

The foam expansion and foam stabilitywere evaluated with 0.5% concentration ofFSGH (Table 6). The foam expansion washigher than previously reported for sole andsquid-skin gelatin hydrolysate at the sameconcentration of hydrolysate [10]. A higheraverage molecular weight for the polypeptidechains and increasing surface hydrophobicity

78 Chiang Mai J. Sci. 2016; 43(1)

are generally positively correlated to foamstability and foaming characteristics thatresult in more incorporation of air and water[25, 26]. Foam stability was monitored for30 min after whipping. The foam stabilitywas in the range of 2.0-6.7% after 1 min ofwhipping and decreased with time (Table 6).The occurring of foam stability, themolecule of protein should form continuousintermolecular polymers enwrapping airbubbles then the important factors for theproduction of stable foams are intermolecularcohesiveness and elasticity. Whereas, a smallermolecule of protein peptides do not havestrength to hold stable foam and results indecreasing of foam stability [25].

The FSGH had a 3.45% emulsioncentrifugation stability (ECS) and a 0.73%emulsion thermal stability (ETS) as show inTable 6. The stability of emulsion containingprotein following homogenization depends

Table 6. Foaming and emulsifying propertiesof FSGH with 0.5% (w/v) concentration.

*Values expressed on average of triplicateanalysis with ± SD.

on the oil droplets absorbing protein on theoil surface, which prevents coalescence ofthe oil droplets [10, 25, 27]. The lowemulsion stability might be due to themolecular weight of the FSGH.

4. CONCLUSIONS

Gelatin from lizardfish scales can beused to produce gelatin hydrolysates withhigh antioxidant activity. Using RSM, theoptimal conditions for FSGH preparationwith high antioxidant activity were foundto be a 3% enzyme-to-substrate ratio at atemperature of 55°C and a hydrolysistime of 3 h. These conditions shouldgive FSGH with predicted values of theDPPH radical scavenging activity of 61.7%,ABTS radical scavenging activity of 485 mMTrolox/g of FSGH, and FRAP assay of9.2 mM Fe2

SO4.7H

2O/g of FSGH. Values

close to these were obtained experimentallywhich confirmed the results predicted bythe model and shows the usefulness ofstatistical modeling. Moreover, FSGH had agood solubility and potentially acceptablefoaming and emulsion properties at lowprotein concentration.

ACKNOWLEDGEMENTS

This research was supported by theASEAN-European University NetworkASEA-Uninet, the Austrian Governmentand the Austrian Academic Exchange Service

AD through a Technology Southeast AsiaGrant. The authors wish to thank KantrangSeafood Co. Ltd., Trang province, Thailandfor supplying the fish scales.

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PropertiesFoam expansionFoam stability(after 1 min)Foam stability(after 5 min)Foam stability(after 10 min)Foam stability(after 20 min)Foam stability(after 30 min)Emulsion centrifugationstability (ECS)Emulsion thermalstability (ETS)

%*8.67±1.156.67±1.15

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