Food Chemistry xxx (2011) xxxxxx

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Indigenous proteases in the skin of unicorn leatherjacket (Alutherus monoceros) and their inuence on characteristic and functional properties of gelatin
Mehraj Ahmad a, Soottawat Benjakul a,, Mahmoudreza Ovissipour b, Thummanoon Prodpran c
a

Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Department of Fisheries, Gorgan Agricultural Sciences and Natural Resources, University of Gorgan, Iran c Department of Material Product Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
b

a r t i c l e

i n f o

a b s t r a c t
Indigenous proteases in the skin of unicorn leatherjacket (Alutherus monoceros) were characterised using autolytic study. Maximised autolysis was found at pH 7 and 50 C. Autolysis was markedly inhibited by 0.04 mM soybean trypsin inhibitor (SBTI), suggesting that heat activated serine protease was predominant in the skin. The impact of indigenous proteases on the properties of gelatin extracted from unicorn leatherjacket skin was investigated. Gelatin was extracted from unicorn leatherjacket skin using distilled water at 50 C for 12 h in the presence and absence of 0.04 mM SBTI. In the presence of SBTI, the degradation was markedly inhibited, but a lower gelatin extraction yield was obtained (P < 0.05). Extracted gelatins contained a1 and a2 chains as the predominant components with some degradation peptides. FTIR spectra indicated a greater loss of molecular order of the triple helix and a higher degradation was found in gelatin extracted in the absence of 0.04 mM SBTI. The net charge of gelatin samples extracted with and without 0.04 mM SBTI became zero at pHs of 8.45 and 7.31, respectively, as determined by f-potential titration. Higher gel strength (320.68 3.02 g) was obtained in gelatin extracted with SBTI, compared with that of gelatin extracted without SBTI (288.63 1.44 g). High emulsifying activity index but lower emulsifying stability index was observed in the former. Therefore, heat-activated serine protease was involved in the degradation of gelatin molecules, thereby affecting the yield, proteinaceous components and properties of gelatin from unicorn leatherjacket skin. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 3 October 2010 Received in revised form 18 November 2010 Accepted 7 January 2011 Available online xxxx Keywords: Unicorn leatherjacket skin Proteolysis Serine protease SBTI Gelatin

1. Introduction Proteolysis induced by heat-activated and heat-stable indigenous proteases associated with skin matrix can contribute to the destabilisation as well as disintegration of collagen structure by disrupting the intra- and intermolecular cross-links (Wu et al., 2008). Collagenases have the unique ability to degrade the major structural extracellular matrix proteins, especially the triple helical strand of type I and type II collagens at Gly775-Leu (Ile)776. After these initial and specic cleavages, 3=4 - and 1=4 -cleavage products are formed, and collagen is spontaneously denatured (helix-to-coil transition) to gelatin (Miller et al., 1976). Collagenases are classied into two major groups, metallocollagenases and serine collagenases (Aoki, Ahsan, Matsuo, Hagiwara, & Watabe, 2003). Furthermore, non-collagenase proteinases can cleave the collagen molecule in the telopeptide region and contribute to hydrolysis of the collagen molecule by disrupting the regions, in which intermolecular cross-links are formed (Bornstein & Traus, 1979).

Corresponding author. Tel.: +66 7428 6334; fax: +66 7455 8866.
E-mail address: soottawat.b@psu.ac.th (S. Benjakul). 0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.01.032

Heat-activated serine protease in bigeye snapper skin was involved in the drastic degradation of the b- and a-chains of the gelatin extracted at 60 C (Intarasirisawat et al., 2007). These enzymes are bound with matrix components such as collagens (Woessner, 1991). The proteolytic degradation of high molecular weight components caused by indigenous proteases during extraction of gelatin at high temperature resulted in adverse effects on gel-forming properties of resulting gelatin (Intarasirisawat et al., 2007). The proteolytic breakdown of collagen structure is most likely related to the disintegration of connective tissues, which has been implicated in quality deterioration of products. Gelatin structures with large amount of high molecular weight components, like c-, b, and a-chains, have been known to possess the maximal functional properties including gelation (Kittiphattanabawon, Benjakul, Visessanguan, & Shahidi, 2010), lm forming ability (Hoque, Benjakul, & Prodpran, 2010), etc. Therefore, it was necessary to produce the gelatin with negligible hydrolysis of peptides for further applications in pharmacy, medicine and food industry. Hence, the use of an appropriate protease inhibitor can be an effective means to obtain the gelatin with limited or negligible hydrolysis of peptides. Nevertheless, no information regarding the characteristics of indigenous proteases in the skin of unicorn

Please cite this article in press as: Ahmad, M., et al. Indigenous proteases in the skin of unicorn leatherjacket (Alutherus monoceros) and their inuence on characteristic and functional properties of gelatin. Food Chemistry (2011), doi:10.1016/j.foodchem.2011.01.032

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leatherjacket (Alutherus monoceros) and their role in properties of gelatin extracted has been reported. Unicorn leatherjacket is an economically important species commonly used for frozen llet production. Thick skin of this species, generated during deskinning process, can serve as the promising raw material for gelatin extraction. Therefore, the objectives of this investigation were to study the autolysis of skin from unicorn leatherjacket and to elucidate their impact on gelatin extraction and characteristics of resulting gelatin. 2. Materials and methods 2.1. Chemicals 2,4,6-Trinitrobenzenesulfonic acid (TNBS), sodium sulphite, Lleucine, bovine serum albumin, ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline monohydrate, ethylene-bis (oxyethylenenitrilo) tetraacetic acid (EGTA), trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane (E-64), iodoacetic acid, pepstatin A, phenylmethanesulfonyl uoride (PMSF), soybean trypsin inhibitor (SBTI), b-mercaptoethanol (b-ME) and type I collagen from calf skin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). FolinCiocalteus phenol reagent and phosphoric acid were purchased from Merck (Darmstadt, Germany). Sodium dodecyl sulphate (SDS), N,N,N0 ,N0 -tetramethyl ethylene diamine (TEMED) and Coomassie blue R-250 were procured from Bio-Rad Laboratories (Hercules, CA, USA). All chemicals were of analytical grade. 2.2. Collection and preparation of sh skin The skin of unicorn leatherjacket (A. monoceros) was obtained from Sea Wealth Frozen Food Co., Ltd., Songkhla, Thailand. Upon arrival to the Department of Food Technology, Prince of Songkla University, Hat Yai, the skin was cleaned and washed with iced tap water (02 C). Prepared skin was then cut into small pieces (0.5 0.5 cm2), placed in polyethylene bags and stored at 20 C until use. The storage time was less than 2 months. 2.3. Pretreatment of unicorn leatherjacket skin Prepared skin was pretreated following the method of Ahmad and Benjakul (2010a), with a slight modication. To remove noncollagenous proteins, the prepared skin was mixed with 0.1 M NaOH at a skin/alkali solution ratio of 1:20 (w/v). The mixture was stirred for 6 h at 4 C using an overhead mechanical stirrer (W20.n, IKA-Werke GmbH & CO.KG, Stanfen, Germany) at a speed of 300 rpm. The alkali solution was changed every 2 h. The samples were then washed with iced tap water until neutral or faintly basic pH was obtained. Thereafter, the prepared skin was swollen by mixing the skins with 0.2 M phosphoric acid at a ratio of 1:10 (w/v). The mixture was stirred for 24 h at 4 C. Finally, the swollen skin was washed thoroughly with iced tap water until neutral or faintly acidic pH of wash water was obtained. The pretreated skin was stored at 20 C until use. 2.4. Autolysis study of pretreated unicorn leatherjacket skin Prior to study, the frozen pretreated skin was powderised in liquid nitrogen using a blender (Model MX-T2GN, National, Taipei, Taiwan). The powder obtained was used for autolysis study. 2.4.1. Temperature prole Autolytic activity was assayed according to the method of Morrissey, Wu, Lin, and An (1993), with a slight modication. Pretreated skin powder (3 g) was added to 9 ml of McIlvaines buffer

(0.2 M Na-phosphate and 0.1 M Na-citrate), pH 7.0. The mixture was homogenised for 2 min at a speed of 11,000 rpm using a homogeniser (Model T25 basic, IKA, Labortechnik, Selangor, Malaysia). The resulting homogenate was incubated at different temperatures (40, 45, 50, 55, 60, 70, 80 and 90 C) for 30 min in a temperature-controlled water bath (Memmert, Schwabach, Germany). Autolytic reaction was terminated by addition of 18 ml of 10% SDS (85 C). The reaction mixture was further incubated at 85 C for 1 h in a temperature-controlled water bath. The supernatant was obtained by centrifuging at 8500g for 15 min using a microcentrifuge (Model MIKRO20, Hettich Zentrifugen, Germany) and was subjected to sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDSPAGE) analysis. Samples were also determined for free amino group content. 2.4.2. pH prole Autolytic activity of pretreated skin was determined at various pHs using different buffers at 50 C for 30 min. Buffers used included 50 mM maleate buffer for pH 1; McIlvaines buffer (0.2 M Na-phosphate and 0.1 M Na-citrate) for pHs 26, 50 mM TrisHCl buffer for pHs 79 and 0.1 M Na2HPO4Na2B4O7 buffer for pH 10. After the incubation at 50 C for 30 min, the autolysis was terminated and was monitored in the same manner as the temperature prole study. 2.4.3. Inhibitor study Pretreated skin powder (0.5 g) was homogenised with 1.5 ml of McIlvaines buffer (pH 7) at a speed of 11,000 rpm for 2 min using a homogeniser. The homogenate was mixed with 2 ml of various protease inhibitors to obtain the different nal concentrations. Those inhibitors included EDTA (10 mM), EGTA (10 mM), iodoacetic acid (10 mM), E-64 (1 mM), pepstatin A (1 mM) and 1,10-phenanthroline (10 mM), PMSF (1 mM) and SBTI (0.04 mM). The mixtures were allowed to stand in ice for 2 h, followed by incubation at optimal temperature (50 C) for 30 min. The reaction was terminated by addition of 2 ml of 10% (w/v) SDS (85 C). After complete solubilisation, the autolytic pattern was then analysed by using SDSPAGE. The control was performed in the same manner, but deionised water was added instead of protease inhibitor solution. 2.4.4. Determination of free amino group content Free amino group content was determined following the method of Benjakul and Morrissey (1997). Properly diluted samples (125 ll) were mixed thoroughly with 2.0 ml of 0.2125 M phosphate buffer, pH 8.2, followed by the addition of 1.0 ml of 0.01% TNBS solution. The mixtures were then placed in a temperature controlled water bath at 50 C for 30 min in the dark. The reaction was terminated by adding 2.0 ml of 0.1 M sodium sulphite. The mixtures were cooled down at room temperature for 15 min. The absorbance was measured at 420 nm using a double beam spectrophotometer (Model UV-1800, Shimadzu, Kyoto, Japan) and free amino group content was expressed in terms of L-leucine. 2.4.5. SDSpolyacrylamide gel electrophoresis (SDSPAGE) Protein patterns were determined using SDSPAGE according to the method of Laemmli (1970) using a 4% stacking gel and a 7.5% separating gel. Samples were mixed at 1:1 (v/v) ratio with the sample buffer (0.5 M TrisHCl, pH 6.8, containing 4% SDS, 20% glycerol). Samples (15 lg) were loaded onto the gel. After electrophoresis using 15 mA/gel (Mini Protein II, Bio-Rad, Hercules, CA, USA), the gel was stained with 0.05% (w/v) Coomassie blue R-250 in 15% (v/v) methanol and 5% (v/v) acetic acid and destained with 30% (v/v) methanol and 10% (v/v) acetic acid. Type I collagen was used as the standard.

Please cite this article in press as: Ahmad, M., et al. Indigenous proteases in the skin of unicorn leatherjacket (Alutherus monoceros) and their inuence on characteristic and functional properties of gelatin. Food Chemistry (2011), doi:10.1016/j.foodchem.2011.01.032

M. Ahmad et al. / Food Chemistry xxx (2011) xxxxxx

2.5. Study on the impact of indigenous proteases on extraction and characteristics of gelatin from unicorn leatherjacket skin To inhibit indigenous serine proteases, the pretreated skin was mixed with 0.04 mM SBTI solution at a pretreated skin/SBTI solution ratio of 1:5 (w/v). The mixture was stirred for 24 h at 4 C using an overhead mechanical stirrer at a speed of 150 rpm. Then, the mixtures in the absence and presence of 0.04 mM SBTI were incubated at 50 C in a temperature-controlled water bath for 12 h with continuous stirring to extract the gelatin. The mixtures were then ltered using two layers of cheesecloth. The ltrates were further ltered using a Whatman No. 4 lter paper (Whatman International, Ltd., Maidstone, England) with the aid of an electric aspirator, (Model VE-11, JEIO TECH, Korea). The resultant ltrate was freeze-dried using a Scanvac Model Coolsafe 55 freeze dryer (Coolsafe, Lynge, Denmark). Gelatins extracted in the absence and presence of 0.04 mM SBTI were referred to as GAI and GPI, respectively. Both gelatin samples were subjected to SDS PAGE as previously described and further analyses were also conducted as follows:

2.5.4. Determination of gel microstructure Microstructures of gelatin gels were visualised using a scanning electron microscope (SEM). Gelatin gels having a thickness of 23 mm were xed with 2.5% (v/v) glutaraldehyde in 0.2 M phosphate buffer (pH 7.2) for 12 h. The samples were then rinsed with distilled water for 1 h and dehydrated in ethanol with a serial concentration of 50%, 70%, 80%, 90% and 100% (v/v). Dried samples were mounted on a bronze stub and sputter-coated with gold (Sputter coater SPI-Module, West Chester, PA, USA). The specimens were observed with a scanning electron microscope (JEOL JSM5800 LV, Tokyo, Japan) at an acceleration voltage of 15 kV. 2.5.5. Determination of emulsifying properties Emulsion activity index (EAI) and emulsion stability index (ESI) of gelatin samples were determined according to the method of Pearce and Kinsella (1978) as modied by Aewsiri, Benjakul, and Visessanguan (2009). Soy bean oil (2 ml) and gelatin solution (1% gelatin, 6 ml) were homogenised using a homogeniser at a speed of 20,000 rpm for 1 min. Emulsions were pipetted out at 0 and 10 min and 100-fold diluted with 0.1% SDS. The mixture was mixed thoroughly for 10 s using a vortex mixer (Scientic Industries, Inc., Bohemia, NY, USA). A500 of the resulting dispersion was measured using a spectrophotometer. EAI and ESI were calculated by the following equation:

2.5.1. Fourier transform infrared spectroscopy (FTIR) FTIR analysis was performed as per the method of Ahmad, Benjakul, and Nalinanon (2010). A Bruker Model EQUINOX 55 FTIR spectrometer (Bruker, Ettlingen, Germany) equipped with a deuterated L-alanine triglycine sulphate (DLATGS) detector was used. The horizontal attenuated total reectance (HATR) accessory was mounted into the sample compartment. The internal reection crystal (Pike Technologies, Madison, WI, USA), made of zinc selenide, had a 45 angle of incidence to the IR beam. Spectra were acquired at a resolution of 4 cm1 and the measurement range was 4000650 cm1 (mid-IR region) at room temperature. Automatic signals were collected in 32 scans at a resolution of 4 cm1 and were normalised against a background spectrum recorded from the clean, empty cell at 25 C. Analysis of spectral data was carried out using the OPUS 3.0 data collection software programme (Bruker, Ettlingen, Germany).

EAI m2 =g 2 2:303 A DF=lC

where A = A500, DF = dilution factor (100), l = path length of cuvette (m), = oil volume fraction and C = protein concentration in aqueous phase (g/m3)

ESI min A0 =A0 A10 Dt

where A0 = A500 at time of 0 min; A10 = A500 at time of 10 min and Dt = 10 min. 2.6. Statistical analyses All experiments were performed in triplicate and a completely randomised design (CRD) was used. Data were presented as means standard deviation and a probability value <0.05 was considered signicant. Analysis of variance (ANOVA) was performed and the mean comparisons were done by T-test. Analysis was performed using SPSS 11.0 for Windows (SPSS Inc., Chicago, IL). 3. Results and discussion 3.1. Effect of temperature and pH on autolysis of pretreated skin The autolytic degradation of pretreated skin at different temperatures, as monitored by free amino group content, is shown in Fig. 1a. The maximal autolytic activity of pretreated skin was observed at 50 C as indicated by the highest free amino group content (P < 0.05). However, a decrease in autolytic activity was found when the temperature was above 50 C, suggesting the thermal denaturation of the indigenous proteases. When the protein patterns of pretreated skin, autolysed at different temperatures, were compared, the highest degradation of b-chain and a-chains was obtained at 50 C (Fig. 1c). This was concomitant with the formation of degradation products with low molecular weight appearing at the dye front. Furthermore, the high molecular weight cross-linked proteins were also degraded at 5070 C. b- and achains constituted as the major proteins in the collagen from unicorn leatherjacket skin (Ahmad & Benjakul, 2010a). The results suggested that the proteases still remained within the skin after pretreatment with alkali and acid for non-collagenous protein removal and swelling processes, respectively. Those heat-activated

2.5.2. f-Potential analysis Gelatin samples were dissolved in distilled water at a concentration of 0.5 mg/ml. The mixtures were stirred at room temperature for 6 h. The f-potential of each sample (20 ml) was measured using a f-potential analyser (ZetaPALS, Brookhaven Instruements Co., Holtsville, NY, USA). f-Potential of samples, adjusted to different pHs with 1.0 M nitric acid or 1.0 M KOH using an autotitrator (BI-ZTU, Brookhaven Instruments Co., Holtsville, New York, USA), was determined. The pI was estimated from pH rendering f-potential of zero.

2.5.3. Determination of gel strength Gels of gelatin were prepared by the method of Fernandez-Daz, 9 Montero, and Gomez-Guillen (2001), with a slight modication. Gelatin samples were dissolved in distilled water (60 C) to obtain the nal concentration of 6.67% (w/v). The solution was stirred until the gelatin was solubilised completely, followed by incubating the samples in a refrigerator at 10 C for 18 h for gel maturation. The dimensions of the sample were 3 cm in diameter and 2.5 cm in height. The gel strength of the samples prepared at 10 C was determined using a Model TA-XT2 Texture Analyser (Stable Micro System, Surrey, UK) with a load cell of 5 kN and equipped with a 1.27 cm diameter at-faced cylindrical Teon plunger. The maximum force (in grams) was recorded when the penetration distance reached 4 mm. The speed of the plunger was 0.5 mm/s.

Please cite this article in press as: Ahmad, M., et al. Indigenous proteases in the skin of unicorn leatherjacket (Alutherus monoceros) and their inuence on characteristic and functional properties of gelatin. Food Chemistry (2011), doi:10.1016/j.foodchem.2011.01.032

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Fig. 1. pH and temperature proles for autolysis of pretreated skin from unicorn leatherjacket. For the temperature prole, autolysis was performed at pH 7 for 30 min at different temperatures. For the pH prole, autolysis was conducted at 50 C for 30 min at different pHs. For Fig. 1c and 1d; I: type I collagen from calf skin. For Fig. 1c; PS: pretreated skin. Numbers denote the temperatures (C). For Fig. 1d; Numbers denote the pHs. Bars represent the standard deviation (n = 3).

proteases were able to cleave c-, b- and a-chains most effectively at 50 C as shown in Fig. 1c. Thus, those proteases more likely contributed to the disintegration of gelatin molecules during the extraction process, which is normally conducted at high temperature. Pretreatment removed not only the proteases to some extent, but also some metallic ions or co-factors required for proteolytic activity of bigeye snapper skin (Intarasirisawat et al., 2007). In the present study, pretreatment could lower the autolysis to some degree, compared with the skin without pretreatment (data not shown). Nevertheless, the remaining proteases were shown to be involved in hydrolysis of collagenous components in the skin. The pH prole for pretreated skin autolysis at 50 C is shown in Fig. 1b. The highest free amino group content was obtained at pH 7. c-, b- and a-chains were almost degraded at pHs 67 with a concomitant formation of low-molecular-weight peptides (Fig. 1d). The results indicated that the neutral proteases were dominant in the pretreated unicorn leatherjacket skin, causing the hydrolysis of collagen molecules. At very acidic or alkaline pHs, the degradation was markedly decreased as evidenced by the low free amino group content and the greater band intensity of b- and a-chains retained. This might be due to the inactivation of the indigenous protease under these conditions due to the conformational changes or ionisation of R-groups of amino acids, especially at the active site of proteases. Although collagen could be solubilised in acidic pH range (Ahmad & Benjakul, 2010a), the degradation was negligible in such a range, especially pH 14. Despite the presence of soluble substrates, no hydrolysis was noticeable. This reconrmed the

inactivation of indigenous protease in an acidic pH range. On the other hand, the pronounced degradation was noticeable at neutral pH, which is commonly used for gelatin extraction.

3.2. Inhibitor study towards autolysis of pretreated skin The effect of various inhibitors on autolysis of unicorn leatherjacket pretreated skin is depicted in Fig. 2. Among all protease inhibitors tested, 0.04 mM SBTI and 1 mM PMSF showed the highest inhibition towards autolysis when tested at pH 7, whilst other inhibitors, including EDTA, EGTA, E-64, iodoacetic acid, pepstatin A and 1,10-phenanthroline, had no inhibitory effect on autolysis. c-, b- and a-chains of pretreated skin were mostly retained in the presence of PMSF, whilst SBTI was found to be the most effective inhibitor towards the degradation of those components. PMSF irreversibly inhibits serine proteases by sulphonylation of the serine residue in the active site of the protease (James, 1978). The active site in each serine protease includes a serine residue, a histidine residue, and an aspartate residue (Johnson, Roderick, & Cook, 2005). Therefore, it can be inferred that a serine protease was the major protease associated with unicorn leatherjacket pretreated skin. In the absence of inhibitors, the pronounced hydrolysis was noticeable as evidenced by the marked decrease in the band intensity of c-, b- and a-chains. Intarasirisawat et al. (2007) reported that serine protease was the major enzyme causing the autolysis of skin from bigeye snapper.

Please cite this article in press as: Ahmad, M., et al. Indigenous proteases in the skin of unicorn leatherjacket (Alutherus monoceros) and their inuence on characteristic and functional properties of gelatin. Food Chemistry (2011), doi:10.1016/j.foodchem.2011.01.032

M. Ahmad et al. / Food Chemistry xxx (2011) xxxxxx

1 2

1 2

PS S50 a

Fig. 2. SDSPAGE pattern of pretreated skin from the unicorn leatherjacket incubated in the presence of various inhibitors: (a) 10 mM EDTA; (b) 10 mM EGTA; (c) 1 mM E-64; (d) 10 mM iodoacetic acid; (e) 10 mM 1,10-phenanthroline; (f) 1 mM pepstatin A; (g) 1 mM PMSF; (h) 0.04 mM SBTI ; I: collagen type I from calf skin. PS: pretreated skin and S50: control (without protease inhibitors). The autolysis was performed at pH 7.0 and 50 C for 30 min.

PS

GAI

GPI

3.3. Yield and characteristics of gelatin from pretreated skin as affected by protease inhibitor 3.3.1. Yield Yields of gelatin extracted from pretreated skin in the absence (GAI) and in the presence of 0.04 mM SBTI (GPI) at 50 C for 12 h are 7.34% and 5.29% (wet weight basis), respectively. When SBTI was incorporated during the extraction, a decrease in the yield was obtained. SBTI most likely inhibited the activity of indigenous serine proteases associated with the skin matrix, thereby preventing the cleavage of peptide chains. This in turn retained the high molecular weight components in the skin matrix, leading to a lower amount of extractable gelatin. The degradation in pretreated skin without SBTI addition, particularly at temperature close to the optimal temperature (50 C), might result in the formation of shorter peptides as well as loosening of the skin matrix. This possibly resulted in the ease of gelatin extraction. During heating at 50 C, hydrogen bonds stabilising triple-helix of mother collagen were destroyed, leading to helix-to-coil transition (Wong, 1989). This resulted in the conversion of collagen to soluble gelatin. Therefore, the addition of protease inhibitor was shown to lower the extraction yield of gelatin from unicorn leatherjacket skin. 3.3.2. Protein pattern Based on protein pattern, GAI contained the lower band intensity of b- and a-chains, in comparison with that found in the pretreated skin and GPI (Fig. 3). The b-chain was almost completely degraded in GAI, whereas, a-chains were retained to some extent. The result suggested that a-chains and cross-linked components of GPI were retained more when SBTI was incorporated during extraction at high temperature. With the extended extraction time (12 h), the remaining collagenolytic protease associated with skin might hydrolyse the collagen molecules progressively in GAI, especially at the optimal temperature (50 C). In addition, proteins with molecular weights lower than a-chains (100 kDa) were also observed in both GAI and GPI, suggesting the degradation of a, band c-components during gelatin extraction caused by thermal hydrolysis or the proteolysis mediated by the remaining indigenous proteases. The result was in agreement with Muyonga, Cole, and Duodu (2004) who reported that gelatin extracted at high temperature contained more peptides with a molecular weight less than the a-chains and a lower proportion of component having a molecular weight greater than the b-chain. The degradation of gelatin from bigeye snapper skin caused by some proteases during extraction process was also reported (Jongjareonrak, Benjakul,

Fig. 3. SDSPAGE pattern of gelatin extracted from the skin of unicorn leatherjacket in the absence (GAI) and in the presence of 0.04 mM of SBTI (GPI). I: collagen type I from calf skin. PS: pretreated skin.

Visessanguan, Prodpran, & Tanaka, 2006). The formation of degradation peptides is associated with the low viscosity, low melting point, low setting point, high setting time, as well as decreased bloom strength of gelatin (Muyonga et al., 2004). Therefore, the addition of SBTI effectively prevented the degradation of b- and a-chains in gelatin from unicorn leatherjacket skin.

3.3.3. FTIR spectra FTIR spectra of GAI and GPI from the skin of unicorn leatherjacket are depicted in Fig. 4a. Spectra of both gelatins had the major peaks in the amide region, but showed slight differences in the spectra. These spectra were in accordance with those reported by Muyonga et al. (2004). GAI and GPI displayed the amide-I bands at the wavenumbers of 1635.89 and 1630.82 cm1, respectively. The amide-I vibration mode resulted primarily from a C@O stretching vibration coupled to contributions from the CN stretch, CCN deformation and in-plane NH bending modes (Bandekar, 1992). The absorption peak at amide-I was characteristic for the coil structure of gelatin (Yakimets et al., 2005). The amide-I peak of GAI appeared at a higher wavenumber, compared to that of GPI. This indicated the greater loss of triple helix and enhanced hydrolysis of collagen caused by heat-activated indigenous proteases in the skin matrix. In addition, GPI showed the shift to a lower wavenumber and lower amplitude for the amide-I peak, compared to GAI. This was probably associated with the higher extent of molecular order due to interaction of C@O with adjacent chains via hydrogen bonds. This indicated that GPI retained more intermolecular cross-links during extraction at high temperature in the presence of SBTI. In the absence of SBTI, the triple helical structure, held by intermolecular hydrogen bonds, was destroyed to the higher extent. The result was in agreement with the higher degradation of GAI, in comparison with GPI (Fig. 3). The characteristic absorption bands of GAI and GPI in the amide-II region were noticeable at the wavenumbers of 1545.82 and 1533.77 cm1, respectively. The amide-II vibration modes are attributed to an out-of-plane combination of CN stretch and inplane NH deformation modes of the peptide group (Bandekar, 1992). A shift to a lower wavenumber and lower amplitude were found in GPI, compared to GAI, indicating that NH was more involved in bonding with the adjacent molecules probably via Hbonding. In addition, amide-III was detected around the wavenumbers of 1236.53 and 1233.74 cm1 for GAI and GPI, respectively.

Please cite this article in press as: Ahmad, M., et al. Indigenous proteases in the skin of unicorn leatherjacket (Alutherus monoceros) and their inuence on characteristic and functional properties of gelatin. Food Chemistry (2011), doi:10.1016/j.foodchem.2011.01.032

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(symmetrical) and 2918.31 cm1 (asymmetrical), representing C H stretching vibrations of the CH2 groups, were found in both GAI and GPI spectra. However, the difference in the spectra between both samples in the region was probably due to the coupling of SBTI with gelatin (Chen et al., 2008; DSouza, Devi, Shridhar, & Naik, 2008). Thus, it can be concluded that the secondary structure of gelatins obtained from the skin of unicorn leatherjacket was affected by the addition of SBTI during extraction process. 3.3.4. f-Potential Zeta-potential (f) representing the surface charge of GAI and GPI at different pH is shown in Fig. 4b. A net charge of zero was obtained at pH 7.31 and 8.45, for GAI and GPI, respectively. At a pH near the isoelectric point, both GAI and GPI had decreased repulsion with increased interaction among gelatin molecules. Protein molecules in an aqueous system have zero net charge at their isoelectric points (pI), in which the positive charges are balanced out by the negative charges (Bonner, 2007). It was found that the f-potential was higher for GPI in the pH range of 48. The differences in f-potential, as well as pI between both gelatins, were most likely due to the differences in the amino acid compositions. Indigenous proteases present in the skin might enhance the extraction efciency, in which a larger content of several peptides could be released. On the other hand, only a small amount of peptides, mainly those with higher MW, was extracted in the presence of SBTI. The result suggested that indigenous proteases involved in gelatin extraction had an impact on pI of the resulting gelatin to some degree.
Fig. 4. Fourier transform infrared spectra (a) and f-potential (b) at different pH of gelatin extracted from the skin of unicorn leatherjacket in the absence (GAI) and presence of 0.04 mM of SBTI (GPI). For Fig. 4b: Bars represent standard deviation (n = 3).

The amide-III represented combination peaks between CN stretching vibrations and NH deformation from amide linkages, as well as absorptions arising from wagging vibrations from CH2 groups of the glycine backbone and proline side-chains (Jackson, Choo, Watson, Halliday, & Mantsch, 1995). The results suggested a disordered molecular structure for both GAI and GPI, probably due to the transformation of a-helical to random coil structure during heating. Those changes were associated with the loss of the triple helix state as a result of denaturation of collagen to gelatin (Muyonga et al., 2004). In addition, absorption bands in GAI and GPI around the wavenumbers of 1080.57 and 1079.13 cm1, respectively, most likely arose from asymmetric stretching vibrations of phosphate groups of phosphorylated proteins coupled to CH2 of the amino acid residues (Jackson et al., 1995). In the present study, phosphoric acid was used for pretreatment of the skin, in which some phosphates were attached to gelatin molecules (Ahmad & Benjakul, 2010b). The amide-A band, arising from the stretching vibrations of the NH group, appeared at 3275.77 and 3276.24 cm1 for GAI and GPI, respectively. Normally, a free NH stretching vibration is found in the range of 34003440 cm1 (Muyonga et al., 2004). When the NH group of a peptide is involved in a hydrogen bond, the position is shifted to lower frequencies (Doyle, Blout, & Bendit, 1975). At the amide-A region, a slightly higher wavenumber was found in GPI, compared to GAI. However, a higher amplitude was found in GAI at the amide-A region, indicating the presence of NH group of shorter peptide fragments. The amide-B was observed at 3075.29 and 3074.28 cm1 for GAI and GPI, respectively, corresponding to an asymmetric stretch vibration of @CH and ANH (Ahmad & Benjakul, 2010b). GPI showed the lower wave3 number of amide-B peak, suggesting the interaction of NH3 group between peptide chains. Peaks at the wavenumber of 2849.24

3.3.5. Gel strength The gel strength of GAI and GPI is shown in Table 1. GPI had a higher gel strength than GAI (P < 0.05). The difference in gel strength might be governed by molecular weight distribution, as well as the aggregation between gelatin molecules. Low molecular weight peptides of GAI might not form the inter-junction zone effectively, leading to a lower aggregation of gelatin needed to form strong gel network. The quality of gelatin is generally determined by the gel strength or bloom value, including low (<150), medium (150220) and high bloom (220300) (Johnston-Bank, 1983). The conguration of polypeptide chains and the way in which the inter-junction was developed to form a stronger network were both crucial for gel formation (Ahmad & Benjakul, 2010b). The gelation of gelatin is considered to be due to a partial recovery of the triple helical structure in gelatin, in which regions rich in Gly-Pro-Hyp are of crucial importance for the stabilisation of the triple helices. In addition, acidic pretreatment also played a role in the determination of gel strength. Phosphate groups used for pretreatment might attach with some amino acids, leading to phosphorylation of both GAI and GPI (Ahmad & Benjakul, 2010b). Phosphorylation may introduce the ionic interaction between phosphate groups and ANH of amino acids, thereby increasing 3 the aggregation (Guo, Li, Zhu, & Zhao, 2005). It was noted that

Table 1 Gel strength, colour and emulsifying properties of gelatin extracted from the skin of unicorn leatherjacket in the absence (GAI) and presence of 0.04 mM of SBTI (GPI). GAI Gel strength (g) EAI (m2/g) ESI (min) L a b 288.63 1.44b 30.21 0.86b 22.62 0.38b 28.92 1.28a 0.83 0.02b 0.20 0.04b GPI 320.68 3.02a 34.32 0.12a 18.91 1.10b 32.54 2.20a 1.43 0.11a 0.81 0.15a

Mean SD (n = 3). Different letters in the same row indicate the signicant differences (p < 0.05).

Please cite this article in press as: Ahmad, M., et al. Indigenous proteases in the skin of unicorn leatherjacket (Alutherus monoceros) and their inuence on characteristic and functional properties of gelatin. Food Chemistry (2011), doi:10.1016/j.foodchem.2011.01.032

M. Ahmad et al. / Food Chemistry xxx (2011) xxxxxx

GPI had a higher gel strength than commercial bovine gelatin (293.22 g) (P < 0.05) (Ahmad & Benjakul, 2010b). In general, sh gelatins have lower hydroxyproline content (about 710% of the total amino acids), compared with that of bovine gelatin (14%) (Nalinanon, Benjakul, Visessanguan, & Kishimura, 2008). The gelling properties of gelatin were also inuenced by the source of raw materials, which vary in proline and hydroxyproline contents (Jongjareonrak, Benjakul, Visessanguan, & Tanaka, 2006). Therefore, the use of SBTI, as a protease inhibitor during the gelatin extraction, markedly improved the gel strength of extracted gelatin. This was mainly due to the maintenance of chain length, which was a prerequisite for better gelation. 3.3.6. Gel colour The colour of gel from both GAI and GPI is shown in Table 1. A higher L-value of GPI gel was observed, compared with GAI (P < 0.05). The higher L-value of GPI was most likely associated with the greater interaction of higher molecular weight peptides,

in which the larger aggregates were formed. A larger aggregate more likely exhibited a greater light scattering effect. However, GPI had a higher b-value (yellowness) and a-value (redness) than those of GAI. Gelatin manufactures generally have a good method to clarify the impurities from the gelatin solution, such as chemical clarication and ltration processes. Generally, the colour did not affect functional properties of gelatin (Ockerman & Hansen, 1988). 3.3.7. Microstructure of gel The microstructures of GAI and GPI gels are illustrated in Fig. 5. Both GAI and GPI gels were sponge or coral-like in structure. GPI gel had a denser and more irregular network of interconnected brous structure with smaller voids, compared with GAI gel. Larger voids were found in GAI and were possibly correlated with low aggregation of peptide chains during gelation. Generally, the arrangement and association of protein molecules in the gel matrix directly contributed to the gel strength of the gelatin (Benjakul, Oungbho, Visessanguan, Thiansilakul, & Roytrakul, 2009). The network of GAI gel with fewer inter-junctions might be disrupted easily by an applied force, resulting in lower gel strength. Therefore, the use of SBTI had an impact on the network of gelatin gel, in which chain length of peptide molecules were retained, and interaction or aggregation could take place more effectively. 3.3.8. Emulsifying properties Emulsion activity index (EAI) and emulsion stability index (ESI) of GAI and GPI are presented in Table 1. At a gelatin concentration of 1%, EAI of GPI was higher than GAI (P < 0.05). Conversely, ESI of GAI was higher than GPI (P < 0.05). This indicated that there was a difference in emulsifying properties between GAI and GPI. GAI contained a higher content of low molecular weight peptides, which had more hydrophilicity and preferably localised in the aqueous phase. As a result, the lower portion of gelatin migrated to the oilwater interface, leading to the low emulsifying property. Nevertheless, emulsion containing GPI was less stable than that with GAI (P < 0.05). Oil-in-water emulsion, prepared with high molecular weight sh gelatin (120 kDa) was more stable than that prepared with low molecular weight sh gelatin (50 kDa). The thickness of adsorbed gelatin lm increased with increasing molecular weight (Ahmad & Benjakul, 2010b). This was associated with the increased stability of emulsions to coalescence during homogenisation (Lobo & Svereika, 2003). In the present study, bulky a-, b- and c-chains prevalent in GPI could not align themselves properly, where a strong lm was formed effectively around an oil droplet. Additionally, GAI with higher degradation might have more charged groups, especially amino or carboxyl groups at the end of peptides, thereby having the ability to facilitate the stabilisation of emulsion via electrostatic repulsion. The stabilisation of emulsion against coalescence/occulation is greatly dependent on the force of electrostatic repulsions between the adsorbed proteins on the interfacial protein lm (Aewsiri et al., 2009). Differences in the intrinsic properties, composition and conformation of gelatins determined the functional properties of gelatin from the skin of two species of bigeye snapper (Benjakul et al., 2009). 4. Conclusion Serine protease was the major enzyme in pretreated skin from unicorn leatherjacket and was involved in the drastic degradation of collagen/gelatin at high temperature, used for gelatin extraction. The addition of 0.04 mM SBTI during extraction could retain the b- and a-chains in resulting gelatin via inhibition of indigenous proteases, in which the gel strength and emulsifying activity were increased. However, it is important to note that the extraction yield was lowered when SBTI was incorporated.

Fig. 5. SEM micrographs of gel from gelatin extracted from the skin of unicorn leatherjacket in the absence (GAI) and presence of 0.04 mM of SBTI (GPI). Magnication: 3500.

Please cite this article in press as: Ahmad, M., et al. Indigenous proteases in the skin of unicorn leatherjacket (Alutherus monoceros) and their inuence on characteristic and functional properties of gelatin. Food Chemistry (2011), doi:10.1016/j.foodchem.2011.01.032

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Acknowledgements The authors would like to express their sincere thanks to Prince of Songkla University and the TRF senior research scholar program for the nancial support.

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Please cite this article in press as: Ahmad, M., et al. Indigenous proteases in the skin of unicorn leatherjacket (Alutherus monoceros) and their inuence on characteristic and functional properties of gelatin. Food Chemistry (2011), doi:10.1016/j.foodchem.2011.01.032