Extraction and Characterization of Type I Collagen from Parrotfish (Scarus sordidus Forsskål, 1775) Scale solubilized with the Aid of Acetic Acid and Pepsin

Waste from marine fish processing is an important source of valuable products. Fish collagen is considered a alternative biomaterial due to its excellent properties, and it is widely used for industrial purposes. Thus, this present study aimed to characterize acid and pepsin-soluble collagens from the waste of parrotfish (Scarus sordidus Forsskål, 1775) scales. The yields (p > 0.05) of acid-soluble collagen (ASC-PFS) and pepsin-soluble collagen (PSC-PFS) were 1.17 g/100 g and 1.00 g/100 g, respectively. Both collagen samples were categorized as type I owing to the presence of two alpha chain subunits (α1 and α1) after being confirmed by a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Under the fourier transform infrared (FTIR) test, the triple helical structure of type I collagens from the ASC-PFS and PSC-PFS was maintained. Moreover, the study of UV visible spectra and X-ray diffraction (XRD) showed the similarity of collagens derived from different fish species, and the thermostability (Tmax) evaluation of all extracted collagens was in the range of 36.22–37.78°C, and their values were comparable to previous research on the fish scale collagens. The effect of various pH and sodium chloride (NaCl) treatments on solubility exhibited that the ASC-PFS and PSC-PFS were highly soluble in an acidic condition (pH < 5.0) and low concentration of sodium chloride (<30 g/L). Taken together, collagens extracted from parrotfish scale waste can be an alternative source for industries.


Introduction
Collagen is the major structural protein in mammals, representing approximately one-third of the total body protein content. Tis fber protein has a specifc right-handed triple helical chain containing three parallel polypeptide (lefthanded) chains [1]. Typically, it is assembled by a Glycine-Xaa-Yaa triplet, where the Xaa and Yaa positions are usually placed by proline and hydroxyproline [2]. It is ubiquitous in the extracellular matrix (ECM) of tissues, where it not only provides strength and structural stability, but also performs highly specialized regulatory functions, particularly during development and repair [3]. To date, almost thirty types of collagen have been investigated, which difer according to their structure of protein, composition of amino acids and molecular attributes. Amongst them, type I is extensively explored and best studied collagen in the felds of medical, nutraceutical, pharmaceutical, cosmetic, and food application due to its biocompatibility, biodegradability and weak antigenicity [4,5]. Traditionally, collagen was made from the skin and bones of mammals, such as cows and pigs [1]. However, use of mammalian collagens raises anxiety among consumers, which is related to some infectious diseases, e.g., foot and mouth disease, transmissible spongiform encephalopathy, and bovine spongiform encephalopathy. Also, poultry-based collagens cause fear due to the incidence of the avian fu virus [6,7]. In another case, some religious groups like Islam and Judaism cannot consume porcine and its derivatives, while bovine is not accepted by Hinduism [8]. In such situations, seafood/fsh by-products (skins, frames, and scales) are believed to be potential sources of collagen due to their high availability, low risk of transmittable disease, and free of religious barriers [9].
Parrotfsh, also known as "Ikan Bayan" in Malaysia, is a tropical fsh species belonging to the family Labridae. Parrotfsh (Scarus sordidus Forsskål, 1775) is commonly recognized by its parrot-like beak of fused teeth, a bluntlyrounded head, and large scales [22]. It is quite popular with consumers due to its high nutritional content. Parrotfsh scale is almost discarded during processing, and to tackle this limitation, transforming it into collagen is a great strategy. Currently, only the skin part of parrotfsh has been extracted as collagen, with a high yield obtained [23]. However, other parts (such as the scale) of that fsh are much less documented. Tis study focused on the collagen extraction derived from the scales of parrotfsh with the addition of an acid solution and the aid of pepsin enzyme. Teir physicochemical properties were also evaluated. Finally, this study could raise the added value of this waste product from fsh and be friendly to the environment. Moreover, it may provide some basic information for further studies.

Extraction of Acid-and Pepsin-Soluble Collagen.
All extraction processes were exhibited in a cool room (4°C). Te method used in this work was slightly modifed from the previous research [24][25][26], as shown in Figure 1. First, dried parrotfsh scales (around 50g) were dissolved in 10 volumes (v/w) of sodium hydroxide (0.1 M) and continuously stirred for 6 h, changing the alkaline solution every 2 h to eliminate undesirable pigment and noncollagenous matter. Te immersed fsh scales were then rinsed with cooled distilled water and neutralized at a neutral condition. Next, 10 volumes of ethylenediaminetetraacetic acid disodium salt (0.5 M) were added to the sample and stirred for 24 h (replacing the solution every 12 h) to demineralize the sample. Te demineralized collagens were washed three times with cold distilled water, and then subjected to acidassisted extraction. A total of ffteen volumes of 0.5 M acetic acid (glacial) were mixed with the pretreated samples and stirred at 500 rpm for 2 days using a homogenizer (IKA ® RW 20 Digital Over Head Stirrer, Selangor, Malaysia). After extraction, the suspended collagens were subjected to fltration using a single layer of cheesecloth. Te fltrate was collected for a further step, while the residue was kept separately in a freezer for further experimentation (pepsinaided extraction). Next, the fltrate (soluble fraction) was precipitated by adding 0.05 M Trizma ® hydrochloride and 2.5 M NaCl. Te precipitated sample was subsequently neutralized (pH 7.0) and then centrifuged for 10 min at 15,000 × g. After centrifugation, two volumes of acetic acid (0.5 M) were dropped on the pellet and mixed thoroughly. Te liquid extracted collagens were then put into a dialysis tubing. Twenty volumes of acetic acid (0.1 M) and cold deionized water were prepared and dialyzed for 3 days. Te dialysate was dried in a Labconco freeze-dryer machine (Kansas City, USA). Te dried collagen was represented acid-soluble collagen of parrotfsh scale (ASC-PFS). In terms of pepsin-assisted extraction, the residue from previous acid extraction was isolated by adding 15 volumes of acetic acid (0.5 M) and bovine pepsin (Himedia, Maharashtra, India) (1.5%, w/w) for two days. After the isolation process, further procedures were similar to the acid extraction process. Te 2 International Journal of Biomaterials freeze-dried sample was known as a pepsin-soluble collagen of parrotfsh scale (PSC-PFS). Both collagen samples were then stored in a freezer until experimentation.

Collagen Analyses
2.3.1. Yield and Colour Analysis. Te yield of parrotfsh scale collagens was stipulated according to the formula described by Matmaroh et al. [24] Yield(%) � Weight of died collagen(ASC∧PSC) Weight of initial dry parrotfish scale × 100. (1) Colour attributes for ASC-PFS and PSC-PFS were assessed through a colorimeter instrument (ColorFlex CX2379, HunterLab, Galveston, TX, USA), as described in a previous study [25]. Te diferences in the color test were presented in the CIELAB or CIE 1976 L * a * b * colour space, where L * is the lightness or brightness, a * is the redness (from green to red), and b * is the yellowness (from blue to yellow). For whiteness index (WI), both ASC and PSC from the parrotfsh scales were determined using a formula stated by Ishamri et al. [27]

UV Absorption Spectrum.
Ultraviolet absorption spectra of ASC-PFS and PSC-PFS from the scales of parrotfsh (Scarus sordidus Forsskål, 1775) were performed under a spectrophotometer of the LAMBDA 25 type (Per-kinElmer, Inc., Waltham, MA, USA). A ffty milligram sample was immersed in 10 mL of AcOH solution (0.5 M) and well mixed. Ten, the mixture was prepared for centrifugation at 8,500 × g for 5 min, and the solubilized collagens were pipetted out into a quartz cell. Te spectra of the extracted collagens were designed at wavelengths from 400 nm to 200 nm, and an acetic acid solution with the same concentration was used for a baseline [28].

Attenuated Total Refectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR).
Te ATR-FTIR spectra of the isolated samples (ASC-PFC and PSC-PFC) were analysed using the FTIR spectrometer (Agilent Cary 630, Cary, NC, USA), and all steps were adopted from the study of Matmaroh et al. [24]. Te dried parrotfsh collagen (20 mg) was placed on the crystal cell of a spectrometer, and the spectra were adjusted between 4,000 nm −1 and 800 cm −1 . All data with signifcant peaks were observed using a software program developed by Agilent Microlab.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE).
Te Mini-PROTEAN Tetra Cell (Bio-Rad Laboratories, Hercules, CA, USA) was used to determine the molecular weight (MW) of the ASC and PSC from the parrotfsh scales, with the established methods from Laemmli [29]. A three milligram of each lyophilized collagen was prepared by mixing with a 5% SDS solution and then put in a water bath (at 85°C for 60 min). Afterwards, the treated collagens were centrifuged at 8,500 × g for 5 min to eliminate insoluble matter. Te supernatant (20 μL) was pipetted into an appropriate centrifuge tube, and then the same volume of sample bufer incorporated, with and without β-mercaptoethanol was added. Te mixed samples were then heated for 5 min at the same temperature and carefully flled carefully in the prepared acrylamide gel (stacking gel: 4% and separating gel: 7.5%). A certain voltage was set at 120 volts for about 1 h. Next, the fxation process was employed to fx the electrophoresed gel, and it was further stained for about 10 min. After staining, the gel was transferred into a destaining container. Te protein marker was used to compare the electrophoretic bands of ASC-PFC and PSC-PFC.

X-Ray Difraction Analysis (XRD).
Analysis of X-ray difraction from the parrotfsh scale collagen prepared by adding acetic acid and pepsin was conducted according to the previous report [30]. Te dried collagen samples were placed into a sample holder and then scanned using an X-ray difraction machine. Te scanning range in both ASC-PFS and PSC-PFS was initiated from 5°to 40°(2θ) with a speed of 0.06°per second, and the current and tube voltage of the XRD apparatus were adjusted to 50 mA and 40 kV, respectively.

Diferential Scanning Calorimetry (DSC).
Te thermal stability test on parrotfsh scales was carried out with a Perkin-Elmer diferential scanning calorimeter (Model DSC7, Norwalk, CA, USA) under a nitrogen atmosphere. Te procedure used in this work was pointed from the study of Kittiphattanabawon et al. [31]. Freeze-dried collagens were prepared for rehydration at a ratio of 1 : 40 (w/v) with distilled water and then incubated for two days in a refrigerator. Afterwards, the prepared samples were accurately weighed (ranging from 5 mg to 10 mg) into an aluminum volatile pan and then tightly sealed with a crimper. Prior to running the samples, a calibration step was performed with an indium. Subsequently, a sealed collagen sample and an empty pan were placed into sample and reference detectors, respectively, and then scanned from 20°C to 45°C at a rate of 1°C per minute. DSC rates were expressed in the maximum transition temperature (T max ) and the total denaturation enthalpy (ΔH).

Solubility Test.
Solubility tests at diferent concentrations of sodium chloride and at various pH treatments were observed in both ASC-PFS and PSC-PFS samples, adopting our previous method [32]. For the sodium chloride assay, the concentrations employed range between 0 g/L and 60 g/L. A total of 5 mL of prepared collagens were pipetted out into fve millilitres of sodium chloride (NaCl) solution and then mixed using a stirrer for 60 min in a chiller. Next, the NaCl-treated collagens were subjected to centrifugation at 10,000 × g for 15 min to separate insoluble samples. Following centrifugation, the protein content of each parrotfsh collagens were analysed based on the established method [33], and a standard protein used in this analysis was bovine serum albumin (BSA). In terms of diferent pH conditions, both collagens were prepared by dissolving in an 0.5 M acetic acid solution and stirred for 4 h in a chiller. Te dissolved collagens were further adjusted at diferent pH values (pH 1.0-9.0) with adding 1 M NaOH and HCl solutions. Next, the treated collagens were stirred for 60 min and centrifuged further at 10,000 × g for 10 min with an Eppendorf centrifuge (Model 5430R, Hamburg, Germany). Te protein concentration of each treated sample was also tested with the Lowry method. Both ASC and PSC from the scales of parrotfsh were used to determine the percentage of relative solubility using the following formula: Relative solubility(%) � Current concentration of protein at current NaCl or pH The highest concentration of protein × 100.  Table 1 shows the yields of ASC and PSC from the parrotfsh scale wastes. Although no signifcant diference (p > 0.05) as observed in both isolated collagen samples, the ASC-PFS showed a higher yield (based on a dry weight basis) than that observed in the PSC-PFS. Both collagens were comparable to the previous study of fsh scale collagens. For instances of ASC and PSC from the lizardfsh (Saurida tumbil) (0.18% and 0.60%) [25], Nile tilapia (Oreochromis niloticus) (0.77% and 0.71%) [34], carp (Cyprinus carpio) (0.97% and 1.37%) [37], and sea bass (Lates calcarifer) (0.38% and 1.06%) [9]. In contrast, the highest yield of fsh collagens (either extracted with the acid solution or aided with the pepsin) was obtained from the skin portion, as reported by numerous research studies, such as sturgeon fsh (Huso huso) (9.98% and 9.08%) [13], sailfsh (Istiophorus platypterus) (5.76% and 2.11%) [38] and Spanish mackerel (Scomberomorous niphonius) (13.68% and 3.49%) [39]. A lower yield in the fsh scale collagens could be due to abundant hydroxyapatite components (Ca 5 (PO 4 ) 3 OH) and many crosslinked areas of fsh scale collagens [40]. Also, the variations in the yields observed in the fsh collagens were might be afected by extraction procedures, tissue composition and structure, and fsh species with diferent sizes and ages [41].

Yield and Colour Attributes of Parrotfsh Collagens.
In the context of colour attributes, collagen is a potential ingredient supplemented in food, cosmetic, pharmaceutical, and medical products. Hence, collagen with a brighter colour is more acceptable because it does not change the original colour of fnal products [42]. Table 1 shows the values of L * , a * , and b * , as well as the whiteness index (WI) in both ASC and PSC derived from the parrotfsh scales (Figures 2(a) and 2(b)). Te results presented indicated that the L * and WI scores of the PSC-PFS sample were significantly higher (p < 0.05) compared to those of the ASC-PFS. Nevertheless, our present study's lightness rate was lower in terms of percentage than that found in the lizardfsh (S. tumbil) scale collagen [25], snakehead (Channa argus) skin collagen treated with hydrogen peroxide (H 2 O 2 ) [36], and also type I collagen from calfskin [7]. Te reason might be due to the lack of treatment during the decolouration process by adding H 2 O 2 a solution as prepared in the previous study for snakehead skin collagen, and the type of fsh scale used in the experimentation. Typically, parrotfsh scales have a colourful pattern, including green, purple, brown, and grey, resulting in the fnal product obtained, as presented in this study (Figure 2(c)).

Protein Profle. Both collagens (ASC-PFS and PSC-PFS)
were almost the same in SDS-PAGE patterns (Figure 3) with presenting two alpha chains (α1 and α2), one β-and c-chains. However, there was a slight diference in molecular weight (MW) of α1 and α2. For the PSC-PFS sample, those chains (α1 � 118.1 kDa and α2 � 107.4 kDa) were lower compared to those (α1 � 123.9 kDa and α2 � 112.7 kDa) of the ASC-PFS. Also, the β-chain of the ASC-PFS sample showed less band intensity than that of the ASC-PFS sample. Tese evidences could be due to the fact that some parts of telopeptide regions in terms of crosslinked components were cleaved by the pepsin during the extraction process, as reported by Khittiphattanabawon et al. [34]. In comparison of the band intensity, especially α1-chain had a twofold increase over that of α2, suggesting that the parrotfsh scale collagen was categorized as type I collagen. Tese obtained data agreed with other fsh collagens, including purplespotted bigeye (Priacanthus tayenus) [10], bigeye tuna (Tunnus obesus) [11], loach (Misgurnus anguillicaudatus) [43], and fying fsh (Cypselurus melanurus) [18], the golden pompano (Trachinotus blochii) [44], and the channel catfsh (Ictalurus punctatus) [45]. Furthermore, under the treatment of nonreducing (without β-ME) and reducing (with β-BE), all extracted collagens showed no diference in electrophoretic patterns, indicating no di-sulfde bond formation in both ASC-and PSC-PFC.

UV Absorption Spectrum.
In general, the signifcant absorption spectrum of collagen can be observed at a wavelength of 210-240 nm [46]. Te obtained results exhibited that the maximum spectra of ASC and PSC derived from the parrotfsh scale were detected at wavelengths of 230 nm and 232 nm, respectively (Figure 4). Tese fndings were in accordance with other experiments in fsh collagen samples, including lizardfsh (S. tumbil) [7], Siberian sturgeon (Acipenser baerii) [14], and miiuy croaker (Miichthys miiuy) [47] and pufer fsh (Lagocephalus inermis) [48]. Moreover, the absorption peaks depicted in this study (both ASC-PFS and PSC-PFS) are related to the functional groups of collagen molecules, such as carboxyl (-COOH), carbonyl (C�O), and amides (CONH 2 ). Meanwhile, for another peak detected in the spectra, there was a low absorption peak at wavelengths of 300−250 nm. Tese peaks represented tryptophan, phenylalanine and tyrosine (aromatic amino acids), as confrmed in all abovementioned references [25,26].

Fourier Transform Infrared Spectroscopy (FTIR).
Te IR spectra of ASC-PFS and PSC-PFS ( Figure 5), and the annotation of each prominent peak in their spectra are also informed ( Table 2). In particular, the amides I−III found in both collagens would be applied to assess the triple helical structure of parrotfsh scale collagens. According to Nikoo et al. [52], using the formula of Δv (v I − v II ), where the diference in wavenumber (cm −1 ) between amides I and II is less than 100 cm −1 , suggesting that the triple helical structure of collagen is preserved. After confrmation, both ASC-PFS and PSC-PFS had the same value of delta v (∆v � 95.05 cm −1 ), and it could be argued that the triple  [7] Values are presented as the mean ± SD from triplicate (n � 3). Means provided in the same column with diferent notation are signifcantly diferent (p < 0.05). helical structure f the parrotfsh scale collagen was maintained. In addition, as proposed by Doyle et al. [49], the triple helical structure of collagen can be verifed using an AIII/A1450 ratio, and the result obtained in this study presented that the triple-helix structure obtained from ASC-PFS and PSC-PFS was intact during the extraction process, as also indicated by their absorption ratio values (∼1.0). Taken together, all the prominent peaks of parrotfsh collagens are in accordance with the previous reports from lizardfsh (S. tumbil) scale collagen [25] Nile tilapia (O. niloticus) scale collagen [34], carp (C. carpio) scale collagen [37], sea bass (L. calcarifer) scale collagen [9], and giant grouper (Epinephelus lanceolatus) scale collagen [53]. Figure 6. Generally, fsh collagen has two signifcant difraction peaks, which are sharp and broad peaks, and these peaks were exhibited in our present study. In comparison to both samples, the difraction peaks were somewhat the same, with the frst peak located at 7.65°and 7.59°, and the second peak at 19.71°and 19.37°, respectively. Tese difraction peaks were typically found in   [44], and lizardfsh (S. tumbil) skin, bone, and scale [7,25,32]. Moreover, to predict the low value of the repetitive spacings,, or d (Å), Zhang et al. [54] reported that the Bragg formula could be used in verifying the d (Å) through d (Å) � alpha/ 2sin theta in which alpha and theta represent the X-ray wavelength (1.54Å) and the Bragg difraction angle, respectively. Te d value of the frst peak was 0.103Å of ASC-PFS and 0.101Å of PSC-PFS. Tis frst peak value describes the range within the molecular chains of the triple helical structure in the collagen molecules. For the second peak, the d values of ASC-and PSC-PFS were 0.260Å and 0.255Å, respectively, refecting the spacing of skeletons. Tese most recent data were consistent with the diameter of a collagen molecule having a triple helical structure and a single left-handed helical chain. Terefore, parrotfsh scalederived ASC and PSC were considered in their native conformations.

Termal Stability Evaluation.
Te DSC results for parrotfsh scale collagens (i.e., ASC-PFS and PSC-PFS) were expressed in the T max and ΔH values, and the obtained results informed that a higher T max (37.78°C) and ΔH (0.35 J/ g) were recorded in the ASC-PFS sample compared to those of the PSC-PFS (T max � 36.22°C and ΔH � 0.02 J/g, respectively) (Figure 7). According to Benjakul et al. [56], collagen with a greater T max value has greater thermal stability due to the presence of imino acids, especially at pyrrolidine rings located in proline and hydroxyproline that were relatively constructed by H bonding via the -OH group of hydroxyproline, assuming that the imino acids are possibly higher in the ASC-PFS sample than that of the PSC-PFS sample.  [31]. In the context of ΔH, PSC-PFS was lower than the ASC-PFS sample, refecting a lower energy required to uncouple collagen alpha chains and convert them into random turns. It might be due to the cleaved telopeptide area by pepsin in the PSC-PFS sample.

Solubility Study. Solubility of the ASC-PFS and
PSC-PFS samples was tested at diferent NaCl concentrations and pH treatments, as presented in Figure 8. In both collagens, a highly soluble (>80%) was observed at the NaCl concentration of 0-20 g/L, while the relative solubility of both ASC-PFS and PSC-PFS declined steadily at the concentration of 30 g/L to 60 g/L. It could be due to the high concentration of sodium chloride added during the precipitation process of the solubilized collagen. In addition to this, an increase in NaCl concentration would enhance the interactions of hydrophobic-hydrophobic amino acids within the polypeptide chains of parrotfsh scale collagen and at the same time increase competition for water, resulting in the high protein precipitation [30]. Our fndings were also supported by other studies on diferent fsh collagens, such as carp (Hypophthalmichthys nobilis) scales [57], spotted golden goatfsh (P. heptacanthus) scales [24], and lizardfsh (S. tumbil) scales [25]. When comparing those two samples used in this study, the PSC-PFS showed more soluble in almost all NaCl treatments than the ASC-PFS sample. It suggests that the peptide at the telopeptide area cleaved by pepsin might contribute to the solubility of pepsin-soluble collagen from parrotfsh scales. In terms of pH treatment, both collagens treated at pH 1.0 to pH 5.0 were soluble, with a relative solubility of more than 60%. Te highest solubility was detected at pH 2.0 and pH 3.0 for ASC-PFS and PSC-PFS, respectively. In contrast, under pH 7.0 and pH 9.0 treatments, all collagen samples decreased sharply, representing below 30% of their relative solubility. As reported in numerous works, for instances: lizardfsh (S. tumbil) scale collagen [25], tilapia (O. niloticus) scale collagen [30], horse mackerel (Trachurus japonicas), scale collagen and grey mullet (Mugil cephalus) scale collagen [18]. It indicates that fsh scale collagens, particularly parrotfsh scale collagens, are unstable in the neutral and alkaline conditions. Te reason could be assumed to be that hydrophobic−hydrophobic interactions occur between the collagen molecules, causing the overall net charge to become zero, especially at the isoelectric point, which usually occurs under neutral conditions [58]. In addition to this, the lowest solubilisation rate was exhibited at pH 7 treatment of ASC-PFS, and it was probably due to the isoelectric point. Overall, the PSC-PFS had higher solubility (particularly under NaCl treatment) because the cleavage of telopeptide areas might afect the protonation of charged amino and carboxyl groups. Tis could infuence the repulsion of molecules associated with the diferent solubilities [24].

Conclusion
Type I collagen from the parrotfsh scales was successfully extracted by acetic acid (ASC-PFS) and with the aid of pepsin (PSC-PFS). Both samples had a high thermal stability, and the yields were relatively higher compared to some previous studies in the fsh scale collagens. Teir structure of triple-helix was maintained during the extraction process upon confrmation by the X-ray difraction and infrared spectroscopy analyses. Tus, collagens from parrotfsh scales could be used as an alternative source of collagen for further application.

Data Availability
Data used to support the fndings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest regarding the publication of this study.