Isolation and Comparative Study on the Characterization of Guanidine Hydrochloride Soluble Collagen and Pepsin Soluble Collagen from the Body of Surf Clam Shell (Coelomactra antiquata)

The aim of this study was to characterize the collagens from the body of surf clam shell (Coelomactra antiquata). Guanidine hydrochloride and pepsin were used to extract collagens. Guanidine hydrochloride soluble collagen (GSC) and pepsin soluble collagen (PSC) were separately isolated from the body of surf clam shell. Results showed that the moisture, protein, carbohydrate, and ash contents of the body of surf clam shell were 82.46%, 11.56%, 3.05%, and 2.38%, respectively, but the fat content was only 0.55%. The yields were 0.59% for GSC and 3.78% for PSC. Both GSC and PSC were composed of α1 and α2 chains and a β chain, however, GSC and PSC showed distinct differences from each other and the type I collagen from grass carp muscle on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). GSC and PSC contained glycine as the major amino acid and had imino acid of 150 and 155 residues/1000 residues, respectively. Fourier transform infrared spectroscopy (FTIR) spectra of GSC and PSC revealed the presence of a triple helix. The GSC appeared to have a dense sheet-like film linked by random-coiled filaments and PSC had fine globular filaments under scanning electron microscopy (SEM). The maximum transition temperature (Tmax) of GSC and PSC was 33.05 °C and 31.33 °C, respectively. These results provide valuable scientific information for the texture study and development of surf clam shell or other bivalve mollusks.


Introduction
Marine-based species comprise approximately one half of the total global biodiversity, and the oceans and aquatic environments in general offer plenty of resources for novel bioactive components. Marine species contain bioactive compounds and much attention has been paid to them, as they play pivotal roles in disease prevention and the maintenance of human health. These marine bioactive compounds exhibit significant biological properties that contribute to their nutraceutical and pharmaceutical potential and are also considered to be safer alternatives to some existing synthetic drugs [1][2][3]. Proteins (including collagen) or peptides are very important bioactive compounds that can be used in food and medicine [4,5].
Collagens, the major component of extracellular matrix (ECM) proteins, are a heterogeneous family of structural proteins representing nearly one-third of the total proteins. So far, approximately 29 types of collagens numbered as I to XXIX have been identified, and each type has considerably different molecular structures, amino acid sequences, and functions [6][7][8]. Most of these collagens have potentially widely commercial applications in food, cosmetic, biomedical, and pharmaceutical (Shanghai, China). Type I collagen from the skeletal muscle of grass carp was prepared in our own laboratory.

Extraction of Guanidine Hydrochloride Soluble Collagen (GSC)
GSC was extracted following the method of Mizuta et al. (1997) with slight modifications [27]. To remove non-collagenous proteins, muscle tissues were homogenized in ten volumes (v/w) of 0.1 M NaOH, and extracted for 24 h with gentle stirring at 4 • C. The residue after alkali extraction (RS-AL) was washed thoroughly with distilled water, and extracted with twenty volumes (v/w) of 50 mM Tris-HCl, pH 7.0, containing 4 M guanidine hydrochloride (G/HCl) for 24 h at 4 • C. The supernatant was collected by centrifugation at 15,000× g, at 4 • C for 30 min, dialyzed against distilled water overnight and then against 0.5 M acetic acid containing 2.0 M NaCl at 4 • C. After centrifugation at 15,000× g for 20 min, the resultant precipitate was collected, dialyzed against distilled water at 4 • C for 48 h, and lyophilized. The preparation obtained was referred to as G/HCl-soluble collagen (GSC).

Extraction of Pepsin Soluble Collagen (PSC)
The insoluble remaining matter after the G/HCl extraction of the RS-AL was washed thoroughly with distilled water and then digested with porcine pepsin in 0.5 M acetic acid at an enzyme/substrate ratio of 1:20 (w/w) for 48 h at 4 • C. After centrifugation at 15,000× g for 20 min, at 4 • C, the supernatant was obtained. NaCl powder was slowly added until the final concentration of 0.6 M was reached. It was then left to stand at 4 • C for 12 h and then centrifuged at 15,000× g for 20 min at 4 • C. The resulting precipitate was dissolved in 0.5 M acetic acid and dialyzed against 0.02 M Na 2 HPO 4 at 4 • C for 48 h. The dialysate was centrifuged at 15,000× g for 20 min at 4 • C. The resulting precipitate was dissolved in 0.5 M acetic acid and dialyzed against distilled water at 4 • C for 48 h. The resulting dialysates was freeze-dried and considered as pepsin soluble collagen (PSC).

Biochemical Compositions Analysis and the Collagen Yield of Surf Clam Shell
The moisture, protein, and ash contents of surf clam shell were determined according to the method of Meng et al. (2007) [28]. The yields of GSC and PSC were calculated based on the dry weight of starting materials. All the experiments were replicated three times.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
SDS-PAGE of collagens from muscle tissues was carried out according to the method of Wu et al. (2014) with slight modifications [29]. Solubilized samples were mixed with one quarter of SDS sample buffer (200 mM Tris-HCl, pH 6.8, containing 8% SDS (w/v), 0.4% bromophenol blue, and 40% (v/v) glycerol), then homogenized and heated in a water bath for 5 min. Samples (12 µg protein/each sample) were loaded onto the polyacrylamide gel (8%), respectively and then electrophoresed at a constant current of 12 mA. After electrophoresis, the gels were stained with 0.1% (w/v) Coomassie Blue R-250 in 50% (v/v) methanol and 7.5% (v/v) acetic acid at 70 • C for 30 min. Finally, the gels were destained with a mixture of 30% (v/v) methanol and 10% (v/v) acetic acid for 30 min twice by gently shaking. The protein molecular weights were calculated based on the unstained protein molecular weight markers (Ferments, Burlington, CA, USA).

Amino Acid Analysis
Amino acid analysis of the GSC and PSC from surf clam shell was performed according to the method of Lin et al. (2017) with slight modifications [30]. GSC and PSC were hydrolyzed respectively in 6 M hydrochloric acid at 110 • C for 24 h in the absence of oxygen. The hydrolysates were analyzed on a Hitachi L-8800 auto amino acid analyzer (Hitachi, Tokyo, Japan) with a mobile phase flow of 0.400 mL min −1 and the flow of ninhydrin solution was set at 0.350 mL min −1 . The content of amino acid was expressed as residues/1000 residues.

Analysis of Fourier Transform Infrared Spectroscopy (FTIR)
Amide band patterning of GSC and PSC was analyzed using a Nicolet AVATAR 360 FTIR spectrometer (Nicolet Co., Madison, WI, USA) according to the method of Lin et al. (2017) with slight modifications [30]. Under drying condition, about 5 mg lyophilized collagen sample and 100 mg potassium bromide (KBr) were ground together using a mortar and pestle. The sample was subjected to a pressure of about 5 × 10 6 Pa in an evacuated die to produce a 13 × 1 mm clear transparent disk. The spectrum was obtained with 32 scans per sample ranging from 4000 to 400 cm −1 . The resulting spectral data was analyzed using ORIGIN 8.0 software (Thermo-Nicolet, Madison, WI, USA).

Scanning Electron Microscopy (SEM)
The morphological characteristics of the pretreated collagens were studied by SEM (Nova NanoSEM 230, Hillsboro, OR, USA) according to the method of Tziveleka et al. (2017) with slight modifications [7]. The samples were mounted on stubs, sputter-coated with gold, and then observed for surface morphology at various magnifications. The SEM observations were made at 15 kV accelerating voltage. A higher vacuum (HV) mode and secondary electron image (SEI) were employed to scan the microscopic images of collagen matrix.

Determination of Denaturation Temperature
GSC and PSC were separately rehydrated by adding deionized water at a solid to solution ratio of 1:40 (w/v). The mixtures were allowed to stand for 2 days at 4 • C prior to analysis. Differential scanning calorimetry (DSC) was performed using a differential scanning calorimeter model DSC 7 (Perkin Elmer, Norwalk, CT, USA) according to the method of Yang et al. (2016) with slight modifications [31]. Calibration was run using the Indium thermogram. The samples (5-10 mg) were accurately weighed into aluminum pans and sealed. The samples were scanned at 1 • C min −1 over the range of 25-50 • C using iced water as the cooling medium. An empty pan was used as the reference. The maximum transition temperature (T max ) was estimated from the thermogram. The total denaturation enthalpy (∆H) was estimated by measuring the area of the DSC thermogram.

Biochemical Compositions
Surf clam shell (Coelomactra antiquata) contained very high moisture content, which was about 82.46 ± 0.12%. The protein, carbohydrate, and ash contents of surf clam shell, which were 11.56 ± 0.08%, 3.05 ± 0.05% and 2.38 ± 0.05%, respectively, were much lower than the moisture content. It is noteworthy that the fat content was very low (only 0.55 ± 0.09%). Tabakaeva et al. (2018) also reported that the two commercially significant edible bivalve mollusk species (Anadara broughtonii and Mactra chinensis) had low fat content, and that protein and carbohydrates were their main components [32].

The Yields of GSC and PSC from Surf Clam Shell
The approximate collagen content of surf clam shell was 4.37 ± 0.04% (on a dry weight basis) of protein for the whole soft body, which was similar to the yields of collagen from Mytilus galloprovincialis and Septifer virgatus [24]. When the GSC and PSC were extracted from the whole soft body of surf clam shell, the respective yields of 0.59 ± 0.03% and 3.78 ± 0.04% (on a dry weight basis) were obtained. So far, few GSC yields have been previously reported. Based on the lyophilized dry weight, the PSC yield of rhizostomous jellyfish (Rhopilema asamushi) was as high as 35.2% [33], whereas the PSC yield of silver carp scales was only 2.32% [34]. Therefore, the collagen yields might be associated with species and preparation methods. Also, seasonal change might also influence the yield of collagen. Olaechea et al. discovered that the collagen content of the foot muscle of the disk abalone (Haliotis discus) showed seasonal change that corresponded well with the meat toughness [35].

Protein Patterns
Electrophoretic patterns of GSC and PSC analyzed by SDS-PAGE under non-reducing condition are shown in Figure 1, along with type I collagen from the muscle of grass carp. It revealed that both of the two collagens comprised at least two different α chains (α 1 and α 2 ) and high-molecular-weight components including β chain. Both GSC and PSC also contained γ chain-sized components to some extent. However, there were considerable differences in the SDS-PAGE patterns, suggesting minor differences in the structure of GSC and PSC. The GSC showed two α chain-sized components (α 1 and α 2 ), with molecular weights estimated to be in the range of 125-135 kDa, and one β chain-sized component with slower mobility than those of the corresponding components in grass carp type I collagen. Compared to GSC, the PSC showed mainly two α chain-sized components (α 1 and α 2 ) with much lower molecular weight components and a β chain-sized component with higher molecular weight. Moreover, the molecular weights of both α chain-sized components and the β chain-sized component of GSC and PSC were higher than that of type I collagen from grass carp muscle. Further, the density of the β chain of GSC was higher than that of PSC, which might indicate that GSC has more intramolecular or intermolecular cross-links than that in PSC. On the contrary, the relative staining intensity of the α 1 and α 2 chains was apparently higher in PSC than that in GSC. This was explained by conversions of some β or γ chain-sized component in the PSC matrix to α-components by the treatment with pepsin. Pepsin cleaves the crosslink containing telopeptide, and the β-chain is converted to two α-chains [36].

Protein Patterns
Electrophoretic patterns of GSC and PSC analyzed by SDS-PAGE under non-reducing condition are shown in Figure 1, along with type I collagen from the muscle of grass carp. It revealed that both of the two collagens comprised at least two different α chains (α1 and α2) and high-molecular-weight components including β chain. Both GSC and PSC also contained γ chain-sized components to some extent. However, there were considerable differences in the SDS-PAGE patterns, suggesting minor differences in the structure of GSC and PSC. The GSC showed two α chain-sized components (α1 and α2), with molecular weights estimated to be in the range of 125-135 kDa, and one β chain-sized component with slower mobility than those of the corresponding components in grass carp type I collagen. Compared to GSC, the PSC showed mainly two α chain-sized components (α1 and α2) with much lower molecular weight components and a β chain-sized component with higher molecular weight. Moreover, the molecular weights of both α chain-sized components and the β chain-sized component of GSC and PSC were higher than that of type I collagen from grass carp muscle. Further, the density of the β chain of GSC was higher than that of PSC, which might indicate that GSC has more intramolecular or intermolecular cross-links than that in PSC. On the contrary, the relative staining intensity of the α1 and α2 chains was apparently higher in PSC than that in GSC. This was explained by conversions of some β or γ chain-sized component in the PSC matrix to α-components by the treatment with pepsin. Pepsin cleaves the crosslink containing telopeptide, and the β-chain is converted to two α-chains [36].
The GSC and PSC extracted from pearl oyster (Pinctada fucata) and Crassostrea gigas exhibited quite a similar pattern [23,24]. However, in our current experiment, the results showed that the SDS-PAGE patterns of the GSC and PSC were considerably different, which was most likely due to the seasons, species, and preparation methods. Also, they were very different from that of collagen from Australasian Snapper (Pagrus auratus) [11], seabass (Lates calcarifer) [20], squid (Todarodes pacificus) [27], and soft-shelled turtle calipash [31].  The GSC and PSC extracted from pearl oyster (Pinctada fucata) and Crassostrea gigas exhibited quite a similar pattern [23,24]. However, in our current experiment, the results showed that the SDS-PAGE patterns of the GSC and PSC were considerably different, which was most likely due to the seasons, species, and preparation methods. Also, they were very different from that of collagen from Australasian Snapper (Pagrus auratus) [11], seabass (Lates calcarifer) [20], squid (Todarodes pacificus) [27], and soft-shelled turtle calipash [31].

Amino Acid Composition
The amino acid compositions of both GSC and PSC from surf clam shell, expressed as residues per 1000 total residues, are shown in Table 1. GSC and PSC were rich in glycine (244 and 254 residues/1000 residues, respectively), which was the major amino acid in collagen. The glycine contents of GSC and PSC were lower than that of PSC from bighead carp (Hypophthalmichthys nobilis) [6], unicorn leatherjacket (Aluterus monocerous) [14], silver carp (Hypophthalmichthys molitrix) [34], and marine crab (Scylla serrate) [26]. Generally, glycine in collagens represents nearly one-third of the total residues and occurs at every third residue in collagens, except for the first 14 amino acid residues from the N-terminus and the first 10 residues from the C-terminus [37]. Glutamic acid contents of GSC and PSC were 118 and 119 residues/1000 residues respectively, thus representing the second most abundant amino acid. Low contents of histidine were found to be 11 and 9 residues/1000 residues in GSC and PSC, respectively. The cysteine contents of GSC and PSC were only 1 and 2 residues/1000 residues, respectively. Table 1. Amino acid composition of guanidine hydrochloride soluble collagen (GSC) and pepsin soluble collagen (PSC) from Coelomactra antiquata (results are expressed as residues/1000 residues). Alanine  70  67  Cystine  1  2  Aspartic acid  81  76  Glutamic acid  118  119  Phenylalanine  17  16  Glycine  244  254  Histidine  11  9  Isoleucine  26  24  Lysine  35  22  Leucine  42  44  Methionine  14  15  Proline  85  86  Arginine  52  65  Serine  55  50  Threonine  35  34  Valine  32  32  Tyrosine  17  16  Hydroxyproline  65  69  Total  1000  1000  Imino acid  150  155 GSC: guanidine hydrochloride soluble collagen; PSC: pepsin soluble collagen.

Amino Acids GSC PSC
The imino acid (proline and hydroxyproline) were unique amino acids found in collagen. Their contents in GSC and PSC were 150 and 155 residues/1000 residues, respectively. However, so far, little information on the imino acid of GSC from bivalve mollusks has been reported. The imino acid content of PSC from surf clam shell was higher than those of the PSC from the fresh body wall of A. leucoprocta (141 residues/1000 residues) [30], and collagen from blue shark cartilage (122 residues/1000 residues) [38], but lower than those of the PSC from the skin of bighead carp (165 residues/1000 residues) [6] and swim bladders of yellowfin tuna (169 residues/1000 residues) [39]. Imino acids contribute to the stability of the helix structure of collagens [40]. In addition, the imino acid content has been known to determine the thermal stability of collagen and the formation of junction zones via hydrogen bondings. Hydroxyproline plays a significant role in stabilizing the triple helical structure by the formation of interchain hydrogen bonds through the hydroxyl group [20]. These results suggested that the slightly different imino acid composition of GSC and PSC from surf clam shell tended to result in the distinct differences in structure and thermal stability.
Marine collagens are a promising source for producing bioactive peptides. Researchers have found some notable activities of marine collagen peptides, such as skin antiaging activity, antioxidant activity, antihypertensive activity, antimicrobial activity, anti-HIV-1, wound healing, and iron-binding [9]. Collagen peptide containing several specific amino acids presented relevant activities. Many researchers have previously found that low molecular weight collagen peptides (di-and tripeptides), especially those with C-terminal Pro or Hyp residues, have exhibited numerous bioactivities including antibacterial, antioxidative, angiotensin converting enzyme (ACE) inhibitory properties, etc. [9]. It is well documented that dietary supplementation with arginin improves glycemic control [41]. GSC and PSC from surf clam shell contained 18 amino acids. Therefore, the GSC and PSC may represent potential resources for producing bioactive peptides.

Fourier Transforms Infrared (FTIR) Spectra of Collagen
FTIR spectra of GSC and PSC exhibited the characteristic peaks of amide A and B as well as amide I, II, and III bands ( Figure 2). FTIR spectra for the GSC and PSC were similar to other collagens [6,39]. The amide A band is associated with the N-H stretching frequency. The amide A band positions of GSC and PSC were found at 3416 and 3374 cm −1 , respectively, which are shown in Figure 2b. The absorption characteristic of amide A is commonly associated with the N-H stretching band and shows the existence of hydrogen bonds. A free N-H stretching vibration occurs in the range of 3400-3440 cm −1 , and when the N-H group of a peptide is involved in hydrogen bonding, the position is shifted to lower frequencies, usually 3300 cm −1 [42]. The result indicated that more N-H groups of GSC were involved in hydrogen bonding than those of PSC. Amide B bands of GSC and PSC were observed at 2925 and 2921 cm −1 , respectively, which represent the asymmetrical stretch of CH 2 [43].
The amide I band positions of GSC and PSC were observed at 1654 and 1667 cm −1 , respectively. The amide I band, with characteristic frequencies in the range from 1600 to 1700 cm −1 , is mainly associated with the stretching vibrations of the carbonyl group (C=O bond) along the polypeptide backbone [38]. Amide II is generally responsible for the combination of the NH in plane bend and the CN stretching vibration [38]. The amide II band of GSC (1540 cm −1 ) was found at a lower wavenumber compared to that of PSC (1556 cm −1 ), suggesting that there was a stronger hydrogen bond in GSC. The absorption between the 1236 and 1452 cm −1 bands (amide III) demonstrated the presence of a helical structure and also suggested the helical arrangement of the two collagens, with the GSC absorption ratio at 1234 cm −1 and the PSC absorption ratio at 1242 cm −1 [30]. Additionally, the absorption peaks around 1451-1462 cm were also found in GSC and PSC. This corresponded well to the pyrrolidine ring vibration of proline and hydroxyproline, as previously described [24].
The FTIR investigations of GSC and PSC from the surf clam shell had some slight differences, which indicated discrepancies in the secondary structures of the collagens. The FTIR spectra were consistent with the results of SDS-PAGE.

Morphological Characterization of Collagens
The morphological structures of the extracted collagens (GSC and PSC) were observed under SEM micro-photography with higher magnification (Figure 3). All the collagens looked like soft white sponge with loose and porous structure by the naked eye. However, the GSC appeared to be a dense irregular sheet-like film linked by random-coiled filaments under SEM, and the surface was partially wrinkled. This was possibly because of dehydration during lyophilizing (Figure 3a), which was in agreement with the skin collagen of Amur sturgeon [21]. Although not well organized, the intersecting sheet-like films were not parallel but entangled in individual bundles in the three-dimensional structure (Figure 3b).
Semblable porous matrix with good interconnectivity in PSC was quite different from GSC (Figure 3c), which was observed to have a complex meshwork form and contact with some fibrils. These collagen fibrils across the porous matrix varied in width and thickness, and intertwined with each other. In addition, the fibrillar appearance observed in PSC changed into an amorphous structure, characterized by a structure composed of strips (Figure 3d). The lyophilized PSC was loose and endowed with uniform and regular alveolate pores due to the evaporation of fluid; also, the pore

Morphological Characterization of Collagens
The morphological structures of the extracted collagens (GSC and PSC) were observed under SEM micro-photography with higher magnification (Figure 3). All the collagens looked like soft white sponge with loose and porous structure by the naked eye. However, the GSC appeared to be a dense irregular sheet-like film linked by random-coiled filaments under SEM, and the surface was partially wrinkled. This was possibly because of dehydration during lyophilizing (Figure 3a), which was in agreement with the skin collagen of Amur sturgeon [21]. Although not well organized, the intersecting sheet-like films were not parallel but entangled in individual bundles in the three-dimensional structure (Figure 3b). other drugs, but also for evaporation of fluid [44]. Architectural features such as porosity, pore size, and specific surface areas are wildly considered as important factors for a biomaterial to understand their biomedical importance [45]. In addition, other architectural features, such as pore shape, pore wall morphology, and interconnectivity of collagen, have also been suggested for use in cell seeding, growth, gene expression, migration, mass transport, and new tissue formation. According to SEM, the extracted collagens form surf clam shell may be used as suitable biomaterials.

Thermal Transition
The maximum transition temperature (Tmax) and enthalpy (ΔH) of GSC and PSC from surf clam shell in deionized water are shown in Figure 4. The Tmax values of GSC and PSC from surf clam shell were measured at 33.05 °C (ΔH = 0.3667 J g −1 ) and 31.33 °C (ΔH = 0.451 J g −1 ), respectively. Thermal stability of collagen was governed by the pyrrolidine rings of proline and hydroxyproline and partially by hydrogen bonding through the hydroxyl group of hydroxyproline [18]. A slightly higher denaturation enthalpy (ΔH) value was found for PSC in comparison to that of GSC. The removal of telopeptides might lead to a more ordered structure of PSC, in which higher energy was required. The presence of imino acids, particularly hydroxyproline in GSC and PSC, might contribute to the stabilization of the triple helix structure through hydrogen bonding in coil-coiled α-chains [17]. The Tmax values of GSC and PSC were higher than those of collagens from carp scales (about 28 °C) and the mesoglea collagen from rhizostomous jellyfish (R. asamushi) (about 28.8 °C) [33], which suggested a higher thermal stability of the former examples. Interestingly, the Tmax of PSC from surf clam shell collagens were discovered to be slightly lower than those of collagen from silver carp scales (35.5 °C) Semblable porous matrix with good interconnectivity in PSC was quite different from GSC (Figure 3c), which was observed to have a complex meshwork form and contact with some fibrils. These collagen fibrils across the porous matrix varied in width and thickness, and intertwined with each other. In addition, the fibrillar appearance observed in PSC changed into an amorphous structure, characterized by a structure composed of strips (Figure 3d). The lyophilized PSC was loose and endowed with uniform and regular alveolate pores due to the evaporation of fluid; also, the pore size of the collagen sponge increased at higher water content during preparation [39]. Additionally, certain areas contained disorganized nanofibers.
Efforts have been made to discover the uses of marine collagen, including possible uses for drug delivery systems, tissue engineering, cosmetics, and nutricosmetics [9]. Uniform and regular network structures of sponges, as drug carriers are propitious, not only for well-proportioned distribution for other drugs, but also for evaporation of fluid [44]. Architectural features such as porosity, pore size, and specific surface areas are wildly considered as important factors for a biomaterial to understand their biomedical importance [45]. In addition, other architectural features, such as pore shape, pore wall morphology, and interconnectivity of collagen, have also been suggested for use in cell seeding, growth, gene expression, migration, mass transport, and new tissue formation. According to SEM, the extracted collagens form surf clam shell may be used as suitable biomaterials.

Thermal Transition
The maximum transition temperature (T max ) and enthalpy (∆H) of GSC and PSC from surf clam shell in deionized water are shown in Figure 4. The T max values of GSC and PSC from surf clam shell were measured at 33.05 • C (∆H = 0.3667 J g −1 ) and 31.33 • C (∆H = 0.451 J g −1 ), respectively. Thermal stability of collagen was governed by the pyrrolidine rings of proline and hydroxyproline and partially by hydrogen bonding through the hydroxyl group of hydroxyproline [18]. A slightly higher denaturation enthalpy (∆H) value was found for PSC in comparison to that of GSC. The removal of telopeptides might lead to a more ordered structure of PSC, in which higher energy was required. The presence of imino acids, particularly hydroxyproline in GSC and PSC, might contribute to the stabilization of the triple helix structure through hydrogen bonding in coil-coiled α-chains [17]. The T max values of GSC and PSC were higher than those of collagens from carp scales (about 28 • C) and the mesoglea collagen from rhizostomous jellyfish (R. asamushi) (about 28.8 • C) [33], which suggested a higher thermal stability of the former examples. Interestingly, the T max of PSC from surf clam shell collagens were discovered to be slightly lower than those of collagen from silver carp scales (35.5 • C) [34]. The thermal stability of collagens not only depend on the imino acid content but also directly correlate with the environmental and body temperatures of fish species [46]. Thus, the thermal properties of GSC and PSC were influenced by the tissues used for collagen extraction compared to mammalian gelatin.  [34]. The thermal stability of collagens not only depend on the imino acid content but also directly correlate with the environmental and body temperatures of fish species [46]. Thus, the thermal properties of GSC and PSC were influenced by the tissues used for collagen extraction compared to mammalian gelatin.

Conclusions
Two different collagens of GSC and PSC were successfully isolated from the surf clam shells. Our results showed that the GSC and PSC had slight differences in molecular weights, amino acid composition, morphological structures, and thermal stability, which implied that GSC and PSC maybe two different kinds of collagens. The properties of GSC and PSC from surf clam shells also showed obvious differences compared to those of fish species. FTIR investigations showed the existence of helical arrangements of the two collagens. Therefore, guanidine hydrochloride and pepsin added extraction could serve as a tool for obtaining collagens without a marked effect on the triple-helical structure. From this study, these results could provide a valuable scientific basis for the study of the texture and development of surf clam shell or other bivalve mollusks. The results also suggest that the collagens can potentially serve as an alternative source of collagen for further application in food, pharmaceutical industries, cosmetic, suitable biomaterial, and other applications.

Conclusions
Two different collagens of GSC and PSC were successfully isolated from the surf clam shells. Our results showed that the GSC and PSC had slight differences in molecular weights, amino acid composition, morphological structures, and thermal stability, which implied that GSC and PSC maybe two different kinds of collagens. The properties of GSC and PSC from surf clam shells also showed obvious differences compared to those of fish species. FTIR investigations showed the existence of helical arrangements of the two collagens. Therefore, guanidine hydrochloride and pepsin added extraction could serve as a tool for obtaining collagens without a marked effect on the triple-helical structure. From this study, these results could provide a valuable scientific basis for the study of the texture and development of surf clam shell or other bivalve mollusks. The results also suggest that the collagens can potentially serve as an alternative source of collagen for further application in food, pharmaceutical industries, cosmetic, suitable biomaterial, and other applications.