Biochemical analysis of collagens from the bone of lizardfish (Saurida tumbil Bloch, 1795) extracted with different acids

Chemicals

Commercial fish collagen hydrolysate (type I collagen) was purchased from SRB Suria Trading (Kedah, Malaysia). Coomassie Blue R-250 (C.I. 42660), sodium dodecyl sulfate (SDS) (8170341000), N,N,N’,N’-tetramethyl ethylene diamine (TEMED) (1107320100), acrylamide (8008300500), Lowry reagent (L3540-25 VL), Folin-Ciocalteu’s phenol reagent (1090010100) and acetic acid (1000632511) were obtained from Merck (Darmstadt, Germany). Lactic acid (CAS 79-33-4) was bought from Bendosen (Kuala Lumpur, Malaysia) and citric acid (C1050-90) was obtained from Systerm (Selangor, Malaysia). Tris(hydroxymethyl) aminomethane hydrochloride (CAS 1185-53-1) and bovine serum albumin (BSA) (A2153-10G) were supplied by the Sigma Chemical Co. (St. Louis, MO, USA). Molecular weight markers using prestained natural protein standards (dual color standards) (1610374) was purchased from BioRad Laboratories (Hercules, CA, USA). Other chemicals used in this study were of analytical grade.

Preparation of lizardfish bone

Fresh lizardfish (S. tumbil) was purchased from a wet market in Kota Kinabalu, Sabah, Malaysia. Samples were placed in an insulated box containing ice at a ratio of fish/ice was 1:2 (w/w) to keep their freshness and delivered to the laboratory within 20 min. After species identification and authentication, the fish samples were weighed (202.58 ± 16.05 g), and the total length was measured (29.86 ± 0.41). The fish was washed with running tap water and subjected to separation of flesh, head, bone, skin and fins using a mechanical deboner machine (SFD-8, Taiwan). For the lizardfish bone collected, an approximate 1.0 × 1.0 cm² sample size was prepared for collagen extraction. Prepared fish bone samples were washed with chilled water and then placed in polyethylene containers. The packed samples were kept in a freezer (−20 °C) until further analysis.

Extraction of acid-soluble collagens

Acid-soluble collagens (ASCs) were carried out according to the method published by Matmaroh et al. (2011) with modifications in demineralization, extraction and centrifugation time. All procedures employed in this extraction process were performed in a chiller (4 °C), and the process is shown in Fig. 1. Thawed fish bones were dissolved with 0.1 M NaOH at a ratio of 1:10 (w/v) for 6 h to remove pigments and non-collagenous proteins, and the solution was changed every 3 h. The treated samples were then washed with cold distilled water until pH 7 was achieved. For the demineralization process, neutral pH samples were added to 0.5 EDTA-2Na solution at pH 7.4 for 48 h at a ratio of 1:10 (w/v), and the solution was changed every 16 h. Next, demineralized samples were washed with cold distilled water three times with continuous stirring for 10 min. and the samples were extracted with different acid solutions, i.e., acetic acid, lactic acid and citric acid, with 0.5 M of each acid used. Acid extraction was conducted by adding acids at a ratio of 1:15 (w/v) for 72 h with continuous stirring. After extraction, treated samples were filtered through 2 layers of cheese cloth. The filtrates were then collected and salted out by adding 2.5 M of NaCl containing 0.05 M Tris (hydroxymethyl) aminomethane (pH 7). Afterwards, the salted-out samples were centrifuged at 15,000× g for 30 min, and the pellets were dissolved in 0.5 M acids at a ratio of 1:5 (w/v). The solubilized samples were dialyzed using a dialysis tubing cellulose membrane (flat width 43 mm, Sigma) in a 20 volume of 0.1 M acetic, lactic and citric acids, respectively for 24 h and the solutions was changed with distilled water for 48 h. The dialyzed samples were lyophilized using a freeze-dryer (Labconco, South Kansas City, KS, USA). ASCs from different acid treatments were then stored in a freezer (−20 °C) until use. For measuring yield, a formula was adopted from Benjakul et al. (2010) based on the initial weight of lyophilized collagen in comparison with the initial weight of fish bone. For hydroxyproline content, all extracted collagens were analyzed using the method developed by Hofman et al. (2011).

Collagen analyses

Color attributes

Color ASCs and commercial fish collagen hydrolysates (CoFC) were determined according to the method reported by Huda et al. (2013) using a colorimeter model ColorFlex CX2379 (HunterLab, Reston, VA, USA). Parameters of color were tested, including lightness (L*), redness (a*) and yellowness (b*). Approximately 500 mg of fiber collagens was placed into a clear glass cup and then inserted into a cell holder of a colorimeter. The whiteness index (WI) was determined according to the study of Briones & Aguilera (2005) using the following formula: ${\displaystyle \text{WI}=100-{\left[{\left(100-L^{\ast }\right)}^2+\left(a^{\ast 2}\right)+\left(b^{\ast 2}\right)\right]}^{0.5}.}$

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) of all ASC and standard samples was performed according to the method from Laemmli (1970) with slight modifications. Approximately 2.5 mg of collagen was dissolved in one mL of SDS (5%), and the mixtures were heated at 85 °C for 1 h in a water bath. Afterward, the heated samples were centrifuged at 8,500× g for 5 min at 25 °C. The supernatants were pipetted and transferred into a new centrifuge tube. The prepared sample buffers (with and without 10% β-mercaptoethanol) were mixed with extracted collagens at a ratio of 1:1 (v/v) and then heated at 85 °C for 3 min. After thermal incubation, around 15 µL of the mixture was loaded into an acrylamide gel containing 5% stacking gel and 12% resolving gel. Next, electrophoresis was performed at a constant voltage of 120 V/gel for approximately 3 h using a Mini Protein II unit (Bio-Rad laboratories, Hercules, CA, USA). Acrylamide gels containing proteins were stained with 0.1% (w/v) Coomassie blue R-250 in 30% (v/v) methanol and 10% (v/v) acetic acid and destained with 30% (v/v) methanol and 10% (v/v) acetic acid. The molecular weight markers were determined using a dual color protein standard (10–250 kDa).

UV absorption spectrum

All ASCs and control sample were scanned based on UV absorption spectra at wavelengths from 400 nm to 200 nm using a UV-Vis spectrophotometer (Agilent Cary 60; Agilent, Santa Clara, CA, USA). This procedure was adopted from Liao et al. (2018). Approximately 10 mg of each lizardfish collagen was dissolved in 0.5 M acetic acid at a ratio of 1:1 (w/v), and the solution was dropped into a quartz cell. The spectral data was directly recorded according to the wavenumber set. The baseline was set with 0.5 M acetic acid solution.

Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy (FTIR) of the lyophilized collagen and CoFC samples was performed using a total reflectance-Fourier transform infrared (ATR-FTIR) (Agilent Cary 630, US) spectrometer according to the protocol from Matmaroh et al. (2011) with modification. One milligram of collagen was placed on a crystal cell. The spectra were adjusted within the wavenumbers of 4,000–600 cm⁻¹ with a resolution of 2 cm⁻¹ for 32 scans against a background spectrum recorded from the clean empty cells at room temperature. The resulting spectra were evaluated using the Agilent Microlab software program.

X-ray diffraction

X-ray diffraction (XRD) of the extracted lizardfish bone collagens with three different acids and the collagen control was carried out using the method of Liao et al. (2018). The lyophilized collagens were scanned using an XRD instrument (Rigaku Smart Lab®, Japan) with copper Kα as a source of X-rays. The tube voltage and current were set at 40 kV and 40 mA, respectively. The scanning range was determined to be between 10° and 50° (2θ) with a speed of 0.06° per second.

Differential scanning colorimetry

Determination of differential scanning colorimetry (DSC) in the lizardfish bone collagens and CoFC was conducted following the method of Sionkowska et al. (2015) using a DSC Q200 (TA Instruments, New Castle, DE, USA). The lyophilized collagens were rehydrated with deionized water at a ratio of 1:40 (w/v). The mixtures were allowed to stand for 2 days in a chiller (4 °C). Before scanning, the samples (3-10 mg) were precisely weighed into aluminum sample pans (PerkinElmer, Waltham, MA, USA) and tightly sealed. The sealed samples were heated from 20 °C to 250 °C in a nitrogen (N₂) stream with a heating rate of 10 °C/min. An empty pan was prepared as the reference. The maximum transition temperature (T_max) was measured from the endothermic peak of the thermogram, while the total denaturation enthalpy (ΔH) was determined by measuring the area of the DSC thermogram.

Solubility evaluation

Solubility profile of collagens was tested with different pH and NaCl concentration prepared according to the methods of Thuy, Okazaki & Osako (2014). In terms of pH treatment, the prepared samples were dissolved in 0.5 M acetic acid solution with continuous stirring at 4 °C for 18 h. The solubilized collagens were then adjusted at different pH values (pH 1-11) with 1 N NaOH and HCl. Each solubility test was prepared approximately eight mL of solubilized samples with a concentration of 3 mg/mL. Distilled water was added to the solutions to obtain a final volume of 10 mL. Next, the collagen solutions were centrifuged at 15,000 rpm for 30 min at 4 °C. For NaCl treatment, five milliliters of solubilized samples were mixed with five mL of NaCl solution. The NaCl concentrations used in this study were in the range of 0–60 g/L. The mixtures were then stirred at 4 °C for 1 h using a magnetic stirrer. Then, the mixtures were centrifuged at 15,000 rpm for 30 min at 4 °C. Protein content in the supernatant was determined by the Lowry method (Lowry et al., 1951) using bovine serum albumin (BSA) as a standard. Relative solubility was calculated using the following equations: ${\displaystyle \text{Relative solubility}\left(\text{\%}\right)=\frac{\text{Current concentration of protein}}{\text{The highest concentration of protein}}\times 100.}$

Statistical analysis

All experiments were performed in triplicate, and the data are expressed as the means ± standard deviation. One way analysis of variance was performed, and mean comparisons were analyzed by Duncan’s multiple range tests using SPSS Statistics version 27.0 (IBM Corp., Armonk, New York).

Yield and hydroxyproline content

Collagen from lizardfish bone was extracted using three different acids (i.e., acetic acid, lactic acid, and citric acid), the yields of collagen samples are presented at Table 1. The results showed a significant difference (P < 0.05) in the yields of collagen, i.e., 1.73%, 1.88%, and 2.59% (based on wet weight of bone), for acetic acid, lactic acid, and citric acid, respectively. The total collagen (mg/g) could be quantified indirectly through hydroxyproline (Hyp) content, as this amino acid is present almost exclusively in collagen. The content of Hyp was initially determined in all extracted samples, and the total collagen was subsequently multiplied by 7.7 as a conversion factor according to the method of Kittiphattanabawon et al. (2005). The results obtained showed that acetic acid-extracted collagen (AaEC) had the highest content of Hyp (98.70 mg/g) compared with lactic acid-extracted collagen (LaEC) (97.10 mg/g) and citric acid-extracted collagen (CaEC) (98.10 mg/g). The AaEC was also higher than that reported from commercial fish collagen hysrolysate (CoFC) (97.30 mg/g). However, those collagen samples were not significantly different (P ≥ 0.05) (Fig. 2). The total collagen of AaEC, LaEC, CaEC and CoFC was 760 mg/g, 747.80 mg/g, 755.20 mg/g and 749.40 mg/g, respectively, and they were not significantly (P ≥ 0.05) different.

Color parameters

Color is one of the most important aspects of collagen as an ingredient in cosmetics, pharmaceuticals and particularly for food production (Sliburyte & Skeryte, 2014). Table 1 shows the color attributes of acid-soluble collagens (ASCs) extracted from lizardfish bone. All ASCs had significantly higher (P < 0.05) L* (lightness) values than commercial fish collagen. Although not significantly different (P ≥ 0.05), the lightness level of AaEC was the highest. On the other hand, the AaEC a* and b* values (P < 0.05) were significantly lower than those of LaEC, CaEC and even CoFC. For the whiteness index (WI), AaEC exhibited the highest (P < 0.05) value compared to LaEC, CaEC and CoFC.

Protein profile

The protein profiles of lizardfish bone collagen extracted with different acids and CoFC sample under nonreducing and reducing conditions are displayed in Fig. 3. The results indicate that all extracted collagen samples contain two different α (α₁ and α₂) chains. The band intensity of α₁ was higher than that of the α₂ chain. Moreover, the intensity of bands in the LaEC and AaEC was greater than that in the CaEC with the same concentration applied. The estimated molecular weights of lizardfish collagens were 126.96 kDa (α₂), 160.81 kDa (α₁), 238.47 kDa (β-chain) and 290.39 kDa (γ chain). Furthermore, the effect of beta-mercaptoethanol (β-ME) (reducing) and no β-ME (non-reducing) additions exhibited similar electrophoretic patterns to all the extracted lizardfish bone collagens. On the other hand, there are no bands found in the commercial fish collagen hydrolysate.

UV absorption spectrum

In general, the triple helical collagen has the characteristic absorption peak at approximately 230 nm (Liao et al., 2018). Figure 4 shows the maximum absorption of lizardfish bone collagens and commercial fish collagen. All the extracted samples (AaEC, LaEC and CaEC) and the CoFC sample had UV maximum absorptions in the range between 231.0 nm and 231.9 nm.

FT-IR

FT-IR spectra peaks and their assignments for the lizardfish bone collagens extracted with acetic, lactic, and citric acids are shown in Fig. 5. The peaks were identified as amide A, amide B and amides I, II, and III (Ahmed, Verma & Patel, 2020). The amide A band reflects N-H stretching paired with H bonds, and several peaks for the acid-extracted samples (AaEC, CaEC, and LaEC) ranged from 3289.46 cm⁻¹ to 3295.05 cm⁻¹, while the commercial fish collagen (CoFC) sample was characterized by the lowest wavenumber for the amide A attribute. The amide B peak positions of CoFC, AaEC, CaEC, and LaEC ranged between 2924.17 cm⁻¹ and 2942.81 cm⁻¹, which represented CH₂₋, CH₃₋, and C-H-asymmetric stretching. For amide I-III bands, peaks were obtained below 1650 cm⁻¹ for all collagen samples.

XRD

All extracted collagen samples showed three diffraction peaks, while one diffraction peak was found in the CoFC sample (Fig. 6). The Bragg 2dsinθ = λ (λ = 0.154 Å) formula was employed to obtain the XRD results. The XRD patterns of AaEC, LaEC and CaEC had diffraction peaks at approximately 7.04°–32.71°, 7.41°–31.74°, and 7.15°–31.70°, respectively. Meanwhile, the commercial fish collagen hydrolysate harbored a diffraction peak at 20.52°. For ASCs, the first diffraction peak (A1) found in this study ranged between 7.04° and 7.41° with d values recorded from 12.49 Å to 12.50 Å. The CaEC sample recorded the greatest d value compared to the LaEC and AaEC samples. The second diffraction peak (A2) for all collagens was located between 19.54° and 21.32° with the CaEC sample showing the smallest d value (4.29 Å) compared with that of LaEC (5.01 Å), AaEC (4.40 Å) and CoFC (4.32 Å). The last diffraction peak, labeled A3, was observed in the range of 31.70°–31.74° with a similar d value (2.82 Å).

Thermal stability

The thermal stability of lizardfish bone collagens and commercial fish collagen hydrolysates was determined using differential scanning calorimetry (DSC). Theoretically, the heat flow was measured between the sample and reference and subsequently provides information regarding the thermal transitions of proteins. The transition temperature value from all collagen samples was determined at the maximum transition point (T_max) or the endothermic peak of transition curves, as presented in Table 2. All extracted collagens (AaEC, LaEC and CaEC) exhibited two noticeable peaks, and the first peak harbored T_max values of approximately 77.92–89.04 °C, which is associated with the release of the water bound in the collagen molecule as well as the degradation of the triple helical structure. Meanwhile, the second peak varied between 174.35 °C and 212.11 °C. This peak presents the melting temperature of the crosslinked collagen parts. In addition to the endothermic peak values, the denaturation enthalpy of the first transformation (ΔH = 169–294.5 J/g) was much higher compared to the second conversion (ΔH = 4.69–91.18 J/g).

Solubility of collagens

The effect of lyophilized collagens and commercial fish collagen hydrolysates treated at different pH levels is shown in Fig. 7A. In general, the extracted collagens were soluble in acidic pH between 1 and 5, with a relative solubility of more than 70%, while CoFC sample was soluble in all pH treatment (>75%). The highest solubility (P < 0.05) for all collagen samples was observed at pH 1. The lowest relative solubility (P < 0.05) for AaEC and LaEC was at neutral pH and for CaEC was at alkaline condition (pH 9). As depicted in Fig. 7B. AaEC, LaEC and CaEC had similar solubility patterns (P < 0.05). Higher solubility was exhibited at low NaCl concentrations ranging between 0 g/L and 20 g/L for all extracted collagens but at all NaCl treatments for the CoFC sample showed higher solubility (>90%). Meanwhile, the solubility sharply decreased (P < 0.05) for lizardfish bone collagens with the addition of more than 30 g/L NaCl.