INTRODUCTION:

Skin is the body's outer layer, which covers the entire external surface. Skin aging involves a combination of internal and external factors. UV radiation is the primary factor in skin aging. UV radiation will generate reactive oxygen species (ROS), which further mediates extracellular matrix degradation, including collagen, hyaluronic acid, elastin, and heparan sulfate—skin aging results in skin dryness, pigmentation, and wrinkles^1,2,3,4. Skin aging is mainly caused by reduced collagen production and increased collagen degradation. Collagen is an abundant structural protein formed in an extracellular matrix with elastin, proteoglycans, and hyaluronate. Collagen consists of several types, including types I and III^5,6.

The research hotspot on antiaging mainly focuses on delaying aging by administering natural or synthetic anti-aging^7,8. One of these is by providing nutritional support to collagen. Collagen peptide is a low molecular weight protein obtained by hydrolyzing collagen with acid, base, enzymatic, or other ways. Collagen peptides have better activity, solubility, and lower allergenic properties than native collagen. Collagen peptide stimulates new collagen synthesis by upregulating gene expression, modification, and degradation of collagen-related gene^9-14.

Collagen can be obtained from cows or pigs. However, the risk of bovine spongiform encephalopathy, foot and mouth disease, and transmissible spongiform encephalopathies has increased marine collagen use. Fish collagen has a molecular weight of about 300 kDa. Fish collagen can be extracted from various sources^15-18. It consists of three polypeptide strands (homotrimeric or heterotrimeric) that are stabilized by hydrogen bonds. The collagen measures about 280nm in length and 1.4 nm in diameter^19-23.

The active form of collagen is peptide. It acts as a ligand in fibroblast cell surface receptors. The binding of peptide collagen in fibroblast receptors will activate MAPK (mitogen-activated protein kinase), TGF- (tissue growth factor), and NF-kb (nuclear factor-kappa beta). This signaling pathway will stimulate the expression of pro-coll1 and inactivation of MMP (matrix metalloprotein)^24,25. The topical administration of protein or peptide is determined by molecular size but also by the three-dimensional structure of it²⁶. The protein or peptide that can be administered topically has a molecular size of 500 Da, log-P 2-3, and water solubility around 1mg/ml²⁷. To increase protein or peptide through topical administration, it could be developed into a drug delivery system^28,29.

Bromelain is one of the proteases in the cysteine protease group found in the entire pineapple. The protease is an enzyme that can break peptide bonds in numerous proteins. Protease will hydrolyze and convert protein into a single amino acid or peptide. The trend of using bromelain is increasing due to its multiple applications in the pharmaceutical and food industry^30,31,32. Moreover, in the context of collagen production, using bromelain will provide halal products for Muslim consumers. Only a few studies have reported on collagen hydrolysis using some protease, and no reports are available on collagen hydrolysis using bromelain (a single enzyme) for fish collagen hydrolysis.

Thermal treatment is one of the standard methods to modify protein. The degree of denaturation is affected by the time and temperature used in the treatment. The different temperatures could alter the protein’s chemical structure³³. Previous studies showed conformational changes or denaturing occurs at 100°C. At higher temperatures, the complete degradation of protein involves dehydration, hydrogen sulfide, ammonia formation, and cleaving of the C-C bond, following decarboxylation and decarbonylation^34,35.

The quantitative analysis of the sample was conducted using the Lowry assay. This assay relies on binding copper ion (Cu²⁺) with protein, forming Cu and protein complex (Shao-Chia Chou and A. Goldstein, n.d.). The color responses of cold-water fish were closer compared to BSA (bovine serum albumin) as standard in the Lowry assay³⁶. The number of cleaved peptide bonds to form protein hydrolysate from thermal and enzymatic hydrolysis is defined as the degree of hydrolysis. There are several methods to determine the degree of hydrolysis, one of which is the trichloroacetic acid (TCA) method. The TCA method measures the amount of TCA-soluble nitrogen³⁷. The protein hydrolysate was characterized using SEM (scanning electron microscope), FTIR (Fourier transform infrared spectroscopy), and DSC (differential scanning calorimetry). SEM uses an energy electron beam to generate information about the sample, including the external morphology (texture). FTIR is used to observe the spectrum of molecular fingerprints and the difference between native and hydrolyzed collagen. DSC is a thermoanalytical technique that compares the material transitions as a function of temperature and time between native and hydrolyzed collagen^38,39.

Our research investigates the most optimal method to produce hydrolyzed collagen. We compare the effectiveness of thermal and enzymatic hydrolysis. The thermal hydrolysis was conducted at various temperatures (50, 60, 70, 80, 90, 100°C) for different time incubation. The enzymatic hydrolysis was performed using bromelain (10 U/ml) at 60°C for 1, 2, 3, and 4-hour incubation. The quantitative analysis was conducted with Lowry. The collagen and hydrolyzed collagen characterization was performed using electrophoresis, SEM, DSC, and FTIR. Our finding showed that thermal hydrolysis will denature collagen, while enzymatic hydrolysis will produce hydrolyzed collagen.

MATERIALS AND METHODS:

Materials:

Bromelain (Merck, Darmstadt, German), fish collagen (Sigma-Aldrich, St. Louis, MO, USA), Ninhidrin (Merck, Darmstadt, German), Trans-4-hydroxy-L-proline (Sigma-Aldrich, St. Louis, MO, USA), 4-(Dimethylamino) benzaldehyde (Sigma-Aldrich, St. Louis, MO, USA), Chloramine T trihydrate (Sigma-Aldrich, St. Louis, MO, USA), Amicon^® ultra centrifugation filters (Merck Millipore, Billericia, MA, USA).

Thermal hydrolysis of collagen:

Fish collagen was dissolved in aquadest at 30 mg/mL. The dissolved fish collagen was then incubated at 50, 60, 70, 80, 90, and 100°C. The incubation was conducted for 1, 2, 3, 4, 5 h. The protein is then stored at 4°C.

Enzymatic hydrolysis of collagen:

Fish collagen was dissolved in aquadest at 30mg/mL. Enzymatic hydrolysis was conducted with bromelain activity of 10U/mL at 60°C for 1, 2, 3, 4h 40, 41. The protein was then stored at 4°C.

Collagen fractionation:

The hydrolysate was fractionated using ultrafiltration with molecular weight cut-off (MWCO) 30 kDa (Amicon®ultra, Merck, German). Amicon®ultra 30 K centrifugal filter unit was pre-washed with 500µl PBS (phosphate buffer saline). The sample was loaded to a centrifugal filter unit and centrifuged at 4x10³g for 5 minutes. The filtrate at the bottom of the centrifugal filter unit contains collagen peptide with a size < 30 kDa, while the supernatant at the top includes peptide > 30 kDa.

Quantitative analysis collagen:

The quantitative protein analysis was conducted using the Lowry method (Lowry et al., 1951) and a spectrophotometer (Agilent Cary UV-Vis, Santa Clara, CA, USA). The collagen in the sample was indirectly quantified based on hydroxyproline (HYP) following the method described previously^42, and the hydroxyproline concentration was calculated with the formula below:

Collagen Concentration (mg/mL) 100

= HYP-Conc (mg/mL) x ----------

13.5

Differential Scanning Calorimetry (DSC):

The native and hydrolyzed samples were thermally analyzed on a DSC Q20 (TA Instruments, DE, USA). The aluminum pan was used to place the sample (1-3 mg), and sample analysis was conducted at 50 to 300°C with a heating rate of 10°C/min. Samples were continuously purged with nitrogen at 50mL/ min.

FTIR spectroscopy:

The IR spectra of the native and hydrolysate collagen were recorded on an FT/IR-6100 type A infrared spectrometer (JASCO, MD, USA) in ATR mode from 4000-700 cm⁻¹ with a resolution of 4 cm⁻¹.

Scanning Electron Microscopy (SEM):

The native and hydrolysate collagen morphology was analyzed using a JEOL JSM-6510 scanning electron microscope (SEM, JEOL Ltd., Tokyo, Japan). All samples were mounted on a double-faced adhesive tape and sputtered with gold (accelerating voltage of 5 kV).

Gel Electrophoresis:

A tricine SDS-PAGE was prepared with two layers of separating gel (10 and 16%) and stacking gel at a concentration of 4%. The sample was prepared by adding loading buffer (Tris-HCl, glycerol, SDS, and bromphenol blue) in a ratio of 1:4. After the gel solidified, the gel was turned at 120 V for 100 minutes (Bio-Rad Power Pac Basic and Mini-Protean tetra cell) alongside with an ultra-low molecular marker. After running, the gel was stained in Coomassie Brilliant Blue R-250 for 2 hours and in a destaining solution for another 2 hours.

RESULT:

Calibration curve for quantitative analysis:

Calibration curves for quantitative collagen analysis were made using BSA (bovine serum albumin) for the Lowry method and hydroxyproline for hydroxyproline hydrolysis analysis. Both calibration curves have R > 0.99.

Figure 1. Calibration curve of A. bovine serum albumin (BSA) with Lowry method B. Hydroxyproline (Hyp)

Thermal and enzymatic hydrolysis:

Thermal hydrolysis was conducted at 50, 60, 70, 80, and 90°C for one-hour incubation. At the same time, enzymatic hydrolysis was conducted with 10 U/ml bromelain at 60°C for 1 to 5-hour incubation—the protein fraction (<30 kDa) was quantified by the Lowry method.

Figure 2. Quantitative assay of protein (<30 kDa) using Lowry method A. After treatment at 50, 60, 70, 80, and 90°C, total protein concentration for an hour B. Total protein concentration after treatment at 100°C for 1, 2, 3,4, and 5 hours.

Degree of hydrolysis:

The degree of hydrolysis was calculated by comparing the lower molecular hydrolyzed protein fraction (<30 kDa) to the initial protein concentration. The protein was quantified using the Lowry method.

Figure 3. Degree of hydrolysis A. thermal hydrolysis at 50, 60, 70, 80, and 90° C for an hour B. enzymatic hydrolysis with bromelain at 60°C for 1, 2, 3, and 4 h incubation

Morphological analysis of collagen and hydrolyzed collagen:

The morphological analysis of native, thermal, and enzymatic hydrolysis of collagen was observed with SEM (scanning electron microscope).

Figure 4. Scanning electron microscope (SEM) of A. Native collagen B. Thermal hydrolysis of collagen C. Enzymatic hydrolysis of collagen

Electrophoresis:

The protein was qualitatively analyzed with Tricine-sodium dodecyl sulfate electrophoresis to compare the native, thermal, and enzymatic hydrolysis of collagen. The presence of a band corresponding to a specific protein or peptide molecular size could be used to observe the hydrolysis process.

Figure 5. Tricine-SDS (sodium dodecyl sulfate) electrophoresis of A. Thermal hydrolysis B. Enzymatic hydrolysis of collagen. M = Marker protein, C = collagen native, B = bromelain, EH = enzymatic hydrolysis

FTIR spectroscopy:

FTIR was used to compare the native and enzymatic hydrolysis of collagen, and a noticeable change at 3400 cm-1 and 1600-1700 cm-1 intensity was observed.

Figure 6. Light-induced Fourier Transform Infra Red (FTIR) difference spectra corresponding to native collagen (blue) and enzymatic hydrolysis of collagen (black)

Differential scanning calorimetry (DSC):

The DSC analysis of native, thermal, and enzymatic collagen hydrolysis showed different thermograms, which suggest a change in physical properties during the process.

Figure 7. Thermogram of differential scanning calorimetry (DSC) of native collagen (red), thermal hydrolysis of collagen (green), and enzymatic hydrolysis with bromelain (blue).

DISCUSSION:

The Lowry assay used the bovine serum albumin (BSA) as a standard. The calibration curve was made at various concentrations (200-800 µg/ml, Fig. 2A). In this study, the total percentage of peptide smaller than 30 kDa is determined by dividing the protein concentration from the total protein (w/w). The small-size peptide makes up around 6.67% of the total protein. The hydroxyproline (Hyp) only occurs in collagen and elastin. Thus, Hyp can be used as a golden standard to quantify the collagen content in the sample. Pure Hyp was used as a standard in the assay (Fig. 2B). According to the assay, the sample contained 50% (w/w) of collagen.

The optimation of thermal treatment for collagen hydrolysis was conducted at 50, 60, 70, 80, 90, and 100°C for an hour. Using these conditions, the protein concentration (<30 kDa) increases by around 273% (17.34 mg/ml) after treatment at 100°C for an hour (Fig. 3A). To increase the peptide fraction (<30 kDa) lower concentration of protein, the thermal treatment was then optimized at 100°C for 1, 2, 3, 4, and 5 h (Fig 3B). After thermal treatment (100°C for 5 hours), the protein concentration increases by 291% (18.2 mg/ml) compared to the initial concentration. However, the different temperature and time incubation did not significantly increase the peptide concentration, as shown by statistical analysis (ANOVA, p < 0.05). The thermal treatment could induce collagen hydrolysis, which is depicted by the increase of lower protein fraction, but the concentration is still minimal. Duncan's analysis proved that differences in time and temperature incubation did not give differences in peptide concentration. Thus, the hydrolysis was optimized with enzymatic hydrolysis.

To compare the effectiveness of thermal hydrolysis, we compare the degree of hydrolysis (Fig.3) with enzymatic hydrolysis. Our result showed that the DH of thermal treatment at 90°C for an hour is 24%; increasing temperature and time incubation did not significantly increase DH (data not shown). The DH of bromelain enzymatic hydrolysis is 60% at 10 U/ml bromelain and incubation at 60°C for an hour. Bromelain-treated collagen showed higher DH. To confirm the result, the hydrolysate was then analyzed with SEM (scanning electron microscope), FTIR (Fourier transform infrared spectroscopy), and DSC (differential scanning calorimetry).

The morphology of samples was observed with SEM (scanning electron microscope), as shown in Figure 4. The morphology of the hydrolyzed collagen can be compared to the native collagen to observe the effectivity of thermal and enzymatic hydrolysis processes. In Fig.4A, we observed an irregular shape of collagen of varying sizes. Thermal treatment results in a sheet-shaped structure with a broader but thinner size (Fig. 4B). This morphology might result from the denaturation process after thermal treatment. SEM analysis shows that thermal treatment mainly does not produce peptides but denaturates collagen. The enzymatic hydrolysis peptide was smaller and thinner than native collagen (Fig. 4C). The smaller peptide was successfully separated After separation with ultrafiltration (30 kDa). The SEM analysis proves enzymatic hydrolysis's effectiveness with bromelain to produce peptide collagen.

Electrophoresis could identify a protein's presence and separate proteins based on physical properties (molecular weight). The native, thermal hydrolysis and enzymatic hydrolysis were resolved at tricine-SDS. The thermal hydrolysis resulted in a similar band pattern to the native (Fig. 5). This result is consistent with our previous observation in which thermal treatment could not hydrolyze collagen. On the other hand, the enzymatic hydrolysis showed no band indicating protein was observed—the difference band pattern between native and enzymatic hydrolysis results from the enzymatic hydrolysis process. The presence band represents the bromelain as a control.

The Fourier transform infrared (FTIR) spectroscopy could be used to analyze small and complex molecules. FTIR has recently been increasingly used to study protein conformational, molecular, and folding (Berthomieu and Hienerwadel, 2009b). The FTIR spectroscopy was used to identify the protein hydrolysis after enzymatic treatment. FTIR spectra of native and enzymatic hydrolysis of collagen showed similar patterns on the fingerprint area of amide I and II (1637 and 1638 cm^-1 for hydrolyzed collagen and 1637 and 1525 for native collagen). Both spectra showed noticeable change (Fig. 6) in the intensity of peak around 3400 cm^-1 (-OH group from amine) and 1600 – 1700 cm^-1 (C=O group for carboxyl).

Differential Scanning Calorimetry (DSC) is a technique used to study the thermodynamics of transitions in biological macromolecules. The DSC of fish collagen, thermal treatment, and enzymatic hydrolysis were analyzed and resulted in Fig 7. The melting temperature of collagen was 90.02 °C, thermal treatment was 100.61°C, and enzymatic hydrolysis was 81.14°C. The thermal properties of the sample were evaluated in dried powder from the hydrolyzed sample (thermal and bromelain hydrolysis) compared to collagen control. Figure 7 shows that the melting temperature reduced from 90.02°C to 81.14°C due to bromelain hydrolysis. Intermolecular helix formation is well known to depend on molecular weight (MW). Thus, the higher the Mw, the higher the Tm values observed. On the other hand, we observed a higher Tm value after thermal treatment. This result is due to the more disorganized structure resulting from thermal treatment.

CONCLUSION:

In this study, heat treatment denatured collagen but did not produce hydrolyzed collagen. Enzymatic hydrolysis using bromelain produces hydrolyzed collagen, and the corresponding results are observed with electrophoresis, SEM, FTIR, and DSC.

CONFLICT OF INTEREST:

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS:

The research was funded by Universitas Airlangga through the Airlangga Research Fund 2023 (Scheme Penelitian Dasar Unggulan, grant No 736/UN3.1.5/PT/2023). The authors gratefully acknowledge the facilities and materials provided by PT Dexa Laboratories of Biomolecular Sciences (DLBS).

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Received on 14.01.2024 Modified on 10.04.2024

Research J. Pharm. and Tech 2024; 17(11):5274-5280.

DOI: 10.52711/0974-360X.2024.00807