Extraction and characterization of biomaterials from pork bones: a potential alternative of pineapple by-product extract for commercial proteases

Extraction and characterization of biomaterials from pork bones: a potential alternative of pineapple by-product extract for commercial proteases | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Extraction and characterization of biomaterials from pork bones: a potential alternative of pineapple by-product extract for commercial proteases Van-Ba Hoa, Won-Seo Park, Jun-Sang Ham, In-Seon Bae This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8217138/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract This study aimed to develop cost-effective methods for extracting biomaterials from bork bones. Collagen and chondroitin sulfate (CS) were extracted from pork bones using a pineapple by-product extract (PE) at 5% (v/w) or commercial proteases (pepsin and Alcalase) under identical conditions. Using PE resulted in a higher collagen yield compared to pepsin (3.77% vs 1.42%). The CS concentration extracted with PE was similar to that extracted with Alcalase (11.98 mg/g vs 11.37 mg/g). Protein pattern analysis showed that collagens extracted using either PE or pepsin were type-1 collagen. 1 H NMR analysis showed that CS extracted using either PE or Alcalase was E-type CS. Among 28 peptides identified, 7 peptides (Gly-Arg, Glu-Gly-Arg, Ala-Val-Gly, Leu-Ala, Leu-Val-Gly, Phe-Ala-Gly-Gly, and Gly-Pro-Leu-Gly-Pro-Hyp-Gly-Ala-Pro-Gly) and 4 peptides (Leu-Pro, Leu-Thr-Gly, Gly-Pro-Leu-Gly-Pro-Hyp-Gly-Ala-Pro-Gly, and Gly-Pro-Ala-Gly-Pro-Val-Gly-Pro-Val-Gly) were found only in the collagen extracted with pepsin and PE, respectively. Notably, peptide hydrolysates from collagen extracted with PE exhibited higher free radical scavenging and ferrous chelating activities compared to those extracted with pepsin. PE emerges as a cost-effective and efficient alternative to commercial enzymes for extracting biomaterials from pork bones. pork bones collagen chondroitin sulfate bioactivity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Pork is the second most-consumed meat type in the world [1]. Bones account for over 10% of each pork carcass, meaning that a significant number of by-products are generated from the pork industry each year. However, only a limited portion of this by-product is directly used for human consumption [2]. In recent decades, advances in technology and material science have shifted toward the comprehensive utilization of these by-products [3]. Collagen and chondroitin sulfate (CS) are two of the most valuable biomaterials found in animal bones. Collagen, accounting for approximately 30% of the total protein in the animal's body, plays a vital role in creating the structure and mechanical properties of cells and connective tissues [3]. Collagen products from animal sources are now widely used in the food and pharmaceutical industries because of their nutritional properties and biological functions [4]. Studies have shown that daily supplementation with collagen significantly improves skin’s health and anti-aging [5]. CS is a type of negatively charged polysaccharide known as a sulfated glycosaminoglycan (GAG). It is abundantly present in the cartilage and extracellular matrix of human and animal joints [6]. CS is composed of hundreds of disaccharide structural units, consisting of glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc). Based on its sulfation pattern, CS is classified into several types, including GlcA-GalNAc-4-O-sulfate (CS-A), GlcA-GalNAc-6-O-sulfate (CS-C), GlcA-2-O-sulfate-GalNAc-6-O-sulfate (CS-D), and GlcA-GalNAc-4,6-O-disulfate (CS-E) [7]. According to the European League Against Rheumatism (EULAR), CS is recommended as a symptomatic slow-acting drug for the treatment of osteoarthritis [8]. In many countries, CS is currently prescribed in combination with glucosamine sulfate to enhance the effectiveness of osteoarthritis treatment [9]. Approximately 80% of commercial CS products on the market are produced in China, primarily sourced from marine organisms [10]. To date, several methods have been developed for extracting collagen and CS from various animal sources. In principle, the extraction process involves the removal of unwanted components, the weakening of chemical bonds, and the breakdown of structural linkages using commercial proteases [6,10,11]. However, these commercial enzymes are often costly, leading to increased extraction expenses and higher prices for the final products. Therefore, it is essential to develop more efficient and cost-effective extraction methods to minimize input costs and reduce the final product price, thereby improving economic returns. Recent studies have shown that plant-derived enzymes can effectively hydrolyze connective tissues and proteins [12]. Pineapple is a rich source of proteases, particularly bromelain, which contains thiol endopeptidases. This protease is abundantly distributed throughout parts of the pineapple plant (e.g., crown, peel, and core) [13]. Bromelain has been widely used for meat tenderization [14]. The pineapple processing industry generates millions of tons of by-products annually. Among these, the core portion (accounting for approximately 16% of the total by-product weight) is a rich source of bromelain [15]. Utilizing these pineapple by-products to convert meat by-products into valuable biomaterials for the food and pharmaceutical industry applications presents a promising strategy to enhance economic returns. This study aimed to develop methods for efficiently utilizing pork bones and characterize the chemical and biological properties of collagen and CS from this by-product. Materials and Methods Materials Pork bones (leg and backbone) were collected from a commercial breed at a processing plant (Wanju, Korea). Acetic acid, NaOH, ethylenediaminetetraacetic acid (EDTA), NaCl, pepsin, Alcalase 2.4L, type-I collagen, chondroitin sulfate, chondroitinase ABC, D-glucuronolactone, DEAE-Sephadex, and chemicals for bioactivity assays: butylated hydroxyanisole (BHA), 2,2-diphenyl-1-picrylhydrazyl (DPPH), etc., were purchased from Sigma-Aldrich (USA). Chemicals used for agarose and SDS-PAGE and agarose electrophoresis were purchased from Bio-Rad (USA). Fresh pineapple fruits were purchased from a local supermarket (Jeonju, Korea). Bone powder preparation The bone samples were removed from fat, lean, and fascia, cut into thin pieces, soaked in liquid nitrogen for 5 min, and ground into fine powder using a grinder (Hanil Electric Co., Korea). The bone powders were dried at 40°C for 24 h before use. Pineapple by-products extract (PE) preparation, and proteolytic activity assay After taking the edible flesh, the by-product (core portion) of the pineapple was collected, ground using a blender, and filtered through a double layer of cheesecloth. The resulting filtrate was centrifuged at 5,000×rpm for 20 min, and the crude extract (referred to as pineapple extract, PE) was collected. Proteolytic activity of PE was assessed using the colorimetric method described by Cruz et al. [16]and Fissore et al. [17] Extraction of collagen Pepsin-soluble collagen (PSC) extraction PSC was extracted following the method of Cao et al. [11] with minor modifications. Briefly, bone samples (100 g) were treated with 900 mL of 0.1 M NaOH for 48 h to remove non-collagenous proteins. After that, the bones were rinsed with tap water for 5 min and drained. The samples were demineralized in 0.25 M EDTA (1:10 w/v ratio) for 2 days, then rinsed with tap water and drained. The demineralized bones were soaked in 0.5 M acetic acid (pH 2.8, 1:10 w/v ratio) for 48 h, followed by the addition of 0.5% pepsin (w/w) for 48 h at 4°C. Following hydrolysis, the samples were centrifuged at 6,000×g for 30 min at 4°C. The supernatant was collected and salted out with NaCl to a final concentration of 2 M/L, and the precipitated collagen was recovered by centrifugation (17,000×g, 50 min, 4°C). The pellets were redissolved in a minimum volume of 0.5 M acetic acid, dialyzed (7 kDa cut-off membrane) against 20 volumes of distilled water for 48 h, and freeze-dried for further use. Pineapple by-products extract soluble collagen (PESC) For PESC extraction, all procedures and conditions were the same as those used for PSC extraction, except that PE was added at 5% (w/v) relative to the sample weight. After hydrolysis, the collagen was collected and precipitated as described above. The extraction yields of PSC and PESC were calculated as the weight of freeze-dried collagen divided by the wet weight of the initial sample, multiplied by 100. Extraction of chondroitin sulfate (CS) The method of Nakano et al. [18] was used for the CS extraction. After washing with tap water and draining, the bone sample (100 g each) was placed in a zipper bag containing five volumes of 0.1 M sodium acetate buffer (pH 8.0) and 2 mL of Alcalase or 5 mL of PE. The sample bags were sealed tightly and incubated in a water bath set at 55°C for 8 h. After extracting, the samples were filtered through cheesecloth, and the filtrates were deproteinized by adding trichloroacetic acid to a final concentration of 7% for 12 h at 4°C. The frozen fat layer on the surface was manually removed, and the samples were centrifuged at 20,000×g for 20 min at 4°C. The supernatant containing CS was collected, precipitated with absolute ethanol to a final concentration of 60% and left overnight. After centrifugation at 3,000×g for 10 min at 10°C, the pellets (crude CS) were collected, freeze-dried, and subjected to further purification. The extraction procedures for the collagen and CS are illustrated in Fig. 1 . Characterization of pork bone collagen Protein pattern of gelatin hydrolysates The protein pattern of collagen was analyzed using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Specifically, the collagen at a concentration of 6 mg/mL was mixed with sample buffer and boiled for 5 min, and 15 µL of each sample was loaded on 4–20% precast gradient gels. The gels were stained with R-250 Coomassie brilliant blue for 1 h and destained for 2 h using the R-250 destaining kit. Amino acid composition The amino acid composition was analyzed after hydrolyzing 50 mg of lyophilized collagen in 5 mL of 6 N HCl at 110°C for 24 h, using a high-speed amino acid analyzer (L-8900, Hitachi Co., Tokyo, Japan). Scanning electron microscopy (SEM) The surface morphology of collagen was examined using an SEM (Supra 40 VP instrument, Germany). Before analysis, an appropriate amount of sample was placed on a sample holder, coated with a thin layer of platinum, and positioned in the specimen chamber. Observations were conducted at an accelerating voltage of 10 kV. Collagen peptide hydrolysates preparation and antioxidant activity assays Collagen hydrolysates were prepared by hydrolyzing the PSC and PESC (100 mg each) with the same PE to produce smaller peptides. Briefly, the sample dissolved in 5 mL of ultrapure water was mixed with 200 µL of PE and hydrolyzed at 50°C for 5 h. After centrifuging at 12,000×g for 10 min at 4°C, the supernatant was collected, freeze-dried, and used for antioxidant assays and peptide sequencing. The DPPH scavenging and ferrous ion chelating assays were used to assess the antioxidant activity of the peptide hydrolysates. The samples and a standard (BHA) were diluted to various concentrations, and the assay procedures were the same as those previously described [19]. The DPPH scavenging activity was calculated using the following formula: Where, A control : absorbance of the control (without test sample); A test : absorbance of the test samples. The Fe 2+ chelating activity was calculated as: 1- (A sample /A control ) x 100. EC 50 values (the effective concentration at which ferrous ions are chelated or DPPH radicals are scavenged by 50%) were also calculated using interpolation from linear regression analysis. Peptide sequencing For sequencing, lyophilized collagen hydrolysates (1 mg) were dissolved in 1 mL of ultrapure water and then filtered through a 0.2 µm syringe filter. Samples (10 µ) were injected into a peptide mapping column (2.1 × 100 mm, 2.7 µm) (Agilent) connected to an ultra-performance liquid chromatography (Vanquish UPLC, Thermo Scientific, USA) coupled with a tandem mass spectrometer (Q Exactive Plus, Thermo Scientific, USA). Peptide separation was performed at 30°C with a flow rate of 100 µL/min using mobile phases A (H₂O/formic acid, 100:0.1 v/v) and B (acetonitrile/formic acid, 100:0.1 v/v). Elution was carried out using a gradient of 2–98% solvent B over 31 min, followed by 2–98% solvent A over 15 min. The mass spectrometry settings included a full scan range of 200 to 1,500 m/z, MS2 fragmentation at 200 m/z, an isolation window of 1.6 m/z, and resolutions of 70,000 and 17,500 for MS and MS2, respectively. Peptides were identified from mass spectra using de novo sequencing software (Thermo FreeStyle, version 1.8). Purification and concentration determination of CS The lyophilized crude CS (dissolved in 2 mL distilled water) was applied to a pre-packed DEAE-Sepharose column, and chromatography was performed using a Gradient Pump Liquid Chromatography (Bio-Rad). Elution was carried out at a flow rate of 1.0 mL/min using a linear gradient of NaCl (50 mM to 2.0 M) in 20 mM Tris-HCl buffer (pH 8.3). Fractions (2 mL each) were collected using a fraction collector (Bio-Rad) and screened for CS using the glucuronic acid carbazole reaction [20]. Fractions containing glucuronic acid were combined, precipitated with alcohol, centrifuged at 3,000×g to recover the CS (as described above), and lyophilized. Finally, the CS concentration (mg/g wet bone sample) was determined using the same procedure (glucuronic acid carbazole reaction), calculated by multiplying the glucuronic acid content by the values of molecular weight difference [21] as follows: In which: 2.593 is the molecular weight of CS ⁄molecular weight of glucuronic acid, and 1.1023 is the molecular weight of glucuronic acid ⁄molecular weight of standard D-glucuronolactone. Chondroitin sulfate migration pattern To confirm the absence of other GAGs (e.g., dermatan sulfate, etc.), the CS samples were digested with chondroitinase ABC before electrophoresis. For digestion, CS samples (50 µg each) were dissolved in 50 µL of Tris-acetate buffer (pH 8.0), and 20 µL of chondroitinase ABC (0.1 mU/µL) was added. The reaction was carried out at 37°C for 5 h. Both non-digested and digested CS samples (10 µL each) were then loaded onto a 0.6% agarose prepared using 0.04 M barium acetate buffer (pH 5.6), and run at 150 mA for 180 min as described by Im et al. [22]. 1 H NMR spectroscopy The structural integrity of CS was examined using a 1 H NMR spectrometer (Bruker, Karlsruhe, Germany) operating at 600 MHz. Approximately 10 mg of lyophilized CS was dissolved in 1 mL of D₂O and analyzed using the instrument. Chemical shifts (ppm) were interpreted with Delta NMR Processing and Control Software (JEOL Delta v6.0, USA). Statistical analysis Statistical analysis was performed using SAS software (version 7.1; SAS Institute Inc., Cary, NC, USA). The General Linear Model procedure was applied, with the extraction method treated as a fixed factor and the measured data as dependent variables. Differences between means were evaluated using Duncan’s multiple range test, with statistical significance set at p < 0.05. Results and Discussion Yield of collagen and CS In this study, the proteolytic activity of PE was tested under the same conditions used for the collagen and CS extraction. The proteolytic activity of PE was 1.18 ± 0.02 and 3.33 ± 0.59 U/mg protein at 4°C and 55°C, respectively, indicating that the activity increased with temperature (Fig. 2 A). Previous studies have also reported that bromelain exhibits activity over a wide range of pH values and temperatures [14]. The extraction with PE resulted in a significantly higher collagen yield (3.77%) compared to the pepsin (1.42%) (p < 0.05) (Fig. 2 B). For collagen extraction, commercial proteases are commonly employed to break intra- and intermolecular covalent cross-links in native collagen, thereby releasing collagen chains. Under the same extraction conditions, bromelain in the PE seemed to exhibit stronger hydrolytic activity, resulting in a higher efficiency than commercial pepsin. Previous studies have shown that bromelain has greater enzymatic activity than pepsin under the same hydrolysis conditions [23]. Cao et al. [11] reported collagen yields of 4–11% from bovine bones extracted with pepsin. Dhakal et al. [24] reported a collagen yield of 9.41% from chicken feet extracted with papain. For CS, similar concentrations (11.37 vs. 11.98 mg/g) were observed for both the CE and PE extraction methods (Fig. 2 C). CS typically exists in the form of proteoglycans within the extracellular matrix, and breaking the covalent bonds between CS and core proteins is essential for its release [7]. In practice, protein hydrolysis using proteases is the most common approach for CS extraction. Nakano et al. [19] extracted CS from chicken biomass using commercial proteases and reported a CS concentration of 213 mg/g sample. Electrophoretic patterns of collagen and CS Figure 3 shows that the collagen extracted using pepsin or PE displayed two distinct bands with molecular weights of approximately 130–120 kDa and 120–110 kDa, corresponding to the α1- and α2-chains of type-I collagen, respectively. This result is consistent with previous findings from various raw material sources extracted using pepsin [11]. The appearance of a few additional bands ranging from 75 to 20 kDa was also observed in the PESC, suggesting that bromelain in PE mainly cleaves interchain cross-links at the telopeptide domain rather than at the α-chains. The electrophoretic pattern of CS is shown in Fig. 4 . Only a single band was observed for each of the CS products extracted using either PE or Alcalase. Their migration pattern was similar to that of the CS standard. Additionally, no bands appeared after the treatment with chondroitinase-ABC. This demonstrates the susceptibility of the CS products to the chondroitinase-ABC. In other words, the CS products extracted from pork bones contain pure GAG. Similarly, Nakano et al. [19] reported the susceptibility of CS products extracted from chicken biomass to chondroitinase-ABC. Scanning electron microscopy of collagen The microstructural properties of the collagen products are presented in Fig. 5 . The PSC exhibited a uniform, porous structure resembling a sponge-like sheet. These features are consistent with previous reports on PSC derived from animal sources [25]. In contrast, the PESC showed a homogeneous, fine, powder-like microstructure, likely resulting from the cleavage of peptide bonds by bromelain. Based on this microstructural result, it can be inferred that the triple-helical structure of collagen molecules was preserved during extraction with pepsin, whereas it was partially disrupted by bromelain in the PE. This is consistent with the SDS-PAGE results (Fig. 4 ). 1 H NMR of CS The spectrum signals mainly in the ranges of 1.8–2.1 ppm and 3.2–4.8 ppm (Fig. 6 ). As previously reported in the literature, signals in the range of 1.9–2.1 ppm correspond to the methyl protons of GalNAc. Signals at approximately 3.3, 3.5, and 3.6 ppm are assigned to the H2, H3, and H5 protons of GlcA, while those at 3.7, 3.8, and 4.05 ppm correspond to the H2, H6, and H4 protons of GalNAc, respectively. Signals at around 4.1 and 4.6 ppm are attributed to the H4 and H6 protons of GalNAc-4SO₄ and GalNAc-6SO₄, respectively [6,22]. The 1 H NMR spectra of CS extracted using either PE or Alcalase showed similar profiles. Both the CS products exhibited characteristic signals at 1.97–2.02, and 3.33, 3.48, and 3.50 ppm, which correspond to the methyl proton of GalNAc, and H2, H3, and H5 protons of GlcA, respectively. Signals corresponding to H2, H6, and H4 protons of GalNAc were observed at 3.76, 4.05, and 3.81 ppm, respectively. Importantly, two signals at approximately 4.15 and 4.63 ppm were detected, corresponding to the H4 and H6 protons of GalNAc-4SO₄ and GalNAc-6SO₄, respectively. Based on these 1 H NMR spectral features, it can be confirmed that the CS extracted from pork bones using PE and Alcalase belongs to the CS-E [8]. Amino acid composition and peptide sequences of pork bone collagen The amino acid composition of PSC and PESC is presented in Table 1 . In both collagen products analyzed, Gly was the most abundant amino acid, accounting for approximately 23.70- 24.17% of the total amino acids, followed by Pro (11.15–13.30%) and Glu (13.39–13.77%). Our findings are consistent with previous reports on collagen extracted from various sources using pepsin [24,25]. Gly, Pro, and Hyp are three of the main amino acids involved in the structural units of collagen molecules [3]. No significant differences in amino acid composition were found between the PSC and PESC (p > 0.05). Table 1 Amino acid composition of pork bone collagen products extracted by pineapple by-product extract or pepsin Items Pineapple byproduct extract soluble collagen (PESC) Pepsin-soluble collagen (PSC) Aspartic acid (Asp) 30.06 ± 0.12 35.20 ± 12.01 Threonine (Thr) 12.08 ± 0.11 13.38 ± 3.02 Serine (Ser) 18.37 ± 0.12 19.07 ± 4.06 Glutamic acid (Glu) 65.31 ± 2.10 69.02 ± 1.82 Proline (Pro) 53.0 ± 5.01 68.0 ± 2.40 Glycine (Gly) 114.01 ± 3.0 122.10 ± 3.90 Alanine (Ala) 49.00 ± 0.14 52.06 ± 15.01 Valine (Val) 12.03 ± 0.11 13.07 ± 4.22 Methionine (Met) 2.08 ± 0.02 1.16 ± 0.23 Isoleucine (Ile) 6.02 ± 0.06 7.08 ± 1.10 Leucine (Leu) 17.01 ± 0.02 18.62 ± 0.67 Tyrosine (Tyr) 10.01 ± 0.14 11.45 ± 1.01 Phenylalanine (Phe) 14.01 ± 0.15 15.08 ± 2.31 Lysine (Lys) 16.0 ± 0.08 18.53 ± 0.50 Histidine (His) 4.06 ± 0.08 4.09 ± 0.24 Arginine (Arg) 52.09 ± 2.22 48.20 ± 1.90 A total of 28 peptides were identified from the collagen hydrolysates (Table 2 ). Amongst, 17 peptides were found in both collagen products, 7 peptides (Gly-Arg, Glu-Gly-Arg, Ala-Val-Gly, Leu-Ala, Leu-Val-Gly, Phe-Ala-Gly-Gly, and Gly-Pro-Leu-Gly-Pro-Hyp-Gly-Ala-Pro-Gly) were found only in the PCS-derived hydrolysate, and 4 peptides (Leu-Pro, Leu-Thr-Gly, Gly-Pro-Leu-Gly-Pro-Hyp-Gly-Ala-Pro-Gly, and Gly-Pro-Ala-Gly-Pro-Val-Gly-Pro-Val-Gly) were found only in the PESC-derived hydrolysate. This demonstrates that the type of enzyme used for the extraction partially influences the type and length of peptides. It was observed that all the peptides contained Gly, Pro, or Hyp, which are the three main amino acids participating in the structural unit of collagen molecules [3]. The amino acid composition and length play a key role in their bioactivities. To date, hydrophobic (Ala, Val, Met, Ile, Leu, Phe, Pro, Trp, Tyr, and Gly) and aromatic amino acids (Phe, Trp, and Tyr) are known to play decisive roles in the bioactivities of peptides [26]. Furthermore, short peptides with 2–20 amino acids typically exhibit stronger biological activity [27]. In this study, all identified peptides exhibited these important characteristics, suggesting their potential bioactivities and applications. Table 2 Peptide sequences derived from pork bone collagen products hydrolyzed with pineapple by-product extract Retention time (min) z m/z Cal. mass (M + H) Sequence † 2.71 1 232.140 232.140 GR * 2.72 1 303.178 303.178 GAR 2.77 1 361.183 361.183 EGR * 3.09 1 260.124 260.124 AOG 3.90 1 246.145 246.145 AVG * 4.07 1 233.149 233.149 LP # 4.43 1 542.258 542.258 AOGPGAG 4.46 1 260.161 260.161 LAG 4.66 1 485.236 485.236 AOGPAG 4.89 1 290.171 290.171 LTG # 5.07 1 203.139 203.139 LA * 6.92 1 302.172 302.172 LOG 8.37 1 288.192 288.192 LVG * 9.53 2 322.672 644.337 LOGERG 10.24 1 351.167 351.167 FAGG * 10.63 1 400.220 400.220 VGPAG 12.57 2 307.658 614.309 GPMGPR 12.65 1 471.257 471.257 AVGPAG 13.07 2 336.168 671.329 GPMGPRG 15.15 2 339.665 678.323 FOGQOT 15.16 2 407.698 814.389 SOGPMGPR 15.93 1 543.279 543.279 LOGPSG 16.95 1 569.294 569.294 LOGPOG 20.31 2 437.228 873.449 FOGPKGPTG 21.90 2 581.815 1162.623 LOGPLGOOGPRG 21.98 1 835.431 835.431 GPLGPOGAPG * 22.05 1 835.431 835.431 GPLGPOGAPG # 22.98 1 807.437 807.437 GPAGPVGPVG # †) Refers to peptide using one-letter code *) Peptide sequences were only found in PSC #) Peptide sequences were only found in PESC Peptide sequences without marks were found in both samples Antioxidant activity of collagen hydrolases Regarding the DPPH assay (Fig. 7 ), the PESC-derived hydrolysate showed stronger activity (EC₅₀ = 0.76 mg/mL) compared to the PSC-derived hydrolysate (EC₅₀ =0.82 mg/mL). Similarly, the PESC-derived hydrolysate also exhibited higher ferrous ion chelating activity (EC₅₀ =0.77 mg/mL) than the PSC-derived counterpart (EC₅₀ =0.83 mg/mL). Nonetheless, the activities of the hydrolysates were still lower than that of the BHA (EC₅₀ =0.65–0.69 mg/mL). The antioxidant activity of protein hydrolysates is significantly influenced by their amino acid composition and peptide length. Specific aromatic and hydrophobic amino acids can interact with free radicals through electron or hydrogen donation, and chelate metal ions [26]. Additionally, peptides made up of hydrophobic amino acids react more easily with fat-soluble free radicals [28]. Peptides with Arg at the C-terminus have also been demonstrated to have strong antioxidant activity [29]. According to the sequencing results (Table 2 ), all the peptides found contain hydrophobic amino acid residues or Arg at the C-terminus. These structural features may be the main mechanisms leading to the antioxidant activities of the PCS- and PESC-derived hydrolysates. Lee et al. [30] reported an EC₅₀ value of 0.632 mg/mL for DPPH scavenging activity of duck skin gelatin hydrolyzed with collagenase. Conclusion In this study, cost-effective methods were developed to extract collagen and CS from pork bones using PE. The efficiency of these methods were compared to that of commercial proteases. The PE demonstrated superior performance, yielding higher amounts of collagen and comparable CS concentration under identical extraction conditions. In the collagen hydrolysates, a large number of peptides with molecular sizes corresponding to 2–12 amino acids were identified. Notably, all of these peptides contained hydrophobic amino acids, which are known to play key roles in bioactivities. The antioxidant activity of peptide hydrolysates derived from PESC was significantly higher than that of PSC. 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Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 25 Dec, 2025 Reviews received at journal 25 Dec, 2025 Reviews received at journal 23 Dec, 2025 Reviews received at journal 14 Dec, 2025 Reviewers agreed at journal 14 Dec, 2025 Reviewers agreed at journal 11 Dec, 2025 Reviewers agreed at journal 11 Dec, 2025 Reviewers invited by journal 07 Dec, 2025 Editor assigned by journal 02 Dec, 2025 Submission checks completed at journal 01 Dec, 2025 First submitted to journal 26 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8217138","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":556562030,"identity":"7e264f66-527c-4185-a0da-cd57e882a087","order_by":0,"name":"Van-Ba Hoa","email":"","orcid":"","institution":"National Institute of Animal Science","correspondingAuthor":false,"prefix":"","firstName":"Van-Ba","middleName":"","lastName":"Hoa","suffix":""},{"id":556562031,"identity":"18922ee3-3d8e-431f-9338-30ef0863464e","order_by":1,"name":"Won-Seo Park","email":"","orcid":"","institution":"National Institute of Animal 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1","display":"","copyAsset":false,"role":"figure","size":399069,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of pork bone collagen and chondroitin sulfate extraction using crude enzyme extract derived from pineapple by-products in comparison with commercial proteases\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8217138/v1/943ac7dacd53168904260400.png"},{"id":97943061,"identity":"2b473257-9338-4390-a457-eaf6c41b6ff3","added_by":"auto","created_at":"2025-12-11 04:41:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":51100,"visible":true,"origin":"","legend":"\u003cp\u003eProteolytic activity of pineapple by-product extract (PE) at 4 and 55 \u003csup\u003eo\u003c/sup\u003eC (A), yield (%) of pork bone collagen extracted using PE and pepsin (B), and concentration (mg/g) of chondroitin sulfate (CS) extracted using commercial enzyme (Alcalase) and PE (C). Different letters (a,b) indicate a significant difference, p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8217138/v1/1d2824d71a81a378ecfd5014.png"},{"id":97943035,"identity":"2f9f93bb-86cd-4715-b5e6-05eea114aa40","added_by":"auto","created_at":"2025-12-11 04:41:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":355742,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE of pork bone (PB) collagen extracted using pineapple extract (PB-PESC) and pepsin (PB-PSC). Type-I std: collagen Type-I standard; marker: high molecular weight protein marker.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8217138/v1/cdeed1e3f7b272e9eb17d96d.png"},{"id":97943033,"identity":"54d37d6b-a234-4ea4-88f7-1c0e562845fc","added_by":"auto","created_at":"2025-12-11 04:41:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":121654,"visible":true,"origin":"","legend":"\u003cp\u003eAgarose electrophoresis of pork bone CS products extracted with pineapple by-product extract (line:1) or Alcalase (line:3), and these two corresponding CS samples were treated with chondroitinase ABC (line: 2 and 4). The non-digested and digested CS samples, or the standard (std) at 1 mg/mL (10 µL each), were applied to 0.6% agarose in 50 mM 1,3-diaminopropane-acetate buffer at 70 mA for 180 min.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8217138/v1/2ebd56230ff2ad6cba8da9c5.png"},{"id":98422111,"identity":"33a78baa-a8bc-4152-bfc5-febacf9986b7","added_by":"auto","created_at":"2025-12-17 16:30:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":460966,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy\u003cstrong\u003e (\u003c/strong\u003eSEM) of pork bone collagen products extracted using pepsin (A) and pineapple by-product extract (B).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8217138/v1/711788ececebc815e9f3c899.png"},{"id":98422842,"identity":"448fdd78-a279-4078-a71d-274d43ea2192","added_by":"auto","created_at":"2025-12-17 16:31:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":303631,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR signals of pork bone chondroitin sulfate products extracted using pineapple by-product extract (A) and Alcalase (B).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8217138/v1/5627d6216e0b15f0973861f1.png"},{"id":98422421,"identity":"c8918b89-176a-44e8-b138-ed3cc247807f","added_by":"auto","created_at":"2025-12-17 16:31:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":285033,"visible":true,"origin":"","legend":"\u003cp\u003eAntioxidant activity (EC\u003csub\u003e50 \u003c/sub\u003evalues) of hydrolysates derived from pork bone pineapple by-product soluble collagen (PESC) and pepsin soluble collagen (PSC), and BHA (butylated hydroxyanisole).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8217138/v1/a921de2312b15515eb089528.png"},{"id":98774755,"identity":"1ae582fc-5113-4d8d-b9cd-7dbaa8e7d115","added_by":"auto","created_at":"2025-12-22 12:13:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2951899,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8217138/v1/014fcabe-3895-4ff2-b40d-e808f8ffbe63.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Extraction and characterization of biomaterials from pork bones: a potential alternative of pineapple by-product extract for commercial proteases","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePork is the second most-consumed meat type in the world [1]. Bones account for over 10% of each pork carcass, meaning that a significant number of by-products are generated from the pork industry each year. However, only a limited portion of this by-product is directly used for human consumption [2]. In recent decades, advances in technology and material science have shifted toward the comprehensive utilization of these by-products [3].\u003c/p\u003e\u003cp\u003eCollagen and chondroitin sulfate (CS) are two of the most valuable biomaterials found in animal bones. Collagen, accounting for approximately 30% of the total protein in the animal's body, plays a vital role in creating the structure and mechanical properties of cells and connective tissues [3]. Collagen products from animal sources are now widely used in the food and pharmaceutical industries because of their nutritional properties and biological functions [4]. Studies have shown that daily supplementation with collagen significantly improves skin\u0026rsquo;s health and anti-aging [5]. CS is a type of negatively charged polysaccharide known as a sulfated glycosaminoglycan (GAG). It is abundantly present in the cartilage and extracellular matrix of human and animal joints [6]. CS is composed of hundreds of disaccharide structural units, consisting of glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc). Based on its sulfation pattern, CS is classified into several types, including GlcA-GalNAc-4-O-sulfate (CS-A), GlcA-GalNAc-6-O-sulfate (CS-C), GlcA-2-O-sulfate-GalNAc-6-O-sulfate (CS-D), and GlcA-GalNAc-4,6-O-disulfate (CS-E) [7]. According to the European League Against Rheumatism (EULAR), CS is recommended as a symptomatic slow-acting drug for the treatment of osteoarthritis [8]. In many countries, CS is currently prescribed in combination with glucosamine sulfate to enhance the effectiveness of osteoarthritis treatment [9]. Approximately 80% of commercial CS products on the market are produced in China, primarily sourced from marine organisms [10]. To date, several methods have been developed for extracting collagen and CS from various animal sources. In principle, the extraction process involves the removal of unwanted components, the weakening of chemical bonds, and the breakdown of structural linkages using commercial proteases [6,10,11]. However, these commercial enzymes are often costly, leading to increased extraction expenses and higher prices for the final products. Therefore, it is essential to develop more efficient and cost-effective extraction methods to minimize input costs and reduce the final product price, thereby improving economic returns.\u003c/p\u003e\u003cp\u003eRecent studies have shown that plant-derived enzymes can effectively hydrolyze connective tissues and proteins [12]. Pineapple is a rich source of proteases, particularly bromelain, which contains thiol endopeptidases. This protease is abundantly distributed throughout parts of the pineapple plant (e.g., crown, peel, and core) [13]. Bromelain has been widely used for meat tenderization [14]. The pineapple processing industry generates millions of tons of by-products annually. Among these, the core portion (accounting for approximately 16% of the total by-product weight) is a rich source of bromelain [15]. Utilizing these pineapple by-products to convert meat by-products into valuable biomaterials for the food and pharmaceutical industry applications presents a promising strategy to enhance economic returns. This study aimed to develop methods for efficiently utilizing pork bones and characterize the chemical and biological properties of collagen and CS from this by-product.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eMaterials\u003c/h2\u003e\n \u003cp\u003ePork bones (leg and backbone) were collected from a commercial breed at a processing plant (Wanju, Korea). Acetic acid, NaOH, ethylenediaminetetraacetic acid (EDTA), NaCl, pepsin, Alcalase 2.4L, type-I collagen, chondroitin sulfate, chondroitinase ABC, D-glucuronolactone, DEAE-Sephadex, and chemicals for bioactivity assays: butylated hydroxyanisole (BHA), 2,2-diphenyl-1-picrylhydrazyl (DPPH), etc., were purchased from Sigma-Aldrich (USA). Chemicals used for agarose and SDS-PAGE and agarose electrophoresis were purchased from Bio-Rad (USA). Fresh pineapple fruits were purchased from a local supermarket (Jeonju, Korea).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eBone powder preparation\u003c/h3\u003e\n\u003cp\u003eThe bone samples were removed from fat, lean, and fascia, cut into thin pieces, soaked in liquid nitrogen for 5 min, and ground into fine powder using a grinder (Hanil Electric Co., Korea). The bone powders were dried at 40\u0026deg;C for 24 h before use.\u003c/p\u003e\n\u003ch3\u003ePineapple by-products extract (PE) preparation, and proteolytic activity assay\u003c/h3\u003e\n\u003cp\u003eAfter taking the edible flesh, the by-product (core portion) of the pineapple was collected, ground using a blender, and filtered through a double layer of cheesecloth. The resulting filtrate was centrifuged at 5,000\u0026times;rpm for 20 min, and the crude extract (referred to as pineapple extract, PE) was collected. Proteolytic activity of PE was assessed using the colorimetric method described by Cruz et al. [16]and Fissore et al. [17]\u003c/p\u003e\n\u003ch3\u003eExtraction of collagen\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003ePepsin-soluble collagen (PSC) extraction\u003c/h2\u003e\n \u003cp\u003ePSC was extracted following the method of Cao et al. [11] with minor modifications. Briefly, bone samples (100 g) were treated with 900 mL of 0.1 M NaOH for 48 h to remove non-collagenous proteins. After that, the bones were rinsed with tap water for 5 min and drained. The samples were demineralized in 0.25 M EDTA (1:10 w/v ratio) for 2 days, then rinsed with tap water and drained. The demineralized bones were soaked in 0.5 M acetic acid (pH 2.8, 1:10 w/v ratio) for 48 h, followed by the addition of 0.5% pepsin (w/w) for 48 h at 4\u0026deg;C. Following hydrolysis, the samples were centrifuged at 6,000\u0026times;g for 30 min at 4\u0026deg;C. The supernatant was collected and salted out with NaCl to a final concentration of 2 M/L, and the precipitated collagen was recovered by centrifugation (17,000\u0026times;g, 50 min, 4\u0026deg;C). The pellets were redissolved in a minimum volume of 0.5 M acetic acid, dialyzed (7 kDa cut-off membrane) against 20 volumes of distilled water for 48 h, and freeze-dried for further use.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003ePineapple by-products extract soluble collagen (PESC)\u003c/h2\u003e\n \u003cp\u003eFor PESC extraction, all procedures and conditions were the same as those used for PSC extraction, except that PE was added at 5% (w/v) relative to the sample weight. After hydrolysis, the collagen was collected and precipitated as described above. The extraction yields of PSC and PESC were calculated as the weight of freeze-dried collagen divided by the wet weight of the initial sample, multiplied by 100.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eExtraction of chondroitin sulfate (CS)\u003c/h3\u003e\n\u003cp\u003eThe method of Nakano et al. [18] was used for the CS extraction. After washing with tap water and draining, the bone sample (100 g each) was placed in a zipper bag containing five volumes of 0.1 M sodium acetate buffer (pH 8.0) and 2 mL of Alcalase or 5 mL of PE. The sample bags were sealed tightly and incubated in a water bath set at 55\u0026deg;C for 8 h. After extracting, the samples were filtered through cheesecloth, and the filtrates were deproteinized by adding trichloroacetic acid to a final concentration of 7% for 12 h at 4\u0026deg;C. The frozen fat layer on the surface was manually removed, and the samples were centrifuged at 20,000\u0026times;g for 20 min at 4\u0026deg;C. The supernatant containing CS was collected, precipitated with absolute ethanol to a final concentration of 60% and left overnight. After centrifugation at 3,000\u0026times;g for 10 min at 10\u0026deg;C, the pellets (crude CS) were collected, freeze-dried, and subjected to further purification. The extraction procedures for the collagen and CS are illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eCharacterization of pork bone collagen\u003c/h3\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eProtein pattern of gelatin hydrolysates\u003c/h2\u003e\n \u003cp\u003eThe protein pattern of collagen was analyzed using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Specifically, the collagen at a concentration of 6 mg/mL was mixed with sample buffer and boiled for 5 min, and 15 \u0026micro;L of each sample was loaded on 4\u0026ndash;20% precast gradient gels. The gels were stained with R-250 Coomassie brilliant blue for 1 h and destained for 2 h using the R-250 destaining kit.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eAmino acid composition\u003c/h2\u003e\n \u003cp\u003eThe amino acid composition was analyzed after hydrolyzing 50 mg of lyophilized collagen in 5 mL of 6 N HCl at 110\u0026deg;C for 24 h, using a high-speed amino acid analyzer (L-8900, Hitachi Co., Tokyo, Japan).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eScanning electron microscopy (SEM)\u003c/h2\u003e\n \u003cp\u003eThe surface morphology of collagen was examined using an SEM (Supra 40 VP instrument, Germany). Before analysis, an appropriate amount of sample was placed on a sample holder, coated with a thin layer of platinum, and positioned in the specimen chamber. Observations were conducted at an accelerating voltage of 10 kV.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eCollagen peptide hydrolysates preparation and antioxidant activity assays\u003c/h2\u003e\n \u003cp\u003eCollagen hydrolysates were prepared by hydrolyzing the PSC and PESC (100 mg each) with the same PE to produce smaller peptides. Briefly, the sample dissolved in 5 mL of ultrapure water was mixed with 200 \u0026micro;L of PE and hydrolyzed at 50\u0026deg;C for 5 h. After centrifuging at 12,000\u0026times;g for 10 min at 4\u0026deg;C, the supernatant was collected, freeze-dried, and used for antioxidant assays and peptide sequencing.\u003c/p\u003e\n \u003cp\u003eThe DPPH scavenging and ferrous ion chelating assays were used to assess the antioxidant activity of the peptide hydrolysates. The samples and a standard (BHA) were diluted to various concentrations, and the assay procedures were the same as those previously described [19]. The DPPH scavenging activity was calculated using the following formula:\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n \u003cp\u003eWhere, A\u003csub\u003econtrol\u003c/sub\u003e: absorbance of the control (without test sample); A\u003csub\u003etest\u003c/sub\u003e: absorbance of the test samples.\u003c/p\u003e\n \u003cp\u003eThe Fe\u003csup\u003e2+\u003c/sup\u003e chelating activity was calculated as: 1- (A\u003csub\u003esample\u003c/sub\u003e /A\u003csub\u003econtrol\u003c/sub\u003e) x 100. EC\u003csub\u003e50\u003c/sub\u003e values (the effective concentration at which ferrous ions are chelated or DPPH radicals are scavenged by 50%) were also calculated using interpolation from linear regression analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003ePeptide sequencing\u003c/h2\u003e\n \u003cp\u003eFor sequencing, lyophilized collagen hydrolysates (1 mg) were dissolved in 1 mL of ultrapure water and then filtered through a 0.2 \u0026micro;m syringe filter. Samples (10 \u0026micro;) were injected into a peptide mapping column (2.1 \u0026times; 100 mm, 2.7 \u0026micro;m) (Agilent) connected to an ultra-performance liquid chromatography (Vanquish UPLC, Thermo Scientific, USA) coupled with a tandem mass spectrometer (Q Exactive Plus, Thermo Scientific, USA). Peptide separation was performed at 30\u0026deg;C with a flow rate of 100 \u0026micro;L/min using mobile phases A (H₂O/formic acid, 100:0.1 v/v) and B (acetonitrile/formic acid, 100:0.1 v/v). Elution was carried out using a gradient of 2\u0026ndash;98% solvent B over 31 min, followed by 2\u0026ndash;98% solvent A over 15 min. The mass spectrometry settings included a full scan range of 200 to 1,500 m/z, MS2 fragmentation at 200 m/z, an isolation window of 1.6 m/z, and resolutions of 70,000 and 17,500 for MS and MS2, respectively. Peptides were identified from mass spectra using de novo sequencing software (Thermo FreeStyle, version 1.8).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003ePurification and concentration determination of CS\u003c/h2\u003e\n \u003cp\u003eThe lyophilized crude CS (dissolved in 2 mL distilled water) was applied to a pre-packed DEAE-Sepharose column, and chromatography was performed using a Gradient Pump Liquid Chromatography (Bio-Rad). Elution was carried out at a flow rate of 1.0 mL/min using a linear gradient of NaCl (50 mM to 2.0 M) in 20 mM Tris-HCl buffer (pH 8.3). Fractions (2 mL each) were collected using a fraction collector (Bio-Rad) and screened for CS using the glucuronic acid carbazole reaction [20]. Fractions containing glucuronic acid were combined, precipitated with alcohol, centrifuged at 3,000\u0026times;g to recover the CS (as described above), and lyophilized. Finally, the CS concentration (mg/g wet bone sample) was determined using the same procedure (glucuronic acid carbazole reaction), calculated by multiplying the glucuronic acid content by the values of molecular weight difference [21] as follows:\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n \u003cp\u003eIn which: 2.593 is the molecular weight of CS \u0026frasl;molecular weight of glucuronic acid, and 1.1023 is the molecular weight of glucuronic acid \u0026frasl;molecular weight of standard D-glucuronolactone.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eChondroitin sulfate migration pattern\u003c/h2\u003e\n \u003cp\u003eTo confirm the absence of other GAGs (e.g., dermatan sulfate, etc.), the CS samples were digested with chondroitinase ABC before electrophoresis. For digestion, CS samples (50 \u0026micro;g each) were dissolved in 50 \u0026micro;L of Tris-acetate buffer (pH 8.0), and 20 \u0026micro;L of chondroitinase ABC (0.1 mU/\u0026micro;L) was added. The reaction was carried out at 37\u0026deg;C for 5 h. Both non-digested and digested CS samples (10 \u0026micro;L each) were then loaded onto a 0.6% agarose prepared using 0.04 M barium acetate buffer (pH 5.6), and run at 150 mA for 180 min as described by Im et al. [22].\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eH NMR spectroscopy\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe structural integrity of CS was examined using a \u003csup\u003e1\u003c/sup\u003eH NMR spectrometer (Bruker, Karlsruhe, Germany) operating at 600 MHz. Approximately 10 mg of lyophilized CS was dissolved in 1 mL of D₂O and analyzed using the instrument. Chemical shifts (ppm) were interpreted with Delta NMR Processing and Control Software (JEOL Delta v6.0, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eStatistical analysis was performed using SAS software (version 7.1; SAS Institute Inc., Cary, NC, USA). The General Linear Model procedure was applied, with the extraction method treated as a fixed factor and the measured data as dependent variables. Differences between means were evaluated using Duncan\u0026rsquo;s multiple range test, with statistical significance set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003eYield of collagen and CS\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eIn this study, the proteolytic activity of PE was tested under the same conditions used for the collagen and CS extraction. The proteolytic activity of PE was 1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 and 3.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59 U/mg protein at 4\u0026deg;C and 55\u0026deg;C, respectively, indicating that the activity increased with temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Previous studies have also reported that bromelain exhibits activity over a wide range of pH values and temperatures [14]. The extraction with PE resulted in a significantly higher collagen yield (3.77%) compared to the pepsin (1.42%) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). For collagen extraction, commercial proteases are commonly employed to break intra- and intermolecular covalent cross-links in native collagen, thereby releasing collagen chains. Under the same extraction conditions, bromelain in the PE seemed to exhibit stronger hydrolytic activity, resulting in a higher efficiency than commercial pepsin. Previous studies have shown that bromelain has greater enzymatic activity than pepsin under the same hydrolysis conditions [23]. Cao et al. [11] reported collagen yields of 4\u0026ndash;11% from bovine bones extracted with pepsin. Dhakal et al. [24] reported a collagen yield of 9.41% from chicken feet extracted with papain.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor CS, similar concentrations (11.37 vs. 11.98 mg/g) were observed for both the CE and PE extraction methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). CS typically exists in the form of proteoglycans within the extracellular matrix, and breaking the covalent bonds between CS and core proteins is essential for its release [7]. In practice, protein hydrolysis using proteases is the most common approach for CS extraction. Nakano et al. [19] extracted CS from chicken biomass using commercial proteases and reported a CS concentration of 213 mg/g sample.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eElectrophoretic patterns of collagen and CS\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that the collagen extracted using pepsin or PE displayed two distinct bands with molecular weights of approximately 130\u0026ndash;120 kDa and 120\u0026ndash;110 kDa, corresponding to the α1- and α2-chains of type-I collagen, respectively. This result is consistent with previous findings from various raw material sources extracted using pepsin [11]. The appearance of a few additional bands ranging from 75 to 20 kDa was also observed in the PESC, suggesting that bromelain in PE mainly cleaves interchain cross-links at the telopeptide domain rather than at the α-chains.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe electrophoretic pattern of CS is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Only a single band was observed for each of the CS products extracted using either PE or Alcalase. Their migration pattern was similar to that of the CS standard. Additionally, no bands appeared after the treatment with chondroitinase-ABC. This demonstrates the susceptibility of the CS products to the chondroitinase-ABC. In other words, the CS products extracted from pork bones contain pure GAG. Similarly, Nakano et al. [19] reported the susceptibility of CS products extracted from chicken biomass to chondroitinase-ABC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eScanning electron microscopy of collagen\u003c/h2\u003e\u003cp\u003eThe microstructural properties of the collagen products are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The PSC exhibited a uniform, porous structure resembling a sponge-like sheet. These features are consistent with previous reports on PSC derived from animal sources [25]. In contrast, the PESC showed a homogeneous, fine, powder-like microstructure, likely resulting from the cleavage of peptide bonds by bromelain. Based on this microstructural result, it can be inferred that the triple-helical structure of collagen molecules was preserved during extraction with pepsin, whereas it was partially disrupted by bromelain in the PE. This is consistent with the SDS-PAGE results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eH NMR of CS\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe spectrum signals mainly in the ranges of 1.8\u0026ndash;2.1 ppm and 3.2\u0026ndash;4.8 ppm (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). As previously reported in the literature, signals in the range of 1.9\u0026ndash;2.1 ppm correspond to the methyl protons of GalNAc. Signals at approximately 3.3, 3.5, and 3.6 ppm are assigned to the H2, H3, and H5 protons of GlcA, while those at 3.7, 3.8, and 4.05 ppm correspond to the H2, H6, and H4 protons of GalNAc, respectively. Signals at around 4.1 and 4.6 ppm are attributed to the H4 and H6 protons of GalNAc-4SO₄ and GalNAc-6SO₄, respectively [6,22]. The \u003csup\u003e1\u003c/sup\u003eH NMR spectra of CS extracted using either PE or Alcalase showed similar profiles. Both the CS products exhibited characteristic signals at 1.97\u0026ndash;2.02, and 3.33, 3.48, and 3.50 ppm, which correspond to the methyl proton of GalNAc, and H2, H3, and H5 protons of GlcA, respectively. Signals corresponding to H2, H6, and H4 protons of GalNAc were observed at 3.76, 4.05, and 3.81 ppm, respectively. Importantly, two signals at approximately 4.15 and 4.63 ppm were detected, corresponding to the H4 and H6 protons of GalNAc-4SO₄ and GalNAc-6SO₄, respectively. Based on these \u003csup\u003e1\u003c/sup\u003eH NMR spectral features, it can be confirmed that the CS extracted from pork bones using PE and Alcalase belongs to the CS-E [8].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eAmino acid composition and peptide sequences of pork bone collagen\u003c/h2\u003e\u003cp\u003eThe amino acid composition of PSC and PESC is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In both collagen products analyzed, Gly was the most abundant amino acid, accounting for approximately 23.70- 24.17% of the total amino acids, followed by Pro (11.15\u0026ndash;13.30%) and Glu (13.39\u0026ndash;13.77%). Our findings are consistent with previous reports on collagen extracted from various sources using pepsin [24,25]. Gly, Pro, and Hyp are three of the main amino acids involved in the structural units of collagen molecules [3]. No significant differences in amino acid composition were found between the PSC and PESC (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAmino acid composition of pork bone collagen products extracted by pineapple by-product extract or pepsin\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eItems\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePineapple byproduct extract soluble collagen (PESC)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePepsin-soluble collagen\u003c/p\u003e\u003cp\u003e(PSC)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAspartic acid (Asp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e30.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e35.20\u0026thinsp;\u0026plusmn;\u0026thinsp;12.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eThreonine (Thr)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e12.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e13.38\u0026thinsp;\u0026plusmn;\u0026thinsp;3.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSerine (Ser)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e18.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e19.07\u0026thinsp;\u0026plusmn;\u0026thinsp;4.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlutamic acid (Glu)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e65.31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e69.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProline (Pro)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e53.0\u0026thinsp;\u0026plusmn;\u0026thinsp;5.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e68.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.40\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlycine (Gly)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e114.01\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e122.10\u0026thinsp;\u0026plusmn;\u0026thinsp;3.90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlanine (Ala)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e49.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e52.06\u0026thinsp;\u0026plusmn;\u0026thinsp;15.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eValine (Val)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e12.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e13.07\u0026thinsp;\u0026plusmn;\u0026thinsp;4.22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMethionine (Met)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsoleucine (Ile)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e6.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e7.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLeucine (Leu)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e17.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e18.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTyrosine (Tyr)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e10.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e11.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhenylalanine (Phe)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e14.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e15.08\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLysine (Lys)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e16.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e18.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHistidine (His)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e4.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e4.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eArginine (Arg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e52.09\u0026thinsp;\u0026plusmn;\u0026thinsp;2.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e48.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eA total of 28 peptides were identified from the collagen hydrolysates (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Amongst, 17 peptides were found in both collagen products, 7 peptides (Gly-Arg, Glu-Gly-Arg, Ala-Val-Gly, Leu-Ala, Leu-Val-Gly, Phe-Ala-Gly-Gly, and Gly-Pro-Leu-Gly-Pro-Hyp-Gly-Ala-Pro-Gly) were found only in the PCS-derived hydrolysate, and 4 peptides (Leu-Pro, Leu-Thr-Gly, Gly-Pro-Leu-Gly-Pro-Hyp-Gly-Ala-Pro-Gly, and Gly-Pro-Ala-Gly-Pro-Val-Gly-Pro-Val-Gly) were found only in the PESC-derived hydrolysate. This demonstrates that the type of enzyme used for the extraction partially influences the type and length of peptides. It was observed that all the peptides contained Gly, Pro, or Hyp, which are the three main amino acids participating in the structural unit of collagen molecules [3]. The amino acid composition and length play a key role in their bioactivities. To date, hydrophobic (Ala, Val, Met, Ile, Leu, Phe, Pro, Trp, Tyr, and Gly) and aromatic amino acids (Phe, Trp, and Tyr) are known to play decisive roles in the bioactivities of peptides [26]. Furthermore, short peptides with 2\u0026ndash;20 amino acids typically exhibit stronger biological activity [27]. In this study, all identified peptides exhibited these important characteristics, suggesting their potential bioactivities and applications.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePeptide sequences derived from pork bone collagen products hydrolyzed with pineapple by-product extract\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRetention time\u003c/p\u003e\u003cp\u003e(min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ez\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003em/z\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCal. mass\u003c/p\u003e\u003cp\u003e(M\u0026thinsp;+\u0026thinsp;H)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSequence\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e232.140\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e232.140\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGR\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e303.178\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e303.178\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGAR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e361.183\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e361.183\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEGR\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e260.124\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e260.124\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAOG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e246.145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e246.145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAVG\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e233.149\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e233.149\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLP\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e542.258\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e542.258\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAOGPGAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e260.161\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e260.161\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e485.236\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e485.236\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAOGPAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e290.171\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e290.171\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLTG\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e203.139\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e203.139\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLA\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e302.172\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e302.172\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLOG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e288.192\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e288.192\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLVG\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e322.672\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e644.337\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLOGERG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e351.167\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e351.167\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFAGG\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e400.220\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e400.220\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eVGPAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e307.658\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e614.309\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGPMGPR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e471.257\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e471.257\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAVGPAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e336.168\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e671.329\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGPMGPRG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e339.665\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e678.323\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFOGQOT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e407.698\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e814.389\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSOGPMGPR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e543.279\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e543.279\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLOGPSG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e569.294\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e569.294\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLOGPOG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e437.228\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e873.449\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFOGPKGPTG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e21.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e581.815\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1162.623\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLOGPLGOOGPRG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e21.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e835.431\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e835.431\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGPLGPOGAPG\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e22.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e835.431\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e835.431\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGPLGPOGAPG \u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e22.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e807.437\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e807.437\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGPAGPVGPVG\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e\u003cb\u003e\u0026dagger;)\u003c/b\u003e\u003c/sup\u003e Refers to peptide using one-letter code\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e*) Peptide sequences were only found in PSC\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e#) Peptide sequences were only found in PESC\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003ePeptide sequences without marks were found in both samples\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eAntioxidant activity of collagen hydrolases\u003c/h2\u003e\u003cp\u003eRegarding the DPPH assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), the PESC-derived hydrolysate showed stronger activity (EC₅₀ = 0.76 mg/mL) compared to the PSC-derived hydrolysate (EC₅₀ =0.82 mg/mL). Similarly, the PESC-derived hydrolysate also exhibited higher ferrous ion chelating activity (EC₅₀ =0.77 mg/mL) than the PSC-derived counterpart (EC₅₀ =0.83 mg/mL). Nonetheless, the activities of the hydrolysates were still lower than that of the BHA (EC₅₀ =0.65\u0026ndash;0.69 mg/mL). The antioxidant activity of protein hydrolysates is significantly influenced by their amino acid composition and peptide length. Specific aromatic and hydrophobic amino acids can interact with free radicals through electron or hydrogen donation, and chelate metal ions [26]. Additionally, peptides made up of hydrophobic amino acids react more easily with fat-soluble free radicals [28]. Peptides with Arg at the C-terminus have also been demonstrated to have strong antioxidant activity [29]. According to the sequencing results (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), all the peptides found contain hydrophobic amino acid residues or Arg at the C-terminus. These structural features may be the main mechanisms leading to the antioxidant activities of the PCS- and PESC-derived hydrolysates. Lee et al. [30] reported an EC₅₀ value of 0.632 mg/mL for DPPH scavenging activity of duck skin gelatin hydrolyzed with collagenase.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, cost-effective methods were developed to extract collagen and CS from pork bones using PE. The efficiency of these methods were compared to that of commercial proteases. The PE demonstrated superior performance, yielding higher amounts of collagen and comparable CS concentration under identical extraction conditions. In the collagen hydrolysates, a large number of peptides with molecular sizes corresponding to 2\u0026ndash;12 amino acids were identified. Notably, all of these peptides contained hydrophobic amino acids, which are known to play key roles in bioactivities. The antioxidant activity of peptide hydrolysates derived from PESC was significantly higher than that of PSC. These findings suggest that PE represents a cost-effective and efficient alternative to commercial enzymes for extracting biomaterials from livestock bones. However, further study is necessary to validate the biological activities of the identified collagen peptides and CS for their prospective use in the functional food and pharmaceutical industries.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study was supported by the 2025-Postdoctoral Fellowship Program of the National Institute of Animal Science (Project No. PJ01748101)\u0026rdquo;, Rural Development Administration, Republic of Korea.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eNot applicable\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eKim SW, Gormley A, Jang KB, Duarte ME. Current status of global pig production: an overview and research trends. 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Identification of novel antioxidant peptides from snakehead (Channa argus) soup generated during gastrointestinal digestion and insights into the anti-oxidation mechanisms. Food Chem. 2020;337:127921.\u003c/li\u003e\n \u003cli\u003eLi B, Chen F, Wang X, Ji B, Wu Y. Isolation and identification of antioxidative peptides from porcine collagen hydrolysate by consecutive chromatography and electrospray ionization-mass spectrometry. Food Chem. 2007;102:1135\u0026ndash;1143.\u003c/li\u003e\n \u003cli\u003eLee SJ, Kim KH, Kim YS, Kim EK, Hwang JW, Lim BO, Moon SH, Jeon BT, Jeon YJ, Ahn CB. Biological activity from the gelatin hydrolysates of duck skin by-products. Process Biochem. 2012;47:1150\u0026ndash;1154\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-science-of-animal-resources","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Science of Animal Resources](https://link.springer.com/journal/44463)","snPcode":"44463","submissionUrl":"https://submission.springernature.com/new-submission/44463/3?","title":"Food Science of Animal Resources","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"pork bones, collagen, chondroitin sulfate, bioactivity","lastPublishedDoi":"10.21203/rs.3.rs-8217138/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8217138/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aimed to develop cost-effective methods for extracting biomaterials from bork bones. Collagen and chondroitin sulfate (CS) were extracted from pork bones using a pineapple by-product extract (PE) at 5% (v/w) or commercial proteases (pepsin and Alcalase) under identical conditions. Using PE resulted in a higher collagen yield compared to pepsin (3.77% vs 1.42%). The CS concentration extracted with PE was similar to that extracted with Alcalase (11.98 mg/g vs 11.37 mg/g). Protein pattern analysis showed that collagens extracted using either PE or pepsin were type-1 collagen. \u003csup\u003e1\u003c/sup\u003eH NMR analysis showed that CS extracted using either PE or Alcalase was E-type CS. Among 28 peptides identified, 7 peptides (Gly-Arg, Glu-Gly-Arg, Ala-Val-Gly, Leu-Ala, Leu-Val-Gly, Phe-Ala-Gly-Gly, and Gly-Pro-Leu-Gly-Pro-Hyp-Gly-Ala-Pro-Gly) and 4 peptides (Leu-Pro, Leu-Thr-Gly, Gly-Pro-Leu-Gly-Pro-Hyp-Gly-Ala-Pro-Gly, and Gly-Pro-Ala-Gly-Pro-Val-Gly-Pro-Val-Gly) were found only in the collagen extracted with pepsin and PE, respectively. Notably, peptide hydrolysates from collagen extracted with PE exhibited higher free radical scavenging and ferrous chelating activities compared to those extracted with pepsin. PE emerges as a cost-effective and efficient alternative to commercial enzymes for extracting biomaterials from pork bones.\u003c/p\u003e","manuscriptTitle":"Extraction and characterization of biomaterials from pork bones: a potential alternative of pineapple by-product extract for commercial proteases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 04:41:26","doi":"10.21203/rs.3.rs-8217138/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-25T22:11:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-25T14:40:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-24T01:17:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-15T00:50:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"236007801709357317541061942161259058476","date":"2025-12-14T13:17:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"153430225007973060189011213888676502193","date":"2025-12-12T00:29:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"235643765411586617963885052659259090418","date":"2025-12-12T00:15:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-07T20:19:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-02T06:52:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-02T01:19:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food Science of Animal Resources","date":"2025-11-27T02:20:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-science-of-animal-resources","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Science of Animal Resources](https://link.springer.com/journal/44463)","snPcode":"44463","submissionUrl":"https://submission.springernature.com/new-submission/44463/3?","title":"Food Science of Animal Resources","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"759460e9-eae4-4317-b85f-02dd2dd2b9bc","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-11T00:53:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-11 04:41:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8217138","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8217138","identity":"rs-8217138","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}