Swim Bladder of Farmed Totoaba macdonaldi: A Source of Value-Added Collagen

Swim Bladder of Farmed Totoaba macdonaldi: A Source of Value-Added Collagen | 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 Swim Bladder of Farmed Totoaba macdonaldi : A Source of Value-Added Collagen Honorio Cruz-López, Sergio Rodríguez-Morales, Luis M Enríquez, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1004119/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Mar, 2023 Read the published version in Marine Drugs → Version 1 posted You are reading this latest preprint version Abstract Purpose Finding strategies to use swim bladder of farmed totoaba ( Totoaba macdonaldi ) is of utmost need to reduce waste. Fish swim bladders are rich in collagen; hence, extracting collagen is a promising alternative with benefits for aquaculture of totoaba and the environment. Methods The elemental biochemical composition of totoaba swim bladders, including proximate composition and amino acid composition were determined. Acid-enzyme solubilisation (PSC) was used to extract collagen from swim bladders and its characteristics were analyzed. The alcalase and papain were used for the preparation of collagen hydrolysates. Results Swim bladders contained 95% protein, 2.4% fat, and 0.8% ash (dry basis). The essential amino acids content was low, but the functional amino acids content was high. The PSC yield was high, 68% (dry weight). The amino acid composition profile, electrophoretic pattern, and structural integrity analyses of the isolated collagen suggested it is typical type-I collagen with high purity. The denaturalization temperature was 34.5 °C, probably attributable to the imino acid content (205 residues/1000 residues). Papain-hydrolysates (<3 kDa) of this collagen exhibited higher radical scavenging activity than Alcalase-hydrolysates. Conclusions Swim bladder from farmed totoaba is an ideal raw material for producing high-quality type-I collagen and a viable alternative to conventional collagen sources. Statement of Novelty To our knowledge, this paper is the first to examine the composition and characteristics of collagen of swim bladder from Totoaba macdonaldi . Although the processing currently wastes bladders, this study showed that they could be a potential source for producing high-quality type-I collagen. Renewable Resources Environmental Engineering Agroecology Totoaba macdonaldi Swim bladder Collagen recovery Collagen hydrolysates By-products Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Totoaba ( Totoaba macdonaldi , Sciaenidae) is a large croaker (up to 2 m long, and 100 kg weight) native to the upper Gulf of California (UGC), Mexico. Once among the most valuable fisheries in the region, T. macdonaldi is currently listed as a critically endangered species according to Mexican law (NOM-059-SEMARNAT-2010) and is included in the CITES Appendix I [1]. Originally developed for ecological purposes (conservation and re-stocking), totoaba culture has attracted the attention of investors due to the high value of its meat [2]. The totoaba aquaculture industry in Mexico was established eleven years ago, and now consists of seven licensed producers registered as Wildlife Conservation Management Units (Unidad de Manejo Ambiental - UMA) for commercial production of totoaba (≈5 kg and 3-years-old) for human consumption [2]. Current market demand is approximately 20 tons per week and is expected to increase annually (UMA-Acuario Oceanico, personal communication). Processing totoaba for meat results in by-products such as swim bladders, the market potential of which is unknown. The dried swim bladder of adult wild totoaba (known as maw ) has characteristics similar to the swim bladder of wild Chinese bahaba ( Bahaba taipingensis ). Both are considered highly nutritious and valuable food in Southeast Asia and China, and they are widely used as tonic foods in traditional Chinese medicine to improve brain function, treat insomnia and dizziness, and support postnatal recovery [3, 4]. The value of totoaba maw in Asian markets is based on bladder size and thickness, and fish age, meaning maws from small totoaba would not be highly valued [4, 5]. However, by-products from the processing of totoaba for meat may contain beneficial compounds (i.e., collagen from the swim bladder) with potential market value. Isolating these would help in processing waste and add value to totoaba production. Collagen is a dominant fibrous protein in connective tissue such as skin, cartilage, bone, and other animal organs [6]. Type I collagen is widely used in the food, cosmetics, pharmaceutical, and tissue engineering industries [7]. Livestock (cattle and pigs) are the primary sources of commercial collagen. However, factors such as fear of disease transmission (zoonoses), as well as the high cost of pure collagen, drive a search for new and safer collagen sources [8, 9]. Aquatic environments are seen as a promising alternative source of collagen because collagen isolated from marine and freshwater fish exhibits weak antigenicity, reduced risk of disease transmission, among other advantages [7]. Collagen has been isolated mostly from fish skin and occasionally from swim bladders. The swim bladder of some fish species is a valuable source of bioactive compounds, mainly collagen [8]. There are various reports on preparation methods for collagen extraction from several marine and freshwater fish species and the biochemical characteristics of the extracted collagen [10–14]. All these collagens are Type I, although significant interspecies variation exists in collagen biochemical properties, including amino acid composition and thermal stability (denaturation temperature). Collagen extraction method efficiency is critical. Extraction with acidic solutions produces lows yields of acid soluble collagen (ASC). Pepsin has been used during the extraction process to increase collagen yield (called pepsin soluble collagen, PSC) and decrease toxicity from telopeptides [15]. For instance, ASC and PSC were extracted from the swim bladder of miiuy croaker ( Miichthys miiuy ) [12] with yields of 1.3% (ASC) and 8.3% (PSC), and catla ( Catla catla ) [8] with yields of 22.2% (ASC) and 62.3% (PSC). Due to its higher yield, the PSC method was used in the present study. Recent studies show that collagen hydrolysates from the swim bladder of the croceine croaker ( Pseudosciaena crocea ) and miiuy croaker exhibit anti-fatigue, anti-amnesic, and antioxidant activities [16, 17]. However, no research has been done to date on the essential biochemical composition of the totoaba swim bladder or the properties of its collagen. The objective of the present study was to isolate collagen from the swim bladder of totoaba and evaluate its composition and properties. Swim bladders were extracted from 3-year-old farmed totoaba, analyzed their biochemical composition, pepsin-soluble collagen isolated from them, and evaluated their physicochemical properties. Tests were also done of DPPH radical-scavenging activity of the collagen and collagen hydrolysates. Materials And Methods Chemicals Pepsin from porcine stomach mucosa, dialysis membrane (14 kDa MWCO), DPPH (2, 2-diphenyl-1-picrylhydrazyl), and type I collagen standard solution from calfskin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alcalase® 2.4 L (proteinase from Bacillus licheniformis ) was donated by Novozymes (Mexico City, Mexico). Papain enzyme from Carica papaya (30,000 U/mg) and Amicon ultrafiltration tubes (3 kDa MWCO) were purchased from Merck Corporation (Burlington, MA, USA). The protein marker and bovine serum albumin standard (2 mg mL -1 ) were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Solvents for amino acid analysis were HPLC grade (T.J. Baker, Chemicals, PA, USA). All other chemicals used in this investigation were analytical grade and used as received. Swim Bladder Collection and Preparation Totoabas were provided by the UMA (UBP) of the Facultad de Ciencias Marinas (FCM), Universidad Autónoma de Baja California (UABC), Mexico, where this study was performed. A total of 24 fish (average body weight = 2.57 ± 0.264 kg; 3-years-old) were randomly sampled from three, eight thousand-liter tanks (i.e. 8 fish/tank). The tanks were supplied with continuous recirculated seawater at a 1.6 L min flow rate. During cultivation the physical and chemical water parameters were monitored twice daily to maintain recommended conditions for totoaba culture. Temperature was controlled at 27 ± 1°C with thermo-control of chillers. Salinity was measured with a refractometer and maintained at an average of 35 ± 0.5‰. Photoperiod was set at a 12:12 light:dark ratio. Oxygen concentration was kept higher than 6 mg L -1 . Before feeding, total ammonia-nitrogen (NH 4 -N) and total nitrite-nitrogen (NO-N) were measured daily with colorimetric test kits (Aquarium Pharmaceutical, Mars, PA, USA), and maintained below 0.2 and 0.1 mg L -1 , respectively. Fish were sedated using clove oil solution (40 mg L -1 ) and euthanized by pithing to avoid distress and suffering following applicable national animal welfare guidelines (NOM-033-ZOO-1995). The fish were dissected to manually remove the swim bladder, which was transported under refrigeration (3 ± 1°C) to the Aquaculture Nutrition Laboratory. Processing byproducts represented 53% of total fish weight, and the swim bladder accounted for approximately 11% of the total byproduct. Blood vessels and residual fat attached to the swim bladder were removed manually, the bladders cleaned, and cut into small pieces (0.4 kg/bag). These pieces were used in the proximate analysis and collagen extraction. Proximate Analysis Swim bladder moisture, protein, and ash contents were quantified using established methods [18]. Moisture content was measured by weight difference after drying (105°C for 12 h), and ash content by combustion in a furnace at 550°C for 12 h. Total lipids were measured using a modification of the Folch extraction method, replacing chloroform with less toxic dichloromethane [19]. Total nitrogen content was measured with the Kjeldahl method in a Vapodest 450 analyzer (Gerhardt Analytical Systems Co., Königswinter, Germany). Crude protein was calculated using a 6.25 conversion factor. Amino Acids Analysis Swim bladder amino acid composition was analyzed with the PicoTag method (Waters Corp., Milford, MA, USA). Samples were hydrolyzed with 6 N hydrochloric acid and 0.1% phenol and incubated in a nitrogen atmosphere at 110°C for 22 h. After hydrolysis, samples and standards were derivatized with phenyl isothiocyanate (PITC) reagent and reconstituted in a sodium phosphate buffer (5 mM, pH 7.4) containing 5% (v/v) acetonitrile. The derivatives were analyzed by reverse-phase chromatography (RP-UHPLC) in an Ultimate 3000 UHPLC system (Thermo Scientific) with Chromeleon software 7.2 (Chromatography Data System). Five µL of the samples were injected into a Pico-Tag ® C18 column (3.9 mm × 150 mm, 4 µm and 60 Å). Separation of the amino acids was done with a binary gradient using AccQ-Tag Eluent as mobile phase A (Waters Inc.) and aqueous acetonitrile as phase B (60% (v/v) in water) in the following gradient mode: 0.0% B at 0.0 min; 46% B for 10 min; then 100% B at 10.5 min; 100% B at 12 min; and returning to 0.0% B at 12.5 min. Total run time was 22 min with a 1.0 mL min flow rate. Column temperature was set at 38°C and UV detection done at 254 nm, which was used for calculation. Amino acid identification and quantitation was performed using a standard amino acid mixture as reference. Swim Bladder Collagen Isolation and Purification Pre-Treatment Collagen extraction was done using the protocol described previously [11, 20]. All pre-treatment steps were done at 4°C under gentle continuous stirring for 12 h. The swim bladders were pre-treated to remove non-collagenous proteins, pigments, fats, and other impurities. Swim bladders were submerged in 0.1 M NaOH kept at a 1:20 ratio (w/v), then washed with cold distilled water until all the alkaline solution was eliminated. The tissue was degreased with 10% (v/v) n -butanol at a 1:20 (w/v) sample/solvent ratio. Both the alkaline wash and degreasing steps were performed by changing solutions at 4 h intervals. Collagen Extraction and Purification The pre-treated swim bladders were digested in 0.5 M acetic acid containing 2% pepsin (w/w) at a 1:40 (w/v) tissue/solution ratio. The mixture was continuously stirred at 4°C for 24 h. After digestion, the viscous extract was filtered with two layers of cheesecloth and precipitated by adding NaCl to a 1.2 M final concentration. The precipitate was collected by centrifuging at 16,000 × g at 4°C for 20 min using a Megafuge 16R centrifuge (Thermo Scientific Co., Waltham, MA, USA), and the resulting pellets dissolved in 0.5 M acetic acid. This solution was purified using a dialysis membrane (14 kDa molecular weight cut-off) against distilled water for 72 h with a change of solution every 4 h. The resulting collagen was lyophilized (Free Zone 2.5L, Labconco Corp., Kansas, MO, USA) and stored at -20°C until further analysis. Collagen yield was calculated based on wet and dry weight of the raw material before and after processing, using Eq. (1). where Wc is the weight of the lyophilized collagen and Wd is the dry weight of the initial swim bladder prior to pre-treatments. Collagen Characterization Amino Acid Composition Amino acid composition of the lyophilized collagen was analyzed as described in section of Amino Acids Analysis. Amino acid quantification was expressed as the number of residues per 1000 total residues. Hydroxylation of proline (Pro) and lysine (Lys) was calculated from the amino acid composition using Eq. (2 and 3): Electrophoretic Pattern Collagen molecular weight (MW) was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the Laemmli method [21]. Briefly, the lyophilized collagen was dissolved in 0.1 M acetic acid and mixed at a 1:2 (v/v) ratio with sample buffer (0.5 M Tris-HCl, pH 6.8, containing 5% SDS, 20% glycerol, 5% β-ME and 0.2% bromophenol blue). The mixed solution was incubated at 95 °C for 5 min. A 10 μL sample was processed with discontinuous polyacrylamide gel electrophoresis (7.5% separator and 4% stacking). A molecular weight protein marker was used to estimate collagen MW. The electrophoresis analysis was run at a 25mA constant current voltage, using a Mini-Protean apparatus (Bio-Rad Laboratories, UK). Protein bands were stained using Coomassie Brilliant Blue R250 solution. Ultraviolet Measurements Spectra measurement was done using a Multiskan GO spectrophotometer (Thermo Scientific). The lyophilized collagen was dissolved in 0.1 M acetic acid at a 0.1 mg mL -1 concentration, under continuous stirring at 4 °C for 12 h. The sample solution was placed in a quartz cell with a 10 mm path length. The UV spectrum was measured at wavelengths between 200-450 nm. The baseline was set with 0.1 M acetic acid, and control standards (bovine serum albumin and collagen type I from calf skin) run under the same conditions. Fourier Transform Infrared (FTIR) Analysis of Functional Groups The FTIR spectra for collagen were measured using a Thermo Nicolet Nexus 670 FTIR spectrometer (Thermo Scientific). Lyophilized collagen (5 mg) was mixed with 100 mg dried potassium bromide (KBr) and compressed under dry conditions. A salt disc was inserted into the sample holder and scanned 40 times from 4000-400 cm −1 with a resolution of 2 cm −1 and compared to a background spectrum recorded from the empty cell at room temperature. The results were plotted between absorbance and wave number (cm −1 ). X-Ray Diffraction (XRD) and Circular Dichroism (CD) The collagen crystal structures were determined using an X-ray diffraction instrument (Bruker D8 Advance DaVinci, Germany) equipped with CuKα radiation (λ = 1.5406 Å), 40 kV tube voltage and 40 mA current. The scans were recorded in the 2θ (2 theta) range between 3 and 60°, at 0.02°/s steps. The CD spectrum was recorded to quantify preservation of the collagen secondary structure. The collagen solution was placed in a quartz cell (10 mm) and CD spectra measured using a spectrometer (J-1500, JASCO, Tokyo, Japan). The lyophilized collagen was dissolved in 0.1 M acetic acid at a 0.1 mg mL -1 concentration and continuously stirred at 4°C for 24 h. Collagen solutions were then placed in a quartz cell with a 10 mm path length. The CD spectra were recorded between 180 and 240 nm at 4°C at a 50 nm/min scan speed with a 0.1 nm interval. Collagen denaturation temperature (T d ) was measured by running a rotatory angle at a fixed 222 nm wavelength, within a 10-50°C temperature range at a 1°C/min heating rate. Protein Solubility Solubility was measured following an established method [22]. Briefly, lyophilized collagen was dissolved in 0.1 M acetic acid to a final concentration of 3 mg mL -1 . The mixture was stirred for 3 h at 4°C and centrifuged at 15,000 × g for 15 min (Megafuge 16R, Thermo Scientific). Supernatant pH was adjusted (1M NaOH or HCl) to obtain a final pH ranging from 2-10 (final volume 5 mL) and centrifuged at 15,000 × g for 15 min at 4°C. Supernatant protein concentration was determined based on the Bradford method. Bovine serum albumin (BSA) was used as the standard and control. Relative solubility was calculated by comparison with the solubility obtained at the pH exhibiting the highest solubility. Zeta Potential Lyophilized collagen was dissolved in 0.1 M acetic acid to a final concentration of 0.1 mg mL −1 and the mixtures continuously stirred at 4°C for 24 h. Collagen solution pH was adjusted to a 2-10 range using NaOH and HCl (1M). One milliliter collagen solution was transferred to a capillary cell, and collagen Zeta (ζ) potential measured using a Zeta potential analyzer (Zetasizer Nano ZS90, Malvern Instr., UK). The isoelectric point was identified. Collagen Hydrolysate Enzymatic Hydrolysis The collagen hydrolysate was prepared by first dissolving the collagen in ultrapure water (1:30, w/v), and denaturing it at 50°C for 10 min in a water bath. Hydrolysis conditions were 50°C and pH 8 for Alcalase ® , and 50°C and pH 7 for papain. Hydrolysis was initiated by adding protease to the mixture at a 2% (w/w) E/S ratio. Enzymatic hydrolysis was done in a water bath at 50°C for 5 h. After incubation, the enzymes were inactivated by heating the sample to 95°C for 10 min, and the undigested collagen precipitated by centrifuging at 10,000 × g for 10 min at 4°C. The supernatant of both hydrolysates was collected and ultrafiltered in an Amicon ultrafiltration unit (Merck Inc., Burlington, MA, USA) with a 3 kDa molecular weight cut-off (MWCO). The ultrafiltered fraction (<3 kDa) was collected, lyophilized, and labelled as Alcalase ® (HCA) or papain (HCP) collagen hydrolysate. Peptide Chromatographic Profile Five microliters of each hydrolysate (3.2 mg mL -1 ) were injected into a BEH300 C18 (5 µm 4.6 × 250 mm column, Waters Inc.) attached to an Ultimate 3000 UHPLC system (Thermo Scientific). Peptides were eluted using water as mobile phase A and acetonitrile as mobile phase B, following a gradient method of 0 to 100% phase B over 35 min with a 1 mL min flow. Column temperature during the run was set at 30°C. The peptides were analyzed at 215 nm. DPPH Radical Scavenging Activity The DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging method was applied according to Lee [23]. Briefly, samples were dissolved in deionized water at 3.2 mg mL -1 , and a 50 µL sample mixed with 50 µL 0.120 mM DPPH in a 96-well microplate. This solution was mixed vigorously and left to stand at room temperature in darkness for 30 min. Sample absorbance was measured at 517 nm using a Multiskan GO microplate spectrophotometer (Thermo Scientific). Ascorbic acid was used as the reference. Percentage DPPH radical scavenging activity was calculated using Eq. (4): Statistical Analysis Data were expressed as the mean ± standard deviation of three replicates. All statistical analyses were run with the STATISTICA software (Version 12, TIBCO Software Inc., Palo Alto, CA, USA). Results And Discussion Swim Bladder Biochemical Composition The swim bladder represented 2% of total body weight in totoaba and contained 67.43 ± 1.24% moisture. Crude protein content (dry weight) was 95.72 ± 1.07%, lipids were 2.46 ± 0.18%, and ash was 0.88 ± 0.06%. Moisture content was low compared to other species: 75.20% in bighead carp ( Hypophthalmichthys nobilis ) [11]; 83.33% in yellowfin tuna ( Thunnus albacares ) [10]; 78.83% in miiuy croaker [12]; and 82.8% in catla [8]. These differences may be attributed to variations in swim bladder water content during tissue processing and storage, as well as biological factors. The low total lipids and minerals contents of the totoaba swim bladder are comparable to those of the miiuy croaker [12]. Protein content was higher than reported for catla (83.0%) [8] and miiuy croaker (90.55%) [12]. Proximate composition in fish can depend on many factors, including seasonal variations in feeding behavior, age, and habitat. Nineteen amino acids were identified in the totoaba swim bladders and the amino acids profile showed collagen to be the predominant protein, which coincides with the swim bladders of other fish species [24]. Glycine was the most abundant amino acid, followed by alanine, proline, arginine, glutamic acid, hydroxyproline, and aspartic acid, which represented 85% of total amino acids (AA). Its amino acids composition showed the totoaba swim bladder to be nutritionally poor since it contained only 12% essential AA compared to 53% conditionally essential and 35% non-essential (35%) amino acids. However, it is rich in functional AA (71%), such as glycine, glutamic acid, aspartic acid, proline, alanine, and arginine, all of which participate in and regulate key metabolic pathways [25]. It also contains high levels of hydrophobic amino acids which are frequently found in antioxidant peptides [26]. Overall, totoaba swim bladder had high protein and low lipids contents, suggesting it potential use in collagen extraction, as a source of functional AA, or a substrate for bioactive peptide production. Collagen Yield Hydrolysis time for PSC extraction can be as long as 72 h [11, 27], so four extraction times were used in the present study (20, 24, 32, and 72 h) (Fig. 1 a). During the extraction process, swim bladder collagen fibers were solubilized entirely in acetic acid upon proteolysis with pepsin (24 h) with good collagen yields (68.18 ± 1.62%, dwb). According to previous studies, intermolecular cross-links in the telopeptide region and triple helices formed via condensation of aldehyde groups cause a decrease in collagen solubility [10], and the pepsin cleaves specifically on the telopeptide region, leading to isolated tropocollagen molecules. For this reason, pepsin is the principal protease used for increasing collagen extraction efficiency and reducing the collagen antigenicity caused by telopeptides [14, 15]. Thus, the PSC yields from totoaba swim bladder are comparable to those of Gulf corvina ( Cynoscion othonopterus ) (69%) [20] and significantly higher than PSC yields from other species: miiuy croaker (8%), yellowfin tuna (12%), giant croaker (15%) ( Nibea japonica ), bester sturgeon (38%) ( Huso x Acipenser ruthenus ), catfish (40%) ( Tachysurus maculatus ), rohu (47%) ( Labeo rohita ), bighead carp (59%) and catla (61%) [8, 10–12, 14, 15, 22, 28]. Interestingly, collagen yield from totoaba and other fish species is lower than the 85.3% (dwb) ASC yield reported for seabass ( Lates calcarifer ) swim bladder [29], suggesting that seabass swim bladder may have less cross-linked collagen fibers. These differences in collagen yield are probably due to extraction conditions, swim bladder firmness (i.e., degree of cross-linking), animal age, nutrition, and development conditions (wild or farmed). Protein content in the totoaba swim bladder collagen (TSBC) was 96.34 ± 1.19%, ash content was 0.83 ± 0.09%, and no fat was detected, which is consistent with collagen from miiuy croaker [12]. Collagen Characterization Amino Acid Composition All collagens have a general (Gly-X-Y)n sequence in their polypeptide chains, so glycine can be expected to be the main amino acid [6]. This agrees with the present results in that TSBC glycine content was 309 /1000 residues, followed by alanine (132 /1000 residues) and proline (122 /1000 residues) (Table 1 ). Low levels of tyrosine, histidine, isoleucine, hydroxylysine, and methionine were observed, and cysteine was not detected, which is reported for collagens [12,25,26]. Aromatic amino acids, mainly tyrosine, are generally found in low concentrations in PSC [22]. Compared to PSC isolated from Gulf corvina and miiuy croaker swim bladders (family, Sciaenidae) [12, 20], the TSBC had higher levels of aspartic acid, glutamic acid, proline, and alanine but lower levels of valine, threonine, isoleucine and leucine (Table 1 ). This variation in amino acid content could be due various factors, such as fish species biology (health state and age), environment (water temperature and feeding), and habitat (wild or farmed). Imino acid (proline and hydroxyproline) content in the TSBC was 205 /1000 residues (Table 1 ), which is consistent with miiuy croaker swim bladder collagen [12]. Imino acid content has been reported to positively affect collagen structural stability because the pyrrolidine ring imposes restrictions on polypeptide chain conformation, thus strengthening the triple helix structure [30, 31]. The degree of hydroxylation of proline (41%) and lysine (16%) also influences collagen self-assembly and stabilization [32]. Table 1. Amino acid composition of swim bladder (composition percentage) and swim bladder collagen from farmed totoaba (residues/1000 residues). Amino Acids Swim Bladder TSBC 1 Gulf corvina PSC 2 PSC Miiuy croaker 3 Asp 5.13 ± 0.05 52 ± 1.48 38 39 Glu 9.61 ± 0.14 101 ± 1.95 79 85 Hyp 5.43 ± 0.05 83 ± 1.43 81 88 Ser 2.02 ± 0.08 23 ± 0.69 34 28 Gly 29.19 ± 0.31 309 ± 3.15 303 334 His 0.47 ± 0.02 5 ± 0.34 7 8 Arg 11.58 ± 0.37 59 ± 4.47 70 55 Thr 1.76 ± 0.06 13 ± 2.62 15 22 Ala 12.26 ± 0.13 132 ± 1.27 118 95 Pro 12.10 ± 0.13 122 ± 1.33 106 112 Tyr 0.47 ± 0.03 2 ± 0.19 2 2 Val 1.58 ± 0.04 16 ± 0.30 20 33 Met 1.36 ± 0.03 7 ± 0.47 9 5 Cys 0.03 ± 0.01 Not detected 0.4 Ile 0.63 ± 0.01 5 ± 0.22 8 13 Leu 1.94 ± 0.05 20 ± 0.08 31 27 Hyl 0.28 ± 0.02 5 ± 0.22 9 6 Phe 1.61 ± 0.03 19 ± 0.54 26 23 Lys 2.52 ± 0.04 31 ± 1.63 44 24 Imino acid 3 205 187 199.5 Degree of Hydroxylation (%) Pro 40.65 Lys 14.43 TSBC 1 : totoaba swim bladder collagen. Pepsin-soluble collagen (PSC) from Gulf corvina ( C. othonopterus ) 2 [20] and miiuy croaker ( M. miiuy ) 3 [12]. Imino acid: Proline + Hydroxyproline. Protein Patterns Electrophoretic analyses of the TSBC showed it to be composed mainly of two different α chains (α1 and α2), in a 2:1 proportion, and a β chain (Fig. 1 b). High molecular weight bands were also observed which correspond to β-chains (dimers) and γ-chains (trimer). This suggests that the TSBC is type I collagen, consisting of heterotrimer ([α1(I)]2α2(I)) chains. Swim bladder from other fish species has been reported to contain type I collagen [10,12,30]. Using the GelAnalyzer software, the apparent molecular weights of the TSBC α1 (142 kDa) and α2 chains (134 kDa) were calculated based on migration distance. The extraction process was clearly effective because the collagen preserved its native structure. Moreover, no low molecular weight (<100 kDa) components were observed, suggesting the pepsin cleaved specifically to the telopeptide region, as previously reported [33]. UV-Vis and FTIR Spectroscopy Maximum absorption for collagen is near 230 nm, due to the peptide bond (R-CONH-R, amide group) of the polypeptide chains [12]. The UV-vis spectrum is therefore an essential parameter for detecting purified collagen [12]. In this spectrum the TSBC exhibited a maximum absorption peak at 228 nm (Fig. 2 a), which was similar to collagen from calf skin, grass carp ( Ctenopharyngodon idella ) [34], and miiuy croaker [12]. As expected, neither the TSBC nor the bovine serum albumin (BSA) reference exhibited a peak at 280 nm; in the TSBC this was due to its low aromatic amino acid (tyrosine and phenylalanine) content (Table 1 ). This result indicates efficient non-collagen protein elimination, and consequent high TSBC purity. In the FTIR spectra, TSBC showed characteristic bands of amide A, B, I, II, and III (Fig. 2 b). Amide absorption bands A and B, which correspond to the stretching vibration of group N-H and asymmetric stretching of CH 2 , were observed in wave numbers 3280 and 3071 cm -1 , respectively. Amide I (C ═ O stretching), amide II (N-H bending and C-N stretching), and amide III (C-N stretching and N-H bending) appeared in frequencies 1629, 1543, and 1237 cm -1 , respectively. The absorption ratio between amide III and the 1454 cm −1 wavelength was 1.05, indicating preservation of the collagen’s triple helix structure. These results coincide with those reported for collagens isolated from other fish species [8, 10–12, 14, 34]. Structural Integrity The TSBC x-ray diffraction (XRD) spectrum exhibited peaks at 7.7° and 20.02° (Fig. 2 c). The former was sharp and corresponded to the triple helix arrangement and distance between molecular chains; the latter was broad and corresponded to the distance between the amino acid residues along the helix [35]. Both peaks were consistent with the characteristic diffraction pattern of the collagen triple helicoidal structure [36]. The circular dichroism (CD) analysis showed the TSBC to have a weak positive absorption peak at 222 nm and a negative one at 197 nm with a crossing point (zero rotation) at 215 nm (Fig. 3 a). This CD spectrum pattern is characteristic of the collagen triple helix structure and consistent with previous reports [28]; the 222 nm peak disappears after thermal denaturation [28, 37]. The results confirm the helix structure of TSBC remained in its native form, and therefore that the isolation process did not affect its molecular integrity. Thermal Behavior Measurements of CD molar ellipticity (θ) as a function of temperature have been used to determine denaturation temperature (Td) [28]. The present CD (222) values decreased by approximately 34.5°C, indicating decomposition of the collagen triple helix structure (Fig. 3 b). Specifically, the intramolecular hydrogen bonds that stabilized the secondary structure of the collagen broke, leading to collapse of the triple helix into a random coil [38]. The present results were similar to those reported for collagen isolated from yellowfin tuna swim bladder (33.9°C) [10] and Gulf corvina (32.5°C) [20]. The Td of TSBC was higher than for collagen from a cold-water fish such as cod (29.6°C) [37], and for a temperate water fish such as miiuy croaker (26.7°C) [12]. Swim bladder collagen from marine fish remains thermostable below 35°C whereas in freshwater fish the threshold is higher: 38°C in grass carp [33] and 39.38°C in catla [8]. Indeed, PSC isolated from the swim bladder of the freshwater fish rohu [14] retains thermal stability at up to 42.16°C, higher than pork skin collagen (37°C) and similar to calfskin collagen [35]. Collagen thermal behavior depends heavily on imino acid content [22, 30], as well as species optimum physiological temperature, which is closely related to its habitat [31, 39]. For the farmed totoaba from UMA, average water temperature is 27 ± 1°C, while under natural conditions surface temperatures in the upper Gulf of California, Mexico, can range from 16 to 31°C on the surface and 13 to 19°C in deep waters (100 to 200 m) [40]. The present TSBC thermal stability result (34.5°C) is probably linked to water temperature in its natural habitat. Possible use of a collagen depends heavily on its thermal stability [41], and the fact that the studied TSBC has thermal stability close to that of terrestrial mammal collagen makes it a promising alternative. Protein Solubility and Zeta Potential Acid pH (2.0-4.0) caused higher solubility in the TSBC, but this parameter decreased from pH 5.0-6.0, resulting in protein precipitation (Fig. 3 c). Collagen solubility was lowest at around pH 6, but increased slightly in the pH 7.0–10.0 range. This may be due to increased repulsion of collagen molecules as the negative charge increases [22]. Similar results have been reported for PSC from the swim bladder of grass carp [34], miiuy croaker [12], Gulf corvina [20] and giant croaker [22]. Zeta potential is a key marker of colloidal dispersion stability and varies in response to pH [12]. As pH increased in the TSBC suspension, the zeta potential progressively decreased from +27 mV (pH 2) to less than -24 mV at pH 10 (Fig. 3 d). At a high magnitude of potential (positive or negative) a solution will resist aggregation, whereas low potential tends to formation of aggregates. For TSBC the zero surface net charge occurred at pH 5.4, this is considered the isoelectric point (pI) and is consistent with the protein solubility results (Fig. 3 c). Since the pI occurred at an acid pH, it may be associated with higher contents of glutamic acid and aspartic acid rather than of basic amino acids, such as histidine, lysine, and arginine (Table 2). The pI value was lower than reported for swim bladder collagen from miiuy croaker (6.85) [12] but similar to that of yellowfin tuna (5.93) [10]. In collagen, the pI is generally closely linked to amino acid composition distribution on its surface. Collagen Hydrolysate Totoaba swim bladder has putative positive therapeutic effects in traditional Chinese medicine [5]. Peptides and collagen from croaker swim bladders have been shown to remove free radicals [12,17,20,23]. The peptide profiles of the hydrolysates produced from the TSBC using Alcalase ® (HCA) and papain (HCP) showed the HCA to have more hydrophilic peptides than the HCP (Fig. 4 a). In contrast, the HCP had more hydrophobic peptides when eluted from 10 to 20 minutes. Protein hydrolysates with antioxidant activity frequently contain mainly hydrophobic amino acids, which play a significant role in free radical elimination [26]. Based on this and the present peptide profiles, the HCP was expected to exhibit higher antioxidant activity than the HCA. The DPPH radical scavenging assay is a popular and efficient way of predicting antioxidant activity since the DPPH radical is more stable than hydroxyl and superoxide radicals [17]. Using the DPPH assay, the antioxidant activity of ultrafiltered fractions (<3 kDa) of the TSBC hydrolysates was tested at 3.2 mg mL -1 . Antioxidant activity was 37% higher ( p <0.05) with the HCP than the HCA, although ascorbic acid far exceeded both (Fig. 4 b). This contrasts with the antioxidant activity results of a study of hydrolysates from the swim bladder of croceine croaker and miiuy croaker in which, at 15-25 mg protein mL -1 , the Alcalase ® hydrolysate had significantly higher activity than hydrolysates prepared with papain, pepsin, neutrase and trypsin [26]. Of note is that, after ultrafiltration, a lower concentration of HCA and HCP (3.2 mg mL -1 ) produced higher antioxidant activity than in the above study. Overall, the present antioxidant activity indicates that this parameter depends strongly on the enzyme used for hydrolysis, suggesting further research is needed to isolate active peptides and clarify their antioxidant activity. Conclusions To our knowledge, this study is the first report on the elemental biochemical composition, isolation, and characteristics of collagen from the swim bladder of farmed totoaba ( T. macdonaldi ). Totoaba swim bladder has high protein and low lipid contents, suggesting it as a possible food supplement with health benefits. Collagen yield was high (68%). The amino acid composition and protein pattern were typical of type-I collagen ([α1(I)]2α2(I)). Structural integrity analyses confirmed that the extraction process used here preserved the collagen native triple helix structure with high purity. The extracted collagen exhibited good thermal stability (34.5°C), which correlated with its imino acid content. Collagen hydrolysate antioxidant activities were influenced by the enzyme employed, highlighting the need for further research on antioxidant peptide purification and identification. The present results constitute a baseline for future studies on the production of bioactive peptides and biomedical applications for this collagen. The results can also help to promote the use of the swim bladder from farmed totoaba as an alternative to conventional collagen sources or as a functional food, which will reduce by-product generation and provide added value to the culture of totoaba. Declarations Acknowledgments Thanks are due Dr. Miguel A. Olvera-Novoa for access to the Aquaculture Nutrition Laboratory, CINVESTAV-Merida; Yadira Cortez-Santiago and Romel Borbon-Ojeda for their assistance with collagen extractions; and César A. Puerto-Castillo for assistance with amino acid analyses. Thanks are also due John Lindsay-Edwards for manuscript review and editing. Author information Affiliations Facultad de Ciencias Marinas, Universidad Autónoma de Baja California (UABC), Carretera Transpeninsular Ensenada - Tijuana No. 3917, Col. Playitas, 22860 Ensenada, Baja California, México. Honorio Cruz-López, Luis M. Enríquez, Conal D. True, and Lus M. López Unidad de Química en Sisal, Facultad de Química, Universidad Nacional Autónoma de México, Puerto de Abrigo S/N, 97356 Sisal, Yucatán, México. Sergio Rodríguez-Morales Facultad de Ciencias de la Ingeniería y Tecnología, Universidad Autónoma de Baja California (UABC), Blvd. Universitario 1000, Unidad Valle de las Palmas, 22260 Tijuana, Baja California, México. Luis Jesús Villarreal-Gómez Centro de Investigación y de Estudio Avanzados del Instituto Politécnico Nacional - Unidad Mérida, Antigua Carretera a Progreso km. 6, 97310 Mérida, Yucatán, México. Leticia Olivera-Castillo 3B’s Research Group, I3B’s – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805-017, Vigo, Guimarães, Portugal Tiago Henriques Silva Corresponding author Correspondence to Lus M. López Funding The research reported here was supported by the Universidad Autónoma de Baja California (UABC), México; the Consejo Nacional de Ciencia y Tecnología (CONACyT) (SAGARPA-CONACYT No. 247698); and fellowship no. 362129 (Honorio Cruz-López). Conflicts of Interest: The authors declare no conflict of interests regarding the information reported in this paper. Ethical approval Fish were handled and treated following the technical specifications for the production, care and use of laboratory animals decreed in the Official Mexican Regulation (NOM-062-ZOO-1999) and according to the ethics statement of the Autonomous University of Baja California (UABC), based on international guidelines. All procedures and experimentation conducted with organisms produced at the UMA (DGVS-CR-IN-1084-B.C./09) are annually reported and evaluated by the General Office of Wildlife (Dirección General de Vida Silvestre - DGVS). 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Supplementary Files Graphicalabstract.CruzLopezHonorio.docx Cite Share Download PDF Status: Published Journal Publication published 08 Mar, 2023 Read the published version in Marine Drugs → Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-1004119","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":63625023,"identity":"faeead55-bc50-4e1c-9842-1a4dd789db39","order_by":0,"name":"Honorio Cruz-López","email":"","orcid":"https://orcid.org/0000-0001-9736-0288","institution":"Universidad Autónoma de Baja California Escuela de Ciencias de la Salud Unidad Valle de las Palmas: Universidad Autonoma de Baja California Facultad de Ciencias Marinas","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Honorio","middleName":"","lastName":"Cruz-López","suffix":""},{"id":63625024,"identity":"4fce370c-2d2c-4246-84c4-49538396be5b","order_by":1,"name":"Sergio Rodríguez-Morales","email":"","orcid":"","institution":"Facultad de Quimica Universidad Nacional Autonoma de Mexico","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sergio","middleName":"","lastName":"Rodríguez-Morales","suffix":""},{"id":63625025,"identity":"a4177cc3-c3e1-4ddf-b2d8-503a39003ef0","order_by":2,"name":"Luis M Enríquez","email":"","orcid":"","institution":"Facultad de Ciencias Marinas Universidad Autonoma de Baja California","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Luis","middleName":"M","lastName":"Enríquez","suffix":""},{"id":63625026,"identity":"6b471f2d-7775-4a12-8995-894de44f3159","order_by":3,"name":"Luis Jesús Villarreal-Gómez","email":"","orcid":"","institution":"Facultad de Ciencias de la Ingieneria y Tecnlogia universidad Autonoma de Baja California","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Luis","middleName":"Jesús","lastName":"Villarreal-Gómez","suffix":""},{"id":63625027,"identity":"8636d342-976b-4fa8-98fa-dcee4b7a3a88","order_by":4,"name":"Conal True","email":"","orcid":"","institution":"Facultad de Ciencias MArinas Universidad Autonoma de Baja California","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Conal","middleName":"","lastName":"True","suffix":""},{"id":63625028,"identity":"766f0470-91f1-4069-84c2-65f8fc4d3967","order_by":5,"name":"Leticia Olivera-Castillo","email":"","orcid":"","institution":"Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Leticia","middleName":"","lastName":"Olivera-Castillo","suffix":""},{"id":63625029,"identity":"85d25f60-c8d9-4e0d-95bb-928f0f0cb2ad","order_by":6,"name":"Tiago Henriques Silva","email":"","orcid":"","institution":"Research institute on Biomaterials, Biodegradables and Biomimetics University of Minho","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tiago","middleName":"Henriques","lastName":"Silva","suffix":""},{"id":63625030,"identity":"adcf7e22-0f8e-4b3c-9bd3-9a2786bf99c0","order_by":7,"name":"Lus M López","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYBACPnYwZcPAIAGi2YjQwsYMptJI13KYJC3Mxx58+HM+sX928wOGD2WHGXRnJBDSwpZuOIPnduKMO8cMGGecO8xgduYAIS08ZtI8ErcTG27kMDDztgG1HG8gQssfg3OJ80Fa/oK0HCboF6AWhoQDiRtAWhiJswXol54DycYbgX452HMunYegX/jZm489+PHHTnbe7eaHD36UWcuZ3Ugg4DLkuAAZz0NQPQNx0TcKRsEoGAUjGgAAv+5ADPhRKyIAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-2371-7218","institution":"Universidad Autonoma de Baja California, Facultad de Ciencias Marinas","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Lus","middleName":"M","lastName":"López","suffix":""}],"badges":[],"createdAt":"2021-10-21 13:34:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1004119/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1004119/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.3390/md21030173","type":"published","date":"2023-03-09T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":15638360,"identity":"1736350b-615e-4703-9fc7-52da861aaa2c","added_by":"auto","created_at":"2021-11-17 16:09:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":100439,"visible":true,"origin":"","legend":"(a) Effect of hydrolysis time on pepsin-soluble collagen yield; (b) SDS-PAGE patterns of collagen from the swim bladder of farmed totoaba (TSBC). HM: high molecular weight marker.","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-1004119/v1/13dc11bfc4ad757aba776fa5.png"},{"id":15638372,"identity":"194115d9-0b7a-47d5-bd2e-da211730b670","added_by":"auto","created_at":"2021-11-17 16:09:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":133659,"visible":true,"origin":"","legend":"(a) Ultraviolet-visible spectra (UV/Vis); (b) Fourier-transform infrared spectra (FTIR); and (c) X-ray diffraction spectra (XRD) of collagen from the swim bladder of farmed totoaba (TSBC).","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-1004119/v1/7948bd091e9cf6a7c1c8b9d3.png"},{"id":15638364,"identity":"7f4e37c0-2181-4e73-961d-cb0406060c00","added_by":"auto","created_at":"2021-11-17 16:09:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":111382,"visible":true,"origin":"","legend":"(a) Circular dichroism (CD) spectra; (b) temperature effect on CD spectra at 221 nm; (c) solubility; and (d) zeta potential of collagen from swim bladder from farmed totoaba (TSBC).","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-1004119/v1/4155e426c514eb5d0b92c6cb.png"},{"id":15638877,"identity":"ef1ea514-bad6-43d5-8705-d93a5d832000","added_by":"auto","created_at":"2021-11-17 16:12:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":65143,"visible":true,"origin":"","legend":"(a) HPLC chromatograms of TSBC hydrolysates produced using Alcalase® (HCA) and papain (HCP); (b) DPPH radical-scavenging assay of collagen hydrolysate (3.2 mg mL-1) from swim bladder of farmed totoaba (TSBC).","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-1004119/v1/875b9238b6fc3b518363e20c.png"},{"id":38493606,"identity":"8591ac25-c110-44c9-bb5e-afa5d551c06f","added_by":"auto","created_at":"2023-06-13 20:34:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":894110,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1004119/v1/d86eae4e-82d8-4e1c-8254-016487e47912.pdf"},{"id":15638375,"identity":"a59e0506-aa83-4982-84f0-4bc50d5c2bd7","added_by":"auto","created_at":"2021-11-17 16:09:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":345329,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.CruzLopezHonorio.docx","url":"https://assets-eu.researchsquare.com/files/rs-1004119/v1/79a36c7b2dd7082d242cedef.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eSwim Bladder of Farmed \u003cem\u003eTotoaba macdonaldi\u003c/em\u003e: A Source of Value-Added Collagen\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTotoaba (\u003cem\u003eTotoaba macdonaldi\u003c/em\u003e, Sciaenidae) is a large croaker (up to 2 m long, and 100 kg weight) native to the upper Gulf of California (UGC), Mexico. Once among the most valuable fisheries in the region, \u003cem\u003eT. macdonaldi\u003c/em\u003e is currently listed as a critically endangered species according to Mexican law (NOM-059-SEMARNAT-2010) and is included in the CITES Appendix I [1]. Originally developed for ecological purposes (conservation and re-stocking), totoaba culture has attracted the attention of investors due to the high value of its meat [2].\u003c/p\u003e \u003cp\u003eThe totoaba aquaculture industry in Mexico was established eleven years ago, and now consists of seven licensed producers registered as Wildlife Conservation Management Units (Unidad de Manejo Ambiental - UMA) for commercial production of totoaba (\u0026asymp;5 kg and 3-years-old) for human consumption [2]. Current market demand is approximately 20 tons per week and is expected to increase annually (UMA-Acuario Oceanico, personal communication). Processing totoaba for meat results in by-products such as swim bladders, the market potential of which is unknown. The dried swim bladder of adult wild totoaba (known as \u003cem\u003emaw\u003c/em\u003e) has characteristics similar to the swim bladder of wild Chinese bahaba (\u003cem\u003eBahaba taipingensis\u003c/em\u003e). Both are considered highly nutritious and valuable food in Southeast Asia and China, and they are widely used as tonic foods in traditional Chinese medicine to improve brain function, treat insomnia and dizziness, and support postnatal recovery [3, 4]. The value of totoaba \u003cem\u003emaw\u003c/em\u003e in Asian markets is based on bladder size and thickness, and fish age, meaning \u003cem\u003emaws\u003c/em\u003e from small totoaba would not be highly valued [4, 5]. However, by-products from the processing of totoaba for meat may contain beneficial compounds (i.e., collagen from the swim bladder) with potential market value. Isolating these would help in processing waste and add value to totoaba production.\u003c/p\u003e \u003cp\u003eCollagen is a dominant fibrous protein in connective tissue such as skin, cartilage, bone, and other animal organs [6]. Type I collagen is widely used in the food, cosmetics, pharmaceutical, and tissue engineering industries [7]. Livestock (cattle and pigs) are the primary sources of commercial collagen. However, factors such as fear of disease transmission (zoonoses), as well as the high cost of pure collagen, drive a search for new and safer collagen sources [8, 9]. Aquatic environments are seen as a promising alternative source of collagen because collagen isolated from marine and freshwater fish exhibits weak antigenicity, reduced risk of disease transmission, among other advantages [7]. Collagen has been isolated mostly from fish skin and occasionally from swim bladders. The swim bladder of some fish species is a valuable source of bioactive compounds, mainly collagen [8]. There are various reports on preparation methods for collagen extraction from several marine and freshwater fish species and the biochemical characteristics of the extracted collagen [10\u0026ndash;14]. All these collagens are Type I, although significant interspecies variation exists in collagen biochemical properties, including amino acid composition and thermal stability (denaturation temperature).\u003c/p\u003e \u003cp\u003eCollagen extraction method efficiency is critical. Extraction with acidic solutions produces lows yields of acid soluble collagen (ASC). Pepsin has been used during the extraction process to increase collagen yield (called pepsin soluble collagen, PSC) and decrease toxicity from telopeptides [15]. For instance, ASC and PSC were extracted from the swim bladder of miiuy croaker (\u003cem\u003eMiichthys miiuy\u003c/em\u003e) [12] with yields of 1.3% (ASC) and 8.3% (PSC), and catla (\u003cem\u003eCatla catla\u003c/em\u003e) [8] with yields of 22.2% (ASC) and 62.3% (PSC). Due to its higher yield, the PSC method was used in the present study. Recent studies show that collagen hydrolysates from the swim bladder of the croceine croaker (\u003cem\u003ePseudosciaena crocea\u003c/em\u003e) and miiuy croaker exhibit anti-fatigue, anti-amnesic, and antioxidant activities [16, 17]. However, no research has been done to date on the essential biochemical composition of the totoaba swim bladder or the properties of its collagen.\u003c/p\u003e \u003cp\u003eThe objective of the present study was to isolate collagen from the swim bladder of totoaba and evaluate its composition and properties. Swim bladders were extracted from 3-year-old farmed totoaba, analyzed their biochemical composition, pepsin-soluble collagen isolated from them, and evaluated their physicochemical properties. Tests were also done of DPPH radical-scavenging activity of the collagen and collagen hydrolysates.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003eChemicals\u003c/h2\u003e\n \u003cp\u003ePepsin from porcine stomach mucosa, dialysis membrane (14 kDa MWCO), DPPH (2, 2-diphenyl-1-picrylhydrazyl), and type I collagen standard solution from calfskin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alcalase\u0026reg; 2.4 L (proteinase from \u003cem\u003eBacillus licheniformis\u003c/em\u003e) was donated by Novozymes (Mexico City, Mexico). Papain enzyme from Carica papaya (30,000 U/mg) and Amicon ultrafiltration tubes (3 kDa MWCO) were purchased from Merck Corporation (Burlington, MA, USA). The protein marker and bovine serum albumin standard (2 mg mL\u003csup\u003e-1\u003c/sup\u003e) were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Solvents for amino acid analysis were HPLC grade (T.J. Baker, Chemicals, PA, USA). All other chemicals used in this investigation were analytical grade and used as received.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003eSwim Bladder Collection and Preparation\u003c/h2\u003e\n \u003cp\u003eTotoabas were provided by the UMA (UBP) of the Facultad de Ciencias Marinas (FCM), Universidad Aut\u0026oacute;noma de Baja California (UABC), Mexico, where this study was performed. A total of 24 fish (average body weight = 2.57 \u0026plusmn; 0.264 kg; 3-years-old) were randomly sampled from three, eight thousand-liter tanks (i.e. 8 fish/tank). The tanks were supplied with continuous recirculated seawater at a 1.6 L min flow rate. During cultivation the physical and chemical water parameters were monitored twice daily to maintain recommended conditions for totoaba culture. Temperature was controlled at 27 \u0026plusmn; 1\u0026deg;C with thermo-control of chillers. Salinity was measured with a refractometer and maintained at an average of 35 \u0026plusmn; 0.5\u0026permil;. Photoperiod was set at a 12:12 light:dark ratio. Oxygen concentration was kept higher than 6 mg L\u003csup\u003e-1\u003c/sup\u003e. Before feeding, total ammonia-nitrogen (NH\u003csub\u003e4\u003c/sub\u003e-N) and total nitrite-nitrogen (NO-N) were measured daily with colorimetric test kits (Aquarium Pharmaceutical, Mars, PA, USA), and maintained below 0.2 and 0.1 mg L\u003csup\u003e-1\u003c/sup\u003e, respectively.\u003c/p\u003e\n \u003cp\u003eFish were sedated using clove oil solution (40 mg L\u003csup\u003e-1\u003c/sup\u003e) and euthanized by pithing to avoid distress and suffering following applicable national animal welfare guidelines (NOM-033-ZOO-1995). The fish were dissected to manually remove the swim bladder, which was transported under refrigeration (3 \u0026plusmn; 1\u0026deg;C) to the Aquaculture Nutrition Laboratory. Processing byproducts represented 53% of total fish weight, and the swim bladder accounted for approximately 11% of the total byproduct. Blood vessels and residual fat attached to the swim bladder were removed manually, the bladders cleaned, and cut into small pieces (0.4 kg/bag). These pieces were used in the proximate analysis and collagen extraction.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003eProximate Analysis\u003c/h2\u003e\n \u003cp\u003eSwim bladder moisture, protein, and ash contents were quantified using established methods [18]. Moisture content was measured by weight difference after drying (105\u0026deg;C for 12 h), and ash content by combustion in a furnace at 550\u0026deg;C for 12 h. Total lipids were measured using a modification of the Folch extraction method, replacing chloroform with less toxic dichloromethane [19]. Total nitrogen content was measured with the Kjeldahl method in a Vapodest 450 analyzer (Gerhardt Analytical Systems Co., K\u0026ouml;nigswinter, Germany). Crude protein was calculated using a 6.25 conversion factor.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec11\"\u003e\n \u003ch2\u003eAmino Acids Analysis\u003c/h2\u003e\n \u003cp\u003eSwim bladder amino acid composition was analyzed with the PicoTag method (Waters Corp., Milford, MA, USA). Samples were hydrolyzed with 6 N hydrochloric acid and 0.1% phenol and incubated in a nitrogen atmosphere at 110\u0026deg;C for 22 h. After hydrolysis, samples and standards were derivatized with phenyl isothiocyanate (PITC) reagent and reconstituted in a sodium phosphate buffer (5 mM, pH 7.4) containing 5% (v/v) acetonitrile. The derivatives were analyzed by reverse-phase chromatography (RP-UHPLC) in an Ultimate 3000 UHPLC system (Thermo Scientific) with Chromeleon software 7.2 (Chromatography Data System). Five \u0026micro;L of the samples were injected into a Pico-Tag\u003csup\u003e\u0026reg;\u003c/sup\u003e C18 column (3.9 mm \u0026times; 150 mm, 4 \u0026micro;m and 60 \u0026Aring;). Separation of the amino acids was done with a binary gradient using AccQ-Tag Eluent as mobile phase A (Waters Inc.) and aqueous acetonitrile as phase B (60% (v/v) in water) in the following gradient mode: 0.0% B at 0.0 min; 46% B for 10 min; then 100% B at 10.5 min; 100% B at 12 min; and returning to 0.0% B at 12.5 min. Total run time was 22 min with a 1.0 mL min flow rate. Column temperature was set at 38\u0026deg;C and UV detection done at 254 nm, which was used for calculation. Amino acid identification and quantitation was performed using a standard amino acid mixture as reference.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\n \u003ch2\u003eSwim Bladder Collagen Isolation and Purification\u003c/h2\u003e\n \u003cp\u003ePre-Treatment\u003c/p\u003e\n \u003cp\u003eCollagen extraction was done using the protocol described previously [11, 20]. All pre-treatment steps were done at 4\u0026deg;C under gentle continuous stirring for 12 h. The swim bladders were pre-treated to remove non-collagenous proteins, pigments, fats, and other impurities. Swim bladders were submerged in 0.1 M NaOH kept at a 1:20 ratio (w/v), then washed with cold distilled water until all the alkaline solution was eliminated. The tissue was degreased with 10% (v/v) \u003cem\u003en\u003c/em\u003e-butanol at a 1:20 (w/v) sample/solvent ratio. Both the alkaline wash and degreasing steps were performed by changing solutions at 4 h intervals.\u003c/p\u003e\n \u003cp\u003eCollagen Extraction and Purification\u003c/p\u003e\n \u003cp\u003eThe pre-treated swim bladders were digested in 0.5 M acetic acid containing 2% pepsin (w/w) at a 1:40 (w/v) tissue/solution ratio. The mixture was continuously stirred at 4\u0026deg;C for 24 h. After digestion, the viscous extract was filtered with two layers of cheesecloth and precipitated by adding NaCl to a 1.2 M final concentration. The precipitate was collected by centrifuging at 16,000 \u0026times; g at 4\u0026deg;C for 20 min using a Megafuge 16R centrifuge (Thermo Scientific Co., Waltham, MA, USA), and the resulting pellets dissolved in 0.5 M acetic acid. This solution was purified using a dialysis membrane (14 kDa molecular weight cut-off) against distilled water for 72 h with a change of solution every 4 h. The resulting collagen was lyophilized (Free Zone 2.5L, Labconco Corp., Kansas, MO, USA) and stored at -20\u0026deg;C until further analysis.\u003c/p\u003e\n \u003cp\u003eCollagen yield was calculated based on wet and dry weight of the raw material before and after processing, using Eq. (1).\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAtoAAABCCAYAAABpRHE0AAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAAAn2SURBVHhe7d2BrdM8GIVhVmCGfwV2YARmYIW7ARuwARMwAQuwABuww/31Io708cl20/YmXNL3kazbOrbjJEU6NWn75lmSJEnSizNoS5IkSTswaEuSJEk7MGhLkiRJOzBoS5IkSTswaEuSJEk7MGhLkiRJOzBoS5IkSTswaEuSJEk7MGhLkiRJOzBoS5IkSTswaEuSJEk7MGhLkiRJOzBoS5IkSTswaEuSJEk7MGhLkiRJOzBoH+jbt2/Pb968+aNQ9wg+ffr0x3G/f//+9xZJkqRzMmgfKEGb0Pla/Pjx4/njx4/PT09Pv2vmPn/+/Kstfe5h0JYkSY/AoH2gWdAmvGall8Lz0ep3Am7fdmvw/f79+/N///131ao6belD31sxZ4O2JEk6O4P2gWZBG4Tl0TZCLWWE9rf6+fPn89u3b68K2UHIpu+tAd+gLUmSHoFB+0CroA1WsnsA/fLly68+PRAzxmycLUb7ugb9KbcwaEuSpEdg0D7QpaCdUF1XitOnh1qCal9RZuWbtqt9BCvS7G+EfV0a5+vXr7+2szJ+LfoZtCVJ0tkZtA90KWijb+dxAnQQsHtQZXuC82wVPDKP0a0fhOx6qwrtUmp7AjZ1BO5r0c+gLUmSzs6gfaAtQbsHXQJpD848r6vRCeMV7Wubim8PYfsI44yC/silY5mhn0FbkiSdnUH7QFuCdg3VrCCnLXW5faSHVOqvCa6MyXgjhOrsB5nPyKVjmaGfQVuSJJ2dQftAW4I2aEPYpV1WsQmmhGDCdw3CeMmg3YN19juy5VhG6GfQliRJZ2fQPtDWoE1wpt1oZZm6fkvILDjP9rO6dQQEa7anzLDNoC1JkjRm0D7Q1qBdQ3VFHWWkh1f21QN5ZB6jH51hjKyir/hhSEmSpDWD9oG2Bm3QrgdewmkP38EtJfRJuRRkZ1/vl9X0Xnpbv95PkiRpzaB9oGuC9t4I1KOwO5tbb0v/Wei/xKAtSZJeM/LQlrxGFprdQYBdg/Y1oTIhdHXbAgcz+2Belw8NPj09/a65HvcyM0b9/uh7vKagPfoJdh7PwnOdM+38CXZJknRGZJRRHiKDjnLorD02BW1CFuGolpreGXy2bYsEUEoNflX2sSVoc+8x7epY9Rhm86NP38YY1I/uZ75WjvM1BG3085Rz1K8B9TkvL3E+2MdLBW3GYbyRejsNcx6hPm1e6g2VJEn6N5F5emhOflvlCfLIKN9dtaKdUDLC4PeEpxxED3kVBz47wJit1CZIERjzuGLsWQAnVN6zghuZx+hC/C0cE8eelf+cn1oy35da4WfMe4M2c6hz7LI9rwOOob92eJ5jq68RSZL0eJIdZlY5NH17jrgqaINBekgipNwbnBJ0EoxGtgRt2vS51JCVE1FDNY/pt8L2S20uyTG+pqD9N4xeQ8H1GV3j2WuMc8l43eh61evO374fxr/3GkuSpH/TpazHtlUOJUf0jHd10M6KZx2InfYEPwvO1KXUbbP2jE19QtDqADH6No1V0J4Fu+6eb9mIHKNBe72i3a8J523Wfha06zWO+g9oFKpH4VuSJD0GMsAqo13KoTVvxtVBG4QUggyBiEH7pBIoKQnOCbj8RQJSnqdP2qMecLavDjBtMmbU+rxRSBvG6+1H6vdGZ7xVGc0z/fr5ejScg1XQBteEc8g5W7XN66iiL3X0reo/EP7269BfG5Ik6TEkO/RFuupS0E6OqG4K2pkMO5vtMKEyYYdQU4NNxkjdqH0f+9IBrn7xkPHYRslJZLw8zv4p1I+wrR7DtbKPe8Y4A87BpaANXiOr641c1yqvrbyWwqAtSZJGkh1eRdBGAk4PM5FQme1ZBe8loXbUvgfeSwc4Cl0znIw6Pv2oW51o6u8JyTnGOgbPz1S2oJ0r2pIk6bV4dUH7UigZBedVSB213ytoJ8RF9p1jYVvfN2jDPtJ+VUbzTL/VeXgEnINVeB5dn1n72TWnrv9jqa+p0euLsVavL0mSdF5kgFVG25JD+/bDgjaTG4WlhJ1R+z7ZSwe4unWkYow67+x7a9C+VfZzzxhnwDmYBecesoNzN+rDuRxdc65fv4a0S/jmb9/PKHxLkqTHQAaY5ROwfZVD6dsz3mFBm3Y8rxNgjASf3j7Pa/DhecpI+qx+TIXxss+KftRnnr1N/TDkrTI/g/a2e7S3mAXtXMe8nmjX98k/llyLXBtJkvSYkh261NeSfBFpw9/qpmTRd9YDDCG1bk9o7RNNiJ617/WX3klg9PV+Qf8a3KsEreyn+9e+3o9zwHX58OHD75o5rgvHvPXn6u/94RrOwUsE7VyvlH7d6zWd7a/2lyRJj42MNsuKK/QZ5bvTpQsO9CVCXMe4t5z4aha0M2dKXTEn1FNXA2QCdMpMfl591Qb9Z9i3oj39bvkp9lXwlSRJ+puuzZK0nWXE0wVtwmn/CfZ7MdaeP8Ge1XJWn+uKOe16e+ZAwKWeleUVLvzo3VXce65u/Wl6g7YkSXrNWNhcZaggYPf/Ua9O+f/lt67SjjDGrSu33SxoM3avJwQnUPf2796923TxL4Xoa9+xjTDG7F3cjEFbkiQ9gtPemHrtfccj996L3M2CNno9jym9PsG/rnwH8yXAsjLOuyv6jtoFQXz2Liz9KatQfMu965fGlCRJOgM/AXagrUE7q9n87e1Zze7ffEI76rONQojm+UzmMnoTkZCdbYRinlN6MM8c+5xWaG/QliRJZ2fQPtDWoM3fPK7hNivWHW0J1nVVmXarWzoYi7FH6Ff3swrlmB3TDO0N2pIk6ewM2gfaErQJs/XWEOrpx/PZBw+p72OubgsB7Rl7hKDNHIJ9Zh4js2Oaob1BW5IknZ1B+0BbgjYht25PwOVe81G/jFlDcILx6gOcjEWbkR6s03YU8sG20dxmaG/QliRJZ2fQPtAqaLOC3FeSQXtu86ir3FXGrPgwJCvaK6tbR8Bc2J5Sg3zHdoO2JEnSnwzaB1oFbYIn2/rtHtQRmmcfNsyYWb1mbIJ2giyBeqT3qxhja3D2w5CSJEljBu0DXQrafTUbl0IpQZdvF6Ev7QjqWa2mfrUSPbuPmzr698Iqd0fAZttotX2G9gZtSZJ0dgbtA62CNivLo3ug6XMpxLK9t9vSj+A8Crz0pXTMu9czxiiArxi0JUnSIzBoH4iQOgvafwNBnFXtHp5HK+tgpbu+GaAf/WcfkpwxaEuSpEdg0D7QawvaYCWdYJ2wPZsj9XXlmuf0W32zyYxBW5IkPQKD9oFeY9AGK9KE6PxcPc+ZZy01GHMPOO2vXcmOPp4kSdIZGbQP9FqD9tEM2pIk6REYtA+UoF0LdY+ANxf1uA3akiTp7AzakiRJ0g4M2pIkSdIODNqSJEnSDgzakiRJ0g4M2pIkSdIODNqSJEnSDgzakiRJ0ot7fv4fwQxboZ+4aucAAAAASUVORK5CYII=\"\u003e\u003c/p\u003e\n \u003cp\u003ewhere Wc is the weight of the lyophilized collagen and Wd is the dry weight of the initial swim bladder prior to pre-treatments.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec13\"\u003e\n \u003ch2\u003eCollagen Characterization\u003c/h2\u003e\n \u003cp\u003eAmino Acid Composition\u003c/p\u003e\n \u003cp\u003eAmino acid composition of the lyophilized collagen was analyzed as described in section of Amino Acids Analysis. Amino acid quantification was expressed as the number of residues per 1000 total residues. Hydroxylation of proline (Pro) and lysine (Lys) was calculated from the amino acid composition using Eq.\u0026nbsp;(2 and 3):\u003c/p\u003e\n \u003cp\u003e\u003cimg 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p59xmge+dm8HyfP1yybhsl1WonYK69zC151Jy8qXQm3uI9mvuormut8o1IX21PVqnnypsnrXHN+XPaTbkY+xRzbxqXbZzjzZ5e0iZfs+gst2WZMc9fl7MuF7Qkm8L/301n1Y2Yd8CuvJefKlStqyBePznSyllFLuxbe3aSnlu0cB7xRUZhDvKMAVPDPeQbYHxehRXcG6ZCiQpo5OPrFJ9mXi4Ha5HKDOOFAAnuvTfD7pp8h+zaHPkwp0qA6yVVCnT7ZKjtaMDZIFLhvdspm67JUMyZQM1RPsSV/Ixj1f8kzxfefT9xXZLl8yBONV157yqTmyX6T8PVz3Hr4OIZv++OOPL58UZGov8A1zXI/WpP0Bl+37yHz5hMI47X/a4zIlw3U4+I35gme3kbnSA9INfKp+yz64PW4fMiRPc9Sf7+mtMIe5pZRSyiO4/aYqpTw9CsanoFKB8ap4gE1gS5sH2R7M8+zjgbq3ecAs6Hc5QB1dCqalWzrULqZgO21LvdIBCt59bfKb63I7vO7z0hbJ9sTG18x43xvJnPZLfS4LOfLxEV/y7GNkn0CG5AFjJ7+oDb/6+PQb89P3Dn2M3yqSlTDX1yLfq022TP51HwJtvo6ULT/JlvQDMF4yaPf5wPhsEykLm+U3fxayR3Pov2Uf9uxDlo9Pv8nXt8Ic349SSinlntx+U5VSnp4pyBcEsKuAmwDXA2bINp+LrNSR4yd9k22yWcE0KKCf1qHgW8E+nz4XnR6sg+Qx158FemhLfE2pF3LeJNtl8El/lmlf5BeX5dC358sck/a5bYDf6M8iGbmnqW/y/Raue4/Jti1bYNoPSDtzXTlvku0yeKY/y7Q+ZNLnspzcE8Gct+7Dnn2pUzbqXc/3/CjMyXe0lFJKuRdNcEu5IAp0p6Ayg2JnCqoli2CXQFfBLiArdaSMSd9kWwbnoIA62wVypSvtzuAePGHJ5AWkL/E1ZdAPOW+S7TL4zPWvcP9P0LfnyxyT9rltgN98fUnuaeqbfL+F695jep+ctAWm/YC0M2XnvEm2y+B5yzYHmSnLyT0RzNFepr1pn9sGe/alTtmodyHf86O4zaWUUsq9aYJbygVRoDsFlRkUO6ugWnOybxqfbZM+2jzwBiUTgjVIDvPpywRF62Ru6qCeOvCH2qakR20K6AVz5MsM+iED/0m2+2XyJeQaYNIHsueIL3n2dyHtc9uA57SFsa7T+/cSqz0mX6xI3UnaAmpzH0CuM2WnnybZvlbkT+te2TvZhE70Mcd1A8+uP+1N+9w22LMv3wPp07vHfOq3wpxcZymllHIvmuCWckEU6E5BZQbFTga4QoFtzlMCID0KiCkKpCd9ss/bGedy0g7qzEmYR7uCeoFs2hWcZ7Au22l3pEftGidSDsg/YpLtvpUM3x/muExHa3F58t2eL2HS5fKYq/3iWTJzjfJx7qnGq9/94eNW5F5vkbqTtEUwx9s1zn2astNPk2zGy3dAv8tgrPvekZ9SnqDPZdPnvnrLPmzZ5+8o6D3Ve+D+cBl7MGflg1JKKeVsvo0WSynfPQp0PahUsOtFgbACVy+e3ICPd1wuAbcHyWqneOAMslFF+hRUU2Q/wbSP9aSEeZ4ECOYoAdA86Uhf5LqwVX0uO21GTtr26dOnf9SZgwzVJc/XSdlLGNwmivvgiC8p6JjW7vMld1oreBs2ocvbqLte5JxB2j3Jnmxxcq8cb2ddqe/XX3/9Rx3ZuSfC2xizRdqUa/I+f0e8/dZ98HGyL9/Rvf1P327h80sppZR70wS3lAui4PTMoHIvUP8oCOKn5JA2T05LKR9DE9xSSimPpAluKRckf32hvIdVEvkMEDjnr17QBLeUj4PvpZ8/TXBLKaU8iia4pZRdSBanJPIZWCWxTXBLKaWUUl6PJrillBH/N323/Hu7R+D/9nHC+1djSimllFLK9WjkV0oppZRSSinlEjTBLaWUUkoppZRyCZrgllJKKaWUUkq5BE1wSymllFJKKaVcgia4pZRSSimllFIuQRPcUkoppZRSSimXoAluKaWUUkoppZRL0AS3lFJKKaWUUsolaIJbSimllFJKKeUSNMEtpZRSSimllHIJmuCWUkoppZRSSrkETXBLKaWUUkoppVyCJrillFJKKaWUUi5BE9xSSimllFJKKZegCW4ppZRSSimllEvQBLeUUkoppZRSyiVogltKKaWUUkop5RI0wS2llFJKKaWUcgma4JZSSimllFJKuQRNcEsppZRSSimlXIImuKWUUkoppZRSLkET3FJKKaWUUkopl6AJbiml3IE///zz8w8//PDl86P5+eefP//4449fa+/jr7/++rKu33///WtLKaWUUsrz0AS3lPJSKEHzQgIosu+nn3762nMcJbeUj05wWRt2nJXgXgHf7zM4W14ppZRS3k4T3FLKS/Lbb799Sfwm+HWSPpLht3LVX3C/d9iPt/yhxRar96iUUkopj6e3cikviv/KqPJKNMF9TfDDmQmufiEvf6PvlQr1Ukop5ZH0Vi7lRVEC9qoB6NEEN/8gQAmr5nvi6MG9ZOR4yHk8a96qnUTK/3o1SRrF60Jt+neynuCmTHC5stfrGgeZ/Ms/Gqd5+YcDrld2rZAOFcdtpbhtQBu+dhnSp7oK44S3ywcgP7te6XT/U3wPyv/vRSmllPJImuCW8qIoMXnVANQTzmSVxGVi5skV8qbklLnSRSEJQo4nnL4HmieUNMoW6q4Xea4X0Om2Mt7H8JzJGOOV2NHnz5orv8ge+YXCGOnk2W2kX/IkQ/Vk8qNsVZKpuaqrn2eK68+1M1bjQTLkX+0VdcZJnmTI/hxfvgW/+LtdSimlPILeyqW8KEpOXjUAVWKyVZTEQCZK9Ml3PDPekzb5V23S52OULDnTvijRYnzul3QruQRPLiFtT9vA56DP5XlCmAmeZKV+zaE97WF8tomUxXplu8sV6deULXsF810G892n8qfaGJv77vqkv3yL+7GUUkp5FL2VS3lRlJi8agC6lZhkEgfylyc26s8kCqbxOYZEzJMnQduUFE5jIZO2nDvpcR2sw98D2UrxZBPSN7lOcH08S5YXt1dk8phMftEc2cmzryXtTV/xTH8W6cnxqW/a1/I3+MX3opRSSnkEvZVLeVGUmLxqALqVmGRSJEh0PPERGu9k4jfp80TQyUROSRUlEzyQLsZhi5IvMelxm7Etk0rX6XPTN7lOcH08TzZPSGfaItIvoDlaM8/+Tqe9mbDyvPUdyPGpb+s9enVyL0oppZRH0Fu5lBdFicmrBqBbiUkmRULtfLrfpiQv2yZ9avN5IB2CxA5bJHPaMyV/nowJTzgdyco5nkRmQpe+yXWC60P+pDsTVTGtD53ow076HemXPTk/7c2EdctnkOPTH+hKm8rf5F6UUkopj6C3cikvylay9ApsJSaZFDm0T30kcS6PBEljkbfSl/MY5wkhyZXrkm1KsITkT4kjbaskkzn5DqDT5TBGCWz6Zi/BhbSLsalTaB0pD5RcesI52eqy0163jWfJzDnyL/Jdn8ar3+W7HeXbvSillFIeQRPcUl4UJSavFoAqQfGSCZIXT24Af2Wb8HnI5BM/61lFyZZQkktx2TzTpoRMe6aSe0ebJ4YgGSqOfJH2YK8SN4r08Kk2yq+//vqPOnNW+rxt5T+R/vI15f7JtmxHRtqLHPeh1j3NBd8Xnlf+V11Jb/kb91EppZTyKJrg3kgGQgqQPpJ7BBHPEpj4ryNvhWBVCcJHgP1vhT241z4oWG8Aehv47Vl9tpc4TrxlTilH6PlSSinlKMTqR+L9I3H1myLv/BP6LFcNmJTcCjbC15p+8LH3QnrODCLuIfMt+C9IRxNcxj1LQKUE0m3XelYJN2vOPhL0e3ynmuC+Dfbj6Pv4SLBJvzwe5ZmT9fL9c+b5orhjgnd/72ylXWOe8ftbSimvis5wYhLB3aEzm+JM45M3Z2ASnn8lyw3aUvw9wtpWl6c44xfHW0Hf2UHqPWS+hVv9ic3PYDek3QRo/lcfMxlh7Or9YuytycseTXBvhz161j/A4/249cxlLY88q8prccb5wvuJHJVE/Xr30ZfnKHXZoXOv730ppTwHnNEev3Be+91Bf8ZeOvtXZ/npCS6oj3IlCCBXCYhognsut/hT790z2M27knZgm74v07tEfWudzL81gdlCgd4z+OvZYW/w1d73/9Ho++Hv1h7st+ac+T6VkvCOrc4Xzrrp+8Q7Of0hkt7bhLOU4vj3gc/U43/YWEop5ePgbM/zOGPh6RwH5q5+dLhLggu6jPxyYwG0UXIxCrYpuQjXJRkKzKSHssruVdJhCfN9vEg5lFWgi430r3SlLPlP8yjg/oCty5gx+MFlSK7qGgMuW3Z6m+RovGymfcv/W+3g/s1+2SH52kvZ4v7UGBV0uv3eDuxV+i9luHzZBszlebXfE/KXywTatC/od5nU1beCMbmO9yCfaZ9LKeVM9s4Xzkg/BzmTVsGKn8uOn6vCz0rk5bnJ+FvO9FJKKfeBM1zx+grO/ylGXsXb8O1tcRAJXQXl6tdlxacuOvWprkBbC+Qyok759OnT/z0jA30a65cY0K66ZArJXJGXIHXGu9Po37sUZd/kbJHrF7kWwbP3JfRjl8akndN8dMtG+Up1xlLXGJ4p6X/6XQ91+vROSK7WmXvishy3Nf0pHQL92CVcH9BPm8vk2edovVoTzxTmolc+0Lr2kM2Jv2PI1jPj3b4VK7kgm7eKfChyf0op5UyOnC+cS5yHnEd+Lid59oPO5ukOQSbwmTbkvVJKKeXxKA7dOos5v7fukemMhzlaPoAullXQr34uLJ512Qja1YZh3q+5urRUzySANod+tfHstsmJk72TgyedfmmuOHpxsmbZKlyXy+DT+5K0UzaIySYfP70cjFfb5AugLf2JLPeRdPM5+Y73wIMa9sJlpu35rmCT191uQb9s11r0bgnaZIf2xv2FjJS7Im1ykEtRPzp8LDZojOuH6T19D5J3dF2llHILR8+XPAcndC47zKMtz3M/g/lMG/JeKaWU8nimc13ofFfJHEQQN099s9QDSLEnI476UazLZCowLZC6ZE+6FJxPhfFcalPfdNmuHIztSnpgK3ERRy/OXBOffklvJToJY3xdkw0+Bj3SKzsyQPDxaSvI/zlv8iV+m8ZC2pov6bQWQTvF94S6+wLol9yVPN/baQ0uYw+XtQfjZAt6NW+SIZ9PvngLkuf+ot7S0tKyVY7C2DyPE84zzjrOI79vk+lcZi5tebf4+cln2rB1r5RSSnkM07k+wZjprAfujSk+P35TBbpYPOlxZDSfjMlgPXFZuny0kEmXgvMV6FvZlqwcfHaCS5vbhDzJny52yaJMmyfo9wt8soF+2Y4s9cm3+dK4TI1x2+X/nDf5krXRlu3CA5D0w7QWl5V7Qrv7AuiX/yZ54HKmNbiMPdKmFYxzn7L2LTvl87QdaN8rOU/y0l+llHIGe+cLZ5KflZxJ010I07kMtPk5Cn6W+rNA1pEzupRSyv1YnesJdwXjMueA6YyHfakLpCwvFlCfjF4F5m6QFqniciddq8Uih75pwbTTnyiZyLVwAfp45O1dipKVawXme7v8wpy01etbMoE+t3Maz7N0ZQCR88HbfK5Q22S3y0eG5OC7yX/0S77rgFxL7ivPLtPtFvRrjnyeY1yu7HFcxh6yeQvGpDy3IdcNR+TewsoXpZRyBlvnC2fbdB9wLuUdBdO5DJyZeZYyjvMS+Ew9eY+UUkp5PIpDPdZdsRrH+T7dM2+OllHil4jQJZSGcKFMlwwgY+uyWeliDu2OZHryKLCN9gnmuc3MS9nomy5kR/NSzyQPkDeNp022ay0r6PPNla58EbRGyRXyo2zQfAp9e3utdo1zOfnS0a89EppHSWSL1oK/fD513xOeZbN0q03keuVf6dC6nJSxRfohod9tFuhVO7pyDG1HbTiC1p17VEopZ3Dm+TKdy5DnLePyjuEslR0690oppXw8nMeZXySr+Ffnv+J3502nvBKlVVldaDlP6MLJwvjsS9ks2Pt9kXtzE7fPkws50Mvk6ByTJS9dYFOnZEebqbm6vJ20i/Gs0dt8nvwx4WuXXmTt+TD1yf9KTCmygXWq7UjyNq3F5VLcR+jWHMlXH8X97/MoItvTx9MeTiAnfQWSJz8lvg85hjbfz/eivZ3svDfsT+73R5B/gPJe2L+j70h5bs5+N16Rs84X5Hhhbxy/p1bfP59fSinlOeCOyHjQ8wVKnvmCucsz/+vnh0IAMRlP22pRV4G1PzLQf9bg+4p7zZfyrOB4OgDey5Tg8n74oUIR6M++t6xPB9dHJ7i8b+9ZxxXhXTjTF/pDqbPANu2ZSv5hWfkbfOXf7TO49TvL/pxtQymllGvBPf6WH3C4Y1Yxy1MkuKsAhcW+ZcHfE1z+j1rjMyeRV/zVS8H4e/eXoPIe/sGuKQCV3at35Yygle/8Rye4oCR3dUC+GuzLmb5gj4+eObyPR98rxrFvZQ0+eu/31GF/bj2HzjgrSimlXJ+tuDM5El9/eIQgIzPYpf2KSU/yyF8fHqnrFnhBnyHZuRfv8fvZQarTBLcJrsOenukLvV9NcB+PvsPv/Z46fGeb4JZSSrkX3DFHYpAj9//TRAgY6+VZk7EzYAO1zntDEiFdzxbEy65X+IOMZ+SMBJfvqfZRCavmU9CRbcz1BNf16X3VPMZq3lY7+Pcq+/XuS77eOU9wJ5kgubJXMvhkvmwC2pEjtE75OvvB9R75LmgsJffIfUBxsEPnqvqlT2tS8fPX+3gW7jvX631etAcr8FH6ZoV8tkLvlIr85HaB7wv4+txf4HXNyf1KvXtrdr+5bkgfag0gu/lknsagz9ekQpvPUxGyGx3+Pmqe6ipH9+mWsaWUUspZrCOEUsqlUbCbAagHuxM5h6A/A33mKjiWPNUVQBOYq4+CDAX1jGWcJxiaJ7vSftWFy3I8kdAY7ADpUF1oDuP92eXzTJE92E7d/SP5AlluD31ed1Z+lK3o8LlKnuinnWfZBPKX5KUvABlaj/RT11gKMt0HOZ6xR0CG5u6htW/h9jrpb8Gz+txfWdec9J/qQnNWIEu2aa58Jf8K1RmvsRRkaA7PuTZfO+O0HmBvqctPkqc59FNE1o+QNpRSSimPYH37llIujQLlDEA94F0VnyM5fAoPtAmKvQ4ejEtfjqFNwbtgngfpCvwzeBcZlGOjy9R8bBBuGzBGa+PZ5eEHXzey3DfISv2uj2cHvdkm6HNZ8hsyUy6kXyfZ1OWP9AWf6VP0q03jff30S5/0S36i+VtFshJ8TP8e0ziXSZ+v1/t49vVTT1nUtT76fa3ak2n9WruDLr07/izwreZMstHv7wf9LoM+3yvZQJv2ysfz7Otnvss/QsospZRSHsF+hFBKuSQKkjMA3UtMpjkEwkoOmO/9k6wcn2NkmwfkgFzaHSUeORYUxKMDpFNkP6QOnyNbKVOwT7uv3dcJvi49T8XtEehL+8XkF/CkhLmesABzZG/6QvWpeL/b6vrkK8YdAX+477ZYrTdJG/j09wRb6c91QPprz3/0SZaXaU20pSyx8pv7298jkfalbupTQe6kM33se3sU5k/rL6WUUu7JfoRQSrkkCpIzAF0F2GKao+Ab6PPAe5JFIK5kbdI3BfCQQTd4kjKBLtmbAbrsxgaHNvpol52O68y1um98neDr0vNR0DnZApNfgDlacyZA4PamL6jneGfyneub9nUL/OG+22K1XpBvBeuWTfp0tA6K+zf9tec/+o6ulTkr36785v7Wu5PrdJluG+R4Z9KZPva9PUraUEoppTyC49FVKeVSKEjOAHQvMVkFrWrPIJig2xMH8LZJn9pyHnWXjz7ZgswpaaBf8nNN1OlDn6NkgbmeFDDe6xn0ywci1y6f86k1ujxgftoDsilhvNaR69MaYJrv9qYvZOvkG5h85/7Q+tKmFehz323BOGRP+H6A1oEdvhfg9VxP+mvPf+hN+cia1jT5DjSWvlwHfdKvNfm7k/a5bUBf2sJ8bJn2irG0Cd/bo6QNpZRSyiNoglvKi6IgOQPQvcRkFbQSYE99CpQlT3opBMwrfTlP4xTU0566JNPRPEqCDNoZ48jGTGg0XrBmT2roc5uY7/2SqzXIZ84qidA6XB72SBbz6NdaJltzPfTLXreNNuQgM+fIvsl39Ln9ko9Mxm8hvUdgXOoG2tNeoE1rc2iTXVq/SH8d9Z+vU2ufQJbLYy2SJd+qDpOtLjvt45k2yZXMnEM/ZbKdNuHyeT4C830NpZRSyiP4Z2RVSnkZFCR7AKokyYsgqM0+TzAUJHub8LlKmmiTDSoZDCvIVpFsBesUBezIVJsH+oCuDMpTtgf+gJ1pD3rdZunR2lXSV8hymylKJnLs5D+R/so1+f65D3JfJ3tBPtx6J2Dy3eR/rQ0ZZ5B2TyV9Avja/SEYKxspegdyzUf9l/uT70/iY9O+fF9k2/Qebe2Ry53mTja7Tyis18dtvaMOY/d8UEoppZxNE9xSXhQFrGcGoGclMmdDIE+5BfyipKJ835CQTYlvuS9NcEsppTyez5//F7Abnn+rN+bnAAAAAElFTkSuQmCC\"\u003e\u003c/p\u003e\n \u003cp\u003eElectrophoretic Pattern\u003c/p\u003e\n \u003cp\u003eCollagen molecular weight (MW) was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the Laemmli method\u0026nbsp;[21]. Briefly, the lyophilized collagen was dissolved in 0.1 M acetic acid and mixed at a 1:2 (v/v) ratio with sample buffer (0.5 M Tris-HCl, pH 6.8, containing 5% SDS, 20% glycerol, 5% \u0026beta;-ME and 0.2% bromophenol blue). The mixed solution was incubated at 95 \u0026deg;C for 5 min. A 10 \u0026mu;L sample was processed with discontinuous polyacrylamide gel electrophoresis (7.5% separator and 4% stacking). A molecular weight protein marker was used to estimate collagen MW. The electrophoresis analysis was run at a 25mA constant current voltage, using a Mini-Protean apparatus (Bio-Rad Laboratories, UK). Protein bands were stained using Coomassie Brilliant Blue R250 solution.\u003c/p\u003e\n \u003cp\u003eUltraviolet Measurements\u003c/p\u003e\n \u003cp\u003eSpectra measurement was done using a Multiskan GO spectrophotometer (Thermo Scientific). The lyophilized collagen was dissolved in 0.1 M acetic acid at a 0.1 mg mL\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003econcentration, under continuous stirring at 4 \u0026deg;C for 12 h. The sample solution was placed in a quartz cell with a 10 mm path length. The UV spectrum was measured at wavelengths between 200-450 nm. The baseline was set with 0.1 M acetic acid, and control standards (bovine serum albumin and collagen type I from calf skin) run under the same conditions.\u003c/p\u003e\n \u003cp\u003eFourier Transform Infrared (FTIR) Analysis of Functional Groups\u003c/p\u003e\n \u003cp\u003eThe FTIR spectra for collagen were measured using a Thermo Nicolet Nexus 670 FTIR spectrometer (Thermo Scientific). Lyophilized collagen (5 mg) was mixed with 100 mg dried potassium bromide (KBr) and compressed under dry conditions. A salt disc was inserted into the sample holder and scanned 40 times from 4000-400 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e with a resolution of 2 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e and compared to a background spectrum recorded from the empty cell at room temperature. The results were plotted between absorbance and wave number (cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e).\u003c/p\u003e\n \u003cp\u003eX-Ray Diffraction (XRD) and Circular Dichroism (CD)\u003c/p\u003e\n \u003cp\u003eThe collagen crystal structures were determined using an X-ray diffraction instrument (Bruker D8 Advance DaVinci, Germany) equipped with CuK\u0026alpha; radiation (\u0026lambda;\u0026thinsp;=\u0026thinsp;1.5406 \u0026Aring;), 40 kV tube voltage and 40 mA current. The scans were recorded in the 2\u0026theta; (2 theta) range between 3 and 60\u0026deg;, at 0.02\u0026deg;/s steps. The CD spectrum was recorded to quantify preservation of the collagen secondary structure. The collagen solution was placed in a quartz cell (10 mm) and CD spectra measured using a spectrometer (J-1500, JASCO, Tokyo, Japan). The lyophilized collagen was dissolved in 0.1 M acetic acid at a 0.1 mg mL\u003csup\u003e-1\u003c/sup\u003e concentration and continuously stirred at 4\u0026deg;C for 24 h. Collagen solutions were then placed in a quartz cell with a 10 mm path length. The CD spectra were recorded between 180 and 240 nm at 4\u0026deg;C at a 50 nm/min scan speed with a 0.1 nm interval. Collagen denaturation temperature (T\u003csub\u003ed\u003c/sub\u003e) was measured by running a rotatory angle at a fixed 222 nm wavelength, within a 10-50\u0026deg;C temperature range at a 1\u0026deg;C/min heating rate.\u003c/p\u003e\n \u003cp\u003eProtein Solubility\u003c/p\u003e\n \u003cp\u003eSolubility was measured following an established method [22]. Briefly, lyophilized collagen was dissolved in 0.1 M acetic acid to a final concentration of 3 mg mL\u003csup\u003e-1\u003c/sup\u003e. The mixture was stirred for 3 h at 4\u0026deg;C and centrifuged at 15,000 \u0026times; g for 15 min (Megafuge 16R, Thermo Scientific). Supernatant pH was adjusted (1M NaOH or HCl) to obtain a final pH ranging from 2-10 (final volume 5 mL) and centrifuged at 15,000 \u0026times; g for 15 min at 4\u0026deg;C. Supernatant protein concentration was determined based on the Bradford method. Bovine serum albumin (BSA) was used as the standard and control. Relative solubility was calculated by comparison with the solubility obtained at the pH exhibiting the highest solubility.\u003c/p\u003e\n \u003cp\u003eZeta Potential\u003c/p\u003e\n \u003cp\u003eLyophilized collagen was dissolved in 0.1 M acetic acid to a final concentration of 0.1 mg mL\u003csup\u003e\u0026minus;1\u003c/sup\u003e and the mixtures continuously stirred at 4\u0026deg;C for 24 h. Collagen solution pH was adjusted to a 2-10 range using NaOH and HCl (1M). One milliliter collagen solution was transferred to a capillary cell, and collagen Zeta (\u0026zeta;) potential measured using a Zeta potential analyzer (Zetasizer Nano ZS90, Malvern Instr., UK). The isoelectric point was identified.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003ch2\u003eCollagen Hydrolysate\u003c/h2\u003e\n \u003cp\u003eEnzymatic Hydrolysis\u003c/p\u003e\n \u003cp\u003eThe collagen hydrolysate was prepared by first dissolving the collagen in ultrapure water (1:30, w/v), and denaturing it at 50\u0026deg;C for 10 min in a water bath. Hydrolysis conditions were 50\u0026deg;C and pH 8 for Alcalase\u003csup\u003e\u0026reg;\u003c/sup\u003e, and 50\u0026deg;C and pH 7 for papain. Hydrolysis was initiated by adding protease to the mixture at a 2% (w/w) E/S ratio. Enzymatic hydrolysis was done in a water bath at 50\u0026deg;C for 5 h. After incubation, the enzymes were inactivated by heating the sample to 95\u0026deg;C for 10 min, and the undigested collagen precipitated by centrifuging at 10,000 \u0026times; g for 10 min at 4\u0026deg;C. The supernatant of both hydrolysates was collected and ultrafiltered in an Amicon ultrafiltration unit (Merck Inc., Burlington, MA, USA) with a 3 kDa molecular weight cut-off (MWCO). The ultrafiltered fraction (\u0026lt;3 kDa) was collected, lyophilized, and labelled as Alcalase\u003csup\u003e\u0026reg;\u003c/sup\u003e (HCA) or papain (HCP) collagen hydrolysate.\u003c/p\u003e\n \u003cp\u003ePeptide Chromatographic Profile\u003c/p\u003e\n \u003cp\u003eFive microliters of each hydrolysate (3.2 mg mL\u003csup\u003e-1\u003c/sup\u003e) were injected into a BEH300 C18 (5 \u0026micro;m 4.6 \u0026times; 250 mm column, Waters Inc.) attached to an Ultimate 3000 UHPLC system (Thermo Scientific). Peptides were eluted using water as mobile phase A and acetonitrile as mobile phase B, following a gradient method of 0 to 100% phase B over 35 min with a 1 mL min flow. Column temperature during the run was set at 30\u0026deg;C. The peptides were analyzed at 215 nm.\u003c/p\u003e\n \u003cp\u003eDPPH Radical Scavenging Activity\u003c/p\u003e\n \u003cp\u003eThe DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging method was applied according to Lee [23]. Briefly, samples were dissolved in deionized water at 3.2 mg mL\u003csup\u003e-1\u003c/sup\u003e, and a 50 \u0026micro;L sample mixed with 50 \u0026micro;L 0.120 mM DPPH in a 96-well microplate. This solution was mixed vigorously and left to stand at room temperature in darkness for 30 min. Sample absorbance was measured at 517 nm using a Multiskan GO microplate spectrophotometer (Thermo Scientific). Ascorbic acid was used as the reference. Percentage DPPH radical scavenging activity was calculated using Eq. (4):\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n \u003cp\u003eData were expressed as the mean \u0026plusmn; standard deviation of three replicates. All statistical analyses were run with the STATISTICA software (Version 12, TIBCO Software Inc., Palo Alto, CA, USA).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results And Discussion","content":"\u003cdiv class=\"Section2\" id=\"Sec17\"\u003e\n \u003ch2\u003eSwim Bladder Biochemical Composition\u003c/h2\u003e\n \u003cp\u003eThe swim bladder represented 2% of total body weight in totoaba and contained 67.43 \u0026plusmn; 1.24% moisture. Crude protein content (dry weight) was 95.72 \u0026plusmn; 1.07%, lipids were 2.46 \u0026plusmn; 0.18%, and ash was 0.88 \u0026plusmn; 0.06%. Moisture content was low compared to other species: 75.20% in bighead carp (\u003cem\u003eHypophthalmichthys nobilis\u003c/em\u003e) [11]; 83.33% in yellowfin tuna (\u003cem\u003eThunnus albacares\u003c/em\u003e) [10]; 78.83% in miiuy croaker [12]; and 82.8% in catla [8]. These differences may be attributed to variations in swim bladder water content during tissue processing and storage, as well as biological factors. The low total lipids and minerals contents of the totoaba swim bladder are comparable to those of the miiuy croaker [12]. Protein content was higher than reported for catla (83.0%) [8] and miiuy croaker (90.55%) [12].\u003c/p\u003e\n \u003cp\u003eProximate composition in fish can depend on many factors, including seasonal variations in feeding behavior, age, and habitat. Nineteen amino acids were identified in the totoaba swim bladders and the amino acids profile showed collagen to be the predominant protein, which coincides with the swim bladders of other fish species [24]. Glycine was the most abundant amino acid, followed by alanine, proline, arginine, glutamic acid, hydroxyproline, and aspartic acid, which represented 85% of total amino acids (AA). Its amino acids composition showed the totoaba swim bladder to be nutritionally poor since it contained only 12% essential AA compared to 53% conditionally essential and 35% non-essential (35%) amino acids. However, it is rich in functional AA (71%), such as glycine, glutamic acid, aspartic acid, proline, alanine, and arginine, all of which participate in and regulate key metabolic pathways [25]. It also contains high levels of hydrophobic amino acids which are frequently found in antioxidant peptides [26]. Overall, totoaba swim bladder had high protein and low lipids contents, suggesting it potential use in collagen extraction, as a source of functional AA, or a substrate for bioactive peptide production.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec18\"\u003e\n \u003ch2\u003eCollagen Yield\u003c/h2\u003e\n \u003cp\u003eHydrolysis time for PSC extraction can be as long as 72 h [11, 27], so four extraction times were used in the present study (20, 24, 32, and 72 h) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). During the extraction process, swim bladder collagen fibers were solubilized entirely in acetic acid upon proteolysis with pepsin (24 h) with good collagen yields (68.18 \u0026plusmn; 1.62%, dwb). According to previous studies, intermolecular cross-links in the telopeptide region and triple helices formed via condensation of aldehyde groups cause a decrease in collagen solubility [10], and the pepsin cleaves specifically on the telopeptide region, leading to isolated tropocollagen molecules. For this reason, pepsin is the principal protease used for increasing collagen extraction efficiency and reducing the collagen antigenicity caused by telopeptides [14, 15]. Thus, the PSC yields from totoaba swim bladder are comparable to those of Gulf corvina (\u003cem\u003eCynoscion othonopterus\u003c/em\u003e) (69%) [20] and significantly higher than PSC yields from other species: miiuy croaker (8%), yellowfin tuna (12%), giant croaker (15%) (\u003cem\u003eNibea japonica\u003c/em\u003e), bester sturgeon (38%) (\u003cem\u003eHuso x Acipenser ruthenus\u003c/em\u003e), catfish (40%) (\u003cem\u003eTachysurus maculatus\u003c/em\u003e), rohu (47%) (\u003cem\u003eLabeo rohita\u003c/em\u003e), bighead carp (59%) and catla (61%) [8, 10\u0026ndash;12, 14, 15, 22, 28]. Interestingly, collagen yield from totoaba and other fish species is lower than the 85.3% (dwb) ASC yield reported for seabass (\u003cem\u003eLates calcarifer\u003c/em\u003e) swim bladder [29], suggesting that seabass swim bladder may have less cross-linked collagen fibers. These differences in collagen yield are probably due to extraction conditions, swim bladder firmness (i.e., degree of cross-linking), animal age, nutrition, and development conditions (wild or farmed). Protein content in the totoaba swim bladder collagen (TSBC) was 96.34 \u0026plusmn; 1.19%, ash content was 0.83 \u0026plusmn; 0.09%, and no fat was detected, which is consistent with collagen from miiuy croaker [12].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec19\"\u003e\n \u003ch2\u003eCollagen Characterization\u003c/h2\u003e\n \u003cp\u003eAmino Acid Composition\u003c/p\u003e\n \u003cp\u003eAll collagens have a general (Gly-X-Y)n sequence in their polypeptide chains, so glycine can be expected to be the main amino acid [6]. This agrees with the present results in that TSBC glycine content was 309 /1000 residues, followed by alanine (132 /1000 residues) and proline (122 /1000 residues) (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Low levels of tyrosine, histidine, isoleucine, hydroxylysine, and methionine were observed, and cysteine was not detected, which is reported for collagens [12,25,26]. Aromatic amino acids, mainly tyrosine, are generally found in low concentrations in PSC [22]. Compared to PSC isolated from Gulf corvina and miiuy croaker swim bladders (family, Sciaenidae) [12, 20], the TSBC had higher levels of aspartic acid, glutamic acid, proline, and alanine but lower levels of valine, threonine, isoleucine and leucine (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). This variation in amino acid content could be due various factors, such as fish species biology (health state and age), environment (water temperature and feeding), and habitat (wild or farmed). Imino acid (proline and hydroxyproline) content in the TSBC was 205 /1000 residues (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), which is consistent with miiuy croaker swim bladder collagen [12]. Imino acid content has been reported to positively affect collagen structural stability because the pyrrolidine ring imposes restrictions on polypeptide chain conformation, thus strengthening the triple helix structure [30, 31]. The degree of hydroxylation of proline (41%) and lysine (16%) also influences collagen self-assembly and stabilization [32].\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1. Amino acid composition of swim bladder (composition percentage) and swim bladder collagen from farmed totoaba (residues/1000 residues).\u003c/strong\u003e\u003c/p\u003e\n \u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAmino Acids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSwim Bladder\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTSBC\u003csup\u003e1\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e\u003cstrong\u003eGulf corvina\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ePSC\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePSC Miiuy croaker\u003csup\u003e3\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eAsp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e5.13 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e52 \u0026plusmn; 1.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eGlu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e9.61 \u0026plusmn; 0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e101 \u0026plusmn; 1.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eHyp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e5.43 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e83 \u0026plusmn; 1.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eSer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e2.02 \u0026plusmn; 0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e23 \u0026plusmn; 0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eGly\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e29.19 \u0026plusmn; 0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e309 \u0026plusmn; 3.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e303\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e334\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eHis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e0.47 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e5 \u0026plusmn; 0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eArg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e11.58 \u0026plusmn; 0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e59 \u0026plusmn; 4.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eThr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e1.76 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e13 \u0026plusmn; 2.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eAla\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e12.26 \u0026plusmn; 0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e132 \u0026plusmn; 1.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003ePro\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e12.10 \u0026plusmn; 0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e122 \u0026plusmn; 1.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e106\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e112\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eTyr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e0.47 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e2 \u0026plusmn; 0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eVal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e1.58 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e16 \u0026plusmn; 0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eMet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e1.36 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e7 \u0026plusmn; 0.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eCys\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e0.03 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003eNot detected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eIle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e0.63 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e5 \u0026plusmn; 0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eLeu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e1.94 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e20 \u0026plusmn; 0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eHyl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e0.28 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e5 \u0026plusmn; 0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003ePhe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e1.61 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e19 \u0026plusmn; 0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eLys\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e2.52 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e31 \u0026plusmn; 1.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eImino acid\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e205\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e187\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e199.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eDegree of Hydroxylation (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.367346938775512%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003ePro\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e40.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.367346938775512%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eLys\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e14.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.367346938775512%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003eTSBC\u003csup\u003e1\u003c/sup\u003e: totoaba swim bladder collagen.\u003csup\u003e\u0026nbsp;\u003c/sup\u003ePepsin-soluble collagen (PSC) from Gulf corvina (\u003cem\u003eC. othonopterus\u003c/em\u003e)\u003csup\u003e2\u003c/sup\u003e [20] and miiuy croaker (\u003cem\u003eM. miiuy\u003c/em\u003e)\u003csup\u003e3\u0026nbsp;\u003c/sup\u003e[12]. Imino acid: Proline + Hydroxyproline.\u003c/p\u003e\n \u003cp\u003eProtein Patterns\u003c/p\u003e\n \u003cp\u003eElectrophoretic analyses of the TSBC showed it to be composed mainly of two different \u0026alpha; chains (\u0026alpha;1 and \u0026alpha;2), in a 2:1 proportion, and a \u0026beta; chain (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). High molecular weight bands were also observed which correspond to \u0026beta;-chains (dimers) and \u0026gamma;-chains (trimer). This suggests that the TSBC is type I collagen, consisting of heterotrimer ([\u0026alpha;1(I)]2\u0026alpha;2(I)) chains. Swim bladder from other fish species has been reported to contain type I collagen [10,12,30]. Using the GelAnalyzer software, the apparent molecular weights of the TSBC \u0026alpha;1 (142 kDa) and \u0026alpha;2 chains (134 kDa) were calculated based on migration distance. The extraction process was clearly effective because the collagen preserved its native structure. Moreover, no low molecular weight (\u0026lt;100 kDa) components were observed, suggesting the pepsin cleaved specifically to the telopeptide region, as previously reported [33].\u003c/p\u003e\n \u003cp\u003eUV-Vis and FTIR Spectroscopy\u003c/p\u003e\n \u003cp\u003eMaximum absorption for collagen is near 230 nm, due to the peptide bond (R-CONH-R, amide group) of the polypeptide chains [12]. The UV-vis spectrum is therefore an essential parameter for detecting purified collagen [12]. In this spectrum the TSBC exhibited a maximum absorption peak at 228 nm (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea), which was similar to collagen from calf skin, grass carp (\u003cem\u003eCtenopharyngodon idella\u003c/em\u003e) [34], and miiuy croaker [12]. As expected, neither the TSBC nor the bovine serum albumin (BSA) reference exhibited a peak at 280 nm; in the TSBC this was due to its low aromatic amino acid (tyrosine and phenylalanine) content (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). This result indicates efficient non-collagen protein elimination, and consequent high TSBC purity.\u003c/p\u003e\n \u003cp\u003eIn the FTIR spectra, TSBC showed characteristic bands of amide A, B, I, II, and III (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). Amide absorption bands A and B, which correspond to the stretching vibration of group N-H and asymmetric stretching of CH\u003csub\u003e2\u003c/sub\u003e, were observed in wave numbers 3280 and 3071 cm\u003csup\u003e-1\u003c/sup\u003e, respectively. Amide I (C ═ O stretching), amide II (N-H bending and C-N stretching), and amide III (C-N stretching and N-H bending) appeared in frequencies 1629, 1543, and 1237 cm\u003csup\u003e-1\u003c/sup\u003e, respectively. The absorption ratio between amide III and the 1454 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e wavelength was 1.05, indicating preservation of the collagen\u0026rsquo;s triple helix structure. These results coincide with those reported for collagens isolated from other fish species [8, 10\u0026ndash;12, 14, 34].\u003c/p\u003e\n \u003cp\u003eStructural Integrity\u003c/p\u003e\n \u003cp\u003eThe TSBC x-ray diffraction (XRD) spectrum exhibited peaks at 7.7\u0026deg; and 20.02\u0026deg; (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec). The former was sharp and corresponded to the triple helix arrangement and distance between molecular chains; the latter was broad and corresponded to the distance between the amino acid residues along the helix [35]. Both peaks were consistent with the characteristic diffraction pattern of the collagen triple helicoidal structure [36]. The circular dichroism (CD) analysis showed the TSBC to have a weak positive absorption peak at 222 nm and a negative one at 197 nm with a crossing point (zero rotation) at 215 nm (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). This CD spectrum pattern is characteristic of the collagen triple helix structure and consistent with previous reports [28]; the 222 nm peak disappears after thermal denaturation [28, 37]. The results confirm the helix structure of TSBC remained in its native form, and therefore that the isolation process did not affect its molecular integrity.\u003c/p\u003e\n \u003cp\u003eThermal Behavior\u003c/p\u003e\n \u003cp\u003eMeasurements of CD molar ellipticity (\u0026theta;) as a function of temperature have been used to determine denaturation temperature (Td) [28]. The present CD (222) values decreased by approximately 34.5\u0026deg;C, indicating decomposition of the collagen triple helix structure (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). Specifically, the intramolecular hydrogen bonds that stabilized the secondary structure of the collagen broke, leading to collapse of the triple helix into a random coil [38]. The present results were similar to those reported for collagen isolated from yellowfin tuna swim bladder (33.9\u0026deg;C) [10] and Gulf corvina (32.5\u0026deg;C) [20]. The Td of TSBC was higher than for collagen from a cold-water fish such as cod (29.6\u0026deg;C) [37], and for a temperate water fish such as miiuy croaker (26.7\u0026deg;C) [12]. Swim bladder collagen from marine fish remains thermostable below 35\u0026deg;C whereas in freshwater fish the threshold is higher: 38\u0026deg;C in grass carp [33] and 39.38\u0026deg;C in catla [8]. Indeed, PSC isolated from the swim bladder of the freshwater fish rohu [14] retains thermal stability at up to 42.16\u0026deg;C, higher than pork skin collagen (37\u0026deg;C) and similar to calfskin collagen [35]. Collagen thermal behavior depends heavily on imino acid content [22, 30], as well as species optimum physiological temperature, which is closely related to its habitat [31, 39]. For the farmed totoaba from UMA, average water temperature is 27 \u0026plusmn; 1\u0026deg;C, while under natural conditions surface temperatures in the upper Gulf of California, Mexico, can range from 16 to 31\u0026deg;C on the surface and 13 to 19\u0026deg;C in deep waters (100 to 200 m) [40]. The present TSBC thermal stability result (34.5\u0026deg;C) is probably linked to water temperature in its natural habitat. Possible use of a collagen depends heavily on its thermal stability [41], and the fact that the studied TSBC has thermal stability close to that of terrestrial mammal collagen makes it a promising alternative.\u003c/p\u003e\n \u003cp\u003eProtein Solubility and Zeta Potential\u003c/p\u003e\n \u003cp\u003eAcid pH (2.0-4.0) caused higher solubility in the TSBC, but this parameter decreased from pH 5.0-6.0, resulting in protein precipitation (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec). Collagen solubility was lowest at around pH 6, but increased slightly in the pH 7.0\u0026ndash;10.0 range. This may be due to increased repulsion of collagen molecules as the negative charge increases [22]. Similar results have been reported for PSC from the swim bladder of grass carp [34], miiuy croaker [12], Gulf corvina [20] and giant croaker [22]. Zeta potential is a key marker of colloidal dispersion stability and varies in response to pH [12]. As pH increased in the TSBC suspension, the zeta potential progressively decreased from +27 mV (pH 2) to less than -24 mV at pH 10 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed). At a high magnitude of potential (positive or negative) a solution will resist aggregation, whereas low potential tends to formation of aggregates. For TSBC the zero surface net charge occurred at pH 5.4, this is considered the isoelectric point (pI) and is consistent with the protein solubility results (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec). Since the pI occurred at an acid pH, it may be associated with higher contents of glutamic acid and aspartic acid rather than of basic amino acids, such as histidine, lysine, and arginine (Table 2). The pI value was lower than reported for swim bladder collagen from miiuy croaker (6.85) [12] but similar to that of yellowfin tuna (5.93) [10]. In collagen, the pI is generally closely linked to amino acid composition distribution on its surface.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec20\"\u003e\n \u003ch2\u003eCollagen Hydrolysate\u003c/h2\u003e\n \u003cp\u003eTotoaba swim bladder has putative positive therapeutic effects in traditional Chinese medicine [5]. Peptides and collagen from croaker swim bladders have been shown to remove free radicals [12,17,20,23]. The peptide profiles of the hydrolysates produced from the TSBC using Alcalase\u003csup\u003e\u0026reg;\u003c/sup\u003e (HCA) and papain (HCP) showed the HCA to have more hydrophilic peptides than the HCP (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). In contrast, the HCP had more hydrophobic peptides when eluted from 10 to 20 minutes. Protein hydrolysates with antioxidant activity frequently contain mainly hydrophobic amino acids, which play a significant role in free radical elimination [26]. Based on this and the present peptide profiles, the HCP was expected to exhibit higher antioxidant activity than the HCA.\u003c/p\u003e\n \u003cp\u003eThe DPPH radical scavenging assay is a popular and efficient way of predicting antioxidant activity since the DPPH radical is more stable than hydroxyl and superoxide radicals [17]. Using the DPPH assay, the antioxidant activity of ultrafiltered fractions (\u0026lt;3 kDa) of the TSBC hydrolysates was tested at 3.2 mg mL\u003csup\u003e-1\u003c/sup\u003e. Antioxidant activity was 37% higher (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) with the HCP than the HCA, although ascorbic acid far exceeded both (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). This contrasts with the antioxidant activity results of a study of hydrolysates from the swim bladder of croceine croaker and miiuy croaker in which, at 15-25 mg protein mL\u003csup\u003e-1\u003c/sup\u003e, the Alcalase\u003csup\u003e\u0026reg;\u003c/sup\u003e hydrolysate had significantly higher activity than hydrolysates prepared with papain, pepsin, neutrase and trypsin [26]. Of note is that, after ultrafiltration, a lower concentration of HCA and HCP (3.2 mg mL\u003csup\u003e-1\u003c/sup\u003e) produced higher antioxidant activity than in the above study. Overall, the present antioxidant activity indicates that this parameter depends strongly on the enzyme used for hydrolysis, suggesting further research is needed to isolate active peptides and clarify their antioxidant activity.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eTo our knowledge, this study is the first report on the elemental biochemical composition, isolation, and characteristics of collagen from the swim bladder of farmed totoaba (\u003cem\u003eT. macdonaldi\u003c/em\u003e). Totoaba swim bladder has high protein and low lipid contents, suggesting it as a possible food supplement with health benefits. Collagen yield was high (68%). The amino acid composition and protein pattern were typical of type-I collagen ([α1(I)]2α2(I)). Structural integrity analyses confirmed that the extraction process used here preserved the collagen native triple helix structure with high purity. The extracted collagen exhibited good thermal stability (34.5\u0026deg;C), which correlated with its imino acid content. Collagen hydrolysate antioxidant activities were influenced by the enzyme employed, highlighting the need for further research on antioxidant peptide purification and identification. The present results constitute a baseline for future studies on the production of bioactive peptides and biomedical applications for this collagen. The results can also help to promote the use of the swim bladder from farmed totoaba as an alternative to conventional collagen sources or as a functional food, which will reduce by-product generation and provide added value to the culture of totoaba.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks are due Dr. Miguel A. Olvera-Novoa for access to the Aquaculture Nutrition Laboratory, CINVESTAV-Merida; Yadira Cortez-Santiago and Romel Borbon-Ojeda for their assistance with collagen extractions; and C\u0026eacute;sar A. Puerto-Castillo for assistance with amino acid analyses. Thanks are also due John Lindsay-Edwards for manuscript review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAffiliations\u003c/p\u003e\n\u003cp\u003eFacultad de Ciencias Marinas, Universidad Aut\u0026oacute;noma de Baja California (UABC), Carretera Transpeninsular Ensenada - Tijuana No. 3917, Col. Playitas, 22860 Ensenada, Baja California, M\u0026eacute;xico.\u003c/p\u003e\n\u003cp\u003eHonorio Cruz-L\u0026oacute;pez, Luis M. Enr\u0026iacute;quez, Conal D. True, and Lus M. L\u0026oacute;pez\u003c/p\u003e\n\u003cp\u003eUnidad de Qu\u0026iacute;mica en Sisal, Facultad de Qu\u0026iacute;mica, Universidad Nacional Aut\u0026oacute;noma de M\u0026eacute;xico, Puerto de Abrigo S/N, 97356 Sisal, Yucat\u0026aacute;n, M\u0026eacute;xico.\u003c/p\u003e\n\u003cp\u003eSergio Rodr\u0026iacute;guez-Morales\u003c/p\u003e\n\u003cp\u003eFacultad de Ciencias de la Ingenier\u0026iacute;a y Tecnolog\u0026iacute;a, Universidad Aut\u0026oacute;noma de Baja California (UABC), Blvd. Universitario 1000, Unidad Valle de las Palmas, 22260 Tijuana, Baja California, M\u0026eacute;xico.\u003c/p\u003e\n\u003cp\u003eLuis Jes\u0026uacute;s Villarreal-G\u0026oacute;mez\u003c/p\u003e\n\u003cp\u003eCentro de Investigaci\u0026oacute;n y de Estudio Avanzados del Instituto Polit\u0026eacute;cnico Nacional - Unidad M\u0026eacute;rida, Antigua Carretera a Progreso km. 6, 97310 M\u0026eacute;rida, Yucat\u0026aacute;n, M\u0026eacute;xico.\u003c/p\u003e\n\u003cp\u003eLeticia Olivera-Castillo\u003c/p\u003e\n\u003cp\u003e3B\u0026rsquo;s Research Group, I3B\u0026rsquo;s \u0026ndash; Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ci\u0026ecirc;ncia e Tecnologia, Zona Industrial da Gandra, 4805-017, Vigo, Guimar\u0026atilde;es, Portugal\u003c/p\u003e\n\u003cp\u003eTiago Henriques Silva\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Lus M. L\u0026oacute;pez\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research reported here was supported by the Universidad Aut\u0026oacute;noma de Baja California (UABC), M\u0026eacute;xico; the Consejo Nacional de Ciencia y Tecnolog\u0026iacute;a (CONACyT) (SAGARPA-CONACYT No. 247698); and fellowship no. 362129 (Honorio Cruz-L\u0026oacute;pez).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interests regarding the information reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFish were handled and treated following the technical specifications for the production, care and use of laboratory animals decreed in the Official Mexican Regulation (NOM-062-ZOO-1999) and according to the ethics statement of the Autonomous University of Baja California (UABC), based on international guidelines. All procedures and experimentation conducted with organisms produced at the UMA (DGVS-CR-IN-1084-B.C./09) are annually reported and evaluated by the General Office of Wildlife (Direcci\u0026oacute;n General de Vida Silvestre - DGVS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eHonorio Cruz-L\u0026oacute;pez: conceptualization, methodology, formal analysis, visualization, writing of original draft. Sergio Rodr\u0026iacute;guez-Morales: supervision, formal analysis, review \u0026amp; editing. Luis Enr\u0026iacute;quez: methodology, review \u0026amp; editing. Luis Jes\u0026uacute;s Villarreal-G\u0026oacute;mez: formal analysis, review \u0026amp; editing. Conal D. True: review \u0026amp; editing, resources. Leticia Olivera-Castillo: formal analysis, methodology, review \u0026amp; editing. Tiago Henriques Silva: formal analysis, review \u0026amp; editing. Lus M. L\u0026oacute;pez: conceptualization, manuscript review \u0026amp; editing, project administration, funding acquisition. All authors have read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eDOF: Norma Oficial Mexicana NOM-169-SEMARNAT-2018. Que establece las especificaciones de marcaje para los ejemplares, partes y derivados de totoaba (Totoaba macdonaldi) provenientes de unidades de manejo para la conservaci\u0026oacute;n de vida silvestre. (2018).\u003c/li\u003e\n \u003cli\u003eLM, Juarez, PA, Konietzko, HM, S.: Totoaba aquaculture and conservation: hope for an endangered fish from Mexico\u0026rsquo;s Sea of Cortez. World Aquac. 47, 30\u0026ndash;38 (2016).\u003c/li\u003e\n \u003cli\u003eLin, S.: Fish air-bladders of commercial value in China. Hong Kong Nat. a Q. Illus. J. Princ. 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Eng. 302, 1600460 (2017).\u0026nbsp;\u003ca href=\"https://doi.org/10.1002/mame.201600460\"\u003ehttps://doi.org/10.1002/mame.201600460\u003c/a\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 2 is not available with this version. \u003c/p\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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Totoaba macdonaldi, Swim bladder, Collagen recovery, Collagen hydrolysates, By-products","lastPublishedDoi":"10.21203/rs.3.rs-1004119/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1004119/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eFinding strategies to use swim bladder of farmed totoaba (\u003cem\u003eTotoaba macdonaldi\u003c/em\u003e) is of utmost need to reduce waste. Fish swim bladders are rich in collagen; hence, extracting collagen is a promising alternative with benefits for aquaculture of totoaba and the environment.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe elemental biochemical composition of totoaba swim bladders, including proximate composition and amino acid composition were determined. Acid-enzyme solubilisation (PSC) was used to extract collagen from swim bladders and its characteristics were analyzed. The alcalase and papain were used for the preparation of collagen hydrolysates.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eSwim bladders contained 95% protein, 2.4% fat, and 0.8% ash (dry basis). The essential amino acids content was low, but the functional amino acids content was high. The PSC yield was high, 68% (dry weight). The amino acid composition profile, electrophoretic pattern, and structural integrity analyses of the isolated collagen suggested it is typical type-I collagen with high purity. The denaturalization temperature was 34.5 °C, probably attributable to the imino acid content (205 residues/1000 residues). Papain-hydrolysates (\u0026lt;3 kDa) of this collagen exhibited higher radical scavenging activity than Alcalase-hydrolysates.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eSwim bladder from farmed totoaba is an ideal raw material for producing high-quality type-I collagen and a viable alternative to conventional collagen sources.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eStatement of Novelty\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eTo our knowledge, this paper is the first to examine the composition and characteristics of collagen of swim bladder from \u003cem\u003eTotoaba macdonaldi\u003c/em\u003e. Although the processing currently wastes bladders, this study showed that they could be a potential source for producing high-quality type-I collagen.\u003c/p\u003e","manuscriptTitle":"Swim Bladder of Farmed Totoaba macdonaldi: A Source of Value-Added Collagen","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-11-17 16:09:15","doi":"10.21203/rs.3.rs-1004119/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"65c9d01d-6824-4e25-bf11-d741c61486c9","owner":[],"postedDate":"November 17th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":8565411,"name":"Renewable Resources"},{"id":8565412,"name":"Environmental Engineering"},{"id":8565413,"name":"Agroecology"}],"tags":[],"updatedAt":"2023-06-13T20:10:07+00:00","versionOfRecord":{"articleIdentity":"rs-1004119","link":"https://doi.org/10.3390/md21030173","journal":{"identity":"marine-drugs","isVorOnly":true,"title":"Marine Drugs"},"publishedOn":"2023-03-09 00:00:00","publishedOnDateReadable":"March 9th, 2023"},"versionCreatedAt":"2021-11-17 16:09:15","video":"","vorDoi":"10.3390/md21030173","vorDoiUrl":"https://doi.org/10.3390/md21030173","workflowStages":[]},"version":"v1","identity":"rs-1004119","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1004119","identity":"rs-1004119","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}