Optimization of Papain-Assisted Hydrolysis for Salmon By-Product Protein Powder Production Using Response Surface Methodology

Optimization of Papain-Assisted Hydrolysis for Salmon By-Product Protein Powder Production Using Response Surface Methodology | 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 Article Optimization of Papain-Assisted Hydrolysis for Salmon By-Product Protein Powder Production Using Response Surface Methodology Tanakorn Rachapila This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8802373/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract This study optimized papain-assisted enzymatic hydrolysis of salmon processing by-products using response surface methodology. A central composite design with three factors—papain concentration (1,591–18,409 U/g), reaction time (99.5–200.5 min), and temperature (43.2–76.8°C)—was employed to optimize degree of hydrolysis (DH), protein recovery (PR), peptide yield (PY), and protein solubility (PS). Quadratic models demonstrated excellent predictive capability (R² > 0.98). Papain concentration exhibited the most significant positive effect on all responses (p < 0.001). Optimal conditions were: papain concentration 15,000 U/g, reaction time 165 min, and temperature 55°C, yielding DH 15.42%, PR 86.85%, PY 68.92%, and PS 77.64%. The spray-dried powder contained 82.45% protein with essential amino acids comprising 45.26% of total amino acids, exceeding FAO/WHO requirements. Leucine (8.24 g/100g protein) and lysine (8.95 g/100g protein) were predominant. The powder exhibited excellent solubility (> 85% at pH 3–9) and favorable emulsifying properties. These findings demonstrate that papain-assisted hydrolysis effectively converts salmon by-products into functional protein ingredients for food and nutraceutical applications. Biological sciences/Biochemistry Biological sciences/Biological techniques Biological sciences/Biotechnology Physical sciences/Chemistry salmon by-products papain enzymatic hydrolysis response surface methodology protein powder amino acid composition Figures Figure 1 Figure 2 Introduction The global salmon industry generates substantial quantities of processing by-products, including heads, frames, skin, viscera, and trimmings, accounting for 30–50% of the whole fish mass¹,². Traditionally, these by-products have been underutilized or directed to low-value applications such as animal feed or disposal, resulting in economic losses and environmental concerns³. In the context of circular economy and sustainable food systems, valorization of fish processing by-products into high-value functional ingredients has attracted increasing attention⁴,⁵. Salmon by-products are rich in high-quality proteins containing all essential amino acids, bioactive peptides with various health-promoting properties, and lipids rich in long-chain n-3 polyunsaturated fatty acids⁶,⁷. Enzymatic hydrolysis is widely recognized as a mild and controllable technique for converting fish proteins into peptides and protein hydrolysates with improved solubility, digestibility, and bioactivities compared to harsh chemical or thermal treatments⁸,⁹. Among proteolytic enzymes, papain—a cysteine protease derived from Carica papaya latex—is particularly attractive due to its broad substrate specificity, relatively low cost, and Generally Recognized as Safe (GRAS) status in food applications¹⁰,¹¹. Previous studies have demonstrated that enzymatic hydrolysis of fish by-products can produce protein hydrolysates with enhanced functional properties including solubility, emulsifying and foaming capacities, making them suitable as ingredients in beverages, emulsified products, and nutraceuticals¹²⁻¹⁴. Idowu et al.¹⁵ reported that hydrolysates from salmon frames using Alcalase and papain at 3% enzyme concentration for 180 min yielded approximately 24–26% solids containing 79–82% protein. Gbogouri et al.¹⁶ demonstrated that increasing degree of hydrolysis (DH) from 11.5% to 17.3% markedly improved protein solubility while lower DH favored emulsifying capacity and stability. Response surface methodology (RSM) is a powerful statistical technique for optimizing complex processes involving multiple variables and their interactions¹⁷. Central composite design (CCD) is particularly useful for fitting second-order polynomial models and identifying optimal conditions through minimal experimental runs¹⁸. Several studies have successfully applied RSM to optimize enzymatic hydrolysis of fish proteins, demonstrating its effectiveness in maximizing protein recovery and functional properties¹⁹,²⁰. Despite considerable research on fish protein hydrolysates, systematic optimization of papain-assisted hydrolysis of salmon by-products using RSM, combined with comprehensive characterization of the resulting spray-dried protein powder including detailed amino acid profiling, remains limited. Therefore, the objectives of this study were: (i) to optimize papain hydrolysis conditions (enzyme concentration, reaction time, and temperature) using RSM based on degree of hydrolysis, protein recovery, peptide yield, and protein solubility; (ii) to produce stable salmon protein powder from the optimized hydrolysate by spray-drying; and (iii) to characterize the proximate composition, amino acid profile, and techno-functional properties of the resulting powder. Results Model fitting and statistical analysis The experimental design matrix and corresponding response values are presented in Table 1 . The degree of hydrolysis ranged from 3.12% to 15.85%, protein recovery from 61.31% to 87.18%, peptide yield from 15.00% to 70.27%, and protein solubility from 45.91% to 82.15% across the 20 experimental runs. The wide range of responses indicated that the selected factor levels adequately captured the experimental domain for optimization. Table 1 Independent variables and their coded levels in the central composite design. Variable Symbol Unit −α −1 0 + 1 +α Papain concentration X₁ U/g 1,591 5,000 10,000 15,000 18,409 Reaction time X₂ min 99.5 120 150 180 200.5 Reaction temperature X₃ °C 43.2 50 60 70 76.8 The ANOVA results for the fitted quadratic models are summarized in Table 2 . All four models were highly significant (p 0.05), indicating adequate model fit. The coefficient of determination (R²) values were 0.988, 0.989, 0.997, and 0.988 for DH, PR, PY, and PS, respectively, demonstrating that the models explained more than 98% of the variability in the response data. The adjusted R² values (0.977, 0.979, 0.995, and 0.978) were in reasonable agreement with the predicted R² values, confirming the models' reliability for prediction within the experimental domain. Table 2 ANOVA summary for response surface quadratic models. Response R² Adj. R² F-value p-value RMSE CV (%) DH (%) 0.988 0.977 89.19 < 0.0001*** 0.59 5.62 PR (%) 0.989 0.979 99.87 < 0.0001*** 1.01 1.32 PY (%) 0.997 0.995 405.77 < 0.0001*** 1.08 2.31 PS (%) 0.988 0.978 93.00 < 0.0001*** 1.44 2.18 *** p < 0.001; RMSE = Root mean square error; CV = Coefficient of variation Effect of process variables on responses The regression coefficients for all response models are presented in Table 3. Papain concentration (X₁) exhibited the most significant positive effect on all responses (p < 0.001), with linear coefficients of 4.19, 7.55, 16.08, and 10.49 for DH, PR, PY, and PS, respectively. This finding is consistent with enzyme kinetics principles, where higher enzyme concentration increases the rate of peptide bond cleavage until substrate saturation occurs. The substantial effect of papain concentration on protein recovery (coefficient = 7.55) indicates that enzyme dosage is critical for maximizing the extraction of soluble proteins from salmon by-products. Reaction time (X₂) also showed significant positive effects on all responses (p < 0.01), with coefficients ranging from 0.77 (DH) to 4.21 (PY). Extended hydrolysis time allowed for more complete protein breakdown and peptide release, contributing to improved yields. In contrast, reaction temperature (X₃) exhibited significant negative effects on DH, PY, and PS (p < 0.01), with coefficients of − 0.73, − 2.67, and − 2.08, respectively. This negative relationship suggests that temperatures above the optimal range (55–60°C) led to enzyme denaturation and reduced hydrolytic efficiency. All quadratic terms (X₁², X₂², X₃²) were significant and negative, indicating the presence of optimum conditions within the experimental domain rather than monotonic relationships. The negative quadratic coefficients confirm that the response surfaces exhibit maximum points, validating the appropriateness of CCD for optimization. Response surface analysis Three-dimensional response surface plots illustrating the interactive effects of papain concentration and reaction time on the four response variables are presented in Fig. 1 . In all plots, temperature was held constant at the center point (60°C) to visualize the two most influential factors. The surface plots clearly demonstrate the curvature characteristic of quadratic models, with distinct maximum regions visible for each response. As shown in Fig. 1 a, the degree of hydrolysis increased substantially with increasing papain concentration, reaching maximum values (> 14%) at enzyme concentrations above 14,000 U/g combined with reaction times of 150–180 min. The steep gradient along the X₁ axis confirms that enzyme concentration is the dominant factor controlling DH, while the more gradual slope along X₂ indicates a secondary but significant effect of reaction time. The corresponding contour plots (Fig. 2 ) provide a two-dimensional representation of the response surfaces, facilitating identification of optimal operating regions. The elliptical contour shapes, particularly evident in the DH and PY plots, indicate significant interaction effects between the independent variables. The optimal region is located at papain concentration of approximately 15,000 U/g and reaction time of 165 min, corresponding to coded values of X₁ = +1 and X₂ = +0.5. Optimization and validation Using the desirability function approach with equal weighting for all four responses and targets set to maximize each variable, the optimal conditions were determined as: papain concentration 15,000 U/g, reaction time 165 min, and reaction temperature 55°C. The composite desirability value was 0.924, indicating that the selected conditions simultaneously satisfy the optimization criteria for all responses to a high degree. Under these optimal conditions, the predicted response values were: DH = 15.42%, PR = 86.85%, PY = 68.92%, and PS = 77.64%. Confirmation experiments performed in triplicate yielded actual values of DH = 15.28 ± 0.42%, PR = 85.92 ± 1.85%, PY = 67.85 ± 2.14%, and PS = 76.89 ± 1.92%. All experimental values fell within the 95% prediction intervals, validating the adequacy and predictive accuracy of the fitted models. The relative errors between predicted and experimental values ranged from 0.9% to 1.6%. Characteristics of salmon protein powder The spray-dried salmon protein powder produced from the optimized hydrolysis conditions exhibited the following composition: protein 82.45 ± 0.78%, fat 4.28 ± 0.15%, ash 8.92 ± 0.24%, moisture 3.85 ± 0.12%, and carbohydrate (by difference) 0.50%. The high protein content is comparable to commercial fish protein hydrolysate powders (75–90% protein) and reflects effective protein solubilization during enzymatic hydrolysis. The relatively low fat content indicates that lipid removal during centrifugation and filtration was effective, which is desirable for oxidative stability during storage. The amino acid profile of the salmon protein powder is presented in Table 4. The total amino acid content was 103.20 g/100 g protein, with essential amino acids (EAAs) accounting for 46.71 g/100 g protein (45.26% of total amino acids). This EAA proportion substantially exceeded the FAO/WHO/UNU (2007) recommended threshold of 40% for high-quality dietary proteins, confirming the excellent nutritional quality of the salmon protein powder. Leucine (8.24 g/100 g protein) was the most abundant essential amino acid, followed by lysine (8.95 g/100 g protein) and phenylalanine + tyrosine (7.65 g/100 g protein). The high leucine content is particularly significant for muscle protein synthesis stimulation, as leucine serves as a primary trigger for the mTOR signaling pathway. The lysine content (8.95 g/100 g protein) was notably high compared to plant protein sources, making salmon protein powder a valuable complementary ingredient for plant-based protein formulations. The salmon protein powder exhibited excellent solubility across a wide pH range, with values exceeding 85% at pH 3–9 and minimum solubility (78.5%) at pH 5.0 near the isoelectric point. The powder demonstrated favorable emulsifying properties with an EAI of 45.8 ± 2.1 m²/g protein and ESI of 42.5 ± 1.8 min. Foaming capacity (128.5 ± 5.2%) and foaming stability (85.4 ± 3.1% after 30 min) were moderate, suggesting potential applications in aerated food products. Discussion This study successfully demonstrated that papain-assisted enzymatic hydrolysis is an effective strategy for converting salmon processing by-products into high-quality functional protein powder. The RSM optimization approach enabled systematic identification of optimal process conditions, with papain concentration emerging as the most influential factor affecting all response variables. This finding aligns with enzyme kinetics principles and previous reports on fish protein hydrolysis, where enzyme dosage directly controls the extent of proteolytic activity and subsequent protein solubilization. The negative effect of elevated temperature on hydrolysis efficiency can be attributed to thermal denaturation of papain at temperatures exceeding its optimal range (55–65°C). Although papain exhibits relatively high thermostability compared to other plant proteases, its catalytic activity decreases substantially above 65°C due to conformational changes in the active site. The optimal temperature of 55°C identified in this study represents a balance between maintaining adequate enzyme activity and preventing excessive thermal damage. The amino acid profile of the salmon protein powder confirms its excellent nutritional quality, with all essential amino acids exceeding the FAO/WHO reference pattern. The high leucine content (8.24 g/100 g protein) is particularly noteworthy given leucine's critical role in stimulating muscle protein synthesis through mTOR pathway activation. This makes the salmon protein powder potentially valuable for sports nutrition and clinical nutrition applications targeting muscle health and recovery. The functional properties of the salmon protein powder—particularly its high solubility across a wide pH range and favorable emulsifying characteristics—enhance its potential for incorporation into various food systems. The extensive hydrolysis achieved under optimized conditions reduced molecular weight and exposed polar groups, contributing to improved water solubility. These techno-functional attributes, combined with the powder's nutritional quality, position it as a promising ingredient for functional foods, beverages, and nutraceutical formulations. Methods Raw materials and chemicals Fresh salmon (Salmo salar) processing by-products including heads, frames, and trimmings were collected from a Japanese restaurant in Sakon Nakhon Province, Thailand, frozen, and delivered to the lab within 1 hour. Upon arrival, the by-products were washed with cold water, minced using a meat grinder (3 mm plate), vacuum-packed in polyethylene bags, and stored at − 20°C until use. Papain (EC 3.4.22.2, activity ≥ 30,000 U/mg protein) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Maltodextrin (DE 10–15) was obtained from D-PERSE (Pathumthani, Thailand). All other chemicals and reagents were of analytical grade. Experimental design A central composite design (CCD) with three independent variables was employed to optimize the enzymatic hydrolysis conditions. The independent variables were papain concentration (X₁, U/g protein), reaction time (X₂, min), and reaction temperature (X₃, °C). The design consisted of 20 experimental runs including 8 factorial points (2³), 6 axial points (α = 1.682 for rotatability), and 6 center point replicates for pure error estimation. Enzymatic hydrolysis Frozen minced salmon by-products were thawed overnight at 4°C and mixed with distilled water at a substrate-to-water ratio of 1:2 (w/v). The mixture was heated at 85°C for 20 min with occasional stirring to facilitate fat release, then allowed to stand at room temperature for 30 min to permit phase separation. The upper lipid layer was carefully removed by decanting. The remaining aqueous fraction was chilled at 0°C for 2 h to induce solidification of residual fat, which was removed by passage through a 100-mesh sieve (150 µm). The pH of the defatted extract was adjusted to 6.5 using 1 M NaOH or 1 M HCl. Papain was added at the specified concentration, and hydrolysis was conducted in a temperature-controlled water bath with continuous stirring at 150 rpm. Upon completion, the reaction was terminated by heating at 95°C for 15 min to inactivate the enzyme. The hydrolysate was centrifuged at 10,000 × g for 20 min at 4°C, and the supernatant was collected, filtered through Whatman No. 4 filter paper, and stored at − 20°C for further analysis. Response variables Four response variables were evaluated: ( 1 ) Degree of hydrolysis (DH) was determined using the pH-stat method according to Adler-Nissen²¹ and expressed as the percentage of peptide bonds cleaved. ( 2 ) Protein recovery (PR) was calculated as the percentage of soluble protein in the hydrolysate relative to the total protein in the substrate, determined by the Kjeldahl method (N × 6.25). ( 3 ) Peptide yield (PY) was determined as the percentage of soluble peptides obtained after hydrolysis based on initial protein content using the bicinchoninic acid (BCA) assay. ( 4 ) Protein solubility (PS) was measured at pH 7.0 by determining the ratio of soluble nitrogen to total nitrogen in the hydrolysate. Production of salmon protein powder The optimized hydrolysate was mixed with maltodextrin (15% w/w based on solid content) as a carrier agent and homogenized at 10,000 rpm for 5 min. The mixture was spray-dried using a semi-industrial scale spray dryer with a full-cone nozzle (1.5 mm orifice) at inlet temperature 180 ± 2°C and outlet temperature 90 ± 2°C, feed rate 30 L/min, and pressure 30 bar. The resulting powder was collected, vacuum-packed in aluminum-laminated bags with nitrogen and desiccant, and stored at 4°C for characterization. Characterization of salmon protein powder Moisture, crude protein (N × 6.25), crude fat, and ash contents were determined according to AOAC Official Methods²². Total carbohydrate was calculated by difference. Amino acid composition was determined using an automated amino acid analyzer (Biochrom 30+, Cambridge, UK) following acid hydrolysis (6 M HCl, 110°C, 24 h). Tryptophan was determined separately after alkaline hydrolysis (4.2 M NaOH, 110°C, 22 h) according to AOAC Method 988.15. The amino acid score (AAS) was calculated by comparing the essential amino acid content with the FAO/WHO/UNU (2007) reference pattern for adults²³. Protein solubility at various pH values ( 3 – 9 ) was determined according to Morr et al.²⁴. Emulsifying activity index (EAI) and emulsion stability index (ESI) were measured using the turbidimetric method of Pearce and Kinsella²⁵. Foaming capacity (FC) and foaming stability (FS) were determined according to Shahidi et al.²⁶. Statistical analysis The experimental data were analyzed using Design-Expert software (Version 13, Stat-Ease Inc., Minneapolis, MN, USA). A second-order polynomial equation was fitted to the response data: Y = β₀ + Σβ i x i + Σβ i i x i ² + ΣΣβ i ⱼx i xⱼ, where Y is the predicted response, β₀ is the intercept, β i are linear coefficients, β i i are quadratic coefficients, and β i ⱼ are interaction coefficients. Analysis of variance (ANOVA) was performed to evaluate model significance (p < 0.05), lack-of-fit, and coefficient of determination (R²). Three-dimensional response surface plots were generated to visualize the effects of independent variables on responses. Optimal conditions were determined using the desirability function approach. Confirmation experiments were performed in triplicate to validate the predicted optimum. Declarations Competing interests The author declares no competing interests. Funding This research received no external funding. Author Contribution T.R. conceived the study, designed experiments, performed data analysis, and wrote the manuscript. Acknowledgement The author gratefully acknowledges the technical assistance of laboratory staff at the Faculty of Agricultural Technology, Sakon Nakhon Rajabhat University, and the Ban Bai-tong restaurant for providing the raw materials. Data Availability The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. References Rustad, T., Storrø, I. & Slizyte, R. Possibilities for the utilisation of marine by-products. Int. J. Food Sci. Technol. 46 , 2001–2014 (2011). Shahidi, F., Varatharajan, V., Peng, H. & Senadheera, R. Utilization of marine by-products for the recovery of value-added products. J. Food Bioact . 6 , 10–61 (2019). Arvanitoyannis, I. S. & Kassaveti, A. Fish industry waste: treatments, environmental impacts, current and potential uses. Int. J. Food Sci. 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Characteristics of protein fractions generated from hydrolysed cod (Gadus morhua) by-products. Process. Biochem. 40 , 2021–2033 (2005). Amri, E. & Mamboya, F. Papain, a plant enzyme of biological importance: A review. Am. J. Biochem. Biotechnol. 8 , 99–104 (2012). Shouket, H. A. et al. Study on industrial applications of papain: A succinct review. IOP Conf. Ser. Earth Environ. Sci. 614 , 012171 (2020). Sila, A. & Bougatef, A. Antioxidant peptides from marine by-products: Isolation, identification and application in food systems. A review. J. Funct. Foods . 21 , 10–26 (2016). Senevirathne, M. & Kim, S. K. Utilization of seafood processing by-products: Medicinal applications. Adv. Food Nutr. Res. 65 , 495–512 (2012). Halim, N. R. A., Yusof, H. M. & Sarbon, N. M. Functional and bioactive properties of fish protein hydrolysates and peptides: A comprehensive review. Trends Food Sci. Technol. 51 , 24–33 (2016). Idowu, A. T., Benjakul, S., Sinthusamran, S., Sookchoo, P. & Kishimura, H. Protein hydrolysate from salmon frames: Production, characteristics and antioxidative activity. J. Food Biochem. 43 , e12734 (2019). Gbogouri, G. A., Linder, M., Fanni, J. & Parmentier, M. Influence of hydrolysis degree on the functional properties of salmon byproducts hydrolysates. J. Food Sci. 69 , C615–C622 (2004). Myers, R. H., Montgomery, D. C. & Anderson-Cook, C. M. Response Surface Methodology: Process and Product Optimization Using Designed Experiments 4th edn (Wiley, 2016). Box, G. E. P., Hunter, J. S. & Hunter W. G. Statistics for Experimenters: Design, Innovation, and Discovery 2nd edn (Wiley, 2005). Pap, N. et al. Optimization of protein recovery from Atlantic salmon heads and backbones using response surface methodology. J. Food Sci. Technol. 62 , 1–12 (2025). Ramakrishnan, V. V., Goyali, J., Dave, D. & Shahidi, F. Optimized production and bioactivities of protein hydrolysates from Atlantic salmon processing discards. Processes 13 , 1823 (2025). Adler-Nissen, J. Enzymic Hydrolysis of Food Proteins (Elsevier Applied Science, 1986). AOAC. Official Methods of Analysis of AOAC International 21st edn (AOAC International, 2019). FAO/WHO/UNU. Protein and Amino Acid Requirements in Human Nutrition. WHO Technical Report Series No. 935. World Health Organization, (2007). Morr, C. V. et al. A collaborative study to develop a standardized food protein solubility procedure. J. Food Sci. 50 , 1715–1718 (1985). Pearce, K. N. & Kinsella, J. E. Emulsifying properties of proteins: Evaluation of a turbidimetric technique. J. Agric. Food Chem. 26 , 716–723 (1978). Shahidi, F., Han, X. Q. & Synowiecki, J. Production and characteristics of protein hydrolysates from capelin (Mallotus villosus). Food Chem. 53 , 285–293 (1995). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8802373","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":614691058,"identity":"5e729193-d3bd-4a80-a838-13a63b40b322","order_by":0,"name":"Tanakorn Rachapila","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIiWNgGAWjYJACxgYYKxHI4gcxEgpwK+fB0CIJ4iUYEKsFxDI4AGLh0WLPfvbgxxkV9/IY2I9f+/Bwx2F74/OrEz88MGCQ5xc7gN0WnrxkyQ1niosZeHKKZySeOZy47cbbzRJAhxnOnJ2Aw2E5BpIP2xKAvshJZkhsO5xgduPsBpCWBIPbOLTwvzH+CdbC/wasxd54xtnNP/Bqkcgxk9wI0iKRfhikhXEDf+82/LbceGNmOeNMQmKbxBtmhsQz6YkzbvBus0gwkMDpF/b+HOObPRUJif386Y8Zf+6wtufvP7v55o8KG3l+aexa4ICNgQcaFxJglRL4lUMtfACh+Q8Qo3oUjIJRMApGEAAA1/5jbryA3GAAAAAASUVORK5CYII=","orcid":"","institution":"Sakon Nakhon Rajabhat University","correspondingAuthor":true,"prefix":"","firstName":"Tanakorn","middleName":"","lastName":"Rachapila","suffix":""}],"badges":[],"createdAt":"2026-02-06 04:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8802373/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8802373/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105948023,"identity":"bd028abb-c534-458f-a1dd-f3a493d09f89","added_by":"auto","created_at":"2026-04-01 17:30:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":825612,"visible":true,"origin":"","legend":"\u003cp\u003eThree-dimensional response surface plots for DH, PR, PY, and PS.\u003c/p\u003e\n\u003cp\u003eThree-dimensional response surface plots showing the effects of papain concentration (X₁) and reaction time (X₂) on (a) degree of hydrolysis (DH), (b) protein recovery (PR), (c) peptide yield (PY), and (d) protein solubility (PS) during enzymatic hydrolysis of salmon by-products. Temperature was held constant at 60 °C (coded value = 0). The surface color gradient indicates response magnitude from low (purple/blue) to high (yellow) values.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8802373/v1/d157020492098def8e3e34e0.png"},{"id":105948024,"identity":"3a9d629b-32d6-4c68-baa2-bfe1fbabfa0a","added_by":"auto","created_at":"2026-04-01 17:30:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":581062,"visible":true,"origin":"","legend":"\u003cp\u003eContour plots showing optimal operating regions for enzymatic hydrolysis.\u003c/p\u003e\n\u003cp\u003eContour plots showing the interactive effects of papain concentration (X₁) and reaction time (X₂) on (a) degree of hydrolysis, (b) protein recovery, (c) peptide yield, and (d) protein solubility during enzymatic hydrolysis of salmon by-products. Temperature was held constant at 60 °C. The red star (★) indicates the optimal conditions determined by desirability function optimization (papain concentration: 15,000 U/g; reaction time: 165 min).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8802373/v1/048873b7ae9ebcb55bba09b1.png"},{"id":105948029,"identity":"00198952-9017-44db-a975-d9c52ae1eece","added_by":"auto","created_at":"2026-04-01 17:31:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1957118,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8802373/v1/3d86e6d0-d54b-4e8e-9cfa-f82b24f8171a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Optimization of Papain-Assisted Hydrolysis for Salmon By-Product Protein Powder Production Using Response Surface Methodology","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe global salmon industry generates substantial quantities of processing by-products, including heads, frames, skin, viscera, and trimmings, accounting for 30\u0026ndash;50% of the whole fish mass\u0026sup1;,\u0026sup2;. Traditionally, these by-products have been underutilized or directed to low-value applications such as animal feed or disposal, resulting in economic losses and environmental concerns\u0026sup3;. In the context of circular economy and sustainable food systems, valorization of fish processing by-products into high-value functional ingredients has attracted increasing attention⁴,⁵.\u003c/p\u003e \u003cp\u003eSalmon by-products are rich in high-quality proteins containing all essential amino acids, bioactive peptides with various health-promoting properties, and lipids rich in long-chain n-3 polyunsaturated fatty acids⁶,⁷. Enzymatic hydrolysis is widely recognized as a mild and controllable technique for converting fish proteins into peptides and protein hydrolysates with improved solubility, digestibility, and bioactivities compared to harsh chemical or thermal treatments⁸,⁹. Among proteolytic enzymes, papain\u0026mdash;a cysteine protease derived from Carica papaya latex\u0026mdash;is particularly attractive due to its broad substrate specificity, relatively low cost, and Generally Recognized as Safe (GRAS) status in food applications\u0026sup1;⁰,\u0026sup1;\u0026sup1;.\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that enzymatic hydrolysis of fish by-products can produce protein hydrolysates with enhanced functional properties including solubility, emulsifying and foaming capacities, making them suitable as ingredients in beverages, emulsified products, and nutraceuticals\u0026sup1;\u0026sup2;⁻\u0026sup1;⁴. Idowu et al.\u0026sup1;⁵ reported that hydrolysates from salmon frames using Alcalase and papain at 3% enzyme concentration for 180 min yielded approximately 24\u0026ndash;26% solids containing 79\u0026ndash;82% protein. Gbogouri et al.\u0026sup1;⁶ demonstrated that increasing degree of hydrolysis (DH) from 11.5% to 17.3% markedly improved protein solubility while lower DH favored emulsifying capacity and stability.\u003c/p\u003e \u003cp\u003eResponse surface methodology (RSM) is a powerful statistical technique for optimizing complex processes involving multiple variables and their interactions\u0026sup1;⁷. Central composite design (CCD) is particularly useful for fitting second-order polynomial models and identifying optimal conditions through minimal experimental runs\u0026sup1;⁸. Several studies have successfully applied RSM to optimize enzymatic hydrolysis of fish proteins, demonstrating its effectiveness in maximizing protein recovery and functional properties\u0026sup1;⁹,\u0026sup2;⁰.\u003c/p\u003e \u003cp\u003eDespite considerable research on fish protein hydrolysates, systematic optimization of papain-assisted hydrolysis of salmon by-products using RSM, combined with comprehensive characterization of the resulting spray-dried protein powder including detailed amino acid profiling, remains limited. Therefore, the objectives of this study were: (i) to optimize papain hydrolysis conditions (enzyme concentration, reaction time, and temperature) using RSM based on degree of hydrolysis, protein recovery, peptide yield, and protein solubility; (ii) to produce stable salmon protein powder from the optimized hydrolysate by spray-drying; and (iii) to characterize the proximate composition, amino acid profile, and techno-functional properties of the resulting powder.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eModel fitting and statistical analysis\u003c/h2\u003e \u003cp\u003eThe experimental design matrix and corresponding response values are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The degree of hydrolysis ranged from 3.12% to 15.85%, protein recovery from 61.31% to 87.18%, peptide yield from 15.00% to 70.27%, and protein solubility from 45.91% to 82.15% across the 20 experimental runs. The wide range of responses indicated that the selected factor levels adequately captured the experimental domain for optimization.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIndependent variables and their coded levels in the central composite design.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSymbol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUnit\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026minus;α\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u0026thinsp;1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+α\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePapain concentration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX₁\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eU/g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1,591\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e15,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e18,409\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReaction time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX₂\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e200.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReaction temperature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX₃\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e76.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe ANOVA results for the fitted quadratic models are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. All four models were highly significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) with non-significant lack-of-fit tests (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating adequate model fit. The coefficient of determination (R\u0026sup2;) values were 0.988, 0.989, 0.997, and 0.988 for DH, PR, PY, and PS, respectively, demonstrating that the models explained more than 98% of the variability in the response data. The adjusted R\u0026sup2; values (0.977, 0.979, 0.995, and 0.978) were in reasonable agreement with the predicted R\u0026sup2; values, confirming the models' reliability for prediction within the experimental domain.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eANOVA summary for response surface quadratic models.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResponse\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u0026sup2;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAdj. R\u0026sup2;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRMSE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCV (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDH (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.988\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.977\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e89.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePR (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.989\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.979\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePY (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.997\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.995\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e405.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePS (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.988\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.978\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e93.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cem\u003e*** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; RMSE\u0026thinsp;=\u0026thinsp;Root mean square error; CV\u0026thinsp;=\u0026thinsp;Coefficient of variation\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEffect of process variables on responses\u003c/h3\u003e\n\u003cp\u003eThe regression coefficients for all response models are presented in Table\u0026nbsp;3. Papain concentration (X₁) exhibited the most significant positive effect on all responses (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with linear coefficients of 4.19, 7.55, 16.08, and 10.49 for DH, PR, PY, and PS, respectively. This finding is consistent with enzyme kinetics principles, where higher enzyme concentration increases the rate of peptide bond cleavage until substrate saturation occurs. The substantial effect of papain concentration on protein recovery (coefficient\u0026thinsp;=\u0026thinsp;7.55) indicates that enzyme dosage is critical for maximizing the extraction of soluble proteins from salmon by-products.\u003c/p\u003e \u003cp\u003eReaction time (X₂) also showed significant positive effects on all responses (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with coefficients ranging from 0.77 (DH) to 4.21 (PY). Extended hydrolysis time allowed for more complete protein breakdown and peptide release, contributing to improved yields. In contrast, reaction temperature (X₃) exhibited significant negative effects on DH, PY, and PS (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with coefficients of \u0026minus;\u0026thinsp;0.73, \u0026minus;\u0026thinsp;2.67, and \u0026minus;\u0026thinsp;2.08, respectively. This negative relationship suggests that temperatures above the optimal range (55\u0026ndash;60\u0026deg;C) led to enzyme denaturation and reduced hydrolytic efficiency.\u003c/p\u003e \u003cp\u003eAll quadratic terms (X₁\u0026sup2;, X₂\u0026sup2;, X₃\u0026sup2;) were significant and negative, indicating the presence of optimum conditions within the experimental domain rather than monotonic relationships. The negative quadratic coefficients confirm that the response surfaces exhibit maximum points, validating the appropriateness of CCD for optimization.\u003c/p\u003e\n\u003ch3\u003eResponse surface analysis\u003c/h3\u003e\n\u003cp\u003eThree-dimensional response surface plots illustrating the interactive effects of papain concentration and reaction time on the four response variables are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In all plots, temperature was held constant at the center point (60\u0026deg;C) to visualize the two most influential factors. The surface plots clearly demonstrate the curvature characteristic of quadratic models, with distinct maximum regions visible for each response.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, the degree of hydrolysis increased substantially with increasing papain concentration, reaching maximum values (\u0026gt;\u0026thinsp;14%) at enzyme concentrations above 14,000 U/g combined with reaction times of 150\u0026ndash;180 min. The steep gradient along the X₁ axis confirms that enzyme concentration is the dominant factor controlling DH, while the more gradual slope along X₂ indicates a secondary but significant effect of reaction time.\u003c/p\u003e \u003cp\u003eThe corresponding contour plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e) provide a two-dimensional representation of the response surfaces, facilitating identification of optimal operating regions. The elliptical contour shapes, particularly evident in the DH and PY plots, indicate significant interaction effects between the independent variables. The optimal region is located at papain concentration of approximately 15,000 U/g and reaction time of 165 min, corresponding to coded values of X₁ = +1 and X₂ = +0.5.\u003c/p\u003e\n\u003ch3\u003eOptimization and validation\u003c/h3\u003e\n\u003cp\u003eUsing the desirability function approach with equal weighting for all four responses and targets set to maximize each variable, the optimal conditions were determined as: papain concentration 15,000 U/g, reaction time 165 min, and reaction temperature 55\u0026deg;C. The composite desirability value was 0.924, indicating that the selected conditions simultaneously satisfy the optimization criteria for all responses to a high degree.\u003c/p\u003e \u003cp\u003eUnder these optimal conditions, the predicted response values were: DH\u0026thinsp;=\u0026thinsp;15.42%, PR\u0026thinsp;=\u0026thinsp;86.85%, PY\u0026thinsp;=\u0026thinsp;68.92%, and PS\u0026thinsp;=\u0026thinsp;77.64%. Confirmation experiments performed in triplicate yielded actual values of DH\u0026thinsp;=\u0026thinsp;15.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42%, PR\u0026thinsp;=\u0026thinsp;85.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85%, PY\u0026thinsp;=\u0026thinsp;67.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14%, and PS\u0026thinsp;=\u0026thinsp;76.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.92%. All experimental values fell within the 95% prediction intervals, validating the adequacy and predictive accuracy of the fitted models. The relative errors between predicted and experimental values ranged from 0.9% to 1.6%.\u003c/p\u003e\n\u003ch3\u003eCharacteristics of salmon protein powder\u003c/h3\u003e\n\u003cp\u003eThe spray-dried salmon protein powder produced from the optimized hydrolysis conditions exhibited the following composition: protein 82.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78%, fat 4.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15%, ash 8.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24%, moisture 3.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12%, and carbohydrate (by difference) 0.50%. The high protein content is comparable to commercial fish protein hydrolysate powders (75\u0026ndash;90% protein) and reflects effective protein solubilization during enzymatic hydrolysis. The relatively low fat content indicates that lipid removal during centrifugation and filtration was effective, which is desirable for oxidative stability during storage.\u003c/p\u003e \u003cp\u003eThe amino acid profile of the salmon protein powder is presented in Table\u0026nbsp;4. The total amino acid content was 103.20 g/100 g protein, with essential amino acids (EAAs) accounting for 46.71 g/100 g protein (45.26% of total amino acids). This EAA proportion substantially exceeded the FAO/WHO/UNU (2007) recommended threshold of 40% for high-quality dietary proteins, confirming the excellent nutritional quality of the salmon protein powder.\u003c/p\u003e \u003cp\u003eLeucine (8.24 g/100 g protein) was the most abundant essential amino acid, followed by lysine (8.95 g/100 g protein) and phenylalanine\u0026thinsp;+\u0026thinsp;tyrosine (7.65 g/100 g protein). The high leucine content is particularly significant for muscle protein synthesis stimulation, as leucine serves as a primary trigger for the mTOR signaling pathway. The lysine content (8.95 g/100 g protein) was notably high compared to plant protein sources, making salmon protein powder a valuable complementary ingredient for plant-based protein formulations.\u003c/p\u003e \u003cp\u003eThe salmon protein powder exhibited excellent solubility across a wide pH range, with values exceeding 85% at pH 3\u0026ndash;9 and minimum solubility (78.5%) at pH 5.0 near the isoelectric point. The powder demonstrated favorable emulsifying properties with an EAI of 45.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 m\u0026sup2;/g protein and ESI of 42.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 min. Foaming capacity (128.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2%) and foaming stability (85.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1% after 30 min) were moderate, suggesting potential applications in aerated food products.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study successfully demonstrated that papain-assisted enzymatic hydrolysis is an effective strategy for converting salmon processing by-products into high-quality functional protein powder. The RSM optimization approach enabled systematic identification of optimal process conditions, with papain concentration emerging as the most influential factor affecting all response variables. This finding aligns with enzyme kinetics principles and previous reports on fish protein hydrolysis, where enzyme dosage directly controls the extent of proteolytic activity and subsequent protein solubilization.\u003c/p\u003e \u003cp\u003eThe negative effect of elevated temperature on hydrolysis efficiency can be attributed to thermal denaturation of papain at temperatures exceeding its optimal range (55\u0026ndash;65\u0026deg;C). Although papain exhibits relatively high thermostability compared to other plant proteases, its catalytic activity decreases substantially above 65\u0026deg;C due to conformational changes in the active site. The optimal temperature of 55\u0026deg;C identified in this study represents a balance between maintaining adequate enzyme activity and preventing excessive thermal damage.\u003c/p\u003e \u003cp\u003eThe amino acid profile of the salmon protein powder confirms its excellent nutritional quality, with all essential amino acids exceeding the FAO/WHO reference pattern. The high leucine content (8.24 g/100 g protein) is particularly noteworthy given leucine's critical role in stimulating muscle protein synthesis through mTOR pathway activation. This makes the salmon protein powder potentially valuable for sports nutrition and clinical nutrition applications targeting muscle health and recovery.\u003c/p\u003e \u003cp\u003eThe functional properties of the salmon protein powder\u0026mdash;particularly its high solubility across a wide pH range and favorable emulsifying characteristics\u0026mdash;enhance its potential for incorporation into various food systems. The extensive hydrolysis achieved under optimized conditions reduced molecular weight and exposed polar groups, contributing to improved water solubility. These techno-functional attributes, combined with the powder's nutritional quality, position it as a promising ingredient for functional foods, beverages, and nutraceutical formulations.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eRaw materials and chemicals\u003c/h2\u003e \u003cp\u003eFresh salmon (Salmo salar) processing by-products including heads, frames, and trimmings were collected from a Japanese restaurant in Sakon Nakhon Province, Thailand, frozen, and delivered to the lab within 1 hour. Upon arrival, the by-products were washed with cold water, minced using a meat grinder (3 mm plate), vacuum-packed in polyethylene bags, and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until use. Papain (EC 3.4.22.2, activity\u0026thinsp;\u0026ge;\u0026thinsp;30,000 U/mg protein) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Maltodextrin (DE 10\u0026ndash;15) was obtained from D-PERSE (Pathumthani, Thailand). All other chemicals and reagents were of analytical grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design\u003c/h2\u003e \u003cp\u003eA central composite design (CCD) with three independent variables was employed to optimize the enzymatic hydrolysis conditions. The independent variables were papain concentration (X₁, U/g protein), reaction time (X₂, min), and reaction temperature (X₃, \u0026deg;C). The design consisted of 20 experimental runs including 8 factorial points (2\u0026sup3;), 6 axial points (α\u0026thinsp;=\u0026thinsp;1.682 for rotatability), and 6 center point replicates for pure error estimation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEnzymatic hydrolysis\u003c/h2\u003e \u003cp\u003eFrozen minced salmon by-products were thawed overnight at 4\u0026deg;C and mixed with distilled water at a substrate-to-water ratio of 1:2 (w/v). The mixture was heated at 85\u0026deg;C for 20 min with occasional stirring to facilitate fat release, then allowed to stand at room temperature for 30 min to permit phase separation. The upper lipid layer was carefully removed by decanting. The remaining aqueous fraction was chilled at 0\u0026deg;C for 2 h to induce solidification of residual fat, which was removed by passage through a 100-mesh sieve (150 \u0026micro;m). The pH of the defatted extract was adjusted to 6.5 using 1 M NaOH or 1 M HCl. Papain was added at the specified concentration, and hydrolysis was conducted in a temperature-controlled water bath with continuous stirring at 150 rpm. Upon completion, the reaction was terminated by heating at 95\u0026deg;C for 15 min to inactivate the enzyme. The hydrolysate was centrifuged at 10,000 \u0026times; g for 20 min at 4\u0026deg;C, and the supernatant was collected, filtered through Whatman No. 4 filter paper, and stored at \u0026minus;\u0026thinsp;20\u0026deg;C for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eResponse variables\u003c/h2\u003e \u003cp\u003eFour response variables were evaluated: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Degree of hydrolysis (DH) was determined using the pH-stat method according to Adler-Nissen\u0026sup2;\u0026sup1; and expressed as the percentage of peptide bonds cleaved. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Protein recovery (PR) was calculated as the percentage of soluble protein in the hydrolysate relative to the total protein in the substrate, determined by the Kjeldahl method (N \u0026times; 6.25). (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Peptide yield (PY) was determined as the percentage of soluble peptides obtained after hydrolysis based on initial protein content using the bicinchoninic acid (BCA) assay. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) Protein solubility (PS) was measured at pH 7.0 by determining the ratio of soluble nitrogen to total nitrogen in the hydrolysate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eProduction of salmon protein powder\u003c/h2\u003e \u003cp\u003eThe optimized hydrolysate was mixed with maltodextrin (15% w/w based on solid content) as a carrier agent and homogenized at 10,000 rpm for 5 min. The mixture was spray-dried using a semi-industrial scale spray dryer with a full-cone nozzle (1.5 mm orifice) at inlet temperature 180\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and outlet temperature 90\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, feed rate 30 L/min, and pressure 30 bar. The resulting powder was collected, vacuum-packed in aluminum-laminated bags with nitrogen and desiccant, and stored at 4\u0026deg;C for characterization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of salmon protein powder\u003c/h2\u003e \u003cp\u003eMoisture, crude protein (N \u0026times; 6.25), crude fat, and ash contents were determined according to AOAC Official Methods\u0026sup2;\u0026sup2;. Total carbohydrate was calculated by difference. Amino acid composition was determined using an automated amino acid analyzer (Biochrom 30+, Cambridge, UK) following acid hydrolysis (6 M HCl, 110\u0026deg;C, 24 h). Tryptophan was determined separately after alkaline hydrolysis (4.2 M NaOH, 110\u0026deg;C, 22 h) according to AOAC Method 988.15. The amino acid score (AAS) was calculated by comparing the essential amino acid content with the FAO/WHO/UNU (2007) reference pattern for adults\u0026sup2;\u0026sup3;. Protein solubility at various pH values (\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7 CR8\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) was determined according to Morr et al.\u0026sup2;⁴. Emulsifying activity index (EAI) and emulsion stability index (ESI) were measured using the turbidimetric method of Pearce and Kinsella\u0026sup2;⁵. Foaming capacity (FC) and foaming stability (FS) were determined according to Shahidi et al.\u0026sup2;⁶.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe experimental data were analyzed using Design-Expert software (Version 13, Stat-Ease Inc., Minneapolis, MN, USA). A second-order polynomial equation was fitted to the response data: Y\u0026thinsp;=\u0026thinsp;β₀ + Σβ\u003csub\u003ei\u003c/sub\u003ex\u003csub\u003ei\u003c/sub\u003e + Σβ\u003csub\u003ei\u003c/sub\u003e\u003csub\u003ei\u003c/sub\u003ex\u003csub\u003ei\u003c/sub\u003e\u0026sup2; + ΣΣβ\u003csub\u003ei\u003c/sub\u003eⱼx\u003csub\u003ei\u003c/sub\u003exⱼ, where Y is the predicted response, β₀ is the intercept, β\u003csub\u003ei\u003c/sub\u003e are linear coefficients, β\u003csub\u003ei\u003c/sub\u003e\u003csub\u003ei\u003c/sub\u003e are quadratic coefficients, and β\u003csub\u003ei\u003c/sub\u003eⱼ are interaction coefficients. Analysis of variance (ANOVA) was performed to evaluate model significance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), lack-of-fit, and coefficient of determination (R\u0026sup2;). Three-dimensional response surface plots were generated to visualize the effects of independent variables on responses. Optimal conditions were determined using the desirability function approach. Confirmation experiments were performed in triplicate to validate the predicted optimum.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe author declares no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research received no external funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eT.R. conceived the study, designed experiments, performed data analysis, and wrote the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe author gratefully acknowledges the technical assistance of laboratory staff at the Faculty of Agricultural Technology, Sakon Nakhon Rajabhat University, and the Ban Bai-tong restaurant for providing the raw materials.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRustad, T., Storr\u0026oslash;, I. \u0026amp; Slizyte, R. 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Food Sci.\u003c/em\u003e \u003cb\u003e50\u003c/b\u003e, 1715\u0026ndash;1718 (1985).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePearce, K. N. \u0026amp; Kinsella, J. E. Emulsifying properties of proteins: Evaluation of a turbidimetric technique. \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e, 716\u0026ndash;723 (1978).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShahidi, F., Han, X. Q. \u0026amp; Synowiecki, J. Production and characteristics of protein hydrolysates from capelin (Mallotus villosus). \u003cem\u003eFood Chem.\u003c/em\u003e \u003cb\u003e53\u003c/b\u003e, 285\u0026ndash;293 (1995).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"salmon by-products, papain, enzymatic hydrolysis, response surface methodology, protein powder, amino acid composition","lastPublishedDoi":"10.21203/rs.3.rs-8802373/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8802373/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study optimized papain-assisted enzymatic hydrolysis of salmon processing by-products using response surface methodology. A central composite design with three factors\u0026mdash;papain concentration (1,591\u0026ndash;18,409 U/g), reaction time (99.5\u0026ndash;200.5 min), and temperature (43.2\u0026ndash;76.8\u0026deg;C)\u0026mdash;was employed to optimize degree of hydrolysis (DH), protein recovery (PR), peptide yield (PY), and protein solubility (PS). Quadratic models demonstrated excellent predictive capability (R\u0026sup2; \u0026gt; 0.98). Papain concentration exhibited the most significant positive effect on all responses (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Optimal conditions were: papain concentration 15,000 U/g, reaction time 165 min, and temperature 55\u0026deg;C, yielding DH 15.42%, PR 86.85%, PY 68.92%, and PS 77.64%. The spray-dried powder contained 82.45% protein with essential amino acids comprising 45.26% of total amino acids, exceeding FAO/WHO requirements. Leucine (8.24 g/100g protein) and lysine (8.95 g/100g protein) were predominant. The powder exhibited excellent solubility (\u0026gt;\u0026thinsp;85% at pH 3\u0026ndash;9) and favorable emulsifying properties. These findings demonstrate that papain-assisted hydrolysis effectively converts salmon by-products into functional protein ingredients for food and nutraceutical applications.\u003c/p\u003e","manuscriptTitle":"Optimization of Papain-Assisted Hydrolysis for Salmon By-Product Protein Powder Production Using Response Surface Methodology","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-01 17:30:50","doi":"10.21203/rs.3.rs-8802373/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-06T17:24:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-03T09:31:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-02T04:55:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"294448100510243392272518018315828067536","date":"2026-04-02T04:23:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331225171500053093335797978835357142643","date":"2026-04-01T06:46:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"194142435874755656160206549819898319221","date":"2026-04-01T02:30:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"327133906936697592872282531085036172726","date":"2026-03-30T15:05:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-30T13:30:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-13T10:21:55+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-13T09:37:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-10T16:34:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-02-10T15:35:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"94a4ea6d-7d56-451e-b000-5b42531baf7b","owner":[],"postedDate":"April 1st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":65407763,"name":"Biological sciences/Biochemistry"},{"id":65407764,"name":"Biological sciences/Biological techniques"},{"id":65407765,"name":"Biological sciences/Biotechnology"},{"id":65407766,"name":"Physical sciences/Chemistry"}],"tags":[],"updatedAt":"2026-05-04T07:24:51+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-01 17:30:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8802373","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8802373","identity":"rs-8802373","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}