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. 2026 Jun 5;37:104078. doi: 10.1016/j.fochx.2026.104078

Deep eutectic solvent-mediated extraction of collagen peptides from sturgeon skin: Molecular-weight control and mechanistic insights

Mingkai Bai b, Ning Wang b, Meichao Zhang c, Zifang Zhao d, Jianwei Yu e, Wenhai She f, Adewale Olusegun Obadina g, Yuanhui Zhao b, Shiyang Li b,⁎, Haohao Wu b,⁎, Ting Zhao a,⁎
PMCID: PMC13273213  PMID: 42317277

Abstract

A choline–oxalic acid deep eutectic solvent (DES) was developed for the molecular-weight-controlled extraction of collagen peptides from sturgeon (Acipenser spp.) skin, a collagen-rich fish-processing by-product containing 78.28% protein and 63.47% collagen. Among three choline-based organic-acid DESs, choline–oxalic acid exhibited the best extraction performance and produced lower-molecular-weight peptide fractions than choline–lactic acid and choline–acetic acid systems. Under the optimized conditions of 70 °C, a solid-to-liquid ratio of 1:80, and 2 h, the extraction rate reached 98.45%. Time-dependent extraction enabled controllable peptide production: a 2 h treatment mainly generated collagen polypeptides in the 1–10 kDa range, whereas extending the reaction to 4 h produced oligopeptides predominantly in the 0.5–1 kDa range. For downstream recovery, isopropanol showed the highest precipitation efficiency, achieving approximately 92% peptide recovery at an extract-to-solvent ratio of 1:6. UV–Vis analysis showed a characteristic collagen peptide absorption band near 230 nm, while FT-IR spectra displayed typical amide bands, including amide I at approximately 1630 cm−1 and amide III at 1240–1300 cm−1, confirming the collagen-derived nature of the products. Molecular simulation further suggested that oxalic acid competed for backbone hydrogen bonding, while chloride ions interacted with hydroxyproline residues, as indicated by a Hyp–Cl− radial distribution peak at 0.328 nm, promoting collagen swelling, triple-helix loosening, and controlled depolymerization. Overall, this DES-based strategy provides a rapid, tunable, and potentially sustainable route for producing collagen peptides from aquatic by-products.

Keywords: Collagen peptides, Deep eutectic solvents, Sturgeon skin, Molecular-weight distribution, Antisolvent precipitation

Graphical abstract

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Highlights

  • •

    A choline–oxalic acid DES extracted collagen peptides from sturgeon skin.

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    Extraction time tuned peptide molecular-weight distribution from 0.5 to 10 kDa.

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    Isopropanol enabled effective recovery of peptides from the DES extract.

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    Spectroscopic and chromatographic analyses confirmed collagen-derived features.

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    Molecular simulation provided insights into controlled DES-assisted depolymerization.

Introduction

Fish processing generates substantial quantities of by-products, such as skin, scales, and bones, which are often discarded or converted into low-value products (Salim et al., 2024). These by-products are rich in bioactive components, particularly collagen, which accounts for 25–35% of total animal protein and represents a valuable raw material for collagen peptide production(Subhan et al., 2015). For religious reasons, Muslims and Jews avoid collagen peptide products derived from pigs, making fish an excellent source of halal collagen peptides (Wang et al., 2023). Moreover, fish collagen peptides exhibit high biosafety, eliminating the risk of diseases transmitted by mammals, such as pigs and cattle, thereby ensuring no threat to human health. Sturgeon skin is particularly suitable for collagen peptide production, as it accounts for approximately 5–7% of body weight and contains about 70% collagen in total skin protein (Atef et al., 2020). Therefore, developing an efficient strategy to convert sturgeon skin into collagen peptides is important for the high-value utilization of aquatic by-products.

Collagen consists of three α-polypeptide chains with repetitive Gly–X–Y sequences that form a characteristic triple-helical structure. Collagen peptides, derived from hydrolyzed collagen or gelatin, are low-molecular-weight bioactive compounds with favorable bioavailability and diverse physiological activities, making them valuable functional ingredients for food, nutraceutical, cosmetic, and biomedical applications (Salim et al. Pozharitskaya et al., 2024). According to molecular weight, collagen peptides can be categorized into oligopeptides and polypeptides; their low molecular weight and relatively linear configurations confer enhanced bioavailability by facilitating efficient intestinal absorption (Lee et al., 2017). Research has demonstrated their efficacy in mitigating obesity, enhancing muscle strength, improving bone density, alleviating joint pain, and promoting skin health (Zdzieblik et al., 2015; Liu et al., 2015; Dobenecker, Böswald, Reese and Hugenberg, 2024; Pozharitskaya et al., 2024; Inoue et al., 2016). In addition, they exhibit antioxidant, antidiabetic, hypotensive, and anti-atherosclerotic activities (O'Keeffe, Norris, Alashi, Aluko and FitzGerald, 2017; Igase et al., 2018). Their growing application potential is reflected by the projected expansion of the global collagen peptides market from USD 699 million in 2023 to USD 922 million by 2028, with a compound annual growth rate of 5.7% (Zhou et al., 2025). In this context, collagen-rich fish skin represents an ideal raw material for producing high-value collagen peptides and offers an effective strategy for the valorization of aquatic by-products.

Collagen peptide production generally involves pretreatment, collagen/gelatin extraction, and subsequent hydrolysis, mainly through chemical or enzymatic methods. Chemical hydrolysis can promote collagen swelling and dissolution, but acid treatment may damage amino acids, corrode equipment, and cause environmental concerns, while alkali treatment often requires long processing times (Fountoulakis & Lahm, 1998). Enzymatic hydrolysis offers higher selectivity and milder conditions, but it is limited by high enzyme cost, long reaction time, and complex purification procedures (Fu et al., 2016; Marcet et al., 2016; Zhang et al., 2013). Furthermore, synergistic or antagonistic interactions among hydrolyzed polypeptides may result in the loss of bioactive peptides during downstream purification (Udenigwe, 2014). Overall, current collagen peptide production still faces challenges related to efficiency, cost, and sustainability, highlighting the need for greener extraction strategies that integrate collagen dissolution, controlled hydrolysis, and simplified separation.

Deep eutectic solvents (DES), emerging as a novel class of eco-friendly solvents, are composed of hydrogen bond acceptors and donors that form homogeneous liquids via extensive intermolecular hydrogen bonding, resulting in melting points far below those of the individual components (Clouthier & Pelletier, 2012; Xu et al., 2023). DES remain liquid over a broad temperature range (0–100 °C), with tunable viscosity even below 273 K, and exhibit excellent stability and biocompatibility (Craveiro et al., 2016). They possess multiple advantages, including facile preparation, high purity, low toxicity, biodegradability, and strong solvating power for both polar and nonpolar compounds. Their ability to dissolve recalcitrant biopolymers and stabilize bioactive compounds through strong hydrogen-bond interactions makes DES particularly attractive as extraction media. The stabilization performance of DES can be further optimized by modulating viscosity and water content, enhancing their suitability for extracting natural bioactive substances. Accordingly, DES have been widely employed as extraction solvents for plant-derived bioactive substances, demonstrating particular efficacy in the recovery of natural compounds.

Although DESs have been explored for extracting natural biomacromolecules, their application in marine collagen peptide production remains limited, and the molecular-weight control and depolymerization mechanism of DES-extracted collagen peptides are still unclear. Therefore, this study aimed to determine whether choline-based organic-acid DESs could efficiently extract collagen peptides from sturgeon skin while regulating their molecular-weight distribution. In this study, we systematically investigated choline-based organic acid DESs for collagen peptide extraction from sturgeon skin. By screening and optimizing DES formulations and extraction parameters, we established an efficient extraction protocol and comprehensively characterized the resulting collagen peptides. This work expands the application of DES in collagen peptide production from marine byproducts and provides an environmentally benign strategy for the high-value utilization of sturgeon skin.

Materials and methods

Materials

Sturgeon skin was provided by Hainan Huayan Fish Skin Processing Co., Ltd. (Haikou, China). Choline chloride (AR, ≥99.0%), lactic acid (AR, ≥99.0%), acetic acid (AR, ≥99.5%), and oxalic acid (AR, ≥99.5%) were obtained from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Coomassie Brilliant Blue, BCA Protein Assay Kit, PBS buffer, sodium hydroxide, and hydroxyproline assay kits were purchased from Sigma-Aldrich Co., Ltd. (Shanghai, China). Electrophoresis-related reagents, including Tris-HCl buffer (pH 6.8), Tris-HCl buffer (pH 8.8), protein loading buffer, and SDS, were acquired from BBI Life Sciences Co., Ltd. (Shanghai, China). Trifluoroacetic acid, serine, sodium tetraborate, o-phthaldialdehyde, and sulfosalicylic acid were procured from Sinopharm Chemical Reagent Co., Ltd. (Chengdu, China). All other chemicals used in this study were of analytical grade.

Composition analysis of sturgeon skin

The moisture, crude protein, crude lipid, and ash contents in sturgeon skin were determined according to AOAC official methods 950.46, 981.10, 960.39, and 920.153, respectively. Hydroxyproline content was determined spectrophotometrically according to GB/T 9695.23–2008, and collagen content was calculated using the corresponding conversion factor.

Synthesis of choline-based organic acid deep eutectic solvents

DES were synthesized using oxalic acid, lactic acid, and acetic acid as hydrogen bond donors (HBDs) and anhydrous choline chloride as the hydrogen bond acceptor (HBA). The HBDs and predried choline chloride were combined at specific molar ratios in Table 1, followed by homogenization under continuous heating at 80 °C in a water bath until a transparent monophasic liquid was formed (Craveiro et al., 2016; Zhang et al., 2021). The viscosity of DESs was measured using Rheometer MCR 301 at room temperature. The detailed compositional parameters of the prepared DES systems are summarized in the accompanying Table 1.

Table 1.

Composition of choline organic acid DES.

DES Hydrogen bond receptor Hydrogen bond donor Molar ratio
Choline chloride -oxalic acid Choline chloride oxalic acid 1:1
Choline chloride - acetic acid acetic acid 1:2
Choline chloride - lactic acid lactic acid 1:1
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Selection and application of choline-organic acid DES systems

A choline-based DES system was systematically selected for collagen extraction according to previously reported DES-assisted extraction methods for collagen peptides and protein-rich biomaterials (Bai et al., 2017; Batista et al., 2022). Precisely 0.3 g of powdered sturgeon skin samples (particle size <100 μm) were weighed using an analytical balance and transferred to airtight glass vials. 30 mL each of choline-oxalic acid DES, choline-acetic acid DES, and choline-lactic acid DES were sequentially introduced into separate reaction vessels. The mixtures were magnetically stirred at 80 °C in a thermostatic water bath for 1 h to facilitate collagen dissolution. After extraction, the slurries were centrifuged at 8000 r/min for 15 min to remove insoluble residues, and the supernatants were collected for yield quantification. The hydroxyproline content in the extracts was determined via UV–Vis spectrophotometry according to the Chinese National Standard, and the extraction efficiency was calculated based on stoichiometric conversion to collagen peptide content (Standard, 2008).

Optimization of the extraction parameters for sturgeon skin collagen peptides

The extraction parameters were optimized using a single-factor experimental design. Temperature, solid-to-liquid ratio, and extraction time were investigated sequentially. While optimizing one factor, the other conditions were kept constant. The optimal conditions were selected based on extraction rate. Data are expressed as means ± standard deviations (n = 3). Different superscript letters represent statistically significant differences (P < 0.05). The extraction rate and collagen peptide purity were calculated as follows. Extraction rate (%) was defined as the ratio of collagen peptides extracted into the DES phase to the total collagen content in the sturgeon skin. Collagen peptide purity (%) was defined as the ratio of collagen peptide content to the total dry weight of the recovered product. Collagen peptide content was calculated from hydroxyproline content using the corresponding collagen conversion factor.

Effect of extraction temperature on collagen peptides yield

The extraction temperature range was selected based on previous studies showing that temperature strongly affects DES viscosity, mass transfer, and collagen dissolution efficiency (Batista et al., 2022; Gaikwad & Kim, 2024). Precisely 0.3 g of pulverized sturgeon skin was weighed using an analytical balance (Mettler Toledo, Switzerland) and transferred to airtight borosilicate glass vials. Preheated choline–oxalic acid DES (24 mL) was added to each vial to give a solid–liquid ratio of 1:80 (w/v), followed by brief vortex mixing to obtain a homogeneous suspension. The capped vials were then placed in a thermostated shaking water bath and incubated at 40, 50, 60, 70, 80, and 90 °C for 2 h with gentle agitation (500 rpm). After incubation, the samples were immediately cooled in an ice–water bath to stop extraction, and any minor loss of water due to evaporation was corrected by adding deionized water to the original volume. The suspensions were centrifuged at 10000 ×g for 20 min at 4 °C (Eppendorf 5810R, Hamburg, Germany), and the supernatants were collected for subsequent determination of extraction rate (E, %) and collagen peptide purity (%) as described below. All temperature treatments were conducted in triplicate.

Effect of solid-liquid ratio on collagen peptides yield

The tested solid-to-liquid ratios were designed according to reported collagen extraction procedures, in which solvent volume is a key factor affecting matrix swelling, solute diffusion, and extraction yield (Gaikwad & Kim, 2024; Jafari et al., 2020). Identical aliquots of powdered sturgeon skin (0.3 g) were accurately weighed into 50 mL screw-cap centrifuge tubes. Preheated choline–oxalic acid DES, prepared as described above, was then added to obtain solid–liquid ratios of 1:40, 1:60, 1:80, 1:100, 1:120, and 1:140 (w/v). The mixtures were vortexed briefly to ensure complete wetting of the skin powder and subsequently extracted in a thermostated shaker at 70 °C for 2 h with continuous agitation (500 rpm). After extraction, the tubes were removed, allowed to cool to room temperature, and centrifuged (10,000 ×g, 20 min). The clear supernatants were collected and appropriately diluted, and collagen peptide concentration was determined using the standardized spectrophotometric assay described above. The extraction rate (E, %) and peptide purity (%) at each solid–liquid ratio were calculated from these measurements. All experiments were performed in triplicate.

Effect of extraction duration on collagen peptides yield

The extraction time range was selected based on previous studies showing that collagen dissolution and peptide formation are time-dependent processes during acid- or DES-assisted extraction (Bai et al., 2017; Jafari et al., 2020). Fixed quantities of powdered sturgeon skin (0.3 g) were accurately weighed into 50 mL screw-cap centrifuge tubes and mixed with 24 mL of preheated choline–oxalic acid DES to obtain a solid–liquid ratio of 1:80 (w/v). The sealed tubes were placed in a thermostated shaking incubator at 70 °C with constant agitation (500 rpm). Sets of tubes were removed after 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 h, immediately cooled in an ice–water bath to quench further extraction, and allowed to reach room temperature. The suspensions were then centrifuged at 10000 ×g for 20 min, and the clear supernatants were collected for determination of collagen peptide concentration using the standardized spectrophotometric assay described above. Extraction rate (E, %) and peptide purity (%) were calculated for each time point to construct the kinetic extraction profile. All duration experiments were conducted in triplicate.

Separation and purification of collagen peptides from sturgeon skin

Organic solvent selection of collagen peptides

The DES-mediated extracts were purified by organic-solvent precipitation according to previously reported antisolvent separation strategies for DES-extracted collagen peptides and food proteins (Bai et al., 2017; Ling & Hadinoto, 2022). Five pre-chilled organic solvents (methanol, ethanol, isopropanol, acetonitrile, and acetone) were systematically evaluated. Aliquots (2 mL) of the crude extract were transferred to glass vials, mixed with 10 mL of each chilled solvent, and incubated at 4 °C for 24 h. The phase separation behavior was qualitatively assessed by visual inspection of the precipitate formation and solution turbidity.

Optimization of the precipitation parameters

Based on preliminary screening, solvents demonstrating effective collagen peptide-DES separation were selected for quantitative analysis. For precipitation rate determination, 2 mL of the sturgeon skin extract was precisely transferred into glass vials, mixed with 10 mL of pre-chilled organic solvent, and incubated at 4 °C. The precipitation rates were measured at specific time intervals (4, 8, 12, 16, 20, and 24 h). Additionally, 2 mL of the extract was mixed with varying ratios of pre-chilled organic solvent (extract-to-solvent ratios of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 and 1:8) and incubated at 4 °C to assess the precipitation efficiency. The supernatants were sampled and transferred to ampoules for hydroxyproline quantification using established hydroxyproline determination methodologies. Precipitation efficiency was calculated from the decrease in hydroxyproline-derived collagen peptide content remaining in the supernatant after antisolvent precipitation. Therefore, this value represents the proportion of collagen-derived material precipitated from the DES extract, rather than the absolute recovery of the final dried peptide product. The precipitation efficiency was calculated based on the measured HYP concentration in the supernatant to identify the optimal precipitation ratio and incubation duration. Subsequently, these parameters were utilized to determine the most effective precipitating reagent through comparative analysis.

Purification of the collagen peptides

After precipitation with isopropanol cooled in an ice bath at an extract-to-solvent ratio of 1:6 at 4 °C for 12 h, the supernatants were decanted, and the peptide-enriched pellets were washed twice with ice-bath-cooled isopropanol. Centrifugation was performed at 5000 ×g for 10 min (4 °C) to ensure complete DES removal. The final precipitates were lyophilized and stored at −80 °C for subsequent characterization.

Nanofiltration purification and residual analysis

After isopropanol precipitation and washing, the peptide-enriched precipitate was redissolved in deionized water and further purified by nanofiltration to remove residual DES components and organic solvent. Nanofiltration was performed using an SF-SA low-pressure membrane performance evaluation system (Hangzhou Saifei Membrane Separation Technology Co., Ltd., Hangzhou, China). The peptide solution was circulated through an organic nanofiltration membrane with a molecular-weight cut-off of 200 Da under an operating pressure of 1 MPa and a circulation flow rate of 40 L/min. During nanofiltration, low-molecular-weight compounds, including oxalic acid, choline chloride, and residual isopropanol, were removed into the permeate, while collagen peptides were retained in the retentate. Filtration was stopped when the retentate volume was concentrated approximately 10-fold. The retentate was then diluted with deionized water and subjected to diafiltration to further remove residual small molecules. The purified peptide solution was finally lyophilized to obtain the collagen peptide product. Residual oxalic acid in the lyophilized product was determined by HPLC with slight modifications according to Yu et al. (2020), and residual isopropanol was determined by GC–MS according to Lim et al. (2024).

Determination of hydrolysis degree of sturgeon skin collagen peptides

The degree of hydrolysis (DH) was quantified using the ortho-phthalaldehyde (OPA) spectrophotometric method. A modified protocol based on Church's methodology for assessing whey protein hydrolysis in dairy systems was implemented (Church et al., 1983). Serine solutions (0–2.0 mM) served as calibration standards, with absorbance measurements conducted at 340 nm using a 96-well microplate reader (BioTek Synergy H1, USA). The DH was calculated as follows:

DH%=Asample−AblankAserine−Ablank×1htotal×100

where htotal represents the total hydrolyzable peptide bonds in sturgeon collagen (determined as 8.6 meq/g via amino acid analysis).

Molecular weight profiling of sturgeon skin collagen peptides via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

The molecular weight distribution of sturgeon skin collagen peptides was analyzed using a discontinuous SDS-PAGE system. The separating gel (12% acrylamide) and stacking gel (5% acrylamide) were polymerized under a nitrogen atmosphere. Samples (20 μg protein/lane) were loaded alongside pre-stained protein markers (10–250 kDa, Thermo Fisher Scientific). Electrophoresis was initiated at a constant voltage of 80 V until the tracking dye (bromophenol blue) migrated to the gel interface, followed by an increase in voltage to 120 V until complete migration to the bottom of the gel. After electrophoresis, the gels were fixed in 40% ethanol/10% acetic acid for 30 min and then stained with Coomassie Brilliant Blue R-250 (0.25% w/v in 40% methanol/10% acetic acid) for 2 h with gentle agitation. Destaining was performed through sequential immersion in a methanol-acetic acid solution (30% methanol/10% acetic acid) until optimal band resolution was achieved. Gel images were captured using a calibrated ChemiDoc system (Bio-Rad Gel Doc XR+) for molecular weight estimation.

Ultraviolet-visible (UV–vis) spectral scanning

The purified product was analyzed using a Shimadzu UV-2550 ultraviolet-visible spectrophotometer (Japan) to preliminarily characterize its composition and structure. The white precipitate obtained via organic solvent precipitation was dissolved in 0.1 M acetic acid. UV–Vis spectral scanning was performed over the wavelength range of 200–400 nm.

Fourier transform infrared (FT-IR) spectroscopy

The lyophilized sample was subjected to FT-IR analysis using a spectrometer operating in the wavenumber range of 4000–500 cm−1. For sample preparation, the lyophilized product was thoroughly homogenized with potassium bromide (KBr) in an agate mortar to obtain a uniform powder. The mixture was then compressed into a transparent pellet using a hydraulic press. A pure KBr pellet was used as the background reference. Spectral data were acquired from 64 cumulative scans, and the transmittance of the pellet was maintained above 90% to ensure measurement accuracy. All the grinding and pellet preparation steps were conducted under infrared illumination to prevent moisture absorption and ensure sample dryness.

Molecular weight profiling of sturgeon skin collagen peptides via high-performance liquid chromatography (HPLC)

The molecular weight distribution of collagen peptides extracted from sturgeon skin was determined using a Shimadzu LC-20 A HPLC system (Kyoto, Japan) following the methodology established by Zhao et al. (2017) (Zhao et al., 2017). Sample preparation involved dissolving the collagen peptides in ultrapure water to a final concentration of 0.5 mg/mL, followed by filtration through a 0.22 μm cellulose acetate syringe filter (Millipore, USA) to remove particulate matter. The chromatographic separation was carried out on a TSK-GEL G2000 SWXL (5 μm, 7.8 × 300 mm) gel filtration column (Tosoh Bioscience, Japan) under isocratic elution conditions. The mobile phase consisted of acetonitrile, ultrapure water, and trifluoroacetic acid (45:55:0.1, v/v/v). A 5 μL sample was injected and eluted at a flow rate of 0.5 mL/min with the column temperature maintained at 30 °C. The UV detection wavelength was measured at 220 nm to monitor peptide bond absorption. System calibration was performed using molecular weight standards (1–20 kDa range) to establish retention time-molecular weight correlations. The molecular-weight ranges of the extracted collagen peptides were estimated from SEC-HPLC retention times based on the calibration curve established using molecular-weight standards.

Statistical analysis

Data processing was performed using SPSS 22.0, and graphs were generated using Origin software. All experiments were conducted in triplicate, and the results are expressed as the mean ± standard deviation. Significant differences between treatments were evaluated using Duncan's multiple range test at a significance level of P < 0.05.

Results and discussion

The biochemical profile of sturgeon skin showed moisture, lipid, and ash contents of 14.12 ± 0.70%, 1.23 ± 0.10%, and 4.62 ± 0.12%, respectively. The total protein content was 78.28%, of which 63.47% was estimated as collagen based on hydroxyproline determination, confirming that collagen was the major protein component of sturgeon skin. These quantitative findings demonstrate the significant potential of sturgeon skin as a premium raw material for collagen peptide extraction, with the exceptionally high collagen proportion serving as a critical biochemical rationale for their industrial utilization.

Selection of optimal deep eutectic solvent system

The three choline chloride-based organic-acid DESs were compared under the same screening condition of 80 °C for 1 h to evaluate their extraction performance and peptide molecular-weight distribution. As shown in Figs. 1a–1c, all three choline chloride-based organic-acid DESs formed clear and homogeneous liquids at room temperature, but their viscosities differed markedly, with the lactic-acid DES exhibiting the highest viscosity, followed by the oxalic-acid DES, and the acetic-acid DES showing the lowest value. This trend agrees with previous reports that choline chloride–lactic acid DESs develop a particularly dense hydrogen-bond network, leading to elevated viscosity compared with other carboxylic-acid-based systems (Pozharitskaya et al., 2024). Viscosity is a key parameter governing mass transfer in DES-based extraction, and overly viscous systems often compromise diffusion and solvent penetration into the matrix (Jablonsky et al., 2018). Among the three DESs, choline–oxalic acid DES showed the highest extraction rate of 58.94%, compared with 36.71% and 15.67% for choline–acetic acid and choline–lactic acid DESs, respectively. (Fig. 1c). Although all the DES systems shared the same hydrogen bond acceptor, differences in the HBDs resulted in variations in acidity, which directly influenced the extraction efficiency. A similar non-monotonic relationship between viscosity and extraction performance has been observed in DES extraction of proteins and phenolic compounds, where optimal yields arise from a balance between solvent strength, acidity, and diffusivity (Wong et al., 2025). The superior performance of the oxalic-acid DES may also be attributed to its stronger acidity and chelating ability, which can more effectively disrupt intermolecular cross-links in collagen, in line with reports that choline chloride–oxalic acid DES efficiently solubilizes keratin and digests fish tissues (Zhang et al., 2021). In parallel, the extracts from the three DES systems were analyzed using high-performance liquid chromatography. For the choline-oxalic acid DES system shown in Fig. 1d, two minor peaks were observed at a retention time of approximately 13.0 min, corresponding to molecular weights below 25 kDa, indicating the presence of low-molecular-weight collagen peptides. Conversely, the lactic acid- and acetic acid-based systems produced peaks in the 25–40 kDa range, reflecting larger macromolecular components. These findings confirm the superior extraction capability of the oxalic acid-based DES for isolating collagen peptides from sturgeon skin. Based on the extraction efficiency, viscosity characteristics, and molecular weight distribution, the choline-oxalic acid DES system was identified as the optimal solvent for extracting collagen peptides from the sturgeon skin.

Fig. 1.

Fig. 1

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Physical state (a), viscosity (b), extraction yield (c), and molecular weight distribution (d) of collagen peptides extracted using choline–oxalic acid, choline–acetic acid, and choline–lactic acid deep eutectic solvents under the screening condition of 80 °C for 1 h. Data are expressed as means ± standard deviations (n = 3). Different superscript letters (a-c) represent statistically significant differences (P < 0.05).

Optimization of choline-oxalic acid DES extraction process

Effect of temperature on extraction rate of sturgeon skin collagen peptides

As shown in Fig. 2a, temperature significantly influenced both extraction rate (E%) and collagen purity in the choline chloride–oxalic acid DES system. Within the range of 40–70 °C, the extraction rate of collagen peptides gradually increased with temperature and reached a maximum of 94.41% at 70 °C, while the collagen peptide purity was 83.67%, indicating that 70 °C provided the best balance between collagen dissolution and controlled depolymerization. This enhancement arises from dual thermal effects: (1) elevated temperature amplifies hydrogen ion activity, intensifying their interaction with imino acid residues to weaken collagen structural integrity; (2) thermal energy reduces the viscosity of the choline-oxalic acid DES, thereby enhancing the solvent fluidity and diffusion efficiency of crushed sturgeon skin within the DES, further increasing the extraction rate (Batista et al., 2022). The purity of the collagen peptides exhibited an initial decline, followed by an increase. At lower temperatures (40–60 °C), the extraction of collagen-derived components appeared to be limited, while partial depolymerization may already have occurred, resulting in a higher proportion of low-molecular-weight peptides. As the temperature increased, the leaching rate surpassed the depolymerization rate, leading to the release of larger collagen fragments and reduced purity. At 90 °C, further disruption of hydrogen bonding within collagen may have promoted the release of smaller peptide fragments, which could account for the observed increase in apparent purity. Based on the extraction rate, purity trends, and molecular weight distribution, the optimal reaction temperature was determined as 70 °C.

Fig. 2.

Fig. 2

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Effects of (a) temperature, (b) liquid-to-solid ratio, and (c) time on the extraction yield of sturgeon skin collagen peptides. Data are expressed as means ± standard deviations (n = 3). Different superscript letters (a-e) represent statistically significant differences (P < 0.05).

Effect of solid-to-liquid ratio on extraction rate of sturgeon skin collagen peptides

As shown in Fig. 2b, increasing the solid-to-liquid ratio from 1:40 to 1:80 improved both the extraction rate and collagen peptide purity, which reached 97.45% and 89.34%, respectively, at 1:80. Further increasing the ratio to 1:100–1:140 did not significantly improve the extraction rate and slightly reduced peptide purity, indicating that 1:80 provided sufficient solvent for collagen dissolution while avoiding unnecessary solvent consumption. Elevated solid-to-liquid ratios enhanced the solvent volume and free hydrogen ion availability, increasing the solute-solvent contact area and diffusion efficiency (Gaikwad & Kim, 2024). This promoted hydrogen ion binding to the imino groups in proline and hydroxyproline, thereby improving the extraction. However, further increases in the ratio (> 1:80) no longer enhanced the diffusion. The purity trends were linked to a limited depolymerization rate dependence on the ratio, with a maximum purity of 1:80. Thus, 1:80 was selected as the optimal ratio for cost-effectiveness.

Effect of time on extraction rate

The extraction time kinetics were evaluated using oxalic acid DES at 70 °C with a 1:80 solid-to-liquid ratio, as shown in Fig. 2c. The extraction rate increased sharply within 2 h, reaching 98.45%, and plateaued thereafter. The purity rose rapidly between 1.5 and 2 h, stabilizing after 2 h, suggesting that collagen dissolution reached near-equilibrium by 2 h and that longer contact primarily removed loosely associated impurities. Within the first 2 h, extraction appeared to dominate over further depolymerization, whereas prolonged treatment beyond 2 h likely promoted additional cleavage of the extracted collagen into smaller peptides. Post-2 h, the collagen was fully leached, and depolymerization dominated, increasing the low-molecular-weight peptide content and purity (Jafari et al., 2020). Taken together, these trends support selecting 70 °C, a solid-to-liquid ratio of 1:80, and 2 h as a practical compromise that affords high extraction rate and high collagen purity while maintaining reasonable solvent consumption and processing time.

Optimization of purification process: Selection and efficacy of precipitants

Collagen polypeptides in sturgeon skin extracts were purified via organic solvent precipitation. As shown in Table 2, methanol, ethanol and isopropanol were fully miscible with the oxalic acid-based DES, forming white precipitates after 24 h of settling, consistent with earlier work showing that short-chain alcohols efficiently precipitate collagen peptides from choline chloride–oxalic acid DES systems (Bai et al., 2017). In contrast, acetonitrile and acetone exhibited immiscibility with the DES. Control experiments with pure DES and these solvents confirmed miscibility, but no precipitation, indicating their specificity for collagen peptide separation. The addition of alcohol-based solvents reduced the solution polarity and disrupted the hydrogen bonds between collagen and the DES, so that the DES dissolves in the organic phase, whereas collagen precipitates. Concurrently, the strong hydration capacity of these alcohols destabilizes the hydration layer on peptide surfaces and promotes aggregation and precipitation, in agreement with mechanistic descriptions of ethanol-precipitated DES-extracted food proteins (Ling & Hadinoto, 2022).

Table 2.

Precipitation efficiency of collagen peptides from the ChCl-OA DES system using different organic solvents.

DES system Organic reagent Extract of sturgeon skin obtained
Choline oxalic acid - DES Methanol Miscible; white precipitate formed upon standing
Ethanol Miscible; white precipitate formed upon standing
Isopropanol Miscible; white precipitate formed upon standing
Acetonitrile Immiscible, no precipitation
Acetone Immiscible, no precipitation
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Determination of the optimal precipitation time

The precipitation rates of methanol, ethanol, and isopropanol in sturgeon skin extracts were compared at different settling times (Fig. 3a). Isopropanol produced the fastest and most extensive precipitation, with the precipitation rate increasing sharply within 12 h and then approaching 93% at 16–24 h, whereas ethanol led to a more gradual increase and methanol gave consistently low precipitation yields throughout the tested period. This kinetic pattern agrees with previous observations for DES-extracted collagen peptides, where the addition of short-chain alcohols causes a rapid rise in peptide recovery during the first few hours followed by a plateau once precipitation equilibrium is reached (Bai et al., 2017). The superior precipitation capability of isopropanol can be attributed to its branched structure, which increases steric hindrance, lowers solvent polarity, and facilitates collagen peptide aggregation. Although the maximum precipitation efficiency was observed at 16–24 h, the additional increase after 12 h was limited, indicating that precipitation had nearly reached equilibrium. Therefore, 12 h was selected as a practical settling time rather than the time giving the absolute maximum precipitation efficiency, balancing precipitation performance, processing time, and operational cost.

Fig. 3.

Fig. 3

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Precipitation rate of sturgeon skin collagen peptides in organic reagent systems with varying (a) precipitation times and (b) solvent ratios. Data are expressed as means ± standard deviations (n = 3). Different superscript letters (a-e) represent statistically significant differences (P < 0.05).

Determination of optimal extract-to-solvent ratio

As shown in Fig. 3b, the precipitation rates improved with increasing solvent ratios in the oxalic acid-based DES system. For all three alcohols, increasing the volume ratio of organic reagent to DES from 1:2 to 1:6 significantly enhanced the precipitation rate (P < 0.05). Isopropanol consistently gave the highest precipitation efficiency, reaching 92% at a ratio of 1:6, whereas ethanol afforded intermediate precipitation efficiency and methanol remained inefficient across the entire range. This value represents the proportion of hydroxyproline-derived collagen material precipitated from the DES extract, rather than the absolute recovery of the final dried peptide product. Therefore, approximately 8% of collagen-derived material may have remained in the supernatant or been lost during separation. Further increasing the organic reagent ratio to 1:7–1:8 did not lead to significant additional precipitation for isopropanol or ethanol, indicating that peptide desolvation and aggregation were essentially complete at 1:6 and that larger solvent volumes mainly increase processing cost without improving yield. This saturation-type response is consistent with general antisolvent-precipitation behavior of proteins, where increasing the alcohol/aqueous phase ratio initially reduces solvent polarity and water activity, promoting hydrophobic association and precipitation, but beyond a critical ratio the driving force for further aggregation becomes negligible (Doan, Tavernier, Okuro and Dewettinck, 2018). Therefore, isopropanol at a 1:6 extract-to-solvent ratio and 12 h settling time was selected for collagen peptide separation, balancing precipitation efficiency, processing time, and operational cost. While a relatively large volume of isopropanol was required for precipitation, isopropanol is generally considered a relatively more acceptable solvent than highly toxic solvents such as methanol or chloroform in industrial solvent selection guides, including the CHEM21 and ACS/GSK solvent selection frameworks (Prat et al., 2016). It is also classified as a Class 3 residual solvent with low toxic potential in the ICH Q3C guideline (ICH, 2021). Due to its low boiling point and high volatility, the isopropanol used in our process can be easily and efficiently recovered and recycled via rotary evaporation in a closed-loop system, thereby minimizing environmental impact and aligning with sustainable engineering practices. Furthermore, to address potential safety concerns associated with oxalic acid-based DES and isopropanol precipitation, the recovered collagen peptides were further purified by nanofiltration. Residual oxalic acid and isopropanol were not detected in the final lyophilized product.

Hydrolysis degree analysis

As shown in Fig. 4a, collagen peptides were obtained after 2 h of reaction between choline-oxalic acid DES and sturgeon skin. The degree of hydrolysis (DH) was measured at 0.5, 1, 1.5, 3, 4, 6, 8, and 10 h. The DH increased sharply during 1–4 h, reflecting intensive cleavage of peptide bonds and a pronounced reduction in collagen molecular weight. After 4 h, the DH curve gradually approached a plateau, and no significant change was observed after 6 h, suggesting that most susceptible sites had been hydrolyzed and the reaction had reached a near-equilibrium state. A similar rapid-then-slow hydrolysis pattern has been reported for acid- or enzyme-assisted collagen degradation, where initial fast attack on accessible helices is followed by a diffusion- and substrate-limited stage with only minor additional peptide scission (Bousopha et al., 2016). Taken together, these results indicate that a reaction time of approximately 4 h is sufficient to achieve an effective and energetically efficient hydrolysis of sturgeon-skin collagen in the DES system.

Fig. 4.

Fig. 4

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Structural and analytical characterization of sturgeon skin collagen peptides: (a) degree of hydrolysis in the choline-oxalic acid DES system; (b) molecular weight distribution of DES-extracted peptides at different extraction times; molecular weight distribution of DES-extracted peptides after 3 h (c) and 4 h (d) determined by gel permeation chromatography; (e) UV − vis spectra; (f) FT-IR analysis identifying secondary structure features. Data are expressed as means ± standard deviations (n = 3). Different superscript letters (a-f) represent statistically significant differences (P < 0.05).

Molecular weight distribution of collagen peptides

As shown in Fig. 4b, the SDS-PAGE analysis at 0.5, 1.0, and 1.5 h reveals the molecular weight profiles of the extracted peptides, demonstrating the rapid fragmentation of sturgeon-skin collagen in the choline-oxalic acid DES. At 0.5 h, a broad smear extending from >70 kDa down to ∼35 kDa is still evident, indicating that native α- and β-chains remain only partially cleaved. By 1 h, the intensity of these high-molecular-weight bands markedly decreases and the staining shifts toward lower-molecular-weight regions, reflecting accumulation of intermediate polypeptides. After 1.5 h, most high-molecular-weight species have disappeared and only a diffuse band below ∼15 kDa is observed, consistent with extensive hydrolysis to low-molecular-weight peptides. However, because most DES-extracted peptides were below 15 kDa, conventional SDS-PAGE showed limited resolution for these small peptides. Therefore, SEC-HPLC was further used to more accurately evaluate the molecular-weight distribution of the extracted collagen peptides. Similar electrophoretic behavior—progressive loss of α-chains and appearance of low-molecular-weight smears with increasing degree of hydrolysis—has been widely reported for fish-skin collagen hydrolysates.

As shown in Figs. 4c–4d, size-exclusion HPLC profiles at 3 and 4 h further corroborate this trend, with progressive shifts of the chromatograms toward longer retention times and narrower peaks, indicating a time-dependent decrease in peptide molecular weight. Both chromatograms are dominated by a single, sharp peak, indicating a relatively narrow molecular-weight distribution of oxalic acid-based DES reaction products at 3 h and 4 h. The major peak shifted from 16.12 min at 3 h to 17.09 min at 4 h, indicating the formation of smaller peptide fragments. Combined with the SDS-PAGE results, the optimized 2-h treatment mainly produced collagen polypeptides in the 1–10 kDa range, whereas extending the reaction to 4 h further enriched oligopeptides predominantly in the 0.5–1 kDa range. These findings indicate that the choline–oxalic acid DES system not only enables efficient collagen extraction but also allows molecular-weight-controlled production of collagen peptides by adjusting the reaction time. These findings also have practical implications for the targeted production of collagen peptide ingredients. The 2 h DES treatment mainly produced collagen polypeptides in the 1–10 kDa range, which may be more suitable for food, nutraceutical, and protein-fortified products requiring moderate peptide size and good processing stability. In contrast, extending the reaction to 4 h enriched smaller oligopeptides, predominantly in the 0.5–1 kDa range, which may be advantageous for applications requiring rapid absorption, high solubility, or improved skin penetration, such as functional beverages and cosmetic formulations (Chen et al., n.d.). Because low-molecular-weight collagen peptides have been reported to show favorable transdermal transport, the 4 h fraction enriched in ˂ 1 kDa peptides may be of interest for cosmetic applications requiring efficient skin penetration (Chai et al., 2010). Therefore, by simply adjusting the extraction time, the choline–oxalic acid DES system provides a practical route to tailor collagen peptide products for different industrial applications.

UV analysis of sturgeon skin collagen peptides

As shown in Fig. 4e, the ethanol-precipitated sturgeon skin collagen peptides display a single intense UV band around 230 nm, closely matching the spectrum of the commercial marine collagen reference (Peptan®). No additional maxima are observed in the 250–280 nm region. An absorption maximum near 220–230 nm is a well-recognized signature of type I collagen and collagen peptides, arising mainly from n → π* transitions of the peptide C Created by potrace 1.16, written by Peter Selinger 2001-2019 O groups associated with abundant glycine, proline, and hydroxyproline residues, whereas aromatic residues that absorb at ∼280 nm are scarce (Dong & Dai, 2022). The coincidence of the 230-nm peak position with reported values for fish collagens and the absence of a 280-nm peak, which would indicate non-collagenous protein impurities, indicate that the DES-extracted, alcohol-precipitated fraction is enriched in collagen-derived peptides and shows spectral characteristics similar to those of the commercial marine collagen reference.

FT-IR analysis of collagen peptides

As shown in Fig. 4f, the FT-IR spectra of DES-extracted sturgeon skin collagen peptides and the commercial marine collagen peptide (Peptan®) exhibit the typical peptide backbone bands of collagen-derived materials. Both samples show an amide B band near 2950 cm−1, assigned to asymmetric CH2 stretching, together with pronounced amide I and amide III bands at ∼1630 cm−1 and 1240–1300 cm−1, respectively, arising from C Created by potrace 1.16, written by Peter Selinger 2001-2019 O stretching and coupled C—N stretching/N–H bending vibrations of the peptide linkage (Muyonga et al., 2004). The amide I maximum around 1630 cm−1, rather than 1650–1660 cm−1 typical of native triple-helical collagen, indicates disruption of the fibrillar helix and formation of gelatin-like collagen peptides while still preserving the characteristic secondary-structure signature of collagen. This interpretation is based on the sensitivity of the amide I band to collagen secondary structure, where a shift toward lower wavenumbers reflects reduced triple-helical order and increased random-coil or gelatin-like conformations. Therefore, the FT-IR spectra suggest that DES treatment loosened the ordered collagen triple helix while retaining characteristic collagen-derived peptide backbone signals. The close correspondence of the amide band positions and profiles between the sturgeon peptide and Peptan® spectra therefore confirms that the DES-extracted product is a collagen-derived polypeptide of comparable structural nature to commercial collagen peptides. Although molecular-weight distribution, UV–Vis, and FT-IR analyses confirmed the collagen-derived nature and preliminary structural features of the DES-extracted peptides, these methods provide limited sequence-level information; therefore, future LC-MS/MS-based peptide sequencing is needed to further identify peptide composition and clarify potential structure–activity relationships.

Mechanism of collagen peptide extraction via acidic choline chloride-based DES

The molecular-weight distribution of collagen peptides can be regulated by DES composition, reaction temperature, and extraction time. Collagen stability is mainly maintained by interchain hydrogen bonding and Hyp-centered hydration, which support the triple-helical conformation(Bella et al., 1995). In ChCl–oxalic acid DES, the MD trajectory shows a progressive and fluctuating increase in RMSD (Fig. 5a), therefore indicating continuous deviation from the initial collagen-like conformation and persistent structural destabilization. Hydrogen-bond analysis further reveals that oxalic acid maintains a stable population of backbone hydrogen bonds (Fig. 5b), consequently demonstrating competitive occupation of backbone acceptor sites that would otherwise participate in interchain coupling. This “hydrogen-bond wedge” effect is consistent with weakening of the native hydrogen-bond network and progressive helix loosening rather than abrupt backbone scission.

Fig. 5.

Fig. 5

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Molecular dynamics simulation of sturgeon skin collagen peptides extraction by choline-oxalic acid DES: (a) Root Mean Square Deviation (RMSD) of the backbone atoms relative to the starting structure; (b) Number of hydrogen bonds formed between Oxalic Acid (OA) and the protein backbone carbonyl groups (Protein-C=O … H-OA) as a function of time; (c) Solvent Accessible Surface Area (SASA) of the peptide; (d) Radial Distribution Functions (RDF) of water around the hydroxyl oxygen of Hyp residues. The peak at 0.188 nm indicates strong hydrogen bonding; (e) RDF of Chloride ions (Cl−) around the hydroxyl oxygen of Hyp residues. The prominent peak at 0.328 nm confirms the formation of a specific Hyp-OH … Cl− interaction geometry; (f) Ramachandran plot of the collagen peptide in the ChCl–OA DES during the final 10 ns of the MD simulation. Each black dot represents the backbone dihedral-angle conformation (φ, ψ) of an individual residue; Conformational changes of the collagen peptide from the initial structure (j) to snapshots in the ChCl–OA DES molecular system at 10–20 ns (h) and 70–90 ns (i), together with the corresponding hydrogen-bonding patterns (partial views) involving surrounding H₂O molecules and Cl− ions.

Importantly, DES does not behave as a homogeneous strongly acidic medium, but instead provides a heterogeneous and dynamically buffered microenvironment at the molecular scale (Spittle et al., 2022). From the MD perspective, oxalic acid molecules are preferentially engaged in hydrogen-bond competition with backbone carbonyls, while choline cations and chloride anions spatially reorganize around exposed peptide segments, forming transient solvation cages. This structured solvation environment may partially shield peptide bonds from nonspecific proton attack, thereby helping to limit excessive hydrolysis and favoring the accumulation of oligopeptides within a defined molecular-weight range.

In parallel, SASA remains elevated with pronounced oscillations (Fig. 5c), thus supporting DES-induced swelling and sustained solvent exposure that enhances accessibility of cleavage-prone regions without triggering catastrophic chain fragmentation. Although Hyp remains tightly hydrated (Fig. 5d), the appearance of a distinct Hyp–Cl− RDF peak at ∼0.328 nm (Fig. 5e) therefore confirms that chloride ions can penetrate the Hyp hydration shell and form Hyp–O–H···Cl− interactions. This electrostatic screening and local steric perturbation undermine water-bridge stabilization, further accelerating helix unwinding while still preserving short peptide segments. The final conformational outcome is captured by the Ramachandran distribution over the last 10 ns (Fig. 5f), which becomes broadened with ψ-axis dispersion away from the narrow PPII-favored region, thus evidencing loss of dihedral confinement and formation of a more disordered, random-coil–like ensemble rather than complete conformational collapse.

These statistical signatures are consistent with time-resolved snapshots, where the peptide transitions from an ordered initial structure (j) to a distorted, expanded conformation at 10–20 ns (h) and a more disrupted rearranged state by 70–90 ns (i), with partial hydrogen-bonding diagrams highlighting extensive solvent-mediated contacts involving H₂O and Cl−. It should be noted that the MD simulation did not directly quantify peptide-bond cleavage or molecular-weight reduction, but provided mechanistic support for the experimentally observed decrease in molecular weight. The increased RMSD and SASA, backbone hydrogen-bond competition, Hyp–Cl− interaction, and broadened Ramachandran distribution suggest that the DES microenvironment loosened the collagen-like structure and increased the exposure of cleavage-prone regions. This structural destabilization could facilitate acidolysis, which is consistent with the SDS-PAGE and SEC-HPLC results showing progressive degradation of high-molecular-weight collagen components into lower-molecular-weight peptides. Collectively, the MD results support a mechanistic interpretation in which oxalic acid contributes to helix weakening through backbone hydrogen-bond competition, while choline chloride helps create a structured solvation environment that modulates local accessibility and depolymerization behavior. Consequently, the triple helix unwinds and swells, exposing susceptible cleavage sites while avoiding excessive depolymerization, thus enabling mild, controllable hydrolysis and efficient extraction of bioactive collagen peptides rather than complete conversion to amino acids under green-processing conditions.

Conclusion

In summary, this study successfully extracted collagen peptides from sturgeon skin, a fishery processing by-product, using a novel choline-oxalic acid-based DES that offers a more rapid and more straightforward alternative to conventional extraction methods. Among the three choline–organic acid DESs evaluated, choline–oxalic acid showed the best extraction performance, reaching an extraction rate above 98% under the optimized conditions. By adjusting the extraction time, the DES system enabled molecular-weight-controlled production of collagen polypeptides (predominantly <10 kDa at 2 h) and collagen oligopeptides (mainly 0.5–1 kDa at 4 h), providing a straightforward means to tailor peptide size distributions for diverse application needs. Isopropanol was identified as the most effective antisolvent for downstream separation, providing high peptide recovery at an extract-to-solvent ratio of 1:6. In addition, molecular simulation provided mechanistic insights into how the acidic DES environment may promote collagen swelling, helix loosening, and controlled depolymerization. Compared with conventional multistep extraction and hydrolysis routes, the proposed process offers a simpler workflow and operates under relatively mild conditions. Future studies should evaluate the bioactivity, digestibility, absorption, and safety of DES-extracted collagen peptides, as well as DES recycling and scale-up feasibility, to support their practical application in food, nutraceutical, and cosmetic products. Overall, choline–oxalic acid DES represents a promising platform for molecular-weight-tunable production of collagen-derived peptides from aquatic by-products, although future studies should further evaluate residual DES components, solvent recovery, DES recycling, product safety, and scale-up feasibility.

CRediT authorship contribution statement

Mingkai Bai: Writing – original draft, Software, Data curation. Ning Wang: Formal analysis, Data curation. Meichao Zhang: Software, Funding acquisition. Zifang Zhao: Validation, Supervision. Jianwei Yu: Project administration. Wenhai She: Visualization. Adewale Olusegun Obadina: Project administration. Yuanhui Zhao: Methodology. Shiyang Li: Methodology, Investigation. Haohao Wu: Writing – review & editing, Visualization, Funding acquisition. Ting Zhao: Supervision, Software, Resources.

Funding

This study was financially supported by Hainan Province's Key Research and Development Project (ZDYF2024XDNY191), Science and Technology Innovation Program of Sanya City (2022KJCX59), Yantai Development Zone Science and Technology Leading Talent Project (2021RC014), Major Science and Technology Project of Haikou City (2023−001), Major Scientific and Technological Innovation Project in Shandong Province (2022CXGC020414), and National Natural Science Foundation of China (32272240).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Contributor Information

Shiyang Li, Email: shiyangli1995@ouc.edu.cn.

Haohao Wu, Email: wuhaohao@ouc.edu.cn.

Ting Zhao, Email: zhaoting@qdu.edu.cn.

Data availability

Data will be made available on request.

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Further reading

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