Effect of a sustained-release sheet loaded with basil essential oil on the preservation of large yellow croaker (Larimichthys crocea)

Effect of a sustained-release sheet loaded with basil essential oil on the preservation of large yellow croaker (Larimichthys crocea) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of a sustained-release sheet loaded with basil essential oil on the preservation of large yellow croaker (Larimichthys crocea) Yun-Fang Qian, Cheng-Jian Shi, Run-Jian Gao, Lu Sun, Sheng-Ping Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5858356/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract To enhance basil essential oil (BEO) stability, a chitosan/sodium alginate-based sustained-release sheet loaded with BEO inclusion complexes (IC) was developed and integrated with a commercial absorbent pad (BC-F/AP). The effects of BC-F/AP on quality maintenance and myofibrillar protein (MP) stability in large yellow croaker ( Larimichthys crocea ) were investigated in comparison with the control (CK), commercial pad (AP), pure BEO, and BEO-IC. The findings showed that the BC-F/AP group suppressed the proliferation of psychrophilic bacteria in fish fillets and minimized cooking loss. On day 10, bacterial counts were approximately 1 log CFU/g lower, and cooking loss was 3.08% less compared to CK. According to experimental results, BC-F/AP group effectively maintained the structure and reduced the degradation and the oxidative aggregation degree of MPs of croaker during storage. The sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results indicated that the BC-F/AP group demonstrated the capability to protect the integrity of MPs. The in vitro incubation experiment involving exudate and MP S revealed that the exudates accelerated the degradation, oxidative aggregation and surface hydrophobicity of MPs. The BC-F/AP group maintained the quality of the large yellow croaker and the integrity of MPs by absorbing exudate and continuously releasing antibacterial and antioxidant active substances. exudate degradation oxidation intrinsic fluorescence intensity surface hydrophobicity sulfhydryl Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Nowadays, food preservation remains a major issue for the global food industry, particularly fish such as large yellow croaker ( Larimichthys crocea ) [ 1 ]. It has important economic value due to its high nutritional value. However, it is prone to spoilage caused by spoilage bacteria, leading to a rapid decline in its quality, which is reflected in the deterioration of texture, odor, and color, during storage and sale [ 2 ]. Among these changes, myofibrillar proteins (MPs) is the most important, which is the major muscle protein, closely related to texture properties, water-holding capacity, and softening [ 3 ]. During MPs degradation, water within the muscle cell matrix transitions from the bound state to free water, resulting in the leakage of exudate [ 4 ]. Recent research indicates that the exudates contain a large number of microorganisms and tissue enzymes that not only change the appearance of fish muscle but also accelerate the textural degradation [ 5 , 6 ]. The application of antimicrobial and antioxidant active substances can be a good choice to delay spoilage. Basil essential oil (BEO), derived from the lamiaceae herb ( Ocimum basilicum ), is classified as Generally Recognized As Safe (GRAS) [ 7 ]. It exhibits antimicrobial and antioxidant properties due to its high content of eugenol, linalool and methyl chavicol [ 8 – 10 ]. However, its strong odor, unstable and hydrophobic properties limit its application in food preservation, not to mention its limited effect on exudates [ 11 ]. As reported, oregano essential oil inhibited the growth of L. monocytogenes in vitro study, but showed limited effectiveness in pork and chicken due to food component interactions [ 12 ]. Smaoui, et al. [ 13 ] incorporated peppermint essential oil into a ground beef mixture but observed a substantial impact on the beef's flavor profile. To overcome the shortcomings of pure EO, embedding in biodegradable polymers is a promising strategy. Currently, polysaccharides and proteins such as chitosan (CS) and sodium alginate (SA) are of interest to researchers for their film-forming and biocompatibility.Sreekanth, et al. [ 14 ] incorporated cinnamon essential oil into chitosan/starch films to reduce microbial counts in beef. Similarly, Cao, et al. [ 15 ] prepared sodium alginate films loaded with oregano essential oil nanoparticles to prolong pork shelf life. CS has excellent film-forming ability and antibacterial properties, but its poor mechanical strength and water barrier performance limit the usage [ 16 , 17 ]. SA exhibits high transparency and flexibility but lacks bioactivity [ 18 ]. Commonly, incorporating encapsulated low-molecular organic active components can improve the mechanical and bioactive characteristics of film [ 19 ]. A novel approach which delays muscle degradation and spoilage in large yellow croaker by absorbing exudates and simultaneously releasing antimicrobial active substances based on the hydrophilic nature is theoretically feasible. However, research in this area is limited, and further studies are required to validate its effects. In this paper, a sustained-release absorbent pad (BC-F/AP) modified with a chitosan/sodium alginate sheet loaded with BEO β-cyclodextrin (β-CD) inclusion complex was prepared. The inhibitory effectiveness of this pad on the bacteria, cooking loss and changes in MPs was evaluated through a series of tests. The degradation degree as well as the oxidation degree of MPs were also analyzed after incubation of exudates. This study aims to provide a sustainable and effective preservation method for aquatic foods. Materials and methods Materials Large yellow croakers were purchased from the local aquatic product market (Shanghai Luchaogang Aquatic Product Market). BEO (purity 99%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. The absorbent pads were purchased from Shandong Debairun Packaging Products Co., Ltd. β-CD (purity 98%) was provided by Shanghai Macklin Biochemical Technology Co., Ltd. CS (Mw ~100000Da, degree of deacetylation: ≥90%), SA (purity: ≥90%, M: G = 1:2) were purchased from Shanghai Sangon Biological Engineering Co., Ltd. Preparation of the inclusion complex The basil essential oil inclusion complex (BEO-IC) was prepared by coprecipitation method [20]. Firstly, β-CD powder (8.6 g) was dissolved in ethanol solution (33%, 100 mL) and stirred at 55 °C for 2.5 h. Subsequently, BEO (1.4 g) was dissolved in ethanol solution (10%, 10 mL) and then slowly added to the β-CD solution. It was stirred at 55 °C for 1.5 h and kept overnight at 4 °C. The inclusion complex was then filtered, freeze-dried for 48 h, and obtained as a powder directly. Preparation of the sustained-release sheet The sustained-release sheet (BC-F) was fabricated using a casting method: Chitosan (CS, 0.9 g) and sodium alginate (SA, 0.1 g) were separately dissolved in 45 mL 2% acetic acid and 5 mL distilled water, respectively. The two solutions were combined and stirred continuously for 30 min, followed by the addition of 0.25 g glycerol as a plasticizer with further stirring for 30 min. Based on pre-optimized parameters, 100 mg BEO-IC was then incorporated into the mixture under 20-min stirring. The homogeneous solution was finally cast onto 160 × 70 mm acrylic plates and dried at 40 °C for 12 h. Preparation of the fish fillets The large yellow croaker was cut into fillets (about 100 g for each piece) and washed with distilled water, then divided into five groups (CK, AP, BEO/AP, BEO-IC/AP, and BC-F/AP) for preservation. The five different groups were treated according to the following procedure. (1) CK: The fish samples were placed in a tray and sealed with food cling wrap. (2) AP: The fish samples were placed in a tray with commercial absorbent pads (160 mm × 70 mm, about 1.80 g, water absorption capacity: about 700%) and sealed with food cling wrap. (3) BEO/AP: BEO (30 mg, equal to the BEO content of 0.5 g BEO-IC) was smeared on the fish samples and then placed in a tray with absorbent pads and sealed with food cling wrap. (4) BEO-IC/AP: BEO-IC (0.5 g) was evenly spread on the tray with absorbent pads, and then the fish samples were placed on it and sealed with food cling wrap. (5) BC-F/AP: The BC-F sheet adhered to the tray with absorbent pads, and the fish samples were placed on it and sealed with food cling wrap. All samples were stored at 4 °C and sampled on days 0, 2, 4, 6, 8, and 10. The experimental design of this study was shown in Fig. 1. Evaluation of fish quality and protein characteristics Psychrophilic bacteria count (PBC) The PBC of the fish samples were tested following the method described by Liu, et al. [21]. Fish samples (5 g) were placed in a homogenization bag containing 45 mL of sterile saline solution (0.85%, m/v). The supernatant (1 mL) was gradient diluted by 9 mL sterilized saline solution. The diluted solution (100 μL) was evenly spread on PCA and incubated at 4 °C for 144 h. Cooking loss The cooking loss of the fish samples was tested according to the method of Radhakrishnan, et al. [22], and was calculated by the following formula (1). Where W 1 and W 2 represent the weight of the fillets before and after cooking, respectively. Extraction of MPs MPs were extracted by the previous method [23]. Fish tissue samples (2 g) were homogenized in 20 mL of Solution A (20 mmol/L Tris-maleate, 0.05 mol/L KCl, pH 7.0) using a homogenizer, followed by centrifugation at 10,000 × g for 15 min at 4 °C. The supernatant was discarded to retain the pelleted fraction. Subsequently, the pellet was resuspended in 20 mL of Solution B (20 mmol/L Tris-maleate, 0.6 mol/L KCl, pH 7.0), re-homogenized, and incubated at 4 °C for 1 h. The MPs was obtained by centrifugation again under the same conditions. Trichloroacetic acid (TCA)-soluble peptides The TCA soluble peptides were tested according to the method of Saengsuk, et al. [24]. The fish samples were mixed with 5% TCA (18 mL) solution, homogenized, and incubated at 4 °C for 1 h before being centrifuged at 10000 g for 15 min at 4 °C. Then, the Lowry method was used to measure the soluble peptide content in the supernatant. Myofibril fragmentation index (MFI) The MFI was tested according to the method of Marino, et al. [25]. The fish sample (1 g) was mixed with the buffer (30 mL, 0.1 M KCl, 7 mM NaH 2 PO 4 , 18 mM Na 2 HPO 4 , and 1 mM EDTA, pH 7.0), followed by centrifugation at 10000 × g for 10 min at 4 °C. The supernatant was discarded, and the pellet was washed at least twice with the buffer. The final protein solution (0.5 mg/mL) was analyzed by measuring absorbance at 540 nm using a spectrophotometer, and the MFI was calculated as absorbance × 200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) The SDS-PAGE profiles of MPs were analyzed referring to the previous method [23]. The MPs solution (2 mg/mL) was mixed with the 2×loading buff in a volume ratio of 1:1 and boiled respectively. A 5% stacking gel and a 10% separating gel were used. The samples (8 μL) and marker (11-245 kDa) were loaded onto the gel for electrophoresis until the samples reached the bottom of the gel. The gel was stained with a staining solution (0.25 g/L Coomassie Brilliant Blue R-250, 25% ethanol, and 8% acetic acid) for 1 h. Then, the gel was decolorized with a decolorizing solution (25% ethanol, 8% acetic acid) until the protein bands were visible. Surface hydrophobicity The surface hydrophobicity of MPs was tested according to the method of Zeng, et al. [26] with slight modification. MPs (1 mg/mL) and bromophenol blue (1 mg/mL, 40 μL) were mixed, incubated, centrifuged (10000 g , 4 °C, 5 min), and the supernatant was measured at 595 nm. Total sulfhydryl content Total sulfhydryl content (μmol/g) in MPs solution was quantified spectrophotometrically at 412 nm using a commercial assay kit (BC1370, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) based on the 5,5'-dithiobis-(2-nitrobenzoic acid)-coupled chromogenic reaction. Particle size distribution and protein turbidity The MPs solution (1 mg/mL) was placed in Zetasizer Nano-ZS respectively (Malvern Instruments, Worcestershire, UK) to detect the particle size distribution. The absorbance of the MPs solution (2 mg/mL) were measured at 360 nm to detect the protein turbidity. Intrinsic fluorescence intensity (IFI) The IFI was tested according to the method of Xu, et al. [27]. The MPs solution (0.1 mg/mL) was scanned by an F-7100 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) to measure the tertiary structure of proteins. Evaluation of the characteristics of exudate and protein characterization of the incubation of exudate with MPs. Extraction of exudate The exudate was extracted by mincing and centrifuging the large yellow croaker meat on days 0, 6, and 10 (0dE, 6dE, and 10dE) respectively, and then it was stored at -80 °C. Evaluation of total protease activity in exudate and protein content of exudate The total protease activity of exudate was assayed following Sriket, et al. [28] with adaptations: Exudate samples (0dE, 6dE, 10dE) were blended with 3 mL pH 5.0 buffer (0.2 M Na 2 HPO 4 /0.1 M citrate) and 1 mL hemoglobin substrate (10 mg/mL), incubated at 50 °C for 15 min. Reactions were terminated with 1 mL 50% TCA, centrifuged (10000 g , 4 °C, 5 min), and soluble peptides in supernatants quantified via Lowry method. Parallel exudate protein quantification employed Coomassie Brilliant Blue assay. Evaluation of protein characteristics of exudate incubated with MPs The exudates (0dE, 6dE, 10dE) from fish were mixed with day-0-extracted MPs (0dCK) at 1:1 (v/v) to generate 0dE+P, 6dE+P, and 10dE+P complexes by incubating at 4 °C for 12 h. The protein content, surface hydrophobicity, IFI, particle size, and SDS-PAGE profiles of the resultant protein solutions were detected using established protocols. Statistical analysis All experiments were conducted in triplicate. Data are expressed as mean ± SD (IBM SPSS Statistics 27), with graphs plotted in Origin 2022. Statistical significance was determined by one-way ANOVA followed by Duncan's multiple comparison test. Significant differences ( P < 0.05) between groups are indicated by distinct superscript letters. Results and discussion Fish quality and protein characteristics Psychrophilic bacteria count (PBC) The initial PBC of the fish sample was 2.80±0.10 log CFU/g (Fig. 2a). With the extension of the storage period, the PBC of all groups showed a significant increase ( P < 0.05). On day 10, the PBC values reached 10.47±0.10, 10.44±0.02, 10.42±0.02, 10.34±0.010, 9.44±0.02 log CFU/g in the CK, AP, BEO/AP, BEO-IC/AP, and BC-F/AP groups, respectively. It indicated that the use of commercial absorbent paper could delay the growth of psychrophilic bacteria, possibly due to its absorption of the exudate from the fish fillets [8]. The BEO/AP treatment groups demonstrated significant bacteriostatic efficacy during the initial 4-day incubation period, as evidenced by suppressed microbial proliferation. The main components of BEO, typically eugenol, linalool, or methyl chavicol, were responsible for its antimicrobial effects [8-10]. However, bacterial counts increased quickly after then, indicating a limited duration of effectiveness for pure BEO. This phenomenon can be primarily attributed to the inherent hydrophobicity and high volatility characteristic of BEO constituents. Furthermore, the complex food matrix containing lipids, proteins, and other organic constituents could potentially compromise the antibacterial efficacy of essential oils [29, 30]. The BEO-IC improved the hydrophobicity and volatility of EO, enabling sustained release [31]. Additionally, it prevented direct contact between EO and fish tissue, thereby reducing EO loss and delaying protein degradation thus suppressing bacterial growth. The BC-F/AP group showed the slowest bacterial growth rate (highest inhibition rate reached 90.66%), aligning with the findings of Ying, et al. [32]. This might result from the larger contact area between the sheet and fish samples in the BC-F/AP group than in the BEO-IC/AP group. Thus, the antibacterial substance in essential oil may be more effectively applied to fish samples, achieving the continuous preservation effect. Meanwhile, CS might also play a role in antibacterial activity [33]. Cooking loss Cooking loss served as a key metric for evaluating fish water-holding capacity (WHC) [34]. The initial cooking loss of the fish sample was 18.04%±0.11%, with the CK group experiencing the highest loss, reaching 35.58%±1.54% during storage (Fig. 2b). This might be attributed to the degradation and oxidation of muscle proteins, the former expanding the inter-bundle myofibril space and the latter forcing water out of the myofilaments [35]. The AP group reduced the cooking loss by absorbing exudates, and the BEO/AP group had lower cooking losses than the AP group in the first four days, while the cooking loss of the BEO-IC/AP group was lower than that of the AP group as a whole. This is consistent with previous research findings [31]. The BC-F/AP group exhibited the lowest cooking loss among all groups, attributed to its synergistic capacity for sustained suppression through exudate absorption and bioactive release, which was going to be proved by the following study. TCA-soluble peptides The initial TCA-soluble peptide content was 0.88±0.02 μmol tyrosine/g, and the CK group showed the most rapid increase, reaching 7.94±0.20 μmol tyrosine/g during storage (Fig. 3a). The increase of TCA-soluble peptide content might be due to protein degradation induced by endogenous and exogenous enzymes (the main factor) [36]. The AP group had lower TCA-soluble peptide content than the CK, and the BEO/AP group had lower content than the AP group in the first four days. The BEO-IC/AP and BC-F/AP groups inhibited the increase of TCA-soluble peptide content during storage, with the BC-F/AP group being the most effective. BEO delayed protein degradation by suppressing endogenous enzyme activity and blocking exogenous enzyme production through its antibacterial and antioxidant properties in the early stage [37]. However, its efficacy gradually diminished over time due to volatilization or binding with fish muscle. In contrast, BEO-IC employed microencapsulation technology to achieve a sustained-release effect, thereby maintaining prolonged protective effects [38]. Furthermore, the BC- F/AP group exhibited the lowest TCA-soluble peptide content, which was attributed to the optimized dispersion homogeneity of BEO-IC and the synergistic antibacterial action of CS [39, 40]. Myofibril fragmentation index (MFI) MFI was often used to characterize the integrity and degradation degree of protein structure [41]. The initial MFI was 45.03±1.77 (Fig. 3b). The MFI values of the CK, AP, BEO/AP, BEO-IC/AP, and BC-F/AP groups reached 231.30±1.92, 222.02±4.58, 239.94±18.38, 197.03±1.43, and 184.48±2.18 respectively during storage. The increase in MFI might be attributed to the action of the endogenous enzymes and microorganisms in fish [42]. This indicated that the BC-F/AP group effectively inhibited protein degradation and maintained the stability of protein structure, in agreement with the results of TCA-soluble peptides. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Changes in SDS-PAGE protein bands are used to reflect protein degradation during storage. The molecular weights of proteins were mainly concentrated in the range of 15-200 kDa, including myosin heavy chain (MHC, ∼200 kDa), actin (∼48 kDa), troponin (∼35 kDa), and tropomyosin (∼32 kDa) [43]. These findings align with parallel trends in protein carbonyl content (PBC), TCA-soluble peptide levels, and myofibrillar fragmentation index (MFI) data. Conversely, the BEO-IC (essential oil-in-cyclodextrin) and BC-F/AP (β-cyclodextrin-fortified active packaging) groups demonstrated preserved MHC structural integrity, owing to the controlled release properties of encapsulated essential oils that ensured sustained antimicrobial efficacy within the muscle matrix. On day 10, the MHC (∼200 kDa) band exhibited substantial degradation with near-complete band disappearance in both the CK and BEO/AP groups, while partial degradation was observed in the AP group (Fig. 3c). This phenomenon was likely due to the volatilization of active components of BEO or binding to fish lipids/proteins. The diminished antimicrobial activity likely permitted microbial proliferation and subsequent secretion of exogenous proteases, which accelerated MHC fragmentation [44]. These findings align with parallel trends in PBC, TCA-soluble peptide levels, and MFI data. Conversely, the BEO-IC and BC-F/AP groups demonstrated preserved MHC structural integrity, owing to the controlled release properties of encapsulated essential oils that ensured sustained antimicrobial efficacy within the muscle matrix. In the CK and BEO/AP groups, the band around 100 kDa disappeared, while it did not change obviously in the other groups. The band around 75 kDa became lighter in color in the BC-F/AP group during storage, but it almost disappeared in the other groups. In the CK and BEO/AP groups, the actin (∼48 kDa) band was degraded and shifted downward, whereas it showed little change in the BEO-IC/AP and BC-F/AP groups. The tropomyosin band (∼32 kDa) was fully degraded in CK, AP, and BEO/AP groups, but persisted in BEO-IC/AP with reduced intensity. In general, the absorbent pads exhibited the following MPs inhibition efficacy ranking: BC-F/AP > BEO-IC/AP > AP > BEO/AP ∼ CK. Surface hydrophobicity The exposure degree of hydrophobic groups in proteins and the alterations in protein structure could be indicated by surface hydrophobicity [45]. The surface hydrophobicity of fresh samples was 2.03±0.54 g BPB/mg protein, and the CK group reached 24.28±0.36 μg BPB/mg protein during storage (Fig. 4a). This might be attributed to the presence of oxidative substances (substantial reactive oxygen species, secondary lipid oxidation products) in the exudate, which facilitated the unfolding of MPs, exposing hydrophobic amino acids and altering their spatial structure [35]. The inhibitory effect of the AP group and the BEO/AP group on the increase in protein surface hydrophobicity was not as significant as BC-F/AP and BEO-IC/AP groups ( P > 0.05). It also confirmed that pure BEO did not display antioxidant activity in food products effectively due to its hydrophobic and volatile characteristics [46]. The BC-F/AP group exhibited the lowest surface hydrophobicity ( P < 0.05), likely due to the more uniform distribution of BEO-IC facilitated by the CS/SA sheet [47]. Total sulfhydryl content Thiol groups are important functional groups of MPs, which can be easily oxidized to form disulfide bonds, causing protein cross-linking and aggregation [48]. The total sulfhydryl content of fresh samples was 134.28±5.56 nmol/mg (Fig. 4b). During storage, the total sulfhydryl content of all groups decreased significantly ( P < 0.05). On the 10th day, the total sulfhydryl contents of the CK, AP, BEO/AP, BEO-IC/AP, and BC-F/AP groups were 29.63±2.94, 33.05±4.78, 32.74±1.05, 43.71±0.12, and 60.41±3.83 nmol/mg, respectively. The BEO-IC/AP and BC-F/AP groups showed significantly higher total sulfhydryl content than other groups ( P < 0.05), peaking in BC-F/AP. This demonstrated that CS/SA sheets incorporating BEO-IC suppressed protein oxidation most effectively, consistent with surface hydrophobicity trends. Turbidity and protein particle size distribution The turbidity of the fresh sample was 0.16 (Fig. 4c). During storage, the turbidity of MPs increased, indicating an increase in protein aggregation. Notably, the protein turbidity in the BEO-IC/AP and BC-F/AP groups was significantly lower than in the other groups ( P < 0.05). More detailed results were confirmed according to the results of protein particle size. The particle size of fresh samples was mainly concentrated around 768 nm. The particle size of all groups was increased during storage (Fig. 5a-b). The phenomenon was due to oxidation exposing hydrophobic groups of the MPs, enhancing protein aggregation through hydrophobic interactions, and sulfhydryl oxidation forming disulfide bonds [49, 50]. BEO-IC/AP and BC-F/AP exhibited reduced protein particle sizes compared to other groups, indicating suppressed oxidation. BC-F/AP demonstrated optimal efficacy, correlating with surface hydrophobicity and total sulfhydryl content trends. Intrinsic fluorescence intensity (IFI) The IFI correlates with tryptophan residue abundance and serves as an indirect indicator of tertiary structural modifications in proteins [51]. Fresh samples showed the highest IFI near 335 nm (Fig. 5c-d). As oxidation induced hydrophobic interactions in the MPs, exposing the tryptophan residues to hydrophilic solvents, thereby reducing the IFI of tryptophan [52]. The IFI of all groups reduced during storage. However, the IFI of the BEO-IC/AP and the BC-F/AP were always more stable than that of other groups, with BC-F/AP achieving peak values. These results confirm their superior efficacy in preserving protein tertiary structure, aligning with the findings on surface hydrophobicity. Analysis of exudate characteristics and in vitro incubation of exudate with MPs Total protease activity and protein content of exudates To demonstrate the effect of exudate on the quality of yellow croaker, the total protease activity and protein content of exudates from croaker fillets on day 0 (0dE), day 6 (6dE), and day 10 (10dE) determined (Fig. 6a). The protein contents and total protease activity of 6dE and 10dE were significantly higher than 0dE ( P 0.05). It is hypothesized that the degradation of fish meat tissue structure leads to the release of intracellular proteases into the exudate [53]. Changes of MPs after incubation with exudate Fig. 6b showed the protein content of MPs before and after incubation with exudates from different days (0dE+P, 6dE+P, and 10dE+P). The 0dE+P group exhibited a higher protein content compared to the initial MPs, equaling the average protein content of MPs and exudate (Fig. 6a). The protein contents in the 6dE+P and 10dE+P were significantly lower than 0dE+P ( P < 0.05) aligning with the results of total protease activity (Fig. 6a). Therefore, the degradation of MPs was highly related with the protease activity of exudates. The SDS-PAGE profile revealed distinct alterations in exudate-MP interactions post-incubation (Fig. 6c). The protein compositions and distributions of exudates on different days were similar (0dE, 6dE, and10dE). A wide range of thick bands appear between 35-48 kDa, and thin and light bands appear near MHC (∼200 kDa), indicating that the exudate was composed of a large number of sarcoplasmic proteins and a small number of MPs [5]. The electrophoretic bands of the mixture samples showed the typical bands of both MPs and exudates with different intensities. After incubation with the exudates, the bands of MPs near the MHC (∼200 kDa), 100 kDa, and 20 kDa became lighter, indicating that the MPs had been degraded. This finding was closely related to the protease activity and microbial content in the exudate, aligning with the research of Zhang, et al. [5]. As shown in Fig. 6d-f, incubation of MPs with the exudate induced three progressive changes: increased protein surface hydrophobicity, attenuated IFI intensity, and enlarged particle sizes. These observations demonstrated oxidation-mediated protein aggregation. These results were consistent with the report by Liu, et al. [35]. Correlation analysis and fish appearance As shown in Fig. 7a, PBC was significantly and positively correlated ( P < 0.05) with cooking loss, TCA-soluble peptide content, MFI, surface hydrophobicity, and turbidity, while it was significantly and negatively correlated with total sulfhydryl content, demonstrating microbial-driven protein degradation/oxidation. Fig. 7b revealed that exudate proteolytic activity positively correlated with exudate protein content and post-incubation myofibrillar protein hydrophobicity ( P < 0.05), yet inversely correlated with post-incubation protein content ( P < 0.05), confirming exudate protease's role in accelerating protein deterioration. Therefore, it is hypothesized that the bacteriostatic pads preserved fish quality by suppressing microbial growth through sustained antimicrobial/antioxidant release and exudate absorption, effectively stabilizing protein structures. Proposed mechanism The experimental findings elucidate how absorbent pads modulate myofibrillar protein (MP) integrity in large yellow croaker through exudate management (Fig. 8a). The untreated samples (CK) accumulated exudates during storage, accelerating myosin heavy chain (MHC) degradation and triggering oxidative protein aggregation via hydrophobicity elevation. The conventional pads (AP) partially absorbed exudates to slow these processes (Fig. 8b), and BEO-IC/AP demonstrated dual functionality – sustained antimicrobial/antioxidant release coupled with exudate adsorption synergistically suppressed MP deterioration (Fig. 8c). The BC-F/AP system achieved optimal performance through enhanced BEO-IC dispersion and the inherent antimicrobial activity of chitosan, collectively minimizing structural degradation and oxidative damage to maintain the integrity of MPs (Fig. 8d). Conclusion In this study, the preservation effects of different absorbent pads (CK, AP, BEO/AP, BEO-IC/AP, and BC-F/AP) on the quality and MPs of large yellow croaker were analyzed. The results indicated that both the BEO-IC/AP and BC-F/AP groups had remarkable and sustainable ability to inhibit bacterial growth and maintain protein integrity, with the BC-F/AP group being the best. The in-vitro experiment indicated that the exudate might be responsible for the degradation, tertiary structure changes and oxidative aggregation of MPs. Therefore, a sustained-release sheet made by CS/SA and BEO combined with water-absorbing pads can be a promising method to maintain the muscle quality of large yellow croaker, not to mention its feasibility of application during packaging processing. However, this study did not detect the of volatile active compounds in BEO and the chemical composition of the exudate, which can be researched further. Future investigations can also prioritize systematic evaluation of sustained-release absorbent pad technologies across varying food matrices to expand its applications. Declarations Author Contributions Y.-F.Q.: Conceptualization, writing - review & editing, funding acquisition, formal analysis, supervision; C.-J.S.: writing - original draft, investigation, validation; R.-J.G.: investigation, data curation; S.L.: investigation; S.-P.Y.: writing - review & editing, project administration, funding acquisition. Acknowledgment We would like to express our sincere gratitude to the College of Food Science and Technology, Shanghai Ocean University for their help in making my experiment successful. Funding This work was financially supported by the National Natural Science Foundation of China (No: 31501551), China Scholarship Council (No: 202008310018), and Special Fund for the Development of Science and Technology of Shanghai Ocean University (No: A2-2006-20-200203). Data availability The data will be available on request. Conflict of interest None. References J. Mao, J. Fu, X. Qi, Y. Chen and B. Zhang, J. Sci. 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Lan and J. Xie, Food Chem. 460 , 140442 (2024).https://doi.org/10.1016/j.foodchem.2024.140442 C. Zhou, D. Pan, Y. Bai, et al., LWT-Food Sci. Technol. 101 , 76-82 (2019).https://doi.org/10.1016/j.lwt.2018.11.026 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 19 May, 2025 Reviews received at journal 17 May, 2025 Reviews received at journal 12 May, 2025 Reviewers agreed at journal 24 Apr, 2025 Reviews received at journal 24 Apr, 2025 Reviewers agreed at journal 23 Apr, 2025 Reviewers agreed at journal 23 Apr, 2025 Reviewers invited by journal 21 Apr, 2025 Submission checks completed at journal 21 Apr, 2025 First submitted to journal 16 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-5858356","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":447565678,"identity":"6f6b6ebf-701e-47f4-91b0-3afd0a9dc5c2","order_by":0,"name":"Yun-Fang Qian","email":"","orcid":"","institution":"Shanghai Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Yun-Fang","middleName":"","lastName":"Qian","suffix":""},{"id":447565679,"identity":"43aba9ce-5ad1-4b88-a51e-5dd28959883f","order_by":1,"name":"Cheng-Jian Shi","email":"","orcid":"","institution":"Shanghai Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Cheng-Jian","middleName":"","lastName":"Shi","suffix":""},{"id":447565680,"identity":"9d5e2bb4-40d9-469c-9eb1-15f9b48bd4e7","order_by":2,"name":"Run-Jian Gao","email":"","orcid":"","institution":"Shanghai Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Run-Jian","middleName":"","lastName":"Gao","suffix":""},{"id":447565681,"identity":"abe882ac-8edb-4b72-89fb-c4cc9d4adcb1","order_by":3,"name":"Lu Sun","email":"","orcid":"","institution":"Shanghai Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Sun","suffix":""},{"id":447565682,"identity":"5801856e-5e19-41cd-8f49-517387cc95c4","order_by":4,"name":"Sheng-Ping Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYPACGwYDMM1GvJY00rUcJkGL/Owew88Fv87bm7P3GDB8KDvMwD+7Ab8WgztnjKVn9t1O3NlzxoBxxrnDDBJ3DhDQIpFjIM3bczvB4EaOATNvG9CFEgkEHDYjx/g3b885e7CWv8RoYbiRYybN8+MA4waQFkZitBjcOVZmzduQnLjhzLGCgz3n0nkkbhBy2Ozmzbd5/tjZGxxv3vjgR5m1HP8MQg6T4DBgYGyDsA8AMQ8B9SAt7A8YGP4QVjcKRsEoGAUjGAAAEcVEgUCABYAAAAAASUVORK5CYII=","orcid":"","institution":"Shanghai Ocean University","correspondingAuthor":true,"prefix":"","firstName":"Sheng-Ping","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-01-19 08:08:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5858356/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5858356/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81536148,"identity":"7498da3b-625c-4f8c-bf3c-3da3dd25457d","added_by":"auto","created_at":"2025-04-28 10:20:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2505406,"visible":true,"origin":"","legend":"\u003cp\u003eFlow chart of the experiment.\u003c/p\u003e","description":"","filename":"Fig.13.png","url":"https://assets-eu.researchsquare.com/files/rs-5858356/v1/4f76ce2cd7566e8363f397c3.png"},{"id":81537673,"identity":"3a8e5f96-6825-4250-8f3d-01406be3f6e7","added_by":"auto","created_at":"2025-04-28 10:36:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2650454,"visible":true,"origin":"","legend":"\u003cp\u003ePBC (\u003cstrong\u003ea\u003c/strong\u003e) and cooking loss (\u003cstrong\u003eb\u003c/strong\u003e) of large yellow croaker treated by different groups respectively.\u003c/p\u003e\n\u003cp\u003eDifferent lowercase letters represent significant differences within groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), and different uppercase letters represent significant differences between different days in the same group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-5858356/v1/817be624f8f66639cf4aeea0.png"},{"id":81536138,"identity":"6b3c22a5-f52b-4f90-bef0-7c6d7e06a445","added_by":"auto","created_at":"2025-04-28 10:20:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9250930,"visible":true,"origin":"","legend":"\u003cp\u003eTCA-soluble peptide content (\u003cstrong\u003ea\u003c/strong\u003e), MFI value (\u003cstrong\u003eb\u003c/strong\u003e), and SDS-PAGE profile (\u003cstrong\u003ec\u003c/strong\u003e) of MPs treated by different groups, respectively (A: BEO/AP, BEO-IC/AP, and C: BC-F/AP).\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-5858356/v1/d99f7a476164b43cacfff595.png"},{"id":81536142,"identity":"5daaffad-b0bb-40d3-976b-6aad4241c128","added_by":"auto","created_at":"2025-04-28 10:20:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2340326,"visible":true,"origin":"","legend":"\u003cp\u003eSurface hydrophobicity (\u003cstrong\u003ea\u003c/strong\u003e), total sulfhydryl content (\u003cstrong\u003eb\u003c/strong\u003e), and turbidity (\u003cstrong\u003ec\u003c/strong\u003e) of MPs treated by different groups on the same day. Different lowercase letters represent significant differences within groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), and different uppercase letters represent significant differences between different days in the same group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-5858356/v1/1a1f68f0f141b40e84d4ecf6.png"},{"id":81536145,"identity":"84ed8f4f-c7dd-4066-88fc-b928ddb1a3dc","added_by":"auto","created_at":"2025-04-28 10:20:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3967180,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size distribution (\u003cstrong\u003ea-b\u003c/strong\u003e) and IFI (\u003cstrong\u003ec-d\u003c/strong\u003e) of MPs treated by different groups respectively.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-5858356/v1/eb19cf85e79359898e7e2901.png"},{"id":81536183,"identity":"575bae56-a5eb-4564-bc49-847619655e8c","added_by":"auto","created_at":"2025-04-28 10:20:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":18699172,"visible":true,"origin":"","legend":"\u003cp\u003eTotal protease activity and protein content (\u003cstrong\u003ea\u003c/strong\u003e) of exudates, protein content (\u003cstrong\u003eb\u003c/strong\u003e), SDS-PAGE profile (\u003cstrong\u003ec\u003c/strong\u003e), surface hydrophobicity (\u003cstrong\u003ed\u003c/strong\u003e), particle size distribution (\u003cstrong\u003ee\u003c/strong\u003e), and IFI (\u003cstrong\u003ef\u003c/strong\u003e) of incubation of exudates with an MPs protein solution (P, 0dE+P, 6dE+P, and 10dE+P).\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-5858356/v1/47cd1e8bcb71f3d764b09ec4.png"},{"id":81536154,"identity":"2537d166-df45-4373-811f-b106158a0b4e","added_by":"auto","created_at":"2025-04-28 10:20:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":17775721,"visible":true,"origin":"","legend":"\u003cp\u003ePearson correlation coefficient heatmap of PBC, cooking loss, TCA-soluble peptide content, MFI, surface hydrophobicity, total sulfhydryl content, and turbidity\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e); exudate protein content, exudate total protease activity, protein content after exudate incubation, and surface hydrophobicity after exudate incubation (\u003cstrong\u003eb\u003c/strong\u003e). Red and blue indicate positive and negative correlations.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-5858356/v1/497abe27b4612217fdddfe9b.png"},{"id":81536164,"identity":"e0c03c14-94fd-4ea7-ae2d-9e8eb43cc990","added_by":"auto","created_at":"2025-04-28 10:20:12","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":23468591,"visible":true,"origin":"","legend":"\u003cp\u003eSimulation of the mechanism of MPs degradation and oxidation (\u003cstrong\u003ea-d\u003c/strong\u003e) induced by CK, AP, LEO-IC/AP, and LC-F/AP groups respectively.\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-5858356/v1/bf1462cbccee97a1d75e34b0.png"},{"id":81538428,"identity":"f190b301-dd18-4826-8c2f-d671ee4a3fd1","added_by":"auto","created_at":"2025-04-28 10:44:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":72624668,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5858356/v1/c982ff3d-a407-4057-8fe8-7545b92ca1e2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of a sustained-release sheet loaded with basil essential oil on the preservation of large yellow croaker (Larimichthys crocea)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNowadays, food preservation remains a major issue for the global food industry, particularly fish such as large yellow croaker (\u003cem\u003eLarimichthys crocea\u003c/em\u003e) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It has important economic value due to its high nutritional value. However, it is prone to spoilage caused by spoilage bacteria, leading to a rapid decline in its quality, which is reflected in the deterioration of texture, odor, and color, during storage and sale [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Among these changes, myofibrillar proteins (MPs) is the most important, which is the major muscle protein, closely related to texture properties, water-holding capacity, and softening [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. During MPs degradation, water within the muscle cell matrix transitions from the bound state to free water, resulting in the leakage of exudate [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Recent research indicates that the exudates contain a large number of microorganisms and tissue enzymes that not only change the appearance of fish muscle but also accelerate the textural degradation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe application of antimicrobial and antioxidant active substances can be a good choice to delay spoilage. Basil essential oil (BEO), derived from the lamiaceae herb (\u003cem\u003eOcimum basilicum\u003c/em\u003e), is classified as Generally Recognized As Safe (GRAS) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. It exhibits antimicrobial and antioxidant properties due to its high content of eugenol, linalool and methyl chavicol [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, its strong odor, unstable and hydrophobic properties limit its application in food preservation, not to mention its limited effect on exudates [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. As reported, oregano essential oil inhibited the growth of \u003cem\u003eL. monocytogenes in vitro\u003c/em\u003e study, but showed limited effectiveness in pork and chicken due to food component interactions [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Smaoui, et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] incorporated peppermint essential oil into a ground beef mixture but observed a substantial impact on the beef's flavor profile. To overcome the shortcomings of pure EO, embedding in biodegradable polymers is a promising strategy.\u003c/p\u003e \u003cp\u003eCurrently, polysaccharides and proteins such as chitosan (CS) and sodium alginate (SA) are of interest to researchers for their film-forming and biocompatibility.Sreekanth, et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] incorporated cinnamon essential oil into chitosan/starch films to reduce microbial counts in beef. Similarly, Cao, et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] prepared sodium alginate films loaded with oregano essential oil nanoparticles to prolong pork shelf life. CS has excellent film-forming ability and antibacterial properties, but its poor mechanical strength and water barrier performance limit the usage [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. SA exhibits high transparency and flexibility but lacks bioactivity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Commonly, incorporating encapsulated low-molecular organic active components can improve the mechanical and bioactive characteristics of film [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. A novel approach which delays muscle degradation and spoilage in large yellow croaker by absorbing exudates and simultaneously releasing antimicrobial active substances based on the hydrophilic nature is theoretically feasible. However, research in this area is limited, and further studies are required to validate its effects.\u003c/p\u003e \u003cp\u003eIn this paper, a sustained-release absorbent pad (BC-F/AP) modified with a chitosan/sodium alginate sheet loaded with BEO β-cyclodextrin (β-CD) inclusion complex was prepared. The inhibitory effectiveness of this pad on the bacteria, cooking loss and changes in MPs was evaluated through a series of tests. The degradation degree as well as the oxidation degree of MPs were also analyzed after incubation of exudates. This study aims to provide a sustainable and effective preservation method for aquatic foods.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLarge yellow croakers were purchased from the local aquatic product market (Shanghai Luchaogang Aquatic Product Market). BEO (purity 99%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. The absorbent pads were purchased from Shandong Debairun Packaging Products Co., Ltd. \u0026beta;-CD (purity 98%) was provided by Shanghai Macklin Biochemical Technology Co., Ltd. CS (Mw ~100000Da, degree of deacetylation: \u0026ge;90%), SA (purity: \u0026ge;90%, M: G = 1:2) were purchased from Shanghai Sangon Biological Engineering Co., Ltd.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003einclusion complex\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe basil essential oil inclusion complex (BEO-IC) was prepared by coprecipitation method [20]. Firstly, \u0026beta;-CD powder (8.6 g) was dissolved in ethanol solution (33%, 100 mL) and stirred at 55 \u0026deg;C for 2.5 h. Subsequently, BEO (1.4 g) was dissolved in ethanol solution (10%, 10 mL) and then slowly added to the \u0026beta;-CD solution. It was stirred at 55 \u0026deg;C for 1.5 h and kept overnight at 4 \u0026deg;C. The inclusion complex was then filtered, freeze-dried for 48 h, and obtained as a powder directly.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of the\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esustained-release sheet\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sustained-release sheet (BC-F) was fabricated using a casting method: Chitosan (CS, 0.9 g) and sodium alginate (SA, 0.1 g) were separately dissolved in 45 mL 2% acetic acid and 5 mL distilled water, respectively. The two solutions were combined and stirred continuously for 30 min, followed by the addition of 0.25 g glycerol as a plasticizer with further stirring for 30 min. Based on pre-optimized parameters, 100 mg BEO-IC was then incorporated into the mixture under 20-min stirring. The homogeneous solution was finally cast onto 160 \u0026times; 70 mm acrylic plates and dried at 40 \u0026deg;C for 12 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of the fish fillets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe large yellow croaker was cut into fillets (about 100 g for each piece) and washed with distilled water, then divided into five groups (CK, AP, BEO/AP, BEO-IC/AP, and BC-F/AP) for preservation. The five different groups were treated according to the following procedure.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(1) CK: The fish samples were placed in a tray and sealed with food cling wrap.\u003c/p\u003e\n\u003cp\u003e(2) AP: The fish samples were placed in a tray with commercial absorbent pads (160 mm \u0026times; 70 mm, about 1.80 g, water absorption capacity: about 700%) and sealed with food cling wrap.\u003c/p\u003e\n\u003cp\u003e(3) BEO/AP: BEO (30 mg, equal to the BEO content of 0.5 g BEO-IC) was smeared on the fish samples and then placed in a tray with absorbent pads and sealed with food cling wrap.\u003c/p\u003e\n\u003cp\u003e(4) BEO-IC/AP: BEO-IC (0.5 g) was evenly spread on the tray with absorbent pads, and then the fish samples were placed on it and sealed with food cling wrap.\u003c/p\u003e\n\u003cp\u003e(5) BC-F/AP: The BC-F sheet adhered to the tray with absorbent pads, and the fish samples were placed on it and sealed with food cling wrap.\u003c/p\u003e\n\u003cp\u003eAll samples were stored at 4 \u0026deg;C and sampled on days 0, 2, 4, 6, 8, and 10.\u0026nbsp;The experimental design of this study was shown in Fig. 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of fish quality and protein characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePsychrophilic bacteria count\u003c/em\u003e\u003cem\u003e\u0026nbsp;(PBC)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe PBC of the fish samples were tested following the method described by Liu, et al. [21]. Fish samples (5 g) were placed in a homogenization bag containing 45 mL of sterile saline solution (0.85%, m/v). The supernatant (1 mL) was gradient diluted by 9 mL sterilized saline solution. The diluted solution (100 \u0026mu;L) was evenly spread on PCA and incubated at 4 \u0026deg;C for 144 h.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCooking loss\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe cooking loss of the fish samples was tested according to the method of Radhakrishnan, et al. [22], and was calculated by the following formula (1).\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" width=\"552\" height=\"70\"\u003e\u003c/p\u003e\n\u003cp\u003eWhere W\u003csub\u003e1\u003c/sub\u003e and W\u003csub\u003e2\u003c/sub\u003e represent the weight of the fillets before and after cooking, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eExtraction of MPs\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMPs were extracted by the previous method [23].\u0026nbsp;Fish tissue samples (2 g) were homogenized in 20 mL of Solution A (20 mmol/L Tris-maleate, 0.05 mol/L KCl, pH 7.0) using a homogenizer, followed by centrifugation at 10,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 15 min at 4 \u0026deg;C. The supernatant was discarded to retain the pelleted fraction. Subsequently, the pellet was resuspended in 20 mL of Solution B (20 mmol/L Tris-maleate, 0.6 mol/L KCl, pH 7.0), re-homogenized, and incubated at 4 \u0026deg;C for 1 h. The MPs was obtained by centrifugation again under the same conditions.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTrichloroacetic acid (TCA)-soluble peptides\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe TCA soluble peptides were tested according to the method of Saengsuk, et al. [24].\u0026nbsp;The fish samples were mixed with 5% TCA (18 mL) solution, homogenized, and incubated at 4 \u0026deg;C for 1 h before being centrifuged at 10000 \u003cem\u003eg\u003c/em\u003e for 15 min at 4 \u0026deg;C. Then, the Lowry method was used to measure the soluble peptide content in the supernatant.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMyofibril fragmentation index (MFI)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe MFI was tested according to the method of Marino, et al. [25].\u0026nbsp;The fish sample (1 g) was mixed with the buffer (30 mL, 0.1 M KCl, 7 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 18 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, and 1 mM EDTA, pH 7.0), followed by centrifugation at 10000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min at 4 \u0026deg;C. The supernatant was discarded, and the pellet was washed at least twice with the buffer. The final protein solution (0.5 mg/mL) was analyzed by measuring absorbance at 540 nm using a spectrophotometer, and the MFI was calculated as absorbance \u0026times; 200.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe SDS-PAGE profiles of MPs were analyzed referring to the previous method [23]. The MPs solution (2 mg/mL) was mixed with the 2\u0026times;loading buff in a volume ratio of 1:1 and boiled respectively. A 5% stacking gel and a 10% separating gel were used. The samples (8 \u0026mu;L) and marker (11-245 kDa) were loaded onto the gel for electrophoresis until the samples reached the bottom of the gel.\u0026nbsp;The gel was stained with a staining solution (0.25 g/L Coomassie Brilliant Blue R-250, 25% ethanol, and 8% acetic acid) for 1 h. Then, the gel was decolorized with a decolorizing solution (25% ethanol, 8% acetic acid) until the protein bands were visible.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSurface hydrophobicity\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe surface hydrophobicity of MPs was tested according to the method of Zeng, et al. [26] with slight modification. MPs (1 mg/mL) and bromophenol blue (1 mg/mL, 40 \u0026mu;L) were mixed, incubated, centrifuged (10000 \u003cem\u003eg\u003c/em\u003e, 4 \u0026deg;C, 5 min), and the supernatant was measured at 595 nm.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTotal sulfhydryl content\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTotal sulfhydryl content (\u0026mu;mol/g) in MPs solution was quantified spectrophotometrically at 412 nm using a commercial assay kit (BC1370, Beijing Solarbio Science \u0026amp; Technology Co., Ltd., Beijing, China) based on the 5,5\u0026apos;-dithiobis-(2-nitrobenzoic acid)-coupled chromogenic reaction.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eParticle size distribution\u003c/em\u003e\u003cem\u003e\u0026nbsp;and protein turbidity\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe MPs solution (1 mg/mL) was placed in Zetasizer Nano-ZS respectively (Malvern Instruments, Worcestershire, UK) to detect the particle size distribution.\u003c/p\u003e\n\u003cp\u003eThe absorbance of the MPs solution (2 mg/mL) were measured at 360 nm to detect the protein turbidity.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIntrinsic fluorescence intensity (IFI)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe IFI was tested according to the method of\u0026nbsp;Xu, et al. [27]. The MPs solution (0.1 mg/mL) was scanned by an F-7100 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) to measure the tertiary structure of proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of the characteristics of exudate and protein characterization of the incubation of exudate with MPs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eExtraction of exudate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe exudate was extracted by mincing and centrifuging the large yellow croaker meat on days 0, 6, and 10 (0dE, 6dE, and 10dE) respectively, and then it was stored at -80 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEvaluation of total protease activity in exudate and protein content of exudate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe total protease activity of exudate was assayed following Sriket, et al. [28] with adaptations: Exudate samples (0dE, 6dE, 10dE) were blended with 3 mL pH 5.0 buffer (0.2 M Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e/0.1 M citrate) and 1 mL hemoglobin substrate (10 mg/mL), incubated at 50 \u0026deg;C for 15 min. Reactions were terminated with 1 mL 50% TCA, centrifuged (10000 \u003cem\u003eg\u003c/em\u003e, 4 \u0026deg;C, 5 min), and soluble peptides in supernatants quantified via Lowry method. Parallel exudate protein quantification employed Coomassie Brilliant Blue assay.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEvaluation of protein characteristics of exudate incubated with MPs\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe exudates (0dE, 6dE, 10dE) from fish were mixed with day-0-extracted MPs (0dCK) at 1:1 (v/v) to generate 0dE+P, 6dE+P, and 10dE+P complexes by incubating at 4 \u0026deg;C for 12 h. The protein content, surface hydrophobicity, IFI, particle size, and SDS-PAGE profiles of the resultant protein solutions were detected using established protocols.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were conducted in triplicate. Data are expressed as mean \u0026plusmn; SD (IBM SPSS Statistics 27), with graphs plotted in Origin 2022. Statistical significance was determined by one-way ANOVA followed by Duncan\u0026apos;s multiple comparison test. Significant differences (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05) between groups are indicated by distinct superscript letters.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cstrong\u003eFish quality and protein characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePsychrophilic bacteria count (PBC)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe initial PBC of the fish sample was 2.80\u0026plusmn;0.10 log CFU/g (Fig. 2a). With the extension of the storage period, the PBC of all groups showed a significant increase (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). On day 10, the PBC values reached 10.47\u0026plusmn;0.10, 10.44\u0026plusmn;0.02, 10.42\u0026plusmn;0.02, 10.34\u0026plusmn;0.010, 9.44\u0026plusmn;0.02 log CFU/g in the CK, AP, BEO/AP, BEO-IC/AP, and BC-F/AP groups, respectively. It indicated that the use of commercial absorbent paper could delay the growth of psychrophilic bacteria, possibly due to its absorption of the exudate from the fish fillets [8]. The BEO/AP treatment groups demonstrated significant bacteriostatic efficacy during the initial 4-day incubation period, as evidenced by suppressed microbial proliferation. The main components of BEO, typically eugenol, linalool, or methyl chavicol, were responsible for its antimicrobial effects [8-10]. However, bacterial counts increased quickly after then, indicating a limited duration of effectiveness for pure BEO. This phenomenon can be primarily attributed to the inherent hydrophobicity and high volatility characteristic of BEO constituents. Furthermore, the complex food matrix containing lipids, proteins, and other organic constituents could potentially compromise the antibacterial efficacy of essential oils [29, 30]. The BEO-IC improved the hydrophobicity and volatility of EO, enabling sustained release [31]. Additionally, it prevented direct contact between EO and fish tissue, thereby reducing EO loss and delaying protein degradation thus suppressing bacterial growth. The BC-F/AP group showed the slowest bacterial growth rate (highest inhibition rate reached 90.66%), aligning with the findings of Ying, et al. [32]. This might result from the larger contact area between the sheet and fish samples in the BC-F/AP group than in the BEO-IC/AP group. Thus, the antibacterial substance in essential oil may be more effectively applied to fish samples, achieving the continuous preservation effect. Meanwhile, CS might also play a role in antibacterial activity [33].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCooking loss\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCooking loss served as a key metric for evaluating fish water-holding capacity (WHC) [34]. The initial cooking loss of the fish sample was 18.04%\u0026plusmn;0.11%, with the CK group experiencing the highest loss, reaching 35.58%\u0026plusmn;1.54% during storage (Fig. 2b). This might be attributed to the degradation and oxidation of muscle proteins, the former expanding the inter-bundle myofibril space and the latter forcing water out of the myofilaments [35]. The AP group reduced the cooking loss by absorbing exudates, and the BEO/AP group had lower cooking losses than the AP group in the first four days, while the cooking loss of the BEO-IC/AP group was lower than that of the AP group as a whole. This is consistent with previous research findings [31]. The BC-F/AP group exhibited the lowest cooking loss among all groups, attributed to its synergistic capacity for sustained suppression through exudate absorption and bioactive release, which was going to be proved by the following study.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTCA-soluble peptides\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe initial TCA-soluble peptide content was 0.88\u0026plusmn;0.02 \u0026mu;mol tyrosine/g, and the CK group showed the most rapid increase, reaching 7.94\u0026plusmn;0.20 \u0026mu;mol tyrosine/g during storage (Fig. 3a). The increase of TCA-soluble peptide content might be due to protein degradation induced by endogenous and exogenous enzymes (the main factor) [36]. The AP group had lower TCA-soluble peptide content than the CK, and the BEO/AP group had lower content than the AP group in the first four days. The BEO-IC/AP and BC-F/AP groups inhibited the increase of TCA-soluble peptide content during storage, with the BC-F/AP group being the most effective. BEO delayed protein degradation by suppressing endogenous enzyme activity and blocking exogenous enzyme production through its antibacterial and antioxidant properties in the early stage [37]. However, its efficacy gradually diminished over time due to volatilization or binding with fish muscle. In contrast, BEO-IC employed microencapsulation technology to achieve a sustained-release effect, thereby maintaining prolonged protective effects [38]. Furthermore, the BC- F/AP group exhibited the lowest TCA-soluble peptide content, which was attributed to the optimized dispersion homogeneity of BEO-IC and the synergistic antibacterial action of CS [39, 40].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMyofibril fragmentation index (MFI)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMFI was often used to characterize the integrity and degradation degree of protein structure [41]. The initial MFI was 45.03\u0026plusmn;1.77 (Fig. 3b). The MFI values of the CK, AP, BEO/AP, BEO-IC/AP, and BC-F/AP groups reached 231.30\u0026plusmn;1.92, 222.02\u0026plusmn;4.58, 239.94\u0026plusmn;18.38, 197.03\u0026plusmn;1.43, and 184.48\u0026plusmn;2.18 respectively during storage. The increase in MFI might be attributed to the action of the endogenous enzymes and microorganisms in fish [42]. This indicated that the BC-F/AP group effectively inhibited protein degradation and maintained the stability of protein structure, in agreement with the results of TCA-soluble peptides.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eChanges in SDS-PAGE protein bands are used to reflect protein degradation during storage. The molecular weights of proteins were mainly concentrated in the range of 15-200 kDa, including myosin heavy chain (MHC, \u0026sim;200 kDa), actin (\u0026sim;48 kDa), troponin (\u0026sim;35 kDa), and tropomyosin (\u0026sim;32 kDa) [43]. These findings align with parallel trends in protein carbonyl content (PBC), TCA-soluble peptide levels, and myofibrillar fragmentation index (MFI) data. Conversely, the BEO-IC (essential oil-in-cyclodextrin) and BC-F/AP (\u0026beta;-cyclodextrin-fortified active packaging) groups demonstrated preserved MHC structural integrity, owing to the controlled release properties of encapsulated essential oils that ensured sustained antimicrobial efficacy within the muscle matrix. On day 10, the MHC (\u0026sim;200 kDa) band exhibited substantial degradation with near-complete band disappearance in both the CK and BEO/AP groups, while partial degradation was observed in the AP group (Fig. 3c). This phenomenon was likely due to the volatilization of active components of BEO or binding to fish lipids/proteins. The diminished antimicrobial activity likely permitted microbial proliferation and subsequent secretion of exogenous proteases, which accelerated MHC fragmentation [44]. These findings align with parallel trends in PBC, TCA-soluble peptide levels, and MFI data. Conversely, the BEO-IC and BC-F/AP groups demonstrated preserved MHC structural integrity, owing to the controlled release properties of encapsulated essential oils that ensured sustained antimicrobial efficacy within the muscle matrix. In the CK and BEO/AP groups, the band around 100 kDa disappeared, while it did not change obviously in the other groups. The band around 75 kDa became lighter in color in the BC-F/AP group during storage, but it almost disappeared in the other groups. In the CK and BEO/AP groups, the actin (\u0026sim;48 kDa) band was degraded and shifted downward, whereas it showed little change in the BEO-IC/AP and BC-F/AP groups. The tropomyosin band (\u0026sim;32 kDa) was fully degraded in CK, AP, and BEO/AP groups, but persisted in BEO-IC/AP with reduced intensity. In general, the absorbent pads exhibited the following MPs inhibition efficacy ranking: BC-F/AP \u0026gt; BEO-IC/AP \u0026gt; AP \u0026gt; BEO/AP \u0026sim; CK.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSurface hydrophobicity\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe exposure degree of hydrophobic groups in proteins and the alterations in protein structure could be indicated by surface hydrophobicity [45]. The surface hydrophobicity of fresh samples was 2.03\u0026plusmn;0.54 g BPB/mg protein, and the CK group reached 24.28\u0026plusmn;0.36 \u0026mu;g BPB/mg protein during storage (Fig. 4a). This might be attributed to the presence of oxidative substances (substantial reactive oxygen species, secondary lipid oxidation products) in the exudate, which facilitated the unfolding of MPs, exposing hydrophobic amino acids and altering their spatial structure [35]. The inhibitory effect of the AP group and the BEO/AP group on the increase in protein surface hydrophobicity was not as significant as BC-F/AP and BEO-IC/AP groups (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05). It also confirmed that pure BEO did not display antioxidant activity in food products effectively due to its hydrophobic and volatile characteristics [46]. The BC-F/AP group exhibited the lowest surface hydrophobicity (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05), likely due to the more uniform distribution of BEO-IC facilitated by the CS/SA sheet [47].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTotal sulfhydryl content\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThiol groups are important functional groups of MPs, which can be easily oxidized to form disulfide bonds, causing protein cross-linking and aggregation [48]. The total sulfhydryl content of fresh samples was 134.28\u0026plusmn;5.56 nmol/mg (Fig. 4b). During storage, the total sulfhydryl content of all groups decreased significantly (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). On the 10th day, the total sulfhydryl contents of the CK, AP, BEO/AP, BEO-IC/AP, and BC-F/AP groups were 29.63\u0026plusmn;2.94, 33.05\u0026plusmn;4.78, 32.74\u0026plusmn;1.05, 43.71\u0026plusmn;0.12, and 60.41\u0026plusmn;3.83 nmol/mg, respectively. The BEO-IC/AP and BC-F/AP groups showed significantly higher total sulfhydryl content than other groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), peaking in BC-F/AP. This demonstrated that CS/SA sheets incorporating BEO-IC suppressed protein oxidation most effectively, consistent with surface hydrophobicity trends.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTurbidity and protein particle size distribution\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe turbidity of the fresh sample was 0.16 (Fig. 4c). During storage, the turbidity of MPs increased, indicating an increase in protein aggregation. Notably, the protein turbidity in the BEO-IC/AP and BC-F/AP groups was significantly lower than in the other groups (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05). More detailed results were confirmed according to the results of protein particle size.\u003c/p\u003e\n\u003cp\u003eThe particle size of fresh samples was mainly concentrated around 768 nm. The particle size of all groups was increased during storage (Fig. 5a-b). The phenomenon was due to oxidation exposing hydrophobic groups of the MPs, enhancing protein aggregation through hydrophobic interactions, and sulfhydryl oxidation forming disulfide bonds [49, 50]. BEO-IC/AP and BC-F/AP exhibited reduced protein particle sizes compared to other groups, indicating suppressed oxidation. BC-F/AP demonstrated optimal efficacy, correlating with surface hydrophobicity and total sulfhydryl content trends.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIntrinsic fluorescence intensity (IFI)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe IFI correlates with tryptophan residue abundance and serves as an indirect indicator of tertiary structural modifications in proteins [51]. Fresh samples showed the highest IFI near 335 nm (Fig. 5c-d). As oxidation induced hydrophobic interactions in the MPs, exposing the tryptophan residues to hydrophilic solvents, thereby reducing the IFI of tryptophan [52]. The IFI of all groups reduced during storage. However, the IFI of the BEO-IC/AP and the BC-F/AP were always more stable than that of other groups, with BC-F/AP achieving peak values. These results confirm their superior efficacy in preserving protein tertiary structure, aligning with the findings on surface hydrophobicity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of exudate characteristics and in vitro incubation of exudate with MPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTotal protease activity and protein content of exudates\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo demonstrate the effect of exudate on the quality of yellow croaker, the total protease activity and protein content of exudates from croaker fillets on day 0 (0dE), day 6 (6dE), and day 10 (10dE) determined (Fig. 6a). The protein contents and total protease activity of 6dE and 10dE were significantly higher than 0dE (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05), and the differences between 6dE and 10dE were not significant (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05). It is hypothesized that the degradation of fish meat tissue structure leads to the release of intracellular proteases into the exudate [53].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eChanges of MPs after incubation with exudate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFig. 6b showed the protein content of MPs before and after incubation with exudates from different days (0dE+P, 6dE+P, and 10dE+P). The 0dE+P group exhibited a higher protein content compared to the initial MPs, equaling the average protein content of MPs and exudate (Fig. 6a). The protein contents in the 6dE+P and 10dE+P were significantly lower than 0dE+P (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05) aligning with the results of total protease activity (Fig. 6a). Therefore, the degradation of MPs was highly related with the protease activity of exudates.\u003c/p\u003e\n\u003cp\u003eThe SDS-PAGE profile revealed distinct alterations in exudate-MP interactions post-incubation (Fig. 6c). The protein compositions and distributions of exudates on different days were similar (0dE, 6dE, and10dE). A wide range of thick bands appear between 35-48 kDa, and thin and light bands appear near MHC (\u0026sim;200 kDa), indicating that the exudate was composed of a large number of sarcoplasmic proteins and a small number of MPs [5]. The electrophoretic bands of the mixture samples showed the typical bands of both MPs and exudates with different intensities. After incubation with the exudates, the bands of MPs near the MHC (\u0026sim;200 kDa), 100 kDa, and 20 kDa became lighter, indicating that the MPs had been degraded. This finding was closely related to the protease activity and microbial content in the exudate, aligning with the research of Zhang, et al. [5]. As shown in Fig. 6d-f, incubation of MPs with the exudate induced three progressive changes: increased protein surface hydrophobicity, attenuated IFI intensity, and enlarged particle sizes. These observations demonstrated oxidation-mediated protein aggregation. These results were consistent with the report by Liu, et al. [35].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCorrelation analysis and fish appearance\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 7a, PBC was significantly and positively correlated (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) with cooking loss, TCA-soluble peptide content, MFI, surface hydrophobicity, and turbidity, while it was significantly and negatively correlated with total sulfhydryl content, demonstrating microbial-driven protein degradation/oxidation. Fig. 7b revealed that exudate proteolytic activity positively correlated with exudate protein content and post-incubation myofibrillar protein hydrophobicity (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), yet inversely correlated with post-incubation protein content (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), confirming exudate protease\u0026apos;s role in accelerating protein deterioration. Therefore, it is hypothesized that the bacteriostatic pads preserved fish quality by suppressing microbial growth through sustained antimicrobial/antioxidant release and exudate absorption, effectively stabilizing protein structures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProposed mechanism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental findings elucidate how absorbent pads modulate myofibrillar protein (MP) integrity in large yellow croaker through exudate management (Fig. 8a). The untreated samples (CK) accumulated exudates during storage, accelerating myosin heavy chain (MHC) degradation and triggering oxidative protein aggregation via hydrophobicity elevation. The conventional pads (AP) partially absorbed exudates to slow these processes (Fig. 8b), and BEO-IC/AP demonstrated dual functionality \u0026ndash; sustained antimicrobial/antioxidant release coupled with exudate adsorption synergistically suppressed MP deterioration (Fig. 8c). The BC-F/AP system achieved optimal performance through enhanced BEO-IC dispersion and the inherent antimicrobial activity of chitosan, collectively minimizing structural degradation and oxidative damage to maintain the integrity of MPs (Fig. 8d).\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, the preservation effects of different absorbent pads (CK, AP, BEO/AP, BEO-IC/AP, and BC-F/AP) on the quality and MPs of large yellow croaker were analyzed. The results indicated that both the BEO-IC/AP and BC-F/AP groups had remarkable and sustainable ability to inhibit bacterial growth and maintain protein integrity, with the BC-F/AP group being the best. The \u003cem\u003ein-vitro\u003c/em\u003e experiment indicated that the exudate might be responsible for the degradation, tertiary structure changes and oxidative aggregation of MPs. Therefore, a sustained-release sheet made by CS/SA and BEO combined with water-absorbing pads can be a promising method to maintain the muscle quality of large yellow croaker, not to mention its feasibility of application during packaging processing. However, this study did not detect the of volatile active compounds in BEO and the chemical composition of the exudate, which can be researched further. Future investigations can also prioritize systematic evaluation of sustained-release absorbent pad technologies across varying food matrices to expand its applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.-F.Q.: Conceptualization, writing - review \u0026amp; editing, funding acquisition, formal analysis, supervision; C.-J.S.: writing - original draft, investigation, validation; R.-J.G.: investigation, data curation; S.L.: investigation; S.-P.Y.: writing - review \u0026amp; editing, project administration, funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our sincere gratitude to the College of Food Science and Technology, Shanghai Ocean University for their help in making my experiment successful.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the National Natural Science Foundation of China (No: 31501551), China Scholarship Council (No: 202008310018), and Special Fund for the Development of Science and Technology of Shanghai Ocean University (No: A2-2006-20-200203).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data will be available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJ. 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Technol. \u003cstrong\u003e101\u003c/strong\u003e, 76-82 (2019).https://doi.org/10.1016/j.lwt.2018.11.026\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-biophysics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Biophysics](https://www.springer.com/journal/11483)","snPcode":"11483","submissionUrl":"https://submission.nature.com/new-submission/11483/3","title":"Food Biophysics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"exudate, degradation, oxidation, intrinsic fluorescence intensity, surface hydrophobicity, sulfhydryl","lastPublishedDoi":"10.21203/rs.3.rs-5858356/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5858356/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo enhance basil essential oil (BEO) stability, a chitosan/sodium alginate-based sustained-release sheet loaded with BEO inclusion complexes (IC) was developed and integrated with a commercial absorbent pad (BC-F/AP). The effects of BC-F/AP on quality maintenance and myofibrillar protein (MP) stability in large yellow croaker (\u003cem\u003eLarimichthys crocea\u003c/em\u003e) were investigated in comparison with the control (CK), commercial pad (AP), pure BEO, and BEO-IC. The findings showed that the BC-F/AP group suppressed the proliferation of psychrophilic bacteria in fish fillets and minimized cooking loss. On day 10, bacterial counts were approximately 1 log CFU/g lower, and cooking loss was 3.08% less compared to CK. According to experimental results, BC-F/AP group effectively maintained the structure and reduced the degradation and the oxidative aggregation degree of MPs of croaker during storage. The sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results indicated that the BC-F/AP group demonstrated the capability to protect the integrity of MPs. The \u003cem\u003ein vitro\u003c/em\u003e incubation experiment involving exudate and MP\u003csub\u003eS\u003c/sub\u003e revealed that the exudates accelerated the degradation, oxidative aggregation and surface hydrophobicity of MPs. The BC-F/AP group maintained the quality of the large yellow croaker and the integrity of MPs by absorbing exudate and continuously releasing antibacterial and antioxidant active substances.\u003c/p\u003e","manuscriptTitle":"Effect of a sustained-release sheet loaded with basil essential oil on the preservation of large yellow croaker (Larimichthys crocea)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-28 10:20:06","doi":"10.21203/rs.3.rs-5858356/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-19T19:07:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-17T16:20:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-12T07:52:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197963895636671017417422299410836488022","date":"2025-04-24T15:07:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-24T04:09:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"138854743395375674712962998422013668935","date":"2025-04-23T11:22:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3347113454749645307052551509390435234","date":"2025-04-23T06:22:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-21T12:37:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-21T05:58:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food Biophysics","date":"2025-03-16T09:03:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-biophysics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Biophysics](https://www.springer.com/journal/11483)","snPcode":"11483","submissionUrl":"https://submission.nature.com/new-submission/11483/3","title":"Food Biophysics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0aaf320a-cde5-41b9-a493-33c9724abb0f","owner":[],"postedDate":"April 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-09T13:38:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-28 10:20:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5858356","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5858356","identity":"rs-5858356","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}