A carrageenan hydrogel loaded with tocopheryl-citral nanoemulsion, method of preparation and use thereof

Abstract

The application belongs to the technical field of bioactive fresh-keeping materials, and particularly relates to a carrageenan hydrogel loaded with tocopherol-citral nanoemulsion, a preparation method and application thereof. The carrageenan hydrogel is obtained by loading the tocopherol-citral nanoemulsion in carrageenan, and the tocopherol-citral nanoemulsion is prepared by emulsifying separation whey protein, deionized water, tocopherol and citral. The carrageenan hydrogel has good antioxidant and antibacterial properties and good biocompatibility, and provides a non-contact active fresh-keeping mode. The hydrogel can play a fresh-keeping role without direct contact with food, continuously fresh-keep through slow release of volatile active ingredients, effectively inhibit lipid oxidation and microbial growth of fresh-cut goldfish during storage, reduce pH, weight loss rate, TBARS value and TVB-N content, maintain product hardness and elasticity, delay quality deterioration, and has application value in the fields of aquatic products, livestock and poultry meat products and fruit and vegetable fresh-keeping.

Description

Technical Field

[0001] This invention belongs to the field of bioactive preservation materials technology, specifically relating to a carrageenan hydrogel loaded with tocopherol-citral nanoemulsion, its preparation method, and its application. Background Technology

[0002] Golden pomfret, with its tender flesh, rich nutrition, and high economic value, has become one of my country's important marine aquaculture fish. However, its muscle has a high water content and is rich in protein and unsaturated fatty acids, making it highly susceptible to microbial growth, lipid oxidation, and protein degradation during storage and distribution, leading to a rapid decline in freshness and a shortened shelf life. Especially during fresh-cut processing, damage to the tissue structure further accelerates oxidation and spoilage, severely limiting its processing utilization and cold chain distribution. Therefore, developing safe, efficient, and sustainable preservation strategies to delay the deterioration of fresh-cut golden pomfret is of great significance for ensuring the stability of the aquatic product supply chain and increasing product added value.

[0003] In recent years, with consumers' increasing demands for food safety, green packaging, and cold chain preservation, active packaging and bio-based preservation materials have become important research directions in the food preservation field due to their good biocompatibility, biodegradability, and environmental friendliness. Existing technologies show that introducing active substances such as natural antioxidants and plant essential oils into packaging materials can inhibit lipid oxidation and microbial growth during food storage, thereby extending product shelf life. For example, existing technologies disclose a composite packaging film loaded with plant essential oil nanoemulsions, achieving food preservation through the slow release of active ingredients. However, in existing active packaging systems, most active components are directly loaded onto the surface of the packaging film, making them prone to volatilization, migration, or rapid release, resulting in insufficient persistence of preservation effects. Some packaging materials have poor mechanical stability and are prone to structural damage under long-term storage or complex environmental conditions. Furthermore, most active packaging relies on direct contact between active substances and the food surface, posing a risk of active ingredient migration into the food, potentially affecting food quality and safety.

[0004] Based on the above findings, this invention provides a carrageenan hydrogel loaded with a tocopherol-citral nanoemulsion and its preparation method. The nanoemulsion uses citral and tocopherol as active components, and carrageenan forms a stable three-dimensional network structure to effectively encapsulate the nanoemulsion. The resulting hydrogel exhibits good gelling properties, structural stability, antioxidant properties, antibacterial properties, and biocompatibility. The method described in this invention is simple, convenient, and easy to operate. It allows for non-contact placement within a packaging system with the food to be preserved, achieving preservation through the release of volatile active ingredients. Summary of the Invention

[0005] To address the aforementioned technical problems, the primary objective of this invention is to provide a carrageenan hydrogel loaded with a tocopherol-citral nanoemulsion. The carrageenan hydrogel is obtained by loading the tocopherol-citral nanoemulsion onto carrageenan, and the tocopherol-citral nanoemulsion is obtained by dissolving tocopherol and citral in whey protein.

[0006] Preferably, the carrageenan concentration is 15-45 mg / mL.

[0007] Preferably, the loading of tocopherol-citral nanoemulsion in the carrageenan hydrogel is 10-50%.

[0008] Preferably, the volume ratio of tocopherol to citral in the tocopherol-citral nanoemulsion is 1:(1-5).

[0009] A second objective of this invention is to provide a method for preparing the carrageenan hydrogel loaded with the tocopherol-citral nanoemulsion, comprising the following steps: (1) Dissolve whey protein in deionized water, add tocopherol and citral, homogenize and sonicate to obtain tocopherol-citral nanoemulsion; (2) Dissolve carrageenan in water and heat at 80-100℃ until completely dissolved; (3) Add the nanoemulsion obtained in step (1) to the carrageenan solution obtained in step (2) and stir until homogeneous; (4) Cool to form a gel, and obtain a nanoemulsion-loaded carrageenan hydrogel.

[0010] Preferably, the homogenization speed in step (1) is 10,000-20,000 rpm and the homogenization time is 1-10 min.

[0011] Preferably, the ultrasonic treatment time in step (1) is 3-10 min.

[0012] A third objective of this invention is to provide the application of the carrageenan hydrogel in the preparation of active preservative materials.

[0013] A fourth objective of this invention is to provide an active preservative material, wherein the active preservative material includes the carrageenan hydrogel.

[0014] Preferably, the active preservative material is used for the preservation of aquatic products, livestock and poultry meat products, or fruit and vegetable products.

[0015] The beneficial effects of the present invention are: (1) The present invention constructs a carrageenan hydrogel loaded with tocopherol-citral nanoemulsion. The nanoemulsion can be dispersed in the three-dimensional network structure of carrageenan to form a stable composite gel system. Compared with pure carrageenan hydrogel, the introduction of an appropriate amount of nanoemulsion can significantly improve the storage modulus, loss modulus and viscosity of the hydrogel, improve its mechanical properties and freeze-thaw stability, and form a more uniform and dense porous network structure, which has good gelling performance and structural stability.

[0016] (2) The carrageenan hydrogel of the present invention has good antioxidant and antibacterial properties. Tocopherol and citral are loaded in the carrageenan network in the form of nanoemulsion, which can achieve uniform dispersion and continuous release of active ingredients. The resulting hydrogel has high ABTS and DPPH free radical scavenging ability and can effectively inhibit the growth of Escherichia coli and Staphylococcus aureus, thereby improving the antioxidant and antibacterial effects of the preservation system.

[0017] (3) The nanoemulsion-loaded carrageenan hydrogel of the present invention uses natural polysaccharide carrageenan, tocopherol and citral as the main raw materials, does not contain chemical crosslinking agents, and has good biocompatibility.

[0018] (4) This invention provides a non-contact active preservation mode. The hydrogel can exert its preservation effect without direct contact with food, and achieves continuous preservation by slow-release of volatile active ingredients. It can effectively inhibit lipid oxidation and microbial growth during the storage of fresh-cut pomfret, reduce pH, weight loss rate, TBARS value and TVB-N content, maintain product hardness and elasticity, and delay quality deterioration. It has application value in the fields of aquatic products, livestock and poultry meat products and fruit and vegetable preservation. Attached Figure Description

[0019] Figure 1 Stability of nanoemulsions;

[0020] Note: Photographs (A), Zeta potential (B), and particle size (C) of nanoemulsions stored at 4 ℃ and 25 ℃ for 1, 7, and 14 days.

[0021] Figure 2 Physicochemical characterization of preservation hydrogels;

[0022] Note: Storage modulus and loss modulus (A); viscosity (B); continuous step strain measurement (C); compressive stress-strain curve (D); swelling (E); freeze-thaw (F).

[0023] Figure 3 SEM image of the preservation hydrogel.

[0024] Figure 4FT-IR spectrum (A); XRD spectrum (B); TGA curve (C); DTG curve (D); ABTS free radical scavenging activity (E); DPPH free radical scavenging activity (F).

[0025] Figure 5 Photographs and bar graphs showing the antibacterial activity of the preservation hydrogel against Escherichia coli and Staphylococcus aureus.

[0026] Figure 6 Photos of fresh-cut pomfret fillets preserved with Control, Cn, TC-Cn4 preservation hydrogel during 10-day storage.

[0027] Figure 7 pH (A), weight loss (B), TBARS (C), and TVB-N (D) of fresh-cut pomfret fillets preserved with Control,Cn,TC-Cn4 preservation hydrogel during 10-day storage. Detailed Implementation

[0028] The scope of protection of the present invention will be described in detail below through specific embodiments. Any technical solutions that can be conceived by those skilled in the art based on the present invention and in combination with common knowledge in the art are within the scope of protection of the present invention.

[0029] It should be noted that, unless otherwise specified, the experimental methods described in the following examples are all conventional methods, and the reagents used are all commercially available.

[0030] In the following examples, TVB-N is an abbreviation for Total Volatile Basic Nitrogen, a core indicator for judging the freshness of animal products such as fish and meat. TVB-N consists of volatile, alkaline nitrogenous substances such as ammonia and amines produced during the decomposition of proteins in meat when it spoils under the action of enzymes and bacteria. A higher TVB-N value indicates more protein degradation, greater loss of nutritional value, and the onset of spoilage.

[0031] In the following examples, TBARS stands for Thiobarbituric Acid Reactive Substances. Like TVB-N, it is an important indicator for evaluating the quality of meat and seafood. However, unlike TVB-N, which reflects protein spoilage, TBARS measures the degree of fat oxidation.

[0032] Example 1: Preparation and Characterization of Nanoemulsions 1. Preparation of nanoemulsions 1.1 Effect of different tocopherol addition amounts on nanoemulsions Take 0.2 g of isolated whey protein and place it in 93 mL of deionized water. Stir at 45 °C for 10 minutes to fully dissolve it in the deionized water. Then homogenize for 3 minutes. During the homogenization process, add 1 mL, 2 mL, and 3 mL of tocopherol and 6 mL, 5 mL, and 4 mL of citral solution, respectively. Finally, sonicate the mixture under ice-water bath conditions.

[0033] The results showed that different amounts of tocopherol significantly affected the stability of the nanoemulsion. When the amounts of tocopherol and citral added were 2 mL and 5 mL, respectively, the resulting nanoemulsion did not show obvious stratification during standing, exhibiting good physical stability; while the emulsions prepared with other ratios all showed varying degrees of stratification, indicating poor emulsion stability. Therefore, the optimal ratio of tocopherol to citral was determined to be 2 mL:5 mL, and subsequent experiments were conducted using this formulation.

[0034] Therefore, the preparation method of the nanoemulsion is as follows: Take 0.2 g of isolated whey protein and place it in 93 mL of deionized water. Stir at 45 °C for 10 minutes to fully dissolve it in the deionized water. Then homogenize for 3 minutes. During homogenization, add 2 mL of tocopherol and 5 mL of citral solution to form a crude emulsion. Finally, sonicate under ice-water bath conditions to obtain the nanoemulsion, named TC.

[0035] 2. Characterization of nanoemulsions The particle size and polydispersity index (PDI) of nanoemulsions were determined at 25 °C using a nanoparticle size analyzer (NANO-ZS90, Malvern Instruments Ltd., UK). Nanoemulsion samples were sealed in glass vials and stored at 4 °C and 25 °C for 0, 7, and 14 days, respectively. Particle size and zeta potential were measured at predetermined time intervals, and appearance was recorded to assess storage stability.

[0036] Experimental results are as follows Figure 1 As shown in Figure A, at 4 °C, the emulsion maintained good stability throughout the storage period, with no obvious stratification observed. However, at 25 °C, the emulsion stability decreased significantly, with slight stratification appearing on day 7 and significant stratification on day 14. These results indicate that the stability of this emulsion is significantly affected by temperature and storage time. Therefore, when further applied to preservation materials, it is more suitable for perishable foods with short storage periods, such as aquatic products.

[0037] The experimental results of zeta potential are as follows: Figure 1As shown in Figure B, the Zeta potentials of the nanoemulsions stored for 0, 7, and 14 days at 4℃ were -21.9, -18.7, and -17.1 mV, respectively; while at 25℃, the corresponding Zeta potentials at these time points were -21.8, -16.5, and -12.78 mV, respectively. Particle size experimental results are shown in Figure B. Figure 1 C shows that at 4 °C, the average particle sizes of the nanoemulsions stored for 0, 7, and 14 days were 215.7, 222.5, and 239.1 nm, respectively, with no significant change. However, at 25 °C, the particle sizes were 209, 238.7, and 269.1 nm, respectively, showing a significant increasing trend with prolonged storage time. These results indicate that lower temperatures help maintain the colloidal stability of the nanoemulsions, while at higher temperatures, droplets are more prone to aggregation or co-aggregation, leading to increased particle size and system instability. This is consistent with... Figure 1 This is consistent with the stratification observed in A.

[0038] Example 2: Preparation and Characterization of Preservative Hydrogel 1. Preparation of Preservative Hydrogels 1.5 g of carrageenan was weighed and dissolved in 50, 45, 40, 35, 30, and 25 mL of deionized water, respectively. The solutions were magnetically stirred at 85 °C for 20 min until completely dissolved. 0.15 g of KCl was added during stirring, followed by 0, 5, 10, 15, 20, and 25 mL of TC, respectively. Stirring continued until the system was homogeneous, resulting in preservation hydrogel samples with nanoemulsion loadings of 0%, 10%, 20%, 30%, 40%, and 50%, named Cn, TC-Cn1, TC-Cn2, TC-Cn3, TC-Cn4, and TC-Cn5. These samples were then photographed.

[0039] 2. Characterization of the preservative hydrogel 2.1 Characterization of mechanical properties Rheological experiments were conducted using a rheometer. A small cylinder was placed at the center of a parallel plate with a diameter of 15 mm. The strain frequency of the rheometer was set to 1%, and the storage modulus G' (Pa) and loss modulus G" (Pa) were measured, along with the viscosity. Compressibility was determined at a constant strain rate of 5% / min at 25 °C. Furthermore, frequency-dependent rheological behavior was determined at a constant strain of 1% under isothermal conditions at 25 °C, as well as continuous step strain measurements were performed under the conditions of low strain (γ=1.0%) and high strain (γ=300%).

[0040] Experimental results of energy storage modulus and loss modulus are as follows: Figure 2As shown in Figure A, at a frequency of 20 Hz, the storage modulus of Cn is 8551 Pa and the loss modulus is 3082 Pa. With the increase of nanoemulsion (TC) addition, the mechanical properties of the preservation hydrogel show a trend of first increasing and then decreasing. Compared with the Cn group, TC-Cn1–TC-Cn4 all increased G' and G" values, indicating that the elasticity and viscosity of the system were enhanced. Among them, TC-Cn3 and TC-Cn4 showed the highest G' values ​​(14200 Pa and 14500 Pa), indicating that their three-dimensional network structure was the most dense and stable. The introduction of an appropriate amount of nanoemulsion helps to form a more uniform network structure and improve the storage modulus. However, when the amount of nanoemulsion added is further increased to a higher level (TC-Cn5), G' and G" decrease significantly. This may be because excessive nanoemulsion leads to a loose gel network structure.

[0041] Viscosity test results are as follows Figure 2 As shown in B, the viscosity of Cn is 67140 mPa. The viscosity of TC-Cn1 is 100,000 mPa. The viscosity of TC-Cn2 is 89570 mPa. The viscosity of TC-Cn3 is 111600 mPa. The viscosity of TC-Cn4 is 109400 mPa. The viscosity of TC-Cn5 is 63420 mPa. The results showed that the viscosity of the preservation hydrogel system first increased and then decreased with increasing nanoemulsion content. Compared with Cn, TC-Cn1–TC-Cn4 significantly increased the system viscosity, with TC-Cn3 and TC-Cn4 reaching 111600 mPa·s and 109400 mPa·s, respectively, indicating that the preservation hydrogel formed a relatively dense and stable three-dimensional network structure. The introduction of an appropriate amount of nanoemulsion improved the anti-flow ability. However, when the nanoemulsion content was further increased to TC-Cn5, the system viscosity decreased significantly (63420 mPa·s), even lower than the control group. This may be due to the loose structure of the system caused by excessive oil phase droplets.

[0042] Continuous step strain measurement and injection experiments were used to further investigate the self-healing and injectability properties of the preservative hydrogel. The experimental results are as follows: Figure 2As shown in Figure C, under high strain conditions (300%), the hydrogel networks of groups Cn, TC-Cn1, TC-Cn2, TC-Cn3, TC-Cn4, and TC-Cn5 were immediately disrupted, causing G' to decrease to approximately 8720, 5627, 9540, 14539, 10240, and 11630 Pa, respectively. When adjusted to a low strain (1%), the G' and G" of the preservation hydrogels almost completely recovered within seconds. The results indicate that the preservation hydrogels possess good self-healing properties.

[0043] The results of the compressive strength test are as follows Figure 2 As shown in Figure D, the maximum compressive strengths of Cn, TC-Cn1, TC-Cn2, TC-Cn3, TC-Cn4, and TC-Cn5 were 56.4 N, 45.6 N, 23.5 N, 58.4 N, 24.6 N, and 33.3 N, respectively. With the introduction of nanoemulsions, the mechanical properties of the preservation hydrogel exhibited a trend of "first decreasing - then increasing - then decreasing again." Among them, TC-Cn3 showed the highest compressive strength (58.4 N), slightly higher than the control group Cn, while TC-Cn2, TC-Cn4, and TC-Cn5 showed significant decreases. The results indicate that the introduction of an appropriate amount of nanoemulsion helps optimize the gel network structure, while excessively low or high amounts weaken its mechanical properties.

[0044] 2.2 Swelling and Freeze-Thaw Cycles The water absorption of the preservative hydrogel was determined by a swelling test. The initial weight of the preservative hydrogel was recorded. Subsequently, the hydrogels were immersed in 3 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate (PB) buffer, and the mass of the hydrogels was measured periodically at time intervals of 4 h, 8 h, 12 h, and 24 h. ), and calculate the swelling rate at different time points.

[0045]

[0046] After recording the initial weight (W1), the preserved hydrogel was placed into 50 mL centrifuge tubes. The hydrogel was then frozen at -18°C for 20 h, followed by thawing at 30°C for 3 h. This freeze-thaw cycle was repeated up to 5 times. After each cycle, the sample was centrifuged at 4000 rpm for 10 min, and the weight of the remaining hydrogel in each tube (W2) was measured. The dehydration shrinkage rate was calculated using the following formula:

[0047] The swelling rate experiment results are as follows Figure 2As shown in Figure E, the swelling rate of each group of preservative hydrogels changed relatively smoothly over 4-24 h, with no significant differences among the groups, and tended to stabilize at 24 h. However, at 24 h, differences emerged among the treatment groups. The Cn group had the highest swelling rate at 148%, while TC-Cn4 and TC-Cn5 had relatively lower rates of 138% and 135%, respectively, showing significant differences. This may be because the hydrophobic components such as tocopherol and citral in the nanoemulsion reduced the overall hydrophilicity of the system, thereby inhibiting further water ingress into the network structure.

[0048] The results of the dehydration shrinkage rate experiment are as follows: Figure 2 As shown in Figure F, the dehydration shrinkage rate of all samples increased with the number of freeze-thaw cycles, indicating that repeated freeze-thaw cycles damage the hydrogel network structure. The Cn group maintained a relatively high dehydration shrinkage rate of 36% after 5 freeze-thaw cycles, indicating that its network structure was relatively loose and its freeze-thaw stability was poor. With the introduction of nanoemulsions, the dehydration shrinkage rates of TC-Cn1 to TC-Cn4 groups decreased significantly, indicating that the addition of an appropriate amount of nanoemulsion helps improve the structural stability of the hydrogel. Among them, the TC-Cn3 and TC-Cn4 groups maintained relatively low dehydration shrinkage rates of 24% and 21% respectively after 5 freeze-thaw cycles, exhibiting the best freeze-thaw resistance. This phenomenon may be related to the dispersion effect of nanoemulsions in the carrageenan network; their presence may play a certain role in filling and supporting the gel network structure, thereby improving the system stability. The introduction of hydrophobic components in the nanoemulsions may affect the water distribution in the system, thereby mitigating the damage to the network structure during freeze-thaw cycles. In addition, there may be some non-covalent interactions between the nanoemulsion and carrageenan, which helps to maintain the stability of the gel network structure.

[0049] 2.3 Scanning Electron Microscopy (SEM) The surface and cross-sectional morphology of the freeze-dried preservation hydrogels were analyzed using field emission scanning electron microscopy. A scanning voltage of 5 kV was set for sample observation. The microscopic results and morphology of all samples were recorded at 25x and 50x magnification.

[0050] Experimental results are as follows Figure 3 As shown, Cn exhibits an irregular fibrous network structure with uneven pore distribution and a relatively loose structure. With increasing nanoemulsion concentration, TC-Cn1 to TC-Cn4 gradually form a more regular and uniform three-dimensional porous network structure. The results indicate that the introduction of an appropriate amount of nanoemulsion helps to regulate the microstructure of carrageenan gel.

[0051] 2.4 Fourier Transform Infrared (FTIR) Fourier transform infrared (FTIR) spectra of the freeze-dried preservation hydrogel were obtained using FTIR spectroscopy. The experimental scanning range was 4000–500 cm⁻¹. 1 The resolution is 4 cm. 1 .

[0052] Experimental results are as follows Figure 4 As shown in Figure A, the molecular structure characteristics of the preservation hydrogel were analyzed using FT-IR. All samples were analyzed at 3200–3400 cm⁻¹. - A broad and strong absorption peak appears at ¹, corresponding to the -OH stretching vibration, mainly originating from the hydroxyl groups and bound water in carrageenan. With the addition of the nanoemulsion, this absorption peak undergoes a slight shift, indicating a change in hydrogen bonding within the system. At 2920 cm⁻¹... - A CH stretching vibration peak was observed near ¹, and its intensity increased slightly with increasing nanoemulsion content, possibly related to the introduction of hydrophobic components into the nanoemulsion. At 1640 cm⁻¹... - ¹ and 1410–1450 cm - The ¹ position corresponds to the C=O stretching vibration and CH bending vibration, respectively. 1200–1000 cm - The region ¹ shows a distinct absorption peak, attributed to the COC and CO stretching vibrations in the carrageenan polysaccharide backbone; approximately 840 cm⁻¹ - The peak at position ¹ represents the characteristic absorption peak of the sulfate ester group. All samples retained the characteristic absorption peak of carrageenan, indicating that the introduction of the nanoemulsion did not disrupt the main structure of the carrageenan.

[0053] 2.5 X-ray diffraction (XRD) XRD patterns of the freeze-dried hydrogels were obtained using an X-ray diffractometer. The samples were irradiated with Cu / Ka at 45 kV and 40 mA and scanned in the 2θ range of 5–80°.

[0054] Experimental results are as follows Figure 4 As shown in Figure B, the Cn sample exhibits typical broad diffraction peaks, indicating that it is primarily amorphous. This phenomenon is common in polysaccharide-based or biopolymer matrices due to the lack of long-range ordered structures. With increasing nanoemulsion loading, no new sharp diffraction peaks were observed in TC-Cn1-TC-Cn5, indicating that the introduction of the nanoemulsion did not lead to the formation of a significant crystal structure. Instead, the diffraction pattern maintained broad peaks, indicating that the system retained its amorphous characteristics. However, the peak broadening changed slightly with increasing nanoemulsion content. This may be related to the influence of the nanoemulsion on the arrangement of carrageenan molecules. Similar phenomena have been reported in biopolymer systems loaded with an oil phase, suggesting that the introduction of the oil phase may affect the polymer chain segment arrangement, thereby altering the material's crystallinity. The results indicate that the introduction of the nanoemulsion did not change the predominantly amorphous structural characteristics of the system.

[0055] 2.6 Thermogravimetric Analysis (TGA) Thermogravimetric analysis was performed on the freeze-dried hydrogel using a thermogravimetric analyzer. The sample was placed in a ceramic crucible and heated from 30 °C to 600 °C at a rate of 10 °C / min under a nitrogen atmosphere.

[0056] Experimental results are as follows Figure 4 As shown in C, 4D, the first stage occurs in the temperature range of 30–100 °C, mainly due to water evaporation. The weight loss rate of Cn is close to 6%, while TC-Cn1 is approximately 3%, TC-Cn2 approximately 4%, TC-Cn3 approximately 5%, and TC-Cn4 and TC-Cn5 both approximately 5%. The second stage occurs in the range of 180–210 °C. TC-Cn3, TC-Cn4, and TC-Cn5 exhibit lower weight loss rates in this range, suggesting that the introduction of nanoemulsions may have improved the thermal stability of the system in this stage. The third stage occurs in the range of 240–260 °C, where the weight loss rate increases sharply. The Cn loss rate is close to 12%, while TC-Cn1 is approximately 18%, and TC-Cn3 and TC-Cn4 reach approximately 25% and 24%, respectively. This stage typically corresponds to the thermal decomposition of the polymer backbone and the rapid decomposition of organic components. The increased weight loss in the nanoemulsion-loaded samples is likely due to the volatilization or thermal decomposition of the introduced oil phase or active substances at high temperatures, thus accelerating the overall mass loss. The fourth stage of degradation occurred in the 400-430℃ range (Cn loss rate close to 3%, TC-Cn4 approximately 5%). This stage is generally related to the further decomposition of carbonized residues. Overall, all samples exhibited good thermal stability. The introduction of the nanoemulsion did not alter the main thermal degradation behavior of the preservation hydrogel. These results indicate that the prepared preservation hydrogel possesses good thermal stability under food preservation and refrigeration application conditions.

[0057] 2.7 Antioxidant activity Take 2.0 g of the preservative hydrogel, cut it into small pieces of approximately 20 mm × 20 mm, and place them in a mixed solution containing 5 mL of deionized water and 5 mL of anhydrous ethanol. Extract on a shaker for 12 h. After extraction, centrifuge the sample at 4000 rpm for 10 min and collect the supernatant. Mix 2.0 mL of the supernatant with 2 mL of 0.17 mM DPPH ethanol solution, react at room temperature in the dark for 30 min, and measure the absorbance of the reaction solution at 517 nm.

[0058] DPPH free radical scavenging activity (%) = (1-A1 / A0)×100% Where A1 is the absorbance of the DPPH solution containing the sample, and A0 is the absorbance of the DPPH solution.

[0059] First, prepare ABTS solution (7.4 mM) and potassium persulfate solution (2.6 mM). Then, mix 5 mL of the ABTS solution with 5 mL of the potassium persulfate solution and let stand overnight at room temperature, protected from light. Before use, further dilute the ABTS mixture with ethanol to achieve an absorbance of 0.7 at 734 nm. Mix 1 mL of the supernatant with 2 mL of the ABTS solution, let stand for 6 minutes, and then measure the absorbance at 734 nm.

[0060] ABTS free radical scavenging activity (%) = (1-B1 / B0)×100% Where B1 is the absorbance of the ABTS solution containing the sample, and B0 is the absorbance of the ABTS working solution.

[0061] The antioxidant activity of the preservation hydrogel was determined by ABTS and DPPH assays. Figure 4 E, 4F). TC exhibited low ABTS radical scavenging activity, approximately 37%, indicating that carrageenan itself has limited antioxidant capacity. The introduction of TC nanoemulsion significantly improved the ABTS radical scavenging rate, with TC-Cn1 and TC-Cn2 reaching approximately 62% and 89%, respectively. With further increases in nanoemulsion content, the ABTS radical scavenging rates of TC-Cn3, TC-Cn4, and TC-Cn5 groups all exceeded 95%, and the differences between groups were not significant (P>0.05). DPPH radical scavenging experimental results ( Figure 4 F) The results showed a similar trend to those obtained by ABTS assay. The Cn group exhibited only about 14% DPPH radical scavenging rate, while the DPPH scavenging activity gradually increased with increasing nanoemulsion loading. The TC-Cn2 and TC-Cn3 groups reached approximately 48% and 53%, respectively, while the TC-Cn4 and TC-Cn5 groups increased to 59% and 61%, significantly higher than the other treatment groups (P<0.05). These results indicate that the hydrophobic active components in the nanoemulsion can effectively exert antioxidant effects.

[0062] 2.8 Antibacterial activity Staphylococcus aureus CMCC 26003 and Escherichia coli ATCC 1122 were diluted with PBS to a concentration of 1 × 10⁻⁶. 6 CFU / mL. Next, 100 μL of Staphylococcus aureus and Escherichia coli suspension (1×10⁻⁶) was added. 6 The samples (CFU / mL) were spread on agar plates. Then, the samples (Cn, TC-Cn1, TC-Cn2, TC-Cn3, TC-Cn4, TC-Cn5) were gently placed on the agar plates and incubated in a 37 °C incubator for 24 h. The colonies on the culture medium were counted and the bacterial activity was calculated.

[0063] Experimental results are as follows Figure 5As shown in Figure A, group Cn did not show significant antibacterial activity against either strain, and the dense colony growth on the plate surface indicated that carrageenan itself lacks antibacterial activity. In contrast, with the increase of TC nanoemulsion loading, a clear antibacterial zone gradually formed around the TC-Cn series preservation hydrogels, indicating that they had a good inhibitory effect on both pathogens. Figure 5 As shown in B, the TC-Cn3, TC-Cn4, and TC-Cn5 groups all significantly reduced the bacterial activity of Escherichia coli. Among them, the TC-Cn4 and TC-Cn5 treatment groups showed the best antibacterial effect, with bacterial activity reduced to 43%, indicating that the high-load nanoemulsion can effectively inhibit the growth of Escherichia coli. Figure 5 C10 showed that *Staphylococcus aureus* also exhibited high sensitivity to the preservation hydrogel, and the bacterial activity gradually decreased with increasing TC addition. The TC-Cn5 group showed the most significant antibacterial effect, with bacterial activity decreasing to 50%. The three-dimensional network structure of carrageenan enabled the sustained release of the active ingredient, thus maintaining a continuous antibacterial effect. Notably, the TC-Cn preservation hydrogel showed good antibacterial activity against both *Escherichia coli* and *Staphylococcus aureus*, with a slightly stronger inhibitory effect against *Escherichia coli*. This may be related to the differences in cell structure and sensitivity to active ingredients among different bacterial species. Overall, the results indicate that the prepared preservation hydrogel has broad-spectrum antibacterial capabilities and can provide effective microbial control for the preservation of seafood.

[0064] Example 3: Evaluation of the effect of preserving fresh-cut golden pomfret 1. Processing of golden pomfret meat Live golden pomfret were euthanized at low temperatures, and the back fillets were removed and rinsed with refrigerated sterile water. In a clean bench, fish fillets of the same size were randomly divided into three groups: ① Control group (no treatment); ② Cn group (Cn preservative hydrogel was placed in the same sealed container as the fish fillets, but not in direct contact); ③ TC-Cn4 group (TC-Cn4 preservative hydrogel was placed in the same sealed container as the fish fillets, also avoiding direct contact, forming a non-contact active preservation system). All samples were stored at 4 ℃, and samples were taken on days 0, 2, 4, 6, 8, and 10 of storage to determine various quality indicators.

[0065] 2. pH and mass loss Each sample was aseptically chopped and placed in 5 times (w / v) of deionized water, then homogenized until the fish meat was completely broken down. The broken sample solution was centrifuged at 10,000 g for 10 minutes using a low-temperature centrifuge, and the supernatant was collected. The pH value was measured using a pH meter.

[0066] The initial mass of the golden pomfret fillets was denoted as m0, and the mass change was recorded every 2 days (denoted as m1). Three repeated measurements were performed for each sample group, and the mass loss rate was calculated.

[0067]

[0068] 3. TVB-N The TVB-N content in fresh-cut golden pomfret was determined according to the Chinese standard (GB 5009.228-2016). Golden pomfret fillet samples were homogenized in deionized water and allowed to stand for 30 minutes. Before distillation, 5 mL of MgO (10 g / L) and 10 mL of deionized water were added to 5 mL of the supernatant. After distillation for 5 minutes, the distillate was titrated with hydrochloric acid (0.001 M) and two drops of a mixed indicator (1% methylene blue and 2% methyl red ethanol solution) to calculate TVB-N.

[0069] 4. TBARS The TBARS value was determined by ultraviolet spectrophotometry. 2 g of chopped fresh-cut pomfret was added to 10 mL of trichloroacetic acid solution (10%, w / v) and centrifuged. Then, 2 mL of the supernatant was mixed with 2 mL of thiobarbituric acid solution (0.67%, w / v). The control group used trichloroacetic acid solution instead of the supernatant. The mixture was boiled for 40 min, rapidly cooled to room temperature, and the absorbance was measured at 532 nm using an ultraviolet spectrophotometer. Results are expressed as mg malondialdehyde (MDA) / kg sample.

[0070] Experimental results are as follows Figure 6As shown, in the early stage of storage (day 0), the samples from each treatment group had a basically consistent appearance, exhibiting the color and structural integrity expected of fresh fish meat. With prolonged storage, the samples from each group gradually showed varying degrees of quality deterioration. By day 4, the control group samples showed obvious yellowing and slight shrinkage; the Cn group showed slight yellowing, while the TC-Cn4 treatment group maintained its original color and showed no obvious shrinkage. By day 8, the control group samples showed significant yellowing and obvious dehydration, which is usually closely related to lipid oxidation and protein denaturation in the fish meat. The Cn group showed a more significant quality decline, but the degree of deterioration was relatively mild; the TC-Cn4 group maintained a better appearance, with lower discoloration and less structural damage, showing a strong ability to maintain quality. By day 10, the control group samples had severely deteriorated, with darker color, drier tissue, and a significant decrease in freshness; the Cn group showed moderate spoilage, while the TC-Cn4 treatment group samples maintained a relatively acceptable appearance. The results showed that, compared with the control group and the Cn group, TC-Cn4 treatment was more effective in delaying the appearance deterioration of fresh-cut pomfret fillets during refrigeration and extending their shelf life. This may be attributed to the fact that the preservative hydrogel effectively inhibits moisture loss and oxidation during storage, thereby maintaining the color and tissue structure stability of the fish.

[0071] During the storage of aquatic products, pH value is an important physicochemical indicator for measuring changes in freshness and the degree of spoilage. For example... Figure 7 Figure A shows the pH trend of fresh-cut golden pomfret fillets in different treatment groups during storage at 4℃. With prolonged storage, the pH value of each group gradually increased. This is mainly attributed to the action of endogenous enzymes and microbial metabolism in the fish meat, leading to protein degradation and the generation of alkaline substances such as ammonia and trimethylamine, thus increasing the system pH. Comparing the treatment groups, the pH value of the TC-Cn4 group was significantly lower than that of the Control and Cn groups, indicating a better inhibitory effect on spoilage. Especially on the 4th day of storage, the pH of the Cn group had increased to 6.88, while the pH of the TC-Cn4 group was 6.77, showing a significant difference from the control group (P<0.05). The results indicate that the TC-Cn4 preservation hydrogel can effectively inhibit protein degradation and microbial growth in fresh-cut golden pomfret fillets during refrigeration, thereby delaying pH increases and maintaining the product's physicochemical stability. This is consistent with its results in maintaining appearance quality, further validating its excellent preservation performance.

[0072] During storage, water evaporation and tissue metabolism are the main factors leading to weight loss in fish. For example... Figure 7As shown in Figure B, from day 2 onwards, the weight loss rate of fresh-cut golden pomfret fillets in all treatment groups began to increase. The Control group reached a weight loss rate of 9.6% on day 8, significantly higher than the Cn group's 7.2% and the TC-Cn4 group's 5.3%. This rapid increase in weight loss rate reflects a decline in the water-retention capacity of the fresh-cut golden pomfret fillets, leading to accelerated quality deterioration. By day 10 of storage, the weight loss rate in the Control group had risen to 13.6%, while the TC-Cn4 group was significantly lower than the other groups, at only 6.6%, nearly 7% lower than the control group. This result indicates that all groups of fresh-cut golden pomfret fillet samples experienced weight loss during storage, but the TC-Cn4 treatment group showed the least weight loss, demonstrating superior water-retention performance.

[0073] During the storage of aquatic products, the TBARS value is an important indicator for measuring the degree of lipid oxidation and quality deterioration. This study observed the changing trends of TBARS values ​​in different treatment groups of golden pomfret fillets during storage at 4°C. The experimental results are as follows: Figure 7 As shown in Figure C, the TBARS values ​​of all treatment groups showed an increasing trend during storage, which is consistent with the findings reported by Javanshir et al. regarding the delay of lipid oxidation in green tiger shrimp by chitosan coatings enhanced with natural active substances. This indicates that the golden pomfret fillets underwent lipid oxidation to varying degrees during storage. After 10 days of storage, the TBARS value of the Control group increased from the initial 0.07 mg MDA / 100g to 1.56 mg MDA / 100g, the TBARS value of the Cn group increased from 0.07 mg MDA / 100g to 1.09 mg MDA / 100g, while the TBARS value of the TC-Cn4 group increased from 0.07 mg MDA / 100g to only 0.97 mg MDA / 100g. The TBARS value of the Control group was consistently higher than that of the Cn and TC-Cn4 groups, indicating that the Cn and TC-Cn4 preservation hydrogels have an inhibitory effect on lipid oxidation. Among them, the TBARS value of the TC-Cn4 treatment group was significantly lower than that of other groups (P<0.05), indicating that it has a stronger inhibitory effect on lipid oxidation.

[0074] Golden pomfret fillets are rich in high-quality protein and free amino acids, but during refrigeration, they are susceptible to degradation by microorganisms and endogenous enzymes, leading to the production of volatile alkaline nitrogenous substances such as ammonia, trimethylamine, and dimethylamine. This results in a continuous increase in TVB-N content. Therefore, TVB-N is often used as an important indicator for evaluating the freshness and spoilage level of aquatic products. Figure 7D). Initially, the TVB-N content of all three groups was 16.58 mg / 100g. With increasing storage time, the TVB-N content of each group showed an increasing trend. After 10 days of storage, the TVB-N content of the Control group increased from 16.58 mg / 100g to 22.55 mg / 100g; the Cn group increased to 20.49 mg / 100g; and the TC-Cn4 group only increased to 18.52 mg / 100g, indicating that both the Cn and TC-Cn4 groups could delay the spoilage of pomfret fillets and inhibit the rate of protein decomposition. The TVB-N value of the TC-Cn4 treatment group was significantly lower than that of the other groups (P<0.05), indicating that it had a stronger inhibitory effect on protein decomposition. This is mainly because citral and tocopherol in the preservation hydrogel can inhibit microbial growth, thereby reducing the reproduction rate of microorganisms in the fish.

[0075] In summary, this invention provides a tocopherol-citral nanoemulsion-supported carrageenan hydrogel and its preparation method. For the first time, tocopherol-citral nanoemulsion is combined with a three-dimensional network structure of carrageenan to construct a non-contact active preservation system with sustained-release function. The prepared composite hydrogel exhibits excellent gelling properties, mechanical properties, freeze-thaw stability, and structural stability. The nanoemulsion is dispersed within the carrageenan network, enabling the continuous release of active ingredients. This hydrogel possesses excellent antioxidant and antibacterial activities, effectively inhibiting the growth of *Escherichia coli* and *Staphylococcus aureus*, reducing lipid oxidation levels, and improving the stability of the preservation system. Furthermore, this invention uses natural carrageenan, tocopherol, and citral as the main raw materials, and the preparation process does not require chemical cross-linking agents, exhibiting good biocompatibility, safety, and environmental friendliness. This invention constructs a non-contact preservation method that achieves continuous preservation through the slow release of volatile active ingredients without direct contact with food. It effectively inhibits microbial growth, lipid oxidation, and protein degradation during the storage of fresh-cut golden pomfret, reducing pH value, weight loss, TBARS value, and TVB-N content, thus maintaining product quality and extending shelf life. Compared with traditional direct-contact active packaging, this invention reduces the risk of active substances migrating into food, improving safety and preservation duration. The nanoemulsion-loaded carrageenan hydrogel is simple to prepare, uses widely available raw materials, is low-cost, and is easy to scale up. It can be widely used in the preservation of aquatic products, livestock and poultry meat products, fruits and vegetables, and other perishable foods, showing good prospects for industrial application and promotional value.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A carrageenan hydrogel loaded with tocopherol-citral nanoemulsion, characterized in that, The carrageenan hydrogel is obtained by loading a tocopherol-citral nanoemulsion onto carrageenan, and the tocopherol-citral nanoemulsion is obtained by dissolving tocopherol and citral in whey protein.

2. The carrageenan hydrogel as described in claim 1, characterized in that, The concentration of carrageenan is 15-45 mg / mL.

3. The carrageenan hydrogel as described in claim 1, characterized in that, The loading of tocopherol-citral nanoemulsion in the carrageenan hydrogel is 10-50%.

4. The carrageenan hydrogel as described in claim 1, characterized in that, The volume ratio of tocopherol to citral in the tocopherol-citral nanoemulsion is 1:(1-5).

5. The method for preparing carrageenan hydrogel loaded with tocopherol-citral nanoemulsion as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) Dissolve whey protein in deionized water, add tocopherol and citral, homogenize and sonicate to obtain tocopherol-citral nanoemulsion; (2) Dissolve carrageenan in water and heat at 80-100℃ until completely dissolved; (3) Add the nanoemulsion obtained in step (1) to the carrageenan solution obtained in step (2) and stir until homogeneous; (4) Cool to form a gel, and obtain a nanoemulsion-loaded carrageenan hydrogel.

6. The preparation method according to claim 5, characterized in that, In step (1), the homogenization speed is 10,000-20,000 rpm and the homogenization time is 1-10 min.

7. The preparation method according to claim 5, characterized in that, The ultrasonic treatment time in step (1) is 3-10 min.

8. The use of carrageenan hydrogel as described in any one of claims 1-4 in the preparation of active preservative materials.

9. An active preservative material, characterized in that, The active preservative material includes the carrageenan hydrogel according to any one of claims 1-4.

10. The active preservative material as described in claim 9, characterized in that, The active preservative material is used for the preservation of aquatic products, livestock and poultry meat products, or fruits and vegetables.

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