A locust bean gum-chitosan-based edible antibacterial coating material and use thereof

Abstract

This invention provides an edible antibacterial coating material based on locust bean gum and chitosan in the field of food antibacterial preservation technology. The preparation involves adding a locust bean gum solution to a chitosan solution and stirring until homogeneous to obtain a locust bean gum-chitosan solution; then adding an ε-polylysine hydrochloride solution to obtain the locust bean gum-chitosan-based antibacterial coating material. This antibacterial coating material can be used for the preservation of chilled meat, significantly inhibiting the growth and reproduction of pathogenic and spoilage-causing microorganisms during storage, improving the sensory characteristics of chilled meat such as color, reducing storage losses, lowering the volatile basic nitrogen content and TBARS value, improving sensory quality, and effectively extending the shelf life of chilled meat. The coating material of this invention is prepared from edible-grade raw materials, possessing both safety and residue-free characteristics, and the preparation process is simple and cost-controllable, showing broad application potential in the entire meat product preservation and distribution chain.

Description

Technical Field

[0001] This invention belongs to the field of food antibacterial and preservation technology, and specifically relates to a method for preparing an edible composite coating using locust bean gum and chitosan as raw materials and its application. Background Technology

[0002] Pork, as the most consumed livestock meat globally, is rich in key nutrients such as protein, fat, and water. However, throughout the entire supply chain, from slaughtering and processing to transportation and storage, it is susceptible to contamination by pathogenic and putrefactive microorganisms such as Escherichia coli, Staphylococcus aureus, Salmonella, and Pseudomonas, accompanied by quality deterioration phenomena such as moisture loss, fat oxidation, and color discoloration. This not only impairs the sensory quality and nutritional function of pork but also significantly increases the risk of foodborne illnesses for consumers. Therefore, developing meat preservation technologies that are both efficient and safe has become one of the core needs that the food industry urgently needs to address.

[0003] Currently, traditional methods of pork preservation still primarily rely on the addition of chemical preservatives such as potassium sorbate and nitrites. While these chemical agents can inhibit the proliferation of harmful microorganisms and extend the shelf life of pork to some extent, long-term excessive use can easily lead to residues in meat products, thus posing potential health risks. Furthermore, they cannot simultaneously address the issues of moisture loss and fat oxidation during pork storage, failing to meet current consumers' core demands for "natural, safe, and high-quality" food, thus limiting their application in the modern food industry. Edible polysaccharide coatings, with their excellent biocompatibility, biodegradability, and safety with no residue, are gradually becoming a key research direction and application hotspot in the food preservation field, replacing chemical preservatives. Chitosan (CS), a cationic polysaccharide obtained from chitin through deacetylation, has advantages such as wide availability, simple preparation, and low application cost. It also possesses excellent film-forming properties and low gas permeability, and has been widely used in the food industry. However, single-component chitosan coatings suffer from drawbacks such as a narrow antibacterial spectrum, limited antibacterial activity, insufficient thermodynamic stability of the coating system, and poor uniformity of adhesion to pork surfaces, making it difficult to meet the technical requirements for long-term meat preservation in actual production. Locust bean gum (LBG), a natural anionic polysaccharide, exhibits significant advantages in inhibiting moisture loss in food due to its strong hydrophilicity and film-forming density. Combining chitosan with LBG can optimize the physicochemical properties and functional efficacy of the coating through their synergistic effect, providing a feasible path to improve pork preservation. Furthermore, ε-polylysine hydrochloride (ε-PLH) is a natural antimicrobial peptide derived from microbial metabolism. Its significant advantages lie in its broad-spectrum antibacterial properties and good thermal stability, allowing it to withstand the mild thermal environments during food processing and storage. By applying this substance to a chilled meat preservation packaging system, and systematically investigating its inhibitory effects on the growth and reproduction of typical spoilage bacteria and pathogenic bacteria in chilled meat under different concentrations and storage conditions, the study aims to clarify the antibacterial action patterns and influencing factors, thus providing a reliable scientific basis for the industrial development and practical application of high-performance antibacterial packaging.

[0004] In summary, current pork preservation technologies suffer from several key challenges. Chemical preservatives pose potential safety risks, while single-component and traditional polysaccharide coatings suffer from weak antibacterial activity, poor system stability, and limited shelf-life. These technological shortcomings have become critical bottlenecks hindering the improvement of pork preservation quality and industrial upgrading. Therefore, there is an urgent need to develop a novel antibacterial coating technology that is edible, possesses excellent antibacterial efficacy, effectively delays moisture loss, fat oxidation, and microbial contamination during pork storage and distribution, and is suitable for industrial production applications. This technology aims to address the core technological pain points in the pork preservation field. Summary of the Invention

[0005] Focusing on the bottlenecks of the existing technologies, this invention discloses a locust bean gum-chitosan-based edible antibacterial material. Relying on the composite synergistic effect of edible locust bean gum and chitosan, and supplemented with functional modifiers for performance optimization, it effectively solves the technical pain points of existing chemical preservatives, such as safety hazards, insufficient antibacterial efficacy of single and traditional compound polysaccharide coatings, poor stability, and limited preservation time, thereby meeting the high-quality preservation requirements in pork storage and distribution.

[0006] Therefore, the present invention provides a locust bean gum-chitosan-based edible antibacterial coating material, the preparation method of which includes the following steps: 1) A chitosan solution was prepared by adding chitosan to an acetic acid-water solution; the mass / volume concentration of chitosan in the chitosan solution was 15 g / L; the volume fraction of acetic acid in the acetic acid-water solution was 1.5%. 2) Sophora japonica gum was added to water to prepare a sophora japonica gum solution with a mass / volume concentration of 10 g / L. 3) Add the locust bean gum solution to the chitosan solution and stir to mix well. The volume ratio of locust bean gum solution to chitosan solution is 1:(1-2). 4) Prepare an ε-polylysine hydrochloride solution and add it to a locust bean gum-chitosan solution. After stirring thoroughly, a locust bean gum-chitosan-based antibacterial coating material is obtained. The mass / volume concentration of the ε-polylysine hydrochloride in the locust bean gum-chitosan solution is 5-15 g / L.

[0007] Further, in step 1), the chitosan solution is placed in a 70°C heat-collecting constant-temperature magnetic stirrer, and the rotation speed is adjusted to 50 rpm. While the solution is being magnetically stirred in a water bath, 0.6% of the solution volume of glycerol is added as a plasticizer to obtain the chitosan solution.

[0008] Further, in step 2), when preparing the locust bean gum solution, it is placed in a 70°C heat-collecting constant-temperature magnetic stirrer, and the speed is adjusted to 50 rpm to obtain the locust bean gum solution.

[0009] Further, in step 3), when preparing the locust bean gum-chitosan solution, the locust bean gum solution is placed in a 70°C heat-collecting constant temperature magnetic stirrer, the speed is adjusted to 50 rpm, and the chitosan solution is added to the locust bean gum solution while the water bath is magnetically stirred; the water bath is magnetically stirred for 2 h, and the locust bean gum-chitosan solution is finally prepared.

[0010] Further, in step 4), when preparing the ε-polylysine hydrochloride solution, the ε-polylysine hydrochloride is dissolved in water, and magnetically stirred at room temperature until completely dissolved, and the stirring speed is adjusted to 30 rpm to obtain the ε-polylysine hydrochloride solution for later use.

[0011] Further, in step 4), the locust bean gum-chitosan solution is placed in a heat-collecting constant temperature magnetic stirrer at 60°C, and the speed is adjusted to 50 rpm. While the solution is being magnetically stirred in a water bath, the prepared ε-polylysine hydrochloride solution is added. After the solution is magnetically stirred in a water bath for 20 minutes, the mixture is ultrasonically treated for 5 minutes to remove air bubbles. Finally, the locust bean gum-chitosan-based antibacterial coating material is prepared.

[0012] The present invention also provides the use of the above-mentioned locust bean gum-chitosan-based antibacterial coating material, which can be used for the preservation of chilled meat. The chilled meat is soaked in the locust bean gum-chitosan-based antibacterial coating material or the locust bean gum-chitosan-based antibacterial coating material is brushed or sprayed onto the surface of the chilled meat to form an antibacterial coating on the surface of the chilled meat.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes a chilled meat preservation material obtained by combining a locust bean gum-chitosan coating loaded with ε-polylysine hydrochloride. Applying this material to chilled meat preservation offers several advantages: First, it improves the color of chilled meat, reduces storage losses, and ensures color stability during storage, thereby enhancing consumer sensory acceptance. Second, it significantly inhibits the growth and reproduction of microorganisms during meat storage, reducing colony counts, volatile basic nitrogen content, and TBARS values, effectively extending the shelf life of meat products. Third, it is suitable for industrial production; the preparation process requires no complex equipment, the steps are simple and controllable, the coating solution can be prepared in batches and can be stably stored at 4°C for 8-10 days, and the impregnation coating method is compatible with pork processing lines, possessing the potential for large-scale application and specifically filling the technological gap in the field of edible coatings for pork preservation.

[0014] The edible coating prepared by this invention can be used as a preservation material for chilled meat. Chitosan (CS), a natural cationic polysaccharide with excellent biocompatibility and biodegradability, forms a more compact composite matrix with locust bean gum (LBG) through intermolecular hydrogen bonding, significantly optimizing the physicochemical stability and interfacial adhesion performance of the single polysaccharide coating. Furthermore, ε-polylysine hydrochloride (ε-PLH) is introduced as a functional antibacterial component. As a natural protein-based broad-spectrum antibacterial agent derived from microbial metabolism, ε-PLH possesses excellent biocompatibility, safety, high efficiency, and environmental friendliness. It can produce a synergistic antibacterial effect with the composite matrix, effectively enhancing the coating's inhibitory efficacy against spoilage and pathogenic bacteria in chilled meat, specifically addressing the inherent defects of single chitosan coatings such as weak antibacterial activity and insufficient stability. In addition, ε-polylysine hydrochloride can produce a synergistic effect when combined with the polysaccharide matrix and other components in the food system. Loading it onto a locust bean gum-chitosan composite carrier to prepare an edible preservation material can further enhance its broad-spectrum antibacterial activity and durability. The preservation coating described in this invention can form a uniform and dense thin film barrier on the surface of chilled meat, which can effectively block surface moisture evaporation and gas exchange, reduce moisture loss and fat oxidation, and fully preserve the original nutritional value of the food, significantly extend the shelf life of chilled meat, and achieve synergistic effect of antibacterial properties and light transmittance, providing a safe and efficient technical solution for high-quality preservation of chilled meat. Attached Figure Description

[0015] Figure 1 This is a schematic diagram illustrating the preparation of an edible antibacterial preservative coating.

[0016] Figure 2 The particle size of the edible antibacterial preservative coating.

[0017] Figure 3 Zeta potential diagram of an edible antibacterial preservative coating.

[0018] Figure 4 pH is the pH value for an edible antibacterial preservative coating.

[0019] Figure 5 Frequency scan G' for edible antibacterial preservative coating.

[0020] Figure 6 Frequency scan G" for edible antibacterial preservative coating.

[0021] Figure 7 The turbidity of the edible antibacterial preservative coating.

[0022] Figure 8 SEM image of the edible antibacterial preservative coating.

[0023] Figure 9 FTIR analysis of the edible antibacterial preservative coating.

[0024] Figure 10 Thermogravimetric analysis diagram of edible antibacterial preservative coating.

[0025] Figure 11 The coating is suitable for use as an edible antibacterial preservative.

[0026] Figure 12 The washability of the edible antibacterial preservative coating.

[0027] Figure 13 The DPPH free radical scavenging ability of the edible antibacterial preservation coating.

[0028] Figure 14 The antibacterial properties of the edible antibacterial preservative coating.

[0029] Figure 15 To investigate the effect of LBS+1ε coating on TVC of chilled meat, the control group was untreated chilled meat, and the experimental group was chilled meat treated with LBS+1ε.

[0030] Figure 16 For the LBS+1ε coating on the color of chilled fresh meat L *The control group consisted of untreated chilled meat, while the experimental group consisted of chilled meat treated with LBS+1ε preservation.

[0031] Figure 17 For the LBS+1ε coating on the color of chilled fresh meat a *The control group consisted of untreated chilled meat, while the experimental group consisted of chilled meat treated with LBS+1ε preservation.

[0032] Figure 18 For the LBS+1ε coating on the color of chilled fresh meat b *The control group consisted of untreated chilled meat, while the experimental group consisted of chilled meat treated with LBS+1ε preservation.

[0033] Figure 19 To investigate the effect of LBS+1ε coating on the texture of chilled meat, the control group consisted of untreated chilled meat, while the experimental group consisted of chilled meat treated with LBS+1ε for preservation.

[0034] Figure 20 To investigate the effect of LBS+1ε coating on the pH of chilled meat, the control group consisted of untreated chilled meat, while the experimental group consisted of chilled meat treated with LBS+1ε for preservation.

[0035] Figure 21 To investigate the effect of LBS+1ε coating on TVB-N in chilled fresh meat, the control group consisted of untreated chilled fresh meat, while the experimental group consisted of chilled fresh meat treated with LBS+1ε.

[0036] Figure 22To investigate the effect of LBS+1ε coating on TBARs of chilled meat, the control group consisted of untreated chilled meat, while the experimental group consisted of chilled meat treated with LBS+1ε.

[0037] Figure 23 To investigate the effect of LBS+1ε coating on the weight loss rate of chilled meat, the control group consisted of untreated chilled meat, while the experimental group consisted of chilled meat treated with LBS+1ε.

[0038] Figure 24 To investigate the effect of LBS+1ε coating on the cooking rate of chilled meat, the control group consisted of untreated chilled meat, while the experimental group consisted of chilled meat treated with LBS+1ε. Detailed Implementation

[0039] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0040] Unless otherwise specified, the reagents and methods used in the following examples were prepared and operated according to conventional methods; the other reagents and raw materials used are all commercially available.

[0041] The indicator bacteria used in this invention, including *Pseudomonas lactis* K-8, *Pseudomonas krusei* C-16, *Salmonella enterica* L1, *Salmonella enterica* N20, *Bacillus subtilis* C2, *Bacillus thaliana* N1, *Staphylococcus aureus* N1, and *Staphylococcus aureus* N2, are all wild-type isolates derived from meat. Example 1

[0042] Coating preparation 1. Preparation of chitosan solution: Chitosan was dissolved in a beaker containing an aqueous acetic acid solution to prepare a chitosan solution with a mass / volume concentration of 15 g / L. The solvent was an aqueous acetic acid solution with a volume concentration of 1.5% (v / v). The beaker was placed in a 70℃ constant temperature magnetic stirrer with the rotation speed adjusted to 50 rpm. While stirring magnetically in a water bath, 6 mL / L (volume fraction 0.6%) of glycerol was added as a plasticizer until the chitosan solution was completely dissolved.

[0043] 2. Preparation of Sophora japonica gum solution: In a beaker, a Sophora japonica gum solution was prepared. The amount of Sophora japonica gum was 10 g / L, and the solvent was distilled water. The beaker was placed in a 70°C heat-collecting constant temperature magnetic stirrer, and the speed was adjusted to 50 rpm until the Sophora japonica gum solution was completely dissolved.

[0044] 3. The preparation method of locust bean gum-chitosan solution is as follows: Place 50 mL of locust bean gum solution in a 70°C heated magnetic stirrer with a heat collector, adjust the speed to 50 rpm, and add 50 mL of chitosan solution to the locust bean gum solution while stirring magnetically in a water bath. Stir magnetically in a water bath until the two are completely mixed, and finally prepare about 100 mL of locust bean gum-chitosan solution.

[0045] 4. Preparation of Sophora japonica gum-chitosan-based antibacterial coating material (1) The preparation method of ε-polylysine hydrochloride solution is as follows: Dissolve 0.5 g, 1 g and 1.5 g of ε-polylysine hydrochloride in 1 mL, 2 mL and 3 mL of distilled water respectively, and stir magnetically at room temperature until completely dissolved. Adjust the speed to 30 rpm to obtain ε-polylysine hydrochloride solution, and put it into an Erlenmeyer flask for later use.

[0046] (2) Place 100 mL of locust bean gum-chitosan mixed solution in a 60°C heat-collecting constant temperature magnetic stirrer, adjust the speed to 50 rpm, and add the prepared ε-polylysine hydrochloride solution while stirring magnetically in a water bath. After stirring magnetically in a water bath for 20 minutes, sonicate the mixed solution for 5 minutes to remove air bubbles, and finally prepare the locust bean gum-chitosan-based antibacterial coating material.

[0047] Sophora japonica gum-chitosan-based antibacterial coating material can be used for the preservation of chilled meat. Specifically, chilled meat is soaked in the Sophora japonica gum-chitosan-based antibacterial coating material or the Sophora japonica gum-chitosan-based antibacterial coating material is brushed or sprayed onto the surface of chilled meat, thereby forming an antibacterial coating on the surface of chilled meat.

[0048] To verify the performance and effectiveness of this material, the following formulations were used in this invention.

[0049] Table 1. Different edible antibacterial coating formulations

[0050] Note: LBG is locust bean gum, CS is chitosan, LBS is a mixture of locust bean gum and chitosan, LBS+0.5ε, LBS+1ε and LBS+1.5ε are 0.5 g, 1 g and 1.5 g of ε-polylysine hydrochloride added to the mixed solution, respectively. Example 2

[0051] Characterization of coatings 1. Particle size and zeta potential of the locust bean gum-chitosan-based antibacterial coating material The particle size and zeta potential of an edible locust bean gum-chitosan-based antibacterial coating material were determined using a Malvern laser particle size analyzer. Measurements were performed at room temperature. The particle size analyzer parameters were set as follows: particle refractive index 1.453, particle absorptivity 0.001, dispersant refractive index 1.330, and sample equilibration time 2 min.

[0052] Depend on Figure 2 It can be seen that LBG has a relatively large particle size, which affects its dispersion rate and uniformity in solution. CS has the next largest particle size. LBS has a smaller particle size than LBG and CS, indicating that mixing refines the particles. After adding ε-polylysine hydrochloride, the particle sizes of LBS + 0.5ε and LBS + 1ε further decreased, because the interaction between ε-polylysine hydrochloride and the system molecules inhibits particle aggregation; the particle size of LBS + 1.5ε increased slightly, possibly because the addition ratio was too high, resulting in excessive intermolecular interactions and causing some particles to agglomerate.

[0053] Figure 3 The results showed that LBG had a negative zeta potential with a large absolute value, indicating that its particle surface carried a large amount of negative charge, resulting in strong electrostatic repulsion between particles and good dispersibility. CS had a positive zeta potential, indicating that its particle surface carried a positive charge and had a certain degree of dispersion stability. LBS had a zeta potential close to 0, indicating weak electrostatic interaction between particles, poor stability, and a tendency to aggregate. The system with added ε-polylysine hydrochloride showed a positive zeta potential that gradually increased, indicating that it increased the positive charge on the particle surface, enhanced electrostatic repulsion, and improved stability.

[0054] 2. pH of Sophora japonica gum-chitosan-based antibacterial coating material The pH of the locust bean gum-chitosan-based antibacterial coating material was measured at room temperature using a pH meter. Figure 4 It is known that locust bean gum is a neutral polysaccharide with a pH close to 7. Chitosan requires acidic conditions to dissolve, therefore the acidity of acetic acid directly causes the system pH to drop to around 3.8. The pH of the LBS group is approximately 4.0, because locust bean gum has a buffering effect on the acidic system of chitosan, but the acidity of chitosan still dominates, with a pH slightly higher than the CS group and much lower than the LBG group. After adding different amounts of ε-polylysine hydrochloride, the pH did not differ significantly from the CS and LBS groups.

[0055] 3. Rheological properties of locust bean gum-chitosan-based antibacterial coating materials The static and dynamic rheological properties of the locust bean gum-chitosan-based antibacterial coating material (coating emulsion) were determined using a rotational rheometer at 25°C. The coating emulsion was dropped onto the center of the test platform of the rheometer, using a 40 mm diameter stainless steel plate clamp at a 4° angle with a 100 μm gap between the plate and the platform. After equilibration for 2 min, the properties were measured at shear rates of 1-300 s⁻¹. -1Rate scanning was performed on the coating emulsion within a certain range to investigate its viscosity behavior.

[0056] The frequency sweep of the coating emulsion was measured in a small-amplitude oscillation mode. The frequency dependence of the coating emulsion was investigated using a 40 mm diameter stainless steel plate with a 100 μm gap between the clamp and the platform, a 1% strain, and a frequency range of 0.10–10 Hz. All measurements were repeated three times at 25 °C, and the values ​​of G′ and G″, representing the storage modulus and loss modulus, were recorded.

[0057] Depend on Figure 5 It is evident that, compared to LBG and CS alone, LBS inherently possesses a better balance between adhesion at low shear and ease of flow at high shear. The addition of ε-polylysine hydrochloride further optimizes the rheological properties. During processing and application, viscosity decreases moderately with increasing shear rate, making the coating material easier to apply evenly to the meat surface and improving ease of handling. Shear force disrupts the newly formed interactions between ε-polylysine hydrochloride and LBS molecules, causing molecular chain untangling and orientation, thus reducing viscosity. However, due to the strong intermolecular interactions, the viscosity decrease is relatively smaller at the same shear rate compared to LBS, and the higher the addition ratio, the more pronounced this phenomenon of relatively slow viscosity decrease due to stronger intermolecular interactions becomes.

[0058] Depend on Figure 6 It is evident that LBG exhibits primarily elastic rheological properties, leaning more towards solid characteristics, resulting in a relatively tough coating that is less prone to viscous deformation. In contrast, CS coatings are relatively softer and more susceptible to viscous deformation. LBS coatings, on the other hand, possess both elasticity and viscosity, exhibiting a relatively balanced performance. Considering both elasticity and viscosity variations, appropriately increasing the amount of ε-polylysine hydrochloride can optimize the coating's mechanical properties. This allows the coating to possess both sufficient elasticity to withstand external impacts and tension without cracking, and appropriate viscosity to better adhere to the object's surface during application, while also adapting to minor surface deformations to some extent without peeling off.

[0059] 4. Turbidity determination of locust bean gum-chitosan-based antibacterial coating material The turbidity of the locust bean gum-chitosan-based antibacterial coating was determined by ultraviolet spectrophotometer with a wavelength of 400-800 nm. The results are expressed as transmittance T and turbidity T of the system. Figure 7The results showed that the light transmittance of the single LBG coating system was poor, while the light transmittance of the coating was significantly improved when chitosan CS and glycerol were added to the system. This phenomenon is attributed to the excellent film-forming properties and gas barrier properties of chitosan itself, while glycerol, as a plasticizer, can effectively improve the flexibility and surface uniformity of the coating. The two work synergistically to optimize the microstructure of the coating, thereby improving its light transmittance. Further introduction of ε-polylysine hydrochloride into the LBS composite system not only endowed the coating with antibacterial function but also simultaneously improved its light transmittance. Within the experimental concentration range, the rule of "the higher the ε-PL addition, the better the light transmittance" was observed, achieving a synergistic effect between the antibacterial and light transmittance properties of the coating.

[0060] 5. Scanning electron microscopy (SEM) characterization of the locust bean gum-chitosan-based antibacterial coating material The locust bean gum-chitosan-based antibacterial coating material was freeze-dried using a vacuum freeze dryer. After freeze-drying, the samples were observed using a GeminiSEM 4800 scanning electron microscope at 2 kV and a magnification of 1000x. Figure 8 It is evident that LBG exhibits numerous wrinkled textures and a relatively loose structure, while CS shows obvious cracks and a lamellar structure. Compared to single polysaccharides, LBS shows significantly improved surface smoothness and a substantial reduction in wrinkles and cracks. At LBS +0.5ε, a fine network structure begins to appear on the surface. The introduction of ε-polylysine hydrochloride increases the intermolecular crosslinking points, and the structure transitions from dense to porous network. When the amount of ε-polylysine hydrochloride added is 1 g, the network structure becomes further enriched and uniform. The crosslinking effect enables the system to form a continuous three-dimensional network, which may have a positive impact on the material's mechanical properties and barrier properties.

[0061] 6. Determination of Infrared Spectroscopy of Sophora japonica Gum-Chitosan-Based Antibacterial Coating Material After freeze-drying the locust bean gum-chitosan-based antibacterial coating material using a vacuum freeze dryer, each sample was compressed into tablets with 100 mg of KBr. Subsequently, Fourier transform infrared (FT-IR) spectroscopy was performed on a Varian 670 spectrometer in attenuated total reflectance mode, recording the spectrum from 400 to 4000 cm⁻¹. -1 The resolution is 2 cm. -1 The scan count was 32. The addition of ε-polylysine hydrochloride introduced amino and peptide bonds, forming new composite structures with locust bean gum and chitosan through electrostatic and hydrogen bonding interactions. With increasing ε-polylysine hydrochloride content, the composite structure gradually transformed from a simple mixture to a complex molecular structure, with intermolecular interactions continuously strengthening and stabilizing. Figure 9 ).

[0062] 7. Determination of TGA in Edible Sophora japonica gum-chitosan-based antibacterial coating material Thermogravimetric analysis (TGA) was performed using a Pyris1 instrument. Appropriate amounts of lyophilized sample (6-10 mg) were placed in an analytical sample aluminum tray, which was then sealed. An unloaded tray served as a control. Nitrogen gas was used for protection at a flow rate of 20 mL / min, and the temperature was uniformly increased from 20°C to 200°C at a rate of 10°C / min. Figure 10 This indicates that after the two polysaccharides are mixed, the intermolecular interactions, such as the binding of chitosan amino or hydroxyl groups with locust bean gum hydroxyl groups, alter the thermal decomposition kinetics, thus adjusting the thermal decomposition mechanism of the composite system. ε-polylysine hydrochloride can regulate the thermal stability of the composite system through its interaction with the polysaccharides, and the effect of 1% on improving thermal stability is more significant.

[0063] 8. Coverage and washability of locust bean gum-chitosan-based antibacterial coating materials The cardboard coating test effectively demonstrates the transparency and uniform coverage of the coating on chilled meat surfaces. To better illustrate the cleaning process of the locust bean gum-based coating, the coating solution was stained with methylene blue. Then, a quartz disc was immersed in the coating solution for 20 seconds and air-dried for 5 minutes. This coating process was repeated to simulate the coating process. Finally, the coating film on the quartz disc was removed by ultrasonic cleaning for 30 seconds. Figure 11 The coated paperboard test clearly demonstrates that after being coated with the methylene blue-stained solution, the paperboard can be completely covered without any defects, indicating that the coating can be evenly applied to the surface of the pork. Furthermore, after the paperboard dries and forms a film, the pattern of the unstained coating on the paperboard is clearly visible, indicating that the coating has excellent transparency and does not affect the aesthetics of the sample. Figure 12 Evidence was provided regarding the cleaning process of the locust bean gum-based barrier coating. After ultrasonic cleaning in water for 30 seconds, the coating (staining) completely dissolved in the water, indicating that the coating can be completely removed by washing before consumption. The uniform coverage and transparency of the coating were tested on the test panel.

[0064] 9. Functional properties of locust bean gum-chitosan-based antibacterial coating materials 9.1 Antioxidant function DPPH was prepared into a 0.065 mmol / L solution with anhydrous ethanol and then transferred to a brown bottle for later use. 200 μL of different concentrations of locust bean gum-chitosan-based antibacterial coating material was pipetted into 3.8 mL of the prepared DPPH solution. After thorough mixing, the mixture was reacted in the dark at room temperature for 30 min. The absorbance was measured at 517 nm using a spectrophotometer. The DPPH scavenging rate was calculated using the following formula.

[0065] C% = {1 - (A1 - A2) / A3} × 100 C%: Indicates clearance rate; A1: Absorbance of 200 μL membrane solution + 3.8 mL DPPH; A2: Absorbance of 200 μL membrane solution + 3.8 mL anhydrous ethanol; A3: Absorbance of 200 μL solvent + 3.8 mL DPPH.

[0066] Depend on Figure 13 It was found that the scavenging activity of locust bean gum (LBG) was approximately 34%, while that of chitosan (CS) was approximately 74%. Locust bean gum has a loose molecular structure and fewer active sites; chitosan, due to its amino and other active groups, exhibits superior scavenging activity. When the two are mixed, the scavenging activity increases to approximately 90%, demonstrating the synergistic effect of locust bean gum and chitosan. Through intermolecular bonding and electrostatic interactions, they not only optimize structural compactness but also increase the exposure of active groups, thereby enhancing oxidative scavenging ability. The scavenging activity increases with the increase in the proportion of ε-polylysine hydrochloride because the amino and carboxyl groups in the molecule can form more cross-links with the polysaccharide, further expanding the three-dimensional network structure and facilitating the function of the active sites.

[0067] 9.2 Antibacterial function Paper discs (6 mm in diameter) coated with a locust bean gum-chitosan-based antibacterial material were placed in an inoculation solution of 0.1 mL 10 6 Bacteria were cultured on soybean casein agar plates containing CFU / mL bacterial suspension at 37°C for 16 h to assess the antibacterial activity of the antibacterial coating. The inhibition zone formed was recorded. Figure 14 The results showed that locust bean gum (LBG) and the locust bean gum-chitosan mixture (LBS) had no antibacterial effect on any of the tested strains. Chitosan (CS) showed only a weak inhibitory effect on *Pseudomonas lactis* K-8 and *Staphylococcus aureus* N2, but no inhibition on the other strains. After the introduction of ε-polylysine hydrochloride, the system exhibited antibacterial activity against multiple strains, with strain specificity and concentration dependence. Example 3

[0068] 1. Raw material pretreatment Pork purchased from the supermarket was processed in a sterile environment. The fascia, excess fat and connective tissue on the surface of the loin were removed, and the meat was cut into pieces of 40±2 g and refrigerated at 4℃ for later use.

[0069] 2. Preservation treatment of chilled meat Pretreated chilled meat was randomly divided into two groups. The treatment group was immersed in a locust bean gum-chitosan-based antibacterial coating material for 30 seconds, then excess coating was dripped off, and the meat was air-dried at 25°C under ventilation. The control group received no treatment. After drying, the coated chilled meat was packaged into sterile sampling bags and stored at 4°C. Samples were taken every two days to measure relevant physiological and quality indicators until the 8th day.

[0070] 3. Changes in total bacterial count (TVC) of pork Under aseptic conditions, weigh 10 g of sample and place it into a sterile bag containing 90 mL of sterile physiological saline. Then homogenize the sample. Perform 10-fold serial dilutions on the homogenate, and take 0.01 mL of each of the three appropriate dilutions. Use TSA plates to assess the total viable bacterial count. Inoculate the sample and incubate at 37°C for 24 hours. The results are expressed as 1 g CFU / g.

[0071] Total bacterial count reflects the degree of microbial contamination in food. Relevant national standards specify the relationship between freshness and total bacterial count for chilled meat: Grade 1 freshness ≤ 4 lg CFU / g; Grade 2 freshness 4-6 lg CFU / g; spoiled meat ≥ 6 lg CFU / g. Figure 15 It can be seen that, regardless of whether the temperature was 37℃ or 4℃, the total bacterial count of both groups of chilled pork increased with the extension of refrigeration time. At 37℃ and 4℃, the initial TVC of chilled pork was 4.69 lg CFU / g and 2.6 lg CFU / g, respectively, indicating that the meat was still fresh. The total bacterial count increased within 0-6 days of storage. At 37℃, after 4 days of storage, the TVC of the control group reached 6.15 lg CFU / g, indicating that the meat had spoiled, while the experimental group reached 4.91 lg CFU / g, indicating that the meat was in a sub-fresh state. Bacterial growth was slow in the 4℃ group, and the overall TVC was much lower than that of the 37℃ group. However, regardless of whether the temperature was 37℃ or 4℃, the total viable bacteria count in the LBS+1ε treatment group was significantly lower than that in the control group at the same time point.

[0072] 4. Determination of pork color After cutting fresh pork samples from both the control and experimental groups into small pieces, the color index of the fresh meat during refrigeration was immediately measured using a CR-400 fully automatic colorimeter and recorded. L *(light-dark), a *(Red-Green) and b * (Yellow-Blue). The color of chilled meat is considered the most important sensory attribute influencing consumers' willingness to buy. L The * value represents the brightness value. L The higher the value, the better the luster of the meat. Figure 16 , 17 18 represents chilled pork from each treatment group during storage. L *value,a *value and b * Changes in values. Among them, the composite treatment group... L The rate of decrease in * values ​​was significantly lower in the combined treatment group than in the control group, and the meat samples in the combined treatment group decreased throughout the entire cold storage period. L The relatively gradual change in the * value indicates that the compound treatment group can slow down the decrease in brightness during the refrigeration of chilled meat, but the compound preservative has no synergistic effect on the brightness of chilled meat. a The * value represents the redness value, which mainly depends on the color and oxidation state of myoglobin in the meat. The two groups of samples were stored for 8 days... a The values ​​were all significantly lower than the initial value on day 0, which may be due to the further oxidation of oxymyoglobin to metmyoglobin. Furthermore, the treatment group... a The values ​​were significantly higher than those of the control group, indicating that the compound natural preservative can prevent oxidation and thus reduce browning caused by oxidation. b * The value represents the yellowness level. b An excessively high * value indicates accelerated oxidation of the meat, leading to the proliferation of bacteria. Figure 16 , 17 As shown in Figures 1 and 18, the yellowness values ​​of both groups of chilled pork gradually increased with the extension of storage time, and the rate of increase in yellowness value of the experimental group was significantly lower than that of the control group. This indicates that the coating can protect the color of chilled pork.

[0073] 5. Determination of the texture of pork Meat samples were tested using a texture analyzer in TPA mode. Parameter settings: probe selected P / 36 R; speed before, during, and after testing all 1.0 mm / s; test interval 5 s; trigger force 5 g; data acquisition rate 400 PPS; strain 75%. The hardness and elasticity of the meat samples were measured.

[0074] Texture properties are important indicators reflecting changes in the tissue structure of chilled meat. Among them, hardness, elasticity, and adhesiveness can well reflect the impact of preservation coatings on the texture properties of chilled pork under refrigeration conditions. Figure 19 As shown, the hardness and elasticity indices generally decreased with prolonged storage time, while the adhesiveness increased. This is because, over time, enzymes with protein-degrading activity are produced in the pork, leading to protein degradation in the muscle, damage to myofibrils, and muscle softening. Furthermore, the control group showed the fastest rate of decrease in these indices, while the experimental group showed a slower rate of decrease. Comprehensive analysis indicates that the composite preservation coating effectively maintains the textural properties of chilled pork, thus contributing to the preservation of its excellent edible quality and commercial value.

[0075] 6. Determination of pH value in pork A portable handheld pH meter was calibrated at two points using a standard solution. The calibrated pH meter was then inserted into a piece of meat, and each piece was measured three times. The average value was taken as the final result. This was in accordance with GB 5009.237-2016, "National Food Safety Standard - Determination of pH Value in Food". pH value can be used as an indicator of meat freshness. Relevant national standards specify the following relationship between meat freshness and pH: Grade 1 freshness: pH 5.80-6.20; Grade 2 freshness: pH 6.30-6.60; Spoiled meat: pH above 6.70. Figure 20 As shown, the pH of chilled pork in all groups initially decreased and then increased throughout the storage period. This is because the fermentation of glycogen and phosphocreatine produces lactic acid and phosphoric acid, increasing acidity and causing the pH value of the chilled pork to be lower than the initial value on day 2. The pH value of all groups gradually increased in the later stages because, during the extended storage process, microorganisms proliferated and produced nitrogenous alkaline substances such as amino acids, ammonia, and trimethylamine through enzymatic reactions. Therefore, the increase in pH value is considered to be a stage of decomposition and spoilage of pork. The pH value of the LBS+1ε group was significantly lower than that of the CK group, indicating that the combination of ε-PL and CS has a synergistic antibacterial and preservative effect, further inhibiting the growth of microorganisms in pork, slowing down the production and accumulation of alkaline substances, thereby controlling the pH rise and achieving the preservation effect.

[0076] 7. Determination of volatile basic nitrogen TVB-N is a commonly used indicator for evaluating meat freshness. The detection method refers to GB5009.228-2016 "National Food Safety Standard - Determination of Volatile Basic Nitrogen in Food". Apply water-soluble gel to the edge of the diffusion dish. Add 1 mL of boric acid solution and 1 drop of mixed indicator to the inner chamber of the dish. Accurately add 1 mL of filtrate to the outer chamber and cover with a ground glass lid. Quickly add 1 mL of saturated potassium carbonate solution through the slit, immediately push the ground glass lid flat to seal the diffusion dish tightly, and gently rotate it in a circular motion on the table to thoroughly mix the sample solution and saturated potassium carbonate solution. Then, incubate at 37°C for 2 hours, cool to room temperature, remove the lid, and titrate with standard hydrochloric acid titration solution (0.01 mol / L). Use a mixed indicator solution of 1 part methyl red ethanol solution and 5 parts bromocresol green ethanol solution; the endpoint color should be purple-red. Use a mixed indicator solution of 2 parts methyl red ethanol solution and 1 part methylene blue ethanol solution; the endpoint color should be blue-purple.

[0077] TVB-N is a core physicochemical indicator for assessing meat freshness. It is defined as the total amount of volatile amines produced by the decomposition of proteins and non-protein nitrogenous compounds in meat under the action of microbial metabolism and enzymatic hydrolysis. Its content directly reflects the degree of spoilage of meat. According to the national standard GB 2707-2016 "National Food Safety Standard for Fresh (Frozen) Livestock and Poultry Products", the TVB-N content of fresh livestock meat shall not exceed 15.0 mg / 100g. Figure 21 The results showed that the TVB-N content in both the control group and the composite coating treatment group increased significantly with prolonged storage throughout the entire cold storage period. This phenomenon is attributed to the increased microbial proliferation and endogenous enzyme activity during storage, which accelerates the decomposition and metabolism of nitrogenous substances in the meat, leading to the accumulation of volatile amines. Specifically, the TVB-N content in the control group reached 15.31 mg / 100g on day 6 of storage, exceeding the national standard limit, indicating that the meat sample had entered the spoilage stage. In contrast, the TVB-N content in the sample treated with the composite preservation coating showed a slow upward trend, reaching 13.75 mg / 100g on day 8, still within the national standard allowable range (close to the limit). Furthermore, throughout the entire storage period, the TVB-N content in the treatment group was significantly lower than that in the control group at all time points. This difference is mainly due to the fact that ε-polylysine hydrochloride (ε-PL) in the composite coating system has a significant inhibitory effect on the growth and reproduction of most spoilage microorganisms. At the same time, the physical barrier effect of the composite coating can slow down the rate of microbial infection and enzymatic reaction, thereby effectively inhibiting the decomposition of nitrogenous substances and the generation of volatile amines in meat products, and ultimately achieving long-term maintenance of the freshness of chilled pork, showing excellent preservation performance.

[0078] 8. Determination of Thiobarbituric Acid Reactive Substances (TBARS) in Pork The effect of packaging film on lipid oxidation during pork storage was assessed by measuring the amount of 2-thiobarbituric acid reactive substances (TBARS). 5 g samples from each sampling time (2 g surface and 3 g interior) were homogenized in 25 mL trichloroacetic acid solution and centrifuged (12,000 rpm). g (5 minutes, 4℃), take 5 mL of the filtrate and add the same volume of 0.02 mol / L 2-thiobarbituric acid solution, heat in a 90℃ water bath for 40 min, remove and cool, 3000 g After centrifugation for 10 min, the supernatant was collected, 5 mL of chloroform was added, and the mixture was allowed to stand for separation. The absorbance of the supernatant was measured at wavelengths of 532 nm and 600 nm.

[0079] TBARS value (mg / kg) = {(A 532 - A 600 ) / 155}× 0.1 × 72.6 × 1000 In the formula: 155 is the molar absorptivity; A 532 and A 600 The absorbance values ​​are 532 nm and 600 nm, respectively.

[0080] The TBARS value is a classic indicator for quantifying the degree of lipid oxidation in meat products. Its value is positively correlated with the level of lipid oxidation, and when the TBARS value is >1 mg / kg, the meat products have undergone obvious oxidative deterioration. Figure 22 The results showed that the TBARS value of the control group pork increased significantly throughout the entire cold storage period. However, the TBARS value of the composite coating group increased at a significantly slower rate, remaining at a low level. The initial TBARS value of the chilled meat was 0.491 mg / kg. By day 6 of storage, the TBARS values ​​of the control group and the composite treatment group reached 1.17 mg / kg and 0.89 mg / kg, respectively. The control group had exceeded the critical value for oxidative deterioration, while the composite treatment group was significantly lower than the control group. The core mechanism of this result is that LBG in the composite coating has excellent oxygen barrier properties. The dense composite matrix formed by LBG and CS can effectively block the contact between external oxygen and the meat surface, reducing the substrate supply for lipid oxidation. Simultaneously, the composite coating can inhibit the growth and reproduction of putrefactive microorganisms on the meat surface, reducing the promoting effect of microbial metabolites on lipid oxidation, thereby synergistically delaying the lipid oxidation process of chilled meat.

[0081] 9. Water-holding capacity of pork The initial mass of the pork is recorded as m1. After the corresponding storage time, the pork is removed, its surface moisture is wiped dry, and its weight is recorded as m2. The weight loss rate is calculated using the following formula: Weight loss rate = {(m1 − m2) / m1} × 100% Cut the chilled fresh meat into pieces of approximately 3 cm × 3 cm × 6 cm, weigh each piece (m1), place them in a cooking bag, remove the air, seal the bag, and heat in a water bath at 85°C for 30 minutes. After removing the bag, cool it to room temperature and weigh it (m2).

[0082] Cooking loss rate = {(m1 - m2) / m1} × 100% Pork contains 70%-75% water, and water-holding capacity, as one of the core indicators for measuring meat quality, refers to the ability of animal muscle to retain moisture throughout the entire chain of processes, including cutting, processing, and subsequent storage. Its quality can be quantitatively assessed through key indicators such as juice loss rate and cooking loss rate. Figure 23It was found that with the extension of storage time, the juice loss rate of pork in both the control group and the treatment group showed an overall increasing trend, and the rate of increase in the early stage of storage (the stage of rapid moisture loss) was significantly higher than that in the later stage (the stage of slower moisture loss). On the 8th day of storage, the juice loss rates of the control group and the treatment group were 2.61% and 1.80%, respectively. The juice loss rate of the treatment group was significantly lower than that of the control group (p<0.05), and the increase in the juice loss rate of the control group throughout the entire storage period was significantly higher than that of the composite coating treatment group. This is mainly attributed to the excellent film-forming properties of the composite coating formed by locust bean gum and chitosan, which can build a physical protective barrier on the surface of chilled meat, effectively preventing the migration of internal moisture to the outside of the meat, thereby reducing juice loss.

[0083] Depend on Figure 24 The results showed that the cooking loss rate of pork in both groups gradually increased with the extension of storage period. On the 8th day of storage, the cooking loss rate of the treatment group (25.44%) was significantly lower than that of the control group (33.46%), indicating that the composite bio-preservation coating can reduce the rate of moisture loss by slowing down the increase in pH value of meat during storage. On the one hand, the composite coating can effectively prevent the evaporation of surface moisture of chilled meat and reduce dry loss; on the other hand, the physical barrier formed by the coating can inhibit the growth and reproduction of microorganisms on the surface of chilled meat, thereby reducing the damage of microbial metabolism to the water-holding capacity of meat, and ultimately achieving efficient retention of moisture in pork.

[0084] The volume ratio of locust bean gum solution to chitosan solution can be 1:2. Appropriately increasing the chitosan solution can still maintain a good antibacterial effect.

Claims

1. A locust bean gum-chitosan-based edible antibacterial coating material, characterized in that, Its preparation method includes the following steps: 1) A chitosan solution was prepared by adding chitosan to an acetic acid-water solution; the mass / volume concentration of chitosan in the chitosan solution was 15 g / L; the volume fraction of acetic acid in the acetic acid-water solution was 1.5%. 2) Sophora japonica gum was added to water to prepare a sophora japonica gum solution with a mass / volume concentration of 10 g / L. 3) Add the locust bean gum solution to the chitosan solution and stir to mix well to obtain the locust bean gum-chitosan solution; the volume ratio of the locust bean gum solution to the chitosan solution is 1:(1-2). 4) Prepare an ε-polylysine hydrochloride solution and add it to a locust bean gum-chitosan solution. After stirring thoroughly, a locust bean gum-chitosan-based antibacterial coating material is obtained. The mass / volume concentration of the ε-polylysine hydrochloride in the locust bean gum-chitosan solution is 5-15 g / L.

2. A sophora japonica gum-chitosan-based edible antibacterial coating material according to claim 1, characterized in that, In step 1), the chitosan solution is placed in a 70°C heat-collecting constant-temperature magnetic stirrer with the speed adjusted to 50 rpm. While the solution is being magnetically stirred in a water bath, 0.6% of the solution volume of glycerol is added as a plasticizer to obtain the chitosan solution.

3. A sophora japonica gum-chitosan-based edible antibacterial coating material according to claim 1, characterized in that, In step 2), when preparing the locust bean gum solution, it is placed in a 70°C heat-collecting constant-temperature magnetic stirrer, and the speed is adjusted to 50 rpm to obtain the locust bean gum solution.

4. A sophora japonica gum-chitosan-based edible antibacterial coating material according to claim 1, characterized in that, In step 3), when preparing the locust bean gum-chitosan solution, the locust bean gum solution is placed in a 70°C heat-collecting constant temperature magnetic stirrer, the speed is adjusted to 50 rpm, and the chitosan solution is added to the locust bean gum solution while the water bath is magnetically stirred; the water bath is magnetically stirred for 2 h to finally prepare the locust bean gum-chitosan solution.

5. A sophora japonica gum-chitosan-based edible antibacterial coating material according to claim 1, characterized in that, In step 4), when preparing the ε-polylysine hydrochloride solution, the ε-polylysine hydrochloride is dissolved in water and magnetically stirred at room temperature until completely dissolved. The stirring speed is adjusted to 30 rpm to obtain the ε-polylysine hydrochloride solution for later use.

6. A sophora japonica gum-chitosan-based edible antibacterial coating material according to claim 1, characterized in that, In step 4), the locust bean gum-chitosan solution is placed in a 60°C heat-collecting constant temperature magnetic stirrer, and the speed is adjusted to 50 rpm. While the solution is being magnetically stirred in a water bath, the prepared ε-polylysine hydrochloride solution is added. After stirring magnetically in a water bath for 20 minutes, the blended solution is ultrasonically treated for 5 minutes to remove air bubbles from the solution, and finally the locust bean gum-chitosan-based antibacterial coating material is prepared.

7. The use of the locust bean gum-chitosan-based edible antibacterial coating material as described in any one of claims 1-6, characterized in that, For the preservation of chilled meat, the chilled meat is soaked in a locust bean gum-chitosan-based antibacterial coating material or the locust bean gum-chitosan-based antibacterial coating material is brushed or sprayed onto the surface of the chilled meat to form an antibacterial coating on the surface of the chilled meat.

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