Preparation method of water-soluble chitosan derivative for chicken preservation

Abstract

The application discloses a preparation method of water-soluble chitosan derivatives which can be used for chicken preservation. The method uses chitosan as raw material, introduces lysine groups through chemical modification, and significantly improves the water solubility and antibacterial activity of chitosan. The specific steps include: after chitosan is reacted with N,N'-dicyclohexyl carbodiimide and orthophosphoric acid, ethanol precipitation and suction filtration are carried out, then the chitosan is refluxed with lysine in PBS buffer (pH 4.5), finally sodium cyanoborohydride reduction, dialysis and freeze-drying are carried out, and water-soluble chitosan derivatives are obtained. The minimum inhibitory concentration (MIC) of the derivatives on staphylococcus aureus and escherichia coli is 3.90 mg / mL, the growth of microorganisms during the storage of chicken can be effectively inhibited, the increase of TVB-N value and pH can be delayed, the weight loss rate can be reduced, and the shelf life can be prolonged. The application solves the problems of poor water solubility and limited application of traditional chitosan, and provides an efficient and safe solution for natural food preservatives.

Description

Technical Field

[0001] This invention belongs to the field of bioactive polysaccharide technology, specifically relating to a method for preparing a water-soluble chitosan derivative that can be used for chicken preservation. Background Technology

[0002] Food preservation is a crucial step in ensuring that food remains fresh, edible, and safe throughout production, transportation, storage, and sales. With increasing consumer demand for food and the continuous expansion of the food industry, coupled with an ever-expanding food supply chain and deepening international trade, food preservation faces numerous challenges. The core role of food preservation technology is becoming increasingly prominent. Its application not only guarantees excellent food quality but also meets consumers' expectations for healthy food, laying a solid foundation for the sustainable development of the food industry. Therefore, finding safe and effective preservation methods is of great significance to the food processing industry and food safety.

[0003] Chitosan, an alkaline polysaccharide obtained by deacetylation of natural chitin, possesses biodegradability, biocompatibility, and antibacterial activity, showing significant potential in food preservation. However, chitosan has extremely poor water solubility, dissolving only in weakly acidic solutions, which greatly limits its large-scale application in chicken preservation—it cannot form a uniform protective film, and its antibacterial components cannot effectively act on the surface and shallow tissues of chicken, thus limiting its preservation effect. Existing chitosan modification methods (such as amidation and esterification) suffer from complex processes, limited improvement in water solubility or antibacterial properties, and are prone to degradation in chicken preservation applications, resulting in insufficient preservation time and difficulty in simultaneously controlling microbial growth and changes in physicochemical indicators. Therefore, developing a chitosan modification technology that is simple to process, cost-effective, and can simultaneously improve water solubility and antibacterial properties, and applying it to chicken preservation to achieve synergistic control of key indicators such as total bacterial count, TVB-N, pH value, and weight loss rate, is of great significance for promoting the commercial application of natural preservatives in the chicken and meat processing industry. Summary of the Invention

[0004] Technical Problem to be Solved: To address the aforementioned technical problems, the present invention aims to provide a method for preparing and applying a water-soluble chitosan derivative for chicken preservation. Using chitosan as a raw material, a chemical reaction is employed to impart two aldehyde active sites to it. Lysine (Lys) is grafted onto the carbon backbone of the chitosan to prepare the chitosan derivative. This chitosan derivative exhibits good antibacterial activity and no significant cytotoxicity, demonstrating promising prospects for development and utilization.

[0005] Technical solution: A method for preparing a water-soluble chitosan derivative that can be used for chicken preservation, comprising the following steps: S1. Chitosan, N,N'-dicyclohexylcarbodiimide and phosphoric acid are mixed and dissolved in dimethyl sulfoxide, and the mixture is stirred and reacted at 25°C for 22-25 h. S2. Pour in excess ethanol for alcohol precipitation, discard the supernatant, filter the precipitate to obtain filter cake; S3. Dissolve the filter cake in excess distilled water, add lysine, adjust the pH to 4.5 with PBS buffer, and reflux in a reflux condenser at 75°C for 7-9 hours to obtain the reflux product; S4. Centrifuge the reflux product, take the supernatant, add sodium cyanoborohydride at 5‰ of the chitosan mass, and react at room temperature for 4-5 hours. S5. Add excess ethanol again for alcohol precipitation, filter and dissolve the filter cake in distilled water, then perform water-soluble dialyzing for 48 hours. S6. After freeze-drying in a freeze dryer for 45-50 hours, a water-soluble chitosan derivative suitable for chicken preservation is obtained.

[0006] Furthermore, the degree of deacetylation of the chitosan in step S1 is ≥85%, and the molecular weight is 50~100 kDa.

[0007] Furthermore, in step S1, the mass-to-volume ratio of chitosan, N,N'-dicyclohexylcarbodiimide, orthophosphoric acid, and dimethyl sulfoxide is 1.00 g: 5.10 g: 0.38 mL: (15~25) mL.

[0008] Furthermore, the stirring rate in step S1 is 500~600 r / min to ensure that the reaction system is mixed uniformly.

[0009] Furthermore, the mass ratio of lysine to chitosan in step S3 is 4.50g:1.00g.

[0010] Furthermore, the centrifugation speed in step S4 is 6000 r / min, and the centrifugation time is 20-22 min.

[0011] Furthermore, the dialysis bag used for dialysis in step S5 has a molecular weight cutoff of 14,000 Da.

[0012] Furthermore, the freeze-drying conditions described in step S6 are: vacuum degree ≤ 10 Pa, temperature ≤ -50℃.

[0013] The present invention also provides a water-soluble chitosan derivative prepared by the above preparation method that can be used for chicken preservation. This derivative contains both amino and carboxyl groups, has significantly improved water solubility, and has excellent antibacterial activity.

[0014] The present invention also provides the application of the above-mentioned water-soluble chitosan derivatives that can be used for chicken preservation in chicken preservation. Beneficial effects

[0015] 1. This invention uses chitosan as a raw material and, through a chemical reaction, enables it to have two active aldehyde sites, and successfully grafts lysine onto the carbon skeleton of chitosan to prepare chitosan derivatives (DLC). 2. The antibacterial mechanism of chitosan relies on the interaction between the positive charge on its C2 amino group and the negative charge on the bacterial cell membrane, thus causing bacterial death and playing an antibacterial role. This invention uses chemical modification to transform the original C2 amino group of chitosan into two aldehyde groups, so that the amino group of lysine undergoes a Schiff base reaction with the aldehyde group and is branched onto the carbon skeleton of chitosan. Due to the special structure of lysine, it contains not only α-NH2 but also ε-NH2. Therefore, it is equivalent to branching two amino groups onto the carbon skeleton of chitosan, which enhances the positive charge of the chitosan derivative and enhances the antibacterial properties of the prepared chitosan derivative. 3. The chitosan derivatives obtained by this invention have enhanced water solubility, good thermal stability, are biodegradable, and have no obvious cytotoxicity; 4. The chitosan derivative obtained by this invention has a good preservation effect on refrigerated chicken, can significantly inhibit the growth of foodborne microorganisms, and has broad-spectrum antibacterial activity against foodborne bacteria, showing good prospects in the field of chicken preservation. Attached Figure Description Figure 1 This is a schematic diagram of the reaction in this invention; Figure 2 Infrared spectra of chitosan, lysine, and the chitosan derivative prepared in Example 1; Figure 3 The 1H NMR spectrum of chitosan and the chitosan derivative prepared in Example 1; Figure 4 Scanning electron microscope images of chitosan and chitosan derivatives prepared in Example 1; Figure 5 The antibacterial effect of chitosan and chitosan derivatives prepared in Example 1 on different bacterial strains is shown in the figure. Figure 6 The effect of the chitosan derivative prepared in Example 1 on the preservation of chicken meat; where A is the TVB content; B is the pH; C is the weight loss rate; and D is the total number of colonies. Detailed Implementation

[0016] This invention proposes a method for preparing a water-soluble chitosan derivative that can be used for chicken preservation. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following will provide a more detailed description of the invention with reference to specific examples. It should be understood that the specific examples described herein are only for explaining the invention and are not intended to limit the invention.

[0017] Example 1 A method for preparing a water-soluble chitosan derivative that can be used for chicken preservation includes the following steps: S1. Take 1.00 g of chitosan with a degree of deacetylation of 90% and a molecular weight of 80 kDa, 5.10 g of N,N'-dicyclohexylcarbodiimide, and 0.38 mL of orthophosphoric acid, mix them, and dissolve them in 20 mL of dimethyl sulfoxide (DMSO). Set the stirring rate to 550 r / min and stir the reaction at 25℃ for 24 h. S2. After the reaction is complete, slowly pour the mixture into 3 times the volume of anhydrous ethanol for alcohol precipitation. After standing for 2.5 h, discard the supernatant and filter to obtain a filter cake. Dissolve the filter cake in 100 mL of distilled water, add 4.50 g of lysine, and adjust the pH of the system to 4.5 with PBS buffer (0.1 mol / L). S3. Transfer the pH-adjusted solution to a reflux condenser and reflux at 75°C for 8 hours; S4. After the reaction is complete, place the reaction solution in a centrifuge and centrifuge at 6000 r / min for 20 min. Take the supernatant. Add 5‰ sodium cyanoborohydride (based on the mass of chitosan) to the supernatant and stir the reaction at room temperature for 4.5 h. S5. After the reaction is complete, pour the solution into 3 times the volume of anhydrous ethanol for alcohol precipitation, filter to obtain a crude filter cake; dissolve the filter cake in 50 mL of distilled water, put it into a dialysis bag with a molecular weight cutoff of 14000 Da, and dialyze with distilled water for 48 h, changing the distilled water every 8 h during the process. S6. After dialysis, the solution is placed in a freeze dryer and freeze-dried for 48 h under a vacuum of 8 Pa and a temperature of -55℃ to obtain the final product.

[0018] Comparative Example 1 The difference between this comparative example and Example 2 is that N,N'-dicyclohexylcarbodiimide and phosphoric acid were not added, but the rest of the steps are the same: S1. Take 1.00 g of chitosan with a degree of deacetylation of 90% and a molecular weight of 80 kDa, dissolve it in 20 mL of dimethyl sulfoxide (DMSO), set the stirring speed to 550 r / min, and stir the reaction at 25°C for 24 h. S2. After the reaction is complete, slowly pour the mixture into 3 times the volume of anhydrous ethanol for alcohol precipitation. After standing for 2.5 h, discard the supernatant and filter to obtain a filter cake. Dissolve the filter cake in 100 mL of distilled water, add 4.50 g of lysine, and adjust the pH of the system to 4.5 with PBS buffer (0.1 mol / L). S3. Transfer the pH-adjusted solution to a reflux condenser and reflux at 75°C for 8 hours; S4. After the reaction is complete, place the reaction solution in a centrifuge and centrifuge at 6000 r / min for 20 min. Take the supernatant. Add 5‰ sodium cyanoborohydride (based on the mass of chitosan) to the supernatant and stir the reaction at room temperature for 4.5 h. S5. After the reaction is complete, pour the solution into 3 times the volume of anhydrous ethanol for alcohol precipitation, filter to obtain a crude filter cake; dissolve the filter cake in 50 mL of distilled water, put it into a dialysis bag with a molecular weight cutoff of 14000 Da, and dialyze with distilled water for 48 h, changing the distilled water every 8 h during the process. S6. After dialysis, the solution was placed in a freeze dryer and freeze-dried for 48 h under a vacuum of 8 Pa and a temperature of -55℃ to obtain the product.

[0019] Performance testing: Infrared spectroscopy measurement Chitosan, lysine, the dialdehyde chitosan derivative (NaIO4-M) prepared in Comparative Example 1, and the chitosan derivative prepared in Example 2 were analyzed using a Nicolet iS10 Fourier transform infrared spectrometer via the potassium bromide pellet method. The scanning range was 500–4000 cm⁻¹. -1 128 scans, 4 cm resolution -1 .

[0020] like Figure 2 As shown in the infrared spectrum of chitosan, the 3200~3500 cm⁻¹ -1 The peak represents the overlapping multiple stretching vibrations of hydroxyl and amino groups in the chitosan molecule, at 2925 cm⁻¹. -1 The weak absorption peak at 1658 cm⁻¹ is attributed to the stretching vibrations of the methylene and methyl groups on the acetyl groups in the chitosan molecule. -1 and 1590 cm -1 These represent the stretching vibration of amide I and the bending vibration of amide II, respectively, 1085 cm. -1 and 1030 cm -1 These are attributed to the characteristic absorption peaks of the C3 and C6 hydroxyl groups of chitosan, respectively. For lysine, the peaks are located at 3200–2500 cm⁻¹. -1 The broad, diffuse peaks appearing in the region represent carboxylic acid groups, which often exist in the form of dimers in solids; these are characteristic peaks of the lysine carboxyl group. (2121 cm⁻¹) -1 The characteristic peak at 3400–3300 cm⁻¹ is the absorption peak of the CH stretching vibration of the amino acid hydrocarbon chain. -1 The peak appearing in the region is an antisymmetric stretching peak of amino NH. 549 cm⁻¹ -1The peak is a planar bending peak of the amino group (NH). For the chitosan derivative modified with lysine, the characteristic absorption peak originally attributed to the C6 hydroxyl group of chitosan disappears. This indicates that, based on the synthetic route diagram, the amino group of lysine is branched to the C6 position of chitosan. The characteristic absorption peaks of the lysine carboxyl group, the planar bending peak of the amino group, and the characteristic absorption peak of the hydrocarbon chain all appear in the target product, thus concluding that the target product was successfully synthesized.

[0021] (2) Nuclear magnetic resonance spectroscopy analysis Chitosan and the water-soluble chitosan derivative prepared in Example 2 of this invention were analyzed using a JNM-ECP600 fully digital nuclear magnetic resonance spectrometer manufactured by JEOL Corporation of Japan. 1 H NMR spectroscopy analysis. The detection temperature was 40℃, and the sample was dissolved in D2O.

[0022] like Figure 3 As shown, the chemical shifts of the sugar backbone in chitosan molecules are mainly between 2.0 and 5.0 ppm. Specifically, the peak at 4.51 ppm corresponds to the hydrogen protons of [H1], the peaks from 3.44 to 4.01 ppm correspond to the hydrogen protons of the chitosan carbon backbone [H3] to [H6], and the peak at 3.03 ppm is attributed to the hydrogen proton bonded to the C2 atom, denoted as [H2]. In lysine-modified chitosan derivatives, due to the bonding of chitosan with the N atom, the C2 and C3 positions of chitosan are transformed into methylene groups, and the 3.03 ppm hydrogen proton bonded to [H2] on the C2 atom disappears. The chemical shifts of these modified derivatives are between 2.7 and 2.9 ppm. These results provide further evidence for the successful preparation of lysine-modified chitosan derivatives.

[0023] (3) Scanning electron microscopy characterization The morphology and microstructure of chitosan and the chitosan derivative prepared in Example 2 were studied using scanning electron microscopy, and the samples were sputtered with gold at an accelerating voltage of 3.0 kV.

[0024] Figure 4 The microstructure of chitosan and lysine-modified chitosan was observed using scanning electron microscopy (SEM). The results showed that the unmodified chitosan surface exhibited a rough, film-like structure, a typical lamellar structure, and a loose texture. In contrast, the lysine-modified chitosan derivative had a smoother and denser surface. This is because the introduction of lysine may increase intermolecular hydrogen bonds, resulting in a more compact surface. Furthermore, a dense, granular structure was observed on the surface, which is likely due to lysine grafting. SEM results further confirmed the successful synthesis of the lysine-modified chitosan derivative.

[0025] Antibacterial activity analysis Two wells were punched in a petri dish containing culture medium. Using a 50-200 μL pipette, 100 μL of diluted Staphylococcus aureus was evenly dropped onto all but one of the wells. A spreader was heated over an alcohol lamp, cooled, and then used to spread the solution evenly. Another plate was prepared using the same method for Staphylococcus aureus to spread Escherichia coli evenly. After drying, the water-soluble chitosan derivative prepared in Example 2 was injected into the wells using a 5-50 μL pipette until filled. The plates were incubated at 37°C for 10-12 h. After incubation, the presence of inhibition zones was observed. The minimum inhibitory concentration (MIC) was determined using a two-fold dilution method and used to perform a minimum inhibitory concentration (MIC) test. The prepared water-soluble chitosan derivative was diluted. 200 μL of 16 mg / mL chitosan was placed in a 96-well plate. 100 μL was then transferred to the next well, and 100 μL of LB broth was added to suspend the solution. This process was repeated until 1 μL of the diluted bacterial culture was added. The plate was then incubated for 12 h. The results were then analyzed using a microplate reader.

[0026] The antibacterial properties of chitosan were enhanced through targeted modification of the chitosan molecule. The C6 hydroxyl group (second only to the C2 amino group in reactivity) exhibits good antibacterial activity during modification. Its antibacterial mechanism primarily relies on the interaction between the positive charge on the C2 amino group and the negative charge on the bacterial cell membrane, leading to bacterial death and thus an antibacterial effect. To further enhance its antibacterial activity, while retaining the C2 amino group, the C6 hydroxyl group was modified. Through an oxidation reaction, the hydroxyl group was dehydrated and oxidized to an aldehyde group. The aldehyde group then reacted with the ε-amino group of lysine via a Schiff base reaction, branching onto the chitosan carbon skeleton. Subsequently, the Schiff base was specifically reduced using sodium cyanoborohydride to obtain a stable compound. This allows the introduction of a carboxyl group at the C6 position of chitosan while retaining the C2 amino group, enhancing its water solubility. Simultaneously, the introduction of an amino group further enhances its antibacterial activity. In summary, the water-soluble chitosan derivative prepared in this invention exhibits clear antibacterial activity and concentration-responsive characteristics. It can inhibit the growth of target bacteria by enhancing cationic interactions to disrupt the bacterial cell membrane.

[0027] (5) Application in chicken The preservation effect of modified chitosan derivatives on chicken was evaluated using four key indicators: volatile basic nitrogen (TVB-N), pH value, weight loss rate, and total bacterial count. All data were based on dynamic monitoring under 4℃ refrigeration conditions. The experimental group (modified chitosan derivatives) and the control group (chitosan) showed significant differences, and the results have clear practical significance.

[0028] TVB-N is a volatile nitrogenous compound produced by the decomposition of meat proteins, and its content directly reflects the degree of spoilage (GB2707-2016 stipulates that TVB-N in fresh poultry meat ≤ 15 mg / 100g). Control group: On day 3, the TVB-N content reached 15.6 mg / 100g, exceeding the national standard limit, indicating the beginning of spoilage in the chicken; on day 5, it rose to 36 mg / 100g, indicating severe spoilage.

[0029] The TVB-N content rose slowly over the first 3 days, and remained below 15 mg / 100g on the 3rd day. It began to rise rapidly on the 4th day, reaching 25 mg / 100g on the 5th day, which was 11 mg / 100g lower than the control group.

[0030] This result indicates that quaternary ammonium salt modified chitosan can effectively inhibit the reproduction of spoilage microorganisms in chicken, slow down the rate of protein decomposition, delay the onset of chicken spoilage by at least 1 day, and significantly extend the shelf life.

[0031] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A method for preparing a water-soluble chitosan derivative for chicken preservation, characterized in that, Includes the following steps: S1. Chitosan, N,N'-dicyclohexylcarbodiimide and phosphoric acid are mixed and dissolved in dimethyl sulfoxide, and the mixture is stirred and reacted at 25°C for 22-25 h. S2. Pour in excess ethanol for alcohol precipitation, discard the supernatant, filter the precipitate to obtain filter cake; S3. Dissolve the filter cake in excess distilled water, add lysine, adjust the pH to 4.5 with PBS buffer, and reflux in a reflux condenser at 75°C for 7-9 hours to obtain the reflux product; S4. Centrifuge the reflux product, take the supernatant, add sodium cyanoborohydride at 5‰ of the chitosan mass, and react at room temperature for 4-5 hours. S5. Add excess ethanol again for alcohol precipitation, filter and dissolve the filter cake in distilled water, then perform water-soluble dialyzing for 48 hours. S6. After freeze-drying in a freeze dryer for 45-50 hours, a water-soluble chitosan derivative suitable for chicken preservation is obtained.

2. The preparation method according to claim 1, characterized in that: The chitosan in step S1 has a degree of deacetylation ≥85% and a molecular weight of 50~100 kDa.

3. The preparation method according to claim 1, characterized in that: The mass-to-volume ratio of chitosan, N,N'-dicyclohexylcarbodiimide, orthophosphoric acid, and dimethyl sulfoxide in step S1 is 1.00 g: 5.10 g: 0.38 mL: (15~25) mL.

4. The preparation method according to claim 1, characterized in that: The stirring rate in step S1 is 500~600 r / min.

5. The preparation method according to claim 1, characterized in that: The mass ratio of lysine to chitosan in step S3 is 4.50 g: 1.00 g.

6. The preparation method according to claim 1, characterized in that: The centrifugation speed in step S4 is 6000 r / min, and the centrifugation time is 20-22 min.

7. The preparation method according to claim 1, characterized in that: The dialysis bag used in step S5 has a molecular weight cutoff of 14,000 Da.

8. A water-soluble chitosan derivative for chicken preservation prepared by the preparation method according to any one of claims 1-7.

9. The application of the water-soluble chitosan derivative for chicken preservation as described in claim 8 in chicken preservation.

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