A composite biological preservative and a preparation method and application thereof

Abstract

This invention discloses a composite biological preservative, its preparation method, and its application, belonging to the field of agricultural product preservation technology. The preservative consists of four food-grade components: chitosan, ε-polylysine, sodium citrate, and glucono-delta-lactone. It constructs a chitosan-based buffer antibacterial film on the surface of agricultural products. This film utilizes the slow-release acidification properties of glucono-delta-lactone and the basic reserve of sodium citrate to form a buffer pair, stabilizing the microenvironment pH within the compatible range (5.5-5.8) for chitosan film formation and ε-polylysine antibacterial activity, achieving synergistic effects among the components. This preservative can extend the shelf life of *Agaricus regia* to over 14 days, and all components are food-grade, safe, and non-toxic, with a simple preparation process.

Description

Technical Field

[0001] This invention relates to the field of preservation technology, and in particular to a composite biological preservative, its preparation method, and its application. Background Technology

[0002] King oyster mushrooms are rich in nutrients, but their high water content (>90%), loose tissue, and lack of cuticle protection make them highly susceptible to browning, dehydration and shrinkage, and microbial infection after harvesting, leading to a rapid decline in commercial value and a short shelf life. Traditional preservation methods, such as refrigeration, modified atmosphere packaging, or the use of chemical preservatives (such as sulfites and sodium hypochlorite), have drawbacks including poor effectiveness, high costs, and potential safety risks related to chemical residues.

[0003] Biological preservation technology has received widespread attention due to its safety and environmental friendliness.

[0004] Chitosan not only possesses antibacterial properties but can also act as a natural film-forming agent, forming a semi-permeable film on the surface of agricultural products to reduce moisture evaporation and respiration intensity, making it a core component of common biological green preservation. However, the acidic solubility requirements of the chitosan film-forming matrix and the pH compatibility with biological antibacterial substances are significant issues. A compromise is usually reached through stepwise processing or sacrificing one function. This invention uses sodium citrate as a pH buffer to stabilize the membrane microenvironment within a weakly acidic range (pH 5.5-5.8), maintaining both the film-forming properties of chitosan and the activity of ε-polylysine. Gluconolactone releases weak acid through slow hydrolysis, assisting in maintaining the acidic environment of the membrane in the early stages of storage and delaying the swelling and inactivation of chitosan. Summary of the Invention

[0005] The main objective of this invention is to propose a composite biological preservative, its preparation method, and its application, aiming to solve the technical problem of poor compatibility between film-forming agents and antibacterial agents in existing biological preservatives.

[0006] To achieve the above objectives, the present invention proposes a composite biological preservative, wherein the preservative comprises chitosan, ε-polylysine, sodium citrate and glucono-delta-lactone.

[0007] In one embodiment, the preservative comprises, on a per 100 mL basis, 0.75% chitosan, 0.01% ε-polylysine, 0.5-2% sodium citrate, and 0.5% glucono-delta-lactone.

[0008] In one embodiment, the chitosan comprises water-soluble chitosan with a degree of deacetylation ≥85% and a viscosity of 50-200 mPa·s.

[0009] In one embodiment, the method for preparing the preservative includes the following steps: S10. Dissolve chitosan in a solvent and stir until completely dissolved to form a colloidal solution; S20. Add gluconolactone to the colloidal solution, stir to dissolve, and adjust and stabilize the pH of the solution to 5.5-5.8 with sodium citrate to obtain the first mixed solution; S30. Mix ε-polylysine with the first mixed solution and dissolve to obtain a second mixed solution; S40. Adjust the volume of the second mixed solution to obtain a preservative.

[0010] This invention provides an application of the preservative described above in the preservation of fungal agricultural products.

[0011] In one embodiment, the fungal agricultural product is *Pleurotus ostreatus*.

[0012] This invention also proposes a method for preserving *Stropharia masticata*, the preservation steps of which include: Treat the large-cap mushrooms with the aforementioned preservative solution and store them at 0-4°C. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0014] Figure 1 This is a schematic diagram showing the mass loss rate of the preservatives prepared in the embodiments of the present invention (groups ck, g1, g2, g3, t1, t2, and t3). Figure 2 This is a schematic diagram of pH value testing of the preservatives prepared in the embodiments of the present invention (groups ck, g1, g2, g3, t1, t2, t3); Figure 3 This is a schematic diagram illustrating the test of malondialdehyde (MDA) content in the preservatives prepared in the embodiments of the present invention (groups ck, g1, g2, g3, t1, t2, and t3). Figure 4 This is a schematic diagram illustrating the antibacterial effect evaluation test of the preservatives prepared in the embodiments of the present invention (groups ck, g1, g2, g3, t1, t2, and t3). Figure 5 Table 2 shows a comparison chart and sensory evaluation score table of the *Agaricus bisporus* samples from group ck and group t1 on day 14 in this embodiment of the invention.

[0015] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0016] In the following testing methods, the physicochemical indicators are measured every 2 days during storage, and the microbiological indicators are measured every 3 days. The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Bio-preservation technology has attracted widespread attention due to its safety and environmental friendliness. Chitosan, as a natural film-forming agent, can form a semi-permeable film on the surface of agricultural products, reducing moisture evaporation and respiration intensity; ε-polylysine, as a natural antimicrobial peptide, can effectively inhibit the growth of Gram-negative bacteria (such as Pseudomonas). However, existing technologies have significant drawbacks: chitosan requires acidic conditions to dissolve, and some bioactive substances are easily inactivated under acidic conditions; direct mixing of the two often leads to precipitation or loss of activity.

[0018] In the field of food preservation, the use of sodium citrate and glucono-delta-lactone alone as food additives has been reported in the literature. For example, sodium citrate is used as a pH buffer and metal ion chelating agent in the preservation of fruits and vegetables, and glucono-delta-lactone is used in certain food preservation systems. The combined use of sodium citrate and glucono-delta-lactone has not been found in any published literature.

[0019] Specifically, the preservative is a film-forming agent mainly composed of chitosan, which forms a semi-permeable film to reduce respiration intensity and moisture evaporation.

[0020] The choice of ε-polylysine over lysozyme is based on the biological characteristics of the dominant spoilage bacteria in *Pleurotus ostreatus*. ε-polylysine targets and destroys *Pseudomonas* (Gram-negative bacteria) that cause spoilage. Post-harvest spoilage of *Pleurotus ostreatus* is mainly caused by *Pseudomonas* spp. Pseudomonas This is caused by Gram-negative bacteria such as lysozyme, which has an outer membrane structure. Lysozyme mainly exerts its bactericidal effect by hydrolyzing the peptidoglycan in the cell wall of Gram-positive bacteria, and is extremely ineffective against Gram-negative bacteria. In contrast, ε-polylysine, as a natural antimicrobial peptide, can disrupt the integrity of the outer membrane of Gram-negative bacteria due to its cationic properties, and has a significant inhibitory effect on Pseudomonas. Moreover, it can exert its efficacy without the need for chelating agents such as EDTA, which is more in line with the requirements of green preservation.

[0021] Chitosan film formation depends on a weakly acidic environment. If the pH of the film microenvironment rises back to the neutral range during storage due to mushroom respiration and metabolism or surface microbial activity, chitosan is prone to deprotonation, leading to film swelling, cracking, and loss of barrier properties. This invention utilizes the slow-release acidification properties of gluconolactone to continuously and controllably release weak acid during storage; simultaneously, sodium citrate is used to construct an alkaline reserve, forming a dynamic buffer pair on the film surface. This system stabilizes the microenvironment pH within the chitosan film-forming range (5.5-5.8), preventing chitosan swelling and failure while maintaining optimal antibacterial activity of ε-polylysine, achieving a synergistic effect of film stability and antibacterial efficacy.

[0022] In some embodiments, the preservative comprises, on a per 100 mL basis, 0.75% chitosan, 0.01% ε-polylysine, 0.5-2% sodium citrate, and 0.5% glucono-delta-lactone. Within the above mass range, the components achieve synergistic effects within the concentration range, avoiding waste and balancing effectiveness, cost, and safety.

[0023] In some embodiments, the chitosan comprises water-soluble chitosan with a degree of deacetylation ≥85% and a viscosity of 50-200 mPa·s. The selection of the above-mentioned water-soluble chitosan ensures the formation of a uniform film under weakly acidic conditions.

[0024] This invention provides a method for preparing a composite biological preservative, comprising the following steps: S10. Dissolve chitosan in a solvent and stir until completely dissolved to form a colloidal solution; S20. Add gluconolactone to the colloidal solution, stir to dissolve, and adjust and stabilize the pH of the solution to 5.5-5.8 with sodium citrate to obtain the first mixed solution; S30. Mix ε-polylysine with the first mixed solution and dissolve to obtain a second mixed solution; S40. The second mixed solution is brought to a constant volume and filtered to obtain a preservative.

[0025] Specifically, step S10 includes: dissolving chitosan in a 1% (v / v) acetic acid solution and stirring at 40-60°C until completely dissolved to form a colloidal solution; Step S20 includes adding gluconolactone to the colloidal solution, stirring to dissolve, slowly adding 10% sodium citrate solution (concentration about 0.5-0.6%) while stirring, adjusting and stabilizing the pH to 5.5-5.8 to obtain the first mixed solution.

[0026] This invention also proposes the application of the preservative in the preservation of fungal agricultural products. This is because fungal agricultural products have loose tissues, high water content, and no outer skin protection, making them extremely prone to water loss, browning, softening, and microbial spoilage. Using this preservative can improve their preservation performance.

[0027] In an embodiment of the present invention, a method for preserving *Stropharia carinata* mushrooms includes the following preservation steps: Mix the preservative as described above with water to obtain a preservative solution; use the preservative solution to spray and treat fungal agricultural products and store them.

[0028] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are merely illustrative of the present invention and are not intended to limit the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0029] Example Experimental materials Giant king oyster mushroom: Harvested from an edible mushroom base in Wuhan, Hubei Province. Fresh fruiting bodies with unopened caps, free from pests and diseases, and uniform in size (cap diameter 5-7 cm) were selected and transported back to the laboratory for processing within 2 hours of harvesting.

[0030] Water-soluble chitosan: purchased from Shanghai Yuanye Biotechnology Co., Ltd., degree of deacetylation 90%, viscosity 100 mPa·s (product number: S11064).

[0031] ε-Polylysine: Purchased from Hebei Yingjun Biotechnology Co., Ltd.

[0032] Sodium citrate: Trisodium citrate dihydrate, food grade, purchased from Hubei Xinrunde Chemical Co., Ltd.

[0033] Gluconolactone: Food grade, purity ≥99%, purchased from Anhui Xingzhou Pharmaceutical and Food Co., Ltd.

[0034] All other reagents were of analytical grade.

[0035] Example 1: Component optimization (ck, g1, g2, g3, t1) Component optimization example 1 (ck, water control): Sterile water was used as the control group; Component optimization example 2 (g1): Chitosan with a concentration of 0.75% was prepared with 1% acetic acid, and the pH was adjusted to 5.6 with 10% sodium citrate and then brought to a final volume. Component optimization example 3 (g2): Chitosan with a concentration of 0.75% was prepared with 1% acetic acid, the pH was adjusted to 5.6 with 10% sodium citrate, and then ε-polylysine with a final concentration of 0.01% was added and the volume was adjusted. Component optimization example 4 (g3): Chitosan with a concentration of 0.75% was prepared with 1% acetic acid, and gluconolactone was added to a final concentration of 0.5%. The pH was adjusted to 5.6 with 10% sodium citrate and then brought to a final volume.

[0036] Total components t1: Chitosan with a concentration of 0.75% was prepared with 1% acetic acid, gluconolactone with a final concentration of 0.5% was added, the pH was adjusted to 5.6 with 10% sodium citrate, and ε-polylysine with a final concentration of 0.01% was added and the volume was brought to a final level.

[0037] The detailed preparation method of t1 (the preparation methods of other implementations are basically the same): Weigh 0.75 g of water-soluble chitosan, add about 85 mL of 1% (v / v) acetic acid solution, and stir magnetically at 50°C (500 rpm) for 30 min to aid dissolution; after cooling to room temperature, add 0.50 g of gluconolactone, and stir at 25°C for 20 min for pre-hydrolysis; slowly add about 5-6 mL of 10% sodium citrate solution (to make the final concentration of sodium citrate about 0.5-0.6%), adjust and stabilize the pH to about 5.6; then add 0.01 g of ε-polylysine (pre-dissolved in a small amount of deionized water), and stir for 10 min; transfer to a 100 mL volumetric flask, and make up to 100 mL with deionized water.

[0038] Example 2: Concentration optimization (t2 and t3) Concentration optimization t2 and t3 involve appropriate optimization of each component in t1.

[0039] The optimized composition of t2 was chitosan (0.5%), gluconolactone (0.3%), and ε-polylysine (0.005%), with a pH of 5.6, and the preparation method was similar to that of t1.

[0040] The optimized concentration of t3 consisted of chitosan (1.5%), gluconolactone (0.8%), and ε-polylysine (0.02%), with a pH of 5.6, and was prepared using a method similar to t1. The performance of the preservatives described in the above embodiments was tested as follows: 1. Quality loss rate Twenty-eight samples of *Agaricus bisporus* were collected, four per group, and marked at the stem. All samples were treated with the same preservative as described in the previous example. The weight of each mycelium was measured every two days, and the cumulative mass loss rate was calculated (mass loss rate = (initial mass - mass after storage) / initial mass × 100%). Depend on Figure 1It was found that all treatment groups significantly delayed the water loss process of the samples. On day 14, the weight loss rate of the CK group reached 49%, while the weight loss rates of the t1, t2, and t3 groups were 39%, 42%, and 37%, respectively. Among them, the t3 group (high concentration group) had the lowest weight loss rate and the best moisturizing effect, which was better than the t1 group; the weight loss rate of the t2 group (low concentration group) was slightly higher than that of the t1 group, but still significantly lower than that of the CK group. In addition, in the component optimization group, the weight loss rate of the g1 group containing only chitosan (approximately 43%) was higher than that of the g2 group (approximately 41%) and the g3 group (approximately 40%) containing ε-polylysine, indicating that the addition of each component contributed to reducing water loss. The weight loss rate of the complete formulation t1 group (39%) was close to that of the g3 group (40%), but significantly better than that of the g2 group (41%) and the g1 group (43%). The t1 group achieved the best balance between moisturizing and antibacterial effects, and its overall preservation performance was better than that of the g2 and g3 groups with any single added component. The results showed that the concentration of each component of the compound biological preservative was positively correlated with the moisturizing effect, and group t1 could achieve a good water retention effect at a moderate concentration.

[0041] 2. pH value measurement The above-mentioned *Stropharia macrocarpa* samples were added to 10 ml of ultrapure water (pH 7), juiced, and allowed to stand for 2 hours. The mixture was then filtered, and the pH value was measured using a pH meter. The test results are as follows: Figure 2 As shown, the pH of the control group (ck) rapidly decreased to around 5.0 on day 8, indicating rapid spoilage of the sample accompanied by microbial fermentation and tissue decomposition. The pH decrease in all treatment groups was significantly slowed due to the chitosan membrane barrier. Among them, group g1, containing only chitosan, had limited pH buffering capacity, with the pH dropping to approximately 5.4 on day 14; group g2, containing ε-polylysine, maintained a pH of around 5.6 on day 14; and group g3, containing gluconolactone, experienced the slowest pH decrease, similar to group t1. The complete formulation of group t1 showed pH buffering stability similar to group g3 throughout the storage period, significantly better than groups g1 and g2. Simultaneously, due to the antibacterial effect of ε-polylysine, group t1 reduced the impact of microbial metabolic acid production on the membrane layer, resulting in superior overall preservation performance compared to groups g2 and g3, which had either component added individually. The pH of group t3 decreased the slowest, remaining above 5.9 on day 14, which was better than group t1 (5.7). The pH of group t2 decreased relatively quickly, reaching approximately 5.5 on day 14, but was still significantly higher than group ck. The results indicate that higher concentrations of the composite biological preservative components have stronger buffering capacity and can more effectively delay sample rancidity.

[0042] 3. Determination of malondialdehyde (MDA) content Malondialdehyde (MDA) is produced due to the peroxidation of lipids in tissue or organ membranes caused by aging or damage under adverse conditions. Its content is closely related to aging and environmental damage. MDA condenses with thiobarbituric acid (TBA) to form a red product with a maximum absorption peak at 532 nm. Colorimetric analysis can estimate the content of lipid peroxidation in a sample. Simultaneously, the absorbance at 600 nm is measured, and the difference between the absorbance at 532 nm and 600 nm is used to calculate the MDA content. The equipment used includes a microplate reader, 96-well plates, a constant temperature water bath, a benchtop centrifuge, adjustable pipettes, a mortar / homogenizer, ice, and distilled water.

[0043] Accurately weigh 0.2 g of the sample to be tested, add 2 mL of 10% trichloroacetic acid and grind into a homogenate, then add 3 mL of 10% trichloroacetic acid and grind further. Centrifuge the homogenate at 4℃ × 12000 rpm for 10 min, collect the supernatant and place it on ice for testing. Take 0.2 mL of the supernatant and transfer it to a centrifuge tube, then add 0.2 mL of 0.6% thiobarbituric acid (dissolved in 10% trichloroacetic acid), mix well, and incubate in a 95℃ water bath for 30 min. Remove and cool on ice, centrifuge at 25℃ and 12000 rpm for 10 min, and take 200 μL of the supernatant into a 96-well plate. Read the absorbance A at 532 nm and 600 nm, respectively, ΔA = A532 - A600. Each treatment is repeated 3 times, where MDA content (nmol / g mass) = [ΔA ÷ (ε × d) × V2 × 10]. 9 ] ÷ (W × V1 ÷ V) = 16.13 × ΔA ÷ W, where V is the total volume of the sample extract (5 mL); V1 is the volume of the sample added to the reaction system (0.2 mL); V2 is the total reaction volume of the sample extract and working solution (0.4 mL); ε is the molar extinction coefficient of MDA (155 × 10⁻⁶). 3 L / mol / cm; W is the sample mass, in g.

[0044] Test results are as follows Figure 3As shown, compared to the control group (ck), the MDA accumulation rate of all treatment groups was slower, especially after day 6. Among them, group t3 consistently had the lowest MDA content, and by day 14, its MDA content was approximately 12% lower than group t1, indicating that the high-concentration component was more effective; group t1 had the second lowest MDA content; group t2 had a higher MDA content than group t1, but was still significantly lower than group ck (by approximately 25% lower than group ck by day 14). Furthermore, among the optimized component groups, group g1, containing only chitosan, had the highest MDA content, while group g3 had a lower MDA content than group g1, and group g2, containing ε-polylysine, also had a lower MDA content than group g1. The complete formulation of group t1 showed better MDA inhibition than groups g2 and g3, which had either component added individually. The results indicate that the concentration of each component in the composite biopreservative is positively correlated with the inhibitory effect on membrane lipid peroxidation, and the synergistic addition of each component plays an important role in delaying membrane lipid peroxidation.

[0045] 4. Evaluation of antibacterial effect: Samples were taken every 3 days, and the total bacterial count was determined by plate counting. Data values ​​(log CFU / ml, mean ± standard deviation, n=3). The specific method is as follows: Weigh 25.0 g of edible fungi sample, cut it into small pieces, add 5 mL of sterile phosphate buffered saline (PBS, pH 7.0), shake and wash to obtain the initial bacterial suspension; the initial bacterial suspension was serially diluted 10-fold (10... -2 -10 -8 Take 200 μl of each dilution and spread it on an agar plate (tryptone 5.0 g / L, yeast extract 2.5 g / L, glucose 1.0 g / L, agar 15.0 g / L), and incubate at 30℃ for 48 h; select plates with colony counts between 30-300 CFU for counting, and calculate the average colony count of the three parallel plates.

[0046] Test results are as follows Figure 4As shown, the control group (ck) experienced the fastest increase in total bacterial count, rising from an initial 3 log(CFU / ml) to 5.2 log(CFU / ml) within 15 days. Among them, group t3 showed the best antibacterial effect, maintaining the lowest total bacterial count throughout the storage period, reaching only 3.8 log(CFU / ml) at 15 days; group t1 was second best, reaching 4.0 log(CFU / ml) at 15 days. In the component optimization group, group g1 (4.6 log CFU / ml), containing only chitosan, showed limited antibacterial effect; group g2 (4.4 log CFU / ml), with the addition of ε-polylysine, exhibited significantly better antibacterial effect than group g1 due to the targeted bactericidal effect of the antimicrobial peptides; group g3 (4.0 log CFU / ml), with the addition of gluconolactone, significantly inhibited microbial proliferation due to the weakly acidic environment of the membrane layer, and the total colony count at 15 days was at the same level as group t1; however, group t1 showed a flatter growth curve in total colony count throughout the entire storage period (days 3-12), and because it also contained ε-polylysine, it had a targeted killing effect on Gram-negative bacteria, resulting in a broader antibacterial spectrum and longer-lasting effect. The complete formulation of group t1 was superior to group g2 in terms of antibacterial durability and overall preservation efficacy, and superior to group g3 in terms of antibacterial stability. The antibacterial effect of group t2 (low concentration group) was weaker than that of group t1, with a concentration of 4.3 log(CFU / ml) at 15 days, but it was still significantly lower than that of group ck. The results indicate that both ε-polylysine and gluconolactone contribute to antibacterial activity, and their synergistic effect enabled group t1 to achieve the best antibacterial effect.

[0047] 5. Appearance Comparison Image of a sample of *Stropharia macrocarpa* (large-cap mushroom) Figure 5 As shown. On day 14, the ck group samples showed obvious browning, dehydration and shrinkage, cap expansion and off-odor, and had lost their commercial value; the t1 group treated samples were generally plump, with white caps and no obvious cap opening, only very slight dehydration, and the preservation effect was good, significantly better than the ck group.

[0048] To further quantify the preservation effect, the sensory quality of *Agaricus bisporus* mushrooms in each treatment group was scored during storage. The scoring method of Meng et al. (2023) was followed (as shown in Table 1). A panel of three testers conducted the evaluation. A 100-point scale was used, and the scores were graded as follows: ≥85 points were Grade 1 (good marketability), 70-84 points were Grade 2 (moderate marketability), and <70 points were considered to have lost commercial value. The sensory scores for each group on day 0, day 7, and day 14 are shown in Table 2.

[0049] Table 1. Sensory Evaluation Criteria for Giant Pleurotus ostreatus

[0050] Table 2 Sensory evaluation scoring table for *Agaricus bisporus* mushrooms treated with different preservatives

[0051] Table 2 shows that on the 7th day of storage, the score of group CK had dropped to 72 points (the critical value for second-grade products), while group t1 maintained a first-grade level of 88 points. By the 14th day, the score of group CK had dropped to 58 points, completely losing its commercial value, while group t1 still maintained a first-grade level of 82 points. Group CK lost its commercial value around the 7th day, while group t1 maintained good marketability until the 14th day. The results indicate that, compared to group CK, the compound biological preservative in group t1 can extend the post-harvest shelf life of *Agaricus bisporus* by about two weeks.

[0052] The above description is merely an exemplary embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification and drawings under the technical concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A composite biological preservative, characterized in that, It includes chitosan, ε-polylysine, sodium citrate, and gluconolactone.

2. The composite biological preservative as described in claim 1, characterized in that, The preservative, calculated in g / 100mL, comprises: 0.75% chitosan, 0.01% ε-polylysine, 0.5-2% sodium citrate, and 0.5% glucono-delta-lactone.

3. The composite biological preservative as described in claim 2, characterized in that, The chitosan includes water-soluble chitosan with a degree of deacetylation ≥85% and a viscosity of 50-200 mPa·s.

4. The method for preparing the composite biological preservative according to any one of claims 1-3, characterized in that, Includes the following steps: S10. Dissolve chitosan in a solvent and stir until completely dissolved to form a colloidal solution; S20. Add gluconolactone to the colloidal solution, stir to dissolve, and adjust and stabilize the pH of the solution to 5.5-5.8 with sodium citrate to obtain the first mixed solution; S30. Mix ε-polylysine with the first mixed solution and dissolve to obtain a second mixed solution; S40. Adjust the volume of the second mixed solution to obtain the preservative.

5. The application of the compound biological preservative as described in any one of claims 1-3 in the preservation of fungal agricultural products.

6. A method for preserving *Stropharia masticata*, characterized in that, Using the compound biological preservative as described in any one of claims 1-3, comprising: mixing the preservative with water to obtain a preservative solution, spraying it onto *Stropharia macrocarpa* mushrooms, and then storing it at 0-4°C.

Images