A stress-resistant, antifreeze, and preservation biofilm formulation and its preparation method

Abstract

This invention relates to the field of agricultural biotechnology, and in particular to a stress-resistant, antifreeze, and preservative biofilm formulation and its preparation method. The stress-resistant, antifreeze, and preservative biofilm formulation is composed of the following raw materials in parts by weight: 13-28 parts film-forming matrix, 5.5-11.5 parts antifreeze active component, 2.51-6.05 parts stress-resistant active component, 7-14 parts crosslinking and plasticizer, 0.1-0.5 parts natural antibacterial agent, and 55-75 parts deionized water. This invention innovatively achieves a four-fold synergy of physical barrier, active stress resistance, efficient antifreeze, and natural antibacterial properties. The film-forming matrix forms a dense and breathable physical barrier, inhibiting moisture loss; the stress-resistant components construct an active stress-resistant system, enhancing the stress resistance of agricultural products; the antifreeze components achieve efficient antifreeze by inhibiting ice crystal formation and stabilizing cell membrane structure; and the natural antibacterial agent specifically inhibits postharvest pathogens such as Penicillium and Botrytis. The four components work synergistically to comprehensively solve multiple problems during the storage of agricultural products.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to a stress-resistant, antifreeze, and preservative biofilm preparation and its preparation method. Background Technology

[0002] Post-harvest preservation and stress protection of agricultural products are crucial aspects of agricultural production and food distribution. Traditional agricultural product preservation technologies mainly rely on physical barrier coatings or cold storage treatments. While these methods inhibit moisture evaporation through physical barriers, they fail to activate the agricultural products' own stress resistance systems, resulting in insufficient protection against environmental stresses such as low temperatures, frost, and drought. Furthermore, most formulations lack antibacterial properties, making it difficult to inhibit post-harvest pathogens such as Penicillium and Botrytis cinerea, leading to frequent disease outbreaks and high spoilage rates during storage. Therefore, it is necessary to design a stress-resistant, freeze-proof, and preservation biofilm formulation. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of existing technologies by proposing a stress-resistant, antifreeze, and preservation biofilm preparation and its preparation method.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: a stress-resistant, antifreeze, and preservation biofilm preparation, wherein the stress-resistant, antifreeze, and preservation biofilm preparation is composed of the following raw materials in parts by weight: 13-28 parts of film-forming matrix, 5.5-11.5 parts of antifreeze active component, 2.51-6.05 parts of stress-resistant active component, 7-14 parts of crosslinking and plasticizer, 0.1-0.5 parts of natural antibacterial agent, and 55-75 parts of deionized water.

[0005] As a further description of the above technical solution: The film-forming matrix is ​​composed of low molecular weight sodium alginate and soluble plant cellulose, wherein the low molecular weight sodium alginate is 10-20 parts and the soluble plant cellulose is 3-8 parts.

[0006] As a further description of the above technical solution: The antifreeze active component consists of an ice-nucleating bacteria-inhibiting oligopeptide fragment and a natural polyol, wherein the ice-nucleating bacteria-inhibiting oligopeptide fragment is 0.5-1.5 parts and the natural polyol is 5-10 parts.

[0007] As a further description of the above technical solution: The natural polyol is one or more of trehalose, mannitol, erythritol, or xylitol.

[0008] As a further description of the above technical solution: The anti-stress active component is composed of L-proline, glutathione and methyl jasmonic acid, wherein the L-proline is 2-5 parts, the glutathione is 0.5-1.0 parts and the methyl jasmonic acid is 0.01-0.05 parts.

[0009] As a further description of the above technical solution: The crosslinking and plasticizer is composed of calcium chloride solution and food-grade glycerin, wherein the calcium chloride solution is 5-10 parts with a concentration of 5-10%, and the food-grade glycerin is 2-4 parts.

[0010] As a further description of the above technical solution: The natural antibacterial agent is thymol or rosemary extract.

[0011] A method for preparing a stress-resistant, freeze-proof, and preservation biofilm formulation, comprising the following steps: S1: Matrix dissolution and pretreatment: The film-forming matrix is ​​added to deionized water and stirred at 300-500 rpm for 30-60 minutes at 60°C to obtain a matrix solution; the mass ratio of the film-forming matrix to deionized water is 1:2-5. S2: Hydration of antifreeze active components: Disperse natural polyols and ice-nucleating bacteria inhibitory oligopeptide fragments in deionized water, and stir slowly at 100-200 rpm for 15-25 minutes at a temperature below 25°C to obtain an antifreeze active dispersion; The mass ratio of the total mass of the natural polyols and ice-nucleating bacteria inhibitory oligopeptide fragments to the mass of deionized water is 1:0.5-1. S3: Mixing of anti-stress active components: Add L-proline and glutathione to the antifreeze active dispersion, stir to dissolve, add methyl jasmonate to aid dissolution, and continue stirring to obtain the anti-stress active solution; S4: High-shear mixing and primary emulsification: The matrix solution and the stress-resistant active solution are injected into the high-shear mixing reactor and sheared and mixed at a shear rate of 8000-12000 rpm for 10-15 minutes to form a primary O / W emulsion; S5: Ultrasonic nanoscale homogenization: The primary emulsion is treated with ultrasound at 20-25kHz and 40-60W / L for 3-8 minutes to obtain a nanoscale homogenized emulsion. S6: pH adjustment and ionic cross-linking activation: Adjust the pH of the homogenized emulsion to 6.0-6.5 with citric acid, add calcium chloride solution dropwise, and stir at 200-300 rpm for 20-30 minutes to initiate the cross-linking reaction and form a composite preparation; S7: Addition of natural antibacterial agents: Add natural antibacterial agents to the compound preparation and stir at a stirring speed of 200-300 rpm for 8-12 minutes to ensure uniform dispersion and obtain a stress-resistant, antifreeze and fresh-preserving biofilm preparation. S8: Sterilization and filling: The stress-resistant, antifreeze and fresh-keeping biofilm preparation is pasteurized at 65°C for 30 minutes, cooled to room temperature after sterilization, sealed and filled to obtain the finished product.

[0012] As a further description of the above technical solution: Prior to step S3, glutathione and methyl jasmonate are micro- or nano-encapsulated using liposomes.

[0013] The present invention has the following beneficial effects: 1. Compared with existing technologies, this invention innovatively achieves a four-fold synergy of physical barrier, active stress resistance, efficient antifreeze, and natural antibacterial properties. The film-forming matrix forms a dense and breathable physical barrier, inhibiting moisture loss; the stress-resistant components construct an active stress-resistant system, enhancing the agricultural product's own stress resistance; the antifreeze components achieve efficient antifreeze by inhibiting ice crystal formation and stabilizing cell membrane structure; and the natural antibacterial agents (thymol and rosemary extract) specifically inhibit post-harvest pathogens such as Penicillium and Botrytis. The synergistic effect of these four components comprehensively solves multiple problems during the storage of agricultural products. Experiments have shown that the formulation of this invention can extend the shelf life of fruits and vegetables to 21-24 days.

[0014] 2. Compared with the prior art, the present invention innovatively introduces an oligopeptide fragment that inhibits ice-nucleating bacteria, thereby reducing ice crystal formation at the source by specifically interfering with the ice-nucleating bacteria's ability to form ice nuclei; at the same time, natural polyols such as trehalose can bind with intracellular water, lower the freezing point of extracellular fluid, stabilize the ice crystal structure, and avoid mechanical damage to the cell membrane by ice crystals.

[0015] 3. Compared with the prior art, the present invention uses a low molecular weight sodium alginate and soluble plant cellulose as a film-forming matrix, which makes the molecular weight moderate and has a certain porosity structure, thus ensuring the air permeability of the membrane.

[0016] 4. Compared with the prior art, the present invention uses micro-nano encapsulation technology to encapsulate glutathione and methyl jasmonate, which can protect the active ingredients from rapid degradation, prolong the slow release period of glutathione and methyl jasmonate on the surface of agricultural products, and significantly enhance the long-term stress resistance of agricultural products. Detailed Implementation

[0017] Example 1 By weight, take 15 parts of low molecular weight sodium alginate, 5 parts of soluble plant cellulose, 1.0 part of ice-nucleating bacteria-inhibiting oligopeptide fragment, 8 parts of trehalose, 3 parts of L-proline, 0.8 parts of glutathione, 0.03 parts of methyl jasmonate, 8 parts of 5% calcium chloride solution, 3 parts of food-grade glycerol, 0.3 parts of thymol, and 70 parts of deionized water.

[0018] Prepare according to the following steps: S1: Matrix dissolution and pretreatment: 15 parts of low molecular weight sodium alginate and 5 parts of soluble plant cellulose were added to 65 parts of deionized water and stirred at 400 rpm for 40 minutes at 60°C to obtain a transparent matrix solution. S2: Hydration of antifreeze active components: Disperse 8 parts of trehalose and 1.0 part of ice-nucleating bacteria inhibitory oligopeptide fragment in 5 parts of deionized water, and stir slowly at 150 rpm for 20 minutes at 20°C to obtain antifreeze active dispersion; S3: Mixing of anti-stress active components: Add 3 parts L-proline and 0.8 parts glutathione to the antifreeze active dispersion and stir for 12 minutes until dissolved; then slowly add 0.03 parts methyl jasmonate with 0.5 parts food-grade ethanol as a dissolving agent and stir for 8 minutes to obtain the anti-stress active solution; S4: High-shear mixing and primary emulsification: The matrix solution and the stress-resistant active solution are injected into a high-shear reactor and sheared at a shear rate of 10,000 rpm for 12 minutes to form a primary emulsion; S5: Ultrasonic nanoscale homogenization: The primary emulsion is ultrasonically treated at 22kHz and 50W / L for 5 minutes to obtain a nanoscale homogenized emulsion with a particle diameter of 100-150nm. S6: pH adjustment and ionic cross-linking activation: Adjust the pH of the homogenized emulsion to 6.2 with citric acid, add 8 parts of 5% calcium chloride solution, and stir at 250 rpm for 25 minutes to initiate the cross-linking reaction and form a composite preparation; S7: Addition of natural antibacterial agent: Add 0.3 parts of thymol to the compound preparation and stir at 250 rpm for 10 minutes to ensure uniform dispersion, so as to obtain a stress-resistant, antifreeze and fresh-keeping biofilm preparation. S8: Sterilization and filling: The stress-resistant, antifreeze, and fresh-preserving biofilm preparation is pasteurized at 65°C for 30 minutes, cooled, and then filled to obtain the finished product.

[0019] Example 2 By weight, take 18 parts of low molecular weight sodium alginate, 6 parts of soluble plant cellulose, 1.2 parts of ice-nucleating bacteria-inhibiting oligopeptide fragment, 9 parts of erythritol, 3.5 parts of L-proline, 0.9 parts of liposome-encapsulated glutathione, 0.04 parts of liposome-encapsulated methyl jasmonate, 9 parts of 8% calcium chloride solution, 3.5 parts of food-grade glycerol, 0.5 parts of thymol, and add deionized water to a total of 73 parts.

[0020] Preparation steps of liposome-encapsulated glutathione: Oil phase preparation: Soybean lecithin and cholesterol were added to anhydrous ethanol and placed in a 50°C constant temperature water bath. The mixture was magnetically stirred (300 rpm) for 18 minutes until the lipids were completely dissolved, forming a clear and transparent oil phase.

[0021] Lipid film formation: The oil phase was transferred to a rotary evaporator, and the temperature was set to 40℃, the rotation speed to 60 rpm, and the vacuum degree to 0.09 MPa. The evaporator was rotated for 25 minutes to completely evaporate the anhydrous ethanol and form a uniform, crack-free lipid film on the inner wall of the flask.

[0022] Core material hydration: Dissolve glutathione and ascorbic acid in PBS buffer and stir for 5 minutes until completely dissolved to obtain an aqueous solution of the core material; pour the aqueous solution of the core material into a flask with a lipid film and place it in a 37°C constant temperature shaking incubator and shake at 150 rpm for 30 minutes to fully hydrate the lipid film and form a milky white crude liposome suspension.

[0023] Ultrasonic disruption and refinement: The crude liposome suspension was transferred to an ultrasonic cell disruptor. The power was set to 250W, the working time was 3 seconds, the interval was 5 seconds, and the process was repeated for 12 minutes to disrupt the large-diameter liposomes and initially reduce the particle size to 300-500nm.

[0024] Extrusion homogenization and size control: The ultrasonically treated suspension is extruded through a high-pressure homogenizer, using polycarbonate films with pore sizes of 400nm, 200nm, and 100nm in sequence, to precisely control the particle size to 100-250nm.

[0025] Purification and impurity removal: Transfer the extruded liposome suspension into a centrifuge tube, centrifuge at 8000 rpm for 10 minutes, discard the bottom precipitate and a small amount of free core material solution in the upper layer, and collect the uniform suspension in the middle layer; wash twice with PBS buffer, centrifuge again to collect, and obtain purified liposome-encapsulated glutathione particles.

[0026] Preparation steps of liposome-encapsulated methyl jasmonate: Oil phase preparation: Soybean lecithin, cholesterol, and methyl jasmonate were added to anhydrous ethanol and stirred in a constant temperature water bath at 50°C for 15 minutes until the lipids and core material were completely dissolved, forming a clear oil phase.

[0027] Lipid film formation: The oil phase was transferred to a rotary evaporator and rotary evaporated for 20 minutes at 40°C, 60 rpm and 0.08 MPa. After the ethanol evaporated, a lipid film containing methyl jasmonic acid was formed on the inner wall of the flask.

[0028] Hydration and dispersion: Pour ascorbic acid into a PBS buffer solution and shake at 150 rpm for 30 minutes in a 37°C constant temperature shaker to hydrate the lipid film and release the liposomes encapsulating methyl jasmonate to form a milky white suspension.

[0029] Ultrasonic disruption: Place the suspension into an ultrasonic cell disruptor, operate at 200W power for 3 seconds, pause for 5 seconds, and circulate for 10 minutes to disrupt large liposome particles and initially refine them to 300-400nm.

[0030] Extrusion diameter control: The ultrasonically treated suspension is extruded through a high-pressure homogenizer and then extruded through 400nm, 200nm, and 100nm polycarbonate films in sequence to control the particle size to 100-200nm.

[0031] Purification: Centrifuge at 8000 rpm for 10 minutes, discard the undissolved lipids at the bottom and the free methyl jasmonate in the upper layer, collect the intermediate suspension, wash once with PBS buffer, centrifuge to collect, and obtain liposome-encapsulated methyl jasmonate particles.

[0032] The preparation steps are as follows: S1: Matrix dissolution and pretreatment: 18 parts of low molecular weight sodium alginate and 6 parts of soluble plant cellulose were added to 65 parts of deionized water and stirred at 450 rpm for 50 minutes at 60°C to obtain a transparent matrix solution. S2: Hydration of antifreeze active components: 1.2 parts of ice-nucleating bacteria inhibitory oligopeptide fragment and 9 parts of erythritol were dispersed in 8 parts of deionized water and stirred at 22°C and 180 rpm for 22 minutes to obtain an antifreeze active dispersion. S3: Mixing of anti-stress active components: Add 3.5 parts of L-proline to the antifreeze active dispersion and stir for 15 minutes; disperse liposome-encapsulated glutathione and liposome-encapsulated methyl jasmonate in 3 parts of deionized water, sonicate for 5 minutes, add to the mixture, and stir for 10 minutes; S4: High-shear mixing and primary emulsification: The matrix solution and the stress-resistant active solution are injected into a high-shear reactor and sheared at a shear rate of 11,000 rpm for 14 minutes to form a primary emulsion; S5: Ultrasonic nanoscale homogenization: The primary emulsion is ultrasonically treated at 24kHz and 55W / L for 6 minutes to obtain a nanoscale homogenized emulsion with a particle diameter of 80-120nm. S6: pH adjustment and ionic cross-linking activation: The pH of the homogenized emulsion was adjusted to 6.3 with citric acid, and 9 parts of 8% calcium chloride solution were added dropwise. The mixture was stirred at 280 rpm for 28 minutes to initiate the cross-linking reaction and form a composite preparation. S7: Addition of natural antibacterial agent: Add 0.5 parts of thymol to the compound preparation and stir at 300 rpm for 11 minutes to ensure uniform dispersion and obtain an anti-freezing and preservation biofilm preparation. S8: Sterilization and filling: The stress-resistant, antifreeze, and fresh-preserving biofilm preparation is pasteurized at 65°C for 30 minutes, cooled, and then filled to obtain the finished product.

[0033] Comparative Example 1: Traditional Paraffin Coating Formulation Take 5 parts by weight of paraffin wax, 2 parts by weight of food-grade glycerin, and 93 parts by weight of deionized water. Heat the paraffin wax to 70°C to melt it, stir evenly, and cool to obtain the paraffin wax coating preparation.

[0034] Comparative Example 2: Single Chitosan Membrane Formulation Take 10 parts by weight of chitosan, 3 parts by weight of food-grade glycerin, and 87 parts by weight of 1% acetic acid solution, and stir until the chitosan is completely dissolved to obtain a chitosan film preparation.

[0035] Test methods (a) Antifreeze effect test Test subjects: Strawberries of uniform ripeness and no damage were randomly divided into 4 groups, with 30 strawberries in each group. These were Example 1-2 groups, Comparative Example 1-2 groups, and a blank control group. Treatment method: All groups were soaked for 3 minutes and then air-dried; the blank control group was not treated. Low temperature stress: Placed in a -3℃ low temperature incubator for 6 hours, then restored to room temperature (25℃). Indicator measurements: Cell membrane permeability was measured after recovery; survival rate and decay rate were measured 3 days after recovery. See Table 1 for details.

[0036] Table 1 shows the test data for antifreeze effect.

[0037] As shown in Table 1, after being subjected to -3℃ low-temperature stress, the strawberry cells in Examples 1-2 showed significantly lower cell membrane permeability than those in the Comparative Examples 1-2 and the blank control group. After 3 days, the survival rate of all groups was above 82%, and the decay rate was below 15%, with Example 2 showing the best results. This indicates that the stress-resistant and antifreeze preservative biofilm formulation of the present invention has a significant antifreeze protective effect compared to traditional paraffin coatings and chitosan membranes. The measurement of cell membrane permeability revealed that the strawberries in the Example groups showed less cell membrane damage after low-temperature stress, indicating that the formulation can effectively stabilize the cell membrane structure and reduce the mechanical damage to cells caused by ice crystals. Furthermore, the survival rate and decay rate data after 3 days further validated its superiority; the strawberries in the Example groups not only had a high survival rate but also a significantly lower decay rate than the Comparative Examples and the blank control group. This fully demonstrates the synergistic effect of the antifreeze and stress-resistant active components in the present invention, providing comprehensive protection for fruits and vegetables under low-temperature conditions. Furthermore, compared with traditional paraffin coatings and single chitosan membranes, the biofilm preparation of the present invention can not only effectively prevent moisture loss, but also has active stress resistance and antibacterial functions, thereby significantly extending the shelf life of fruits and vegetables.

[0038] (II) Preservation and antibacterial effect test Test subjects: Citrus fruits of uniform maturity and free from pests and diseases were randomly divided into 4 groups of 20 fruits each, namely Example 1-2 groups, Comparative Example 1-2 groups, and a blank control group. Treatment method: Examples 1-2 and Comparative Examples 1-2 were treated with foliar spraying at a rate of 80 mL / m². 2 Air dry naturally; the blank control group was left untreated. Storage conditions: Store at room temperature (25℃, relative humidity 60%), and perform regular testing; Indicator measurements: On day 21 of storage, moisture loss rate, fruit firmness (texture analyzer, puncture depth 5 mm), and soluble solids content (handheld refractometer) were measured; shelf life (number of days stored until 20% decay rate) was recorded; after 21 days of storage, pathogen infection rate (percentage of fruit infected with Penicillium and Botrytis) was measured. Specific data are shown in Table 2. Table 2 shows the test data for preservation and antibacterial effects.

[0039] As shown in Table 2, after 21 days of storage at room temperature, the citrus fruits in Examples 1-2 exhibited a moisture loss rate of less than 10%, maintained a firmness above 11.5 N, had a soluble solids content above 12.8%, a shelf life of 21-24 days, and a pathogen infection rate of less than 12%, significantly superior to Comparative Examples 1-2. This demonstrates that the stress-resistant and antifreeze preservation biofilm formulation of this invention has superior preservation and antibacterial effects compared to traditional paraffin coatings and chitosan films. The citrus fruits in the Example groups showed a low moisture loss rate during storage, indicating that the biofilm formulation effectively locks in internal fruit moisture and reduces evaporation. Simultaneously, the maintenance of firmness demonstrates the protective effect of the formulation on fruit texture, preventing softening caused by moisture loss and cell wall degradation. The high level of soluble solids content further proves the good retention effect of internal nutrients in the fruit. The significant extension of shelf life and the substantial reduction in pathogen infection rate reflect the outstanding performance of the biofilm formulation in inhibiting microbial infection and delaying fruit spoilage. Compared with traditional paraffin coatings and single chitosan membranes, this invention, through the synergistic effect of multiple functions, not only improves the preservation effect but also enhances the antibacterial ability, providing a more reliable solution for the long-term storage of fruits and vegetables.

[0040] (III) Stress Resistance Test Test subjects: Tomato seedlings of uniform growth, randomly divided into 4 groups of 15 seedlings each, namely Example 1-2 groups, Comparative Example 1-2 groups, and a blank control group; Treatment method: Examples 1-2 and Comparative Examples 1-2 were treated with foliar spraying at a rate of 60 mL / m². 2 The blank control group was sprayed with an equal amount of deionized water. Drought stress: Stop watering on the second day after treatment and continue drought stress for 7 days; Index determination: After the stress period ended, chlorophyll content (SPAD method), malondialdehyde (MDA) content (thiobarbituric acid method), and leaf proline content (ninhydrin colorimetric method) were measured. See Table 3 for specific data.

[0041] Table 3 shows the test data for stress resistance effect.

[0042] Table 3 shows that under drought stress, the chlorophyll and proline content of tomato seedlings in Examples 1-2 were significantly higher than those in Comparative Examples 1-2 and the blank control group. The MDA content of tomato seedlings in Examples 1-2 was significantly lower than that in the control group 1-2. This indicates that the formulation of this invention can enhance plant stress resistance through a dual mechanism of exogenous protection and endogenous activation. Glutathione and methyl jasmonate exert their effects in the plant through complex metabolic pathways, further verifying their importance in exogenous protection. The experimental results show that the chlorophyll content of tomato seedlings in the Example groups remained at a high level after drought stress, indicating that the formulation can effectively delay leaf senescence and maintain photosynthetic efficiency. Meanwhile, the significant increase in L-proline content, as an important osmotic regulator, also reflects the enhanced adaptability of plants under adverse conditions. Furthermore, the decrease in MDA content directly reflects the reduction in cell membrane lipid peroxidation, indicating that the formulation can effectively inhibit the damage of reactive oxygen species to cell structure. Comprehensive analysis of the experimental data shows that the biofilm formulation of this invention not only forms an external protective barrier but also comprehensively enhances the drought resistance of plants by activating their endogenous stress-resistance mechanisms. This dual-action mechanism provides new technical support for coping with extreme climatic conditions in agricultural production and has broad application prospects.

[0043] In summary, the stress-resistant, antifreeze, and preservation biofilm formulation of the present invention achieves a synergistic effect of physical barrier, active stress resistance, efficient antifreeze, and natural antibacterial properties through reasonable component ratio and optimized preparation process. It has excellent comprehensive performance, is safe and environmentally friendly, and has broad application prospects in bio-agriculture.

[0044] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A stress-resistant, antifreeze, and preservative biofilm preparation, characterized in that, The stress-resistant, antifreeze, and preservation biofilm preparation is composed of the following raw materials in parts by weight: 13-28 parts of film-forming matrix, 5.5-11.5 parts of antifreeze active component, 2.51-6.05 parts of stress-resistant active component, 7-14 parts of crosslinking and plasticizer, 0.1-0.5 parts of natural antibacterial agent, and 55-75 parts of deionized water.

2. The anti-freezing and preservation biofilm preparation according to claim 1, characterized in that: The film-forming matrix is ​​composed of low molecular weight sodium alginate and soluble plant cellulose, wherein the low molecular weight sodium alginate is 10-20 parts and the soluble plant cellulose is 3-8 parts.

3. The anti-freezing and preservation biofilm preparation according to claim 1, characterized in that: The antifreeze active component consists of an ice-nucleating bacteria-inhibiting oligopeptide fragment and a natural polyol, wherein the ice-nucleating bacteria-inhibiting oligopeptide fragment is 0.5-1.5 parts and the natural polyol is 5-10 parts.

4. The stress-resistant, antifreeze, and preservation biofilm preparation according to claim 3, characterized in that: The natural polyol is one or more of trehalose, mannitol, erythritol, or xylitol.

5. The anti-freezing and preservation biofilm preparation according to claim 1, characterized in that: The anti-stress active component is composed of L-proline, glutathione and methyl jasmonic acid, wherein the L-proline is 2-5 parts, the glutathione is 0.5-1.0 parts and the methyl jasmonic acid is 0.01-0.05 parts.

6. The anti-freezing and preservation biofilm preparation according to claim 1, characterized in that: The crosslinking and plasticizer consists of a calcium chloride solution and food-grade glycerin, wherein the calcium chloride solution comprises 5-10 parts with a concentration of 5%-10%, and the food-grade glycerin comprises 2-4 parts.

7. The anti-freezing and preservation biofilm preparation according to claim 1, characterized in that: The natural antibacterial agent is thymol or rosemary extract.

8. A method for preparing a stress-resistant, antifreeze, and preservative biofilm formulation, characterized in that, The preparation of the stress-resistant, antifreeze, and preservation biofilm formulation according to any one of claims 1-6 comprises the following steps: S1: Matrix dissolution and pretreatment: The film-forming matrix is ​​added to deionized water and stirred at 300-500 rpm for 30-60 minutes at 60°C to obtain a matrix solution; the mass ratio of the film-forming matrix to deionized water is 1:2-5. S2: Hydration of antifreeze active components: Disperse natural polyols and ice-nucleating bacteria inhibitory oligopeptide fragments in deionized water, and stir slowly at 100-200 rpm for 15-25 minutes at a temperature below 25°C to obtain an antifreeze active dispersion; The mass ratio of the total mass of the natural polyols and ice-nucleating bacteria inhibitory oligopeptide fragments to the mass of deionized water is 1:0.5-1. S3: Mixing of anti-stress active components: Add L-proline and glutathione to the antifreeze active dispersion, stir to dissolve, add methyl jasmonate to aid dissolution, and continue stirring to obtain the anti-stress active solution; S4: High-shear mixing and primary emulsification: The matrix solution and the stress-resistant active solution are injected into the high-shear mixing reactor and sheared and mixed at a shear rate of 8000-12000 rpm for 10-15 minutes to form a primary O / W emulsion; S5: Ultrasonic nanoscale homogenization: The primary emulsion is treated with ultrasound at 20-25kHz and 40-60W / L for 3-8 minutes to obtain a nanoscale homogenized emulsion. S6: pH adjustment and ionic cross-linking activation: Adjust the pH of the homogenized emulsion to 6.0-6.5 with citric acid, add calcium chloride solution dropwise, and stir at 200-300 rpm for 20-30 minutes to initiate the cross-linking reaction and form a composite preparation; S7: Addition of natural antibacterial agents: Add natural antibacterial agents to the compound preparation and stir at a stirring speed of 200-300 rpm for 8-12 minutes to ensure uniform dispersion and obtain a stress-resistant, antifreeze and fresh-preserving biofilm preparation. S8: Sterilization and filling: The stress-resistant, antifreeze and fresh-keeping biofilm preparation is pasteurized at 65°C for 30 minutes, cooled to room temperature after sterilization, sealed and filled to obtain the finished product.

9. The method for preparing a stress-resistant, antifreeze, and preservative biofilm formulation according to claim 8, characterized in that: Prior to step S3, glutathione and methyl jasmonate are micro- or nano-encapsulated using liposomes.

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