A method for applying plant exosome loaded natural antibacterial substances to food preservation

Abstract

The application provides a food preservation method using plant exosome loaded with natural antibacterial substances, relates to the field of food preservation and biological materials, and comprises the following steps: loading natural antibacterial substances and the plant exosome under preset loading conditions to obtain a plant exosome loaded with natural antibacterial substances; and applying the plant exosome loaded with natural antibacterial substances to the food preservation method, wherein the natural antibacterial substances are loaded into the plant exosome, the obtained composite is dispersed in a dispersion medium for food surface treatment to obtain a preservation preparation, and the preservation preparation is applied to the surface of food to be preserved to form a preservation layer, so that the natural antibacterial substances are stably loaded, uniformly distributed and continuously delivered on the surface of the food, the problems of volatilization, inactivation and short action time of the natural antibacterial substances during use are reduced, the food preservation effect is more stable, and the shelf life is prolonged.

Description

Technical Field

[0001] This invention relates to the field of food preservation and biomaterials technology, specifically to a method for using plant exosomes loaded with natural antibacterial substances for food preservation. Background Technology

[0002] During storage, transportation, and sales, food is frequently affected by microbial growth, respiration and metabolism, water loss, oxidation, and changes in external temperature and humidity, subsequently leading to spoilage, color fading, softening, and flavor degradation. Food preservation technology has therefore continuously developed and formed various application pathways. Existing technologies mainly include low-temperature preservation, modified atmosphere packaging, vacuum packaging, edible coatings, preservative treatment, and preservation of natural active ingredients. Low-temperature preservation focuses on inhibiting microbial growth and slowing down food metabolism; modified atmosphere packaging and vacuum packaging focus on regulating the packaging environment, reducing oxygen content, and delaying quality changes; edible coatings are mostly applied to the surface of food, forming an insulating layer and carrying some... For food preservation, preservative treatments often rely on antibacterial components such as sorbates and benzoates to maintain storage stability. Natural active ingredient preservation primarily utilizes plant essential oils, polyphenols, flavonoids, organic acids, and plant extracts to inhibit spoilage and pathogenic bacteria. In recent years, delivery systems such as liposomes, nanoemulsions, microcapsules, and bio-based nanoparticles have also entered the food preservation field. These carriers mainly undertake the functions of encapsulating, protecting, and releasing active substances. Plant-derived nanovesicles have also begun to enter the research field. Related research mainly focuses on active ingredient delivery, membrane structure protection, and the development of natural materials. These technologies together constitute the main technological foundation of the current food preservation field.

[0003] The core deficiency of existing technologies lies in the fact that there is still a lack of a carrier system for natural antimicrobial substances in food preservation applications that combines the characteristics of natural source, stable encapsulation, and continuous delivery. After natural antimicrobial substances enter the food system, they are prone to uneven dispersion, activity decay, and short duration of action, making it difficult to maintain stable food preservation effects. Existing technologies also cannot simultaneously take into account natural properties, antimicrobial performance, and practical application requirements. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for using plant exosomes loaded with natural antibacterial substances for food preservation. The technical problem this invention aims to solve is: how to address the issues of insufficient stable loading, insufficient continuous delivery, and poor stability of preservation effects of natural antibacterial substances in food preservation applications by loading natural antibacterial substances onto plant exosomes and applying them to the surface of food.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for using plant exosomes loaded with natural antibacterial substances for food preservation, comprising: S1. Plant exosomes are obtained by extraction and purification of edible plant tissues; S2. The natural antibacterial substance is loaded and combined with the plant exosomes under a preset loading condition to obtain a plant exosome loaded with natural antibacterial substance complex; S3. Disperse the plant exosome-loaded natural antibacterial substance complex in a food surface treatment dispersion medium to prepare a preservative preparation; S4. Apply the plant exosome-loaded natural antibacterial substance preservative to the surface of the food to be preserved, and form a preservation layer containing the plant exosome-loaded natural antibacterial substance complex on the surface of the food to be preserved. S5. Store the food to be preserved after S4 under preservation conditions.

[0006] Preferably, the edible plant tissue is selected from garlic bulbs, mint leaves, and oregano leaves.

[0007] Preferably, the extraction and purification include: homogenizing the edible plant tissue, centrifuging it at a relative centrifugal force of 300×g-3000×g for 10min-30min, taking the supernatant, filtering it through a microporous membrane of 0.22μm-0.8μm, centrifuging it at a relative centrifugal force of 100000×g-150000×g for 30min-180min, collecting the precipitate and resuspending it to obtain the plant exosomes.

[0008] Preferably, the natural antibacterial substance is selected from thymol, carvacrol, and cinnamaldehyde.

[0009] Preferably, the preset loading conditions are as follows: plant exosomes and natural antibacterial substances are mixed at a mass ratio of 1:0.05-1:1, incubated at 4℃-25℃ for 30min-240min, subjected to ultrasonic treatment for 1min-10min, and then centrifuged to remove the unloaded natural antibacterial substances to obtain a plant exosome-loaded natural antibacterial substance complex.

[0010] Preferably, the dispersion medium for food surface treatment includes deionized water and chitosan, wherein the mass percentage of chitosan in the dispersion medium for food surface treatment is 0.1%-3.0%.

[0011] Preferably, the application in S4 is carried out by spraying, with a spraying amount of 0.5 mL to 5 mL per 100 cm² of food surface.

[0012] Preferably, the preservation and storage conditions are: storage temperature of 0℃-15℃ and relative humidity of 60%-95%.

[0013] This invention provides a method for using plant exosomes loaded with natural antibacterial substances for food preservation. It has the following beneficial effects:

[0014] This method of loading natural antibacterial substances onto plant exosomes for food preservation involves preparing a preservative by loading natural antibacterial substances onto plant exosomes and dispersing the resulting complex in a food surface treatment dispersion medium. The preservative is then applied to the surface of the food to be preserved to form a preservation layer. This method achieves stable loading, uniform distribution, and continuous delivery of natural antibacterial substances on the food surface, mitigating the problems of easy volatility, easy inactivation, and short action time of natural antibacterial substances during use. This results in a more stable food preservation effect and an extended shelf life.

[0015] Plant exosomes are extracted from edible plant tissues, and thymol, carvacrol, or cinnamaldehyde are used as natural antibacterial substances. The mixture is compounded with a food surface treatment dispersion medium containing chitosan and applied to the surface of the food to be preserved by spraying. This method improves the surface adhesion and utilization rate of natural antibacterial substances and helps maintain the integrity and continuity of the food surface preservation layer, thus taking into account natural properties, antibacterial performance, and practical application needs. Attached Figure Description

[0016] Figure 1 This is an overall flowchart of the method of the present invention; Figure 2 This is a schematic diagram of the plant exosome extraction and loading composite of the present invention; Figure 3 This is a schematic diagram illustrating the preparation, application, and storage of the preservative agent of the present invention. Detailed Implementation

[0017] The technical solutions in the embodiments of the present invention have been clearly and completely described. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0018] Example 1 like Figure 1-3 As shown, this embodiment of the invention provides a method for using plant exosomes loaded with natural antibacterial substances for food preservation, comprising: S1. Plant exosomes were obtained by extraction and purification of edible plant tissues. The edible plant tissue was garlic bulb. The extraction and purification process included: homogenizing the edible plant tissues, centrifuging at a relative centrifugal force of 300×g for 10 min, collecting the supernatant, filtering it through a 0.8 μm microporous membrane, centrifuging at a relative centrifugal force of 100000×g for 30 min, collecting the precipitate and resuspending it to obtain plant exosomes.

[0019] S2. A natural antibacterial substance and plant exosomes were loaded and compounded under preset loading conditions to obtain a plant exosome-loaded natural antibacterial substance complex. The natural antibacterial substance was thymol. The preset loading conditions were as follows: the plant exosomes and the natural antibacterial substance were mixed at a mass ratio of 1:0.05, incubated at 4°C for 30 min, sonicated for 1 min, and then centrifuged to remove the unloaded natural antibacterial substance, thus obtaining the plant exosome-loaded natural antibacterial substance complex.

[0020] S3. A preservative preparation is prepared by dispersing a plant exosome-loaded natural antibacterial complex in a food surface treatment dispersion medium. The food surface treatment dispersion medium includes deionized water and chitosan, with chitosan comprising 0.1% by mass.

[0021] S4. Apply a preservative agent containing plant exosomes loaded with natural antibacterial substances to the surface of the food to be preserved, forming a preservative layer containing a complex of plant exosomes loaded with natural antibacterial substances on the surface of the food. S4 is applied by spraying at a rate of 0.5 mL per 100 cm² of food surface.

[0022] S5. Store the food to be preserved after treatment in S4 under the following preservation conditions: storage temperature 0℃, relative humidity 60%.

[0023] Under relatively mild separation, loading, formulation, and application conditions, the extraction of plant exosomes from garlic bulbs, the loading and compounding of thymol, the preparation of preservative agents, and the construction of a food surface preservative layer can still be completed, forming a complete food preservation process. This embodiment corresponds to a lower loading level and a lighter surface treatment intensity, making it suitable for food preservation scenarios that are sensitive to treatment conditions, have fragile surface tissues, and are easily affected by excessive spraying. It is also suitable for application scenarios with high requirements for process intensity and raw material dosage control.

[0024] Example 2 This invention provides a method for using plant exosomes loaded with natural antibacterial substances for food preservation, comprising: S1. Plant exosomes were obtained by extraction and purification of edible plant tissues. The edible plant tissue was peppermint leaves. The extraction and purification process included: homogenizing the edible plant tissues, centrifuging at a relative centrifugal force of 1650×g for 20 min, collecting the supernatant, filtering it through a 0.51 μm microporous membrane, centrifuging at a relative centrifugal force of 125000×g for 105 min, collecting the precipitate and resuspending it to obtain plant exosomes.

[0025] S2. A natural antibacterial substance and plant exosomes were loaded and compounded under preset loading conditions to obtain a plant exosome-loaded natural antibacterial substance complex. The natural antibacterial substance was carvacrol. The preset loading conditions were as follows: the plant exosomes and natural antibacterial substance were mixed at a mass ratio of 1:0.53, incubated at 14.5℃ for 135 min, sonicated for 5.5 min, and then centrifuged to remove the unloaded natural antibacterial substance, thus obtaining the plant exosome-loaded natural antibacterial substance complex.

[0026] S3. A preservative preparation is prepared by dispersing a plant exosome-loaded natural antibacterial complex in a food surface treatment dispersion medium. The food surface treatment dispersion medium includes deionized water and chitosan, with chitosan comprising 1.55% by mass.

[0027] S4. Apply a preservative agent containing plant exosomes loaded with natural antibacterial substances to the surface of the food to be preserved, forming a preservative layer containing a complex of plant exosomes loaded with natural antibacterial substances on the surface of the food. S4 is applied by spraying, with a spraying volume of 2.75 mL per 100 cm² of food surface.

[0028] S5. Store the food to be preserved after treatment in S4 under the following preservation conditions: storage temperature 7.5℃, relative humidity 77.5%.

[0029] The separation of exosomes from peppermint leaves, the loading and compounding of carvacrol, the formation of the preservative formulation, its application to the food surface, and subsequent storage exhibit a good connection between these steps. The processes are well-coordinated, achieving both the carrier-based processing of the active ingredients and the formation of a relatively stable surface preservative layer. This embodiment corresponds to the implementation under conventional preservation conditions and is applicable to fresh-cut fruits and vegetables, as well as other food preservation scenarios requiring a balance between formulation stability, surface coverage, and ease of handling. It demonstrates good versatility and representativeness.

[0030] Example 3 This invention provides a method for using plant exosomes loaded with natural antibacterial substances for food preservation, comprising: S1. Plant exosomes were obtained by extraction and purification of edible plant tissues. The edible plant tissue was oregano leaf. The extraction and purification process included: homogenizing the edible plant tissues, centrifuging at a relative centrifugal force of 3000×g for 30 min, collecting the supernatant, filtering it through a 0.22 μm microporous membrane, centrifuging at a relative centrifugal force of 150000×g for 180 min, collecting the precipitate and resuspending it to obtain plant exosomes.

[0031] S2. The natural antibacterial substance and plant exosomes were loaded and compounded under preset loading conditions to obtain a plant exosome-loaded natural antibacterial substance complex. The natural antibacterial substance was cinnamaldehyde. The preset loading conditions were as follows: the plant exosomes and the natural antibacterial substance were mixed at a mass ratio of 1:1, incubated at 25°C for 240 min, ultrasonicated for 10 min, and then centrifuged to remove the unloaded natural antibacterial substance, thus obtaining the plant exosome-loaded natural antibacterial substance complex.

[0032] S3. A preservative preparation is prepared by dispersing a plant exosome-loaded natural antibacterial complex in a food surface treatment dispersion medium. The food surface treatment dispersion medium includes deionized water and chitosan, with chitosan comprising 3.0% by mass.

[0033] S4. Apply a preservative agent containing plant exosomes loaded with natural antibacterial substances to the surface of the food to be preserved, forming a preservative layer containing a complex of plant exosomes loaded with natural antibacterial substances on the surface of the food. S4 is applied by spraying, with a spraying volume of 5 mL per 100 cm² of food surface.

[0034] S5. Store the food to be preserved after treatment in S4 under the following preservation conditions: storage temperature 15℃, relative humidity 95%.

[0035] Under high levels of separation, loading, formulation, and application conditions, it is still possible to complete the separation of plant exosomes derived from oregano leaves, cinnamaldehyde complexation, preservative formulation construction, and food surface layering treatment, forming a surface preservation system with high coverage. This embodiment corresponds to a high loading level, strong surface treatment intensity, and large formulation dosage, and is suitable for food preservation scenarios with high surface contamination risk, strict preservation requirements, and the need for sufficient surface coverage. It is also suitable for application scenarios with high requirements for the integrity and long-lasting effect of the preservation layer.

[0036] Example 4 In this embodiment, fresh-cut fruits and vegetables from the same batch were selected as samples to be preserved and divided into experimental groups A, B, and C according to the same specifications, with three parallel samples in each group. All three groups used a preservative preparation prepared by loading thymol onto plant exosomes derived from garlic bulbs, which was then sprayed onto the sample surface for storage treatment. The three groups were used to compare the effects of parameter changes on the implementation process and preservation results.

[0037] 1. Comparison of plant exosome preparation Experimental group A first used relatively mild pretreatment conditions to homogenize garlic bulbs and then perform preliminary centrifugation with a low relative centrifugal force. The supernatant was then filtered through a microporous membrane with a larger pore size. Subsequently, the precipitate was collected and resuspended under low-level ultracentrifugation conditions to obtain plant exosomes. The initial centrifugation conditions were 300×g for 10 min with a filtration pore size of 0.8 μm, and the secondary centrifugation conditions were 100000×g for 30 min.

[0038] Experimental group B improved the pretreatment intensity and subsequent separation level. First, it was centrifuged at 1650×g for 20min to remove coarse particulate impurities, then filtered through a 0.51μm microporous membrane, and then centrifuged at 125000×g for 105min to bring the separation conditions of the obtained plant exosomes to an intermediate level.

[0039] Experimental group C further enhanced the separation process. After centrifugation at 3000×g for 30 min, a 0.22μm microporous membrane was used for finer filtration, and then centrifugation at 150000×g for 180 min was performed to complete the collection and resuspension of the precipitate.

[0040] The separation conditions in experimental group A were relatively mild, and the impurity removal intensity was low. The separation intensity and filtration accuracy of experimental group B were at an intermediate level, balancing separation efficiency and operational intensity. The separation conditions in experimental group C were the strongest, and the filtration accuracy was the highest, resulting in the most effective purification and sedimentation collection of the raw material extract.

[0041] 2. Comparison of thymol-loaded composites After obtaining plant exosomes from garlic bulbs, experimental group A used a lower loading ratio and a shorter compounding time to mix plant exosomes with thymol at a mass ratio of 1:0.05, incubate at 4℃ for 30 min, then perform ultrasonic treatment for 1 min, and finally remove unloaded thymol by centrifugation to obtain the complex.

[0042] Experimental group B no longer used the low temperature and short time method, but increased the mass ratio to 1:0.53 and adjusted the incubation conditions to 14.5℃ and 135min, and extended the ultrasonic time to 5.5min, so that the loading process was in an intermediate state.

[0043] Experimental group C used a higher feeding ratio and a longer reaction time. Plant exosomes and thymol were mixed at a ratio of 1:1, incubated at 25°C for 240 min, and then subjected to 10 min of ultrasonic treatment. Afterwards, unloaded components were removed by centrifugation to obtain the loaded complex.

[0044] In experimental group A, the thymol dosage was relatively low and the loading time was short. In experimental group B, the dosage and loading time were both at intermediate levels, resulting in more balanced loading conditions. In experimental group C, the dosage, incubation time, and ultrasonic time were all at their highest, leading to the most complete compounding process. The differences among the three groups in this step were mainly reflected in the progressively increasing loading and compounding intensity.

[0045] 3. Comparison of preservative formulations After the loading and compounding were completed, the three groups added the obtained garlic bulb-derived plant exosomes loaded with thymol to a food surface treatment dispersion medium to prepare a preservative, but the level of chitosan added to the dispersion medium was different.

[0046] Experimental group A used a lower concentration of chitosan, with a chitosan mass percentage of 0.1% in the dispersion medium. The resulting system was relatively thin and suitable for lighter surface treatment.

[0047] Experimental group B increased the chitosan content to 1.55%, which brought the film-forming and adhesion capabilities of the dispersion medium to an intermediate level.

[0048] Experimental group C further increased the chitosan content to 3.0%, corresponding to a higher concentration of film-forming system.

[0049] Experimental group A had lower dispersion medium viscosity and lower film-forming component content, resulting in a lighter formulation. Experimental group B had a moderate chitosan level, balancing flowability and film-forming properties. Experimental group C had the highest chitosan content, resulting in a formulation with stronger film-forming tendency and surface adhesion. In this step, the three groups showed a change in formulation concentration and film-forming ability from low to high.

[0050] 4. Comparison of surface application processes After the preservative was prepared, all three groups treated the surface of fresh-cut fruits and vegetables by spraying, but the amount applied per unit area was different.

[0051] Experimental group A used a light spraying method, spraying 0.5 mL of preservative per 100 cm² of food surface to form a thin preservative layer on the sample surface.

[0052] Experimental group B increased the spraying amount to 2.75 mL per 100 cm² of food surface to obtain more adequate surface coverage.

[0053] Experimental group C used a larger spraying volume, applying 5 mL of preservative per 100 cm² of food surface to form a thicker coating layer on the sample surface.

[0054] Experimental group A had the smallest application rate per unit area, resulting in lighter surface coverage. Experimental group B had a moderate spraying rate, providing more thorough surface coverage. Experimental group C had the largest spraying rate, achieving the highest level of coverage and accumulation of the preservative on the food surface. These three groups demonstrated differences in surface treatment from light to heavy coverage in this step.

[0055] 5. Comparison of storage conditions After spraying, experimental group A stored the samples under low temperature and low relative humidity conditions, with a storage temperature of 0℃ and a relative humidity of 60%. Experimental group B used intermediate storage conditions, with a temperature of 7.5℃ and a relative humidity of 77.5%. Experimental group C was stored under high temperature and high relative humidity conditions, with a temperature of 15℃ and a relative humidity of 95%.

[0056] Experimental group A had the strongest inhibitory storage environment and the lowest environmental load. Experimental group B was in an intermediate storage state with relatively balanced environmental conditions. Experimental group C had higher storage temperature and humidity, and the samples faced the greatest external preservation pressure. The three groups in this step constituted a comparison of storage conditions from low to high environmental load.

[0057] 6. Detection and Comparison Methods Samples were taken from experimental groups A, B, and C at the same storage time point to test the total bacterial count, weight loss rate, hardness retention rate, color difference value, and sensory status, and each item was compared.

[0058] First, experimental group A and experimental group B were compared to analyze the changes in each detection index after the parameter was increased from the lower limit to the median value. Then, experimental group B and experimental group C were compared to analyze the changes in each detection index after the parameter was increased from the median value to the upper limit. Finally, experimental group A, experimental group B and experimental group C were comprehensively compared to evaluate the impact of different parameter combinations on the food preservation treatment effect.

[0059] In summary, experimental groups A, B, and C formed a continuous gradient, which can be used to verify the effect of different parameter values ​​on the food preservation treatment results.

[0060] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for using plant exosomes loaded with natural antibacterial substances for food preservation, characterized in that, include: S1. Plant exosomes are obtained by extraction and purification of edible plant tissues; S2. The natural antibacterial substance is loaded and combined with the plant exosomes under a preset loading condition to obtain a plant exosome loaded with natural antibacterial substance complex; S3. Disperse the plant exosome-loaded natural antibacterial substance complex in a food surface treatment dispersion medium to prepare a preservative preparation; S4. Apply the plant exosome-loaded natural antibacterial substance preservative to the surface of the food to be preserved, and form a preservation layer containing the plant exosome-loaded natural antibacterial substance complex on the surface of the food to be preserved. S5. Store the food to be preserved after S4 under preservation conditions.

2. The method for using plant exosomes loaded with natural antibacterial substances for food preservation according to claim 1, characterized in that: The edible plant tissues are selected from garlic bulbs, mint leaves, and oregano leaves.

3. The method for using plant exosomes loaded with natural antibacterial substances for food preservation according to claim 1, characterized in that: The extraction and purification process includes: homogenizing the edible plant tissue, centrifuging it at a relative centrifugal force of 300×g-3000×g for 10-30 min, collecting the supernatant, filtering it through a microporous membrane of 0.22μm-0.8μm, centrifuging it at a relative centrifugal force of 100000×g-150000×g for 30-180 min, collecting the precipitate and resuspending it to obtain the plant exosomes.

4. The method for using plant exosomes loaded with natural antibacterial substances for food preservation according to claim 1, characterized in that: The natural antibacterial substances are selected from thymol, carvacrol, and cinnamaldehyde.

5. The method for using plant exosomes loaded with natural antibacterial substances for food preservation according to claim 1, characterized in that: The preset loading conditions are as follows: plant exosomes and natural antibacterial substances are mixed at a mass ratio of 1:0.05-1:1, incubated at 4℃-25℃ for 30min-240min, subjected to ultrasonic treatment for 1min-10min, and then centrifuged to remove the unloaded natural antibacterial substances to obtain a plant exosome-loaded natural antibacterial substance complex.

6. The method for using plant exosomes loaded with natural antibacterial substances for food preservation according to claim 1, characterized in that: The dispersion medium for food surface treatment includes deionized water and chitosan, wherein the mass percentage of chitosan in the dispersion medium for food surface treatment is 0.1%-3.0%.

7. The method for using plant exosomes loaded with natural antibacterial substances for food preservation according to claim 1, characterized in that: The application described in S4 is by spraying, with a spraying volume of 0.5 mL to 5 mL per 100 cm² of food surface.

8. The method for using plant exosomes loaded with natural antibacterial substances for food preservation according to claim 1, characterized in that: The preservation and storage conditions are: storage temperature of 0℃-15℃ and relative humidity of 60%-95%.

Images