Bacillus velezensis P15 fruit and vegetable fresh-keeping agent based on site competition mechanism and use method of bacillus velezensis P15 fruit and vegetable fresh-keeping agent

Abstract

The invention relates to the technical field of biological control, and discloses a bacillus velezensis P15 fruit and vegetable fresh-keeping agent based on a site competition mechanism, the active ingredient of the fresh-keeping agent is fermentation liquor of bacillus velezensis P15, the preservation number of the bacillus velezensis P15 strain is China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.31995. According to the preservative and the method, the bacillus velezensis P15 fermentation liquor is used as an active ingredient, the preservative is free of pathogenicity, the product can be degraded, and the health and environmental risks of chemical preservatives are avoided; the raw materials are low-cost food-grade substances, cold chain or high-airtight packaging is not needed, spraying / soaking can be carried out, and the cost is greatly reduced; the strain inhibits pathogenic bacteria by means of site competition with the antibacterial substance, and the film-forming agent locks water and controls respiration, so that the fresh-keeping period is prolonged, the loss is reduced, and the method is suitable for various fruits and vegetables, is suitable for normal temperature and low temperature, and is simple to operate and easy to popularize.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, specifically to a Bacillus vesicles P15 fruit and vegetable preservative based on a site competition mechanism and its application method. Background Technology

[0002] As people's living standards improve, their demands for food safety and quality are also increasing. The freshness and shelf life of fruits and vegetables have become a focus of consumer attention. However, after harvesting, fruits and vegetables are prone to spoilage and quality decline due to physiological activities and microbial influences, leading to economic losses and food safety issues. Traditional preservation methods such as low-temperature refrigeration, chemical preservative treatment, and modified atmosphere packaging can delay spoilage, but they also pose potential health risks, environmental burdens, and high costs associated with chemical preservatives. Therefore, we propose a Bacillus baileyi P15 fruit and vegetable preservative based on a site competition mechanism and its application method to address the aforementioned problems. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a Bacillus baileyi P15 fruit and vegetable preservative based on a site competition mechanism and its application method. This solves the problem that fruits and vegetables are prone to spoilage and quality decline after harvesting due to physiological activities and microbial influences, leading to economic losses and food safety issues. Traditional preservation methods such as low-temperature refrigeration, chemical preservative treatment, and modified atmosphere packaging can delay fruit and vegetable spoilage, but they suffer from potential health risks, environmental burdens, and high costs associated with chemical preservatives.

[0004] To achieve the above objectives, the present invention is implemented through the following technical solution: a Bacillus vesicle P15 fruit and vegetable preservative based on a site competition mechanism, wherein the active ingredient of the preservative is the fermentation broth of Bacillus vesicle P15, and the Bacillus vesicle P15 strain has the accession number of China General Microbiological Culture Collection Center, accession number CGMCC NO.31995.

[0005] Preferably, the preservative is a liquid preparation composed of fermentation broth, or a composite liquid preparation containing the fermentation broth and supplemented with nutrients and film-forming agents.

[0006] Preferably, the nutrient is selected from one of sucrose, glucose, peptone, and yeast extract.

[0007] Preferably, the film-forming agent is selected from sodium alginate, chitosan, pullulan, and sodium carboxymethyl cellulose.

[0008] Preferably, the viable bacterial concentration of Bacillus vesiculus P15 in the preservative is 1×10⁻⁶. 7 CFU / mL to 1×10 9CFU / mL; the concentration of the film-forming agent is 0.1%-3.0% (w / v).

[0009] This invention provides a method for using Bacillus belye P15 fruit and vegetable preservative based on a site competition mechanism, comprising the aforementioned fruit and vegetable preservative, characterized by the following steps:

[0010] S1. Activate and ferment Bacillus vesiculosus P15 to obtain fermentation broth; use the fermentation broth directly or combine it with nutrients and film-forming agents to prepare a preservative working solution;

[0011] S2. After harvesting fruits and vegetables and before storage, the preservative working solution is evenly applied to the surface of the fruits and vegetables by spraying, soaking or coating.

[0012] S3. The treated fruits and vegetables are stored under suitable conditions. The Bacillus berberis P15 inhibits the growth of pathogens on the surface of the fruits and vegetables through a site competition mechanism, thereby achieving preservation.

[0013] Preferably, in step S1, the fermentation time is 18-36 hours.

[0014] Preferably, in step S2, the preservative working solution is applied until a liquid film is evenly covered on the surface of the fruits and vegetables, with no obvious dripping.

[0015] Preferably, the suitable conditions are ambient temperature of 10-25°C or low-temperature refrigeration of 0-4°C.

[0016] Preferably, the fruits and vegetables include apples, pears, grapes, peaches, bananas, and strawberries.

[0017] Beneficial effects

[0018] This invention provides a Bacillus baileyi P15 fruit and vegetable preservative based on a site competition mechanism and its application method. Compared with the prior art, it has the following advantages:

[0019] This fruit and vegetable preservative based on a site competition mechanism, Bacillus vesicle P15, and its application method, utilizes Bacillus vesicle P15 fermentation broth as its active ingredient. It is non-pathogenic and the product is biodegradable, avoiding the health risks and environmental pollution associated with chemical preservative residues. The raw materials are low-cost, food-grade substances, requiring no cold chain or airtight packaging. Operation involves only spraying and soaking, significantly reducing costs and solving the high-cost problem of traditional methods. The strain inhibits pathogens through site competition and antimicrobial substances, while the film-forming agent reduces moisture loss and moderately controls respiration. This dual action delays spoilage, extends shelf life, and reduces economic losses. It is suitable for various fruits and vegetables such as apples and pears, and is applicable at both room and low temperatures. The operation is simple and easy to promote, overcoming the limitations of traditional methods. Attached Figure Description

[0020] Figure 1 The images show the colony, cell morphology, and Gram staining of Bacillus belyssus P15 of this invention; A shows the colony of Bacillus belyssus P15, and B shows the cell morphology and Gram staining.

[0021] Figure 2 This is a flowchart illustrating the application method of Bacillus vesiculosus P15 fruit and vegetable preservative based on a site competition mechanism according to the present invention. Detailed Implementation

[0022] 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 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.

[0023] like Figure 1 As shown:

[0024] Example 1:

[0025] A fruit and vegetable preservative based on a site competition mechanism using Bacillus velezensis P15 is proposed. The active ingredient of the preservative is the fermentation broth of Bacillus velezensis P15. The Bacillus velezensis P15 strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China), with accession number CGMCC. No. 31995, classified and named Bacillus velezensis, with a preservation date of September 18, 2024, has the following characteristics: Gram-positive, rod-shaped, capable of forming spores, highly resistant to adverse conditions (able to withstand a certain range of temperature and pH changes), easily colonizes on the surface of fruits and vegetables without pathogenicity, and competes with pathogenic microorganisms for nutrient sites and attachment sites on the surface of fruits and vegetables by secreting antibacterial substances (such as lipopeptides and polypeptide compounds), thereby inhibiting the growth of pathogenic microorganisms, and is harmless to humans and the environment;

[0026] The preservative comes in two formulations, which can be selected according to the actual application scenario:

[0027] It consists only of the fermentation broth of Bacillus vesiculosus P15, with a simple composition, making it suitable for short-term storage or scenarios where the preservative composition is extremely limited.

[0028] It is formulated from Bacillus vesiculosus P15 fermentation broth, nutrients, and film-forming agents. Among them, the nutrients provide continuous nutritional support for Bacillus vesiculosus P15, prolonging its survival and colonization time on the surface of fruits and vegetables; the film-forming agent forms a breathable and edible protective film on the surface of fruits and vegetables, reducing water loss and invasion of external pathogens, while providing a stable colonization environment for the strain.

[0029] Nutrients are selected from sucrose, glucose, peptone, and yeast extract. All of the above substances are food-grade raw materials with high safety: sucrose and glucose can be directly utilized by Bacillus belye P15 as a carbon source to maintain the strain's metabolic activities; peptone and yeast extract are rich in amino acids, vitamins, and minerals, providing the strain with nitrogen sources and growth factors to promote strain reproduction;

[0030] The film-forming agent is selected from one of sodium alginate, chitosan, pullulan, and sodium carboxymethyl cellulose. All of the above film-forming agents possess good biocompatibility and biodegradability: sodium alginate is a natural polysaccharide extracted from brown algae, exhibiting good film-forming properties and certain antibacterial activity; chitosan, derived from animal shells, not only forms a breathable membrane but also inhibits the growth of some pathogenic microorganisms; pullulan, produced by fermentation of *Brachystomata*, produces a membrane with high transparency and flexibility, without affecting the appearance of fruits and vegetables; sodium carboxymethyl cellulose is a common food additive, offering low film-forming cost and improving the water solubility and stability of the membrane.

[0031] The viable bacterial concentration of Bacillus vesiculus P15 in the preservative was 1×10⁻⁶. 7 CFU / mL to 1×10 9 CFU / mL; the concentration of the film-forming agent is 0.1%-3.0% (w / v), by selecting 1×10 8 The viable bacterial concentration of CFU / mL, compared to 1×10 7 The CFU / mL has increased tenfold, allowing the strain to colonize the surface of fruits and vegetables faster and in greater numbers. It can quickly occupy the nutrient and physical sites on the fruit and vegetable surface, forming a competitive suppression against pathogenic microorganisms. Even when faced with fruits and vegetables with high initial microbial load, it can effectively inhibit the growth of pathogens and avoid the problems of weak competition and fluctuating preservation effect at low concentrations.

[0032] Compared to 1×10 8 CFU / mL, 1×10 8 The CFU / mL live bacteria concentration reduces the strain culture quantity by 90%, and the amount of culture medium, fermentation time and energy consumption required during fermentation are all significantly reduced, which can significantly reduce the raw material and total production cost of the preservative. At the same time, there is no need to optimize the fermentation process in order to pursue a high concentration, which reduces the complexity of the process and the production risk.

[0033] The core function of film-forming agents is to form a continuous, breathable protective film on the surface of fruits and vegetables, reducing moisture loss, blocking the invasion of external pathogens, and providing a stable colonization environment for Bacillus belye P15. The concentration of film-forming agents is 0.1%-3.0% (w / v, i.e., 0.1g-3.0g of film-forming agent per 100mL of preservative). When the concentration of film-forming agents is lower than 0.1%, it is difficult to form a complete and continuous protective film on the surface of fruits and vegetables, and it cannot effectively reduce moisture loss and pathogen invasion. When the concentration is higher than 3.0%, the film layer is too thick, the breathability is poor, and it is easy to cause oxygen deficiency on the surface of fruits and vegetables, triggering anaerobic respiration. At the same time, the film layer is prone to cracking and peeling, affecting the preservation effect and appearance. Therefore, 0.1%-3.0% is limited to the optimal concentration range of film-forming agents.

[0034] It should be noted that for detailed methods of isolation and screening of Bacillus belyss P15, please refer to the patent literature, publication number: CN119162042A.

[0035] like Figure 2 As shown:

[0036] Example 2:

[0037] This invention provides a method for using Bacillus belye P15 fruit and vegetable preservative based on a site competition mechanism, comprising the following steps:

[0038] S1. Take the freeze-dried powder of Bacillus belye P15 (preservation number CGMCCNO.31995) and inoculate it into LB liquid medium (10g tryptone, 10g NaCl, 10g yeast extract, 1L deionized water, pH 7.0). Activate the culture by shaking in a shaker at 25-37℃ and 150-220rpm for 12-16 hours to obtain the seed culture. Inoculate the seed culture at a rate of 1%-5% into fermentation medium (20g / L glucose, 15g / L peptone, 5g / L yeast extract, 2g / L potassium dihydrogen phosphate, 0.5g / L magnesium sulfate, pH 7.0-7.2). Ferment and culture at 30-37℃ and 180-220rpm for 18-36 hours until the viable concentration of Bacillus belye P15 in the fermentation broth reaches 1×10⁻⁶. 7 -1×10 9 CFU / mL, stop culturing and obtain fermentation broth;

[0039] If preparing a basic liquid formulation, the above fermentation broth can be used directly as the preservative working solution; if preparing a compound liquid formulation, nutrients and film-forming agents are added to the fermentation broth in proportion, and stirred at room temperature for 10-15 minutes until the nutrients and film-forming agents are completely dissolved to obtain the preservative working solution.

[0040] S2. Within 24 hours of harvesting fruits and vegetables (at this time, respiration has not yet increased dramatically, the number of surface microorganisms is low, and the preservation effect is optimal), apply the preservative working solution to the surface of the fruits and vegetables by spraying, soaking, or coating until a continuous liquid film is evenly covered on the surface without significant dripping. If the liquid film is incomplete, uncovered areas are susceptible to pathogen infection; if the liquid film is too thick and drips, it not only wastes the preservative but may also form liquid accumulation on the surface of the fruits and vegetables, which can actually promote microbial growth. The specific application method can be adjusted according to the type of fruits and vegetables.

[0041] It can be done by spraying, soaking, and coating. Specifically, spraying is suitable for fruits and vegetables with hard skin that are not easily damaged, such as apples, pears, and peaches. It uses a manual sprayer or automatic spraying equipment to spray evenly at a distance of 20-30cm from the surface of the fruits and vegetables. It is highly efficient and suitable for large-scale processing.

[0042] Soaking method: Suitable for small fruits and vegetables such as strawberries and grapes. Gently soak the fruits and vegetables in the preservative working solution for 10-30 seconds, then remove them and drain off the excess liquid. Be careful to control the soaking time to avoid the fruits and vegetables absorbing too much water and rotting.

[0043] Coating method: Suitable for fruits and vegetables with soft peels, such as bananas. Use a soft brush or sponge to gently apply the coating to avoid mechanical damage to the peel.

[0044] S3. Store the treated fruits and vegetables under suitable conditions, such as room temperature storage at 10-25℃ or low temperature refrigeration at 0-4℃.

[0045] This method utilizes Bacillus vesiculosus P15 fermentation broth as its active ingredient. This broth is non-pathogenic and its products are biodegradable, avoiding the health risks and environmental pollution associated with chemical preservative residues. The raw materials are low-cost, food-grade substances, eliminating the need for cold chain or airtight packaging. Operations only require spraying and soaking, significantly reducing costs and solving the high-cost problem of traditional methods. The strain inhibits pathogens through site competition and antimicrobial substances, while the film-forming agent reduces moisture loss and moderately controls respiration. This dual action delays spoilage, extends shelf life, and reduces economic losses. It is suitable for various fruits and vegetables such as apples and pears, and can be used at both room and low temperatures. The method is simple to operate and easy to promote, overcoming the limitations of traditional methods.

[0046] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are 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 Bacillus vesicle P15 fruit and vegetable preservative based on a site competition mechanism, characterized in that: The active ingredient of the preservative is the fermentation broth of Bacillus vesicularis P15, and the preservation number of the Bacillus vesicularis P15 strain is China General Microbiological Culture Collection Center, preservation number CGMCC NO.31995.

2. The Bacillus vesicle P15 fruit and vegetable preservative based on site competition mechanism according to claim 1, characterized in that: The preservative is a liquid preparation composed of fermentation broth, or a composite liquid preparation containing the fermentation broth and supplemented with nutrients and film-forming agents.

3. The Bacillus vesicle P15 fruit and vegetable preservative based on a site competition mechanism according to claim 2, characterized in that: The nutrients are selected from one of sucrose, glucose, peptone, and yeast extract.

4. The Bacillus vesicle P15 fruit and vegetable preservative based on a site competition mechanism according to claim 2, characterized in that: The film-forming agent is selected from one of sodium alginate, chitosan, pullulan, and sodium carboxymethyl cellulose.

5. The Bacillus vesicle P15 fruit and vegetable preservative based on site competition mechanism according to claim 1, characterized in that: The preservative contains Bacillus vesiculosus P15 at a viable concentration of 1×10⁻⁶. 7 CFU / mL to 1×10 9 CFU / mL; the concentration of the film-forming agent is 0.1%-3.0% (w / v).

6. A method for using Bacillus vesicles P15 fruit and vegetable preservative based on a site competition mechanism, comprising the fruit and vegetable preservative as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Activate and ferment Bacillus vesiculosus P15 to obtain fermentation broth; use the fermentation broth directly or combine it with nutrients and film-forming agents to prepare a preservative working solution. S2. After harvesting fruits and vegetables and before storage, the preservative working solution is evenly applied to the surface of the fruits and vegetables by spraying, soaking or coating. S3. The treated fruits and vegetables are stored under suitable conditions. The Bacillus berberis P15 inhibits the growth of pathogens on the surface of the fruits and vegetables through a site competition mechanism, thereby achieving preservation.

7. The method of using the Bacillus vesicles P15 fruit and vegetable preservative based on a site competition mechanism according to claim 6, characterized in that: In S1, the fermentation culture time is 18-36 hours.

8. The method of using the Bacillus vesicularis P15 fruit and vegetable preservative based on the site competition mechanism according to claim 6, characterized in that: In step S2, the preservative working solution is applied until a liquid film is evenly covered on the surface of the fruits and vegetables, with no obvious dripping.

9. The method of using the Bacillus vesicularis P15 fruit and vegetable preservative based on the site competition mechanism according to claim 6, characterized in that: The suitable conditions are ambient temperature of 10-25℃ or low-temperature refrigeration of 0-4℃.

10. The method of using the Bacillus vesicularis P15 fruit and vegetable preservative based on the site competition mechanism according to claim 6, characterized in that: The fruits and vegetables mentioned include apples, pears, grapes, peaches, bananas, and strawberries.

Images