Formula and preparation method of novel antifungal compound biopesticide bactericidal preservative

Abstract

The invention discloses a novel compound biological preservative formula for resisting pathogenic fungi, which consists of 10mg / L of thiabendazole and 10mg / L of berberine in a mass ratio of one of 1: 1, 1: 2, 1: 4, 1: 8 and 1: 16. In the formula, berberine and thiabendazole synergistically resist corrosion, so that the dosage of thiabendazole can be reduced, healthy corrosion prevention of fruits, vegetables and agricultural products is facilitated, and the formula is beneficial to the ecological environment and human health and also has the effect of resisting pathogenic fungi.

Description

Technical Field

[0001] The invention belongs to the field of antiseptic fungicides, and particularly relates to a formula and a preparation method of an antifungal compounded biopesticide fungicide preservative formed by compounding berberine with a benzimidazole fungicide, thiabendazole. Background Art

[0002] The issue of preserving fruits and vegetables after harvest has long been a bottleneck restricting the development of the fruit and vegetable industry. Due to their high water content and active metabolism, fruits and vegetables are extremely susceptible to microbial infection, water loss and wilting, and physiological aging during post-harvest transportation, storage, and marketing, resulting in significant losses. This not only wastes resources but also impacts the economic benefits and sustainable development of the fruit and vegetable industry. Therefore, the development of efficient fruit and vegetable preservation technologies is urgent. While common fruit and vegetable preservation methods, such as refrigeration and controlled atmosphere (CA) preservation, can extend the shelf life of fruits and vegetables to a certain extent, they are associated with high equipment investment, high operating costs, and limited applicability. For example, refrigeration requires low-temperature equipment, while CA preservation has strict requirements on ambient gas composition and complex equipment. In contrast, spray-based biological preservatives can be used at room temperature or relatively mild conditions, eliminating the need for specialized, large-scale equipment, offering a more flexible and economical option for fruit and vegetable preservation.

[0003] In the field of fruit and vegetable preservation, the development and application of safe and efficient preservation technologies are required to reduce chemical residues and ensure that consumers can consume safe and healthy fruits and vegetables. This provides guidance for the research and development of new compound biological preservatives, encouraging researchers to develop green, environmentally friendly, and residue-free preservation products. Thiabendazole, a commonly used chemical pesticide, is effective against a variety of fruit and vegetable diseases and can effectively inhibit the growth and reproduction of pathogens. However, long-term use of thiabendazole alone leads to the development of pathogen resistance and the escalating dosage, which not only increases costs but also exacerbates the risk of pesticide residues, posing a potential threat to the environment and human health. Combining botanical active ingredients with thiabendazole can achieve complementary advantages. The antibacterial and antioxidant properties of botanical active ingredients, combined with thiabendazole's high inhibitory ability against specific pathogens, can comprehensively inhibit microbial growth and effectively delay the aging of fruits and vegetables. For example, the combination of carvacrol and thiabendazole, two plant-derived active ingredients, greatly enhances the inhibitory effect on common fruit and vegetable pathogenic fungi such as Botrytis cinerea, significantly extending the shelf life of fruits and vegetables, reducing losses, and maintaining the freshness and quality of fruits and vegetables.

[0004] According to surveys, the majority of preservatives used in the market are traditional chemical preservatives, such as benzoic acid and its salts, and sorbic acid and its salts. These can, to a certain extent, inhibit microbial growth and extend the shelf life of fruits and vegetables. However, long-term use of these preservatives carries numerous risks. First, chemical preservative residues on the surface of fruits and vegetables may pose a potential threat to human health, such as interfering with normal metabolism and damaging liver and kidney function. Furthermore, some chemical preservatives are difficult to degrade and continue to accumulate in the environment, polluting soil, water, and other ecological environments.

[0005] The use of thiabendazole preservative in the market is mature. Although its preservative effect is significant, excessive use can be toxic to fish, pollute ponds and water sources, affect the environment and destroy the ecology. At the same time, its residue on fruits can endanger human health. The present invention compounds thiabendazole and berberine, studies their antibacterial effect on pathogenic fungi, finds the optimal compounding ratio of thiabendazole and berberine through a large number of experiments, and mixes them to produce a high-efficiency, low-toxicity, and green biological preservative. The present invention can reduce the amount of thiabendazole used, reduce environmental pollution, protect human health, and achieve better antibacterial effect. It is of great significance. At the same time, the raw materials are easily available in the market and the cost is low, the production process is simple, the market potential is large, the economic and social benefits are significant, and the present invention has a very broad application prospect and can be easily promoted in the market.

[0006] Thiabendazole, also known as Tekodol, is a benzimidazole fungicide. Its mechanism of action is to bind to pathogenic microtubules' β-tubulin, inhibiting its assembly with α-tubulin or causing the depolymerization of pre-assembled microtubules. This process disrupts normal spindle formation, ultimately preventing mitosis in fungal cells, and is effective against a wide range of fungal diseases. Thiabendazole is a systemic, broad-spectrum fungicide used in agriculture to control a variety of diseases caused by ascomycetes, deuteromycetes, and basidiomycetes on crops, fruit trees, and cash crops. Its excellent control effectiveness is particularly evident against typical fungal diseases such as wheat smut, rice blast, citrus penicillium, and tomato gray mold. Furthermore, thiabendazole demonstrates unique efficacy in fruit preservation. It can be used during storage on a variety of fruits, including citrus, lychees, and pears, effectively controlling fungal diseases and extending their shelf life. Summary of the Invention

[0007] A novel anti-pathogenic fungus compound biological preservative formula is composed of 10 mg / L thiabendazole and 10 mg / L berberine, wherein the mass ratio of thiabendazole to berberine is one of 1:1, 1:2, 1:4, 1:8, and 1:16.

[0008] A method for preparing a novel anti-pathogenic fungus compound biological preservative is disclosed. 10 mg / L thiabendazole and 10 mg / L berberine are compounded at mass ratios of 1:1, 1:2, 1:4, 1:8, and 1:16.

[0009] More preferably, thiabendazole and berberine are compounded in a mass ratio of 1:1.

[0010] More preferably, the preparation method of 10 mg / L berberine is as follows: dissolve 2.5 mg of berberine raw powder in hot water, dilute to 250 ml, and prepare a 10 mg / L berberine solution.

[0011] Further preferably, the preparation method of 10 mg / L thiabendazole is as follows: dissolve 2.5 mg of thiabendazole original drug in an appropriate amount of methanol, draw the dissolved thiabendazole-methanol mixture with a syringe, add it into a needle filter for filtration and sterilization, and dilute it with water to 250 ml to prepare a 10 mg / L thiabendazole solution.

[0012] Berberine, also known as berberine, is the primary component of the traditional Chinese medicine Coptis chinensis (Coptis chinensis) and exhibits significant antibacterial properties. Berberine belongs to the quaternary ammonium alkaloid class. The antibacterial mechanism of quaternary ammonium antimicrobial agents lies primarily in their positively charged nitrogen cations, which adsorb to the negatively charged bacterial cell surface. This adsorption process interferes with enzyme activity on the bacterial membrane surface and in the cytoplasm, thereby hindering normal cellular metabolism and ultimately leading to apoptosis. In this way, quaternary ammonium antimicrobial agents effectively inhibit bacterial growth and reproduction. Berberine exhibits broad antibacterial properties. Its unique multi-target antibacterial mechanism enables it to effectively inhibit fungal growth while reducing the risk of fungal resistance. This property gives berberine a unique advantage in the antibacterial field, providing a new option for antimicrobial pesticides. Berberine's inhibitory effects on plant pathogenic fungi have been reported. For example, research by Ruan Yuan et al. showed that at a concentration of 100 μg / mL, berberine exhibited inhibitory effects exceeding 75% against watermelon wilt (Fusarium oxysporum), tomato early blight (Alternaria solani), and apple leaf spot (Alternaria alternata), demonstrating significant antifungal activity. Based on multi-site field efficacy trials, Ling Shipeng et al. demonstrated that a 0.5% berberine solution diluted 400-fold achieved over 75% efficacy against pumpkin powdery mildew, pepper blight, and lettuce sclerotinia rot, demonstrating its significant disease control efficacy. Based on antifungal activity tests, Qu Huan et al. demonstrated that berberine hydrochloride exhibited an extremely high inhibition rate of 99.9% against corn leaf blight at a concentration of 100 μg / mL. Research by Tan Ting et al. showed that berberine exhibited an 89.23% inhibition rate against Fusarium oxysporum at a concentration of 4 mg / mL. Lin Bo et al. found that the minimum inhibitory concentration (MIC) of berberine hydrochloride against the growth of Penicillium digitatum was 0.16 g / L, while the minimum fungicidal concentration (MFC) was 0.32 g / L. This finding reveals the effective concentration range of berberine hydrochloride for inhibiting and killing Penicillium digitatum. Studies have shown that berberine hydrochloride inhibits spore germination and hyphal growth of Penicillium digitatum and can also delay the progression of green mold disease in citrus fruit. Its antibacterial mechanism may involve affecting the permeability and energy metabolism of mycelial cells. In the research of Wang Minggen et al., berberine solution was made into corresponding drug-sensitive tablets, which showed antibacterial activity against Aspergillus glaucoides and Aspergillus niger. With the increase of concentration, this antibacterial activity was significantly enhanced.Wang Xin et al. used the filter paper method to determine the antibacterial activity of berberine. Berberine has a broad antimicrobial spectrum, exhibiting strong inhibitory activity against Gram-negative Escherichia coli and Gram-positive Bacillus subtilis and Staphylococcus aureus, but exhibiting slightly less potent inhibitory activity against Aspergillus niger. Fu Weihui et al. combined berberine with carbendazim, demonstrating a synergistic effect at a berberine-to-carbendazim ratio of 3657:1. This suggests that the addition of a trace amount of carbendazim to berberine can enhance its antibacterial effect. Li Pengyu et al. conducted field efficacy trials and found that a 1000-fold dilution of 10% berberine wettable powder had a 71.7% efficacy against gray mold. Combining berberine with Sophora flavescens, calamine, sodium nitrophenolate, and brassinolide all enhanced berberine's efficacy against gray mold in tomatoes. Therefore, berberine has a broad and significant inhibitory and even long-lasting effect against a variety of common plant pathogenic fungi. Compared to other drugs, berberine is less likely to induce microbial resistance and can significantly enhance its antibacterial effect when used in combination with other agents.

[0013] The present invention enables berberine and thiabendazole to simultaneously play an antiseptic role, and can reduce the amount of thiabendazole used, making fruits, vegetables, and agricultural products healthier during the antiseptic process, having certain benefits for the natural ecological environment and human health, and being more popular among people. It ensures the development of green agriculture and is in line with my country's policy of adhering to the path of green development.

[0014] The present invention is a novel anti-pathogenic fungus composite biological preservative mainly based on the plant active extract berberine and the chemical pesticide thiabendazole, which can effectively preserve fruits, vegetables and agricultural products. It can replace the chemical preservatives circulating on the market in the preservation and freshness of fruits, vegetables and agricultural products. Compared with using chemical preservatives alone to preserve fruits, vegetables and agricultural products, it can have a more efficient effect and can extend the storage time of fruits, vegetables and agricultural products, while promoting the development of green biological pesticides.

[0015] The plant active extract berberine of the present invention has significant antibacterial effects. Berberine belongs to the quaternary ammonium alkaloid class. The antibacterial mechanism of quaternary ammonium antimicrobial agents lies primarily in their positively charged nitrogen cations, which can adsorb to the negatively charged bacterial cell surface. This adsorption process interferes with enzyme activity on the bacterial membrane surface and in the cytoplasm, thereby hindering the normal metabolic processes of the cells and ultimately leading to apoptosis. In this way, quaternary ammonium antimicrobial agents effectively inhibit the growth and reproduction of bacteria. Berberine has a wide range of antibacterial properties. Its unique multi-target antibacterial mechanism enables it to effectively inhibit fungal growth while reducing the risk of fungal resistance. This characteristic gives berberine a unique advantage in the antibacterial field and provides a new option for antibacterial preservatives. Studies have shown that berberine has a wide range of significant inhibitory and even killing effects on a variety of common plant pathogenic fungi, with long-lasting effects. Compared with other drugs, berberine is less likely to induce drug resistance in microorganisms, and when used in combination with other agents, it can significantly enhance its antibacterial effects.

[0016] Thiabendazole is a low-toxic fungicide. Experimental data show that it is mildly irritating to rabbit eyes. If it accidentally comes into contact with the eyes, the eyelids should be lifted and rinsed with running water. If you feel unwell, you should seek medical attention immediately. If you accidentally inhale it into the mouth, you should quickly leave the scene to a place with fresh air. Keep your respiratory tract open. If the situation is serious, you should seek medical attention immediately. The present invention mixes berberine and thiabendazole in a certain ratio to produce a high-efficiency, low-toxic, green biopreservative, which reduces the use of thiabendazole and thus reduces the harm caused by thiabendazole to the ecological environment and humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The inhibition rate of different thiabendazole concentrations against Aspergillus niger is shown in the figure. As the figure shows, the inhibition rate gradually increases with increasing thiabendazole concentration, but does not significantly increase after a certain concentration. The figure also shows that thiabendazole has a good inhibitory effect on Aspergillus niger at concentrations of 10 mg / L and 20 mg / L. The optimal inhibitory concentration of thiabendazole is 10 mg / L.

[0018] Figure 2 The figure shows the inhibitory effect of different concentrations of thiabendazole on Aspergillus niger. The inhibitory effect significantly increases with increasing thiabendazole concentration. As shown in the figure, the inhibitory effect is good at concentrations of 10 mg / L and 20 mg / L, so the optimal inhibitory effect is achieved at 10 mg / L. The optimal inhibitory concentration of thiabendazole is 10 mg / L.

[0019] Figure 3 The following table shows the inhibition rate of berberine against Aspergillus niger at different concentrations. As can be seen from the figure, the inhibition rate gradually increases with increasing berberine concentration. The figure also shows that berberine has a good bactericidal effect at a concentration of 10 mg / L. The optimal inhibitory concentration of berberine is 10 mg / L.

[0020] Figure 4 The figure shows the antibacterial effect of berberine at different concentrations against Aspergillus niger. As the berberine concentration increases, the antibacterial effect significantly improves. As shown in the figure, the antibacterial effect is best at a concentration of 10 mg / L, so the optimal antibacterial effect is achieved at a concentration of 10 mg / L. The optimal antibacterial concentration of berberine is 10 mg / L.

[0021] Figure 5 The antibacterial effect of a 1:1 mixture of thiabendazole and berberine against Aspergillus niger, Aspergillus niger, Penicillium italium, and the pathogenic bacteria causing tea leaf blight is shown in the figure. As can be seen from the figure, a 1:1 mixture of thiabendazole and berberine has a significant inhibitory effect on the mycelial growth of Aspergillus niger, Aspergillus niger, Penicillium italium, and the pathogenic bacteria causing tea leaf blight.

[0022] Figure 6 、 Figure 7 The strawberries in the control group were severely rotten and moldy, and the mold grew vigorously.

[0023] Figure 8 、 Figure 9 The strawberries in the treatment group with the optimal combination ratio of thiabendazole and berberine were relatively fresh and free of rot and mold.

[0024] Figure 10 、 Figure 11 The beans in the control group turned yellow and had mold growing on both ends.

[0025] Figure 12 、 Figure 13 The beans in the treatment group with the best combination ratio of thiabendazole and berberine had a good overall effect except that the beans turned a little yellow, and the rot was not as serious as in the control group. DETAILED DESCRIPTION

[0026] Laboratory Facilities: All experiments were conducted at the Jiangsu Provincial Key Laboratory of Medicinal Plant Biotechnology. This laboratory, which boasts an engineering and technology center and a large fleet of advanced reaction instruments and testing equipment, provides a robust hardware foundation for research and is well-suited for conducting indoor antibacterial experiments.

[0027] In this experiment, thiabendazole and berberine were combined to study their antibacterial effects on pathogenic fungi. Through a large number of experiments, the optimal combination ratio of thiabendazole and berberine was found, in order to obtain a biological preservative that can reduce the dosage of thiabendazole, reduce environmental pollution, protect human health, and achieve better antibacterial effects.

[0028] Complete the extraction and screening of plant active ingredients and other plant-derived fungicides, conduct a large number of antibacterial experiments, screen out plant active ingredients that can be compounded with thiabendazole, conduct compounding experiments, and design a compound green biological preservative.

[0029] Conduct greenhouse experiments and finally analyze the experimental data and conduct industrial research.

[0030] 1. Equipment: analytical balance, electronic balance, weighing paper, graduated cylinder, beaker, glass rod, culture dish, sterilized bamboo stick, alcohol lamp, constant temperature incubator, autoclave, clean bench, fume hood, rubber gloves, pipette, sterilized hole punch;

[0031] Thiabendazole (provided by Jiangsu Noen Crop Science Co., Ltd.); Berberine (purchased online, produced by Shanghai Hulian Biopharmaceutical Co., Ltd.); Potato dextrose agar (PDA) medium (provided by Jiangsu Key Laboratory of Medicinal Plant Biotechnology);

[0032] Aspergillus niger ATCC 1015 was provided by COFCO Biotechnology Co., Ltd. The strain source is referenced from published literature: Liu Menghan. A culture medium and culture method for the expanded culture of Aspergillus niger [J]. Contemporary Chemical Industry, 2024, 53(01): 167-170+175. A sweet potato long-beaked shell fungus (Cordyceps militaris) (sample number: CF1.01127) was provided by Xuzhou Academy of Agricultural Sciences, Jiangsu Province. The strain source is based on the published literature Li Tengjie, Liang Sunyan, Guo Jianheng, et al. Study on the inhibitory effect of 2-methylbutyric acid, a volatile of Bacillus tequila XK29, on sweet potato long-beaked shell fungus [J]. Acta Microbiologica Sinica, 2022, 62(12): 5018-5028. DOI: 10.13343 / j.cnki.wsxb.20220238. A strain of Penicillium italicum was isolated by our research group from diseased citrus fruits. The isolation method was based on previously published literature. (Mao Jiali. Identification and functional analysis of the CYP51B gene promoter, a target enzyme of a highly resistant strain of Penicillium citrinum. Central China Normal University, 2021. DOI: 10.27159 / d.cnki.ghzsu.2018.000350.) A tea leaf blight pathogen was isolated by our research group from fresh diseased tea leaves. The isolation method was based on a published article: Qi Li. Inhibitory effects of free aroma and glycoside aroma precursors on tea leaf pathogenic fungi[D]. Anhui Agricultural University, 2009. Currently, the four strains are backed up in Room 404, Key Laboratory of Medicinal and Edible Plant Biotechnology, Jiangsu Normal University, No. 101, Shanghai Road, Tongshan District, Xuzhou City, Jiangsu Province (Email: 1290835974@qq.com).

[0033] 1. Prepare PDA culture medium (1) PDA formula: 200g potato, 20g glucose, 20g agar, 5g peptone, 3g potassium dihydrogen phosphate, 1.5g magnesium sulfate, 1000ml water, natural pH (2) Preparation steps of PDA culture medium a. Ingredients for cooking juice Place 1000ml of water in a pot, bring to a boil, add 200g of peeled and sliced ​​potatoes and cook for 20-30 minutes until the chips are soft but not mushy. Filter through eight layers of gauze and take the filtrate. b. Agar dissolution and hydration Add the potato filtrate back to the pot, add water to 1000ml, and continue to heat and boil. Add agar, continue to simmer until the agar is completely dissolved, and add water again to 1000ml; c. Drug dissolution After the agar is dissolved, add 20g glucose, 3g potassium dihydrogen phosphate, 1.5g magnesium sulfate, and 5g peptone; d. Sterilize after packaging Pour the prepared culture medium into a clean wide-mouth bottle, the volume should not exceed 1 / 2 of the triangular flask, and do not tighten the bottle cap (otherwise the pressure will increase during heating and the bottle cap will be easily washed off). After packaging, place it in a portable high-pressure sterilizer, tighten the lid, and exhaust the cold air in the pot. When the temperature rises to between 122-124℃, start the timer for 30 minutes, and the sterilization is complete; e. Pour the plate and cool When the culture medium in the Erlenmeyer flask drops to about 50°C, pour it into the plate (if the temperature is too high, condensation water will easily occur, leading to bacterial contamination; if the temperature is too low, the culture medium will solidify).

[0034] Note: The culture dish should be sterilized by ultraviolet light in a transfer window for 30 minutes before use. Then, it should be placed on a sterile clean bench.

[0035] 2. Prepare PDA containing single and compound medicines (1) Formula and preparation of botanical fungicide berberine: In this experiment, berberine raw powder was dissolved in hot water. 2.5 mg of berberine raw powder was dissolved in hot water and diluted to 250 ml to prepare a 10 mg / L berberine solution.

[0036] (2) Formula and preparation of the chemical pesticide thiabendazole: Weigh 2.5 g of thiabendazole and dissolve it in an appropriate amount of methanol solution. After the thiabendazole is completely dissolved, use a syringe to draw the dissolved thiabendazole-methanol mixture and add it to a needle filter for filtration and sterilization. The filtered liquid is directly poured into a sterilized centrifuge tube and then diluted with distilled water to 250 ml to prepare a 10 mg / L thiabendazole solution.

[0037] (4) Prepare plates containing thiabendazole at different concentration gradients. Take five 40 ml portions of sterilized PDA medium, add thiabendazole stock solution, and use a pipette to draw 200 l, 400 l, 800 l, 1600 l, and 3200 l of the stock solution into 40 ml PDA, respectively. Pour 3 plates for each gradient, each plate containing approximately 12 ml, for a total of 15 plates.

[0038] (5) Prepare plates containing berberine at various concentrations. Use a 1000-μl pipette in a clean bench to pipette 667 μl, 1333 μl, 2667 μl, 5333 μl, and 10666 μl of berberine solution into five 40-ml portions of inactivated PDA. Pour 40 ml of PDA into five plates, with approximately 12 ml per plate.

[0039] (6) Formula and preparation of the compound of berberine and thiabendazole: After finding the optimal antibacterial concentration of thiabendazole and berberine, at this concentration, compound them according to the mass ratio of thiabendazole to berberine of 1:1, 1:2, 1:4, 1:8, and 1:16. Appropriately reducing the concentration of thiabendazole and using a compound of thiabendazole and berberine with a reduced content can also achieve the antibacterial effect of thiabendazole alone.

[0040] 3. Inoculation Sterilization: Place the prepared culture medium, pathogens, and punch into a clean bench, irradiate with UV light for 30 minutes, and blow dry for 15 minutes; 4. Punching Use a 6mm puncher to punch out fungus cakes on Aspergillus niger, Penicillium italicum, Aspergillus niger and tea leaf blight pathogen strains respectively; 5. Inoculation Burn the tip of a sterilized bamboo stick back and forth under the outer flame of an alcohol lamp, wait for a few seconds to cool, pick up the beaten cake and place it on a PDA medium. Inoculate three plates of each of Aspergillus niger, Penicillium italicum, Aspergillus niger, and Tea Leaf Blight pathogen on the PDA medium containing different agents. 6. Cultivation Press gently with a bamboo stick, with the mycelium facing downward, seal the container, place it in a constant temperature incubator, incubate for 12-24 hours, then invert and incubate for 7 days.

[0041] After conducting numerous compounding experiments and analyzing the inhibition rates, we determined that a 1:1 ratio of thiabendazole to berberine is most effective against fungi such as Aspergillus niger and Penicillium italicum. Based on this ratio, we formulated our thiabendazole-berberine compound formulation. This formulation was then used in greenhouse experiments on fruit.

[0042] 1. Purpose of the experiment The inhibitory effects of thiabendazole, berberine and the combination of thiabendazole-berberine on pathogenic fungi were studied and their inhibition rates were calculated.

[0043] 2 Test methods (1) Three groups of antibacterial experiments were conducted on Aspergillus niger using 1.25 mg / L, 2.5 mg / L, 5 mg / L, 10 mg / L, and 20 mg / L thiabendazole. The diameters of Aspergillus niger colonies were measured on the first, third, and fifth days. The inhibition rates of the three groups of experiments were calculated and the average values ​​were obtained. An inhibition rate table was prepared. The lowest concentration of thiabendazole with the best inhibition rate was 10 mg / L. Figure 1 .

[0044] (2) Three groups of antibacterial experiments were conducted on Aspergillus niger using 0.625 mg / L, 1.25 mg / L, 2.5 mg / L, 5 mg / L, and 10 mg / L berberine. The diameters of Aspergillus niger colonies were measured on the first, third, and fifth days. The inhibition rates of the three groups of experiments were calculated and the average value was obtained. The lowest concentration of berberine with the best inhibition rate was 10 mg / L. A table of inhibition rates was prepared. Figure 2 .

[0045] (3) After finding the optimal inhibitory concentration of thiabendazole and berberine, five groups of antibacterial experiments were conducted on Aspergillus niger, Penicillium italicum, Aspergillus serratus, and Tea Leaf Blight pathogens at these concentrations using a mass ratio of 1:1, 1:2, 1:4, 1:8, and 1:16. The colony diameters of Aspergillus niger, Penicillium italicum, Aspergillus serratus, and Tea Leaf Blight pathogens were measured on the first, second, third, fourth, and fifth days. The inhibition rates of the three groups of experiments were calculated and the average value was obtained.

[0046] (4) Use 10 mg / L of thiabendazole to conduct antibacterial experiments on Aspergillus niger, Penicillium italicum, Aspergillus niger, and tea leaf blight pathogens, measure the colony diameters of various bacteria, calculate the inhibition rates of various bacteria, and prepare an inhibition rate table. Figure 3 .

[0047] 3 Test results After conducting numerous compounding experiments and analyzing the inhibition rates, we determined that a 1:1 ratio of thiabendazole to berberine was most effective against fungi such as Aspergillus niger, Penicillium italicum, Aspergillus niger, and tea leaf blight. Based on this ratio, we formulated our thiabendazole-berberine compound formulation. This formulation was then used in greenhouse experiments on fruit.

[0048] 1. Purpose of the experiment The use of new anti-pathogenic fungi compound biological preservatives can reduce the impact of harmful fungi on fruits and vegetables during the storage process, thereby achieving the effect of sterilization and preservation, reducing adverse effects on the human body, and extending their shelf life.

[0049] 2 Test conditions 2.1 Selection of trial subjects Test subjects: beans, strawberries 2.2 Environmental conditions The experiment was conducted at the Jiangsu Provincial Key Laboratory of Medicinal Plant Biotechnology. This laboratory boasts an engineering technology center equipped with a large number of advanced laboratory instruments and testing equipment, providing a strong hardware foundation for scientific research. It also boasts a large number of greenhouses, providing the necessary conditions for conducting greenhouse experiments.

[0050] 3 Experimental design and arrangement (1) Turn on the ultra-clean workbench and irradiate with ultraviolet light for half an hour; (2) Wash and dry the fresh beans and strawberries you bought; (3) Prepare the reagents on the clean bench; Thiabendazole and berberine are respectively compounded in an optimal ratio to form a compound reagent, and the compound reagent is dispensed into small spray bottles.

[0051] (4) Wash and dry the beans and strawberries purchased, and divide them into a treatment group and a control group. Spray the same dose of the compound reagent on the corresponding fruits and vegetables, and do not treat the other group. The two groups will form a mutual control. The fruits and vegetables will be cultured in a greenhouse. The treatment group and the control group will each be repeated three times. After one week, observe the results and calculate the good fruit rate and weight loss rate and take the average value.

[0052] 4 Experimental Results 4.1 Experimental results Figure 6-Figure 13 .

[0053] Figure 6 、 Figure 7 The strawberries in the control group were severely rotten and moldy, and the mold grew vigorously. Figure 8 、 Figure 9 The strawberries in the treatment group with the optimal combination ratio of thiabendazole and berberine were significantly different between the two groups. The rot and mold of the strawberries in the control group were more serious than those in the treatment group. Figure 10 、 Figure 11 The beans in the control group turned yellow and had mold growing on both ends of the beans. Figure 12 、 Figure 13 The beans in the treatment group with the optimal combination ratio of thiabendazole and berberine showed good overall effect except that some beans in the two groups turned yellow and moldy, and the rot was not as serious as that in the control group.

[0054] 4.2 Index determination method (1) Calculation of weight loss rate Weight loss rate refers to the weight loss of fruit during storage due to water evaporation or other reasons.

[0055] Weight loss rate (%) = (fruit mass before storage - fruit mass after storage) × 100% / (fruit mass before storage) The following are the average weight loss rates of the control group and the treatment group:

[0056] The weight loss rate of the treatment group was significantly lower than that of the control group, indicating that the preservation effect was significant, and the combined effect was equivalent to that of using thiabendazole alone.

[0057] (2) Calculation of good fruit rate Good fruit rate (%) = (number of good fruits × 100%) / number of samples The following are the average good fruit rates of the control group and the treatment group:

[0058] The good fruit rate of the compound group was significantly higher than that of the control group, indicating that the preservation effect was significant.

[0059] 4.2 Security The tested fruit and vegetable preservatives had no phytotoxicity symptoms on beans and strawberries, and the applicators did not feel any discomfort after applying the pesticides.

Claims

1. A novel anti-pathogenic fungus compound biological preservative formula, characterized in that: It is composed of 10 mg / L thiabendazole and 10 mg / L berberine, and the mass ratio of thiabendazole to berberine is one of 1:1, 1:2, 1:4, 1:8, and 1:

16.

2. A method for preparing a novel anti-pathogenic fungus compound biological preservative, characterized in that: The mass ratios of 10 mg / L thiabendazole and 10 mg / L berberine were 1:1, 1:2, 1:4, 1:8 and 1:

16.

3. The preparation method of the novel anti-pathogenic fungus compound biological preservative according to claim 2, characterized in that: Thiabendazole and berberine are compounded in a mass ratio of 1:

1.

4. The preparation method of the novel anti-pathogenic fungus compound biological preservative according to claim 2, characterized in that: The preparation method of 10 mg / L berberine is as follows: dissolve 2.5 mg of berberine powder in hot water, dilute to 250 ml, and prepare a 10 mg / L berberine solution.

5. The preparation method of the novel anti-pathogenic fungus compound biological preservative according to claim 2, characterized in that: The preparation method of 10 mg / L thiabendazole is as follows: dissolve 2.5 mg of thiabendazole original drug in an appropriate amount of methanol, draw the dissolved thiabendazole-methanol mixture with a syringe, add it into a needle filter for filtration and sterilization, and dilute it with water to 250 ml to prepare a 10 mg / L thiabendazole solution.

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