Use of fatty acid polyol esters pefa in the inhibition of harmful fungi

Abstract

The application provides a use of a fatty acid polyol ester (PEFA) in inhibiting harmful fungi. The fatty acid polyol ester is a glycolipid composed of a polyol head and (R)-3-hydroxy fatty acid and has different degrees of acetylation. The (R)-3-hydroxy fatty acid is C14, C16 or C18. The harmful fungi include Aspergillus flavus and Candida albicans. The application further prepares a nanoemulsion therefrom, which is composed of 12-12.8 parts by weight of a surfactant, 4-3.2 parts by weight of a cosurfactant, 4-5 parts by weight of the PEFA and an appropriate amount of water; the amount of the water is an amount for supplementing the nanoemulsion to 100 parts by weight. The PEFA is prepared into the nanoemulsion, which can significantly increase the dispersion stability thereof in water, thereby improving the inhibiting effect on the harmful fungi and having obvious practical application value and economic benefit prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and relates to new uses of fatty acid polyol esters in resisting harmful fungi, and to methods and applications for preparing nanoemulsions using the aforementioned fatty acid polyol esters. Background Technology

[0002] Harmful fungal contamination severely impacts the storage and use of agricultural products, traditional Chinese medicine products, and food. The prevention and control of harmful fungi permeates agriculture, industry, and the traditional Chinese medicine sector. Currently, the main methods for controlling harmful fungi include chemical antifungal agents, physical antibacterial agents, biological antibiotics, and antifungal microorganisms. However, chemical antifungal agents can produce toxic side effects, physical antibacterial agents require stringent conditions, and biological antibiotics often lead to drug resistance. In this context, research on antifungal microorganisms has opened up new avenues for biological antibacterial action, with their fermentation products, such as sophorolipids and cellobioses, exhibiting good antibacterial activity.

[0003] Aspergillus flavus is a fungus that infects grains and cash crops (such as corn, peanuts, and beans). During storage and transportation, it produces large amounts of aflatoxin, a highly toxic and carcinogenic compound, causing severe economic losses. Furthermore, aflatoxin produced by Aspergillus flavus (AFB1) is extremely toxic, 10 times more toxic than potassium cyanide and 68 times more toxic than arsenic. It has been proven to have carcinogenic, teratogenic, and mutagenic effects, and is a major carcinogen for liver cancer. Livestock and poultry consuming aflatoxin-contaminated feed suffer poisoning, disease, and even death, causing enormous economic losses to the livestock industry. Dairy cows consuming aflatoxin-contaminated feed experience reduced milk production, impaired liver function, and weakened immunity; the toxin also transfers to milk, posing a serious threat to human health. In addition, aflatoxin has very stable physicochemical properties; industrial processing, daily cooking, and pasteurization cannot effectively remove it, allowing it to persist in the food chain and pose a long-term threat to public health.

[0004] Candida albicans, also known as white yeast, is a fungus commonly found in the oral cavity, upper respiratory tract, intestines, and vagina of healthy individuals. Under normal circumstances, Candida albicans exists in small numbers in a healthy body and does not cause disease. However, when the body's immune function / general defense is weakened, or when the balance of normal flora is disrupted, it can easily proliferate and change its growth pattern (budding hyphae) to invade cells and cause disease. After infecting the body, Candida albicans can invade multiple tissues and organs throughout the body, causing significant harm and a high mortality rate. In severe cases, it can penetrate the host's epithelial barrier and invade deep tissues, causing systemic infection through its bloodstream and endangering the host's life. Currently, clinical treatment for vulvovaginal Candida albicans mainly uses azole drugs such as fluconazole and miconazole nitrate. However, with the abuse and improper use of azole drugs, the proportion of Candida albicans with drug resistance has increased, and the toxic side effects of these drugs have also increased.

[0005] The inventors' previous research discovered a deep-sea red yeast strain, XY11, that specifically accumulates polyolesters of fatty acids (PEFA) extracellularly. PEFA is a novel, low-solubility glycol ester surfactant, primarily composed of two key components linked by carboxyl-terminal esterification: a polyol head group with varying degrees of acetylation and an acetylated 3-hydroxy fatty acid. The polyol head group is typically d-arabinitol or d-mannitol, and the 3-hydroxy fatty acid is mainly a C16:0 or C18:0 saturated fatty acid. Currently, there are no reports on the inhibitory effects of PEFA on harmful fungi. Summary of the Invention

[0006] Based on the current state of antifungal microorganisms in the prior art, this application provides the use of fatty acid polyol ester (PEFA) in inhibiting harmful fungi, and further prepares it into a nanoemulsion for use as an antibacterial agent, thereby improving its antibacterial effect.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] The application of fatty acid polyol esters (PEFAs) in inhibiting harmful fungi, wherein the fatty acid polyol esters are glycolipids composed of a polyol head and (R)-3-hydroxy fatty acid, and are acetylated to varying degrees. The (R)-3-hydroxy fatty acid is C14, C16, or C18. The harmful fungi include Aspergillus flavus and Candida albicans.

[0009] The inventors unexpectedly discovered during their research that PEFA not only possesses surface activity but also exhibits certain antifungal activity against pathogenic fungi. Therefore, PEFA can be used to inhibit harmful fungi. Further research by the inventors revealed that the mechanism by which PEFA inhibits these harmful fungi includes inducing apoptosis and necrosis of the fungal cells. As a novel antifungal agent, PEFA has great commercial potential in the field of inhibiting fungal pathogens. This invention is applicable to the use of PEFA as a bacteriostatic agent in the inhibition of pathogenic fungi, altering the cell membrane permeability of target fungi and inducing apoptosis in the strain, thereby triggering apoptosis and necrosis of the pathogenic fungi, thus achieving the effect of inhibiting pathogenic fungi.

[0010] Preferably, the fatty acid polyol ester is prepared into a nanoemulsion. The nanoemulsion, by weight, comprises 12-12.8 parts by weight of surfactant, 4-3.2 parts by weight of co-surfactant, 4-5 parts by weight of PEFA, and an appropriate amount of water; the amount of water is used to bring the nanoemulsion to 100 parts by weight. The surfactant is Tween-80, Tween-20, polyoxyethylene 35 castor oil, hydrogenated castor oil CO-40, or polyglycerol-6-octanoate. The co-surfactant is 1,2-propanediol, glycerol, or n-butanol.

[0011] The nanoemulsion was prepared by the following method: 12-12.8 parts by weight of surfactant, 4-3.2 parts by weight of co-surfactant, and 4-5 parts by weight of PEFA were weighed and transferred to a reaction apparatus. The mixture was stirred at room temperature at a speed of 400-600 r / min until homogeneous. Then, water was added dropwise to the mixture until 100 parts by weight were reached, and stirring continued until the system was homogeneous and transparent, thus obtaining the nanoemulsion. Since PEFA is a sugar ester surfactant with low solubility in water and cannot form a stable solution, the inventors discovered that preparing PEFA into a nanoemulsion can significantly increase its dispersion stability in water, thereby enhancing its effect in inhibiting harmful fungi, demonstrating obvious practical application value and economic benefits.

[0012] More preferably, the PEFA is produced by fermentation using the deep-sea red yeast strain XY11. The deep-sea red yeast strain XY11 is a strain that specifically accumulates polyolesters of fatty acids (PEFA) extracellularly, as discovered in the inventors' previous research. Using this strain for PEFA fermentation yields a crude product yield as high as 43.8 g / L, with a high substrate conversion rate, thus reducing production costs.

[0013] Compared with the prior art, the technical effects of the present invention are as follows:

[0014] (1) This invention provides a novel use of PEFA for inhibiting pathogenic fungi, which is significantly different from its known use as a surfactant and is non-obvious.

[0015] (2) This invention provides a nanoemulsion prepared using PEFA. The preparation of the nanoemulsion significantly improves the dispersion stability of PEFA in aqueous solution and enhances the antibacterial effect, which has great practical application value.

[0016] (3) The nanoemulsion described in this invention is not only environmentally friendly and biodegradable, but also low in cost and easy to use. It has a good control effect on pathogenic fungi such as Aspergillus flavus and Candida albicans, and has considerable economic benefits. Attached Figure Description

[0017] Appendix Figure 1 The inhibition zone of PEFA against pathogenic fungi (a. Aspergillus flavus, b. Candida albicans) as described in Example 3.

[0018] Appendix Figure 2 The results of the Annexin V-FITC / PI double staining experiment of PEFA and hydroxy fatty acids on Candida albicans described in Example 5 are shown.

[0019] Appendix Figure 3 The Tyndall effect of the PEFA nanoemulsion described in Example 6;

[0020] Appendix Figure 4 The image shown is a transmission electron microscope (TEM) image of the PEFA nanoemulsion described in Example 6.

[0021] Appendix Figure 5 The particle size and PDI results of the PEFA nanoemulsion and PEFA micelles described in Example 13 are shown. Detailed Implementation

[0022] The present invention will now be described in further detail with reference to specific embodiments. It should be understood that the specific embodiments described in the following embodiments of the present invention are merely illustrative examples of specific implementations of the present invention and are intended to explain the present invention, and do not constitute a limitation thereof.

[0023] Unless otherwise specified, the experimental conditions in the following examples are generally in accordance with conventional conditions or the conditions recommended by the reagent company. Unless otherwise specified, the reagents and consumables used in the following examples are commercially available. The experimental water is pure water. Unless otherwise specified, the methods used are conventional processes in the field.

[0024] Example 1: Production of PEFA by fermentation of deep-sea red yeast strain XY11

[0025] (1) The red yeast was activated on YPD solid medium for 2 days. The YPD solid medium was prepared as follows: 20 g glucose, 10 g yeast powder, 20 g peptone, and 20 g agar were added to the solid medium; 1 L of double-distilled water was used for preparation, and the mixture was autoclaved at 115 ℃ for 30 min. The red yeast strain has been disclosed in invention patent application 2022113701201 (depository unit: China Center for Type Culture Collection, deposit number: CCTCC NO: M 2021518, deposit date: May 19, 2021).

[0026] (2) Single colonies were transferred to 5 ml of YPD liquid medium and cultured at 28°C and 180 rpm for 16 h. Then, they were transferred to 50 mL of glucose oil-producing medium and cultured at 28°C and 180 rpm for 7 days. The YPD liquid medium was prepared as follows: 20 g glucose, 10 g yeast extract, 20 g peptone; prepared with 1 L double-distilled water and autoclaved at 115°C for 30 min. The glucose oil-producing medium was prepared as follows: 140 g glucose, 1 g peptone, 1 g yeast extract, 7 g potassium dihydrogen phosphate, 0.25 g disodium hydrogen phosphate, 0.15 g magnesium sulfate heptahydrate; prepared with 1 L double-distilled water and autoclaved at 115°C for 30 min.

[0027] (3) After 7 days of shaking culture, the fermentation broth was transferred to a 50 mL centrifuge tube. PEFA was extracted from the fermentation broth with 15 mL of ethyl acetate. After centrifugation at 8000 rpm for 5 min, 10 mL of the organic phase containing PEFA was transferred to a 30 mL flat-bottomed oil bottle. Ethyl acetate was evaporated by rotary evaporator at 80 °C and 160 rpm. The flat-bottomed flask was then transferred to an 80 °C oven for 1 h to obtain the product PEFA.

[0028] Example 2: PEFA Structural Characterization Analysis

[0029] (1) HPLC analysis of the PEFA polyol head

[0030] PEFA was alkaline hydrolyzed using 2 mL of 2.0 M KOH, dried in an oven at 80 °C for 3-4 hours, and fatty acids were removed using chloroform. The supernatant was collected after separation and pH was adjusted to neutral. HPLC analysis of the sample was performed using a Thermo UltiMate 3000 system equipped with an evaporative light scattering detector (SEDERE L T-ELSD Shodex 80) and an Asahipak NH2P-504E column. The analysis revealed that the PEFA polyol head consisted of arabinitol and mannitol.

[0031] (2) Gas phase analysis of PEFA fatty acids

[0032] Impurities were washed with saturated sodium chloride. After mixing with chloroform in PEFA, sulfuric acid / methanol was added to methylate the fatty acids. Following washing with sodium chloride solution, hexane was added for extraction to remove impurities. The fatty acid components were then analyzed using an Agilent 7890A-5975C gas chromatograph-mass spectrometer (GC-MS). The results are shown in Table 1.

[0033] Table 1 Fatty acid composition of PEFA

[0034] 3-OH-C14:0 3-OH-C16:0 3-OH-C18:0 3.1% 75.8% 21.1%

[0035] As shown in Table 1, the product obtained after fermentation of glucose and xylose is fatty acid polyol ester (PEFA). The main components of PEFA are hydroxy fatty acids and polyol heads. Therefore, it can be seen that the PEFA produced by fermentation of the red yeast strain in Example 1 is structurally similar to the PEFA produced by fermentation of red yeast reported in the literature, and there is no essential difference.

[0036] Example 3: Analysis of the anti-aspergillus flavus and Candida albicans activity of PEFA.

[0037] The biological activity of PEFA has not been studied to date; this invention investigates its antifungal activity. Specifically, a cultured suspension of *Aspergillus flavus* and *Candida albicans* was evenly spread onto YPD solid medium. After the bacterial suspension dried, 100 μL of PEFA solution dissolved in DMSO (10 mg / mL) was added dropwise to the center of the medium, with DMSO serving as a negative control. The mixture was incubated at 28°C for 24 h, and the results were observed. Figure 1 As shown. By Figure 1 It can be seen that PEFA can significantly inhibit the growth of Aspergillus flavus and Candida albicans, indicating that it has antifungal activity.

[0038] Next, the inventors used *Candida albicans* as an example to study the antifungal mechanism of PEFA. First, PEFA was hydrolyzed to prepare its 3-hydroxy fatty acid component. Then, Annexin V-FITC / PI double staining experiments were used to study PEFA and its 3-hydroxy fatty acid component separately. The specific mechanism is as follows: dual labeling with Annexin V-FITC and PI distinguishes between live cells, apoptotic or necrotic cells, and dead cells. Specifically, PI causes cells with altered cell membrane permeability to emit red fluorescence, Annexin V-FITC causes apoptotic cells to emit green fluorescence, necrotic cells emit both red and green fluorescence, and normal cells do not fluoresce. The results are as follows: Figure 2 As shown. By Figure 2The results showed that untreated *Candida albicans* cells did not exhibit red or green fluorescence under a fluorescence microscope, indicating they were normal cells. *Candida albicans* cells treated with PEFA showed both red and green fluorescence under a fluorescence microscope, indicating altered cell membrane permeability and apoptosis. The 3-hydroxy fatty acid-treated group showed only green fluorescence and only apoptosis. This demonstrates that PEFA can induce cell necrosis in pathogenic fungi by utilizing fungal cell membrane permeability, and can also induce apoptosis through its 3-hydroxy fatty acid structure. In conclusion, PEFA is a novel antifungal agent with multiple functions, a finding that significantly differs from its known use as a surfactant.

[0039] Example 4: Determination of the minimum inhibitory concentration (MIC) of PEFA against Aspergillus flavus and Candida albicans

[0040] The minimum inhibitory concentration (MIC) of PEFA against *Candida albicans* and *Aspergillus flavus* was determined using the Beyotime C0009S kit. Specifically, PEFA solutions of different concentrations, pre-dissolved in DMSO, were added to the culture systems of *Aspergillus flavus* and *Candida albicans*. The absorbance at 530 nm was measured according to the kit instructions, and the inhibition rate was calculated. The results are shown in Table 2. Table 2 shows that the minimum inhibitory concentration of PEFA against *Aspergillus flavus* was 6 mg / mL, and the MIC against *Candida albicans* was 7 mg / mL, indicating that PEFA has a good antibacterial effect.

[0041] Table 2. Minimum inhibitory concentrations (MICs) of Aspergillus flavus and Candida albicans

[0042] Aspergillus flavus Candida albicans MIC 6 mg / ml 7 mg / ml

[0043] However, because the hydrophobic ends of PEFA glycolipid molecules are acetylated arabinitol and mannitol, their water solubility is poor. Therefore, they need to be dissolved in DMSO beforehand during the dissolution process. Even so, the final PEFA aqueous solution has poor stability. Experiments showed that the PEFA solution became turbid after being left at room temperature for 24 hours. The inventors analyzed that this turbidity was due to PEFA aggregation, which limits the application of PEFA in antibacterial applications.

[0044] Example 5: Selection of Surfactants and Co-surfactants in the Preparation of PEFA Nanoemulsions

[0045] Nanoemulsions are thermodynamically stable, isotropic, transparent or semi-transparent homogeneous dispersions that spontaneously form from water, oil, surfactants, and co-surfactants, with particle sizes ranging from 1 to 100 nm. They offer advantages such as good stability, simple preparation, and improved drug bioavailability. The preparation of PEFA nanoemulsions has not yet been studied.

[0046] This embodiment first used different types of surfactants: Tween-80, Tween-20, polyoxyethylene 35 castor oil, hydrogenated castor oil CO-40, polyglycerol-6-octanoate, polyglycerol-6-didecanoate, polyglycerol-2-seboleate, Tween-80 and Span-80, and Tween-20 and Span-80. 12 parts by weight of surfactant were accurately weighed and thoroughly mixed with 4.5 parts by weight of PEFA. The mixture was stirred with a thermostatic magnetic stirrer, and distilled water was added dropwise to 100 parts by weight while stirring. The emulsion was observed, and the formation of nanoemulsions was determined using the evaluation criteria for nanoemulsions. The evaluation criteria were: ① particle size between 10-100 nm; ② clear and transparent or translucent appearance; ③ stable without stratification after high-speed centrifugation; ④ Tyndall effect after parallel light incidence. The particle size and PDI value of the nanoemulsion were measured using a Malvern particle size analyzer, and the presence of the Tyndall effect was observed by parallel laser illumination. The results are detailed in Table 3. As shown in Table 3, polyglycerol-6-didecanoate, polyglycerol-2-soleate, Tween-80 and Span-80, and Tween-20 and Span-80 cannot form nanoemulsions when used as surfactants, while surfactants Tween-80, Tween-20, hydrogenated castor oil CO-40, and polyglycerol-6-octanoate can form nanoemulsions.

[0047] Table 3 Screening of Surfactants

[0048] Surfactant type Phenomenon description Particle size (nm) PDI value Tween-80 Can generate the Tyndall effect 17.64±2.09 0.18±0.01 Tween-20 Can generate the Tyndall effect 26.87±6.73 0.18±0.03 Polyoxyethylene 35 castor oil Can generate the Tyndall effect 12.16±1.78 0.22±0.01 Hydrogenated castor oil CO-40 Can generate the Tyndall effect 45.69±23.55 0.45±0.02 Polyglycerol-6-octanoate Can generate the Tyndall effect 37.30±33.28 0.68±0.03 Polyglycerol-6-didecanoate The Tyndall effect cannot be formed. / / Polyglycerol-2-fold oleate The Tyndall effect cannot be formed. / / Tween-80 and Span-80 The Tyndall effect cannot be formed. / / Tween-20 and Span-80 The Tyndall effect cannot be formed. / /

[0049] Table 4 Screening of co-surfactants

[0050] Co-surfactant type Phenomenon description Particle size (nm) PDI value Glycerol Can generate the Tyndall effect 13.03±0.07 0.24±0.02 1,2-Propanediol Can generate the Tyndall effect 16.99±0.06 0.23±0.01 n-Butanol Can generate the Tyndall effect 15.45±1.03 0.31±0.04

[0051] Then, with polyoxyethylene castor oil as the surfactant, 12 parts by weight of the surfactant, 4.5 parts by weight of PEFA, and 4 parts by weight of the co-surfactant were mixed using different types of co-surfactants: 1,2-propanediol, glycerol, and n-butanol. Distilled water was added dropwise to 100 parts by weight while stirring. The emulsion was observed, and the formation of nanoemulsions was determined using the evaluation criteria for nanoemulsions. The particle size and PDI value of the nanoemulsions after the addition of co-surfactants were measured. The results are detailed in Table 4. As shown in Table 4, when co-surfactants were added to the nanoemulsion system, and the co-surfactants were 1,2-propanediol, glycerol, and n-butanol, the standard deviation of the nanoemulsion particle size was smaller, the particle size distribution was more uniform, the system was more stable, and the PDI value was less than 0.3.

[0052] Example 6: Preparation and Characterization of PEFA Nanoemulsion

[0053] Nanoemulsions were prepared using the surfactant and co-surfactant screened in Example 5. 12 parts by weight of polyoxyethylene castor oil, 4 parts by weight of glycerol, and 4.5 parts by weight of PEFA were added to a beaker. The beaker was placed on a thermostatic magnetic stirrer and stirred thoroughly at 600 r / min until fully mixed. 79.5 parts by weight of distilled water were added dropwise to the beaker, and stirring continued until a homogeneous and transparent liquid was obtained. The Tyndall effect was observed when the nanoemulsion was irradiated with a laser pointer in parallel. Figure 3 Then, the particle size and PDI value of the nanoemulsion were measured using a Malvern particle size analyzer, which were 12.16±1.78 and 0.22±0.01, respectively, indicating that the particle size was small and uniform. Finally, a drop of the prepared PEFA nanoemulsion was taken and a copper mesh was immersed in it. The copper mesh was removed and dried on filter paper. A drop of phosphotungstic acid (2%, w / v) was dropped on the copper mesh, and after standing at room temperature for 2 min, it was stained. After drying, the diameter of the PEFA nanoemulsion particles was observed to be 11-14 nm using a transmission electron microscope. Figure 4 The results are roughly the same as those measured by the Malvern particle size analyzer. These results indicate that these conditions can yield good PEFA nanoemulsions.

[0054] Example 7: Preparation and Characterization of PEFA Nanoemulsions

[0055] This embodiment is based on Example 6, but the preparation conditions are changed to 12.4 parts by weight of Tween-80, 4 parts by weight of glycerol, and 4 parts by weight of PEFA, with distilled water added dropwise to 100 parts by weight while stirring at 400 rpm. The characterization results are shown in Table 5, which indicate that nanoemulsions can be formed.

[0056] Example 8: Preparation and Characterization of PEFA Nanoemulsion

[0057] This embodiment is based on Example 6, but the preparation conditions were changed to 12.8 parts by weight of Tween-20, 3.4 parts by weight of 1,2-propanediol, and 4.5 parts by weight of PEFA, with distilled water added dropwise to a total of 100 parts by weight while stirring at 400 rpm. The characterization results are shown in Table 5, which indicate that nanoemulsions can be formed.

[0058] Example 9: Preparation and Characterization of PEFA Nanoemulsion

[0059] This embodiment is based on Example 6, but the preparation conditions are changed to 12.2 parts by weight of polyoxyethylene 35 castor oil, 3.6 parts by weight of n-butanol, and 5 parts by weight of PEFA, with distilled water added dropwise to 100 parts by weight while stirring at 500 rpm. The characterization results are shown in Table 5, which indicate that nanoemulsions can be formed.

[0060] Example 10: Preparation and Characterization of PEFA Nanoemulsions

[0061] This embodiment is based on Example 6, but the preparation conditions are changed to 12 parts by weight of Tween-80, 3.8 parts by weight of 1,2-propanediol, and 5 parts by weight of PEFA, with distilled water added dropwise to a total of 100 parts by weight while stirring at 600 rpm. The characterization results are shown in Table 5, which indicate that nanoemulsions can be formed.

[0062] Example 11: Preparation and Characterization of PEFA Nanoemulsion

[0063] This embodiment is based on Example 6, but the preparation conditions are changed to 12.8 parts by weight of Tween-20, 3.2 parts by weight of glycerol, and 4.5 parts by weight of PEFA, with distilled water added dropwise to a total of 100 parts by weight while stirring at 600 rpm. The characterization results are shown in Table 5, which indicate that nanoemulsions can be formed.

[0064] Example 12: Preparation and Characterization of PEFA Nanoemulsion

[0065] This embodiment is based on Example 6, but the preparation conditions are changed to 12.4 parts by weight of polyoxyethylene 35 castor oil, 4 parts by weight of n-butanol, and 4 parts by weight of PEFA, with distilled water added dropwise to 100 parts by weight while stirring at 500 rpm. The characterization results are shown in Table 5, which indicate that nanoemulsions can be formed.

[0066] Table 5. Nanoemulsions prepared in Examples 7-12 and their characterization results.

[0067] Nanoemulsion system Phenomenon description Particle size (nm) PDI value Example 7 Can generate the Tyndall effect 14.56±0.06 0.21±0.02 Example 8 Can generate the Tyndall effect 14.67±0.35 0.29±0.05 Example 9 Can generate the Tyndall effect 12.67±0.37 0.15±0.03 Example 10 Can generate the Tyndall effect 23.03±0.09 0.27±0.02 Example 11 Can generate the Tyndall effect 16.53±1.54 0.42±0.01 Example 12 Can generate the Tyndall effect 17.46±0.70 0.65±0.03

[0068] Example 13: Stability determination of PEFA nanoemulsion

[0069] The stability tests of the nanoemulsions prepared in Examples 6-12 were consistent. Example 6 will be used as an example for illustration. The nanoemulsions prepared in Example 6 were placed at room temperature for 3, 7, 14, 21, and 28 days, and their appearance, particle size, and PDI value were observed and measured. The results... Figure 5 As shown. By Figure 5 It can be seen that PEFA nanoemulsion remains clear and transparent within 28 days, and can maintain a small particle size (12-13 nm) and a low PDI (less than 0.30).

[0070] The inventors prepared PEFA micelles as a control. The preparation of these micelles required first dissolving them in DMSO, then dispersing an appropriate amount in water. The final concentration of PEFA was determined to be 10 mg / mL, and the micelle particle size was 128.93 ± 0.65 nm. After standing for one day, the mixture became turbid, and the micelle particle size was 564 ± 65 nm. This indicates that the preparation of PEFA nanoemulsions significantly improved the dispersion stability of PEFA in water.

[0071] Example 14: Determination of the minimum inhibitory concentration (MIC) of PEFA nanoemulsion against two pathogenic fungi

[0072] The stability tests of the nanoemulsions prepared in Examples 6-12 were consistent. Example 6 will be used as an example for illustration. The PEFA nanoemulsion prepared in Example 6 was diluted at different ratios, and the inhibitory effect of PEFA on *Candida albicans* and *Aspergillus flavus* was determined using the Beyotime C0009S-kit according to the method in Example 4. The results are shown in Table 6.

[0073] Table 6 shows that the MIC of PEFA nanoemulsion against Aspergillus flavus was 1.2 mg / ml, and the MIC against Candida albicans was 1.5 mg / ml. These results indicate that the minimum inhibitory concentration (MIC) of PEFA nanoemulsion was significantly lower than that of PEFA in Example 4. This suggests that PEFA nanoemulsion exhibits a more significant antifungal effect, presumably due to its smaller particle size, better dispersibility, and higher effective concentration.

[0074] Table 6. Minimum Inhibitory Concentrations (MICs) of Aspergillus flavus and Candida albicans

[0075] Aspergillus flavus Candida albicans MIC 1.2 mg / ml 1.5 mg / ml

[0076] In summary, this application first provides a novel use of PEFA for inhibiting pathogenic fungi, a use that is significantly different from its known use as a surfactant and is therefore non-obvious. Building upon this novel use, this application provides nanoemulsions prepared using PEFA, thereby significantly improving the storage stability of PEFA and enhancing its antibacterial effect. Specifically, the MIC of the PEFA nanoemulsion against Aspergillus flavus is 1.2 mg / mL, and against Candida albicans is 1.5 mg / mL, indicating significant practical application value. Furthermore, the PEFA yield can reach 43.8 g / L, thus the use of PEFA nanoemulsions for antifungal purposes shows considerable economic potential.

[0077] It should be noted that the above description of the invention is based on specific and effective embodiments, but does not constitute a limitation on the invention. Furthermore, the specific implementation methods of the invention are not limited to the content described herein. Any designs and embodiments derived by those skilled in the art without departing from the concept of the invention and the appended claims should be covered within the patent scope of this invention.

Claims

1. The application of fatty acid polyol esters (PEFA) in inhibiting harmful fungi, characterized by: The fatty acid polyol ester is a glycolipid substance composed of a polyol head and (R)-3-hydroxy fatty acid; the (R)-3-hydroxy fatty acid is C14, C16 or C18; the harmful fungi are Aspergillus flavus and Candida albicans; the PEFA is produced by fermentation using deep-sea red yeast strain XY11.

2. The application according to claim 1, characterized in that: The fatty acid polyol ester was prepared into a nanoemulsion.

3. The application according to claim 2, characterized in that: The nanoemulsion, by weight, consists of 12-12.8 parts by weight of surfactant, 4-3.2 parts by weight of co-surfactant, 4-5 parts by weight of PEFA, and an appropriate amount of water; the amount of water used is to bring the nanoemulsion to 100 parts by weight.

4. The application according to claim 3, characterized in that: The surfactant is Tween-80, Tween-20, polyoxyethylene 35 castor oil, hydrogenated castor oil CO-40, or polyglycerol-6-octanoate.

5. The application according to claim 4, characterized in that: The co-surfactant is 1,2-propanediol, glycerol, or n-butanol.

6. The application according to any one of claims 2-5, characterized in that: The nanoemulsion was prepared by the following method: 12-12.8 parts by weight of surfactant, 4-3.2 parts by weight of co-surfactant and 4-5 parts by weight of PEFA were weighed and transferred to a reaction apparatus. The mixture was stirred at room temperature at a speed of 400-600 r / min until the three were mixed evenly. Then, water was added dropwise to the bottle to make up 100 parts by weight. The mixture was stirred until the system was uniform and transparent, thus obtaining the nanoemulsion.

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