Preparation methods and applications of cyclic (isoluene-isoluene) dipeptides

Abstract

This invention discloses a method for preparing cyclic (isoluene-isoluene) dipeptides and their applications, belonging to the field of biotechnology. The structure of the synthesized compound is disclosed by NMR identification, and the detection method is disclosed by liquid chromatography. Practical applications have shown that this compound promotes plant growth, increases crop yield, enhances crop resistance to plant viruses, and improves crop antibacterial function. Furthermore, field experiments have demonstrated its ability to regulate plant growth and its good antiviral efficacy.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to the preparation method and application of cyclic (isoluene-isoluene) dipeptides. Background Technology

[0002] Plants live in complex environments, facing various potential pathogens and beneficial microorganisms. Plant pathogens directly threaten crop growth and yield, leading to reduced yields or even crop failure. Traditional chemical pesticides are widely used worldwide for the prevention and control of crop diseases; however, the extensive use of chemical pesticides has resulted in harmful pesticide residues and environmental pollution, declining crop quality, and threats to human health. Therefore, there is an urgent need to develop new, green, efficient, and safe strategies to increase crop yields and improve the current situation.

[0003] The article "Research Progress on Brassinolide and ZNC Immunoinducer" points out that ZNC immunoinducer is a secondary metabolite derived from the fermentation of plant endophytic fungi, specifically a secondary metabolite extracted from the mycelium of the endophytic fungus *Paecilomyces variotii* isolated and purified from wild sea buckthorn plants. Patent CN109824757 discloses a cyclic (isoluene-isoluene) dipeptide and its preparation method. A strain of *Paecilomyces variotii* SJ1 isolated from wild sea buckthorn is fermented, separated, and extracted to obtain an extract, which is ZNC. After rotary evaporation and concentration, the extract is separated by silica gel column chromatography with different solvent gradients, and finally purified by HPLC. The purified sample is then collected and freeze-dried to obtain the single compound product, cyclic (isoluene-isoluene) dipeptide.

[0004] The patent also provides applications of cyclic (isoluene-isoluene) dipeptide in promoting crop seed germination, improving crop salt tolerance, inducing crop flowering, resisting cucumber downy mildew, and enhancing crop frost resistance.

[0005] However, there is currently no in-depth research on the source and application of this substance. Summary of the Invention

[0006] In view of the shortcomings of the prior art, this invention provides a method for preparing cyclic (isoluene-isoluene) dipeptides and their applications. The technical solution implemented by this invention is as follows:

[0007] As a first aspect of the present invention, a method for preparing a cyclic (isoluene-isoluene) dipeptide is provided, wherein the cyclic (isoluene-isoluene) dipeptide is obtained by fermentation, isolation, and purification of *Purpureocillium lilacinum*, the strain of which was deposited on May 27, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 41240.

[0008] Furthermore, the preparation method of the cyclic (isole-isole) dipeptide includes: after the *Paecilomyces lilacinus* is cultured on a PDA plate, sterile water is added, spore hyphae and spores are scraped to prepare a bacterial suspension, filtered to prepare a spore suspension, and cultured in a culture medium at a temperature of 25℃~35℃ and a pH of 5~7. After the spore suspension is cultured in the culture medium, the culture medium is centrifuged, the mycelium is collected by filtration, and dried to constant weight.

[0009] The mycelium was mixed with ethanol, ultrasonically vibrated, and the broken bacterial solution was vacuum filtered. The filtrate was collected as the extract. The extract was concentrated by rotary evaporation and then freeze-dried to produce freeze-dried powder.

[0010] As a second aspect of the invention, it is to provide the application of cyclic (isoluene-isoluene) dipeptide in promoting the growth of rice.

[0011] Furthermore, the application in promoting rice growth includes promoting the growth of the above-ground parts of rice and promoting the growth of the roots of rice.

[0012] Furthermore, the concentration of the cyclo(isoleucine-isoleucine) dipeptide sprayed is 5–20 nm / mL.

[0013] As a third aspect of the invention, it is to provide the application of cyclic (isoluene-isoluene) dipeptide in promoting the synthesis of organic matter in rice.

[0014] Furthermore, the aforementioned applications in promoting the synthesis of organic matter in rice include increasing the synthesis of soluble protein, dry matter, nitrogen accumulation, starch, and free amino acids.

[0015] As a fourth aspect of the invention, it is to provide the use of cyclic (isoluene-isoluene) dipeptide in promoting tobacco resistance to PVX infection.

[0016] Furthermore, the cyclic (isolure-isolure) dipeptide is used to promote tobacco's resistance to PVX infection, with a concentration of 5–20 ng / mL.

[0017] As a fifth aspect of the invention, it is to provide the use of cyclic (isoluene-isoluene) dipeptide in Arabidopsis thaliana's resistance to infection by pstDC3000 bacteria.

[0018] Furthermore, regarding the application of the cyclic (isoleucine-isoleucine) dipeptide in Arabidopsis thaliana's resistance to PSTDC3000 infection, the cyclic (isoleucine-isoleucine) dipeptide is dissolved in an organic solvent and then diluted with water to the concentration used for spraying, with a concentration of 5-20 ng / mL.

[0019] As a sixth aspect of the invention, it is to provide the application of cyclic (isoluene-isoluene) dipeptide in increasing maize yield and in increasing the protein and starch content of maize.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] (1) This invention provides a microbial-based preparation method for cyclic (isoluene-isoluene) dipeptides, which are prepared by fermentation, separation and purification of *Purpureocillium lilacinum*, providing a new approach for the preparation of cyclic dipeptides;

[0022] (2) The present invention has found that the compound can promote plant growth, increase crop yield, improve crop resistance to plant viruses, and improve crop antibacterial function. In field experiments, it was found that the compound can regulate plant growth and has a good antiviral effect.

[0023] (3) This invention provides the application of cyclic (isoluene-isoluene) dipeptide in promoting rice growth and increasing maize yield, especially promoting the growth of rice aboveground parts and rice roots, promoting the synthesis of rice organic matter, and increasing the protein and starch content of maize; in addition, cyclic (isoluene-isoluene) dipeptide can promote tobacco resistance to PVX infection and Arabidopsis resistance to pstDC3000 infection. Attached Figure Description

[0024] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0025] Figure 1 This is the negative ion mass spectrometry chromatogram from the structure identification experiment of the compound cyclic (isolus-isolus) dipeptide in Example 3;

[0026] Figure 2 This is a positive ion mass spectrometry chromatogram from the structure identification experiment of the compound cyclic (isolus-isolus) dipeptide in Example 3;

[0027] Figure 3This is the liquid chromatogram used in the identification experiment of the cyclic (isolus-isolus) dipeptide structure of the compound in Example 3;

[0028] Figure 4 The image shows the 1H NMR spectrum of the compound cyclic (isolus-isolus) dipeptide structure in Example 3.

[0029] Figure 5 The liquid chromatogram of the synthesized sample in Example 4;

[0030] Figure 6 This is the mass spectrum of Example 4b.

[0031] Figure 7 The mass spectrum shown is c in Example 4;

[0032] Figure 8 The effect of cyclic (isoluene-isoluene) dipeptide on rice seedling growth under different nitrogen nutrition conditions in Example 5 (28d);

[0033] Figure 9 The effect of cyclic (isoluene-isoluene) dipeptide on rice seedling growth under different nitrogen nutrition conditions in Example 5 (35d);

[0034] Figure 10 The effect of cyclic (isoluene-isoluene) dipeptide on the underground roots of rice under different nitrogen nutrition conditions in Example 5 (35d);

[0035] Figure 11 The fluorescence of tobacco leaves under 365nm ultraviolet light in Example 6;

[0036] Figure 12 The relative expression level of the virus in tobacco leaves under different drug treatments in Example 6 is OD450.

[0037] Figure 13 The infection of Arabidopsis thaliana leaves by pstDC3000 bacteria after spraying different concentrations of the compound in Example 7;

[0038] Figure 14 The image shows the effect of the compound in Example 7 spraying Arabidopsis thaliana leaves for 5 days on the resistance to infection by pstDC3000 bacteria. Detailed Implementation

[0039] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0040] *Paecilomyces lilacinus* is an endoparasitic fungus widely distributed throughout the world, possessing advantages such as high efficacy, broad host range, and ease of cultivation. Its metabolites can produce physiologically active substances such as indoleacetic acid, cytokinins, and various biological enzymes, stimulating crop growth, promoting the growth of plant vegetative organs, and also promoting seed germination and growth.

[0041] This invention describes the fermentation, isolation, and purification of *Purpureocillium lilacinum* (a strain deposited on May 27, 2024, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, accession number CGMCC No. 41240), followed by mass spectrometry and NMR analysis, which identified it as a cyclic (isoleucine-isoleucine) dipeptide. *Purpureocillium lilacinum* has low nutritional requirements and can grow not only on various conventional culture media but also in various natural substrates, such as agricultural by-product waste, residues, plant leaves, or plant extracts.

[0042] Natural cyclic dipeptides are mainly derived from extracts of mycelium.

[0043] Example 1: Isolation of the natural compound cyclic (isoluene-isoluene) dipeptide

[0044] The components of the culture medium used in the examples are as follows:

[0045] PDA medium: 150-250g potato, 20-25g agar powder, 15-25g glucose, 1000mL distilled water; autoclave at 121℃ for 20min before use.

[0046] After culturing the tested strain on PDA plates for 7 days, 10 mL of sterile water was added, and spores and spores were scraped to prepare a bacterial suspension. The suspension was then filtered through double-layered gauze to prepare a spore suspension, and the concentration of the spore suspension was adjusted to 1 × 10⁻⁶. 9 Units / mL are available for use.

[0047] The adjusted spore suspension was transferred to PDA medium and cultured at 25℃~35℃ and pH 5~7 for 5 days. After 5 days of culture, the culture was centrifuged at 8000r / min for 20min, the mycelium was collected by filtration, and dried at 60℃ to constant weight.

[0048] Mycelium was mixed with 30% ethanol at a mass ratio of 3:7, and ultrasonically vibrated for 2 hours at a power of 680W with a frequency of 4 seconds of operation followed by 6 seconds of rest. The fragmented bacterial solution was then vacuum filtered, and the filtrate was collected as the extract. The extract was concentrated by rotary evaporation and then freeze-dried to obtain a freeze-dried powder.

[0049] The lyophilized powder was dissolved in 30% ethanol and then separated using a Shimadzu high-performance liquid chromatograph (HPLC) with an Inertsil ODS-3 column (4.6 × 250 mm, 5 μm). Elution was performed using an acetonitrile-water system at a flow rate of 1 mL / min and a UV detection wavelength of 200 nm. The mobile phase ratio was water:acetonitrile = 80:20. 500 μg of the sample was dissolved in 300 μl of 30% ethanol and injected for detection. The eluent was collected, lyophilized, purified by HPLC, and analyzed by NMR. The cyclic dipeptide compound was identified as cyclo(Ile-Ile), with the following structure:

[0050]

[0051] Example 2: Synthesis of the compound cyclic (isoluene-isoluene) dipeptide

[0052] If a large quantity of compounds is required, chemical synthesis is still necessary. This invention provides a method for synthesizing cyclic (isoluene-isoluene) dipeptide compounds.

[0053] Take L-isoleucine (10g, 0.0762mol, Mw: 131.17) and heat it to reflux in toluene at 20V, about 110℃. Use a pre-set water separator to perform water separation every 1 hour and maintain the reflux reaction for 6 to 10 hours.

[0054] After the reaction was completed, the temperature was lowered to about 75°C, 10g of anhydrous ethanol was added, the mixture was stirred and dispersed, and then the temperature was lowered to 0-10°C for crystallization for 1-4 hours. The mixture was filtered, dried, and the product was 8.96g of white solid, which was the cyclic (isolus-isolus) dipeptide compound with a yield of about 50%.

[0055] The selection and optimization of suitable reaction solvents and the operation of appropriate reaction temperatures; increasing the amount of solvent will make the reaction system more conducive to intermolecular cyclization reactions; water separation can accelerate the reaction, shorten the reaction time, and reduce side reactions and impurity formation; thanks to the optimization of the reaction degree above, a higher yield of product can be obtained with simple post-processing.

[0056] Example 3: Identification of the structure of the compound cyclic (isoluene-isoluene) dipeptide

[0057] Instrument model: Waters SQ-D-2;

[0058] Testing conditions:

[0059] Liquid phase:

[0060] Injection volume: 2 μL;

[0061] Column temperature: 30℃;

[0062] Mobile phase: A - 0.1% formic acid water, B - acetonitrile, 0 min A: 90%, 10 min A: 10%, 12-15 min A: 90%;

[0063] Running time: 15 minutes;

[0064] Detection wavelength: 210nm;

[0065] Flow rate: 1 mL / min;

[0066] Chromatographic column: ZORBAX SB-C18 4.6*150mm 3.5μm;

[0067] Mass spectrometry section:

[0068] Ion source: API-ES, positive and negative ion detection mode;

[0069] Capillary voltage: 3.0kV;

[0070] Tapered hole voltage: 40V;

[0071] Solvent removal temperature: 450℃;

[0072] Flow rate: 650 L / Hr.

[0073] Mass spectrometry analysis confirmed the mass spectrum of the synthesized compound, the cyclic (isoluene-isoluene) dipeptide. The negative ion mass spectrometry chromatogram is shown below. Figure 1 As shown, the positive ion chromatogram is as follows: Figure 2 As shown, the liquid chromatogram is as follows: Figure 3 As shown.

[0074] HNMR testing conditions:

[0075] Nuclear magnetic resonance spectrometer model: Bruker 400M

[0076] Dissolve 5 mg of sample in 0.6 mL of DMSO and detect the proton NMR spectrum using 400 M NMR.

[0077] Based on the mass spectrometry results, the isomer spectra of the cyclic dipeptide compound are as follows: Figure 4 As shown.

[0078] Example 4: Detection of the synthesized sample of the compound cyclic (isoluene-isoluene) dipeptide

[0079] Weigh 5 mg of the synthesized compound sample, dissolve it in ethanol and bring the volume to 5 mL. After mixing, take 1 mL of the solution and pass it through a 0.22 μm organic filter membrane. Then, perform the liquid chromatography analysis according to the following reference conditions.

[0080] Liquid chromatography reference conditions:

[0081] Column: Inertsil ODS-3, 5μm, 4.6×250mm

[0082] Mobile phase: water: acetonitrile = 80:20

[0083] Ultraviolet detector wavelength: 200nm

[0084] Column temperature: 40℃

[0085] Flow rate: 1 mL / min

[0086] Chromatogram as shown Figure 5 As shown, the area normalization method was used to detect and analyze the synthesized sample: the content of cyclic (isolus-isolus) dipeptide was in the range of 90-95%.

[0087] Further mass spectrometry analysis of the samples revealed that b and c are isomers of a.

[0088] b-mass spectrometry results are as follows Figure 6 As shown, the c-mass spectrum is as follows Figure 7 As shown.

[0089] This invention provides the anti-drought, antibacterial, growth-promoting, and antiviral effects of the compound cyclo(isoluene-isoluene) dipeptide.

[0090] Example 5: Growth-promoting effect of the compound cyclic (isoluene-isoluene) dipeptide on rice

[0091] 5.1 Effects of compounds on rice growth under different nitrogen nutrition conditions

[0092] This experiment consisted of 12 treatments, each repeated 9 times. The results are shown in Tables 1, 2, and 3.

[0093] Table 1. Seedling Stage Experimental Treatment Design Table

[0094]

[0095] Table 2 Effects of compounds on rice seedling growth under different nitrogen nutrition conditions (aboveground parts) (14–35 days)

[0096]

[0097]

[0098] The results showed that the compound promoted the growth of rice aboveground parts under different nitrogen concentrations. Based on relevant growth indicators, it was found that after dissolving the compound in a small amount of dimethyl sulfoxide and then diluting it with water to the desired concentration for spraying, the rice plant height, leaf area, stem diameter, and SPAD were significantly higher than the control group after 35 days. Furthermore, the growth-promoting effect on rice seedlings increased with increasing compound concentration. Under nitrogen nutrient conditions of 80 mg / L, the compound concentration of 20 ng / mL showed the most significant growth-promoting effect on rice aboveground parts (P < 0.05), followed by 5 ng / mL and 10 ng / mL. Figure 8 ,9).

[0099] Table 3. Effects of cyclic (isoluene-isoluene) dipeptide on the underground roots of rice under different nitrogen nutrition conditions (underground parts)

[0100] deal with Fresh weight (g) Dry weight (g) Root length (cm) Number of roots (bars) N20-H0 1.7675 0.16 9.9125 16.06 N20-H5 2.9994 0.22 10.8813 19.81 N20-H10 3.0763 0.22 12.5313 19.88 N20-H20 3.2069 0.25 12.7438 22.06 N40-H0 2.1088 0.17 11.7500 17.63 N40-H5 3.1819 0.20 12.4436 23.19 N40-H10 3.6044 0.25 13.3250 25.19 N40-H20 4.4125 0.31 13.6563 24.56 N80-H0 2.8319 0.23 13.5000 16.69 N80-H5 5.2963 0.31 13.4438 18.81 N80-H10 4.7713 0.42 13.6625 20.88 N80-H20 5.4963 0.43 14.3063 21.13

[0101] The results showed that the compound promoted root growth in rice under different nitrogen nutrient conditions, and all treatment groups were significantly better than the control group (P < 0.05). The best root-promoting effect was achieved with a nitrogen nutrient concentration of 80 mg / L and a compound concentration of 20 ng / mL. Figure 10 ).

[0102] 5.2 Effects of compounds on organic matter synthesis in rice plants under different nitrogen nutrition conditions

[0103] The results showed that the compound effectively promoted the synthesis of organic matter in rice under various treatments. Under different nitrogen nutrient conditions (20, 40, and 80 mg / L), the overall trend of soluble protein, dry matter, total nitrogen, total nitrogen accumulation, and free amino acid synthesis was significantly higher in the CK treatment group than in the control group (P < 0.05). At a nitrogen nutrient concentration of 80 mg / L, the compound had a significant effect on the synthesis of soluble protein, dry matter, total nitrogen, total nitrogen accumulation, and free amino acids in rice plants, promoting an increase in the synthesis of soluble protein, dry matter, nitrogen accumulation, starch, and free amino acids.

[0104] Table 4. Effects of compounds on organic matter synthesis in rice plants under different nitrogen nutrition conditions.

[0105]

[0106] As shown in Table 4, the results showed that the compound effectively promoted the synthesis of organic matter in rice under different nitrogen nutrient conditions (20, 40, and 80 mg / L). After spraying the compound, the overall trend of soluble protein, dry matter, total nitrogen, total nitrogen accumulation, and free amino acid synthesis was significantly higher than that of the control group (P < 0.05). Among them, at a nitrogen nutrient concentration of 80 mg / L, the compound had a more significant effect on the synthesis of soluble protein, dry matter, total nitrogen, total nitrogen accumulation, and free amino acids in rice plants, promoting the increase of soluble protein, dry matter, nitrogen accumulation, starch, and free amino acid synthesis.

[0107] This experiment preliminarily explored the effects of compounds on the growth of rice seedlings under different nitrogen nutrition conditions. Relevant growth indicators and physicochemical indicators were measured, and the results showed that the compounds had a good growth-promoting effect on rice.

[0108] Example 6: Effects of the compound on tobacco virus

[0109] Test of effective concentration of compound against PVX in tobacco

[0110] This experiment consisted of 8 treatment groups, with each treatment repeated 5 times. The pesticide solution was sprayed onto the leaves using a foliar spraying method. The treatment methods are shown in Table 5.

[0111] Table 5. Drug Treatment Table

[0112]

[0113] The virus was inoculated into three true leaves below the leaves of the tobacco system using a friction inoculation method. On the 5th day after inoculation, the fluorescence of the leaves was observed under 365nm ultraviolet light. After sampling the diseased leaves, they were stored in a refrigerator at -80℃.

[0114] The virus content in the leaves of each treatment group was detected using a potato virus enzyme-linked immunosorbent assay kit. The results showed that, compared to the control (CK), the fluorescence intensity on the leaves of all treatment groups was significantly reduced after spraying the compound. Figure 11 ).

[0115] Different concentrations of the compound had a certain influence on the tobacco's resistance to PVX. As the compound concentration increased, the tobacco's resistance to PVX first increased and then slightly decreased, with the best effect observed at 5–20 ng / mL. Figure 12 The inhibition rates were 68.74%, 69.93%, 70.62%, and 71.53%, respectively. The antiviral infection ability decreased at 50 ng / mL, with an inhibition rate of 53.13%. When the compound concentration was 100 ng / mL, the inhibition rate was 29.56% (Table 6).

[0116] Table 6 Inhibition rate of PVX by different drug treatments

[0117]

[0118] Note: Lowercase letters in the table indicate that the difference is significant at 5%.

[0119] The results showed that the compound had a good promoting effect on tobacco's resistance to PVX infection, with the most significant effect observed at concentrations of 5–20 ng / mL.

[0120] Example 7: Antibacterial function of the compound

[0121] Effects of compounds on Arabidopsis thaliana resistance to PSTDC3000 infection

[0122] pstDC3000 vaccination and disease investigation

[0123] Each extended leaf of Arabidopsis thaliana was inoculated with PSTDC3000 bacterial suspension using a percolation inoculation method, with four Arabidopsis thaliana plants per replicate. After incubation in a 24℃ artificial climate chamber for 16 / 8 hours for 3–5 days, samples were taken for analysis based on disease incidence. The calculation formula is: CFU / g = colony count × 10⁻⁶. 6 ×100 / g

[0124] The infection status of Arabidopsis thaliana leaves by pstDC3000 bacteria after spraying different concentrations of the compound (5 days) Figure 13 As shown.

[0125] Table 7. Effects of compounds on Arabidopsis thaliana's resistance to pstDC3000 (control efficacy / %)

[0126]

[0127]

[0128] The results, as shown in Table 7, indicate that the compounds applied to Arabidopsis leaves for 5 days provided some resistance to infection by pstDC3000 bacteria. Figure 14 However, different concentrations of the compound showed varying abilities to resist pstDC3000 infection in Arabidopsis thaliana. The best effect was observed at a concentration of 20 ng / mL, resulting in the lowest leaf bacterial content and a control efficacy of 66.68%.

[0129] Example 8: Field Trial of Compounds

[0130] The experiment was conducted in Tianbao Fourth Village, Tianbao Town, Tai'an City, Shandong Province (117°21′20″E, 35°56′36″N), an area with a warm temperate semi-humid continental monsoon climate. The tested soil was brown soil, classified as common simple leached soil in the Chinese soil classification system. The tested maize variety was Zhengdan 958, with a growth period of 96–125 days and a 100-kernel weight of approximately 33.0 g. The tested traditional fertilizers were urea (N 46%), superphosphate (P2O5 16%), and potassium sulfate (K2O5 1%). The experiment included five treatments, each replicated three times. The plot area was 20 m². 2 (5m × 4m), 3 replicates. The maize field experiment adopted a randomized block design, as shown in the table. Maize was planted by direct seeding, with a row spacing of 60cm and a plant spacing of 20cm. The experimental design and results are shown in Tables 8 and 9.

[0131] Table 8 Experimental Design

[0132]

[0133] Table 9. Effects of soluble compounds on maize plant height.

[0134]

[0135] Data analysis showed that for cyclodipeptide soluble concentrates, when the effective ingredient dosage was between 0.02 and 0.033 mg / kg, the weight of 100 tablets ranged from 25.41 to 27.20 g, showing a significant increasing trend compared to the blank control.

[0136] When the effective ingredient dosage of cyclic dipeptide soluble concentrate is between 0.02 and 0.033 mg / kg, the yield per mu of corn plot is between 560 and 610 kg, with a yield increase rate between 18.6% and 29.2%. Compared with the control group, spraying cyclic dipeptide soluble concentrate can significantly increase the yield per mu of corn.

[0137] Table 10. Maize Quality Analysis

[0138]

[0139] In terms of quality, protein and starch content are important indicators for evaluating the nutritional value of corn. Table 10 shows that the addition of cyclic dipeptide soluble concentrate improved the protein and starch content of corn, with starch increasing by 0.98%–1.05% and protein by 4.52%–5.85%. This effectively enhances the nutritional value of corn and meets the market demand for high-nutrition corn.

[0140] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing cyclic (isoluene-isoluene) dipeptide, characterized in that, The cyclic (isoleucine-isoleucine) dipeptide was obtained from the fermentation, isolation and purification of *Paecilomyces lilacinus*. The *Paecilomyces lilacinus* strain was deposited on May 27, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 41240.

2. The method for preparing the cyclic (isoluene-isoluene) dipeptide according to claim 1, characterized in that, The preparation method of the cyclic (isolure-isolure) dipeptide includes: after the *Pursola lilacinus* is cultured on a PDA plate, sterile water is added, spore hyphae and spores are scraped to prepare a fungal suspension, filtered to prepare a spore suspension, and the spore suspension is cultured in a culture medium at a temperature of 25℃~35℃ and a pH of 5~7. After the culture medium is centrifuged, the mycelium is collected by filtration and dried to constant weight. The mycelium was mixed with ethanol, ultrasonically vibrated, and the broken bacterial solution was vacuum filtered. The filtrate was collected as the extract. The extract was concentrated by rotary evaporation and then freeze-dried to produce freeze-dried powder.

3. Application of cyclic (isoleucine-isoleucine) dipeptide in Arabidopsis thaliana's resistance to infection by pstDC3000 bacteria.

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