Preparation and application of a bidirectional fermentation extract of *Bacillus thuringiensis* and traditional Chinese medicine

Abstract

This invention discloses a method for preparing a bidirectional fermentation extract of *Bacillus thuringiensis* and traditional Chinese medicine, and the application of this extract in inhibiting plant pathogenic fungi, belonging to the fields of microbial fermentation technology and biopesticides. This invention significantly enhances the antibacterial activity of the extract through bidirectional fermentation of *Bacillus thuringiensis* and traditional Chinese medicine, achieving an inhibition rate of up to 100%, comparable to the positive control carbendazim at the same concentration. The active ingredients are enhanced: the content of total terpenes, total polyphenols, and total flavonoids is significantly increased after fermentation. It is green and safe: derived from natural traditional Chinese medicine and fungi, with no chemical residues, and is environmentally friendly.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology and biopesticides. Specifically, it relates to a method for preparing a bidirectional fermentation extract of *Bacillus thuringiensis* and traditional Chinese medicine, and the application of the extract in inhibiting plant pathogenic fungi. Background Technology

[0002] Plant pathogenic fungi are the main cause of crop diseases, accounting for 70% to 80% of all plant diseases and posing a serious threat to food security. For example, major fungal diseases such as wheat stripe rust and Fusarium head blight can lead to a 20% to 50% reduction in crop yield, or even total crop failure, resulting in an annual crop yield loss of 10% to 20%. For a long time, agricultural production has relied on chemical pesticides for disease control. While these methods are effective quickly, they easily lead to a series of prominent problems: First, the disorderly use of chemical pesticides can harm beneficial microorganisms and natural enemies of pests in the field, disrupting the ecological balance and potentially exacerbating the damage caused by harmful organisms; second, pathogens easily develop resistance, reducing control effectiveness and leading to a continuous increase in pesticide use; furthermore, pesticide residues can pollute the environment and affect human and animal health through the food chain. Therefore, developing safer and more sustainable alternative control products remains a top priority.

[0003] Traditional Chinese medicine (TCM) is characterized by low toxicity, low residue, and friendliness to non-target organisms and the environment. It contains various active ingredients such as polysaccharides, alkaloids, and flavonoids, exhibiting broad-spectrum antibacterial potential. However, direct extraction and utilization of TCM suffers from limitations such as low dissolution rates of active ingredients, complex structures that hinder absorption, and unstable efficacy. Two-way fermentation technology is one effective strategy to address these issues, stemming from the combination of traditional TCM processing methods and modern biotechnology. This technology uses specific TCM herbs as a medicinal matrix and co-cultivates them with beneficial microorganisms possessing clearly defined biological activities. During this process, the growth and metabolic activities of the microorganisms can transform the macromolecules of the TCM, improving the extraction rate and bioavailability of active ingredients, and even generating new active compounds. Simultaneously, certain components in the TCM (such as polysaccharides) can also promote the growth and metabolism of the microorganisms, forming a bidirectional, beneficial "novel fermentation process." Compared to traditional processes, this technology offers advantages in production efficiency, cost control, and process controllability, providing a new direction for the creation of new TCM drugs.

[0004] White sac rake tooth fungus ( Irpex lacteusAs a common wood-rotting fungus, its fermentation extract has been shown to possess various biological activities, including antibacterial and anti-inflammatory properties. However, its application in the bidirectional fermentation of traditional Chinese medicine (TCM), particularly for the inhibition of plant pathogenic fungi, remains a blank area of ​​research. Existing TCM-derived antibacterial agents are mostly limited to extracts from single medicinal materials, lacking large-scale, targeted production processes that achieve synergistic effects through specific microbial fermentation. Therefore, this invention develops a bidirectional fermentation extract based on a combination of *Bacillus thuringiensis* and selected TCM herbs, which is expected to significantly improve the antibacterial activity and stability of the final product. This innovation not only overcomes many drawbacks of chemical pesticides and aligns with the trend of pesticide reduction and greening, but also provides a feasible path to solve the problem of unstable effects from direct application of TCM. Such research has significant application value for promoting the development of biological pesticides, ensuring the quality and safety of agricultural products, and promoting sustainable agricultural development. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, the present invention aims to provide a method for preparing a bidirectional fermentation extract of *Bacillus thunbergii* and traditional Chinese medicine, and the application of this extract in inhibiting plant pathogenic fungi. By employing bidirectional fermentation technology, the antibacterial activity of traditional Chinese medicine is enhanced, overcoming the drawbacks of chemical pesticides.

[0006] To solve the above problems, the technical solution adopted by the present invention is as follows: A method for preparing a bidirectional fermentation extract of *Bacillus thunbergii* and traditional Chinese medicine includes the following steps: Strain activation: Inoculate *Bacillus thunbergii* into a culture medium and incubate at 25°C for 5 days to obtain a solid strain; Preparation of Chinese herbal medicine culture medium: Pulverize the Chinese herbal medicine into powder that can pass through a No. 6 sieve. Add 15g-25g of Chinese herbal medicine powder according to the size of the culture medium plate. Then add distilled water and adjust the moisture content of the culture medium plate to 40%-65%. Autoclave at 121℃ for 1 hour. Two-way fermentation: In a clean bench, solid inoculum blocks are inoculated into a traditional Chinese medicine culture medium and cultured statically at 28-30℃ for 10-40 days to obtain fermented microbial material; Extraction preparation: The fermentation bacteria were dried, 10 times their weight of ethanol were added, and the mixture was ultrasonically extracted for 30 minutes. After filtration, the extract was concentrated to obtain the bidirectional fermentation extract.

[0007] Furthermore, the Chinese medicinal herbs mentioned include oregano and scutellaria.

[0008] Furthermore, when adding ethanol for extraction, the volume fraction of ethanol is 70%.

[0009] On the other hand, the application of the *Bacillus thunbergii* and the bidirectional fermentation extract of traditional Chinese medicine obtained by the above preparation method in plant antibacterial agents.

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention significantly enhances the antibacterial activity of the extract through bidirectional fermentation of *Bacillus thunbergii* and traditional Chinese medicine, achieving an antibacterial rate of up to 100%, comparable to the positive control carbendazim at the same concentration. The active ingredients are enhanced: the content of total terpenes, total polyphenols, and total flavonoids is significantly increased after fermentation. It is green and safe: derived from natural traditional Chinese medicine and fungi, with no chemical residues, and is environmentally friendly. Attached Figure Description

[0011] Figure 1 The changes in antibacterial activity of the fermentation extracts in Examples 1-2 at different fermentation stages are shown.

[0012] Figure 2 The inhibitory effects of fermentation extracts from Examples 1-2 on the mycelial growth of six plant pathogens at different fermentation stages were investigated.

[0013] Figure 3 The polyphenol content of the fermentation extracts in Examples 1-2 at different fermentation stages.

[0014] Figure 4 The content of flavonoids in the fermentation extracts of Examples 1-2 at different fermentation stages.

[0015] Figure 5 The content of terpenoids in the fermentation extracts of Examples 1-2 at different fermentation stages. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Example 1: A bidirectional fermentation extract of *Bacillus thunbergii* and traditional Chinese medicine was prepared according to the following steps: First, inoculate *Bacillus thunbergii* into PDA medium and incubate it in a constant temperature incubator at 25°C for 5 days in the dark to obtain a solid culture.

[0018] Next, the traditional Chinese medicine culture medium was prepared. First, the oregano was pulverized and sieved until all the powder could pass through a No. 6 sieve (i.e., a No. 6 mesh sieve with a mesh size of 0.150 mm ± 0.006 mm). After pulverization, the powder was loaded into culture medium plates. In this example, the weight of the powder added was 20 g / plate. Then, distilled water was added to adjust the moisture content of the culture medium in the plates to 65%. Finally, the plates were autoclaved at 121°C for 1 hour.

[0019] After the Chinese medicine culture medium is sterilized, it is taken out and cooled to room temperature. It is then inoculated with a solid inoculum block (7 mm) of *Bacillus thunbergii* and cultured at 25°C for 10 days to obtain fermented mycelium.

[0020] The dried fermentation average was extracted three times with 95% ethanol (each time for 3 days), with the ethanol volume fraction being 70% during the extraction. After extraction, the extract was filtered to obtain the extract solution, which was then concentrated under reduced pressure using a rotary evaporator to obtain the concentrate. The concentrate solution was first dried in a forced-air drying oven, and then dried in a vacuum freeze dryer. After thorough drying, it was weighed and the extraction rate was calculated to obtain the extract with 95% ethanol concentration, i.e., the bidirectional fermentation extract.

[0021] Example 2: A bidirectional fermentation extract of *Bacillus thunbergii* and traditional Chinese medicine was prepared according to the following steps: First, inoculate *Bacillus thunbergii* into PDA medium and incubate it in a constant temperature incubator at 25°C for 5 days in the dark to obtain a solid culture.

[0022] Next, the traditional Chinese medicine culture medium is prepared. First, the Scutellaria baicalensis is pulverized and sieved until all the powder can pass through a No. 6 sieve. After pulverization, the powder is loaded into culture medium plates. In this example, the weight of the powder added is 20g / plate. Then, distilled water is added to adjust the moisture content of the culture medium in the plates to 65%. Finally, the plates are autoclaved at 121°C for 1 hour.

[0023] After the Chinese medicine culture medium is sterilized, it is taken out and cooled to room temperature. It is then inoculated with a solid inoculum block (7 mm) of *Bacillus thunbergii* and cultured at 25°C for 30 days to obtain fermented mycelium.

[0024] The dried fermentation average was extracted three times with 95% ethanol (each time for 3 days), with the ethanol volume fraction being 70% during the extraction. After extraction, the extract was filtered to obtain the extract solution, which was then concentrated under reduced pressure using a rotary evaporator to obtain the concentrate. The concentrate solution was first dried in a forced-air drying oven, and then dried in a vacuum freeze dryer. After thorough drying, it was weighed and the extraction rate was calculated to obtain the extract with 95% ethanol concentration, i.e., the bidirectional fermentation extract.

[0025] Experimental Example 1: Determination of Antibacterial Activity Test samples: Fermentation extracts from Examples 1 and 2, plant pathogenic fungi: *Cephalotaxus fortunei* (the pathogen of maize large leaf spot). Exserohilum turcicum Alternaria zeylanica ( Alternaria alternata Fusarium pseudoverticum ( Fusarium verticillioides Fusarium graminearum ( Fusarium graminearum ), ginseng root rot pathogen ( Fusarium solani ), ginseng rust pathogen ( Cylindrocarpon destructans ).

[0026] Test method: Different fermentation extracts were dissolved in methanol and added to PDA medium that had been autoclaved at 121°C for 30 min and cooled to about 50°C to prepare a drug-containing medium with a final concentration of 15 mg / mL. After solidification, the medium was made into drug-containing plates.

[0027] The treatment group was a PDA medium containing bidirectional fermentation extracts with the same final concentration but different fermentation times, while the control group had no solute added but an equal amount of solvent (methanol) added. Each treatment was repeated three times.

[0028] Take activated plant pathogen agar plates, punch holes using a sterilized punch (5 mm inner diameter), inoculate the center of the agar plate, and incubate in the dark at 25℃. Observe and record the mycelial growth status daily until the mycelium in the blank control group completely covers the plate. The in vitro antibacterial activity against plant pathogenic fungi was determined using the mycelial growth rate method, and the colony diameter was determined using the streak method. The inhibition rate of the ethanol extract is calculated using the following formula: ;

[0029] Results of antibacterial activity assay as follows Figure 1 and Figure 2 As shown, in Figure 2 The vertical columns represent the different bacterial species inoculated: A is the pathogen of maize leaf spot; B is Alternaria zei; C is Fusarium verticillata; D is Fusarium graminearum; E is the pathogen of ginseng root rot; and F is the pathogen of ginseng rust rot. The horizontal columns represent the drug-containing culture media with different additives: a is the 0-day extract; b is the 10-day extract; c is the 20-day extract; d is the 30-day extract; e is the 40-day extract; f is methanol; and g is carbendazim.

[0030] Experimental Example 2: Determination of Total Polyphenol Content: Test samples: Fermentation extracts from Examples 1 and 2.

[0031] Test method: A standard curve was plotted using the Folin-Ciocalteu colorimetric method, followed by content determination. Accurately weigh 0.100 g of gallic acid standard, dissolve and dilute with 75% ethanol to obtain a standard solution with a mass concentration of 0.02 mg / mL. Take 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mL of the gallic acid standard solution and place them into 15 mL capped centrifuge tubes, then add distilled water to make up to 3 mL. Add 1 mL of Folin-Ciocalteu reagent and 6 mL of 10% Na₂CO₃ solution (10 g of sample dissolved in distilled water and then diluted to volume in a 100 mL volumetric flask) to each tube, and then dilute to 10 mL with 75% ethanol. React in a 30℃ water bath for 1 h. 200 μL of the reaction liquid was taken from centrifuge tubes and placed into 96-well plates. The absorbance was measured at 750 nm. The above was repeated 3 times and the average value was calculated. A standard curve was plotted with gallic acid mass (mg) on ​​the x-axis and absorbance value on the y-axis to obtain the regression equation.

[0032] Determination of total polyphenol content: Take 0.5 mL of sample solution (concentration as above, 0.5 mg / mL) into 15 mL capped centrifuge tubes, perform the color reaction as described above, and measure the absorbance value. Substitute the absorbance value into the regression equation to calculate the mass of total polyphenols in the extract. The total polyphenol content is calculated using the following formula: ;

[0033] The results of the experiment are as follows Figure 3 As shown in the figure, the vertical axis represents the total polyphenol content, and the horizontal axis represents the fermentation extracts with different fermentation days.

[0034] Experimental Example 3: Determination of Total Flavonoid Content: Test samples: Fermentation extracts from Examples 1 and 2.

[0035] Test method: A standard curve was plotted using the NaNO2-Al(NO3)3-NaOH colorimetric method, followed by content determination. Accurately weigh 0.100 g of rutin standard, dissolve and dilute with 75% ethanol solution to obtain a standard solution with a mass concentration of 0.02 mg / mL. Take six 10 mL test tubes and accurately measure 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mL of gallic acid standard solution into 15 mL capped centrifuge tubes. Add 75% ethanol to a final volume of 5 mL, mix well, and then add 0.3 mL of 5% NaNO2 solution (5 g of sample dissolved in distilled water and diluted to volume in a 100 mL volumetric flask). Mix well and let stand for 5 min. Add 0.3 mL of 10% Al(NO3)3 solution to each tube, mix well, and let stand for 6 min. Add 4 mL of 1 mol / L NaOH solution to each tube, then add 75% ethanol solution to a final volume of 10 mL, mix well, and let stand for 10 min. 200 μL of the reacted liquid was pipetted into a 96-well plate from a centrifuge tube, and the absorbance was measured at 510 nm. This was repeated three times, and the average value was calculated. A standard curve was plotted with rutin mass (mg) on ​​the x-axis and absorbance on the y-axis to obtain the regression equation. Total flavonoid content determination: 0.5 mL of sample solution (concentration as above, 0.5 mg / mL) was added to a 15 mL capped centrifuge tube. After the colorimetric reaction was performed as described above, the absorbance was measured. Substituting the absorbance value into the regression equation, the mass of total flavonoids in the extract could be calculated. The total flavonoid content was calculated using the following formula: ;

[0036] The test results are as follows Figure 4 As shown in the figure, the vertical axis represents the total flavonoid content, and the horizontal axis represents the fermentation extracts with different fermentation days.

[0037] Experimental Example 4: Determination of Total Terpenoid Content: Test samples: Fermentation extracts from Examples 1 and 2.

[0038] Test method: Weigh 2 mg of oleanolic acid standard and prepare a standard solution with a concentration of 0.2 mg / mL using methanol. Weigh 5 mg of the dried ethanol extract and prepare a sample solution with a concentration of 0.5 mg / mL using methanol. Take 0.1, 0.2, 0.3, 0.4, and 0.5 mL of the oleanolic acid standard solution and place them in 15 mL capped centrifuge tubes respectively. Evaporate the solvent in a water bath, add 0.2 mL of freshly prepared 5% vanillin-acetic acid solution and 0.8 mL of perchloric acid, mix well, and place in a 60℃ water bath for 15 min. Immediately after removal, place in an ice-water bath for 5 min, add 5 mL of acetic acid solution, mix well, and let stand for 10 min. Use the methanol reagent without oleanolic acid as a blank control. 200 μL of the reacted liquid was pipetted into a 96-well plate from a centrifuge tube, and the absorbance was measured at 560 nm. This was repeated three times, and the average value was calculated. A standard curve was plotted with the mass of oleanolic acid (mg) on ​​the x-axis and the absorbance on the y-axis to obtain the regression equation. Sample content determination: 0.5 mL of the prepared sample solution was taken, and after color development according to the method described above for preparing the standard curve, the absorbance was measured at 560 nm. The absorbance value was substituted into the regression equation to calculate the mass of total terpenoids in the extract. The total terpenoid content was calculated using the following formula: ;

[0039] The test results are as follows Figure 5 As shown in the figure, the vertical axis represents the total terpenoid content, and the horizontal axis represents the fermentation extracts with different fermentation days.

[0040] Based on the above experimental results, it can be demonstrated that this method significantly enhances the antibacterial activity of the extract through bidirectional fermentation of Bacillus subtilis and traditional Chinese medicine, with an antibacterial rate of up to 100%, which is comparable to the positive control carbendazim at the same concentration. The active ingredients are enhanced: the content of total terpenes, total polyphenols and total flavonoids is significantly increased after fermentation. It is green and safe: it is derived from natural traditional Chinese medicine and fungi, with no chemical residues and is environmentally friendly.

Claims

1. A preparation method of a bidirectional fermentation extract of white-cystid Cladogamus and traditional Chinese medicine, characterized in that, It comprises the following steps: Strain activation: inoculate the white cyst harpia in culture medium, cultivate for 5 days at 25℃, and obtain solid strain; Preparation of Chinese medicine medium: crush the Chinese medicine to pass through No. 6 sieve, add 15g-25g of Chinese medicine powder according to the size of the medium plate, then add distilled water and adjust the water content of the medium plate to 40%-65%, and autoclave at 121℃ for 1 hour; Bidirectional fermentation: inoculate the solid strain block in the Chinese medicine medium in a super-clean bench, and culture at 28-30℃ for 10-40 days to obtain fermented fungus; Preparation of extract: dry the fermented fungus, add ethanol for extraction, filter the extracted extract, and concentrate to obtain bidirectional fermentation extract.

2. The preparation method of the bidirectional fermented extract of the white-cystid Armillaria according to claim 1, characterized in that, The Chinese medicine comprises oregano and radix scrophulariae.

3. The preparation method of the bidirectional fermented extract of Armillaria gallica and traditional Chinese medicine according to claim 1, characterized in that, The volume fraction of ethanol is 70% when added for extraction.

4. Application of the white cyst harpia bidirectional fermentation extract with Chinese medicine in a plant bacteriostatic agent, which is prepared by the preparation method according to any one of claims 1-3.

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