A cda enzyme-producing bacillus and application thereof to promoting agarwood resinosis

Abstract

The application discloses a CDA enzyme-producing bacillus, and a culture method of the bacillus, which comprises the following steps: (1) using a fermentation medium containing MgSO4, (NH4)2SO4 and chitin to culture the CDA enzyme-producing bacillus for 168 hours; (2) using an LB medium to activate the CDA enzyme-producing bacillus, picking a single colony after the strain grows into a colony, inoculating the single colony into a new LB medium, and culturing for 2-3 days to obtain a seed liquid; (3) transferring the obtained seed liquid into a new fermentation medium to carry out large-scale culture, and carrying out low-speed centrifugation on the bacterial liquid after culturing for 3 days; and (4) pouring out part of the culture medium to obtain a concentrated bacterial liquid. The bacterial liquid can be used for promoting the linyang of agarwood. The CDA enzyme-producing bacillus obtained by purifying, fermenting and activating the bacillus GXU-B2 can be injected into the xylem of agarwood by means of liquid static drop, and the CDA enzyme and the bacillus can stimulate the defense system of the aquilaria plant to promote the linyang of agarwood, and the linyang is deep and uniform in the xylem.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to a CDA-producing Bacillus and its application in promoting agarwood formation. Background Technology

[0002] In recent years, naturally occurring agarwood has been unable to meet market demand, while agarwood cultivation and induction techniques have become increasingly sophisticated. Artificial methods for promoting resin formation have seen significant development, including mechanical damage, biological inoculants, chemical inducers, combinations of biological and chemical inducers, and in vitro plant tissue culture for resin production. Compared with traditional methods, biological inoculants are more effective at inducing agarwood formation.

[0003] Common biological inoculation methods include solid inoculation and liquid inoculation. Solid inoculation involves directly inserting mycelia into artificially drilled holes, resulting in high-quality resin but low yield. Liquid inoculation mainly has two methods: one is liquid injection, which involves directly inoculating the tree with mycelial solution using a syringe after drilling. This method uses a smaller amount of mycelial solution, but the quality of resin formation needs improvement. The other is liquid infusion, which involves slowly injecting mycelial solution into the vascular bundles of the Aquilaria plant using an infusion device. The mycelial solution flows and distributes throughout the tree through the vascular bundles, promoting resin formation throughout the entire plant; hence, it is also known as the "whole-body resin formation method." This invention studies the use of the liquid infusion method to inject mycelial solution to enrich the strains of Aquilaria resin-forming bacteria. Summary of the Invention

[0004] The main objective of this invention is to provide a CDA-producing Bacillus and its application in promoting agarwood formation.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A CDA-producing Bacillus, wherein the method for culturing the Bacillus includes the following steps:

[0007] (1) The strains obtained by screening and purification were cultured for CDA production for 168 h in a fermentation medium containing MgSO4, (NH4)2SO4 and chitin.

[0008] (2) Activate the CDA-producing Bacillus using LB medium. After the strain grows, pick a single colony and inoculate it into LB medium for 2-3 days to obtain seed culture.

[0009] (3) Transfer the seed culture obtained in step (2) into a new fermentation medium containing MgSO4, (NH4)2SO4 and chitin for expansion culture. After culturing for 3 days, centrifuge the bacterial culture at low speed.

[0010] (4) Pour off a portion of the culture medium from the bacterial solution after low-speed centrifugation to obtain concentrated bacterial solution.

[0011] The Bacillus sp. is deposited at the China Center for Type Culture Collection (CCTCC) under the name Bacillus GXU-B2 (CCTCC M 20231974) and the date of deposit is October 20, 2023.

[0012] Furthermore, in steps (1) and (3), the inoculum amount of the fermentation medium and PDB medium is 5%. If the inoculum amount is too large, it will cause insufficient dissolved oxygen and affect product synthesis; if the inoculum amount is too small, it will prolong the culture time and reduce the productivity of the fermenter.

[0013] Furthermore, in step (1), the fermentation temperature of the fermentation medium is 37°C, the initial pH is 7, and the shaking speed is 180 r / min. The fermentation temperature and initial pH are different from the fermentation conditions of general fermentation media. The main reason is that under these fermentation conditions, the enzyme production of Bacillus is optimal.

[0014] Furthermore, the fermentation medium comprises the following components and concentrations: MgSO4 concentration of 0.20%, (NH4)2SO4 concentration of 2.15%, chitin concentration of 1.55%, potato starch concentration of 1.00%, cassava starch concentration of 1.00%, corn starch concentration of 1.00%, soybean meal concentration of 1.25%, and MnSO4 concentration of 0.15%.

[0015] Furthermore, in step (3), the speed of low-speed centrifugation is 180 r / min. Low-speed centrifugation can precipitate the bacterial strains in the bacterial solution, remove the supernatant, and retain the concentrated bacteria.

[0016] The application of the bacterial solution obtained from the cultivation of Bacillus of the present invention in promoting the formation of agarwood.

[0017] Furthermore, the concentration of the bacterial solution is OD600 = 0.3, and the concentration of the bacterial solution directly affects the agarwood formation effect.

[0018] Furthermore, the agarwood mentioned is Aquilaria sinensis.

[0019] The advantages of this invention are as follows: The CDA-producing Bacillus GXU-B2 obtained by purification, fermentation and activation is injected into the xylem of agarwood by liquid droplet method. The CDA enzyme and Bacillus can stimulate the defense system of Aquilaria plants to promote the formation of resin in agarwood, and the resin formation penetrates into the xylem evenly. Attached Figure Description

[0020] Figure 1 This is an optical microscope image of Bacillus spores GXU-B2 that produce CDA enzyme;

[0021] Figure 2This is a Gram staining image of GXU-B2, a spore-forming CDA enzyme;

[0022] Figure 3 This is a scanning electron microscope image of Bacillus spores GXU-B2, which produces CDA enzyme;

[0023] Figure 4 This is a phylogenetic tree diagram of the CDA-producing Bacillus GXU-B2 based on the 16 rRNA gene;

[0024] Figure 5 This is the enzyme activity curve of Bacillus spores GXU-B2, which produces CDA enzyme;

[0025] Figure 6 This is a comparison chart of carbon source utilization results for Bacillus spores GXU-B2 that produce CDA enzyme;

[0026] Figure 7 This is a comparison chart of nitrogen source utilization results for Bacillus spores GXU-B2 that produce CDA enzyme;

[0027] Figure 8 This is a comparison chart of inorganic salt utilization results of Bacillus spores GXU-B2 that produce CDA enzyme;

[0028] Figure 9 These are contour plots and response surface plots of Bacillus GXU-B2, which produces CDA enzyme;

[0029] Figure 10 This is a photo of the inoculation site of CDA-producing Bacillus GXU-B2 via intravenous drip injection;

[0030] Figure 11 This is a picture of the resin formation of Aquilaria sinensis four months after inoculation with CDA-producing Bacillus GXU-B2. Detailed Implementation

[0031] To make the objectives, technical solutions, and beneficial effects of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, so as to facilitate understanding by those skilled in the art.

[0032] The culture media used in this embodiment are LB medium and fermentation medium.

[0033] LB medium (liquid): 1% peptone, 0.5% yeast extract, 1% NaCl; LB medium (solid): 1% peptone, 0.5% yeast extract, 1% NaCl, 1.8% agar powder, pH natural, autoclaved at 121℃ before use.

[0034] Fermentation medium: MgSO4 concentration 0.20%, (NH4)2SO4 concentration 2.15%, chitin concentration 1.55%, potato starch concentration 1.00%, cassava starch concentration 1.00%, corn starch concentration 1.00%, soybean meal concentration 1.25%, MnSO4 concentration 0.15%, with the remainder being water.

[0035] The Bacillus used in this embodiment was deposited at the China Center for Type Culture Collection on October 20, 2023, with the deposit name Bacillus GXU-B2 (Bacillus sp.) and the accession number CCTCC M20231974.

[0036] A CDA-producing Bacillus, characterized in that the cultivation method of the Bacillus includes the following steps:

[0037] (1) The strains obtained by screening and purification were cultured for CDA production for 168 h in a fermentation medium containing MgSO4, (NH4)2SO4 and chitin. The fermentation temperature was 37℃, the initial pH was 7, and the shaking speed was 180 r / min.

[0038] (2) Activate the CDA-producing Bacillus using LB medium. After the strain grows, pick a single colony and inoculate it into a new LB medium and culture for 2-3 days to obtain seed culture.

[0039] (3) Transfer the seed culture obtained in step (2) into a new fermentation medium containing MgSO4, (NH4)2SO4 and chitin for expansion culture. After 3 days of culture, centrifuge the bacterial culture at a low speed of 180 r / min.

[0040] (4) Pour off a portion of the culture medium from the bacterial solution after low-speed centrifugation to obtain concentrated bacterial solution.

[0041] In steps (1) and (3), the inoculum amount of the fermentation medium and PDB medium is 5%.

[0042] The screened and purified bacterial strains were observed under Gram staining and scanning electron microscopy on LB medium to analyze their physiological and biochemical characteristics, such as... Figure 1-3 As shown. From Figure 1-3 The colony morphology of Bacillus GXU-B2 can be obtained as follows: single colonies are irregular in shape, with a smooth, raised, moist, translucent, white, and glossy surface. The colony periphery secretes a large amount of transparent mucus, which has a viscous feel. After prolonged incubation, the colony surface becomes dry, dull, wrinkled, and not raised, making it difficult to pick up.

[0043] The whole genome DNA of the screened and purified bacterial strains was extracted and amplified by PCR using universal primers 1492R (5'-GGTTACCTTGTTACGACTT-3') and 27F (5'-AGAGTTTGATCCTGGCTCAG-3') for the 16S rRNA gene. The amplified products were then sequenced. The sequencing data were aligned online using NCBI BLAST to analyze sequence homology. Closely related sequences were downloaded, and the sequence files were imported into MEGA X software. A phylogenetic tree was constructed using maximum likelihood (ML) analysis. Figure 4 As shown.

[0044] The enzyme production curve of the screened and purified strain was determined. The strain in the exponential growth phase was transferred to fermentation medium (100 mL in a 250 mL Erlenmeyer flask). Three samples were taken every 12 hours. Finally, CDA activity was measured using the p-nitroacetanilide method. The results are shown below. Figure 5 As shown.

[0045] Culture medium optimization experiments were conducted to optimize the CDA production of Bacillus GXU-B2. The effects of different carbon sources, nitrogen sources, and inorganic salts on CDA production by Bacillus GXU-B2 were investigated. Carbon sources used included glucose, sucrose, maltose, chitin powder, corn starch, lactose, potato starch, and cassava starch. Nitrogen sources included peptone, NH4Cl, (NH4)2SO4, KNO3, NaNO3, urea, soybean meal, and beef extract. Inorganic salts included MgSO4, CaCl2, MnSO4, ZnSO4, NaCl, FeSO4, KCl, and CuSO4. The experimental procedure involved inoculating the exponential growth phase strain (5% inoculum) into 100 mL / 250 mL fermentation medium, with three replicates per group. CDA enzyme activity was measured using the p-nitroacetanilide method after culturing, and the experiment was repeated three times. The experiment revealed that the utilization rates of carbon sources by Bacillus GXU-B2, from highest to lowest, were: potato starch > corn starch > chitin > cassava starch > lactose > maltose > glucose = sucrose; the utilization rates of nitrogen sources, from highest to lowest, were: soybean meal > (NH4)2SO4 = beef extract > urea > NH4Cl > KNO3 > NaNO3 = peptone; and the utilization rates of inorganic salts, from highest to lowest, were: CaCl2 > NaCl > FeSO4 > MnSO4 > CuSO4 > MgSO4 > ZnSO4 > KCl. Figure 6-8 As shown.

[0046] Based on the utilization rates of carbon, nitrogen, and inorganic salts by Bacillus GXU-B2, the Plackett-Burman experimental design using Design-Expert software was used to determine the top three factors with the most significant impact on CDA yield from eight factors. The four carbon sources (potato starch, chitin, cassava starch, and corn starch), two nitrogen sources (soybean meal and (NH4)2SO4), and two inorganic salts (MnSO4 and MgSO4) with the best performance in single-factor optimization experiments were selected as optimization factors, with CDA activity as the response value for screening. Three parallel groups were set up, and the experiment was repeated three times. The results showed that MgSO4, (NH4)2SO4, and chitin were the main factors affecting CDA accumulation (p < 0.05), as shown in Figure 1 and Table 2.

[0047] Table 1. Plackett-Burman design and results for Bacillus GXU-B2

[0048]

[0049] Table 2. Analysis of Variance for Plackett-Burman Design of Bacillus GXU-B2

[0050]

[0051] Based on the magnitude and direction of the effect values ​​of the significantly influencing factors in the Plackett-Burman experiment, a steepest climbing experiment was conducted. The steepest climbing experiment used the gradient of experimental values ​​as the climbing direction, thus enabling a rapid and economical approach to the optimal enzyme production region. This experiment was conducted in triplicate, with three replicates. The experimental results showed that Bacillus GXU-B2 exhibited maximum enzyme activity at concentrations of 1.70% chitin, 1.80% (NH4)2SO4, and 0.20% MgSO4, as shown in Table 3.

[0052] Table 3. Experimental Design and Results of Steepest Climbing Experiment with Bacillus GXU-B2

[0053]

[0054] Based on the Plackett-Burman design and the steepest ascent experiment results, three factors were selected, with three levels for each factor (MgSO4, (NH4)2SO4, and chitin). The fermentation medium was further optimized using the Box-Behnken design in Design-Expert software, and the experimental data were fitted to obtain the model equation. Experimental results showed that when the concentration of MgSO4 was 0.20%, the concentration of (NH4)2SO4 was 2.15%, and the concentration of chitin was 1.55%, the maximum enzyme activity of 2.805 U / mL was obtained, as shown in Table 4. The obtained regression equation is: Y = 2.7391 - 0.1173A + 0.1551B + 0.0605C - 0.2005AB + 0.0340AC - 0.6545BC - 0.2667A² - 0.3424B² - 0.7056C², with a model correlation coefficient R² = 0.9832, a correction coefficient R²Agj = 0.9531, and a CV = 6.07%. This indicates that the regression equation can simulate and predict the actual results of the fermentation experiment relatively well.

[0055] The GXU-B2 regression model (p < 0.01) was highly significant, and the model lack-of-fit term (p = 0.1823) was not significant, indicating that the model's predicted values ​​have a good fit with the actual values, as shown in Table 5.

[0056] Table 4. Box-Behnken Experimental Design and Results for Bacillus GXU-B2

[0057]

[0058]

[0059] Table 5. Box-Behnken design regression analysis of Bacillus GXU-B2.

[0060]

[0061] Observe the contour plot and response surface of Bacillus GXU-B2 Figure 9(In the figure, AC is the contour plot of the Box-Behnken experimental design, and DF is the response surface plot of the Box-Behnken experimental design.) It was found that the interaction between (NH4)2SO4 and chitin was significant (p < 0.05), while the interaction between MgSO4 and chitin was not significant (p > 0.05). Therefore, the optimal fermentation conditions for Bacillus GXU-B2 are: MgSO4 concentration of 0.20%, (NH4)2SO4 concentration of 2.15%, and chitin concentration of 1.55%. Thus, the optimal fermentation medium composition and concentrations were determined as follows: MgSO4 concentration 0.20%, (NH4)2SO4 concentration 2.15%, chitin concentration 1.55%, potato starch concentration 1.00%, cassava starch concentration 1.00%, corn starch concentration 1.00%, soybean meal concentration 1.25%, and MnSO4 concentration 0.15%.

[0062] The bacterial solution obtained in this embodiment can promote agarwood formation. The specific operation method is as follows:

[0063] (1) Select 10 Aquilaria sinensis trees aged 7-10 years with no obvious damage to the surface of the trunk;

[0064] (2) Drill holes at different heights on the trunk of the Aquilaria sinensis tree with an electric drill. The hole diameter is 3mm and the hole depth is 5cm.

[0065] (3) Dilute the obtained bacterial solution to a final concentration of OD600 = 0.3, and then put the diluted bacterial solution into the inoculation device;

[0066] (4) Pressurize the inoculation bottle of the inoculation device to 1.5 MPa and finally suspend it on the selected white sandalwood.

[0067] The inoculation device includes an inoculation bottle, an infusion tube, a flow switch, and an inoculation head. The bottom of the inoculation bottle is connected to the infusion tube, which is equipped with the flow switch and the inoculation head. After suspending the inoculation device, the inoculation head is inserted into the hole drilled in the agarwood. Figure 10 As shown.

[0068] Four months after inoculation, the bark of the inoculated Aquilaria sinensis tree is cut open until the discolored wood is visible. The tree body after the bark has been removed is photographed and analyzed. The discolored portion is excavated until the tree itself contains little or no discolored tissue. The obtained tissue is photographed and the area of ​​resin formation is measured. Figure 11 As shown in the figure. The results indicate that the Aquilaria sinensis tissue inoculated with Bacillus GXU-B2 exhibits extensive resin formation, with the discoloration extending deep into the xylem and exhibiting a darker color. The discolored area is evenly distributed from the perforated area to the edge of the discolored region.

[0069] Although the specific embodiments of the present invention have been described and illustrated in detail above, it should be noted that various changes and modifications can be made to the above embodiments without departing from the spirit of the present invention and the scope set forth in the appended claims.

Claims

1. The application of a CDA-producing Bacillus bacterial suspension in promoting agarwood formation, wherein the agarwood is Aquilaria sinensis, and the Bacillus was deposited at the China Center for Type Culture Collection on October 20, 2023, with accession number CCTCC NO: M20231974, characterized in that... The method for culturing Bacillus includes the following steps: (1) The CDA-producing Bacillus was cultured for 168 h in a fermentation medium containing MgSO4, (NH4)2SO4 and chitin to produce CDA enzyme; (2) Activate the Bacillus obtained in step (1) using LB medium. After the strain grows, pick a single colony and inoculate it into a new LB medium and culture it for 2-3 days to obtain seed liquid. (3) Transfer the seed culture obtained in step (2) into a new fermentation medium containing MgSO4, (NH4)2SO4 and chitin for expansion culture. After culturing for 3 days, centrifuge the bacterial culture at low speed. (4) Pour out part of the culture medium after low-speed centrifugation to obtain concentrated bacterial solution.

2. The application according to claim 1, characterized in that, In step (1), the inoculum size of the fermentation medium is 5%; in step (2), the inoculum size of the LB medium is 5%.

3. The application according to claim 1, characterized in that, The fermentation temperature of the fermentation medium in step (1) is 37°C, the initial pH is 7, and the shaking speed is 180 r / min.

4. The application according to claim 1, characterized in that, The fermentation medium consists of MgSO4, (NH4)2SO4, chitin, potato starch, cassava starch, corn starch, soybean meal, and MnSO4.

5. The application according to claim 1, characterized in that, In step (3), the speed of low-speed centrifugation is 180 r / min.

6. The application according to claim 1, characterized in that, The OD600 of the Bacillus was 0.3.

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