Bacillus capable of producing CDA enzyme and application thereof in promoting agilawood formation

Abstract

The invention discloses bacillus capable of producing CDA enzyme. A culture method of the bacillus comprises the following steps: (1) carrying out CDA enzyme production culture on a strain obtained by screening and purifying by using a fermentation culture medium containing MgSO4, (NH4) 2SO4 and chitin for 168 hours; (2) activating bacillus capable of producing CDA enzyme by using an LB culture medium, selecting a single bacterial colony after the bacterial strain grows out the bacterial colony, inoculating the single bacterial colony into a new LB culture medium, and culturing for 2-3 days to obtain a seed solution; (3) transferring the obtained seed solution into a new fermentation culture medium for enlarged culture, and centrifuging the bacterial solution at a low speed after culturing for 3 days; and (4) pouring out part of the culture medium from the bacterial liquid subjected to low-speed centrifugation to obtain concentrated bacterial liquid. The bacterial liquid can be used for promoting agilawood formation of agilawood. The bacillus GXU-B2 is purified, fermented and activated to obtain the CDA enzyme-producing bacillus, the CDA enzyme-producing bacillus is injected into the xylem of the agilawood through a liquid intravenous drip method, the CDA enzyme and the bacillus can stimulate a defense system of agilawood plants to promote agilawood formation of the agilawood, and the agilawood is deeply and uniformly formed into the xylem.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a CDA enzyme-producing bacillus and an application thereof in promoting the formation of agarwood. Background Art

[0002] In recent years, naturally produced agarwood cannot meet the market demand for agarwood, and the cultivation and induction technology of agarwood has become increasingly advanced. The technology of artificially promoting agarwood formation has made great progress, including mechanical damage, biological inoculants, chemical inducers, a combination of biological and chemical inducers, and in vitro plant tissue culture to produce agarwood. Compared with traditional methods, biological inoculants can more effectively induce agarwood to form.

[0003] Common biological inoculation methods include solid inoculation and liquid inoculation. Solid inoculation refers to directly connecting the hyphae into the holes drilled by humans. Although the quality of the agarwood is good, the yield is low. There are two main methods of liquid inoculation. The first is the liquid injection method, which refers to the use of a syringe to directly inoculate the bacterial solution into the tree after the hole is punched. This method uses less bacterial solution, but the quality of the agarwood needs to be improved; the second is the liquid static drip method, which refers to the use of an infusion device to slowly inject the bacterial solution into the duct of the Agarwood plant. The bacterial solution flows through the duct and is distributed to the tree, promoting the formation of agarwood on the whole plant, so it is also called the "whole body agarwood method". The present invention studies the use of liquid static drip method to inject bacterial solution to enrich the strains of agarwood. Summary of the invention

[0004] The main purpose of the present invention is to provide a Bacillus sp. producing CDA enzyme and its application in promoting the formation of agarwood.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A CDA enzyme-producing bacillus, wherein the culturing method of the bacillus comprises the following steps:

[0007] (1) The screened and purified strains were cultured for CDA enzyme production for 168 h using a fermentation medium containing MgSO4, (NH4)2SO4, and chitin;

[0008] (2) Activating the CDA enzyme-producing Bacillus using LB medium, picking a single colony after the strain grows out and inoculating it into LB medium for 2-3 days to obtain a seed solution;

[0009] (3) transferring the seed solution obtained in step (2) into a new fermentation medium containing MgSO4, (NH4)2SO4, and chitin for expansion culture, and centrifuging the bacterial solution at a low speed after culturing for 3 days;

[0010] (4) After low-speed centrifugation, part of the culture medium is poured out to obtain a concentrated bacterial solution.

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

[0012] Furthermore, in step (1) and step (3), the inoculation amount of the bacterial strain in the fermentation medium and the PDB medium is 5%. Too large an inoculation amount will cause insufficient dissolved oxygen and affect product synthesis; too small an inoculation amount will prolong the culture time and reduce the productivity of the fermenter.

[0013] Furthermore, the fermentation temperature of the fermentation medium in step (1) is 37° C., the initial pH value is 7, and the shaking speed is 180 r / min, wherein the fermentation temperature and the initial pH value are different from the general fermentation conditions of the fermentation medium, mainly because the enzyme production of Bacillus is optimal under this fermentation condition.

[0014] Furthermore, the components and concentrations of the fermentation medium are: MgSO4 concentration of 0.20%, (NH4)2SO4 concentration of 2.15%, chitosan 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, the rotation speed of the low-speed centrifugation in step (3) is 180 r / min. The strains in the bacterial solution can be precipitated by low-speed centrifugation, the supernatant can be removed, and the concentrated bacteria can be retained.

[0016] The bacterial liquid obtained during the culture process of the bacillus of the present invention is used 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 effect of agarwood formation.

[0018] Furthermore, the agarwood is white agarwood.

[0019] The advantages of the present invention are as follows: the Bacillus GXU-B2 of the present invention is purified, fermented and activated to obtain the CDA enzyme-producing Bacillus, which is injected into the xylem of agarwood through a liquid static drip method; the CDA enzyme and the Bacillus can stimulate the defense system of the Aquilaria plant to promote the formation of agarwood, and the agarwood is formed evenly in the xylem. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is an optical microscopic image of Bacillus sporogenes GXU-B2 producing CDA enzyme;

[0021] Figure 2is the Gram staining image of Bacillus sporogenes GXU-B2 producing CDA enzyme;

[0022] Figure 3 is a scanning electron micrograph of Bacillus sporogenes GXU-B2 producing CDA enzyme;

[0023] Figure 4 This is the phylogenetic tree of CDA enzyme-producing Bacillus sporogenes GXU-B2 based on 16 rRNA genes;

[0024] Figure 5 is the enzyme activity curve of Bacillus sporogenes GXU-B2 producing CDA enzyme;

[0025] Figure 6 This is a comparison chart of the carbon source utilization rate of the CDA enzyme-producing Bacillus GXU-B2;

[0026] Figure 7 This is a comparison chart of nitrogen source utilization by Bacillus sporogenes GXU-B2 producing CDA enzyme;

[0027] Figure 8 This is a comparison chart of the utilization rate of inorganic salts by Bacillus sporogenes GXU-B2 producing CDA enzyme;

[0028] Fig. 9 are the contour plots and response surface plots of Bacillus GXU-B2 producing CDA enzyme;

[0029] Fig.10 This is a picture of the site of intravenous injection inoculation of Bacillus GXU-B2 producing CDA enzyme;

[0030] Fig.11 This is a picture of the fragrance formation of Aucklandia odorifera four months after inoculation with Bacillus GXU-B2 that produces CDA enzyme. DETAILED DESCRIPTION

[0031] In order to make the objectives, technical solutions and beneficial effects of the present invention more clear, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings to facilitate understanding by technicians.

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

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

[0034] Fermentation medium: MgSO4 concentration 0.20%, (NH4)2SO4 concentration 2.15%, chitosan 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%, and the remaining ingredients are water.

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

[0036] A CDA enzyme-producing bacillus, characterized in that the culturing method of the bacillus comprises the following steps:

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

[0038] (2) Activating the CDA enzyme-producing Bacillus using LB medium, picking a single colony after the strain grows out and inoculating it into a new LB medium for 2-3 days to obtain a seed solution;

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

[0040] (4) After low-speed centrifugation, part of the culture medium is poured out to obtain a concentrated bacterial solution.

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

[0042] The screened and purified strains were observed by Gram staining and scanning electron microscopy on LB medium to analyze their physiological and biochemical characteristics, such as Figure 1-3 As shown. Figure 1-3 It can be seen that the colony morphology of Bacillus GXU-B2 is: the single colony is irregular in shape, with a smooth, raised, moist, translucent, white, and shiny surface. A large amount of transparent mucus is secreted around the colony, which feels sticky. After the culture time is extended, the surface of the colony becomes dry, dull, wrinkled, not raised, and difficult to pick up.

[0043] The whole genome DNA of the strains was extracted from the screened and purified strains, and PCR amplification was performed using the universal primers 1492R (5'-GGTTACCTTGTTACGACTT-3') and 27F (5'-AGAGTTTGATCCTGGCTCAG-3') of the 16SrRNA gene, and the amplified products were sequenced. The sequencing data were blasted online in NCBI to analyze the homology between sequences, download the sequence information with close genetic relationship, import the sequence file into MEGA X software, and use the maximum likelihood method (ML) to construct a phylogenetic tree, such as Figure 4 shown.

[0044] The enzyme production curve of the screened and purified strains was measured, and the strains in the exponential growth phase were transferred to the fermentation medium (100 mL of liquid in a 250 mL triangular bottle). Three bottles were sampled every 12 hours, and finally the CDA activity was measured using the p-nitroacetanilide method. The results are as follows: Figure 5 shown.

[0045] The medium optimization experiment was conducted on the CDA enzyme produced by Bacillus GXU-B2, and the effects of different carbon sources, nitrogen sources and inorganic salts on the CDA enzyme produced by Bacillus GXU-B2 were studied. In the experiment, the carbon sources used were glucose, sucrose, maltose, powdered chitin, corn starch, lactose, potato starch, and cassava starch; the nitrogen sources used were peptone, NH4Cl, (NH4)2SO4, KNO3, NaNO3, urea, soybean meal, and beef extract; the inorganic salts used were MgSO4, CaCl2, MnSO4, ZnSO4, NaCl, FeSO4, KCl, and CuSO4. The experimental process is to inoculate the bacteria in the exponential growth phase (inoculation volume of 5%) into 100mL / 250mL fermentation medium, and each group has 3 parallel groups. After cultivation, the CDA enzyme activity is measured using the p-nitroacetanilide method, and the experiment is repeated 3 times. Through the experiment, it can be known that the utilization rate of carbon sources by Bacillus GXU-B2 from high to low is: potato starch> corn starch> chitin> cassava starch> lactose> maltose> glucose= sucrose; the utilization rate of nitrogen sources from high to low is: soybean meal> (NH4)2SO4= beef extract> urea> NH4Cl> KNO3> NaNO3= peptone; the utilization rate of inorganic salts from high to low is: CaCl2> NaCl> FeSO4> MnSO4> CuSO4> MgSO4> ZnSO4> KCl, such as Figure 6-8 shown.

[0046] According to the utilization rate of carbon source, nitrogen source and inorganic salt by Bacillus GXU-B2, the Plackett-Burman experimental design of 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, corn starch), two nitrogen sources (soybean meal, (NH4)2SO4), and two inorganic salts (MnSO4, MgSO4) with the best effect in the single factor optimization experiment were selected as optimization factors, and the CDA activity was used 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 of Bacillus GXU-B2

[0048]

[0049] Table 2 Analysis of variance of Plackett-Burman design for Bacillus GXU-B2

[0050]

[0051] The steepest climbing experiment was carried out based on the effect size and direction of the significant influencing factors in the Plackett-Burman test results. The steepest climbing experiment used the gradient of the experimental value as the climbing direction, which could quickly and economically approach the optimal enzyme production area. This experiment set up 3 groups of parallels and repeated the experiment 3 times. The experimental results showed that Bacillus GXU-B2 obtained the maximum enzyme activity at a concentration of 1.70% chitin, 1.80% (NH4)2SO4, and 0.20% MgSO4, as shown in Table 3.

[0052] Table 3 Design and results of the steepest climbing experiment of Bacillus GXU-B2

[0053]

[0054] According to the Plackett-Burman design and the results of the steepest climbing experiment, three factors were selected, and three levels were selected for each factor (MgSO4, (NH4)2SO4), chitin). The fermentation medium was further optimized using the Box-Behnken design of the Design-Expert software, and the model equation was obtained by fitting the experimental data. The 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 2.805U / 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, model correlation coefficient R2=0.9832, correction coefficient R2Agj=0.9531, CV=6.07%, indicating that the regression equation can better simulate and predict the actual results of the fermentation experiment.

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

[0056] Table 4 Bacillus GXU-B2 Box-Behnken test design and results

[0057]

[0058]

[0059] Table 5 Analysis of variance of Box-Behnken regression design for Bacillus GXU-B2

[0060]

[0061] Observe the contour map and response surface of Bacillus GXU-B2 Fig. 9(AC in the figure is the contour map of the Box-Behnken experimental design, and DF is the response surface map of the Box-Behnken experimental design) It was found that the interaction between (NH4)2SO4 and chitin was significant (p < 0.05), and the interaction between MgSO4 and chitin was not significant (p > 0.05). Therefore, the optimal fermentation conditions for Bacillus GXU-B2 are when the concentration of MgSO4 is 0.20%, the concentration of (NH4)2SO4 is 2.15%, and the concentration of chitosan is 1.55%. Therefore, the optimal fermentation medium composition and concentration were determined: MgSO4 concentration of 0.20%, (NH4)2SO4 concentration of 2.15%, chitosan 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%.

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

[0063] (1) 10 Aquilaria sinensis trees aged 7 to 10 years were selected, with no obvious damage to the trunk surface;

[0064] (2) Use an electric drill to drill holes at different heights on the trunk of the Aquilaria sinensis tree, with a hole diameter of 3 mm and a hole depth of 5 cm;

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

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

[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, and the infusion tube is provided with a flow switch and an inoculation head. After the inoculation device is hung, the inoculation head can be inserted into the hole punched on the white agarwood. Fig.10 shown.

[0068] Four months after inoculation, the bark of the inoculated Aquilaria sinensis tree was scratched until discolored wood was seen. The tree body after the bark was scratched was photographed for analysis, and the discolored part was dug out until the tree body itself contained no or a small amount of discolored tissue. The obtained tissue was photographed and the range of the agarwood was measured, such as Fig.11 The results showed that the Aquilaria sinensis tissue inoculated with Bacillus GXU-B2 had a large area of ​​fragrance, the color change range penetrated deep into the wood, the color was darker, and the color change part was evenly distributed from the punching point to the color change edge.

[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 may be made to the above embodiments without departing from the spirit of the present invention and the scope of the appended claims.

Claims

1. A Bacillus sp. producing CDA enzyme, characterized in that: The method for culturing the bacillus comprises the following steps: (1) The screened and purified strains were cultured for 168 h to produce CDA enzyme using a fermentation medium containing MgSO4, (NH4)2SO4, and chitin; (2) Activating the CDA enzyme-producing Bacillus using LB medium, picking a single colony after the strain grows out and inoculating it into a new LB medium for 2-3 days to obtain a seed solution; (3) transferring the seed solution obtained in step (2) into a new fermentation medium containing MgSO4, (NH4)2SO4, and chitin for expansion culture, and centrifuging the bacterial solution at a low speed after culturing for 3 days; (4) After low-speed centrifugation, part of the culture medium is poured out to obtain a concentrated bacterial solution.

2. The CDA enzyme-producing Bacillus according to claim 1, characterized in that: In the steps (1) and (2), the bacterial inoculation amount of the fermentation medium and the LB medium is 5%.

3. The CDA enzyme-producing Bacillus 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 CDA enzyme-producing Bacillus according to claim 1, characterized in that: The components and concentrations of the fermentation medium are: MgSO4 concentration of 0.20%, (NH4)2SO4 concentration of 2.15%, chitosan 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%.

5. The CDA enzyme-producing Bacillus according to claim 1, characterized in that: The rotation speed of the low-speed centrifugation in step (3) is 180 r / min.

6. Use of the bacterial liquid obtained according to claim 1 in promoting the formation of agarwood.

7. The use of the bacterial liquid in promoting the formation of agarwood according to claim 6, characterized in that: The concentration of the bacterial solution is OD600=0.

3.

8. The use of the bacterial liquid in promoting the formation of agarwood according to claim 6, characterized in that: The agarwood is white agarwood.

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