Method for increasing fermentation yield of phytophthora nicotianae JH-11-1 and application

Abstract

The invention relates to a method for improving the fermentation yield of a strain JH-11-1 and application of the method. Through systematic single-factor and multi-factor optimization experiments, an optimal fermentation process parameter combination including an optimal carbon and nitrogen source, an optimal fermentation temperature, optimal culture time, an optimal pH value, an optimal rotating speed and the like is determined; the optimized combination can obviously improve the mycelium biomass, so that the thallus concentration in the fermentation liquor reaches a relatively high level, a reliable scheme is provided for solving the yield bottleneck in industrial production, the components of the culture medium are cheap and easy to obtain, the fermentation period is reasonably controlled, the production cost is effectively reduced, and the method is suitable for industrial production. The invention successfully develops a fermentation process which is efficient, low in cost and suitable for industrialization of the JH-11-1 strain, and lays a solid foundation for large-scale industrial fermentation and field popularization and application.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a method and application for increasing the fermentation yield of Phytophthora tobaccoii JH-11-1. Background Technology

[0002] Fermentation refers to the process by which microorganisms are cultivated on a large scale using aerobic or anaerobic respiration, accumulating their secreted metabolites. In industry, to improve the level of the target product during fermentation, comprehensive optimization of the process is typically achieved through several aspects, including optimizing the fermentation medium, fermentation environmental conditions, and cultivation methods.

[0003] Phytophthora nicotianae strain JH-11-1 is a weakly pathogenic Phytophthora strain screened in our laboratory. Previous indoor pot and field trials have shown that the shake-flask fermented suspension of strain JH-11-1 can infect tobacco plants and effectively induce disease resistance, exhibiting good control of tobacco black shank disease. It also has a certain growth-promoting effect, effectively promoting tobacco growth (see patent CN119709422A). Conventional field application involves dilution with an aqueous solution. Therefore, obtaining a large mycelial yield through shake-fermentation is currently an important issue in fermentation research.

[0004] The low potency and high production cost of microbial agents prevent current technologies from meeting the needs of industrial production and large-scale field application, hindering their transformation from laboratory research to industrial production and large-scale field application. Therefore, there is an urgent need in this field to develop a fermentation optimization method tailored to the functional characteristics of strain JH-11-1, capable of simultaneously and efficiently improving its biomass and functional potency, in order to overcome the industrialization bottleneck of its transition from "laboratory results" to "field products". Summary of the Invention

[0005] This invention addresses the gaps in existing technologies by providing a method and application for increasing the fermentation yield of Phytophthora tobaccoensis JH-11-1. By optimizing the fermentation conditions of the strain, the optimal carbon and nitrogen source, optimal fermentation temperature, optimal fermentation time, optimal pH value, optimal rotation speed, and optimal liquid volume are determined to achieve the optimal fermentation combination, which can effectively reduce economic costs.

[0006] Specifically, the present invention provides a method for increasing the fermentation yield of Phytophthora tobaccoensis JH-11-1, the method comprising: culturing Phytophthora tobaccoensis JH-11-1 at a culture temperature of 28~32℃ and a shaking speed of 90~120rpm / min for 7~14 days.

[0007] Furthermore, the method further includes: inoculating Phytophthora tobaccoensis JH-11-1 into a culture medium with a pH of 6-7 and a volume of 120-200 mL.

[0008] Furthermore, the fermentation medium for Phytophthora tobaccoii JH-11-1 is oat medium.

[0009] Furthermore, the purified Phytophthora tobaccoii JH-11-1, after 7 days of culture, was inoculated into oat medium at pH 6 at an inoculation amount of 14% (V / V) and cultured for 7 days at a temperature of 28℃ and a shaking speed of 120 r / min.

[0010] Furthermore, the optimal process parameters are: cultured for 15 days at a culture temperature of 32℃ and a shaking speed of 90 r / min.

[0011] Furthermore, the maximum mycelial dry weight of Phytophthora tobaccois JH-11-1 obtained by the above-mentioned optimal process can reach 0.469 g / 100 mL.

[0012] Furthermore, the *Phytophthora indicum* JH-11-1 was deposited at the China Center for Type Culture Collection on October 11, 2024, with accession number CCTCC NO: M 20242162.

[0013] This invention also provides the application of the above method in increasing the fermentation yield of Phytophthora tobaccoii JH-11-1.

[0014] Beneficial Effects: Compared with the prior art, the fermentation optimization method for Phytophthora tobaccois strain JH-11-1 provided by this invention has the following significant advantages and beneficial effects:

[0015] 1. Significantly improved fermentation efficiency and biomass: Through systematic single-factor and multi-factor optimization experiments, this invention has established the optimal combination of fermentation process parameters, including the optimal carbon and nitrogen source, optimal fermentation temperature, optimal culture time, optimal pH value, optimal rotation speed, and optimal liquid volume. This optimized combination can significantly improve mycelial biomass and achieve a high level of cell concentration in the fermentation broth, providing a reliable solution to the production bottleneck in industrial production.

[0016] 2. Good process reproducibility and easy to scale up production: The fermentation process parameters provided by this invention are clear and the operation process is standardized. It has excellent reproducibility and stability, and it is easy to scale up from laboratory shake flask scale to fermenter scale, breaking the technical barrier from "laboratory results" to "industrial products".

[0017] 3. Production costs are effectively reduced and economic benefits are outstanding: The fermentation culture medium components determined by this invention are inexpensive and readily available, and the fermentation cycle is reasonably controlled. While achieving high biomass, it effectively reduces the raw material and operating costs per unit yield, laying an economic foundation for large-scale industrial fermentation and field application.

[0018] 4. Broad application prospects: This invention has successfully developed a high-efficiency, low-cost fermentation process suitable for the industrialization of strain JH-11-1, which not only greatly promotes the productization process of this biocontrol agent, but also provides a useful reference for the development of fermentation processes for other similar microbial preparations. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This illustrates the effect of different fermentation times on mycelial biomass in embodiments of the present invention.

[0021] Figure 2 This illustrates the effect of different fermentation broth pH values ​​on mycelial biomass in embodiments of the present invention.

[0022] Figure 3 This illustrates the effect of different fermentation temperatures on mycelial biomass in embodiments of the present invention.

[0023] Figure 4 This illustrates the effect of different rotation speeds on mycelial biomass in embodiments of the present invention.

[0024] Figure 5 This illustrates the effect of different liquid volumes on mycelial biomass in embodiments of the present invention. Detailed Implementation

[0025] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art. Unless specifically stated, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless specifically stated, the reagents and materials used in the following embodiments are commercially available.

[0026] Example 1: Optimization of Single-Factor Fermentation Conditions

[0027] The *Phytophthora indicum* strain JH-11-1 used in this invention was obtained through previous screening in our laboratory and was deposited at the China Center for Type Culture Collection on October 11, 2024, with accession number CCTCC NO: M 20242162 (see patent CN119709422A).

[0028] This embodiment uses a single-factor method to optimize the fermentation conditions of this bacterium. Fermentation time, pH value of fermentation broth, temperature, shaking speed, and liquid volume are selected as key factors. Each factor is tested with gradient levels. All experimental groups have three replicates. Mycelial biomass is used as the evaluation index, as detailed below:

[0029] (1) Effect of different culture times on mycelial biomass: After 7 days of purification and culture, strain JH-11-1 was inoculated into oat liquid medium with a volume of 200 mL and an inoculum size (V / V) of 14%. The culture temperature was 28℃, the shaking speed was 160 r / min, and the culture time ranged from 3 to 28 days, with a gradient of 7 days. The dry weight of the mycelium was weighed, and the effect of culture time on mycelial biomass was compared. The results are as follows: Figure 1 As shown in the figure. Through the design of this experiment, the results showed that, within the cultivation period of 3 to 28 days, the mycelial biomass yield of JH-11-1 was the highest at 7 days, which was 0.2520 ± 0.00318 g / 100 mL, and was suitable for the growth of strain JH-11-1.

[0030] (2) Effect of different fermentation broth pH on mycelial biomass: After 7 days of purification and culture, strain JH-11-1 was inoculated into oat liquid medium at a volume of 200 mL, with an inoculum size (V / V) of 14%. The culture temperature was 28℃, the shaking speed was 160 r / min, and the culture time was 7 days. The pH of the fermentation broth ranged from 5 to 9, with each 1 representing a gradient. The dry weight of the mycelium was measured, and the effect of fermentation broth pH on mycelial biomass was compared. The results are as follows: Figure 2 As shown.

[0031] Based on the experimental design and analysis, the results show that, among different fermentation broth pH values, the highest mycelial biomass yield of JH-11-1 was 0.2182±0.0707g / 100mL at pH 6, which is suitable for the growth of strain JH-11-1.

[0032] (3) Effect of different temperatures on mycelial biomass: After 7 days of purification and culture, strain JH-11-1 was inoculated into oat liquid medium (200 mL, inoculum size (V / V) 14%), cultured for 7 days at a shaking speed of 160 r / min, and at temperatures ranging from 22 to 34 °C in increments of 3 °C. Mycelial dry weight was measured, and the effect of culture temperature on mycelial biomass was compared. Results are as follows: Figure 3 As shown.

[0033] Based on the experimental design and analysis, the results show that, among different temperatures, the highest mycelial biomass yield of JH-11-1 is 0.1880±0.0251 g / 100 mL at 28℃, which is suitable for the growth of strain JH-11-1.

[0034] (4) Effect of different rotation speeds on mycelial biomass: After 7 days of purification and culture, strain JH-11-1 was inoculated into oat liquid medium (200 mL, inoculum size (V / V) 14%), cultured for 7 days at 28℃. The shaking speed ranged from 120 to 200 rpm, with increments of 20 rpm. Mycelial dry weight was measured, and the effect of rotation speed on mycelial biomass was compared. The results are as follows: Figure 4 As shown.

[0035] Based on the experimental design and analysis, the results show that, among different rotation speeds, the highest mycelial biomass yield of JH-11-1 is 0.3213±0.035 g / 100mL when the rotation speed is 120rpm / min, which is suitable for the growth of strain JH-11-1.

[0036] (5) Effect of different liquid volumes on mycelial biomass: After 7 days of purification and culture, strain JH-11-1 was inoculated into oat liquid medium at an inoculation rate (V / V) of 14%. The culture time was 7 days, the culture temperature was 28℃, the shaking speed was 160 r / min, and the liquid volume ranged from 120 to 200 mL in increments of 20 mL. The dry weight of the mycelium was measured, and the effect of liquid volume on mycelial biomass was compared. The results are as follows: Figure 5 As shown.

[0037] Based on the experimental design and analysis, the results show that, with a liquid volume of 200 mL, the mycelial biomass yield of JH-11-1 is the highest, at 0.1943±0.0287 g / 100 mL, which is suitable for the growth of strain JH-11-1.

[0038] Example 2: Optimization of fermentation process for Phytophthora tobacco strain JH-11-1

[0039] (1) Plackett-Burman (PB) design: After determining the temperature, rotation speed, pH, liquid volume and time in the fermentation medium, the parameters of each condition of the fermentation medium are mainly evaluated using the Plackett-Burman design of the Design Expert software.

[0040] Table 1. Results of the Plackeet-Burman experiment

[0041]

[0042] Table 2 Regression analysis results of the Plackeet-Burman experimental design

[0043]

[0044] The Plackett-Burman experiment was used to screen out the main influencing factors of temperature, time, rotation speed, liquid volume, and pH. The effects of culture medium fermentation conditions on the fermentation yield of JH-11-1 were, in descending order: temperature > culture time > rotation speed > pH > liquid volume. Treatment 7 (temperature 31℃, time 14 days, rotation speed 100 rpm, liquid volume 160 mL, pH 9) showed the highest mycelial dry weight. Regression analysis revealed that the main factors affecting the mycelial dry weight of the JH-11-1 fermentation broth were temperature, shaking time, and rotation speed.

[0045] (2) Steepest climb test: In order to further analyze the effects of three parameters, namely temperature, shaking time and rotation speed, on the dry weight of mycelium in JH-11-1 fermentation broth, the steepest climb test was conducted using response surface methodology for the three factors.

[0046] Table 3. Experimental Design and Results for the Steepest Climb

[0047]

[0048] As shown in Table 3 above, the fermentation broth of strain JH-11-1 had the highest mycelial dry weight in treatment group 2 (i.e., temperature 32℃, rotation speed 90 rpm / min, time 15 d). This value was used as the center point for response surface methodology (RSM). Further RSM was conducted using a Box-Behenken design with three levels for temperature, rotation speed, and time. Seventeen treatments were set with 32℃, 90 rpm, and 15 d as baselines to explore the effects of different temperatures, rotation speeds, and times on mycelial dry weight.

[0049] Table 4 Box-Behnken Design Coding Level Table

[0050]

[0051] Table 5. Box-Behnken central combination experimental design and results

[0052]

[0053] Table 6. Regression Analysis Results of Box-Behnken Experimental Design

[0054]

[0055] The results showed that, using the mycelial dry weight of strain JH-11-1 as the response value, the regression equation could be obtained based on the results of the central composite experiment and the variance test:

[0056] Y=0.443+0.0005A-0.000875B-0.000875C-0.00175AB-0.00125AC-0.047BC-0.21775A 2 -0.0265B 2 -0.0235C 2

[0057] Y represents the response value, A represents temperature, B represents time, and C represents rotational speed. The correlation coefficient R0 is also relevant. 2 =0.99, indicating that this equation can effectively simulate the results of the central composite experiment.

[0058] It can be confirmed that the optimal process parameters for culturing strain JH-11-1 are: culture temperature 32℃, rotation speed 90rpm / min, culture time 15d, and the maximum mycelial dry weight obtained by this process can reach 0.469g / 100mL.

[0059] In summary, this invention screened different carbon and nitrogen sources to identify the most suitable carbon and nitrogen sources for the growth of strain JH-11-1, finding sucrose to be the optimal carbon source and peptone to be the optimal nitrogen source. Further, through single-factor experiments on five fermentation conditions—culture temperature, culture time, shaking speed, pH of the fermentation broth, and volume—the optimal culture medium conditions for strain JH-11-1 growth were determined to be: culture temperature 28℃, culture time 7 days, shaking speed 120 rpm / min, pH 6, and volume 200 mL. Further Plackett-Burman experiments were conducted to identify the main influencing factors of temperature, time, shaking speed, volume, and pH. The results showed that the influence of culture medium fermentation conditions on the fermentation yield of JH-11-1 was in the order of temperature > culture time > shaking speed > pH > volume. The main factors affecting the mycelial dry weight of the JH-11-1 fermentation broth were temperature, culture time, and shaking speed. To further analyze the effects of three parameters—temperature, shaking time, and rotation speed—on the mycelial dry weight of JH-11-1 fermentation broth, a steepest climb experiment was conducted using response surface methodology (RSM) to assess these three factors. The results showed that treatment 2 (temperature 32℃, rotation speed 90 rpm / min, and time 15 days) produced the highest mycelial dry weight of JH-11-1 fermentation broth, and this value was used as the center point for the RSM analysis. Further RSM analysis confirmed that the optimal process parameters for effectively increasing the yield of JH-11-1 strain were: a culture temperature of 32℃, a rotation speed of 90 rpm / min, and a culture time of 15 days. This optimized combination significantly increases mycelial biomass, and the culture medium components are inexpensive and readily available. The reasonable control of the fermentation cycle effectively reduces production costs. Therefore, this invention successfully develops a high-efficiency, low-cost fermentation process suitable for the industrialization of JH-11-1 strain, laying a solid foundation for large-scale industrial fermentation and field application.

[0060] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for increasing the fermentation yield of Phytophthora tobaccoii JH-11-1, characterized in that, The method includes: culturing Phytophthora tobaccoensis JH-11-1 at a culture temperature of 28~32℃ and a shaking speed of 90~120rpm / min for 7~14 days.

2. The method according to claim 1, characterized in that, The method further includes inoculating Phytophthora tobaccoensis JH-11-1 into a culture medium with a pH of 6-7 and a volume of 120-200 mL.

3. The method according to claim 1, characterized in that, The fermentation medium for Phytophthora tobaccoii JH-11-1 is oat medium.

4. The method according to claim 1, characterized in that, After 7 days of purification and culture, *Phytophthora indicum* JH-11-1 was inoculated into oat medium at pH 6 with a volume of 200 mL and an inoculation amount of 14% (V / V). The medium was cultured at 28℃ and a shaking speed of 120 r / min for 7 days.

5. The method according to claim 4, characterized in that, The optimal process parameters are: culture at a temperature of 32℃ and a shaking speed of 90 r / min for 15 days.

6. The method according to claim 1, characterized in that, The *Phytophthora indicum* JH-11-1 was deposited at the China Center for Type Culture Collection on October 11, 2024, with accession number CCTCC NO: M 20242162.

7. The application of the method as described in any one of claims 1-6 in increasing the fermentation yield of Phytophthora tobaccois JH-11-1.

Images