Rhizopus arrhizus JHK31, fungicide and application of rhizopus arrhizus JHK31 in sophora flavescens fermentation
By using the fermentation process of Rhizobium sausage JHK31, the oxidized maltine in Sophora is converted into Maltine, which solves the problem of high production cost of Maltine, and achieves efficient and economical maltine production, which promotes its widespread application in biopesticides.
Patent Information
- Application Number
- CN202510301048.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-14
- Publication Date
- 2025-06-10
AI Technical Summary
In the prior art, the production cost of matrine is relatively high, and the lack of efficient microorganisms to ferment malpine is limited, which limits its promotion and application.
A strain of Rhizobacterium seldomegum JHK31 and its fungal agent are provided. The oxidized maltine in Sophora is converted into matrine through fermentation, which improves the yield of maltine and reduces production costs.
Through the fermentation process of Rhizobium Leukorma JHK31, the yield of maltine is increased by 615%, reducing production costs, solving the problem of high production costs of maltine, and promoting its widespread application in biopesticides.
Smart Images
Figure CN120118749A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microbial fermentation, and particularly relates to a Rhizopus arrhizus JHK31, a bacterial agent thereof, and their application in Sophora flavescens fermentation. Background Art
[0002] Matrine, as a natural, low-toxic, low-residue, and environmentally friendly plant-derived biopesticide, has a broad-spectrum insecticidal effect on various pests such as diamondback moths, fall armyworms, and nematodes, and has very broad application prospects. Matrine is mainly derived from Sophora flavescens. In the prior art, it can be obtained by ethanol extraction. However, the content of matrine in Sophora flavescens is only about 0.1%. A large amount of Sophora flavescens raw materials are consumed in the extraction process, and there are also losses in the extraction process, resulting in a relatively high cost of extracting matrine from Sophora flavescens, which limits the further popularization and application of matrine. In the prior art, the yield of some alkaloids can usually be increased through microbial fermentation. However, there is a lack of corresponding microorganisms in the prior art to efficiently ferment Sophora flavescens. Therefore, the production cost of matrine cannot be reduced to a greater extent through microbial fermentation. Summary of the Invention
[0003] Aiming at the deficiencies in the prior art, the present invention provides a Rhizopus arrhizus JHK31, a bacterial agent thereof, and their application in Sophora flavescens fermentation, which solves the problem of relatively high production cost of matrine in the prior art.
[0004] According to an embodiment of the present invention, a Rhizopus arrhizus JHK31 is provided. Rhizopus arrhizus JHK31 has been deposited at the China Center for Type Culture Collection on May 10, 2023, with the deposit number CCTCC NO: M2023722. The provided Rhizopus arrhizus JHK31 can convert oxymatrine in Sophora flavescens into matrine through fermentation, thereby increasing the yield of matrine and further reducing the production cost of matrine.
[0005] A bacterial agent containing the above Rhizopus arrhizus JHK31 is also provided.
[0006] The above Rhizopus arrhizus JHK31 and the bacterial agent containing Rhizopus arrhizus JHK31 can both be used for fermenting Sophora flavescens to increase the content of matrine.
[0007] Compared with the prior art, the present invention has the following beneficial effects:
[0008] A strain of Rhizopus arrhizus JHK31 and a bacterial agent containing Rhizopus arrhizus JHK31 are provided. They can both convert oxymatrine into matrine through fermentation, thereby increasing the yield of matrine, reducing the production cost of matrine, solving the problem of the relatively high production cost of matrine in the prior art, indirectly reducing the production cost of matrine biological pesticides, and enabling the application and promotion of matrine in matrine fermentation to be carried out more widely. BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Figure 1 It is the colony morphology of the strain JHK31 in the embodiment of the present invention;
[0010] Figure 2 It is the content of matrine alkaloids after fermentation of the Rhizopus arrhizus JHK31 strain in the embodiment of the present invention;
[0011] Figure 3 It is the chromatogram in the embodiment of the present invention;
[0012] Figure 4 It is the microscopic examination image (10×) of the Rhizopus arrhizus JHK31 strain in the embodiment of the present invention;
[0013] Figure 5 It is the result of the single-factor experiment of matrine fermentation in the embodiment of the present invention;
[0014] In the above-mentioned drawings: DETAILED DESCRIPTION OF THE EMBODIMENTS
[0015] The technical solutions in the present invention will be further described below in conjunction with the drawings and embodiments.
[0016] All kinds of experimental operations involved in the specific embodiments are conventional technologies in the art. For the parts not specifically annotated in this article, those of ordinary skill in the art can refer to various common reference books, scientific and technological literatures, or relevant specifications, manuals, etc. before the filing date of this invention application for implementation.
[0017] A strain of Rhizopus arrhizus JHK31 has been deposited at the China Center for Type Culture Collection (Wuhan University, Wuhan, Hubei, China) on May 10, 2023, with the deposit number CCTCC NO: M2023722. The preservation center completed the detection on May 17, 2023, and the result was survival. The Rhizopus arrhizus JHK31 was initially screened by the pressure selection method using matrine as the sole carbon source from the rotten dead wood, fallen leaves, or humus soil with perennial piled wood in the woods of Jiaxing University (formerly Jiaxing College), and the strains that can quickly ferment matrine were isolated and purified by plate. This strain of Rhizopus arrhizus JHK31 can also be made into a bacterial agent.
[0018] The Rhizopus arrhizus strain JHK31 and the microbial agent containing the same can both convert oxymatrine in Sophora flavescens (the content of oxymatrine in Sophora flavescens is much higher than that of matrine, and the content of oxymatrine is as high as more than 3%) into matrine, thereby improving the yield of matrine.
[0019] In a more specific scheme:
[0020] Weigh a certain amount of the sample and place it in 100 mL of sterile water. Shake it on a shaker at 200 r / min for 1 h, let it stand for 10 min, take 8 mL of the supernatant and inoculate it into a fermenter containing 8 g of Sophora flavescens powder. Mix well and incubate at a constant temperature of 28 °C for several days. Observe the growth status of the colonies in the fermenter every day, select the fermenter with the fastest colony growth rate and the best state, pick the bacterial plaque and purify and isolate it on a PDA solid medium. Repeatedly streak and purify the isolated single colonies to obtain pure strains. Through the above screening, 1 strain of dominant strain was obtained, numbered JHK31, and the colony morphology is as Figure 1 shown.
[0021] Furthermore, after activating the Rhizopus arrhizus strain JHK31, make it into a spore suspension, control the spore concentration at 0.5 - 0.6×10 8 cells / mL. Take 1 mL and inoculate it into a Sophora flavescens matrix (8 g of Sophora flavescens and 7 mL of sterile water). After mixing, ferment at a constant temperature of 28 °C for 7 d, and make 3 replicates. Use the Sophora flavescens matrix without inoculating the strain as a control. After the fermentation is completed, take 1 g of the fermentation product and put it into a three-necked flask, add 30 mL of deionized water, extract it at room temperature of 25 °C for 30 min, centrifuge the extract at 8000 r / min for 6 min, take the supernatant, and perform HPLC detection to determine the contents of matrine and oxymatrine. The results are as Figure 2 、 3 shown. It can be seen from the results that the content of the total alkaloids in Sophora flavescens after fermentation by the Rhizopus arrhizus strain JHK31 decreased by 8.62% compared with unfermented Sophora flavescens. It may be that there are other transformation pathways during the fermentation process. However, the content of matrine has increased greatly, and it has increased by 615% compared with the unfermented control group, indicating that microbial fermentation can efficiently promote the directional transformation of oxymatrine into matrine.
[0022] Furthermore, observe the colonies of the Rhizopus arrhizus strain JHK31 to obtain colony characteristics, and further perform staining microscopy by the cover slip method to obtain the morphological characteristics of the strain. The results are as Figure 4As shown. The strain grows rapidly and can cover the entire plate in 48 hours. The hyphae are white in the early stage, the colony is loose and cotton candy-like, and the stolons spread strongly to the plate cover, and black sporangia are formed in the later stage. Under a microscope, there are creeping hyphae and rhizoctonia. The hyphae are transparent and without septa. Sporangia grow at the rhizoctonia. The round sporangia are located at the top of the sporangia. They are brown when mature. The spores are round and dark brown. It is initially judged to be Rhizopus.
[0023] Furthermore, the purified 18S rDNA sequence of the Rhizopus arrhizus strain JHK31 was compared by BLAST to obtain similarity information of the strain, as shown in Table 1. The strain was identified as Rhizopus arrhizus.
[0024] Table 1 Blast results of Rhizopus arrhizus JHK31 strain
[0025]
[0026] Fermentation process of directed conversion of matrine by Rhizopus arrhizus JHK31:
[0027] Grind Sophora flavescens into powder and pass through a 40-mesh sieve. Inoculate Rhizopus arrhizus JHK31 into PDA medium (potato dextrose agar medium: 200 g potato, 20 g glucose, 18 g agar, 1000 ml distilled water), culture at 28°C for 2 days, pick spores and inoculate into sterile water, dilute with sterile water to a spore suspension of 0.5-0.6×108 / ml, and use it immediately.
[0028] Accurately weigh 8g of Sophora flavescens powder, and under the fixed conditions of 2% carbon source (glucose) and fermentation temperature of 28°C, investigate the effects of inoculation amount (2%, 4%, 6%, 8%, 10%), carbon source (0%, 2%, 4%, 6%, 8%), solid-liquid ratio (0.7, 0.8, 0.9, 1, 1.1), and fermentation time (3d, 5d, 7d, 9d, 11d) on the yield of matrine after fermentation. Three parallel experiments were conducted for each factor, and the average value was recorded. The results are shown in the figure. Figure 5 shown.
[0029] From the results of the above single-factor experiments, the optimal inoculation amount is 6%, the glucose content is 4%, the ratio of material to liquid is 1.1:1, and the fermentation days are 7 days. When the inoculation amount is too low, the biomass of the strain is low and the fermentation effect is poor, affecting the release and transformation of matrine. When the inoculation amount is too high, it may have an adverse effect on the bacteria in terms of dissolved oxygen, accumulation of metabolic wastes, and competition for nutrients, thus affecting the yield of matrine. When adding glucose as a carbon source, because an appropriate amount of carbon source can promote the growth of Rhizopus arrhizus JHK31, the yield of matrine is significantly increased. However, too high a carbon source may inhibit the degradation of matrine cellulose, thus reducing the yield of matrine. The ratio of material to liquid shows an upward trend directly between 0.8 and 1.1. In solid-state fermentation, too high a ratio of material to liquid will cause the matrine powder to agglomerate, the air permeability to become poor, and the growth of the strain to be uneven, thus reducing the yield of matrine. Within the range of 11 days of fermentation, the yield of matrine shows an upward trend with the extension of fermentation time. When the fermentation time is too long, with the increase of metabolites, changing the fermentation environment may lead to the transformation of matrine through other pathways, and the yield will instead decrease.
[0030] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.
Claims
1. A strain of Rhizopus arrhizus JHK31, characterized in that The Rhizopus arrhizus JHK31 (Rhizopus arrhizus JHK31) was deposited in the China Center for Type Culture Collection on May 10, 2023, with the deposit number being CCTCCNO: M2023722.
2. A bacterial agent, characterized in that Contains the Rhizopus arrhizus JHK31 as claimed in claim 1.
3. The application of Rhizopus arrhizus JHK31 as claimed in claim 1 in Sophora flavescens fermentation, characterized in that, Fermentation of Sophora flavescens to increase the content of matrine.
4. The application of Rhizopus arrhizus JHK31 as claimed in claim 3 in Sophora flavescens fermentation, characterized in that, Convert oxymatrine to matrine.
5. The use of the microbial agent as claimed in claim 2 in the fermentation of Sophora flavescens, characterized in that: Fermentation of Sophora flavescens to increase the content of matrine.
6. The use of the microbial agent as claimed in claim 5 in the fermentation of Sophora flavescens, characterized in that: Convert oxymatrine to matrine.