A strain of aerophilic Bacillus and its application in the preparation of sclareol

Abstract

The present application belongs to the field of microbial fermentation technology, and specifically relates to a strain of aerophilic Bacillus and its application in the preparation of sclareol. Bacillus aerophilus , deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on January 13, 2025, with the deposit number: CGMCC No.33380; the address of the deposit unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; fermenting sugarcane juice with it can obtain clarysol; after subsequent extraction, it can be used for industrial production.

Description

Technical Field

[0001] The present application belongs to the field of microbial fermentation technology, and specifically relates to an aerophilic Bacillus and its application in the preparation of sclareol. Background Art

[0002] Sclareol is a diterpenoid compound with the chemical formula C 20 H 36 O2 is an essential chemical substance for plant growth, development and general metabolism, and is a secondary metabolite of plants. The growth environment of sclareol is limited, and the planting area is relatively small, resulting in a limited amount of plants available for the extraction of sclareol; the process of extracting sclareol from sclareol is relatively complicated, which is not only time-consuming and labor-intensive, but also requires high equipment and technology; ecological factors such as climate change and environmental pollution may affect the growth and yield of sclareol, thereby affecting the supply of sclareol. Therefore, some manufacturers are unwilling to invest a lot of resources in its large-scale production.

[0003] Sclareol can be used in cosmetics, health products, flavors and fragrances, and pesticides. Sclareol also has great application value in medicine and has high economic value. Sclarea alcohol can alleviate TGF-β1-induced LX-2 hepatic stellate cell activation and CCl4-induced mouse liver fibrosis (Song Anning, Zhang Tiantian, Zheng Shanshan, et al. Sclarea alcohol inhibits hepatic stellate cell activation and liver fibrosis by regulating TGF-β / Smad signaling pathway [J / OL]. World Science and Technology-Modernization of Traditional Chinese Medicine, 1-9 [2025-01-04].); KULKARNI et al. also proved that sclarea alcohol exhibits the most effective antibacterial activity (KULKARNI RR, SHURPALI K, PURANIK VG, et al. Antimycobacterial labdanediterpenes from Leucas stelligera [J]. J Nat Prod, 2013, 76(10), 1836-1841.); and sclarea alcohol can also be used as a precursor to synthesize natural active substances that are difficult to obtain, such as Widendiol A (inhibits the activity of acetylcholinesterase), (+) -Coranarian A (anti-angiogenic activity), (+) -Pupehenone (anti-tuberculosis activity), etc. (Zhu Bofeng, Li Xiaoqin, Wu Jingshuai, et al. Primary and secondary hydroxylation modification of clarysol by Cunninghamella elegans AS3.2028 [J]. Journal of Shenyang Pharmaceutical University, 2018, 35(04): 325-328.). Summary of the invention

[0004] In view of the problem of low sclareol production at present, the present invention provides an aerophilic Bacillus, which can be used to ferment sugarcane juice to obtain sclareol; the fermentation process is simple and easy to industrialize.

[0005] The technical solution of this application is as follows:

[0006] In a first aspect of the present invention, a strain of aerophilic Bacillus is protected, wherein the aerophilic Bacillus is Bacillus aerophilus , deposited in the General Microbiology Center of China Microorganism Culture Collection on January 13, 2025, with the deposit number: CGMCC No. 33380; the address of the deposit unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] Another aspect of the present invention is to protect the use of the above-mentioned aerophilic Bacillus in the preparation of sclareol.

[0008] Preferably, aerophilic Bacillus is used to ferment sugarcane juice to increase the sclareol content.

[0009] Preferably, yeast BY4741 and aerophilic Bacillus are used to ferment sugarcane juice to prepare sclareol. The yeast BY4741 is a commercial yeast.

[0010] Preferably, the aerophilic Bacillus is added in the form of a preparation; the preparation is prepared by the following steps: inoculating the aerophilic Bacillus into a culture medium, and culturing at 35° C.-40° C. and 200 rpm-240 rpm for 24 h-48 h.

[0011] Preferably, the aerophilic Bacillus is inoculated into LB liquid culture medium at an inoculation amount of ≥1%.

[0012] This application screened out a strain of aerophilic Bacillus from the soil of sugarcane fields Bacillus aerophilus The selected strains were subjected to colony PCR using bacterial universal primers 27F and 1492R. The PCR products were sent to Qingdao Weilai Company for sequencing. BLAST was used to compare the 16S rRNA sequences on NCBI, and the homology was 99%. The strain was identified as Bacillus aerophilus. Bacillus aerophilus It grows in LB solid medium, and its colony characteristics are: milky white, plump, round and convex. The deposit information is as follows:

[0013] Deposit date: January 13, 2025;

[0014] Deposit number: CGMCC No. 33380;

[0015] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0016] Classification name: Aerophilic Bacillus Bacillus aerophilus .

[0017] Beneficial effects of the present invention

[0018] An aerophilic Bacillus strain was isolated from the soil of sugarcane field Bacillus aerophilus ; It is used in the fermentation of sugarcane juice, and the content of sclareol and / or ethyl caproate and / or ethyl vanillate can be increased by controlling the fermentation conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Aerophilic Bacillus Bacillus aerophilus Colony diagram. DETAILED DESCRIPTION

[0020] The following are specific implementations of the present invention, and in conjunction with the accompanying drawings, the technical solution of the present invention is further described, but the present invention is not limited to these embodiments.

[0021] Example 1 Isolation and identification of aerophilic Bacillus.

[0022] 1. Isolation of strains

[0023] The specific steps are as follows:

[0024] Dig about 10.0g of surface soil (0-10cm), put it in a clean plastic bag, spread the soil and let it air dry naturally at room temperature, then sieve out large particles through a 2mm sieve and retain fine soil. Take 1.0g of dry soil sample, add it to 9ml of sterile water, mix well and dilute it 1:10, then take 50 μL of the dilution and evenly inoculate it on the LB solid culture medium. Place the inoculated culture medium in an incubator at 35℃ and culture it for 24 hours, and obvious colony formation is observed. Select the dominant colony, transfer it to a new LB solid culture medium by streak inoculation method, and continue to culture it at 35℃ for 24 hours. The observation results show that the colonies are milky white, plump, round and convex, such as Figure 1 shown.

[0025] 2. Identification of strains

[0026] The specific steps are as follows:

[0027] 1. The obtained strain was amplified using universal bacterial primers 27F, whose nucleotide sequence is shown in SEQ ID No. 1 (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R, whose nucleotide sequence is shown in SEQ ID No. 2 (5′-CTACGGCTACCTTGTTACGA -3′) to amplify the 16S r RNA gene of the strain.

[0028] 2. Use 30 μL reaction mixture for PCR, select Taq DNA Polymerase (Takara) enzyme, and the PCR conditions are pre-denaturation at 95°C for 5 min; 35 cycles of amplification at 95°C for 30 s; 58°C for 30 s; and 72°C for 1 min 30 s to obtain PCR products.

[0029] 3. The PCR product obtained in step 2 was analyzed by 1% agarose gel electrophoresis and sent to Qingdao Weilai Company for sequencing.

[0030] 4. The sequence was compared with the previously published bacterial 16S rRNA sequence in the NCBI database, and the strain homology was 99%. The strain was identified as Aerophilic Bacillus Bacillus aerophilus , and is deposited in the China General Microbiology Center (CGMCC) with the deposit number: CGMCC No. 33380.

[0031] The 16S rRNA sequence of the strain is shown in SEQ ID No.3:

[0032]

[0033] Example 2

[0034] A method for preparing sclareol, comprising the following specific steps:

[0035] 1. Inoculate the aerophilic Bacillus into LB liquid culture medium at a 1% inoculation rate and culture at 35°C and 200 rpm. 8 CFU / mL, stop culturing and obtain the bacterial agent.

[0036] 2. Adjust the sugar content of 2L sugarcane juice to 20 °Bx (20℃), sterilize, and ferment at 18℃, 23℃, and 28℃ using yeast BY4741, and add the aerophilic Bacillus inoculum in step 1 at 2% of the total fermentation liquid volume; when the sugar content no longer decreases, the fermentation ends.

[0037] 3. In step 2, the addition amount of yeast BY4741 is 1 mg / L, and it is added to the sugarcane juice after being activated with warm water.

[0038] The results of fermentation at different temperatures are shown in Table 1.

[0039] Table 1 Comparison of the contents of sclareol, ethyl hexanoate and ethyl vanillate at different fermentation temperatures in Example 2

[0040]

[0041] Example 3

[0042] A method for preparing sclareol, comprising the following specific steps:

[0043] 1. Inoculate the aerophilic Bacillus into LB liquid culture medium at a 1% inoculation rate and culture at 35°C and 200 rpm. 8 CFU / mL, stop culturing and obtain the bacterial agent.

[0044] 2. Adjust the sugar content of 2L sugarcane juice to 20 °Bx (20℃), sterilize, and ferment at 28℃ using yeast BY4741. Add the aerobic Bacillus inoculum in step 1 at 1% of the total fermentation liquid volume. When the sugar content no longer decreases, the fermentation ends.

[0045] 3. In step 2, the addition amount of yeast BY4741 is 1 mg / L, and it is added to the sugarcane juice after being activated with warm water.

[0046] Example 4

[0047] A method for preparing sclareol, comprising the following specific steps:

[0048] 1. Inoculate the aerophilic Bacillus into LB liquid culture medium at a 1% inoculation rate and culture at 35°C and 200 rpm. 8 CFU / mL, stop culturing and obtain the bacterial agent.

[0049] 2. Adjust the sugar content of 2L sugarcane juice to 20 °Bx (20℃), sterilize, and ferment at 28℃ using yeast BY4741. Add the aerobic Bacillus inoculum in step 1 at 4% of the total fermentation liquid volume. When the sugar content no longer decreases, the fermentation ends.

[0050] 3. In step 2, the addition amount of yeast BY4741 is 1 mg / L, and it is added to the sugarcane juice after being activated with warm water.

[0051] Example 5

[0052] A method for preparing sclareol, comprising the following specific steps:

[0053] 1. Inoculate the aerophilic Bacillus into LB liquid culture medium at a 1% inoculation rate and culture at 35°C and 200 rpm. 8 CFU / mL, stop culturing and obtain the bacterial agent.

[0054] 2. Adjust the sugar content of 2L sugarcane juice to 20 °Bx (20℃), sterilize it, add the aerophilic Bacillus inoculum in step 1 according to 2% of the total fermentation liquid volume, and ferment it at 28℃; when the sugar content no longer decreases, the fermentation is over.

[0055] Comparative Example 1 was fermented without adding aerophilic Bacillus agent.

[0056] A method for preparing sclareol, comprising the following specific steps:

[0057] 1. Bring 2L of sugarcane juice to 20°Bx (20°C), sterilize it, and ferment it at 28°C using yeast BY4741. When the sugar content no longer decreases, the fermentation is over.

[0058] 2. In step 1, the addition amount of yeast BY4741 is 1 mg / L, and it is activated with warm water and then added to the sugarcane juice.

[0059] Implementation effect example

[0060] The fermentation broths after fermentation in Examples 2-5 and Comparative Example 1 were subjected to GC-MS headspace solid phase microextraction to determine the contents of sclareol, ethyl hexanoate, and ethyl vanillate. The determination method was:

[0061] Add 1.5 g NaCl to a 20 mL screw-cap bottle, take 8 mL of fermentation broth into the screw-cap bottle, and sonicate for 15 min. Add 2-octanol as an internal standard, sonicate again for 10 min, and perform GC-MS headspace solid phase microextraction.

[0062] GC conditions: RTX-WAX capillary column (30m×0.25μm×0.25mm); carrier gas: high-purity helium; splitless injection, injection port temperature: 250°C; column temperature: programmed temperature: 40°C for 3 min, increased to 130°C at 2°C / min, increased to 220°C at 10°C / min, and maintained for 4 min.

[0063] MS conditions: transfer line temperature 250 °C; ion source temperature 230 °C; quadrupole temperature 150 °C; electromagnetic source EI; ion source EI; ionization energy 70 eV; scanning mass range 15~500 amu; mass spectrometry scanning mode: full scan (SCAN).

[0064] Data processing: The detected volatile components were analyzed by mass spectrometry and searched against the NIST2020 spectral library, with a matching factor greater than 80; the content was calculated using the internal standard method.

[0065] The contents of sclareol, ethyl caproate and ethyl vanillate in Examples 2-5 and Comparative Example 1 are compared, and the contents are shown in Table 2.

[0066] Table 2 Comparison of the contents of sclareol, ethyl hexanoate and ethyl vanillate in the examples and the comparative examples

[0067]

[0068] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the formula and preparation process of the present invention may be subject to various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A strain of aerophilic Bacillus, characterized in that: The aerophilic bacillus is aerophilic bacillus Bacillus aerophilus , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 13, 2025, with the deposit number: CGMCC No.33380; the address of the deposit unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the aerophilic Bacillus according to claim 1 in the preparation of sclareol.

3. The use according to claim 2, characterized in that: The aerophilic bacillus described in claim 1 is used to ferment sugarcane juice to prepare sclareol.

4. The use according to claim 2, characterized in that: The yeast BY4741 and the aerophilic bacillus described in claim 1 are used to ferment sugarcane juice to prepare sclareol.

5. The use according to claim 2, characterized in that: The aerophilic bacillus is added in the form of a preparation; the preparation is prepared by the following steps: inoculating the aerophilic bacillus into a culture medium, and culturing at 35° C.-40° C. and 200 rpm-240 rpm for 24 h-48 h.

6. The use according to claim 5, characterized in that: The aerophilic bacillus was inoculated into LB liquid culture medium at an inoculation amount of ≥1%.

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