Bacillus for degrading sclareol and application

Abstract

The invention belongs to the field of bioengineering, and relates to an engineering strain, in particular to a bacillus for degrading sclareol and application. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) The bacillus for degrading the sclareol can convert the ambergris intermediate product sclareol into a Yunxiang substance, the degradation rate of the sclareol within 72 h is 65.99% or above, and when the bacillus is used for fermenting tobacco leaves, the quality of the tobacco leaves can be improved, and the sensory quality of cigarettes can be improved.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, relates to an engineering strain, and specifically relates to a bacillus strain capable of degrading sclareol and application thereof. Background Art

[0002] Sclareol, also known as sclareol, is a diterpenoid compound. It has a similar aroma to ambergris, which is long-lasting and delicate, and is an ideal raw material for synthetic ambergris products. As an intermediate for synthetic ambergris and an important ingredient in the preparation of spices, sclareol is mainly used in the synthesis of natural ambergris substitutes such as sclareolide and ambroxol, and can also be used in the preparation of spices. It has important application value in the pharmaceutical and cosmetic industries.

[0003] Sclareol plays an important role as the middle ring of a series of ambergris products. Sclareol can be converted into compounds with ambergris aroma such as hard-tail aldehydes, ambroxol, ambroxol, and ambroxan through oxidation, cyclization, and degradation. At present, the production of sclareol is mostly extracted and purified from plants, and there are also methods to produce sclareol by constructing brewer's yeast. For the synthesis of ambergris products, sclareol is mostly used as a raw material for chemical oxidation with H2O2 and KMnSO4, defatting, and LiAlH4 reduction to synthesize ambergris products such as ambroxol and ambroxol. Summary of the invention

[0004] The purpose of the present invention is to provide a bacillus for degrading sclareol and its application, so as to solve the technical problem of sclareol degradation.

[0005] To achieve the above objectives, this application is implemented through the following technical solutions:

[0006] A bacillus strain capable of degrading sclareol is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, and its deposit number is CGMCC NO.30671.

[0007] Furthermore, Bacillus is a Gram-positive bacterium, 10-15 μm × 25-30 μm in size, short rod-shaped, with flagella, and appears as yellow round spots on the plate, solid and opaque, with smooth surroundings.

[0008] Furthermore, the method for culturing Bacillus is to inoculate the Bacillus bacterial solution into the culture medium at an inoculation rate of 1%, and the culture conditions are pH 4-6, temperature 28-35°C, rotation speed 120-170 rpm, and culture for 24 hours.

[0009] Furthermore, the culture medium formula consists of 1 g / L K2HPO4, 0.5 g / L MgSO4•7H2O, 0.005 g / L FeSO4•7H2O, 0.5 g / L NaCl, 0.65 g / L KH2PO4, 0.001 g / L MnSO4, 0.5 g / L (NH4)2•SO4, 0.1g / L CaCl2•2H2O, 0.005 g / L Na2MoO4•2H2O and 1.0 g / L clarysol.

[0010] An application, using any of the above-mentioned Bacillus species capable of degrading sclareol as a member for degrading sclareol.

[0011] Furthermore, the Bacillus that degrades clarysol is used in the fermentation of tobacco leaves.

[0012] Furthermore, the degradation rate of sclareol by Bacillus degrading sclareol was above 65.99% within 72 h.

[0013] The beneficial effects of the present invention are:

[0014] The sclareol-degrading Bacillus of the present invention can convert sclareol, an intermediate product of ambergris, into a sclareol-friendly substance, and the degradation rate of sclareol within 72 hours is above 65.99%. The use of the Bacillus in tobacco leaf fermentation is helpful to improve the quality of tobacco leaves and the sensory quality of cigarettes. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a plate map of the purified strain.

[0016] Figure 2 This is an electron microscope photo of the strain.

[0017] Figure 3 This is the degradation curve of clarysol by Bacillus.

[0018] Figure 4 Phylogenetic tree of Bacillus. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is described in detail below in conjunction with the accompanying drawings. The following embodiments are merely exemplary and can only be used to explain and illustrate the technical solution of the present invention, but cannot be construed as limiting the technical solution of the present invention.

[0020] The Bacillus pantoea piersonii involved in this application, which degrades clarysol, was deposited on May 17, 2024 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC NO.30671.

[0021] The formula of the carbon-deficient sclareol-rich culture medium involved in the present application consists of 1 g / L K2HPO4, 0.5 g / L MgSO4•7H2O, 0.005 g / L FeSO4•7H2O, 0.5 g / L NaCl, 0.65 g / L KH2PO4, 0.001 g / L MnSO4, 0.5 g / L (NH4)2•SO4, 0.1 g / L CaCl2•2H2O, 0.005 g / L Na2MoO4•2H2O and 1.0 g / L sclareol.

[0022] The formula of the bacterial LB culture medium involved in the present application consists of 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl and 1 L of distilled water, with a pH of 7.0.

[0023] Screening of sclareol-degrading bacteria:

[0024] Weigh 10 g of fresh soil, divide it into quarters, put it into a 250 mL Erlenmeyer flask, add 100 mL of sterile water and soak it overnight, take 10 mL and centrifuge it, dissolve the precipitate with an equal amount of sterile water, transfer 150 μL and add it to 15 mL of LB medium, and culture it at 30 °C and 150 r / min for two days to obtain the bacterial source.

[0025] The bacterial source obtained by enrichment culture is gradiently diluted on the carbon-deficient perilla alcohol-rich medium, and the bacteria of different shapes and sizes are picked out and cultured separately. The obtained bacteria are fermented in the carbon-deficient perilla alcohol-rich medium, and the GC-MS detection is compared with the GC-MS detection chart of the blank control to observe the degradation products of perilla alcohol and the changes in their content. The bacterial source that can degrade perilla alcohol is selected, and 1 mL is transferred from it for gradient dilution in sequence. The two concentration gradients of 10~4 and 10~6 are selected for plate coating on the carbon-deficient perilla alcohol-rich medium. Figure 1 Place the coated plate in an incubator at 30°C for 2 to 4 days, select colonies with good morphology, and repeatedly streak and purify them until no other colonies appear on the plate. Then examine them under a microscope. Figure 2 As shown, it can be confirmed as pure culture.

[0026] Preparation of seed solution:

[0027] Inoculate the Bacillus stored in the slant into 30 mL of LB medium and culture for 12 h before use.

[0028] Bacterial degradation characteristics of cis-abietanol:

[0029] The Bacillus seed liquid was inoculated into a 1 g / L carbon-deficient perilla alcohol medium at a 1% inoculation rate and cultured for 72 h at a temperature of 30 ° C and a shaking speed of 150 rpm. A blank control was also performed. Samples were taken every 6 h, and 2 mL was sampled each time. The content of cis-abirol in the fermentation broth was determined by HPLC.

[0030] After 72 h of culture, the residual concentration of sclareol in the original 1 mg / mL culture medium was 0.34 mg / mL, while that of the control was 1 mg / mL.

[0031] Preparation before sample measurement:

[0032] The inoculated fermentation medium was used as the experimental group, and the uninoculated fermentation medium was used as the control group. Sampling was performed every 6 h. 2 mL of fermentation broth was added with an equal volume of analytical grade dichloromethane. The mixture was vortexed and extracted at 2500 r / min for 10 min. The mixture was centrifuged at 10000 r / min for 10 min. The lower organic phase was taken and the above steps were repeated 3 times. The organic phases were combined, dried with anhydrous sodium sulfate, allowed to stand overnight, concentrated, and filtered through a 0.45 um organic filter membrane before GC-MS detection of content changes.

[0033] The results of the physicochemical tests on the degradation bacteria are as follows Figure 3 As shown, the residual amount of sclareol gradually decreased with the passage of culture time and reached a stable level at 34.01%. The test results proved that the bacterial degradation rate of sclareol was 65.99%.

[0034] Sclareol has a similar aroma to ambergris, which is long-lasting and delicate, and is an ideal raw material for synthetic ambergris products. As an intermediate for synthetic ambergris and an important ingredient in the preparation of fragrances, sclareol is mainly used in the synthesis of natural ambergris substitutes such as sclareolide and ambroxol, and can also be used in the preparation of fragrances. It has important application value in the pharmaceutical and cosmetic industries.

[0035] The aging of tobacco leaves after redrying is a very important step, which has a great impact on the quality of tobacco leaves. Due to the presence of unique lysine in oriental tobacco, studies have shown that in the early and middle stages of oriental tobacco aging, the lysine compounds and cypermethrin compounds in the tobacco leaves increase significantly, and after aging, the characteristic aroma component of oriental tobacco, ambergris, is significantly increased. Therefore, the rational conversion of ambergris intermediate clarysol into ambergris substances is helpful to improve the quality of tobacco leaves and the sensory quality of cigarettes.

[0036] During the aging period of oriental tobacco leaves, if substances such as afir alcohol can be converted into ambergris flavor substances, the aroma of tobacco leaves can be increased. However, the aging period is relatively long, generally 24 to 36 months, and this process will undoubtedly increase the production cost of tobacco leaves. Bacillus can degrade more than 65.99% of clarysol within 72 hours, indicating that the bacteria can be used for tobacco leaf fermentation in the future.

[0037] The above description is only a part of the specific embodiments of the present application and is not intended to limit the present application. For relevant technical personnel in the field, the present application has various changes and variations. Any modification and replacement made within the relevant principles and implementation methods of the present application should be included in the scope of protection of the present application.

Claims

1. A Bacillus strain capable of degrading sclareol, characterized in that: This strain is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, and its deposit number is: CGMCC NO.30671.

2. The Bacillus for degrading sclareol according to claim 1, characterized in that: Bacillus is a Gram-positive bacterium, 10-15 μm × 25-30 μm in size, short rod-shaped, with flagella, and appears as yellow round spots on the plate, solid and opaque with smooth surroundings.

3. The Bacillus for degrading sclareol according to claim 1, characterized in that: The culture method of Bacillus is to inoculate the Bacillus bacterial liquid into the culture medium at a 1% inoculation rate, the culture conditions are pH 4-6, temperature 28-35°C, rotation speed 120-170 rpm, and culture for 24 hours.

4. The Bacillus for degrading sclareol according to claim 3, characterized in that: The culture medium formula consists of 1 g / L K2HPO4, 0.5 g / L MgSO4•7H2O, 0.005 g / L FeSO4•7H2O, 0.5 g / L NaCl, 0.65 g / L KH2PO4, 0.001 g / L MnSO4, 0.5 g / L (NH4)2•SO4, 0.1 g / L CaCl2•2H2O, 0.005 g / L Na2MoO4•2H2O and 1.0 g / L clarysol.

5. An application, characterized in that: Use of the sclareol-degrading Bacillus according to any one of claims 1 to 4 as a sclareol-degrading agent.

6. The use according to claim 5, characterized in that: Bacillus that degrades clarysol is used in tobacco fermentation.

7. The use according to claim 5, characterized in that: The degradation rate of sclareol by Bacillus degrading sclareol was over 65.99% within 72 hours.

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