Bacillus for degrading cis-abienol to generate sclareol and application thereof

Abstract

The invention belongs to the field of bioengineering, and particularly relates to a bacillus for degrading cis-abienol to generate sclareol and application of the bacillus. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.30672, and the strain is classified and named as bacillus sp. The bacillus disclosed by the invention can convert a Labdan substance cis-abienol into an ambergris substance sclareol, and is used for fermenting tobacco leaves, so that the tobacco leaves are fermented, and the yield of the tobacco leaves is increased. 41.9% or above of cis-abienol can be degraded within 48 h, the quality of 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 capable of degrading cis-abietanol to generate sclareol and application thereof. Background Art

[0002] Terpenoid compounds in tobacco are not only of great physiological significance to tobacco growth and development, but also of great significance to the formation of tobacco quality. Diterpenoid compounds such as cedarwood and lysine are important substances in tobacco leaf secretions. They contribute greatly to the aroma quality of tobacco and have an important influence on the aroma style characteristics of cigarette smoke. Ceramics are commonly found in common cultivated tobacco such as flue-cured tobacco, burley tobacco, and oriental tobacco; while lysine compounds are only found in oriental tobacco and a small number of cigar varieties. During the process of tobacco leaf conditioning and aging, lysine compounds will undergo oxidation, degradation, and transformation, and can form a variety of lysine degradation compounds. Among them, the most important degradation compound is ambroxan, which has a strong ambergris aroma and plays an important role in increasing the aroma and taste of cigarettes.

[0003] Cis-abietanol is the main lysine diterpenoid substance in tobacco. It is mainly found in oriental tobacco and cigars and is the main component of glandular hair secretions on the surface of tobacco. Cis-abietanol is an important aroma precursor. During the preparation of tobacco leaves, it is converted into substances with a pleasant ambergris aroma such as ambroxan and ambergris lactone through oxidation and degradation. In addition to generating aroma substances, the presence of abietanol can also make tobacco resistant to insects, diseases and other microorganisms. For example, cis-abietanol can be used as an inducer to effectively prevent and control the occurrence of tobacco bacterial wilt. In addition, it has been reported that cis-abietanol has the effect of resisting bacterial growth and infection. Therefore, cis-abietanol has a good prospect in medicine and chemical biology.

[0004] The intermediate product of cis-abieol degradation contains sclareol, which has a similar aroma to ambergris, with a long-lasting and delicate aroma, and is an ideal raw material for synthetic ambergris products. As an intermediate for synthetic ambergris and an important component for preparing 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. Summary of the invention

[0005] The purpose of the present invention is to provide a bacillus for degrading cis-abietanol to generate sclareol and its application, so as to solve the technical problem of cis-abietanol degradation.

[0006] To achieve the above objectives, this application is implemented through the following technical solutions:

[0007] A Bacillus sp. strain is disclosed which can degrade cis-abietanol to generate sclareol. The strain is currently deposited in the General Microbiological Center of China Microbiological Culture Collection Administration. Its deposit number is CGMCC NO.30672 and its classification name is Bacillus sp.

[0008] Furthermore, the Bacillus is a Gram-negative bacterium, and under 15k-fold electron microscope observation, its size is 6-8 μm×15-18 μm, it is short rod-shaped, has no flagella, and appears as white round spots on the plate.

[0009] 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.

[0010] 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, 0.1 g / L CaCl2•2H2O, 0.005 g / L Na2MoO4•2H2O and 0.5 g / L cis-abietanol.

[0011] An application is the use of any of the above-mentioned Bacillus sp. for degrading cis-abieol to produce sclareol as an application for degrading cis-abieol.

[0012] Furthermore, the Bacillus is used for fermentation of tobacco leaves.

[0013] Furthermore, the degradation rate of cis-abietanol by the Bacillus is above 41.9%.

[0014] The beneficial effects of the present invention are:

[0015] The bacillus of the present invention can convert the lysine substance cis-abietanol into the ambergris substance sclareol, and can be used in the fermentation of tobacco leaves, and can degrade more than 41.9% of the cis-abietanol within 48 hours, which is helpful to improve the quality of tobacco leaves and the sensory quality of cigarettes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a plate map of the purified strain.

[0017] Figure 2 This is an electron microscope photo of the strain.

[0018] Figure 3This is the degradation curve of cis-abietanol by Bacillus.

[0019] Figure 4 Phylogenetic tree of Bacillus. DETAILED DESCRIPTION

[0020] 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.

[0021] The Bacillus sp. involved in the present application, which degrades cis-abietanol to produce clarysol, was deposited on May 17, 2024 at the General Microbiological Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC NO.30672, and the classification name: Bacillus sp.

[0022] The reagents, equipment, etc. involved in this application can be obtained through commercial channels.

[0023] The carbon-deficient and rich cis-abietanol culture medium formula of 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 0.5 g / L cis-abietanol.

[0024] Screening of cis-abietanol-degrading bacteria involved in this application:

[0025] 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.

[0026] The bacterial source obtained by enrichment culture is gradiently diluted on a carbon-deficient cis-abietanol-rich medium, and bacteria of different shapes and sizes are picked out and cultured separately. The obtained bacteria are fermented in a carbon-deficient cis-abietanol-rich medium, and the GC-MS detection is compared with the GC-MS detection chart of the blank control to see the degradation product of cis-abietanol, and the degradation content of cis-abietanol is detected by HPLC. Select the bacterial source that can degrade cis-abietanol, and then transfer 1 mL from it for gradient dilution in sequence, and select the two concentration gradients of 10~4 and 10~6 for plate coating on a carbon-deficient cis-abietanol-rich medium. Figure 1 Place the coated plate in an incubator at 30°C for 2-4 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.

[0027] Preparation of seed solution:

[0028] Inoculate the Bacillus stored in the slant into 30 mL of LB medium and culture for 12 h before use. The formula of bacterial LB medium is: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 1 L distilled water, pH to 7.0.

[0029] Bacterial degradation characteristics of cis-abietanol:

[0030] The Bacillus seed liquid was inoculated into a carbon-deficient medium containing 0.5 g / L cis-abirol at a 1% inoculation rate and cultured for 48 h at a temperature of 30 °C and a shaking speed of 150 rpm. A blank control was also performed. Samples were taken every 4 h, and 2 mL was sampled each time. The content of cis-abirol in the fermentation broth was determined by HPLC.

[0031] After 48 h of culture, the residual concentration of cis -abietanol in the original 0.5 mg / mL carbon-deficient and rich cis -abietanol culture medium was 0.29 mg / mL, and that of the control was 0.5 mg / mL.

[0032] Preparation before sample measurement:

[0033] 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 4 hours. 2 mL of fermentation broth was added with an equal volume of analytical grade dichloromethane, vortexed for 10 min, 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, and allowed to stand overnight. The sample solution was blown with nitrogen to evaporate the dichloromethane, and the liquid mobile phase was added. After passing through a 0.45 um organic filter membrane, the content change was detected by HPLC.

[0034] The results of the physicochemical tests on the degradation bacteria are as follows Figure 3 As shown, the residual amount of cis-abietanol gradually decreased with the culturing time and reached a stable level at 58.1%. The test results proved that the bacterial degradation rate of cis-abietanol was 41.9%.

[0035] 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.

[0036] During the tobacco leaf curing and aging process, most of the abirol, styrol and lisdendial are oxidized and degraded, and the degradation products are mainly ambroxan, ambergris lactone, γ-bicyclic homofarnesal and dehydroambergris lactone. These compounds have a strong ambergris aroma and play a very important role in increasing the flavor of cigarettes. Therefore, the rational conversion of the lisdendial substance abirol into ambergris substances will help improve the quality of tobacco leaves and the sensory quality of cigarettes.

[0037] During the aging period of oriental tobacco leaves, if substances such as abirol 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 41.9% of cis-abirol within 48 hours, indicating that the bacteria can be used for tobacco leaf fermentation in the future.

[0038] 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 sp. that degrades cis-abietanol to produce sclareol, characterized in that: The strain is currently deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number: CGMCC NO.30672, and the classification name: Bacillus sp.

2. The Bacillus for degrading cis-abieol to produce sclareol according to claim 1, characterized in that: The bacillus is a Gram-negative bacterium. Under 15k-fold electron microscope observation, its size is 6-8 μm×15-18 μm, it is short rod-shaped, has no flagella, and is white round spots on the plate.

3. The Bacillus for degrading cis-abieol to produce sclareol according to claim 1, characterized in that: The culture method of Bacillus is to inoculate the Bacillus 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 cis-abieol to produce 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 0.5 g / L cis-abietanol.

5. An application, characterized in that: Use of the Bacillus sp. capable of degrading cis-abieol to produce sclareol according to any one of claims 1 to 4 as a molecule for degrading cis-abieol.

6. The use according to claim 5, characterized in that: The bacillus is used for fermentation of tobacco leaves.

7. The use according to claim 5, characterized in that: The degradation rate of cis-abietanol by the bacillus is above 41.9%.

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