Bacillus for degrading cis-abienol and application

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to bacillus for degrading cis-abienol and application. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) NO. The bacillus can convert the unique Labyman substance cis-abienol existing in the aromatic tobacco into the ambergris substance, the degradation rate is 37.65%, so that the fragrance of the tobacco leaves is increased, and the bacillus is used for fermentation of the tobacco leaves, so that the quality of the tobacco leaves is improved, and the sensory quality of cigarettes is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and relates to an engineering strain, in particular to a bacillus strain capable of degrading cis-abietanol and application thereof. Background Art

[0002] Diterpenoids 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 impact 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. The lysine aroma precursor specific to oriental tobacco and cigars, abirol (generally rarely found in flue-cured tobacco), plays an important role in the aroma characteristics of tobacco. Its degradation produces substances with a strong ambergris aroma, which is very effective in increasing the aroma and taste of cigarettes and is often used in tobacco extracts.

[0003] Abiesol is mainly derived from oriental tobacco and cigar tobacco. It is a natural lysine diterpenoid compound, and its structure is mainly cis-abiesol. Abiesol is an important aroma precursor in tobacco. Its degradation products include clarysol, ambroxol, ambroxol lactone, ambroxan and other important aroma components that can coordinate with the aroma of tobacco, mellow the smoke, and increase the original aroma of tobacco. Therefore, how to effectively utilize the degradation and transformation of abiesol to produce tobacco aroma components during tobacco preparation, aging and processing, and fully tap the potential of tobacco itself, is of great value for improving the quality and use value of tobacco leaves, increasing the aroma of cigarettes, highlighting the style characteristics of cigarettes and improving the safety of cigarettes. Summary of the invention

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

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

[0006] A bacillus strain capable of degrading cis-abietanol is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, and its deposit number is CGMCC NO.30673.

[0007] Furthermore, the strain was observed to be a Gram-positive bacterium under a microscope, with a size of 10-15 μm × 25-30 μm, a straight rod shape, flagella, and an irregular ring shape on the plate, with a solid center and edges and a transparent ring in the middle.

[0008] Furthermore, the method for culturing Bacillus is to inoculate the Bacillus bacterial solution into the culture medium at an inoculum 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 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.

[0010] An application, use of any of the above-mentioned Bacillus for degrading cis-abietanol.

[0011] Furthermore, the Bacillus is used for fermentation of tobacco leaves.

[0012] Furthermore, the degradation rate of cis-abietanol by the Bacillus was 37.65%.

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

[0014] The Bacillus of the present application can convert the unique lysine substance abietol present in oriental tobacco into the ambergris substance, thereby increasing the aroma of tobacco leaves, helping 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 Microscope photograph of the bacteria.

[0017] Figure 3 This is the degradation curve of cis-abietanol 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 of this application, Latin name: Bacillus sp., was deposited on May 17, 2024 at the General Microbiology Center of China National Microbiological Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, No. CGMCC NO.30673.

[0021] The bacillus of the present application is a Gram-positive bacterium observed under a microscope, with a size of 10-15 μm×25-30 μm, a straight rod shape, flagella, and an irregular ring shape on a plate, with a solid center and edges and a transparent ring in the middle.

[0022] Screening of Bacillus in this application:

[0023] Weigh 10 g of fresh soil, quarter it, put it into a 250 mL Erlenmeyer flask, add 100 mL of sterile water and soak it overnight, take 10 mL and centrifuge it, re-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.

[0024] The bacterial source obtained by enrichment culture is diluted in gradient on the inorganic salt solid medium containing cis-abirol, and the bacteria of different shapes and sizes are picked out and cultured separately. The obtained bacteria are fermented in the cis-abirol inorganic salt 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-abirol, and the degradation content of cis-abirol is detected by HPLC. The bacterial source that can degrade cis-abirol 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 cis-abirol-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.

[0025] Preparation of seed solution:

[0026] 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: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 1 L distilled water, pH to 7.0.

[0027] Bacterial degradation characteristics of cis-abietanol:

[0028] The Bacillus seed liquid was inoculated into the inorganic salt medium containing 0.5 g / L cis-abirol at a 1% inoculation rate, and cultured for 48 hours at a temperature of 30°C and a shaking speed of 150 rpm. A blank control was also performed. Samples were taken every 4 hours, and 2 mL was sampled each time. The content of cis-abirol in the fermentation broth was determined by HPLC.

[0029] The culture medium used in this 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.

[0030] After 48 h of culture, the residual concentration of cis-abieol in the original 0.5 mg / mL culture medium was 0.316 mg / mL, and the control was 0.5 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 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.

[0033] 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 63.35%. The test results proved that the bacterial degradation rate of cis-abietanol was 36.65%.

[0034] 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 for degrading cis-abirol, characterized in that: The strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, and its deposit number is CGMCC NO.30673.

2. The Bacillus for degrading cis-abirol according to claim 1, characterized in that: This species is a Gram-positive bacterium observed under a microscope. It is 10-15 μm × 25-30 μm in size, straight rod-shaped, with flagella, and irregular ring-shaped on the plate. The center and edges are solid, and the middle is a transparent circle.

3. The Bacillus for degrading cis-abirol 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 cis-abirol according to claim 3, characterized in that: The culture medium 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 according to any one of claims 1 to 4 for degrading cis-abietanol.

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 37.65%.

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