Bacillus strain for degrading cis-abienol and application thereof

Abstract

The application belongs to the technical field of bioengineering, and particularly relates to a bacillus strain capable of degrading cis-abienol and application thereof. The strain has a preservation number of CGMCC NO.30673. The bacillus strain of the application can convert the unique lycianthes material cis-abienol existing in oriental tobacco into a ambergris material, and the degradation rate is 37.65%, so as to increase the aroma of tobacco leaves, and the fermentation of tobacco leaves helps to improve the quality of tobacco leaves and improve the sensory quality of cigarettes.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to an engineered bacterial strain, specifically a Bacillus strain that degrades cis-fixing alcohol and its applications. Background Technology

[0002] Diterpenoids such as cephalans and lipatanilides are important substances in tobacco leaf secretions, contributing significantly to tobacco aroma quality and influencing the aroma characteristics of cigarette smoke. Cephalans are commonly found in common cultivated tobaccos such as flue-cured, burley, and aromatic tobaccos; while lipatanilides are only found in aromatic tobaccos and a small number of cigar varieties. The lipatanilide aroma precursor firol (generally rare in flue-cured tobacco), specific to aromatic tobaccos and cigars, plays a crucial role in tobacco aroma characteristics. Its degradation produces substances with a strong ambergris aroma, which is very effective in enhancing cigarette aroma and flavor, and is often used in tobacco extracts.

[0003] Taxyl alcohol, primarily derived from aromatic tobacco and cigar tobacco varieties, is a natural lysine diterpenoid compound with a predominantly cis-taxyl alcohol structure. Taxyl alcohol is an important aroma precursor in tobacco, and its degradation and transformation products include various important aroma components such as perillyl alcohol, ambroxol, ambroxol lactone, and ambroxol, which can harmonize with tobacco aroma, smooth the smoke, and enhance the inherent aroma of tobacco. Therefore, effectively utilizing the degradation and transformation of taxyl alcohol to produce tobacco aroma components during tobacco curing, aging, and processing, and fully exploring the inherent potential of tobacco, is of great value for improving tobacco leaf quality and usability, enhancing cigarette aroma, highlighting cigarette style characteristics, and improving cigarette safety. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Bacillus that degrades cis-fixine and its application, in order to solve the technical problem of cis-fixine degradation.

[0005] To achieve the above objectives, this application employs the following technical solution:

[0006] A strain of Bacillus sp. that degrades cis-fiberol has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 30673.

[0007] Furthermore, microscopic observation revealed that this strain is a Gram-positive bacterium, measuring 10–15 μm × 25–30 μm in size, with straight rod-like shapes and flagella. On a plate, it appears as an irregular ring, with a solid center and edges and a transparent ring in the middle.

[0008] Furthermore, the method for culturing Bacillus involves inoculating the Bacillus bacterial suspension into the culture medium at an inoculum volume of 1%, and culturing under the following conditions: pH 4–6, temperature 28–35℃, rotation speed 120–170 rpm, 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-fiberol.

[0010] One application is the use of any of the above-mentioned Bacillus species as a degrader of cis-haystol.

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

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

[0013] The beneficial effects of this invention are:

[0014] The Bacillus strain of this application can convert the unique lysine in aromatic tobacco, fir alcohol, into ambergris, thereby increasing the aroma of tobacco leaves, improving the quality of tobacco leaves, and enhancing the sensory quality of cigarettes. Attached Figure Description

[0015] Figure 1 This is a plate image showing the purification of the bacterial strain.

[0016] Figure 2 These are microscope images of the bacterial strain.

[0017] Figure 3 This is a graph showing the degradation curve of cis-fiberol by Bacillus subtilis.

[0018] Figure 4 This is a phylogenetic tree of Bacillus. Detailed Implementation

[0019] The technical solution of the present invention will be described in detail below with reference to 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, and should not be construed as limiting the technical solution of the present invention.

[0020] The Bacillus sp. of this application was deposited on May 17, 2024, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO.30673.

[0021] The Bacillus species described in this application are Gram-positive bacteria as observed under a microscope. They are 10–15 μm × 25–30 μm in size, straight rod-shaped, and have flagella. On a plate, they are irregularly circular, 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, and place it in a 250 mL Erlenmeyer flask. Add 100 mL of sterile water and soak overnight. Take 10 mL and centrifuge. Redissolve the precipitate with an equal volume of sterile water. Transfer 150 μL to 15 mL of LB medium and incubate for two days at 30 ℃ and 150 r / min to obtain the inoculum.

[0024] The bacterial source obtained through enrichment culture was serially diluted on an inorganic salt solid medium containing cis-fixine. Cells of different shapes and sizes were selected and cultured separately. The resulting cells were fermented in cis-fixine inorganic salt medium, and the degradation products of cis-fixine were analyzed by GC-MS and compared with those of a blank control. The degradation content of cis-fixine was determined by HPLC. Bacterial sources capable of degrading cis-fixine were selected, and 1 mL of each was serially diluted to 10⁻⁴ and 10⁻⁶ concentrations. These concentrations were then plated on carbon-deficient cis-fixine-enriched medium. Figure 1 As shown. The plated plates were placed in an incubator and incubated at 30°C for 2–4 days. Well-morphologically sound colonies were selected and repeatedly streaked for isolation and purification until no other colonies appeared on the plate. The plates were then examined under a microscope. Figure 2 As shown, this confirms that it is a pure culture.

[0025] Preparation of seed solution:

[0026] The Bacillus spores preserved in the agar slant were inoculated into 30 mL of LB medium and cultured for 12 h before use. The formulation of bacterial LB medium was: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 1 L distilled water, pH adjusted to 7.0.

[0027] Bacterial degradation properties of cis-fiberol:

[0028] Bacillus seed culture was inoculated at a rate of 1% into a medium containing 0.5 g / L cis-fiber alcohol and cultured for 48 h at 30°C with a shaking speed of 150 rpm. A blank control was also performed. Samples were taken every 4 h, 2 mL each time, and the content of cis-fiber alcohol 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-fiberol.

[0030] After 48 hours of culture, the residual concentration of cis-fiberol in the original 0.5 mg / mL culture medium was 0.316 mg / mL, while 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. Samples were taken every 4 hours for analysis. 2 mL of fermentation broth was taken, and an equal volume of analytical grade dichloromethane was added. The mixture was vortexed for 10 min, centrifuged at 10000 r / min for 10 min, and the lower organic phase was collected. 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 then purged with nitrogen to volatilize the dichloromethane. The sample was then added to the liquid mobile phase, filtered through a 0.45 μm organic filter membrane, and the content change was detected by HPLC.

[0033] The physicochemical test results of the degrading bacteria are as follows: Figure 3 As shown, the residual amount of cis-taperidine gradually decreased with the passage of culture time, and reached a stable level at 63.35%. The test results proved that the bacterial degradation rate of cis-taperidine was 36.65%.

[0034] The above description is merely a partial embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations are possible for those skilled in the art. Any modifications and substitutions made within the principles and implementation methods of this application should be included within the scope of protection of this application.

Claims

1. A Bacillus sp. strain that degrades cis-fixyl pine resin, characterized in that, This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 30673.

2. The Bacillus sp. that degrades cis-fixyl as described in claim 1, characterized in that, This bacterial strain is observed under a microscope to be a Gram-positive bacterium, measuring 10–15 μm × 25–30 μm in size. It is straight rod-shaped with flagella, and appears as an irregular ring on a plate, with a solid center and edges and a transparent ring in the middle.

3. The Bacillus sp. that degrades cis-fixyl iron according to claim 1, characterized in that, The method for culturing Bacillus involves inoculating the Bacillus bacterial suspension into the culture medium at an inoculum volume of 1%, and culturing under the following conditions: pH 4–6, temperature 28–35℃, rotation speed 120–170 rpm, for 24 hours.

4. The Bacillus sp. that degrades cis-fixyl iron 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-fiberol.

5. The application of a Bacillus sp. that degrades cis-fixyl benzoate, characterized in that, The use of the Bacillus species of any one of claims 1 to 4 as a degrader of cis-hawthorn alcohol.

6. The application of the Bacillus sp. that degrades cis-fixyl alcohol according to claim 5, characterized in that, The Bacillus species are used for the fermentation of tobacco leaves.

7. The application of the Bacillus sp. that degrades cis-fixyl alcohol according to claim 5, characterized in that, The degradation rate of cis-fiberol by the Bacillus was 36.8%.

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