Pantoea capable of degrading sclareol and application

Abstract

The invention belongs to the field of bioengineering, and relates to an engineering strain, in particular to pantoea for degrading sclareol and application. The preservation number of the pantoea is CGMCC (China General Microbiological Culture Collection Center) NO.30674 According to the pantoea, an ambergris intermediate product sclareol can be reasonably converted into ambergris substances, substances such as abienol and the like can be converted into ambergris aroma substances in fermentation of tobacco leaves, and 52.11% or above of sclareol can be degraded by the pantoea within 72 h, so that the aroma of the tobacco leaves is increased, and the aroma of the tobacco leaves is improved. 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 Pantoea strain capable of degrading sclareol and an 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 in tobacco is degraded to produce cyclic ethers with 2 carbon atoms reduced, 1,2,5,9-tetramethyl-2-hydroxydecahydronaphthalene, 2-hydroxybutyric acid ketone, and sclareolide. Sclareolide is reduced to another very important substance, sclareol. At present, for the production of sclareol, most of the extraction and purification of sclareol is from plants, and there are also engineering bacteria of brewer's yeast to produce sclareol. At present, there are few studies on tobacco diterpenoids in China, especially on the degradation mechanism of such compounds in tobacco. Therefore, the study of the biodegradation of sclareol will help lay the foundation for the biotransformation of tobacco diterpenoids in the later stage. Summary of the invention

[0004] The purpose of the present invention is to provide a Pantoea strain capable of degrading sclareol and its application, so as to solve the technical problem of sclareol degradation in the above-mentioned background technology.

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

[0006] A strain of Pantoea that degrades clarysol is currently deposited in the China General Microorganism Collection Center with a deposit number of CGMCC NO.30674.

[0007] Furthermore, the Pantoea is a Gram-positive bacterium, with a size of 0.5-1 μm×1-3 μm, a straight rod shape, flagella, a beige color on a plate, a rough surface, an irregular shape, thick in the middle and thin at the edges.

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

[0009] Furthermore, the formula of 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, 0.1g / L CaCl2•2H2O, 0.005 g / L Na2MoO4•2H2O and 1.0 g / L clarysol.

[0010] An application, using any of the above-mentioned Pantoea for degrading sclareol as a sclareol-degrading agent.

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

[0012] Furthermore, the degradation rate of sclareol by Pantoea is above 52.11%.

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

[0014] The Pantoea involved in the present invention can reasonably convert the ambergris intermediate product sclareol into ambergris substances. When used in the fermentation of tobacco leaves, it can convert substances such as afir alcohol into ambergris flavor substances. The Pantoea of ​​the present application can degrade more than 52.11% of sclareol within 72 hours, 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 sclareol by Pantoea.

[0018] Figure 4 This is the phylogenetic tree of Pantoea. 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 present application relates to Pantoea that degrades clarysol, which was deposited in the China General Microorganism Collection Center on May 17, 2024, with the address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC NO.30674.

[0021] In the present application, the formula of the carbon-deficient perilla alcohol-rich culture medium is involved, which is composed 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 perilla alcohol.

[0022] The formulation of the bacterial LB medium consists of 10 g tryptone, 5 g yeast extract, 10 g NaCl, 1 L distilled water, and the pH is adjusted to 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 2-4 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 sclareol:

[0029] The seed liquid of Pantoea was inoculated into a medium containing 1 g / L carbon-deficient perilla alcohol 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 0.479 mg / mL, while 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 47.89%. The test results showed that the bacterial degradation rate of sclareol was 52.11%.

[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 use of lysine absolute oil in tobacco flavors can make up for the aroma of tobacco, correct the smell, enhance the taste, and improve the smoking quality. Due to the presence of unique lysine substances 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 are significantly increased, and after aging, the characteristic aroma components of oriental tobacco, ambergris substances, are 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 aroma 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 52.11% of clary alcohol 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 Pantoea strain that degrades sclareol, characterized in that: The Pantoea is currently deposited in the China General Microorganism Collection Center with the collection number: CGMCC NO.30674.

2. The Pantoea for degrading sclareol according to claim 1, characterized in that The Pantoea is a Gram-positive bacterium, with a size of 0.5-1 μm×1-3 μm, a straight rod shape, flagella, a beige color on a plate, a rough surface, an irregular shape, thick in the middle and thin at the edges.

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

4. The Pantoea for degrading sclareol according to claim 3, characterized in that The formula of 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, 0.1g / L CaCl2•2H2O, 0.005 g / L Na2MoO4•2H2O and 1.0 g / L clarysol.

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

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

7. The use according to claim 5, characterized in that: The degradation rate of sclareol by Pantoea is above 52.11%.

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