Bacillus for producing acetoin and application thereof

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to bacillus for producing acetoin and application of the bacillus, the bacillus is classified and named as Bacillus sp, the strain number is H64, the bacillus is preserved in China General Microbiological Culture Collection Center on September 11, 2024, and the preservation number is CGMCC No. 31938. The bacillus screened by the invention can grow in the coating liquid of the reconstituted tobacco, and the coating liquid can be directly used as a nutrient source to ferment and produce acetoin.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and specifically relates to an acetoin-producing bacillus and application thereof. Background Art

[0002] Acetoin (also known as 3-hydroxy-2-butanone, 3-hydroxybutanone or methylacetyl carbinol) is a edible flavoring with a special creamy aroma. It is mainly used to enhance the taste of food. Its unique aroma can bring a unique flavor and experience to the product. It is currently widely used in industries such as tobacco, alcohol and cosmetics.

[0003] In the tobacco industry, acetoin is mainly used as a flavoring agent to add creamy aroma and taste to cigarettes, improve the taste of tobacco products, balance and soften smoke. At the same time, acetoin also helps to reduce the bitterness and irritation produced when cigarettes are burned, bringing smokers a more pleasant smoking experience. It has now become one of the indispensable important additives in tobacco products. Studies have found that acetoin in tobacco is mainly produced by microorganisms on the surface of tobacco leaves during the alcoholization process of tobacco leaves, using the glucose of the tobacco leaves themselves as a carbon source for fermentation. It is an important aroma component of cigarettes.

[0004] As an important flavoring auxiliary material in cigarette products, the aroma components of reconstituted tobacco have a huge impact on the overall smoking quality of cigarettes. In the prior art, reconstituted tobacco is a product made by reorganizing tobacco stems, broken tobacco pieces, tobacco dust or low-grade tobacco leaves by physical or chemical methods and adding a coating liquid. The main aroma components in tobacco raw materials such as tobacco stems, broken tobacco pieces, tobacco dust or low-grade tobacco leaves are transferred to the extract by extraction means, and the extract is concentrated to obtain the coating liquid. According to the preparation process of reconstituted tobacco coating liquid, the reconstituted tobacco coating liquid contains rich nutrients (such as glucose, fructose, amino acids, trace elements, etc.), and is a good raw material for microbial aroma production. However, since the coating liquid is a concentrated liquid, its osmotic pressure is high and the content of toxic substances such as nicotine is relatively high, which has high requirements on microbial fermentation technology and the tolerance of corresponding microorganisms. Therefore, directly using the sugar in the reconstituted tobacco coating liquid as a carbon source and adopting microbial fermentation technology to produce reconstituted tobacco coating material containing acetoin can establish a new way to improve the quality of reconstituted tobacco, which is of great research significance.

[0005] The present invention is proposed to solve at least one of the above problems. Summary of the invention

[0006] The present invention aims to provide an acetoin-producing Bacillus sp., which can synthesize acetoin by fermentation with reconstituted tobacco leaf coating liquid as raw material, thus creating a new approach for improving the quality of reconstituted tobacco leaves.

[0007] The technical solution adopted by the present invention is:

[0008] A strain of Bacillus producing acetoin, named Baclicus sp, with strain number H64, was deposited in the General Microbiology Center of China Microorganism Culture Collection on September 11, 2024, with deposit number CGMCC No.31938, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0009] The bacillus of the present invention is separated from tobacco leaves in the middle stage of alcoholization, is a gram-positive bacillus with spores, and forms small opaque colonies on the surface of LB solid culture medium, with wrinkles and irregular edges.

[0010] The 16S rDNA sequence (as shown in SEQ ID NO. 1) obtained by PCR with universal primers of the Bacillus of the present invention is 99% similar to the 16S rDNA sequence of other Bacillus, but different.

[0011] The upstream primer of the universal primer PCR is fD1: 5'-agagtttgatcctggctcag-3' (as shown in SEQ ID NO.2);

[0012] Downstream primer rP2: 5'-cggctaccttgttacgactt-3' (as shown in SEQ ID NO. 3).

[0013] The present invention also provides the use of the acetoin-producing Bacillus in the fermentation production of acetoin.

[0014] The present invention further provides the use of acetoin-producing Bacillus in enhancing the flavor and quality of reconstituted tobacco leaves.

[0015] The present invention also provides a method for preparing a coating material containing acetoin using the above-mentioned Bacillus, comprising the following steps:

[0016] Step (1), inoculating a purified single colony of Bacillus H64 into a sterile LB medium at 37°C, and culturing until the OD600 is 2.0-3.0 to obtain a seed solution;

[0017] Step (2), inoculating the Bacillus seed liquid obtained by culture in step (1) into the reconstituted tobacco leaf coating liquid at an inoculation rate of 1-3% (v / v), and culturing at 30-37° C. and 180 rpm for 4-12 hours to obtain the coating liquid containing acetoin.

[0018] The present invention also provides a reconstituted tobacco leaf, wherein the substrate of the reconstituted tobacco leaf is coated with the above-mentioned coating liquid containing acetoin. The specific preparation process is to treat the fermented coating liquid obtained in step (2) at 80-120 degrees Celsius and then coat it on the reconstituted tobacco leaf substrate to obtain the reconstituted tobacco leaf.

[0019] The prepared reconstituted tobacco leaves can be cut into shreds to obtain filamentous reconstituted tobacco leaves after being conditioned at a humidity of 50-60%, a temperature of 20-25° C., and a balancing time of more than 24 hours.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The Bacillus obtained by screening in the present invention can grow in the coating liquid of reconstituted tobacco leaves, and can ferment and produce acetoin directly using the coating liquid as a nutrient source.

[0022] 2. The acetoin produced by the Bacillus fermentation can be retained in the coating material of the reconstituted tobacco leaves to directly enhance the flavor and quality of the reconstituted tobacco leaves, or it can be used as a food additive after purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure is a mass spectrometry detection result diagram of acetoin obtained by fermentation with Bacillus sp. of the present invention. DETAILED DESCRIPTION

[0024] The present invention is further described below by way of examples, which are not limited to the examples. Experimental methods without specific conditions specified in the examples are generally carried out under conventional conditions and conditions described in the manual, or under conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all commercially available unless otherwise specified.

[0025] Example 1 Screening of Bacillus H64

[0026] (1) The culture medium used in this example is as follows:

[0027] The identification solid culture medium was: tryptone 3.0 g / L, beef extract 2.0 g / L, NaCl 6.0 g / L, MgSO4·7H2O, 2.0 g / L, reconstituted tobacco leaf coating liquid 5.0 g / L, agar 15.0 g / L, pH 7.0.

[0028] The identification liquid culture medium is: tryptone 3.0g / L, beef extract 2.0g / L, NaCl 6.0g / L, MgSO4·7H2O, 2.0g / L, reconstituted tobacco leaf coating liquid 5.0g / L, pH 7.0.

[0029] The product identification liquid culture medium is: 20 mL of reconstituted tobacco leaf coating liquid. The coating liquid is tested to have a sugar content of 200 mg / g, a nitrogen content of about 5 mg / g, an organic acid content of 50 mg / g, a total amino acid content of about 6 mg / g, and a metal ion content of 0.55 mg / g.

[0030] (2) The specific screening method is as follows;

[0031] Take 2.0 g of tobacco leaves in the middle stage of alcoholization and place them in 50 mL of sterile water and activate them at 37°C for 12 hours. Then use a sterile pipette to draw 1 mL from this test tube and add it to another test tube containing 9 mL of sterile water. Mix well. Repeat this process to make 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Mixed bacterial cultures at different dilutions.

[0032] The diluted mixed bacterial liquid of different concentrations was spread on the above identification solid culture medium, and a single colony was picked after culturing for 24 hours, and purified by plate streaking method until a single colony was obtained.

[0033] A total of 10 strains were obtained after screening. The 10 strains were inoculated into the above-mentioned screening liquid culture medium, and cultured at 37°C, 180rpm for 8-12h to obtain 10 seed solutions, and then the 10 seed solutions were inoculated into the product identification liquid culture medium at an inoculation rate of 1%, and the corresponding fermentation liquid was obtained after fermentation for 12-18 hours, and the acetoin content was determined by colorimetry.

[0034] Colorimetric determination method of acetoin content:

[0035] 2 mL of fermentation broth was diluted 20 times with pure water, and then placed in a certain reaction system (400 μL of fermentation broth, 360 μL of 0.5% creatine solution and 360 μL of 5% 1-naphthol solution) at 60°C for 15 min. The OD530 value was determined. The value was between 0.1 and 1.0 each time. If the value was not within the accurate range, the dilution factor was readjusted to make it within the accurate range. A standard curve was drawn corresponding to 0, 1, 2, 4, 8, and 10 mg / L acetoin standard samples, and the acetoin content was calculated using the standard curve.

[0036] The best strain obtained after screening was named H64.

[0037] Example 2 Identification of strain H64

[0038] A single colony of the optimal strain H64 obtained in Example 1 was inoculated into LB liquid culture medium, and after 12 hours, the culture was centrifuged to extract genomic DNA, and then the DNA was electrophoresed using agarose gel electrophoresis with a concentration of 1% (w / v). The sample containing genomic DNA was subjected to PCR gene amplification.

[0039] The upstream primer of PCR was fD1: 5'-agagtttgatcctggctcag-3' (as shown in SEQ ID NO.2);

[0040] Downstream primer rP2: 5'-cggctaccttgttacgactt-3' (as shown in SEQ ID NO. 3).

[0041] The PCR reaction system is as follows: 0.5 μL of genomic DNA of the strain is added, the final concentration of the primer is 0.4 μM, the DNA polymerase is about 2.5 U, the total reaction volume is 50 μL, and the remaining volume is made up with sterile water.

[0042] The reaction conditions of PCR were as follows: denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute, for a total of 30 cycles.

[0043] The PCR product was about 1.5 kb. It was subjected to 1% agarose gel electrophoresis, stained with 0.01% ethidium bromide, and observed under an ultraviolet analyzer. The product with a bright band at about 1.5 kb was sequenced, and the sequencing result is shown in SEQ ID NO.1.

[0044] The 16S rDNA sequence of the strain of the present invention was compared with the 16S rDNA sequence published in GenBank. The 16S rDNA sequence was similar to but not completely identical to the 16S rDNA sequences of other Bacillus strains. The strain was initially identified as Bacillus, and the strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.31938.

[0045] Example 3 Production of coating liquid containing acetoin by fermentation of reconstituted tobacco leaf coating liquid

[0046] The strain H64 was scratched with an inoculation loop and inoculated into a test tube containing a culture medium, wherein the culture medium was an LB culture medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 20 g / L glucose) supplemented with 2 mL of glucose, and cultured at 180 rpm for 12 hours to activate Bacillus H64. The activated strain was inoculated into 20 mL of reconstituted tobacco leaf coating liquid at an inoculum amount of 2%, and cultured in a shaking incubator at 180 r / min and 37° C. for 18 hours to obtain a fermentation liquid, i.e., 20 mL of a coating liquid containing acetoin. The acetoin yield was then determined by the colorimetric method in Example 1, and the final acetoin yield was 0.084 g.

[0047] Among them, the main components of the reconstituted tobacco leaf coating liquid used in this embodiment are: sugar content is 200 mg / g, nitrogen content is about 5 mg / g, organic acid content is 50 mg / g, total amino acid content is about 6 mg / g, and metal ion content is 0.55 mg / g.

[0048] Qualitative detection of acetoin: The fermentation broth was centrifuged at 12000r / min for 10min, filtered through a 0.22μm membrane, and then qualitatively detected by GC-MS. An Agilent HP-INNOWax capillary column (30m×0.32mm, 0.25μm.) was used, and the temperature program was 50℃ for 2 minutes, then 10℃ / min to 240℃, and then maintained at 240℃ for 3 minutes. The mass spectrum obtained is as follows Figure 1 As shown, it was determined to be acetoin after comparison with the standard library.

[0049] Application Example 1

[0050] 20 mL of the coating liquid containing acetoin obtained in Example 3 was treated at 120 degrees for 5 minutes, and then coated on a 7 g blank substrate of reconstituted tobacco leaves. After natural drying, the reconstituted tobacco leaves were cut into shreds to obtain filamentous reconstituted tobacco leaves. The filamentous reconstituted tobacco leaves were then placed in a constant temperature and humidity chamber with a humidity of 55% and a temperature of 25°C, and then taken out after an equilibrium time of 25 hours. The acetoin content therein was measured to be 125 μg / g.

[0051] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

[0052] SEQ ID NO.1

[0053]

[0054] SEQ ID NO.2 agagtttgat cctggctcag 20 SEQ ID NO.3 cggctacctt gttacgactt 20

Claims

1. A strain of Bacillus producing acetoin, which is classified and named Bacillus sp., with strain number H64, and was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on September 11, 2024, with the deposit number CGMCC No.31938.

2. Use of the acetoin-producing Bacillus according to claim 1 in the fermentation production of acetoin.

3. Use of the acetoin-producing Bacillus according to claim 1 in enhancing the flavor and quality of reconstituted tobacco leaves.

4. A method for preparing a coating material containing acetoin using the Bacillus according to claim 1, characterized in that: The method comprises the following steps: Step (1), inoculating Bacillus H64 into LB medium, and culturing until OD600 is 2.0-3.0 to obtain seed solution; Step (2), inoculating the Bacillus seed liquid obtained by culture in step (1) into the reconstituted tobacco leaf coating liquid at an inoculation rate of 1-3% (v / v), and culturing at 30-37° C. and 180 rpm for 4-12 hours to obtain the coating liquid containing acetoin.

5. A reconstituted tobacco leaf coated with the acetoin-containing coating liquid according to claim 4.

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