Bacillus cereus H5 and application thereof

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to bacillus cereus H5 and application thereof, the bacillus cereus H5 has been preserved in China General Microbiological Culture Collection Center on September 11, 2024, and the preservation number is CGMCC No.31937. The bacillus cereus H5 has been applied to the field of microbial fermentation. The bacillus cereus H5 obtained through screening can grow in reconstituted tobacco coating liquid, and the coating liquid can be directly used as a nutrient source for fermentation production of propyl acetate.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to Bacillus cereus H5 and application thereof. Background Art

[0002] Reconstituted tobacco is a product made by reorganizing tobacco stems, broken tobacco flakes, tobacco dust or low-grade tobacco leaves by physical or chemical methods and adding coating liquid. Reconstituted tobacco is widely added to cigarette products because it can reduce the tar content of cigarettes and improve the smoking safety of tobacco products. Among them, by adjusting the content of each aroma component in the coating liquid, the purpose of improving the overall aroma of reconstituted tobacco leaves or giving reconstituted tobacco leaves a special aroma can be achieved.

[0003] The unadjusted coating liquid is a product obtained by transferring the main aroma components such as sugar, nitrogen, and organic acid in the tobacco raw material to the extract by means of extraction, and then concentrating the extract. However, the unadjusted coating material often has the problem that the aroma characteristics are not prominent and the aroma type is single. For this reason, the technicians mainly change the content of each flavor component in the coating liquid by directly adding tobacco flavors and spices to improve the suction quality of reconstituted tobacco leaves and enrich the aroma type of reconstituted tobacco leaves. However, according to the preparation process of the reconstituted tobacco leaf coating liquid, the reconstituted tobacco leaf coating liquid contains rich nutrients (such as glucose, fructose, amino acids, trace elements), which is a good microbial aroma raw material, but because the coating liquid is a concentrated liquid, its osmotic pressure is large, and the content of toxic substances such as nicotine is relatively high, and there are high requirements for microbial fermentation technology and the tolerance of corresponding microorganisms. Therefore, the technology of directly using microbial fermentation technology to adjust the content of each flavor component in the coating liquid has not been reported.

[0004] Among them, propyl acetate (also known as propyl acetate, n-propyl acetate or n-propyl acetate) has a soft fruity aroma and is often added to the coating liquid as a fruity flavor to give the reconstituted tobacco leaves a fruity aroma. The existing technology mainly changes the content of propyl acetate in the coating liquid of the reconstituted tobacco leaves by direct addition, and there is no report on the method of directly producing propyl acetate using the coating liquid as a raw material by microbial fermentation.

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

[0006] The invention provides a strain of Bacillus cereus H5, which can directly ferment and produce propyl acetate with a coating liquid as a 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] The first aspect of the present invention provides a Bacillus cereus H5, which has been deposited in the General Microbiology Center of the China Culture Collection Administration on September 11, 2024, with a deposit number of CGMCC No. 31937, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0009] The Bacillus cereus H5 of the present invention is a Gram-positive bacillus with spores, producing mesophytic or sub-mesophytic round or columnar spores, and the spore capsules have no obvious swelling. The size of the bacteria observed under a microscope is 1.0-1.2 μm x 3.0-5.0 μm. On the surface of LB solid culture medium, the bacteria form round or nearly round, soft, non-pigmented, slightly shiny white colonies (candle-like color). The 16SrDNA sequence (as shown in SEQ ID NO.1) obtained by PCR with universal primers of the above strain has 99% similarity with the 16SrDNA sequence of Bacillus cereus DXFD1, but is different.

[0010] Wherein, the upstream primer of the above PCR is fD1:5'-agagtttgatcctggctcag-3' (as shown in SEQ ID NO.2);

[0011] Downstream primer rP2: 5'-cggctaccttgttacgactt-3' (as shown in SEQ ID NO. 3).

[0012] The second aspect of the present invention provides a use of the Bacillus cereus H5 described in the first aspect in the fermentation production of propyl acetate.

[0013] The third aspect of the present invention provides a use of the Bacillus cereus H5 described in the first aspect in the production of propyl acetate by fermentation using reconstituted tobacco leaf coating liquid as raw material.

[0014] The fourth aspect of the present invention provides a method for synthesizing propyl acetate using the Bacillus cereus H5 described in the first aspect, wherein the method uses the reconstituted tobacco leaf coating liquid as a raw material and specifically comprises the following steps:

[0015] Step (1), inoculate a single colony of Bacillus cereus H5 into sterilized LB liquid medium and culture until OD 600 2.8-4.2, to obtain seed solution;

[0016] Step (2), inoculating the seed solution prepared in step (1) into the reconstituted tobacco leaf coating solution at an inoculation rate of 1-5%, culturing in a shaker at 30-37° C. and 180 rpm for 8-24 hours to prepare a coating solution containing propyl acetate.

[0017] The present invention also provides a reconstituted tobacco leaf, wherein the substrate of the reconstituted tobacco leaf is coated with the coating liquid containing propyl acetate as described above. 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.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The Bacillus cereus H5 screened by the present invention can grow in the coating liquid of reconstituted tobacco leaves, and can ferment and produce propyl acetate directly using the coating liquid as a nutrient source.

[0020] 2. The propyl acetate produced by the fermentation of Bacillus cereus H5 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

[0021] Figure 1 This is a diagram showing the mass spectrometry detection results of propyl acetate obtained by fermentation with Bacillus cereus H5 of the present invention. DETAILED DESCRIPTION

[0022] 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.

[0023] Example 1 Screening of Bacillus cereus H5

[0024] (1) The culture medium used in this embodiment is as follows:

[0025] Solid screening medium: tryptone 5g / L, NaCl 3g / L, MgSO4·7H2O, 8g / L, tobacco chips 50g / L, agar 20g / L;

[0026] Liquid screening medium: tryptone 5g / L, NaCl 3g / L, MgSO4·7H2O, 8g / L, tobacco chips 50g / L.

[0027] (2) The specific screening method is as follows:

[0028] Take a tobacco leaf sample in the middle stage of aging and dissolve it in 5 mL of water for 1 g of sample. Place the water sample at room temperature for 24 hours to activate it. 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 Samples at different dilutions.

[0029] Take different concentrations of diluted sample bacterial solution and spread it on the above solid screening medium. After culturing for 36 hours, pick a single colony and purify it on the same solid medium until a single colony is obtained.

[0030] The purified single colony was inoculated into liquid screening medium, cultured at 37°C and 180 rpm for 48 h, and subjected to GC-MS detection. Finally, a strain capable of producing high amounts of propyl acetate was obtained and named H5.

[0031] Example 2 Identification of strain H5

[0032] (1) The culture medium and materials used in this example are as follows:

[0033] LB medium is: yeast powder 5g / L, peptone 10g / L, sodium chloride 4g / L, pH 7.0; wherein the LB medium needs to be sterilized at 120°C for 15 minutes before use.

[0034] LB solid medium: add 20 g / L agar to the above LB medium.

[0035] TAE: 50x TEA buffer containing 2M tris-acetate, 0.05M EDTA, pH 8.3

[0036] Agarose electrophoresis gel: 1x TAE electrophoresis buffer was added with 1.0% (w / v) agarose gel.

[0037] (2) The specific identification methods are as follows:

[0038] The screened bacteria were cultured in LB liquid medium, and after 12 hours, centrifuged to extract genomic DNA, and then the DNA was electrophoresed using the agarose gel electrophoresis described above. The samples containing genomic DNA were 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: add 0.5 μl of the strain genomic DNA to adjust to an appropriate concentration, the primer concentration is 0.4 μM, the dNTP concentration is 0.2 mM, the DNA polymerase is about 2.5 U, and the total reaction volume is 50 μl.

[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 sequencing result after sequencing the PCR product is shown in SEQ ID NO.1. The 16S rDNA sequence of the strain was homologously compared with the 16S rDNA sequence published in GenBank. The 16S rDNA sequence was similar to but not identical to the 16S rDNA sequence of Bacillus cereus. The strain was preliminarily identified as Bacillus cereus. The strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number: CGMCC No.31937.

[0044] Example 3 Producing a coating liquid containing propyl acetate by fermentation of a reconstituted tobacco leaf coating liquid

[0045] The strain was streaked with an inoculation loop and inoculated into 5 mL of LB liquid culture medium, and cultured at 37° C. and 120 rpm for 12 hours to activate Bacillus cereus H5. A culture medium with the reconstituted tobacco leaf coating liquid as a carbon source was taken, and the activated Bacillus cereus H5 was inoculated at an inoculum amount of 2%, and inoculated into a 250 mL shaking flask containing 50 mL of the culture medium with the reconstituted tobacco leaf coating liquid as a nutrient source, and cultured in a shaking incubator at 120 r / min at 37° C. After culturing for 48 hours, the obtained fermentation liquid was a coating material containing 20 μg / g of propyl acetate.

[0046] 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.

[0047] Detection of propyl acetate: Take the above fermentation broth and centrifuge it at 15000r / min for 5min, filter it with 0.22μm membrane, and use GC-MS for qualitative detection. Use Agilent HP-INNOWax capillary column (30m×0.32mm, 0.25μm.), the temperature program is 40℃ for 6 minutes, then 5℃ / min program temperature increase to 100℃, 15℃ / min program temperature increase to 240℃ for 3 minutes. The injection port temperature is 250℃, the ion source temperature is 230℃, and the mass spectrum is as follows: Figure 1 As shown, after comparison with the standard library, it was found to be propyl acetate.

[0048] 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.

[0049] SEQ ID NO.1

[0050]

[0051] SEQ ID NO.2agagtttgat cctggctcag 20SEQ ID NO.3cggctacctt gttacgactt20.

Claims

1. A Bacillus cereus H5, characterized in that The Bacillus cereus H5 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on September 11, 2024, with the deposit number CG MCC No.31937.

2. Use of the Bacillus cereus H5 according to claim 1 in the fermentation production of propyl acetate.

3. Use of the Bacillus cereus H5 according to claim 1 in the fermentation production of propyl acetate using reconstituted tobacco leaf coating liquid as raw material.

4. A method for synthesizing propyl acetate using the Bacillus cereus H5 of claim 1, characterized in that: The method uses the reconstituted tobacco leaf coating liquid as a raw material and specifically comprises the following steps: Step (1), inoculate Bacillus cereus H5 into LB liquid medium and culture until OD 600 to 2.8-4.2, to obtain seed solution; Step (2), inoculating the seed solution prepared in step (1) into the reconstituted tobacco leaf coating solution at an inoculation rate of 1-5%, culturing at 30-37° C. and 180 rpm for 8-24 hours to prepare a coating solution containing propyl acetate.

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

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