Bacillus velezensis and application thereof

Abstract

The invention discloses a bacillus velezensis strain and an application of the bacillus velezensis strain. The preservation number of the bacillus velezensis strain is CCTCC (China Center for Type Culture Collection) NO: M20231614. The bacillus velezensis BLS-2 can be used for fermenting the cigar tobacco leaves to decompose starch and fibers, the problems of bitterness, acerbity and the like caused by high content of macromolecular substances in cigars are solved, the quality of the cigars is further improved, and the bacillus velezensis BLS-2 has better colonization capability on the cigar tobacco leaves and can be applied to the cigar tobacco leaves. The effect can be continuously achieved after spraying.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a Velezella sp. strain and application thereof. Background Art

[0002] Cigars refer to cigarette products made from cigar tobacco leaves. Compared with tobacco leaves used for cigarettes, cigar tobacco leaves have a relatively more fragrant aroma and higher smoke concentration. Starch and cellulose are important components of tobacco leaves, which affect the quality of cigars. In burning cigarettes, starch affects the burning speed and integrity of tobacco shreds, and also affects the aroma reaction of tobacco shreds due to the unpleasant burnt smell produced when starch burns; at the same time, cellulose combustion produces a pungent smoke smell and tastes bitter. Therefore, proper degradation of starch and cellulose is the key to improving the quality of tobacco leaves. Fermentation is an important process to improve the physical and chemical properties and quality of cigar tobacco leaves, and it can also reduce irritating and disgusting odors. The flavor of cigar tobacco leaves determines the quality of finished cigar tobacco leaves, and the enhancement of flavor generally relies on microbial fermentation. In the traditional fermentation process, the biochemical reaction is changed and the quality of tobacco leaves is improved by changing the fermentation conditions such as temperature, humidity, time, and initial moisture content of tobacco leaves. However, the effect of traditional fermentation methods on improving sensory quality is limited. For most cigar tobacco leaves, the defects such as insufficient aroma value and quality, and confusion of the main aroma cannot be ignored. Therefore, adding beneficial microorganisms to degrade the content of macromolecular substances such as starch and cellulose in tobacco leaves during the fermentation process plays an important role in improving the quality of cigars and forming a unique flavor.

[0003] Currently, the publicly available Bacillus Velezii is mostly used in the fields of disease control and growth promotion of agricultural crop spiders, and there are no reports of its use in degrading starch, cellulose, etc. in tobacco leaves to improve tobacco leaf quality. Summary of the invention

[0004] In order to overcome the problems existing in the prior art, the present invention provides a strain of Bacillus Velezii and application thereof.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions:

[0006] The first aspect of the present invention provides a strain of Bacillus velezensis, wherein the deposit number of the Bacillus velezensis is CCTCCNO: M20231614.

[0007] The second aspect of the present invention provides a bacterial agent, wherein the active ingredient of the bacterial agent is the Bacillus Velezii described in the first aspect.

[0008] The third aspect of the present invention provides the use of the Bacillus Velez subtilis described in the first aspect and the bacterial agent described in the second aspect in the fermentation of cigar tobacco leaves for starch and fiber decomposition.

[0009] Through the above technical solutions, the present invention can at least achieve the following beneficial effects:

[0010] First, Bacillus velezensis CCTCC NO.M20231614 provided by the present invention has multiple functions during the fermentation process of cigars. It has been confirmed that Bacillus velezensis BLS-2 forms a clear zone on the culture medium containing starch and cellulose, indicating that this strain can produce both amylase and cellulase. At the same time, it has been confirmed that when this strain is sprayed during the cigar fermentation process, it can effectively degrade the starch content in cigar tobacco leaves. The starch degradation rate is 37.7% after 18 days of tobacco leaf fermentation and 43.5% after 35 days of tobacco leaf fermentation. Adding this strain during the cigar tobacco leaf fermentation process can improve problems such as bitterness and astringency caused by high macromolecule substance content in cigars, thereby improving the quality of cigars.

[0011] Second, Bacillus velezensis BLS-2 provided by the present invention is a strain isolated from fermented Dominican tobacco leaves. Compared with strains isolated from other environments and samples and many strains artificially cultivated by genetic engineering modification or induced mutation and other methods in the prior art, Bacillus velezensis BLS-2 has better colonization ability for cigar tobacco leaves, which is conducive to its continuous exertion of effects after spraying. At the same time, as a smoking product, cigars are more green and safe with native bacteria.

[0012] Third, the Bacillus velezensis provided by the present invention is propagated in nutrient broth as a culture medium and then centrifuged. The centrifuged bacterial cells can be diluted with mineral water and then used for spraying. The ratio of mineral water to fermentation supernatant is 1:1. It has the characteristics of wide raw material sources, low cost, environmental friendliness, and simple preparation method. At the same time, this method of adding the strain will not introduce other substances to affect the fermentation process except for the strain itself; cigar fermentation is a relatively complex process, and adding any additional substance is likely to affect its flavor and tobacco leaf quality. Therefore, this method helps to improve the quality of cigar tobacco leaves. Brief Description of the Drawings

[0013] Figure 1 is the phylogenetic tree of strain BLS-2 in the present invention;

[0014] Figure 2 is the physical diagram of the research experiment on the starch-degrading and fiber-degrading abilities of the strain in the present invention. The left diagram in the figure is the physical diagram of starch degradation, and the right diagram is the physical diagram of fiber degradation;

[0015] Figure 3 is the starch degradation situation of the strain during the fermentation process of Yunxue No. 39 cigar tobacco leaves; Biological Deposit

[0016] The Bacillus velezensis provided by the present invention, with the strain number BLS-2, is taxonomically named: Bacillus velezensis BLS-2. It was deposited at the China Center for Type Culture Collection on September 5, 2023. The address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The strain deposit number is CCTCC NO: M20231614. Detailed implementation manners

[0017] Unless otherwise specified, the materials and reagents used in the present invention are commercially available.

[0018] In the present invention, Bacillus velezensis BLS-2 and Bacillus velezensis CCTCC NO: M20231614 are the same strain, and they have the same meaning. Their names (numbers) can be used interchangeably.

[0019] Example 1

[0020] This example is used to illustrate the isolation, identification, and preservation of Bacillus velezensis BLS-2 provided by the present invention.

[0021] (I) Strain isolation and purification

[0022] During the research process, the inventors isolated a strain numbered BLS-2 from the fermented Dominican cigar tobacco leaves provided by the Yuxi Company of Yunnan Tobacco Company using the dilution plating method.

[0023] (II) Strain identification

[0024] 1. Molecular identification

[0025] 16S rDNA was used for gene sequencing to construct a phylogenetic tree. First, the strain was subjected to PCR amplification, and the PCR amplification product was extracted for sequencing. The sequencing results are shown as SEQ ID NO.1 in Table 1 below. The sequencing results were input into the NCBI website for data comparison to find the sequences related to this bacterial strain and calculate the similarity between them; finally, the MEGA.11.0 software was used to construct a phylogenetic tree, and the phylogenetic tree is as shown in the appendix Figure 1 It was identified that the strain isolated in the present invention is Bacillus velezensis of the genus Bacillus.

[0026] Table 1 Nucleotide sequence table

[0027]

[0028]

[0029] (3) Research on the enzyme-producing ability of the strain 1. Starch-degrading ability of the strain

[0030] The purified BLS-2 was inoculated into a nutrient broth medium for strain cultivation. The cultivated strain was then inoculated again into a starch medium, and the starch medium was placed upside down in an incubator at 28°C for 2 - 3 days. After the strain grew, it was stained with Lugol's iodine solution. The starch medium contains soluble starch, and soluble starch turns blue when it meets iodine. After turning blue, observe whether there is an aperture around the strain. If there is a transparent aperture around the strain, it indicates that the strain produces amylase, and the strain has produced amylase to decompose the surrounding starch. Observe the aperture production situation and measure the aperture diameter and colony diameter. Each treatment was set with three replicates. The amylase production situation of strain BLS-2 is as shown in the appendix. Figure 2 as follows.

[0031] Starch medium: 10 g soluble starch, 5 g peptone, 2.5 g sodium chloride, 2.5 g beef extract, 7.5 - 10 g agar powder, 500 mL distilled water, pH: 7.0 - 7.2. Sterilize with an autoclave for half an hour at a temperature of 121°C.

[0032] Lugol's iodine solution: 0.5 g iodine, 1.0 g potassium iodide, 150 mL distilled water. Sterilize with an autoclave for half an hour at a temperature of 121°C.

[0033] 2. Cellulose-degrading ability of the strain

[0034] After the purified BLS-2 was inoculated on an LB plate and cultured for 2 days, a small amount of colonies was picked up with a sterile inoculation loop and inoculated onto a carboxymethyl cellulose sodium medium (cellulase-producing differential medium) for culture. It was placed upside down in an incubator at 28°C for 2 - 3 days. Observe the aperture production situation and measure the aperture diameter and colony diameter. Each treatment was set with three replicates. The cellulase production situation of strain BLS-2 is as shown in the appendix. Figure 2 as follows.

[0035] Carboxymethyl cellulose sodium medium: 0.04 g congo red, 8 g CMC-Na, KH 2 PO 4 1 g, 15 g agar, 100 mL potato juice, made up to 1 L with distilled water, pH value 7.0. Sterilize with an autoclave for half an hour at a temperature of 121°C. It is used to screen microorganisms with the function of degrading pectin.

[0036] (4) Strain preservation

[0037] Bacillus velezensis, strain number BLS-2, its taxonomic name is: Bacillus velezensis BLS-2, which was deposited at the China Center for Type Culture Collection on September 5, 2023. The address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, and the deposit number is CCTCC NO: M20231614.

[0038] Example 2

[0039] Amylolytic ability of strain BLS-2

[0040] 1. Preparation of fermentation aid for cigar tobacco

[0041] Materials: 1 g of syrup, 10 mL of apple cider vinegar, 10 g of cinnamon, 100 g of tobacco midrib, 100 mL of sugarcane wine (above 50 degrees), 100 mL of ultrapure water.

[0042] (1) Mix syrup and apple cider vinegar in a ratio of 1:10 to obtain stock solution A;

[0043] (2) Mix cinnamon and tobacco midrib evenly with water and heat to boiling, with a ratio of 1:10. Take the supernatant, and filter it after the temperature drops to obtain stock solution B;

[0044] (3) Mix the above stock solution A, stock solution B and sugarcane wine in a ratio of 1:10:10 to obtain a mixed solution, and mix evenly to obtain the fermentation stock solution;

[0045] (4) Mix the above fermentation stock solution and water evenly at a ratio of 1:50 to obtain the fermentation aid.

[0046] 2. Fermentation test of cigar tobacco

[0047] (1) Inoculate strain BLS-2 in the frozen tube into three bottles of 200 mL NB culture medium, shake at 180 r / min at 28 °C for 48 h. After centrifuging the bacterial solution at 4000 rpm for 20 min, dissolve the centrifuged cells in 450 mL of glucose water as the strain fermentation solution for standby.

[0048] (2) Fermentation of cigar tobacco

[0049] Select Yunxue No. 39 cigar tobacco leaves with uniform size, no mechanical damage and no mold infection for fermentation. The CK group was sprayed with 300 mL of fermentation aid and 300 mL of glucose solution, and the treatment group was sprayed with 300 mL of fermentation aid and 300 mL of strain fermentation broth. Each treatment had 5 kg of tobacco leaves, and each treatment was repeated three times. The tobacco leaves were fermented in a fermentation box at a temperature of 35 °C and a relative humidity of 75% for 35 days. Samples were taken and the starch content of the tobacco leaves was measured on the 18th and 35th days of fermentation. After 18 days of tobacco leaf fermentation, the starch content of the CK group was 41.4 mg / g, the starch content of the treatment group was 25.8 mg / g, and the starch degradation rate was 37.7%; after 35 days of tobacco leaf fermentation, the starch content of the CK group was 37.5 mg / g, the starch content of the treatment group was 21.2 mg / g, and the starch degradation rate was 43.5%. The effect of strain BLS-2 on degrading the starch content of fermented cigar tobacco leaves is as shown in Figure 3 shown.

[0050] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in terms of form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A strain of Bacillus Velez, characterized in that: The deposit number of the Bacillus velezensis is CCTCC NO: M20231614.

2. A bacterial agent, the active ingredient of which is the Bacillus Velezii described in claim 1.

3. Use of the Bacillus Velezii according to claim 1 or the bacterial agent according to claim 2 in the fermentation of cigar tobacco leaves to degrade starch and defibrinate.

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