Salt-tolerant bacillus and application thereof

Abstract

The invention provides a salt-tolerant bacillus and application thereof, and belongs to the technical field of microorganisms, the salt-tolerant bacillus is named as the salt-tolerant bacillus CC and preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M 20251509. The salt-tolerant bacillus CC provided by the invention is a strain separated from tobacco leaves, and has better colonization ability and environmental adaptability; and meanwhile, the compound enzyme has pectinase producing activity, can reduce pectin to 17.2% in the cigar tobacco leaf fermentation process, remarkably reduces the bitter taste of cigars, can improve the fragrance and flavor of cigars, reduces the generation of unpleasant odor and irritant substances, remarkably improves the sensory quality score of cigars, and improves the overall quality of tobacco leaves.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a salt-tolerant Bacillus and application thereof. BACKGROUND

[0002] In recent years, with the rapid development of China's economy and the continuous improvement of the consumption level of residents, the market demand for cigar tobacco shows a significant growth trend. Cigar tobacco is a kind of cigarette product, which refers to a smoking cigarette entirely made of tobacco leaves. Pectin is a complex polysaccharide substance, mainly existing in the primary cell wall and intercellular layer of higher plants. The content and degradation degree of pectin in tobacco directly affect the processing performance and combustion characteristics of tobacco leaves. High pectin content will increase the stickiness of tobacco leaves and affect the rolling process, and pectin combustion will produce methanol, affecting the safety of smoking. Studies have shown that high content of pectin substances will cause incomplete combustion of tobacco and produce methanol, which is further oxidized into formaldehyde, formic acid and the like, adversely affecting the smoking and safety of tobacco. Therefore, the content of pectin has become an important indicator for evaluating the quality of tobacco and tobacco products, and reducing the pectin content in tobacco leaves is of great significance to improve the quality of tobacco.

[0003] The main processes of cigar production include cultivation, airing, fermentation, rolling and aging, and fermentation is the key step to improve the quality of cigar tobacco leaves. During the natural fermentation process of tobacco leaves, the pectin degradation efficiency is low, which often leads to quality problems such as green and bitter taste, and further highlights the important role and application potential of microbial fermentation technology in improving the quality of domestic cigar. SUMMARY

[0004] In view of the problems in the prior art, the present application provides a salt-tolerant Bacillus and its application. The present application uses high-quality cigar tobacco leaves and cigarette sticks as the screening material of beneficial strains, and a salt-tolerant Bacillus capable of efficiently degrading pectin is screened therefrom to degrade pectin in tobacco leaves, thereby further improving the quality and safety of tobacco leaves.

[0005] To achieve the above-mentioned purpose, the present application solves its technical problems through the following technical solutions:

[0006] The application provides a Bacillus halotolerans, and the strain number is CC, the taxonomic name is Bacillus halotolerans CC, and the Bacillus halotolerans CC is preserved in the China Center for Type Culture Collection on July 2, 2025, and the address is No. 299, Bajiyi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, and the preservation number is CCTCC NO: M 20251509.

[0007] The application provides a bacterial agent, and the active ingredient of the bacterial agent is the Bacillus halotolerans CC.

[0008] The application provides the application of the Bacillus halotolerans CC or the bacterial agent in the fermentation of pectin, the increase of total nitrogen, the increase of total sugar, the increase of reducing sugar, the increase of free amino acid and the reduction of nicotine content.

[0009] Further, the fermentation method of the cigar leaf is that bacterial liquid prepared from the Bacillus halotolerans CC and fermentation aids are sprayed on the surface of the cigar leaf to perform fermentation.

[0010] Further, the preparation material of the fermentation aid comprises 1 g of sugar syrup, 10 mL of apple vinegar, 10 g of cassia, 100 g of tobacco main veins, 100 mL of 50-degree sugarcane wine and 100 mL of ultrapure water.

[0011] The preparation method of the fermentation aid is as follows.

[0012] Step (1) mixing the sugar syrup and the apple vinegar to obtain a stock solution A;

[0013] Step (2) uniformly mixing the cassia and the tobacco main veins with ultrapure water and heating to boiling, taking supernatant, and filtering after temperature drop to obtain a stock solution B;

[0014] Step (3) mixing the stock solution A, the stock solution B and the sugarcane wine to obtain a mixed solution, and uniformly mixing to obtain a fermentation stock solution;

[0015] Step (4) additionally taking ultrapure water, uniformly mixing the fermentation stock solution and the ultrapure water at a ratio of 1:50 to obtain the fermentation aid.

[0016] Further, the preparation method of the bacterial liquid is as follows.

[0017] Step (1) inoculating the Bacillus halotolerans CC in the LB liquid medium, and culturing at 30 DEG C and 180 r / min for 24 h to obtain the first generation culture;

[0018] Step (2) re-inoculate the first generation culture into LB liquid medium at an inoculation amount of 2%, and culture at 30 DEG C and 180 r / min for 24 h to obtain the second generation culture;

[0019] Step (3) centrifuge the second generation culture at 5000 r / min for 15 min, discard the supernatant, retain the bacterial body, and rinse with 0.05% glucose solution to prepare a bacterial liquid, so that the effective viable bacterial number of Bacillus CC in the bacterial liquid is not less than 1.0 x 10 8 CFU / mL.

[0020] Further, the volume ratio of the bacterial liquid to the fermentation aid in the third aspect is 1:1.

[0021] Further, the fermentation temperature in the third aspect is 35 DEG C, the humidity is 75%, and the fermentation time is 35 d.

[0022] The fourth aspect of the present application provides an application of the Bacillus CC in the first aspect or the bacterial agent in the second aspect in changing the microbial community structure of cigar tobacco leaves, and the change of the microbial community structure includes: the abundance of Bacillota and Staphylococcus is increased; the abundance of Chryseobacterium and Rhodococcus is decreased; the abundance of Ascomycota is decreased, the abundance of Basidiomycota is increased, the abundance of Aspergillus and Cladosporium in Ascomycota is decreased, and the abundance of Wallemia is increased, and the cigar tobacco leaf is Yunxue No. 39, and the part is middle tobacco.

[0023] Through the above technical solution, the present application can at least achieve the following beneficial effects:

[0024] Firstly, the Bacillus CC provided by the present application is a strain isolated from tobacco leaves, and compared with strains isolated from other environments and samples and strains artificially cultivated by many genetic engineering modification or induced mutation methods in the prior art, the beneficial microorganism obtained has better colonization ability and environmental adaptability for tobacco leaves, is beneficial to continuously play a role, and the original strain is more green and safe.

[0025] Secondly, the salt-tolerant Bacillus CC provided by the application can directly observe transparent circles around the colonies on a pectinase production medium, indicating that the strain has pectinase activity; in terms of pectin content, the addition of the strain significantly reduces the pectin content in tobacco leaves to 17.2%, rapidly degrades pectin in tobacco leaves, reduces the accumulation of degradation products in tobacco leaves, significantly reduces the bitterness of cigars, and improves the overall quality of tobacco leaves.

[0026] Thirdly, in the cigar tobacco leaves fermented by the salt-tolerant Bacillus CC provided by the application, the total nitrogen content reaches about 4.34%, which is higher than that of CK; the addition of the salt-tolerant Bacillus CC strain increases the total sugar and reducing sugar contents in tobacco leaves, which is significantly higher than that of the CK group, indicating that the salt-tolerant Bacillus CC promotes the hydrolysis of polysaccharides and the accumulation of sugars, which is beneficial to improve the flavor of tobacco leaves. The free amino acid content of the tobacco leaves added with the salt-tolerant Bacillus CC strain is higher than that of the CK group. The nicotine content of the tobacco leaves added with the salt-tolerant Bacillus CC strain is 2.9% lower than that of the CK group. At the same time, the sensory quality score of the fermented cigar tobacco is improved compared with the CK group.

[0027] Fourthly, in the cigar tobacco leaves fermented by the salt-tolerant Bacillus CC provided by the application, the addition of the salt-tolerant Bacillus CC affects the microbial community structure and regulates the metabolic relationship between microorganisms, realizes microecological regulation and metabolic chain transmission, thereby adapting to the complex chemical environment of fermented tobacco leaves and improving the quality of tobacco leaves. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Figure is the transparent circle result of strain CC on the preliminary screening medium;

[0029] Figure 2 Figure is the glucose standard curve;

[0030] Figure 3 Figure is the pectinase enzyme activity graph of each strain;

[0031] Figure 4 Figure is the single colony morphology and electron microscope graph of strain CC, wherein the left graph is the colony morphology graph of salt-tolerant Bacillus CC; and the right graph is the electron microscope graph of salt-tolerant Bacillus CC;

[0032] Figure 5 Figure is the phylogenetic tree of strain CC based on 16S rDNA gene sequence;

[0033] Figure 6 Figure is the growth curve graph of salt-tolerant Bacillus CC;

[0034] Figure 7 Figure is the acid-base environment tolerance result of salt-tolerant Bacillus CC;

[0035] Figure 8 Results of tolerance of Bacillus halotolerans CC to temperature;

[0036] Figure 9 Results of tolerance of Bacillus halotolerans CC to bile salt;

[0037] Figure 10 Figure of content difference of pectin, total nitrogen, total sugar, free amino acid, nicotine and reducing sugar of different treatment groups;

[0038] Figure 11 Figure of relative abundance of bacterial community of different treatment cigar at the level of door;

[0039] Figure 12 Figure of relative abundance of bacterial community of different treatment cigar at the level of door;

[0040] Figure 13 Figure of relative abundance of fungal community of different treatment cigar at the level of door;

[0041] Figure 14 Figure of relative abundance of fungal community of different treatment cigar at the level of door.

[0042] Biological preservation

[0043] The Bacillus halotolerans provided by the application has a strain number of CC, and its taxonomic name is Bacillus halotolerans CC. The Bacillus halotolerans CC has been preserved in the China Center for Type Culture Collection on July 2, 2025, and is located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, and has a strain preservation number of CCTCC NO: M 20251509. DETAILED DESCRIPTION

[0044] The cigar leaf sample used for isolating microorganisms is collected from a cigar leaf curing workshop in Hekou, Mosha Town, Xingping Yi and Dai Autonomous County, Yuxi City, Yunnan Province, and the varieties are Yunxue No. 1 and Yunxue No. 39. In addition, high-quality finished cigars from Cuba, the Dominican Republic and Nicaragua are purchased, and all samples are taken back to the laboratory for microbial isolation and culture. The tobacco variety used in the fermentation test is Yunxue No. 39, and the part is the middle tobacco.

[0045] The culture media used in the test are specific nutrient combinations for culturing and propagating microorganisms, which are selected according to the required microorganism type and test purpose. The culture media involved in the test are shown in Table 1:

[0046] Table 1 Required culture medium for the test

[0047]

[0048] Unless otherwise indicated, the materials, reagents used in the present application are commercially available.

[0049] In the present application, the salt-tolerant Bacillus CC, the salt-tolerant Bacillus CCTCC NO: M 20251509 and the strain CC are the same strain, and the three have the same meaning, and their names (numbers) can be used interchangeably.

[0050] In the present application, the data processing uses Excel 2019 software to statistically analyze the strain HC value, conventional indicators of cigar tobacco, and sequencing data. SPSS 27.0 software is used to analyze the conventional indicators of cigar tobacco, and the alpha diversity index is analyzed by One-way ANOVA combined with LSD test to process the difference significance between strains. All data are plotted using Excel 2019 software and Origin 2021 software. The difference of microbial groups after fermentation is processed by Shanghai Meiji Pharmaceutical Technology Co., Ltd. Biological information analysis cloud platform.

[0051] Example 1: Isolation, screening, identification and evaluation of strains

[0052] 1. Isolation of strains

[0053] Microorganisms were isolated from Yunxue No. 1, Yunxue No. 39 cigar tobacco leaves and Cuban, Dominican, Nicaraguan finished high-quality cigar tobacco. The dilution plating method was used. The tobacco leaves and cigarettes were cut into pieces with sterile scissors, 2 g of sample was taken into a mortar and 10 mL of sterile water was added for grinding. 1 mL of the grinding liquid was taken into a sterile test tube, 10 mL of sterile water was added to the 10 mL mark, the cap was covered, and it was shaken at 30°C for 30 min. 1 mL of the supernatant was gradient diluted to 10 3 , 10 4 , 10 5 times liquid, 100 μL of the diluted liquid was added to NA, NB, PDA, LB medium for uniform coating, each gradient was repeated three times, and it was cultured in a 30°C constant temperature incubator for 3 days to obtain single colonies.

[0054] 2. Preliminary screening of strains

[0055] The single colonies of different morphologies and colors on the above culture medium were collected with a inoculating loop, and purified on NA culture medium. The purified single colonies were inoculated into pectinase separation medium, pectinase screening medium, and pectinase-producing bacteria selection medium, and incubated in a 30°C constant temperature incubator for 48 hours. The transparent circle around the colonies was directly observed on the pectinase-producing medium, and the pectinase separation and screening medium was dyed with Lugol's iodine solution first, and then the transparent circle around the bacterial colonies was observed. The colony diameter d (mm) and the transparent circle diameter D (mm) around the colony were measured by cross method according to the growth rate of the colony, and the HC value (HC=D / d) was used to preliminarily determine the pectinolytic ability of the strain.

[0056] After 112 strains of bacteria were inoculated on the pectinase separation medium, pectinase screening medium, and pectinase-producing bacteria medium with pectin as the only carbon source, and incubated in a 30°C constant temperature incubator for 48 hours, the transparent circle around the colonies was directly observed on the pectinase-producing medium, and the pectinase separation and screening medium was dyed with Lugol's iodine solution first, and then the transparent circle around the bacterial colonies was observed. 15 strains of bacteria that could produce transparent circles were preliminarily screened, and 4 strains with a D / d value greater than or equal to 1.6 mm were selected and named LY, SF, NY, and CC for re-screening. The transparent circle results of strain CC on the preliminary screening medium are shown in Figure 1 The preliminary screening results of the 4 pectinase-producing strains are shown in Table 2.

[0057] Table 2 Preliminary screening results of pectinase-producing strains

[0058]

[0059] 3 Re-screening of strains

[0060] The strains that produced transparent light circles in the preliminary screening were inoculated into 150 mL of LB liquid culture medium, and incubated at 30°C and 180 r / min for 48 hours. Then the culture was centrifuged at 4°C and 10,000 r / min for 10 minutes, and the supernatant was used to determine the pectinase activity. The specific operation steps are as follows:

[0061] Preparation of DNS solution:

[0062] Take 3,5-dinitrosalicylic acid 3.15 g, add 500 mL of deionized water, stir, heat in 45°C water bath, and gradually add 100 mL of 0.2 g / mL sodium hydroxide solution while constantly stirring, pay attention to the solution temperature not more than 48°C. In turn add 91.0 g of potassium sodium tartrate tetrahydrate, 2.50 g of phenol and 2.50 g of anhydrous sodium sulfite. Continue to heat in 45°C water bath, while adding water 300 mL, stirring until completely dissolved. After cooling to room temperature, use water to constant volume to 1 L, filter with filter paper. The filtrate is stored in a brown bottle, keep away from light, store at room temperature for 7 days before use.

[0063] Preparation of glucose standard curve:

[0064] Prepare citric acid monohydrate-disodium hydrogen phosphate buffer solution of pH 6.0: take 3.672 g of citric acid (C6H807·H2O), 57.1 g of disodium hydrogen phosphate (Na2HPO4·2H2O), respectively, dissolve in 400 mL of distilled water, after completely dissolved, compound the solution, and adjust the pH to 6 with 0.1 mol / L hydrochloric acid or sodium hydroxide, constant volume to 1 L.

[0065] Prepare 1.0 mg / mL glucose standard solution: take 0.1 g of glucose and dissolve in pH 6.0 buffer solution, constant volume to 100 mL. Add 0, 0.5, 1, 2, 3, 4, 5 mL of 1 mg / mL glucose solution to 7 groups of colorimetric tubes respectively, and constant volume to 10 mL, then add 4 mL of DNS solution respectively, shake well, cool immediately after boiling water bath for 5 min, measure the absorbance at 540 nm, take the glucose content as the abscissa, and the absorbance OD 540 nm as the ordinate, draw the standard curve, the glucose standard curve is shown in Figure 2 , the linear regression equation is y=0.1119x-0.0232, R 2 =0.9975, indicating good linear fitting degree, which can be used to calculate the pectinase activity.

[0066] Determination of pectinase activity by DNS method:

[0067] Preparation of crude enzyme solution: take the culture at 4°C, centrifuge at 10000 r / min for 10 min, and the obtained supernatant is the crude enzyme solution.

[0068] Take 5 mL of 10 g / L pectin solution in a colorimetric tube, heat in 50°C water bath for 5 min, add 4 mL of buffer solution, then add 1 mL of diluted enzyme solution, react in 50°C water bath for 30 min. Take 2 mL of reaction solution and add to another colorimetric tube, add 2 mL of distilled water, then add 4 mL of DNS solution, cool immediately after boiling water bath for 5 min, and finally constant volume to 25 mL. Measure the OD value of the solution at 540 nm by spectrophotometer. The control is the crude enzyme solution inactivated by boiling water bath. Each treatment is repeated three times.

[0069] Enzyme activity definition: 1 unit of enzyme activity (U / mL) is defined as the amount of enzyme that catalyzes the hydrolysis of pectin to produce 1 μg of galacturonic acid in 1 minute under specific conditions.

[0070] The amount of galacturonic acid (W) = the amount of glucose x 194 / 180 x 1000

[0071] Pectinase enzyme activity (U / mL) = W x 5N / T

[0072] In the formula: N: dilution multiple of crude enzyme solution T: reaction time

[0073] 1000: 1 mg = 1000 μg 5: take 1 / 5 of the reaction solution (i.e. 2 mL)

[0074] The measured enzyme activity of pectinase produced by each strain is shown in Table 2. Figure 3 The results show that the strain CC has the highest enzyme activity, so strain CC is selected for subsequent experiments.

[0075] 4 Identification of the strain

[0076] 4.1 Morphological identification of the strain

[0077] The strain CC with the highest enzyme activity obtained by re-screening was inoculated on a nutrient agar (NA) plate for plate coating operation. The plate was placed in a 30°C incubator for 48 hours of culture, and the morphological characteristics of single colonies were observed and recorded, including colony size, shape, color, edge, surface texture and transparency, etc. Then, single colonies were picked from the plate, and samples were prepared for scanning electron microscopy (SEM) observation to further analyze the surface microstructure of the bacterial cells. The morphology of single colonies and electron micrographs of strain CC are shown in Table 4. Figure 4 The colonies of strain CC on the nutrient agar plate were white, and the surface colony morphology was round, with a raised middle, a neat edge, and smoothness. Under electron microscopy scanning, they were rod-shaped and smooth.

[0078] 4.2 Physiological and biochemical identification

[0079] The physiological and biochemical identification of the strain was carried out according to the Common Bacteria System Identification Manual and the Bergey's Bacteria Identification Manual, including Gram staining test, starch hydrolysis test, hydrogen peroxide test, methyl red test, V-P test, citric acid decomposition test, hydrogen sulfide test, indole test, and contact enzyme test. The results of physiological and biochemical tests of strain CC are shown in Table 3.

[0080] Table 3 Results of physiological and biochemical tests of the strain

[0081]

[0082] Note: +: positive, -: negative.

[0083] The results indicate that strain CC is a Gram-positive bacterium. A positive starch hydrolysis test demonstrates its ability to produce amylase, which breaks down starch into maltose. A positive catalase test indicates the production of catalase, which decomposes hydrogen peroxide into water and oxygen. A negative methyl red test indicates the absence of acidic products. A positive VP test indicates the production of acetylmethylethanol. A negative citric acid decomposition test indicates the inability to utilize citrate as a carbon source. A negative indole test indicates the inability to decompose tryptophan to produce indole. A positive catalase test indicates the production of an enzyme that catalyzes the decomposition of H₂O₂. A hydrogen sulfide test indicates the production of hydrogen sulfide. Based on the morphological identification and physiological and biochemical tests, strain CC can be preliminarily identified as belonging to the genus *Bacillus*.

[0084] 4.3 Molecular biological identification

[0085] Genomic DNA of the target bacteria was extracted using the Solarbio Bacterial Genomic DNA Extraction Kit. PCR amplification of the genomic DNA of pectin-degrading bacteria was performed using universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') for the bacterial 16S rRNA gene. The PCR reaction mixture consisted of a total volume of 25 µl, 12.5 µl of 2×Taq PCR Master Mix, 1 µl of 27F primer, 1 µl of 1429R primer, 1 µl of template DNA, and deionized water (H2O) to a final volume of 25 µl. PCR amplification conditions were as follows: 95℃ for 2 min, 95℃ for 30 s, 57℃ for 30 s, and 72℃ for 30 s, for a total of 35 cycles, followed by a final extension at 72℃ for 5 min. PCR products were detected by 2% agarose gel electrophoresis and observed using a gel imaging system. The amplified products were then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID NO.1.

[0086] SEQ ID NO.1:

[0087] TTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGT

[0088] GTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTA

[0089] CTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTG

[0090] AGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGT

[0091] TCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGAT

[0092] TTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGA

[0093] GTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGG

[0094] GACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACC

[0095] TGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGA

[0096] TGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGC

[0097] TCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCG

[0098] ACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGG

[0099] GCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCA

[0100] GGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTT

[0101] ACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGC

[0102] ATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTC

[0103] CCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGA

[0104] CTTAAGGAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGC

[0105] TTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTT

[0106] TCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTACTTGTTCT

[0107] TCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGC

[0108] GTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCT

[0109] CCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTC

[0110] TCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCATTACCTCACCAACTA

[0111] GCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTA

[0112] TGTTTGAACCATGCGGTTCAAACAAGCATCCGGTATTAGCCCCGGTTTCC

[0113] CGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGT

[0114] CCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTC.

[0115] The sequencing results were input into the NCBI (http: / / www.ncbi.nlm.nih.gov) database for sequence alignment analysis by BLAST. The 16S rDNA gene sequences of strains with high similarity and high homology were selected as references. The MEGA11 software was used to construct a phylogenetic tree by the neighbor-joining method. The phylogenetic tree of strain CC based on 16S rDNA gene sequences is shown in Figure 5 Strain CC clusters together with Bacillus halotolerans, and the homology between the 16S rDNA gene sequence and Bacillus halotolerans strain Z66 is 97%. Therefore, strain CC is preliminarily identified as Bacillus halotolerans in the genus Bacillus, named Bacillus halotolerans CC, and its taxonomic name is Bacillus halotolerans CC. It was preserved in the China Center for Type Culture Collection on July 2, 2025, at address No. 299, Bajiyi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, with a strain preservation number of CCTCC NO: M 20251509.

[0116] 4.4 Growth curve

[0117] Bacillus halotolerans CC strain was inoculated into a triangular flask containing LB liquid medium and cultured at 30°C and 180 r / min for 24 h to prepare a seed solution. The seed solution was inoculated into a liquid medium with a volume of 150 mL of LB liquid medium at a 2% inoculation amount. The absorbance of the bacterial solution at 600 nm was measured every 4 hours using a spectrophotometer, with uninfected LB medium as a blank control. The growth curve was plotted with time as the horizontal coordinate and OD600 as the vertical coordinate. The growth curve of Bacillus halotolerans CC is shown in Figure 6 The growth curve can be roughly divided into four stages: lag phase, logarithmic growth phase, stationary phase, and decline phase. From 0 to 10 h, the strain was just inoculated into the culture medium and needed to adapt to the new environment, synthesize metabolic intermediates such as enzymes and coenzymes, but with the extension of time, the cell number increased slowly. From 10 to 24 h, after the preparation of the lag phase, it entered the rapid growth stage, the metabolism was vigorous, and the cell number increased logarithmically. From 24 to 34 h, it entered the stationary phase, the growth rate of microorganisms gradually slowed down, the cell number reached the maximum, and the growth was relatively stable. After 34 h, it gradually entered the decline phase.

[0118] 4.5 Growth under different pH conditions

[0119] The bacterial liquid of salt-tolerant Bacillus CC strain was inoculated into LB liquid medium with pH values of 2, 3, 5, 7, 8, and 10 at an inoculation amount of 2%, and the LB liquid medium with pH value of 7 was used as a control. Each group was cultured at 30°C and 180 r / min for 24 h. The OD600 values of each treatment were determined by using a spectrophotometer, and the survival rate of the strain under different pH conditions was calculated according to the OD600 value of the control group (survival rate = OD600 value of treated bacterial liquid / OD600 value of untreated bacterial liquid x 100%). The tolerance of salt-tolerant Bacillus CC strain to acid and alkaline environments is shown in FIG. 2. Figure 7 As shown in FIG. 2, when the pH value was 2-3 in the acidic environment, the survival rate of salt-tolerant Bacillus CC was about 3.5%. As the pH value increased, the survival rate also increased. When the pH value was 5, the survival rate of salt-tolerant Bacillus CC was relatively high, reaching 95.6%. When the pH value continued to increase to 7, the survival rate of the strain decreased sharply. When the pH value increased to 10, the survival rate of the strain was about 1%. This indicates that salt-tolerant Bacillus CC can tolerate slightly acidic and slightly alkaline environments and has strong environmental adaptability.

[0120] 4.6 Growth at different temperatures

[0121] The bacterial liquid of salt-tolerant Bacillus CC strain was inoculated into LB liquid medium at an inoculation amount of 2%. The medium was placed in a water bath at temperatures of 20°C, 30°C, 40°C, 50°C, 70°C, and 100°C for 5 min, and then immediately cooled with cold water to room temperature. The LB liquid medium with a temperature of 30°C was used as a control. Each group was cultured at 30°C and 180 r / min for 24 h. The OD600 values of each treatment were determined by using a spectrophotometer, and the survival rate of the strain under different pH conditions was calculated according to the OD600 value of the control group (survival rate = OD600 value of treated bacterial liquid / OD600 value of untreated bacterial liquid x 100%). The tolerance of salt-tolerant Bacillus CC strain to temperature is shown in FIG. 3. Figure 8 As shown in FIG. 3, when the water bath temperature increased to 40°C, the survival rate of salt-tolerant Bacillus CC was 73.33%. As the temperature further increased, the survival rate of salt-tolerant Bacillus CC decreased significantly. When the temperature was 70°C, the survival rate of salt-tolerant Bacillus CC was 10%. When the temperature reached 100°C, the survival rate of the strain was 9.09%. This indicates that salt-tolerant Bacillus CC has strong tolerance to high temperature.

[0122] 4.7 Determination of salt tolerance

[0123] The bacterial liquid of salt-tolerant Bacillus CC strain was inoculated into LB liquid medium at an inoculation amount of 2%, and the medium was placed in LB liquid medium with bile salt concentrations of 0.1%, 0.3%, 0.5%, and 1%, respectively. The LB liquid medium with only the strain inoculated and without bile salt was used as a control. Each group was cultured at 30°C and 180 r / min for 24 h. The OD600 values of each treatment were measured using a spectrophotometer, and the survival rate of the strain under different pH conditions was calculated based on the OD600 value of the control group (survival rate = OD600 value of treated bacterial liquid / OD600 value of untreated bacterial liquid × 100%). The tolerance of salt-tolerant Bacillus CC strain to bile salt is shown in Table 1. Figure 9 As shown in Table 1, the survival rate of salt-tolerant Bacillus CC gradually decreased with the increase of bile salt concentration. When the bile salt concentration was 0.1%, the survival rate of salt-tolerant Bacillus CC was 80.91%. When the bile salt concentration was 0.3%, the survival rate of salt-tolerant Bacillus CC was 51.82%. When the bile salt concentration was 0.5%, the survival rate of salt-tolerant Bacillus CC was 30.61%. When the bile salt concentration was 1.0%, the survival rate of salt-tolerant Bacillus CC was about 1%. This indicates that salt-tolerant Bacillus CC has a certain tolerance to bile salt.

[0124] 4.8 Safety evaluation of pectolytic Bacillus CC strain

[0125] The paper disc diffusion method (Kirby-Bauer, K-B method) was used to determine the drug sensitivity of the strain. First, the salt-tolerant Bacillus CC strain was diluted and spread on nutrient agar plates, and then the plates were incubated at 30°C for 24 h. Four to five single colonies were picked from the cultured nutrient plates and transferred to sterile saline test tubes containing 2-3 mL of sterile saline (saline was used to dilute the bacterial body). The bacterial suspension was stirred with a loop to ensure uniform dispersion in the saline, forming a uniform suspension. Finally, the turbidity of the bacterial suspension was compared with the standard turbidity tube, and the single colony or sterile saline was adjusted to match the standard turbidity tube to ensure that the concentration of the bacterial suspension (about 1.0 × 10 8 CFU / mL) met the experimental requirements.

[0126] Take 100 μl of bacterial suspension with a spreader evenly on MH agar medium, and use sterile forceps to tightly attach antibiotic paper to the medium, 4 kinds of drug sensitive paper are attached to each medium, each kind of drug sensitive paper is tested 3 times in parallel, and the plate is placed in a 35 °C incubator and cultured for 24 h. The drug sensitive paper used in this study includes streptomycin and amikacin of aminoglycosides, erythromycin of macrolides, penicillin and ampicillin of β-lactams, chloramphenicol of chloramphenicol, ciprofloxacin and levofloxacin of fluoroquinolones, ceftriaxone and ceftriaxone of cephalosporins, lincomycin of lincosamides, and vancomycin of glycopeptides. According to the standards of the American Clinical and Laboratory Standards Institute (Clinical laboratory standard institute, CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST), the sensitivity of the target strain to the used antibiotics is judged according to the bacteriostatic circle produced by different antibiotics, the drug resistance of the strain is judged, and the sensitivity is divided into sensitive (S), resistant (R) and intermediate (I). The results of antibiotic sensitivity test are shown in Table 4:

[0127] Table 4 Results of antibiotic sensitivity test

[0128]

[0129] It is shown that the salt-tolerant Bacillus CC strain exhibits sensitivity to most of the tested antibiotics, especially in aminoglycosides, macrolides and fluoroquinolones, and only exhibits intermediate or resistance in some antibiotics, which preliminarily proves that the use of the salt-tolerant Bacillus CC strain in cigar fermentation is safe.

[0130] Example Two Cigar Fermentation Test

[0131] 1 Test Design

[0132] The test is designed for 2 treatments, treatment one: control group (CK): normal fermentation (spray 500 ml 0.05% glucose solution + fermentation aid, the volume ratio of glucose solution and fermentation aid is 1:1), treatment two: addition of salt-tolerant Bacillus CC strain (500 mL bacterial suspension + fermentation aid, the volume ratio of bacterial suspension and fermentation aid is 1:1).

[0133] Preparation of cigar leaf fermentation aid:

[0134] Materials: sugar syrup 1 g, apple vinegar 10 mL, cassia 10 g, tobacco vein 100 g, 50 ° sugarcane wine 100 mL, ultrapure water 100 mL.

[0135] The preparation method is: (1) mixing the sugar syrup and apple vinegar to obtain a stock solution A; (2) mixing the cassia bark and tobacco main vein with ultrapure water and heating to boiling, taking the supernatant, and filtering after the temperature drops to obtain a stock solution B; (3) mixing the stock solution A, the stock solution B and the sugarcane wine to obtain a mixed solution, and uniformly mixing to obtain a fermentation stock solution; (4) additionally taking ultrapure water, uniformly mixing the fermentation stock solution with the ultrapure water at a ratio of 1:50 to obtain a fermentation aid.

[0136] The salt-tolerant Bacillus CC strain separated from the surface of tobacco leaves was inoculated in LB liquid medium and cultured at 30°C with 180r / min shaking for 24h to obtain the first generation culture; the first generation culture was inoculated again in LB liquid medium at an inoculation amount of 2% and cultured at 30°C with 180r / min shaking for 24h to obtain the second generation culture; the second generation culture was centrifuged at 5000r / min for 15min, and the supernatant was discarded, and the bacterial cells were reserved and washed with 500mL of 0.05% glucose solution as a suspension for standby use (the bacterial cell concentration was about 1.0×10 8 CFU / mL).

[0137] The same variety and the same part of cigar tobacco leaves were selected for fermentation. Four stacks of 5kg of cigar tobacco leaves were stacked in a fermentation box with a temperature of 35°C and a humidity of 75% for fermentation, and sampling was performed after fermentation was completed (35 days). The collected samples were numbered, and the changes of various compounds and the enrichment of microorganisms in the fermentation process were analyzed by determining the non-targeted metabolomics and microbial diversity of the fermented tobacco leaves, and then the quality recovery of cigar tobacco was analyzed.

[0138] 2Physicochemical component analysis of fermented cigar tobacco

[0139] The fermented cigar tobacco leaves were removed from the main vein part, and the samples were ground into powder with liquid nitrogen, and then stored in a-80°C ultra-low temperature refrigerator. The six conventional indexes of total pectin (Pectin), total nitrogen (TN), total sugar (TS), free amino acid (FAA), nicotine (NIC) and reducing sugar (RS) were determined.

[0140] (1) Determination of total pectin content

[0141] Operation steps: after the sample is ground into powder with liquid nitrogen, about 0.1 g sample is weighed, 1 mL of anhydrous ethanol is added, and after homogenization, it is extracted at 90°C for 30 min, and after cooling, it is centrifuged at 5000g, 25°C for 10 min, and the supernatant is removed. The precipitate is washed with 1 mL of 80% ethanol, and the supernatant is removed by centrifugation. After repeating the operation twice, 1 mL of distilled water is added to the precipitate, which is placed in a 50°C constant temperature water bath for 30 min, and after cooling, it is centrifuged at 8000g, 25°C for 10 min, and the supernatant is taken for determination of soluble pectin. 0.5 mL of 0.5 mol / L H2SO4 is added to the precipitate, mixed well, and then immersed in a boiling water bath for 1 h. After cooling, it is centrifuged at 8000g, 25°C for 10 min, and the supernatant is taken for determination of insoluble pectin. 50 μL of the sample solution is taken in a 1.5 mL centrifuge tube, 0.3 mL of concentrated sulfuric acid is added, mixed well, and placed in a 90°C water bath for 10 min. After cooling, 50 μL of carbazole-ethanol solution is added, mixed well, and placed for 30 min. Then 200 μL is taken and placed in a 96-well plate, and the absorbance value A at 525 nm is read.

[0142] Standard curve drawing: 1 mg / mL galacturonic acid standard solution is diluted into 1.00, 0.80, 0.60, 0.40, 0.20, 0.10, 0.05, 0.00 mg / mL gradient solutions, and the sample is detected at the same time. The absorbance value is measured at a wavelength of 525 nm. The standard curve equation between the absorbance value and the detection concentration is established.

[0143] Result calculation: soluble pectin (mg / g) = C soluble × V soluble / M × F

[0144] Insoluble pectin (mg / g) = C insoluble × V insoluble / M × F

[0145] Total pectin (mg / g) = soluble pectin + insoluble pectin

[0146] In the formula: C - sample concentration (mg / mL) calculated from the standard curve

[0147] V soluble - total volume of soluble pectin extract (1 mL)

[0148] V insoluble - total volume of insoluble pectin extract (0.5 mL)

[0149] M - sample mass (g)

[0150] F - dilution factor

[0151] (2) Determination of total nitrogen content

[0152] Operation steps: take 0.25g sample, wrap with non-waste weighing paper, put into digestion tube, add 3g potassium sulfate as catalyst, then add 8.0mL concentrated sulfuric acid along the bottle wall. Digest at 420℃ for 1 hour until the solution is clear and transparent. After digestion, the digestion tube is taken out and cooled to room temperature, and the digestion tube is placed on the Kjeldahl apparatus for titration.

[0153] Result calculation: N%=1.401xCx(V-V0) / W

[0154] In the formula: C-standard hydrochloric acid molar concentration (mol / L)

[0155] W-sample mass (g)

[0156] V-sample titration standard hydrochloric acid consumption (mL)

[0157] V0-blank sample titration standard hydrochloric acid consumption (mL)

[0158] (3) Determination of total sugar content

[0159] Operation steps: The sample is ground into powder with liquid nitrogen, 0.1g sample is weighed, 2.5mL 10% hydrochloric acid is added, and the homogenate is ground, boiled in a water bath for 1h, 1.5mL 30% NaOH solution is added after cooling, and the volume is made up to 10mL with distilled water, centrifuged at 8000g for 10min at room temperature, 50µL supernatant is taken in a 1.5ml centrifuge tube, 50µLDNS is added, mixed well, boiled in a water bath for 5min, cooled to room temperature, then 200µL distilled water is added, mixed well, and 200µL is taken to a 96-well plate to measure the absorbance value at 540nm.

[0160] Preparation of standard curve: dilute 5mg / mL glucose standard solution to 1.0, 0.8, 0.4, 0.3, 0.2, 0.1mg / ml gradient solution, measure at the same time as the sample, and draw the standard curve according to the concentration and absorbance value.

[0161] Result calculation: total sugar (mg / g)=CxV / MxF

[0162] In the formula: C-sample concentration (mg / mL) calculated from sample absorbance value in standard curve

[0163] V-sample extraction volume (10mL)

[0164] M-sample mass (g)

[0165] F-dilution factor

[0166] (4) Determination of reducing sugar content

[0167] Operation steps: after the sample is ground into powder by liquid nitrogen, about 0.1 g of sample is weighed, 0.5 mL of distilled water is added, and after homogenization, it is placed in a water bath at 80℃ for 40 min, and oscillated from time to time. After cooling, centrifugation is performed at 8000 g and 25℃ for 10 min, and the supernatant is taken for measurement. 50 μL of the sample solution is taken, 50 μL of DNS reagent is added, and after mixing, it is placed in a boiling water bath for 5 min. After cooling, 200 μL of distilled water is added, and after mixing, 200 μL is taken in a 96-well plate, and the absorbance at 540 nm is read.

[0168] Standard curve drawing: same as (3)

[0169] Result calculation: reducing sugar (mg / g) = C x V / W x F

[0170] In the formula: C - the content of reducing sugar calculated from the standard curve (mg / mL)

[0171] V - extraction volume (0.5 mL)

[0172] M - sample mass (g)

[0173] F - dilution factor

[0174] (5) Determination of free amino acid content

[0175] Operation steps: the sample is ground into powder by liquid nitrogen, about 0.1 g of sample is weighed, 1 mL of 10% acetic acid solution is added, and after homogenization, it is tightly covered and placed in a boiling water bath for extraction for 15 min; after cooling, centrifugation is performed at 10000 rpm and 4℃ for 10 min, and the supernatant is taken for measurement. In an EP tube, 10 μL of sample solution, 120 μL of acetic acid-sodium acetate buffer solution, 100 μL of 3% indanone solution and 10 μL of 0.3% ascorbic acid solution are added in sequence. After mixing, the bottle cap is tightly covered, and it is placed in a boiling water bath for 15 min. After cooling, it is repeatedly inverted several times, centrifuged at 8000 rpm for 5 min, and the supernatant is taken for measurement of absorbance at 570 nm.

[0176] Standard curve drawing: 5 mg / mL leucine standard solution is diluted with distilled water to 0.50, 0.40, 0.30, 0.20, 0.10, 0.05, 0.00 mg / mL standard solutions of different concentrations, and the absorbance at different concentrations is measured according to the operation of the sample solution. The standard curve is drawn according to the relationship between concentration and absorbance.

[0177] Result calculation: amino acid (mg / g) = C x V / M x F

[0178] In the formula: C - the content of amino acid calculated from the standard curve (mg / mL)

[0179] V - extraction volume (1 mL)

[0180] M - sample mass (g)

[0181] F - dilution factor

[0182] (6) Determination of nicotine content

[0183] Operation steps: After the sample powder, take about 0.2g sample in the digestion tube, add 20g sodium chloride and 2g of sodium hydroxide solid reagent, add 30mL of pure water, immediately put it into the semi-automatic Kjeldahl nitrogen determination instrument, distill for about 6 minutes, use the 250mL flask containing 10mL 1mol / L sulfuric acid solution as the receiver, the end of the condenser tube should be immersed in the sulfuric acid solution. Collect 220~230mL distillate, remove the flask while flushing the end of the condenser tube with pure water. The liquid in the flask is quickly diluted to 250ml capacity bottle with pure water. Remove a certain volume (usually 10mL) of distillate to a 50mL capacity bottle, dilute to the mark with 0.025mol / L sulfuric acid solution. With 0.025mol / L sulfuric acid solution as reference, determine the absorbance of the solution at 236nm, 259nm and 282nm with spectrophotometer. (If the absorbance at 259nm exceeds 0.7, a smaller volume of distillate should be taken to re-dilute and determine the absorbance at the three wavelengths)

[0184] Result calculation: N%=1.059* (A259nm-0.5*(A236nm+A282nm))*250*F / (m*34.3*10)

[0185] In the formula: A - the measured value of absorbance at each wavelength,

[0186] 1.059×[A259nm-0.5×(A236nm+A282nm)] - corrected absorbance at 259nm

[0187] m - sample mass (g)

[0188] F - dilution factor

[0189] After fermentation (35 days), the pectin, total nitrogen (TN), total sugar (TS), free amino acid (FAA), nicotine (NIC), and reducing sugar (RS) of the cigar tobacco leaves were analyzed. The results of the difference analysis of the physicochemical components among different treatment groups are as follows Figure 10As shown, in terms of pectin content, the addition of salt-tolerant Bacillus CC strain significantly reduced the pectin content in tobacco leaves to about 17.2%, which was significantly lower than the 20.8% of the CK treatment group. This indicates that the CC strain has strong pectin degradation ability. In terms of total nitrogen content, the total nitrogen content of tobacco leaves fermented by adding salt-tolerant Bacillus CC strain reached about 4.34%, which was significantly higher than that of CK. In terms of total sugar and reducing sugar, the addition of salt-tolerant Bacillus CC strain significantly increased the total sugar and reducing sugar content in tobacco leaves, which was significantly higher than that of the CK group. This indicates that salt-tolerant Bacillus CC not only degrades pectin, but also promotes the hydrolysis of polysaccharides and the accumulation of sugars, which is beneficial to improve the flavor of tobacco leaves. In terms of free amino acids, the free amino acid content of the treatment with salt-tolerant Bacillus CC strain was higher than that of the CK group. In terms of nicotine content, the nicotine content of the tobacco leaves with the addition of salt-tolerant Bacillus CC strain was significantly lower than that of the CK group of 2.9%. These changes indicate that the addition of salt-tolerant Bacillus CC strain helps to improve the aroma and flavor of cigar tobacco, and reduces the generation of unpleasant odors and irritating substances.

[0190] 3 Sensory quality evaluation of fermented cigar

[0191] At the end of 35 days of fermentation, five sensory evaluation experts were invited to evaluate the appearance quality and internal sensory quality of the four treatments. The scoring method was the full score method. The scoring standards are shown in Tables 5 and 6:

[0192] Table 5 Appearance scoring standards

[0193]

[0194] Table 6 Internal sensory scoring standards

[0195]

[0196] The sensory quality of the fermented tobacco leaves was evaluated, and the appearance score is shown in Table 7, and the internal sensory score is shown in Table 8:

[0197] Table 7 Appearance score

[0198]

[0199] Table 8 Internal sensory score

[0200]

[0201] It can be seen that the sensory quality score of the treatment with the addition of salt-tolerant Bacillus CC strain is higher than that of the CK treatment.

[0202] 4 Microbial diversity index analysis of fermented cigar

[0203] The sample was taken out from the ultra-low temperature freezer at -80°C, 5 g of the sample was weighed into a conical flask containing sterile water, ultrasonic for 10 min, vortex for 1 min, the washed sample was taken out, and the washing liquid was passed through a 0.22 μm filter membrane. The filtered membrane was quickly frozen with liquid nitrogen and transferred to a -80°C refrigerator for storage. The filter membrane was sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for total genomic DNA extraction of microbial community, 1% agarose gel electrophoresis was used to detect the quality of the extracted genomic DNA, and NanoDrop2000 was used to determine the DNA concentration and purity. Using the extracted DNA as a template, the upstream primer 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and the downstream primer 806R (5'-GGACTACHVGGGTWTCTAAT-3') carrying the Barcode sequence were used to amplify the 16S rRNA gene V3-V4 variable region by PCR. The PCR product was recovered using a 2% agarose gel, and the DNA gel recovery and purification kit was used for product purification, and the recovered product was detected and quantified using Qubit 4.0. The fastp (https: / / github.com / OpenGene / fastp, version 0.19.6) software was used for quality control of double-end raw sequencing sequences, and the FLASH (http: / / www.cbcb.umd.edu / software / flash, version 1.2.11) software was used for splicing, and the high-throughput sequencing data analysis was completed. The Meiji Shengxin cloud platform was used to analyze the microbial diversity abundance and community composition difference of the flattened data.

[0204] The relative abundance of bacterial communities in different treated cigar tobaccos at the phylum level is shown in Figure 11 As shown in the figure, Bacillota, Bacteroidota, Actinomycetota, Pseudomonadota and Deinococcota are the dominant phyla in the bacterial community. At the phylum classification level, the CK group is mainly composed of Bacillota, Bacteroidota, Actinomycetota and Pseudomonadota, among which Bacillota shows ultra-high abundance, and compared with it, the CC group has a significant increase, and Bacteroidota has a significant decrease in abundance in the CC treatment group. The relative abundance of bacterial communities in different treated cigar tobaccos at the genus level is shown in Figure 12As shown, the CK group was mainly composed of Staphylococcus, Chryseobacterium, Rhodococcus, Bacillus, and Micrococcus, among which Staphylococcus showed an extremely high abundance. Compared with the CK group, the abundance of Staphylococcus in the tobacco leaves treated with the addition of CC strain increased, while the abundance of Chryseobacterium and Rhodococcus decreased. The relative abundance of fungal communities in different treatments of cigar tobacco at the level of phylum is shown in Figure 4. Figure 13 As shown, at the level of phylum classification, Ascomycota and Basidiomycota were the main dominant fungal phyla of all treatments. Among them, Ascomycota was the dominant fungal phylum with the highest relative abundance in all treatments. After the addition of CC strain fermentation, the abundance of Ascomycota decreased significantly, while the abundance of Basidiomycota increased significantly. The relative abundance of fungal communities in different treatments of cigar tobacco at the level of genus is shown in Figure 5. Figure 14 As shown, the difference was more obvious at the level of genus classification. For example, the top six genera in terms of relative abundance were Aspergillus, Cladosporium, Wallemia, Alternaria, Penicillium, and Candida. Compared with the CC treatment, the abundance of Aspergillus and Cladosporium decreased, while the relative abundance of Wallemia increased. The abundance of Alternaria decreased under the treatment of CC strain. The abundance of Penicillium decreased under the treatment of CC strain. The abundance of Candida increased under the treatment of CC strain. In summary, the addition of CC strain fermentation can change the microbial community structure of tobacco leaves and create a new fermentation environment.

[0205] Although the specific embodiments of the present application are described in detail with reference to the accompanying drawings, it should not be understood as limiting the scope of protection of the patent. Various modifications and variations made by those skilled in the art within the scope described in the claims are still within the scope of protection of the patent.

Claims

1. A strain of Bacillus halodurans (Bacillus halodurans) having the characteristics of Bacillus halotolerans , wherein The taxonomic name of the salt-tolerant Bacillus is: Bacillus halodurans CC Bacillus halotolerans CC, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20251509.

2. An inoculant characterized in that, The active ingredient of the bacterial agent is the salt-tolerant Bacillus CC of claim 1.

3. The salt-tolerant Bacillus CC of claim 1 or the bacterial agent of claim 2 is applied to ferment pectin, increase total nitrogen, increase total sugar, increase reducing sugar, increase free amino acid and reduce nicotine content of cigar tobacco leaves.

4. Use according to claim 3, characterized in that, The fermentation method of the cigar tobacco leaves is to spray the bacterial liquid prepared by the salt-tolerant Bacillus CC of claim 1 and fermentation aids on the surface of the cigar tobacco leaves for fermentation.

5. Use according to claim 4, characterized in that, The preparation materials of the fermentation aids include: 1g of sugar syrup, 10mL of apple vinegar, 10g of cassia, 100g of tobacco main vein, 100mL of 50° sugar cane wine and 100mL of ultrapure water. The fermentation aid preparation method is as follows: Step (1) mix the sugar syrup and apple vinegar to obtain a stock solution A; Step (2) mix the cassia and tobacco main vein with ultrapure water and heat to boiling, take the supernatant, and filter after the temperature drops to obtain a stock solution B; Step (3) mix the stock solution A, stock solution B and sugar cane wine to obtain a mixed solution, and mix uniformly to obtain a fermentation stock solution; Step (4) additionally take ultrapure water, mix the fermentation stock solution with the ultrapure water at a ratio of 1:50 to obtain the fermentation aid.

6. Use according to claim 3, characterized in that, The bacterial liquid preparation method is as follows: Step (1) inoculate the salt-tolerant Bacillus CC of claim 1 in LB liquid medium, and culture at 30℃, 180r / min shaking for 24h to obtain the first generation culture; Step (2) inoculate the first generation culture in LB liquid medium again at an inoculation amount of 2%, and culture at 30℃, 180r / min shaking for 24h to obtain the second generation culture; Step (3) centrifuge the second generation culture at 5000 r / min for 15 min, discard the supernatant, retain the bacterial bodies, rinse with 0.05% glucose solution to prepare a bacterial solution, so that the effective viable bacterial number of Bacillus CC in the bacterial solution is not less than 1.0 x 10 8 CFU / mL.

7. Use according to claim 3, characterized in that, The volume ratio of the bacterial liquid to the fermentation aid is 1:

1.

8. Use according to claim 3, characterized in that, The fermentation temperature is 35℃, the humidity is 75%, and the fermentation time is 35d.

9. Use of Bacillus halodurans CC of claim 1 or inoculant of claim 2 in cigar leaf fermentation to alter microbial community structure, the altering microbial community structure comprising: Bacillus phylum ( Bacillota ) and Staphylococcus spp. Staphylococcus The abundance of *Cryptobacter* increased; *Cryptobacter* genus ( Chryseobacterium ) and Rhodococcus spp. ( Rhodococcus The abundance of Ascomycota decreased; Ascomycota The abundance of Basidiomycetes ( ) decreased, and the abundance of Basidiomycetes ( ) decreased. Basidiomycota The abundance of Aspergillus ( ) increased, and the abundance of Aspergillus in the Ascomycota phylum increased. Aspergillus ) and Cladosporium ( Cladosporium The abundance of ) decreased, and the abundance of the genus *Bacillus* ( Wallemia The abundance increased, and the cigar tobacco variety was Yunxue 39, with the part being the middle part of the tobacco.

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