CN114276959B

CN114276959B - Bacillus altitudinis and application thereof in improving cigar quality

Info

Publication number: CN114276959B
Application number: CN202111637094.XA
Authority: CN
Inventors: 秦艳青; 刘甜甜; 郭仕平; 金垚; 雷云康; 吴重德; 何正川; 杨富; 张瑞娜; 杨吉光; 罗辉; 吕吉; 曾旸; 谭舒; 阳苇丽; 谢良文; 杨兴有; 赵宇
Original assignee: Sichuan University
Current assignee: Sichuan University; China National Tobacco Corp Sichuan Branch
Priority date: 2021-12-29
Filing date: 2021-12-29
Publication date: 2023-01-24
Anticipated expiration: 2041-12-29
Also published as: CN114276959A

Abstract

The invention discloses a Bacillus altitudinis and an application thereof in improving cigar quality, wherein the Bacillus altitudinis (Bacillus altitudinis) is named as Bacillus altitudinis DMNJLTT1 and is preserved in China center for type culture collection, the preservation address is No. 299 in the eighth way in Wuchang district in Wuhan city, hubei province, the preservation center of Wuhan university, the preservation date is 2021 year, 11 months and 05 days, and the preservation number is CCTCCNO: M20211370. The strain (Bacillus altitudinis DMNJLTT 1) has protease production activity, can improve neutral aroma components of cigar leaves, improves aroma, and has excellent effect on cigar quality improvement; meanwhile, the bacillus altitudinis is used for the development of subsequent related products, and has academic research significance and practical application value.

Description

Bacillus altitudinis and application thereof in improving cigar quality

Technical Field

The invention relates to the technical field of biology, in particular to bacillus altitudinis and application thereof in improving cigar quality.

Background

Chinese cigar domestic raw material development has accumulated to a certain extent, but as the starting is late, the whole research foundation is weak, the domestic cigar tobacco raw material has certain difference with foreign high-quality production areas in aspects of appearance, quality, aroma and the like, most domestic raw materials can only be used as low-grade cigar cores, and the domestic middle-high-end cigar brand raw materials basically depend on import.

Cigars are special tobacco products formed by rolling tobacco leaves after airing, fermenting and aging, can be divided into cigar coats, cigar sleeves and cigar cores according to the application of the tobacco leaves in cigar products, and have the characteristics of strong fragrance, small tar content, strong strength and the like, so the cigar is widely popular with consumers. The main components in tobacco are carbohydrates and nitrogen-containing compounds. The former mainly comprises components such as saccharides and starch, the latter mainly comprises nicotine and protein, and the content of the protein in the tobacco leaves is closely related to the quality of the tobacco. When the protein content is too high, odor and offensive odor are generated during combustion, and the smoking quality is seriously influenced. Therefore, the protein substances of the domestic cigar tobacco leaves need to be moderately lightened, so that the smoking quality of the domestic cigars is improved.

Disclosure of Invention

In view of the above-mentioned disadvantages, the present invention provides a bacillus altitudinis and its application in improving cigar quality. The strain has protease activity, can improve neutral aroma components of cigar tobacco leaves, improves aroma, and has excellent effect on improving cigar quality.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a bacillus altitudinis (A), (B), (C) and (D)Bacillus altitudinis) Is named asBacillus altitudinis DMNJLTT1 is preserved in China center for type culture Collection, the preservation address is the eight-channel 299 Wuhan university school in Wuchang district (the first attached small opposite side of Wuhan university) in Wuhan city, hubei province, the preservation date is 2021, 11 and 05 days, and the preservation number is CCTCCNO: M20211370.

The invention also provides the bacillus altitudinis (b) ((b))Bacillus altitudinis ) Application of DMNJLTT1 in improving cigar quality.

The improvement of the cigar quality comprises the improvement of neutral aroma components of cigar tobacco leaves, the improvement of aroma and the like; the neutral aroma components of the cigar tobacco mainly comprise nicotine, terpenoid, aldehyde ketone, carotenoid degradation products and the like.

The invention also provides an enzyme preparation for improving the quality of cigars, and the active component of the enzyme preparation comprises the bacillus altitudinis (A), (B) and (C)Bacillus altitudinis ）DMNJLTT1。

The invention also provides a compound microbial preparation for improving the quality of cigars, and the compound microbial preparationThe active ingredient comprises the bacillus altitudinis (A), (B), (C) and (C)Bacillus altitudinis ）DMNJLTT1。

The invention also provides a compound microbial preparation for producing protease, and the active ingredients of the compound microbial preparation comprise the bacillus altitudinis (A), (B) and (C)Bacillus altitudinis ）DMNJLTT1。

In summary, the invention has the following advantages:

1. the invention provides a bacillus altitudinis (A)Bacillus altitudinis ) DMNJLTT1 and application thereof in improving cigar quality. The strain has protease activity, can improve neutral aroma components of cigar tobacco leaves, improves aroma, and has excellent effect on improving cigar quality; simultaneously the Bacillus altitudinis (A), (B), (C) and (C)Bacillus altitudinis ) DMNJLTT1 is used for the development of subsequent related products, such as an enzyme preparation and a compound microbial preparation, and the neutral aroma components of the added bacterial preparation and the non-added bacterial preparation are analyzed by gas chromatography-mass spectrometry, so that the method contributes to the mechanism of improving the quality of the cigar products after protein degrading microorganisms are added, and has academic research significance and practical application value.

2. In the present invention, bacillus altitudinis (A), (B) and (C)Bacillus altitudinis ) The application result of DMNJLTT1 in improving the cigar quality is verified, gas chromatography-mass spectrometry combined analysis used in few researches is selected, the analysis result is more scientific and representative, the neutral aroma components of the cigar leaves can be analyzed through the gas chromatography-mass spectrometry combined analysis, and the result is more convincing.

Drawings

FIG. 1 is a single colony of a part of the strain isolated and purified according to the present invention;

FIG. 2 is a graph showing the results of casein hydrolysis experiments with DMNJLTT1 strain according to the present invention;

FIG. 3 is a graph of a standard tyrosine curve according to the present invention;

FIG. 4 is a colony morphology of DMNJLTT1 strain of the present invention;

FIG. 5 is a gram stain of the DMNJLTT1 strain of the present invention;

FIG. 6 is a biological scanning electron microscope image of the DMNJLTT1 strain of the present invention;

FIG. 7 is a graph showing the result of the electrophoresis in the present invention;

FIG. 8 is a diagram showing the comparison and identification results in the present invention;

FIG. 9 is a phylogenetic tree diagram of the DMNJLTT1 strain of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

Thus, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 Bacillus altitudinis: (Bacillus altitudinis ) Isolation, purification, screening and identification of DMNJLTT1

In this example, unless otherwise specified or specified, the experimental procedures or reagents may be those conventionally used in the art or those commercially available may be used. In the embodiment, culturable microorganisms are separated and purified from the surface of high-quality cigar tobacco leaves by a traditional separation method, functional microorganisms for degrading proteins are screened out and applied to different cigars, and the aroma and the quality of the cigar tobacco leaves can be improved, and the method comprises the following specific steps:

1. separating and purifying

(1) Screening raw materials: from the surface of high-quality cigar tobacco leaves (core, sleeve and coat);

(2) Separating and purifying the culture medium: 10% tobacco extract medium (1L): 100 ml of tobacco extract, 9 g of tryptone, 9 g of sodium chloride, 4.5 g of yeast extract and 20 g of agar, natural pH, temperature at room temperature the preparation of the tobacco extract: 15 Boiling cigar tobacco leaf and 400 ml distilled water for 30min, cooling at room temperature, and filtering with two layers of gauze to obtain boiling water extract of tobacco leaf;

(3) Preparing a bacterial suspension: balancing the moisture of tobacco leaves in an oven at 40 ℃ for 3 hours, taking 1 g of middle tobacco leaves, cutting the middle tobacco leaves under the aseptic condition by using sterilized scissors, soaking the cut tobacco leaves in 100 mL of sterile water, performing shaking culture at 37 ℃ and 200 r/min for more than 30min, filtering a culture solution by using sterile single-layer gauze, centrifuging for 15min at 9000 r/min, discarding the supernatant, and re-suspending the precipitate by using 5mL of sterile water to obtain an original bacterial suspension;

(4) Primary separation and purification: diluting the original bacterial liquid by 0, 10, 100, 1000, 10000 and 100000 times, respectively sucking 100 μ L of the diluted bacterial liquid, uniformly coating the diluted bacterial liquid on a solid culture medium of a tobacco water extract, and culturing for 48h at 37 ℃; the obtained colonies were cultured and purified by plating until single colonies were obtained.

As a result, 57 strains with different morphologies were isolated and named DMNJLTT1-DMNJLTT57, and the single colony patterns of the isolated and purified partial strains are shown in FIG. 1.

2. Screening of protein degrading functional bacteria and determination of protease activity

(1) Functional screening of 57 strains of bacteria separated and purified from tobacco leaves: because the existence of macromolecular substance protein in the tobacco leaves has negative influence on the smoking characteristics of the cigar products, the degraded macromolecular substance protein is selected as a screening standard, a screening culture medium is determined to be a casein degradation culture medium, 57 strains of bacteria are subjected to dot-panel experiments in the casein culture medium, and the growth condition of the strains and the size of a proteolysis transparent circle are observed.

(2) Degradation characteristics and protease activity of protein degradation functional bacteria

1. Protease screening solid medium: 10.0 g/L of casein, 5.0 g/L of yeast extract, 4.0 g/L of NaCl, 20 g/L of agar powder and pH 7.2;

2. tobacco extract liquid medium (1L): 100 ml of tobacco extract, 9 g of tryptone, 9 g of sodium chloride and 4.5 g of yeast extract;

3. and (3) measuring the protein degradation capability of the strain: selecting a single colony, inoculating the single colony in a protease screening solid culture medium, performing inverted culture for 48 hours at 37 ℃, measuring the diameter (D) of the colony and the diameter (D) of a degradation ring by using a ruler, and calculating the degradation index I of the degradation protein of the strain;

4. preparing a crude enzyme solution: inoculating the strain with strong protein degradation capability into tobacco leaf extract liquid culture medium, and culturing at 37 deg.C to obtain strain OD ₆₆₀ When the activity is between 0.8 and 1.2, the activity is used for subsequent determination of the protease activity;

5. and (3) determining the activity of the protease: centrifuging 5ml of crude enzyme solution at 4 deg.C at 8000 r/min for 5min, removing precipitate, collecting supernatant, adding 50 ml of phosphate buffer, shaking, diluting properly to obtain protease solution, and performing ultraviolet spectrophotometry at 660 nm.

The result shows that the strain DMNJLTT1 has the best degradation effect on casein hydrolysis, the strain with the strongest degradation capability has a casein hydrolysis experiment as shown in figure 2, and the degradation index colony diameter and the transparent ring diameter D/D reach 3.955; the highest enzyme activity of the strain DMNJLTT1 is 577.15U; in addition, the tyrosine standard curve is shown in FIG. 3.

3. Physiological and biochemical experiment and molecular biological identification of protein degrading functional bacteria DMNJLTT1

(1) Physiological and biochemical forms

After the plate streaking separation, the colony color is white and yellowish as a whole, the single colony is smooth, the central bulge is white and rough, and the edge is smooth, as shown in fig. 4 and table 1.

Note: "+" indicates positive, and "-" indicates negative

The bacillus is observed by microscopic examination and is in a right cylindrical shape, most of the thallus is straight, and the few thallus is bent, and two ends of the thallus are blunt and round. The gram-positive bacteria were purple when observed by microscopic examination after gram staining, as shown in FIG. 5.

(2) Bacteria scanning electron microscope observation

Pretreatment of a bacterial sample: centrifuging the bacterial liquid at 3000 r/min for 10min, and discarding the supernatant. 2.5 percent of glutaraldehyde is injected, and the mixture is kept stand for more than 2 hours for fixation. After fixation, the bacteria were centrifuged, the supernatant discarded, resuspended in distilled water, repeated 3 times, and finally resuspended in distilled water. Dehydrating with 30%, 50%, 70%, 80%, 90% gradient concentration ethanol, standing for 10min in each gradient, dehydrating with pure ethanol for 3 times, 30min each time, then replacing ethanol with pure tert-butanol for 3 times, 30min each time, finally sucking the mixed bacteria-tert-butanol suspension liquid to drop on a sample table covered with a cover glass, and placing in a freeze dryer for vacuum drying, wherein the result is shown in fig. 6.

(3) Molecular biological identification

The experimental steps are as follows:

1. DNA extraction

The TSINGKE plant DNA extraction kit (universal type) is used, and the specific steps are as follows: (1) Placing Spin Column in Collection Tube, adding 250 μ l Buffer BL, and centrifuging at 12000 rpm/min for 1 min to activate silica gel membrane; (2) The dried tissue (not more than 20 mg) was sampled and ground thoroughly with liquid nitrogen. Grinding, placing in a 1.5 ml centrifuge tube, adding 400 μ l Buffer gP1, vortex oscillating for 1 min, water bathing at 65 ℃ for 10-30 min, taking out, reversing and mixing uniformly for full cracking; (3) Adding 150 μ l Buffer gP2, vortex oscillating for 1 min, ice-cooling for 5 min; (4) Centrifuging at 12000 rpm/min for 5min, and transferring the supernatant into a new centrifuge tube; (5) Adding absolute ethanol with the same volume as the supernatant, immediately and fully oscillating and uniformly mixing, transferring all liquid into Spin Column, centrifuging at 12,000 rpm/min for 30 s, and discarding waste liquid; (6) Adding 500 μ l Buffer Pw (absolute ethanol is added before use) into Spin Column, centrifuging at 12000 rpm/min for 30 s, and discarding the waste liquid; (7) Adding 500 μ l of Wash Buffer (added with absolute ethyl alcohol before use) into Spin Column, centrifuging at 12000 rpm/min for 30 s, and discarding the waste liquid; (8) repeating the operation step 7; (9) Putting Spin Column back into Collection Tube, centrifuging at 12,000 rpm/min for 2 min, uncovering and air drying for 1 min; (10) The Spin Column is taken out and put into a clean centrifuge tube, 50-100 μ l of TE Buffer (preheated TE Buffer at 65 ℃) is added at the center of the adsorption film, and the adsorption film is placed at 20-25 ℃ for 2 min and centrifuged at 12,000 rpm/min for 2 min.

2. PCR amplification

(1) The bacterial species identification universal primer is shown as follows:

the extracted DNA sample is diluted in proper amount and used as PCR template for amplification by Scienda 1 XTSE 101 gold Mix.

3. Electrophoretic detection

The amplified PCR products were subjected to agarose gel electrophoresis (2 ul sample +6ul bromophenol blue) at 300V for 12 min to obtain an identification gel pattern, as shown in FIG. 7.

4. Comparison and identification

(1) The sequencing results were spliced with ContigExpress and the parts that were not aligned on both ends were removed.

(2) The assembled sequences were aligned in NCBI database (blast. NCBI. Nlm. Nih. Gov), and the alignment results are shown in fig. 8.

(3) Selecting the most homologous species

Through the above identification experiments, DMNJLTT1 was determined to be Bacillus, geobacillus, (A) Bacillus altitudinisBacillus altitudinis) Is named asBacillus altitudinisThe phylogenetic tree of DMNJLTT1 is shown in FIG. 9, and the sequence is shown in SEQ ID No. 1.

Example 2 Bacillus altitudinis: (A)Bacillus altitudinis ) Performance detection and verification experiment of DMNJLTT1

1. Gas chromatography-mass spectrometry analysis of neutral volatile components in control group and experimental group of cigar tobacco leaves

Application example: after the bacterial strain is made into bacterial suspension, the bacterial suspension is added to the surface of a domestic assorted Chinese cigar leaf, the tobacco leaf is cultured in an incubator at 37 ℃ for 24 hours, the volatile components before and after the cultivation are respectively tested, and the change of the aroma component content, such as the change of neophytadiene and aldehyde ketone compounds, is observed.

1. Sample source: tobacco leaf in the middle of cigar sheath of assorted 37025

2. Sample pretreatment: control group of animals370252525 drying cigar case and cigar case of experimental group at 40 deg.C, grinding into powder, and sieving with 40 mesh sieve. Accurately weighing 0.5 g of tobacco powder, adding 1mL of sterilized tobacco leaf extract liquid culture medium into a control group, and adding 1mL of Bacillus altitudinis (OD 660 of 0.8-1.2) into an experimental group in a 20 mL headspace bottleBacillus altitudinis ) Culturing the DMNJLTT1 bacterial suspension at 30 ℃ for 7d a week, dripping 10 ul of phenethyl acetate internal standard on the inner side of a headspace bottle, sealing, and then placing on a solid phase microextraction heating table for balancing at 70 ℃ for 20 min; and (3) inserting the aged extraction head into a headspace bottle, sampling the headspace (the extraction time is 30min at the extraction temperature of 70 ℃), pulling out the extraction head from the headspace bottle, immediately inserting the extraction head into a sample inlet of a gas chromatograph, analyzing the extraction head for 5min at 250 ℃, and performing GC/MS detection.

3. Chromatographic analysis conditions: a chromatographic column: DB-5ms (30 m.times.0.25 mm.times.0.25 μm), carrier gas: high-purity helium, flow rate of 1.5 mL/min, sample injection amount: 1.μ L, injection port temperature: 250. DEG C, sample injection mode: the split ratio is 10: 1. temperature rising procedure: the initial temperature was 60 ℃ for 2 min, and the temperature was raised to 240 ℃ at 4 ℃/min for 5 min.

4. Mass spectrometry conditions: an ion source: EI source, ionization voltage 70 eV, ion source temperature 300 ℃, transmission line temperature: 250. temperature, solvent delay time 3 min, scanning mode: full scan range: 33-350 amu

5. The instrument comprises the following steps: the manufacturer ThermoFisher semer fei, usa, model DSQ9000, triple quadrupole gas-mass spectrometer.

The results show that, as shown in table 2, the abscissa is the component, the main volatile components of tobacco leaves are tobacco alkaloids, olefins, esters, aldehydes and ketones, heterocycles, alkanes and alcohols, and the specific principle is as follows:

1) The tobacco alkaloid has special contribution to color and flavor of tobacco leaves, including nicotine, diene nicotine and the like, has no special flavor, and several pyridine compounds formed by high-temperature decomposition have fragrance.

2) The ester component has aroma group, has sweet taste, fruit taste or wine flavor, and can coordinate with tobacco leaf aroma to make contribution, the dihydroactinidiolide has coumarin-like aroma, methyl nonanoate can increase tobacco leaf aroma concentration, and the methyl tetradecanoate can make tobacco leaf taste and aroma more mellow.

3) Terpenoids important in tobacco, such as neophytadiene, phytol, menthol, and cembrane, can increase the taste and aroma of tobacco leaf, and have a pleasant odor, which can be degraded and converted into aroma components, and photooxidation of neophytadiene to produce phytofuran

4) Carbonyl in the aldehyde ketone molecular structure is a fragrant group, so that the aldehyde ketone has elegant fragrance, the solanone can smooth the tobacco leaves, the ionone can increase the sweet taste of the tobacco leaves, and the decanal can increase the faint scent of the lemon.

5) The influence of volatile acid on the fragrance of tobacco leaves, the isovaleric acid and the (+/-) -3-hydroxy lauric acid have obvious fragrance of aromatic tobacco, and have good effects on taste and fragrance.

After 7 days of fermentation at 30 ℃, the results of the control group and the experimental group show that:

1. the content of nitrogen-containing compounds (nicotine, myosmine and 2,3' -bipyridine) in the experimental group is obviously lower than that in the control group, which verifies the functional characteristics of the Geobacillus altissima, namely the degradation of protein nitrogen-containing compounds.

2. 43 volatile components are detected in the control group, 77 volatile components are detected in the experimental group, and the aroma substances of the cigar leaves are obviously increased after the fermentation of the bacillus plateau, such as phytofuran, ethyl palmitate, linalool, geranylgeraniol, (+/-) -3-hydroxy lauric acid, citrus ketone and other components.

3. Terpenes, esters, aldehydes and ketones and acids tend to increase integrally.

4. Solanone, hexahydropseudoionone, 4,7, 9-megastigmatrien-3-one, citrus ketone, neophytadiene, cembrane, cedrene, 2-hexyl-1, 1-dicyclopropane-2-methyl octanoate, and 17-octadecynoic acid are increased. Geranylacetone was slightly reduced, but the overall aldone content was increased.

TABLE 2 Bacillus altitudinis (A)Bacillus altitudinis ) Composition of volatile matter content of cigar tobacco before and after fermentation of DMNJLTT1 suspension at 30 ℃ for 7 days of assorted 37025s

In summary, the present invention provides a Bacillus altitudinis for the first time (Bacillus altitudinis ) DMNJLTT1 and application thereof in improving cigar quality. The strain has protease activity, can improve neutral aroma components of cigar tobacco leaves, improves aroma, and has excellent effect on improving cigar quality. The method mainly comprises the following steps: the obtained highland Bacillus (A), (B)Bacillus altitudinis ) OD prepared from DMNJLTT1 ₆₆₀ Uniformly spraying the bacterial suspension between 0.8 and 1.6 on the surface of an assorted 3702525 cigar leaf, fermenting for 7 days at the temperature of 30 ℃, processing a sample by adopting headspace solid phase microextraction, performing semi-quantitative analysis on an internal standard phenethyl acetate, and analyzing the content change of neutral aroma components (mainly nicotine, terpenoid, aldonic and carotenoid degradation products and the like) of the cigar leaf by adopting a gas chromatography-mass spectrometer.

The foregoing is merely exemplary and illustrative of the present invention and it is within the purview of one skilled in the art to modify or supplement the embodiments described or to substitute similar ones without the exercise of inventive faculty, and still fall within the scope of the claims.

Sequence listing

<110> Sichuan university

<120> Bacillus altitudinis and application thereof in improving cigar quality

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1378

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

acctcaccga cttcgggtgt tgcaaactct cgtggtgtga cgggcggtgt gtacaaggcc 60

cgggaacgta ttcaccgcgg catgctgatc cgcgattact agcgattcca gcttcacgca 120

gtcgagttgc agactgcgat ccgaactgag aacagatttg tgggattggc taaaccttgc 180

ggtctcgcag ccctttgttc tgtccattgt agcacgtgtg tagcccaggt cataaggggc 240

atgatgattt gacgtcatcc ccaccttcct ccggtttgtc accggcagtc accttagagt 300

gcccaactga atgctggcaa ctaagatcaa gggttgcgct cgttgcggga cttaacccaa 360

catctcacga cacgagctga cgacaaccat gcaccacctg tcactctgtc cccgaaggga 420

aagccctatc tctagggttg tcagaggatg tcaagacctg gtaaggttct tcgcgttgct 480

tcgaattaaa ccacatgctc caccgcttgt gcgggccccc gtcaattcct ttgagtttca 540

gtcttgcgac cgtactcccc aggcggagtg cttaatgcgt tagctgcagc actaaggggc 600

ggaaaccccc taacacttag cactcatcgt ttacggcgtg gactaccagg gtatctaatc 660

ctgttcgctc cccacgcttt cgctcctcag cgtcagttac agaccagaga gtcgccttcg 720

ccactggtgt tcctccacat ctctacgcat ttcaccgcta cacgtggaat tccactctcc 780

tcttctgcac tcaagtttcc cagtttccaa tgaccctccc cggttgagcc gggggctttc 840

acatcagact taagaaaccg cctgcgagcc ctttacgccc aataattccg gacaacgctt 900

gccacctacg tattaccgcg gctgctggca cgtagttagc cgtggctttc tggttaggta 960

ccgtcaaggt gcaagcagtt actcttgcac ttgttcttcc ctaacaacag agctttacga 1020

tccgaaaacc ttcatcactc acgcggcgtt gctccgtcag actttcgtcc attgcggaag 1080

attccctact gctgcctccc gtaggagtct gggccgtgtc tcagtcccag tgtggccgat 1140

caccctctca ggtcggctac gcatcgtcgc cttggtgagc cgttacctca ccaactagct 1200

aatgcgccgc gggtccatct gtaagtgaca gccgaaaccg tctttcatcc ttgaaccatg 1260

cggttcaagg aactatccgg tattagctcc ggtttcccgg agttatccca gtcttacagg 1320

caggttaccc acgtgttact cacccgtccg ccgctaacat ccgggagcaa gctccctt 1378

Claims

1. A kind of Bacillus altitudinis (A)Bacillus altitudinis) Is characterized by being namedBacillus altitudinisDMNJLTT1 is preserved in China center for type culture Collection, the preservation address is No. 299 of eight branches in Wuchang district, wuhan city, hubei province, the preservation date is 2021 year, 11 month and 05 day, and the preservation number is CCTCC NO: M20211370.

2. The method of claim 1 Bacillus altitudinis (A), (B) and (C)Bacillus altitudinis ) Application of DMNJLTT1 in improving cigar quality.

3. A complex microbial preparation for improving cigar quality, wherein the active ingredient of the complex microbial preparation comprises the bacillus altitudinis (Bacillus altitudinis) as claimed in claim 1Bacillus altitudinis ）DMNJLTT1。

4. A complex microbial preparation producing protease, characterized in that the active ingredient of the complex microbial preparation comprises Bacillus altitudinis (Bacillus altitudinis) of claim 1Bacillus altitudinis ）DMNJLTT1。