CN113215062A - Bacillus amyloliquefaciens, and acquisition method and application thereof - Google Patents
Bacillus amyloliquefaciens, and acquisition method and application thereof Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
- A24B15/18—Treatment of tobacco products or tobacco substitutes
- A24B15/20—Biochemical treatment
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention discloses bacillus amyloliquefaciens, an acquisition method and application thereof, belonging to the technical field of tobacco processing; the bacillus amyloliquefaciens is preserved in Guangdong province microbial strain preservation center, and the preservation number is GDMCC No: 61457. the strain provided by the invention is derived from microorganisms on the surface of flue-cured tobacco, and the prepared biological agent can act on tobacco leaves and can not introduce exogenous microorganisms; the strains can secrete high-activity amylase, can degrade starch substances in the tobacco leaves, increase the sweet feeling of the tobacco leaves and improve the quality of the tobacco leaves.
Description
Technical Field
The invention relates to the technical field of tobacco processing, in particular to bacillus amyloliquefaciens, an acquisition method and application thereof.
Background
Tobacco leaves are a commodity with higher economic value and are the main raw materials of the cigarette industry. In recent years, along with the development of the cigarette industry and the proposal of Chinese style cigarettes, higher requirements are put forward on the quality of tobacco leaves, and in addition to the mildew prevention and insect killing work of the tobacco leaves in the aspect of tobacco leaf storage, the alcoholization quality of the tobacco leaves is improved to the greatest extent by taking the improvement of the alcoholization quality of the tobacco leaves as the center, the optimal tobacco leaf fragrance is obtained, the miscellaneous gas and the irritation of the tobacco leaves are reduced, and the tobacco leaves are used in a proper alcoholization period, so that the efficiency of the quality of the tobacco leaves is exerted to the greatest extent.
The tobacco mellowing is also called as aging, is a mild and slow tobacco fermentation method, and is an important means for improving the internal quality of the tobacco and the availability of the tobacco. The tobacco mellowing is a process of continuously decomposing and converting the components contained in the tobacco leaves in a storage mode, and mainly shows the change of chemical components of the tobacco leaves, such as the degradation of macromolecular substances, browning reaction, the loss of low-molecular irritant components and the like. The tobacco leaf aroma and taste improvement agent is mainly characterized in that macromolecular compounds without aroma characteristics, such as starch, reducing sugar, protein, amino acid, nicotine, higher fatty acid, aroma precursor and the like, are decomposed and converted to form small molecular compounds with aroma characteristics, such as volatile acid, volatile carbonyl compounds and various volatile aroma components, so that the aroma and taste quality of tobacco leaves are obviously improved. Because the reaction generated in the tobacco mellowing process is complex, the existing theoretical research has not yet elucidated the tobacco mellowing mechanism thoroughly. From the current theoretical research, it is generally believed that there are 3 main actions in the tobacco mellowing process, namely, the actions of chemical catalysis, microorganisms and enzymes.
The alcoholization process of the tobacco leaves is divided into two modes of natural alcoholization and artificial alcoholization according to whether human intervention is carried out or not. The artificial alcoholization (fermentation) is a method for increasing aroma and improving quality of tobacco leaves by exerting the function of strengthening microbial strains under the conditions of proper temperature and humidity according to the quality characteristics of the tobacco leaves. Compared with natural alcoholization, the alcoholization process can be accelerated, alcoholization time is shortened, and tobacco quality is improved in a targeted manner. Tobacco researchers have done a lot of work on artificial alcoholization using biological agents, separating and screening microorganisms with certain functions from a tobacco leaf ecosystem, preparing into microbial inoculum after amplification culture, and spraying the microbial inoculum on the surface of tobacco leaves to play a role. The biological agent prepared by the screened microbial strains is expected to carry out artificial alcoholization on the tobacco leaves, the original quality defects of the tobacco leaves are changed by utilizing the function of the enhanced microbial inoculum, and the applicability and the utilization rate of the raw materials are improved.
The invention obtains a strain of microorganism from alcoholized tobacco leaves by screening, and the microorganism is acted on the tobacco leaves at the initial stage of alcoholization and the inferior tobacco leaves which can not meet the use requirements after being alcoholized for two years under proper conditions, so that the degradation of starch in the tobacco leaves can be promoted, the accumulation of reducing sugar can be obtained, the fragrance of the tobacco leaves is enriched, and the quality of the tobacco leaves is improved.
Disclosure of Invention
The first purpose of the invention is to provide a bacillus amyloliquefaciens capable of promoting the degradation of starch in tobacco leaves, obtaining the accumulation of reducing sugar, enriching the fragrance of the tobacco leaves and improving the quality of the tobacco leaves. The second purpose of the invention is to provide an acquisition method of the bacillus amyloliquefaciens. The third purpose of the invention is to provide the application of the bacillus amyloliquefaciens in tobacco leaf production. The fourth purpose of the invention is to provide the application of the bacillus amyloliquefaciens in the tissue fermentation of tobacco strains
The invention realizes the purpose through the following technical scheme:
the first object of the present invention is achieved by: a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens strain) has the similarity percentage (Per. Ident) of 99.86 percent with Bacillus amyloliquefaciens. Is separated from tobacco plant tissues and has been preserved in Guangdong provincial microorganism culture collection (GDMCC for short, address: No. 59 building 5 of Middleya 100 institute of Middleya, Guangdong provincial microorganism research institute, postal code: 510070) at 6.4.6.2021, with the preservation number being GDMCC No: 61457.
the second object of the present invention is achieved by: a method for obtaining Bacillus amyloliquefaciens specifically comprises the following steps:
(1) collecting microorganisms on the surface of the tobacco leaf:
selecting 10-50 g of a mildew-free, worm-eating-free and nondestructive tobacco leaf sample from an alcoholization box, immersing the sample into 0.85% physiological saline water containing 100-200 ml, and oscillating the sample for 2-4 hours at the room temperature and at 200-220 rpm; filtering with two layers of sterile gauze, and collecting filtrate containing microorganisms on the surface of the tobacco leaves;
(2) microorganism primary screen with starch degradation capability
Performing gradient centrifugation on the filtrate under the action of 500-10000 g of centrifugal force to remove tobacco leaf raw material fragments and macromolecular impurities, and finally washing with 0.85% physiological saline, and performing heavy suspension precipitation to obtain a bacterial suspension of microorganisms on the surface of the tobacco leaf;
(3) isolation of microorganisms having starch degrading ability:
uniformly coating 100-200 mu L of the bacterial liquid on LB and solid culture media containing tobacco leaf extracting solutions respectively, and culturing at constant temperature of 30-37 ℃ for 2-3 days until the number and the types of grown bacterial colonies are unchanged; employing the microbial selection System QPixTM400(Molecular Devices) are aseptically transferred into a 250mL shake flask, and after 24 hours of culture, the mixture is centrifuged at 3000rpm for 10min, and the supernatant is taken and the amylase activity of each strain is measured.
(4) Purification and strain identification
Inoculating the screened strain with higher amylase capability into an LB liquid medium, carrying out shake culture at 30-40 ℃ for 24-36 h, centrifuging fermentation liquor at 8000-10000 g and 4 ℃ for 5-10 min, removing supernatant, collecting bacterial sludge, and washing the bacterial sludge for 2-3 times by using sterile 0.85% physiological saline; the bacterial genome extraction kit is adopted to extract the thallus DNA, the products obtained by 16s rDNA PCR amplification are sequenced after being checked by gel electrophoresis, and then compared in an NCBI (National Center for Biotechnology Information, https:// www.ncbi.nlm.nih.gov /) database, the strain is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens strain).
The third object of the present invention is achieved by: an application of bacillus amyloliquefaciens in tobacco production specifically comprises the following steps:
(1) biological preparation
Inoculating the screened strain into an LB liquid culture medium containing a tobacco leaf extracting solution, carrying out shaking culture at 37 ℃ for 24h, centrifuging a fermentation liquid at 10000g and 4 ℃ for 10min, removing a supernatant, collecting bacterial sludge, and washing the bacterial sludge with sterile 0.85% physiological saline for 2-3 times; adding a proper amount of trehalose and a sucrose solution protective agent, performing vacuum freeze concentration, preparing microbial inoculum dry powder, and preserving at low temperature for later use;
(2) application of biological agent in tobacco mellowing
Mixing biological preparation with sterile water to obtain 1 × 10 mixture8～10Uniformly spraying the cfu/mL bacterial suspension onto the surface of the tobacco leaves according to the addition of 20-30%, culturing for 12-48 h at 30-40 ℃ and 70-80% humidity, taking out the tobacco leaves, placing the tobacco leaves in an oven, drying the tobacco leaves at 80-90 ℃ until the water content is 13-14%, and stopping the continuous metabolism of the bacterial strains;
(3) evaluation of tobacco leaf quality
And (3) moisture balance: keeping the alcoholized tobacco leaves at the temperature of 22 ℃ and the humidity of 65% for balancing the moisture for 48 h;
the sensory quality of the tobacco leaves is evaluated by professional smokers according to YC/T138-1998 tobacco and tobacco product sensory evaluation method and YC/T496-2014 cigarette sensory comfort evaluation method.
The fourth object of the present invention is achieved by: an application of Bacillus amyloliquefaciens in the tissue fermentation of tobacco plants. The tobacco plant tissue is the leaf and/or stem of flue-cured tobacco.
The invention also provides a biological agent, which comprises the bacillus amyloliquefaciens.
The invention has the beneficial effects that:
the strain provided by the invention is derived from microorganisms on the surface of flue-cured tobacco, and the prepared biological agent can act on tobacco leaves and can not introduce exogenous microorganisms;
the strain has the advantages of high growth speed, difficult infectious microbes infection, strong capability of enduring harsh conditions, easy culture and the like.
The strain can secrete high-activity amylase, can degrade starch substances in the tobacco leaves, increases the sweet feeling of the tobacco leaves, and improves the quality of the tobacco leaves;
the main component of the biological agent is a microbial strain, the preparation process of the strain is simple and easy to operate, and the production cost is reduced; the acting time of the strain is 12-48 h, which is shorter than that of a single strain preparation reported to be used for artificial alcoholization;
the biological agent is used for artificially fermenting tobacco leaves, can improve the quality of the tobacco leaves to a certain extent by absorbing energy through sensory products, can increase the sweet taste of the tobacco leaves, enrich the fragrance, increase the plump feeling of smoke and reduce the irritation of the tobacco leaves to a certain extent.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
A strain of Bacillus amyloliquefaciens strain is deposited in Guangdong province microorganism strain collection center, and the collection number is GDMCC No: 61457.
the strain is derived from microorganisms on the surface of flue-cured tobacco, and the prepared biological agent can not introduce exogenous microorganisms when acting on tobacco leaves; can secrete high-activity amylase, can degrade starch substances in the tobacco leaves, increase the sweet feeling of the tobacco leaves and improve the quality of the tobacco leaves;
the method for obtaining the strain specifically comprises the following steps:
(1) collecting microorganisms on the surface of the tobacco leaf:
selecting 10-50 g of a mildew-free, worm-eating-free and nondestructive tobacco leaf sample from an alcoholization box, immersing the sample into 0.85% physiological saline water containing 100-200 ml, and oscillating the sample for 2-4 hours at the room temperature and at 200-220 rpm; filtering with two layers of sterile gauze, and collecting filtrate containing microorganisms on the surface of the tobacco leaves;
(2) microorganism primary screen with starch degradation capability
Performing gradient centrifugation on the filtrate under the action of 500-10000 g of centrifugal force to remove tobacco leaf raw material fragments and macromolecular impurities, and finally washing with 0.85% physiological saline, and performing heavy suspension precipitation to obtain a bacterial suspension of microorganisms on the surface of the tobacco leaf;
(3) isolation of microorganisms having starch degrading ability:
uniformly coating 100-200 mu L of the bacterial liquid on LB and solid culture media containing tobacco leaf extracting solutions respectively, and culturing at constant temperature of 30-37 ℃ for 2-3 days until the number and the types of grown bacterial colonies are unchanged; employing the microbial selection System QPixTM400(Molecular Devices) are aseptically transferred into a 250mL shake flask, and after 24 hours of culture, the mixture is centrifuged at 3000rpm for 10min, and the supernatant is taken and the amylase activity of each strain is measured.
(4) Purification and strain identification
Inoculating the screened strain with higher amylase capability into an LB liquid medium, carrying out shake culture at 30-40 ℃ for 24-36 h, centrifuging fermentation liquor at 8000-10000 g and 4 ℃ for 5-10 min, removing supernatant, collecting bacterial sludge, and washing the bacterial sludge for 2-3 times by using sterile 0.85% physiological saline; the bacterial genome extraction kit is adopted to extract the thallus DNA, the products obtained by 16s rDNA PCR amplification are sequenced after being checked by gel electrophoresis, and then compared in an NCBI (National Center for Biotechnology Information, https:// www.ncbi.nlm.nih.gov /) database, the strain is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens strain).
The application of the strain in tobacco processing specifically comprises the following steps:
(1) biological preparation
Inoculating the screened strain into an LB liquid culture medium containing a tobacco leaf extracting solution, carrying out shaking culture at 37 ℃ for 24h, centrifuging a fermentation liquid at 10000g and 4 ℃ for 10min, removing a supernatant, collecting bacterial sludge, and washing the bacterial sludge with sterile 0.85% physiological saline for 2-3 times; adding a proper amount of trehalose and a sucrose solution protective agent, performing vacuum freeze concentration, preparing microbial inoculum dry powder, and preserving at low temperature for later use;
(2) application of biological agent in tobacco mellowing
Mixing biological preparation with sterile water to obtain 1 × 10 mixture8～10Uniformly spraying the cfu/mL bacterial suspension onto the surface of the tobacco leaves according to the addition of 20-30%, culturing for 12-48 h at 30-40 ℃ and 70-80% humidity, taking out the tobacco leaves, placing the tobacco leaves in an oven, drying the tobacco leaves at 80-90 ℃ until the water content is 13-14%, and stopping the continuous metabolism of the bacterial strains;
(3) evaluation of tobacco leaf quality
And (3) moisture balance: keeping the alcoholized tobacco leaves at the temperature of 22 ℃ and the humidity of 65% for balancing the moisture for 48 h;
the sensory quality of the tobacco leaves is evaluated by professional smokers according to YC/T138-1998 tobacco and tobacco product sensory evaluation method and YC/T496-2014 cigarette sensory comfort evaluation method.
The following describes embodiments of the present invention in more detail with reference to specific examples:
example 1
Obtaining, identifying and preserving strains:
separating alcoholized tobacco leaf microorganisms:
(1) collecting microorganisms on the surface of the tobacco leaf:
from different positions in the alcoholization tank, 10g of a mildew-free, worm-eating-free and non-destructive tobacco leaf sample is selected by a five-point sampling method and immersed in a 250mL conical flask containing 200 mL of 0.1M and 0.85% physiological saline, and oscillated at 220rpm and 30 ℃ for 3 hours. Filtering with two layers of sterile gauze, and collecting filtrate containing microorganisms on the surface of tobacco leaf.
(2) And (3) separating microorganisms: and uniformly coating 100-200 mu L of the bacterial liquid on LB and solid culture media containing tobacco leaf extracting solutions respectively, and culturing at constant temperature of 30-37 ℃ for 2-3 days until the number and the types of grown bacterial colonies are unchanged. Employing the microbial selection System QPixTM400(Molecular Devices) were aseptically transferred into 250mL shake flasks, incubated for 24h, centrifuged at 3000rpm for 10min, and the supernatant was collected. The amylase activity of each strain was determined according to the experimental method. Washing thallus precipitate, adding sterile water to obtain bacterial suspension, separating and purifying strains by flat plate, culturing to obtain bacterial suspension, mixing with 50% glycerol at a ratio of 1:1, and preservingPlacing in an ultra-low temperature refrigerator at-80 deg.C for use.
Screening a culture medium: peptone 10g/L, NaCl 5g/L, yeast extract 5g/L, pH 7.2. Sterilizing other components in the culture medium at 121 deg.C for 20min, cooling, and rapidly adding xanthophyll solution into the culture medium before inoculation.
Purification and strain identification
Inoculating the screened strain with higher starch degradation capability into an LB liquid medium, carrying out shaking culture at 30 ℃ for 24h, centrifuging the fermentation liquor at 10000g and 4 ℃ for 10min, discarding the supernatant, collecting bacterial sludge, and washing the bacterial sludge with sterile 0.85% physiological saline for 2-3 times. Bacterial genome extraction kit is adopted to extract thallus DNA which is used as PCR amplification template. The 16s rDNA universal primer 27F (5 'AGAGTTTGATCCTGGCTCAG 3'), 1492R (5 'TACGGCTACCTTGTTACGACTT 3'), pre-denatured at 94 ℃ for 5min, denatured at 94 ℃ for 30s, annealed at 55 ℃ for 30s, extended at 72 ℃ for 90s, 30 cycles, annealed at 72 ℃ for 10min, amplified, and the obtained product was sent to Shanghai Biotechnology engineering company for sequencing after passing the gel electrophoresis test, and then compared in NCBI (National Center for Biotechnology Information, https:// www.ncbi.nlm.nih.gov /) database, and the strain was Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) with a similar percentage to Bacillus amyloliquefaciens (Per.Ident) of 99.86%. Is preserved in the Guangdong provincial microorganism culture collection (with the preservation number of GDMCC No: 61457).
Example 2
Preparation of biological agent and application of strain in tobacco leaf production:
biological preparation
Inoculating the screened strain into an LB liquid culture medium containing a tobacco leaf extracting solution, carrying out shaking culture at 37 ℃ for 24h, centrifuging a fermentation liquid at 10000g and 4 ℃ for 10min, removing a supernatant, collecting bacterial sludge, and washing the bacterial sludge with sterile 0.85% physiological saline for 2-3 times. And adding a proper amount of trehalose and a sucrose solution protective agent, performing vacuum freezing concentration, preparing the microbial inoculum dry powder, and preserving at low temperature for later use.
Enzyme production performance test of strain
The secretion of starch degrading enzymes by the strain was determined. The bacterial strain is found to have the capability of degrading protein and can secrete amylase with higher enzyme activity.
TABLE 1 enzyme-producing ability test of strains
| Class of enzyme | Amylase (U/g. h) |
| Enzyme activity | 92.36±2.01 |
Artificially alcoholized tobacco leaves without alcoholization
Diluting the biological preparation with sterile water to thallus concentration of 1 × 1010And (3) uniformly spraying the cfu/mL of the bacterial strain onto the surface of the tobacco leaves according to the addition of 20%, uniformly mixing, culturing for 24 hours in a temperature and humidity control incubator at 37 ℃ and 70% of humidity, taking out the tobacco leaves, placing the tobacco leaves in an oven, drying the tobacco leaves at 90 ℃ until the moisture is 13-14%, and stopping the continuous metabolism of the bacterial strain. Spraying the same amount of sterile water on the tobacco leaves, and fermenting under the same culture condition to obtain an experimental control sample.
Evaluating the tobacco leaf quality:
and (3) moisture balance: and (3) placing the alcoholized tobacco leaves under the conditions of temperature of 22 ℃ and humidity of 65% for balancing moisture for 48 h.
The sensory quality of tobacco leaves is evaluated by 7 professional smokers according to YC/T138-1998 tobacco and tobacco product sensory evaluation method, YC/T496-2014 cigarette sensory comfort evaluation method and related enterprise single material tobacco and evaluation standards. According to the result of the tobacco smoking, the original tobacco smoking is characterized in that the tobacco smoking has obvious irritation and miscellaneous gas, the fragrance is faint, but the fresh and sweet feeling is still good, the whole tobacco smoking has certain sweetness, the irritation and miscellaneous gas of the smoke are reduced after the tobacco smoking is treated by the biological agent, the fragrance concentration is increased, the full feeling is improved, the quality of the tobacco can be better promoted, and the alcoholization time of the tobacco can be shortened.
Example 3
Use of the biological agent in tobacco production (omitted procedure same as in example 2):
the biological agent prepared in example 2 is used for artificially alcoholizing inferior tobacco leaves (strong flavor tobacco leaves which are naturally alcoholized for 2 years and still have the quality not reaching the minimum use requirement). The alcoholization condition is to dilute the biological preparation with sterile water to the thallus concentration of 1 × 108 ～10And (3) uniformly spraying the cfu/mL of the strain onto the surface of the tobacco leaves according to the addition of 20%, uniformly mixing, culturing for 48 hours in a temperature and humidity control incubator at the temperature of 37 ℃ and the humidity of 70%, taking out the tobacco leaves, placing the tobacco leaves in an oven, drying the tobacco leaves at the temperature of 80-90 ℃ until the moisture is 13-14%, and stopping the continuous metabolism of the strain. Spraying the same amount of sterile water on the tobacco leaves, and fermenting under the same culture condition to obtain an experimental control sample. And evaluating the quality of the tobacco leaves. The original tobacco leaf product has the overall characteristics of obvious stimulation to the throat, obvious miscellaneous gas and weak sweet feeling. After being treated by biological agent, the sweetness can be obviously increased, the fragrance fullness is increased, and the irritation of tobacco leaves is reduced.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims. It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition. In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Sequence listing
<110> limited liability company for tobacco industry in Sichuan
<120> Bacillus amyloliquefaciens, obtaining method and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1457
<212> DNA
<213> Bacillus amyloliquefaciens (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
gccaggggcg ggggtgctaa tacatgcaag tcgagcggac agatgggagc ttgctccctg 60
atgttagcgg cggacgggtg agtaacacgt gggtaacctg cctgtaagac tgggataact 120
ccgggaaacc ggggctaata ccggatggtt gtttgaaccg catggttcag acataaaagg 180
tggcttcggc taccacttac agatggaccc gcggcgcatt agctagttgg tgaggtaacg 240
gctcaccaag gcgacgatgc gtagccgacc tgagagggtg atcggccaca ctgggactga 300
gacacggccc agactcctac gggaggcagc agtagggaat cttccgcaat ggacgaaagt 360
ctgacggagc aacgccgcgt gagtgatgaa ggttttcgga tcgtaaagct ctgttgttag 420
ggaagaacaa gtgccgttca aatagggcgg caccttgacg gtacctaacc agaaagccac 480
ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt ccggaattat 540
tgggcgtaaa gggctcgcag gcggtttctt aagtctgatg tgaaagcccc cggctcaacc 600
ggggagggtc attggaaact ggggaacttg agtgcagaag aggagagtgg aattccacgt 660
gtagcggtga aatgcgtaga gatgtggagg aacaccagtg gcgaaggcga ctctctggtc 720
tgtaactgac gctgaggagc gaaagcgtgg ggagcgaaca ggattagata ccctggtagt 780
ccacgccgta aacgatgagt gctaagtgtt agggggtttc cgccccttag tgctgcagct 840
aacgcattaa gcactccgcc tggggagtac ggtcgcaaga ctgaaactca aaggaattga 900
cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta 960
ccaggtcttg acatcctctg acaatcctag agataggacg tccccttcgg gggcagagtg 1020
acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1080
gagcgcaacc cttgatctta gttgccagca ttcagttggg cactctaagg tgactgccgg 1140
tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat gacctgggct 1200
acacacgtgc tacaatggac agaacaaagg gcagcgaaac cgcgaggtta agccaatccc 1260
acaaatctgt tctcagttcg gatcgcagtc tgcaactcga ctgcgtgaag ctggaatcgc 1320
tagtaatcgc ggatcagcat gccgcggtga atacgttccc gggccttgta cacaccgccc 1380
gtcacaccac gagagtttgt aacacccgaa gtcggtgagg taacctttag gagccagccg 1440
ccgaaggtga acagagg 1457
Claims (5)
1. A strain of Bacillus amyloliquefaciens strain is deposited in Guangdong province microorganism strain collection center, and the collection number is GDMCC No: 61457.
2. the method for obtaining Bacillus amyloliquefaciens according to claim 1, which comprises the following steps:
(1) collecting microorganisms on tobacco leaf surface
Selecting 10-50 g of a mildew-free, worm-eating-free and nondestructive tobacco leaf sample from an alcoholization box, immersing the sample into 0.85% physiological saline water containing 100-200 ml, and oscillating the sample for 2-4 hours at the room temperature and at 200-220 rpm; filtering with two layers of sterile gauze, and collecting filtrate containing microorganisms on the surface of the tobacco leaves;
(2) microorganism primary screen with starch degradation capability
Performing gradient centrifugation on the filtrate under the action of 500-10000 g of centrifugal force to remove tobacco leaf raw material fragments and macromolecular impurities, and finally washing with 0.85% physiological saline, and performing heavy suspension precipitation to obtain a bacterial suspension of microorganisms on the surface of the tobacco leaf;
(3) isolation of microorganisms having starch-degrading ability
Uniformly coating 100-200 mu L of the bacterial liquid on LB and solid culture media containing tobacco leaf extracting solutions respectively, and culturing at constant temperature of 30-37 ℃ for 2-3 days until the number and the types of grown bacterial colonies are unchanged; employing the microbial selection System QPixTM400(Molecular Devices) are aseptically transferred into a 250mL shake flask, and after 24 hours of culture, the mixture is centrifuged at 3000rpm for 10min, and the supernatant is taken and the amylase activity of each strain is measured.
(4) Purification and strain identification
Inoculating the screened strain with higher amylase capability into an LB liquid medium, carrying out shake culture at 30-40 ℃ for 24-36 h, centrifuging fermentation liquor at 8000-10000 g and 4 ℃ for 5-10 min, removing supernatant, collecting bacterial sludge, and washing the bacterial sludge for 2-3 times by using sterile 0.85% physiological saline; and extracting thallus DNA by using a bacterial genome extraction kit, carrying out gel electrophoresis inspection on a product obtained by 16s rDNA PCR amplification, and sequencing after the product is qualified.
3. The application of the bacillus amyloliquefaciens as claimed in claim 1 in tobacco leaf production is characterized by comprising the following steps of:
(1) biological preparation
Inoculating the screened strain into an LB liquid culture medium containing a tobacco leaf extracting solution, carrying out shaking culture at 37 ℃ for 24h, centrifuging a fermentation liquid at 10000g and 4 ℃ for 10min, removing a supernatant, collecting bacterial sludge, and washing the bacterial sludge with sterile 0.85% physiological saline for 2-3 times; adding a proper amount of trehalose and a sucrose solution protective agent, performing vacuum freeze concentration, preparing microbial inoculum dry powder, and preserving at low temperature for later use;
(2) application of biological agent in tobacco mellowing
Mixing biological preparation with sterile water to obtain 1 × 10 mixture8～10Uniformly spraying the cfu/mL bacterial suspension onto the surface of the tobacco leaves according to the addition of 20-30%, culturing for 12-48 h at 30-40 ℃ and 70-80% humidity, taking out the tobacco leaves, placing the tobacco leaves in an oven, drying the tobacco leaves at 80-90 ℃ until the water content is 13-14%, and stopping the continuous metabolism of the bacterial strains;
(3) evaluation of tobacco leaf quality
And (3) moisture balance: keeping the alcoholized tobacco leaves at the temperature of 22 ℃ and the humidity of 65% for balancing the moisture for 48 h;
the sensory quality of the tobacco leaves is evaluated by professional smokers according to YC/T138-1998 tobacco and tobacco product sensory evaluation method and YC/T496-2014 cigarette sensory comfort evaluation method.
4. Use of the bacillus amyloliquefaciens according to claim 1 in fermentation of tobacco plant tissue.
5. A biological agent comprising the Bacillus amyloliquefaciens of any one of claims 1-4.
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