CN111616403B - Biological agent for accelerating tobacco mellowing and improving tobacco quality and application thereof - Google Patents
Biological agent for accelerating tobacco mellowing and improving tobacco quality and application thereof Download PDFInfo
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Abstract
The invention discloses a biological agent for accelerating tobacco mellowing and improving the quality of tobacco and application thereof, belonging to the technical field of tobacco mellowing. The invention separates and obtains a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) capable of producing a plurality of enzymes from alcoholized tobacco leaves. The strain is suitable for fermentation of flue-cured tobacco, is beneficial to promoting the alcoholization process of tobacco leaves with different flavors and qualities, improves the quality and shortens the alcoholization time. The strain is easy to culture, has high growth speed, can tolerate causticity conditions, has high activity, can produce various enzymes, and has the characteristics of capability of degrading high molecular substances and the like. By properly adjusting the conditions of the biological preparation for treating the tobacco leaves, the effects of reducing the irritation of the tobacco leaves, weakening the miscellaneous gas, increasing the sweet taste and the like can be obtained.
Description
Technical Field
The invention relates to a biological agent for accelerating tobacco mellowing and improving the quality of tobacco and application thereof, belonging to the technical field of tobacco mellowing.
Background
Tobacco mellowing (also known as aging) is an important step in the cigarette industry. The quality of the newly-harvested tobacco leaves in the current year has certain defects, such as heavy green miscellaneous gas, covering fragrance with miscellaneous gas, high irritation and rough smoke, and particularly, the low-grade tobacco leaves have bad feelings of bitterness, acridness, astringency and the like. In order to improve the quality of tobacco leaves and to increase the usability and applicability of tobacco leaves for the manufacture of cigarettes, it is necessary to carry out an alcoholization treatment of the tobacco leaves. The tobacco alcoholization is to make the tobacco undergo a series of biochemical reactions under the combined action of microorganisms, enzymes and chemical components by a storage mode, for example, macromolecular compounds without aroma characteristics such as starch, protein, pectin, cellulose, lignin, nicotine, aroma precursor (terpenes) and the like undergo decomposition and conversion to form small molecular compounds with aroma characteristics such as reducing sugar, organic acid, amino acid, carbonyl compound and the like, thereby promoting the improvement of the aroma and the smoking quality of the tobacco. However, the efficiency of natural alcoholization of tobacco leaves in industry is low, and the time is usually as long as 2-3 years, so that the inventory management cost is increased, the turnover period of the tobacco leaves is long, the mobile capital occupation is large, and the quality of some inferior tobacco leaves after alcoholization still can not meet the production and use requirements. In this regard, the artificial alcoholization is being paid increasing attention by researchers and industries.
The high content of high molecular substances in the tobacco leaves often affects the quality of the tobacco leaves, for example, when the content of starch is high, the burning speed and completeness of the tobacco leaves are affected, and a certain burnt flavor is generated; when the protein content is higher, the odor of burning feather can be generated during burning, and the feather is spicy and bitter; and the high content of the pectin substances in the tobacco leaves can increase the generation amount of tar in the tobacco leaves and increase the content of harmful substances such as formaldehyde, acetaldehyde and the like in the smoke. Meanwhile, water-soluble sugar, reducing sugar, amino acid and other micromolecule substances generated by degrading starch, pectin, protein and other substances can balance acid and alkali taste, can promote the generation of Maillard reaction products under certain conditions, reduce the irritation of smoke and contribute to the taste and fragrance of the smoke. Therefore, the microorganism or commercial hydrolase preparation containing the corresponding degrading enzyme is used alone or in combination, which is beneficial to promoting the alcoholization process. A composite tobacco quality-enhancing agent containing bacillus megatherium and bacillus subtilis, and additionally containing saccharifying enzyme and protease is prepared by Zhao Mingzhi et al in 2006; panjiahua and the like in 2012 research a composite microbial inoculum matched by lactobacillus plantarum and acetobacter thermoaceti; 2011 person of Yi et al uses a bacterial agent of bacillus which can produce amylase at high yield; in 2006, plum Lin et al, and the aged, developed Bacillus subtilis and Bacillus altitudinis inoculants suitable for upper tobacco leaves. Although related researches are more and have various characteristics, the methods have different effects, and the problems of complex preparation process of the microbial inoculum, high production cost, small feasibility of industrial application and operation, low universality on different tobacco varieties and the like exist.
Disclosure of Invention
In order to solve the current situations of low efficiency, long time, poor treatment effect on inferior and inferior tobacco leaves and the like of natural alcoholization of the tobacco leaves at present, the invention screens and obtains a strain capable of producing a plurality of enzymes such as amylase, saccharifying enzyme, protease, pectinase and the like from alcoholized tobacco leaves, the comprehensive capability of producing the enzymes is strong, and the degradation effect of polymer substances in a balanced manner by matching other microbial agents or enzyme preparations in the preparation of biological preparations is omitted; the strain has good universality on quality improvement of tobacco leaves, and can have good quality improvement effect on the tobacco leaves at the initial stage of alcoholization and the tobacco leaves which can not meet the use requirement after alcoholization for two years.
The invention provides a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), which is preserved in Guangdong province microorganism strain preservation center at 23.3.2020, with the preservation number of GDMCC No. 60982, and the preservation address of No. 59 building 5 of Michelia Tokyo 100, Guangzhou city.
The invention provides a biological agent, which is prepared from the bacillus amyloliquefaciens.
The invention provides a preparation method of the biological agent, which comprises the steps of inoculating the bacillus amyloliquefaciens into an LB liquid culture medium containing a tobacco leaf extracting solution, and carrying out shake culture at 35-38 ℃ for 20-25 h until the concentration of a bacterial solution is 1.0 multiplied by 107～1.0×1010cfu/mL, centrifuging, removing the supernatant, collecting bacterial sludge, washing the bacterial sludge for 2-3 times by using sterile 0.85% physiological saline, adding trehalose with the final concentration of 3-7% (v/v) and sucrose solution with the final concentration of 4-8% (v/v) as a protective agent, and performing vacuum freeze concentration to prepare microbial inoculum dry powder, namely the biological preparation.
The invention provides a method for accelerating tobacco leaf purification, which is characterized in that after being diluted by water, a biological agent is added, or bacillus amyloliquefaciens liquid is sprayed on the surface of tobacco leaves, and the tobacco leaves are cultured for 20-25 hours.
In one embodiment of the present invention, the biological preparation is diluted with water to a cell concentration of 1.0X 107～1.0×1010cfu/mL。
In one embodiment of the invention, the biological agent is diluted and sprayed onto the surface of tobacco leaves according to the proportion of 10-25% (w/w).
In one embodiment of the invention, tobacco leaves are cultured in an environment with the temperature of 35-40 ℃ and the humidity of 65-75%.
In one embodiment of the invention, the tobacco leaves comprise fen-flavor non-alcoholized tobacco leaves, or inferior Luzhou-flavor, or inferior mid-flavor tobacco leaves.
The invention also provides a method for improving the fragrance of tobacco leaves, which is to dilute the biological agent by adding water or spray the bacillus amyloliquefaciens bacterial liquid on the surfaces of the tobacco leaves and culture for 12-25 hours.
The invention also protects the application of the bacillus amyloliquefaciens, or the biological agent, or the method for accelerating the purification of the tobacco leaves in the alcoholization of the tobacco leaves.
The invention has the beneficial effects that:
(1) the strain provided by the invention is derived from an ecological system of alcoholized tobacco leaves, and the prepared biological agent acts on the tobacco leaves and does not introduce exogenous microorganisms;
(2) the bacillus amyloliquefaciens has the advantages of high strain growth speed, difficult infectious microbe infection, strong capability of enduring harsh conditions and easy culture; the tobacco leaf flavoring agent can secrete amylase, glucoamylase, protease, pectinase and other enzymes, the enzyme activities of the amylase, the glucoamylase, neutral protease and acid protease can respectively reach 90.92U/g.min, 38.52U/g.h, 64.33U/g.h and 25.27U/g.h, the degradation of various high molecular substances in the tobacco leaf can be realized, and the generation of rich small-molecular fragrant substances is facilitated;
(3) the acting time of the strain is 12-48 h, which is shorter than that of a similar single strain preparation reported;
(4) the bacillus amyloliquefaciens can be suitable for different tobacco leaves, and the evaluation of aroma components in alcoholized tobacco leaves is analyzed through sensory product smoking and HS-SPME-GC/MS technology, so that the effects of improving the quality of the tobacco leaves, reducing the irritation and impurity gas of the tobacco leaves and increasing the sweet taste are achieved, and the increase of various volatile components in the tobacco leaves is facilitated.
Biological material preservation
Bacillus amyloliquefaciens, which is classified and named as Bacillus amyloliquefaciens, is preserved in Guangdong province microorganism strain preservation center in 3-23 days 2020, and has the preservation number of GDMCC No:60982, the preservation address is No. 59 building 5 of No. 100 Dazhong Jie-Lu-100 Guangzhou city.
Detailed Description
The embodiments of the present invention will be described in further detail with reference to examples. The examples are intended to illustrate the invention, but are not intended to limit the scope of the invention.
And (3) amylase activity determination: determining the activity of amylase by using an iodine color development method, wherein the activity of the amylase is defined as water enzyme extract of each gram of dry tobacco leaves, 1mg of soluble starch solution is hydrolyzed in 1min at the temperature of 60 ℃, the activity is defined as an enzyme activity unit and is calculated as follows: u/g.min.
And (3) assaying the activity of the saccharifying enzyme: the activity of the carbohydrase is determined by a 3, 5-dinitrosalicylic acid (DNS) color development method, the enzyme activity is defined as water enzyme extraction liquid per gram of dry tobacco leaves, under the conditions of 40 ℃ and pH4.6, 1 hour of soluble starch is hydrolyzed to generate 1mg of glucose, and the enzyme activity is defined as an enzyme activity unit and is calculated as follows: u/g.h.
And (3) enzyme activity determination of pectinase: measuring enzyme activity of pectinase by a DNS (domain name system) color development method, wherein the enzyme activity is defined as water enzyme extract of each gram of dry tobacco leaves, and hydrolyzing the pectin solution for 1h at 50 ℃ to generate 1mg of galacturonic acid, which is defined as an enzyme activity unit and is calculated as follows: u/g.h.
And (3) measuring the enzyme activity of neutral protease: neutral protease enzyme activity is determined by adopting a forskolin phenol method, the enzyme activity is defined as water enzyme extract of each gram of dry tobacco leaves, and 1 mu g of tyrosine generated by hydrolyzing casein every hour at 40 ℃ under p H7.2.2 is defined as 1 enzyme activity unit. The method comprises the following steps: u/g.h.
And (3) measuring the enzyme activity of the acid protease: the process differs from the neutral protease in that the pH of the enzyme is changed to 3.
Determining volatile and semi-volatile aroma components in the alcoholized tobacco by using an HS-SPME-GC/MS technical platform:
(1) sample treatment: the alcoholized tobacco leaves are treated for 90s by a grinder with 70Hz and sieved by a 60-mesh sieve. Accurately weighing 2.000g of tobacco powder, filling the tobacco powder into a 20mL headspace bottle, adding 1 mu L of tritiated naphthalene-dichloroethane internal standard solution, fully and uniformly mixing, and then covering a magnetic headspace bottle cover to be detected. And (3) dividing the alcoholized tobacco leaf sample to be tested and the reference sample into 6 parallel samples in each group.
(2) Headspace solid phase microextraction: adopting a divinylbenzene/activated carbon/polydimethylsiloxane DVB/CAR/PDMS solid phase micro-extraction fiber head with the film thickness of 50/30 mu m; incubation temperature: extraction time at 60 ℃: and (3) 30 min.
(3) GC/MS chromatographic conditions: a Thermo Trace1310-ISQ GC/MS gas chromatography/mass spectrometer equipped with an autosampler was used. A highly inert cross-linked weakly polar HP-5 capillary column (60 m.times.0.25 mm.times.0.25 μm) was used;
(4) and (3) analyzing a aroma detection result: and the detection result is subjected to data processing, and the influence of the biological agent on volatile aroma components in the tobacco leaves after the tobacco leaves are processed is contrastively analyzed.
Example 1: isolation and characterization of strains
The method comprises the following steps: separating alcoholized tobacco leaf microorganisms:
the tobacco leaves used for screening the strains are from Liangshan in Sichuan and are provided by tobacco industry Limited liability company in Sichuan.
(1) Collecting microorganisms on the surface of the tobacco leaf:
a mildew-free, worm-eating-free, non-destructive tobacco sample 10g was selected from the alcoholizing chamber and immersed in a 250mL Erlenmeyer flask containing 200 mL of 0.1M, pH7.2 sterile phosphate buffer and shaken at 220rpm and 30 ℃ for 2 hours. Filtering with two layers of sterile gauze, and collecting filtrate containing microorganisms on the surface of tobacco leaf.
(2) And (3) separating microorganisms: diluting the above bacterial liquid, uniformly coating 100 μ L of the diluted bacterial liquid on LB solid culture medium containing tobacco leaf extract, and culturing at 37 deg.C for 1 day until the number and kinds of the grown bacterial colony are unchanged. Transferring the strain to a new LB plate by using a sterile inoculating needle, culturing at the constant temperature of 37 ℃ for 12-14 h, picking out a single colony from the plate to the new LB plate, and continuously culturing for 2-3 generations in such a way until a stable single colony strain is obtained.
Step two: screening of target Strain
(1) Primary screening by a flat plate: 2% (v/v) soluble starch, 1% (v/v) pectin and 2% (v/v) casein were added to LB basal mediumRespectively obtaining primary screening culture mediums capable of producing amylase, pectin degrading enzyme and protein degrading enzyme. Pure culture of each isolated strain to 1.0 × 106～1.0×108cfu/mL bacterial solution is dibbled to a primary screening flat plate, and the capability of each bacterial strain for degrading insoluble high molecular substances to form transparent rings is inspected.
(2) Enzyme activity detection and re-screening:
culturing the strain capable of forming a large transparent ring in LB culture medium at 37 deg.C for 1 day, and measuring the activity of amylase, diastase, pectinase and protease to examine the enzyme production.
Selecting strains with more enzyme-producing species and higher enzyme activity, wherein the enzyme-producing conditions of the strains are shown in table 1.
TABLE 1 enzyme production of preferred strains
Step three: identification of strains
And (2) screening strains, inoculating the strains into an LB liquid culture medium containing a tobacco leaf extracting solution, carrying out shaking culture at 37 ℃ for 24 hours to obtain a bacterial liquid, centrifuging the bacterial liquid at 10000g and 4 ℃ for 10 minutes, removing supernatant, collecting bacterial sludge, and washing the bacterial sludge for 2-3 times by using sterile 0.85% physiological saline. Bacterial genome extraction kit is adopted to extract thallus DNA which is used as PCR amplification template.
PCR amplification was performed using 16s rDNA universal primers 27F and 1492R: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 90s, 30 cycles, and annealing at 72 deg.C for 10 min.
27F：5’AGAGTTTGATCCTGGCTCAG 3’，(SEQ ID NO.2)
1492R：5’TACGGCTACCTTGTTACGACTT 3’，(SEQ ID NO.3)。
The obtained PCR product was qualified by gel electrophoresis, sent to Shanghai Biotech engineering company for sequencing, and then compared in NCBI (National Center for Biotechnology Information, https:// www.ncbi.nlm.nih.gov /) database to identify Bacillus amyloliquefaciens (SEQ ID NO. 1) with a percentage of similarity of 99.52% to NC-014551.1.
Step four: strain preservation
The strain is sent to the Guangdong province microorganism strain preservation center for preservation.
Example 2: preparation of biological agents
The strain obtained in example 1 was inoculated into LB liquid medium containing tobacco extract (10 g of 40 mesh tobacco powder added to 100mL of pH7.2 phosphate buffer solution, extracted at room temperature for 1 hour, filtered with gauze and stored at low temperature for further use), and cultured with shaking at 37 ℃ for 24 hours until the bacterial liquid concentration was 1.0X 107～1.0×1010cfu/mL, centrifuging the fermentation liquor at 10000g and 4 ℃ for 10min, removing the supernatant, collecting bacterial sludge, and washing the bacterial sludge for 2-3 times by using sterile 0.85% physiological saline. And adding trehalose with the final concentration of 3-7% (w/w) and sucrose solution with the final concentration of 4-8% (w/w) as protective agents, performing vacuum freeze concentration, preparing microbial inoculum dry powder, and preserving at 4 ℃ for later use.
Example 3: application of biological agent to non-alcoholized fen-flavor tobacco leaves
1. Biological agent alcoholized tobacco leaf
The biological preparation prepared in example 2 was diluted with sterile water to a cell concentration of 1.0X 107～1.0×1010And (3) after cfu/mL, uniformly spraying the mixture to the surface of the tobacco leaves according to the addition amount of 10% (w/w), uniformly mixing, culturing for 24 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% (w/w), and stopping the continuous metabolism of the bacterial strains.
Spraying the same amount of sterile water on the tobacco leaves, and fermenting under the same culture condition to obtain an experimental control sample.
2. 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 standards YC/T138-1998 tobacco and tobacco product-sensory evaluation method and YC/T496-2014 cigarette sensory comfort evaluation method. The specific evaluation results are shown in Table 2.
TABLE 2 Change in sensory quality of tobacco leaf before and after treatment
According to the result of the tobacco smoking, the original tobacco smoking is characterized by obvious irritation and offensive odor, faint aroma, fresh and sweet taste and certain sweetness. After being treated by the biological agent, the smoke becomes soft and exquisite, the fragrance becomes richer, a little more honey and sweet fragrance is added, the sweet feeling is increased, the quality of the tobacco leaves can be better promoted to be improved, and the alcoholization time of the tobacco leaves can be shortened.
3. Detecting and analyzing aroma components of tobacco leaves:
and (3) adopting an HS-SPME-GC/MS technical platform to measure volatile and semi-volatile aroma components in the alcoholized tobacco leaves.
(1) Sample treatment: treating alcoholized tobacco leaves with a grinder at 70Hz for 90s, and sieving with a 60-mesh sieve; accurately weighing 2.000g of tobacco powder, filling the tobacco powder into a 20mL headspace bottle, adding 1 mu L of tritiated naphthalene-dichloroethane internal standard solution, fully and uniformly mixing, and then covering a magnetic headspace bottle cover to be detected. Grouping alcoholized tobacco leaf samples to be tested and control samples, wherein each group comprises 6 parallel samples;
(2) headspace solid phase microextraction: adopting a divinylbenzene/activated carbon/polydimethylsiloxane DVB/CAR/PDMS solid phase micro-extraction fiber head with the film thickness of 50/30 mu m; incubation temperature: extraction time at 60 ℃: 30 min;
(3) GC/MS chromatographic conditions: adopting a Thermo Trace1310-ISQ GC/MS gas chromatography/mass spectrometer with an autosampler; a highly inert cross-linked weakly polar HP-5 capillary column (60 m.times.0.25 mm.times.0.25 μm) was used;
(4) and (3) analyzing a detection result: and after the detection result data are processed, the change conditions of volatile aroma components in the tobacco leaves are processed by using the bacillus amyloliquefaciens biological agent through comparative analysis.
As shown in Table 3, the contents of volatile substances such as alcohol, phenols, esters, carbonyls, heterocycles and the like in the treated tobacco leaves are all improved compared with the control group, wherein the contents of the carbonyls and the heterocycles are respectively improved by 35.7% and 35.3% compared with the control group.
As shown in Table 4, substances which have promotion effects on the flavor, such as rich flowery odour, faint scent and the like, of the treated tobacco leaves are all improved compared with a control, but the content of high molecular substances which are not beneficial to the flavor or do not contribute much to the flavor is reduced, so that the bacterium can degrade and convert the high molecular substances, promote the formation of small molecular flavor substances and has a good effect of improving the quality of the tobacco leaves.
TABLE 3 analysis of the content of volatile substances before and after tobacco treatment (μ g/100g dry basis)
Note: the control is tobacco leaves which are not treated by the bacillus amyloliquefaciens, and the treated tobacco leaves are tobacco leaves which are treated by the bacillus amyloliquefaciens
TABLE 4 analysis of the content of important volatile substances before and after the tobacco treatment (. mu.g/100 g dry basis)
Note: NA: indicating the amount of undetected material; the flavor characteristics of the substances are evaluated by referring to Shihong, tobacco aroma science, Beijing: chinese agricultural press 2011.
TABLE 5 content of six conventional substances in tobacco leaves before and after treatment
Through content measurement of six conventional substances in the tobacco leaves, the total sugar content of the treated tobacco leaves is greatly increased by 39.3 percent and 36.9 percent respectively compared with a control; although the total plant alkali and the total nitrogen content are also increased, the sugar-alkali ratio, the sugar-nitrogen ratio, the chlorine content and the potassium content are proper, and the method plays an important role in balancing the tobacco fragrance and increasing the tobacco fragrance concentration.
Example 4: application of biological agent in improving quality of inferior strong aromatic tobacco leaves
The specific implementation manner is the same as that of example 3, and the differences are as follows: selecting inferior tobacco leaves (strong aromatic tobacco leaves of which the quality does not reach the minimum use requirement in 2 years of natural alcoholization) to carry out artificial alcoholization.
The alcoholization condition is to dilute the biological preparation with sterile water to a thallus concentration of 1.0 × 106～1.0×108And (3) after cfu/mL, uniformly spraying the mixture to the surface of the tobacco leaves according to the addition amount of 20% (w/w), 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.
Evaluating the quality of tobacco leaves:
the reference tobacco product is characterized by faint aroma, obvious earthy smell and woody smell, weak sweet feeling, certain concentration of smoke, certain concentration but not obvious strength and obvious stimulation to the throat. After being treated by the biological agent, the fragrance concentration is increased, the strength is slightly reduced, the penetrating feeling is increased, and the effects of better maintaining the charm and the texture are achieved. The specific absorption results are shown in Table 6.
As shown in Table 7, the content of volatile substances such as alcohol, phenols, acids, esters, carbonyls, heterocycles and alkanes in the treated tobacco leaves is increased compared with that in the control group, wherein the content of the acids is increased by 24.5 percent compared with that in the control group.
As shown in Table 8, substances which have promotion effects on flavor, such as rich flowery odour and faint scent, of the treated tobacco leaves are all improved compared with a control, but the content of high molecular substances which are not beneficial to flavor or do not contribute much to flavor is reduced, so that the bacterium can degrade and convert the high molecular substances, promote the formation of small molecular flavor substances, and has a good effect of improving the quality of the tobacco leaves.
TABLE 6 change in sensory quality of tobacco leaf before and after treatment
TABLE 7 analysis of the content of volatile substances before and after tobacco treatment (μ g/100g dry basis)
Note: the control is tobacco leaves which are not treated by the bacillus amyloliquefaciens, and the treated tobacco leaves are tobacco leaves which are treated by the bacillus amyloliquefaciens
TABLE 8 analysis of the content of important volatile substances before and after the tobacco treatment (. mu.g/100 g dry basis)
Note: NA: indicating the amount of undetected material; the evaluation of the flavor of each position refers to Shihong, etc., tobacco aroma, Beijing: chinese agricultural press 2011.
TABLE 9 content of six conventional substances in tobacco leaves before and after treatment
According to the content measurement of six conventional substances in the tobacco leaves, the content of the total sugar in the treated tobacco leaves is increased, and the tobacco leaves have good promotion effects on balancing the fragrance of the tobacco leaves and increasing the concentration of the tobacco fragrance.
Example 5: application of biological agent in improving quality of inferior middle-flavor tobacco leaves
The specific implementation manner is the same as that of example 3, and the differences are as follows: selecting inferior tobacco leaves (middle-flavor tobacco leaves which are naturally alcoholized for 2 years and still have the quality not reaching the minimum use requirement) to carry out artificial alcoholization.
The alcoholization condition is to dilute the biological preparation with sterile water to a thallus concentration of 1.0 × 107～1.0×1010And (3) evenly spraying the cfu/mL of the strain onto the surface of the tobacco leaves according to the addition amount of 20% (w/w), evenly 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.
The reference tobacco product is characterized by full aroma amount and good texture, but has large smoke concentration, obvious strength, large stimulation, poor taste comfort, slightly obvious alkaline aroma and obvious tongue surface residue.
After being treated by the biological agent, the effects of better improving the strength and the irritation, better taste comfort, better quality retention and the like can be achieved. The results of the specific absorption are shown in Table 10.
TABLE 10 change in sensory quality of tobacco products before and after tobacco treatment
As shown in table 11, the contents of volatile substances such as alcohols, phenols, esters, carbonyls, heterocycles, and alkanes were all increased in the treated tobacco leaves compared to the control group, wherein the carbonyls and heterocycles were increased by 21.9% and 25.2% respectively.
TABLE 11 analysis of the content of volatile substances before and after tobacco treatment by classification (μ g/100g dry basis)
Note: the control is tobacco leaves which are not treated by the bacillus amyloliquefaciens, and the treated tobacco leaves are tobacco leaves which are treated by the bacillus amyloliquefaciens
As shown in table 12, substances having flavor-promoting effects such as rich flowery flavor and faint scent of the treated tobacco leaves are all improved compared with the control, but the contents of high molecular substances which are not beneficial to flavor or do not contribute much to flavor are reduced, which indicates that the bacterium can degrade and convert the high molecular substances, promote the formation of small molecular flavor substances, and has a good effect of improving the quality of the tobacco leaves.
TABLE 12 analysis of the content of important volatile substances before and after tobacco treatment (mg/100g dry basis)
Note: NA: indicating the amount of undetected material; the evaluation of the flavor of each position refers to Shihong, etc., tobacco aroma, Beijing: chinese agricultural press 2011.
TABLE 13 content of six conventional substances in tobacco leaves before and after treatment
According to the content measurement of six conventional substances in the tobacco leaves, the content of the total sugar in the treated tobacco leaves is increased, and the tobacco leaves have good promotion effects on balancing the fragrance of the tobacco leaves and increasing the concentration of the tobacco fragrance.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
CHINA TOBACCO SICHUAN INDUSTRIAL Co.,Ltd.
<120> biological agent for accelerating tobacco mellowing and improving tobacco quality and application thereof
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1457
<212> DNA
<213> Bacillus amyloliquefaciens
<400> 1
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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
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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
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<400> 2
agagtttgat cctggctcag 20
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence
<400> 3
tacggctacc ttgttacgac tt 22
Claims (10)
1. A strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is preserved in Guangdong province microorganism culture collection center at 3-23 days 2020, and the preservation number is GDMCC No. 60982.
2. A biological agent, wherein the biological agent is produced from the bacillus amyloliquefaciens of claim 1.
3. The method for producing a biological agent according to claim 2, wherein the Bacillus amyloliquefaciens is cultured until a cell concentration of the bacterial liquid reaches 1.0X 107～1.0×1010cfu/mL, centrifuging, removing the supernatant, collecting bacterial sludge, washing the bacterial sludge with normal saline for 2-3 times, adding a protective agent, and performing vacuum freezing concentration to prepare microbial inoculum dry powder, namely the biological preparation.
4. The method of claim 3, wherein the protective agent is trehalose and sucrose.
5. A method for accelerating tobacco mellowing, which is characterized in that the method is to add the bacterial liquid of the bacillus amyloliquefaciens of claim 1 to the surface of tobacco; or diluting the biological agent of claim 2 with water and adding the diluted biological agent to the surface of tobacco leaves.
6. The method according to claim 5, wherein the Bacillus amyloliquefaciens is added at a bacterial liquid concentration of 1.0 x 107～1.0×1010cfu/mL, or diluting the biological preparation with water until the thallus concentration is 1.0 × 107～1.0×1010cfu/mL。
7. The method according to claim 6, wherein the bacterial liquid or the diluted biological agent is added to the surface of the tobacco leaf according to the mass ratio of 10-25%.
8. The method according to claim 5, wherein the tobacco leaves are cultured in an environment of 30 to 40 ℃ and 60 to 80% humidity.
9. A method for improving the flavor of tobacco leaves, which is characterized in that the bacterial liquid of the bacillus amyloliquefaciens of claim 1 is added to the surfaces of the tobacco leaves; or diluting the biological agent of claim 2 with water and adding the diluted biological agent to the surface of tobacco leaves.
10. Use of a bacillus amyloliquefaciens according to claim 1, or a biological agent according to claim 2, or a method according to any one of claims 5 to 9 for alcoholizing tobacco leaves.
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| CN113215062A (en) * | 2021-06-15 | 2021-08-06 | 四川中烟工业有限责任公司 | Bacillus amyloliquefaciens, and acquisition method and application thereof |
| CN114747797A (en) * | 2022-04-20 | 2022-07-15 | 四川中烟工业有限责任公司 | Method for directionally promoting sugar conversion of cigar by utilizing biological fermentation quality-enhancing agent |
| CN115404180A (en) * | 2022-06-26 | 2022-11-29 | 上海龙殷生物科技有限公司 | Bacillus fragrans and application thereof in tobacco |
| CN117701437B (en) * | 2023-12-14 | 2024-10-18 | 湖北省烟草科学研究院 | Bacillus subtilis for degrading starch in tobacco leaves and application thereof |
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