CN105543122B - Composite microbial inoculum and application thereof in tobacco mellowing - Google Patents
Composite microbial inoculum and application thereof in tobacco mellowing Download PDFInfo
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- CN105543122B CN105543122B CN201510866196.7A CN201510866196A CN105543122B CN 105543122 B CN105543122 B CN 105543122B CN 201510866196 A CN201510866196 A CN 201510866196A CN 105543122 B CN105543122 B CN 105543122B
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Abstract
The invention relates to a composite microbial inoculum which is prepared by respectively culturing bacillus subtilis, bacillus licheniformis and bacillus amyloliquefaciens and mixing the bacillus subtilis, the bacillus licheniformis and the bacillus amyloliquefaciens in proportion. The bacillus subtilis and the bacillus licheniformis are separated from the Maotai distiller's yeast. The invention relates to the development of the function of the Maotai wine aroma-producing microorganisms and the improvement of the tobacco quality, applies the Maotai wine yeast microorganism fermentation product to the tobacco fermentation for the first time, can increase the variety of high-quality microorganisms in the tobacco fermentation and the variety of aroma substances through the activity of the aroma-producing microorganisms or the addition of metabolites thereof, and has the effect of enhancing the aroma similar to the addition of the Maotai wine, thereby improving the alcoholization quality of the tobacco.
Description
Technical Field
The invention relates to a composite microbial inoculum and application thereof in tobacco mellowing, belonging to the technical field of tobacco flavoring treatment.
Background
In the tobacco mellowing and fermenting stage, specific microorganisms are added, so that the mellowing and fermenting time of the tobacco can be effectively shortened, the contents of main chemical components in the tobacco (nicotine content, protein, amino acid content and TSNA) can be regulated and controlled, and the content of tobacco fragrance precursors can be increased.
Many research results show that the internal quality of the tobacco can be obviously improved and the miscellaneous gas and the irritation of the tobacco can be reduced by improving the microbial fermentation conditions of the tobacco or adding various substances. For example, Seaaich et al reported that spraying a spore suspension of Alternaria alternata 6 days before cigarettes could greatly improve the aroma of tobacco leaves. Wentiligy and other researches find that certain yeast strains can induce tobacco leaves to generate attractive fragrance after being inoculated on alcoholized tobacco leaves for 3 days. The research of Zhao MingQin and the like finds that the tobacco leaves treated by the microorganisms have obviously improved aroma quality and reduced miscellaneous gas and irritation. CN104207324A discloses a Bacillus subtilis with a preservation number of CCTCC M2013150, which is inoculated in tobacco leaves and fermented, and the result shows that the content of aroma substances in the tobacco leaves can be improved. However, the above method has a limited improvement effect.
Disclosure of Invention
The invention aims to provide a composite microbial inoculum and application thereof in tobacco mellowing. The composite microbial inoculum is used for tobacco mellowing, so that the content of aroma substances of the tobacco can be increased, the mellowing quality of the tobacco can be improved, and a foundation is laid for developing the tobacco with unique style and characteristics.
In order to achieve the purpose, the invention adopts the following technical scheme:
a composite microbial inoculum is prepared by mixing Bacillus subtilis, Bacillus licheniformis and Bacillus amyloliquefaciens; wherein the viable count of Bacillus subtilis is not less than 109cfu/ml, viable count of Bacillus licheniformis not less than 109cfu/ml, viable count of Bacillus amyloliquefaciens not less than 109cfu/ml。
Wherein, the bacillus subtilis and the bacillus licheniformis are both separated from the Maotai distiller's yeast; the Bacillus subtilis is identified as Bacillus subtilis MTLB01(Bacillus subtilis MTLB01), the preservation number is CCTCC NO: M2015575, the preservation date is 2015, 9 and 25 days, and the Bacillus subtilis is preserved in China center for type culture Collection (CCTCC for short, address: Wuhan university in Wuhan, China, zip code: 430072);
the Bacillus licheniformis is identified as Bacillus licheniformis MTLB02(Bacillus licheniformis MTLB02), the preservation number is CCTCC NO: M2015576, the preservation date is 2015, 9 and 25 days, and the Bacillus licheniformis is preserved in China center for type culture Collection (CCTCC for short, address: Wuhan university, Wuhan, China, Zip code: 430072).
The bacillus amyloliquefaciens used in the invention can be selected from conventional bacillus amyloliquefaciens understood by a person skilled in the art, such as those purchased commercially or cultured by oneself.
In a preferred embodiment of the present invention, the number of viable bacteria of Bacillus subtilis in the complex microbial agent is 109cfu/ml, viable count of Bacillus licheniformis 109cfu/ml, viable count of Bacillus amyloliquefaciens 109cfu/ml。
In the composite microbial inoculum, the bacillus subtilis and the bacillus licheniformis are obtained by the following steps:
1) inoculating black part of Maotai wine yeast into PDA plate culture medium, culturing at 48-50 deg.C for 23-24 hr, selecting single colony, and performing conventional purification culture;
2) inoculating the purified bacterial liquid into a wheat culture medium, and performing gradient culture at 37 ℃, 46 ℃ and 55 ℃ for 6 d; wherein the culture is carried out at 37 ℃ for 2 days, at 46 ℃ for 2 days and at 55 ℃ for 2 days;
3) and (3) screening bacillus subtilis and bacillus licheniformis which produce more substances with sauce fragrance from the mixed strains obtained by culturing in the step 2).
The preparation method of the PDA culture medium comprises the following steps: weighing 200g of potato, cleaning, peeling, chopping, adding 1000mL of ddH2Boiling O for 30min, filtering with four layers of gauze, adding 20g glucose and 15-20g agar, dissolving, sterilizing at 121 deg.C for 30min, pouring onto flat plate, and cooling.
The formula of the wheat culture medium is as follows: crushing wheat into half 100g of wheat grains and half of wheat flour, adding 50% water, sterilizing at 121 deg.C for 30min, cooling, and scattering.
The invention also provides a preparation method of the composite microbial inoculum, which comprises the steps of respectively inoculating the bacillus subtilis, the bacillus licheniformis and the bacillus amyloliquefaciens in a PDA culture medium, culturing for 22-24h at 36-37 ℃, collecting bacterial liquid, and mixing according to a proportion.
The invention also provides application of the composite microbial inoculum in the tobacco alcoholization fermentation process.
The invention also provides a tobacco alcoholization treatment method, which sprays the composite microbial inoculum on the surface of tobacco leaves to carry out alcoholization fermentation treatment. Wherein the spraying amount is 1 percent, namely 1mL of bacterial liquid/kg of tobacco leaves; the alcoholization fermentation conditions are as follows: the temperature is 38-40 deg.C, and the time is 23-25 days.
The invention relates to the development of the function of the Maotai wine aroma-producing microorganisms and the improvement of the tobacco quality, applies the Maotai wine yeast microorganism fermentation product to the tobacco fermentation for the first time, can increase the variety of high-quality microorganisms in the tobacco fermentation and the variety of aroma substances through the activity of the aroma-producing microorganisms or the addition of metabolites thereof, and has the effect of enhancing the aroma similar to the addition of the Maotai wine, thereby improving the alcoholization quality of the tobacco.
Drawings
FIG. 1 is a graph showing the total amount of aroma substances at different fermentation times.
FIG. 2 is a graph showing the content of neophytadiene at different fermentation times.
FIG. 3 is a graph showing how much megastigmatrienone content was measured at various fermentation times.
FIG. 4 is a graph of the content of dihydroactinidiolide in different fermentation times.
FIG. 5 is a graph showing solanone content at different fermentation times.
FIG. 6 is a graph showing the content of beta-carotene degradation products at different fermentation times.
FIG. 7 is a graph of the content of degradation products of lutein at different fermentation times.
FIG. 8 is a graph showing the volatile ketone content at different fermentation times.
FIG. 9 is a graph showing the content of semi-volatile ketones at different fermentation times.
FIG. 10 is a graph showing the volatile aldehyde content at different fermentation times.
FIG. 11 is a graph showing the content of linear fatty acid esters at different fermentation times.
FIG. 12 is a graph of heterocyclic content for different fermentation times.
FIG. 13 is a graph showing the volatile alcohol content at different fermentation times.
FIG. 14 is a graph showing the linear fatty acid content at different fermentation times.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1 preparation of Complex microbial inoculum
1.1 preparation of the culture Medium
1) Preparation of PDA liquid culture medium: weighing 200g of potato, washing, peeling, chopping, adding 1000mL of ddH2Boiling O for 30min, filtering with four layers of gauze, adding 20g glucose, dissolving, sterilizing at 121 deg.C for 30min, and cooling.
2) Preparing a wheat culture medium: crushing wheat into half 100g of wheat grains and half of wheat flour, adding 50% water, sterilizing at 121 deg.C for 30min, cooling, and scattering.
1.2 preparation of Complex microbial inoculum
1) Separation of bacillus subtilis and bacillus licheniformis
Inoculating the black part of the Maotai-chung koji into a PDA (PDA dextrose agar) plate culture medium, culturing for 24h at 50 ℃, selecting a single colony for purification culture, adding the separated purified bacterial liquid into a wheat culture medium, and culturing for 6d according to the temperature gradient of 37 ℃, 46 ℃ and 55 ℃, wherein the culture is carried out for 2 days at 37 ℃, 2 days at 46 ℃ and 2 days at 55 ℃; and (3) screening strains (namely bacillus subtilis and bacillus licheniformis) which produce more sauce flavor substances from the cultured mixed strains to serve as flavor-producing strains.
Wherein, the preservation bacterial number of the bacillus subtilis is as follows: CCTCC M2015575, and the preservation bacterial number of the bacillus licheniformis is: CCTCC M2015576.
2) Preparation of complex microbial inoculum
Respectively inoculating Bacillus subtilis, Bacillus licheniformis and Bacillus amyloliquefaciens in PDA culture medium, culturing at 37 deg.C for 24 hr, collecting bacterial liquid, and counting viable bacteria by 109cfu/ml、109cfu/ml、109And (5) mixing cfu/ml to obtain the composite microbial inoculum.
Example 2 alcoholizing fermentation treatment of tobacco leaves
A3C2 (middle leaf) in the area of Guizhou province in Qianxuan is selected for testing, tobacco leaves are pretreated by adopting a simultaneous distillation extraction method, the composite microbial inoculum prepared in the example 1 is sprayed on the tobacco leaves according to the inoculation amount of 1 percent (1mL bacterial liquid/kg tobacco leaves), and the alcoholization fermentation treatment is carried out for 25 days at the temperature of 40 ℃.
The control example was sprayed with an equal amount of water.
Analysis of Experimental Effect
The tobacco leaves treated in example 2 and the tobacco leaves treated in the comparative example were analyzed, and the contents of main neutral aroma components, aroma precursor components and sensory quality were compared at different fermentation times.
Wherein, the chromatographic analysis condition and the qualitative and quantitative analysis method of the aroma substances are as follows:
chromatographic conditions are as follows: chromatographic column DB-5MS, 30 mm × 0.25 μm, column flow rate of 1.0mL/min, temperature programming, keeping at 40 deg.C for 5min, heating at 5 deg.C/min to 280 deg.C for 10min, sample injection amount of 1 μ L, and split ratio of 30: 1.
Mass spectrum conditions: solvent delay 3min, transmission line temperature 280 ℃, ionization mode: EI, ionization energy 70eV, quadrupole temperature 150 ℃, ion source temperature 230 ℃, mass spectrometry scan range: 35-550 amu.
And (3) identifying sensory quality: and (3) smoking the tobacco leaves at different fermentation times, and identifying the samples according to the quality, the quantity, the taste, the miscellaneous gas, the irritation and the indexes of the fragrance.
1. Neutral aroma detection
TABLE 1 content of main neutral aroma components (μ g/g)
And (4) conclusion:
(1) total amount of aroma substances: as can be seen from FIG. 1, for the middle tobacco leaf, the fermentation time was 10 days at 40 ℃, the composite bacteria > control (water was added); fermenting for 15 days, and comparing (adding water) > composite bacteria; fermenting for 20 days, and comparing (adding water) > composite bacteria; fermentation time 25 days, complex bacteria > control (water added). Therefore, the total amount of aroma substances is more after the tobacco leaves are alcoholized and fermented for 10 days and 25 days at the temperature of 40 ℃.
(2) A new phytodiene: as can be seen from FIG. 2, for the middle tobacco leaf, the fermentation time is 10 days at 40 ℃, and the composite bacteria are compared (added with water); fermenting for 15 days, and comparing (adding water) > composite bacteria; fermenting for 20 days, and comparing (adding water) > composite bacteria; the middle tobacco leaves with 25 days of fermentation time, the composite bacteria are compared (added with water). Therefore, at 40 ℃, the fermentation time is 25 days, and the addition of the composite microbial inoculum is beneficial to the improvement of the accumulation amount of the new phytodiene.
(3) Megastigmatrienone and dihydroactinidiolide: as can be seen from FIGS. 3 and 4, the middle tobacco leaves were fermented at 40 ℃ for 10 days in the presence of the control (water) complex bacteria; fermenting for 15 days, and comparing (adding water) > composite bacteria; fermenting for 20 days, and comparing (adding water) > composite bacteria; fermentation time 25 days, complex bacteria > control (water added). Therefore, at 40 ℃, the fermentation time is 25 days, and the addition of the compound microbial inoculum is beneficial to the accumulation of the contents of megastigmatrienone and dihydroactinidiolide.
(4) Solanone: as can be seen from FIG. 5, for the middle tobacco leaf, the fermentation time was 10 days at 40 ℃ and the control (water addition) > complex bacteria; fermentation time 15 days, complex bacteria > control (water addition); fermenting for 20 days, and comparing (adding water) > composite bacteria; fermentation time 25 days, complex bacteria > control (water added). Therefore, at 40 ℃, the fermentation time is 15 days and 25 days, and the addition of the compound microbial inoculum is beneficial to the accumulation of the solanone content.
2. Aroma precursor detection
TABLE 2 Main fragrance precursor Classification (μ g/g)
And (4) conclusion:
(5) beta-carotene degradation products, lutein degradation products, volatile aldehydes, volatile ketones, volatile alcohols, straight chain fatty acid esters, semi-volatile ketones, and heterocycles: as can be seen from FIGS. 6-13, for the middle tobacco leaves, the fermentation time was 10 days, 15 days and 20 days at 40 ℃ and the control (water addition) > complex bacteria; fermentation time 25 days, complex bacteria > control (water added). Therefore, at 40 ℃ and within 25 days of fermentation time, the addition of the composite microbial inoculum is beneficial to improving the accumulation of beta-carotene degradation products, lutein degradation products, volatile aldehydes, volatile ketones, volatile alcohols, straight chain fatty acid esters, semi-volatile ketones and heterocyclic substances.
(6) Straight chain fatty acids: as can be seen from FIG. 14, for the middle tobacco leaves, the fermentation time was 10 days, 15 days and 20 days at 40 ℃, the composite bacteria > control (water was added); and when the fermentation time reaches 25 days, the control (water is added) > composite bacteria. It can be seen that at 40 ℃, the fermentation time is 10 days, 15 days and 20 days, and the accumulation of the composite bacteria treated straight chain fatty acid is more.
3. Sensory quality detection
The tobacco leaf samples treated by the compound bacteria at 40 ℃ and the control (water added) samples treated by the compound bacteria at different alcoholization times are subjected to sensory evaluation and quality identification, and the results are shown in Table 3.
TABLE 3 comparative sensory quality analysis
| Treatment of | Time of fermentation | Quality of fragrance | Amount of fragrance | Tasty flavor | Miscellaneous qi | |
| Control | ||||||
| 10 | ||||||
| Compound bacterium | ||||||
| 10 days | + | - | + | |||
| |
15 | |||||
| Compound bacterium | ||||||
| 15 days | + | + | ||||
| |
20 | |||||
| Compound bacterium | ||||||
| 20 days | + | + | + | + | ||
| |
25 | |||||
| Compound bacterium | ||||||
| 25 days | + | + | + | + | + |
As can be seen from Table 3, the smoking quality of the tobacco leaves treated by adding the compound bacteria is generally better than that of the tobacco leaves treated by adding water, the alcoholization time is 15 days, and the miscellaneous gas and the irritation of the tobacco leaves are obviously improved; the quality, quantity, taste, miscellaneous gas and irritation of tobacco leaf fragrance can be improved after 20 days; the alcoholization time is 25 days, and the sensory smoking quality of the tobacco leaves reaches the best.
In conclusion, at 40 ℃, the fermentation time is 25 days, the composite microbial inoculum is added for treatment, and the content and the total amount of several main neutral aroma substances in the tobacco leaves are more than those of the control (water is added) treatment. The main aroma substance precursors are analyzed in a classified manner, the fermentation time is 10 days, 15 days and 20 days, except for the straight-chain fatty acid, the content change trends of the beta-carotene degradation product, the lutein degradation product, the volatile aldehyde, the volatile ketone, the volatile alcohol, the straight-chain fatty acid ester, the semi-volatile ketone and the heterocyclic are basically consistent, namely the fermentation time is about 25 days, and the content of each substance is the most treated by compound bacteria.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (3)
1. A tobacco alcoholization treatment method is characterized in that a compound microbial inoculum is sprayed on the surface of tobacco leaves to carry out alcoholization fermentation treatment; the spraying amount of the composite microbial inoculum is 1mL of bacterial liquid/kg of tobacco leaves; wherein:
the alcoholization fermentation conditions are as follows: the temperature is 38-40 ℃, and the time is 23-25 days;
the composite microbial inoculum is prepared by mixing bacillus subtilis, bacillus licheniformis and bacillus amyloliquefaciens; wherein the viable count of Bacillus subtilis is not less than 109cfu/ml, viable count of Bacillus licheniformis not less than 109cfu/ml, viable count of Bacillus amyloliquefaciens not less than 109cfu/ml；
Wherein the bacillus subtilis and the bacillus licheniformis are separated from the Maotai distiller's yeast;
the Bacillus subtilis was identified as Bacillus subtilis (B.) (Bacillus subtilis) MTLB01M2015575 with the preservation number of CCTCC NO;
the Bacillus licheniformis is identified as Bacillus licheniformis (B.) (Bacillus licheniformis) MTLB02The preservation number is CCTCC NO: M2015576.
2. The tobacco mellowing method according to claim 1, wherein the number of viable bacteria of Bacillus subtilis in the complex microbial agent is 109cfu/ml, viable count of Bacillus licheniformis 109cfu/ml, viable count of Bacillus amyloliquefaciens 109cfu/ml。
3. The tobacco mellowing method according to claim 1 or 2, characterized in that the complex microbial inoculum is obtained by a method comprising: respectively inoculating Bacillus subtilis, Bacillus licheniformis and Bacillus amyloliquefaciens in PDA culture medium, culturing at 36-37 deg.C for 22-24 hr, collecting bacterial liquid, and mixing at a certain proportion.
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| CN107080280B (en) * | 2017-03-24 | 2018-07-13 | 贵州大学 | A kind of method and cigarette preparing Sauce flavor cigarette using bacillus amyloliquefaciens |
| CN107467707B (en) * | 2017-08-11 | 2020-07-14 | 陕西中烟工业有限责任公司 | Method for improving quality of tobacco sheets by papermaking method by adopting aromatized composite microbial preparation |
| CN107354109B (en) * | 2017-08-11 | 2020-11-06 | 陕西中烟工业有限责任公司 | Aroma-imparting compound microbial preparation for rapid fermentation of tobacco leaves and application thereof |
| 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 |
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