CN117530484A - Method for fermenting tobacco leaves by enrichment strain of tobacco source culture medium - Google Patents
Method for fermenting tobacco leaves by enrichment strain of tobacco source culture medium Download PDFInfo
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- CN117530484A CN117530484A CN202311408622.3A CN202311408622A CN117530484A CN 117530484 A CN117530484 A CN 117530484A CN 202311408622 A CN202311408622 A CN 202311408622A CN 117530484 A CN117530484 A CN 117530484A
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- 241000208125 Nicotiana Species 0.000 title claims abstract description 210
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 210
- 239000001963 growth medium Substances 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 37
- 239000007788 liquid Substances 0.000 claims abstract description 70
- 230000001580 bacterial effect Effects 0.000 claims abstract description 54
- 238000009630 liquid culture Methods 0.000 claims abstract description 41
- 238000000855 fermentation Methods 0.000 claims abstract description 25
- 230000004151 fermentation Effects 0.000 claims abstract description 25
- 238000012258 culturing Methods 0.000 claims abstract description 21
- 239000002994 raw material Substances 0.000 claims abstract description 21
- 230000001954 sterilising effect Effects 0.000 claims abstract description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000012153 distilled water Substances 0.000 claims abstract description 15
- 238000001914 filtration Methods 0.000 claims abstract description 13
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- 238000005507 spraying Methods 0.000 claims abstract description 9
- 238000003756 stirring Methods 0.000 claims abstract description 9
- 238000002156 mixing Methods 0.000 claims abstract description 5
- 239000002609 medium Substances 0.000 claims description 15
- 230000001953 sensory effect Effects 0.000 abstract description 18
- 239000007789 gas Substances 0.000 abstract description 11
- 235000019504 cigarettes Nutrition 0.000 abstract description 4
- 230000000813 microbial effect Effects 0.000 abstract description 4
- 239000002068 microbial inoculum Substances 0.000 description 15
- 238000011156 evaluation Methods 0.000 description 13
- 241000609458 Corynespora Species 0.000 description 10
- 244000063299 Bacillus subtilis Species 0.000 description 8
- 235000014469 Bacillus subtilis Nutrition 0.000 description 8
- 244000005700 microbiome Species 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 5
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 5
- 241000407778 Bacillus subtilis TO-A Species 0.000 description 4
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- 125000003118 aryl group Chemical group 0.000 description 3
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- 239000012880 LB liquid culture medium Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
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- 238000011282 treatment Methods 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 241001508813 Clavispora lusitaniae Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
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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
Abstract
The invention provides a method for fermenting tobacco leaves by using tobacco source culture medium enrichment strain, which utilizes tobacco leaves to be tested to prepare tobacco source liquid culture medium to enrich target strains, and the enriched bacterial liquid is subjected to spraying fermentation on the tobacco leaves to be tested. The method specifically comprises the following steps: adding tobacco leaf raw materials to be tested into distilled water, uniformly mixing and stirring, placing the mixture in a constant-temperature shaking table, sufficiently shaking, filtering, and sterilizing the filtered liquid under high pressure to obtain a tobacco source liquid culture medium; inoculating the purified target strain into the tobacco source liquid culture medium, and culturing in a constant-temperature shaking table to obtain enriched bacterial liquid; spraying the enriched bacterial liquid on tobacco leaves to be tested according to a proportion; fermenting the sprayed tobacco leaves to be tested. The invention can reduce bad smell generated when the conventional LB, YPD and other liquid culture mediums enrich strains, further eliminate miscellaneous gases brought to tobacco leaves when the bacterial liquid is sprayed to the tobacco leaves, and achieve the purposes of improving the quality of microbial fermentation tobacco leaves and improving the sensory quality of cigarettes.
Description
Technical Field
The invention relates to the technical field of tobacco mellowing, in particular to a method for fermenting tobacco leaves by enriching strains in a tobacco source culture medium.
Background
The tobacco mellowing can improve the appearance of tobacco, coordinate the internal chemical components of tobacco, make the aroma exposed, reduce the green miscellaneous gas, and have more mellow taste, thus being an important link of the tobacco raw material guarantee of the cigarette industry enterprises. The fermentation and alcoholization of flue-cured tobacco leaves are an important biochemical process for improving the quality of the tobacco leaves, and the mechanism of the fermentation and alcoholization is mainly divided into 3 major categories of enzyme action, microorganism action and chemical action. The microorganisms on the surfaces of the tobacco leaves can synthesize and secrete a plurality of extracellular hydrolytic enzymes, and the enzymes can accelerate the degradation of macromolecular substances such as starch, pectin, cellulose, protein, lignin and the like, convert the macromolecular substances into small molecular compounds, coordinate chemical components, reduce pungency and miscellaneous gases, increase the content of aroma compounds and finally improve the quality of the tobacco.
With the continuous deep research on tobacco microorganism, exogenous addition of microorganism or other beneficial microorganism in tobacco is considered to shorten tobacco alcoholization period, increase tobacco aroma, improve tobacco quality and improve tobacco safety. The exogenous microorganism needs to be enriched with a liquid medium during the process of being made into a microbial inoculum. Liquid culture media conventionally used are LB (bacterial) and YPD (fungal, yeast) culture media, or bacterial cells obtained by resuspension centrifugation with sterile water to obtain bacterial liquid. However, the microbial agent enriched and cultured by LB and YPD has mixed gas of a culture medium, and residual smell of a liquid culture medium exists when sensory evaluation is carried out after tobacco leaf spraying, so that the sensory quality of the tobacco leaf is affected. The bacteria obtained by resuspension of the bacteria with sterile water can not achieve ideal tobacco leaf fermenting effect due to slow growth of the bacterial strain caused by lack of nutrient substances.
Therefore, the invention designs a method for fermenting tobacco leaves by enriching strains in a grass-source culture medium.
Disclosure of Invention
The invention provides a method for fermenting tobacco leaves by using a tobacco source culture medium enrichment strain, and aims to solve the problems of miscellaneous gases and residual smell of a culture medium caused by conventional LB and YPD liquid culture medium enrichment strains and bacterial liquid fermentation of tobacco leaves.
In order to achieve the above purpose, the embodiment of the invention provides a method for fermenting tobacco leaves by using a tobacco source culture medium enrichment strain, which is characterized in that a tobacco source liquid culture medium is prepared from tobacco leaves to be tested, target strains are enriched, and the enriched bacterial liquid is subjected to spraying fermentation on the tobacco leaves to be tested. The method specifically comprises the following steps: adding tobacco leaf raw materials to be tested into distilled water, uniformly mixing and stirring, placing the mixture in a constant-temperature shaking table, sufficiently shaking, filtering, and sterilizing the filtered liquid under high pressure to obtain a tobacco source liquid culture medium; inoculating the purified target strain into the tobacco source liquid culture medium, and culturing in a constant-temperature shaking table to obtain enriched bacterial liquid; spraying the enriched bacterial liquid on tobacco leaves to be tested according to a proportion; fermenting the sprayed tobacco leaves to be tested. The invention can reduce bad smell generated when the conventional LB, YPD and other liquid culture mediums enrich strains, further eliminate miscellaneous gases brought to tobacco leaves when the bacterial liquid is sprayed to the tobacco leaves, and achieve the purposes of improving the quality of microbial fermentation tobacco leaves and improving the sensory quality of cigarettes.
The embodiment of the invention provides a method for fermenting tobacco leaves by enriching strains in a tobacco source culture medium, which comprises the following steps:
s1, adding tobacco leaf raw materials to be tested into distilled water, mixing and stirring uniformly, placing the mixture in a constant-temperature shaking table, sufficiently shaking, filtering, and sterilizing the filtered liquid under high pressure to obtain a tobacco source liquid culture medium;
s2, inoculating the purified and streaked target strain into the tobacco source liquid culture medium, and culturing in a constant-temperature shaking table to obtain enriched bacterial liquid;
s3, spraying the enriched bacterial liquid on tobacco leaves to be tested according to a proportion;
s4, fermenting the sprayed tobacco leaves to be tested.
Preferably, the tobacco leaf raw material to be tested in the step S1 is 50g and distilled water is 1L.
Preferably, the tobacco source liquid medium concentration in step S1 is 50g/L.
Preferably, in step S1, the stirring speed is 200r/min and the stirring temperature is 30 ℃.
Preferably, the constant temperature shaking table in step S1 is oscillated at 200r/min for 30min at 30 ℃.
Preferably, the culture conditions in step S2 are: culturing in a constant temperature shaking table at 30 ℃ and 200r/min for 24-48 h.
More preferably, the culture conditions in step S2 are: culturing in a shaking table at 30deg.C and 200r/min for 36 hr.
Preferably, in the step S3, the enriched bacterial liquid and the tobacco leaves to be tested are sprayed according to the proportion of 4 percent.
Preferably, the fermentation conditions in step S4: fermenting for 2-5 days at 25 ℃ and humidity of 30%.
More preferably, the fermentation conditions in step S4: fermenting at 25deg.C and humidity of 30% for 2 days.
The scheme of the invention has the following beneficial effects:
(1) Compared with the conventional liquid culture mediums for enriching strains, such as LB, YPD and the like, tobacco leaves are adopted as raw materials of the enrichment culture medium, namely, tobacco leaves which need to be treated are adopted as raw materials for preparing the liquid enrichment culture medium in the earlier stage, and the adaptation and domestication process is provided for the strains as a whole.
(2) The method disclosed by the invention creates a specific growth environment for the strain to be tested in advance, adapts to the growth environment of the later-stage sprayed tobacco leaves in the strain enrichment link, and avoids the problems that the growth environment is changed, the strain cannot grow and the microbial inoculum effect is reduced when the later-stage sprayed tobacco leaves.
(3) The components in the tobacco source enrichment medium provide nutrients for the strain required by growth.
(4) The invention uses tobacco source as enrichment culture medium, solves the problem that the sensory quality is affected by non-tobacco miscellaneous gas generated by conventional culture medium.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved by the present invention more apparent, the following detailed description will be made with reference to specific embodiments.
Unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present invention.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
Aiming at the problems of miscellaneous gases and residual odor of a culture medium caused by conventional LB and YPD liquid culture medium enrichment strains and bacterial liquid fermentation of tobacco leaves, the invention provides a method for fermenting tobacco leaves by using a tobacco source culture medium enrichment strain. The method utilizes tobacco leaves to be tested to prepare a tobacco source liquid culture medium to enrich target strains, and the enriched bacterial liquid is subjected to spraying fermentation on the tobacco leaves to be tested. The method specifically comprises the following steps: adding tobacco leaf raw materials to be tested into distilled water, uniformly mixing and stirring, placing the mixture in a constant-temperature shaking table, sufficiently shaking, filtering, and sterilizing the filtered liquid under high pressure to obtain a tobacco source liquid culture medium; inoculating the purified target strain into the tobacco source liquid culture medium, and culturing in a constant-temperature shaking table to obtain enriched bacterial liquid; spraying the enriched bacterial liquid on tobacco leaves to be tested according to a proportion; fermenting the sprayed tobacco leaves to be tested. The invention can reduce bad smell generated when the conventional LB, YPD and other liquid culture mediums enrich strains, further eliminate miscellaneous gases brought to tobacco leaves when the bacterial liquid is sprayed to the tobacco leaves, and achieve the purposes of improving the quality of microbial fermentation tobacco leaves and improving the sensory quality of cigarettes.
The constant temperature shaking table of the embodiment of the invention is ZHWY211B, which is manufactured by Shanghai Zhi Cheng analysis instruments; all tobacco leaves to be tested were: alcoholizing for 17 months, wherein the production place is Shandong, the variety is NC55, the grade is C4F tobacco, the strain to be tested is 2 strains, and the strain information is shown in Table 1:
TABLE 1 information on strains to be tested
| Strain | Seed species |
| Strain 1 to be tested | Bacillus subtilis (Bacillus subtilis) |
| Strain 2 to be tested | Saccharomyces cerevisiae (Clavispora lusitaniae) |
The following will be described by way of specific examples.
Example 1
Inoculating bacillus subtilis to an LB liquid medium, and fermenting Shandong NC 55C 4F tobacco leaves, wherein the method comprises the following steps:
(1) Preparing an LB liquid culture medium, and the formula: 10g/L tryptone, 5g/L, naCl g/L yeast extract.
(2) Inoculating the bacillus subtilis streaked on the flat plate into the LB liquid culture medium in the step (1), and culturing in a shaking table at 30 ℃ and 200r/min until the strain grows in a logarithmic phase, namely OD600 = 2.2, so as to obtain bacterial liquid.
(3) 4g of the bacterial liquid (concentration: 4%) obtained in the step (2) was sucked up, and 100g of Shandong NC 55C 4F tobacco leaves were sprayed with the bacterial liquid.
(4) And (3) placing the tobacco leaves treated by the microbial inoculum in the step (3) in a constant temperature and humidity incubator with temperature and humidity for fermentation for 48 hours.
(5) And (3) carrying out sensory evaluation on the tobacco leaves fermented in the step (4).
Example 2
Inoculating bacillus subtilis to a Shandong NC 55C 4F tobacco leaf culture medium, and fermenting Shandong NC 55C 4F tobacco leaves, wherein the method comprises the following steps of:
(1) 50g of Shandong NC 55C 4F tobacco leaf raw material which is alcoholized for 17 months is weighed by a balance with the precision of 0.01g, and is added into a conical flask with 1L of sterilized distilled water after being sheared by a sterilizing scissors, and is uniformly shaken for 30min under the conditions of a constant temperature shaking table with the temperature of 30 ℃ and the speed of 200 r/min.
(2) Filtering the tobacco leaves in the mixture in the step (1) by using gauze, and taking out the remaining liquid after autoclaving at 121 ℃ for 15min to obtain the sterile tobacco source liquid culture medium.
(3) Inoculating the bacillus subtilis streaked on the plate into the tobacco source liquid culture medium in the step (2), and culturing in a shaking table at 30 ℃ and 200r/min until the strain grows in the logarithmic phase, namely OD600 = 2.2, so as to obtain bacterial liquid.
(4) 4g of the bacterial liquid (concentration: 4%) obtained in the step (3) was sucked up, and 100g of Shandong NC 55C 4F tobacco leaves were sprayed with the bacterial liquid.
(5) And (3) placing the tobacco leaves treated by the microbial inoculum in the step (4) into a constant temperature and humidity incubator with temperature and humidity for fermentation for 48 hours.
(6) And (5) performing sensory evaluation on the tobacco leaves fermented in the step (5).
Example 3
Inoculating bacillus subtilis to a strong-flavor tobacco leaf culture medium, and fermenting Shandong NC 55C 4F tobacco leaves, wherein the method comprises the following steps of:
(1) 50g of C2F tobacco leaf raw material with the grade of K326 in Hunan state with the alcoholization of 12 months is weighed by a balance with the precision of 0.01g, cut by a pair of sterilizing scissors, added into a conical flask with 1L of sterilizing distilled water, and shaken uniformly for 30min under the condition of a constant temperature shaking table at 30 ℃ and 200 r/min.
(2) Filtering the tobacco leaves in the mixture in the step (1) by using gauze, and taking out the remaining liquid after autoclaving at 121 ℃ for 15min to obtain the sterile tobacco source liquid culture medium.
(3) Inoculating the bacillus subtilis streaked on the plate into the tobacco source liquid culture medium in the step (2), and culturing in a shaking table at 30 ℃ and 200r/min until the strain grows in the logarithmic phase, namely OD600 = 2.2, so as to obtain bacterial liquid.
(4) 4g of the bacterial liquid (concentration: 4%) obtained in the step (3) was sucked up, and 100g of Shandong NC 55C 4F tobacco leaves were sprayed with the bacterial liquid.
(5) And (3) placing the tobacco leaves treated by the microbial inoculum in the step (4) into a constant temperature and humidity incubator with temperature and humidity for fermentation for 48 hours.
(6) And (5) performing sensory evaluation on the tobacco leaves fermented in the step (5).
Example 4
Inoculating bacillus subtilis to a medium-flavor tobacco leaf culture medium, and fermenting Shandong NC 55C 4F tobacco leaves, wherein the method comprises the following steps of:
(1) 50g of C2F tobacco leaf raw material with the K326 grade of Guizhou Zunyi for 12 months is weighed by a balance with the precision of 0.01g, cut by a pair of sterilizing scissors, added into a conical flask with 1L of sterilizing distilled water, and shaken uniformly for 30min under the condition of a constant temperature shaking table at 30 ℃ and 200 r/min.
(2) Filtering the tobacco leaves in the mixture in the step (1) by using gauze, and taking out the remaining liquid after autoclaving at 121 ℃ for 15min to obtain the sterile tobacco source liquid culture medium.
(3) Inoculating the bacillus subtilis streaked on the plate into the tobacco source liquid culture medium in the step (2), and culturing in a shaking table at 30 ℃ and 200r/min until the strain grows in the logarithmic phase, namely OD600 = 2.2, so as to obtain bacterial liquid.
(4) 4g of the bacterial liquid (concentration: 4%) obtained in the step (3) was sucked up, and 100g of Shandong NC 55C 4F tobacco leaves were sprayed with the bacterial liquid.
(5) And (3) placing the tobacco leaves treated by the microbial inoculum in the step (4) into a constant temperature and humidity incubator with temperature and humidity for fermentation for 48 hours.
(6) And (5) performing sensory evaluation on the tobacco leaves fermented in the step (5).
Example 5
Inoculating bacillus subtilis to a faint scent tobacco leaf culture medium, and fermenting Shandong NC 55C 4F tobacco leaves, wherein the method comprises the following steps of:
(1) 50g of C2F tobacco leaf raw material with the grade of Yunnan Yuxi K326 which is alcoholized for 12 months is weighed by a balance with the precision of 0.01g, cut by a pair of sterilizing scissors, added into a conical flask with 1L of sterilizing distilled water, and shaken for 30min under the condition of a constant temperature shaking table with the temperature of 30 ℃ and 200 r/min.
(2) Filtering the tobacco leaves in the mixture in the step (1) by using gauze, and taking out the remaining liquid after autoclaving at 121 ℃ for 15min to obtain the sterile tobacco source liquid culture medium.
(3) Inoculating the bacillus subtilis streaked on the plate into the tobacco source liquid culture medium in the step (2), and culturing in a shaking table at 30 ℃ and 200r/min until the strain grows in the logarithmic phase, namely OD600 = 2.2, so as to obtain bacterial liquid.
(4) 4g of the bacterial liquid (concentration: 4%) obtained in the step (3) was sucked up, and 100g of Shandong NC 55C 4F tobacco leaves were sprayed with the bacterial liquid.
(5) And (3) placing the tobacco leaves treated by the microbial inoculum in the step (4) into a constant temperature and humidity incubator with temperature and humidity for fermentation for 48 hours.
(6) And (5) performing sensory evaluation on the tobacco leaves fermented in the step (5).
Example 6
The corynespora viticola is inoculated to YPD liquid culture medium, and the Shandong NC 55C 4F tobacco leaves are fermented, and the method comprises the following steps:
(1) Preparing YPD liquid culture medium, and the formula: 20g/L of tryptone, 10g/L of yeast extract and 20g/L of glucose.
(2) Inoculating the corynespora viticola streaked on the plate into the YPD liquid culture medium in the step (1), and culturing in a shaking table at 30 ℃ and 200r/min until the strain growth log phase is OD600 = 2.2 to obtain bacterial liquid.
(3) 4g of the bacterial liquid (concentration: 4%) obtained in the step (2) was sucked up, and 100g of Shandong NC 55C 4F tobacco leaves were sprayed with the bacterial liquid.
(4) And (3) placing the tobacco leaves treated by the microbial inoculum in the step (3) in a constant temperature and humidity incubator with temperature and humidity for fermentation for 48 hours.
(5) And (3) carrying out sensory evaluation on the tobacco leaves fermented in the step (4).
Example 7
The corynespora vitis is inoculated to a Shandong NC 55C 4F tobacco leaf culture medium, and the Shandong NC 55C 4F tobacco leaf is fermented, and the method comprises the following steps:
(1) 50g of Shandong NC 55C 4F tobacco leaf raw material which is alcoholized for 17 months is weighed by a balance with the precision of 0.01g, and is added into a conical flask with 1L of sterilized distilled water after being sheared by a sterilizing scissors, and is uniformly shaken for 30min under the conditions of a constant temperature shaking table with the temperature of 30 ℃ and the speed of 200 r/min.
(2) Filtering the tobacco leaves in the mixture in the step (1) by using gauze, and taking out the remaining liquid after autoclaving at 121 ℃ for 15min to obtain the sterile tobacco source liquid culture medium.
(3) Inoculating the corynespora viticola streaked on the plate into the tobacco source liquid culture medium in the step (2), and culturing in a shaking table at 30 ℃ and 200r/min until the strain grows in logarithmic phase, namely OD600 = 2.2, so as to obtain bacterial liquid.
(4) 4g of the bacterial liquid (concentration: 4%) obtained in the step (3) was sucked up, and 100g of Shandong NC 55C 4F tobacco leaves were sprayed with the bacterial liquid.
(5) And (3) placing the tobacco leaves treated by the microbial inoculum in the step (4) into a constant temperature and humidity incubator with temperature and humidity for fermentation for 48 hours.
(6) And (5) performing sensory evaluation on the tobacco leaves fermented in the step (5).
Example 8
Inoculating the corynespora vinifera yeast into a strong-flavor tobacco leaf culture medium, and fermenting Shandong NC 55C 4F tobacco leaves, wherein the method comprises the following steps of:
(1) 50g of C2F tobacco leaf raw material with the grade of K326 in Hunan state with the alcoholization of 12 months is weighed by a balance with the precision of 0.01g, cut by a pair of sterilizing scissors, added into a conical flask with 1L of sterilizing distilled water, and shaken uniformly for 30min under the condition of a constant temperature shaking table at 30 ℃ and 200 r/min.
(2) Filtering the tobacco leaves in the mixture in the step (1) by using gauze, and taking out the remaining liquid after autoclaving at 121 ℃ for 15min to obtain the sterile tobacco source liquid culture medium.
(3) Inoculating the corynespora viticola streaked on the plate into the tobacco source liquid culture medium in the step (2), and culturing in a shaking table at 30 ℃ and 200r/min until the strain grows in logarithmic phase, namely OD600 = 2.2, so as to obtain bacterial liquid.
(4) 4g of the bacterial liquid (concentration: 4%) obtained in the step (3) was sucked up, and 100g of Shandong NC 55C 4F tobacco leaves were sprayed with the bacterial liquid.
(5) And (3) placing the tobacco leaves treated by the microbial inoculum in the step (4) into a constant temperature and humidity incubator with temperature and humidity for fermentation for 48 hours.
(6) And (5) performing sensory evaluation on the tobacco leaves fermented in the step (5).
Example 9
Inoculating the corynespora vinifera yeast into an intermediate-flavor tobacco leaf culture medium, and fermenting Shandong NC 55C 4F tobacco leaves, wherein the method comprises the following steps of:
(1) 50g of C2F tobacco leaf raw material with the K326 grade of Guizhou Zunyi for 12 months is weighed by a balance with the precision of 0.01g, cut by a pair of sterilizing scissors, added into a conical flask with 1L of sterilizing distilled water, and shaken uniformly for 30min under the condition of a constant temperature shaking table at 30 ℃ and 200 r/min.
(2) Filtering the tobacco leaves in the mixture in the step (1) by using gauze, and taking out the remaining liquid after autoclaving at 121 ℃ for 15min to obtain the sterile tobacco source liquid culture medium.
(3) Inoculating the corynespora viticola streaked on the plate into the tobacco source liquid culture medium in the step (2), and culturing in a shaking table at 30 ℃ and 200r/min until the strain grows in logarithmic phase, namely OD600 = 2.2, so as to obtain bacterial liquid.
(4) 4g of the bacterial liquid (concentration: 4%) obtained in the step (3) was sucked up, and 100g of Shandong NC 55C 4F tobacco leaves were sprayed with the bacterial liquid.
(5) And (3) placing the tobacco leaves treated by the microbial inoculum in the step (4) into a constant temperature and humidity incubator with temperature and humidity for fermentation for 48 hours.
(6) And (5) performing sensory evaluation on the tobacco leaves fermented in the step (5).
Example 10
Inoculating the corynespora vinifera yeast into a faint scent tobacco leaf culture medium, and fermenting Shandong NC 55C 4F tobacco leaves, wherein the method comprises the following steps of:
(1) 50g of C2F tobacco leaf raw material with the grade of Yunnan Yuxi K326 which is alcoholized for 12 months is weighed by a balance with the precision of 0.01g, cut by a pair of sterilizing scissors, added into a conical flask with 1L of sterilizing distilled water, and shaken for 30min under the condition of a constant temperature shaking table with the temperature of 30 ℃ and 200 r/min.
(2) Filtering the tobacco leaves in the mixture in the step (1) by using gauze, and taking out the remaining liquid after autoclaving at 121 ℃ for 15min to obtain the sterile tobacco source liquid culture medium.
(3) Inoculating the corynespora viticola streaked on the plate into the tobacco source liquid culture medium in the step (2), and culturing in a shaking table at 30 ℃ and 200r/min until the strain grows in logarithmic phase, namely OD600 = 2.2, so as to obtain bacterial liquid.
(4) 4g of the bacterial liquid (concentration: 4%) obtained in the step (3) was sucked up, and 100g of Shandong NC 55C 4F tobacco leaves were sprayed with the bacterial liquid.
(5) And (3) placing the tobacco leaves treated by the microbial inoculum in the step (4) into a constant temperature and humidity incubator with temperature and humidity for fermentation for 48 hours.
(6) And (5) performing sensory evaluation on the tobacco leaves fermented in the step (5).
The results of the sensory evaluation of the Shandong NC 55C 4F tobacco leaves after fermentation of the microbial inoculum of examples 1 to 10 are carried out by using the Shandong NC 55C 4F tobacco leaves as a control, and the specific evaluation results are shown in Table 2.
TABLE 2 Effect of different treatments on the sensory quality of tobacco leaves
The effect of the different treatments on the organoleptic quality of the tobacco leaves is shown in table 2. The tobacco quality was improved by using the tobacco source liquid medium (example 2, example 7), the medium prepared from the aromatic tobacco raw material (example 3, example 8) and the medium prepared from the intermediate aromatic tobacco raw material (example 4, example 9) as compared with the CK control, whereas the tobacco quality was not improved by using the aromatic tobacco medium (example 5, example 10) and the LB (example 1), YPD (example 6) as compared with the CK control. Wherein, no matter the type of the strain to be tested, the tobacco is fermented by adopting the microbial inoculum prepared by the enrichment strain of the tobacco source liquid culture medium, and the tobacco has the lowest miscellaneous gas and the best quality. And the microbial inoculum prepared by enriching strains through LB and YPD culture mediums has the most miscellaneous gases after fermenting tobacco leaves, and the quality of the tobacco leaves is the worst.
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A method for fermenting tobacco leaves by enriching strains in a tobacco source culture medium, which is characterized by comprising the following steps:
s1, adding tobacco leaf raw materials to be tested into distilled water, mixing and stirring uniformly, placing the mixture in a constant-temperature shaking table, sufficiently shaking, filtering, and sterilizing the filtered liquid under high pressure to obtain a tobacco source liquid culture medium;
s2, inoculating the purified and streaked target strain into the tobacco source liquid culture medium, and culturing in a constant-temperature shaking table to obtain enriched bacterial liquid;
s3, spraying the enriched bacterial liquid on tobacco leaves to be tested according to a proportion;
s4, fermenting the sprayed tobacco leaves to be tested.
2. The method for fermenting tobacco leaves by using the enriched strain of tobacco source culture medium according to claim 1, wherein the tobacco leaf raw material to be tested in the step S1 is 50g and distilled water is 1L.
3. The method for fermenting tobacco leaves by using a tobacco source medium-enriched strain according to claim 1, wherein the concentration of the tobacco source liquid medium in the step S1 is 50g/L.
4. The method for fermenting tobacco leaves by using a strain enriched in a tobacco source medium according to claim 1, wherein the stirring speed in the step S1 is 200r/min and the stirring temperature is 30 ℃.
5. The method for fermenting tobacco leaves by using the enriched strain of tobacco source culture medium according to claim 1, wherein the constant temperature shaking table in the step S1 is oscillated for 30min under the condition of 200r/min at the temperature of 30 ℃.
6. The method for fermenting tobacco leaves by using a tobacco source medium-enriched strain according to claim 1, wherein the culturing conditions in the step S2 are as follows: culturing in a constant temperature shaking table at 30 ℃ and 200r/min for 24-48 h.
7. The method for fermenting tobacco leaves by using a tobacco source medium-enriched strain according to claim 6, wherein the culturing conditions in the step S2 are as follows: culturing in a shaking table at 30deg.C and 200r/min for 36 hr.
8. The method for fermenting tobacco leaves by using the enriched strain of the tobacco source culture medium according to claim 1, wherein the enriched bacterial liquid and the tobacco leaves to be tested in the step S3 are sprayed according to the proportion of 4%.
9. The method for fermenting tobacco leaves by using a tobacco source medium-enriched strain according to claim 1, wherein the fermentation conditions in step S4: fermenting for 2-5 days at 25 ℃ and humidity of 30%.
10. The method for fermenting tobacco leaves by using a tobacco source medium-enriched strain according to claim 9, wherein the fermentation conditions in step S4: fermenting at 25deg.C and humidity of 30% for 2 days.
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