CN116042475A - Enterobacter huoshanense enzyme preparation, preparation method and application thereof in tobacco shred treatment - Google Patents
Enterobacter huoshanense enzyme preparation, preparation method and application thereof in tobacco shred treatment Download PDFInfo
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Abstract
The invention discloses an enterobacter cholerae enzyme preparation, a preparation method and application thereof in tobacco shred treatment. The enterobacter cholerae enzyme preparation comprises enterobacter cholerae, wherein the enterobacter cholerae is (Enterobacter hormaechei) F34 and is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 4 months and 29 days in 2022, and the preservation number is: CGMCC No.24827. The enterobacter cholerae enzyme preparation can obviously improve the smoking quality of low-grade tobacco shreds within 4 hours. The treatment time is short, long-time fermentation is avoided, and the aroma quality and the aroma quantity are quickly and efficiently improved, the stimulation is reduced, the taste of the low-grade tobacco shreds is improved, and meanwhile, the sweetness is increased. The application of the enterobacter cholerae enzyme preparation finds a new way for the treatment of tobacco shreds, especially low-grade tobacco shreds, and greatly reduces the stock of the low-grade tobacco shreds.
Description
Technical Field
The invention relates to the technical field of tobacco, in particular to an enterobacter cholerae enzyme preparation, a preparation method and application thereof in tobacco shred treatment.
Background
The enterobacter cholerae is a straight bar bacterium, gram-negative, periphytic flagella exercise and facultative anaerobic. Easy to grow on common culture medium, ferment glucose, produce acid and gas, positive VP experiment and negative MR experiment, and can generally use citrate and malonate as unique C source and energy source without producing H from thiosulfate 2 S, S. Most strains can slowly liquefy gelatin without producing deoxyribonuclease.
Scientific researches find that the known escherichia coli group has multiple functions of producing 2, 3-butanediol and penicillin, degrading carotene, polysaccharide, reducing sugar and the like in the aspect of chemical application, and a part of bacteria produce biological enzymes capable of producing beta-carotene into beta-ionone. In the aspect of biology, the research shows that the enterobacter cholerae can play a role in promoting growth by accelerating the secretion of thymus hormone, can promote the proliferation of drosophila intestinal cells, and can obviously promote the growth of drosophila. In the aspect of reducing harm, screening of a strain of enterobacter cholerae which can be used for polyethylene degradation has been reported. From this, the group of enterobacteria cholerae can provide help to scientific research in a number of aspects, and has great industrial application potential.
At present, the problem of large stock quantity of low-grade tobacco leaves in the tobacco industry generally exists, and in order to reduce the stock quantity, the ratio of the low-grade tobacco leaves in a cigarette formula can be further improved, but the smoking quality of the low-grade tobacco leaves is also reduced, so that in order to avoid the situation, a method for rapidly and stably improving the internal quality of the low-grade tobacco leaves is required to be sought in the industry.
Biological enzymes have been widely used in various industries such as food, agriculture and medicine because of their advantages of mild conditions, simple operation, safety and effectiveness. And the degradation of various substances in the tobacco leaf preparation and alcoholization processes all need the participation of enzymes, so the enzymes are critical to the formation of tobacco leaf quality and flavor. And as the enzyme is a catalyst of the same kind of substances, the same kind of reaction is mainly catalyzed, and the catalyst has the outstanding characteristics of substrate specificity, high catalytic reaction efficiency, mild acting conditions and the like, so the catalyst has wide application prospect in tobacco preparation, and the catalyst can effectively regulate chemical components in tobacco, degrade macromolecular substances and increase fragrance components by utilizing biological enzyme catalytic conversion, thereby being an effective means for improving the quality of the tobacco. In addition, studies on the synergistic addition of enzyme preparations to tobacco additives, enzyme preparations to flavor plants, and enzyme preparations to microorganisms to tobacco and tobacco flakes are also under great progress. Different types of enzymes are added into tobacco in a matched manner for catalytic conversion, so that macromolecular compound substances can be well degraded into micromolecular aroma substances, and meanwhile, the action efficiency of a single enzyme can be increased. Therefore, the use of biological enzymes to improve the intrinsic chemical composition of tobacco leaves has been one of the hot spots in the tobacco industry in recent years.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an enterobacter cholerae enzyme preparation, a preparation method and application thereof in tobacco shred treatment, which overcomes the defects in the prior art.
The technical problems to be solved by the invention are realized by the following technical scheme:
an enterobacter cholerae enzyme preparation comprising enterobacter cholerae, wherein the enterobacter cholerae is (Enterobacter hormaechei) F34 and deposited in the China general microbiological culture collection center, month 29 of 2022, with accession number: cgmccno.24827, preservation address: beijing, china.
Preferably, in the above technical solution, the method for obtaining the escherichia coli thallus includes the following steps:
1) The formula of the culture medium comprises: 10g/L NaCl, 10g/L tryptone, 5g/L yeast extract;
2) Strain activation: activating and culturing the enterobacter cholerae to obtain an enterobacter cholerae activating solution;
3) And (3) treatment: inoculating the activated bacterial liquid obtained in the step 2) into the culture medium obtained in the step 1) according to the volume ratio of 1% to form a treatment liquid, wherein the treatment culture condition after inoculation is the same as the activation culture condition, and the culture time is 6-12 hours;
4) Centrifuging the treatment solution obtained in the step 3) for 10min at the temperature of 8000r/min by using an ultralow temperature high-speed centrifuge at the temperature of 4 ℃, and obtaining a precipitate which is thalli, and storing the thalli at the low temperature of minus 30 ℃ for later use.
Preferably, in the above technical solution, the strain activation conditions in step 2) are: shake culturing at 25-35 deg.c and pH 5-7 and 100-150 r/min for 12-24 hr.
The preparation method of the enterobacter cholerae enzyme preparation comprises the following steps:
1) Crushing of the bacterial cells: weighing a proper amount of bacteria according to the weight of m: v=1:15 (g/ml) in 0.015M PBS buffer, crushing with an ultrasonic cell crusher for 10min each time, crushing twice, centrifuging at 4deg.C and 10000r/min for 15min after cell crushing, centrifuging, and collecting supernatant;
2) Salting out enzyme protein: to the resulting supernatant was added PBS: (NH 4) 2 SO 4 Adding saturated ammonium sulfate solution at a ratio of 1:2, standing at low temperature for 3 hr, centrifuging at 4deg.C and 10000r/min for 15min,centrifuging, pouring out supernatant, and temporarily placing the obtained precipitate in a refrigerator at-30deg.C;
3) Salt ion dialysis: dissolving the precipitate by using 15mmol/L PBS solution according to 15 times volume, placing into a dialysis bag, then placing the dialysis bag into a 1000ml beaker filled with 50mmol/L PBS solution, stirring at low temperature, and changing water every 2 hours for three times for 8 hours to obtain the enterobacter cholerae enzyme preparation.
A tobacco product having both aroma and taste upon smoking.
An application of an enterobacter cholerae enzyme preparation in tobacco shred treatment.
Preferably, in the above technical solution, the method includes the following steps: spraying Shi Huoshi enterobacter enzyme preparation into tobacco shreds according to enzyme adding amount of 100mg/g, adding 12% water to the sprayed tobacco shreds, placing into a 37 ℃ incubator for 4 hours, drying the water for 1min, and rolling into cigarettes.
Preferably, in the above technical scheme, the cut tobacco is low-grade cut tobacco.
The technical scheme of the invention has the following beneficial effects:
the enterobacter cholerae enzyme preparation can obviously improve the smoking quality of low-grade tobacco shreds within 4 hours. The treatment time is short, long-time fermentation is avoided, and the aroma quality and the aroma quantity are quickly and efficiently improved, the stimulation is reduced, the taste of the low-grade tobacco shreds is improved, and meanwhile, the sweetness is increased. The application of the enterobacter cholerae enzyme preparation finds a new way for the treatment of tobacco shreds, especially low-grade tobacco shreds, and greatly reduces the stock of the low-grade tobacco shreds.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention.
FIG. 1 is a plate diagram of Enterobacter cholerae.
FIG. 2 is an electron micrograph of Enterobacter cholerae.
FIG. 3 is a graph of an analysis of the phylogenetic tree of Enterobacter cholerae.
FIG. 4 is a gel diagram of an Enterobacter cholerae enzyme preparation.
FIG. 5 is a GC-MS diagram of the major components of cut tobacco treated with an enterobacter cholerae enzyme preparation.
Detailed Description
Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that: the relative arrangement of the components and steps, numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless it is specifically stated otherwise.
EXAMPLE 1 Enterobacter cholerae
An enterobacter cholerae enzyme preparation, the enterobacter cholerae enzyme preparation comprising enterobacter cholerae, wherein the enterobacter cholerae is (Enterobacter hormaechei) F34 and is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the month 29 of 2022, and the preservation number is: CGMCC No.24827.
The above-mentioned strain of Enterobacter cholerae is a gram-negative bacterium, and is yellow and circular in shape (as shown in FIG. 1) on a plate.
Experiment preparation:
preparing seed liquid: the enterobacter cholerae stored in the freezing tube is inoculated into 30mL of LB culture medium according to the inoculation amount of 1 percent, and is cultured for 12-24 hours for standby.
Disclosed formulation of bacterial LB medium: 10g tryptone, 5g yeast extract, 10g NaCl, distilled water 1L, pH 7.0.
EXAMPLE 2 preparation of bacterial cells
The preparation method of the enterobacter cholerae thallus comprises the following steps:
1) The formula of the culture medium comprises: 10g/L NaCl, 10g/L tryptone, 5g/L yeast extract;
2) Strain activation: activating and culturing the enterobacter cholerae to obtain an enterobacter cholerae activating solution; preferably, the strain activation conditions are: shake culturing for 12-24 hours at 25-35 ℃ and pH 5-7 at 100-150 r/min;
3) And (3) treatment: inoculating the activated bacterial liquid obtained in the step 2) into the culture medium obtained in the step 1) according to the volume ratio of 1% to form a treatment liquid, wherein the treatment culture condition after inoculation is the same as the activation culture condition, and the culture time is 6-12 hours;
4) Centrifuging the treatment solution obtained in the step 3) for 10min at the temperature of 8000r/min by using an ultralow temperature high-speed centrifuge at the temperature of 4 ℃, and obtaining a precipitate which is thalli, and storing the thalli at the low temperature of minus 30 ℃ for later use.
Example 3 preparation of enzyme preparation
The preparation method of the enterobacter cholerae enzyme preparation comprises the following steps:
1) Crushing of the bacterial cells: weighing a proper amount of bacteria according to the weight of m: v=1:15 (g/ml) in 0.015M PBS buffer, crushing with an ultrasonic cell crusher for 10min each time, crushing twice, centrifuging at 4deg.C and 10000r/min for 15min after cell crushing, centrifuging, and collecting supernatant;
2) Salting out enzyme protein: to the resulting supernatant was added PBS: (NH 4) 2 SO 4 Adding saturated ammonium sulfate solution at a ratio of (1:2), standing at low temperature for 3 hr, centrifuging at 4deg.C and 10000r/min for 15min, centrifuging, pouring out supernatant, and placing the obtained precipitate in a refrigerator at-30deg.C;
3) Salt ion dialysis: dissolving the precipitate by using 15mmol/L PBS solution according to 15 times volume, placing into a dialysis bag, then placing the dialysis bag into a 1000ml beaker filled with 50mmol/L PBS solution, stirring at low temperature, and changing water every 2 hours for three times for 8 hours to obtain the enterobacter cholerae enzyme preparation.
Example 4 application of preparation of Enterobacter cholerae enzyme preparation in tobacco shred
The application of the enterobacter cholerae enzyme preparation in tobacco shred treatment comprises the following steps:
spraying Shi Huoshi enterobacter enzyme preparation into tobacco shreds according to enzyme adding amount of 100mg/g, adding 12% water to the sprayed tobacco shreds, placing into a 37 ℃ incubator for 4 hours, drying the water for 1min, and rolling into cigarettes. The absorption was evaluated after 2d equilibration of water. The tobacco shred is low-grade tobacco shred.
Example 5 sample measurement
Pulverizing the processed tobacco shred sample into powder, and removing and sieving non-broken sundries in the tobacco powder by using a 40mm sample separating sieve. 30g of tobacco dust is weighed and placed in a 1000mL round bottom flask, 400mL of purified water and zeolite are added to the flask, and the mixture is shaken well. 100mL of methylene chloride was accurately measured in a 250mL round bottom flask and placed in a 60℃water bath, and the time was counted for 2.5h as the aqueous phase was refluxed for the first time. Each group was made in parallel 6 groups.
And (3) extracting the obtained extract by acid and alkali, adding 20g of anhydrous sodium sulfate into the extract after the extraction is finished, and refrigerating and standing overnight. To the extract was added 100. Mu.L of 2, 6-dichlorotoluene as an internal standard, and the mixture was concentrated to 1mL by atmospheric distillation for GC/MS detection.
The GC-MS detection method comprises the following steps:
chromatographic conditions: HP-5MS 5% Phenyl Methyl Silox (model 30 m. Times.0.25 mm. Times.0.25 μm);
the temperature of the sample inlet is 280 ℃;
high-purity helium is used as carrier gas, and the flow rate is 1mL/min;
programming temperature: the initial temperature is 50 ℃, the temperature is kept for 4min, and then the temperature is increased to 240 ℃ at a heating rate of 2 ℃/min;
not split;
the sample injection amount is 1 mu L;
GC-MS used full scan mode.
Mass spectrometry conditions:
the temperature of the transmission line is 280 ℃;
the temperature of the ion source is 280 ℃, and the temperature of the quadrupole rods is 150 ℃;
the ionization mode is electron bombardment (EI), and the electron energy is 70eV;
the solvent delay time is 8min; scanning mass range (m/z): 35-550 mu.
Detection result:
the aroma components in the tobacco shreds are detected and treated as shown in table 1, and the results prove that: the content of delta-elemene, thirty-heptanol, 3-furfural, lagenal, 5-hydroxy-3-methyl-1-indanone, kaurenal, 13-epi-minool, 3, 5-dihydroxytoluene, 4-cyclopentene-1, 3-dione, 3, 4-diethylbiphenyl, methyl linoleate, dibutyl phthalate, methyl stearate, 2-acetylpyrrole, propiolactone and dihydroactinolide is improved.
TABLE 1 data sheet of neutral aroma composition change of tobacco shreds after treatment with Enterobacter cholerae
And (3) detecting the effect brought by adding the enzyme preparation into the tobacco leaves, selecting 15 tobacco production personnel for quality control, and summarizing the quality control evaluation of the tobacco production personnel as shown in the following table 2.
TABLE 2 comparison of tobacco shred smoking evaluation effects before and after treatment with Enterobacter cholerae
The result shows that the tobacco shred treated by the enterobacter cholerae enzyme preparation has obviously improved aroma quality and aroma quantity, and the sweetness is improved; the taste is obviously improved. Especially for low-grade tobacco shreds, the fragrance and taste improvement amplitude of the low-grade tobacco shreds is far more than expected, so that a new method for rapidly and stably improving the internal quality of the low-grade tobacco shreds is provided, and the method can greatly reduce the inventory of the low-grade tobacco shreds.
Although the present invention has been described with reference to the above embodiments, it should be understood that the present invention is not limited thereto, and that various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.
Claims (8)
1. An enterobacter cholerae enzyme preparation, wherein the enterobacter cholerae enzyme preparation comprises enterobacter cholerae, the enterobacter cholerae is (Enterobacter hormaechei) F34, and the enterobacter cholerae is deposited in the China general microbiological culture Collection center (China general microbiological culture Collection center) at 4 months 29 of 2022, and has the deposit number: CGMCC No.24827.
2. The enterobacter cholerae enzyme preparation according to claim 1, wherein the method for obtaining enterobacter cholerae cells comprises the following steps:
1) The formula of the culture medium comprises: 10g/L NaCl, 10g/L tryptone, 5g/L yeast extract;
2) Strain activation: activating and culturing the enterobacter cholerae to obtain an enterobacter cholerae activating solution;
3) And (3) treatment: inoculating the activated bacterial liquid obtained in the step 2) into the culture medium obtained in the step 1) according to the volume ratio of 1% to form a treatment liquid, wherein the treatment culture condition after inoculation is the same as the activation culture condition, and the culture time is 6-12 hours;
4) Centrifuging the treatment solution obtained in the step 3) for 10min at the temperature of 8000r/min by using an ultralow temperature high-speed centrifuge at the temperature of 4 ℃, and obtaining a precipitate which is thalli, and storing the thalli at the low temperature of minus 30 ℃ for later use.
3. The enterobacter johnsonii enzyme preparation of claim 2, wherein the strain activation conditions in step 2) are: shake culturing at 25-35 deg.c and pH 5-7 and 100-150 r/min for 12-24 hr.
4. A method for preparing an enterobacter johnsonii enzyme preparation according to any one of claims 1 to 3, comprising the steps of:
1) Crushing of the bacterial cells: weighing a proper amount of bacteria according to the weight of m: v=1:15 (g/ml) in 0.015M PBS buffer, crushing with an ultrasonic cell crusher for 10min each time, crushing twice, centrifuging at 4deg.C and 10000r/min for 15min after cell crushing, centrifuging, and collecting supernatant;
2) Salting out enzyme protein: to the resulting supernatant was added PBS: (NH 4) 2 SO 4 Adding saturated ammonium sulfate solution at a ratio of (1:2), standing at low temperature for 3 hr, centrifuging at 4deg.C and 10000r/min for 15min, centrifuging, pouring out supernatant, and placing the obtained precipitate in a refrigerator at-30deg.C;
3) Salt ion dialysis: dissolving the precipitate by using 15mmol/L PBS solution according to 15 times volume, placing into a dialysis bag, then placing the dialysis bag into a 1000ml beaker filled with 50mmol/L PBS solution, stirring at low temperature, and changing water every 2 hours for three times for 8 hours to obtain the enterobacter cholerae enzyme preparation.
5. A tobacco product having both aroma and taste upon smoking, characterized in that the tobacco product comprises the enterobacter cholerae enzyme preparation of any one of claims 1-3, and the enterobacter cholerae enzyme preparation prepared by the preparation method of claim 4.
6. The application of the enterobacter cholerae enzyme preparation in tobacco shred treatment is characterized in that the enterobacter cholerae enzyme preparation is prepared by the preparation method of claim 4 according to any one of the enterobacter cholerae enzyme preparation activities of claims 1-3.
7. Use of an enterobacter cholerae enzyme preparation according to claim 6, in tobacco shred processing, comprising the steps of: spraying Shi Huoshi enterobacter enzyme preparation into tobacco shreds according to enzyme adding amount of 100mg/g, adding 12% water to the sprayed tobacco shreds, placing into a 37 ℃ incubator for 4 hours, drying the water for 1min, and rolling into cigarettes.
8. The use of an enterobacter cholerae enzyme preparation according to claim 7, wherein the cut tobacco is a low-grade cut tobacco.
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| CN117126767A (en) * | 2023-06-28 | 2023-11-28 | 安徽省农业科学院烟草研究所 | Potassium-dissolving growth-promoting enterobacter cholerae and microbial inoculum and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117126767A (en) * | 2023-06-28 | 2023-11-28 | 安徽省农业科学院烟草研究所 | Potassium-dissolving growth-promoting enterobacter cholerae and microbial inoculum and application thereof |
| CN117126767B (en) * | 2023-06-28 | 2024-03-29 | 安徽省农业科学院烟草研究所 | Potassium-dissolving growth-promoting enterobacter cholerae and microbial inoculum and application thereof |
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