CN111575321B - Rhizosphere pseudoriver bacillus tobacco fermentation product, preparation method and application thereof - Google Patents
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Abstract
The invention discloses a preparation method of rhizosphere pseudoriver bacillus tobacco leavening, which comprises the following steps: activating rhizosphere pseudoriver bacillus C1-9 strain; preparing a rhizosphere Pseudohelobacter C1-9 seed culture solution; thirdly, fermenting the tobacco by utilizing rhizosphere Pseudohelobacter C1-9 to obtain tobacco fermentation liquor; fourthly, extracting the tobacco fermentation liquor to obtain the rhizosphere pseudoriver bacillus tobacco fermentation product. The invention also discloses the rhizosphere pseudoriver bacillus tobacco fermentation product and application of the rhizosphere pseudoriver bacillus tobacco fermentation product in cigarettes. The rhizosphere pseudoriver bacillus tobacco fermentation product can obviously improve the smoking quality of cigarettes.
Description
Technical Field
The invention belongs to the technical field of tobacco flavors and fragrances, and particularly relates to a fermentation product of rhizosphere Pseudohelobacter pseudohelenium C1-9 of tobacco, a preparation method and application thereof.
Background
The essence and the spice have important positions in the industries of food, feed, cosmetics, chemical industry, pharmacy and the like, and the sources of the essence and the spice mainly comprise two modes of natural and chemical synthesis. Wherein, the essence and flavor synthesized by chemistry accounts for about 85 percent of the whole product, but the essence and flavor synthesized by chemistry has the defects of more by-products, low yield, high cost, environmental pollution and the like. Along with the advocated green industry, natural essence and spice are more and more regarded by people, and mainly come from animals and plants, but the animal and plant spice resources are limited, the extraction cost is high, and the increasing market demand is far from being met. The perfume prepared by the microorganism is a perfume which is produced by taking natural raw materials as starting materials and using a fermentation technology, an enzyme technology and the like and also belongs to a natural perfume; and the spice prepared by the microorganism has the advantages of short production period, no influence by weather, mild reaction conditions, less three wastes formed after product separation, environmental friendliness and the like, so that the spice can be used as a substitute for aroma-producing plants.
The tobacco industry is an important consumer product manufacturing industry, and the improvement of cigarette safety through tar reduction and harm reduction has become a common target of the tobacco industry. However, the problem of insufficient cigarette aroma components is more prominent along with the reduction of the tar content of the cigarettes. In order to make up for the aroma and taste of cigarettes, the addition of tobacco flavors and fragrances becomes an effective means for improving the decisive factor of the product style and the aroma quality of the cigarettes. The method for obtaining the tobacco flavor and fragrance with various style characteristics and good safety performance also becomes one of important directions for the research and development of tobacco products.
The flavor and fragrance for tobacco are classified into flavors derived from tobacco itself, natural flavors other than tobacco, and artificially synthesized flavors according to the source. The natural spice prepared by the microorganisms is one of the raw material sources of the cigarette spice emerging in recent years, and the microbial spice prepared by the microorganisms has the advantages of easily obtained raw materials, mild conditions, high selectivity and the like. But the types of dominant strains which can be used for producing the aroma by microorganisms are not many at present, so that the screening and the use of the new strains for preparing the tobacco flavor and aroma by fermenting the tobacco have practical significance.
Disclosure of Invention
The invention aims to provide a rhizosphere pseudoriver bacillus tobacco fermentation product, a preparation method and application thereof. The preparation method of the rhizosphere pseudoriver bacillus tobacco leavening comprises the steps of fermenting tobacco by utilizing microorganism rhizosphere pseudoriver bacillus C1-9 with a fragrance producing function, and obtaining the rhizosphere pseudoriver bacillus C1-9 leavening through preparation processes such as heating reflux extraction, ethanol redissolution and the like, wherein the species and the content of aroma components are obviously improved, and the rhizosphere pseudoriver bacillus tobacco leavening has the effects of enriching cigarette fragrance, enabling the tobacco fragrance to be prominent and full and enhancing the style characteristics of the cigarette when being used in tobacco shreds.
The technical scheme of the invention is as follows:
all percentages used in the present invention are mass percentages unless otherwise indicated.
The invention discloses a preparation method of rhizosphere pseudoriver bacillus tobacco leavening, which comprises the following steps:
activating rhizosphere pseudoriver bacillus C1-9 strain;
② preparing a culture solution of rhizosphere Pseudohelobacter C1-9 seeds;
thirdly, fermenting the tobacco by utilizing rhizosphere Pseudohelobacter C1-9 to obtain tobacco fermentation liquor;
fourthly, extracting the tobacco fermentation liquor obtained in the third step to obtain the rhizosphere pseudomonas tobacco fermentation product.
Preferably, the activation of rhizosphere pseudoriver bacillus C1-9 strain comprises the following steps: sterilizing the culture medium at 121 deg.C for 25 min, placing into slant, inoculating Pseudohelobacter rhizogenes C1-9 strain, and culturing at 28 deg.C for 1 week to obtain test tube strain; the culture medium is R2A agar culture medium.
Preferably, the step of preparing the rhizosphere Pseudohelobacter C1-9 seed culture solution comprises the following specific steps: sterilizing a seed culture medium at 121 ℃ for 25 minutes, selecting part of mycelia obtained in the step I from slant culture, inoculating the mycelia into a seed solution, and performing shake culture at 30 ℃ for 48 hours to obtain a seed culture solution; the seed culture medium is R2A liquid culture medium.
Preferably, the fermentation step of the third step is: pulverizing tobacco into tobacco powder, adding water, and mixing; and then adding a sugar source and inorganic salt into the tobacco powder, sterilizing the tobacco powder for 25 minutes at 121 ℃, inoculating the culture solution of the rhizosphere Pseudohelobacter C1-9 seeds obtained in the step II according to 5-20% of the weight of the tobacco powder, performing shake culture at 30 ℃, and fermenting for 24-48 hours to obtain the tobacco fermentation liquor. The tobacco may be tobacco leaf or leaf stem.
Preferably, the sugar source is one or a mixture of glucose, lactose and fructose, and the adding amount of the sugar source is 1.0-5.0 wt% of the mixed solution.
Preferably, the inorganic salt is KH2PO4、(NH4)2SO4、MgSO4、Fe3(SO4)2、NaCl、CaCl2The addition amount of the mixture of one or more of the components is 0.1 to 2.0 weight percent of the mixed solution.
Preferably, the step of extracting the tobacco fermentation liquor in the step (iv) is as follows: heating, refluxing and extracting the tobacco fermentation liquor obtained in the step (III) at 60-80 ℃ for 2-4 h, then carrying out suction filtration under the condition that the filter mesh number is 150-250 meshes, and concentrating the obtained filtrate at 45-60 ℃ under the condition of 60-100 kPa to obtain a tobacco fermentation concentrated extract; adding 85-95 wt% ethanol in an amount which is 3-5 times the weight of the obtained tobacco fermentation concentrated extract, mixing, and then placing under a freezing condition of-10 ℃ for settling for 8-12 hours to obtain supernatant and precipitate; taking supernatant and concentrating the supernatant at 45-65 ℃ under the condition of 60-90 kPa until the density is 1.0-1.5 g/cm3And obtaining the rhizosphere pseudoriver bacillus tobacco fermentation product.
The second aspect of the invention discloses a rhizosphere pseudoriver bacillus tobacco fermentation product prepared by the preparation method.
The third aspect of the invention discloses the application of the rhizosphere Pseudohelobacter tobacco fermentation product in improving the smoking quality of cigarettes.
Preferably, diluting the rhizosphere Pseudohelobacter tobacco fermentation product by 3-5 times of propylene glycol or ethanol, and spraying the product onto cut tobacco of cigarette in a spraying manner; the addition amount of the rhizosphere pseudoriver bacillus tobacco fermentation product is 0.2-0.5 wt% of the mass of the cigarette tobacco shreds.
The microorganism rhizosphere pseudoriver bacillus C1-9 is obtained by separating from rhizosphere soil of tobaccos in Yunnan province, morphological, physiological and biochemical properties and 16S rDNA analysis are carried out on the microorganism rhizosphere pseudoriver bacillus C1-9, and an identification result shows that the microorganism rhizosphere pseudoriver bacillus C1-9 is a new strain, is classified and named as rhizosphere pseudoriver bacillus (Latin name: Rivibacter soli) in microbiology, and is prepared from the following biological materials (strains): c1-9, which has been preserved in China general microbiological culture Collection center (CGMCC for short) in 2019, 8 and 9 months, wherein the preservation number is CGMCC No.1.13864, and the preservation unit is as follows: china academy of sciences microbiological research institute, the preservation address is: xilu No.1 Hospital No. 3, Beijing, Chaoyang, Beicheng.
The rhizosphere Pseudohelobacter C1-9 strain has the main morphological characteristics and physiological and biochemical characteristics as follows: the growth is good on most culture media, and on an R2A agar culture medium, the colony has neat edges and a convex middle part and is light yellow; starch hydrolysis, cellulose hydrolysis, catalase, acid phosphatase, trypsin, tween 20, 40, 60 and 80 hydrolysis are positive, oxidase, casein hydrolysis, alpha-galactosidase and urease are negative. Fructose, lactose, citric acid, cellobiose, fucose, sweetening alcohol, melibiose, alanine, arginine, aspartic acid can be used. The polar lipid component of cell membrane mainly comprises phosphatidyl glycerol, diphosphatidyl glycerol and phosphatidyl ethanolamine, and the respiratory quinone of cell is ubiquinone-8 (Q-8).
The invention has the following beneficial effects:
1. the method utilizes the new microbial strain rhizosphere pseudoriver bacillus C1-9 to ferment the tobacco for the first time, and the obtained rhizosphere pseudoriver bacillus tobacco fermented product has obviously improved aroma component types and contents; the tobacco shred composition is applied to the tobacco shred of the cigarette, and has the effects of enriching the cigarette fragrance, enabling the tobacco fragrance to be prominent and full and enhancing the style characteristic of the cigarette. The rhizosphere pseudoriver bacillus tobacco fermentation product can obviously improve the smoking quality of cigarettes when being applied to the cigarettes.
2. The preparation process of the rhizosphere pseudoriver bacillus tobacco fermentation product has no harmful organic solvent, the preparation process is safe and environment-friendly, the raw materials are easy to obtain, the cost is low, and the industrial production is convenient to realize. The preparation method of the rhizosphere pseudoriver bacillus tobacco fermentation product has good application value.
Drawings
FIG. 1 is a GC-MS spectrum of a tobacco fermentation product of Pseudohelobacter rhizogenes prepared in example 1.
FIG. 2 is a GC-MS spectrum of a control tobacco extract prepared in example 2.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1
1. Activation of rhizosphere Pseudohelobacter C1-9 strain
The culture medium is a slant preservation culture medium which is an R2A agar culture medium, and the formula of the culture medium is as follows: 0.5g of glucose, 0.5g of yeast extract, 0.5g of peptone, 0.5g of acid hydrolyzed casein, 0.5g of soluble starch, 0.3g of sodium pyruvate, 0.3g of dipotassium phosphate, 0.05g of magnesium sulfate, 15g of agar and distilled water to reach the volume of 1000mL and the pH value of 7.2. Sterilizing the culture medium at 121 deg.C for 25 min, placing on slant, inoculating Pseudohelobacter rhizogenes C1-9 strain, and culturing at 28 deg.C for 1 week to obtain test tube strain.
2. Preparation of seed culture solution of rhizosphere Pseudohelobacter C1-9
A seed culture medium is adopted, the seed culture medium is an R2A liquid culture medium, and the formula is as follows: 0.5g of glucose; 0.5g of yeast extract; peptone 0.5 g; acid hydrolyzed casein 0.5 g; 0.5g of soluble starch; 0.3g of sodium pyruvate; dipotassium phosphate 0.3 g; magnesium sulfate 0.05 g; 1000mL of distilled water, pH 7.2. And (2) sterilizing the culture medium at 121 ℃ for 25 minutes, picking part of mycelia from the test tube inclined plane in the step (1), inoculating the mycelia into seed liquid, and performing shake culture at 30 ℃ for 48 hours to obtain liquid strains.
3. Fermenting tobacco by utilizing rhizosphere pseudoriver bacillus C1-9
Crushing the aired tobacco leaves into tobacco powder with the particle size of 50 meshes, adding water with the mass of 4 times of the tobacco powder, and uniformly mixing; adding glucose, lactose and fructose in an amount of 1 wt% and KH in an amount of 0.1 wt% to the above mixture2PO4、NaCl、MgSO4And after uniform mixing, sterilizing at 121 ℃ for 25 minutes, inoculating the obtained rhizosphere Pseudohelobacter C1-9 seed culture solution according to 5% of the mass of the tobacco powder, and performing shake culture at 30 ℃ for fermentation for 48 hours to obtain tobacco powder fermentation liquor.
4. Extracting and refining tobacco powder fermentation liquor
A. Heating reflux extraction: heating and refluxing the tobacco powder fermentation liquor obtained in the step (3) at 60 ℃ for extraction for 3 hours; then carrying out suction filtration while the solution is hot, wherein the filtration mesh number is 200 meshes; concentrating the filtrate at 50 deg.C under 80kPa until the volume is not changed to obtain concentrated extractive solution of fermented tobacco powder.
B. Ethanol redissolution: and C, adding 95% ethanol with the mass of 4 times of that of the fermented tobacco powder concentrated extract obtained in the step A, fully stirring and dissolving, and then placing under a freezing condition of-10 ℃ for settling for 12 hours to obtain supernatant and precipitate.
C. Concentration: collecting supernatant, and concentrating at 50 deg.C under 90kPa to density of 1.2g/cm3The tobacco fermented product of the rhizosphere pseudoriver bacillus is obtained, and the physicochemical indexes of the tobacco fermented product are shown in the table 1.
TABLE 1 physicochemical indexes of rhizosphere Pseudoriver bacillus tobacco fermented product
| Serial number | Detecting items | The result of the detection |
| 1 | Appearance and clarity | Reddish brown liquid, clear |
| 2 | Relative density (g/cm)3) | 1.2036±0.008 |
| 3 | Refractive index | 1.3681±0.008 |
| 4 | Total amount of volatile component (wt%) | 25.7±5.0 |
| 5 | Degree of miscibility in ethanol (25 ℃ C.) | Insoluble in water, soluble in 60-95 wt% ethanol |
| 6 | Pb | <5mg/kg |
| 7 | As | <1mg/kg |
| 8 | Coumarin compound | Undetected |
| 9 | Safrole | Not detected out |
5. Application of rhizosphere pseudoriver bacillus tobacco fermentation product
Dissolving the obtained rhizosphere pseudohelobacter tobacco fermentation product accounting for 0.3 percent of the mass of the cigarette tobacco shreds by using propylene glycol 5 times of the mass of the tobacco shreds, spraying the tobacco shreds onto the cigarette tobacco shreds in a spraying manner, putting the tobacco shreds into a constant-temperature and constant-humidity box, and balancing for 48 hours under the conditions that the humidity is 60 +/-2 percent and the temperature is 22 +/-2 ℃; then rolling into cigarette for smoke evaluation.
Example 2
1. Preparation of control tobacco extract and control cigarette
Crushing tobacco leaves into powder with the particle size of 50 meshes, adding 4 times of water, uniformly mixing to replace tobacco powder fermentation liquor, and preparing a control tobacco extract and a control cigarette thereof according to the steps 4-5 in the embodiment 1.
2. Sensory evaluation of cigarettes
The tobacco fermented product containing rhizosphere pseudomonas in example 1 is named as a test cigarette, the tobacco fermented product containing no tobacco extract is named as a blank cigarette, and the tobacco fermented product containing the control tobacco extract prepared in the embodiment is named as a control cigarette; the test pieces were handed to professional smokers for sensory comparison and smoking, and the results are shown in Table 2. According to the smoking evaluation result, the control cigarette has certain cream fragrance and faint scent when being smoked, but the aroma texture is slightly thin, the smoke scent is not ideal, and the irritation is slightly improved; when a test cigarette containing the rhizosphere pseudohelobacter tobacco fermentation product is smoked, the test cigarette has special flower fragrance, fruit fragrance and sweet fragrance besides cream fragrance and faint scent, the fragrance is strong and natural, the fragrance is more stereoscopic and full, the smoke irritation is also obviously reduced, and the aftertaste is cleaner. Therefore, the rhizosphere pseudoheusjaponicus tobacco fermentation product can obviously improve the cigarette smoking quality.
TABLE 2 cigarette evaluation results
3. Volatile aroma component comparative analysis and evaluation of rhizosphere pseudoriver bacillus tobacco fermentation product
GC-MS analyses were carried out on the tobacco fermentation product of Pseudohelobacter rhizogenes prepared in example 1 and the control tobacco extract prepared in example 2, respectively, and the GC-MS spectra of both were shown in FIGS. 1 and 2, wherein the volatile substances are shown in Table 3. As can be seen from table 3, the rhizosphere pseudomonas tobacco fermented product prepared by rhizosphere pseudomonas fermentation has significantly increased types and contents of volatile substances, such as cyclopentanone, benzyl alcohol, 2-acetyl pyrrole, phenethyl alcohol, octanoic acid, phenethyl formate, gamma-nonalactone, (E) -beta-damascenone, dihydroactinidiolide, megastigmatrienone, and the like, compared with the control tobacco extract of example 2. The aroma threshold of the aroma substances is low, and the slight increase of the content can promote the strong degree of the aroma of the tobacco to be obviously improved. The rhizosphere pseudoriver bacillus tobacco fermentation product greatly improves the cigarette smoking quality.
TABLE 3 comparison of aroma components of tobacco fermentates of rhizosphere Pseudohelobacter rhizogenes of example 1 with control tobacco extracts of example 2
Note: "/" indicates no detection.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A preparation method of a rhizosphere Pseudohelobacter tobacco fermentation product is characterized by comprising the following steps:
activating a rhizosphere Pseudohelobacter (Rivibacter soli) C1-9 strain; the preservation number of the rhizosphere Pseudoriver bacillus (Rivibacter soli) is CGMCC No. 1.13864;
preparing a rhizosphere Pseudohelobacter C1-9 seed culture solution;
thirdly, fermenting the tobacco by utilizing rhizosphere pseudoriver bacillus C1-9 to obtain tobacco fermentation liquor;
fourthly, extracting the tobacco fermentation liquor obtained in the third step to obtain the rhizosphere pseudomonas tobacco fermentation product.
2. The preparation method of claim 1, wherein the activation of rhizosphere pseudoriver bacillus C1-9 strain comprises the following steps: sterilizing the culture medium at 121 deg.C for 25 min, placing into slant, inoculating Pseudohelobacter rhizogenes C1-9 strain, and culturing at 28 deg.C for 1 week to obtain test tube strain; the culture medium is R2A agar culture medium.
3. The preparation method of claim 1, wherein the step of preparing the culture solution of the rhizosphere Pseudohelobacter C1-9 seeds comprises the following steps: sterilizing a seed culture medium at 121 ℃ for 25 minutes, selecting part of mycelia obtained in the step I from slant culture, inoculating the mycelia into a seed solution, and performing shake culture at 30 ℃ for 48 hours to obtain a seed culture solution; the seed culture medium is R2A liquid culture medium.
4. The method for preparing according to claim 1, characterized in that the fermentation step of step (c) is: pulverizing tobacco into tobacco powder, adding water, and mixing; and then adding a sugar source and inorganic salt into the tobacco powder, sterilizing the tobacco powder for 25 minutes at 121 ℃, inoculating the culture solution of the rhizosphere Pseudohelobacter C1-9 seeds obtained in the step II according to 5-20% of the weight of the tobacco powder, performing shake culture at 30 ℃, and fermenting for 24-48 hours to obtain the tobacco fermentation liquor.
5. The method according to claim 4, wherein the sugar source is a mixture of one or more of glucose, lactose and fructose, and the amount of the sugar source added is 1.0 to 5.0 wt% of the mixed solution.
6. The process according to claim 4, wherein the inorganic salt is KH2PO4、(NH4)2SO4、MgSO4、Fe3(SO4)2、NaCl、CaCl2The addition amount of the mixture of one or more of the components is 0.1 to 2.0 weight percent of the mixed solution.
7. The preparation method according to claim 1, wherein the step of extracting the tobacco fermentation broth comprises the steps of: heating and refluxing the tobacco fermentation liquor obtained in the step (III) at 60-80 ℃ for 2-4 h, then carrying out suction filtration under the condition that the filtration mesh number is 150-250 meshes, and concentrating the obtained filtrate at 45-60 ℃ under 60-100 kPa to obtain a tobacco fermentation concentrated extract; adding 85-95 wt% ethanol in an amount which is 3-5 times the weight of the obtained tobacco fermentation concentrated extracting solution, mixing, and then placing under a freezing condition of-10 ℃ for settling for 8-12 hours to obtain supernatant and precipitate; taking supernatant and concentrating the supernatant at 45-65 ℃ under the condition of 60-90 kPa until the density is 1.0-1.5 g/cm3And obtaining the rhizosphere pseudoriver bacillus tobacco fermentation product.
8. A tobacco fermentation product of rhizosphere Pseudoheobacter obtained by the method according to claim 1.
9. Use of the rhizosphere pseudoheobacter tobacco ferment of claim 8 for improving the smoking quality of a cigarette.
10. The use according to claim 9, wherein the rhizosphere pseudomonas tobacco fermentation product is diluted by 3-5 times of propylene glycol or ethanol and then sprayed onto cut tobacco of cigarettes in a spraying manner; the addition amount of the rhizosphere pseudoriver bacillus tobacco fermentation product is 0.2-0.5 wt% of the mass of the cigarette tobacco shreds.
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