阅读说明：本技术 一种根际假河杆菌烟草发酵物、其制备方法及用途 (Rhizosphere pseudoriver bacillus tobacco fermentation product, preparation method and application thereof ) 是由 段焰青 高莉 杜刚 李源栋 董立民 陈兴 蒋举兴 郭青 于 2020-05-19 设计创作，主要内容包括：本发明公开了一种根际假河杆菌烟草发酵物的制备方法,包括以下步骤：①将根际假河杆菌C1-9菌种活化；②制备根际假河杆菌C1-9种子培养液；③利用根际假河杆菌C1-9对烟草进行发酵,得到烟草发酵液；④提取烟草发酵液,得到根际假河杆菌烟草发酵物。本发明还公开了所述根际假河杆菌烟草发酵物及用于卷烟中的用途。本发明的根际假河杆菌烟草发酵物能显著提升卷烟抽吸品质。(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.)

1. A preparation method of a rhizosphere Pseudohelobacter tobacco fermentation product is characterized by comprising 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 obtained in the third step to obtain the rhizosphere pseudomonas tobacco fermentation product.

2. The preparation method of claim 1, wherein the step of activating rhizosphere Pseudohelobacter 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 according to claim 1, wherein 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 wt% 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 of claim 1, wherein the step ④ of extracting the tobacco fermentation liquid comprises the steps of heating and refluxing the tobacco fermentation liquid obtained in the step ③ at 60-80 ℃ for 2-4 hours, performing suction filtration with a filter mesh number of 150-250 meshes, concentrating the obtained filtrate at 45-60 ℃ under 60-100 kPa to obtain a tobacco fermentation concentrated extract, adding 85 wt% -95 wt% of ethanol with the mass being 3-5 times of that of the tobacco fermentation concentrated extract, mixing, settling at-10 ℃ for 8-12 hours under a freezing condition to obtain a supernatant and a precipitate, concentrating the supernatant at 45-65 ℃ under 60-90 kPa to a density of 1.0-1.5 g/cm to obtain a supernatant and a precipitate³And 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.

Technical Field

The invention belongs to the technical field of tobacco flavors and fragrances, and particularly relates to a fermentation product of rhizosphere pseudomonas pseudoheuchensis C1-9 of tobacco, a preparation method and application thereof.

Background

The essences and flavors play an important role in the industries of food, feed, cosmetics, chemical industry, pharmacy and the like, and the sources of the essences and flavors mainly comprise two modes of natural and chemical synthesis. Wherein, the chemically synthesized essence accounts for about 85 percent of the whole product, but the chemically synthesized essence has the defects of more byproducts, low yield, high cost, environmental pollution and the like. Along with the advocation of the green industry, natural flavors and fragrances are more and more emphasized by people, the natural flavors and fragrances are mainly derived from animals and plants, but the resources of the animal and plant fragrances are limited, the extraction cost is high, and the market demand which is increased day by day is far not 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 perfume prepared by the microorganism has the advantages of short production period, no influence of climate, mild reaction condition, less three wastes formed after the product is separated, environmental protection and the like, so that the perfume can be used as a substitute for fragrant plants.

The tobacco industry is an important consumer product manufacturing industry, and the improvement of cigarette safety through tar and harm reduction has become a common target in 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 acquisition of tobacco flavors and fragrances with different style characteristics and good safety performance also becomes one of the important directions for the research and development of tobacco products.

The flavors and fragrances 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 available 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 pseudoriver bacillus rhizosphere C1-9 with a fragrance producing function, and obtaining the rhizosphere pseudoriver bacillus C1-9 leavening through preparation processes of 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;

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 obtained in the third step to obtain the rhizosphere pseudomonas tobacco fermentation product.

Preferably, the step I of activating the rhizosphere Pseudohebacillus C1-9 strain comprises the following specific 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 ④ of extracting the tobacco fermentation liquid comprises the steps of heating and refluxing the tobacco fermentation liquid obtained in the step ③ at the temperature of 60-80 ℃ for 2-4 hours, then performing suction filtration under the condition that the filter mesh number is 150-250 meshes, concentrating the obtained filtrate at the temperature of 45-60 ℃ and under the condition of 60-100 kPa to obtain a tobacco fermentation concentrated extracting solution, adding 85 wt% -95 wt% of ethanol with the mass being 3-5 times of that of the tobacco fermentation concentrated extracting solution into the obtained tobacco fermentation concentrated extracting solution, mixing the mixture, then performing sedimentation at the temperature of-10 ℃ for 8-12 hours under the condition of refrigeration to obtain supernatant and precipitate, concentrating the supernatant at the temperature of 45-65 ℃ and under the condition of 60-90 kPa to obtain the supernatant with the density of 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, an identification result shows that the microorganism rhizosphere pseudoriver bacillus C1-9 is a new strain, the microorganism classification is named as rhizosphere pseudoriver bacillus (Latin name: Rivibacter sp.), and the reference biological materials (strains) are as follows: 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: the institute of microbiology of the Chinese academy of sciences, the preservation address is: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.

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 cigarette tobacco shred, 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 mentioned 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.