Aroma-producing paenibacillus and application thereof

Abstract

The invention discloses an aroma-producing paenibacillus sp., which is paenibacillus sp. SI8, is preserved in China General Microbiological Culture Collection Center (CGMCC), and has a preservation number of CGMCC No.1. 19443. The 16S rDNA sequence of the paenibacillus is as shown in SEQ ID NO.1. The invention also discloses a preparation method of the aroma-producing paenibacillus sp. The strain can be converted into micromolecular aroma substances by utilizing dextrin, cane sugar, fructose, maltose, trehalose and the like, and the biological spice prepared from the strain is used for flavoring cigarettes and has an obvious effect of increasing special sour and sweet vowels of smoke. The strain is easy to store, the fermentation process is simple and easy to control, the cost is low, large-scale industrial production is convenient to realize, and the strain has good development and application prospects. The strain is easy to preserve and activate, the fermentation process is easy to control, the fermentation spice quality is stable, the cost is low, and large-scale production and application are easy.

Description

Technical Field

[0001] The invention relates to the cross-technical field of microbial fermentation technology and tobacco flavors and fragrances, and in particular to an aroma-producing bacillus strain and application thereof. Background Art

[0002] Tobacco is one of the important pillars of my country's finance and has made great contributions to my country's social and economic development. To advance my country's tobacco process and achieve sustainable development of the cigarette industry, we must take the road of tobacco technology innovation. Flavoring and adding ingredients are key links in the cigarette process and are one of the effective means to effectively improve the smoking quality of cigarettes and form a unique style of cigarettes. At present, most of the tobacco flavors added to my country's tobacco are extracted by traditional methods and then blended. Although the production operation is simple, it also has disadvantages such as monotonous aroma and unclear effect of improving cigarette smoking.

[0003] There are many kinds of microorganisms. For a long time, people have used pure culture technology to investigate, study and develop microorganisms in the environment. Using microorganisms to ferment aroma raw materials can increase the content of various aroma substances in traditional extracted spices and enhance the richness of aroma. With the development of science and technology, the importance of microbial resources in scientific research and bio-industry has become increasingly prominent. However, there are relatively few types of industrial aroma-producing microorganisms, and the types of fermented spices are limited. There is an urgent need to find and screen new microbial strains that can ferment and produce special aromas to expand the richness of tobacco spices and meet the needs of developing special cigarettes. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an aroma-producing Bacillus and its application. The Bacillus SI8 can utilize dextrin, sucrose, fructose, maltose, trehalose and the like to convert into small molecule aroma substances. The biological flavor prepared by the Bacillus SI8 is used in cigarette flavoring, which has an obvious effect on increasing the special sweet and sour tone of smoke.

[0005] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0006] A strain of aroma-producing Paenibacillus sp., wherein the Paenibacillus sp. is Paenibacillus sp. SI8, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on March 8, 2022, with the deposit number CGMCC No.1.19443.

[0007] Preferably, in the above technical solution, the 16S rDNA sequence of the Paenibacillus is as shown in SEQ ID NO.1.

[0008] A method for preparing biological flavors by fermenting Paenibacillus sp., wherein the Paenibacillus sp. is Paenibacillus sp. SI8 according to any one of claims 1 to 2, and the method comprises the following steps:

[0009] (1) activating and expanding the culture of Paenibacillus SI8 and inoculating it into a fermentation medium, and culturing it in a shake flask at 28° C. for 72 hours to obtain a fermentation broth;

[0010] (2) extracting the seed solution with ethyl acetate for multiple times and combining the extracts;

[0011] (3) Concentrate to obtain a clear light brown liquid, which is the bio-flavor.

[0012] Preferably, in the above technical solution, in step (1), the activation and expansion culture of Paenibacillus SI8 comprises the following steps:

[0013] (11) Test tube slant culture:

[0014] A slant preservation medium was used, the medium was R2A agar medium, the medium was sterilized at 121°C for 25 minutes, placed into a slant, inoculated with Bacillus subtilis SI8 strain, and cultured at 28°C for 1 week to obtain a test tube strain;

[0015] (12) Seed culture:

[0016] Using a seed culture medium, sterilize the culture medium at 121° C. for 25 minutes, pick a portion of mycelium from the test tube slope in step (11) and inoculate it into the seed liquid, and culture it in a shake flask at 28° C. for 48 hours to obtain a liquid strain;

[0017] (13) Fermentation culture:

[0018] The liquid bacteria in step (12) were added to the fermentation medium at an inoculation rate of 10%, and cultured in a shake flask for 72 hours at a culture temperature of 28° C. to obtain a fermentation liquid.

[0019] Preferably, in the above technical solution, the formula of the R2A agar medium is: 0.5 g glucose, 0.5 g yeast extract, 0.5 g peptone, 0.5 g acid hydrolyzed casein, 0.5 g soluble starch, 0.3 g sodium pyruvate, 0.3 g dipotassium hydrogen phosphate, 0.05 g magnesium sulfate, 15 g agar, distilled water to 1000 mL, pH 7.2.

[0020] Preferably, in the above technical solution, the formula of the seed culture medium is: dextrin 120g, soybean powder 40g, yeast extract 2g, tryptophan 0.5g, β-alanine 5g, magnesium sulfate 0.5g, ammonium phosphate 0.2g, distilled water to 1000mL, pH 7.2.

[0021] Preferably, in the above technical solution, in step (1), the fermentation culture specifically comprises:

[0022] Take 5 L of fermentation medium, sterilize the medium at 121° C. for 30 min, add the liquid strain in step (2) to the fermentation medium at a 10% inoculation rate, culture in a shake flask for 72 h at a culture temperature of 28° C., and obtain a fermentation broth.

[0023] Preferably, in the above technical solution, the formula of the fermentation medium is: 10 g soybean powder; 10 g glucose; 3 g peptone; 2.5 g sodium chloride; 2 g calcium carbonate; 1000 mL distilled water, pH 7.2.

[0024] A method for preparing biological flavors by fermenting bacillus sp. and an application of biological flavors in cigarettes.

[0025] Preferably, in the above technical solution, the biological flavoring is used to increase the sweet and sour tone of the cigarette.

[0026] The above technical solution of the present invention has the following beneficial effects:

[0027] (1) The new strain SI8 of the present application can utilize dextrin, sucrose, fructose, maltose, trehalose, etc. to convert into small molecule aroma substances. Morphological research, physiological and biochemical analysis, and molecular biological identification show that the strain is a new species of microorganism belonging to Paenibacillus, and the microbiological classification is named Paenibacillus SI8. The biological flavor prepared by fermentation has a special sweet and sour aroma. It is used in cigarette flavoring, which has a significant effect on increasing the special sweet and sour tone of smoke and promoting the production of body fluids. It can expand the richness of tobacco flavors and further meet the development needs of special cigarettes.

[0028] (2) The strain is easy to preserve and activate, the fermentation process is easy to control, the fermented flavor has stable quality and low cost, and is easy to mass produce and apply. It has broad application prospects in the field of tobacco flavors and fragrances. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0030] Figure 1 This is an electron microscope photo of Paenibacillus SI8 of the present invention grown on R2A medium;

[0031] Figure 2 This is a phylogenetic tree constructed based on the 16S rDNA gene sequence of Paenibacillus sp. SI8 and some related strains of the present invention. DETAILED DESCRIPTION

[0032] 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 components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention unless otherwise specifically stated.

[0033] Strain information: Paenibacillus sp. SI8 strain, deposit number: CGMCC No.1.19443, deposit date: March 8, 2022, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC), address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. This strain has the ability to decompose glucose, mannose, maltose, and gluconate to produce a series of small molecule aroma components.

[0034] Example 1 Isolation of Bacillus SI8

[0035] Tobacco rhizosphere soil samples were collected from the tobacco planting base in Kunming, Yunnan, and sealed in plastic bags and stored at 4°C for later use. Accurately weigh 10 g of soil sample, add 90 mL of sterile water, shake on a shaker at 28°C and 180 rpm for 30 min; dilute with sterile water to a concentration of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Times, take 0.2mL of each of the five concentrations of bacterial solution and spread it on LB plate culture medium, set up 3 parallel tests for each concentration of bacterial solution, culture at 28℃ for 48h, pick different single colonies from the plate of each soil sample, purify them on LB plate culture medium, add glycerol according to 20% of the volume of the bacterial solution, and store them in a -80℃ refrigerator for later use.

[0036] The isolated microorganisms were inoculated into LB liquid culture medium, and cultured in a shaking incubator at 28°C and 160 rpm until the bacterial liquid concentration reached OD = 2.0, which was used as seed liquid for later use. The seed liquid of each strain to be tested was inoculated into the screening medium at 1%, and cultured in a shaking incubator at 28°C and 160 rpm for 2-4 days. The appearance and aroma changes of the fermentation liquid were observed every day, and the microorganisms that could produce pleasant aroma were screened and numbered SI8.

[0037] Example 2 Identification of Paenibacillus SI8

[0038] 1. Morphological characteristics of strains

[0039] The isolated and purified strain SI8 was subjected to morphological, physiological and biochemical properties and 16S rDNA analysis, and the identification results showed that it belonged to Paenibacillus sp. and was named Paenibacillus sp. SI8 in microbiological classification.

[0040] Electron microscopic photograph of Bacillus SI8 on R2A medium Figure 1 shown.

[0041] 2. Physiological and biochemical characteristics of strains

[0042] Bacillus SI8 grows well on most culture media. On R2A agar medium, the colonies have neat edges, convex in the middle, and are milky white and moist. SI8 can grow at 10℃-45℃, with the best growth temperature at 28-30℃; the growth pH is pH 7.0-8.0; it grows well in the salinity range of 0-1% (w / v). The tea phenol-AS-BI-phosphohydrolase is positive, and it can utilize a variety of carbon sources such as dextrin, sucrose, fructose, maltose, trehalose, etc. The main fatty acids are (≥5.0%) anteiso-C 15:0 (63.8%),iso-C 16:0 (10.2%), C 16:0 (7.6%) and anteiso-C 17:0 (5.6%).

[0043] 3. Molecular taxonomic characteristics of bacterial species

[0044] The 16S rDNA partial sequence of Paenibacillus SI8 is shown in the accompanying figure. The sequence was compared with the known sequences in the GenBank database by BLAST analysis, and the 16S rDNA gene sequences of related species were obtained from the database to construct a phylogenetic tree. Figure 2 Comparative analysis revealed that the Paenibacillus sp. SI8 of the present invention had a different taxonomic status from the reference strains. The comprehensive morphological, physiological and biochemical, cytochemical and phylogenetic analysis showed that the Paenibacillus sp. SI8 of the present invention was a new species, named Paenibacillus sp. SI8.

[0045] The accession number of the 16S rDNA gene nucleotide sequence of Paenibacillus SI8 in the GenBank database is OL307708, and the deposit number of the General Microbiological Center of the China Microbiological Culture Collection Administration is CGMCC NO.1.19443. The phylogenetic tree constructed from the 16S rDNA gene sequence of Paenibacillus SI8 and related species is shown in the figure. Figure 2 shown.

[0046] Example 3 Application of Paenibacillus SI8 in the fermentation preparation of biological flavors

[0047] 1. Cultivation of Paenibacillus sp. SI8

[0048] (1) Test tube slant culture

[0049] A slant preservation medium was used, the medium was R2A agar medium, the formula of the medium was: 0.5g glucose, 0.5g yeast extract, 0.5g peptone, 0.5g acid hydrolyzed casein, 0.5g soluble starch, 0.3g sodium pyruvate, 0.3g dipotassium hydrogen phosphate, 0.05g magnesium sulfate, 15g agar, distilled water to 1000mL, pH 7.2. The medium was sterilized at 121°C for 25 minutes, placed into a slant, inoculated with Bacillus subtilis SI8 strain, and cultured at 28°C for 1 week to obtain a test tube strain;

[0050] (2) Seed culture

[0051] A seed culture medium is used, and the formula of the seed culture medium is: dextrin 120g, soybean powder 40g, yeast extract 2g, tryptophan 0.5g, β-alanine 5g, magnesium sulfate 0.5g, ammonium phosphate 0.2g, distilled water to 1000mL, pH 7.2. The culture medium is sterilized at 121°C for 25 minutes, a portion of mycelium is picked from the inclined surface of the test tube in step (1) and inoculated into the seed liquid, and cultured in a shake flask at 28°C for 48 hours to obtain a liquid strain;

[0052] (3) Fermentation culture

[0053] Take 5L of fermentation medium, the formula of which is: 10g soybean powder; 10g glucose; 3g peptone; 2.5g sodium chloride; 2g calcium carbonate; 1000mL distilled water, pH 7.2. Sterilize the medium at 121°C for 30min, add 10% of the liquid strain in step (2) to the fermentation medium, and culture in a shake flask for 72h at a culture temperature of 28°C to obtain a fermentation liquid.

[0054] 2. Preparation of fermentation extract bio-flavors

[0055] The fermentation broth was extracted with 3L of ethyl acetate for three consecutive times, the extracts were combined, and concentrated under reduced pressure at 50°C and 0.06MPa to obtain a clear light brown liquid with a special sweet and sour aroma, which was the desired biological flavor.

[0056] 3. Evaluation of bio-flavors

[0057] 3.1 Evaluation of physical and chemical indicators

[0058] The obtained fermented tobacco flavor is a clear light brown liquid. The physical and chemical properties of the product are shown in Table 1.

[0059] Table 1 Physicochemical indicators of biological flavors fermented by strain SI8

[0060]

[0061] 3.2 Cigarette flavoring experiment

[0062] Take 0.05g of fermented tobacco flavor, dilute it 10 times with water, and evenly spray it on 50g of the group without flavor leaves, put it in a clean sealed bag, leave it at room temperature for 2 hours, and then dry the tobacco in a 120℃ oven to make the moisture content reach 11%-13%. Put the treated tobacco and the control into a constant temperature and humidity chamber, balance it at a temperature of 22℃ and a humidity of 60% for 24 hours, and roll it into cigarettes by hand, and conduct sensory evaluation by a smoking evaluation team composed of smoking evaluation experts.

[0063] Evaluations show that fermented spices have a special sweet and sour aroma, which has a significant effect on increasing the characteristic tone of smoke, highlighting the sweet and sour characteristics, and having a good sense of body fluid. It also improves the texture and permeability of the smoke. It is recommended to use them in the cigarette adding stage at a dosage of 5-10g / box.

[0064] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is limited by the claims and their equivalents.

Claims

1. A strain of aroma-producing Bacillus, characterized in that: The Bacillus sp. is Paenibacillus sp. SI8, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on March 8, 2022, with the deposit number CGMCC No.1.19443.

2. The aroma-producing Paenibacillus according to claim 1, characterized in that The 16S rDNA sequence of the Paenibacillus is shown as SEQ ID NO.

1.

3. A method for preparing biological flavors by fermenting with Paenibacillus sp., characterized in that: The Paenibacillus sp. is the Paenibacillus sp. SI8 according to any one of claims 1 to 2, and the method comprises the following steps: (1) activating and expanding the culture of Paenibacillus SI8 and inoculating it into a fermentation medium, and culturing it in a shake flask at 28° C. for 72 hours to obtain a fermentation broth; (2) extracting the fermentation broth with ethyl acetate for multiple times and combining the extracts; (3) Concentrate to obtain a clear light brown liquid, which is the bio-flavor.

4. The method for preparing biological flavors by fermenting with Paenibacillus according to claim 3, characterized in that: In step (1), the activation and expansion culture of Paenibacillus sp. SI8 includes the following steps: (11) Test tube slant culture: A slant preservation medium was used, the medium was R2A agar medium, the medium was sterilized at 121°C for 25 minutes, placed into a slant, inoculated with Bacillus subtilis SI8 strain, and cultured at 28°C for 1 week to obtain a test tube strain; (12) Seed culture: Using a seed culture medium, sterilize the culture medium at 121° C. for 25 minutes, pick a portion of mycelium from the test tube slope in step (11) and inoculate it into the seed liquid, and culture it in a shake flask at 28° C. for 48 hours to obtain a liquid strain; (13) Fermentation culture: The liquid bacteria in step (12) was added to the fermentation medium at an inoculation rate of 10%, and cultured in a shake flask for 72 hours at a culture temperature of 28° C. to obtain a fermentation liquid.

5. The method for preparing biological flavors by fermenting with Paenibacillus according to claim 4, characterized in that: The formula of the R2A agar medium is: 0.5 g glucose, 0.5 g yeast extract, 0.5 g peptone, 0.5 g acid hydrolyzed casein, 0.5 g soluble starch, 0.3 g sodium pyruvate, 0.3 g dipotassium hydrogen phosphate, 0.05 g magnesium sulfate, 15 g agar, distilled water to 1000 mL, pH 7.

2.

6. The method for preparing biological flavors by fermenting with Paenibacillus according to claim 4, characterized in that: The formula of the seed culture medium is: 120 g dextrin, 40 g soybean powder, 2 g yeast extract, 0.5 g tryptophan, 5 g β-alanine, 0.5 g magnesium sulfate, 0.2 g ammonium phosphate, distilled water to 1000 mL, pH 7.

2.

7. The method for preparing biological flavors by fermenting with Paenibacillus according to claim 4, characterized in that: The fermentation culture is specifically as follows: Take 5 L of fermentation medium, sterilize the medium at 121° C. for 30 min, add the liquid strain in step (12) to the fermentation medium at a 10% inoculation rate, and culture in a shake flask for 72 h at a culture temperature of 28° C. to obtain a fermentation broth.

8. The method for preparing biological flavors by fermenting with Paenibacillus according to claim 7, characterized in that: The formula of the fermentation medium is: 10 g soybean powder; 10 g glucose; 3 g peptone; 2.5 g sodium chloride; 2 g calcium carbonate; 1000 mL distilled water, pH 7.

2.

9. Use of the biological flavor prepared by the method for preparing biological flavor by fermentation with Paenibacillus according to any one of claims 3 to 8 in cigarettes.

10. The use according to claim 9, characterized in that: The biological flavor is used to increase the sweet and sour tone of the cigarette.

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