Preparation method and application of burley tobacco fermented flavor

Abstract

The present invention provides a preparation method and application of burley tobacco fermented flavors, relates to a preparation and extraction method of tobacco flavors, and belongs to the field of tobacco technology. The preparation method comprises the following steps: (1) preparation of tobacco streptomyces N35 bacterial agent; (2) using burley tobacco powder as a substrate, using actinomycete tobacco streptomyces N35 bacterial agent and bacterial rhizosphere chitonella yh7-1 bacterial agent to carry out joint fermentation treatment to obtain fermented burley tobacco; (3) subjecting the fermented burley tobacco to ethanol reflux extraction, filtering and concentrating to obtain burley tobacco fermented flavors. In the patent of the present invention, actinomycete tobacco streptomyces N35 and bacterial rhizosphere chitonella yh7-1 play a synergistic role, can effectively degrade cellulose, protein, etc. in burley tobacco, and release special aroma substances. The flavors prepared by fermenting burley tobacco are applied to cigarette tobacco shreds, which can make the aroma harmonious and full, reduce irritation, soften the smoke, make up for the original aroma of tobacco, and strengthen the style characteristics of tobacco leaves.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco flavors and fragrances, and particularly relates to a preparation method and application of burley tobacco fermentation tobacco flavors. Background Art

[0002] Tobacco is a spice used in cigarette products. It has a natural and harmonious aroma, which can make up for the original aroma of tobacco and strengthen the style characteristics of tobacco leaves. It is an irreplaceable spice in the process of tobacco flavoring. Tobacco extracts usually contain a certain amount of protein, pectin, hemicellulose, lignin, etc. These substances will be decomposed to produce some components during the smoking process of cigarettes. These components will increase the irritation and impurities of cigarette smoking. Therefore, it is of great significance to use microbial fermentation to degrade protein and cellulose to produce aroma precursors.

[0003] Actinomycetes are an important microbial resource. They have a complex secondary metabolic system and can produce a variety of secondary metabolites with novel chemical structures and significant biological activities. Currently, actinomycetes are mostly used in antibiotic research. There are also studies on the use of actinomycetes to degrade organic compounds such as cellulose to carry out biological treatment of environmental pollution. Therefore, the research and development of soil actinomycete resources has extremely high scientific value, economic value and social value.

[0004] In order to solve the above problems, the present invention is proposed. Summary of the invention

[0005] Burley tobacco is one of the important raw materials for blended cigarettes, and has the characteristics of strong strength, rich and full aroma, strong irritation, heavy smell, etc. The present invention utilizes the degradation effect of actinomycetes on cellulose, lignin, etc., combined with bacterial fermentation of burley tobacco, and reduces the irritation of smoking by degrading macromolecular substances. In addition, some small molecular substances have special aromas, which can soften the smoke and have an improvement effect on the quality of cigarette products.

[0006] The purpose of the present invention is to provide a preparation method of burley tobacco fermented flavor with soft smoke and its application in cigarettes in view of the characteristics of burley tobacco flavor such as strong irritation and heavy impurities. The method uses burley tobacco as raw material, processes it by using tobacco Streptomyces N35 and rhizospheric Chitonella yh7-1 synergistic fermentation process, and then obtains a burley tobacco fermented flavor with good safety through solvent extraction process. The burley tobacco fermented flavor is applied to cigarette shreds, and the aroma is harmonious, the irritation is reduced, the smoke is soft, the original aroma of tobacco can be compensated, and the style characteristics of tobacco leaves can be strengthened. A method for preparing a flavor that can significantly improve the smoking quality of cigarettes is provided.

[0007] *Unless otherwise specified, all percentages used in the present invention are by mass.

[0008] The first aspect of the present invention relates to a method for preparing burley tobacco fermented flavor, which comprises the following steps:

[0009] (1) Preparation of Streptomyces nicotianae N35 inoculum and Rhizobacterium oxydontum yh7-1 inoculum;

[0010] (2) using burley tobacco powder as a substrate, mixing an actinomycete Streptomyces nicotianae N35 agent and a rhizospheric Chidonobacterium yh7-1 agent, and subjecting them to a combined solid-state fermentation treatment to obtain fermented burley tobacco;

[0011] (3) subjecting the fermented burley tobacco to ethanol reflux extraction, filtering and concentrating to obtain the burley tobacco fermentation flavor.

[0012] Preferably, step (1) specifically comprises: inoculating the liquid strain of Streptomyces nicotianae N35 into the fermentation medium at an inoculum rate of 5-35%, and shaking and culturing at 15-55° C. for 3-15 days to obtain a culture solution; centrifuging the culture solution, washing the precipitate with sterile water, and finally shaking evenly with sterile water, and diluting 5-20 times to obtain the Streptomyces nicotianae N35 bacterial agent.

[0013] The liquid strain of rhizospheric Bacillus subtilis YH7-1 is inoculated into a fermentation medium at an inoculation rate of 5 to 30%, and cultured at 10 to 50° C. with shaking for 3 to 10 days to obtain a culture solution; the culture solution is centrifuged, the precipitate is washed with sterile water, and finally, it is shaken evenly with sterile water and diluted 3 to 15 times to obtain a rhizospheric Bacillus subtilis YH7-1 bacterial agent.

[0014] The above-mentioned tobacco Streptomyces N35, whose microbiological classification is named tobacco Streptomyces N35, Latin name: Streptomyces tabaci N35, has been deposited in the General Microbiological Center of China Microorganism Culture Collection Administration on October 13, 2021, with the deposit number CGMCC 4.7701. Address: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0015] Rhizospheric Chthonobacterium yh7-1, whose microbiological classification is named as Rhizospheric Chthonobacterium yh7-1, Latin name: Chthonobacter rhizosphaerae yh7-1, has been deposited in the General Microbiological Center of China Microorganism Culture Collection Administration Committee on May 27, 2020, with the deposit number of CGMCC 1.17236. For the isolation, cultivation and identification of Rhizospheric Chthonobacterium yh7-1, reference can be made to the patent application filed by the applicant on March 1, 2021, with application number 202110226320.9, entitled A Rhizospheric Chthonobacterium and Its Isolation Method and Application, and all the descriptions of Rhizospheric Chthonobacterium yh7-1 in the patent are incorporated into this application.

[0016] The main morphological characteristics and physiological and biochemical properties of the tobacco streptomyces N35 strain separated by the present invention are: it grows well on most culture media, forms branched base hyphae and aerial hyphae on R2A culture media, and the colonies are white. Casein hydrolysis, cellulose hydrolysis, Tween 20, 40, 60 and 80 hydrolysis are positive, starch hydrolysis is negative, and no indole is produced. It can utilize alpha-lactose, maltose, cellobiose, trehalose, mannose, fructose, sorbitol, galactose, rhamnose and inositol. The polar lipid components of the cell membrane mainly include phosphatidylethanolamine, phosphatidylinositol and an unknown phospholipid, and the respiratory quinone of the cell is methyl menaquinone-9 (MK-9 (H8)).

[0017] The nucleotide sequence of the 16S rDNA gene of the microorganism Streptomyces nicotianae N35 isolated by the present invention is shown in the sequence table. The sequence has been submitted to the international nucleotide sequence database (GenBank) with the sequence accession number: MT598006.

[0018] Preferably, step (2) specifically comprises: crushing the dried burley tobacco leaves into powder with a particle size of 30-80 meshes, balancing the moisture content to 10%-13%. Spraying the tobacco Streptomyces N35 and the rhizospheric Chitonella yh7-1 bacterial agent on the burley tobacco powder in a ratio of 1:1, spraying 10-50 mL of the bacterial agent per 100 g of burley tobacco powder, mixing, placing the treated burley tobacco powder in a constant temperature and humidity chamber at 22°C and 60% for fermentation for 48-96 hours to obtain fermented burley tobacco.

[0019] Preferably, step (3) specifically comprises: adding 5 to 10 times the mass of fermented burley tobacco to 80% to 95% ethanol, extracting under hot reflux at 50 to 80° C. for 1 to 3 hours, filtering and concentrating the resulting clear liquid to obtain burley tobacco fermentation flavor.

[0020] The second aspect of the present invention provides an application of the burley tobacco fermentation flavor obtained by the preparation method described in the first aspect of the present invention, wherein the burley tobacco fermentation flavor is applied to cigarette shreds to obtain a cigarette containing the burley tobacco fermentation flavor.

[0021] Preferably, the burley tobacco fermentation flavoring prepared by the preparation method described in the first aspect of the present invention is diluted 3 to 5 times by mass with propylene glycol or ethanol in an amount of 0.3 to 0.6% relative to the mass of cigarette tobacco, and then sprayed onto the cigarette tobacco to obtain cigarettes containing burley tobacco fermentation flavoring.

[0022] A third aspect of the present invention provides a burley tobacco fermented flavoring cigarette, which comprises the burley tobacco fermented flavoring prepared by the preparation method described in the first aspect of the present invention.

[0023] The present invention has the following beneficial effects:

[0024] 1. The present invention uses the actinomycete Streptomyces nicotianae N35 agent and the rhizosphere bacteria Chitonella yh7-1 agent to jointly ferment burley tobacco powder to prepare burley tobacco fermented flavors. The actinomycete Streptomyces nicotianae N35 and the rhizosphere bacteria Chitonella yh7-1 play a synergistic role, which can effectively degrade cellulose, protein, etc. in burley tobacco and release special aroma substances. The flavors prepared by fermenting burley tobacco are applied to cigarette shreds, which can make the aroma harmonious and full, reduce irritation, soften the smoke, make up for the original aroma of tobacco, and strengthen the style characteristics of tobacco leaves.

[0025] 2. The present invention improves the quality of burley tobacco flavors and is applied in cigarette processing, enriching the sources and types of tobacco flavors and fragrances, and meeting the personalized and diversified needs of consumers. The entire preparation process does not introduce harmful organic solvents, the obtained flavors and fragrances and processing technology are environmentally friendly and safe, and the raw materials are easily available, the cost is low, and it is easy to realize industrial production, and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is an electron microscope photo of the tobacco Streptomyces N35 of the present invention on R2A medium;

[0027] Figure 2 This is a phylogenetic tree constructed based on the 16SrDNA gene sequence of the tobacco Streptomyces N35 and some related strains of the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0029] Example 1

[0030] Isolation, cultivation and identification of Streptomyces nicotianae N35.

[0031] 1. Isolation of Streptomyces nicotianae N35

[0032] Tobacco 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 30°C and 180 rpm for 30 min; dilute with sterile water to a concentration of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5Times, take 0.2mL of each of the five concentrations of bacterial solution and spread it on R2A plate culture medium, set up three parallel tests for each concentration of bacterial solution, and culture at 30℃ 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.

[0033] The isolated microorganisms were inoculated into LB liquid medium, and cultured at 30°C and 160rpm on a shaker until the bacterial liquid concentration reached OD = 2.0, which was used as seed liquid for later use. 1% of the seed liquid of each strain to be tested was inoculated into the screening medium, and cultured at 30°C and 160rpm on a shaker for 3-5 days. The appearance and aroma changes of the fermentation liquid were observed every day, and the microorganisms that can produce aroma were screened and numbered L4-6.

[0034] 2. Identification of Streptomyces nicotianae N35

[0035] The isolated and purified strain N35 was subjected to morphological, physiological and biochemical properties and 16SrDNA analysis, and the identification results showed that it belonged to Streptomyces. Its microbiological classification was named tobacco Streptomyces N35 (Latin name: Streptomyces tabaciN35).

[0036] The electron microscopic photograph of Streptomyces nicotianae N35 on R2A medium is attached. Figure 1 shown.

[0037] Streptomyces nicotianae N35 grows well on most culture media, forms branched basal hyphae and aerial hyphae on R2A medium, and the colonies are white. Casein hydrolysis, cellulose hydrolysis, Tween 20, 40, 60 and 80 hydrolysis are positive, starch hydrolysis is negative, and no indole is produced. It can utilize α-lactose, maltose, cellobiose, trehalose, mannose, fructose, sorbitol, galactose, rhamnose, and inositol. The polar lipid components of the cell membrane mainly include phosphatidylethanolamine, phosphatidylinositol and an unknown phospholipid, and the respiratory quinone of the cell is menaquinone-9 (MK-9 (H8)).

[0038] The physiological and biochemical characteristics of tobacco Streptomyces N35 are shown in Table 1:

[0039] Table 1 Physiological and biochemical characteristics of Streptomyces nicotianae N35

[0040] experiment Growth response experiment Growth response a-Lactose + + Tween 20, 40, 60 and 80 + + Indole production - - Casein + + Cellulose hydrolysis + + β-Cyclodextrin - - Starch hydrolysis - - D-Mannose + +

[0041] Note: "+" indicates a positive result, "-" indicates a negative result

[0042] The carbon and nitrogen utilization of tobacco Streptomyces N35 is shown in Table 2:

[0043] Table 2 Carbon and nitrogen source utilization of Streptomyces nicotianae N35

[0044] Carbon source utilization result Carbon or nitrogen source utilization result sucrose - Citric Acid - maltose + benzoic acid - Trehalose + Inositol + Cellobiose + L-Aspartic Acid + Xylitol - L-Histidine + Raffinose - Tryptophan - Melibiose - Proline +

[0045] Note: "+" indicates a positive result, "-" indicates a negative result

[0046] The partial sequence of 16S rDNA of Streptomyces nicotianae N35 is shown in the figure caption. 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 tobacco Streptomyces N35 of the present invention and the strain (Streptomyces caldifontis CPCC 204147 T ) are closely related, forming an independent branch on the phylogenetic tree, and the comprehensive characteristics of morphology, physiological and biochemical characteristics, cytochemistry and phylogenetic analysis show that there is an obvious gap between the two, indicating that the tobacco Streptomyces N35 of the present invention is a new species, named Streptomyces tabaci N35.

[0047] The accession number of the 16S rDNA gene nucleotide sequence of Streptomyces nicotianae N35 in the GenBank database is MT598006, and the deposit number of the General Microbiological Center of the China Microbiological Culture Collection Administration is CGMCC 4.7701. The phylogenetic tree constructed from the 16S rDNA gene sequence of Streptomyces nicotianae N35 and related species is shown in the attached figure. Figure 2 shown.

[0048] Example 2

[0049] 1. Test tube slant culture

[0050] A slant preservation medium is used, the medium is R2A agar medium, and the formula of the medium is: 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 is sterilized at 121°C for 25 minutes, placed into a slant, inoculated with tobacco soil Dai's bacteria L4-6 strain, and cultured at 28°C for 1 week to obtain a test tube strain.

[0051] 2. Seed Culture

[0052] 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 the culture is shaken at 28°C for 36 hours to obtain a liquid strain.

[0053] 3. Preparation of inoculants

[0054] (1) Inoculate the liquid strains of Streptomyces nicotianae N35 and Rhizobacterium oxysporum YH7-1 into the fermentation medium at an inoculation rate of 10%, and culture with shaking at 28° C. for 6 days to obtain a fermentation liquid, that is, Streptomyces nicotianae N35 and Rhizobacterium oxysporum YH7-1 bacterial agents.

[0055] The culture medium formula is: 10 g soybean powder; 10 g glucose; 3 g peptone; 2.5 g sodium chloride; 2 g calcium carbonate; 500 mL distilled water, pH 7.0, to obtain the fermentation medium.

[0056] 2. Burley tobacco fermentation

[0057] The moisture content of burley tobacco powder was balanced to 10%, and 20 mL of tobacco Streptomyces N35 and rhizospheric Chitonella yh7-1 inoculants were sprayed on every 100 g of pseudo-gentiana powder. The mixture was mixed well, and the treated burley tobacco powder was placed in a constant temperature and humidity chamber at 22° C. and 60% for fermentation for 24 hours to obtain fermented burley tobacco.

[0058] 3. Preparation of burley tobacco fermentation flavor

[0059] Take the fermented burley tobacco, add 3kg of 95% ethanol, and heat reflux extract at 70℃ for 3h; filter the hot reflux extract while hot, and filter the mesh size to 200 mesh; continue to concentrate the clear liquid obtained by filtration at 45℃ and 80kPa to a density of 1.3g / cm 3 The fermentation extract is the burley tobacco fermentation flavor.

[0060] 4. Application of Burley Tobacco Flavors

[0061] Take 0.4% of burley tobacco flavoring by weight of cigarette tobacco, dissolve it in 4 times the amount of propylene glycol, spray it on the cigarette tobacco, put it in a constant temperature and humidity chamber, balance it for 48 hours under the conditions of humidity 60%±2 and temperature 22℃±2, and roll it into cigarette sticks, which are the test cigarettes containing burley tobacco fermentation flavoring.

[0062] 5. Evaluation of Burley Tobacco Flavors

[0063] The test cigarettes and blank cigarettes were handed over to professional smokers to evaluate the aroma style of the cigarettes with reference to YC / T 497-2014 "Sensory Evaluation Method for Chinese Cigarette Style". The aroma style, comfort characteristics and smoke characteristics were evaluated and compared. The results are shown in Table 3. Compared with the blank cigarettes, the test cigarettes have fuller, rounder and more coordinated smoke, less irritation, and softer smoke, which significantly improves the smoking quality of the cigarettes.

[0064] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

[0065] Table 3 Sensory evaluation of burley tobacco fermentation flavors

[0066]

[0067] List of attached documents:

[0068] The partial sequence of 16S rDNA of Streptomyces nicotianae N35 is as follows:

[0069]

Claims

1. A method for preparing burley tobacco fermented flavor, characterized in that: It includes the following steps: (1) Preparation of Streptomyces nicotianae N35 inoculum and Rhizobacterium oxydontum yh7-1 inoculum; (2) using burley tobacco powder as a substrate, and using an actinomycete Streptomyces nicotianae N35 agent and a rhizospheric bacterial agent Chidonobacterium yh7-1 to perform a combined fermentation treatment on the powder to obtain fermented burley tobacco; (3) subjecting the fermented burley tobacco to ethanol reflux extraction, and obtaining the burley tobacco fermented flavor by filtering and concentrating; The step (1) specifically comprises: inoculating a liquid strain of Streptomyces nicotianae N35 into a fermentation medium at an inoculum rate of 5 to 35%, and culturing at 15 to 55° C. for 3 to 15 days to obtain a culture solution; centrifuging the culture solution, washing the precipitate with sterile water, and finally shaking the culture solution evenly with sterile water, and diluting the culture solution by 5 to 20 times to obtain a Streptomyces nicotianae N35 bacterial agent; The liquid strain of rhizosphere Chitonbacterium yh7-1 is inoculated into the fermentation medium at an inoculation rate of 5 to 30%, and the culture is shaken at 10 to 50° C. for 3 to 10 days to obtain a culture solution; the culture solution is centrifuged, the precipitate is washed with sterile water, and finally, the culture solution is shaken evenly with sterile water and diluted 3 to 15 times to obtain the rhizosphere Chitonbacterium yh7-1 bacterial agent; Step (2) specifically comprises: crushing the dried burley tobacco leaves into powder with a particle size of 30 to 80 meshes, and balancing the moisture content to 10% to 13%; applying tobacco Streptomyces N35 and rhizospheric Chitonella yh7-1 bacterial agent in a ratio of 1:1 on the burley tobacco powder, spraying 10 to 50 mL of the bacterial agent on every 100 g of burley tobacco powder, mixing, and placing the treated burley tobacco powder in a constant temperature and humidity chamber at 22° C. and 60% for fermentation for 48 to 96 hours to obtain fermented burley tobacco; Step (3) specifically comprises: adding 5 to 10 times the mass of fermented burley tobacco into 80% to 95% ethanol, performing heat reflux extraction at 50 to 80° C. for 1 to 3 hours, filtering and concentrating the obtained clear liquid to obtain burley tobacco fermentation flavor.

2. The method for preparing burley tobacco fermented flavor according to claim 1, characterized in that: Step (1) Streptomyces tabaci N35, whose microbiological classification is named Streptomyces tabaci N35, Latin name: Streptomyces tabaci N35, has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on October 13, 2021, and the deposit number is CGMCC 4.7701; Chthonobacter rhizosphaerae yh7-1, whose microbiological classification is named Chthonobacter rhizosphaerae yh7-1, Latin name: Chthonobacter rhizosphaerae yh7-1, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 27, 2020, with the deposit number CGMCC 1.17236.

3. An application of burley tobacco fermented flavor obtained by the preparation method according to any one of claims 1 to 2, characterized in that: The burley tobacco fermentation flavor is applied to cigarette shreds to obtain cigarettes containing burley tobacco fermentation flavor.

4. The use according to claim 3, characterized in that: The burley tobacco fermentation flavor is diluted 3 to 5 times by weight with propylene glycol or ethanol in an amount of 0.3 to 0.6% relative to the weight of cigarette shreds, and then sprayed on the cigarette shreds in a spraying manner to obtain cigarettes containing burley tobacco fermentation flavor.

5. A burley tobacco fermented flavored cigarette, characterized in that: It comprises the burley tobacco fermented flavor prepared by the preparation method according to any one of claims 1 to 2.

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