CN117568246A - Geobacillus producing aroma and application thereof in tobacco products - Google Patents
Geobacillus producing aroma and application thereof in tobacco products Download PDFInfo
- Publication number
- CN117568246A CN117568246A CN202410056331.0A CN202410056331A CN117568246A CN 117568246 A CN117568246 A CN 117568246A CN 202410056331 A CN202410056331 A CN 202410056331A CN 117568246 A CN117568246 A CN 117568246A
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- CN
- China
- Prior art keywords
- tobacco
- fragrance
- fermentation
- aroma
- geobacillus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
- A24B15/18—Treatment of tobacco products or tobacco substitutes
- A24B15/20—Biochemical treatment
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention relates to a bacillus altitudinarian and application thereof in tobacco products, belonging to the technical field of microorganisms. The strain has the advantages of strong tolerance, high growth speed, high safety, excellent stress resistance, mildew resistance and alcoholization acceleration, can generate 33 characteristic aroma substances, can directionally improve the flavor quality of tobacco products, and has good application prospect. The cigar intensified fermentation production shows that the fermentation process uses the geobacillus for producing aroma, the fermentation time can reach the standard of quality smoking within 30 days, the fermentation time is shortened, the aroma styles of the cigar tobacco leaf such as nut aroma, sweet aroma, flower aroma and the like are prominent after the fermentation is finished, the cigar style is revealed, the smoking sensory quality is obviously higher than that of unreinforced fermentation tobacco leaf, and the main chemical components meet the requirements of cigar national standards.
Description
Technical Field
The application relates to the technical field of microorganisms, in particular to a bacillus cereus strain and application thereof in tobacco products.
Background
With the development of the tobacco industry and the improvement of the living standard of people, consumers more pursue pleasure, smoke, flavor and quality are better, and tobacco products with lower toxicity of harmful substances and more comfortable smoking sensory experience have to develop in the future. Tobacco is a commercial crop grown widely worldwide, and a large class includes flue-cured tobacco and cigars. Cigars are used as a special tobacco product with mellow fragrance, full taste, strong strength and low nicotine content. The domestic cigar becomes a new economic growth point in the tobacco industry, and is an important way for promoting the upgrading of traditional cigarettes, extending the industrial chain and promoting the high-quality development of tobacco. Domestic cigars have been developed rapidly in recent years, but the stability of tobacco fermentation quality and flavor characteristics are still in need of improvement compared with foreign cigar producing areas such as copaiba and Brazil. The problems of long fermentation period, complex process, unstable fermentation quality and the like of cigar tobacco leaves are the main problems in production.
The bacillus bacteria are found to be key microorganisms in tobacco leaves such as cigars and the like in the current research. The bacillus is a bacterium which can generate stress-resistant spores and has stronger environmental adaptability and tolerance, has high growth speed and higher safety, most bacillus bacteria have no toxicity, can secrete a plurality of enzymes such as protease with strong activity, can exist in a sporophyte form in the life process, has the characteristics of high temperature resistance, saline-alkali resistance, extrusion resistance and the like, and is considered as one of the most beneficial microorganisms with application development potential. The breeding and the strengthening fermentation of the strain with specific functions are used for improving the flavor quality of tobacco products such as domestic cigars and the like, so that the method is an effective and feasible means. Microbial fermentation is a key to the development of flavor quality in tobacco products such as cigars and plays a key role in the development of typical style characteristics. The microorganism strengthening technology and in-situ regulation are applied in a plurality of industries, and good effects are achieved.
The application of bacillus in tobacco products has been reported in related researches, in the patent application of CN202111518952.9, the inventor utilizes bacillus to prepare sauce sweet tobacco, in the patent application of CN202210927374.2, the inventor utilizes bacillus arvensis YNLTT1 with protease production activity for improving neutral aroma components of cigar tobacco, in the patent application of CN 202210731668.8, the inventor utilizes bacillus strain to accelerate tobacco alcoholization and improve quality grade of tobacco, in the patent application of CN202210707244.8, the inventor utilizes tea aroma microorganism-bacillus cereus to ferment cigar tobacco, cigar tea aroma is obvious, sucking is more mellow and irritation is obviously reduced, and in the patent application of CN201811352803.8, the inventor utilizes bacillus pumilus to ferment alcoholization of cigar tobacco, and the quality of cigar tobacco is better improved. In the patent application of CN202211469364.5, the inventor performs cigar fermentation by separating and screening bacillus beleiensis from cigar tobacco leaves to obviously reduce nitrosamine peculiar to tobacco and improve the safety of the tobacco. In recent years, research on bacillus highland has been mainly focused on the fields of feed, medicine, textiles, aquatic products, sewage treatment, plant protection, and the like. As a novel biocontrol microbial factor, a great deal of researches and patents prove that the bacillus highland is paid attention to the aspects of plant disease prevention and control, plant growth promotion and the like. The bacillus highland also has related researches in tobacco products, in patent application CN202011326947.3, the inventor utilizes bacillus highland for cigar mildew biocontrol control, in patent application NC 202111637094.X, the inventor utilizes bacillus highland with protease activity for cigar to promote neutral aroma components of cigar tobacco leaves, promote aroma and improve cigar quality, and in patent application CN202210816889.5, CN201911143373.3 and CN201911143374.8, the inventor utilizes bacillus highland to reduce tobacco-specific nitrosamines in tobacco. In addition, bacillus altitudinalis plays an important role in foods as a fermenting microorganism and a probiotic, and in patent application publication No. CN201210144483.3, the inventor uses bacillus altitudinalis to produce alkaline protease, and applies to soybean peptide production.
Although the application of bacillus strains in foods and tobacco products has been reported, related researches on the application of bacillus cereus in tobacco products such as flue-cured tobacco leaves, tobacco flavors and cigar tobacco leaves, in retrievable researches and patents of bacillus cereus applied to the related fields of cigar and other tobacco products, no in-situ bacillus cereus strain capable of directionally improving aroma and simultaneously resisting high temperature, nicotine and mildew and producing aroma and application thereof on the premise of ensuring the original flavor quality are not available at present.
The applicant used the same method to screen cigar tobacco for Bacillus macerans (Bacillus velezensis) JYX-B-7 and Bacillus macerans (Bacillus altitudinis) MT1-B-6, both having similar properties and aroma components. The invention requests to protect the bacillus cereus (Bacillus altitudinis) strain MT1-B-6 and the application thereof in tobacco products, and has important significance for improving the flavor quality and shaping the typical style characteristics of the tobacco products such as domestic cigars and the like.
Disclosure of Invention
In order to solve the problems, the bacillus cereus capable of directionally improving the flavor quality is not available in the production of tobacco products such as cigars. The invention provides a bacillus altitudinis strain with the functions of producing fragrance, preventing mildew and accelerating alcoholizationBacillus altitudinis) And their use in tobacco products.
The invention provides a bacillus altitudinis strain for producing fragranceBacillus altitudinis) The strain MT1-B-6 is preserved in China Center for Type Culture Collection (CCTCC) NO: m20232099, the preservation address is university of Wuhan in Wuhan, china.
In one embodiment of the invention, the monoclonal colony of the geobacillus cereus is characterized by being round, white, opaque, smooth in surface and provided with bulges.
The first object of the present invention is to provide geobacillus cereus MT1-B-6, which has been preserved in China Center for Type Culture Collection (CCTCC) at the year 2023, month 11, and the preservation number CCTCC NO: m20232099, the preservation address is university of Wuhan in Wuhan, china.
In one embodiment of the invention, the geobacillus cereus MT1-B-6 has the fermentation characteristics of high temperature resistance, nicotine resistance, mildew resistance, aroma production and the like.
In one embodiment of the invention, the bacillus cereus is producedBacillus altitudinis) MT1-B-6 has a good temperature tolerance range, and the strain still has a good growth condition in the range of 15-45 ℃, so that the requirements of the whole fermentation production stage of cigar tobacco leaves can be met.
In one embodiment of the present invention, the aroma substances include ketones and fried flower fragrances having creamy, sweet, fruity, camphor/peppermint fragrances, chocolate, creamy pyrazines, and aldehydes having bitter almond, rose, citrus, strong greasy, fruity fragrances.
In one embodiment of the invention, the cream, sweet, fruit, camphor/peppermint flavored ketones include acetoin, 2, 3-butanedione, isophorone, hydroxyacetone, geranylacetone, 6-methyl-2-heptanone, and the like.
In one embodiment of the invention, the fried-flower aroma, chocolate aroma, creamy-flavor pyrazines comprise 2, 5-dimethylpyrazine.
In one embodiment of the present invention, the bitter almond, rose, citrus, strong greasy, fruity aldehydes include 2, 4-dimethylbenzaldehyde, nonanal, decanal, octanal, and the like.
The second object of the invention is to provide the application of the bacillus cereus (Bacillus altitudinis) strain MT1-B-6 in preparing tobacco products.
In one embodiment of the invention, the tobacco product is tobacco flavor, tobacco extract, flue-cured tobacco leaf or cigar tobacco leaf, etc.
In one embodiment of the invention, the tobacco product is a tobacco flavor using the geobacillus cereus (Bacillus altitudinis) strain MT1-B-6 as a flavoring agent.
In one embodiment of the invention, the tobacco product is a tobacco extract, and the tobacco extract uses the geobacillus cereus (Bacillus altitudinis) strain MT1-B-6 as a fermentation inoculant.
In one embodiment of the invention, the tobacco product is a cigarette tobacco leaf, and the cigarette tobacco leaf uses the geobacillus cereus (Bacillus altitudinis) MT1-B-6 strain to be directly added to tobacco leaf or used as a tobacco mellowing agent to be sprayed on the surface of the tobacco leaf to be mellowed for accelerating the tobacco mellowing or being sprayed on the leaf in a threshing and redrying stage or being sprayed on cut tobacco in a tobacco shredding stage.
In one embodiment of the invention, the tobacco product is cigar tobacco leaves, and the cigar tobacco leaves are subjected to enhanced fermentation by using the bacillus cereus (Bacillus altitudinis) MT1-B-6 strain to directly act on or act as an enhanced ferment, and are sprayed on or added to the surface of tobacco leaves to be fermented in an atomization manner;
the reinforced fermentation process comprises the following steps: fermenting for 0-5 days, at 30 ℃ and relative humidity of 60-80%, fermenting for 5-15 days, at 35 ℃ and relative humidity of 60-80%, fermenting for 15-25 days, at 40 ℃ and relative humidity of 60-80%, fermenting for 25-30 days, at 45 ℃ and relative humidity of 60-80%.
Compared with the prior art, the beneficial effects that above-mentioned at least one technical scheme that this description embodiment adopted can reach include at least:
(1) The invention provides a bacillus altitudinarian production MT1-B-6, which has a good temperature tolerance range, has a good growth condition within a temperature range of 15-45 ℃ and can meet the requirements of cigar tobacco fermentation production.
(2) The geobacillus cereus MT1-B-6 can generate 33 characteristic aroma substances in a culture medium fermentation broth, and has cream aroma, sweet aroma, fruit aroma, camphor/peppermint aroma ketones (acetoin, 2, 3-butanedione, isophorone, hydroxyacetone, geranylacetone, 6-methyl-2-heptanone and the like), stir-fried flower aroma, chocolate aroma, cream aroma pyrazines (2, 5-dimethylpyrazine), bitter almond aroma, roses, citrus aroma, strong grease flavor, fruit aroma aldehydes (2, 4-dimethylbenzaldehyde, nonanal, decanal, octanal and the like).
(3) When the bacillus altitudinariacus MT1-B-6 for producing cigar is used for enhancing fermentation production, compared with normal fermentation, the bacillus altitudinariacus MT1-B-6 can improve the aroma, smoke and aftertaste of tobacco products, improve the uniformity of tobacco leaves, accelerate cigar alcoholization, and after fermentation, the cigar tobacco leaves have prominent aroma styles such as nut aroma, sweet aroma, flower aroma and the like, and the cigar styles are revealed.
Drawings
FIG. 1 shows the geobacillus cereus of the present inventionBacillus altitudinis) Colony morphology of MT 1-B-6.
Description of the embodiments
The detection of main chemical components of flue-cured tobacco and cigar tobacco adopts a continuous flow analyzer: total sugar, reducing sugar, total nitrogen, total plant alkali, potassium and chlorine. The bacillus cereus fermentation liquid and the volatile flavor substances of the tobacco leaves are detected by gas chromatography-mass spectrometry (GC-MS). Cigar sensory evaluation refers to tobacco industry standards and related industry enterprise standards.
Specific embodiments of the present invention are described below with reference to the accompanying drawings. The experimental methods used in the implementation examples are all conventional methods unless otherwise specified; materials, reagents and the like used, unless otherwise indicated, are all commercially available.
PBS buffer: 8.0g of sodium chloride, 0.2g of potassium chloride, 1.44g of disodium hydrogen phosphate and 0.24g of monopotassium phosphate are weighed and dissolved in 800mL of distilled water, the solution is regulated to 7.4 by HCl, and finally distilled water is added to fix the volume to 1L, so that the 0.01M PBS buffer solution can be obtained.
LB medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride, water to 1000 mL,2% agar, autoclaved at 121℃for 20 min, and cooled for later use.
Preparation of tobacco leaching liquor culture medium: 20.0 Cutting cigar leaf, adding 500mL distilled water, shaking and extracting at 200 rpm for 30-60 min, filtering, sterilizing at 121deg.C for 20 min, and sterilizing to obtain tobacco extractive solution.
1. Sample pretreatment
Taking tobacco leaves, finished cigarettes and cigar tobacco leaves of different sources as strain screening sources, temporarily storing samples at-4 ℃ before experiments, taking 5 g samples with sterile scissors on an ultra-clean workbench, cutting the samples, placing the cut samples into triangular flasks with sterilized PBS or 0.9% physiological saline in advance, and placing the triangular flasks into a shaking table at 37 ℃ for culturing at 200 rpm for 30 min.
2. Dilution coating plate
Diluting the supernatant of the sample in step 1 to 10 -1 ，10 -2 ，10 -3 Then respectively coating on LB plates, inverting the coated plates, and culturing at 37 ℃ in a constant temperature and humidity incubator for 24-48h until monoclonal colonies appear on the plates.
3. Three-zone scribe line separation
And (3) selecting different monoclonal colonies in the step (2) according to morphological characteristic differences, carrying out three-region lineation on a new LB plate, inverting the lineated plate, and culturing at 37 ℃ in a constant temperature and humidity incubator for 24-48h until the monoclonal colonies appear on the plate.
4. Purification and preservation
Repeating the step 3 for 2-3 times until the plate has only single-morphology monoclonal colony, inoculating the monoclonal colony into liquid LB culture medium, placing in a shaking table at 37 ℃ and 200 rpm for culturing for 24-48 hours, then uniformly mixing the bacterial liquid and sterilized 30% concentration glycerol in an equal proportion, and preserving in a freezing tube at-80 ℃ after numbering.
5. Identification of strains
Taking the purified monoclonal colony in the step 4, extracting DNA (deoxyribonucleic acid) in sterile water by boiling at high temperature, carrying out sample feeding sequencing after amplification, sequencing the 16S rRNA sequence of MT1-B-6 by using 16S rRNA as shown in SEQ ID NO.1, comparing the homology of the analytical strain with bacillus altitudinalis, bacillus aerosporus and bacillus pumilus in an NCBI database to be 100%, and failing to determine the bacillus species level, and further carrying out specific gene sequencingphoPRSequencing, the 16S rRNA sequence of MT1-B-6 is shown as SEQ ID NO.2, and the homology of the strain to the Geobacillus altitudinalis is 99.15% in NCBI database comparison analysis, so that the strain can be identified as Geobacillus altitudinalis. The strain is preserved in 2023, 11 and 01 days and preserved in China center for type culture Collection, and the preservation number is CCTCC NO: m20232099, the preservation address is university of Wuhan in Wuhan, china.
(1) Isolation and activation of strains
And (3) streaking and separating the bacillus altitudinalis strain preserved in the glycerol pipe to obtain a monoclonal colony, and picking the monoclonal colony to be inoculated into a LB liquid medium of 100 mL to activate bacillus altitudinalis strain fermentation broth.
(2) Determination of volatile flavour substances
The volatile flavor substances of the strain fermentation broth of the Geobacillus strain are determined by adopting a headspace solid-phase microextraction-gas chromatography-mass spectrometry technology, 6mL of the supernatant of the fermentation broth after centrifugation is added into a 20 mL sample bottle, 2g of sodium chloride and 10 mu L of 2-octanol (0.822 mg/L) are added as internal standards, and sample injection detection is carried out after extraction.
(3) Volatile flavour analysis
Qualitative analysis is carried out on volatile flavor substances in the fermentation liquor by using an NIST14 database to determine the category of the volatile flavor compounds, semi-quantitative analysis is carried out on the basis of an internal standard to determine the relative content of main flavor compounds, and the results of main characteristic aroma substances of the fermentation liquor of the geobacillus cereus strain MT1-B-6 are as follows:
acetoin, CAS number 513-86-0, intense creamy;
2-methylbutyric acid, CAS number 116-53-0, has fragrance of fruit, cheese, acidic dairy products;
2, 3-butanedione, CAS number 431-03-8, sweet, milk;
2-phenethyl alcohol, CAS number 60-12-8, rose fragrance;
isobutyric acid, CAS number 79-31-2, has the flavor of dairy fermentation, weak fruit flavor and has the bottom note of fruit flavor;
2, 4-dimethylbenzaldehyde, CAS number 15764-16-6, bitter almond flavor;
dibutyl phthalate, CAS number 84-74-2, aromatic;
isoamyl alcohol, CAS number 123-51-3, has a fusel, savory and spicy taste, and has a body, ether, banana fragrance;
2, 3-butanediol, CAS number 24347-58-8, sweet;
isooctyl salicylate, CAS number 118-60-5, weak floral note;
4-methylpentanoic acid, CAS number 646-07-1, cheese-like aroma;
acetophenone, CAS number 98-86-2, almond flavor;
4-vinylguaiacol, CAS number 7786-61-0, baking flavor, nutty flavor, fruity flavor, and spicy flavor;
caproic acid, CAS number 142-62-1, fat odor;
isophorone, CAS number 78-59-1, camphor/mint flavor;
nonanal, CAS number 124-19-6, has fragrance of roses, oranges and the like, and has strong grease smell;
hydroxyacetone, CAS number 116-09-6, has flavor;
2, 5-dimethyl pyrazine, CAS number 123-32-0, with a pungent, floral aroma and chocolate, creamy smell;
ethyl palmitate, CAS No. 628-97-7, ethyl palmitate gas;
benzoic acid, CAS number 99-86-5, citrus and lemon-like aromas;
isooctanol, CAS number 104-76-7, has sweet and light floral notes;
4-vinyl phenol, CAS No. 2628-17-3, has mold and meat aroma, and has sweet aroma and aroma of vanilla bean extract when diluted;
decanal, CAS number 112-31-2, orange and orange aroma;
lauric acid, CAS number 143-07-7 with a light smell of bay oil or soap;
octanal, CAS number 124-13-0, fruit flavor;
pelargonic acid, CAS number 112-05-0, light fats and coconut aroma;
3-acetylpyridine, CAS number 350-03-8, peanut and nut fragrance;
geranylacetone, CAS No. 3796-70-1, has fresh, clear, light floral aroma, slightly sweet-rose aroma;
lauryl alcohol, CAS number 112-53-8, floral fragrance;
6-methyl-2-heptanone, CAS number 928-68-7, with a characteristic odor;
1-octen-3-ol, CAS number 3391-86-4, mushroom, lavender, rose, and hay aromas;
menthol, CAS number 1490-04-6, peppermint fragrance;
fatty alcohol, CAS number 36653-82-4, greasy taste.
Centrifuging the fermentation liquid of the bacillus cereus obtained in advance according to the step 1 in the embodiment 2, re-dissolving and uniformly mixing thalli with sterile water, inoculating seed liquid of a strain to be tested in a tobacco leaching liquid culture medium according to the proportion of 5% -10%, placing the seed liquid in a shaking table for fermentation at 37 ℃ and 200r/min for 2 d, preparing a fermentation liquid, inoculating sterile water as a blank control group, adding 2 times of ethanol with the volume fraction of 75% into the fermentation liquid for heating reflux extraction after fermentation, repeating the operation for 3 times, filtering, taking supernatant, and distilling off the ethanol by a rotary evaporator at 60 ℃ to obtain the spice for the tobacco.
Weighing tobacco flavor according to the mass fraction of 0.01% -0.20%, dissolving with 5 mL purified water, uniformly spraying on 100g tobacco with a micro sprayer, taking purified water as blank control, balancing in environment with the temperature of 20.0+ -1.0deg.C and the relative humidity of 60% for 36-48 h to obtain the cigarette, performing sensory comprehensive description on the cigarette by a professional evaluation group by adopting a whole circulation method, and performing sensory evaluation according to the sensory quality evaluation standard of the cigarette, and judging by taking aroma quality, aroma quantity, miscellaneous gas, irritation, strength and aftertaste as main indexes. The result shows that the spice prepared by the bacillus cereus strain can well improve the quality of cigarettes, increase the aroma, reduce miscellaneous gases and irritation and enhance the aftertaste.
The method comprises the steps of obtaining a bacillus alpina fermentation liquid in advance according to the mode of step 1 in example 2, centrifuging the bacillus alpina fermentation liquid, then re-dissolving and uniformly mixing thalli with sterile water, selecting 200 g commercial tobacco extract as a raw material, placing the raw material in a 500mL triangular shake flask, inoculating 5% -10% of the bacillus alpina fermentation liquid, culturing and fermenting for 5-10 days at the temperature of a constant temperature and humidity incubator at 37 ℃, after fermentation, dissolving and diluting the obtained tobacco extract with sterile water after fermentation, spraying the diluted tobacco extract onto mixed leaf group tobacco shreds according to the additive amount of 0.05-0.1%, and evaluating the flavor quality according to the method of evaluating and sucking by a professional Commission according to the sensory evaluation method of tobacco additives.
Centrifuging the fermentation liquid of Geobacillus fragrans obtained in advance in the step 1 of example 2, redissolving and mixing the thallus with sterile water, wherein the concentration of the bacterial liquid is 10 6 -10 8 CFU/mL is sprayed on tobacco leaves according to the moisture content of the tobacco leaves in the tobacco leaf mass fraction ratio of 1% -10% in the procedure of heating and humidifying the tobacco leaves, the moisture is supplemented, the moisture content is 20% after vacuum moisture regaining, then the temperature is balanced by 4-6 h at 40 ℃, the moisture content is reduced to 12% after redrying at 100-120 ℃, and the tobacco leaves after redrying are cut into shreds and rolled into sample cigarettes for smoking assessment. The result shows that the bacillus cereus strain for producing the aroma can well improve the quality of cigarettes when being used for threshing and redrying tobacco leaves of the cigarettes, has balanced smoke, increased aroma richness, reduced miscellaneous gases and irritation and enhanced aftertaste in the oral cavity and the nasal cavity.
(1) Preparation of enhanced ferment
According to example 2Step 1, obtaining a fermentation liquor of the geobacillus cereus in advance, centrifuging the fermentation liquor, re-dissolving and uniformly mixing thalli by using sterile water, wherein the concentration of the fermentation liquor is 10 6 -10 8 CFU/mL。
(2) Enhanced addition of ferment
The method is characterized in that the method is used in a cigar tobacco fermentation stage after airing or agricultural fermentation according to the proportion of 1% -10% of the mass fraction of tobacco leaves, the moisture content of the tobacco leaves after adding is 20% -25%, and specifically, the method adopts the steps of adding in the initial fermentation stage (adding in 0 days of fermentation, BV-0), adding in the early fermentation stage (adding in 5 days of fermentation, BV-5), adding in the middle fermentation stage (adding in 15 days of fermentation, BV-15) and adding in the later fermentation stage (adding in 25 days of fermentation, BV-25), and uniformly adding the reinforced ferment on the tobacco leaves by directly spraying or pressurized atomization.
(3) Process for preparing starter for cigar intensified fermentation
Wrapping the tobacco leaves in the step (2) by using gunny bags in a constant temperature and humidity fermentation box for stacking and variable temperature fermentation, wherein the fermentation process comprises the following steps: fermenting for 0-5 days (temperature 30 ℃ C., relative humidity 60% -80%), fermenting for 5-15 days (temperature 35 ℃ C., relative humidity 60% -80%), fermenting for 15-25 days (temperature 40 ℃ C., relative humidity 60% -80%), fermenting for 25-30 days (temperature 45 ℃ C., relative humidity 60% -80%); the control was no starter added.
(4) Cigar tobacco leaf main chemical component and sensory evaluation
After 30 days of fermentation, the cigar tobacco leaves of the reinforced fermentation group are uniform in appearance color, brown to tan in color, mature in maturity, sufficient in oil content, thin in leaf, good in integrity and uniform, and basically free of mildew, and the control group is slightly mildewed and green spots, mainly brown in color and insufficient in maturity; the main chemical component results show that the total nitrogen, total sugar, reducing sugar content and total plant alkali content of the reinforced fermentation group are lower than those of a control, other indexes have no obvious difference, the total nitrogen content is between 2.76 and 3.42 percent, the total sugar content is between 0.45 and 0.63 percent, the reducing sugar content is between 0.18 and 0.29 percent, the chloride ion content is between 0.43 and 0.69 percent, the total plant alkali content is between 2.11 and 2.83 percent, and the potassium ion content is between 4.45 and 6.62 percent; the smoking evaluation result shows that the fermentation degree of cigar tobacco obtained by adopting the fermentation processes of (2) and (3) is far higher than that of a comparison (table 1), the quality of the cigar tobacco subjected to enhanced fermentation (normal fermentation; fermentation 5d addition, fermentation 15d addition and fermentation 25d addition) is higher than that of the comparison, the whole cigar tobacco is characterized by taking nut aroma, baking aroma, sweet aroma as a main component, costustoot, flower aroma, cocoa aroma and sweet aroma as auxiliary components, and the aroma of cigar tobacco after the fermentation is finished, such as nut aroma, sweet aroma, flower aroma and the like is outstanding compared with the comparison style, the cigar style is in good condition, the aroma quantity is increased, the taste is pure and comfortable, and the smoke and the aftertaste are increased.
TABLE 1 evaluation of sensory quality of added cigar tobacco leaves by different addition fermentation stages of Geobacillus cescens strain MT1-B-6
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While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. The geobacillus cereus strain MT1-B-6 is characterized in that the geobacillus cereus strain MT1-B-6 is classified and named asBacillus altitudinisMT1-B-6 is preserved in China Center for Type Culture Collection (CCTCC) at the preservation number of M20232099 in the year 2023, month 11 and day 01.
2. The geobacillus cereus strain MT1-B-6 of claim 1, wherein the geobacillus cereus is obtained by screening cigar tobacco leaves and has high temperature resistance, nicotine resistance, mildew resistance and aroma fermentation characteristics.
3. The geobacillus cereus strain MT1-B-6 with fragrance of claim 2, wherein the fragrance comprises ketones with creamy, sweet, fruity, camphor/mint fragrance, fried flower fragrance, chocolate fragrance, creamy pyrazines and aldehydes with bitter almond fragrance, roses, citrus fragrance, strong oil smell, fruit fragrance.
4. A strain of geobacillus cereus MT1-B-6 with a fragrance as claimed in claim 3, wherein the ketones with creamy, sweet, fruity, camphor/peppermint fragrance comprise acetoin, 2, 3-butanedione, isophorone, hydroxyacetone.
5. A geobacillus cereus strain MT1-B-6 with a fragrance as claimed in claim 3, wherein the fried-flower fragrance, chocolate fragrance, creamy-fragrant pyrazines comprise 2, 5-dimethyl pyrazine.
6. A geobacillus cereus strain MT1-B-6 with a fragrance as claimed in claim 3, wherein the aldehydes with a strong oil smell and fruit smell include 2, 4-dimethylbenzaldehyde, nonanal, decanal and octanal.
7. Use of a geobacillus cereus strain MT1-B-6 according to any one of claims 1-6 for the preparation of tobacco products.
8. The use according to claim 7, wherein the tobacco product is any one of a tobacco flavor, a tobacco extract, a tobacco leaf, or a cigar leaf.
9. The use according to claim 8, characterized in that: the tobacco product is cigar tobacco, and the cigar tobacco preparation method is characterized in that the bacillus cereus MT1-B-6 strain directly acts on or is used as a strengthening ferment and is sprayed on or added on the surface of cigar tobacco to be fermented in a pressurized atomization mode for strengthening fermentation.
10. The use according to claim 9, characterized in that: the reinforced fermentation conditions are as follows: fermenting for 0-5 days, fermenting at 30 ℃ and relative humidity of 60%, fermenting for 5-15 days, fermenting at 35 ℃ and relative humidity of 60% -80%, fermenting for 15-25 days, fermenting at 40 ℃ and relative humidity of 60% -80%, fermenting for 25-30 days, fermenting at 45 ℃ and relative humidity of 60% -80%.
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| CN116083268A (en) * | 2022-08-03 | 2023-05-09 | 四川大学 | Bacillus aryabhattai and application thereof in improving cigar fragrance |
| CN116083268B (en) * | 2022-08-03 | 2024-04-09 | 四川大学 | Bacillus aryabhattai and application thereof in improving cigar fragrance |
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