Paenibacillus parabens and application thereof
The treatment of cigar leaves through Bacillus parabens fermentation has solved the problem of improving the fermentation quality of Chinese cigars, and the aroma and permeability of tobacco leaves have been enhanced, the miscellaneous air is reduced, and the quality is significantly improved.
Patent Information
- Application Number
- CN202510556701.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-29
- Publication Date
- 2025-07-08
AI Technical Summary
There is room for improvement in the fermentation quality and flavor of Chinese cigars compared with traditional cigar origins. The existing technology lacks effective microbial resources to apply to cigar leaf fermentation to improve its quality.
Tobacco leaves were fermented by Metabacillus iocasae, and the bacterial fermentation broth and tobacco leaves were cultured and mixed. The fermentation conditions included 28℃~32℃, 180rpm~220rpm, 20h~28h, and the liquid culture medium was 0.1%~10% peptone, 0.01%~5% yeast powder, 0.1%~10% sodium chloride, pH 6.8~7.2, fermentation time was 20 days~30 days, and relative humidity was 70%~75%.
It significantly increases the content of aroma-causing ingredients such as chlorophyll and carotenoid degradation products in tobacco leaves, reduces the irritation of miscellaneous air, increases the aroma and aroma permeability, and improves the overall quality of tobacco leaves.
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Figure CN120272379A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of microbiology, and particularly to a Metabacillus and its application. Background Art
[0002] With the acceleration of the globalization process, the sales volume of the global cigar market has shown a rapid growth trend in recent years. Chinese cigars are in a stage of rapid development, and significant breakthroughs have been made in aspects such as cigar culture dissemination, raw materials, and technology. However, compared with the products from traditional cigar-producing regions such as Cuba and Brazil, there is still room for improvement in the fermentation quality and flavor of Chinese cigars.
[0003] Research shows that microorganisms play a crucial role in the fermentation process of cigar tobacco. Microbial fermentation can effectively degrade macromolecular substances in cigar tobacco leaves that release irritating odors during incomplete combustion, such as proteins, starches, celluloses, etc., and at the same time produce various volatile aromatic organic compounds, such as alcohols, aldehydes, ketones, etc. This process is a key means to improve the industrial usability of cigar tobacco leaf raw materials.
[0004] Therefore, by means of biotechnology, exploring new microbial resources and applying them to strengthen the fermentation process of cigar tobacco leaves is of great significance for improving the quality of cigar tobacco leaves. Summary of the Invention
[0005] The present application mainly provides a Metabacillus strain, which can stably produce flavor substances. When used for fermenting tobacco leaves, it can significantly reduce irritation and off-odors, improve the aroma quantity and transparency, thereby enhancing the usability of tobacco leaves.
[0006] The present application achieves the above object through the following technical solutions:
[0007] In a first aspect of the present application, there is provided a Metabacillus iocasae with a preservation number of CGMCC NO.32323.
[0008] In a second aspect of the present application, there is provided a biological preparation, which includes one or more of the above Metabacillus, the culture of the above Metabacillus, the lysate of the above Metabacillus, and the extract of the above Metabacillus.
[0009] In a third aspect of the present application, there is provided a method for preparing a tobacco product, including the following steps:
[0010] Fermenting tobacco leaves with the above Metabacillus or the above biological preparation to prepare a tobacco product.
[0011] In some of these embodiments, the variety of the tobacco product includes one or more of cigar tobacco leaves, flue-cured tobacco, and sun-cured tobacco.
[0012] In some of these embodiments, the fermentation treatment includes the following steps:
[0013] Cultivate the Bacillus paralicheniformis, collect the culture solution to obtain a bacterial fermentation broth; and
[0014] Mix the bacterial fermentation broth and the tobacco leaves for fermentation treatment.
[0015] In some of these embodiments, the cultivation conditions include: placing the Bacillus paralicheniformis in a sterilized liquid medium and culturing it at 28°C to 32°C and 180 rpm to 220 rpm for 20 h to 28 h;
[0016] Optionally, the nutrients in the liquid medium include: 0.1% (w / v) to 10% (w / v) peptone, 0.01% (w / v) to 5% (w / v) yeast powder, 0.1% (w / v) to 10% (w / v) sodium chloride, and the pH of the liquid medium is 6.8 to 7.2.
[0017] In some of these embodiments, the cell concentration in the bacterial fermentation broth is 10 6 cells / mL to 10 8 cells / mL.
[0018] In some of these embodiments, the volume-mass ratio of the bacterial fermentation broth to the tobacco leaves is 10 mL to 30 mL: 100 g.
[0019] In some of these embodiments, the temperature of the fermentation treatment is 30°C to 35°C, the relative humidity of the fermentation treatment is 70% to 75%, and the time of the fermentation treatment is 20 days to 30 days.
[0020] The fourth aspect of the present application provides a tobacco product, which is prepared by fermentation with the above-mentioned Bacillus paralicheniformis and / or the above-mentioned biological agent, or is prepared by the above-mentioned preparation method.
[0021] The present application provides a Bacillus paralicheniformis strain that can stably produce flavor substances. By applying the Bacillus paralicheniformis to the enhanced fermentation of tobacco leaves, compared with the control group fermented with sterile water, the Bacillus paralicheniformis fermentation group can significantly increase the contents of chlorophyll degradation products (such as phytol), carotenoid degradation products (such as dihydroactinidiolide 5,6,7,7a-tetrahydro-4,7,7a-trimethyl-2-(4H)-benzofuranone; β-damascenone; 3,5,5-trimethyl-4-(3-oxobutyl)cyclohex-2-en-1-one; β-apo-13-carotene; squalene; stigmasterol), and cembranoid degradation products (such as isosclerone) and other aroma components. At the same time, the miscellaneous gas irritation of the Bacillus paralicheniformis enhanced fermentation group is significantly reduced, and the caramel-like sweetness, baking aroma, and woody aroma increase. The smoke is more mellow, and the aroma quantity increases. These results indicate that the application of Bacillus paralicheniformis not only enhances the aroma characteristics of tobacco leaves but also helps to improve the overall quality of tobacco leaves, providing an effective biotechnology means for the tobacco fermentation process. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.
[0023] Figure 1 It is the electrophoretic detection diagram of the amplification product in Example 1 of the present application;
[0024] Figure 2 It is the comparison diagram of the quality characteristics of tobacco products in Example 1 and Comparative Example 1 of the present application;
[0025] Figure 3 It is the comparison diagram of the aroma characteristics of tobacco products in Example 1 and Comparative Example 1 of the present application;
[0026] Figure 4 It is the comparison diagram of the biochemical index results of tobacco products in Example 1 and Comparative Example 1 of the present application.
[0027] The Bacillus paralicheniformis provided by the present application was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on October 24, 2024. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC NO. 32323. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0028] To make the above objects, features, and advantages of the present application more apparent and understandable, a detailed description of the specific embodiments of the present application is provided. Many specific details are set forth in the following description to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present application. Therefore, the present application is not limited by the specific embodiments disclosed below.
[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in this application can be obtained through the market or prepared by existing methods.
[0030] Unless otherwise stated or there is a contradiction, the terms or phrases used herein have the following meanings:
[0031] As used herein, the terms "and / or", "or / and", "and / or" include any one of two or more related listed items, as well as any and all combinations of the related listed items. The said any and all combinations include any two related listed items, any more related listed items, or the combination of all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are used to connect at least three items, it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, D, that is, it includes the combination of any two or any three of A, B, C, D, and also includes the combination of the four items A, B, C, D (that is, the technical solution connected by "logical AND").
[0032] In this application, when it comes to "multiple", "multiple kinds", "multiple times", "multiple elements", etc., unless otherwise specifically defined, it means greater than 2 or equal to 2 in quantity. For example, "one or more kinds" means one kind or greater than or equal to two kinds.
[0033] In this application, "further", "even further", "especially", etc. are used for descriptive purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.
[0034] In this application, "optionally", "optional", and "option" mean that something is either present or absent, that is, any one of two alternative scenarios of "present" or "absent". If the term "optional" appears multiple times in a technical solution, and there is no special indication, no contradiction, or mutual restriction relationship, then each "optional" is independent of the others.
[0035] In this application, for technical features described in an open-ended manner, they include closed technical solutions composed of the listed features, as well as open technical solutions that include the listed features.
[0036] In this application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical values are considered continuous within the above numerical intervals, and include the two numerical endpoints of the numerical range (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, if t is an integer selected from 1 - 10, it means that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this article should be understood to include any and all sub-ranges subsumed therein.
[0037] For the temperature parameters in this application, unless otherwise specifically defined, both constant temperature treatment is allowed, and fluctuations within a certain temperature range are also allowed. It should be understood that the so-called constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.
[0038] In this application, both %(w / w) and wt% represent weight percentages, %(v / v) refers to volume percentages, and %(w / v) refers to mass-volume percentages.
[0039] As a type of bacteria with relatively strong tolerance, Bacillus has the characteristics of fast growth rate and high safety. Most Bacillus bacteria are non-toxic and can secrete various enzymes such as proteases with relatively strong activity. In addition, during its life cycle, it can exist in the form of spores and has excellent properties such as high temperature resistance, salt and alkali tolerance, and extrusion resistance.
[0040] The technical personnel in this application have isolated Parabacillus on the surface of cigar tobacco leaves during long-term research. This strain can produce protease and amylase, and at the same time can also generate volatile aromatic organic compounds. However, as of now, there has been no relevant report on its application in cigar tobacco leaf fermentation.
[0041] Based on this, an embodiment of the present application provides a strain of Metabacillus iocasae, which was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on October 24, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC NO. 32323.
[0042] This strain of Metabacillus can stably produce flavor substances. By applying Metabacillus to the enhanced fermentation of tobacco leaves, compared with the control group fermented with sterile water, the Metabacillus fermentation group can significantly increase the contents of chlorophyll degradation products (such as phytol), carotenoid degradation products (such as dihydroactinidiolide 5,6,7,7a-tetrahydro-4,7,7a-trimethyl-2-(4H)-benzofuranone; β-damascenone; 3,5,5-trimethyl-4-(3-oxobutyl)cyclohex-2-en-1-one; β-apo-13-carotene; squalene; stigmasterol), and cembranoid degradation products (such as isosclerone) and other aroma components. At the same time, the irritation of miscellaneous odors in the Metabacillus enhanced fermentation group is significantly reduced, the caramel-like, baking, and woody aromas increase, the smoke is more mellow, and the aroma quantity increases. These results indicate that the application of Metabacillus not only enhances the aroma characteristics of tobacco leaves but also helps to improve the overall quality of tobacco leaves, providing an effective biotechnological means for the tobacco fermentation process.
[0043] In some of these embodiments, the above-mentioned Metabacillus is screened from cigar tobacco leaves.
[0044] An embodiment of the present application also provides a biological preparation, which includes one or more of the above-mentioned Metabacillus, the culture of the above-mentioned Metabacillus, the lysate of the above-mentioned Metabacillus, and the extract of the above-mentioned Metabacillus.
[0045] Another embodiment of the present application also provides a method for preparing a tobacco product, including the following steps: fermenting tobacco leaves with the above-mentioned Metabacillus or the above-mentioned biological preparation to prepare a tobacco product.
[0046] In some of these embodiments, the varieties of the above-mentioned tobacco products include one or more of cigar tobacco leaves, flue-cured tobacco, and sun-cured tobacco.
[0047] The method for preparing a tobacco product in the present application has a simple process, is safe and non-toxic, and is suitable for industrial promotion and application.
[0048] In some of these embodiments, the above-mentioned fermentation treatment includes steps S10 to S20.
[0049] Step S10: Cultivate the above-mentioned Metabacillus under suitable conditions, collect the culture solution, and obtain a bacterial fermentation broth.
[0050] Step S20: Mix the above bacterial fermentation broth and tobacco leaves for fermentation treatment.
[0051] In some of these embodiments, the above culturing conditions include: placing the above Bacillus paralicheniformis in a sterilized liquid medium and culturing it at 28°C to 32°C and 180 rpm to 220 rpm for 20 h to 28 h.
[0052] In a specific example, the above culturing conditions include: placing the above Bacillus paralicheniformis in a sterilized liquid medium and culturing it at 30°C and 200 rpm for 24 h.
[0053] In some of these embodiments, the nutrients in the above liquid medium include: 0.1% (w / v) to 10% (w / v) peptone, 0.01% (w / v) to 5% (w / v) yeast powder, 0.1% (w / v) to 10% (w / v) sodium chloride, and the pH of the above liquid medium is 6.8 to 7.2.
[0054] It can be understood that peptone contains rich amino acids, vitamins and other growth factors required for the growth of microorganisms; yeast powder can provide high-quality protein, complete amino acids and essential precursor substances for the growth of strains; sodium chloride can be used as an inorganic salt to promote the basic metabolism of microorganisms, affect the synthesis of metabolites, and at the same time has a buffering effect on the pH value of the medium, and can maintain the pH value required for the growth of microorganisms.
[0055] In a specific example, the nutrients in the above liquid medium include 1% (w / v) peptone, 0.5% (w / v) yeast powder, 1% (w / v) sodium chloride, and the pH of the above liquid medium is 7.0.
[0056] In some of these embodiments, the cell concentration in the above bacterial fermentation broth is 10 6 cells / mL to 10 8 cells / mL. As an example, the concentration of the bacterial solution can be 10 6 cells / mL, 2×10 6 cells / mL, 3×10 6 cells / mL, 4×10 6 cells / mL, 5×10 6 cells / mL, 6×10 6 cells / mL, 7×10 6 cells / mL, 8×10 6 cells / mL, 9×10 6 cells / mL, 10 7 cells / mL, 2×10 7 cells / mL, 3×10 7 cells / mL, 4×107 CFU / mL, 5×10 7 CFU / mL, 6×10 7 CFU / mL, 7×10 7 CFU / mL, 8×10 7 CFU / mL, 9×10 7 CFU / mL, 10 8 CFU / mL, or any value within the range formed by any two of the above point values.
[0057] In some embodiments, in step S10, after the step of collecting the culture medium, it further includes: diluting the culture medium so that the cell concentration after dilution is 10 6 CFU / mL to 10 8 CFU / mL.
[0058] Optionally, the dilution factor is 5 to 50 times.
[0059] In some embodiments, the volume-mass ratio of the above bacterial fermentation broth to the above tobacco leaves is 10 mL to 30 mL: 100 g.
[0060] In some embodiments, the temperature of the above fermentation treatment is 30°C to 35°C. As an example, the temperature of the fermentation treatment can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or any value within the range formed by any two of the above point values.
[0061] In some embodiments, the relative humidity of the above fermentation treatment is 70% to 75%. As an example, the relative humidity of the fermentation treatment can be 70%, 71%, 72%, 73%, 74%, 75%, or any value within the range formed by any two of the above point values.
[0062] In some embodiments, the time of the above fermentation treatment is 20 days to 30 days. As an example, the time of the fermentation treatment can be 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or any value within the range formed by any two of the above point values.
[0063] Another embodiment of the present application also provides a tobacco product, which is prepared by fermenting with the above-mentioned Bacillus subtilis and / or the above-mentioned biological agent, or by the above-mentioned preparation method.
[0064] For the above-mentioned tobacco product, the irritation of miscellaneous odors is significantly reduced, the caramel-like fragrance, baking fragrance, and woody fragrance are increased, the smoke is more mellow, and the aroma amount is increased.
[0065] The present application will be further described below in conjunction with specific examples and comparative examples, but it should not be construed as a limitation on the protection scope of the present application. For the raw materials involved in the following specific examples, unless otherwise specified, they can all be obtained commercially. For the instruments used, unless otherwise specified, they can all be obtained commercially. For the processes involved, unless otherwise specified, they are all conventional selections of those skilled in the art.
[0066] The present application will be further described below in conjunction with specific examples and comparative examples, but it should not be construed as a limitation on the protection scope of the present application. For the raw materials involved in the following specific examples, unless otherwise specified, they can all be obtained commercially. For the instruments used, unless otherwise specified, they can all be obtained commercially. For the processes involved, unless otherwise specified, they are all conventional selections of those skilled in the art.
[0067] Example 1
[0068] I. Strain isolation, screening and identification
[0069] (1) Sample pretreatment
[0070] Take cigar tobacco leaves as the source for strain screening. Before the experiment, store the samples at -4°C temporarily. Use sterile scissors to take 5 g of the samples in a laminar flow hood, cut them into pieces and place them in a triangular flask pre-filled with sterilized phosphate buffer. The phosphate buffer specifically contains 8 g of sodium chloride (NaCl), 0.2 g of potassium chloride (KCl), 1.44 g of disodium hydrogen phosphate (Na2HPO4), and 0.24 g of potassium dihydrogen phosphate (KH2PO4) per 1 L. Place the triangular flask on a shaker and culture it at 37°C and 200 rpm for 30 min.
[0071] (2) Dilution coating on plates
[0072] Dilute the supernatant of the sample in step (1) to 10 -1 、10 -2 and 10 -3 , and then coat them on NB plates respectively. Invert the coated plates and culture them in a constant temperature and humidity incubator at 30°C for 24 h to 28 h until monoclonal colonies appear on the plates.
[0073] (3) Three-zone streaking isolation
[0074] According to the morphological characteristics differences, pick different monoclonal colonies in step 2 and perform three-zone streaking on a new NB plate. Invert the streaked plate and culture it in a constant temperature and humidity incubator at 30°C for 24 h to 28 h until monoclonal colonies appear on the plate. The colonies of the strain SC-6 are round (diameter 1 - 3 mm), beige, opaque and flat; and physiological and biochemical tests are carried out on the strain:
[0075] 1) Detect the protease activity of the strain. Use skim milk powder as the substrate. The specific medium components are shown in Table 1. After the strain is cultured on the plate for 24 h, observe whether there is a clear zone.
[0076] Table 1
[0077]
[0078] 2) Detect the amylase activity of the strain. Use starch as the substrate. The specific medium components are shown in Table 2. After the strain is cultured on the plate for 24 h, add 5 ml of iodine solution to the screening plate and stain for 2 min, then observe whether there is a clear zone.
[0079] Table 2
[0080]
[0081] 3) Detect the cellulase activity of the strain. Use sodium carboxymethyl cellulose as the substrate. The specific medium components are shown in Table 3. After the strain grows for 24 h, stain with 1% Congo red solution prepared with 95% absolute ethanol for 10 - 15 min, and then decolorize with 1 M NaCl solution and observe the size of the clear zone.
[0082] Table 3
[0083]
[0084] The specific results of the physiological and biochemical indexes are shown in Table 4.
[0085] Table 4
[0086]
[0087] Among them, "-" indicates negative; "+" indicates positive.
[0088] (4) Purification and preservation
[0089] Repeat step 3 for 2 - 3 times of purification until there is only a monoclonal colony with a single morphological characteristic on the plate. Then inoculate the monoclonal colony into a liquid medium, place it on a shaker at 30 °C and 200 rpm for 24 h - 28 h. Then, proportionally absorb the bacterial liquid and the sterilized 30% glycerol and mix them evenly in a cryotube, and preserve them at -80 °C.
[0090] (5) Strain identification
[0091] Take the purified monoclonal colonies in step (4) in sterile water, extract DNA by boiling at high temperature, send the sample for sequencing after amplification, and use the primers shown in 27F: 5´-AGTTTGATCMTGGCTCAG-3´; 1492R: 5´-GGTTACCTTGTTACGACTT-3´ for sequencing detection. The electrophoresis detection map of the PCR amplification product is as follows Figure 1 shown; the 16s sequence is as shown in SEQ ID NO.1. Further sequence comparison found that: the homology between the analyzed strain and Bacillus paralicheniformis is 100%, and it can be identified as Bacillus paralicheniformis. Among them, Metabacillus iocasae and Priestia megaterium are the names of the same strain at different times. This Bacillus paralicheniformis was deposited in the China General Microbiological Culture Collection Center on October 24, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC NO.32323. The specific 16s sequence shown in SEQ ID NO.1 is as follows
[0092]
[0093] II. Fermentation Method of Cigar Tobacco Leaves
[0094] (1) Inoculate Bacillus paralicheniformis preserved with glycerol into a liquid medium for fermentation culture. The liquid volume in the shake flask is 20%, and the inoculation amount is 2%. Incubate at 30°C and 200 rpm in a constant temperature and humidity incubator for 24 h. Among them, the components of the liquid medium include: 1% (w / v) peptone, 0.5% (w / v) yeast powder, 1% (w / v) sodium chloride, and 97.5% (v / v) distilled water, with a pH of 7.0.
[0095] (2) Dilute the above culture solution with water so that the concentration of the thalli after dilution is 10 6 ~10 8 cells / mL to obtain a fermentation broth.
[0096] (3) Inoculate the fermentation broth onto the rehydrated cigar tobacco leaves at a ratio of 20%.
[0097] (4) Put the inoculated tobacco leaves into a jute bag and place them in a constant temperature and humidity fermentation chamber for fermentation treatment. The temperature of the fermentation treatment is 35°C, the relative humidity is 75%, and the fermentation time is 21 days.
[0098] Test:
[0099] (1) Dry the fermented cigar tobacco leaf samples at 45°C, pass them through a 0.25 mm sieve, and determine the contents of total sugar, alkaloids, and aroma components by gas chromatography - mass spectrometry (GC - MS).
[0100] (2) Roll the fermented tobacco leaf samples into single - filler cigars with a length of 110 mm and a diameter of 14 mm, cure them in a Binder constant temperature and humidity box at 18°C and a relative humidity of 65% for 1 month for sensory evaluation, and organize a sensory quality evaluation expert group to conduct sensory quality evaluation.
[0101] The sensory quality evaluation standard refers to the product technical standard of Great Wall Cigar Factory, "Sensory Evaluation Method for the Style Characteristics and Quality of Chinese Cigar 'Mellow Sweet Aroma' Style" QJ / 08.J.6005 - 2020 A for the sensory quality evaluation standard.
[0102] Comparative Example 1
[0103] The fermentation method of cigar tobacco leaves in Comparative Example 1 is basically the same as that in Example 1, except that: pure water is inoculated onto the surface of the rehydrated cigar tobacco leaves at a volume ratio of 20%.
[0104] Other steps and parameter conditions are the same as those in Example 1.
[0105] The results of the aroma component content after the fermentation of cigar tobacco leaves in the comparative examples are shown in Table 5 below.
[0106] Table 5
[0107]
[0108] The sensory quality scoring results of the above-mentioned examples and comparative examples are shown in Table 6 and Table 7 below. Figure 2 Fig. for comparing the quality characteristics of the tobacco products of Example 1 and Comparative Example 1; Figure 3 Fig. for comparing the aroma characteristics of Example 1 and Comparative Example 1.
[0109] Table 6
[0110]
[0111] Table 7
[0112]
[0113] The detection results of the biochemical indexes of the above-mentioned examples and comparative examples are shown in Table 8 below. Figure 4 Fig. for comparing the biochemical indexes in Example 1 and Comparative Example 1.
[0114] Table 8
[0115]
[0116] Experiments show that compared with fermenting cigar tobacco leaves with sterile water in Comparative Example 1, the total sugar in cigar tobacco leaves fermented with Bacillus paralicheniformis decreased by 70.8%, and the total alkaloids decreased by 21%. In terms of aroma components, compared with Comparative Example 1, the degradation products of chlorophyll (such as phytol), the degradation products of carotenoids (such as dihydroactinidiolide 5,6,7,7a-tetrahydro-4,7,7a-trimethyl-2-(4H)-benzofuranone; β-damascenone; 3,5,5-trimethyl-4-(3-oxobutyl)cyclohex-2-en-1-one; β-apo-13-carotene; squalene; stigmasterol), the degradation products of cembranes (such as isolongifolene) and other aroma components increased by 14.4%. The smoking evaluation results also show that the miscellaneous gas irritation in the group fermented with Bacillus paralicheniformis was significantly reduced, and the caramel-like fragrance, baking fragrance, and woody fragrance increased. The smoke was more mellow, and the aroma quantity increased.
[0117] Comparative Example 2
[0118] The fermentation method of cigar tobacco in comparative example 2 is basically the same as that in example 1, except that the tobacco leaves are inoculated with different strains, specifically Bacillus agni with a deposit number of CCTCCNo: M2022956. In the invention, the alkaloid content increases instead of decreases. In example 1 of the present application, the enhanced fermentation with Bacillus parasitica can effectively reduce the alkaloid content and optimize the taste of cigar tobacco.
[0119] Comparative Example 3
[0120] The fermentation method of cigar tobacco leaves in Comparative Example 3 is basically the same as that in Example 1, except that the tobacco leaves are inoculated with different strains, specifically lysine Bacillus fusiformis with a deposit number of CGMCC No. 31713. The content of flavor substances produced is somewhat different from that in Example 1 of the present application, and the content of flavor substances produced after the parabacillus in Example 1 of the present application is higher.
[0121] Comparative Example 4
[0122] The fermentation method of cigar tobacco leaves in Comparative Example 4 is basically the same as that in Example 1, except that the tobacco leaves are inoculated with different strains, specifically Bacillus subtilis with a deposit number of CCTCCNO: M20211370. The strain in Example 1 of the present application has amylase activity in addition to protease activity, and the types of flavor substances are more varied.
[0123] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0124] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.
Claims
1. A Metabacillus iocasae, characterized in that, The preservation number is CGMCC No. 32323.
2. A biological preparation, characterized in that, The biological agent includes one or more of the Bacillus paralicheniformis described in claim 1, the culture of the Bacillus paralicheniformis described in claim 1, the lysate of the Bacillus paralicheniformis described in claim 1, and the extract of the Bacillus paralicheniformis described in claim 1.
3. A method for preparing a tobacco product, characterized in that, It includes the following steps: Using the Bacillus paralicheniformis described in claim 1 or the biological agent described in claim 2 to ferment tobacco leaves to prepare tobacco products.
4. The method for preparing a tobacco product according to claim 3, characterized in that, The varieties of the tobacco products include one or more of cigar tobacco leaves, flue-cured tobacco, and sun-cured tobacco.
5. The application according to any one of claims 3 to 4, characterized in that The fermentation treatment includes the following steps: Culturing the Bacillus paralicheniformis, collecting the culture solution to obtain a bacterial fermentation broth; and Mixing the bacterial fermentation broth and the tobacco leaves for fermentation treatment.
6. The application according to claim 5, characterized in that, The culturing conditions include: placing the Bacillus paralicheniformis in a sterilized liquid medium and culturing it at 28°C to 32°C and 180 rpm to 220 rpm for 20 h to 28 h; Optionally, the nutrients in the liquid medium include: 0.1% (w / v) to 10% (w / v) peptone, 0.01% (w / v) to 5% (w / v) yeast powder, 0.1% (w / v) to 10% (w / v) sodium chloride, and the pH of the liquid medium is 6.8 to 7.
2.
7. The application according to claim 5, characterized in that The cell concentration in the bacterial fermentation broth is 10 6 cells / mL to 10 8 cells / mL.
8. The application according to claim 5, characterized in that, The volume-mass ratio of the bacterial fermentation broth to the tobacco leaves is 10 mL to 30 mL: 100 g.
9. The application according to any one of claims 3 to 4, 6 to 8, characterized in that The temperature of the fermentation treatment is 30°C to 35°C, the relative humidity of the fermentation treatment is 70% to 75%, and the time of the fermentation treatment is 20 days to 30 days.
10. A tobacco product, characterized in that, It is prepared by fermentation with the Bacillus paralicheniformis described in claim 1 and / or the biological agent described in claim 2, or is prepared by the preparation method described in any one of claims 3 to 9.