A strain, biological agent and its application and tobacco leaf alcoholization method

Abstract

The present invention discloses a strain, a biological agent and its application and a tobacco leaf alcoholization method, and relates to the field of tobacco. The strain of the present invention: Bacillus sp. GYCH1; deposit number: CCTCC M 20221805; deposit date: November 15, 2022; deposit unit: China Center for Type Culture Collection. The biological agent of the present invention includes yeast, crystalline trehalose and aged tobacco powder. The tobacco leaf alcoholization method of the present invention: the bacterial suspension is sprayed on the surface of the tobacco leaf to obtain a pretreated tobacco leaf; the pretreated tobacco leaf is anaerobically fermented in an oxygen-free atmosphere to obtain a fermented tobacco leaf 1; the fermented tobacco leaf 1 is aerobically fermented under aerobic conditions to obtain a fermented tobacco leaf 2; the fermented tobacco leaf 2 is baked and balanced to obtain an alcoholized tobacco leaf. The Bacillus of the present invention has a stronger ability to resist extreme conditions, and the improvement of the alcoholization formula and its aerobic and anaerobic treatment processes improves the alcoholization efficiency, alcoholization quality and comprehensive effect of tobacco leaves.

Description

Technical Field

[0001] The invention relates to the field of tobacco, and in particular to a strain, a biological preparation and application thereof, and a tobacco leaf aging method. Background Art

[0002] The production of cigarette raw materials often forms different raw material characteristics due to differences in region, climate, variety and cultivation technology. In order to meet the demand of cigarette manufacturers for raw materials, human factors are often added in the tobacco aging process. Enzymes, bacterial agents and flavors and fragrances are often added to accelerate or improve the aging speed of tobacco and improve quality. The species involved in the disclosed tobacco leaf alcoholizing bacterial agents mainly include: lactic acid bacteria, Bacillus saltitudinis, saccharomyces, Aspergillus, Arthrobacter, Alcaligenes, Trichoderma, Eurotiales, Pseudomonas fluorescens, Bacillus cereus, Aspergillus niger, etc.; the disclosed tobacco leaf alcoholizing enzymes mainly include: saccharifying enzymes, amylases, cellulases, lipases, proteases, pectinases, ligninases, laccases, etc. Adding artificial non-natural flavors will bring certain harm to human health, while the matching of natural flavors is extremely demanding and it is difficult to meet the growing market demand; therefore, screening and optimizing tobacco aging microorganisms is still the first choice to shorten the aging cycle, improve aging efficiency, improve aging quality, and control aging costs.

[0003] In the prior art, the strains of alcoholizing bacteria are isolated from rhizosphere soil of tobacco, endophytes of tobacco plants and the surface of vegetative bodies of fresh tobacco plants. Baked leaves or tobacco shreds are not suitable habitats for the isolated strains. These strains have good alcoholizing effects under laboratory control conditions, but due to their poor adaptability to baked tobacco leaves, the application process operation is relatively harsh, the alcoholizing effect is unstable, and it is difficult to achieve an ideal state.

[0004] In the prior art, tobacco alcoholization bacteria agents and enzyme agents also have different carriers, which more or less have a certain impact on the alcoholization results. Among them, enzyme agents also have defects such as single alcoholization products and easy to form enrichment effects, which are prone to negative effects of one thing and another, resulting in unstable alcoholization effects and difficult to control alcoholization process technology. This is also one of the reasons why many tobacco alcoholization preparations are difficult to promote and apply in actual production. Bacteria agents, enzyme agent carriers or additionally added flavoring substances will have an adverse effect on the characteristics and style of the cigarette raw materials after alcoholization. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is that the existing tobacco aging efficiency is low and the aging quality needs to be further improved, and further provides a strain, a biological agent and its application and a tobacco aging method.

[0006] On the one hand, the present invention provides a strain, the taxonomic name of the strain: Bacillus sp. GYCH1; preservation number: CCTCC M 20221805; preservation date November 15, 2022; preservation unit: China Center for Type Culture Collection (CCTCC), and the preservation address is Wuhan University, Wuhan, China.

[0007] The Bacillus sp. GYCH1 provided by the present invention can produce spores and has a stronger ability to resist extreme conditions. When the conditions are suitable, the strain is activated quickly and has vigorous metabolic growth; when there are adverse conditions, spores will be formed again, making the bacteria in a dormant or semi-dormant state. Whether it is in the change of conditions during the alcoholization process or in the storage as a bacterial agent, it has significant advantages.

[0008] On the other hand, the present invention provides a biological preparation comprising the above-mentioned strain.

[0009] Optionally, the biological agent includes yeast (Wickerhamomyces sp.), trehalose and / or aged tobacco powder.

[0010] Optionally, in parts by weight, the weight of the Bacillus is 15-20 parts, the weight of the yeast is 10-15 parts, the weight of the trehalose is 5-8 parts, and the weight of the aged tobacco powder is 65-70 parts.

[0011] Optionally, the weight portion of the bacillus is 20 parts, the weight portion of the yeast is 10 parts, the weight portion of the trehalose is 5 parts, and the weight portion of the aged tobacco powder is 65 parts;

[0012] Optionally, the viable count of the Bacillus is 3×10 8 -5×10 8 cfu / g;

[0013] Optionally, the viable count of the yeast is 2×10 8 -3×10 8 cfu / g;

[0014] Optionally, the trehalose is crystalline trehalose;

[0015] Optionally, the aged tobacco powder is prepared from flue-cured tobacco K326.

[0016] The trehalose in the above-mentioned biological preparation formula provided by the present invention has unique biological functions, can effectively maintain the stability and integrity of intracellular biofilms, proteins and active peptides in adverse conditions, and is praised as the sugar of life. It has the function of ensuring the activity of the strains and natural epiphytes of aged tobacco leaves in the formula. It can stabilize macromolecular substances and can play a certain role in correcting the flavor of aged tobacco leaves.

[0017] The aged tobacco powder (K326) introduced into the above-mentioned biological preparation formula provided by the present invention as a strain carrier can not only avoid the introduction of exogenous substances that interfere with the quality of aged tobacco leaves, but more importantly, can supplement the natural dominant bacteria on mature aged tobacco leaves during the aging process; in addition, it also facilitates the commercialization and application operation of the bacterial agent in the formula.

[0018] In another aspect, the present invention provides a method for preparing a biological agent, the method comprising: culturing the Bacillus to obtain a fermentation liquid 1, centrifuging the fermentation liquid 1, and collecting the bacterial cells 1.

[0019] The method further comprises: culturing the yeast to obtain a fermentation liquid 2, centrifuging the fermentation liquid 2 to collect bacterial cells 2; grinding and sieving the cured tobacco leaves to collect aged tobacco powder; and mixing the bacterial cells 1, the bacterial cells 2, the trehalose and the aged tobacco powder to obtain the biological preparation.

[0020] Optionally, the Bacillus is cultured in LB liquid culture medium at a temperature of 36°C-38°C and a rotation speed of 180-200 r / min on a shaking table; the OD of the fermentation liquid 1 is 600 When the value is 2-4, centrifugal separation is performed for 2-5 minutes at room temperature and a rotation speed of 4000-6000r / min.

[0021] Optionally, the temperature of the shaking culture is 37° C., and the rotation speed is 180 r / min; when the OD value of the fermentation liquid 1 is 3, centrifugation is performed at room temperature and a rotation speed of 4000 r / min for 5 minutes.

[0022] Optionally, the yeast is cultured in a PDA liquid medium under a shaking table at a temperature of 29°C-31°C and a rotation speed of 180-200 r / min; the OD of the fermentation liquid 2 is 600 When the value is 2-4, centrifugal separation is performed for 2-3 minutes at room temperature and a rotation speed of 4000-6000r / min.

[0023] Optionally, the yeast is cultured in a shaking incubator at a temperature of 30° C. and a rotation speed of 180 r / min; when the OD value of the fermentation liquid 2 is 3, it is centrifuged at room temperature and a rotation speed of 4000 r / min for 3 minutes.

[0024] In another aspect, the present invention provides the use of the above-mentioned strain or the above-mentioned biological preparation or the biological preparation prepared by the above-mentioned method in tobacco leaf alcoholization.

[0025] In another aspect, the present invention provides a tobacco leaf alcoholization method, the alcoholization method comprising: mixing the above biological agent or the biological agent prepared by the above method with water to prepare a bacterial suspension;

[0026] The bacterial suspension is sprayed on the surface of the tobacco leaves to be alcoholized to obtain pretreated tobacco leaves;

[0027] The pretreated tobacco leaves are subjected to anaerobic fermentation in a closed oxygen-free atmosphere to obtain fermented tobacco leaves 1;

[0028] The fermented tobacco leaves 1 are aerobically fermented under closed aerobic conditions to obtain fermented tobacco leaves 2;

[0029] The fermented tobacco leaves 2 are baked and balanced to obtain alcoholized tobacco leaves.

[0030] Optionally, the oxygen-free atmosphere is a nitrogen atmosphere.

[0031] Optionally, the water is sterile water; the bacterial suspension is prepared by mixing 1 part by weight of the biological agent with 95-105 parts by volume of the sterile water, and the ratio of parts by weight to parts by volume is g / mL or kg / L; and / or

[0032] 4 parts by volume of the bacterial suspension are sprayed on the surface of 45-55 parts by weight of the tobacco leaves to be aged, wherein the ratio of the parts by weight to the parts by volume is g / mL or kg / L; and / or

[0033] The anaerobic fermentation is carried out at a temperature of 31°C-37°C and a relative humidity of 75%-85% for 44-52 hours; the aerobic fermentation is carried out in natural air at a temperature of 31°C-37°C and a relative humidity of 75%-85% for 20-28 hours; and / or

[0034] The baking temperature is 100° C.-110° C., and the baking process is terminated when the moisture content of the fermented tobacco leaves 2 is controlled at 10%-14%; and / or

[0035] The balance means that the cured tobacco leaves are balanced for 20-28 hours under the conditions of a temperature of 20° C.-30° C. and a relative humidity of 55%-65%.

[0036] Optionally, the anaerobic fermentation is carried out at a temperature of 32° C. and a relative humidity of 80% for 48 hours; the aerobic fermentation is carried out at a temperature of 32° C. and a relative humidity of 80% for 24 hours;

[0037] The baking temperature is 105° C.; the baking process is terminated when the moisture content of the fermented tobacco leaves 2 is 12%;

[0038] The equilibrium temperature is 26° C., the relative humidity is 60%, and the equilibrium time is 24 hours.

[0039] Equilibrium: refers to the process in which tobacco is left to stand under certain conditions but chemical changes continue to occur inside the tobacco. It is a professional term in the tobacco industry.

[0040] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0041] The bacillus (Bacillus sp.GYCH1) provided by the invention is a superior strain separated and screened from alcoholized tobacco leaves, and the bacillus and yeast (Wickerhamomyces sp.) have high amylase and protease production activities respectively. When used in combination in the alcoholization process of tobacco leaves, in addition to the tobacco leaves themselves promoting the degradation and transformation of substances, the bacillus can also produce active substances such as nystatin and gramicidin during the growth process, and these active substances have an inhibitory effect on molds and pathogenic bacteria in the tobacco leaf fermentation process, and have a promoting effect on other tobacco leaf epiphytic bacteria; the two strains of bacteria both have facultative anaerobic characteristics, especially the aerobic respiration carried out by the yeast under aerobic conditions and the anaerobic respiration carried out under anaerobic conditions, and the degradation and transformation types of macromolecular organic substances and the types of products formed under aerobic and anaerobic conditions are all different, and the two strains of bacteria used in combination on tobacco leaves can achieve a more comprehensive and balanced alcoholization effect.

[0042] The formula and aging process of the biological microbial agent of the present invention are easy to understand, the tobacco leaf aging effect is excellent, and it has enlightenment and application value for scientific research and cigarette production.

[0043] Through continuous exploration and optimization, the present invention has found an effective tobacco aging formula and its aerobic and anaerobic treatment processes, enriching the diversity of tobacco aging methods and approaches. The improvements in the above two aspects improve the tobacco aging efficiency, aging quality and comprehensive tobacco aging effect, and also improve the production efficiency of cigarettes and the flexibility of production arrangements, and are highly feasible in practice.

[0044] Bacillus preservation certification information provided by the present invention:

[0045] Taxonomic name: Bacillus sp. GYCH1; Deposit number: CCTCC M20221805; Deposit date November 15, 2022; Deposit unit: China Center for Type Culture Collection (CCTCC); Deposit address: Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0047] Figure 1 It is a curve diagram of enzyme production activity of strain GYCH1 and strain GYCH2 provided in the embodiment of the present invention;

[0048] Figure 2A This is a colony characteristic diagram of the strain GYCH1 provided in an embodiment of the present invention;

[0049] Figure 2B This is a spore characteristic diagram of strain GYCH1 provided in an embodiment of the present invention;

[0050] Figure 2C This is an electron microscope image of the ultrastructural features of the strain GYCH1 provided in the embodiment of the present invention;

[0051] Figure 3 It is a schematic diagram of the phylogenetic tree of the 16S rRNA sequence of the strain GYCH1 provided in the embodiment of the present invention;

[0052] Figure 4 It is a schematic diagram of the phylogenetic tree of the gyrB sequence of the strain GYCH1 provided in the embodiment of the present invention. DETAILED DESCRIPTION

[0053] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0054] The yeast (Wickerhamomyces sp.) in the following examples was purchased from Zhengzhou Qiluo Biotechnology Co., Ltd., and the yeast is referred to as GYCH2 in the present invention.

[0055] Example 1 (strain GYCH1)

[0056] 1. Isolation of strain GYCH1

[0057] 1.1 Culture medium

[0058] Separation medium: 300mL tobacco leaf water extract (20g tobacco leaf sample is added to 400mL distilled water and boiled for 30min, and then filtered through two layers of gauze after cooling to obtain tobacco leaf extract), 700mL distilled water, 0.3g beef extract, 1.0g peptone, 0.5g sodium chloride, 1.5g agar, natural pH, sterilized at 120℃ high pressure steam for 30min. Separation method: Take 5g of aged tobacco leaf crushed sample (passed through 40 mesh sieve) and put it into a sterilized triangular flask, add 100mL sterile water, shake for 5min, let it stand for 5min, take 100μL supernatant and apply it on a solid separation medium plate, and after constant temperature culture at 37℃ for 48h, pick the colony and streak it for purification, and store the purified strain under 15% glycerol and minus 20℃ for standby.

[0059] 1.2 Acquisition of strain GYCH1

[0060] Through isolation and cultivation in separation medium, a strain with typical characteristics and vigorous growth was purified by streaking and numbered GYCH1.

[0061] 2. Determination of enzyme activity of strain GYCH1 (Bacillus) and strain GYCH2 (yeast)

[0062] 2.1 Culture medium and assay method

[0063] (Bacillus) amylase production fermentation medium: 3g soluble starch, 10g yeast powder, 0.5g potassium dihydrogen phosphate, 0.5g dipotassium hydrogen phosphate, 0.01g ferrous sulfate, 0.5g magnesium sulfate, 1000ml distilled water, pH7.0.

[0064] (Yeast) protease fermentation medium: 3g beef extract, 5g peptone, 5g NaCl, 15g skimmed milk powder, 1000mL distilled water, pH 7.0.

[0065] Determination method: strains GYCH1 and GYCH2 were inoculated into 50 mL of different enzyme production culture medium in a conical flask, respectively, and cultured at 35°C and 160 r / min in a shaking incubator for 16 h. Then 6 mL was transferred to a 1000 mL conical flask containing 75 mL of enzyme production culture medium and cultured in a shaking incubator for 48 h under the same conditions. Samples were taken every 4 h during the fermentation. The fermentation supernatant was the crude enzyme solution, that is, the crude enzyme solution of Bacillus and the crude enzyme solution of yeast were obtained respectively. The dinitrosalicylic acid colorimetric (DNS) method was used to determine the amylase activity of the crude enzyme solution of Bacillus; the Folin-phenol method was used to determine the protease activity of yeast (Jiang Yongmei. Microbial Breeding Experiment, Science Press, 2012), and the inoculated sterile water (CK) was used as the control.

[0066] 2.2 Enzyme activity of GYCH1 and GYCH2

[0067] Depend on Figure 1 It can be seen that in the fermentation enzyme production process of GYCH1 strain and GYCH2 strain, the amylase activity of Bacillus GYCH1 strain reached a maximum of 4.8U / ml when the fermentation was 30h, and it increased exponentially from 6 to 12h, and then slowly decreased after 30h; the protease activity of yeast GYCH2 strain reached a maximum of 2.8U / mL when the fermentation was 36h, and it gradually increased in the early stage of fermentation, and then slowly decreased after 36h; in comparison, the peak value of enzyme production activity of the two strains was similar, and the overall fermentation enzyme production characteristics were relatively consistent, which was conducive to the alcoholization of tobacco leaves and the coordination of control conditions.

[0068] 2.3 Polyphasic identification of strain GYCH1

[0069] Identification method: The purified strain GYCH1 was inoculated into LB medium and cultured at 37°C for 2 days. After typical colonies were formed, morphological characteristics were observed and physiological and biochemical tests were performed.

[0070] Molecular identification: The genomic DNA of the strain was extracted using TaKaRa MiniBEST Bacteria Genomic DNA ExtractionKitVer.3.0;

[0071] PCR primers for 16S rRNA:

[0072] 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, (SEQ ID NO.1)

[0073] 1492R: 5′-GGTTACCTTGTTACGACTT-3′, (SEQ ID NO. 2)

[0074] gyrB gene PCR amplification primers:

[0075] F: 5′-GAGGTCGTCATGACCCAGCA-3′, (SEQ ID NO.3)

[0076] R: 5′-GTCTTGGTCTGGCCCTCGAACTG-3′, (SEQ ID NO.4)

[0077] The PCR amplification systems are:

[0078] Ddw (ultra light water) 14.25 μl 10×PCR Buffer 2.0 μl dNTP mixture 0.5 μl Taq polymerase 0.25 μl Primer 1 (forward primer) 1.0 μl Primer 2 (reverse primer) 1.0 μl

[0079] PCR reaction program: 95°C pre-denaturation for 5 min, cycle parameters: 95°C denaturation for 1 min, 52°C annealing for 45 s, 72°C extension for 1.5 min, 35 cycles, 72°C extension for 10 min at the end of the cycle, and finally incubation at 4°C. PCR products were detected by 0.8% agarose gel electrophoresis, and products with clear bands were selected for purification.

[0080] The PGR amplification products were detected by electrophoresis and sequenced to obtain the gene sequence (sequencing by Beijing Aoke Dingsheng Biotechnology Co., Ltd.), and an evolutionary tree was constructed after homology comparison.

[0081] Identification results:

[0082] (1) General morphological characteristics

[0083] The colony is wrinkled, slightly convex, opaque, earthy yellow, without capsule, and with flagella; Gram-positive, the size of the spore is (0.4-0.7) μm × (1-1.5) μm, located in the center of the bacteria or slightly off; the size of the bacteria is (0.4-0.8) μm × (1.0-2.5) μm, as shown in the electron microscope picture Figure 2A-2C shown.

[0084] (2) Physiological and biochemical characteristics

[0085] The results of physiological and biochemical characteristics of strain GYCH1 are shown in Table 1.

[0086] Table 1. Physiological and biochemical analysis of strain GYCH1

[0087] Measurement items GYCH1 Measurement items GYCH1 Gelatin hydrolysis test + glucose + VP determination + Xylose + Methyl red + lactose + Nitrate reduction - maltose - Catalase + fructose + Citric acid utilization + Indole test - Sportiness + Protease + Inositol - Lipase + Sorbitol - Amylase - Mannitol + Indole + sucrose + Hydrogen sulfide production -

[0088] Note: “+” means positive, “-” means negative.

[0089] (3) Molecular identification of strain GYCH1

[0090] Using the genomic DNA of strain GYCH1 as a template, the sizes of 16S rRNA and gyrB amplified by PGR were in line with expectations after electrophoresis detection. The gene sequences of 16S rRNA (1428 bp) and gyrB (969 bp) were obtained after sequencing of the PGR product. The phylogenetic tree was constructed using MEGA 6.0. The results showed that the 16S rRNA sequence of strain GYCH1 was clustered in the same branch with Bacillus altitudinis (NR042337.1 Bacillus altitudinis 41KF2b), with a similarity of 96%. Figure 3 As shown in the figure, the gyrB sequence of strain GYCH1 is clustered in the same branch with Bacillus altitudinis JSCX-1 (KU955327.1), and the similarity is only 93%. Figure 4As shown; the sequence clustering analysis results of 16S rRNA and gyrB of strain GYCH1 did not meet the general requirement of more than 98% similarity to identify the species. Combined with the morphological and physiological and biochemical characteristics, the strain was identified as a Bacillus strain, tentatively named Bacillus sp. GYCH1, and the strain was preserved with a taxonomic name of Bacillus sp. GYCH1, with a preservation number of CCTCC M 20221805; the preservation date is November 15, 2022; the preservation unit is China Center for Type Culture Collection (CCTCC); the preservation address is Wuhan University, Wuhan, China.

[0091] 16S rRNA sequence of strain GYCH1 (SEQ ID NO.5):

[0092] ATACATGCAAGTCGAGCGGACAGAAGGGAGCTTGCTCCCGGATGTTAGCGGCGGACGGGTGAGTA

[0093] ACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGAGCTAATACCGGATAGTTCC

[0094] TTGAACCGCATGGTTCAAGGATGAAAGACGGTTTCGGCTGTCACTTACAGATGGACCCGCGGCGCATT

[0095] AGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCA

[0096] CACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGA

[0097] AAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAA

[0098] GAACAAGTGCAAGAGTAACTGCTTGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTG

[0099] CCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGG

[0100] CGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAAACTT

[0101] GAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTTGAGGAACACCA

[0102] GTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATT

[0103] AGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTCTTAGGGGGTTTCCGCCCCTTAGTGC

[0104] TGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGAC

[0105] GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTT

[0106] GACATCCTCTGACAACCCTAGAGATAGGGCTTTCCCTTCGGGGACAGAGTGACAGGTGGTGCATGGTT

[0107] GTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGC

[0108] CAGCATTCAGTTCGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAA

[0109] ATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCTGCGAGACC

[0110] GCAAGGTTTAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAA

[0111] GCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCG

[0112] CCCGTCACACCACGAGAGTTTGCAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCCGAAGG。

[0113] gyrB sequence of strain GYCH1 (SEQ ID NO.6):

[0114] AACGCGGTGTTCCAGTTGGAGATTTAGAGATCATTGGTGAAACAGATGTAACAGGGACAACCACT

[0115] CATTTTGTGCCAGATCCAGAAATTTTCACTGAAACCATTGAATTTGATTACGACACACTTGCTAACCG

[0116] TGTACGTGAGTTAGCTTTCTTAACAAAAGGCGTCAACATCATCATTGAAGACTTACGTGAAGGCAAAG

[0117] AGCGAAGAAACGAATACTGCTATGAAGGCGGTATTAAGAGCTATGTAGAACATGTGAATCGCTCGAAG

[0118] GAAGTCGTTCATGAAGAACCAGTTTACATCGAGGGTGAAAAAGACGGAATCACCGTTGAAGTTGCACT

[0119] GCAATACAACGATTCCTATACAAGCAATATTTATTCTTTCGCCAATAACATCAACACATATGAAGGCG

[0120] GATCACACGAAGCTGGCTTTAAAACCGGTCTGACGCGTGTCATCAATGATTATGCTCGTAAAAAATGGC

[0121] GTATTCAAAGATGGAGACTCGAATTTGAGCGGTGAAGATGTACGAGAAGGCATAACAGCCATTATCTC

[0122] TATCAAACATCCAGACCCTCAATTCGAAGGACAAACGAAGACAAAGCTCGGTAACTCAGAAGCAAGAA

[0123] CCATTACCGACTCCCTCTTCTCCGAAGCACTTGAGAAATTCCCTCTTAGAGAACCCTGATGCTGCAAAG

[0124] AAAATTGTGGAGAAAGGTGTGATGGCAGCTCGTGCAAGAATGGCTGCCAAAAAGGCACGTGAGCTGAC

[0125] AAGACGTAAAAGTGCACTGGAAGTCTCTAGCTTGCCTGGGAAACTGGCAGACTGTTCTTCTAAAGATC

[0126] CTTCCATCTCTGAGCTTTATATCGTAGAGGGAGATTCTGCGGGCGGATCTGCTAAGCAAGGTCGTGAT

[0127] CGAATTTCCAAGCGATCTTACCGTTAAGAGGGAAGATCCTAAACGTTGAAAAAGCCCGACTAGATAAAATTTTATCTAACAACGAGG.

[0128] The following Examples 2 to 4 use the strain obtained in Example 1 to prepare biological preparations.

[0129] Example 2 (Biological Agent 1)

[0130] A. The Bacillus sp. GYCH1 obtained in Example 1 was cultured in LB liquid medium at 37°C and 180 r / min shaking. The fermentation broth OD 600 When the optical density (measured at 600 nm) value was 3, centrifuge at 4000 r / min for 5 min at room temperature and collect 20 g of cells. The number of viable cells was 3×108 -5×10 8 cfu / g;

[0131] B. PDA liquid medium was used to culture yeast (Wickerhamomyces p. GYCH2) at 30°C and 180 r / min in a shaking incubator. The fermentation liquid OD 600 When the optical density (measured at 600 nm) value was 3, 10 g of cells were collected by centrifugation at 4000 r / min for 3 min at room temperature, and the number of viable cells was 2 × 10 8 -3×10 8 cfu / g;

[0132] C. Crystalline trehalose: purchased from the market (Linyuan trehalose), weigh 5 g;

[0133] D. Aged tobacco powder: K326 flue-cured tobacco leaves (provided by Guangxi Zhongyan, commercially available) were selected, fully ground by a pulverizer, passed through a 200-mesh standard sieve, and 65 g of fine powder was collected;

[0134] The prepared components A, B, C and D are mixed to obtain biological preparation 1.

[0135] Example 3 (Biological Agent 2)

[0136] The difference between Example 3 and Example 2 is that 15 g of Bacillus cells, 15 g of yeast cells, 8 g of crystalline trehalose, and 70 g of aged tobacco powder are mixed to obtain biological agent 2.

[0137] Example 4 (Biological Agent 3)

[0138] The difference between Example 4 and Example 2 is that 18 g of Bacillus cells, 13 g of yeast cells, 6 g of crystalline trehalose, and 68 g of aged tobacco powder are mixed to obtain biological agent 3.

[0139] Example 5 utilizes the biological agent 1 prepared in the above Example 2 to age tobacco leaves.

[0140] Example 5 (Tobacco Leaf Aging)

[0141] 1 g of tobacco alcoholization bacterial agent (i.e., biological agent 1 prepared in Example 2) was weighed, added to 99 mL of sterile water and fully mixed to prepare a 1% bacterial suspension; 4 mL of the bacterial suspension was evenly sprayed on 50 g of tobacco shreds (K326, B2F, provided by Guangxi Zhongyan, commercially available), and sterile gloves were worn to stir and ensure uniform spraying, and sterile water treatment was set as a control; the treated tobacco shreds were transferred to an anaerobic incubator, and nitrogen (equivalent to atmospheric pressure) was injected to replace the air in the container, and fermented for 48 hours at a temperature of 32° C. and a relative humidity of 80%; then the nitrogen was released, natural air was put into the air, and fermentation continued for 24 hours at 32° C. and a relative humidity of 80% under aerobic conditions; after the fermentation was completed, the tobacco shreds were baked at 105° C., the tobacco shreds were stirred every 1 min, and the baking was terminated when the moisture mass percentage of the tobacco shreds was 12%, and finally the tobacco shreds were balanced at 26° C. and a relative humidity of 60% for 24 hours to fully release and volatilize the impurities, and the entire fermentation process was terminated to obtain alcoholized tobacco shreds 1.

[0142] Example 6 (Tobacco Leaf Aging)

[0143] The difference between Example 6 and Example 5 is that the anaerobic fermentation is carried out at a temperature of 37°C and a relative humidity of 85% for 50 hours; the aerobic fermentation is carried out at a temperature of 35°C and a relative humidity of 85% for 28 hours; after the fermentation is completed, the tobacco is baked at 110°C, the tobacco is turned and stirred every 1 minute, and the baking is terminated when the moisture mass percentage of the tobacco is 10%. Finally, the tobacco is balanced at 20°C and a relative humidity of 55% for 28 hours to fully release and volatilize the impurities, and the entire fermentation process is terminated to obtain alcoholized tobacco 2.

[0144] Example 7 (Tobacco Leaf Aging)

[0145] The difference between Example 7 and Example 5 is that anaerobic fermentation is performed at a temperature of 35°C and a relative humidity of 75% for 44 hours; aerobic fermentation is continued at a temperature of 27°C and a relative humidity of 75% for 20 hours; after the fermentation is completed, the tobacco is baked at 100°C, the tobacco is turned and stirred every 1 minute, and the baking is terminated when the moisture mass percentage of the tobacco is measured to be 14%. Finally, the tobacco is balanced at 30°C and a relative humidity of 65% for 20 hours to allow the impurities to be fully released and volatilized, and the entire fermentation process is terminated to obtain alcoholized tobacco 3.

[0146] Comparative Example 1 (Tobacco Leaf Aging)

[0147] The difference between Comparative Example 1 and Example 5 is that in Comparative Example 1, an equal volume of sterile water is used to replace the 1% bacterial suspension to alcoholize the shredded tobacco, and other conditions remain unchanged, thereby obtaining alcoholized shredded tobacco 4.

[0148] The alcoholization quality effects of alcoholized tobacco 1 of Example 5, alcoholized tobacco 2 of Example 6, alcoholized tobacco 3 of Example 7 and alcoholized tobacco 4 of Comparative Example 1 were verified:

[0149] The test process was repeated three times and the average value was taken; after the aging was completed, the quality of the aging tobacco was measured and evaluated.

[0150] Quality index determination method: The total alkaloids were determined according to the standard YC / T 160-2002; the chlorine, total sugar, reducing sugar, nicotine and potassium contents in tobacco leaves were determined according to the standard YC / T217-2002. The determination results are shown in Table 2.

[0151] Smoking evaluation method: According to the single-material tobacco smoking evaluation standard, a corresponding smoking evaluation table (scoring on a 10-point scale) is set. The main smoking evaluation indicators are: aroma volume (A), richness (B), pleasantness (C), permeability (D), fineness (E), continuity (F), clumping (G), softness (H), concentration (I), sweetness (J), miscellaneous smell (K), stimulation (L) and aftertaste (M). The smoking evaluation results are shown in Table 3. The larger the number in Table 3, the better the flavor of a certain flavor in the tobacco leaf, and the smaller the number, the worse the flavor. The full score is 10 points.

[0152] Table 2. Chemical composition analysis data of alcoholized tobacco leaves

[0153] Variety and grade deal with Nicotine / % Total sugar / % Reducing sugar / % Potassium / % chlorine / % K326, B2F Alcoholized tobacco 4 (sterile water) 4.00 22.19 19.87 1.16 0.73 K326, B2F Alcoholized tobacco 1 (biological agent 1) 3.82 25.36 22.59 1.47 0.61 K326, B2F Alcoholized tobacco 2 (biological agent 1) 3.85 25.12 22.05 1.42 0.65 K326, B2F Alcoholized tobacco 3 (biological agent 1) 3.86 25.05 22.08 1.40 0.64

[0154] Table 3. Record of results of alcoholized tobacco leaf smoking test

[0155]

[0156] It can be seen from the measurement data in Table 2 that compared with the effect of alcoholizing tobacco shreds with clear water, after alcoholizing tobacco leaves using the biological preparation 1 of the present invention and aerobic-anaerobic process combined with the biological preparation 1 of the present invention, the nicotine and chlorine harmful substance contents of the tobacco shreds after alcoholization are significantly reduced; the contents of total sugar, reducing sugar and potassium beneficial substances are significantly increased, indicating that the alcoholization effect of the tobacco shreds is excellent.

[0157] From the results of the smoking evaluation in Table 3, it can be seen that compared with the effect of alcoholized tobacco shreds with clear water, after alcoholization of tobacco leaves by the combination of the biological preparation 1 of the present invention and its aerobic-anaerobic process, the alcoholized tobacco shreds have obvious improvements in flavors and flavors such as aroma, richness, pleasantness, permeability, fineness, continuity, clumping, softness, concentration, sweetness, miscellaneous smell, stimulation and aftertaste, indicating that the alcoholization effect of the alcoholized tobacco shreds is excellent. Combining the chemical analysis in Table 2 and the flavor evaluation results in Table 3, it can be seen that the tobacco shreds alcoholized by the biological preparation of the present invention can be evaluated as high-quality tobacco shreds.

[0158] The Bacillus, yeast, crystalline trehalose and aged tobacco powder selected in the biological preparation formula of the present invention can promote the aging of tobacco leaves and balance the aging effect by using the two strains in combination; the aged tobacco powder is used as a carrier to avoid the introduction of exogenous substances to interfere with the quality of aging tobacco leaves, and can also supplement the natural dominant bacteria of mature aged tobacco leaves during the aging process; the above-mentioned innovative biological preparation formula of the present invention can improve and optimize the aging quality and comprehensive effect of tobacco leaves.

[0159] The present invention innovatively adopts an aerobic-anaerobic process to carry out an alcoholization process on tobacco leaves, which can achieve a higher alcoholization quality while controlling the entire alcoholization process time, thereby improving the alcoholization effect and alcoholization efficiency.

[0160] The present invention adopts a preferred biological preparation formula and an optimized alcohol fermentation process, and further improves the tobacco alcoholization quality effect and alcoholization efficiency through improvements in two aspects, and can be promoted for use.

[0161] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A Bacillus sp. GYCH1, with the deposit number being CCTCC M 20221805.

2. A biological agent, characterized in that: The biological agent comprises the Bacillus according to claim 1.

3. A biological agent according to claim 2, characterized in that: The biological agent comprises yeast (Wickerhamomyces sp.), trehalose and / or aged tobacco powder.

4. A biological agent according to claim 3, characterized in that: In parts by weight, the composition comprises: 15-20 parts by weight of the bacillus, 10-15 parts by weight of the yeast, 5-8 parts by weight of the trehalose, and 65-70 parts by weight of the aged tobacco powder.

5. A biological agent according to claim 4, characterized in that: The weight proportion of the bacillus is 20 parts, the weight proportion of the yeast is 10 parts, the weight proportion of the trehalose is 5 parts, and the weight proportion of the aged tobacco powder is 65 parts.

6. A biological agent according to claim 5, characterized in that: The viable count of the bacillus is 3×10 8 -5×10 8 cfu / g; And / or, the viable count of the yeast is 2×10 8 -3×10 8 cfu / g; and / or, the trehalose is crystalline trehalose; And / or, the aged tobacco powder is prepared from flue-cured tobacco K326.

7. The method for preparing a biological agent according to any one of claims 3 to 6, characterized in that: The method comprises: The bacillus is cultured to obtain a fermentation liquid 1, and the fermentation liquid 1 is centrifuged to collect the bacterial cells 1.

8. The method for preparing a biological agent according to claim 7, characterized in that: The method further comprises: The yeast is cultured to obtain a fermentation liquid 2, and the fermentation liquid 2 is centrifuged to collect bacterial cells 2; Grinding and sieving the cured tobacco leaves to collect aged tobacco powder; The bacterial cell 1, the bacterial cell 2, the trehalose and the aged tobacco powder are mixed to obtain the biological preparation.

9. The method for preparing a biological agent according to claim 8, characterized in that: The bacillus was cultured in LB liquid culture medium at a temperature of 36°C-38°C and a rotation speed of 180-200 r / min on a shaking table; the OD of the fermentation liquid 1 was 600 When the value is 2-4, centrifugal separation is performed at room temperature and a speed of 4000-6000r / min for 2-5min; And / or, the yeast is cultured in a PDA liquid medium at a temperature of 29°C-31°C and a rotation speed of 180-200 r / min on a shaking table; the OD of the fermentation broth 2 is 600 When the value is 2-4, centrifugal separation is performed for 2-3 minutes at room temperature and a rotation speed of 4000-6000r / min.

10. Use of the biological preparation prepared by the method for preparing a bacillus according to claim 1, a biological preparation according to any one of claims 2 to 6, or a biological preparation according to any one of claims 7 to 9 in tobacco alcoholization.

11. A tobacco leaf aging method, characterized in that: The alcoholization method comprises: A biological agent according to any one of claims 2 to 6 or a biological agent prepared by the method for preparing a biological agent according to any one of claims 7 to 9 is mixed with water to prepare a bacterial suspension; The bacterial suspension is sprayed on the surface of the tobacco leaves to be alcoholized to obtain pretreated tobacco leaves; The pretreated tobacco leaves are subjected to anaerobic fermentation in a closed oxygen-free atmosphere to obtain fermented tobacco leaves 1; The fermented tobacco leaves 1 are aerobically fermented under closed aerobic conditions to obtain fermented tobacco leaves 2; The fermented tobacco leaves 2 are baked and balanced to obtain alcoholized tobacco leaves.

12. A tobacco leaf aging method according to claim 11, characterized in that: The water is sterile water; the bacterial suspension is prepared by mixing 1 part by weight of the biological agent with 95-105 parts by volume of the sterile water, and the ratio of parts by weight to parts by volume is g / mL or kg / L; and / or 4 parts by volume of the bacterial suspension are sprayed on the surface of 45-55 parts by weight of the tobacco leaves to be aged, and the ratio of the parts by weight to the parts by volume is g / mL or kg / L; the anaerobic fermentation is carried out at a temperature of 31°C-37°C and a relative humidity of 75%-85% for 44-52 hours; the aerobic fermentation is carried out in natural air at a temperature of 31°C-37°C and a relative humidity of 75%-85% for 20-28 hours; and / or The baking temperature is 100° C.-110° C., and the baking process is terminated when the moisture content of the fermented tobacco leaves 2 is controlled at 10%-14%; and / or The balance means that the cured tobacco leaves are balanced for 20-28 hours under the conditions of a temperature of 20° C.-30° C. and a relative humidity of 55%-65%.

Images