Mixed bacterial liquid for improving cigar tobacco leaf quality and application thereof and fermentation method for improving cigar tobacco leaf quality

Abstract

The present invention relates to the field of tobacco technology, and in particular to a mixed bacterial solution for improving the quality of cigar tobacco leaves, an application thereof, and a fermentation method for improving the quality of cigar tobacco leaves. The present invention provides a mixed bacterial solution for improving the quality of cigar tobacco leaves, the mixed bacterial solution comprising two or more of a bacterial solution of Bacillus amyloliquefaciens GL0525, a bacterial solution of Bacillus amyloliquefaciens SS0813, and a bacterial solution of Bacillus velez MZ1030. The present invention also provides a fermentation method for improving the quality of cigar tobacco leaves, which can effectively enhance the flavor and quality of cigar tobacco leaves, eliminate the impurities of cigar tobacco leaves, and improve and enhance the smoking taste of cigar tobacco leaves.

Description

Technical Field

[0001] The invention relates to the technical field of tobacco, and in particular to a mixed bacterial liquid for improving the quality of cigar tobacco leaves, an application thereof, and a fermentation method for improving the quality of cigar tobacco leaves. Background Art

[0002] Domestic cigar tobacco fermentation is a key step in the cigar production process, which is of great significance to improving the quality and flavor of cigars. In China, with the gradual popularization of cigar culture and the development of the domestic cigar industry, the fermentation technology of cigar tobacco is also constantly improving. However, this process still faces a series of technical and industrial challenges.

[0003] The fermentation of cigar tobacco is a very complex biochemical process that needs to be carried out under controlled conditions for a long time. To ensure that each batch of tobacco can achieve the ideal fermentation effect, the control of the fermentation process is extremely high. Slight changes in factors such as temperature, humidity, and time may have a significant impact on the quality of the final tobacco. Despite technological advances, it is still a challenge for domestic cigar tobacco to maintain quality consistency and achieve standardized production during the fermentation process. There may be quality fluctuations between different batches of tobacco and tobacco from different production bases, which poses an obstacle to improving the competitiveness of domestic cigars in the international market.

[0004] In order to further improve the fermentation efficiency and tobacco leaf quality, continuous technological innovation and R&D work is needed. This includes in-depth research on microbial activities during the fermentation process, optimization of fermentation conditions, and improvement of tobacco leaf quality evaluation methods after fermentation. The mid-to-high-end handmade cigars on the market are usually "purely natural" and do not contain flavors and spices, so it is difficult to improve product quality through normal flavoring methods in the tobacco industry.

[0005] Based on this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a mixed bacterial liquid for improving the quality of cigar tobacco leaves, its application and a fermentation method for improving the quality of cigar tobacco leaves. The fermentation method can effectively enhance the flavor and quality of cigar tobacco leaves, eliminate the impurities of cigar tobacco leaves, and improve and enhance the smoking taste of cigar tobacco leaves.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The invention provides a mixed bacterial liquid for improving the quality of cigar tobacco leaves. The mixed bacterial liquid comprises two or more of a bacterial liquid of Bacillus amyloliquefaciens GL0525, a bacterial liquid of Bacillus amyloliquefaciens SS0813 and a bacterial liquid of Bacillus velez MZ1030.

[0009] Preferably, when the mixed bacterial solution includes Bacillus argyi GL0525 bacterial solution and Bacillus amyloliquefaciens SS0813 bacterial solution, the volume ratio of the Bacillus argyi GL0525 bacterial solution to the Bacillus amyloliquefaciens SS0813 bacterial solution is 0.5-1.5:0.5-1.5;

[0010] The OD600 of the Bacillus argyi GL0525 bacterial solution is 0.5 to 1.5;

[0011] The OD600 of the Bacillus amyloliquefaciens SS0813 bacterial liquid is 0.5-1.5.

[0012] Preferably, when the mixed bacterial solution includes Bacillus argyi GL0525 bacterial solution and Bacillus velezii MZ1030 bacterial solution, the volume ratio of the Bacillus argyi GL0525 bacterial solution to the Bacillus velezii MZ1030 bacterial solution is 0.5-1.5:0.5-1.5;

[0013] The OD600 of the Bacillus argyi GL0525 bacterial solution is 0.5 to 1.5;

[0014] The OD600 of the Bacillus Velez subtilis MZ1030 bacterial solution is 0.5-1.5.

[0015] Preferably, when the mixed bacterial solution includes Bacillus amyloliquefaciens SS0813 bacterial solution and Bacillus velez MZ1030 bacterial solution, the volume ratio of the Bacillus amyloliquefaciens SS0813 bacterial solution to the Bacillus velez MZ1030 bacterial solution is 0.5-1.5:0.5-1.5;

[0016] The OD600 of the Bacillus amyloliquefaciens SS0813 bacterial solution is 0.5 to 1.5;

[0017] The OD600 of the Bacillus Velez subtilis MZ1030 bacterial solution is 0.5-1.5.

[0018] Preferably, when the mixed bacterial solution includes Bacillus arguingensis GL0525 bacterial solution, Bacillus amyloliquefaciens SS0813 bacterial solution and Bacillus velezii MZ1030 bacterial solution, the volume ratio of the Bacillus arguingensis GL0525 bacterial solution, the Bacillus amyloliquefaciens SS0813 bacterial solution and the Bacillus velezii MZ1030 bacterial solution is 0.5-1.5:0.5-1.5:0.5-1.5;

[0019] The OD600 of the Bacillus argyi GL0525 bacterial solution is 0.5 to 1.5;

[0020] The OD600 of the Bacillus amyloliquefaciens SS0813 bacterial solution is 0.5 to 1.5;

[0021] The OD600 of the Bacillus Velez subtilis MZ1030 bacterial solution is 0.5-1.5.

[0022] The invention provides application of the mixed bacterial liquid in improving the quality of cigar tobacco leaves.

[0023] The present invention provides a fermentation method for improving the quality of cigar tobacco leaves, comprising the following steps:

[0024] Mixing cigar tobacco leaves with the mixed bacterial solution to obtain a fermentation raw material; fermenting the fermentation raw material to obtain fermented cigar tobacco leaves;

[0025] The mixed bacterial liquid is the mixed bacterial liquid;

[0026] The fermentation is a variable temperature and humidity cyclic fermentation; fermentation is performed at a temperature of 32-38° C. and a humidity of 65-75% for 45-50 hours, and fermentation is performed at a temperature of 42-48° C. and a humidity of 80-90% for 45-50 hours as one cycle, and the cycle is repeated 8-10 times.

[0027] Preferably, the mass ratio of the cigar tobacco leaves to the mixed bacterial solution is 1:0.20-0.25.

[0028] The invention also provides application of the fermentation method in improving the quality of cigar tobacco leaves.

[0029] Beneficial effects of the present invention:

[0030] 1. Compared with other fermentation methods, the fermentation method provided by the present invention uses mixed bacterial liquids of microorganisms of different proportions during the fermentation process, so that the native microbial community in the cigar tobacco leaves changes, the dominant microbial flora is enriched in the cigar tobacco leaves, and a series of biochemical changes in the internal substances of the tobacco leaves are promoted, thereby promoting the improvement of the quality of the cigar tobacco leaves;

[0031] 2. The fermentation method of the present invention can effectively improve the smoking quality of tobacco leaves and reduce irritation and impurities;

[0032] 3. The variable temperature and humidity cyclic fermentation method of the present invention can better promote the activity of dominant microorganisms, improve the quality of tobacco leaves, make the combustibility of tobacco leaves more stable, and make the color more uniform, thereby achieving the purpose of improving the intrinsic quality of tobacco leaves and enhancing industrial availability. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The content of carotenoid degradation products in cigar tobacco leaves before and after mixed bacterial liquid fermentation;

[0034] Figure 2 The content of phenylalanine degradation products in cigar tobacco leaves before and after mixed bacterial fermentation;

[0035] Figure 3is the content of ceprecane degradation products in cigar tobacco leaves before and after mixed bacterial liquid fermentation;

[0036] Figure 4 The content of chlorophyll degradation products in cigar tobacco leaves before and after mixed bacterial liquid fermentation;

[0037] Figure 5 The content of volatile alkaloids in cigar tobacco leaves before and after mixed bacterial fermentation;

[0038] Figure 6 It is the content of other volatile flavor metabolites in cigar tobacco leaves before and after mixed bacterial liquid fermentation.

[0039] Biological Deposit Description

[0040] Bacillus amyloliquefaciens SS0813 is deposited in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. The deposit date is December 1, 2023, and the deposit number is: CCTCCNO: M20232418.

[0041] Bacillus velezensis MZ1030 is deposited in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. The deposit date is December 1, 2023, and the deposit number is: CCTCC NO: M20232416.

[0042] Priestia aryabhattai GL0525 is deposited in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. The deposit date is December 1, 2023, and the deposit number is: CCTCC NO: M20232417. DETAILED DESCRIPTION

[0043] The invention provides a mixed bacterial liquid for improving the quality of cigar tobacco leaves. The mixed bacterial liquid comprises two or more of a bacterial liquid of Bacillus amyloliquefaciens GL0525, a bacterial liquid of Bacillus amyloliquefaciens SS0813 and a bacterial liquid of Bacillus velez MZ1030.

[0044] In the present invention, the preparation method of the Bacillus arginii GL0525 bacterial liquid is as follows: inoculating and culturing the Bacillus arginii GL0525 to obtain a seed liquid; inoculating the seed liquid into an LB liquid culture medium at an inoculation rate of 1% to 5% (v / v), and culturing at 35 to 39° C. for 12 to 14 hours to obtain the Bacillus arginii GL0525 bacterial liquid;

[0045] The inoculation amount is preferably 3%; the culture temperature is preferably 37° C.; and the culture time is preferably 13 h.

[0046] In the present invention, the preparation method of the Bacillus amyloliquefaciens SS0813 bacterial liquid is as follows: inoculating and culturing the Bacillus amyloliquefaciens SS0813 to obtain a seed liquid; inoculating the seed liquid into an LB liquid culture medium at an inoculation rate of 1% to 5% (v / v), and culturing at 35 to 39° C. for 12 to 14 hours to obtain a Bacillus amyloliquefaciens SS0813 bacterial liquid;

[0047] The inoculation amount is preferably 3%; the culture temperature is preferably 37° C.; and the culture time is preferably 13 h.

[0048] In the present invention, the preparation method of the Bacillus Velez MZ1030 bacterial liquid is as follows: inoculating and culturing the Bacillus Velez MZ1030 to obtain a seed liquid; inoculating the seed liquid into an LB liquid culture medium at an inoculation amount of 1% to 5% (v / v), and culturing at 35 to 39° C. for 12 to 14 hours to obtain the Bacillus Velez MZ1030 bacterial liquid;

[0049] The inoculation amount is preferably 3%; the culture temperature is preferably 37° C.; and the culture time is preferably 13 h.

[0050] In the present invention, when the mixed bacterial solution includes Bacillus arfaecium GL0525 bacterial solution and Bacillus amyloliquefaciens SS0813 bacterial solution, the volume ratio of the Bacillus arfaecium GL0525 bacterial solution to the Bacillus amyloliquefaciens SS0813 bacterial solution is 0.5-1.5:0.5-1.5, preferably 1:1;

[0051] The OD600 of the Bacillus argyi GL0525 bacterial solution is 0.5-1.5, preferably 1.0;

[0052] The OD600 of the Bacillus amyloliquefaciens SS0813 bacterial solution is 0.5-1.5, preferably 1.0.

[0053] In the present invention, when the mixed bacterial solution includes Bacillus arginense GL0525 bacterial solution and Bacillus velezii MZ1030 bacterial solution, the volume ratio of the Bacillus arginense GL0525 bacterial solution to the Bacillus velezii MZ1030 bacterial solution is 0.5-1.5:0.5-1.5, preferably 1:1;

[0054] The OD600 of the Bacillus argyi GL0525 bacterial solution is 0.5-1.5, preferably 1.0;

[0055] The OD600 of the Bacillus Velez subtilis MZ1030 bacterial solution is 0.5-1.5, preferably 1.0.

[0056] In the present invention, when the mixed bacterial solution includes Bacillus amyloliquefaciens SS0813 bacterial solution and Bacillus velez MZ1030 bacterial solution, the volume ratio of the Bacillus amyloliquefaciens SS0813 bacterial solution to the Bacillus velez MZ1030 bacterial solution is 0.5-1.5:0.5-1.5, preferably 1:1;

[0057] The OD600 of the Bacillus amyloliquefaciens SS0813 bacterial solution is 0.5 to 1.5, preferably 1.0;

[0058] The OD600 of the Bacillus Velez subtilis MZ1030 bacterial solution is 0.5-1.5, preferably 1.0.

[0059] In the present invention, when the mixed bacterial solution includes Bacillus arguingensis GL0525 bacterial solution, Bacillus amyloliquefaciens SS0813 bacterial solution and Bacillus velez MZ1030 bacterial solution, the volume ratio of the Bacillus arguingensis GL0525 bacterial solution, the Bacillus amyloliquefaciens SS0813 bacterial solution and the Bacillus velez MZ1030 bacterial solution is 0.5-1.5:0.5-1.5:0.5-1.5, preferably 1:1:1;

[0060] The OD600 of the Bacillus argyi GL0525 bacterial solution is 0.5-1.5, preferably 1.0;

[0061] The OD600 of the Bacillus amyloliquefaciens SS0813 bacterial solution is 0.5 to 1.5, preferably 1.0;

[0062] The OD600 of the Bacillus Velez subtilis MZ1030 bacterial solution is 0.5-1.5, preferably 1.0.

[0063] In the present invention, Bacillus arguta is a type of acidothermic bacteria that can produce spores. In the fermentation process of cigar tobacco leaves, microbial activity is a key link, which affects the quality, flavor and combustion performance of tobacco leaves. Although Bacillus arguta is not one of the most important microorganisms in cigar tobacco leaf fermentation, under certain conditions, Bacillus arguta may occupy a dominant position in the tobacco leaf fermentation process, especially in a high temperature and acidic environment;

[0064] Bacillus amyloliquefaciens is a bacterium widely used in industry and agriculture. It can produce a large number of enzymes, such as amylase and protease, which are very useful in the food industry and other applications. During the fermentation of cigar tobacco leaves, Bacillus amyloliquefaciens can help decompose large molecular organic substances in tobacco leaves, such as protein and cellulose, and convert them into small molecular substances, thereby improving the aroma and taste of tobacco leaves. Bacillus amyloliquefaciens participates in this process through the enzymes it produces (such as proteases), which helps to improve the fermentation effect of tobacco leaves;

[0065] Bacillus Velez is a bacterium widely found in the natural environment. It can produce a large number of secondary metabolites, such as antibiotics and enzymes, which have potential application value in agriculture and food industry. Bacillus Velez can produce a variety of enzymes, such as proteases and amylases, which help to form aroma components and degrade undesirable components during tobacco aging. In addition, Bacillus Velez can also produce antibacterial substances, such as lipopeptide antibiotics, which can inhibit the growth of harmful microorganisms during tobacco fermentation and protect tobacco from microbial contamination.

[0066] The invention is based on the purely natural fermentation characteristics of handmade cigars. Bacillus amyloliquefaciens has the activity of producing protease, which can be used to enhance the neutral aroma components of cigar tobacco leaves. Bacillus amyloliquefaciens can produce a large amount of amylase, protease and the like, which can help decompose macromolecular organic substances in tobacco leaves, such as protein and cellulose, and convert them into small molecular substances, thereby enhancing the aroma and taste of tobacco leaves and reducing irritation and foreign smell. Bacillus velez can produce a variety of enzymes, which are helpful for the formation of aroma components and the degradation of undesirable components during the aging of tobacco leaves. In addition, Bacillus velez can also produce antibacterial substances, such as lipopeptide antibiotics, which can inhibit the growth of harmful microorganisms during the fermentation of tobacco leaves, protect tobacco leaves from microbial contamination, and achieve synergistic fermentation of microorganisms, thereby achieving the improvement of quality and aroma of cigar tobacco leaves.

[0067] The invention provides application of the mixed bacterial liquid in improving the quality of cigar tobacco leaves.

[0068] The present invention provides a fermentation method for improving the quality of cigar tobacco leaves, comprising the following steps:

[0069] Mixing cigar tobacco leaves with the mixed bacterial solution to obtain a fermentation raw material; fermenting the fermentation raw material to obtain fermented cigar tobacco leaves;

[0070] The mixed bacterial liquid is the mixed bacterial liquid;

[0071] The fermentation is a variable temperature and humidity cyclic fermentation; fermentation is performed at a temperature of 32-38° C. and a humidity of 65-75% for 45-50 hours, and fermentation is performed at a temperature of 42-48° C. and a humidity of 80-90% for 45-50 hours as one cycle, and the cycle is repeated 8-10 times.

[0072] The variable temperature and variable humidity cyclic fermentation is preferably performed at 35° C. and 70% humidity for 48 hours, and at 45° C. and 85% humidity for 48 hours as one cycle, and the cycle is repeated 9 times.

[0073] In the present invention, artificial variable temperature and humidity fermentation is a more complex and sophisticated cigar tobacco processing method. By accurately controlling the changes in temperature and humidity during the fermentation process, the chemical component conversion in the tobacco is further optimized, thereby improving the quality of the tobacco and enhancing its aroma. This method plays an important role in the production and quality improvement of cigar tobacco aroma substances. Variable temperature and humidity fermentation promotes the organic substances (such as sugars and proteins) in the tobacco to be more effectively converted into flavor compounds such as phenols, aldehydes, esters and other volatile compounds through microbial action and non-biological reactions. By controlling the temperature and humidity conditions during the fermentation process, the generation of specific aroma components in the tobacco can be directional to a certain extent, so that the cigar shows a richer and more complex flavor level. In addition, variable temperature and humidity fermentation helps to more effectively reduce harmful components in tobacco, such as nicotine and certain acidic substances, making the tobacco smoother and reducing the irritation during smoking. Appropriate humidity control has an important influence on the combustibility of tobacco leaves. Moderate humidity can ensure that the tobacco leaves burn more smoothly during smoking and enhance the smoking experience. Appropriate temperature and humidity conditions can improve the quality of tobacco leaves and promote the activities of beneficial microorganisms (such as Bacillus agni, Bacillus amyloliquefaciens and Bacillus velezii), while inhibiting the growth of microorganisms that may cause the quality of tobacco leaves to decline, and further improving the quality of tobacco leaves through the optimization of microbial communities. The variable temperature and humidity fermentation environment promotes the enhanced activity of microorganisms that produce beneficial enzymes, such as proteases and cellulases, which help decompose macromolecular substances in tobacco leaves and promote the formation of aroma substances. This fermentation treatment technology plays an important role in improving the quality of cigar tobacco leaves, optimizing their flavor characteristics and accelerating the maturation process.

[0074] In the present invention, the mass ratio of the cigar tobacco leaves to the mixed bacterial solution is 1:0.20-0.25, preferably 1:0.225.

[0075] In the present invention, the cigar tobacco leaves need to be rehumidified before being mixed with the mixed bacterial solution to make the moisture content reach 28% to 30%, and then the moisture is balanced under the conditions of temperature 35° C. and humidity 75%.

[0076] The invention also provides application of the fermentation method in improving the quality of cigar tobacco leaves.

[0077] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0078] Example 1 A mixed bacterial solution for improving the quality of cigar tobacco leaves

[0079] Bacillus arginii GL0525 and Bacillus amyloliquefaciens SS0813 were inoculated and cultured separately to obtain seed liquid; the seed liquid was inoculated into LB liquid culture medium at an inoculation amount of 3% (v / v), and cultured at 37°C for 13 hours to obtain Bacillus arginii GL0525 bacterial liquid and Bacillus amyloliquefaciens SS0813 bacterial liquid; the Bacillus arginii GL0525 bacterial liquid and the Bacillus amyloliquefaciens SS0813 bacterial liquid were diluted to OD600 of 1.0 respectively, and then mixed at a volume ratio of 1:1 to obtain a mixed bacterial liquid.

[0080] Example 2 A mixed bacterial solution for improving the quality of cigar tobacco leaves

[0081] Bacillus arginii GL0525 and Bacillus velezii MZ1030 are inoculated and cultured respectively to obtain seed liquid; the seed liquid is inoculated into LB liquid culture medium at an inoculation amount of 3% (v / v), and cultured at 37°C for 13 hours to obtain Bacillus arginii GL0525 bacterial liquid and Bacillus velezii MZ1030 bacterial liquid; the Bacillus arginii GL0525 bacterial liquid and the Bacillus velezii MZ1030 bacterial liquid are diluted to OD600 of 1.0 respectively, and then mixed at a volume ratio of 1:1 to obtain a mixed bacterial liquid.

[0082] Example 3 A mixed bacterial solution for improving the quality of cigar tobacco leaves

[0083] Bacillus amyloliquefaciens SS0813 and Bacillus velezii MZ1030 are inoculated and cultured respectively to obtain seed liquid; the seed liquid is inoculated into LB liquid culture medium at an inoculation amount of 3% (v / v), and cultured at 37°C for 13 hours to obtain Bacillus amyloliquefaciens SS0813 bacterial liquid and Bacillus velezii MZ1030 bacterial liquid; the Bacillus amyloliquefaciens SS0813 bacterial liquid and the Bacillus velezii MZ1030 bacterial liquid are diluted to OD600 of 1.0 respectively, and then mixed at a volume ratio of 1:1 to obtain a mixed bacterial liquid.

[0084] Example 4 A mixed bacterial solution for improving the quality of cigar tobacco leaves

[0085] Bacillus argais GL0525, Bacillus amyloliquefaciens SS0813 and Bacillus velez MZ1030 are inoculated and cultured respectively to obtain seed liquid; the seed liquid is inoculated into LB liquid culture medium at an inoculation amount of 3% (v / v), and cultured at 37°C for 13 hours to obtain Bacillus argais GL0525 bacterial liquid, Bacillus amyloliquefaciens SS0813 bacterial liquid and Bacillus velez MZ1030 bacterial liquid; the Bacillus argais GL0525 bacterial liquid, the Bacillus amyloliquefaciens SS0813 bacterial liquid and the Bacillus velez MZ1030 bacterial liquid are diluted to OD600 of 1.0 respectively, and then mixed at a volume ratio of 1:1:1 to obtain a mixed bacterial liquid.

[0086] Example 5 A fermentation method for improving the quality of cigar tobacco leaves

[0087] (1) After the cigar tobacco leaves are rehumidified to a moisture content of 29%, the moisture content is balanced in a constant temperature and humidity chamber (37°C, 75%);

[0088] (2) spraying the mixed bacterial liquid uniformly onto the cigar tobacco leaves treated in step (1) (the mass of the mixed bacterial liquid sprayed is 23% of the mass of the cigar tobacco leaves), so that each tobacco leaf is completely covered with a layer of water film but no water is dripping, thereby obtaining a fermentation raw material; and then placing the fermentation raw material in a variable temperature and humidity fermentation box, wherein the temperature is 35° C. and the humidity is 70% and the fermentation is carried out for 48 hours, and the temperature is 45° C. and the humidity is 85% and the fermentation is carried out for 48 hours as one cycle, and the cycle is repeated 9 times to obtain fermented cigar tobacco leaves.

[0089] Experimental Example 1 Sensory Quality Evaluation

[0090] The mixed bacterial liquids described in Examples 1 to 4 were combined with the fermentation method described in Example 5 as experimental groups, namely, mixed bacterial liquid 1 treatment group, mixed bacterial liquid 2 treatment group, mixed bacterial liquid 3 treatment group, and mixed bacterial liquid 4 treatment group; the mixed bacterial liquid described in Example 5 was replaced with water as a water-added control group.

[0091] The sensory quality of the fermented cigar tobacco leaves obtained from the mixed bacterial liquid 1 treatment group, the mixed bacterial liquid 2 treatment group, the mixed bacterial liquid 3 treatment group, the mixed bacterial liquid 4 treatment group and the water-added control group were evaluated respectively.

[0092] The sensory evaluation method or standard for fermented cigar tobacco is the "Cigar Style Characteristics Evaluation Method".

[0093] Table 1 Sensory quality evaluation results

[0094]

[0095] As can be seen from Table 1, the fermentation method provided by the present invention can effectively ferment cigar tobacco leaves through "pure natural" microbial enrichment without introducing exogenous chemical components, effectively improve the smoking quality of tobacco leaves, reduce irritation and impurities, and achieve quality improvement and flavor enhancement of cigar tobacco leaves.

[0096] Experimental Example 2 Determination of the content of volatile flavor metabolites before and after fermentation

[0097] The mixed bacterial liquids described in Examples 1 to 4 were combined with the fermentation method described in Example 5 as experimental groups, namely, mixed bacterial liquid 1 treatment group, mixed bacterial liquid 2 treatment group, mixed bacterial liquid 3 treatment group, and mixed bacterial liquid 4 treatment group; the mixed bacterial liquid described in Example 5 was replaced with water as a water-added control group;

[0098] Weigh the cigar tobacco leaves before fermentation and fermented cigar tobacco leaves in the water-added control group and the mixed bacterial solution 1 to 4 treatment groups as test samples;

[0099] 0.5 g of sample was weighed and added to a 20 mL headspace bottle containing 8 mL of saturated sodium chloride solution and 1 μL of 2-phenylethyl acetate internal standard solution; the headspace bottle was then placed in a 70°C water bath and stirred for continuous incubation for 20 min; the SPME needle was inserted into the headspace bottle, the extraction needle was pushed out, and the volatiles were extracted and adsorbed for 35 min, and then immediately analyzed by GC-MS for 5 min; in an HP-5MS capillary column (30 m×0.25 mm×0.25 μm), volatile organic compounds were separated under the following GC conditions: injection port temperature, 250°C; carrier gas, helium (purity 99.99%); flow rate 0.8 mL / min; the sample was injected without splitting. The temperature program was carried out under the following conditions: the starting temperature was 60°C, maintained for 2 min, then heated to 180°C at a rate of 3°C / min, maintained for 2 min, then heated to 260°C at a rate of 6°C / min, and maintained at 260°C for 2 min; the MS conditions were as follows: ionization energy was 70 eV; the ion source temperature was 230°C; the quadrupole mass detector temperature was 150°C; and the mass scanning range was 35-450 m / z.

[0100] The contents of carotenoid degradation products, phenylalanine degradation products, cedarwood degradation products, chlorophyll degradation products, volatile alkaloids, and other volatile flavor metabolites in the samples are as follows: Figures 1 to 6 As shown, mixed bacterial liquid 1 is mixed bacterial liquid 1 treatment group, mixed bacterial liquid 2 is mixed bacterial liquid 2 treatment group; mixed bacterial liquid 3 is mixed bacterial liquid 3 treatment group; mixed bacterial liquid 4 is mixed bacterial liquid 4 treatment group.

[0101] from Figure 1 It can be seen that the content of carotenoid degradation products in the water-added control group after fermentation was significantly reduced; for the samples treated with mixed culture solution 1, mixed culture solution 2 and mixed culture solution 3, the content of such volatile aroma components after fermentation also showed a slight downward trend. In the samples treated with mixed culture solution 4, the content of carotenoid degradation products after fermentation showed an increasing trend.

[0102] from Figure 2 It can be seen that the content of phenylalanine degradation products in the water-added control group showed a downward trend after fermentation; while for the samples treated with mixed bacteria liquid 1, mixed bacteria liquid 2, mixed bacteria liquid 3 and mixed bacteria liquid 4, the content of phenylalanine degradation products after fermentation showed a significant increasing trend.

[0103] from Figure 3It can be seen that the content of cedar degradation products in the cigar tobacco leaves of the water-added control group after fermentation was significantly reduced; for the samples treated with mixed bacterial liquid 1, mixed bacterial liquid 2 and mixed bacterial liquid 3, the content of such volatile aroma components also showed a downward trend after fermentation. In the samples treated with mixed bacterial liquid 4, the content of cedar degradation products after fermentation showed a slight increasing trend.

[0104] from Figure 4 It can be seen that the content of chlorophyll degradation products in cigar tobacco leaves of the water-added control group after fermentation was slightly reduced; while for the samples treated with mixed bacterial liquid 1, mixed bacterial liquid 2 and mixed bacterial liquid 3, the content of such volatile aroma components after fermentation also showed a certain downward trend. Only in the sample treated with mixed bacterial liquid 4, the content of chlorophyll degradation products after fermentation showed an increasing trend.

[0105] from Figure 5 It can be seen that the content of volatile alkaloids in the samples of the water-added control group, mixed bacteria solution 1, mixed bacteria solution 2, mixed bacteria solution 3 and mixed bacteria solution 4 treatment groups showed a significant downward trend after fermentation.

[0106] from Figure 6 It can be seen that the contents of other volatile flavor metabolites in the samples of the water control group, mixed bacteria liquid 1, mixed bacteria liquid 2, mixed bacteria liquid 3 and mixed bacteria liquid 4 treatment groups showed a significant upward trend after fermentation; and in the samples treated with mixed bacteria liquid 4, the increasing trend of the contents of other volatile flavor metabolites after fermentation was the most obvious.

[0107] From the analysis results of aroma components, it can be seen that the mixed bacterial medium is added during the fermentation of cigar tobacco leaves. The contents of common aroma components such as carotenoid degradation products, phenylalanine degradation products and cypermethrin degradation products in cigar tobacco leaves treated with mixed bacterial liquid 4 are increased compared with the control group and other treatment groups after fermentation. In addition, the volatile alkaloids after fermentation are significantly reduced compared with the control group. These substances are reported to have a certain effect in reducing the irritation of tobacco leaves and improving the aroma quality of tobacco leaves. The addition of mixed bacterial liquid 4 introduces a variety of enzymes and antibacterial substances such as amylase and protease into the cigar tobacco leaves, protects the tobacco leaves from mold contamination, promotes microbial cooperative fermentation, and increases the content of various flavor substances. The introduction of mixed bacterial liquid 4 can significantly increase the richness of the aroma of tobacco leaves, weaken the bitterness and ammonia taste of tobacco leaves, increase the sweet and burnt aroma, and improve the smoke characteristics and combustion characteristics.

[0108] Example 6 A fermentation method for improving the quality of cigar tobacco leaves

[0109] (1) After the cigar tobacco leaves are rehumidified to a moisture content of 30%, the moisture content is balanced in a constant temperature and humidity chamber (37°C, 75%);

[0110] (2) spraying the mixed bacterial liquid uniformly onto the cigar tobacco leaves treated in step (1) (the mass of the mixed bacterial liquid sprayed is 25% of the mass of the cigar tobacco leaves), so that each tobacco leaf is completely covered with a layer of water film but no water is dripping, thereby obtaining a fermentation raw material; and then placing the fermentation raw material in a variable temperature and humidity fermentation box, wherein the temperature is 38° C. and the humidity is 75% for fermentation for 50 h, and the temperature is 48° C. and the humidity is 90% for fermentation for 50 h as one cycle, and the cycle is repeated 8 times to obtain fermented cigar tobacco leaves.

[0111] Example 7 A fermentation method for improving the quality of cigar tobacco leaves

[0112] (1) After the cigar tobacco leaves are rehumidified to a moisture content of 28%, the moisture content is balanced in a constant temperature and humidity chamber (37°C, 75%);

[0113] (2) spraying the mixed bacterial liquid uniformly onto the cigar tobacco leaves treated in step (1) (the mass of the mixed bacterial liquid sprayed is 20% of the mass of the cigar tobacco leaves), so that each tobacco leaf is completely covered with a layer of water film but no water is dripping, thereby obtaining a fermentation raw material; and then placing the fermentation raw material in a variable temperature and humidity fermentation box, wherein the temperature is 32° C. and the humidity is 65%, and the fermentation is carried out for 45 hours. The temperature is 42° C. and the humidity is 80%, and the fermentation is carried out for 45 hours as one cycle. The cycle is repeated 10 times to obtain fermented cigar tobacco leaves.

[0114] It can be seen from the above embodiments that the present invention provides a mixed bacterial liquid for improving the quality of cigar tobacco leaves, its application and a fermentation method for improving the quality of cigar tobacco leaves. The fermentation method can effectively enhance the flavor and quality of cigar tobacco leaves, eliminate the impurities of cigar tobacco leaves, and improve and enhance the smoking taste of cigar tobacco leaves.

[0115] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mixed bacterial liquid for improving the quality of cigar tobacco, characterized in that: The mixed bacterial solution includes two or more of the bacterial solution of Bacillus amylopectin GL0525, the bacterial solution of Bacillus amyloliquefaciens SS0813 and the bacterial solution of Bacillus velez MZ1030; The Bacillus arginii GL0525 bacterial liquid is prepared from Bacillus arginii GL0525. Priestia aryabhattai ) GL0525, deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China, on December 1, 2023, with the deposit number: CCTCC NO: M20232417; The Bacillus amyloliquefaciens SS0813 bacterial liquid is prepared from Bacillus amyloliquefaciens SS0813. Bacillus amyloliquefaciens )SS0813, deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China, on December 1, 2023, with the deposit number: CCTCC NO:M20232418; The Velez Bacillus MZ1030 bacterial liquid is prepared from Velez Bacillus MZ1030. Bacillus velezensis )MZ1030, deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China. The deposit date is December 1, 2023, and the deposit number is: CCTCC NO:M20232416.

2. The mixed bacterial solution according to claim 1, characterized in that: When the mixed bacterial solution includes Bacillus argyi GL0525 bacterial solution and Bacillus amyloliquefaciens SS0813 bacterial solution, the volume ratio of the Bacillus argyi GL0525 bacterial solution to the Bacillus amyloliquefaciens SS0813 bacterial solution is 0.5-1.5:0.5-1.5; The OD600 of the Bacillus argyi GL0525 bacterial solution is 0.5-1.5; The OD600 of the Bacillus amyloliquefaciens SS0813 bacterial solution is 0.5-1.

5.

3. The mixed bacterial solution according to claim 1, characterized in that: When the mixed bacterial solution includes Bacillus argyi GL0525 bacterial solution and Bacillus velezii MZ1030 bacterial solution, the volume ratio of the Bacillus argyi GL0525 bacterial solution to the Bacillus velezii MZ1030 bacterial solution is 0.5-1.5:0.5-1.5; The OD600 of the Bacillus argyi GL0525 bacterial solution is 0.5-1.5; The OD600 of the Bacillus Velez MZ1030 bacterial solution is 0.5-1.

5.

4. The mixed bacterial solution according to claim 1, characterized in that: When the mixed bacterial solution includes Bacillus amyloliquefaciens SS0813 bacterial solution and Bacillus velezii MZ1030 bacterial solution, the volume ratio of the Bacillus amyloliquefaciens SS0813 bacterial solution to the Bacillus velezii MZ1030 bacterial solution is 0.5-1.5:0.5-1.5; The OD600 of the Bacillus amyloliquefaciens SS0813 bacterial solution is 0.5-1.5; The OD600 of the Bacillus Velez MZ1030 bacterial solution is 0.5-1.

5.

5. The mixed bacterial solution according to claim 1, characterized in that: When the mixed bacterial solution includes Bacillus arfaecium GL0525 bacterial solution, Bacillus amyloliquefaciens SS0813 bacterial solution and Bacillus velezii MZ1030 bacterial solution, the volume ratio of the Bacillus arfaecium GL0525 bacterial solution, the Bacillus amyloliquefaciens SS0813 bacterial solution and the Bacillus velezii MZ1030 bacterial solution is 0.5-1.5:0.5-1.5:0.5-1.5; The OD600 of the Bacillus argyi GL0525 bacterial solution is 0.5-1.5; The OD600 of the Bacillus amyloliquefaciens SS0813 bacterial solution is 0.5-1.5; The OD600 of the Bacillus Velez MZ1030 bacterial solution is 0.5-1.

5.

6. Use of the mixed bacterial liquid according to any one of claims 1 to 5 in improving the quality of cigar tobacco leaves.

7. A fermentation method for improving the quality of cigar tobacco, characterized in that: The steps include: Mixing cigar tobacco leaves with the mixed bacterial solution to obtain a fermentation raw material; fermenting the fermentation raw material to obtain fermented cigar tobacco leaves; The mixed bacterial solution is the mixed bacterial solution according to any one of claims 1 to 5; The fermentation is a variable temperature and humidity cyclic fermentation; fermentation for 45 to 50 hours at a temperature of 32 to 38° C. and a humidity of 65 to 75%, and fermentation for 45 to 50 hours at a temperature of 42 to 48° C. and a humidity of 80 to 90% is one cycle, and the cycle is repeated 8 to 10 times.

8. The fermentation method according to claim 7, characterized in that The mass ratio of the cigar tobacco leaves to the mixed bacterial solution is 1:0.20-0.

25.

9. Use of the fermentation method according to any one of claims 7 to 8 in improving the quality of cigar tobacco leaves.

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