CN118308259A - Mixed bacterial liquid for improving cigar tobacco quality, application thereof and fermentation method for improving cigar tobacco quality - Google Patents
Mixed bacterial liquid for improving cigar tobacco quality, application thereof and fermentation method for improving cigar tobacco quality Download PDFInfo
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Abstract
The invention relates to the technical field of tobacco, in particular to a mixed bacterial liquid for improving cigar tobacco quality, application thereof and a fermentation method for improving cigar tobacco quality. The invention provides a mixed bacterial liquid for improving cigar tobacco quality, which comprises two or more than two of bacillus aryabhattai GL0525 bacterial liquid, bacillus amyloliquefaciens SS0813 bacterial liquid and bacillus bailii MZ1030 bacterial liquid. The invention also provides a fermentation method for improving the quality of cigar tobacco leaves, which can effectively carry out flavoring and quality improvement on the cigar tobacco leaves, eliminate the miscellaneous gas of the cigar tobacco leaves and improve and enhance the taste of the cigar tobacco leaves.
Description
Technical Field
The invention relates to the technical field of tobacco, in particular to a mixed bacterial liquid for improving cigar tobacco quality, application thereof and a fermentation method for improving cigar tobacco quality.
Background
The fermentation of domestic cigar tobacco leaves is a key step in the cigar production process, and has important significance for 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 leaves is also advancing continuously. However, this process still faces a number of technical and industrialization challenges.
Fermentation of cigar tobacco leaves is a very complex biochemical process that requires long time runs under controlled conditions. Ensures that each batch of tobacco leaves can achieve ideal fermentation effect, and has extremely high control requirement on the fermentation process. Small variations in temperature, humidity, time, etc. may have a significant impact on the quality of the final tobacco. Despite technological advances, maintaining quality consistency and achieving standardized production of domestic cigar tobacco leaves during fermentation remains a challenge. Quality fluctuations may exist between different batches of tobacco leaves, different production bases of tobacco leaves, which constitute an obstacle to improving the competitiveness of domestic cigars in the international market.
In order to further improve the fermentation efficiency and tobacco quality, technical innovation and development work are required continuously. This includes intensive research of microbial activity during fermentation, optimization of fermentation conditions, and improvement of tobacco quality assessment methods after fermentation. The middle-high grade full-manual cigars on the current market are usually 'purely natural', and no essence and spice are added, so that the quality of products is difficult to improve by the normal flavoring means of the tobacco industry.
Based on this, the present invention has been proposed.
Disclosure of Invention
The invention aims to provide a mixed bacterial liquid for improving the quality of cigar tobacco leaves, application thereof and a fermentation method for improving the quality of cigar tobacco leaves, wherein the fermentation method can effectively carry out aroma enhancement and quality improvement on cigar tobacco leaves, eliminate the miscellaneous gas of cigar tobacco leaves and improve and enhance the taste of cigar tobacco leaves.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides a mixed bacterial liquid for improving cigar tobacco quality, which comprises two or more than two of bacillus aryabhattai GL0525 bacterial liquid, bacillus amyloliquefaciens SS0813 bacterial liquid and bacillus bailii MZ1030 bacterial liquid.
Preferably, when the mixed bacterial liquid comprises bacillus aryabhattai GL0525 bacterial liquid and bacillus amyloliquefaciens SS0813 bacterial liquid, the volume ratio of the bacillus aryabhattai GL0525 bacterial liquid to the bacillus amyloliquefaciens SS0813 bacterial liquid is 0.5-1.5:0.5-1.5;
OD600 of the bacillus aryabhattai GL0525 bacterial liquid is 0.5-1.5;
The OD600 of the bacillus amyloliquefaciens SS0813 bacterial liquid is 0.5-1.5.
Preferably, when the mixed bacterial liquid comprises bacillus aryabhattai GL0525 bacterial liquid and bacillus berryis MZ1030 bacterial liquid, the volume ratio of the bacillus aryabhattai GL0525 bacterial liquid to the bacillus berryis MZ1030 bacterial liquid is 0.5-1.5:0.5-1.5;
OD600 of the bacillus aryabhattai GL0525 bacterial liquid is 0.5-1.5;
the OD600 of the bacillus bailii MZ1030 bacterial liquid is 0.5-1.5.
Preferably, when the mixed bacterial liquid comprises bacillus amyloliquefaciens SS0813 bacterial liquid and bacillus bailii MZ1030 bacterial liquid, the volume ratio of the bacillus amyloliquefaciens SS0813 bacterial liquid to the bacillus bailii MZ1030 bacterial liquid is 0.5-1.5:0.5-1.5;
OD600 of the bacillus amyloliquefaciens SS0813 bacterial liquid is 0.5-1.5;
the OD600 of the bacillus bailii MZ1030 bacterial liquid is 0.5-1.5.
Preferably, when the mixed bacterial liquid comprises bacillus aryabhattai GL0525 bacterial liquid, bacillus amyloliquefaciens SS0813 bacterial liquid and bacillus bailii MZ1030 bacterial liquid, the volume ratio of the bacillus aryabhattai GL0525 bacterial liquid, the bacillus amyloliquefaciens SS0813 bacterial liquid and the bacillus bailii MZ1030 bacterial liquid is 0.5-1.5:0.5-1.5;
OD600 of the bacillus aryabhattai GL0525 bacterial liquid is 0.5-1.5;
OD600 of the bacillus amyloliquefaciens SS0813 bacterial liquid is 0.5-1.5;
the OD600 of the bacillus bailii MZ1030 bacterial liquid is 0.5-1.5.
The invention provides application of the mixed bacterial liquid in improving the quality of cigar tobacco leaves.
The invention provides a fermentation method for improving cigar tobacco quality, which comprises the following steps:
mixing cigar tobacco leaves with the mixed bacterial liquid to obtain fermentation raw materials; fermenting the fermentation raw material to obtain fermented cigar tobacco leaves;
the mixed bacterial liquid is the mixed bacterial liquid;
the fermentation is variable temperature and humidity cyclic fermentation; fermenting for 45-50 h at the temperature of 32-38 ℃ and the humidity of 65-75%, fermenting for 45-50 h at the temperature of 42-48 ℃ and the humidity of 80-90% for 8-10 times.
Preferably, the mass ratio of the cigar tobacco leaves to the mixed bacterial liquid is 1:0.20-0.25.
The invention also provides application of the fermentation method in improving the quality of cigar tobacco leaves.
The invention has the beneficial effects that:
1. Compared with other fermentation modes, the fermentation mode provided by the invention utilizes the microbial mixed bacterial liquid with different proportions in the fermentation process, so that the original microbial community in cigar tobacco leaves is changed, dominant microbial flora is enriched in the cigar tobacco leaves, a series of biochemical changes of the tobacco leaf internal substances are promoted, and the quality of the cigar tobacco leaves is promoted;
2. the fermentation method can effectively improve the smoking quality of tobacco leaves and reduce the irritation and miscellaneous gases;
3. The temperature-changing and humidity-changing circulating fermentation method can promote the activity of dominant microorganisms, improve the quality of tobacco leaves, ensure that the combustibility of the tobacco leaves is more stable and the color is more uniform, and further achieve the purposes of improving the internal quality of the tobacco leaves and improving the industrial availability.
Drawings
FIG. 1 shows carotenoid degradation product content in cigar tobacco before and after fermentation of mixed bacterial solution;
FIG. 2 shows phenylalanine degradation product content in cigar tobacco before and after fermentation of mixed bacterial solution;
FIG. 3 shows the cembrane degradation product content of cigar leaves before and after fermentation of the mixed bacterial liquid;
FIG. 4 shows chlorophyll degradation product content in cigar leaves before and after fermentation of the mixed bacterial solution;
FIG. 5 shows the volatile alkaloid content in cigar leaves before and after fermentation of the mixed bacterial solution;
FIG. 6 shows the content of other volatile flavor metabolites in cigar leaves before and after fermentation of the mixed liquor.
Description of biological preservation
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SS0813, deposited at China center for type culture Collection, having a date of deposit of 2023, 12 months and 01 days, university of Wuhan, china, accession number: CCTCC NO: M20232418.
Bacillus belicus (Bacillus velezensis) MZ1030 deposited at China center for type culture Collection, with a date of deposit of 2023, 12 months and 01 days at university of Wuhan, china, with a deposit number: CCTCC NO: M20232416.
Bacillus aryabhattai (Priestia aryabhattai) GL0525 deposited at China center for type culture Collection, having a accession number of China, university of Wuhan, and having a accession date of 2023, 12 and 01: CCTCC NO: M20232417.
Detailed Description
The invention provides a mixed bacterial liquid for improving cigar tobacco quality, which comprises two or more than two of bacillus aryabhattai GL0525 bacterial liquid, bacillus amyloliquefaciens SS0813 bacterial liquid and bacillus bailii MZ1030 bacterial liquid.
In the invention, the preparation method of the bacillus aryabhattai GL0525 bacterial liquid comprises the following steps: inoculating and culturing bacillus aryabhattai GL0525 to obtain seed liquid; inoculating the seed solution into LB liquid culture medium according to the inoculum size of 1% -5% (v/v), and culturing for 12-14 h at 35-39 ℃ to obtain bacillus aryabhattai GL0525 bacterial solution;
The inoculum size is preferably 3%; the culture temperature is preferably 37℃and the culture time is preferably 13 hours.
In the invention, the preparation method of the bacillus amyloliquefaciens SS0813 bacterial liquid comprises the following steps: inoculating and culturing bacillus amyloliquefaciens SS0813 to obtain seed liquid; inoculating the seed solution into LB liquid culture medium according to the inoculum size of 1% -5% (v/v), and culturing for 12-14 h at 35-39 ℃ to obtain bacillus amyloliquefaciens SS0813 bacterial solution;
The inoculum size is preferably 3%; the culture temperature is preferably 37℃and the culture time is preferably 13 hours.
In the invention, the preparation method of the bacillus belicus MZ1030 bacterial liquid comprises the following steps: inoculating and culturing bacillus bailii MZ1030 to obtain seed liquid; inoculating the seed solution into LB liquid culture medium according to the inoculum size of 1% -5% (v/v), and culturing for 12-14 h at 35-39 ℃ to obtain bacillus bailii MZ1030 bacterial solution;
The inoculum size is preferably 3%; the culture temperature is preferably 37℃and the culture time is preferably 13 hours.
In the invention, when the mixed bacterial liquid comprises bacillus aryabhattai GL0525 bacterial liquid and bacillus amyloliquefaciens SS0813 bacterial liquid, the volume ratio of the bacillus aryabhattai GL0525 bacterial liquid to the bacillus amyloliquefaciens SS0813 bacterial liquid is 0.5-1.5:0.5-1.5, preferably 1:1;
The OD600 of the bacillus aryabhattai GL0525 bacterial liquid is 0.5-1.5, preferably 1.0;
The OD600 of the bacillus amyloliquefaciens SS0813 bacterial liquid is 0.5-1.5, preferably 1.0.
In the invention, when the mixed bacterial liquid comprises the bacillus aryabhattai GL0525 bacterial liquid and the bacillus berryis MZ1030 bacterial liquid, the volume ratio of the bacillus aryabhattai GL0525 bacterial liquid to the bacillus berryis MZ1030 bacterial liquid is 0.5-1.5:0.5-1.5, preferably 1:1;
The OD600 of the bacillus aryabhattai GL0525 bacterial liquid is 0.5-1.5, preferably 1.0;
the OD600 of the bacillus bailii MZ1030 bacterial liquid is 0.5-1.5, preferably 1.0.
In the invention, when the mixed bacterial liquid comprises bacillus amyloliquefaciens SS0813 bacterial liquid and bacillus bailii MZ1030 bacterial liquid, the volume ratio of the bacillus amyloliquefaciens SS0813 bacterial liquid to the bacillus bailii MZ1030 bacterial liquid is 0.5-1.5:0.5-1.5, preferably 1:1;
the OD600 of the bacillus amyloliquefaciens SS0813 bacterial liquid is 0.5-1.5, preferably 1.0;
the OD600 of the bacillus bailii MZ1030 bacterial liquid is 0.5-1.5, preferably 1.0.
In the invention, when the mixed bacterial liquid comprises bacillus aryabhattai GL0525 bacterial liquid, bacillus amyloliquefaciens SS0813 bacterial liquid and bacillus bailii MZ1030 bacterial liquid, the volume ratio of the bacillus aryabhattai GL0525 bacterial liquid, the bacillus amyloliquefaciens SS0813 bacterial liquid and the bacillus bailii MZ1030 bacterial liquid is 0.5-1.5:0.5-1.5, preferably 1:1:1;
The OD600 of the bacillus aryabhattai GL0525 bacterial liquid is 0.5-1.5, preferably 1.0;
the OD600 of the bacillus amyloliquefaciens SS0813 bacterial liquid is 0.5-1.5, preferably 1.0;
the OD600 of the bacillus bailii MZ1030 bacterial liquid is 0.5-1.5, preferably 1.0.
Bacillus arvensis is a type of acidocaldarius capable of producing spores in the invention, and microbial activity is a key ring in the fermentation process of cigar tobacco leaves, and affects the quality, flavor and combustion performance of the tobacco leaves. While bacillus arvensis is not one of the most predominant microorganisms in cigar leaf fermentation, bacillus arvensis may dominate the leaf fermentation process under certain conditions, particularly in high temperature and acidic environments;
Bacillus amyloliquefaciens, a bacteria widely used in industrial and agricultural fields, is capable of producing a large amount of enzymes such as amylase, protease, etc., which are very useful in the food industry and other applications. In the cigar tobacco fermentation process, the bacillus amyloliquefaciens can help to decompose macromolecular organic substances in tobacco, such as protein and cellulose, and convert the macromolecular organic substances into micromolecular substances, so that the aroma and the taste of the tobacco are improved. The bacillus amyloliquefaciens participates in the process through the generated enzyme (such as protease), so that the fermentation effect of tobacco leaves is improved;
Bacillus beleiensis is a bacterium widely existing in natural environments, and can produce a large amount of secondary metabolites, such as antibiotics, enzymes and the like, which have potential application values in agriculture and food industry. Bacillus beleiensis is capable of producing a variety of enzymes, such as proteases, amylases, etc., that contribute to the formation of aroma components and the degradation of undesirable components during tobacco aging. In addition, bacillus beleiensis can also produce antibacterial substances, such as lipopeptides antibiotics, which can inhibit the growth of harmful microorganisms in the tobacco fermentation process and protect the tobacco from microbial contamination;
According to the purely natural fermentation characteristics of the handmade cigars, the bacillus arvensis has the activity of producing protease, can be used for improving neutral aroma components of cigar tobacco leaves, can produce a large amount of amylase, protease and the like, can help to decompose macromolecular organic substances in the tobacco leaves, such as protein and cellulose, and convert the macromolecular organic substances into micromolecular substances, thereby improving the aroma and taste of the tobacco leaves and reducing the irritation and miscellaneous gases; bacillus belicus is capable of producing a variety of enzymes that contribute to the formation of aroma components and the degradation of undesirable components during tobacco aging; in addition, bacillus beleiensis can also produce antibacterial substances, such as lipopeptid antibiotics, which can inhibit the growth of harmful microorganisms in the tobacco fermentation process, protect the tobacco from microbial contamination, and realize the quality improvement and aroma enhancement of cigar tobacco through the synergistic fermentation of the microorganisms.
The invention provides application of the mixed bacterial liquid in improving the quality of cigar tobacco leaves.
The invention provides a fermentation method for improving cigar tobacco quality, which comprises the following steps:
mixing cigar tobacco leaves with the mixed bacterial liquid to obtain fermentation raw materials; fermenting the fermentation raw material to obtain fermented cigar tobacco leaves;
the mixed bacterial liquid is the mixed bacterial liquid;
the fermentation is variable temperature and humidity cyclic fermentation; fermenting for 45-50 h at the temperature of 32-38 ℃ and the humidity of 65-75%, fermenting for 45-50 h at the temperature of 42-48 ℃ and the humidity of 80-90% for 8-10 times.
The temperature-changing and humidity-changing cyclic fermentation is preferably carried out for 48h at 35 ℃ and 70% humidity, and 48h at 45 ℃ and 85% humidity, and the cycle is 9 times.
In the invention, the artificial temperature-changing and humidity-changing fermentation is a more complex and fine cigar tobacco processing method, and the chemical component conversion in tobacco leaves is further optimized by accurately controlling the temperature and humidity change in the fermentation process, so that the quality of the tobacco leaves is improved and the fragrance of the tobacco leaves is enhanced. The variable temperature and humidity fermentation promotes the organic matters (such as sugar and protein) in the tobacco leaves to be more effectively converted into flavor compounds such as phenols, aldehydes, esters and other volatile compounds through the microbial action and the non-biological reaction. By controlling the temperature and humidity conditions in the fermentation process, the generation of specific aroma components in tobacco leaves can be directionally influenced to a certain extent, so that cigars exhibit more rich and complex flavor layers. In addition, the variable-temperature and variable-humidity fermentation is beneficial to more effectively reducing harmful components in tobacco leaves, such as nicotine and certain acidic substances, so that the tobacco leaves are smoother, and the irritation during smoking is reduced. Proper humidity control has an important influence on the combustibility of tobacco leaves, and moderate humidity can ensure that the tobacco leaves burn more stably during smoking, so that smoking experience is improved. The proper temperature and humidity conditions can promote the improvement of tobacco quality, promote the activity of beneficial microorganisms (such as bacillus aryabhattai, bacillus amyloliquefaciens, bacillus bailii and the like), inhibit the growth of microorganisms possibly causing the degradation of the tobacco quality, and further promote the tobacco quality through the optimization of microbial communities. The variable temperature and humidity fermentation environment promotes the enhancement of the microbial activities of beneficial enzymes, such as protease, cellulase and the like, which help to decompose macromolecular substances in tobacco leaves and promote the formation of aroma substances. The fermentation treatment technology plays an important role in improving the quality of cigar tobacco leaves, optimizing the flavor characteristics of cigar tobacco leaves and accelerating the ripening process.
In the invention, the mass ratio of the cigar tobacco leaves to the mixed bacterial liquid is 1:0.20-0.25, preferably 1:0.225.
In the invention, before the cigar tobacco leaves are mixed with the mixed bacterial liquid, the cigar tobacco leaves are required to be remoistened, and after the water content reaches 28% -30%, the water content is balanced under the conditions of the temperature of 35 ℃ and the humidity of 75%.
The invention also provides application of the fermentation method in improving the quality of cigar tobacco leaves.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1 Mixed bacterial liquid for improving cigar tobacco quality
Inoculating and culturing bacillus aryabhattai GL0525 and bacillus amyloliquefaciens SS0813 respectively to obtain seed liquid; inoculating the seed solution into LB liquid culture medium according to the inoculum size of 3% (v/v), and culturing at 37 ℃ for 13 hours to obtain bacillus aryabhattai GL0525 bacterial solution and bacillus amyloliquefaciens SS0813 bacterial solution; and respectively diluting the bacillus aryabhattai GL0525 bacterial liquid and the bacillus amyloliquefaciens SS0813 bacterial liquid to an OD600 of 1.0, and then mixing according to a volume ratio of 1:1 to obtain mixed bacterial liquid.
Example 2 Mixed bacterial liquid for improving cigar tobacco quality
Inoculating and culturing bacillus aryabhattai GL0525 and bacillus berryis MZ1030 respectively to obtain seed liquid; inoculating the seed solution into LB liquid culture medium according to the inoculum size of 3% (v/v), and culturing at 37 ℃ for 13h to obtain bacillus aryabhattai GL0525 bacterial solution and bacillus bailii MZ1030 bacterial solution; and respectively diluting the bacillus aryabhattai GL0525 bacterial liquid and the bacillus berryis MZ1030 bacterial liquid to an OD600 of 1.0, and then mixing according to a volume ratio of 1:1 to obtain a mixed bacterial liquid.
Example 3 Mixed bacterial liquid for improving cigar tobacco quality
Inoculating and culturing bacillus amyloliquefaciens SS0813 and bacillus bailii MZ1030 respectively to obtain seed liquid; inoculating the seed solution into LB liquid culture medium according to the inoculum size of 3% (v/v), and culturing at 37 ℃ for 13h to obtain bacillus amyloliquefaciens SS0813 bacterial solution and bacillus bailii MZ1030 bacterial solution; and respectively diluting the bacillus amyloliquefaciens SS0813 bacterial liquid and the bacillus bailii MZ1030 bacterial liquid to an OD600 of 1.0, and then mixing according to a volume ratio of 1:1 to obtain mixed bacterial liquid.
Example 4 Mixed bacterial liquid for improving cigar tobacco quality
Inoculating and culturing bacillus aryabhattai GL0525, bacillus amyloliquefaciens SS0813 and bacillus bailii MZ1030 respectively to obtain seed liquid; inoculating the seed solution into LB liquid culture medium according to the inoculum size of 3% (v/v), and culturing at 37 ℃ for 13h to obtain bacillus aryabhattai GL0525 bacterial solution, bacillus amyloliquefaciens SS0813 bacterial solution and bacillus bailii MZ1030 bacterial solution; and respectively diluting the bacillus aryabhattai GL0525 bacterial liquid, the bacillus amyloliquefaciens SS0813 bacterial liquid and the bacillus berryis MZ1030 bacterial liquid to an OD600 of 1.0, and then mixing according to a volume ratio of 1:1:1 to obtain mixed bacterial liquid.
Example 5 fermentation method for improving cigar tobacco quality
(1) Conditioning cigar tobacco leaves to make the water content 29%, and balancing the water content in a constant temperature and humidity box (37 ℃ and 75%);
(2) Uniformly spraying the mixed bacterial liquid into the cigar tobacco leaves treated in the step (1) (the sprayed mass of the mixed bacterial liquid is 23% of the mass of the cigar tobacco leaves), and completely covering each piece of tobacco leaves with a layer of water film without dripping water to obtain fermentation raw materials; and placing the fermentation raw materials into a variable temperature and humidity fermentation box, wherein the fermentation is carried out for 48 hours at the temperature of 35 ℃ and the humidity of 70%, and the fermentation is carried out for 48 hours at the temperature of 45 ℃ and the humidity of 85%, and the fermentation is carried out for 9 times, so as to obtain the fermented cigar tobacco leaves.
Experimental example 1 sensory quality evaluation
The mixed bacterial solutions described in examples 1 to 4 and the fermentation method described in example 5 were used as experimental groups, respectively, a mixed bacterial solution 1 treatment group, a mixed bacterial solution 2 treatment group, a mixed bacterial solution 3 treatment group, and a mixed bacterial solution 4 treatment group; the mixed bacterial liquid described in example 5 was replaced with water as a water-added control group.
And respectively carrying out sensory quality evaluation on the fermented cigar tobacco obtained by the mixed bacteria liquid 1 treatment group, the mixed bacteria liquid 2 treatment group, the mixed bacteria liquid 3 treatment group, the mixed bacteria liquid 4 treatment group and the water adding control group.
The sensory evaluation method or standard of the fermented cigar tobacco leaf is cigar style characteristic evaluation method.
TABLE 1 sensory quality evaluation results
As can be seen from Table 1, the fermentation mode provided by the invention can effectively ferment cigar tobacco leaves through the enrichment of purely natural microorganisms on the premise of not introducing exogenous chemical components, effectively improve the smoking quality of the tobacco leaves, reduce the irritation and miscellaneous gases, and realize the quality improvement and aroma enhancement of the cigar tobacco leaves.
Experimental example 2 determination of volatile flavor metabolite content before and after fermentation
The mixed bacterial solutions described in examples 1 to 4 and the fermentation method described in example 5 were used as experimental groups, respectively, a mixed bacterial solution 1 treatment group, a mixed bacterial solution 2 treatment group, a mixed bacterial solution 3 treatment group, and a mixed bacterial solution 4 treatment group; the mixed bacterial liquid described in the example 5 is replaced by water to be used as a water adding control group;
Respectively weighing cigar tobacco leaves before fermentation and fermented cigar tobacco leaves in the water adding control group and the mixed bacteria liquid 1-4 treatment groups as detection samples;
0.5g of the sample was weighed into a headspace bottle containing 8mL of saturated sodium chloride solution and 1. Mu.L of an internal standard solution of 2-phenylethyl acetate in 20 mL; then placing the headspace bottle in a water bath with the temperature of 70 ℃ and stirring for continuous incubation for 20min; inserting the SPME needle tube into a headspace bottle, pushing out an extraction needle, extracting and adsorbing volatile matters for 35min, and immediately analyzing by GC-MS for 5min; volatile organic compounds were separated in an HP-5MS capillary column (30 m. Times.0.25 mm. Times.0.25 μm) under the following GC conditions: the temperature of the sample inlet is 250 ℃; carrier gas, helium (99.99% purity); the flow rate is 0.8mL/min; the sample is introduced without splitting. Wherein the temperature-raising program is performed under the following conditions: the initial temperature is 60 ℃, kept for 2min, then heated to 180 ℃ at a rate of 3 ℃/min, kept for 2min, then heated to 260 ℃ at a rate of 6 ℃/min, and kept at 260 ℃ for 2min; the MS conditions were as follows: ionization electric energy is 70eV; the ion source temperature is 230 ℃; the temperature of the quadrupole mass detector is 150 ℃; the mass scanning range is 35-450 m/z.
The contents of carotenoid degradation products, phenylalanine degradation products, cembrane degradation products, chlorophyll degradation products, volatile alkaloids and other volatile flavor metabolites in the sample are shown in the figures 1-6, wherein the mixed bacteria liquid 1 is a mixed bacteria liquid 1 treatment group, and the mixed bacteria liquid 2 is a mixed bacteria liquid 2 treatment group; the mixed bacteria liquid 3 is a mixed bacteria liquid 3 treatment group; the mixed bacteria liquid 4 is a mixed bacteria liquid 4 treatment group.
As can be seen from fig. 1, the water-added control group showed a significant decrease in the carotenoid degradation product content after fermentation; for the samples treated by the mixed bacteria liquid 1, the mixed bacteria liquid 2 and the mixed bacteria liquid 3, the content of the volatile aroma components after fermentation also shows a slight decrease trend, and the content of carotenoid degradation products after fermentation shows an increase trend in the samples treated by the mixed bacteria liquid 4.
From FIG. 2, it can be seen that the phenylalanine degradation product content of the water-added control group after fermentation is in a decreasing trend; and the content of the phenylalanine degradation products after fermentation in the samples treated by the mixed bacteria liquid 1, the mixed bacteria liquid 2, the mixed bacteria liquid 3 and the mixed bacteria liquid 4 shows a tendency of obviously increasing.
As can be seen from fig. 3, the water-added control group showed a significant decrease in the cembrane degradation products in the cigar leaves after fermentation; for the samples treated by the mixed bacteria liquid 1, the mixed bacteria liquid 2 and the mixed bacteria liquid 3, the content of the volatile aroma components also shows a decreasing trend after fermentation, and the content of the degradation products of the cembrane after fermentation shows a slight increasing trend in the samples treated by the mixed bacteria liquid 4.
As can be seen from fig. 4, the water-added control group has slightly reduced chlorophyll degradation product content in the cigar tobacco leaves after fermentation; for the samples treated by the mixed bacteria liquid 1, the mixed bacteria liquid 2 and the mixed bacteria liquid 3, the content of the volatile aroma components after fermentation also shows a certain decreasing trend, and the content of chlorophyll degradation products after fermentation only shows an increasing trend in the samples treated by the mixed bacteria liquid 4.
From fig. 5, it can be seen that the volatile alkaloid content after fermentation is significantly reduced in the water-added control group, or in the samples of the mixed bacteria liquid 1, mixed bacteria liquid 2, mixed bacteria liquid 3 and mixed bacteria liquid 4 treatment group.
From fig. 6, it can be seen that the samples of the water-added control group, the mixed bacteria liquid 1, the mixed bacteria liquid 2, the mixed bacteria liquid 3 and the mixed bacteria liquid 4 treated group all show obvious rising trends of the contents of other volatile flavor metabolites after fermentation; and in the sample treated by the mixed bacteria liquid 4, the increasing trend of the content of other volatile flavor metabolites after fermentation is most obvious.
As can be seen from the analysis results of the aroma components, the mixed bacteria medium is added in the cigar tobacco fermentation process, and the contents of the common aroma components such as carotenoid degradation products, phenylalanine degradation products, cembrane degradation products and the like in the cigar tobacco treated by the mixed bacteria liquid 4 are increased compared with the contents of the cigar tobacco treated by the control group and other treatment groups, and in addition, the contents of the fermented volatile alkaloids are obviously reduced compared with the control group, so that the substances are reported to have certain effects in reducing the irritation of the tobacco, improving the aroma quality of the tobacco and the like. The addition of the mixed bacterial liquid 4 introduces various enzymes and antibacterial substances such as rich amylase, protease and the like into the cigar tobacco leaves, protects the tobacco leaves from being polluted by mould, promotes the synergistic fermentation of microorganisms and increases the content of various flavor substances. The introduction of the mixed bacteria liquid 4 can obviously increase the aroma richness of the tobacco leaves, weaken the bitter taste and ammonia taste of the tobacco leaves, increase sweet and sweet fragrance and improve the smoke characteristic and combustion characteristic.
Example 6 fermentation method for improving cigar tobacco quality
(1) Conditioning cigar tobacco leaves to make the water content 30%, and balancing the water content in a constant temperature and humidity box (37 ℃ and 75%);
(2) Uniformly spraying the mixed bacterial liquid into the cigar tobacco leaves treated in the step (1) (the sprayed mass of the mixed bacterial liquid is 25% of the mass of the cigar tobacco leaves), and completely covering each piece of tobacco leaves with a layer of water film without dripping water to obtain fermentation raw materials; and placing the fermentation raw materials into a variable temperature and humidity fermentation box, wherein the fermentation is carried out for 50 hours at the temperature of 38 ℃ and the humidity of 75%, and the fermentation is carried out for 50 hours at the temperature of 48 ℃ and the humidity of 90%, and the fermentation is carried out for 8 times, so as to obtain the fermented cigar tobacco leaves.
Example 7A fermentation method for improving cigar tobacco quality
(1) Conditioning cigar tobacco leaves to a water content of 28%, and balancing water in a constant temperature and humidity box (37 ℃ and 75%);
(2) Uniformly spraying the mixed bacterial liquid into the cigar tobacco leaves treated in the step (1) (the sprayed mass of the mixed bacterial liquid is 20% of the mass of the cigar tobacco leaves), and completely covering each piece of tobacco leaves with a layer of water film without dripping water to obtain fermentation raw materials; and placing the fermentation raw materials into a variable temperature and humidity fermentation box, wherein the fermentation is carried out at the temperature of 32 ℃ and the humidity of 65% for 45 hours, the fermentation at the temperature of 42 ℃ and the humidity of 80% for 45 hours is carried out for one cycle, and the cycle is carried out for 10 times, so as to obtain the fermented cigar tobacco leaves.
According to the embodiment, the mixed bacterial liquid for improving the quality of cigar tobacco leaves, the application of the mixed bacterial liquid and the fermentation method for improving the quality of cigar tobacco leaves are provided, and the fermentation method can effectively carry out aroma enhancement and quality improvement on cigar tobacco leaves, eliminate the miscellaneous gas of cigar tobacco leaves and improve and enhance the taste of cigar tobacco leaves.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (9)
1. The mixed bacterial liquid for improving the quality of cigar tobacco leaves is characterized by comprising two or more than two of bacillus aryabhattai GL0525 bacterial liquid, bacillus amyloliquefaciens SS0813 bacterial liquid and bacillus bailii MZ1030 bacterial liquid.
2. The mixed bacterial liquid according to claim 1, wherein when the mixed bacterial liquid comprises bacillus aryabhattai GL0525 bacterial liquid and bacillus amyloliquefaciens SS0813 bacterial liquid, the volume ratio of the bacillus aryabhattai GL0525 bacterial liquid to the bacillus amyloliquefaciens SS0813 bacterial liquid is 0.5-1.5:0.5-1.5;
OD600 of the bacillus aryabhattai GL0525 bacterial liquid is 0.5-1.5;
The OD600 of the bacillus amyloliquefaciens SS0813 bacterial liquid is 0.5-1.5.
3. The mixed bacterial liquid according to claim 2, wherein when the mixed bacterial liquid comprises bacillus aryabhattai GL0525 bacterial liquid and bacillus berryis MZ1030 bacterial liquid, the volume ratio of the bacillus aryabhattai GL0525 bacterial liquid to the bacillus berryis MZ1030 bacterial liquid is 0.5-1.5:0.5-1.5;
OD600 of the bacillus aryabhattai GL0525 bacterial liquid is 0.5-1.5;
the OD600 of the bacillus bailii MZ1030 bacterial liquid is 0.5-1.5.
4. The mixed bacterial liquid according to claim 3, wherein when the mixed bacterial liquid comprises bacillus amyloliquefaciens SS0813 bacterial liquid and bacillus bailii MZ1030 bacterial liquid, the volume ratio of the bacillus amyloliquefaciens SS0813 bacterial liquid to the bacillus bailii MZ1030 bacterial liquid is 0.5-1.5:0.5-1.5;
OD600 of the bacillus amyloliquefaciens SS0813 bacterial liquid is 0.5-1.5;
the OD600 of the bacillus bailii MZ1030 bacterial liquid is 0.5-1.5.
5. The mixed bacterial liquid according to claim 4, wherein when the mixed bacterial liquid comprises bacillus aryabhattai GL0525 bacterial liquid, bacillus amyloliquefaciens SS0813 bacterial liquid and bacillus bailii MZ1030 bacterial liquid, the volume ratio of the bacillus aryabhattai GL0525 bacterial liquid, the bacillus amyloliquefaciens SS0813 bacterial liquid and the bacillus bailii MZ1030 bacterial liquid is 0.5-1.5:0.5-1.5;
OD600 of the bacillus aryabhattai GL0525 bacterial liquid is 0.5-1.5;
OD600 of the bacillus amyloliquefaciens SS0813 bacterial liquid is 0.5-1.5;
the OD600 of the bacillus bailii MZ1030 bacterial liquid is 0.5-1.5.
6. Use of the mixed liquor according to any one of claims 1 to 5 for improving the quality of cigar tobacco.
7. A fermentation method for improving cigar tobacco quality, comprising the steps of:
mixing cigar tobacco leaves with the mixed bacterial liquid to obtain fermentation raw materials; fermenting the fermentation raw material to obtain fermented cigar tobacco leaves;
the mixed bacterial liquid is the mixed bacterial liquid according to any one of claims 1 to 5;
the fermentation is variable temperature and humidity cyclic fermentation; fermenting for 45-50 h at the temperature of 32-38 ℃ and the humidity of 65-75%, fermenting for 45-50 h at the temperature of 42-48 ℃ and the humidity of 80-90% for 8-10 times.
8. The fermentation method according to claim 7, wherein the mass ratio of the cigar tobacco to the mixed bacterial liquid is 1:0.20-0.25.
9. Use of the fermentation process of any one of claims 7 to 8 for improving the quality of cigar tobacco leaves.
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