CN118235879A - Method for fermenting cigar tobacco leaves by utilizing bacillus, microbial inoculum for fermenting cigar tobacco leaves and application of microbial inoculum - Google Patents

Abstract

The invention provides a method for fermenting cigar tobacco leaves by using bacillus, a microbial inoculum for fermenting cigar tobacco leaves and application thereof, and belongs to the technical field of tobacco fermentation. Inoculating and culturing bacillus to obtain seed liquid, inoculating the seed liquid into a culture medium containing fermented grains according to an inoculum size of 1% -5% (v/v), and culturing at 30-40 ℃ for 12-14 h to obtain bacillus liquid; spraying bacillus liquid to cigar tobacco leaves, and fermenting at variable temperature to balance water. The bacillus is bacillus amyloliquefaciens SS0813, bacillus bailii MZ1030 or bacillus aryabhattai GL0525. The invention combines the strains with variable temperature fermentation to treat cigar tobacco leaves, can stably improve the content of various aroma substances in the cigar tobacco leaves, obviously reduce the pungent aroma components such as nicotine and the like, and ensure that the cigar is more mellow in smoking.

Description

Method for fermenting cigar tobacco leaves by utilizing bacillus, microbial inoculum for fermenting cigar tobacco leaves and application of microbial inoculum

Technical Field

The invention belongs to the technical field of tobacco fermentation, and particularly relates to a method for processing cigar tobacco leaves by utilizing bacillus fermentation, a microbial inoculum for processing cigar tobacco leaves by fermentation and application thereof.

Background

Compared with imported cigar tobacco leaves, the sensory quality of the domestic cigar tobacco leaves has a certain gap, and the domestic cigar tobacco leaves are mainly characterized in that the richness of the aroma is low, the miscellaneous gas is heavy, the oral cavity residue is obvious, the sweet feeling is weak, and the like, so that the domestic and foreign cigar markets are occupied by the imported cigars all the year round. In order to improve the smoking quality of domestic cigar tobacco leaves, cigar factories change the physicochemical properties of the tobacco leaves through fermentation technology, thereby improving the fragrance and taste of the tobacco leaves and reducing the irritation and miscellaneous gases.

In the fermentation process of cigar tobacco leaves, oxidation, microbial action and enzyme action are the process mechanisms for changing the quality of cigar cigarettes, and the introduction of fermentation medium can influence the quality of cigar cigarettes by influencing the growth and metabolism of microorganisms. However, the research on cigar fermentation process at home and abroad is mainly focused on optimization of fermentation process, and the research on cigar tobacco microorganism strengthening medium is less, so that microorganism strains for practical cigar production are scarce.

At present, a constant temperature fermentation method is generally adopted for microbial enrichment fermentation of cigar tobacco leaves, but in the natural fermentation process, the change of the internal temperature of the fermentation is generally accompanied by succession of microbial communities, and the constant temperature fermentation method is not beneficial to propagation, growth and metabolism of microorganisms.

The fermented grains are used as main bodies of the fermentation of the white spirit and are also main carriers of fermentation microorganisms, the diversity and the composition of microbial communities in the fermented grains are closely related to the flavor development substances of the white spirit, and the fermented grains can generate various volatile flavor components such as esters, alcohols, acids and the like in the fermentation process, thereby providing important contribution to the modeling of the flavor of the white spirit. The cigar with the bouquet flavor obtained by utilizing the microbial fermentation technology has important value for enriching cigar products. However, at present, few researches on improving the bouquet flavor of cigar tobacco leaves by utilizing microbial fermentation are reported. Therefore, providing a microbial strain that can enhance the bouquet flavor of cigar tobacco leaves and a fermentation method is a technical problem that needs to be solved in the art.

Disclosure of Invention

In view of the above, the invention aims to provide a method for fermenting cigar tobacco leaves by using bacillus, a microbial inoculum for fermenting cigar tobacco leaves and application thereof, so as to solve the problems of low fragrance and richness, heavy miscellaneous gas, obvious oral cavity residue, weak sweet feeling and the like of the existing cigar tobacco leaves.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for processing cigar tobacco leaves by utilizing bacillus fermentation, which comprises the following steps:

1) Inoculating the bacillus seed liquid into a culture medium containing fermented grains according to an inoculum size of 1% -5% (v/v), and culturing for 12-14 h at 30-40 ℃ to obtain bacillus liquid;

2) Spraying bacillus liquid to cigar tobacco leaves, and fermenting at variable temperature to balance water.

Preferably, the bacillus is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SS0813, bacillus bailii (Bacillus velezensis) MZ1030 or bacillus aryabhattai (Priestia aryabhattai) GL0525;

the bacillus amyloliquefaciens SS0813 is deposited in China center for type culture Collection of university of Wuhan with the deposit number: CCTCC NO: M20232418;

The bacillus belicus MZ1030 is deposited with the university of armed university chinese typical culture collection with the deposit number: CCTCC NO: M20232416;

The bacillus aryabhattai GL0525 is deposited with the China center for type culture Collection of university of Wuhan with the deposit number: CCTCCNO: M20232417.

Preferably, the variable temperature combined fermentation is carried out for 1 to 6 fermentation periods, wherein the 1 fermentation period is divided into three fermentation stages, the temperature of the first fermentation stage is 20 to 40 ℃, the humidity is 65 to 75 percent, and the time is 42 to 54 hours; the temperature of the second fermentation stage is 41-55 ℃, the humidity is 80-90%, and the time is 42-54 h; the temperature of the third fermentation stage is 20-40 ℃, the humidity is 65-75%, and the time is 42-54 h.

Preferably, the temperature of the first fermentation stage in the temperature-changing combined fermentation process of the bacillus amyloliquefaciens SS0813 or the bacillus bailii MZ1030 is 25 ℃, the humidity is 70%, and the time is 48 hours; the temperature of the second fermentation stage is 50 ℃, the humidity is 85%, and the time is 48 hours; the temperature of the third fermentation stage is 25 ℃, the humidity is 70%, and the time is 48 hours;

The temperature of the first fermentation stage in the temperature-changing combination fermentation process of the bacillus aryabhattai GL0525 is 35 ℃, the humidity is 70%, and the time is 48 hours; the temperature of the second fermentation stage is 45 ℃, the humidity is 85%, and the time is 48 hours; the temperature of the third fermentation stage is 35 ℃, the humidity is 70%, and the time is 48 hours.

Preferably, the culture medium containing fermented grains comprises the following components: 15-25 g/L of fermented grains, 3-8 g/L of glucose and the balance of water.

Preferably, the water content of the cigar tobacco leaves after the bacillus bacterial liquid is sprayed is 25-35%, and the water content of the cigar tobacco leaves after the water is balanced is 19-21%.

Preferably, the inoculation amount of the bacillus liquid is 20% -30%.

Preferably, the OD value of the bacillus bacteria liquid is 0.5-1.5.

The invention also provides a microbial inoculum for fermenting cigar tobacco leaves, which comprises bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SS0813, bacillus bailii (Bacillus velezensis) MZ1030 or bacillus aryabhattai (Priestia aryabhattai) GL0525;

the bacillus amyloliquefaciens SS0813 is deposited in China center for type culture Collection of university of Wuhan with the deposit number: CCTCC NO: M20232418;

The bacillus belicus MZ1030 is deposited with the university of armed university chinese typical culture collection with the deposit number: CCTCC NO: M20232416;

The bacillus aryabhattai GL0525 is deposited with the China center for type culture Collection of university of Wuhan with the deposit number: CCTCCNO: M20232417.

The invention also provides the application of the method or the microbial inoculum in reducing the irritation and miscellaneous gases of cigar tobacco leaves and improving the fragrance of the cigar tobacco leaves.

Compared with the prior art, the invention has the following beneficial effects:

The cigar tobacco is fermented by using the bouquet microorganism, namely bacillus amyloliquefaciens SS0813, bacillus bailii MZ1030 or bacillus aryaensis GL0525, so that the cigar tobacco with obvious bouquet can be obtained. Cigar tobacco leaves fermented by the bacillus amyloliquefaciens SS0813, the bacillus bailii MZ1030 or the bacillus aryaensis GL0525 are obvious in bouquet, aroma components are increased, the pungent aroma components such as nicotine are obviously reduced, and the cigar is more mellow to suck.

Compared with constant-temperature fermentation, the temperature-variable combined fermentation can obviously improve the content of aroma components of cigar tobacco leaves, and simultaneously obviously improve the irritation and miscellaneous gases on the premise of keeping the aroma richness and the aroma quantity to the greatest extent. In the early stage of fermentation, low-temperature fermentation is adopted to facilitate microorganism growth and metabolism and provide precursor conditions for subsequent secretion of enzyme activity and generation of aroma components, and high-temperature high-humidity fermentation is adopted in the middle stage of fermentation to facilitate enzyme activity to degrade proteins, starch, alkaloids and the like to generate micromolecular saccharides and volatile nitrogen-containing compounds, so that the irritation and miscellaneous gases of tobacco leaves are reduced, and simultaneously, maillard reaction can be promoted to generate pyrazine, furan and other aroma components by high-temperature fermentation. The temperature and humidity in the later fermentation stage are reduced again, so that the phenomenon of core burning of a fermented tobacco pile is avoided, and the quality of cigar tobacco leaves is damaged.

Drawings

FIG. 1 is a photograph of colony morphology of strain SS0813, strain MZ1030 and strain GL0525 on a plate;

FIG. 2 is a gram of strain SS0813, strain MZ1030 and strain GL 0525;

FIG. 3 is a scanning electron micrograph of strain SS0813, strain MZ1030 and strain GL 0525;

FIG. 4 shows the carotenoid degradation product content of cigar leaves after different treatments before and after fermentation;

FIG. 5 shows phenylalanine-converted product content in cigar tobacco leaves after and before fermentation;

FIG. 6 shows the content of degradation products of cembrane compounds in cigar leaves after different treatments before and after fermentation;

FIG. 7 shows chlorophyll degradation product content in cigar leaves after different treatments before and after fermentation;

FIG. 8 shows the content of volatile nicotine substances in cigar leaves after different treatments before and after fermentation;

Fig. 9 shows the content of other volatile flavour metabolites in cigar leaves under different treatments before and after fermentation.

Description of biological preservation

The bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SS0813 provided by the invention is preserved in China center for type culture Collection of university of Wuhan, and the preservation number is: the CCTCC NO is M20232418, the preservation time is 2023, 12 months and 01 days, and the preservation address is No.299 of Wuhan, hubei province.

The bacillus belicus (Bacillus velezensis) MZ1030 provided by the invention is preserved in China center for type culture Collection of university of Wuhan, and the preservation number is: the CCTCC NO is M20232416, the preservation time is 2023, 12 months and 01 days, and the preservation address is No. 299 of Wuhan, hubei province.

The bacillus aryabhattai (Priestia aryabhattai) GL0525 provided by the invention is preserved in China center for type culture Collection of university of Wuhan, and the preservation number is: the CCTCC No. M20232417 has the preservation time of 2023, 12 months and 01 days, and the preservation address is No. 299 of Wuhan, hubei province.

Detailed Description

The invention provides a method for processing cigar tobacco leaves by utilizing bacillus fermentation, which comprises the following steps:

1) Inoculating the bacillus seed liquid into a culture medium containing fermented grains according to an inoculum size of 1% -5% (v/v), and culturing for 12-14 h at 30-40 ℃ to obtain bacillus liquid;

2) Spraying bacillus liquid to cigar tobacco leaves, and fermenting at variable temperature to balance water.

In the invention, bacillus seed liquid is inoculated into a culture medium containing fermented grains according to the inoculum size of 1% -5% (v/v), and is cultured for 12-14 h at 30-40 ℃ to obtain bacillus bacterial liquid. The bacillus is preferably bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SS0813, bacillus bailii (Bacillus velezensis) MZ1030 or bacillus aryabhattai (Priestia aryabhattai) GL0525; the bacillus amyloliquefaciens SS0813 is deposited in China center for type culture Collection of university of Wuhan with the deposit number: CCTCC NO: M20232418; the bacillus belicus MZ1030 is deposited with the university of armed university chinese typical culture collection with the deposit number: CCTCCNO: M20232416; the bacillus aryabhattai GL0525 is deposited with the China center for type culture Collection of university of Wuhan with the deposit number: CCTCCNo: m20232417; the seed liquid is obtained by inoculating bacillus to an LB culture medium for culture, wherein the culture condition is preferably 32-42 ℃, and the rotation speed is 200-240 rpm for 10-15 h; further preferably 34-40 ℃, and the rotating speed is 210-230 rpm, and culturing for 11-14 h; the inoculation amount of the seed liquid is preferably 2-4%; the culture medium containing fermented grains comprises the following components: 15-25 g/L of fermented grains, 3-8 g/L of glucose and the balance of water; the consumption of the fermented grains is preferably 18-22 g/L; the dosage of the glucose is preferably 4-7 g/L; the culture temperature is preferably 32-38 ℃, and the culture time is preferably 12.5-13.5 h.

In the invention, bacillus bacterial liquid is sprayed to cigar tobacco leaves, and the water is balanced after temperature-changing combined fermentation is carried out. The OD value of the bacillus liquid is preferably 0.5-1.5, and more preferably 0.8-1.2; the inoculation amount of the bacillus liquid is preferably 20-30% (v: volume of bacillus liquid mL/m: mass of cigar tobacco leaves g), and more preferably 22-28%; the water content of cigar tobacco leaves after the bacillus bacterial liquid is sprayed is preferably 25-35%, and more preferably 28-32%; the variable temperature combined fermentation is carried out for 1 to 6 fermentation periods, wherein 1 fermentation period is divided into three fermentation stages, the temperature of the first fermentation stage is preferably 20 to 40 ℃, more preferably 22 to 38 ℃, the humidity is preferably 65 to 75%, more preferably 67 to 72%, and the time is preferably 42 to 54 hours, more preferably 45 to 51 hours; the temperature in the second fermentation stage is preferably 41 to 55 ℃, more preferably 43 to 52 ℃, the humidity is preferably 80 to 90%, more preferably 82 to 88%, the time is preferably 42 to 54 hours, more preferably 45 to 51 hours; the temperature in the third fermentation stage is preferably 20 to 40 ℃, more preferably 22 to 38 ℃, the humidity is preferably 65 to 75%, more preferably 67 to 72%, the time is preferably 42 to 54 hours, more preferably 45 to 51 hours; the temperature of the first fermentation stage in the temperature-changing combination fermentation process of the bacillus amyloliquefaciens SS0813 or the bacillus bailii MZ1030 is 25 ℃, the humidity is 70%, and the time is 48 hours; the temperature of the second fermentation stage is 50 ℃, the humidity is 85%, and the time is 48 hours; the temperature of the third fermentation stage is 25 ℃, the humidity is 70%, and the time is 48 hours; the temperature of the first fermentation stage in the temperature-changing combination fermentation process of the bacillus aryabhattai GL0525 is 35 ℃, the humidity is 70%, and the time is 48 hours; the temperature of the second fermentation stage is 45 ℃, the humidity is 85%, and the time is 48 hours; the temperature of the third fermentation stage is 35 ℃, the humidity is 70%, and the time is 48 hours; the water content of the cigar tobacco leaves after balancing the water is preferably 19-21%, and more preferably 19.5-20.5%.

The invention also provides a microbial inoculum for fermenting cigar tobacco leaves, which comprises bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SS0813, bacillus bailii (Bacillus velezensis) MZ1030 or bacillus aryabhattai (Priestia aryabhattai) GL0525; the bacillus amyloliquefaciens SS0813 is deposited in China center for type culture Collection of university of Wuhan with the deposit number: CCTCC NO: M20232418; the bacillus belicus MZ1030 is deposited with the university of armed university chinese typical culture collection with the deposit number: CCTCCNO: M20232416; the bacillus aryabhattai GL0525 is deposited with the China center for type culture Collection of university of Wuhan with the deposit number: CCTCCNO: m20232417.

The invention also provides the application of the method or the microbial inoculum in reducing the irritation and miscellaneous gases of cigar tobacco leaves and improving the fragrance of the 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

Isolation and identification of the species:

1. Separation of strains: the bacillus amyloliquefaciens SS0813, bacillus bailii MZ1030 or bacillus aryaensis GL0525 are obtained by separation and purification in fermented grains. The method comprises the following steps:

(1) 1-5g of fermented grains are taken and placed in 50-100mL of sterile water, and the fermented grains are oscillated for 20-40min under the conditions of 30-40 ℃ and 120-220r/min to obtain bacterial suspension. 1mL of the bacterial suspension is taken and placed in a 9mL sterile water test tube, so as to obtain 10 ^-1 of diluted bacterial suspension, and the diluted bacterial suspension of 10 ^-2、10-3、10^-4、10^-5、10^-6 is prepared in sequence by analogy. The diluted bacterial suspension of 0.1mL 10 ^-4-10^-6 is sucked and evenly coated on beef extract peptone culture medium and is inversely cultured for 24 hours in a 30 ℃ incubator.

(2) According to different morphological characteristics of the bacterial colonies on the dilution coating plate, selecting single bacterial colonies with good growth vigor and dominant position for microscopic examination, carrying out repeated streak purification on the bacterial colonies subjected to microscopic examination, and then inoculating the bacterial colonies into a corresponding plate culture medium for preservation at 4 ℃ for standby identification.

2. Morphological identification

Selecting single bacterial colony after multiple times of purification for gram staining, and primarily judging that the bacterial colony is gram-positive bacillus when the bacterial colony is obviously blue-purple and short-rod-shaped under a microscope; further picking single bacterial colony after multiple times of purification, carrying out overnight enrichment culture, centrifuging bacterial suspension to obtain precipitated bacterial colony, carrying out freeze-drying treatment on the precipitated bacterial colony, observing bacterial by using a scanning electron microscope, and determining that the bacterial colony under the scanning electron microscope presents an obvious short rod-shaped structure and is gram-positive bacillus (shown in figures 1, 2 and 3).

3. Physiological and biochemical test identification

And selecting a single colony after multiple purification for a physiological and biochemical test, and further determining that the strain is bacillus according to a physiological and biochemical test result table 1.

TABLE 1 physiological and biochemical test results of strains SS0813, MZ1030 and GL0525

4. Molecular biological identification

After culturing bacillus amyloliquefaciens strain SS0813, bacillus belicus strain MZ1030 and bacillus aryaensis GL0525 in beef extract peptone culture medium for 48 hours, extracting bacterial group DNA, carrying out PCR amplification on bacterial genome DNA after extraction, recovering PCR products by using AxyPrep DNA gel recovery kit, taking the PCR products after purification of each strain, and carrying out DNA sequencing by using a sequencer ABI3730-XL (as shown in table 2).

TABLE 2 16S rDNA sequences of the strains

And (3) comparing the spliced sequence file with data in an NCBI 16S database by using an NCBI Blast program to obtain species information with the maximum sequence similarity with a species to be detected, and identifying SS0813, MZ1030 and GL0525 strains as bacillus amyloliquefaciens, bacillus bailii and bacillus aryabhattai respectively.

Example 2

Bacillus amyloliquefaciens SS0813, bacillus bailii MZ1030 or Bacillus aryaensis GL0525 are respectively inoculated into LB broth culture medium, and are cultured for 12 hours overnight at 37 ℃ and 220rpm to obtain seed liquid, and then the seed liquid is transferred into high-quality cigar tobacco fermentation culture medium (20 g/L of fermented grains, 5g/L of glucose and the balance of water, and sterilized for 30 minutes at 115 ℃) to obtain bacterial liquid at 37 ℃.

The bacterial liquid of bacillus amyloliquefaciens SS0813, the bacterial liquid of bacillus bailii MZ1030 or the bacterial liquid of bacillus aryabhattai GL0525 are uniformly diluted to 1.0OD, and then are respectively and uniformly sprayed on cigar tobacco leaves to be fermented in 30 percent of inoculum size (mL/g). The temperature and humidity in constant temperature fermentation are 35 ℃ and 75%, each fermentation period is 6d, and 5 fermentation periods are required to pass through.

The bacterial solution of Bacillus amyloliquefaciens SS0813, bacillus bailii MZ1030 or Bacillus aryabhattai GL0525 was uniformly diluted to 1.0OD, and then uniformly sprayed onto cigar tobacco leaves with 30% inoculum size (mL/g), respectively. The temperature of the first fermentation stage of the bacillus amyloliquefaciens SS0813 or the bacillus bailii MZ1030 in the variable-temperature fermentation is 25 ℃, the humidity is 70%, and the time is 48 hours; the temperature of the second fermentation stage is 50 ℃, the humidity is 85%, and the time is 48 hours; the temperature of the third fermentation stage is 25 ℃, the humidity is 70%, and the time is 48 hours; the temperature of the first fermentation stage of the bacillus aryabhattai GL0525 is 35 ℃, the humidity is 70%, and the time is 48 hours; the temperature of the second fermentation stage is 45 ℃, the humidity is 85%, and the time is 48 hours; the temperature of the third fermentation stage is 35 ℃, the humidity is 70%, and the time is 48 hours; each fermentation period was 6d, requiring a total of 5 fermentation periods.

After the fermentation is finished, the volatile aroma components of cigar tobacco leaves under constant temperature and variable temperature fermentation treatment are subjected to HS-SPME-GC-MS (headspace solid-phase microextraction-gas chromatography mass spectrometry), and the results are shown in Table 3.

TABLE 3 analysis results of volatile aroma components of temperature-constant and temperature-variable fermented cigar leaves under different treatments

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As can be seen from the analysis results of the volatile aroma components of the cigar samples, the Bacillus amyloliquefaciens SS0813, the Bacillus bailii MZ1030 and the Bacillus aryabhattai GL0525 are enriched and cultured in the cigar tobacco fermentation process, and the fermentation mode of temperature-varying fermentation is adopted, so that the enrichment and culture treatment of the Bacillus amyloliquefaciens SS0813, the Bacillus bailii MZ1030 and the Bacillus aryabhattai GL0525 can influence the quality of tobacco, the aroma quantity is sufficient, the irritation and the miscellaneous gas are obviously reduced, and the volatile aroma components are rich. This further illustrates that the combined use of enrichment culture treatment and temperature swing fermentation of Bacillus amyloliquefaciens SS0813, bacillus bailii MZ1030 and Bacillus aryabhattai GL0525 has better technical effects.

Example 3

Bacillus amyloliquefaciens SS0813, bacillus bailii MZ1030 or Bacillus aryaensis GL0525 are respectively inoculated into LB broth culture medium, and are cultured for 12 hours overnight at 37 ℃ and 220rpm to obtain seed liquid, and then the seed liquid is transferred into fermented grain fermentation culture medium (20 g/L of fermented grain, 5g/L of glucose and the balance of water, and sterilized for 30 minutes at 115 ℃) to obtain bacterial liquid at 37 ℃.

The bacterial solutions of bacillus amyloliquefaciens SS0813, bacillus bailii MZ1030 or bacillus aryaensis GL0525 are uniformly diluted to 1.0OD, and then uniformly sprayed on cigar tobacco leaves respectively with 30% of inoculum size (mL/g). The temperature of the first fermentation stage of the bacillus amyloliquefaciens SS0813 or the bacillus bailii MZ1030 in a single fermentation period in the variable-temperature fermentation is 25 ℃, the humidity is 70%, and the time is 48 hours; the temperature of the second fermentation stage is 50 ℃, the humidity is 85%, and the time is 48 hours; the temperature of the third fermentation stage is 25 ℃, the humidity is 70%, and the time is 48 hours. The temperature of the first fermentation stage of the bacillus aryabhattai GL0525 is 35 ℃, the humidity is 70%, and the time is 48 hours; the temperature of the second fermentation stage is 45 ℃, the humidity is 85%, and the time is 48 hours; the temperature of the third fermentation stage is 35 ℃, the humidity is 70%, and the time is 48 hours. A total of 1 fermentation cycle is required.

Fermentation WAs performed with reference to treatment with a fermented grain medium (FT, fermented grain 20g/L, glucose 5g/L, balance water, 115 ℃ C. Sterilized for 30 min) and ultra pure Water (WA) without addition of Bacillus amyloliquefaciens SS0813, bacillus bailii MZ1030 or Bacillus aryabhattai GL0525, and after the fermentation WAs completed, HS-SPME-GC-MS (headspace solid phase microextraction-gas chromatography mass spectrometry) analysis WAs performed on volatile aroma components of cigar tobacco fermentation processes under different fermentation treatments, and the results are shown in Table 4.

Table 41 analysis results of volatile aroma components of cigar tobacco leaves under different fermentation treatment in fermentation periods

As is clear from Table 4, cigar leaves fermented by Bacillus amyloliquefaciens SS0813, bacillus belicus MZ1030 and Bacillus aryaensis GL0525 have faint scent, increased aldehyde ketone substances of licorice fragrance, prominent cigar leaf style, slightly reduced nicotine content, slightly reduced irritation of the obtained cigar leaf, and increased balance of aroma-causing components.

Example 4

The fermentation period in example 3 was replaced with 2 fermentation periods in total, and the other treatments were the same, and the experimental results are shown in table 5.

Table 52 results of analysis of volatile aroma components of cigar leaves under different fermentation treatment in fermentation periods

As is clear from Table 5, the cigar leaves fermented by Bacillus amyloliquefaciens SS0813, bacillus bailii MZ1030 and Bacillus aryabhattai GL0525 were slightly reduced in nicotine content, and the obtained cigar leaves were slightly reduced in irritation and further increased in balance of aroma-generating components.

Example 5

The fermentation cycle in example 3 was replaced with a total of 3 fermentation cycles, all other treatments being identical. The experimental results are shown in Table 6.

Table 6 3 analysis results of volatile aroma components of cigar tobacco leaves under different fermentation treatment in fermentation periods

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As is clear from Table 6, the cigar leaves fermented by Bacillus amyloliquefaciens SS0813, bacillus belicus MZ1030 and Bacillus aryaensis GL0525 have slightly increased aldehyde and ketone substances with faint scent and licorice scent, the nicotine content is reduced, the irritation of the obtained cigar tobacco leaves is reduced, and the balance and coordination of aroma-causing components are obviously increased.

Example 6

The fermentation cycle in example 3 was replaced with a total of 4 fermentation cycles, all other treatments being identical. The experimental results are shown in Table 7.

Table 7 4 analysis results of volatile aroma components of cigar tobacco leaves under different fermentation treatment in fermentation periods

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As is clear from Table 7, cigar leaves fermented by Bacillus amyloliquefaciens SS0813, bacillus belicus MZ1030 and Bacillus aryaensis GL0525 have the advantages of obviously increasing aldehyde ketone substances with faint scent and licorice scent, obviously highlighting the style of cigar leaves, reducing the nicotine content, reducing the irritation of the obtained cigar leaves, and further obviously increasing the balance and coordination of aroma components.

Example 7

The fermentation cycle in example 3 was replaced with a total of 5 fermentation cycles, all other treatments being identical. The experimental results are shown in Table 8.

Table 85 results of analysis of volatile aroma components of cigar leaves under different fermentation treatment in fermentation periods

As can be seen from Table 8, cigar leaves fermented by Bacillus amyloliquefaciens SS0813, bacillus belicus MZ1030 and Bacillus aryaensis GL0525 have the advantages of obviously increasing aldehyde ketone substances with faint scent and licorice scent, obviously highlighting the style of cigar leaves, obviously reducing the nicotine content, obviously reducing the irritation of the obtained cigar leaves, and further obviously increasing the balance and coordination of aroma components.

Example 8

The fermentation cycle in example 3 was replaced with a total of 6 fermentation cycles, all other treatments being identical. The experimental results are shown in Table 9.

Table 96 results of analysis of volatile aroma components of cigar tobacco leaves under different fermentation treatment of fermentation periods

As is clear from Table 9, cigar leaves fermented by Bacillus amyloliquefaciens SS0813, bacillus belicus MZ1030 and Bacillus aryaensis GL0525 have the advantages of obviously increasing aldehyde ketone substances with faint scent and licorice scent, obviously highlighting the style of cigar leaves, obviously reducing the nicotine content, obviously reducing the irritation of the obtained cigar leaves, and further obviously increasing the balance and coordination of aroma-causing components.

Example 9

The cigar tobacco leaves after 6 fermentation periods and the cigar tobacco leaves before fermentation in the example 8 are subjected to qualitative and quantitative analysis on aroma components of the tobacco leaves by using an Agilent8890-7000D gas chromatograph-mass spectrometer.

The specific detection method comprises the following steps: the pretreatment of the fermented cigar tobacco sample adopts a headspace solid-phase microextraction method, 0.5g of cigar tobacco sample is weighed and added into a headspace bottle containing 8mL of saturated sodium chloride solution and 1 mu L of 2-phenethyl acetate internal standard solution. The headspace vial was then placed in a 70 ℃ water bath and incubated with stirring for 20 minutes. After stabilization, the extraction needle was pushed out, the adsorbed volatiles were extracted for 35 minutes, and then immediately resolved by GC-MS for 5 minutes. In an HP-5MS capillary column (30 m 0.25mm 0.25 μm), VOCs were separated under the following GC conditions: the temperature of the sample inlet is 250 ℃; carrier gas, helium (99.99% purity); the flow rate was 0.8mL/min. The sample is introduced without splitting. The temperature-raising program was carried out under the following conditions: the initial temperature was 60℃for 2min, then at a rate of 3℃per minute to 180℃for 2min, then at a rate of 6℃per minute to 260℃and 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 ℃; mass scan range is 35-450m/z.

The qualitative and quantitative conditions of the aroma substances are as follows: the GC-MS data file was deconvolved using Quantitative Analysis b.09.00 to identify isolated components of the compound. The deconvolved components are then compared to the mass spectra of the target compounds and the retention time of the normal paraffins is used as a reference. These compounds were initially identified by reference to retention times provided in the NIST 20 database. Volatile compounds were quantified using an internal standard method using phenethyl 2-acetate at a concentration of 128.75. Mu.g/. Mu.L as an internal standard.

Experimental results: as shown in fig. 4-9. From the analysis of fig. 4-9, it can be seen that the aroma components of the tobacco leaves in different treatment groups are measured together to obtain six major components of carotenoid degradation products, phenylalanine conversion products, cembrane degradation products, chlorophyll degradation products, volatile alkaloids and other volatile flavor metabolites, wherein the carotenoid substances or degradation products thereof can provide sweet aroma, fruit aroma, flower aroma and costustoot in the combustion and smoking process of the tobacco leaves, the phenylalanine conversion products are important components of the aroma, the tobacco flower aroma, nut aroma and fruit taste can be increased, the main degradation products of cembrane degradation are solanone, the cembrane degradation products have unique aroma, the volatile alkaloids have significant influence on the flavor and smoking experience of cigar tobacco leaves, the overall flavor, the strength and the satisfaction of cigar are significant, and the chlorophyll and degradation products thereof have faint scent, can reduce the irritation of smoke, improve the aroma and the taste of the smoke and make the smoke softer.

As can be seen from the above examples, when the bacillus amyloliquefaciens SS0813, the bacillus belicus MZ1030 or the bacillus aryabhattai GL0525 are added and combined with the variable-temperature fermentation treatment, the content of various aroma-causing substances in cigar tobacco leaves can be stably improved, the content of the pungent aroma components such as nicotine and the like is obviously reduced, and the cigar is more mellow in smoking. Provides technical reference for improving and enhancing the comprehensive quality and industrial availability of domestic cigar tobacco raw materials.

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 (10)

1. A method for fermenting cigar tobacco leaves by using bacillus, which is characterized by comprising the following steps:

1) Inoculating the bacillus seed liquid into a culture medium containing fermented grains according to an inoculum size of 1% -5% (v/v), and culturing for 12-14 h at 30-40 ℃ to obtain bacillus liquid;

2) Spraying bacillus liquid to cigar tobacco leaves, and fermenting at variable temperature to balance water.

2. The method of claim 1, wherein the bacillus is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SS0813, bacillus bailii (Bacillus velezensis) MZ1030, or bacillus alnicosum (Priestia aryabhattai) GL0525;

the bacillus amyloliquefaciens SS0813 is deposited in China center for type culture Collection of university of Wuhan with the deposit number: CCTCC NO: M20232418;

The bacillus belicus MZ1030 is deposited with the university of armed university chinese typical culture collection with the deposit number: CCTCC NO: M20232416;

The bacillus aryabhattai GL0525 is deposited with the China center for type culture Collection of university of Wuhan with the deposit number: CCTCCNO: M20232417.

3. The method according to claim 1, wherein the temperature-variable combined fermentation is carried out for 1 to 6 fermentation periods, the 1 fermentation period is divided into three fermentation stages, the temperature of the first fermentation stage is 20 to 40 ℃, the humidity is 65 to 75%, and the time is 42 to 54 hours; the temperature of the second fermentation stage is 41-55 ℃, the humidity is 80-90%, and the time is 42-54 h; the temperature of the third fermentation stage is 20-40 ℃, the humidity is 65-75%, and the time is 42-54 h.

4. The method according to claim 3, wherein the temperature of the first fermentation stage in the temperature swing combination fermentation process of bacillus amyloliquefaciens SS0813 or bacillus bailii MZ1030 is 25 ℃, the humidity is 70%, and the time is 48 hours; the temperature of the second fermentation stage is 50 ℃, the humidity is 85%, and the time is 48 hours; the temperature of the third fermentation stage is 25 ℃, the humidity is 70%, and the time is 48 hours;

The temperature of the first fermentation stage in the temperature-changing combination fermentation process of the bacillus aryabhattai GL0525 is 35 ℃, the humidity is 70%, and the time is 48 hours; the temperature of the second fermentation stage is 45 ℃, the humidity is 85%, and the time is 48 hours; the temperature of the third fermentation stage is 35 ℃, the humidity is 70%, and the time is 48 hours.

5. The method according to claim 1, wherein the fermented grain-containing medium comprises the following components: 15-25 g/L of fermented grains, 3-8 g/L of glucose and the balance of water.

6. The method of claim 1, wherein the water content of the cigar leaf after the bacillus bacteria liquid is sprayed is 25% -35%, and the water content of the cigar leaf after the water is balanced is 19% -21%.

7. The method of claim 1, wherein the bacillus bacteria solution is inoculated in an amount of 20% to 30%.

8. The method of claim 1, wherein the bacillus bacteria liquid has an OD value of 0.5 to 1.5.

9. A microbial agent for fermenting cigar tobacco leaves, which is characterized in that the microbial agent comprises bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SS0813, bacillus bailii (Bacillus velezensis) MZ1030 or bacillus aryabhattai (Priestia aryabhattai) GL0525;

the bacillus amyloliquefaciens SS0813 is deposited in China center for type culture Collection of university of Wuhan with the deposit number: CCTCC NO: M20232418;

The bacillus belicus MZ1030 is deposited with the university of armed university chinese typical culture collection with the deposit number: CCTCC NO: M20232416;

The bacillus aryabhattai GL0525 is deposited with the China center for type culture Collection of university of Wuhan with the deposit number: CCTCCNo: m20232417.

10. Use of the method of any one of claims 1 to 8 or the microbial agent of claim 9 for reducing cigar leaf irritation and miscellaneous gases and improving cigar leaf aroma.