A method for preparing roasted soybean extract for pretreatment of cigar-type reconstituted tobacco raw materials, its products and applications

Abstract

This invention relates to a method for preparing a soybean extract for the pretreatment of cigar-type reconstituted tobacco raw materials, as well as its products and applications. The method includes the following steps: roasting soybean raw materials at 150-210℃ for 5-10 minutes; then roasting at 220-250℃ for 7-12 minutes; and finally roasting at 260-300℃ for 12-20 minutes to obtain roasted soybeans; pulverizing the roasted soybeans and then hot-extracting them to obtain an extract; and then subjecting the extract to sedimentation to remove impurities. In the soybean extract obtained by this method, large molecular carbon and nitrogen compounds such as starch decompose upon heating to generate a large number of small molecular sugars and amino acids, and react to produce a large number of aroma precursors such as glycoamino condensates. These aroma precursors serve as substrates for microorganisms during tobacco fermentation, making the nutty, roasted, sweet, and bean aromas of the fermented tobacco more prominent, further enhancing the flavor and aroma of the fermented tobacco.

Description

Technical Field

[0001] This invention belongs to the field of tobacco processing technology, specifically relating to a method for preparing roasted soybean extract for pretreatment of cigar-type reconstituted tobacco raw materials, its products, and applications. Background Technology

[0002] With the continuous development of the domestic economy and the rapid growth of emerging consumer groups, cigar consumption is gradually gaining popularity in economically developed regions and tourist cities in China. Although the domestic cigar market has initially taken shape, it is still in its initial stage, with small sales volume and a low level of development. Domestic cigar brands are still mainly low-end and lack competitiveness. There are currently no reconstituted tobacco products specifically designed for semi-machine-made cigars, machine-made cigars, and cigar-style flue-cured cigars. Cigar raw materials for reconstituted tobacco are relatively scarce; therefore, some Burley tobacco fragments are used as raw materials for reconstituted tobacco to enhance its aroma's "body" and "base" notes.

[0003] Burley tobacco, originally from the United States, has a relatively thick midrib, high moisture content, thin and light leaves, a loose but not coarse tissue structure, high elasticity, high filling power, strong smoldering and fire retention, and good absorption capacity. It is mainly used as a raw material for blended cigarettes. Although the production and industrial use of Burley tobacco is relatively inexpensive, its low sugar content and excessively high nitrogen compound content result in a strong ammonia and bitter taste when smoked, leading to cautious use in actual industrial production. Therefore, how to consume the ammonia, protein, and excessive alkaline compounds in Burley tobacco, remove the spiciness and irritation, enhance the natural harmony of the aroma, and compensate for the natural bean-like characteristics of tobacco, thereby realizing its application in cigar-type reconstituted tobacco, is of great practical significance.

[0004] Soybeans are an annual herbaceous plant, and their extracts contain the original aroma components of soybeans. These extracts offer advantages such as a naturally harmonious aroma, complementing the bean aroma of tobacco and highlighting its unique characteristics, making them a valuable flavoring ingredient for cigarette flavoring and the development of new tobacco products. While soybeans themselves contain abundant components, current soybean extract products are relatively complex in composition, resulting in relatively low utilization rates in cigarettes. Therefore, improving the effective components in soybean extracts to achieve cost-effective improvement of tobacco leaf quality is of great practical significance. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing roasted soybean extract, as well as its products and applications.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing roasted soybean extract for pretreatment of cigar-type reconstituted tobacco raw materials, the preparation method comprising the following steps:

[0008] (1) First bake the soybean raw material at 150-210℃ for 5-10 minutes, then bake at 220-250℃ for 7-12 minutes, and finally bake at 260-300℃ for 12-20 minutes to obtain baked soybeans;

[0009] (2) After grinding the roasted soybeans, hot extraction was carried out to obtain the extract;

[0010] (3) The extract is subjected to sedimentation to remove impurities, and the roasted soybean extract is obtained.

[0011] The 150-210℃ range can be selected from 150℃, 160℃, 170℃, 180℃, 190℃, 200℃, 210℃, etc., and the 5-10min range can be selected from 5min, 6min, 7min, 8min, 9min, 10min, etc.

[0012] The 220-250℃ range can be selected from 220℃, 230℃, 240℃, 250℃, etc., and the 7-12min range can be selected from 7min, 8min, 9min, 10min, 11min, 12min, etc.

[0013] The 260-300℃ range can be selected from 260℃, 270℃, 280℃, 290℃, 300℃, etc., and the 12-20min range can be selected from 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min, 20min, etc.

[0014] Other specific point values ​​within the above range can be selected, and will not be elaborated on here.

[0015] The method for preparing roasted soybean extract disclosed in this invention creatively employs a segmented roasting process for pretreatment of soybean raw materials. This process causes the large-molecule carbon and nitrogen compounds such as starch in soybeans to decompose upon heating, generating a large number of small-molecule sugars and amino acids. These react to produce a large number of aroma precursors, such as glycoamino condensates. These aroma precursors serve as substrates for microorganisms during tobacco fermentation, resulting in more prominent nutty, roasted, sweet, and bean aromas in the fermented tobacco leaves, further enhancing their flavor and aroma. Compared to single-stage roasting, segmented roasting better increases the richness of aroma precursors, preserving not only small-molecule aroma substances but also flavor peptides, leading to a more significant improvement in flavor and aroma.

[0016] Preferably, the moisture content of the soybean raw material in step (1) is 10-15%, such as 10%, 11%, 12%, 13%, 14%, 15%, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0017] Preferably, the moisture content of the roasted soybeans is 3%-7%, such as 3%, 4%, 5%, 6%, 7%, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0018] Preferably, the roasted soybeans in step (2) are ground to obtain soybean powder with a mesh size of 200-300 (e.g., 200 mesh, 220 mesh, 240 mesh, 260 mesh, 280 mesh, 300 mesh, etc.). The material temperature during grinding is below 45℃ (e.g., 45℃, 42℃, 40℃, 38℃, 36℃, 34℃, 32℃, 30℃, etc.). Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0019] Preferably, the extractant for the hot extraction is water.

[0020] Preferably, the mass ratio of roasted soybeans to extractant is 1:(3-5), such as 1:3, 1:3.5, 1:4, 1:4.5, 1:5, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0021] Preferably, the temperature of the hot extraction is 80-100℃ (e.g., 80℃, 82℃, 84℃, 86℃, 88℃, 90℃, 92℃, 94℃, 96℃, 98℃, 100℃, etc.), and the hot extraction time is 10-30min (e.g., 10min, 12min, 14min, 16min, 18min, 20min, 25min, 27min, 30min, etc.). Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0022] Preferably, the hot extraction is accompanied by a stirring process, with a stirring speed of 600-800 rpm, such as 600 rpm, 620 rpm, 640 rpm, 680 rpm, 700 rpm, 720 rpm, 740 rpm, 760 rpm, 780 rpm, 800 rpm, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0023] Preferably, step (3) involves sedimentation and impurity removal at a temperature of 4-7℃ (e.g., 4℃, 5℃, 6℃, 7℃, etc.) for a sedimentation time of 30-45 min (e.g., 30 min, 32 min, 34 min, 36 min, 38 min, 40 min, 42 min, 45 min, etc.), followed by collection of the supernatant. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0024] Preferably, after sedimentation and impurity removal, the mixture is further concentrated at a temperature of 60-65℃, such as 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0025] Preferably, the density of the concentrated roasted soybean extract is 1.0-1.3 g / cm³. 3 For example, 1.0 g / cm³ 3 1.1g / cm 3 1.2g / cm 3 1.3g / cm 3 Other specific point values ​​within this range can also be selected, and will not be elaborated on here.

[0026] In a second aspect, the present invention provides a roasted soybean extract prepared according to the preparation method described in the first aspect.

[0027] Thirdly, the present invention provides the application of the roasted soybean extract according to the second aspect in tobacco fermentation.

[0028] Fourthly, the present invention provides a method for fermenting tobacco leaves, the fermentation method comprising the following steps:

[0029] The compound enzyme preparation, the roasted soybean extract described in the second aspect, and the tobacco raw material are mixed and fermented.

[0030] The compound enzyme preparation is obtained by fermentation of compound bacteria, which includes Bacillus velezensis DF-08 strain with accession number CCTCCNo: M 2023456 and Pantoea sp. B-3 strain with accession number CCTCCNO: M 20221165.

[0031] This invention creatively combines soybean extract and a compound enzyme preparation to enhance tobacco leaf quality through a fermentation process. During fermentation, the soybean extract organically integrates with tobacco leaf components, creating a unique environment that increases the number and diversity of microbial communities on the tobacco leaf surface. This provides a substrate for microbial catalysis of the degradation of macromolecules such as starch, protein, and pectin into smaller molecules, thus improving tobacco leaf quality. Furthermore, this invention creatively obtains specific compound enzyme preparations by culturing and fermenting strains DF-08 and B-3. These enzymes degrade macromolecular organic compounds within the tobacco leaves, producing aroma compounds. This increases the aroma content of the tobacco leaves while reducing off-flavors and irritation, significantly improving sensory quality. It also effectively reduces the sugar content in the tobacco leaves, increasing smoke concentration and body, resulting in a more prominent sweet, woody, and bean-like aroma in Burley tobacco, a full-bodied smoke, and a high smoke concentration and richness.

[0032] Preferably, the preparation method of the compound enzyme preparation includes the following steps:

[0033] The activated compound bacteria were inoculated into a culture medium for fermentation to obtain a fermentation broth. The fermentation broth was centrifuged, and the supernatant was collected to obtain the compound enzyme preparation.

[0034] Specifically, DF-08 and B-3 strains were inoculated into the culture medium and activated and cultured sequentially to obtain seed liquid. The DF-08 seed liquid and B-3 strain seed liquid were inoculated into the culture medium at a volume ratio of (2-3):(2-3) for expansion culture to obtain fermentation broth. The fermentation broth was centrifuged, and the supernatant was taken to obtain the compound enzyme preparation.

[0035] Among them, (2-3) can be selected from 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, etc. Other specific point values ​​within this range can be selected, which will not be elaborated here.

[0036] Preferably, the fermentation temperature after mixing with tobacco raw materials is 35-45℃ (e.g., 35℃, 36℃, 37℃, 38℃, 39℃, 10℃, 41℃, 42℃, 43℃, 44℃, 45℃, etc.), the fermentation humidity is 60-70% (60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, etc.), and the fermentation time is 72-168h (72h, 80h, 85h, 90h, 95h, 100h, 120h, 140h, 160h, 168h, etc.). Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0037] Preferably, the dry weight of the roasted soybean extract is 5-15% of the dry weight of the tobacco raw material, such as 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0038] Preferably, the dry weight of the compound enzyme preparation is 0.01-1% of the dry weight of the tobacco raw material, such as 0.01%, 0.05%, 0.1%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0039] Preferably, the fermentation process is followed by inactivation treatment.

[0040] Preferably, the inactivation treatment temperature is 65-95℃ (e.g., 65℃, 67℃, 69℃, 71℃, 73℃, 75℃, 80℃, 85℃, 90℃, 95℃, etc.), and the inactivation treatment time is 8-15min (e.g., 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, etc.). Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0041] Fifthly, the present invention provides a cigar-shaped reconstituted tobacco leaf, the cigar-shaped reconstituted tobacco leaf comprising tobacco leaves treated according to the fermentation treatment method described in the fourth aspect.

[0042] Preferably, the tobacco leaves are Burley tobacco.

[0043] Preferably, the fermented tobacco leaves in the cigar-shaped reconstituted tobacco leaves have a mass percentage of 20-30%, such as 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0044] Preferably, the method for preparing cigar-shaped reconstituted tobacco leaves includes: mixing fermented tobacco leaves with other tobacco raw materials (flue-cured tobacco stems, flue-cured tobacco fragments, etc.), and then preparing cigar-shaped reconstituted tobacco leaves through processes such as extraction, solid-liquid separation, papermaking, coating, and drying.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The method for preparing roasted soybean extract disclosed in this invention creatively employs a segmented roasting process for pretreatment of soybean raw materials. This process causes the large-molecule carbon and nitrogen compounds such as starch in soybeans to decompose upon heating, generating a large number of small-molecule sugars and amino acids. These react to produce a large number of aroma precursors, such as glycoamino condensates. These aroma precursors serve as substrates for microorganisms during tobacco fermentation, resulting in more prominent nutty, roasted, sweet, and bean aromas in the fermented tobacco leaves, further enhancing their flavor and aroma. Compared to single-stage roasting, segmented roasting better increases the richness of aroma precursors, preserving not only small-molecule aroma substances but also flavor peptides, leading to a more significant improvement in flavor and aroma.

[0047] The Bacillus velezensis DF-08 strain involved in this invention is classified and named Bacillus velezensis DF-08. It is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO: M 2023456, deposited on April 3, 2023, and located at Wuhan University, Wuhan, China.

[0048] The Pantoea B-3 strain involved in this invention is classified and named Pantoea sp. B-3. It is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20221165, deposited on July 25, 2022, and located at Wuhan University, Wuhan, China. Attached Figure Description

[0049] Figure 1 This is a diagram illustrating the aroma characteristics of the cigar-shaped reconstituted tobacco prepared in Test Example 6. Detailed Implementation

[0050] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0051] Unless otherwise specified, the reagents and consumables used in the following embodiments were purchased from conventional reagent manufacturers in the art; unless otherwise specified, the experimental methods and techniques used were conventional methods and techniques in the art.

[0052] The DF-08 strain involved in the following examples is classified as Bacillus velezensis DF-08, deposited on April 3, 2023, with accession number CCTCC NO: M 2023456.

[0053] The strain B-3 involved in the following examples is classified as Pantoea sp. B-3, deposited on July 25, 2022, with accession number CCTCC NO: M 20221165.

[0054] The NA medium used in the following examples is: 3.0 g / L beef extract, 10.0 g / L peptone, 5.0 g / L NaCl, 20 g / L agar, pH = 7.9, sterilized at 121°C for 20 min; liquid NA medium is: 3.0 g / L beef extract, 10.0 g / L peptone, 5.0 g / L NaCl, pH = 6.9, sterilized at 121°C for 20 min.

[0055] Example 1

[0056] This embodiment provides a method for preparing roasted soybean extract:

[0057] (1) Select soybeans that are uniform in size, free from mold, and have a moisture content of 12%. Bake the soybeans at 200℃ for 8 minutes until they turn golden yellow. Bake the golden yellow soybeans at 240℃ for 9 minutes until they turn brownish-black. Bake the brownish-black soybeans at 270℃ for 15 minutes until they turn brownish-black. The moisture content of the baked soybeans is 5%.

[0058] (2) The roasted soybeans were pulverized under controlled temperature. The pulverization mesh size was 250 mesh and the material temperature was 40℃ during pulverization to obtain roasted soybean ultrafine powder. The roasted soybean ultrafine powder was extracted by stirring in water at 90℃. The oven-dry mass ratio of roasted soybeans to water was 1:4. The extraction time was 11 min and the stirring speed was 700 rpm to obtain the extract.

[0059] (3) The obtained extract was sealed and allowed to settle at 5°C for 37 minutes. The supernatant was then collected, and the settled impurities were removed. The collected supernatant was then concentrated at 62°C to obtain roasted soybean extract with a density of 1.18 g / cm³. 3 .

[0060] Example 2

[0061] This embodiment provides a method for preparing roasted soybean extract:

[0062] (1) Select soybeans that are uniform in size, free from mold, and have a moisture content of 10%. Bake the soybeans at 160℃ for 10 minutes until they turn golden yellow. Bake the golden yellow soybeans at 220℃ for 12 minutes until they turn brownish-black. Bake the brownish-black soybeans at 260℃ for 19 minutes until they turn brownish-black. The moisture content of the baked soybeans is 6%.

[0063] (2) The roasted soybeans were pulverized under controlled temperature. The pulverization mesh size was 200 mesh and the material temperature was 45℃ during pulverization to obtain roasted soybean ultrafine powder. The roasted soybean ultrafine powder was extracted by stirring in water at 100℃. The oven-dry mass ratio of roasted soybeans to water was 1:5. The extraction time was 12 min and the stirring speed was 600 rpm to obtain the extract.

[0064] (3) The obtained extract was sealed and allowed to settle at 7°C for 30 minutes. The supernatant was then collected, and the settled impurities were removed. The collected supernatant was then concentrated at 60°C to obtain roasted soybean extract with a density of 1.15 g / cm³. 3 .

[0065] Example 3

[0066] This embodiment provides a method for preparing roasted soybean extract:

[0067] (1) Select soybeans that are uniform in size, free from mold, and have a moisture content of 14%. Bake the soybeans at 210℃ for 6 minutes until they turn golden yellow. Bake the golden yellow soybeans at 250℃ for 7 minutes until they turn brownish-black. Bake the brownish-black soybeans at 300℃ for 12 minutes until they turn brownish-black. The moisture content of the baked soybeans is 4%.

[0068] (2) The roasted soybeans were pulverized under controlled temperature. The pulverization mesh size was 300 mesh and the material temperature was 37℃ during pulverization to obtain roasted soybean ultrafine powder. The roasted soybean ultrafine powder was extracted by stirring in water at 80℃. The oven-dry mass ratio of roasted soybeans to water was 1:3. The extraction time was 10 min and the stirring speed was 800 rpm to obtain the extract.

[0069] (3) The obtained extract was sealed and allowed to settle at 4°C for 45 minutes. The supernatant was then collected, and the settled impurities were removed. The collected supernatant was then concentrated at 65°C to obtain roasted soybean extract with a density of 1.20 g / cm³. 3 .

[0070] Comparative Example 1

[0071] This comparative example provides a method for preparing roasted soybean extract, which differs from Example 1 only in that the roasting step (1) is not performed, and the soybean raw material is directly subjected to temperature-controlled pulverization in step (2), while other conditions remain unchanged.

[0072] Comparative Example 2

[0073] This comparative example provides a method for preparing roasted soybean extract, which differs from Example 1 only in step (1), selecting soybeans that are uniform in size, free from mold, and have a moisture content of 12%, and roasting the soybeans at 50°C until the moisture content of the soybeans is 5%, while keeping other conditions unchanged.

[0074] Comparative Example 3

[0075] This comparative example provides a method for preparing roasted soybean extract, which differs from Example 1 only in step (1), selecting soybeans that are uniform in size, free from mold, and have a moisture content of 12%, and roasting the soybeans at 200°C until the moisture content of the soybeans is 5%, while keeping other conditions unchanged.

[0076] Comparative Example 4

[0077] This comparative example provides a method for preparing roasted soybean extract, which differs from Example 1 only in step (1), selecting soybeans that are uniform in size, free from mold, and have a moisture content of 12%, and roasting the soybeans at 240°C until the moisture content of the soybeans is 5%, while keeping other conditions unchanged.

[0078] Comparative Example 5

[0079] This comparative example provides a method for preparing roasted soybean extract, which differs from Example 1 only in step (1), selecting soybeans that are uniform in size, free from mold, and have a moisture content of 12%, and roasting the soybeans at 270°C until the moisture content of the soybeans is 5%, while keeping other conditions unchanged.

[0080] Comparative Example 6

[0081] This comparative example provides a method for preparing roasted soybean extract, which differs from Example 1 only in step (1). Select soybeans that are uniform in size, free from mold, and have a moisture content of 12%. Roast the soybeans at 200°C for 8 minutes, and then roast them at 270°C until the moisture content of the soybeans is 5%. All other conditions remain unchanged.

[0082] Preparation Example 1

[0083] This preparation example provides a method for preparing a complex enzyme preparation:

[0084] (1) Activation of bacterial strains: DF-08 strain and B-3 strain were placed on NA medium and cultured in a biochemical climate chamber at 30°C for 42 hours.

[0085] (2) Preparation of seed culture: In the ultra-clean workbench, scrape off the bacterial cells obtained in step (1) and inoculate them into test tubes (18×180mm) containing 5mL of liquid NA medium. Culture them overnight in a shaker at 30℃ and 180rpm to obtain DF-08 seed culture and B-3 seed culture.

[0086] (3) Expanded culture: The DF-08 seed liquid and B-3 strain seed liquid obtained in step (2) were mixed at a volume ratio of 1:1, and then transferred to a sterile conical flask (250 mL) containing 100 mL liquid NA medium at a ratio of 5 mL bacterial suspension / 100 mL NA medium. The mixture was cultured at 30 °C and 180 rpm until OD600 = 1.8.

[0087] (4) Preparation of compound enzyme preparation: Centrifuge the bacterial solution in step (3) at 8000 rpm and 4℃ for 10 min, take the supernatant and freeze-dry it to obtain the compound enzyme freeze-dried agent.

[0088] Comparative Preparation Example 1

[0089] This comparative preparation example provides a method for preparing a compound enzyme solution. The only difference between this method and preparation example 1 is that in step (3), the expansion culture is inoculated only with DF-08 seed culture, while all other conditions remain unchanged.

[0090] Comparative Preparation Example 2

[0091] This comparative preparation example provides a method for preparing a compound enzyme solution. The only difference between this method and preparation example 1 is that in step (3), the expansion culture is inoculated only with B-3 seed culture, while all other conditions remain unchanged.

[0092] Comparative preparation example 3

[0093] This comparative preparation example provides a method for preparing a compound enzyme solution, which differs from Preparation Example 1 only in that the DF-08 strain is replaced by a commercially available Bacillus belyssus SHBCC D12248 strain of equal mass.

[0094] Application Example 1

[0095] This application example provides a method for fermenting tobacco leaves:

[0096] (1) Weigh 100g of Burley tobacco leaf raw material (moisture content is 12%), weigh 8.2g of roasted soybean extract prepared in Example 1 (dry weight), dilute with water and spray evenly on the surface of tobacco leaves, dilute 0.5g of compound enzyme freeze-drying agent prepared in Example 1 with water and add it to the tobacco leaves sprayed with soybean extract (moisture content of tobacco leaves after treatment is 18%).

[0097] (2) Fermented for 120 hours at a temperature of 43℃ and a humidity of 67%. After fermentation, the mixture was treated at 80℃ for 10 minutes to inactivate the inoculum and the moisture content was adjusted to 12%.

[0098] Application Example 2

[0099] This application example provides a method for fermenting tobacco leaves:

[0100] (1) Weigh 100g of Burley tobacco leaf raw material (moisture content is 12%), weigh 6.25g of roasted soybean extract prepared in Example 2 (dry weight), dilute with water and spray evenly on the surface of tobacco leaves, add 0.3g of compound enzyme freeze-drying agent prepared in Example 1 (dry with water) to the tobacco leaves sprayed with soybean extract (moisture content of tobacco leaves after treatment is 15%).

[0101] (2) Fermented for 144 hours at a temperature of 40℃ and a humidity of 70%. After fermentation, the mixture was treated at 65℃ for 15 minutes to inactivate the inoculum and the moisture content was adjusted to 12%.

[0102] Application Example 3

[0103] This application example provides a method for fermenting tobacco leaves:

[0104] (1) Weigh 100g of Burley tobacco leaf raw material (moisture content is 12%), weigh 10g of dry weight of roasted soybean extract prepared in Example 3, dilute with water and spray evenly on the surface of tobacco leaves, dilute 0.1g of compound enzyme freeze-drying agent prepared in Example 1 and add it to the tobacco leaves sprayed with soybean extract (moisture content of tobacco leaves after treatment is 20%).

[0105] (2) Fermented for 96 hours at 45°C and 65% humidity. After fermentation, the mixture was inactivated at 95°C for 8 minutes and the moisture content was adjusted to 12%.

[0106] Comparative Application Examples 1-6

[0107] This comparative application example provides four methods for fermenting tobacco leaves. The only difference between this method and Application Example 1 is that the roasted soybean extract obtained in Example 1 is replaced by roasted soybean extract obtained in Comparative Examples 1-6 in equal mass, while all other conditions remain unchanged.

[0108] Compare and contrast with example 7-9

[0109] This comparative application example provides three methods for fermenting tobacco leaves. The only difference between this method and application example 1 is that the compound enzyme preparation obtained in preparation example 1 is replaced with the enzyme preparation obtained in comparative preparation examples 1-3, while all other conditions remain unchanged.

[0110] Comparative Application Example 10

[0111] This comparative application example provides a method for fermenting tobacco leaves:

[0112] (1) Weigh 100g of Burley tobacco raw material (moisture content is 12%), dilute 0.5g of the compound enzyme freeze-drying agent prepared in Preparation Example 1 with water and spray it onto the tobacco leaves (moisture content of the tobacco leaves after treatment is 18%).

[0113] (2) Fermented for 120 hours at a temperature of 43℃ and a humidity of 67%. After fermentation, the mixture was treated at 70℃ for 10 minutes to inactivate the inoculum and the moisture content was adjusted to 12%.

[0114] Comparative Application Example 11

[0115] This comparative application example provides a method for improving the flavor of tobacco leaves by using roasted soybean extract as a tobacco flavoring agent:

[0116] Weigh 100g of Burley tobacco raw material (moisture content of 12%), weigh 8.2g of roasted soybean extract prepared in Example 1 (dry weight), dilute with water and spray evenly on the surface of tobacco leaves (moisture content of tobacco leaves after treatment is 12%).

[0117] Test Example 1

[0118] This test case evaluates the sensory quality of Burley tobacco leaves treated with test cases 1-3 and comparative application case 1-11:

[0119] A blank control group was set up, and an equal amount of purified water was sprayed evenly onto the surface of the tobacco leaves using a sprayer at a ratio of 10% of the tobacco leaf weight.

[0120] After balancing the moisture content of the treated Burley tobacco leaves from Application Examples 1-3, Comparative Application Examples 1-11, and the blank control group, they were rolled into single-material cigarettes using a hand-rolling machine and placed in a constant temperature and humidity chamber (temperature 20℃, relative humidity 65%) for 48 hours before sensory quality evaluation was conducted.

[0121] Sensory quality evaluation was conducted in accordance with the relevant evaluation criteria of YC / T415-2011 (Sensory Evaluation Method for Tobacco Products). Eight indicators, including aroma quality, aroma quantity, off-flavors, concentration, irritation, smoothness, aftertaste and strength, were scored (see Table 1). The sensory evaluation scores are shown in Table 2.

[0122] Table 1

[0123]

[0124] Table 2

[0125]

[0126] As shown in Table 2, compared with the blank control group, the treated tobacco leaves showed improvements in aroma quality, aroma quantity, off-flavors, irritation, aftertaste, strength, and smoothness.

[0127] Comparing Application Example 1 with Comparative Application Examples 1-6, it can be seen that the segmented roasting process provided by the present invention can significantly improve the quality of tobacco leaves compared with one-time roasting and other segmented roasting methods, and shows significant improvement in the aroma quality and aroma quantity of tobacco leaves.

[0128] Comparing Application Example 1 with Comparative Application Examples 7-9, it can be seen that the aroma and irritation scores of the mixed-strain fermentation enzyme preparation are significantly improved compared with those of the single-strain fermentation enzyme preparation.

[0129] Comparing Application Example 1 with Comparative Application Examples 10-11, it can be seen that soybean extract and compound enzyme preparation have a synergistic effect in improving the quality of tobacco leaves.

[0130] Test Example 2

[0131] The tobacco leaves treated with the blank control group, application example 1, and comparative application examples 4, 7, 8, and 10 were dried at 50°C, pulverized, and then passed through a 60-mesh sieve for the determination of major chemical components.

[0132] Detection of main chemical components: The determination of total sugar, reducing sugar (mass fraction method), nicotine, chlorine, potassium, total nitrogen, and protein was carried out in accordance with tobacco industry standards YC / T 159-2019 "Determination of water-soluble sugars in tobacco and tobacco products - Continuous flow method", YC / T 468-2021 "Determination of total alkaloids in tobacco and tobacco products - Continuous flow method (potassium thiocyanate)", YC / T 162-2011 "Determination of chlorine in tobacco and tobacco products - Continuous flow method", YC / T 217-2007 "Determination of potassium in tobacco and tobacco products - Continuous flow method", YC / T161-2002 "Determination of total nitrogen in tobacco and tobacco products - Continuous flow method", and YC / T 166-2003 "Determination of total protein content in tobacco and tobacco products".

[0133] Ethanol extraction was used, and starch content was determined by anthrone colorimetric method, referring to "Wang Ruixin, Han Fugen, Yang Suqin, et al. Chemical Quality Analysis Methods for Tobacco [M]. Zhengzhou: Henan Science and Technology Press, 1990".

[0134] Cellulose was determined according to the tobacco industry standard YC / T 347-2010 "Determination of Neutral Detergent Fiber, Acid Detergent Fiber and Acid-washed Lignin in Tobacco and Tobacco Products - Detergent Method"; pectin was determined according to YC / T 346-2010 "Determination of Pectin in Tobacco and Tobacco Products - Ion Chromatography".

[0135] Each sample was repeated 3 times, and the results are shown in Table 3.

[0136] Table 3

[0137]

[0138]

[0139] As shown in Table 3, compared with the blank control group, the application of Example 1, which uses soybean extract and microbial enzymes to co-ferment the tobacco leaves, can effectively reduce the content of macromolecules such as protein, starch, cellulose and pectin in Burley tobacco leaves, reduce the content of nitrogenous compounds, and increase the content of total sugar, reducing sugar, potassium and other substances, resulting in a more balanced chemical composition.

[0140] Test Example 3

[0141] This test case examined the microbial diversity of tobacco leaves treated with the blank control group, Application Example 1, and Comparative Application Examples 10-11.

[0142] Using EZNA Soil DNA was extracted using the kit and amplified by PCR. DNA purity was assessed using a NanoDrop 2000 micro-spectrophotometer. After passing the purity test, the V3–V4 variable regions of the bacteria were amplified using 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACNNGGGTATC TAAT-3'). The PCR products were recovered using 2% agarose gel, mixed, and sent to the Illumina MiSeq platform for gene sequencing. Each sample was performed in triplicate. Bacterial community composition was determined using high-throughput sequencing. The top 10 bacterial genera in terms of relative abundance in the bacterial community of the treated tobacco leaf samples were the dominant genera: *Chloroplast*, *Pseudomonas*, *Bacillus*, *Pantoea*, *Methylobacterium-Methylorubrum*, *Sphingomonas*, *Staphylococcus*, *Acinetobacter*, *Enterobacter*, and *Aureimonas*. Compared with the control group, the relative abundance of *Pseudomonas*, *Bacillus*, *Pantoea*, *Methylobacterium*, *Sphingomonas*, and *Staphylococcus* increased in Example 1, while the relative abundance of *Enterobacter* and *Aureimonas* showed a decreasing trend.

[0143] Table 4

[0144] Blank control group <![CDATA[1.2999±0.0797 b ]]> <![CDATA[0.7131±0.0032 a ]]> <![CDATA[426.4408±56.1289 b ]]> <![CDATA[402.1444±78.8179 b ]]> Application Example 1 <![CDATA[1.5641±0.0651 a ]]> <![CDATA[0.6121±0.0054 a ]]> <![CDATA[654.4541±84.1659 a ]]> <![CDATA[564.0578±65.5106 b ]]> Comparative Application Example 10 <![CDATA[1.4526±0.0561 b ]]> <![CDATA[0.6724±0.0042 b ]]> <![CDATA[526.5241±57.1689 b ]]> <![CDATA[485.0985±75.4587 b ]]> Comparative Application Example 11 <![CDATA[1.4245±0.0365 b ]]> <![CDATA[0.6412±0.0041 b ]]> <![CDATA[564.2541±54.1259 b ]]> <![CDATA[508.2541±71.5147 b ]]>

[0145] Alpha diversity analysis can reflect the diversity and richness of the microbial community in a sample. ACE and Chao indices reflect species richness; a higher index indicates greater species richness. Shannon and Simpson indices are commonly used to reflect community diversity; a higher Shannon index indicates greater species diversity, while a higher Simpson index indicates the opposite. Significant differences were observed in all treated tobacco leaf samples (p > 0.05).

[0146] As shown in Table 4, the tobacco leaves treated with the co-fermentation of soybean extract and compound enzyme preparation in Example 1 had the highest Shannon, ACE, and Chao indices and the lowest Simpson index, indicating that the species richness and community diversity of the tobacco leaf samples increased after co-fermentation with soybean extract and compound enzyme preparation.

[0147] Test Example 4

[0148] This test case analyzed cell wall material in tobacco leaves treated with the blank control group, Application Example 1, and Comparative Application Examples 10-11.

[0149] Cellulose and lignin were determined according to the tobacco industry standard YC / T 347-2010 "Determination of Neutral Detergent Fiber, Acid Detergent Fiber and Acid-washed Lignin in Tobacco and Tobacco Products - Determination by Detergent Method"; pectin was determined according to YC / T346-2010 "Determination of Pectin in Tobacco and Tobacco Products - Determination by Ion Chromatography". Each sample was repeated 3 times. The results are shown in Table 5.

[0150] Table 5

[0151] Blank control group 13.65 8.65 8.87 Application Example 1 10.57 6.44 6.54 Comparative Application Example 10 11.17 6.87 6.99 Comparative Application Example 11 12.56 7.98 7.85

[0152] As shown in Table 5, the tobacco leaves treated with the synergistic fermentation of soybean extract and compound enzyme preparation in Application Example 1 showed a significant reduction in cellulose, pectin and lignin content.

[0153] Test Example 5

[0154] This test example performs various enzyme activity assays on the composite enzyme preparations obtained in Preparation Example 1 and Comparative Preparation Examples 1-3:

[0155] The determination of protease activity shall be performed in accordance with GB / T 23527-2009.

[0156] The activities of xylanase, amylase, pectinase and cellulase were determined using the DNS method, referring to the methods described in "Lai Guodong, Qin Changsheng, Zhao Danyang et al. Isolation and screening of cellulose-degrading strains [J]. Forestry and Environmental Science, 2021, 37(04): 24-32." and "Wang Xiaodan, Guo Liqiong, Zhao Lichao et al. Methods for determining xylanase activity and definition of enzyme activity units [J]. Food and Fermentation Industries, 2009, 35(09): 128-31."

[0157] The determination of peroxidase activity, manganese peroxidase activity and laccase activity was performed in accordance with the method described in "Guo Lingling, Jiang Zhiyang, Tao Shuyu et al. Screening and degradation effect of heat-resistant lignin-degrading bacteria [J]. Journal of Microbiology, 2022, 42(04): 64-9."

[0158] The determination of nicotine dehydrogenase activity was based on the method described in “Ma Lin, Zhang Junsong, Zeng Xiaoying, et al. Study on fermentation conditions of nicotine dehydrogenase-producing strain Arthrobacter Z3 [J]. Tobacco Science and Technology, 2007, (10): 60-3.”

[0159] The method for determining tannin enzyme activity was based on the method described in “Zhang Mengmeng, Liu Qian, Pan Yong, et al. Screening, identification and fermentation process optimization of tannin-degrading bacteria in cigar tobacco leaves [J]. Tobacco Science and Technology, 2023, 56(07): 32-40.”

[0160] The method for determining β-carotene enzyme activity was based on the method described in “Li Jinpeng, Qi Xiaoqin, Liu Jianhua, et al. Enzymatic properties of β-carotene degrading enzymes in Kuterella foenum-graecum [J]. China Food Additives, 2017, (04): 59-66.”

[0161] As shown in Table 6, the total enzyme activity of Preparation Example 1 was significantly higher than that of other Preparation Examples, especially the enzyme activities of xylanase and amylase were significantly increased compared with other Preparation Examples.

[0162] Table 6

[0163] neutral protease 15.2746 12.3458 16.4881 8.7435 alkaline protease 28.0768 19.5458 29.0689 10.4362 acidic protease 8.3073 2.6554 4.0045 3.5706 Cellulase 2.3375 1.6361 2.1358 2.1980 Xylanase 28.2277 1.6611 1.7642 2.2182 pectinase 2.9145 2.3324 2.4434 2.7226 amylase 32.1083 7.1869 6.9965 7.1313 Lignin peroxidase 0.1023 2.4271 0.1625 0.1726 Lignin manganese peroxidase 0.0201 0.0302 0.0503 0.0604 Lignin Laccase 0.0198 0.0102 0.0132 0.0122 Tanninase 3.9144 5.6358 2.2314 11.9728 Nicotinic enzyme 1.0956 1.2351 2.4000 7.5667 β-carotene enzyme 0.0012 0.0103 0.0027 0.0067 Total enzyme activity 122.4001 56.7122 67.7615 56.8118

[0164] Test Example 6

[0165] This test case evaluates the sensory quality of Burley tobacco processed into cigar-shaped reconstituted tobacco.

[0166] The preparation method is as follows:

[0167] 1) Mix burley tobacco, cigar tobacco fragments, flue-cured tobacco stems and tobacco fragments to obtain a mixed raw material. Add 8% wood pulp fiber by mass, mix evenly, and then add 7 times the mass of water of the mixture. Extract at 60℃ for 40 minutes and perform solid-liquid separation to obtain residue and extract. Repeat the above steps and extract the residue again under the same conditions to obtain residue and extract.

[0168] 2) Combine the residues from both extractions, pulp them, control the freeness of the pulp to 32°SR, and form paper with a basis weight of 45g / m³. 2 film base;

[0169] 3) Combine the extracts from both extractions and concentrate them to a density of 1.180 g / cm³. 3 The concentrated tobacco extract was used as the coating liquid, and the preparation temperature was 44.0℃;

[0170] 4) The substrate is dip-coated with a coating liquid using a dip-coating process, with a coating coverage of 40%.

[0171] 5) Dry the coated reconstituted tobacco leaves at 105℃ until the moisture content is 11% and the oven-dry weight is 105 g / m³. 2 .

[0172] 0 Burley tobacco (unprocessed) 20%, cigar scraps 30%, flue-cured tobacco stems 30%, flue-cured tobacco scraps 20% 1# Burley tobacco (Application Example 1) 20%, cigar scraps 30%, flue-cured tobacco stems 30%, flue-cured tobacco scraps 20% 2# Burley tobacco (Application Example 1) 25%, cigar scraps 25%, flue-cured tobacco stems 30%, flue-cured tobacco scraps 20% 3# Burley tobacco (Application Example 1) 30%, cigar scraps 20%, flue-cured tobacco stems 30%, flue-cured tobacco scraps 20%

[0173] Referring to "YC / T 498-2014 Sensory Evaluation Method for Reconstituted Tobacco (Papermaking Method)", the prepared reconstituted tobacco product was shredded, rolled into cigarette samples, and nine experts with sensory evaluation qualifications were invited to conduct a sensory quality evaluation. The results are shown in Table 7 and... Figure 1 As shown.

[0174] Table 7

[0175]

[0176]

[0177] Depend on Figure 1As shown in Table 7, the cigar-shaped reconstituted tobacco prepared using Burley tobacco fermented in Application Example 1 exhibited superior sensory quality compared to the control group at 20%, 25%, and 30% of the tobacco used, particularly in terms of bean aroma and flavor. At a 25% addition rate, the reconstituted tobacco showed more pronounced cigar-like characteristics, with good aroma clarity and comfort. While the aroma intensity was slightly lower at 30%, the overall sensory quality remained good. At a 30% addition rate, the reconstituted tobacco still exhibited strong cigar-like characteristics, with good aroma integration and richness, and a full-bodied aroma. However, the comfort level was slightly lower compared to the 25% addition rate, but the overall sensory quality remained good. This significantly increased the application ratio of Burley tobacco in cigar-shaped reconstituted tobacco, reduced the proportion of cigar-shaped raw materials used, and further highlighted the cigar-like style characteristics of the reconstituted tobacco.

[0178] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0179] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0180] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for fermenting tobacco leaves in cigar-shaped reconstituted tobacco, characterized in that, The fermentation treatment method includes the following steps: The compound enzyme preparation, roasted soybean extract and tobacco raw materials are mixed and fermented; The compound enzyme preparation is obtained by fermentation of compound bacteria, which includes Bacillus velezensis DF-08 strain with preservation number CCTCC No: M 2023456 and Pantoea sp. B-3 strain with preservation number CCTCC NO: M20221165. The roasted soybean extract was prepared by the following method, the method comprising: (1) First bake the soybean raw material at 150-210℃ for 5-10 minutes, then bake at 220-250℃ for 7-12 minutes, and finally bake at 260-300℃ for 12-20 minutes to obtain baked soybeans; (2) After grinding the roasted soybeans, hot extraction was performed to obtain the extract; (3) The extract was subjected to sedimentation to remove impurities, and the roasted soybean extract was obtained; The tobacco leaves are Burley tobacco.

2. The fermentation treatment method according to claim 1, characterized in that, The moisture content of the soybean raw material in step (1) is 10-15%.

3. The fermentation treatment method according to claim 1, characterized in that, The moisture content of the roasted soybeans in step (1) is 3%-7%.

4. The fermentation treatment method according to claim 1, characterized in that, In step (2), the roasted soybeans are ground to obtain soybean powder with a mesh size of 200-300. The material temperature during grinding is below 45℃.

5. The fermentation treatment method according to claim 1, characterized in that, The extractant used in step (2) for hot extraction is water.

6. The fermentation treatment method according to claim 5, characterized in that, The oven-dry weight ratio of the roasted soybeans to the extractant is 1:(3-5).

7. The fermentation treatment method according to claim 1, characterized in that, The hot extraction temperature is 80-100℃, and the hot extraction time is 10-30 minutes.

8. The fermentation treatment method according to claim 1, characterized in that, The hot extraction process is accompanied by stirring, with a stirring speed of 600-800 rpm.

9. The fermentation treatment method according to claim 1, characterized in that, Step (3) involves sedimentation and impurity removal, which includes sealing and sedimentation at a temperature of 4-7°C for 30-45 minutes, followed by collection of the supernatant.

10. The fermentation treatment method according to claim 9, characterized in that, After sedimentation and impurity removal, the mixture is further concentrated at a temperature of 60-65℃.

11. The fermentation treatment method according to claim 10, characterized in that, The density of the concentrated roasted soybean extract is 1.0-1.3 g / cm³. 3 .

12. The fermentation treatment method according to claim 1, characterized in that, The preparation method of the compound enzyme preparation includes the following steps: The activated compound bacteria were inoculated into a culture medium for fermentation to obtain a fermentation broth. The fermentation broth was centrifuged, and the supernatant was collected to obtain the compound enzyme preparation.

13. The fermentation treatment method according to claim 1, characterized in that, The fermentation temperature after mixing with tobacco raw materials is 35-45℃, the fermentation humidity is 60%-70%, and the fermentation time is 72-168h.

14. The fermentation treatment method according to claim 1, characterized in that, The dry weight of the roasted soybean extract is 5-15% of the dry weight of the tobacco raw material.

15. The fermentation treatment method according to claim 1, characterized in that, The dry weight of the compound enzyme preparation is 0.01-1% of the dry weight of the tobacco raw material.

16. The fermentation treatment method according to claim 1, characterized in that, The fermentation process is followed by inactivation treatment.

17. The fermentation treatment method according to claim 16, characterized in that, The inactivation treatment temperature is 65-95℃, and the inactivation treatment time is 8-15 minutes.

18. A cigar-shaped reconstituted tobacco leaf, characterized in that, The cigar-shaped reconstituted tobacco leaves include tobacco leaves treated by the fermentation process according to any one of claims 1-17; The tobacco leaves are Burley tobacco.

19. The cigar-shaped reconstituted tobacco leaf according to claim 18, characterized in that, The fermented tobacco leaves constitute 20-30% of the mass of the cigar-shaped reconstituted tobacco leaves.

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