A Korean bacillus and its culture and use

Abstract

The present application relates to the field of microbial technology. Specifically, it relates to a Korean Bacillus and a culture thereof, and the use of the Korean Bacillus and the culture in processing tobacco raw materials or preparing tobacco products or in improving the sensory quality of tobacco smoking. The present application also relates to a method for processing tobacco raw materials or preparing tobacco products, or a method for improving the sensory quality of tobacco products when smoking.

Description

Technical Field

[0001] The present application relates to the field of microbial technology. Specifically, it relates to a Korean Bacillus and a culture thereof, and the use of the Korean Bacillus and the culture in processing tobacco raw materials or preparing tobacco products or in improving the sensory quality of tobacco smoking. The present application also relates to a method for processing tobacco raw materials or preparing tobacco products, or a method for improving the sensory quality of tobacco products when smoking. Background Art

[0002] Korean Bacillus is a type of rod-shaped bacteria with yellow colonies and endospores. It was first isolated from willow root tissue by Korean scholars Lim Jee-Min and others in 2006 and identified as a new species in the genus Bacillus, named Bacillus koreensis.

[0003] With the development of genomics, Canadian scholars recently reclassified the genus Bacillus based on CSIs after a comprehensive comparison of the genomic systems of 300 Bacillus species. Based on this, Korean Bacillus is independent of the genus Bacillus in terms of genetic evolution. In the current taxonomy, Korean Bacillus is officially renamed Priestia koreensis and belongs to the genus Priestia sp. The new classification method has been adopted by authoritative organizations such as NCBI and DSMZ.

[0004] Korean Bacillus is rich in secondary metabolites and has great potential for development and application. However, there is currently little research on the development and application of Korean Bacillus. Among domestic patent applications, only the Hubei Institute of Ecological and Environmental Sciences (patent number CN202111068096.1) disclosed the use of Korean Bacillus LQB126 to prepare immobilized microbial agents and use them to control heavy metal pollution.

[0005] Therefore, technicians in this industry need to study Korean Bacillus and its applications to better utilize the advantages of Korean Bacillus. Summary of the invention

[0006] In order to solve the above problems, in a first aspect, the present application provides a Korean Bacillus (Bacillus koreensis), which is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No.24131 or CGMCC No.24132.

[0007] In a second aspect, the present application provides a culture of Korean Bacillus, comprising Korean Bacillus or a lysate (eg, enzymolysate) or a metabolite thereof.

[0008] In certain embodiments, the culture includes amylases, cellulases, and / or pectinases produced by Bacillus koreana.

[0009] In certain embodiments, the culture includes amylase, cellulase, and pectinase produced by Bacillus koreana.

[0010] In certain embodiments, the enzymatic hydrolysate is a product obtained by treating Korean Bacillus with an enzyme (eg, protease, lysozyme).

[0011] In certain embodiments, the culture includes the Korean Bacillus with a deposit number of CGMCC No. 24131 described in the first aspect, the Korean Bacillus with a deposit number of CGMCC No. 24132, or any combination thereof.

[0012] In certain embodiments, the culture is a bacterial broth of Bacillus koreana.

[0013] In certain embodiments, the culture is obtained by culturing Korean Bacillus in a bacterial liquid culture medium.

[0014] In certain embodiments, the bacterial culture medium includes nutrient-providing substances, inorganic salts and water.

[0015] In certain embodiments, the bacterial culture medium comprises peptone, yeast extract, sodium chloride and water.

[0016] In certain embodiments, the bacterial culture medium comprises 5-15 g / L of peptone, 1-10 g / L of yeast extract, 5-15 g / L of sodium chloride and water.

[0017] In certain embodiments, the bacterial culture medium comprises 10 g / L of peptone, 5 g / L of yeast extract, 10 g / L of sodium chloride and water.

[0018] In certain embodiments, the culture is a crude enzyme solution prepared from the bacterial solution.

[0019] In certain embodiments, the crude enzyme solution is obtained by fermenting the bacterial solution.

[0020] In certain embodiments, the crude enzyme solution is obtained by inoculating the bacterial solution into a bacterial solution culture medium for cultivation, and then inoculating into a fermentation medium for cultivation.

[0021] In certain embodiments, the crude enzyme solution is obtained by the following method: culturing Korean Bacillus in the bacterial culture medium, then transplanting it to the bacterial culture medium at a rate of 5-15‰ (v / v) (e.g., 5‰, 10‰, 15‰) for cultivation, and then transplanting it to a fermentation medium at a rate of 40-60‰ (v / v) (e.g., 40‰, 45‰, 50‰, 55‰, 60‰) for fermentation culture.

[0022] In certain embodiments, the fermentation medium comprises: peptone, beef extract, sodium chloride, anhydrous calcium chloride, soluble starch and water.

[0023] In certain embodiments, the fermentation medium comprises: 5-15 g / L of peptone, 1-5 g / L of beef extract, 1-10 g / L of sodium chloride, 0.1-1 g / L of anhydrous calcium chloride, 1-5 g / L of soluble starch and water.

[0024] In certain embodiments, the fermentation medium comprises: 10 g / L of peptone, 3 g / L of beef extract, 5 g / L of sodium chloride, 0.5 g / L of anhydrous calcium chloride, 2 g / L of soluble starch and water.

[0025] In certain embodiments, the bacterial culture medium and / or the fermentation medium further comprises an organosilicon defoaming agent.

[0026] In certain embodiments, the bacterial culture medium and / or the fermentation medium further comprises 0.1-5 g / L of an organosilicon defoaming agent.

[0027] In certain embodiments, the bacterial culture medium and / or the fermentation medium further comprises 1 g / L of an organosilicon defoaming agent.

[0028] In certain embodiments, the culture medium is a refined enzyme liquid prepared from the crude enzyme liquid.

[0029] In certain embodiments, the refined enzyme solution is obtained by filtering and concentrating the crude enzyme solution.

[0030] In certain embodiments, the refined enzyme solution is obtained by filtering the crude enzyme solution through a ceramic membrane, filtering through an ultrafiltration membrane and concentrating the crude enzyme solution.

[0031] In certain embodiments, the refined enzyme solution is obtained by filtering the crude enzyme solution through a ceramic membrane with a pore size of 200 nm, then filtering through an ultrafiltration membrane with a molecular weight cutoff of 150 KDa, and then concentrating through an ultrafiltration membrane with a molecular weight cutoff of 10 KDa.

[0032] In certain embodiments, the culture is a solid enzyme preparation prepared from the refined enzyme liquid.

[0033] In certain embodiments, the solid enzyme preparation is obtained by drying the purified enzyme solution.

[0034] In certain embodiments, the solid enzyme preparation is obtained by adding skimmed milk powder and / or trehalose to the refined enzyme solution and then drying it.

[0035] In certain embodiments, the solid enzyme preparation is obtained by adding 20-40‰ (e.g., 20‰, 30‰, 40‰) (m / v) of skimmed milk powder and 1-5‰ (e.g., 1‰, 2‰, 3‰, 4‰, 5‰) (m / v) of trehalose to the refined enzyme solution, stirring and dissolving, and then drying to a moisture content of 9-10% (e.g., 9%, 9.2%, 9.4%, 9.6%, 9.8%, 10%).

[0036] In a third aspect, the present application provides the use of the Korean Bacillus described in the first aspect or the culture described in the second aspect in processing tobacco raw materials or preparing tobacco products, or in improving the smoking sensory quality of tobacco products.

[0037] In certain embodiments, the tobacco raw material is selected from raw tobacco, strip tobacco, or any combination thereof.

[0038] In certain embodiments, the tobacco raw material is redried tobacco strips.

[0039] In certain embodiments, the tobacco product is selected from a cigarette, an electronic cigarette, or any combination thereof.

[0040] The reason why the Korean Bacillus described in the first aspect or the culture described in the second aspect can improve the smoking sensory quality of tobacco products may be that the various macromolecular degradation complex enzymes produced by the growth and fermentation of Korean Bacillus YL-2 can degrade the macromolecular substances in the cell walls of tobacco shreds, reduce impurities and irritation, and are beneficial to the release and accumulation of tobacco aroma precursor substances, thereby improving the smoking sensory quality of tobacco leaves.

[0041] In a fourth aspect, the present application provides a method for processing tobacco raw materials or preparing tobacco products, or a method for improving the smoking sensory quality of tobacco products, the method comprising: applying the Korean Bacillus described in the first aspect or the culture described in the second aspect to tobacco.

[0042] In certain embodiments, the administering is selected from spraying, painting or soaking.

[0043] In certain embodiments, the tobacco raw material is selected from raw tobacco, strip tobacco, or any combination thereof.

[0044] In certain embodiments, the tobacco raw material is redried tobacco strips.

[0045] In certain embodiments, the tobacco product is selected from a cigarette, an electronic cigarette, or any combination thereof.

[0046] In certain embodiments, when the culture is a bacterial liquid of Korean Bacillus, the method comprises: spraying the bacterial liquid on redried tobacco strips, and fermenting the redried tobacco strips.

[0047] In certain embodiments, the method comprises: spraying the bacterial liquid at an amount of 0.02-0.06 mL / g on the redried tobacco strips, and drying the redried tobacco strips at 30-50° C. and 50-70% relative humidity for 5-10 days.

[0048] In certain embodiments, the method comprises: spraying the bacterial liquid at an amount of 0.04 mL / g on shredded redried tobacco strips, and keeping the redried tobacco strips at 40° C. and 60% relative humidity for 7 days.

[0049] In certain embodiments, when the culture is a crude enzyme solution, the method comprises: spraying the crude enzyme solution on redried tobacco strips, and incubating the redried tobacco strips.

[0050] In certain embodiments, the method comprises: spraying the crude enzyme solution at 8-12 U / Kg on redried tobacco strips, keeping the redried tobacco strips at 20-30°C and 70-90% relative humidity for 3-7 days, and then inactivating the crude enzyme solution.

[0051] In certain embodiments, the method comprises: spraying the crude enzyme solution at an amount of 10U / Kg on shredded redried tobacco strips, and placing the redried tobacco strips at 25°C and 80% relative humidity for 5 days; then placing them at 60-80°C for 15-30 minutes, and then placing them at 37°C and 60% relative humidity overnight.

[0052] In certain embodiments, when the culture is a refined enzyme liquid, the method comprises: spraying the refined enzyme liquid on redried tobacco strips, and incubating the redried tobacco strips.

[0053] In certain embodiments, the method comprises: spraying the refined enzyme solution at 80-120 U / Kg on redried tobacco strips, keeping the redried tobacco strips at 20-30°C and 50-70% relative humidity for 6-10 days, and then inactivating the refined enzyme solution.

[0054] In certain embodiments, the method comprises: spraying the refined enzyme solution at an amount of 100 U / Kg on shredded redried tobacco strips, keeping the redried tobacco strips at 25°C and 60% relative humidity for 8 days, then placing them at 60-80°C for 15-30 minutes, and then placing them at 37°C and 60% relative humidity overnight.

[0055] In certain embodiments, when the culture is a solid enzyme preparation, the method comprises: preparing the solid enzyme preparation into a solution and spraying the solution on redried tobacco strips, and incubating the redried tobacco strips.

[0056] In certain embodiments, the method comprises: configuring the solid enzyme preparation into a solution and spraying it on redried tobacco strips at an amount of 80-120 U / Kg, drying the redried tobacco strips at 30-50°C and 40-60% relative humidity for 1-5 hours, and then inactivating the enzyme solution.

[0057] In certain embodiments, the method comprises: configuring the solid enzyme preparation into a solution by adding water, and spraying the solution on the cut redried tobacco strips at an amount of 100 U / Kg, and then placing the redried tobacco strips at 40°C and 50% relative humidity for 3 hours, and then placing them at 60-80°C for 15-30 minutes, and then placing them at 37°C and 60% relative humidity overnight.

[0058] Definition of terms

[0059] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the biochemical, microbiological and other operating steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0060] As used herein, the term "Korean Bacillus (Bacillus koreensis)" refers to a class of yellow, endospore-forming rod-shaped bacteria of bacterial colonies. With the development of genomics, Canadian scholars recently reclassified Bacillus based on CSIs by comprehensively comparing the genomic systems of 300 Bacillus species. Accordingly, Korean Bacillus is independent of Bacillus on the genetic evolution branch, and is officially renamed as Korean Priestia (Priestiakoreensis), and is simultaneously classified under Priestia (Priestia sp.). Authoritative organizations such as NCBI and DSMZ have adopted new classification methods, but what is still recorded in some published materials is the former name "Korean Bacillus" of "Korean Priestia". Therefore, in the text, Korean Bacillus (Bacillus koreensis) and Korean Priestia (Priestia koreensis) both represent the same bacteria and can be used interchangeably.

[0061] As used herein, the term "enzyme activity" refers to the activity of an enzyme, and may also be referred to as "enzyme activity" or "enzyme activity", which all represent the same meaning and are interchangeable. Generally speaking, the higher the enzyme activity, the stronger the ability of the corresponding enzyme to catalyze a chemical reaction. Methods for determining enzyme activity are known to those skilled in the art, and may be determined by physical methods (e.g., polarimetry, refractometry, specific gravity) or chemical methods (e.g., Fehling's method, potassium permanganate method, DNS colorimetry).

[0062] In some embodiments, the amount of enzyme required to hydrolyze 1 micromole of substrate in 1 minute is defined as 1 enzyme activity unit U. In some embodiments, the amount of enzyme required to hydrolyze 1 μg of glucose or galacturonic acid produced by 1.5% soluble starch, 1% sodium carboxymethyl cellulose and 1% pectin in 1 minute is defined as 1 enzyme activity unit U.

[0063] As used herein, the term "enzyme hydrolysate of Korean Bacillus" refers to a product obtained after enzymatic degradation of Korean Bacillus. In certain embodiments, the enzyme hydrolysate of Korean Bacillus is a product obtained by enzymatically treating a bacterial solution of Korean Bacillus.

[0064] As used herein, the term "tobacco (Nicotiana tabacum L.)" is a plant of the genus Nicotiana in the Solanaceae family, and is the raw material for industrial tobacco products (e.g., cigarettes, electronic cigarettes). Tobacco is widely cultivated in provinces and regions in northern and southern China. It is a thermophilic crop and is sensitive to temperature. Different temperature conditions have a greater impact on the quality and yield of tobacco.

[0065] As used herein, the term "fermentation" refers to the process of preparing microbial cells themselves, or direct metabolites or secondary metabolites by means of the life activities of microorganisms under aerobic or anaerobic conditions. As used herein, the term "fermentation broth" refers to the culture broth in which microorganisms are cultured during the fermentation process.

[0066] As used herein, the term "culture" refers to a cell mass or growth of a microorganism within a certain period of time. In certain embodiments, the microorganism is Korean bacillus. In certain embodiments, the culture is included in the Korean bacillus obtained by culturing and breeding under certain conditions. In certain embodiments, the culture includes a culture medium grown with Korean bacillus. In certain embodiments, the culture includes a metabolite (e.g., amylase, cellulase and / or pectinase) of Korean bacillus.

[0067] Advantageous Effects of the Invention

[0068] Compared with the prior art, this application uses Korean Bacillus to prepare 4 kinds of cultures, namely, Korean Bacillus bacterial liquid, crude enzyme liquid, refined enzyme liquid and solid enzyme preparation. After experimental testing, the sensory quality of tobacco smoking was improved after these four cultures were used to treat tobacco. In addition, the enzyme activities of amylase, cellulase and pectinase in crude enzyme liquid, refined enzyme liquid and solid enzyme preparation were tested, and it was found that the enzymes in the crude enzyme liquid already had a higher enzyme activity, while the enzyme activity of the refined enzyme liquid was an order of magnitude higher than that of the crude enzyme liquid, and the enzyme activity of the solid enzyme preparation was another order of magnitude higher than that of the refined enzyme liquid! The solid enzyme preparation is prepared by dehydrating the refined enzyme liquid, so it not only has a very high enzyme activity, but is also more conducive to industrial transportation and storage.

[0069] Embodiments of the present invention will be described in detail below in conjunction with examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. According to the following detailed description of preferred embodiments, various objects and advantages of the present invention will become apparent to those skilled in the art.

[0070] Notes on the Deposit of Biological Materials

[0071] Bacillus koreensis YL-2 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It has the deposit number CGMCC No. 24131, and the deposit date is December 20, 2021.

[0072] Bacillus koreensis YLA-2 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It has the deposit number CGMCC No. 24132, and the deposit date is December 20, 2021. DETAILED DESCRIPTION

[0073] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0074] Unless otherwise specified, the experiments and methods described in the embodiments are carried out basically according to conventional methods well known in the art and described in various references. For example, conventional techniques such as biochemistry, microbiology used in the present invention can be found in METHODS IN ENZYMOLOGY series (Academic Publishing Company).

[0075] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0076] Example 1. Treatment of B3F redried tobacco strips with Korean Bacillus YL-2 bacterial solution

[0077] Take the rejuvenated Korean Bacillus YL-2 bacterial plate, inoculate it into a bottling volume of 100 / 250mL seed culture medium (seed culture medium formula is: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, pH 7.0), culture it in a constant temperature shaker at 37°C and 180rpm for 24h, prepare a seed solution with an OD600 of 1.724, and adjust the OD600 of the seed solution to 1.0 with pure water. Take B3F redried tobacco strips, cut them into shreds, weigh 50g of tobacco shreds, and evenly apply 2mL of the diluted seed solution to the tobacco shreds through an atomizer at a spray rate of 0.04mL / g, and ferment them for 7d at 40°C and a relative humidity of 60%.

[0078] After fermentation, the unfermented tobacco was used as the blank control group. The tobacco samples of the blank control group and the experimental group were rolled into cigarettes of basically the same length, thickness and tightness, and the moisture was balanced at 22°C and 60% relative humidity. After 24 hours, 7 judges with provincial or above cigarette sensory evaluation qualifications conducted a smoking sensory quality evaluation. Specifically, the 7 judges scored the cigarettes with reference to the evaluation indicators in Table 1, among which concentration and strength were descriptive indicators and were not included in the total score. The total sensory quality score = (flavor score × 0.6 + sweetness × 0.4) × 0.4 + [(flavor quality × 0.4 + aroma volume × 0.3 + permeability × 0.1 + miscellaneous gas × 0.2) × 0.55 + (fineness × 0.4 + softness × 0.3 + roundness × 0.3) × 0.2 + (irritation × 0.3 + dryness × 0.3 + aftertaste × 0.4) × 0.25] × 0.6. The calculated total sensory quality scores are recorded in Table 3.

[0079] Table 1. Scoring standards for sensory evaluation indicators

[0080]

[0081] Example 2. Treatment of B3F redried tobacco strips with crude enzyme solution of Korean Bacillus YL-2

[0082] The Korean Bacillus YL-2 strain plate with good subculture and rejuvenation was inoculated into a seed culture medium with a filling volume of 700 / 2000mL (the seed culture medium formula is: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, pH 7.0), and cultured in a constant temperature shaker at 30°C and 180rpm for 30h to prepare a first-level seed solution with an OD600 of 1.821; the first-level seed solution was transplanted into a seed culture medium with a filling volume of 70 / 200L (the seed culture medium formula is: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, silicone defoamer 1g / L, pH7.0) at a rate of 10‰ (v / v), the culture temperature was set to 40°C, the agitator speed was set to 150rpm, the ventilation volume was set to 0.6vvm, the tank pressure was set to 0.04MPa, and the O The D600 value of the secondary seed liquid was 1.932; the secondary seed liquid was transferred to a fermentation medium with a filling volume of 1400 / 2000L (the fermentation medium formula was: peptone 10g / L, beef extract 3g / L, sodium chloride 5g / L, anhydrous calcium chloride 0.5g / L, soluble starch 2g / L, silicone defoamer 1g / L, pH7.0) at a rate of 50‰ (v / v), the fermentation temperature was set to 40°C, the agitator speed was 120rpm, the ventilation volume was 0.8vvm, the tank pressure was 0.04MPa, and the YL-2 fermentation crude enzyme solution was prepared after fermentation for 30h.

[0083] DNS method was used to prepare 1.5% soluble starch (pH 5.0), 1% sodium carboxymethyl cellulose (pH 5.0), 1% pectin (pH 9.4) The enzymatic activity of the YL-2 crude enzyme solution was determined by reacting the reaction substrate at 50°C. The amount of enzyme required to hydrolyze the above substrate in 1 minute to produce 1 μg of glucose or galacturonic acid was defined as 1 enzyme activity unit U; the crude enzyme solution was diluted with pure water to a 250U / mL working solution, 50g of B3F redried tobacco was cut and weighed, and 2mL of the working solution was evenly applied to the tobacco at a spraying rate of 10U / Kg of tobacco using an atomizer, and incubated at 25°C and a relative humidity of 80% for 5h. After the incubation, it was placed in a 75°C oven for 20min to inactivate the enzyme, and then placed at 37°C and a relative humidity of 60% for equilibration overnight. The blank control group was sprayed with an equal amount of pure water and the same operation as above was performed. The sensory quality of the suction was evaluated according to the experimental scheme described in Example 1, and the calculated total score of the sensory quality was recorded in Table 3.

[0084] Example 3. Treatment of B3F redried tobacco strips with Korean Bacillus YL-2 refined enzyme solution

[0085] 1200L of the YL-2 crude enzyme solution of Example 2 was filtered through a 200nm pore size ceramic membrane to collect the permeate, and the permeate was filtered twice using an ultrafiltration membrane with a molecular weight cutoff of 150KDa to collect the permeate, and the permeate was finally concentrated using an ultrafiltration membrane with a molecular weight cutoff of 10KDa (the above membrane filtration operations all used a cold water system to control the feed liquid temperature to <10°C, and the 200nm ceramic membrane and 150KDa ultrafiltration membrane were filtered using an equal volume of pure water to improve the recovery rate), and concentrated to a final volume of 60L (concentrated 20 times), and the concentrated solution was collected as the YL-2 refined enzyme solution.

[0086] The DNS method was used to determine the enzyme activity of the YL-2 refined enzyme solution; the refined enzyme solution was diluted with pure water to a 250U / mL working solution, 50g of B3F redried tobacco was cut and weighed, and 2mL of the working solution was evenly applied to the tobacco using an atomizer at a spray rate of 100U / Kg. The mixture was incubated at 25°C and a relative humidity of 60% for 8h. After the incubation, the mixture was placed in a 75°C oven for 20min to inactivate the enzyme, and then equilibrated overnight at 37°C and a relative humidity of 60%. The same operation as above was performed on a blank control group sprayed with an equal amount of pure water. The sensory quality of the suction was evaluated according to the experimental scheme described in Example 1, and the calculated total sensory quality score was recorded in Table 3.

[0087] Example 4. Treatment of B3F redried tobacco strips with Korean Bacillus YL-2 solid enzyme preparation

[0088] Take 50L of the YL-2 refined enzyme solution of Example 3, add 1500g of skimmed milk powder at 30‰ (m / v), add 100g of trehalose at 2‰ (m / v), stir and dissolve at low temperature, and then use spray drying at an inlet air temperature of 120°C and an outlet air temperature of 70°C to prepare a YL-2 solid enzyme preparation with a moisture content of 9.2%.

[0089] The DNS method was used to determine the enzyme activity of the YL-2 solid enzyme preparation; the solid enzyme preparation was prepared into a 400U / mL working solution using pure water, 50g of B3F redried tobacco was cut and weighed, and 2.5mL of the working solution was evenly applied to the tobacco using an atomizer at a spray rate of 100U / Kg. The mixture was incubated at 40°C and a relative humidity of 50% for 3h. After the incubation, the mixture was placed in an oven at 75°C for 20min to inactivate the enzyme, and then equilibrated overnight at 37°C and a relative humidity of 60%. An equal amount of pure water was sprayed according to the same operation as above as a blank control group. The sensory quality of the suction was evaluated according to the experimental scheme described in Example 1, and the calculated total sensory quality score was recorded in Table 3.

[0090] The results of enzyme activity determination in three cultures (crude enzyme solution, refined enzyme solution and solid enzyme preparation) prepared by Korean Bacillus YL-2 are shown in Table 2. The enzyme activities of the crude enzyme solution, refined enzyme solution and solid enzyme preparation were tested. The results showed that the amylase, cellulase and pectinase in the crude enzyme solution already had high enzyme activities, while the enzyme activities of the three enzymes in the refined enzyme solution were one order of magnitude higher than those of the crude enzyme solution, and the enzyme activities of the three enzymes in the solid enzyme preparation were one order of magnitude higher than those of the refined enzyme solution! The solid enzyme preparation is prepared by dehydrating the refined enzyme solution, so it not only has very high enzyme activity, but is also more conducive to industrial transportation and storage.

[0091] Table 2 Results of enzyme activity determination in Korean Bacillus YL-2 enzyme solution

[0092]

[0093]

[0094] The results of tobacco leaf smoking sensory quality are shown in Table 3. The tobacco leaf smoking sensory quality is the ultimate embodiment of tobacco leaf quality. The results show that fermenting tobacco shreds with Korean Bacillus YL-2 or treating tobacco shreds with YL-2 crude enzyme solution, refined enzyme solution, and solid enzyme preparation can effectively improve the smoking sensory quality of tobacco shreds.

[0095] Table 3 Results of sensory quality evaluation of B3F treated with Korean Bacillus YL-2

[0096]

[0097] In addition, the applicant also conducted relevant experiments using the Korean Bacillus YLA-2 strain according to the experimental scheme and steps recorded in Examples 1-4, and obtained the enzyme activity determination results of the enzymes in the three cultures (crude enzyme solution, refined enzyme solution and solid enzyme preparation) prepared by Korean Bacillus YL-2, as well as the sensory quality results of tobacco leaves smoking of the four cultures (bacterial solution, crude enzyme solution, refined enzyme solution and solid enzyme preparation). The experimental results obtained are similar to those of Korean Bacillus YL-2.

[0098] In summary, the four cultures of Korean Bacillus can improve the smoking sensory quality of tobacco leaves. Among them, the crude enzyme solution, refined enzyme solution and solid enzyme preparation of Korean Bacillus significantly improved the enzyme activity.

[0099] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A Korean Bacillus ( Bacillus koreensis ) YL-2, the Korean Bacillus YL-2 was deposited in the China General Microbiological Culture Collection Center on December 20, 2021, with the deposit number CGMCC No.24131.

2. A culture of Korean Bacillus, comprising the Korean Bacillus YL-2 according to claim 1.

3. The culture of claim 2, further comprising amylase, cellulase and / or pectinase produced by Bacillus koreana.

4. The culture according to claim 2, wherein The culture is a bacterial liquid of Korean Bacillus YL-2.

5. The culture according to claim 2, which is obtained by culturing Korean Bacillus YL-2 in a bacterial liquid culture medium.

6. The culture according to claim 2, wherein The culture medium is a crude enzyme solution prepared from Korean Bacillus YL-2 bacterial solution.

7. The culture according to claim 6, wherein the crude enzyme solution is obtained by fermenting the bacterial solution.

8. The culture according to claim 6, wherein the crude enzyme solution is obtained by inoculating the bacterial solution into a bacterial solution medium for cultivation, and then inoculating the culture medium for cultivation.

9. The culture according to claim 6, wherein the crude enzyme solution is obtained by the following method: culturing Korean Bacillus YL-2 in a bacterial culture medium, then transplanting it to the bacterial culture medium at a concentration of 5-15‰ (v / v) for cultivation, and then transplanting it to a fermentation medium at a concentration of 40-60‰ (v / v) for fermentation culture.

10. The culture of claim 8, wherein the fermentation medium comprises: Peptone, beef extract, sodium chloride, anhydrous calcium chloride, soluble starch and water.

11. The culture of claim 8, wherein the fermentation medium comprises: 5-15g / L peptone, 1-5g / L beef extract, 1-10g / L sodium chloride, 0.1-1g / L anhydrous calcium chloride, 1-5g / L soluble starch and water.

12. Use of the Korean Bacillus YL-2 according to claim 1 or the culture according to any one of claims 2 to 11 in processing tobacco raw materials or preparing tobacco products, or in improving the smoking sensory quality of tobacco products.

13. The use according to claim 12, wherein the tobacco raw material is selected from raw tobacco, strip tobacco, or any combination thereof.

14. The use according to claim 12, wherein the tobacco raw material is redried tobacco strips.

15. The use according to claim 12, wherein the tobacco product is selected from cigarettes, electronic cigarettes, or any combination thereof.

16. A method for processing tobacco raw materials or preparing tobacco products, or a method for improving the sensory quality of tobacco products when smoked, the method comprising: The Korean Bacillus YL-2 of claim 1 or the culture of any one of claims 2 to 11 is applied to tobacco.

17. The method of claim 16, wherein the applying is selected from spraying, painting or soaking.

18. The method according to claim 16, wherein the tobacco raw material is selected from raw tobacco, strip tobacco, or any combination thereof.

19. The method according to claim 16, wherein the tobacco raw material is redried tobacco strips.

20. The method of claim 16, wherein the tobacco product is selected from a cigarette, an electronic cigarette, or any combination thereof.

21. The method of claim 16, when the culture is a bacterial liquid of Korean Bacillus YL-2, the method comprises: The bacterial liquid is sprayed on the redried tobacco strips, and the redried tobacco strips are fermented.

22. The method of claim 16, when the culture is a crude enzyme solution, the method comprising: The crude enzyme solution is sprayed on the redried tobacco strips, and the redried tobacco strips are incubated.

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