Bacillus subtilis, fermentation composition, fermentation method, cellulase preparation, preparation method, tobacco composition and tobacco product

Abstract

The invention belongs to the field of microorganisms, and particularly relates to bacillus subtilis with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.24134. The invention also relates to a fermentation composition, a fermentation method, a cellulase preparation and a preparation method thereof, a tobacco composition and a tobacco product. The cellulase produced by fermentation of the bacillus subtilis is high in enzyme activity and purity, and is beneficial to decomposition of excessive cellulose after being added into tobacco raw materials, introduction of exogenous breath is reduced, and the sensory quality of tobacco products is improved.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and specifically relates to Bacillus subtilis, a fermentation composition, a fermentation method, a cellulase preparation and a preparation method thereof, a tobacco composition and a tobacco product. Background Art

[0002] Cellulose is one of the main components of tobacco leaf cell walls. A moderate amount of cellulose helps tobacco burn, but excessive cellulose can cause negative sensory effects such as irritating burning smell and choking sensation. Cellulase is a series of enzymes that can degrade cellulose, mainly composed of exo-β-glucanase, endo-β-glucanase and β-glucosidase. Adding a moderate amount of cellulase during cigarette processing can reduce the cellulose content in tobacco, thereby improving the sensory quality of cigarettes.

[0003] However, there are many types of cellulases, and different cellulases have different abilities to degrade tobacco cellulose. Moreover, after cellulases degrade the cellulose in tobacco, they burn together with the tobacco during smoking. Different cellulases may produce different flavors through combustion and thermal cracking. Therefore, whether cellulases can adapt to tobacco leaves has become a difficult problem that needs to be overcome.

[0004] On the other hand, commercially available cellulase is expensive, and the price per gram of cellulase with higher purity is usually more than 100 yuan, which cannot be used on a large scale; food-grade cellulase is relatively cheap, but the purity and enzyme activity are low, and it is easy to introduce foreign smell when used in tobacco, which is also not suitable for the tobacco industry. Therefore, it is urgent to obtain low-priced and high-purity cellulase preparations.

[0005] Bacillus subtilis is a probiotic in the food and feed industry. It has the potential to produce cellulase and is relatively safe. However, the enzymatic activity of cellulase produced by Bacillus subtilis is currently low and is not suitable for use in tobacco processing. Summary of the invention

[0006] One of the purposes of the present invention is to provide a Bacillus subtilis fermentation-produced cellulase with high enzymatic activity and purity, which is added to tobacco raw materials to help decompose excessive cellulose and / or reduce the introduction of exogenous odor into the tobacco raw materials, thereby improving the sensory quality of tobacco products. On this basis, the present invention also provides a fermentation composition, a fermentation method, a cellulase preparation and a preparation method thereof, a tobacco composition and a tobacco product.

[0007] To achieve the above-mentioned purpose, the first aspect of the present invention provides a Bacillus subtilis, whose preservation number is CGMCC No. 24134, the preservation date is December 20, 2021, and the preservation unit is the General Microbiology Center of China National Microbiological Culture Collection Administration (CGMCC).

[0008] The second aspect of the present invention provides a fermentation composition, comprising Bacillus subtilis or its bacterial solution and culture medium; wherein the Bacillus subtilis comprises one or more of the Bacillus subtilis of the first aspect of the present invention, Bacillus subtilis (CGMCC No. 1.8886) and Bacillus subtilis (CGMCC No. 1.4468).

[0009] Bacillus subtilis (CGMCC No. 1.8886) refers to Bacillus subtilis with a deposit number of CGMCC No. 1.8886, which was purchased from the General Microbiological Center of China National Committee for the Preservation and Administration of Microbiological Cultures, and has been disclosed in Chinese patent document CN117448419A.

[0010] Bacillus subtilis (CGMCC No. 1.4468) refers to Bacillus subtilis with a deposit number of CGMCC No. 1.4468, which was purchased from the General Microbiology Center of China National Microbiological Culture Collection Administration.

[0011] In any embodiment of the second aspect of the present invention, the culture medium comprises a nitrogen source, an inorganic salt, an optional carbon source, an optional thickener and optional water.

[0012] In any embodiment of the second aspect of the present invention, the nitrogen source, inorganic salt, carbon source and thickener are all common substances in the art, and these substances can be purchased commercially on the market.

[0013] In any embodiment of the second aspect of the present invention, the nitrogen source includes one or more of an organic nitrogen source and an inorganic nitrogen source; preferably, the nitrogen source includes one or more of peptone, yeast extract, soybean cake powder and ammonium sulfate.

[0014] In any embodiment of the second aspect of the present invention, the inorganic salt includes one or more of inorganic sodium salt, inorganic potassium salt, inorganic magnesium salt, inorganic manganese salt and their hydrates; preferably, the inorganic salt includes one or more of sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate, potassium dihydrogen phosphate, manganese sulfate and their hydrates.

[0015] In any embodiment of the second aspect of the present invention, the carbon source comprises an organic carbon source; preferably, the carbon source comprises one or more of beef extract and wheat straw powder.

[0016] In any embodiment of the second aspect of the present invention, the thickener comprises one or more of sodium carboxymethyl cellulose and agar.

[0017] In any embodiment of the second aspect of the present invention, the fermentation composition is any one of the following:

[0018] 1) The culture medium comprises: peptone, yeast extract or beef extract, sodium carboxymethyl cellulose, and sodium chloride;

[0019] 2) The culture medium comprises: wheat straw powder, peptone, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, sodium chloride, and ammonium sulfate;

[0020] 3) The culture medium comprises: wheat straw powder, peptone, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, manganese sulfate monohydrate, soybean cake powder, and yeast extract.

[0021] In any embodiment of the second aspect of the present invention, the fermentation composition comprises any one of the following:

[0022] In item 1), the culture medium comprises: 8-12 parts by weight (e.g., 8, 9, 10, 11, 12 parts by weight) of peptone, 2-6 parts by weight (e.g., 2, 3, 4, 5, 6 parts by weight) of yeast extract or beef extract, 3-7 parts by weight (e.g., 3, 4, 5, 6, 7 parts by weight) of sodium carboxymethyl cellulose, and 8-12 parts by weight (e.g., 8, 9, 10, 11, 12 parts by weight) of sodium chloride;

[0023] In item 2), the culture medium comprises: 3-7 parts by weight (e.g., 3, 4, 5, 6, 7 parts by weight) of wheat straw powder, 0.1-0.5 parts by weight (e.g., 0.1, 0.2, 0.3, 0.4, 0.5 parts by weight) of peptone, 0.05-0.2 parts by weight (e.g., 0.05, 0.08, 0.1, 0.12, 0.15, 0.16, 0.18, 0.19, 0.2 parts by weight) of 0.01-0.05 weight part (e.g., 0.01, 0.02, 0.03, 0.04, 0.05 weight part) of dipotassium hydrogen phosphate, 0.01-0.05 weight part (e.g., 0.01, 0.02, 0.03, 0.04, 0.05 weight part) of magnesium sulfate heptahydrate, 0.3-0.7 weight part (e.g., 0.3, 0.4, 0.5, 0.6, 0.7 weight part) of sodium chloride, 0.1-0.5 weight part (e.g., 0.1, 0.2, 0.3, 0.4, 0.5 weight part) of ammonium sulfate;

[0024] In item 3), the culture medium comprises: 3-7 parts by weight (e.g., 3, 4, 5, 6, 7 parts by weight) of wheat straw powder, 8-12 parts by weight (e.g., 8, 9, 10, 11, 12 parts by weight) of peptone, 4-8 parts by weight (e.g., 4, 5, 6, 7, 8 parts by weight) of dipotassium hydrogen phosphate, 0.5-1.8 parts by weight (e.g., 0.5, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.7, 1.8 parts by weight) of magnesium sulfate heptahydrate, 1-5 parts by weight (e.g., 1, 2, 3, 4, 5 parts by weight) of potassium dihydrogen phosphate, 0.1-0.3 parts by weight (e.g., 0.1, 0.15, 0.2, 0.25, 0.3 parts by weight) of manganese sulfate monohydrate, 1-3 parts by weight (e.g., 1, 1.5,

[0025] 1.8, 2, 2.3, 2.5, 2.7, 3 parts by weight) of soybean cake powder, 0.2-0.6 parts by weight (e.g. 0.2, 0.3, 0.4, 0.5, 0.6 parts by weight) of yeast extract.

[0026] In any embodiment of the second aspect of the present invention, the culture medium includes or does not include Tween-80, preferably does not include Tween-80.

[0027] In any embodiment of the second aspect of the present invention, the fermentation composition further comprises an insoluble adsorbent.

[0028] In any embodiment of the second aspect of the present invention, the ratio of the insoluble adsorbent to the culture medium is greater than 0 and less than or equal to 0.01 g / mL, for example, 0.001 g / mL, 0.002 g / mL, 0.003 g / mL, 0.004 g / mL, 0.005 g / mL, 0.006 g / mL, 0.008 g / mL, 0.009 g / mL, and 0.01 g / mL.

[0029] In any embodiment of the second aspect of the present invention, the insoluble adsorbent is selected from one or more of glass balls, anion exchange fillers, cation exchange fillers, and macroporous adsorption resins, and can be a macroporous adsorption resin.

[0030] In any embodiment of the second aspect of the present invention, the insoluble adsorbent is selected from one or more of glass balls, cross-linked fiber DE-52, cross-linked fiber CM-52, macroporous adsorption resin D301, and anion exchange resin 717, and can be macroporous adsorption resin D301.

[0031] In any embodiment of the second aspect of the present invention, the insoluble adsorbent is an unused insoluble adsorbent or a recycled insoluble adsorbent.

[0032] In any embodiment of the second aspect of the present invention, the insoluble adsorbent is recovered 1-4 times, for example 1, 2, 3, or 4 times.

[0033] In any embodiment of the second aspect of the present invention, the volume ratio of the Bacillus subtilis liquid to the culture medium is 0.5%-3%, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%.

[0034] The third aspect of the present invention provides a fermentation method, comprising the following steps:

[0035] Bacillus subtilis or its bacterial solution is inoculated into a culture medium and cultured at 30° C. to 40° C. (e.g., 30° C., 32° C., 35° C., 37° C., 38° C., 39° C., 40° C.) for 1 to 120 hours (e.g., 1 hour, 5 hours, 10 hours, 12 hours, 15 hours, 20 hours, 24 hours, 26 hours, 28 hours, 30 hours, 33 hours, 36 hours, 38 hours, 40 hours, 45 hours, 48 ​​hours, 50 hours, 52 hours, 55 hours, 56 hours, 58 hours, 60 hours, 63 hours) , 65 hours, 67 hours, 70 hours, 72 hours, 75 hours, 78 hours, 80 hours, 82 hours, 84 hours, 86 hours, 88 hours, 90 hours, 92 hours, 94 hours, 96 hours, 98 hours, 100 hours, 103 hours, 105 hours, 107 hours, 108 hours, 110 hours, 112 hours, 115 hours, 118 hours, 120 hours) to obtain a fermentation product; wherein, the Bacillus subtilis or its bacterial solution and the culture medium are as described in the second aspect of the present invention.

[0036] In any embodiment of the third aspect of the present invention, the volume ratio of the Bacillus subtilis liquid to the culture medium is 0.5%-3%, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%.

[0037] In any embodiment of the third aspect of the present invention, the culture is carried out under stirring conditions; optionally, the stirring speed is 100-200 rpm, for example, 100 rpm, 120 rpm, 130 rpm, 150 rpm, 160 rpm, 180 rpm, 200 rpm.

[0038] In any embodiment of the third aspect of the present invention, the culture is carried out in a fermenter; wherein the ventilation rate of the fermenter is 0.1-0.5vvm, for example 0.1vvm, 0.2vvm, 0.3vvm, 0.4vvm, 0.5vvm.

[0039] In any embodiment of the second aspect or the third aspect of the present invention, the Bacillus subtilis liquid is prepared by the following steps:

[0040] Rejuvenate Bacillus subtilis and isolate single colonies;

[0041] Inoculate a single colony into a seed culture medium and culture at 30-40° C. (e.g., 30° C., 32° C., 35° C., 37° C., 38° C., 39° C., 40° C.) to obtain a bacterial solution;

[0042] Optionally, a single colony is inoculated in 30-100 mL (e.g., 30 mL, 40 mL, 50 mL, 70 mL, 80 mL, 90 mL, 100 mL) of seed culture medium;

[0043] Optionally, the culture is performed for 8-20 hours, for example, 8, 9, 10, 12, 14, 15, 16, 18, 20 hours;

[0044] Optionally, the culture is cultured under stirring conditions, preferably with a stirring speed of 100-200 rpm.

[0045] A fourth aspect of the present invention provides a method for preparing a cellulase preparation, comprising the following steps:

[0046] Fermentation is carried out according to the fermentation method of the third aspect of the present invention;

[0047] The fermentation product was separated into a clear liquid.

[0048] In any embodiment of the fourth aspect of the present invention, the method further comprises:

[0049] The clear liquid is sterilized, impurities removed, and concentrated to obtain a concentrated liquid.

[0050] In any embodiment of the fourth aspect of the present invention, the method further comprises: mixing the concentrated liquid with a filler and drying.

[0051] In any embodiment of the fourth aspect of the present invention, a 150-250 nm (eg, 150 nm, 180 nm, 200 nm, 230 nm, 250 nm) ceramic membrane is used for sterilization.

[0052] In any embodiment of the fourth aspect of the present invention, a 100-200 kD (e.g., 100 kD, 120 kD, 150 kD, 170 kD, 180 kD, 200 kD) ultrafiltration membrane is used to remove impurities, and the molecular weight of the impurities is greater than 100-200 kD.

[0053] In any embodiment of the fourth aspect of the present invention, 5-15 kD (e.g., 5 kD, 7 kD, 8 kD, 9 kD, 10 kD, 12 kD, 14 kD, 15 kD) ultrafiltration membrane is used for concentration.

[0054] In any embodiment of the fourth aspect of the present invention, the filler is a filler commonly used in the art.

[0055] The fifth aspect of the present invention provides a cellulase preparation, which is a clear liquid, a concentrated liquid or a dried product obtained by the method of the fourth aspect of the present invention.

[0056] In any embodiment of the fifth aspect of the present invention, the enzyme activity of the cellulase preparation is 30-82 U / mL (e.g., 30 U / mL, 33 U / mL, 35 U / mL, 36 U / mL, 37 U / mL, 38 U / mL, 40 U / mL, 42 U / mL, 44 U / mL, 45 U / mL, 47 U / mL, 49 U / mL, 50 U / mL, 52 U / mL, 53 U / mL, 55 U / mL, 57 U / mL). L, 58U / mL, 59U / mL, 60U / mL, 62U / mL, 64U / mL, 65U / mL, 67U / mL, 68U / mL, 69U / mL, 70U / mL, 72U / mL , 73U / mL, 74U / mL, 75U / mL, 76U / mL, 77U / mL, 78U / mL, 79U / mL, 80U / mL, 81U / mL, 82U / mL) or 1.2×10 4 -2×10 4 U / g (e.g. 1.2×10 4 U / g, 1.4×10 4 U / g, 1.5×10 4 U / g, 1.6×10 4 U / g, 1.8×10 4 U / g, 1.9×10 4 U / g, 2.0×10 4 U / g).

[0057] In any embodiment of the fifth aspect of the present invention, the specific enzyme activity of the cellulase preparation is 0.2-0.5 U / μg, such as 0.2 U / μg, 0.3 U / μg, 0.4 U / μg, 0.5 U / μg.

[0058] In any embodiment of the fifth aspect of the present invention, the OD600 of the cellulase preparation is 1.2-2.5, for example, 1.2, 1.4, 1.5, 1.6, 1.8, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5.

[0059] The sixth aspect of the present invention provides a tobacco composition, which comprises tobacco raw material and the cellulase preparation according to the fifth aspect of the present invention.

[0060] In any embodiment of the sixth aspect of the present invention, the mass ratio of the enzyme activity of the cellulase preparation to the tobacco raw material is 0.01-0.05 U / g, for example, 0.01 U / g, 0.02 U / g, 0.03 U / g, 0.04 U / g, 0.05 U / g.

[0061] In any embodiment of the sixth aspect of the present invention, the tobacco raw material includes one or more of shredded tobacco, tobacco leaves, and tobacco dust.

[0062] The seventh aspect of the present invention provides a tobacco product, comprising the tobacco composition of the sixth aspect of the present invention; optionally, the tobacco product is a cigarette.

[0063] The present invention achieves one or more of the following beneficial effects:

[0064] (1) The cellulase preparation produced by fermentation of Bacillus subtilis (CGMCC No. 24134) of the present invention has high enzyme activity and high purity, and is added to tobacco raw materials to help decompose excess cellulose and / or reduce the introduction of exogenous odor, thereby improving the sensory quality of tobacco products.

[0065] (2) The fermentation composition of the present invention is suitable for preparing cellulase preparations. The prepared cellulase preparations have high enzyme activity and high purity. Adding the preparations into tobacco products can reduce the introduction of exogenous odors and improve the sensory quality.

[0066] (3) The fermentation method of the present invention is suitable for preparing cellulase preparations, which can further improve the enzymatic activity and purity of the cellulase preparations, and can improve the sensory quality of tobacco products by adding the cellulase preparations into the tobacco products.

[0067] (4) Adding an insoluble adsorbent to the fermentation composition of the present invention is beneficial to promoting the production of cellulase, thereby increasing the enzymatic activity of the cellulase preparation. Adding the cellulase preparation to tobacco products is beneficial to improving the sensory quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0069] Figure 1 It is the morphological characteristics of Bacillus subtilis (CGMCC No.24134);

[0070] Figure 2 This is the gene sequence matching result of Bacillus subtilis (CGMCC No.24134). DETAILED DESCRIPTION

[0071] The embodiments of the present invention will be described clearly and completely below in conjunction with the examples. Obviously, the described examples are only some examples of the present invention, rather than all examples. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the examples in the present invention, all other examples obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0072] Bacillus subtilis YL-1 was screened from YLC1Y-2019 tobacco leaves in Yongding, Longyan, Fujian. It was deposited in the General Microbiology Center (CGMCC) of the China Culture Collection Administration on December 20, 2021, with the deposit number CGMCC No. 24134. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101.

[0073] Example 1: Identification of Bacillus subtilis (CGMCC No. 24134)

[0074] (1) Morphological characteristics of Bacillus subtilis (CGMCC No. 24134)

[0075] like Figure 1 As shown, the colony morphology of the strain verified by streaking is grayish white, with a dry and raised surface, which can be seen to collapse and form wrinkles, and the edges are irregular and burr-like.

[0076] (2) Molecular biological identification of Bacillus subtilis (CGMCC No. 24134)

[0077] The universal primers were synthesized by the solid phase phosphoramidite triester method, and the primer sequences were:

[0078] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No. 1)

[0079] 1492R: 5′-TACGGTTACCTTGTTACGACTT-3′ (SEQ ID No. 2)

[0080] Using total DNA of Bacillus subtilis (CGMCC No. 24134) as template [extracted by bacterial DNA kit of Bioengineering (Shanghai) Co., Ltd.] and 27F / 1492R as primers, 16S rDNA sequence was amplified. The PCR reaction system was as follows:

[0081] The total volume of the system is 25 μL: 2× Rapid Taq MasterMix 12.0 μL, 27F and 1492R primers 1 μL each, DNA 1 μL, ddH2O 10 μL.

[0082] PCR reaction program: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 15 s, for a total of 32 cycles; extension at 72°C for 5 min.

[0083]

[0084] After NCBI gene sequence alignment ( Figure 2 ), the strain had the highest similarity with Bacillus subtilis and was therefore identified as Bacillus subtilis.

[0085] Example 2: Different fermentation media for cellulase production

[0086] (1) Materials:

[0087] Peptone was purchased from Guangdong Huankai Microbiological Technology Co., Ltd., catalog number: 050160A;

[0088] Yeast extract was purchased from Guangdong Huankai Microbiological Technology Co., Ltd., catalog number: 050090A;

[0089] Sodium carboxymethyl cellulose was purchased from Aladdin Reagent (Shanghai) Co., Ltd., catalog number: C104985;

[0090] Beef extract was purchased from Guangdong Huankai Microbiological Technology Co., Ltd., catalog number: 050060;

[0091] Bean cake powder was purchased from Beijing Solebow Technology Co., Ltd., item number: FA0180;

[0092] Wheat straw powder is obtained by grinding mature harvested wheat straw to 100 mesh.

[0093] (2) Culture medium preparation:

[0094] LB solid medium: 10.0 g of peptone, 5.0 g of yeast extract, 10.0 g of sodium chloride, 20.0 g of agar, 1000 mL of distilled water, pH = 7.0;

[0095] Seed liquid culture medium: 10.0 g of peptone, 5.0 g of yeast extract, 10.0 g of sodium chloride, mixed with 1000 mL of distilled water, pH of the culture medium = 7.0;

[0096] Fermentation medium A: 10.0 g of peptone, 5.0 g of yeast extract, 5.0 g of sodium carboxymethyl cellulose, 10.0 g of sodium chloride, 1000 mL of distilled water, and the pH of the medium was 7.0;

[0097] Fermentation medium B: 10.0 g of peptone, 5.0 g of yeast extract, 5.0 g of sodium carboxymethyl cellulose, 10.0 g of sodium chloride, 10.0 g of Tween-80, 1000 mL of distilled water, and the pH of the medium was 7.0;

[0098] Fermentation medium C: 10.0 g of peptone, 3.0 g of beef extract, 5.0 g of sodium carboxymethyl cellulose, 10.0 g of sodium chloride, 10.0 g of Tween-80, 1000 mL of distilled water, and the pH of the medium was 7.0;

[0099] Fermentation medium D: 5.0 g wheat straw powder, 0.3 g peptone, 0.1 g potassium hydrogen phosphate, 0.03 g magnesium sulfate heptahydrate, 0.5 g sodium chloride, 0.3 g ammonium sulfate, add 1000 mL of distilled water and mix, the pH of the medium is 7.0;

[0100] Fermentation medium E: 5.0 g wheat straw powder, 10.0 g peptone, 6.0 g dipotassium hydrogen phosphate, 1.0 g magnesium sulfate heptahydrate, 3.0 g potassium dihydrogen phosphate, 0.2 g manganese sulfate monohydrate, 2.0 g soybean cake powder, 0.4 g yeast extract, 1000 mL of distilled water, the pH of the medium was 7.0.

[0101] (3) Fermentation of cellulase

[0102] The Bacillus subtilis (CGMCC No. 24134) bacterial solution stored in a cryotube at -80°C was placed in LB solid medium and rejuvenated at 37°C for 24 h. A single colony was scraped into a 250 mL conical flask containing 50 mL of seed liquid medium, and cultured at 37°C and 180 r / min for 12 h to obtain a seed liquid, which was inoculated into a 250 mL conical flask containing 100 mL of fermentation medium at a 1 volume % inoculum, and fermented at 37°C and 150 r / min. During the fermentation process, 5 mL of fermentation supernatant was taken at 0, 12, 24, 36, 48, 60, 72, 84, 96, and 110 h (the fermentation liquid was centrifuged at 10000 r / min for 10 min to take the supernatant) to detect the enzyme activity of cellulase. The method for detecting the enzyme activity of cellulase refers to the Chinese patent document CN117247866A. The highest enzyme activity and its corresponding peak time were determined according to the change trend of the cellulase enzyme activity, as shown in Table 1.

[0103] (4) Application in cigarettes:

[0104] 1.25U of cellulase was sprayed on every 50g of tobacco shreds. After 4h of enzymatic reaction at room temperature (25℃~30℃), the shreds were dried at 135℃ for 70s. After cooling to room temperature, the shreds were made into cigarettes for sensory evaluation. Among them, the sensory evaluation method refers to the Chinese patent document CN117247866A, and the comprehensive sensory score is the average of the sensory evaluation scores of each dimension. The bacterial count is characterized by OD600: 0.2mL of supernatant was taken and the absorbance at 600nm was measured in an ELISA instrument. The blank group did not add the crude enzyme solution, and an equal amount of distilled water was used instead when spraying the shredded tobacco. The results are shown in Table 1.

[0105] Table 1 Effects of various fermentation media on cellulase production by Bacillus subtilis and its effects on cigarette sensory perception

[0106]

[0107]

[0108] From Table 1, we can see that:

[0109] The fermentation cycle of fermentation medium A, C, and E is shorter, which is 48 hours, and the enzyme activity is higher, which has obvious advantages in the production process of cellulase.

[0110] Compared with the blank group with only water added, the fermentation supernatant (crude enzyme solution) corresponding to fermentation medium A and fermentation medium D has an improving effect on the sensory properties of tobacco.

[0111] Tween-80 is the difference between fermentation medium A and fermentation medium B. By comparing the fermentation effects of these two culture media, it can be seen that Tween-80 not only inhibits the growth of Bacillus subtilis (CGMCC No.24134), but also inhibits the production of cellulase and has a negative effect on the sensory improvement of tobacco.

[0112] The difference between fermentation medium B and fermentation medium C is that the former uses yeast extract, while the latter uses beef extract. By comparing the fermentation effects of the two culture media, it can be seen that beef extract promotes the production of cellulase by Bacillus subtilis (CGMCC No. 24134).

[0113] Compared with fermentation medium D, fermentation medium E contains more nitrogen sources (soybean cake powder, yeast extract) and metal ions (potassium, magnesium, manganese), which has a better effect on promoting the production of cellulase, but the rich nitrogen source has a negative effect on the sensory properties of tobacco. Fermentation medium E has high enzyme activity but poor sensory properties. Overall, fermentation medium A is the optimal medium.

[0114] The cellulase activity of the Bacillus subtilis (CGMCC No. 24134) of the present invention measured in fermentation medium A and fermentation medium E is significantly higher than the cellulase activity of the recombinant strain in the literature [Kong Mengmeng, Lu Peng, Chen Qiansi, et al. Construction of cellulase-producing engineering strains and their application in aging tobacco leaves [J]. Progress in Biotechnology, 2024, 14(2): 263-270].

[0115] Example 3: Comparison of enzyme production capacity of different strains and their application effects in cigarettes

[0116] The fermentation medium A of Example 2, the inoculation and fermentation method of Example 2 were used to produce enzymes, and the cellulase activity of the supernatant after the fermentation (48h) and its effect on the sensory evaluation of tobacco were detected according to the method of Example 2, wherein the blank group did not add the crude enzyme solution, and an equal amount of distilled water was used instead when spraying the tobacco. The strains compared were Bacillus subtilis (CGMCC No. 1.8886), Bacillus subtilis (CGMCC No. 1.4468), and Bacillus subtilis (CGMCC No. 24134) of the present invention, and the results are shown in Table 2.

[0117] Table 2 Comparison of enzyme production of different Bacillus subtilis and their application effects in cigarettes

[0118]

[0119] As shown in Table 2, the three enzyme-added groups have better sensory effects than the blank group with water added, and the Bacillus subtilis (CGMCC No. 24134) of the present invention has a better effect of producing cellulase than the other two strains of Bacillus subtilis, and has the best sensory improvement effect on cigarettes. Among the three strains, the Bacillus subtilis (CGMCC No. 24134) of the present invention is the best strain.

[0120] Example 4: Different insoluble adsorbents for enzyme production by Bacillus subtilis (CGMCC No. 24134) and their applications Effect comparison

[0121] The insoluble adsorbents in the following experimental groups were washed according to their respective instructions before use, and after being dried with hot air at 40°C, the required mass was weighed and added to the prepared fermentation medium for sterilization.

[0122] Control group: original fermentation medium A;

[0123] Experimental group 1: 0.2 g of 1 mm diameter glass beads (purchased from Shanghai Yuanye, product number H23362-100g) were added to every 100 mL of fermentation medium A;

[0124] Experimental group 2: 0.2 g of cross-linked fiber DE-52 (purchased from Shanghai Yuanye, product number S14024-100g) was added to every 100 mL of fermentation medium A;

[0125] Experimental group 3: 0.2 g of cross-linked fiber CM-52 (purchased from Shanghai Yuanye, product number S14020-100g) was added to every 100 mL of fermentation medium A;

[0126] Experimental group 4: 0.2 g of macroporous adsorption resin D301 (purchased from Shanghai Yuanye, product number S14020-100g) was added to every 100 mL of fermentation medium A;

[0127] Experimental group 5: 0.2 g of anion exchange resin 717 (purchased from Shanghai Yuanye, product number S14158-1 kg) was added to every 100 mL of fermentation medium A.

[0128] Bacillus subtilis (CGMCC No. 24134) was used as the production strain, and the above-mentioned fermentation medium was used to produce enzymes in the inoculation and fermentation method of Example 2. The bacterial count after fermentation (48h), the cellulase activity and specific enzyme activity of the supernatant, and the effect of the crude enzyme solution on the sensory evaluation of tobacco were detected according to the method of Example 2. The blank group did not add crude enzyme solution, and an equal amount of distilled water was used instead when spraying tobacco. Specific enzyme activity = cellulase activity of fermentation supernatant / protein content of fermentation supernatant, specific enzyme activity unit: U / μg. Protein content determination method reference [Liao Kuanqi, Ding Ning, Nie Lixuan, et al. Optimization of ultrasound-assisted protein extraction process of Bacillus subtilis fermentation waste residue by response surface method [J]. Biochemical Engineering, 2024, 10(04): 19-23+29].

[0129] The experimental results are shown in Table 3.

[0130] Table 3 Comparison of the effects of different insoluble adsorbents on cellulase production and application

[0131]

[0132] It can be seen from the above table:

[0133] Judging from the OD600 values, except for DE-52 which has an inhibitory effect on bacterial growth, other insoluble adsorbents have little effect on the bacterial count.

[0134] The enzyme production, specific enzyme activity and the effect of crude enzyme solution on tobacco sensory organs of experimental group 1 were similar to those of the control group, indicating that glass beads could not change the enzyme production performance of the strain. Cross-linked fiber DE-52 and anion exchange resin 717 were anion exchange fillers, which had an adsorption effect on anions in the fermentation broth, and cross-linked fiber CM-52 was a cation exchange filler, which had an adsorption effect on cations in the fermentation broth. However, based on the results of the control group, experimental group 2, experimental group 3 and experimental group 5, these three fillers were not conducive to the production of cellulase by the bacteria, and it may be that the adsorbent fillers would cause the loss of nutrients in the fermentation broth. Compared with the control group, the macroporous adsorption resin D301 of experimental group 4 could increase the enzyme activity of cellulase in the fermentation broth, and the specific enzyme activity was also increased. The higher the specific enzyme activity, the higher the purity of cellulase in the crude enzyme solution, indicating that the macroporous adsorption resin D301 could inhibit the accumulation of impurities in Bacillus subtilis (CGMCC No. 24134) during the fermentation process.

[0135] The effects of macroporous adsorption resin D301 with different recycling times on enzyme production of Bacillus subtilis (CGMCC No. 24134) were investigated. Influence

[0136] Recover D301 to prepare fermentation medium: After the fermentation of experimental group 4 is completed, D301 is intercepted with a 100-mesh sieve. The intercepted D301 is washed according to its instructions. After washing, 5 times the volume of deionized water is added and pasteurized at 80°C for 30 min, and then stored at 4°C. Before use, it is dried with hot air at 40°C, and then 0.2 g is weighed and added to 100 mL of fermentation medium A for sterilization.

[0137] Bacillus subtilis (CGMCC No. 24134) was used as the production strain, and the fermentation medium prepared by the above-mentioned recycled D301 was used to produce enzymes in the inoculation and fermentation manner of Example 2, and the cellulase activity in the supernatant after the fermentation was completed (48 h) was detected according to the method of Example 2.

[0138] A total of 4 recyclings were performed, and the results are shown in Table 4.

[0139] Table 4 Effects of different recycling times of macroporous adsorption resin D301 on cellulase production

[0140] Recycling times 0 1 2 3 4 Enzyme activity (U / mL) 78.0 77.5 75.2 72.4 64.5

[0141] As shown in Table 4, the macroporous adsorption resin D301 has the application prospect of recycling and reuse in promoting the production of cellulase by Bacillus subtilis (CGMCC No. 24134), but the enzyme activity decreased significantly after three times of recycling, and it is recommended to recycle no more than three or four times.

[0142] Study on the effect of the addition amount of macroporous adsorption resin D301 on the enzyme production of Bacillus subtilis (CGMCC No. 24134)

[0143] 0, 0.2 g, 0.4 g, 0.6 g, 0.8 g, and 1.0 g of macroporous adsorption resin D301 (purchased from Shanghai Yuanye, product number S14020-100 g) were added to every 100 mL of fermentation medium A, respectively, and then sterilized together.

[0144] Bacillus subtilis (CGMCC No. 24134) was used as the production strain, the above fermentation medium was used, and the enzyme was produced by the inoculation and fermentation method of Example 2. The cellulase activity in the supernatant at the end of fermentation (48 h) was detected according to the method of Example 2.

[0145] The results are shown in Table 5.

[0146] Table 5 Effect of different addition amounts of macroporous adsorption resin D301 on cellulase production

[0147]

[0148] It can be seen from Table 5 that when the addition amount of the macroporous adsorption resin D301 exceeds 0.8 g, the enzymatic activity of cellulase is lower than that of the control group.

[0149] Investigation of the application of macroporous adsorption resin D301 in the fermentation of two other strains of Bacillus subtilis

[0150] 0.2 g of macroporous adsorption resin D301 (purchased from Shanghai Yuanye, product number S14020-100 g) was added to every 100 mL of fermentation medium A, and then sterilized together.

[0151] Bacillus subtilis (CGMCC No. 1.8886) and Bacillus subtilis (CGMCC No. 1.4468) were used as production strains, and the above-mentioned fermentation medium was used to produce enzymes in the inoculation and fermentation method of Example 2, and the cellulase activity and specific enzyme activity in the supernatant after the fermentation (48 h) were detected according to the method of Example 2. Fermentation medium A without D301 was used as the control group, and the enzyme activity increment and specific enzyme activity increment were calculated, and the increment represented the test data of the experimental group minus the test data of the control group.

[0152] The results are shown in Table 6.

[0153] Table 6 Fermentation effect of macroporous adsorption resin D301 on different Bacillus subtilis

[0154]

[0155] As shown in Table 6, the macroporous adsorption resin D301 can promote the production of cellulase by Bacillus subtilis (CGMCC No. 1.8886) and Bacillus subtilis (CGMCC No. 1.4468) and improve the enzymatic activity and specific enzymatic activity of cellulase in the fermentation supernatant, and the purity of the cellulase is higher.

[0156] Example 5: Pilot production and application of cellulase

[0157] Although the sensory scores of experimental group 4 and control group in Table 3 are equal, the fermentation supernatant (crude enzyme solution) of the two groups has different effects on the sensory style of cigarettes. The control group has a slightly sweet and burnt aroma, while the experimental group 4 has a slightly sweet and refreshing aroma. From an application perspective, both groups are suitable for scale-up production.

[0158] Bacillus subtilis (CGMCC No. 24134) was used as the production strain, and the fermentation medium formulas of the control group and the experimental group 4 in Example 4 were selected respectively, and fermentation was performed in a fermenter. The seed liquid was prepared by shaking a flask with the seed liquid medium of Example 2, and inoculated into a 100L fermenter at a ratio of 1 volume %, with a liquid volume of 60L, a fermentation time of 15h, a temperature of 37°C, a rotation speed of 150r / min, and a ventilation volume of 0.3vvm; after the fermentation in the fermenter, the sterilization was performed by filtering with a 200nm ceramic membrane, and the sterilized clear liquid was filtered through a 150kD ultrafiltration membrane to remove impurities with a molecular weight greater than 150kD, and then a 10kDa ultrafiltration membrane was used for interception to obtain a cellulase concentrate with a molecular weight of 10 to 150kDa, and skimmed milk powder was added to the cellulase concentrate, the mass of the skimmed milk powder being 3% of the mass of the cellulase concentrate, and then spray-dried to obtain enzyme powder.

[0159] According to the method of Example 2, the enzyme preparations were respectively tested for enzyme activity and sprayed on tobacco leaves for sensory evaluation. The blank group did not add the crude enzyme solution, and an equal amount of distilled water was used instead when spraying the tobacco leaves. The experimental results are shown in Table 7.

[0160] Table 7 Enzyme activity and application of different cellulase preparations

[0161] Group Enzyme activity (U / g) Comprehensive sensory score Blank Group 0 5.5 Control group <![CDATA[1.1×10 4 ]]> 7 Experimental Group 4 <![CDATA[1.6×10 4 ]]> 7

[0162] The enzyme powders produced in the pilot test of the control group and the experimental group 4 both have a sensory improvement effect on cigarettes and have application value. Compared with the control group, the enzyme production of the experimental group 4 increased.

[0163] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Bacillus subtilis, its preservation number is CGMCC No.24134, the preservation date is December 20, 2021, and the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC).

2. A fermentation composition comprising Bacillus subtilis or its bacterial solution and a culture medium; wherein: The Bacillus subtilis includes one or more of the Bacillus subtilis described in claim 1, Bacillus subtilis (CGMCC No. 1.8886) and Bacillus subtilis (CGMCC No. 1.4468).

3. The fermented composition according to claim 2, wherein The culture medium comprises a nitrogen source, an inorganic salt, an optional carbon source, an optional thickening agent and optional water.

4. The fermentation composition according to claim 3, characterized in that One or more of the following: (1) The nitrogen source includes one or more of an organic nitrogen source and an inorganic nitrogen source; preferably, the nitrogen source includes one or more of peptone, yeast extract, soybean cake powder and ammonium sulfate; (2) The inorganic salt includes one or more of an inorganic sodium salt, an inorganic potassium salt, an inorganic magnesium salt, an inorganic manganese salt and hydrates thereof; preferably, the inorganic salt includes one or more of sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate, potassium dihydrogen phosphate, manganese sulfate and hydrates thereof; (3) The carbon source includes an organic carbon source; preferably, the carbon source includes one or more of beef extract and wheat straw powder; (4) The thickener comprises one or more of sodium carboxymethyl cellulose and agar.

5. The fermentation composition according to any one of claims 2 to 4, which is any one of the following: 1) The culture medium comprises: Peptone, yeast extract or beef extract, sodium carboxymethylcellulose, sodium chloride; 2) The culture medium comprises: wheat straw powder, peptone, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, sodium chloride, and ammonium sulfate; 3) The culture medium comprises: wheat straw powder, peptone, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, manganese sulfate monohydrate, soybean cake powder, and yeast extract.

6. The fermented composition according to claim 5, characterized in that Any of the following: In item 1), the culture medium comprises: 8-12 parts by weight of peptone, 2-6 parts by weight of yeast extract or beef extract, 3-7 parts by weight of sodium carboxymethyl cellulose, and 8-12 parts by weight of sodium chloride; In item 2), the culture medium comprises: 3-7 parts by weight of wheat straw powder, 0.1-0.5 parts by weight of peptone, 0.05-0.2 parts by weight of dipotassium hydrogen phosphate, 0.01-0.05 parts by weight of magnesium sulfate heptahydrate, 0.3-0.7 parts by weight of sodium chloride, and 0.1-0.5 parts by weight of ammonium sulfate; In item 3), the culture medium comprises: 3-7 parts by weight of wheat straw powder, 8-12 parts by weight of peptone, 4-8 parts by weight of dipotassium hydrogen phosphate, 0.5-1.8 parts by weight of magnesium sulfate heptahydrate, 1-5 parts by weight of potassium dihydrogen phosphate, 0.1-0.3 parts by weight of manganese sulfate monohydrate, 1-3 parts by weight of soybean cake powder, and 0.2-0.6 parts by weight of yeast extract.

7. The fermented composition according to any one of claims 2 to 6, wherein The culture medium may or may not include Tween-80.

8. The fermented composition according to any one of claims 2 to 7, wherein The fermentation composition also includes an insoluble adsorbent; Optionally, the ratio of the insoluble adsorbent to the culture medium is greater than 0 and less than or equal to 0.01 g / mL; Optionally, the insoluble adsorbent is selected from one or more of glass balls, anion exchange fillers, cation exchange fillers, and macroporous adsorption resins, and more preferably macroporous adsorption resins; Optionally, the insoluble adsorbent is selected from one or more of glass balls, cross-linked fiber DE-52, cross-linked fiber CM-52, macroporous adsorption resin D301, anion exchange resin 717, and more preferably macroporous adsorption resin D301.

9. The fermented composition according to claim 8, wherein The insoluble adsorbent is an unused insoluble adsorbent or a recycled insoluble adsorbent; Optionally, the insoluble adsorbent is recovered 1-4 times.

10. The fermented composition according to any one of claims 2 to 9, wherein The volume ratio of the Bacillus subtilis liquid to the culture medium is 0.5%-3%.

11. A fermentation method comprising the following steps: Inoculate Bacillus subtilis or its bacterial solution into a culture medium, and culture at 30°C-40°C for 1-120 hours to obtain a fermentation product; wherein, The Bacillus subtilis or its bacterial solution and the culture medium are as described in any one of claims 2 to 10.

12. The fermentation method according to claim 11, characterized in that One or more of the following: The volume ratio of the Bacillus subtilis liquid to the culture medium is 0.5%-3%; Cultivate under stirring conditions; The method is cultured in a fermenter; wherein the ventilation volume of the fermenter is 0.1-0.5 vvm.

13. A method for preparing a cellulase preparation, comprising the following steps: Fermentation is carried out according to the fermentation method of claim 11 or 12; The fermentation product was separated into a clear liquid.

14. The method according to claim 13, further comprising: sterilizing, removing impurities and concentrating the clear liquid to obtain a concentrated liquid; Optionally, the method further comprises: mixing the concentrated liquid with a filler and drying.

15. A cellulase preparation, which is a clear liquid, a concentrated liquid or a dried product obtained by the method of claim 13 or 14.

16. The cellulase preparation according to claim 15, characterized in that One or more of the following: The enzymatic activity of the cellulase preparation is 30-82U / mL; The specific enzyme activity of the cellulase preparation is 0.2-0.5 U / μg; The OD600 of the cellulase preparation is 1.2-2.

5.

17. A tobacco composition comprising tobacco raw material and the cellulase preparation according to claim 15 or 16; Optionally, the mass ratio of the enzyme activity of the cellulase preparation to the tobacco raw material is 0.01-0.05 U / g; Optionally, the tobacco raw material includes one or more of shredded tobacco, tobacco leaves, and tobacco dust.

18. A tobacco product comprising the tobacco composition according to claim 17; optionally, the tobacco product is a cigarette.

Images