Bacillus subtilis sodium nitrate fermentation method and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus subtilis as well as a composition, a culture or a leavening agent containing the bacillus subtilis. The invention also designs a method for preparing the fermentation liquor or the use liquor of the bacillus subtilis, the fermentation liquor or the use liquor obtained by the method, and application of the fermentation liquor or the use liquor in regulation and / or degradation of beta-carotene. The invention also relates to a method for processing tobacco raw materials or preparing tobacco products, and tobacco leaves or tobacco products treated by the method.

Description

Technical Field

[0001] This application relates to the field of microbial technology, specifically to Bacillus subtilis, and compositions, cultures, or fermentation agents containing the same. This application also provides methods for preparing a fermentation broth or a working liquid of Bacillus subtilis, a fermentation broth or working liquid obtained by the methods, and the use of the fermentation broth or working liquid for regulating and / or degrading β-carotene. This application also provides methods for processing tobacco raw materials or preparing tobacco products, as well as tobacco leaves or tobacco products treated by the methods. Background Art

[0002] β-carotene is one of the most stable natural pigments. It can generate C9-C13 norisoprenoid aroma compounds by breaking double bonds at different positions. These compounds have a significant impact on food aroma due to their low sensory threshold. β-carotene degradation products can enrich aroma and improve aroma quality. Increasing the amount of β-carotene degradation products in tobacco leaves can impart unique aroma characteristics and enhance tobacco quality.

[0003] β-carotene can be degraded through numerous pathways, with biodegradation playing a dominant role. This degradation occurs primarily through the secretion of highly specific microbial exoenzymes or the production of metabolites through their own growth and metabolism, leading to the degradation of β-carotene and its conversion into small-molecule flavor compounds. To further enhance food flavor, extensive research has been conducted on enhancing the volatile aroma compounds produced by β-carotene degradation through fermentation and application of bacterial strains.

[0004] For example, patent publication number CN114774374A discloses a thermostable carotenoid-degrading enzyme, its preparation method, and application, including: fermenting a Bacillus subtilis strain to prepare a crude enzyme solution for degrading carotenoids; degrading β-carotene using the crude enzyme solution; and fermenting the bacterial suspension with tobacco leaves to obtain tobacco leaves containing β-carotene degradation products. However, after multiple fermentation adjustments, the β-carotene degradation rate of this Bacillus subtilis strain still does not exceed 65% at most (see the Examples section of the disclosure, particularly Figures 1 to 9). Therefore, there is still significant room for improvement in its degradation rate.

[0005] For example, the patent document with publication number CN109266624A discloses a method for preparing a carotenoid-degrading enzyme, which uses a fermentation crude enzyme liquid of Kurthia sp to treat wolfberry residue or wolfberry pulp, thereby increasing the volatile aroma components of wolfberry wine and improving the quality of wolfberry wine. These technical means are all based on the characteristics of enzyme production by functional strains to degrade β-carotene, but the enzyme in the crude enzyme liquid fermented by the strain may be inactivated by temperature, pH or inhibitors (such as phenols, nicotine), resulting in unstable treatment effects. In industrial production, the enzyme reaction conditions need to be controlled, which increases costs. The small molecule metabolites in the non-enzyme filtrate are generally more stable, less affected by the environment, and have stronger process adaptability. However, at present, there are no reports on the use of the non-enzyme filtrate after the fermentation of the strain for the tobacco fermentation process to improve the efficiency of β-carotene degradation and improve the quality of tobacco leaves.

[0006] Therefore, technicians in this industry need to conduct research on methods for biodegrading β-carotene in order to efficiently degrade β-carotene and improve the sensory quality of tobacco leaves. Summary of the Invention

[0007] In order to solve the above problems, the inventors of this application conducted extensive research and regulated the fermentation of Bacillus subtilis by sodium nitrate, which significantly shortened the time for the fermentation liquid to degrade β-carotene and significantly increased the degradation rate of β-carotene, thereby effectively improving the sensory quality of tobacco leaves.

[0008] In a first aspect, the present application provides a Bacillus subtilis, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 32633.

[0009] In a second aspect, the present application provides a composition comprising the Bacillus subtilis described in the first aspect.

[0010] In certain embodiments, the composition further comprises a microorganism selected from the group consisting of bacteria, fungi (eg, yeast), or any combination thereof.

[0011] According to the information disclosed in the prior art, those skilled in the art have the corresponding ability to select one or more suitable microorganisms of other species and use them in combination with the Bacillus subtilis without generating antagonistic effects.

[0012] In certain embodiments, the microorganism is a probiotic.

[0013] In certain embodiments, the probiotic is selected from probiotic bacteria, yeast, or any combination thereof.

[0014] As used herein, the term "probiotic bacteria" is defined as any non-pathogenic bacteria that, when administered in sufficient amounts as live bacteria to a host, can have a beneficial effect on the health of the host.

[0015] In certain embodiments, the bacteria is selected from the group consisting of Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof.

[0016] In certain embodiments, the composition further comprises other microorganisms for fermentation, such as yeast.

[0017] In a third aspect, the present application provides a culture or fermentation agent comprising the Bacillus subtilis according to the first aspect or the composition according to the second aspect.

[0018] In certain embodiments, the culture or starter further comprises sodium nitrate.

[0019] In certain embodiments, the culture or starter further comprises components for growing and / or fermenting Bacillus subtilis (eg, fermentation medium).

[0020] In certain embodiments, the fermentation medium further comprises one or more selected from the group consisting of a carbon source, a nitrogen source, an inorganic salt, a growth factor (eg, a metal ion), a buffer, and water.

[0021] In certain embodiments, the fermentation medium comprises or consists of the following components: K2HPO4, MgSO4·7H2O, NaNO3, FeSO4·7H2O, KCl, sucrose, yeast powder and water.

[0022] In certain embodiments, the fermentation medium comprises or consists of the following components: K2HPO4 0.1-1 g, MgSO4·7H2O 0.1-1 g, NaNO3 1-5 g, FeSO4·7H2O 0.001-0.1 g, KCl 0.1-1 g, sucrose 10-20 g, and yeast powder 1-5 g.

[0023] In certain embodiments, the fermentation medium comprises or consists of the following components: K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, NaNO3 3 g, FeSO4·7H2O 0.01 g, KCl 0.5 g, sucrose 15 g, yeast powder 2 g and water.

[0024] In certain embodiments, the fermentation medium comprises or consists of the following components: sucrose, NaNO3, K2HPO4, MgSO4.7H2O, FeSO4.7H2O, KCl and water.

[0025] In certain embodiments, the fermentation medium comprises or consists of the following components: sucrose 10-20 g, NaNO3 1-5 g, K2HPO4 0.1-1 g, MgSO4·7H2O 0.1-1 g, FeSO4·7H2O 0.001-0.1 g, KCl 0.1-1 g and water.

[0026] In certain embodiments, the fermentation medium comprises or consists of the following components: sucrose 15 g, NaNO 3 3 g, K 2 HPO 4 0.5 g, MgSO 4 ·7H 2 O 0.5 g, FeSO 4 ·7H 2 O 0.01 g, KCl 0.5 g and water.

[0027] In a fourth aspect, the present application provides a method for preparing a fermentation broth or a use liquid of Bacillus subtilis, the method comprising:

[0028] (1) Providing the Bacillus subtilis described in the first aspect, the composition described in the second aspect, or the culture or fermentation agent described in the third aspect;

[0029] (2) inoculating the Bacillus subtilis into a fermentation medium under conditions suitable for fermentation to obtain a fermentation broth; wherein the fermentation medium contains sodium nitrate;

[0030] Optionally, the method further comprises:

[0031] (3) The fermentation liquid is centrifuged to remove the bacterial cells to obtain a working liquid.

[0032] In certain embodiments, in step (1), a bacterial solution of Bacillus subtilis is provided.

[0033] In certain embodiments, the bacterial solution of Bacillus subtilis is activated, and then a single activated colony is picked and inoculated into a culture medium for cultivation to obtain the bacterial solution of Bacillus subtilis.

[0034] In certain embodiments, in step (2), the fermentation medium further comprises one or more selected from the group consisting of: a carbon source, a nitrogen source, an inorganic salt, a growth factor (eg, a metal ion), a buffer, and water.

[0035] In certain embodiments, the fermentation medium comprises or consists of the following components: K2HPO4, MgSO4·7H2O, NaNO3, FeSO4·7H2O, KCl, sucrose, yeast powder and water.

[0036] In certain embodiments, the fermentation medium comprises or consists of the following components: K2HPO4 0.1-1 g, MgSO4·7H2O 0.1-1 g, NaNO3 1-5 g, FeSO4·7H2O 0.001-0.1 g, KCl 0.1-1 g, sucrose 10-20 g, and yeast powder 1-5 g.

[0037] In certain embodiments, the fermentation medium comprises or consists of the following components: K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, NaNO3 3 g, FeSO4·7H2O 0.01 g, KCl 0.5 g, sucrose 15 g, yeast powder 2 g and water.

[0038] In certain embodiments, the fermentation medium comprises or consists of the following components: sucrose, NaNO3, K2HPO4, MgSO4.7H2O, FeSO4.7H2O, KCl and water.

[0039] In certain embodiments, the fermentation medium comprises or consists of the following components: sucrose 10-20 g, NaNO3 1-5 g, K2HPO4 0.1-1 g, MgSO4·7H2O 0.1-1 g, FeSO4·7H2O 0.001-0.1 g, KCl 0.1-1 g and water.

[0040] In certain embodiments, the fermentation medium comprises or consists of the following components: sucrose 15 g, NaNO 3 3 g, K 2 HPO 4 0.5 g, MgSO 4 ·7H 2 O 0.5 g, FeSO 4 ·7H 2 O 0.01 g, KCl 0.5 g and water.

[0041] In certain embodiments, in step (2), the seed solution obtained in step (1) is inoculated into the fermentation medium at an inoculum amount of 1-20% (v / v) (e.g., 1% (v / v), 5% (v / v), 10% (v / v), 15% (v / v), 20% (v / v)).

[0042] In certain embodiments, in step (2), the seed solution obtained in step (1) is inoculated into the fermentation medium at an inoculation amount of 10% (v / v).

[0043] In certain embodiments, the method is accomplished by the following steps:

[0044] (a) Preparation of Bacillus subtilis seed solution: Activate the preserved Bacillus subtilis culture in TSB solid medium, and inoculate a single colony of the activated strain into TSB liquid medium and culture for 12 h;

[0045] (b) Fermentation broth preparation: The seed solution obtained in step (a) was inoculated into the fermentation medium at a 10% (v / v) inoculation rate and cultured at 37° C. and 180 rpm for 3 h;

[0046] (c) Fermentation broth treatment: The fermentation broth obtained in step (b) was centrifuged for 10 minutes, and the supernatant was collected in an ultrafiltration centrifuge tube, and centrifuged for 20 minutes. The liquid after centrifugation was collected as the working liquid.

[0047] In certain embodiments, the TSB liquid culture medium formula is: 30 g of tryptone soy broth medium powder and 1000 mL of distilled water.

[0048] In certain embodiments, the TSB solid culture medium formula is: 30 g of tryptone soy broth medium powder, 1000 mL of distilled water and 20.0 g of agar powder.

[0049] In a fifth aspect, the present application provides a fermentation liquid or a use liquid obtained by the method described in the fourth aspect.

[0050] In a sixth aspect, the present application provides use of the Bacillus subtilis as described above, or the composition as described above, or the culture or fermentation agent as described above, or the fermentation broth or use liquid as described above, in regulating and / or degrading β-carotene (for example, use in regulating and / or degrading β-carotene in tobacco raw materials or tobacco products).

[0051] In certain embodiments, the tobacco raw material is selected from raw tobacco, strip tobacco, or any combination thereof.

[0052] In certain embodiments, the tobacco raw material is redried tobacco strips.

[0053] In certain embodiments, the tobacco product is selected from a cigarette, an electronic cigarette, or any combination thereof.

[0054] In a seventh aspect, the present application provides 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: applying the Bacillus subtilis as described above, or the composition as described above, or the culture or fermentation agent as described above, or the fermentation liquid or use liquid as described above to the tobacco raw materials or tobacco products.

[0055] In certain embodiments, the tobacco feedstock or tobacco product is cultured or fermented after administration.

[0056] In certain embodiments, the administering is selected from spraying, painting, or soaking.

[0057] In certain embodiments, the tobacco raw material is selected from raw tobacco, strip tobacco, or any combination thereof.

[0058] In certain embodiments, the tobacco product is selected from a cigarette, an electronic cigarette, or any combination thereof.

[0059] In an eighth aspect, the present application provides a treated tobacco raw material or tobacco product, which is treated by the method as described above, or treated by the Bacillus subtilis as described above, or the composition as described above, or the culture or fermentation agent as described above, or the fermentation liquid or use liquid as described above.

[0060] In certain embodiments, the fermentation broth or use liquid, Bacillus subtilis, composition or culture or starter are applied to tobacco raw material or tobacco product and fermented to obtain the treated tobacco raw material or tobacco product.

[0061] In certain embodiments, the administering is selected from spraying, painting, or soaking.

[0062] In certain embodiments, the tobacco raw material is selected from raw tobacco, strip tobacco, or any combination thereof.

[0063] In certain embodiments, the tobacco product is selected from a cigarette, an electronic cigarette, or any combination thereof.

[0064] Definition of terms

[0065] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the biochemical, microbiological, and other procedures used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0066] As used herein, the term "Bacillus subtilis" is a Gram-positive bacterium belonging to the genus Bacillus. It is a rod-shaped, aerobic or facultative anaerobic bacterium, and its most notable feature is that it can form highly resistant endospores (spores) under harsh environments (such as nutrient deficiency, high temperature, dryness, etc.), thus having extremely strong survival ability. Typically, the cells of Bacillus subtilis are straight rod-shaped (about 2-5 μm long) and move by flagella. Bacillus subtilis is widely present in soil, rotting vegetation, air and water, and is an important component of natural microbial communities.

[0067] As used herein, the term "tobacco (Nicotiana tabacum L.)" refers to a plant of the genus Nicotiana in the Solanaceae family, used as the raw material for industrial cigarettes. Tobacco is widely cultivated in provinces across northern and southern China. It is a thermophilic crop and is sensitive to temperature. Different temperature conditions significantly affect tobacco quality and yield.

[0068] As used herein, the term "tobacco leaf" refers to the leaf blade of the plant Nicotiana tabacum. As used herein, the term "tobacco stem" refers to the thick, hard veins on a tobacco leaf.

[0069] As used herein, the term "fermentation" refers to the process of producing microbial cells themselves, or direct metabolites or secondary metabolites, through 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.

[0070] As used herein, the term "fermentation medium" refers to a synthetic mixture that provides essential nutrients and a growth environment for microorganisms, cells, or enzymes, and is used to support the growth, metabolism, and synthesis of target products (e.g., antibiotics, enzymes, organic acids, vaccines, etc.) of the target organism during the fermentation process. In certain embodiments, the microorganism is Bacillus subtilis. In certain embodiments, the microorganism is Bacillus subtilis Y-46.

[0071] The components of the fermentation medium include, but are not limited to: carbon sources (carbon elements that provide energy and synthesize the cytoskeleton, such as glucose, sucrose, starch, and glycerol); nitrogen sources (nitrogen-containing substances such as proteins and nucleic acids, such as yeast, peptone, ammonium sulfate, soybean meal, and urea); inorganic salts (to maintain osmotic pressure, enzyme activity, and cell structure, such as phosphate (K2HPO4), magnesium sulfate (MgSO4), and sodium chloride); growth factors (to assist metabolism, such as vitamins, amino acids, biotin, thiamine, and trace metal ions such as Fe2 + ,Zn2 + ); buffer (to stabilize pH, e.g., CaCO3, phosphate buffer).

[0072] Advantageous Effects of the Invention

[0073] Compared with existing technologies, the Bacillus subtilis and fermentation method of the present application can complete the degradation of β-carotene in the fermentation broth in a short period of time (for example, a degradation rate of almost 100% is achieved in just 3 hours of fermentation), significantly outperforming other Bacillus subtilis and other fermentation methods. Furthermore, a comparison of sensory evaluations of tobacco produced using different fermentation methods showed that the comprehensive sensory score of tobacco produced using the fermentation method of the present application was significantly improved, and the aroma quality of the flue-cured tobacco was enhanced, the aroma richness was increased, and the smoke was smooth and fine.

[0074] Therefore, the Bacillus subtilis and fermentation method of the present application have broad prospects in the application of preparing Bacillus subtilis fermentation broth and improving the aroma of tobacco leaves.

[0075] Below in conjunction with embodiment, embodiment of the present invention is described in detail, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, rather than to limit the scope of the 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.

[0076] Notes on the Deposit of Biological Materials

[0077] Bacillus subtilis y-47 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. 32633 and the deposit date is November 14, 2024. DETAILED DESCRIPTION

[0078] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0079] Unless otherwise specified, otherwise carry out the experiment and method described in embodiment basically according to conventional methods well known in the art and described in various references.For example, conventional techniques such as employed biochemistry, microbiology among the present invention can be found in " Methods in Enzymology " (METHODS IN ENZYMOLOGY) series (Academic Publishing Company).

[0080] In addition, if specific conditions are not specified in the examples, the experiments were performed under conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially. It is understood that the examples describe the present invention by way of example and are not intended to limit the scope of the present invention. All publications and other references mentioned herein are incorporated herein by reference in their entirety.

[0081] Example 1. Isolation and identification of strains

[0082] The present invention separates multiple bacterial strains from tobacco leaf extract from Luoyuan, Fujian Province, and screens out a Bacillus subtilis from them, which is named Bacillus subtilis Y-47.

[0083] Specifically, the strain was cultured for 48-72 hours, and a single colony was picked. Microscopically, the spores were oval to columnar, located in the center or slightly off the center of the bacterium, and the bacterium did not expand after spore formation. The colony was round, with slightly irregular edges, a slightly wrinkled and dry surface, an opaque milky white color, a hard texture, and easy to pick. The colony had a slightly raised center and relatively flat edges. Furthermore, a single clone of the strain was picked for 16S rRNA sequencing. The sequencing results showed that the strain isolated in this application was Bacillus subtilis.

[0084] In summary, the present application obtained a new strain of Bacillus subtilis y-47 and deposited it.

[0085] Example 2. Comparison of degradation of β-carotene

[0086] 1. Comparison of β-carotene degradation after fermentation in different culture media

[0087] In this embodiment, the degradation rate of β-carotene is used as an indicator to determine the formula of the Bacillus subtilis fermentation medium. The method of the present invention for fermenting Bacillus subtilis using sodium nitrate specifically comprises the following steps:

[0088] (1) Seed preparation: Take an appropriate amount of bacterial suspension from the glycerol tube and spread it on TSB solid medium. Activate and incubate twice at 30°C. Pick a single activated colony and inoculate it into TSB liquid medium. Incubate at 30°C and 180 rpm for 12 h. TSB medium: 30 g TSB powder, 1000 mL distilled water, pH = 7. For solid medium, add 20 g agar powder.

[0089] (2) Preparation of fermentation filtrate: The seed solution was inoculated into the fermentation medium at a 10% (v / v) inoculum. Fermentation was carried out at 37°C and 180 rpm for 3 h. The supernatant was then centrifuged at 4°C and 10,000 rpm for 10 min to obtain the fermentation supernatant. The fermentation supernatant was placed in a 10 kDa ultrafiltration centrifuge tube and centrifuged at 4°C and 8,000 rpm for 20 min to obtain a non-enzyme filtrate with a molecular weight of less than 10 kDa.

[0090] While maintaining carbon source supply and other conditions unchanged, the nitrogen source and metal ion content of the β-carotene fermentation medium were varied. Four fermentation medium groups were set up, all pre-added with 10 mg / L of β-carotene. The medium was inoculated with Bacillus subtilis Y-47 seed liquid and fermented for 12 hours. The β-carotene content was measured every 3 hours (where β-carotene content = 10 mg / L × (1 - β-carotene degradation rate); the β-carotene degradation rate of the strain was determined spectrophotometrically, with the unfermented medium serving as the blank control and the fermentation supernatant of the fermentation with the added bacteria serving as the experimental group. After fermentation, the absorbance at a wavelength of 460 nm was measured for both the experimental and control groups. The degradation rate (R) was calculated as follows: R = (A1 - A2 / A0) × 100%, where A1 is the absorbance of the control group after fermentation; A2 is the absorbance of the experimental group after fermentation; and A0 is the absorbance of the control group at a wavelength of 460 nm before fermentation). In addition, a medium A containing sodium nitrate that was not fermented (not inoculated with Bacillus subtilis Y-47 seed liquid) was set as a blank medium CK, and the β-carotene content was measured after 12 hours.

[0091] Culture medium A: K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, NaNO3 3 g, FeSO4·7H2O 0.01 g, KCl 0.5 g, sucrose 15 g, yeast powder 2 g, β-carotene reagent 7.5 mL, and distilled water 1000 mL.

[0092] Compared to medium A, medium B lacks a nitrogen source (yeast powder). The specific ingredients are as follows: sucrose 15g, NaNO3 3g, K2HPO4 0.5g, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g, KCl 0.5g, β-carotene reagent 7.5mL, and distilled water 1000mL.

[0093] Compared with medium A, medium C lacks NaNO3. The specific ingredients are as follows: sucrose 15g, yeast powder 2g, K2HPO4 0.5g, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g, KCl 0.5g, β-carotene reagent 7.5mL, and distilled water 1000mL.

[0094] Compared to Medium A, Medium D lacks inorganic salts, growth factors (e.g., metal ions), and buffers. The specific ingredients are as follows: sucrose 15 g, yeast powder 2 g, β-carotene reagent 7.5 mL, and distilled water 1000 mL.

[0095] Table 1 Degradation of β-carotene by strain y-47 in different culture media

[0096]

[0097]

[0098] As can be seen from Table 1, after 12 hours, the degradation rate of β-carotene in the blank medium CK (i.e., non-fermented medium A) was only 14.4%, indicating that the β-carotene degradation was caused by the fermentation of Bacillus subtilis Y-47.

[0099] Furthermore, after 3 hours of fermentation in medium A, Bacillus subtilis Y-47 achieved a β-carotene degradation rate of 99.2%, and after 9 hours of fermentation in medium B, the β-carotene degradation rate was 99.6%. Both mediums A and B were able to achieve nearly complete degradation of β-carotene in a relatively short fermentation time, and both mediums A and B contained sodium nitrate.

[0100] However, after 12 hours of fermentation in medium C without sodium nitrate, β-carotene degradation by Bacillus subtilis Y-47 was still not significant, with a β-carotene degradation rate of less than 30%. These results demonstrate that the addition of sodium nitrate to the fermentation medium plays a crucial role in β-carotene degradation by Bacillus subtilis Y-47. Furthermore, fermentation medium D, lacking several essential components for Bacillus subtilis fermentation, essentially rendered Bacillus subtilis Y-47 incapable of fermentation. Consequently, the β-carotene degradation rate was virtually identical to that of the CK medium.

[0101] In summary, combined with the time and cost advantages, culture medium A was used as the β-carotene-lowering fermentation medium for subsequent experiments.

[0102] 2. Comparison of β-carotene degradation by different strains

[0103] This example uses the β-carotene degradation rate as an indicator to determine the β-carotene degradation rate of different Bacillus subtilis strains. The strains tested were Bacillus subtilis Y-47, as well as Bacillus subtilis E4-7 and E6-12 (the isolation and identification procedures for these two strains were the same as in Example 1). The specific experimental procedures are as described above, and the fermentation medium used was Medium A, which showed the best results.

[0104] Table 2. Degradation of β-carotene by different Bacillus subtilis strains

[0105]

[0106] The experimental results showed that after 3 hours of fermentation, the β-carotene degradation rate of Bacillus subtilis E4-7 and 6-12 was much lower than that of Bacillus subtilis Y-47; after 12 hours of fermentation, the β-carotene degradation rate of Bacillus subtilis E4-7 and 6-12 was still around 40%, far lower than that of Bacillus subtilis Y-47. In summary, even though the tested strains all belonged to Bacillus subtilis and used the same fermentation medium, the β-carotene degradation ability of Bacillus subtilis Y-47 of the present application was still significantly better than that of other strains (degradation speed was fast and the degradation rate reached almost 100%). Therefore, the Bacillus subtilis Y-47 of the present application is particularly suitable for cultivation or fermentation to degrade β-carotene.

[0107] Example 3. Application of sodium nitrate in regulating non-enzyme filtrate in flue-cured tobacco

[0108] The filtrate of the fermentation medium without sodium nitrate (fermentation medium C in Example 1) was used as treatment 1, and the filtrate of the fermentation medium with sodium nitrate (fermentation medium A in Example 1) was used as treatment 2. Both were diluted 10 times, and 2.5 mL of treatment 1 and treatment 2 samples were taken.

[0109] Use a portable spray gun to evenly spray each solution onto 50g of Fujian Nanping flue-cured tobacco B03-2020. Place the leaves sprayed with the different filtered solutions in a marked ziplock bag and ferment at 25°C for 4 hours. After fermentation, spread the tobacco leaves on a tray and dry them at 135°C for 70 seconds. After drying, place the tobacco leaves in a constant temperature and humidity chamber at 22±1°C and 60±2% relative humidity for 48 hours to equilibrate moisture.

[0110] 1g (±0.1g) of tobacco was made into cigarette sticks, randomly numbered, and distributed to 10 professional smoking evaluators. The aroma quality, aroma volume, fineness, and other items of each cigarette were evaluated and scored with reference to the "YC / T 497-2014 Sensory Evaluation Method for Chinese Cigarette Style".

[0111] Table 3 Sensory evaluation of tobacco leaf samples

[0112]

[0113] It can be seen from Table 3 that compared with treatment 1 in which the fermentation medium did not contain sodium nitrate, treatment 2 in which the fermentation medium contained sodium nitrate had better smoking effect, improved aroma quality, increased aroma richness, and better smoke fineness.

[0114] Gas chromatography-mass spectrometry (GC-MS) was used to determine the content of β-carotene degradation products in fermented flue-cured tobacco from Treatments 1 and 2. 0.5 g of fermented flue-cured tobacco cuts were weighed and placed in a sample vial. 10 μL of a 10 mg / mL 2-octanol solution was added as an internal standard. The helium flow rate was 1.0 mL / min. The column oven temperature was maintained at 40°C for 5 min, then increased at 3°C / min to 120°C, then increased at 6°C / min to 240°C, held at 240°C for 5 min, and then post-run at 240°C for 5 min. The ion source temperature was 230°C, and the quadrupole temperature was 150°C. The electron impact ion energy was 70 eV, and mass spectra were acquired in full scan mode from 35 to 450 m / z.

[0115] Table 4 Content of β-carotene degradation products in flue-cured tobacco under different treatments

[0116]

[0117] Table 4 shows that, compared with Treatment 1, the contents of β-carotene degradation products, except dihydroactin and β-cyclocitral, increased significantly in flue-cured tobacco leaves treated with Treatment 2. Specifically, compared with Treatment 1, the content of 6-methyl-5-hepten-2-one increased by 1.72 times, the content of (E,E)-2,4-heptadienal increased by 2.25 times, and the content of β-damascenone increased by 1.54 times. The results showed that treatment 2, i.e., the filtrate of the sodium nitrate fermentation medium of Bacillus subtilis y-47, could effectively promote the degradation of β-carotene in tobacco leaves after being applied to flue-cured tobacco. The increase in the content of these β-carotene degradation products gave the flue-cured tobacco a unique aroma characteristic, such as 6-methyl-5-hepten-2-one with lemongrass, (E,E)-2,4-heptadienal with green and spicy aroma, β-damascenone with rich rose aroma, and megastigmatrienone which could enhance the dry fragrance and sweetness of tobacco leaves, thereby softening the flue-cured tobacco smoke and improving the quality of flue-cured tobacco.

[0118] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A Bacillus subtilis strain, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 32633.

2. A composition comprising the Bacillus subtilis according to claim 1; Preferably, the composition further comprises other microorganisms, for example, probiotics; Preferably, the composition further comprises other microorganisms for fermentation, such as yeast.

3. A culture or fermentation agent comprising the Bacillus subtilis according to claim 1 or the composition according to claim 2; Preferably, the culture or fermentation agent further comprises sodium nitrate; Preferably, the culture or fermentation agent further comprises components for culturing and / or fermenting Bacillus subtilis (e.g., fermentation medium); Preferably, the fermentation medium further comprises one or more selected from the group consisting of: a carbon source, a nitrogen source, an inorganic salt, a growth factor (e.g., a metal ion), a buffer, and water; Preferably, the fermentation medium comprises or consists of the following components: K2HPO4, MgSO4·7H2O, NaNO3, FeSO4·7H2O, KCl, sucrose, yeast powder and water; Preferably, the fermentation medium comprises or consists of the following components: K2HPO4 0.1-1g, MgSO4·7H2O 0.1-1g, NaNO3 1-5g, FeSO4·7H2O 0.001-0.1g, KCl 0.1-1g, sucrose 10-20g, yeast powder 1-5g; Preferably, the fermentation medium comprises or consists of the following components: K2HPO4 0.5g, MgSO4·7H2O 0.5g, NaNO3 3g, FeSO4·7H2O 0.01g, KCl 0.5g, sucrose 15g, yeast powder 2g and water; Preferably, the fermentation medium comprises or consists of the following components: sucrose, NaNO3, K2HPO4, MgSO4·7H2O, FeSO4·7H2O, KCl and water; Preferably, the fermentation medium comprises or consists of the following components: sucrose 10-20 g, NaNO3 1-5 g, K2HPO4 0.1-1 g, MgSO4·7H2O 0.1-1 g, FeSO4·7H2O 0.001-0.1 g, KCl 0.1-1 g and water; Preferably, the fermentation medium comprises or consists of the following components: sucrose 15 g, NaNO3 3 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, KCl 0.5 g and water.

4. A method for preparing a fermentation broth or a use liquid of Bacillus subtilis, the method comprising: (1) Providing the Bacillus subtilis according to claim 1, the composition according to claim 2, or the culture or fermentation agent according to claim 3; (2) inoculating the Bacillus subtilis into a fermentation medium under conditions suitable for fermentation to obtain a fermentation broth; wherein the fermentation medium contains sodium nitrate; Optionally, the method further comprises: (3) The fermentation liquid is centrifuged to remove the bacterial cells to obtain a working liquid.

5. The method according to claim 4, wherein In step (1), a bacterial solution of Bacillus subtilis is provided; Preferably, the bacterial liquid of Bacillus subtilis is activated, and then a single activated colony is picked and inoculated into a culture medium for cultivation to obtain the bacterial liquid of Bacillus subtilis.

6. The method according to claim 4 or 5, wherein In step (2), the fermentation medium further comprises one or more selected from the group consisting of: a carbon source, a nitrogen source, an inorganic salt, a growth factor (e.g., a metal ion), a buffer, and water; Preferably, the fermentation medium comprises or consists of the following components: K2HPO4, MgSO4·7H2O, NaNO3, FeSO4·7H2O, KCl, sucrose, yeast powder and water; Preferably, the fermentation medium comprises or consists of the following components: K2HPO4 0.1-1g, MgSO4·7H2O 0.1-1g, NaNO3 1-5g, FeSO4·7H2O 0.001-0.1g, KCl 0.1-1g, sucrose 10-20g, yeast powder 1-5g; Preferably, the fermentation medium comprises or consists of the following components: K2HPO4 0.5g, MgSO4·7H2O 0.5g, NaNO3 3g, FeSO4·7H2O 0.01g, KCl 0.5g, sucrose 15g, yeast powder 2g and water; Preferably, the fermentation medium comprises or consists of the following components: sucrose, NaNO3, K2HPO4, MgSO4·7H2O, FeSO4·7H2O, KCl and water; Preferably, the fermentation medium comprises or consists of the following components: sucrose 10-20 g, NaNO3 1-5 g, K2HPO4 0.1-1 g, MgSO4·7H2O 0.1-1 g, FeSO4·7H2O 0.001-0.1 g, KCl 0.1-1 g and water; Preferably, the fermentation medium comprises or consists of the following components: sucrose 15 g, NaNO3 3 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, KCl 0.5 g and water.

7. The method according to any one of claims 4 to 6, wherein In step (2), the seed solution obtained in step (1) is inoculated into the fermentation medium at an inoculum amount of 1-20% (v / v) (e.g., 1% (v / v), 5% (v / v), 10% (v / v), 15% (v / v), 20% (v / v)); Preferably, in step (2), the seed liquid obtained in step (1) is inoculated into the fermentation medium at an inoculation amount of 10% (v / v).

8. The method according to any one of claims 4 to 7, wherein The method is completed by the following steps: (a) Preparation of Bacillus subtilis seed solution: Activate the preserved Bacillus subtilis culture in TSB solid medium, and inoculate a single colony of the activated strain into TSB liquid medium and culture for 12 h; (b) Fermentation broth preparation: The seed solution obtained in step (a) was inoculated into the fermentation medium at a 10% (v / v) inoculation rate and cultured at 37° C. and 180 rpm for 3 h; (c) Fermentation broth treatment: The fermentation broth obtained in step (b) was centrifuged for 10 minutes, and the supernatant was collected in an ultrafiltration centrifuge tube and centrifuged for 20 minutes. The liquid after centrifugation was collected as the working liquid; Preferably, the TSB liquid culture medium formula is: 30 g of tryptone soy broth medium powder and 1000 mL of distilled water; Preferably, the TSB solid culture medium formula is: 30 g of tryptone soy broth medium powder, 1000 mL of distilled water and 20.0 g of agar powder.

9. The fermentation liquid or use liquid obtained by the method according to any one of claims 4 to 8.

10. Use of the Bacillus subtilis according to claim 1, or the composition according to claim 2, or the culture or starter according to claim 3, or the fermentation liquid or used liquid according to claim 9, in regulating and / or degrading β-carotene (e.g., in regulating and / or degrading β-carotene in tobacco raw materials or tobacco products); Preferably, the tobacco raw material is selected from raw tobacco, strip tobacco, or any combination thereof; Preferably, the tobacco raw material is redried tobacco strips; Preferably, the tobacco product is selected from cigarettes, electronic cigarettes, or any combination thereof.

11. 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: applying the Bacillus subtilis according to claim 1, the composition according to claim 2, the culture or starter according to claim 3, or the fermentation liquid or use liquid according to claim 9 to tobacco raw materials or tobacco products; Preferably, the tobacco raw material or tobacco product is cultured or fermented after administration; Preferably, the applying is selected from spraying, painting or soaking; Preferably, the tobacco raw material is selected from raw tobacco, strip tobacco, or any combination thereof; Preferably, the tobacco product is selected from cigarettes, electronic cigarettes, or any combination thereof.

12. A treated tobacco raw material or tobacco product, which has been treated by the method according to any one of claims 4 to 8, or treated with the Bacillus subtilis according to claim 1, the composition according to claim 2, the culture or starter according to claim 3, or the fermentation liquid or used liquid according to claim 9; Preferably, the fermentation liquid or use liquid, Bacillus subtilis, composition or culture or starter is applied to tobacco raw material or tobacco product and fermented to obtain the treated tobacco raw material or tobacco product; Preferably, the applying is selected from spraying, painting or soaking; Preferably, the tobacco raw material is selected from raw tobacco, strip tobacco, or any combination thereof; Preferably, the tobacco product is selected from cigarettes, electronic cigarettes, or any combination thereof.

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