Isatchenkia orientalis and application thereof

Abstract

The invention discloses issatchenkia orientalis and application thereof. Relates to the technical field of fermentation. The invention provides a specific strain and application thereof in reconstituted tobacco. According to the tobacco leaf extracting solution fermented by the bacterial strain, the content of esters such as 2, 2, 4-trimethylpentanediol isobutyl ester, 2, 2, 4-trimethyl-1, 3-pentanediol monoisobutyrate, tributyl phosphate and methyl palmitate is remarkably increased, eight novel ester substances with the characteristic aroma such as fruit aroma and rosin are newly generated, and the tobacco leaf extracting solution has the advantages that the content of the esters such as the 2, 2, 4-trimethylpentanediol isobutyl ester, the 2, 2, 4-trimethyl-1, 3-pentanediol monoisobutyrate, the tributyl phosphate and the methyl palmitate is increased; and the whole fragrance style is obviously enhanced. It is shown that the aroma-producing issatchenkia orientalis IO-1 fermented tobacco leaf extracting solution has good aroma-producing capacity, and through the combined effect of enzymolysis and fermentation, the extracting solution is expected to play an important application value in the field of reconstituted tobacco leaf raw material quality improvement.

Description

Technical Field

[0001] This invention relates to the field of fermentation technology, and more specifically to an Oriental Isaac yeast and its applications. Background Technology

[0002] The cigarette industry generates a large amount of byproducts such as tobacco stems, tobacco leaf fragments, and tobacco dust every year. Directly discarding or incinerating these byproducts would lead to resource waste and environmental pollution. The current treatment method is usually to process them into reconstituted tobacco sheets using the papermaking process for use in cigarette blending. However, these products generally suffer from defects such as a prominent woody aroma, insufficient sweetness, significant off-flavors, and poor smoking comfort, which seriously restricts quality upgrades and is currently the industry's main technical bottleneck.

[0003] Existing improvement technologies mostly focus on optimizing physicochemical processes and adjusting raw material ratios. While these technologies offer some improvement, they suffer from two major limitations: limited enhancement of sensory quality and increased process complexity and production costs. In the tobacco industry, biocatalysis technology, which uses microorganisms or their enzymes to target and degrade macromolecules and harmful components, promoting coordinated material transformation and achieving "quality enhancement and aroma enhancement," has significant advantages and value. However, nicotine and alkaloids in tobacco typically strongly inhibit the activity of conventional strains, and the industry lacks specialized microbial resources with strong tolerance and aroma-producing functions, hindering the application of biotechnology. Therefore, there is an urgent need to develop specialized functional strains that are highly adaptable to the tobacco environment to achieve a significant improvement in the quality of reconstituted tobacco leaves.

[0004] Oriental Isaac yeast has been reported to be used in the fermentation of baijiu (Chinese liquor), where it can lead to changes in the content of volatile flavor compounds in the mash.

[0005] Therefore, whether or not a *Issa yeast of the Orient* and its application can be provided to overcome the above-mentioned technical deficiencies is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides *Issatchenkia orientalis* and its applications. The core strategy of this invention is to discover and apply highly efficient aroma-producing microorganisms. Specifically, a yeast strain with significant aroma-enhancing potential was successfully isolated, purified, and screened from the surface of tobacco leaves, and identified as *Issatchenkia orientalis* by molecular biology. Addressing the characteristics of tobacco raw material components (such as fiber structure and limited available sugars), this invention creatively introduces a microbial enzymatic hydrolysis pretreatment process. Through systematic screening, the optimal scheme for preparing tobacco leaf extract using a complex enzyme solution of *Aspergillus brunneoviolaceus* Ab-10 and *Penicillium oxalicum* MCAX was determined, aiming to fully release flavor precursors such as sugars and significantly improve the accessibility of fermentation substrates. Based on this, key fermentation process parameters (such as temperature and pH) of the *Issatchenkia orientalis* strain in tobacco leaf extract were optimized through single-factor experiments. Experiments confirmed that under optimized fermentation conditions, this strain can efficiently utilize the components in tobacco leaf extract to produce rich and harmonious aroma compounds.

[0007] This invention utilizes gas chromatography-mass spectrometry (GC-MS) to analyze the aroma components in fermentation products, aiming to verify the effectiveness of microbial fermentation technology in improving the quality of low-grade tobacco raw materials. The research results of this invention are expected to provide a reference for improving the quality of low-grade tobacco raw materials using microbial fermentation technology.

[0008] Preservation Information: Issatchenkia orientalis IO-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The accession number is CGMCC: NO.34940, and the deposit date is June 18, 2025.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] An Oriental Issatchenkia IO-1, with accession number CGMCC34940.

[0011] The present invention also provides the above-mentioned Oriental Isaac's yeast IO-1 for use as a fermentation product of tobacco leaves or tobacco waste.

[0012] The present invention also provides a bacterial agent containing the above-mentioned *Issa mesasura* IO-1.

[0013] The present invention also provides the application of the above-mentioned *Issa mesasura* IO-1 in the preparation of tobacco products.

[0014] Preferred: For use in fermenting tobacco leaves or tobacco waste.

[0015] The present invention also provides a method for improving the quality of tobacco leaves or tobacco waste using the above-mentioned *Issa mesasura* IO-1, comprising the following steps:

[0016] (1) Preparation of tobacco leaf extract: including adding Penicillium oxalate MACX enzyme solution and Aspergillus purpureus Ab-10 enzyme solution to tobacco leaves or tobacco waste;

[0017] (2) The Oriental Isaac yeast 10-1 described in claim 1 is inoculated into a fermentation medium containing tobacco extract for fermentation culture.

[0018] Preferred step (1) is as follows: 10g of tobacco leaves or tobacco waste are added, and the amounts of Penicillium oxalate MACX enzyme solution and Aspergillus purpureus Ab-10 enzyme solution are both 2mg protein / g tobacco leaves. Then, it is mixed with 30mL of sterile water and 100μL of chloramphenicol, sealed, and placed in a shaker at 50℃ and 150rpm. After 24h, it is centrifuged to obtain tobacco leaf extract.

[0019] Preferred conditions for inoculation in step (2): pH 6, temperature 24℃, fermentation time 24h.

[0020] Preferred: The fermentation medium consists of 2% tobacco extract, 0.1% ammonium sulfate, 0.1% potassium dihydrogen phosphate and 0.1% magnesium sulfate.

[0021] As can be seen from the above technical solution, compared with the prior art, this invention discloses an *Isaiah oryzae* strain and its application, achieving the following technical effects: This invention screened an aroma-producing *Isaiah oryzae* strain IO-1 from tobacco leaves. This strain can utilize soluble sugars and proteins in tobacco leaf extracts for growth and aroma production. Through single-factor experiments, the fermentation conditions were optimized, determining the optimal conditions for strain IO-1 to be a temperature of 24℃ and a pH of 6. Enzymatic hydrolysis of tobacco leaves was performed using a combination of enzyme solutions from *Aspergillus purpureus* Ab-10 and *Penicillium oxalate* MCAX, followed by inoculation with strain IO-1 and fermentation under optimal conditions for 24 hours. The enzymatic degradation-increased aroma-enhancing biocatalytic technology of this invention significantly alters the composition of aromatic substances in tobacco leaf extracts, particularly significantly increasing the content of alcohols and esters. Specifically, the content of phenylethanol, a key aroma-producing substance with a rose aroma, increased dramatically from 1587.30 μg / L to 21016.82 μg / L, an increase of 12.24 times. The content of esters such as 2,2,4-trimethylpentanediol isobutyl ester, 2,2,4-trimethyl-1,3-pentanediol monoisobutyrate, tributyl phosphate, and methyl palmitate was significantly increased. Three new aroma components were also detected: isobutanol, 1-(2-butoxyethoxy)-ethanol, and dodecanol. Furthermore, eight new ester aromatic compounds were detected (methyl acetate, isoamyl acetate, methyl hexanoate, methyl salicylate, phenylethylvalerate, octyl lactone, decyl lactone, and methyl dehydroabirate), with the most significant increase in the variety of ester compounds. This indicates that the aroma-producing *Issaccharomyces orientalis* IO-1 fermented tobacco extract possesses excellent aroma-producing capabilities and, through the combined action of enzymatic hydrolysis and fermentation, holds significant application value in improving the quality of reconstituted tobacco raw materials. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 The attached figure is a morphological diagram of the strain provided by the present invention.

[0024] Figure 2 The attached figure is a phylogenetic tree diagram provided by the present invention.

[0025] Figure 3 The attached figure shows the growth of IO⁻¹ under different culture media pH and temperature conditions provided by this invention.

[0026] Figure 4The attached figure shows the reducing sugar concentration in the extract of different enzyme solutions provided by the present invention after enzymatic hydrolysis of broken tobacco leaves for 24 hours. Among them, Tr is Trichoderma reesei QM9414 enzyme solution; Po is Penicillium oxalate MCAX enzyme solution; and Ab is Aspergillus purpureus Ab-10 enzyme solution. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] This invention discloses an Oriental Isaac's yeast and its applications.

[0029] Instruments, materials and reagents

[0030] tobacco leaf.

[0031] YPD liquid medium consists of 1% yeast extract, 2% peptone, and 2% glucose. YPD solid medium is prepared by adding 2% agar powder to this mixture.

[0032] Seed culture medium: 2% wheat bran, 1% peptone, 1% glucose, 0.2% ammonium sulfate, 0.3% potassium dihydrogen phosphate and 0.05% magnesium sulfate.

[0033] Enzyme-producing culture medium: 3% wheat bran, 3% microcrystalline cellulose, 1.5% soybean meal, 0.2% ammonium sulfate, 0.5% potassium dihydrogen phosphate and 0.05% magnesium sulfate.

[0034] Fermentation medium: 2% tobacco extract, 0.1% ammonium sulfate, 0.1% potassium dihydrogen phosphate and 0.1% magnesium sulfate.

[0035] Raw materials not mentioned in the examples are conventional materials, and methods not mentioned are conventional methods, which will not be described further here.

[0036] Example 1

[0037] Isolation and screening of aroma-producing microorganisms

[0038] Add 0.5g of tobacco leaves to 50mL of sterile physiological saline, incubate at 25℃ and 150rpm for 3h with shaking, then centrifuge. Discard most of the supernatant, and mix the precipitate with the remaining supernatant (about 3mL) thoroughly to obtain a suspension. Spread 100μL of the appropriately diluted suspension evenly onto YPD solid medium and incubate overnight at 30℃. On the second day, observe the size, morphology, and color of the colonies. Select single colonies with different morphologies and streak them on YPD solid plates. Finally, pick single colonies for enrichment culture, collect the cells by centrifugation, add 30% glycerol, and freeze at -20℃ for later use.

[0039] After activating the isolated microbial strains, they were inoculated into YPD liquid medium at a 1% inoculum and cultured at 30°C and 200 rpm for 24 hours. The strains with prominent aromas were then selected for identification by smell.

[0040] Strain identification

[0041] Colony morphology observation: Dip a sterile inoculation loop into the bacterial solution and streak it on a YPD solid plate. Observe the colony morphology after 24 hours.

[0042] Cell morphology observation: Take a certain amount of bacterial culture and adjust OD 600 The concentration was 0.2. 100 μL was inoculated into 3 mL of LYPD liquid culture medium and cultured at 28 °C and 200 rpm for 24 h. After that, an appropriate amount of bacterial solution was taken to make a pellet and the cell morphology of the bacteria was observed under a 40x objective.

[0043] Species identification: DNA was extracted from yeast culture and amplified using primers listed in Table 1. The PCR products were verified by 1% agarose gel electrophoresis and then sent to Qingdao NIO Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with sequences in the NCBI database using BLAST. The identification results were derived based on the species information with the highest sequence similarity, and a phylogenetic tree was constructed based on the neighbor-linking method.

[0044] Table 1 Primers for 26S rDNA amplification and sequencing

[0045]

[0046] Experimental results:

[0047] Isolation, screening and identification of aroma-producing microorganisms

[0048] Three microorganisms were isolated from tobacco leaves and named IO-1, IO-2, and IO-3, respectively. After fermentation in YPD liquid medium for 24 hours, the fermentation broth was initially smelled. Both IO-1 and IO-2 exhibited aromas, with IO-2 showing a weaker, fresher fragrance, while IO-1 had a stronger fruity aroma (see Table 2). Therefore, IO-1 was selected for further research and patent preservation procedures.

[0049] Table 2

[0050]

[0051] Single colonies obtained after streaking IO⁻¹ onto YPD solid medium and incubating at 30°C for 24 hours are shown below. Figure 1 (Left image) shows pale yellow, round cells with a small diameter and smooth, regular edges. (Right image) Under the microscope, they appear as irregular oval or sausage-shaped single cells. 10⁻¹ genomic units were extracted, amplified, and sequenced. The sequencing results were compared with sequences in the NCBI database using BLAST, and a phylogenetic tree was constructed (see...). Figure 2 Ultimately, IO-1 was identified as Issatchenkia orientalis.

[0052] Example 2

[0053] Fermentation conditions optimization

[0054] Fermentation temperature and pH were optimized. Fermentation temperatures were set at 20℃, 24℃, 28℃, 32℃, and 36℃, with YPD liquid medium pH values ​​of 4, 5, 6, 7, and 8. When a single parameter was changed, the remaining parameters were set to either a temperature of 24℃ or a medium pH of 6. The fermentation time was 24 hours. Initial OD values ​​were measured at different temperatures and pH values. 600 and OD after 24 hours of culture 600 Analyze the growth capacity of the bacteria. Each group was divided into three replicates.

[0055] Experimental results:

[0056] Optimal fermentation conditions for the strain

[0057] The optimal temperature and pH for the growth of strain IO-1 in YPD liquid medium were analyzed, using OD0.1 as the metric. 600 The value is used to judge the growth status of the strain. For example... Figure 3 As shown in a) and b), under the culture conditions of 28℃, the culture medium pH was set to 4, 5, 6, 7, and 8, respectively. The results showed that IO⁻¹ had the highest cell concentration at pH 6.0, at which point OD₂O₃ was highest. 600 The value was 4.84. Under conditions of pH 6.0 in the culture medium, and with culture temperatures set at 20, 24, 28, 32, and 36℃, it was found that the cell growth rate first increased and then decreased with increasing culture temperature. The IO⁻¹ cell growth rate was fastest at 24℃. 600 The value was 3.95. Therefore, the optimal fermentation conditions for IO⁻¹ were finally determined to be 24°C and a culture medium pH of 6.

[0058] Example 3

[0059] Saccharification and fermentation

[0060] Enzyme solution preparation: Aspergillus purpureus Ab-10 (preservation number CGMCCNo.41105), Penicillium oxalate MCAX (from an engineered strain for improving hemicellulase production constructed in our laboratory, patent application number CN202011525326.8, authorization announcement number CN 112695022 B), and Trichoderma reesei were used.

[0061] After overnight culture in seed culture medium, QM9414 (ATCC 26921) was transferred to 50 mL of enzyme-producing culture medium at a 10% inoculum and cultured at 30 °C and 200 rpm for 5 days. The fermentation broth was filtered through a 0.22 μm filter membrane, and the crude enzyme solution was concentrated using an ultrafiltration tube. The protein concentration was determined by the Bradford method for subsequent enzymatic hydrolysis experiments.

[0062] Tobacco leaf extracts were prepared by enzymatic hydrolysis of broken tobacco leaves using different combinations of Penicillium oxalate MCAX enzyme solution, Aspergillus purpureus Ab-10 enzyme solution, and Trichoderma reesei QM9414 enzyme solution as shown in Table 3. The reducing sugar content in the extracts was determined by the DNS method.

[0063] The results are as follows Figure 4 Among the selected enzymes, the combination of *Penicillium oxalate* MCAX enzyme solution and *Aspergillus purpureus* Ab-10 enzyme solution showed the best enzymatic hydrolysis effect on broken tobacco flakes, with the highest reducing sugar yield (60.66 g / L) after 24 hours of enzymatic hydrolysis. Therefore, the extract obtained by combining *Penicillium oxalate* MCAX enzyme solution and *Aspergillus purpureus* Ab-10 enzyme solution was selected for subsequent fermentation experiments.

[0064] Table 3 Different enzyme solutions for enzymatic hydrolysis of broken tobacco leaves

[0065]

[0066] Based on the above experimental results, further experiments were conducted:

[0067] Preparation of tobacco leaf extract: 10g of crushed tobacco leaves were added, and 2mg protein / g tobacco leaf was added to both *Penicillium oxalate* MACX enzyme solution and *Aspergillus purpureus* Ab-10 enzyme solution. This mixture was combined with 30mL of sterile water and 100μL of chloramphenicol, sealed with aluminum foil, and placed in a shaker at 50℃ and 150rpm for 24 hours. After centrifugation, the tobacco leaf extract was obtained. Fermentation medium was prepared from the extract and dispensed into 10 vials. The pH was adjusted to the optimal pH for the bacteria and then sterilized. The selected strain (*Issatchenkia orientalis* IO-1) was inoculated into YPD liquid test tubes at a 1% (v / v) ratio for activation until OD... 600When the pH was 0.2, the culture medium was inoculated into the optimal pH (pH 6) fermentation medium and cultured at the optimized optimal temperature (24℃). After 24 hours of fermentation, the changes in aroma compounds in the fermentation broth were detected.

[0068] GC-MS aroma component analysis

[0069] Take 500 μL of fermentation broth into a headspace vial, and add 5 μL of 100 μg / mL 2-methyl-3-heptanone as an internal standard. Tighten the cap and mix well. Insert a 50 / 30 μm DVB / CAR / PDMS extraction head into the headspace vial 1 cm below the liquid surface, and place it in an 80℃ oven for extraction for 60 min. Remove the vial and immediately insert it into the gas chromatograph injection port (temperature 230℃) for analysis for 5 min.

[0070] The chromatographic column was a DB-5MS capillary column (30m × 0.25mm × 0.25μm). Temperature conditions: initial column temperature was set at 45℃ and held for 3 min; then increased to 180℃ at 3℃ / min and held for 1 min; finally increased to 250℃ at 5℃ / min and held for 5 min, for a total of 68 min. Carrier gas: high-purity helium, column flow rate 1 mL / min, injection port temperature 230℃. Mass spectrometry conditions: EI source, 70 eV, ion source temperature 230℃, mass transfer line temperature 270℃, quadrupole temperature 150℃, mass spectrometry scan range 35-550 m / z.

[0071] Experimental results:

[0072] Analysis of aroma-producing substances in fermented tobacco leaf extract

[0073] Under optimal fermentation conditions, the types and contents of aroma components in the IO-1 fermented tobacco extract were determined by GC-MS. The aroma components of the fermentation extract were classified according to functional groups, mainly into ketones, alcohols, acids, aldehydes, esters, phenols, and other aroma substances.

[0074] The results showed that after fermentation by strain IO-1, the content of aroma substances such as alcohols and esters in the extract increased compared with the control extract, with increases of 541.74% and 30.44%, respectively.

[0075] As shown in Table 4, the content of three alcohols in the fermented tobacco extract was significantly increased compared to the control group. For example, the content of phenethyl alcohol, a key aroma compound with a rose-like fragrance, increased dramatically from 1587.30 μg / L to 21016.82 μg / L. Three new aroma components were also added: isobutanol, 1-(2-butoxyethoxy)-ethanol, and dodecanol. Isoamyl alcohol increased by 8276.90%, exhibiting an apple brandy aroma. Linalool and phenethyl alcohol increased by 164.18% and 1224.06%, respectively, each possessing fruity, woody, and rose-like aromas. Furthermore, the content of eight alcohols in the fermented extract, including diacetone alcohol and 2-ethylhexanol, showed a decreasing trend compared to the control extract.

[0076] Table 4. Alcohol content in fermentation extract (μg / L)

[0077]

[0078]

[0079] As shown in Table 5, the content of four esters in the fermentation extract was significantly increased compared to the control group, and eight new aroma components were added: methyl acetate, isoamyl acetate, methyl hexanoate, methyl salicylate, phenylethyl valerate, octyl acetate, decyl acetate, and methyl dehydroabirate. Among these, isoamyl acetate, a unique component of the IO-1 fermentation extract, has a banana aroma; methyl hexanoate has a strong floral and fruity aroma; methyl salicylate has a sweet and refreshing wintergreen oil aroma; octyl acetate has a coconut aroma; decyl acetate has a strong coconut and peach aroma; and methyl dehydroabirate has a woody pine aroma. Furthermore, the content of six esters in the fermentation extract, including ethyl acetate and isobutyl acetate, showed a decreasing trend compared to the control extract.

[0080] Table 5. Esters in the fermentation extract (μg / L)

[0081]

[0082]

[0083]

[0084] In summary, the examples demonstrated effective enzymatic hydrolysis of broken tobacco leaves by comparing different enzyme combinations, yielding a tobacco extract rich in reducing sugars. Simultaneously, a novel strain of *Issatchenkia orientalis* was screened from tobacco leaves. This strain not only effectively utilizes the nutrients in the tobacco extract for fermentation and aroma production but also exhibits the potential to create unique aroma profiles such as fruity and floral notes. Furthermore, this yeast possesses good acid and heat resistance and maintains excellent fermentation characteristics under multiple stress conditions.

[0085] The IO-1 strain identified in this invention exhibited a significant aroma-enhancing effect after fermenting tobacco leaf extract. Specifically, the content of phenethyl alcohol, an important tobacco aroma compound that imparts a soft, delicate rose scent, increased dramatically from 1587.30 μg / L to 21016.82 μg / L, a 12.24-fold increase. Furthermore, the fermentation extract also showed newly added isobutanol, 1-(2-butoxyethoxy)-ethanol, and dodecanol, while the content of other higher fatty alcohols such as isoamyl alcohol and linalool also increased significantly. These substances typically function as unique trace components of flavor, and most possess specific aroma profiles.

[0086] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0087] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of *Issatchenkia orientalis* IO-1, characterized in that, The accession number is: CGMCC 34940.

2. The fermentation product of the Oriental Isaac's yeast 10-1 according to claim 1 for tobacco leaves or tobacco waste.

3. An inoculum containing the *Issa mesasura* 10-1 as described in claim 1.

4. The use of the Oriental Isaac's yeast IO-1 as described in claim 1 in the preparation of tobacco products.

5. The application as described in claim 4, characterized in that, Used for fermenting tobacco leaves or tobacco waste.

6. A method for improving the quality of tobacco leaves or tobacco waste using the *Issa mesasura* 10-1 as described in claim 1, characterized in that, Includes the following steps: (1) Preparation of tobacco leaf extract: including adding Penicillium oxalate MACX enzyme solution and Aspergillus purpureus Ab-10 enzyme solution to tobacco leaves or tobacco waste; (2) The Oriental Isaac yeast 10-1 of claim 1 is inoculated into a fermentation medium containing the tobacco extract for fermentation culture.

7. The method as described in claim 6, characterized in that, Step (1) is as follows: 10g of tobacco leaves or tobacco waste are added, and the amounts of Penicillium oxalate MACX enzyme solution and Aspergillus purpureus Ab-10 enzyme solution are both 2mg protein / g tobacco leaves. Then, it is mixed with 30mL of sterile water and 100μL of chloramphenicol, sealed, and placed in a shaker at 50℃ and 150rpm. After 24h, it is centrifuged to obtain tobacco leaf extract.

8. The method as described in claim 6, characterized in that, Step (2) Inoculation conditions: pH 6, temperature 24℃, fermentation time 24h.

9. The method as described in claim 6, characterized in that, The fermentation medium consists of: 2% tobacco extract, 0.1% ammonium sulfate, 0.1% potassium dihydrogen phosphate, and 0.1% magnesium sulfate.

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