Pichia pastoris ZK01 and application thereof

Abstract

The invention belongs to the technical field of microbial fermentation, and relates to pichia pastoris ZK01 and application thereof. The classification name of the pichia pastoris ZK01 is pichia pastoris, the pichia pastoris ZK01 is preserved in the China General Microbiological Culture Collection Center on March 7, 2025, and the preservation number of the pichia pastoris ZK01 is CGMCC No.33761. The pichia pastoris ZK01 is named as pichia pastoris ZK01. The pichia pastoris ZK01 is named as pichia pastoris ZK01. The preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain is screened from naturally alcoholized tobacco leaves, can be used for synthesizing 2-phenethyl alcohol by taking L-phenylalanine as a substrate, has good tolerance to metabolites, can grow in an environment containing 5g / L of 2-phenethyl alcohol, and can be used for industrial production of 2-phenethyl alcohol. Meanwhile, the strain can also be used in a tobacco alcoholization process, and 2-phenethyl alcohol produced by metabolism of the strain can endow alcoholized tobacco with unique fragrance similar to rose fragrance, so that the tobacco quality is effectively improved, and the fragrance level and sensory experience of the tobacco are enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Pichia yeast strain ZK01 and an application thereof. Background Art

[0002] 2-Phenylethanol, also known as β-phenylethanol, phenylethyl alcohol, or benzyl alcohol, is a colorless, viscous liquid at room temperature with a sweet, rose-like fragrance. It is soluble in water and glycerin and miscible in alcohol and ether. It is primarily used in flavoring foods such as honey, bread, peaches, and berries, and is widely used as a flavoring in soaps and cosmetics.

[0003] The main methods for producing 2-phenylethanol include chemical synthesis and biosynthesis. Biosynthesis has the advantages of low energy consumption and low environmental pollution, and can meet the growing market demand for "natural" products. Among the microorganisms used for biosynthesis of 2-phenylethanol, yeast is an important type of production strain. Currently, the yeast that can produce 2-phenylethanol is mostly Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), these natural strains generally have poor tolerance to 2-phenylethanol, which to some extent limits their application in the production of high-concentration 2-phenylethanol. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a strain of Pichia pastoris ( Pichia sp. ) ZK01 and its application, the strain can produce high 2-phenylethanol and has good tolerance to 2-phenylethanol.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a strain of Pichia pastoris ZK01, which is classified as Pichia pastoris ( Pichia sp. ), deposited in the General Microbiology Center of China Culture Collection Administration on March 7, 2025, with the deposit number CGMCC No.33761; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] Its 26S rDNA (as shown in SEQ ID No. 1) is as follows: .

[0007] The present invention provides the Pichia yeast ( Pichia sp. ZK01 is derived from naturally aged tobacco leaves. This strain grows well in temperatures ranging from 20-35°C (optimum temperature 30°C) and a pH range of 3.0-7.0 (optimum pH 5.5), and can even grow in an environment with 5 g / L of 2-phenylethanol. The optimal conditions for catalyzing the synthesis of 2-phenylethanol from L-phenylalanine are temperatures of 28-30°C and a pH of 4.5-6.0. After 72 hours of fermentation in a medium containing L-phenylalanine, the strain achieved a 2-phenylethanol yield of 333.81 mg / L. This strain demonstrates both efficient biosynthesis and product tolerance, demonstrating promising potential for industrial application.

[0008] In a second aspect, the present invention further provides a microbial agent comprising the above-mentioned Pichia pastoris ZK01 and a biologically acceptable microbial agent carrier and / or excipient.

[0009] The microbial preparation provided herein may contain only Pichia pastoris ZK01 or a combination of fermentation microorganisms containing Pichia pastoris ZK01; it may also include other substances required for preparation, such as carriers, nutrients, protective agents, and fillers. Formulations of the microbial preparation include, but are not limited to, suspensions, powders, and granules.

[0010] In a third aspect, the present invention further provides the use of the above-mentioned Pichia yeast ZK01 or the above-mentioned bacterial agent in a tobacco aging process.

[0011] The Pichia yeast ZK01 provided by the present invention can be used for tobacco alcoholization, and the 2-phenylethanol produced by its metabolism can impart rose fragrance to the alcoholized tobacco, thereby improving tobacco quality and enhancing the coordination of tobacco aroma.

[0012] In a fourth aspect, the present invention further provides a fermentation product of the above-mentioned Pichia pastoris ZK01 strain, wherein the fermentation product is produced by fermentation of the above-mentioned Pichia pastoris ZK01 strain or a combination of microorganisms comprising the above-mentioned Pichia pastoris ZK01.

[0013] The fermentation products are substances generated by the metabolic activities of Pichia pastoris ZK01 under suitable fermentation conditions, including primary metabolites and secondary metabolites of raw materials after microbial metabolism, such as Pichia pastoris ZK01 cells, fermentation broth, fermentation products, etc.

[0014] Preferably, the fermentation product is a fermentation broth; and the culture medium of the fermentation broth contains L-phenylalanine.

[0015] In a fifth aspect, the present invention also provides a method for preparing 2-phenylethanol, comprising the following steps: taking the activated Pichia yeast ZK01 or the above-mentioned bacterial agent and inoculating it into a fermentation medium containing L-phenylalanine; and the initial OD value of the fermentation medium after inoculation is 600 The pH value is 0.5-0.8, and the culture is carried out at a temperature of 28-30°C for 24-96 hours to obtain a fermentation liquid.

[0016] Preferably, the fermentation medium comprises 30 g / L glucose, 0.5 g / L MgSO4·7H2O, 2 g / L K2HPO4, 6.7 g / L YNB, 4 g / L L-phenylalanine, and a pH of 5.5-6.5.

[0017] Preferably, the initial OD of the fermentation medium after inoculation is 600 The concentration of 0.5 was set, and the culture was carried out at a temperature of 30°C for 72 h to obtain the fermentation liquid.

[0018] Preferably, the fermentation product is alcoholized tobacco.

[0019] Aged tobacco (fermented tobacco) refers to tobacco intermediate products that undergo long-term physical, chemical and biological transformation of tobacco raw materials (such as tobacco leaves, tobacco shreds, tobacco stems, etc.) through a naturally or artificially controlled storage environment (such as temperature, humidity regulation, microbial action, etc.) to improve their sensory quality, chemical composition coordination and processing suitability.

[0020] More preferably, the fermentation product is alcoholized tobacco leaves or alcoholized tobacco shreds.

[0021] The fermentation substrate for alcoholized tobacco leaves or cut tobacco is pre-treated tobacco leaves or cut tobacco. The type and quality of the tobacco leaves or cut tobacco, as well as the inoculum amount of Pichia yeast ZK01 or the aforementioned inoculum, can be adjusted according to actual needs to optimize the fermentation effect and the quality of the target product.

[0022] In a sixth aspect, the present invention also provides a method for preparing alcoholized tobacco leaves, comprising the following steps: taking the activated Pichia yeast ZK01 or the above-mentioned bacterial agent and inoculating it on the balanced tobacco leaves, with the inoculation amount being 10 5 -10 7 CFU / kg, 19-34℃, culture until the target alcoholization effect is achieved.

[0023] Whether the target aging effect has been achieved can be determined by testing its appearance quality (such as color, oil content, leaf integrity, etc.), chemical composition (such as total sugar, nicotine, total nitrogen, etc.), and sensory evaluation quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a plate colony image of Pichia pastoris ZK01 strain; Figure 2 This is an optical microscope image of Pichia pastoris ZK01 strain; Figure 3 is the phylogenetic tree of Pichia pastoris ZK01 strain; Figure 4 The GC-MS results of the Pichia pastoris ZK01 fermentation broth in Example 1 are as follows; Figure 5 GC-MS results of the standard product in Example 1; Figure 6 Tolerance test of Pichia pastoris ZK01 to 2-phenylethanol (a is the growth of the strain at 0 g / L 2-phenylethanol; b is the growth of the strain at 1 g / L 2-phenylethanol; c is the growth of the strain at 2 g / L 2-phenylethanol; d is the growth of the strain at 3 g / L 2-phenylethanol; e is the growth of the strain at 4 g / L 2-phenylethanol; f is the growth of the strain at 5 g / L 2-phenylethanol); Figure 7The growth of Pichia pastoris ZK01 under different culture conditions (a is the growth at different temperatures; b is the growth at different pH); Figure 8 The production of 2-phenylethanol by Pichia pastoris ZK01 under different culture conditions (a is the change in production at different temperatures; b is the change in production at different pH); Figure 9 The growth and metabolism of the Pichia pastoris strain ZK01 when producing 2-phenylethanol in Example 2; Figure 10 The 2-phenylethanol production capacity of the Pichia pastoris strain ZK01 in Example 2. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0026] The strains used in the following examples of the present invention are derived from the following: The Pichia pastoris ZK01 strain was deposited at the General Microbiology Center of the China Culture Collection Administration on March 7, 2025, with the deposit number CGMCC No. 33761; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0027] The culture medium used in the following examples of the present invention is as follows: YEPD medium: 10 g yeast extract powder, 20 g peptone, 20 g glucose, 1000 mL distilled water, pH 6.0; 20 g agar should also be added to prepare YEPD solid medium. When preparing YEPD solid medium containing streptomycin, add a sufficient amount of streptomycin solution before pouring the plates to a final concentration of 100 mg / mL.

[0028] Fermentation medium: 30 g glucose, 0.5 g MgSO4·7H2O, 2 g K2HPO4, 6.7 g YNB, 4 g L-phenylalanine, 1000 mL distilled water, pH 4.5-6.0; L-phenylalanine was added to the culture medium after filtration and sterilization.

[0029] The tobacco leaves and aged tobacco leaves used in the following examples of the present invention were provided by Hebei China Tobacco Industry Co., Ltd.

[0030] Unless otherwise specified, other reagents and materials in the examples of the present invention are commercially available.

[0031] Example 1 This example provides a Pichia yeast strain that produces 2-phenylethanol. The strain was isolated from aged tobacco leaves (provided by Hebei China Tobacco Industry Co., Ltd.).

[0032] 1. Isolation and purification Accurately weigh 5 g of aged tobacco leaves, chop them with sterile scissors, and place them in a 250 mL Erlenmeyer flask containing 100 mL of 0.85% sterile saline. Shake the flask at 30°C and 150 rpm for 30 min. Then, filter the tobacco leaves through sterile gauze to obtain the bacterial suspension. Spread an appropriate amount of the bacterial suspension onto a YEPD solid plate (containing 100 mg / mL streptomycin) in triplicate. Incubate the plate in a constant-temperature incubator at 30°C for 72 h. Select colonies of appropriate size and streak them for isolation and purification. Repeat this process several times until a single colony is obtained. Store the isolated and purified strain in a -80°C freezer.

[0033] Screening of aroma-producing strains All the isolated strains were inoculated into YEPD liquid culture medium and cultured at 30°C and 200 rpm for 24 h to obtain activated bacterial liquid; the activated bacterial liquid was inoculated into 50 mL fermentation medium (pH 6.0) at a 1% inoculum volume (v / v) and cultured at 30°C and 200 rpm for 36 h to obtain fermentation liquid. The fermentation liquid was identified by olfaction and fermentation liquid with obvious floral aroma was screened.

[0034] The fermentation broths with a distinct floral aroma were collected and centrifuged to obtain the supernatant. The supernatant was extracted with an equal volume of ethyl acetate. The extract was then concentrated to approximately 1 mL using a rotary evaporator. The 2-phenylethanol content was measured using LC-MS. Based on the 2-phenylethanol content in the fermentation broth, a high-yielding strain, ZK01, was screened. The fermentation broth of strain ZK01 had a rose aroma. The results are shown in Table 1. Figure 4 .

[0035] LC-MS measurement method: Benzyl alcohol was used as the standard (LC-MS results are shown in Figure 5 Chromatographic conditions: chromatographic column: ZORBAX Eclipse XDB-C18; mobile phase: ultrapure water (containing 1% formic acid, phase A), methanol (phase B); gradient elution program: 5% B from 0 to 3 min, linearly increased from 5% B to 40% B from 3 to 10 min, linearly increased from 40% B to 90% B from 10 to 15 min, maintained at 90% B for 15 to 20 min, decreased to 5% B from 20 to 21 min, and balanced for 5 min; injection mode was automatic; injection volume: 2 μL; flow rate: 1.0 mL / min; mass spectrometry conditions were: ESI (electrospray ionization source, positive ion mode), SIM scanning mode, spray voltage: 4000-4500 V.

[0036] 3. Identification of Pichia pastoris ZK01 strain 3.1 Colony and bacterial morphology of strain ZK01 The colony is milky white, dull, with fine notches on the edge. Figure 1 Microscopic morphology of bacteria: cells are oval or oblong, existing singly, see Figure 2 .

[0037] The strain was preliminarily identified as Pichia pastoris ( Pichia sp. ).

[0038] 3.2 Molecular biological identification of strain ZK01 The screened strains were inoculated into YEPD liquid medium and cultured at 30°C for 48 h. The bacteria were collected and genomic DNA was extracted using a yeast genome extraction kit (DP307). Subsequently, the extracted DNA templates were amplified by PCR using universal primers.

[0039] The primer sequences are as follows: NL1: 5'-GCATATCAATAAGCGGAGGAAAAG-3'; NL4: 5'-GGTCCGTGTTTTCAAGACGG-3'.

[0040] PCR amplification conditions were as follows: pre-denaturation at 94°C for 4 min; 32 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; and a final extension at 72°C for 10 min.

[0041] The obtained PCR products were detected by agarose gel electrophoresis, and the PCR products that met the target fragment size (544 bp) were sent to Shanghai Sangon Biotechnology Co., Ltd. for 26S rDNA sequence sequencing.

[0042] The 26S rDNA sequence of strain ZK01 was Blast aligned on the Genbank website, and the phylogenetic tree was constructed using MEGA software. The results showed that ( Figure 3 ), the strain was on the same branch as Pichia pastoris and was identified as Pichia pastoris ( Pichia sp. ).

[0043] In summary, combined with 26S rDNA sequence analysis and physiological and biochemical identification results, strain ZK01 was identified as Pichia pastoris ( Pichia sp. ), named Pichia pastoris ZK01.

[0044] Pichia pastoris ZK01 was deposited in the General Microbiology Center of China Culture Collection Administration on March 7, 2025, with the deposit number CGMCC No. 33761; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0045] 4. Tolerance of Pichia pastoris ZK01 to 2-phenylethanol The biomass (OD 600 The tolerance to 2-phenylethanol was evaluated by PCR, and Saccharomyces cerevisiae BY4742 and Pichia pastoris GS115 were used as control strains for comparative analysis.

[0046] Pichia pastoris ZK01 (ZK01), Saccharomyces cerevisiae BY4742 (BY4742) and Pichia pastoris GS115 (GS115) were inoculated into 20 mL YEPD liquid medium, and cultured at 30°C and 200 rpm for 24 h until the logarithmic growth phase (OD 600 ≈2.0), and activated bacterial solution was obtained; the three strains were inoculated into 50 mL YEPD liquid culture medium containing different concentrations of 2-phenylethanol (0-5 g / L), and the initial OD of the culture medium after inoculation was controlled. 600 The culture was shaken at 30°C and 200 rpm for 96 h, and samples were taken every 24 h to measure the OD 600 The results were as follows: Figure 6 shown.

[0047] One of the main factors limiting the biosynthesis of 2-phenylethanol by yeast is the inhibitory effect of 2-phenylethanol on cells. The better the tolerance of yeast to 2-phenylethanol, the higher its ability to convert and synthesize 2-phenylethanol. By observing the growth of the strains in YEPD liquid culture medium with different concentrations of 2-phenylethanol, the experimental results showed that when cultured normally in YEPD liquid culture medium without 2-phenylethanol, the maximum bacterial concentrations of ZK01, BY4742 and GS115 were not much different, and the bacterial concentrations all reached the maximum after 72 hours of culture; when 2-phenylethanol was contained in the YEPD liquid culture medium, BY4742 showed extreme intolerance, and the bacterial concentration could only fluctuate around 3 at any 2-phenylethanol concentration between 1-5 g / L; GS115, which is also a Pichia yeast like ZK01, had a slightly lower bacterial concentration than ZK01 when the 2-phenylethanol concentration was less than or equal to 3 g / L and the culture time was the same, but GS115 showed obvious intolerance when the 2-phenylethanol concentration reached 4 g / L or above; the Pichia yeast ZK01 provided by the present invention had an OD value of 72 hours when the 2-phenylethanol concentration was less than or equal to 4. 600The value is about 6, and it can still grow under the condition of 5 g / L 2-phenylethanol. The bacterial concentration is significantly better than that of the other two bacteria. It can be seen that the Pichia pastoris ZK01 provided by the present invention has excellent tolerance to 2-phenylethanol.

[0048] 5. Culture Conditions The experiment found that the growth temperature of Pichia yeast ZK01 is 20-35℃, the suitable growth temperature range is 25-30℃ (the optimum temperature is 30℃); the growth pH is 3.0-7.0, the suitable growth pH range is 4.5-6.0 (the optimum pH is 5.5), the results are shown in Figure 7 The optimum conditions for the synthesis of 2-phenylethanol using L-phenylalanine as a precursor are temperature 28-30°C (optimum 30°C) and pH 4.5-6.0 (optimum pH 5.5). Figure 8 .

[0049] Example 2 This example studies the ability of Pichia pastoris strain ZK01 to produce 2-phenylethanol and its growth and metabolism. The specific steps are as follows: A single colony of Pichia pastoris ZK01 was picked and inoculated into 20 mL of YEPD liquid medium, and cultured at 37°C and 200 rpm for 24 h until the logarithmic growth phase (OD 600 ≈2.0), to obtain seed solution; the seed solution was inoculated into the fermentation medium (pH 5.5), and the OD of the fermentation medium after inoculation was 600 The biomass (OD 600 ), glucose, 2-phenylethanol, ethanol and phenylalanine contents were analyzed, and the results are shown in Figure 9-10 .

[0050] Depend on Figure 9-10 As shown, Pichia pastoris ZK01 consumed glucose and phenylalanine, metabolizing them to produce alcohol, 2-phenylethanol, and phenylpyruvate. During fermentation, the biomass of ZK01 increased with fermentation time, but growth slowed between 24 and 48 hours, presumably due to the dual inhibitory effects of ethanol and 2-phenylethanol. Ethanol is a common byproduct of yeast, and the synergistic inhibitory effect of ethanol and 2-phenylethanol on cells is stronger than that of either alone. With increasing culture time, the biomass of Pichia pastoris ZK01 increased again and remained stable, indicating that the strain has a certain tolerance to both ethanol and 2-phenylethanol. The trend in 2-phenylethanol production also mirrored the biomass of the strain. The 2-phenylethanol content reached its peak at 333.81 mg / L at 72 hours of fermentation.

[0051] Example 3 This embodiment provides a Pichia pastoris ZK01 bacterial powder, which is composed of Pichia pastoris ZK01, trehalose, and skim milk powder, and is specifically prepared by the following steps: (1) Preparation of protective agent: Dissolve trehalose and skim milk powder in water and mix well so that the mass percentage concentration of trehalose in the obtained mixture is 10% and the mass percentage concentration of skim milk powder is 12%.

[0052] (2) Pichia pastoris ZK01 was picked from the glycerol tube stored at -80℃ and inoculated into YEPD solid medium. It was cultured at 30℃ for 2-3 days and activated twice. A single colony was picked and inoculated into YEPD liquid medium. It was cultured at 30℃ and 200 rpm for 18 hours. The cells were counted by hemocytometer and cultured until the bacterial concentration reached 1×10 8 CFU / mL, and the seed solution of strain ZK01 was obtained.

[0053] (3) The ZK01 seed solution was inoculated into YEPD liquid medium at a 3% inoculum (v / v) and cultured at 30°C and 0 rpm for 18 h to obtain the ZK01 fermentation broth. The supernatant was removed after centrifugation at 4°C and 6000 rpm for 10 min, and the culture was washed three times with sterile saline to collect the bacterial sludge. The bacterial sludge was mixed with the protective agent at a ratio of 1:1 and pre-frozen at -20°C for 12 h. After it was completely frozen, the sample was quickly transferred to a vacuum freeze dryer and freeze-dried (-60°C) for 24 h to obtain freeze-dried bacterial powder.

[0054] Example 4 This embodiment provides a method for producing 2-phenylethanol.

[0055] (1) Pick a single colony of Pichia pastoris ZK01 or a frozen strain of Pichia pastoris ZK01 and inoculate it into YEPD liquid medium. Culture the medium at 30°C and 200 rpm for 24 h to obtain a seed culture medium.

[0056] (2) The activated bacterial solution was inoculated into the YEPD liquid culture medium. The initial OD value of the YEPD liquid culture medium after inoculation was 600 The activated bacterial solution was obtained by culturing at 30°C and 200 rpm for 24 h.

[0057] (3) The activated bacterial solution was inoculated into the fermentation medium (pH 6.0). The initial OD value of the fermentation medium after inoculation was 600 The mixture was cultured at 30 °C and 200 rpm for 72 h to obtain the fermentation broth.

[0058] (4) The fermentation broth was centrifuged at 5500G for 5 minutes to remove the bacterial cells and obtain a supernatant. The supernatant was mixed with ethyl acetate at a volume ratio of 1:2, and after thorough oscillation, the mixture was centrifuged at 5000G for 5 minutes to separate the aqueous and organic phases. The organic phase was separated and then the ethyl acetate solvent was removed from the organic phase by vacuum distillation at 50°C using a rotary evaporator to obtain crude 2-phenylethanol.

[0059] Example 5 This example provides the use of Pichia yeast ZK01 in the preparation of aged tobacco leaves.

[0060] Take the activated Pichia yeast ZK01 sludge, add it into sterile water and mix well to prepare 5×10 4 40 mL of ZK01 inoculum was evenly sprayed onto 200 g of balanced C2F tobacco leaves. Stirring was performed while wearing sterile gloves to ensure even application. This was used as the sample. A sterile water treatment was also used as the control (CK). The sample and control were fermented at 32°C, 80% CO2, and constant humidity for 30 days.

[0061] After fermentation, the tobacco leaves were cured at 105°C, stirred every minute, and moisture was measured. Curing was terminated when the moisture content reached 12%. Finally, the leaves were equilibrated at 26°C and 60% relative humidity for 24 hours to allow for the release and volatilization of impurities. After aging, the quality of the aging leaves was measured and evaluated. The total alkaloids, chlorine, total sugars, reducing sugars, nicotine, and potassium content of the tobacco leaves were determined according to relevant industry standards. A corresponding smoking evaluation scale (scoring on a 10-point scale) was developed based on the single-ingredient smoking evaluation standards. The main evaluation indicators include aroma volume (A), aroma quality (B), strength (C), aftertaste (D), smoothness (E), softness (F), strength (G), sweetness (H), impurities (I), and pungency (L).

[0062] Table 1 Chemical composition content of fermented tobacco leaves

[0063] Table 2 Statistics of smoking evaluation of fermented tobacco leaves

[0064] Treating tobacco leaves with the Pichia yeast ZK01 strain significantly optimized their chemical composition and improved their sensory quality. After fermentation, the total sugar, reducing sugar, and total alkaloid contents in the leaves decreased significantly, while nicotine and potassium levels remained within optimal ranges and chlorine content slightly changed. This improved smoking quality and met industry standards, demonstrating that the strain can effectively regulate the balance of key chemical components in tobacco leaves. Sensory evaluation revealed that the fermented tobacco leaves performed exceptionally well in key indicators such as aroma quality, concentration, and finesse. Off-flavors and irritation were significantly reduced, resulting in a more harmonious and mellow overall quality and improved aroma harmony. This suggests that fermentation converts more sugars and amino acids in the tobacco leaves into flavor compounds, resulting in a richer aftertaste.

[0065] In summary, the present invention provides Pichia pastoris ( Pichia sp. ZK01 combines efficient 2-phenylethanol synthesis with significant product tolerance. Furthermore, it maintains good growth in the presence of both ethanol and 2-phenylethanol. This strain can be used for industrial production of 2-phenylethanol and in the production of alcoholized tobacco to improve tobacco quality.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Pichia yeast strain ZK01, characterized in that: Its taxonomic name is Pichia pastoris ( Pichia sp. ), deposited in the General Microbiology Center of China Culture Collection Administration on March 7, 2025, with the deposit number CGMCC No. 33761; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A bacterial agent, characterized in that The invention comprises the Pichia yeast ZK01 according to claim 1 and a biologically acceptable microbial agent carrier and / or auxiliary material.

3. Use of the Pichia yeast ZK01 according to claim 1 or the bacterial agent according to claim 2 in a tobacco alcoholization process.

4. The fermentation product of the Pichia pastoris ZK01 strain according to claim 1, characterized in that The fermentation product is produced by fermenting the Pichia pastoris ZK01 strain according to claim 1 or a microbial combination comprising the Pichia pastoris ZK01 strain according to claim 1.

5. The fermentation product according to claim 4, characterized in that The fermentation product is a fermentation broth; the culture medium of the fermentation broth contains L-phenylalanine.

6. A method for preparing 2-phenylethanol, characterized in that, The following steps are involved: The activated Pichia yeast ZK01 according to claim 1 or the bacterial agent according to claim 2 is inoculated into a fermentation medium containing L-phenylalanine. The initial OD value of the fermentation medium after inoculation is 600 The pH value is 0.5-0.8, and the culture is carried out at a temperature of 28-30°C for 24-96 hours to obtain a fermentation liquid.

7. The method for preparing 2-phenylethanol according to claim 6, wherein The fermentation medium comprises 30 g / L glucose, 0.5 g / L MgSO4·7H2O, 2 g / L K2HPO4, 6.7 g / L YNB, 4 g / L L-phenylalanine, and a pH of 5.5-6.

5.

8. The method for preparing 2-phenylethanol according to claim 6, wherein The initial OD of the fermentation medium after inoculation 600 The concentration of 0.5 was set, and the culture was carried out at a temperature of 30°C for 72 h to obtain the fermentation liquid.

9. The fermentation product according to claim 4, characterized in that The fermentation product is alcoholized tobacco.

10. A method for preparing alcoholized tobacco leaves, characterized in that: The following steps are involved: The activated Pichia yeast ZK01 according to claim 1 or the bacterial agent according to claim 2 was inoculated on the balanced tobacco leaves, with an inoculation rate of 10 5 -10 7 CFU / kg, 19-34℃, culture until the target alcoholization effect is achieved.

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