Pichia pastoris JSL-7 and its application

Abstract

This invention relates to the field of microbial technology, and particularly to Pichia pastoris JSL-7 and its applications. The JSL-7 strain of this invention was isolated from cattle. Testing showed that this strain exhibits good antibacterial effects against Escherichia coli, Staphylococcus aureus, and / or Shigella flexneri. Furthermore, this strain is non-toxic in vivo, possesses good bile salt and acid resistance, and can colonize well in the animal's gastrointestinal tract, exhibiting good beneficial bacteria properties. In addition to its beneficial bacteria properties, analysis of its metabolites revealed that this strain can produce menthol without genetic modification, a characteristic not found in existing strains. Furthermore, through optimization of the fermentation medium, the optimal fermentation medium for the preparation of menthol by this strain was determined. This discovery can effectively shorten the production time of menthol, simplify the production process, and provide new strains and ideas for the future fermentation production of menthol.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbiology, in particular to Pichia pastoris JSL-7 and its application. BACKGROUND

[0002] Pichia pastoris belongs to the methylotrophic yeasts of the genus Saccharomyces. Pichia ethanolica Compared with other yeasts, the most remarkable feature of Pichia pastoris is its methylotrophy, which means it can use simple compounds such as methanol as carbon source and energy for growth. Saccharomycetaceae This feature is mainly realized by the metabolic pathway catalyzed by alcohol oxidase, and the promoter of this pathway (such as AOX1 promoter) is very strong and is often used for efficient expression of foreign genes in yeast.

[0003] Therefore, Pichia pastoris has great potential in the field of biotechnology, especially in the expression of heterologous proteins. Pichia ethanolica Like its "cousin" Pichia pastoris, it may have the ability to efficiently secrete proteins and be suitable for high-density fermentation, which is very advantageous for the industrial production of foreign proteins (such as industrial enzyme preparations or biological drugs).

[0004] Menthol is a natural organic compound widely present in plants such as mint, and is widely used in medicine, food and daily chemical products due to its unique cooling sensation. The current methods for obtaining menthol are mainly extraction and synthesis: extraction is mainly from the leaves and stems of plants in the genus Mentha (such as the Labiatae plant Mentha haplocalyx). Through steam distillation, menthol crude oil is obtained, and further purification processes such as freezing crystallization are used to obtain it. In addition, it can also be prepared by chemical synthesis. There are also studies on the production of menthol by microbial fermentation, mainly through the construction of engineered bacteria to modify microorganisms to produce menthol. The commonly used menthol engineering bacteria strains are mainly Escherichia coli and yeast, and there is no related report on the synthesis of menthol by wild Escherichia coli and yeast. In the prior art, yeast mainly metabolizes menthol through two technical ideas: one is to introduce the metabolic pathway of menthol to convert sugars into menthol, and the other is to introduce an esterase metabolic system to convert menthol esters into menthol. No matter which technical approach, genetic engineering of yeast is required, which requires complex modification methods and must have high professional knowledge.

[0005] Therefore, if a yeast strain with good antibacterial and probiotic properties can be screened from nature, and can also produce menthol through fermentation metabolism, the scope of probiotic applications of the strain will be greatly expanded, and the ability to produce menthol by fermentation will be enhanced. Summary of the Invention

[0006] In view of the above, it is necessary to screen yeast strains from nature that have good antibacterial and probiotic properties and can also produce menthol through fermentation metabolism. This would greatly expand the scope of probiotic applications of such strains and enhance the ability to produce menthol using fermentation.

[0007] To achieve the above objectives, this invention has screened out a new strain: Pichia pastoris (ethanol). Pichia ethanolica JSL-7, its classification name is: Pichia ethanolica JSL-7, Chinese classification name: Pichia pastoris JSL-7, accession number GDMCC NO: 65730; this strain is deposited at Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on January 8, 2025.

[0008] The present invention also includes the ethanol Pichia pastoris ( Pichia ethanolica Application of JSL-7 in the preparation of bile salt-resistant and / or acid-resistant drugs.

[0009] The present invention also includes the ethanol Pichia pastoris ( Pichia ethanolica Application of JSL-7 in the production of menthol.

[0010] Furthermore, the fermentation medium for menthol production consists of a 10% (v / v) sugar-plant mixture and a 90% (v / v) YPD liquid medium; the sugar-plant mixture is prepared by mixing peppermint extract, lemongrass extract, 100 g / L galactose solution, and 100 g / L glucose solution in a volume ratio of 2-4:2-3:3-5:3-5.

[0011] Furthermore, the sugar-plant mixture is prepared by mixing peppermint extract, lemongrass extract, 100 g / L galactose solution and 100 g / L glucose solution in a volume ratio of 2:3:5:5.

[0012] Furthermore, the preparation method of the peppermint extract is as follows: fresh peppermint is crushed and mixed with water at a solid-liquid mass ratio of 1:1, then boiled and kept boiling for 5 minutes, filtered, and cooled to room temperature to obtain the peppermint extract; the preparation method of the lemongrass extract is as follows: fresh lemongrass is crushed and mixed with water at a solid-liquid mass ratio of 1:1, then boiled and kept boiling for 5 minutes, filtered, and cooled to room temperature to obtain the lemongrass extract.

[0013] The present invention also includes the ethanol Pichia pastoris ( Pichia ethanolica The application of JSL-7 in the preparation of antibacterial agents, wherein the antibacterial agent inhibits Escherichia coli (Escherichia coli). Escherichia coli Staphylococcus aureus Staphylococcus aureus ) and / or Shigella flexneri ( Shigella flexneri ).

[0014] The present invention also includes a method for applying the described ethanol-based Pichia pastoris (Pichia pastoris). Pichia ethanolica The method for producing menthol using JSL-7 is as follows: Pichia pastoris (ethanol-producing yeast) Pichia ethanolica JSL-7 was inoculated into the fermentation medium, and the fermentation broth containing menthol was obtained after the fermentation was completed.

[0015] The present invention has the following beneficial effects: The strain JSL-7 of the present invention was isolated from cattle. Testing showed that this strain has good antibacterial effects against Escherichia coli, Staphylococcus aureus, and / or Shigella flexneri. Furthermore, this strain has no in vivo toxicity, good bile salt and acid resistance, and can colonize well in the animal's gastrointestinal tract, exhibiting good beneficial bacteria properties. In addition to its beneficial bacteria properties, analysis of its metabolites revealed that this strain can produce menthol without genetic modification, a characteristic not possessed by strains in the prior art. Moreover, through optimization of the fermentation medium, we determined the optimal fermentation medium for the preparation of menthol by this strain. This discovery can effectively shorten the production time of menthol, simplify the production process, and provide new strains and ideas for the future fermentation production of menthol. Attached Figure Description

[0016] Figure 1 This is a colony morphology diagram of strain JSL-7.

[0017] Figure 2 The image shows the results of the hemolytic activity test for strain JSL-7.

[0018] Figure 3 The image shows the growth characteristics of strain JSL-7.

[0019] Figure 4 The figure shows the acid production characteristics of strain JSL-7.

[0020] Figure 5 The image shows the metabolites of strain JSL-7.

[0021] Information on the preservation of biological materials

[0022] The strain information preserved in this application is: Pichia pastoris (ethanol) Pichia ethanolica JSL-7, its classification name is:Pichia ethanolica JSL-7, Chinese classification name: Pichia pastoris JSL-7, accession number GDMCCNO: 65730; this strain is deposited at Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on January 8, 2025. Detailed Implementation

[0023] All features disclosed in this specification, or steps in all methods or processes disclosed herein, may be combined in any way, except for mutually exclusive features and / or steps.

[0024] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0025] Example 1

[0026] This example demonstrates the isolation and identification of the bacterial strain.

[0027] Isolation and purification of strains: A total of 67 rumen and rectal contents were collected from healthy cattle (Jersey cattle, Murrah buffalo, Nereid buffalo, Mediterranean buffalo, Weizhou yellow cattle, and Nandan yellow cattle). These were placed in sterile cryovials containing glycerol and transported back to the laboratory at -80°C for later use in liquid nitrogen. The rumen and rectal contents stored at -80°C (thawed at low temperature) were serially diluted 10-fold in sterile PBS in a sterile operating table, and thoroughly shaken (vortexed) to form a 1:10 sample homogenate. Using a 1 ml sterile pipette or micropipette, the 1:10 sample homogenate was slowly injected along the tube wall into a sterile test tube containing 9 ml of sterile PBS / sterile saline (ensuring the pipette tip did not touch the diluent). The test tube was shaken or a second sterile pipette was used to repeatedly pipette and agitate until thoroughly mixed, thus preparing a 1:10 sample homogenate. Take another 1ml sterile pipette or micropipette tip and, following the above procedure, homogenize the sample in 10-fold increments, using a different 1ml sterile pipette or tip for each increment. Select 3-4 appropriate dilutions and spread 100µl onto YPD agar plates or NA agar plates containing chloramphenicol (final concentration 25μg / ml). Use a spreader to carefully and quickly spread the inoculum onto the agar surface (clockwise or counterclockwise), ensuring the spreader does not touch the edge of the plate. After spreading, let the plates stand for 10 minutes to allow complete absorption of the inoculum into the medium. Invert the plates and incubate at 39℃ for 3-7 days. Select single colonies of yeast and bacteria for isolation and purification. Store the purified strains in 20% glycerol at -80℃. A total of 265 strains were isolated and purified.

[0028] 2. Screening for EPEC-resistant strains: The purified strains were screened for EPEC resistance using the following method.

[0029] ①EPEC resuscitation: Enteropathogenic Escherichia coli ( Escherichia coli EPEC (strain number: BNCC340977) (Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains) was used. After sterilizing the surface of the ampoule, it was opened in a biosafety cabinet, and 0.5 ml of sterile water was poured into the ampoule to fully dissolve the bacterial powder. 200 μl of the solution was then spread onto a nanoplatelet and incubated at 37°C for two generations. Single colonies were then picked to prepare a bacterial suspension for later use. Activated EPEC was stored in 20% glycerol at -80°C.

[0030] ② Screening: Take 10 μL of suspected yeast culture (bacteria) and streak a straight line on the surface of a YPD(NA) agar plate; incubate the YPD(NA) agar plate at 39℃ for 60 h; after incubation, take a sample diluted to 5 × 10⁻⁶ μL. 6 10 μL of CFU / ml EPEC bacterial suspension was streaked onto a YPD(NA) agar plate. The streak line was perpendicular to the line forming the suspected yeast (bacterial) colony, and the streak line did not touch the existing colonies of the suspected yeast (bacteria). After streaking, the YPD(NA) agar plate was incubated at 37℃ (the optimal growth temperature for EPEC). After 24 hours, the antibacterial effect on the suspected yeast (bacteria) was observed. A total of 15 strains with EPEC inhibition were screened.

[0031] ③ Screening for hemolytic activity of strains: 10 μL of suspected yeast (bacterial) bacterial suspension was inoculated onto YPD(NA) agar plates containing 7% (v / v) defibrinated sheep blood (Shanghai Yuanye Biotechnology Co., Ltd., catalog number: MP20026). The plates were incubated at 39℃ for 48 h, and the formation of hemolytic zones around the colonies was observed to determine whether the screened antibacterial strains exhibited hemolytic activity. 7 out of 15 strains showed hemolysis; the 8 strains without hemolytic zones were selected for further research.

[0032] 3. Strain Identification: Eight strains resistant to EPEC were identified using the following method: Genomic DNA was extracted from the eight strains, and the ITS and 16S regions of the genomic DNA were simultaneously amplified. Agarose gel electrophoresis revealed that the ITS sequence PCR product was approximately 500 bp, consistent with the size of the yeast ITS region sequence. Electrophoresis of the 16S sequence PCR product showed that the bacteria amplified a single target band, with the 16S region PCR product being approximately 1100 bp. After successful sequencing of the amplified products, BLAST homology comparison was performed on the obtained sequences using NCBI. The results are shown in the table below. The ITS region showed that five strains were similar to... Pichia ethanolicaThe similarity rates were 99.76%, 99.76%, 99.55%, 100.00%, and 99.54%, respectively. The 16S region showed that the three strains were similar to... Enterobacter quasihormaechei , Bacillus safensis , Pseudocitrobacter faecalis The similarity rates were 99.79%, 100.00%, and 99.79%, respectively (see Table 1 for details).

[0033]

[0034] Note: * in the table represents BLAST based on ITS or 16S region sequences.

[0035] Morphological identification: Strain JSL-7 was inoculated onto YPD agar plates and cultured for 24 hours. The resulting colony morphology is as follows: Figure 1 As shown in the figure, the colony is round, white, 1.5–3.5 mm in diameter, with irregular edges, thick and moist, and easy to pick up, which is consistent with the colony morphology of yeast. Combined with the molecular identification results in Table 1, the classification and naming of strain JSL-7 were determined. Pichia ethanolica .

[0036] Example 2

[0037] The selected yeast strains were tested for tolerance to bile salts, artificial gastric juice, and intestinal juice.

[0038] ① Bile salt tolerance assessment: The survival rate of the screened yeast strains was determined after incubation at 39°C for 3 hours in a 0.3% bile salt environment. The purified strains were inoculated into sterile YPD liquid medium and cultured at 39°C and 160 rpm for 24 hours. After centrifugation at 8000 rpm for 10 minutes at 4°C, the bacterial sludge was collected, washed with sterile PBS, and the bacterial concentration was adjusted to approximately 5 × 10⁻⁶. 7 CFU / mL. Centrifuge 1 mL of the above bacterial suspension (8000 rpm, 10 min), discard the supernatant, add 1 mL of YPD liquid medium containing 0.3% ox bile salts, and incubate at 39°C for 3 h. Perform viable cell counts on the 0 h and 3 h suspensions using the plate count method, and record the results as N. 0h and N 3h And use the formula: (N) 3h / N 0h The survival rate of the strain was calculated by multiplying the result by 100%, and the results are shown in Table 2.

[0039]

[0040] In the table, different letters in the superscript of the same column indicate significant differences (P < 0.05), while the same letter indicates no significant differences (P > 0.05). The same applies to the following tables.

[0041] As shown in Table 2, strain JSL-7 can survive well under 0.3% bile salt conditions, with a survival rate as high as 79.11%, which is significantly higher than other yeast strains of the same species screened in the same batch.

[0042] ② Artificial gastric juice tolerance assessment: After purification, the screened yeast was inoculated into sterile YPD medium and cultured at 39℃ and 160 r / min for 24 h. The culture was then centrifuged at 4℃ and 8000 r / min for 10 min, and the bacterial sludge was collected. The sludge was washed with sterile PBS, and the bacterial concentration was adjusted to approximately 5 × 10⁻⁶. 7 CFU / mL. Centrifuge 1 mL of the above bacterial suspension (8000 rpm, 10 min), discard the supernatant, add 1 mL of artificial gastric fluid (pH 1.5 and 3.0, respectively), and incubate at 39 °C for 3 h. Perform viable cell counts on the 0 h and 3 h suspensions using the plate count method, and record the results as N. 0h and N 3h And use the formula: (N) 3h / N 0h The survival rate of the strains was calculated by multiplying the result by 100% and the result is shown in Table 3.

[0043]

[0044] As shown in Table 3, strain JSL-7 can survive well in artificial gastric fluid at pH 3.0 with a survival rate of 151.91%, and in artificial gastric fluid at pH 1.5 with a survival rate of 58.10%, which is significantly higher than other yeast strains of the same species screened in the same batch.

[0045] ③ Artificial intestinal fluid tolerance assessment: After purification, the screened yeast was inoculated into sterile YPD medium and cultured at 39℃ and 160 r / min for 24 h. The culture was then centrifuged at 4℃ and 8000 r / min for 10 min to collect the bacterial sludge. The sludge was washed with sterile PBS, and the bacterial concentration was adjusted to approximately 5 × 10⁻⁶. 7 CFU / mL. Centrifuge 1 mL of the above bacterial suspension (8000 rpm, 10 min), discard the supernatant, add 1 mL of artificial intestinal fluid, and incubate at 39°C for 3 h. Perform viable cell counts on the 0 h and 3 h suspensions using the plate count method, and record the results as N. 0h and N 3h And use the formula: (N) 3h / N 0h The survival rate of the strains was calculated by multiplying the result by 100% and the result is shown in Table 4.

[0046]

[0047] As shown in Table 4, strain JSL-7 can survive well in artificial intestinal fluid, with a survival rate of 121.27%, which is significantly higher than other yeast strains of the same species screened in the same batch.

[0048] Example 3

[0049] This embodiment performs a comprehensive evaluation of the probiotic properties of the screened yeast strains.

[0050] ① Hydrophobicity of the strains: The surface hydrophobicity of the strains was evaluated using the microbial adherent hydrocarbon method (BATH), which involves collecting bacterial cells and resuspending them in PBS to adjust the OD of the bacterial solution. 600 The initial OD value was 0.4. 3 ml of the bacterial suspension was added to 0.6 ml of xylene and vortexed for 60 seconds. The two phases were then separated at 39°C for 30 minutes. The aqueous phase was removed and the OD value was measured. 600 The hydrophobicity of the strain is calculated using the formula [(1-ODtreatment) / ODinitial]×100% (denoted as ODtreatment).

[0051] ② Strains' self-aggregation ability: Collect bacterial cells and resuspend them in PBS to adjust the OD of the bacterial solution. 600 The value was 0.4 (denoted as ODinitial). After thoroughly mixing 3 ml of suspension, incubate at 39°C for 2 hours. After incubation, carefully remove 1 ml of bacterial solution from the upper region and measure the OD. 600 The self-aggregation ability of the strain was calculated using the formula [(1-ODtreatment) / ODinitial]×100%, and the results are shown in Table 5.

[0052]

[0053] As shown in Table 5, strain JSL-7 exhibited higher self-aggregation ability and hydrophobicity than other yeast strains. Self-aggregation ability and hydrophobicity are related to the adhesion and proliferation of the strain in intestinal epithelial cells. The self-aggregation ability of probiotics allows them to reach higher concentrations in the intestine, which is beneficial for adhesion and resistance to harsh environments. High hydrophobicity facilitates the interaction between probiotics and intestinal epithelial cells; the stronger the hydrophobicity of the strain, the easier it is to suspend in nonpolar solvents. A self-aggregation value ≥ 60% is considered strong, between 30% and 60% indicates moderate self-aggregation ability, and below 30% indicates weak self-aggregation ability. A hydrophobicity value ≥ 70% is considered highly hydrophobic, between 40% and 70% indicates moderate hydrophobicity, and < 40% is considered low hydrophobicity.

[0054] ③ Co-aggregation ability of bacterial strains and pathogenic bacteria: The overnight cultured screening strains and EPEC suspension were centrifuged (4℃, 8000×g, 10min), washed twice with sterile PBS solution (pH 6.7), and finally resuspended in PBS solution to achieve a final bacterial concentration of 1×10⁻⁶. 8CFU / mL. Take 1.5 mL each of the screening strain suspension and the pathogen suspension, mix well, vortex for 10 s, and incubate at 39°C for 2 h. Simultaneously prepare suspensions containing only the screening strain or only the pathogen, mix well, vortex for 10 s, and incubate at 39°C for 2 h. Carefully aspirate the top layer of liquid (generally 10% of the total volume) and measure the absorbance at 600 nm. The ability of the screening strain to co-agglutinate the pathogen is expressed by the following formula.

[0055] Coagulation (%) = [1 - Amix / (Aprobiotic + Apathogen)] × 100, where Amix, Aprobiotic, and Apathogen represent the absorbance values ​​of the screened strain, EPEC, and their mixture after incubation at 39°C for 2 hours, respectively. The copolymerization results are shown in Table 6.

[0056]

[0057] As shown in Table 6, strain JSL-7 has a higher copolymerization ability than other yeast strains.

[0058] The probiotic properties of yeast strains were comprehensively evaluated, and the evaluation criteria are shown in Table 7.

[0059]

[0060] The scoring results are shown in Table 8.

[0061]

[0062] As shown in Table 8, among the yeast strains screened in the same batch, strain JSL-7 had the highest overall score, indicating that strain JSL-7 has good probiotic potential.

[0063] Example 4

[0064] This example demonstrates the drug susceptibility and toxicity tests of strain JSL-7.

[0065] ① Antifungal drug susceptibility: The antifungal drug susceptibility test of strain JSL-7 was conducted using the yeast-like fungal susceptibility test kit REF 14 204 (bioMérieux France). The results are shown in Table 9.

[0066]

[0067] Note: MIC represents the minimum inhibitory concentration; sensitivity: S represents sensitive; I represents intermediate; R represents resistant.

[0068] As shown in Table 9, strain JSL-7 showed no resistance to 5-fluorocytosine, amphotericin B, fluconazole, itraconazole, or voriconazole.

[0069] ② Subacute oral toxicity test of strain JSL-7: 24 5-week-old SPF-grade BALB / c mice were acclimatized for 7 days and randomly divided into two groups. The control group was administered sterile PBS by gavage at 0.1 ml / 10 g body weight, while the treatment group was administered a high-dose ethanol-concentrated Pichia pastoris JSL-7 bacterial suspension (suspension concentration 1.75 × 10⁻⁶) by gavage. 9 CFU / ml). Mice were allowed free access to food and water during the experiment. Mice were administered the drug via gavage for 28 consecutive days, and the results were as follows: There were no statistically significant differences in body weight, feed intake, feed conversion ratio, blood physiological and biochemical indicators, and organ indices between the treated and control groups. Hematologic and endoscopic examinations (H&E) of important organs such as the liver, kidneys, spleen, lungs, heart, brain, testes, epididymis, ileum, and colon in both the experimental and treated groups showed no pathological damage. Therefore, strain JSL-7 has no subacute toxicity to the body, and its NOAEL (no visible adverse effect dose) is 1.75 × 10⁻⁶. 10 CFU / (kg.WB) / day.

[0070] ③ Hemolytic activity test of strain JSL-7: 10 μL of JSL-7 bacterial culture was inoculated onto a YPD agar plate containing 7% (v / v) defibrinated sheep blood (Shanghai Yuanye Biotechnology Co., Ltd., catalog number: MP20026). The plate was incubated at 39℃ for 48 h. The presence or absence of a hemolytic zone around the colony was observed to determine whether the JSL-7 strain exhibited hemolytic activity. The results are as follows: Figure 2 As shown: There is no hemolytic zone around the colony of strain JSL-7, indicating that strain JSL-7 is non-hemolytic.

[0071] Example 5

[0072] This example is a study on the growth characteristics, acid production characteristics and metabolites of strain JSL-7.

[0073] ① Growth curve and acid production characteristics of strain JSL-7: Strain JSL-7 was cultured overnight to OD 600 Approximately 1.0 μL was used as the seed culture, and 1% was inoculated into 50 mL shake tubes containing YPD medium. The culture was incubated at 39°C and 160 rpm, and samples were taken at 0, 2, 4, 6, 9, 12, 18, 24, 34, 48, and 60 h to determine the pH and OD of the bacterial culture. 600 Three replicates were performed at each time point to establish growth and acid production curves for strain JSL-7. Specific results are as follows: Figure 3 and Figure 4 As shown, Figure 3 The growth curve of strain JSL-7 is shown in the figure. As can be seen from the figure, after 40 hours, the OD of strain JSL-7... 600There was almost no increase, meaning that the market entered a stable period after 40 hours; Figure 4 The figure shows the acid production curve of strain JSL-7. As can be seen from the figure, the pH value gradually decreased to 4.5 with the extension of fermentation time. No significant change was observed after 34 hours, and the trend of change is close to that of the growth curve.

[0074] ② Metabolites of strain JSL-7: To study the metabolites of strain JSL-7, the fermentation broth after 48 hours of fermentation (stationary phase) was selected for metabolite analysis: strain JSL-7 was cultured overnight to OD... 600 Approximately 1.0 μL was used as the seed culture, and 1% was inoculated into 50 mL shake tubes containing YPD medium. The culture was incubated at 39℃ and 160 rpm, and samples were taken at 48 h (stationary phase). The 48 h fermentation broth of strain JSL-7 was used as the experimental sample, and the original culture medium as the control sample. Six replicates were made for both the experimental and control samples, and samples were stored at -80℃. Metabolites from the original culture medium (0 h) and the 48 h (stationary phase) fermentation broth of strain JSL-7 were detected and identified using liquid chromatography-tandem mass spectrometry (LC-MS / MS), and differential metabolites were further analyzed. Upregulated differential metabolites identified in positive and negative ion modes were screened using PLS-DA VIP>1, p<0.05, and FC>2 (fold change, FC being the fold change). A total of 547 significantly upregulated differential metabolites (i.e., metabolites that significantly accumulated in the fermentation broth) were screened, as detailed below. Figure 5 As shown, Figure 5 This is a graph showing the metabolites of strain JSL-7, including lipids and lipid-like molecules (2,2-dimethyl-3-(2-methylprop-1-en-1-yl)cyclopropanecarboxylic acid, menthol, acetylcarnitine, (2E,4E)-N-isobutyl-6-(2-thienyl)hex-2,4-dieneamide, α-linolenic acid, γ-linolenic acid, Δ¹ 7 -Octadecanoic acid, etc.) accounted for 19.56%, organic acids and their derivatives accounted for 18.83%, organic heterocyclic compounds accounted for 15.36%, benzene derivatives accounted for 8.59%, phenylpropanoids and polyketides accounted for 6.95%, organic oxygen compounds accounted for 4.20%, alkaloids and their derivatives accounted for 2.93%, lignans, neolignans and related compounds accounted for 0.73%, nucleosides, nucleotides and their analogues accounted for 0.73%, organic nitrogen compounds accounted for 0.18%, and organic sulfur compounds accounted for 0.18%.

[0075] Example 6

[0076] This example is a study on the production of menthol by strain JSL-7.

[0077] In Example 5, during the detection of metabolites, we found that strain JSL-7 can produce menthol. Although there are reports of yeast producing menthol in the prior art, we found that the prior art mainly uses two approaches through genetic engineering: one is to introduce a menthol metabolic pathway to convert sugars into menthol, and the other is to introduce an esterase metabolic system to convert menthol esters into menthol. Currently, there are no reports of wild-type yeast strains producing menthol. However, the strain JSL-7 of this application can produce menthol in its metabolites in YPD liquid medium, which contains about 2% glucose. We speculate that strain JSL-7 may naturally possess a menthol metabolic system that can convert sugars into menthol. Therefore, the applicant used different sugar sources as fermentation media to produce menthol to determine the possibility of producing menthol using wild-type strain JSL-7. The specific scheme is as follows.

[0078] Single colonies of strain JSL-7 and control strain 4 (strain 4 in Table 1 of Example 1, which is of the same species as strain JSL-7) were obtained by inoculating YPD plates. After incubation at 39°C and 200 rpm for 24 h, the inoculum was added to YPD liquid medium at a mass percentage of 1%. After incubation at 39°C and 200 rpm for 16 h, different sugar solutions from Table 10 were added. The sugar solution content was 10% of the total liquid fermentation medium (i.e., the sugar solution volume percentage was 10%, and the YPD liquid medium volume percentage was 90%). Fermentation continued for another 80 h, for a total fermentation time of 96 h. After fermentation, the bacterial culture was broken up with glass beads and an equal volume of ethyl acetate. The supernatant ethyl acetate layer was then collected by high-speed centrifugation and analyzed by gas chromatography-mass spectrometry (GC-MS) to determine the menthol yield. The results are shown in Table 10.

[0079]

[0080] As shown in Table 10, the *Pichia pastoris* strain JSL-7 can convert sugar into menthol and ferment it to produce menthol. However, the same strain, *Pichia pastoris* strain 4, cannot convert sugar into menthol and cannot ferment it to produce menthol. This indicates that although strain JSL-7 and strain 4 are of the same species, due to differences in their metabolic systems, strain JSL-7 may inherently possess a menthol metabolic system capable of converting sugar into menthol, a function not found in other wild-type *Pichia pastoris* strains. Furthermore, in terms of yield, galactose and glucose are the optimal sugars for this strain to metabolize and produce menthol, while sucrose cannot produce menthol.

[0081] In existing technologies, menthol is mainly extracted from mint plants. Therefore, we considered co-fermenting mint plants with strain JSL-7 to study the possibility of increasing menthol production, as detailed below.

[0082] Step 1: Preparation of peppermint plant extracts. Specific methods are as follows: ① Peppermint extract: Crush fresh peppermint leaves and mix with water at a solid-liquid mass ratio of 1:1. Boil and maintain boiling for 5 minutes. Filter and cool to room temperature to obtain peppermint extract. ② Thyme extract: Crush fresh thyme leaves and mix with water at a solid-liquid mass ratio of 1:1. Boil and maintain boiling for 5 minutes. Filter and cool to room temperature to obtain thyme extract. ③ Lemongrass extract: Crush fresh lemongrass leaves and mix with water at a solid-liquid mass ratio of 1:1. Boil and maintain boiling for 5 minutes. Filter and cool to room temperature to obtain lemongrass extract.

[0083] Step 2: Mix 10% (v / v) of the extract from Step 1 with 90% (v / v) of YPD liquid medium, inoculate with strain JSL-7 and control strain 4 (strain 4 in Table 1 of Example 1, which is the same species as strain JSL-7), and ferment for 96 h. After fermentation, take the bacterial solution, add glass beads and an equal volume of ethyl acetate to break it up, then centrifuge at high speed and take the upper ethyl acetate layer for gas chromatography-mass spectrometry to detect the yield of menthol. The results are shown in Table 11.

[0084]

[0085] As shown in Table 11, strain 4 still failed to produce menthol during fermentation. Even with the addition of peppermint plant extracts, menthol was not detected in the fermentation broth. This indicates that after peppermint plants are extracted by boiling and then fermented with yeast, menthol cannot be produced if the yeast itself does not have menthol-related metabolic genes. In contrast, with the addition of peppermint plants, peppermint extract and lemongrass extract of strain JSL-7 increased the menthol content of the fermentation product, while thyme extract inhibited the menthol content. This is most likely because thyme extract inhibited the growth of yeast strain JSL-7, leading to a decrease in menthol production.

[0086] Therefore, based on the sugars in Table 10, we considered preparing a fermentation medium by mixing peppermint extract, lemongrass extract, galactose, and glucose with YPD liquid medium to produce menthol. To this end, we improved the raw material ratio of the fermentation medium through orthogonal experiments. The specific scheme was as follows: peppermint extract, lemongrass extract, 100 g / L galactose solution, and 100 g / L glucose solution were mixed according to the volume ratios in Tables 12 and 13. Then, the mixture was added to YPD liquid medium to obtain the fermentation medium. The volume percentage of the mixture was 10%, and the volume percentage of YPD liquid medium was 90%. After fermentation, the menthol content was measured, and the results are shown in Table 13.

[0087]

[0088] The orthogonal analysis of the above proportions and the results are shown in Table 13.

[0089]

[0090] As shown in Table 13, the menthol yields of experiments 3, 5, 6, 8, and 9 were all superior to the optimal results of the single-factor experiments: 6.02 mg / L (menthol yield of peppermint extract in Table 11). The menthol yields of the other experimental groups were all below 6.02 mg / L. Considering the optimal results of the single-factor experiments, we selected the optimal raw material ratio for experiments 3, 5, 6, 8, and 9 to prepare the fermentation medium: a volume ratio of peppermint extract, lemongrass extract, 100 g / L galactose solution, and 100 g / L glucose solution of 2-4:2-3:3-5:3-5. Among these experiments, experiment 3 had the highest yield. Therefore, we believe the optimal volume ratio of peppermint extract, lemongrass extract, 100 g / L galactose solution, and 100 g / L glucose solution in the fermentation medium is 2:3:5:5.

[0091] Based on the range, the volume percentage of lemongrass extract > the volume percentage of 100 g / L glucose solution > the volume percentage of 100 g / L galactose solution > the volume percentage of peppermint extract. This indicates that the fermentation medium with the greatest impact on menthol yield is the volume percentage of lemongrass extract, followed by the volume percentages of 100 g / L glucose solution and 100 g / L galactose solution, while the volume percentage of peppermint extract has the least impact.

[0092] Therefore, considering all factors, the optimal method for producing menthol using strain JSL-7 fermentation is as follows: After activating strain JSL-7 by inoculating it onto YPD agar plates, single colonies of strain JSL-7 are obtained. These single colonies are then inoculated into the fermentation medium at a mass ratio of 1%, and cultured at 39℃ and 200 rpm for 96 h. After fermentation, the bacterial solution is broken up with glass beads and an equal volume of ethyl acetate. The resulting ethyl acetate layer is then collected by high-speed centrifugation and analyzed by gas chromatography-mass spectrometry (GC-MS) to determine the menthol yield. The fermentation medium is prepared by mixing peppermint extract, lemongrass extract, 100 g / L galactose solution, and 100 g / L glucose solution in a volume ratio of 2-4:2-3:3-5:3-5. A 10% (v / v) volume fraction of this mixture is then thoroughly mixed with 90% (v / v) YPD liquid culture medium.

[0093] The preparation method of peppermint extract is as follows: fresh peppermint is crushed and mixed with water at a solid-liquid mass ratio of 1:1. After boiling, the mixture is kept boiling for 5 minutes. The mixture is then filtered and cooled to room temperature to obtain peppermint extract. The preparation method of lemongrass extract is as follows: fresh lemongrass is crushed and mixed with water at a solid-liquid mass ratio of 1:1. After boiling, the mixture is kept boiling for 5 minutes. The mixture is then filtered and cooled to room temperature to obtain lemongrass extract.

[0094] Example 7

[0095] This example is a study on the antibacterial activity of strain JSL-7.

[0096] In screening strains, this application uses strains that can inhibit Escherichia coli (Escherichia coli). Escherichia coli With EPEC as the primary target, antibacterial activity is the evaluation index for bacterial strains. To investigate the superior antibacterial performance of strain JSL-7 against other pathogens, we considered studying the antibacterial effect of strain JSL-7 against other pathogens. Using laboratory-preserved pathogens as samples, we employed the perforated agar diffusion method to determine the antibacterial activity of strain JSL-7 against Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus ), Shigella flexneri ( Shigella flexneri ), Enterobacter aerogenes ( Enterobacter aerogenes Yersinia enterocolitica ( ) Yersinia enterocolitica The antibacterial activity of the sample was measured in triplicate; the results are shown in Table 14.

[0097]

[0098] Note: “—” in the table indicates no inhibitory effect.

[0099] Table 14 shows that strain JSL-7 is effective against Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus ) and / or Shigella flexneri ( Shigella flexneri It has antibacterial effects, particularly against Enterobacter aerogenes (Enterobacter). Enterobacter aerogenes ) and Yersinia enterocolitica ( Yersinia enterocolitica ) and no antibacterial effect; combined with the experimental results of Example 1, we know that strain JSL-7, in addition to being effective against Staphylococcus aureus ( Staphylococcus aureus ) and / or Shigella flexneri ( Shigella flexneri In addition to its antibacterial effect, it is also effective against Escherichia coli (Escherichia coli). Escherichia coli It also has antibacterial effects; therefore, strain JSL-7 can inhibit Escherichia coli (Escherichia coli). Escherichia coli Staphylococcus aureus Staphylococcus aureus ) and / or Shigella flexneri ( Shigella flexneri ) growth.

[0100] In summary, the *Pichia pastoris* JSL-7 strain, screened by the applicant, exhibits excellent antibacterial effects against *Escherichia coli*, *Staphylococcus aureus*, and / or *Shigella flexneri*. Furthermore, this strain is non-toxic in vivo, possesses good bile salt and acid resistance, and can colonize well in the animal gut, demonstrating excellent beneficial bacteria properties. This strain can produce menthol without genetic modification. Through optimization of the fermentation medium, we have also determined the optimal fermentation medium for the preparation of menthol using this strain. The fermentation process described in this application can effectively shorten the menthol production time and simplify the production process, providing new strains and ideas for future fermentation-based menthol production.

[0101] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. Pichia pastoris (Muts) JSL-7, characterized in that, Pichia ethanolica ) JSL-7, characterized in that, The Pichia pastoris (P. pastoris) Pichia ethanolica The accession number of JSL-7 is GDMCC NO: 65730.

2. Use of Pichia ethanolica JSL-7 as claimed in claim 1 for the production of menthol, wherein, The fermentation medium for producing menthol is composed of 10% sugar-plant mixed solution by volume and 90% YPD liquid medium by volume; the sugar-plant mixed solution is obtained by mixing menthol extract, citronella extract, 100 g / L galactose solution and 100 g / L glucose solution in a volume ratio of 2-4:2-3:3-5:3-5. The preparation method of the menthol extract is as follows: fresh mint is crushed and mixed with water in a solid-liquid mass ratio of 1:1, then boiled and kept boiling for 5 min, filtered and cooled to room temperature to obtain the menthol extract; the preparation method of the citronella extract is as follows: fresh citronella is crushed and mixed with water in a solid-liquid mass ratio of 1:1, then boiled and kept boiling for 5 min, filtered and cooled to room temperature to obtain the citronella extract.

3. Use according to claim 2, characterized in that, The sugar-plant mixed solution is obtained by mixing menthol extract, citronella extract, 100 g / L galactose solution and 100 g / L glucose solution in a volume ratio of 2:3:5:

5.

4. The Pichia pastoris (P. pastoris) of claim 1, wherein the P. pastoris is P. pastoris GS 115. Pichia ethanolica ) JSL-7 for use in the preparation of an antibacterial agent, characterized in that, The pathogenic bacteria inhibited by the bacteriostatic agent are Escherichia coli (Escherichia coli ), Staphylococcus aureus ( Staphylococcus aureus ) and / or Shigella flexneri ( Shigella flexneri ).

5. A process for the production of menthol using Pichia pastoris (P. pastoris) JSL-7 as claimed in claim 1. Pichia ethanolica ) wherein the process for the production of menthol using Pichia pastoris (P. pastoris) JSL-7 as claimed in claim 1. The method is: inoculating Pichia pastoris (JSL-7) into YPD fermentation medium, and obtaining the fermentation liquor containing menthol after fermentation. Pichia ethanolica ) JSL-7 into YPD fermentation medium, and obtaining the fermentation liquor containing menthol after fermentation.

Images