Kluyveromyces marxianus swu-392 and application thereof

Abstract

The application discloses a strain of Kluyveromyces marxianus SWU-392 and an application thereof, and belongs to the technical field of microorganisms. In view of the fact that Gastrodia elata is limited in food application due to the presence of adverse flavor substances such as p-methylphenol and hexanal, and the prior art cannot simultaneously improve flavor and retain active ingredients, the yeast with a preservation number of GDMCC No: 67309 is applied to fermentation of Gastrodia elata, adverse substances such as p-methylphenol and aldehydes in Gastrodia elata are effectively degraded through a process of constant-temperature solid-state fermentation at 30 DEG C for 4 days, the content of ester and other floral and fruity flavor substances is significantly increased, and the contents of Gastrodia elata elements and p-hydroxybenzyl alcohol are slightly increased. The process is mild and safe, no chemical additives are added, the strain has strong adaptability, and is easy to be industrialized and enlarged, so that the flavor and efficacy of Gastrodia elata are simultaneously optimized, and a green and efficient technical scheme is provided for food application of Gastrodia elata.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and particularly relates to a Kluyveromyces marxianus SWU-392 and an application thereof. BACKGROUND

[0002] As a substance with both food and traditional Chinese medicine properties, Gastrodia elata has great development potential in the food field due to its multiple pharmacological activities such as improving cognitive impairment and immune regulation. However, the para-methylphenol and other substances contained in Gastrodia elata itself can produce a special flavor similar to horse urine, and there are also aldehyde substances such as hexanal with a halitosis smell, which seriously restricts its food application and industrialization process.

[0003] In the prior art, aroma-producing yeasts have been used to improve the flavor of foods such as acid fish, sea buckthorn juice and fermented grains, to enrich the aroma levels and mask the unpleasant odor by producing esters, alcohols and other flavor substances. However, there is currently no related research on the application of aroma-producing yeasts to improve the flavor of Gastrodia elata, and there is a lack of green, safe and efficient regulation technology for the special unpleasant flavor of Gastrodia elata. At the same time, the existing fermentation technology often has difficulty in balancing the retention and improvement of active ingredients when improving the flavor of foods, and how to optimize the flavor of Gastrodia elata while maintaining or even improving the content of core active ingredients such as gastrodin and p-hydroxybenzyl alcohol has become a technical problem to be solved. SUMMARY

[0004] To solve the above technical problems, the present application provides a Kluyveromyces marxianus, and the preservation number of the Kluyveromyces marxianus is GDMCC No: 67309.

[0005] The present application also provides an application of the above-mentioned Kluyveromyces marxianus in improving the flavor of Gastrodia elata.

[0006] Further, the application is any one of the following:

[0007] (1) increasing the content of gastrodin or p-hydroxybenzyl alcohol in Gastrodia elata;

[0008] (2) degrading para-methylphenol or aldehyde unpleasant flavor substances in Gastrodia elata.

[0009] The present application also provides an application of the above-mentioned Kluyveromyces marxianus in preparing Gastrodia elata products.

[0010] Further, the application includes fermentation of Gastrodia elata slurry by the above-mentioned Kluyveromyces marxianus.

[0011] Further, the Gastrodia elata is Gastrodia elata f. rubra.

[0012] Furthermore, the fermentation conditions are 29~31℃.

[0013] Furthermore, the fermentation time is 3-5 days.

[0014] The present invention also provides a method for preparing Gastrodia elata products using the above-mentioned Kluyveromyces marxianus, comprising the following steps: fermenting Gastrodia elata slurry using the above-mentioned Kluyveromyces marxianus at a fermentation temperature of 29~31℃ for 3~5 days.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) Highly targeted: For the first time, Kluyveromyces martensii was applied to the fermentation of Gastrodia elata. The selected SWU-392 strain can accurately target and degrade undesirable flavor substances in Gastrodia elata, with a methylphenol removal rate of 22.04% and a significant reduction in aldehyde content.

[0017] (2) Significantly enhanced flavor: After fermentation, the number of volatile flavor compounds in Gastrodia elata increased from 25 to 57, and the total amount of esters was about 32.5 times that of the unfermented group, forming a pleasant flavor profile dominated by floral and fruity aromas and sweet aromas. Electronic nose principal component analysis showed significant differences from the unfermented group.

[0018] (3) Good retention of active ingredients: After fermentation, the content of gastrodin increased from 0.14 mg / g to 0.22 mg / g, and p-hydroxybenzyl alcohol increased from 5.07 mg / g to 5.69 mg / g, achieving dual optimization of flavor and efficacy.

[0019] (4) The process is mild and safe: solid-state fermentation is adopted, with a fermentation temperature of 30℃ and a time of 4 days. No chemical additives are required, which is in line with the concept of green food processing. The strain has certain salt, acid and ethanol tolerance, strong adaptability and easy industrial scale-up.

[0020] (5) Excellent strain performance: The selected Kluyveromyces martensii SWU-392 has an ester production capacity of 1466.29±71.53mg / L, which is much higher than that of conventional aroma-producing yeasts, and its flavor improvement efficiency is outstanding.

[0021] Biological preservation instructions for Kluyveromyces martensii SWU-392:

[0022] Preservation institution: Guangdong Provincial Center for Microbial Culture Collection;

[0023] Accession number: GDMCC No: 67309;

[0024] Deposit date: November 17, 2025;

[0025] Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou;

[0026] Taxonomic name: Kluyveromyces marxianus. Attached Figure Description

[0027] Figure 1 The results show the ester production capacity of multiple yeast strains in Example 1.

[0028] Figure 2 The types of volatile aroma compounds in the fermented Gastrodia elata by the aroma-producing strain in Example 1 are identified.

[0029] Figure 3 The results of electronic nose analysis of the aroma-producing strain fermented Gastrodia elata in Example 1 are shown in Figure a: electronic nose radar diagram; b: principal component analysis diagram.

[0030] Figure 4 The changes in the contents of gastrodin and p-hydroxybenzyl alcohol in the aroma-producing strain SWU-392 before and after fermentation in Example 1 are shown.

[0031] Figure 5 The images show the colony morphology and cell morphology of the aroma-producing strain SWU-392 in Example 1, where a: colony morphology; b: cell morphology.

[0032] Figure 6 This is the phylogenetic tree of Kluyveromyces marxianus SWU-392 from Example 1.

[0033] Figure 7 The growth curve of the aroma-producing strain SWU-392 in Example 1 is shown.

[0034] Figure 8 The results of the tolerance test for the aroma-producing strain SWU-392 in Example 1 are shown, where a: glucose concentration; b: NaCl concentration; c: ethanol volume fraction; d: acetic acid volume fraction.

[0035] In the above figure, 392 represents the SWU-392 strain. Detailed Implementation

[0036] Example 1

[0037] 1. Materials and Methods

[0038] 1.1 Test Materials

[0039] 1.1.1 Gastrodia elata raw material

[0040] The black-red Gastrodia elata was collected in Qijiang District, Chongqing on December 30, 2024. After the sample arrived at the laboratory, it was immediately rinsed with clean water to remove the attached soil and surface impurities. After drying, it was vacuum-sealed in bags and stored at -20℃ for later use.

[0041] 1.1.2 Source of strains

[0042] The 26 strains used in this experiment (numbered 118, 112, 162, 442, 222, 448, 412, 168, 228, 56, 372, 82, 510, 224, 312, SWU-392, YA12, YA9, YA6, YA5, YA3, Y9, YO3, YO8, T31, T41) were all obtained from the laboratory strain bank.

[0043] 1.1.3 Reagents and Instruments

[0044] Main reagents: 0.1 mol / L NaOH standard titration solution and 0.1 mol / L HCl standard titration solution were purchased from Sinopharm Chemical Reagent Co., Ltd.; glucose, maltose, trehalose, fructose, sucrose, and lactose were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; potassium nitrate and sodium nitrate were purchased from Chengdu Kelong Chemical Co., Ltd.; beef extract, peptone, and yeast extract were purchased from Beijing Aoboxing Biotechnology Co., Ltd.

[0045] YPD liquid culture medium: 20 g / L peptone, 20 g / L glucose, 10 g / L yeast extract, 1000 mL pure water, sterilized at 121℃ for 15 min.

[0046] Gastrodia elata culture medium: Grind black Gastrodia elata into a pulp (without adding water) and sterilize at 121℃ for 15 min.

[0047] Major instruments and equipment: LHS-150CLY incubator, Shanghai Qixin Scientific Instruments Co., Ltd.; ISQ 7610 GC-MS, Thermo Fisher Scientific, USA; electronic nose, Shanghai Baosheng Industrial Development Co., Ltd.; sterilizer, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory.

[0048] 1.2 Test Methods

[0049] 1.2.1 Screening of aroma-producing yeasts

[0050] 1.2.1.1 Strain activation

[0051] The strain, preserved in glycerol tubes at -80℃, was thawed at room temperature. After complete thawing, it was inoculated into YPD liquid medium at a 2% (v / v) inoculum and cultured at 30℃ with shaking for 48 hours. Then, the activated strain was inoculated into YPD solid medium for purification 3-4 times to obtain single colonies.

[0052] 1.2.1.2 Preliminary screening of aroma-producing strains

[0053] The aroma-producing strains were initially screened using an olfactory method. The specific procedure was as follows: Single colonies were picked, and each purified strain was inoculated onto YPD solid medium and incubated at 30°C inverted for 48 hours. The presence and aroma characteristics of each strain were observed, and strains with strong and pleasant aromas were selected for subsequent experimental studies. Results were categorized as "-" (no aroma detected), "+" (slight aroma), "++" (moderate aroma), and "+++" (strong aroma).

[0054] 1.2.1.3 Re-screening of aroma-producing strains

[0055] The ester-producing capacity of the aroma-producing strain was determined using total ester content as an indicator. Following the method of CHEN et al., with appropriate modifications, the specific procedure was as follows: The aroma-producing strain was inoculated into 150 mL of YPD medium at a 2% (v / v) inoculation rate and cultured at 30℃ for 72 h. After fermentation, 25 mL of the fermentation broth was transferred to a 250 mL Erlenmeyer flask, 100 mL of distilled water and 2 drops of phenolphthalein indicator were added, and titrated with 0.1 mol / L NaOH standard titration solution until a faint red color appeared. Then, 10 mL of 0.1 mol / L NaOH standard titration solution was accurately added, and the saponification reaction was carried out at 40℃ for 24 h. Back titration was then performed with 0.1 mol / L HCl standard titration solution until the red color completely disappeared, and the volume of HCl consumed was recorded as V1. Uninoculated YPD medium was used as a blank control, and the volume of HCl consumed was recorded as V0. The total ester content was calculated using the following formula:

[0056] (1)

[0057] Where: X, total ester content (calculated as ethyl acetate), mg / L; C, actual concentration of HCl standard titration solution, mol / L; V0, volume of HCl standard titration solution consumed by the blank control, mL; V1, volume of HCl standard titration solution consumed by the aroma-producing strain fermentation broth, mL; 88, molar mass fraction of ethyl acetate (g / mol); 25, volume of fermentation broth, mL; 1000, unit conversion factor.

[0058] 1.2.2 Analysis of volatile flavor compounds in fermented Gastrodia elata by aroma-producing strains

[0059] Adjust the bacterial concentration to 1.1 × 10⁻⁶. 7CFU / mL was inoculated at a rate of 2% (v / w) into a 100 mL blue-capped bottle containing 50 g of Gastrodia elata culture medium and fermented at 30 ℃ for 4 days. The volatile flavor compounds of the aroma-producing strain fermented Gastrodia elata were determined by HS-SPME-GC-MS. 5 g of sample was accurately weighed into a 20 mL headspace vial, 2.0 mL of saturated NaCl solution was added, followed by the addition of internal standard solution (10.0 µL 2-octanol, 15.0 µg / mL). The headspace vial was placed in an 80 ℃ incubator for 20 min to equilibrate. Extraction was performed at 80 ℃ for 40 min using SPME fiber (DVB / CWR / PDMS, 50 / 30 µm × 1 cm), followed by desorption at 250 ℃ for 2 min at the injection port.

[0060] Gas chromatography conditions: TR-FFAP capillary column (30 m × 0.25 mm × 0.25 µm); splitless; gas chromatograph temperature program set as follows: initial temperature 40℃, hold for 3 min, increase to 100℃ at a rate of 10℃ / min, hold for 2 min, then increase to 160℃ at a rate of 3℃ / min, hold for 5 min, and finally increase to 230℃ at a rate of 13℃ / min, hold for 10 min; helium (99.999% purity) as carrier gas, flow rate 1 mL / min.

[0061] Mass spectrometry conditions: ion source temperature 250 °C; transfer line temperature 240 °C; quadrupole temperature 150 °C; electron impact ionization 70 eV; scan range 40 to 400 m / z.

[0062] Qualitative and quantitative analysis: Compounds with a match ≥800 were retained for qualitative analysis using retention index and the NIST20 mass spectrometry library. The internal standard method was used, and the content of each compound was calculated based on the peak area ratio of the target compound to the internal standard. Each sample group was tested in triplicate.

[0063] 1.2.3 Electronic nose

[0064] The injection needle was directly inserted into a sealed sample vial containing the sample, and the sample was allowed to equilibrate at room temperature for 30 minutes before measurement using an electronic nose. Measurement conditions: sampling time 1 second / group; sensor self-cleaning time 90 seconds; sensor zeroing time 5 seconds; sample preparation time 5 seconds; injection flow rate 1 L / min; analysis sampling time 80 seconds. The maximum value of the response curve was selected for analysis.

[0065] 1.2.4 Determination of Gastrodin and p-hydroxybenzyl alcohol content

[0066] The determination of gastrodin and p-hydroxybenzyl alcohol content was carried out in accordance with the Chinese Pharmacopoeia.

[0067] 1.2.5 Identification of aroma-producing strains

[0068] 1.2.5.1 Morphological observation

[0069] (1) Observation of colony characteristics: The purified aroma-producing strains were inoculated onto YPD plates and cultured at 30 ℃ for 48 h. The colony characteristics were observed and recorded.

[0070] (2) Cell morphology observation: Pick a single colony from the YPD plate and observe the cell morphology under an optical microscope.

[0071] 1.2.5.2 Physiological and Biochemical Tests

[0072] Physiological and biochemical tests of aroma-producing strains were conducted with reference to the "Handbook of Characteristics and Identification of Yeasts".

[0073] 1.2.5.3 Molecular biological identification

[0074] Molecular biological identification was performed on the selected aroma-producing strains. The purified strains were sequenced by Panrui Cloud Intelligence Technology (Zhengzhou) Co., Ltd. (e-test), and the sequencing results were submitted to the National Center for Biotechnology Information (NCBI). Homology comparison was performed using the basic local alignment search tool (BLAST), and a phylogenetic tree was constructed in MEGA 12.0 software using the neighbor-joining (NJ) method.

[0075] 1.2.6 Growth characteristics and tolerance of aroma-producing strains

[0076] 1.2.6.1 Growth curve of aroma-producing strains

[0077] The selected aroma-producing strains were inoculated into YPD liquid medium at a rate of 2% (v / v) and cultured at 30 ℃ with shaking at 180 r / min. OD was measured at 2-h intervals. 560 value.

[0078] 1.2.6.2 Tolerance test of aroma-producing strains

[0079] The aroma-producing strain was inoculated at a 2% (v / v) in YPD liquid medium containing different mass concentrations of glucose (0 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L, 500 g / L, 600 g / L, 700 g / L), NaCl (0 g / L, 20 g / L, 40 g / L, 60 g / L, 80 g / L, 100 g / L, 120 g / L, 140 g / L), different volume fractions of ethanol (0%, 2%, 4%, 6%, 8%, 10%, 12%, 14%), and acetic acid (0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%), and cultured at 30℃ and 180 r / min for 48 h. After cultivation, the OD of the fermentation broth was measured using uninoculated culture medium of the corresponding concentration as a blank control. 560 value.

[0080] 1.2.7 Data Processing

[0081] Statistical analysis of the data was performed using Excel 2019 and IBM SPSS 26.0; bar charts, line charts, radar charts, and principal component analysis plots were generated using Origin 2024; and phylogenetic trees were generated using MEGA 12.0.

[0082] 2 Results and Analysis

[0083] 2.1 Screening of aroma-producing strains

[0084] 2.1.1 Initial screening results of aroma-producing strains

[0085] Nine strains with distinct and pleasant aromas were selected by olfactory screening, as shown in Table 1.

[0086] Table 1. Screening results using the olfactory method

[0087]

[0088] Note: "++" represents a strong aroma, "++" represents a medium aroma, and "+" represents a slight aroma.

[0089] 2.1.2 Results of secondary screening of aroma-producing strains

[0090] To further screen for strains with excellent ester-producing capabilities, nine aroma-producing strains initially screened by the olfactory method were inoculated into ester-producing culture media. The total ester content of the fermentation broth was determined using the saponification method, and the ester-producing capabilities of each aroma-producing strain were compared. The results are as follows: Figure 1 As shown. By Figure 1It was found that strains SWU-392 and 168 had significantly higher ester production capacity than the other seven strains (p < 0.05). Strain SWU-392 had the highest total ester content, reaching (1466.29 ± 71.53) mg / L; while strain YA6 had the second highest ester production capacity, reaching (989.45 ± 180.00) mg / L. Therefore, strains SWU-392, 168, and YA6 were selected for subsequent research on improving the flavor of Gastrodia elata.

[0091] 2.2 Analysis of volatile flavor compounds in fermented Gastrodia elata by aroma-producing strains

[0092] 2.2.1 Analysis of the types of volatile flavor compounds

[0093] To further screen aroma-producing strains that effectively enhance the unique flavor of *Gastrodia elata*, this study used HS-SPME-GC-MS technology to systematically analyze the volatile flavor components of *Gastrodia elata* fermented with three strains: 168, SWU-392, and YA6. Figure 2 Fermentation significantly enriched the variety of volatile aroma compounds in *Gastrodia elata*. A total of 70 volatile flavor compounds with aroma characteristics were detected, including 25 in the unfermented group, 44 in group 168, 57 in group SWU-392, and 52 in group YA6. Compared with the unfermented group, each fermented group showed a significant increase in the number of flavor compounds such as alcohols, esters, ketones, and acids. Group YA6 had the most alcohol compounds (12), the highest among all strains; group 392 had 9 acid compounds, higher than other strains. Notably, ketones were not detected in the unfermented group, while they were produced in all fermented groups. Esters mainly originated from the biosynthesis of aroma-producing strains or from esterification reactions of acids and alcohols catalyzed by lipases. In summary, strain SWU-392 has a relatively outstanding ability to produce volatile flavor compounds, producing 17 kinds of esters, the most among the three strains, reflecting the extensiveness and diversity of the ester synthesis metabolic pathways of this strain.

[0094] 2.2.2 Analysis of Volatile Flavor Compound Content

[0095] Further analysis was conducted on the volatile flavor components of *Gastrodia elata* fermented by various strains. Table 2 shows that phenols were the most abundant volatile flavor compounds before and after fermentation. Specifically, p-methylphenol, reported as one of the sources of the "horse urine" odor in *Gastrodia elata*, decreased by 19.96%, 22.04%, and 19.08% respectively after fermentation by strains 168, SWU-392, and YA6, indicating that these strains may have the ability to metabolize p-methylphenol. In addition, aldehydes, some of which originate from lipid oxidation and may produce a "rancid" odor, such as hexanal, (E)-2-octenal, and (E,E)-2,4-decadienal, also showed a significant decrease in content after fermentation. These aldehydes typically have extremely low sensory thresholds and significantly affect flavor quality; their reduction contributes to the improvement of the overall flavor of *Gastrodia elata*. Alcohols were the second most abundant volatile flavor compounds after fermentation, showing a significant difference compared to the unfermented group (p<0.05). Notably, the alcohol content in group SWU-392 (692.14 µg / kg) was significantly higher than that in group 168 (410.47 µg / kg) and group YA6 (224.82 µg / kg), with isoamyl alcohol and phenethyl alcohol being the main components. Isoamyl alcohol primarily exhibited fruity, floral, and malty aromas, while phenethyl alcohol mainly presented rose and honey aromas. Esters are the most abundant volatile flavor compounds, and group SWU-392 demonstrated outstanding ester production capacity in both variety and content, with its total amount (482.76 µg / kg) being approximately 32.5 times that of the unfermented group and significantly higher than other fermented groups. Among them, the content of 2-phenylethyl acetate, which has sweet, rose, and apple aroma characteristics, was as high as 353.76 µg / kg, approximately 31 times and 30 times that of groups 168 and YA6, respectively, and this substance was not detected in the unfermented group. Acids are precursors for the synthesis of esters. The acid content of the SWU-392 and 168 groups was significantly higher than that of the unfermented group and the YA6 group. This was mainly due to the accumulation of some medium and long chain fatty acids, such as palmitic acid and dodecanoic acid.

[0096] By comprehensively comparing the ester production capacity and volatile flavor composition characteristics of strains 168, SWU-392 and YA6, strain SWU-392 showed significant advantages in both the diversity of ester types and the richness of flavor components, indicating that this strain has excellent aroma-producing characteristics and flavor regulation capabilities.

[0097] Table 2. Content of volatile aroma compounds in fermented Gastrodia elata by aroma-producing strains

[0098]

[0099]

[0100]

[0101]

[0102]

[0103] Note: "-" indicates that the substance was not detected; the significance of differences between groups in the table is indicated by different lowercase letters, and the specific meanings of the lowercase letters are as follows: if two groups are labeled with the same letter (e.g., both are labeled 'a'), it means that there is no statistically significant difference between them; if two groups are labeled with different letters (e.g., 'a' and 'b'), it means that there is a statistically significant difference between them; if two groups are labeled with one letter (e.g., 'a' and 'ab', 'b' and 'bc'), even if there are different letters, it means that there is no significant difference between them.

[0104] 2.3 Electronic nose analysis

[0105] Electronic nose analysis of the fermented Gastrodia elata by aroma-producing strains can effectively distinguish the differences in overall flavor among fermentation products of different strains. The results are as follows: Figure 3 As shown. Figure 3 The radar chart for group a visually illustrates the differences in response intensity of *Gastrodia elata* fermented by different aroma-producing strains on 10 sensors (S1-S10). From the sensor response values, after fermentation by aroma-producing strains 168, SWU-392, and YA6, the response values ​​of sensors S1, S4, S5, S6, and S7, which are sensitive to short-chain alkanes, ammonia-based aromatic components, alcohols, ketones, aldehydes, and benzene-based aromatic components, significantly increased. Furthermore, strain SWU-392 showed higher response values ​​in sensors S1, S4, S5, S6, and S7 than the other groups. However, sensors S3, S8, S9, and S10 showed no significant response, indicating that very little ammonia, sulfides, and low-carbon hydrocarbons were produced after fermentation.

[0106] Principal component analysis (PCA) showed that PC1 and PC2 contributed 77.3% and 16.1% respectively, with a cumulative contribution of 93.4%, indicating that it effectively reflected the differences between groups. Figure 3 As shown in b, the unfermented group was far from the fermented groups 168, SWU-392, and YA6, indicating that the flavor of *Gastrodia elata* undergoes significant changes after fermentation by aroma-producing strains. Groups 168 and YA6 were closer together, suggesting similar flavors after fermentation. However, group SWU-392 was far from the other groups, indicating a more significant flavor change after fermentation with strain SWU-392. This may be due to the production of more volatile flavor components such as esters and alcohols during SWU-392 fermentation, thus affecting its flavor characteristics.

[0107] Considering the ester production capacity and volatile flavor compounds of the strain, SWU-392 was ultimately selected as the target strain for subsequent experiments.

[0108] 2.4 Content of Gastrodin and p-hydroxybenzyl alcohol

[0109] Gastrodin and p-hydroxybenzyl alcohol are the main active components of Gastrodia elata. The contents of gastrodin and p-hydroxybenzyl alcohol in Gastrodia elata before and after fermentation by the optimal aroma-producing strain SWU-392 were determined, and the results are as follows: Figure 4 As shown in the figure, the contents of gastrodin and p-hydroxybenzyl alcohol both increased after fermentation, rising from 0.14 mg / g and 5.07 mg / g before fermentation to 0.22 mg / g and 5.69 mg / g, respectively, but the increase was limited. This phenomenon may be related to the dynamic transformation between gastrodin and its aglycone, p-hydroxybenzyl alcohol, during fermentation: gastrodin can be hydrolyzed to p-hydroxybenzyl alcohol under the action of β-glucosidase, while p-hydroxybenzyl alcohol can also be resynthesized into gastrodin through glycosylation under certain conditions. The two are in a dynamic equilibrium of interconversion, resulting in insignificant changes in content.

[0110] 2.5 Analysis of the identification results of aroma-producing strains

[0111] 2.5.1 Morphological observation results

[0112] Colony and cell morphology of strain SWU-392 on YPD solid medium are shown in the figure. Figure 5 .like Figure 5 As shown in a, the colony is milky white, round, with a smooth surface, neat edges, a viscous and opaque texture, and is easy to pick up. Microscopic observation (…) Figure 5 b) It can be seen that its cells are oval and reproduce by budding.

[0113] 2.5.2 Results of Physiological and Biochemical Tests

[0114] The physiological and biochemical test results of strain SWU-392 are shown in Table 3. As can be seen from Table 3, this strain can utilize glucose, fructose, sucrose and lactose, but cannot utilize maltose and trehalose; it can utilize organic nitrogen sources such as yeast extract, beef extract and peptone, but cannot utilize inorganic nitrogen sources such as sodium nitrate and ammonium sulfate; the glucose fermentation test result was positive, it could not produce starch-like compounds, and it had no urease activity.

[0115] Table 3. Physiological and biochemical test results of aroma-producing strain SWU-392

[0116]

[0117] 2.5.3 Molecular biological identification results

[0118] Phylogenetic trees were constructed using the NJ method in MEGA 12 software. The results are shown in [Figure number missing]. Figure 6 .Depend on Figure 6It can be seen that strain SWU-392 is on the same branch as Kluyveromyces marxianus and has a high degree of similarity and is most closely related. Based on morphological observation and physiological and biochemical test results, strain SWU-392 was identified as Kluyveromyces marxianus.

[0119] 2.6 Analysis of growth characteristics and tolerance of aroma-producing strains

[0120] 2.6.1 Growth curve of aroma-producing strain SWU-392

[0121] The growth curve of aroma-producing strain SWU-392 is as follows: Figure 7 As shown, it can be observed that the strain is in the lag phase from 0 to 6 h, with slow growth; it enters the logarithmic growth phase from 6 to 18 h, with vigorous growth; and then reaches the stationary phase from 18 to 24 h, where its biomass tends to stabilize.

[0122] 2.6.2 Tolerance analysis of aroma-producing strain SWU-392

[0123] The environmental tolerance results of strain SWU-392 are as follows: Figure 8 As shown, the increased osmotic pressure caused by high sugar concentrations inhibits yeast cell growth and metabolic behavior, leading to slowed or stopped fermentation. Figure 8 As shown in Figure a, the strain reached its optimal growth state at a glucose concentration of 200 g / L; when the glucose concentration exceeded 300 g / L, the high glucose environment caused osmotic pressure stress on the cells, leading to a significant inhibition of growth. However, when the glucose concentration was increased to 700 g / L, although the biomass of strain SWU-392 decreased, it could still maintain growth, indicating that this strain possesses the characteristic of tolerating higher glucose concentrations. Figure 8 b shows that when the NaCl concentration is between 0 and 100 g / L, the OD of strain SWU-392 increases with increasing NaCl concentration. 560 The value showed a continuous downward trend; when the NaCl concentration exceeded 120 g / L, the growth of strain SWU-392 almost stopped. Appropriate amounts of ethanol have a certain promoting effect on yeast growth, but excessively high concentrations will produce a toxic effect and inhibit its growth. Figure 8 c indicates that strain SWU-392 grows well when the ethanol volume fraction is between 0% and 4%; when the ethanol volume fraction is between 4% and 6%, OD... 560 The value dropped sharply; and when the ethanol volume fraction exceeded 8%, strain SWU-392 almost stopped growing. Acetic acid can inhibit the growth and fermentation of microorganisms. Figure 8As can be seen from d, the growth of strain SWU-392 was gradually inhibited as the volume fraction of acetic acid increased; however, when the volume fraction of acetic acid reached 0.7%, the strain was still able to maintain a certain biomass, indicating that it has a certain degree of tolerance to acetic acid.

[0124] 3. Conclusions and Discussion

[0125] This study aimed to screen for an aroma-producing strain that could effectively improve the unique flavor of Gastrodia elata. Through olfactory analysis, total ester content determination, volatile flavor component analysis, and the use of an electronic nose, along with a series of physiological and biochemical tests and growth characteristic analyses, an aroma-producing strain, identified as Kluyveromyces marxianus, SWU-392, was ultimately selected. This strain exhibited superior ester production capacity and higher levels and types of volatile flavor components in fermented Gastrodia elata compared to other fermentation groups. Specifically, in the SWU-392 fermentation group, the content of p-methylphenol, a representative component of the "horse urine smell," and aldehydes with a "spicy" smell (including hexanal, (E)-2-octenal, and (E,E)-2,4-decadienal) were significantly reduced, effectively alleviating the unpleasant odor of Gastrodia elata. Simultaneously, the content of alcohols, represented by isobutanol and phenylethyl alcohol, and esters, represented by 2-phenylethyl acetate, significantly increased, giving fermented Gastrodia elata a pleasant aroma characterized primarily by "floral and fruity" and "sweet" notes. The accumulation of these beneficial flavor components not only enriched the aroma profile of fermented Gastrodia elata but may also have effectively masked residual unpleasant odors. Furthermore, after fermentation, the levels of gastrodin and p-hydroxybenzyl alcohol, the main active components of Gastrodia elata, were also increased.

[0126] This study successfully screened a flavor-producing strain, SWU-392, suitable for fermenting Gastrodia elata, providing a theoretical basis for improving the flavor and quality of Gastrodia elata. However, the metabolic regulatory mechanism of strain SWU-392 on special flavor components during the fermentation of Gastrodia elata remains unclear. Therefore, future research should combine metabolomics to further analyze the key metabolic pathways and regulatory networks of its unpleasant odor substances, so as to promote the application of this strain in the development of Gastrodia elata foods.

[0127] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Kluyveromyces marxianus, characterized by, The preservation number of the Kluyveromyces martensii is GDMCC No: 67309.

2. The application of Kluyveromyces martensii as described in claim 1 in improving the flavor of Gastrodia elata, characterized in that, The gastrodia elata slurry was fermented using the Kluyveromyces Marcius as described in claim 1.

3. The application according to claim 2, characterized in that, The application is any one of the following: (1) Increase the content of gastrodin or p-hydroxybenzyl alcohol in Gastrodia elata; (2) Degrading p-methylphenol or aldehydes in Gastrodia elata; the aldehydes are hexanal, (E)-2-octenal and (E,E)-2,4-decadienal.

4. The application of Kluyveromyces martensii as described in claim 1 in the preparation of Gastrodia elata products, characterized in that, The gastrodia elata slurry was fermented using the Kluyveromyces Marcius as described in claim 1.

5. The application according to claim 4, characterized in that, The application includes fermenting Gastrodia elata slurry using the Kluyveromyces Marcius as described in claim 1.

6. The application according to claim 5, characterized in that, The gastrodia elata mentioned is dark red gastrodia elata.

7. The application according to claim 6, characterized in that, The fermentation conditions are 29~31℃.

8. The application according to claim 7, characterized in that, The fermentation time is 3-5 days.

9. A method for preparing Gastrodia elata products using Kluyveromyces martensii as described in claim 1, characterized in that, Includes the following steps: The gastrodia elata slurry was fermented using the Kluyveromyces martensii described in claim 1 at a fermentation temperature of 29-31°C for 3-5 days.

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