Application of cyberlindnera jadinii in the production of xinfengguo ma xiang liquor

Abstract

This invention belongs to the field of yeast screening and application technology, and particularly relates to a strain of *Saccharomyces cerevisiae* HM-02 and its application in *Xanthoceras sorbifolium* malt-flavored baijiu. This invention uses HM-02 to ferment *Xanthoceras sorbifolium* cake, which significantly reduces the fat content of *Xanthoceras sorbifolium* and increases the antioxidant level and viable count of HM-02 in the fermentation broth. By adding *Xanthoceras sorbifolium* cake fermentation broth during the stacking stage of the brewing process, followed by fermentation in tanks, distillation, storage, and blending, *Xanthoceras sorbifolium* malt-flavored baijiu can be prepared. Results show that HM-02 in the *Xanthoceras sorbifolium* cake fermentation broth can survive in the mash, significantly increasing the abundance of *Saccharomyces cerevisiae* and *Weissella* species in the mash, decreasing the abundance of acid-tolerant lactic acid bacteria and *Saccharomyces cerevisiae*, increasing the content of aroma components such as β-phenylethanol in the *Xanthoceras sorbifolium* malt-flavored baijiu, increasing pleasant aroma components such as 2,3-butanediol and methyl heptanoate, and reducing the content of undesirable flavors.

Description

Technical Field

[0001] This invention belongs to the field of yeast screening and application technology, and in particular relates to a type of yeast called Jedings-Ceberlindner and its application in a type of baijiu (Chinese liquor) with the aroma of Sapindus mukorossi. Background Technology

[0002] Xanthoceras sorbifolia Bunge is a woody oilseed crop unique to my country. Its seeds are rich in oils, proteins, flavonoids, polyphenols, and terpenes, among other active ingredients, and have been increasingly explored for application in the food and health food industries in recent years. Xanthoceras sorbifolia cake, the residue left after physical pressing of the fruit, can be used to develop specialty baijiu (Chinese white liquor). This not only expands the application scenarios of the raw material but also endows the product with a unique flavor and health benefits. Wheat-aroma baijiu is a type of baijiu brewed primarily from wheat. Its core technological characteristic is that wheat accounts for no less than 50% of the grain, resulting in a product with a rich wheat aroma.

[0003] *Xanthoceras sorbifolium* cake is characterized by its high fiber and oil content, and contains active ingredients such as flavonoids and polyphenols. The aroma of *Xanthoceras sorbifolium* cake is complex, exhibiting a unique fruity and woody aroma mixed with a musty and rancid smell. Preliminary exploratory experiments showed that when *Xanthoceras sorbifolium* cake is directly added to the fermenting mash, the liquor retains its unique fruity and woody aroma but also exhibits a noticeable rancid taste. It is speculated that the rancid taste is mainly due to the excessive oxidation of oils, producing hydroperoxides, which then decompose to produce small molecules such as aldehydes, ketones, and acids. Yeast is an important fungus in the baijiu brewing process. During fermentation, yeast produces alcohol and carbon dioxide through metabolism. Simultaneously, yeast can absorb and utilize fatty acids as its sole carbon source. Furthermore, some yeasts can also utilize their powerful lipid metabolism function to produce free fatty acids, demonstrating a strong ability to regulate lipid metabolism. Additionally, by adding exogenous microorganisms to the mash, it is possible to improve the flavor of the liquor by altering the composition of the mash's microbial community.

[0004] Therefore, this invention proposes to screen a yeast strain to ferment *Xanthoceras sorbifolium* cake first, and then add the fermented *Xanthoceras sorbifolium* broth to the mash. By improving the fatty acid composition of *Xanthoceras sorbifolium*, the composition of the mash microbial community is improved, thereby improving the flavor of the liquor. This invention demonstrates that using *Saccharomyces cerevisiae* HM-02 (isolated from a malt-flavored fermentation pit) to first ferment *Xanthoceras sorbifolium* cake, and then adding the fermented *Xanthoceras sorbifolium* broth to the baijiu brewing process, the results show that the exogenously added HM-02 can survive in the mash and further improve the composition and flavor of the liquor by changing the microbial community of the mash, eliminating unpleasant odors, and realizing the preparation of *Xanthoceras sorbifolium* malt-flavored baijiu. This further promotes the development and utilization of *Xanthoceras sorbifolium* resources and enriches the composition of malt-flavored baijiu. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Cyberlindnera jadinii (HM-02) and its application in a malty-flavored baijiu made from Sapindus mukorossi fruit.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A strain of Cyberlindnera jadinii (HM-02) was isolated from a wheat-flavored fermentation pit and deposited at the China Center for Type Culture Collection (CCTCC) in Wuhan, China on May 30, 2025, with accession number CCTCC NO: M20251229.

[0008] Furthermore, the nucleotide sequence of the 18S rDNA of this *Gynedilum crenenalis* HM-02 is shown in SEQ ID NO: 1.

[0009] Another objective of this invention is to provide an application of *Cyberlindnera jadinii* (HM-02) for preparing HM-02 *Xanthoceras sorbifolium* fermentation broth. The specific preparation method is as follows: *Xanthoceras sorbifolium* cake (the cake remaining after physical pressing of *Xanthoceras sorbifolium*) is added to distilled water at a solid-liquid ratio of 1:5. After sterilization at 121°C for 30 minutes, HM-02 seed liquid (8.01g CFU / mL viable bacteria) is inoculated at a 1% inoculation ratio. After fermentation at 35°C and 200 rpm for 72 hours, the fatty acid content in the *Xanthoceras sorbifolium* cake decreases from 12.02% to 3.1%, the viable bacteria count reaches 7.101g CFU / mL, and the in vitro antioxidant indicators (PTIO, ABTS, DPPH, and FRAP values) in the fermentation broth are significantly improved.

[0010] Another objective of this invention is to further apply the HM-02 *Xanthoceras sorbifolium* fermentation liquid prepared above to the preparation of *Xanthoceras sorbifolium* malt-flavored baijiu. The *Xanthoceras sorbifolium* fermentation liquid is added to the stacking step at a ratio of 5% to 10%, and after fermentation, distillation, storage, and blending, *Xanthoceras sorbifolium* malt-flavored baijiu can be prepared. Mechanistic studies have shown that *Cyberlindnera jadinii* HM-02 in the fermentation broth of *Xanthoceras sorbifolium* cake can survive in the mash and significantly increases the abundance of *Cyberlindnera jadinii* and *Weissella* in the fermented mash, while decreasing the abundance of *Lactobacillus acetotolerans* and *Saccharomyces cerevisiae*. This further increases the content of aroma components such as β-phenylethanol in *Xanthoceras sorbifolium* wheat-flavored baijiu, and increases pleasant aroma components such as 2,3-butanediol (CAS: 513-85-9, fruity and creamy aroma) and methyl heptanoate (CAS: 106-73-0, grassy and honey aroma); while decreasing the content of undesirable flavors such as 2-vinyl-6-methylpyrazine (CAS: 13925-09-2, smoky flavor) and 1H-pyrrole-2-carboxaldehyde (CAS: 1003-29-8, musty flavor).

[0011] The advantages of this invention are:

[0012] Adding *Xanthoceras sorbifolium* cake directly to the fermentation mash results in a liquor with a distinct rancid flavor. This invention first uses *Cyberlindnera jadinii* (Jardin's *Cyberlindnera jadinii* HM-02) to prepare a *Xanthoceras sorbifolium* cake fermentation broth. After fermentation, the fatty acid content of the *Xanthoceras sorbifolium* cake decreased from 12.02% to 3.1%, the viable cell count in the fermentation broth reached 7.101 g CFU / mL, and the in vitro antioxidant indicators (PTIO, ABTS, DPPH, and FRAP values) were significantly improved. Furthermore, by adding the *Xanthoceras sorbifolium* cake fermentation broth during the stacking process, the *Cyberlindnera jadinii* HM-02 in the fermentation broth can survive in the mash, and the concentration of *Cyberlindnera* in the post-fermentation mash is significantly increased. The abundance of *Jadinii* and *Weissella* was reduced, while the abundance of *Lactobacillus acetotolerans* and *Saccharomyces cerevisiae* was decreased. This increased the content of aroma components such as β-phenylethanol in *Xanthoceras sorbifolium* wheat-flavored baijiu, and increased pleasant aroma components such as 2,3-butanediol (CAS: 513-85-9, fruity and creamy aroma) and methyl heptanoate (CAS: 106-73-0, grassy and honey aroma). It also reduced the content of undesirable flavors such as 2-vinyl-6-methylpyrazine (CAS: 13925-09-2, smoky flavor) and 1H-pyrrole-2-carboxaldehyde (CAS: 1003-29-8, musty flavor). Attached Figure Description

[0013] Figure 1 It is a phylogenetic tree of yeast Y5.

[0014] Figure 2 This is the relative abundance composition (Top 10) of bacteria and fungi in fermented mash for 30 days in this invention; where Figure A represents the relative abundance bar chart of bacterial composition and Figure B represents the relative abundance bar chart of fungal composition.

[0015] Figure 3 This is a Pearson correlation analysis diagram of differential microorganisms and differential flavor components; Note: The values ​​in the squares represent the Pearson correlation coefficients, * indicates P < 0.05, and ** indicates P < 0.01. Detailed Implementation

[0016] Example

[0017] A species of Cyberlindnera jadinii HM-02, isolated from malt-flavored fermented mash, was deposited at the China Center for Type Culture Collection (CCTCC) in Wuhan, China on May 30, 2025, with accession number CCTCM20251229.

[0018] Furthermore, the nucleotide sequence of the 18S rDNA of Cyberlindnera jadinii HM-02 is shown in SEQ ID NO: 1.

[0019] Another objective of this invention is to provide an application of *Cyberlindnera jadinii* (HM-02) for preparing HM-02 *Xanthoceras sorbifolium* fermentation broth. The specific preparation method is as follows: *Xanthoceras sorbifolium* cake is mixed with distilled water at a solid-liquid ratio of 1:5, sterilized at 121°C for 30 minutes, and then inoculated with HM-02 seed culture (8.01g CFU / mL viable bacteria) at a volume ratio of 5%. After fermentation at 35°C and 120 rpm for 72 hours, the fatty acid content in the *Xanthoceras sorbifolium* cake decreases from 12.02% to 3.1%, the viable bacteria count reaches 7.11g CFU / mL, and the in vitro antioxidant indicators (PTIO, ABTS, DPPH, and FRAP values) in the fermentation broth are significantly improved.

[0020] The HM-02 *Xanthoceras sorbifolium* fermentation liquid is further applied in the preparation of *Xanthoceras sorbifolium* malt-flavored baijiu. The specific procedure involves adding the *Xanthoceras sorbifolium* fermentation liquid at a ratio of 5% to 10% to the stacking process, followed by fermentation, distillation, storage, and blending to produce *Xanthoceras sorbifolium* malt-flavored baijiu. Mechanistic studies have shown that *Cyberlindnera jadinii* HM-02 in the fermentation broth of *Xanthoceras sorbifolium* cake can survive in the mash and significantly increases the abundance of *Cyberlindnera jadinii* and *Weissella* in the fermented mash, while decreasing the abundance of *Lactobacillus acetotolerans* and *Saccharomyces cerevisiae*. This further increases the content of aroma components such as β-phenylethanol in *Xanthoceras sorbifolium* wheat-flavored baijiu, and increases pleasant aroma components such as 2,3-butanediol (CAS: 513-85-9, fruity and creamy aroma) and methyl heptanoate (CAS: 106-73-0, grassy and honey aroma); while decreasing the content of undesirable flavors such as 2-vinyl-6-methylpyrazine (CAS: 13925-09-2, smoky flavor) and 1H-pyrrole-2-carboxaldehyde (CAS: 1003-29-8, musty flavor).

[0021] Experimental Example

[0022] 1.1 Experimental Materials

[0023] The *Xanthoceras sorbifolium* cake, the cake remaining after physical pressing of *Xanthoceras sorbifolium* seeds, was provided by Henan Langxiaopu Biotechnology Co., Ltd. Grains such as sorghum and wheat were purchased from local farmers' markets. The medium-temperature Daqu (fermentation starter) was provided by Shandong Xuxu Distillery Starter Co., Ltd. Yeast strains (Y1-Y6) were isolated and preserved from wheat-flavored mash of Henan Suitang Winery Co., Ltd. by the Microbial Resource Development and Utilization Laboratory of Huanghuai University.

[0024] 1.2 Purification of Yeast

[0025] Six yeast strains were activated and then inoculated onto YPD solid medium using the dilution-spreading method: 1% yeast extract, 2% peptone, 2% glucose, and 2% agar powder. The medium was incubated at 30°C for 24 hours. After incubation, single colonies were picked and inoculated onto YPD liquid medium, and cultured at 30°C with shaking at 200 rpm for 24 hours. The viable count was determined using the plate count method, and the viable count was adjusted to 1.0 × 10⁻⁶ using sterilized YPD liquid medium. 9 CFU / mL was used as a seed culture and stored at 4°C for later use.

[0026] 1.3 Fermentation of *Sapindus mukorossi* cake

[0027] After drying the *Xanthoceras sorbifolium* cake to constant weight at 65℃, it was pulverized through a 120-mesh sieve. 10g of the pulverized cake was added to an Erlenmeyer flask, and distilled water was added at a solid-liquid ratio of 1:5. The pH was adjusted to 6.0–6.5, and the flask was sterilized at 121℃ for 30 minutes. After cooling, seed culture of different yeasts was inoculated at a volume ratio of 5%, and the flask was incubated at 35℃ and 120 rpm for 72 hours. After incubation, the pH and viable cell count were measured. The fermentation broth was centrifuged at 3000 rpm for 5 minutes, and the solid residue and supernatant were collected separately. The solid residue was dried at 65℃ and stored for later use, while the supernatant was stored at 4℃ for later use.

[0028] 1.3.1 Determination of pH and viable cell count in fermentation broth

[0029] The pH of the fermentation broth was measured using an electronic pH meter. Viable cell counts were determined using the plate dilution method: after 72 hours of fermentation, the broth was serially diluted 10-fold with sterile water, plated onto YPD solid plates, and incubated at 30°C for 48 hours. Colony counts were then calculated, and results were expressed as 1g CFU / mL. Yeast strain Y5 was screened based on the viable cell count for subsequent experiments.

[0030] 1.3.2 Changes in pH and viable cell count during fermentation of *Xanthoceras sorbifolium* cake by yeast Y5

[0031] Y5 yeast was used to ferment *Xanthoceras sorbifolium* cake, following the same experimental method as described in 1.3. Samples were taken at 24h, 48h, and 72h of fermentation to determine pH and viable cell count (method as described in 1.4). After fermentation, the fermentation broth was centrifuged at 8000 rpm for 5 min at 4℃, and the supernatant was collected as the post-fermentation sample. The solid residue was dried at 65℃ to constant weight for nutrient composition analysis.

[0032] 1.3.3 Evaluation of in vitro antioxidant indices in the supernatant of yeast Y5 fermentation broth

[0033] After inoculating the seed culture with *Xanthoceras sorbifolium* cake, the samples were immediately centrifuged. Samples that did not ferment were considered to have fermented for 0 hours. Two supernatant samples, a VC solution (0.1 mg / mL), and a control group of sterilized PDA liquid culture medium, along with a total of four sample solutions, were used to evaluate their in vitro antioxidant properties.

[0034] (1) PTIO (2-phenyl-4,4,5,5-tetramethylimidazolin-3-oxy-1-oxygen, PTIO) free radical scavenging test: Weigh 0.3 g of PTIO solid and dissolve it in 2000 mL of buffer solution to prepare a PTIO standard solution. Add 1.6 mL of the standard solution to the reactor, and add different volumes of sample solution (0.3, 0.6, 0.9, 1.2 and 1.5 mL), and replenish with buffer solution to a total reaction volume of 3.6 mL. Incubate at 37℃ for 2 h, and then measure the absorbance value A at 557 nm. A0 represents the absorbance value without sample, which should be between 0.2 and 0.6. A represents the absorbance value with different volumes of sample added. PTIO scavenging rate (%) = (A0-A) / A0 × 100%. The removal rate was calculated using a 0.1 mg / mL VC solution as a reference solution, and the results were expressed as the amount of VC equivalent to each gram of slag (mg VCE / g).

[0035] (2) ABTS (2,2-Azinobis-(3-ethylbenzthiazoline-6-sulphonate), ABTS) free radical scavenging test: Dissolve 7 mM ABTS in buffer solution, add 2.45 mM potassium persulfate, and mix thoroughly with an equal volume of ABTS and potassium persulfate. After thorough mixing, allow the solution to stand for 12 h in the dark at room temperature to generate stable ABTS. + Solution. Dilute with anhydrous ethanol and adjust the absorbance value so that the solution reaches 0.70 ± 0.02 at a wavelength of 734 nm. This solution yields the standard ABTS. + Working solution. The absorbance value of the sample is A, and the absorbance value without sample is A0. Add the sample solution (0.035, 0.070, 0.105, 0.140 mL) and 3.2 mL of ABTS to the test tubes sequentially. + The working solutions were mixed, and anhydrous ethanol was added to bring the total reaction volume to 3.6 mL. The reaction was carried out at room temperature for 6 min. ABTS removal rate = (A0-A) / A0 × 100%. The removal rate of 0.1 mg / mL VC was also determined, and the results were expressed as the amount of VC equivalent to each g of slag (mg VCE / g).

[0036] (3) DPPH (1,1-Diphenyl-2-picrylhydrazylradical, DPPH) free radical scavenging test: Dissolve 1 mg of DPPH powder in 24 mL of anhydrous ethanol to prepare a stock solution. Take 1 mL of the stock solution and dilute it with 0.5 mL of 95% ethanol, and measure the absorbance value A (0.6-1.0) at 519 nm. Add different volumes of the test sample solution (0.3, 0.6, 0.9, 1.2, 1.5 mL) and 3 mL of working solution to the reaction system in sequence, and finally make up the volume to 4.5 mL with ethanol, mix well, react at room temperature for 30 minutes, and measure the absorbance value A of each group at 519 nm. The absorbance value of the control group without sample is A0. DPPH scavenging rate = (A0-A) / A0×100%. The scavenging rate of 0.1 mg / mL VC was also measured, and the results were expressed as the amount of VC equivalent to each g of slag (mgVCE / g).

[0037] (4) FRAP (Ferricion Reducing Antioxidant Power) Assay: FRAP working solution is prepared fresh for each use. Preparation of FRAP working solution: Mix 2 mL of 20 mmol / L FFeCl3 solution, 2 mL of 10 mmol / L TPTZ solution, and 20 mL of 0.3 M acetate buffer. Mix 0.1 mL of the sample to be tested with 2.4 mL of FRAP working solution, incubate at 37℃ for 10 min, and then measure the absorbance at 593 nm. Replace the sample with a 0.1–1.6 mmol / L FeSO4 standard solution and measure the absorbance using the same method. The linear regression equation is obtained as: Y = 0.0606x + 0.0241, where Y is the absorbance value and X is the FeSO4 concentration (mmol / L). Substitute the sample absorbance value into the regression equation to calculate the FRAP value, in mmol / L FFeSO4, which represents the antioxidant capacity of the sample equivalent to the reducing power of FeSO4 at the same molar concentration.

[0038] 1.3.4 Analysis of Antioxidant Components in Fermentation Residue of Yeast Y5

[0039] After drying the residues before and after fermentation at 65℃ to constant weight, nutritional composition analysis was performed. Crude protein was determined using GB / T6432-201 (7.2 total content method), crude fat using GB / T6433-2006, crude ash using GB / T6433-2007, crude fiber using the filter bag method (GB / T6434-20226), and starch using GB / T20194-2018.

[0040] 1.3.518S rRNA strain identification

[0041] Genomic DNA was extracted using a fungal genomic DNA extraction kit. Fungal PCR amplification of the V4 variable region of the 18S rRNA gene was performed using primer pair 547F (5'-CAGCASCYGCGGTAATTCC-3') and V4 R (5'-ACTTTCGTTCTTGATYRA-3'). The PCR products were quantified and homogenized using agarose gel electrophoresis. A DNAPCR-Free sample preparation kit was used to construct a library, which was then sequenced using the NovaSeq 600 platform. The 18S rRNA data was uploaded to the NT (Nutrient Sequence Database) database, and a phylogenetic tree was constructed using MEGA 7.0 software.

[0042] 1.4 Preparation of Xanthoceras sorbifolium liquor

[0043] Step 1: Raw Material Pretreatment. The fermentation raw material ratio is: wheat 50%–70%, sorghum 20%–35%, and rice, glutinous rice, and corn added at 5%–20%. Sorghum is crushed using a roller crusher, with the powder passing through a 1.2mm sieve controlled at 25%–35%. Corn and wheat are not crushed. After weighing the sorghum, corn, and wheat according to the ratio, the grains are moistened at high temperature. 50%–70% hot water (70℃–80℃) is added by weight, stirred evenly, and piled up for moistening for 24 hours to allow the grains to fully absorb water. During the moistening process, the mixture is turned over every 6 hours to ensure uniform moistening. The rice husks are steamed in an open container for at least 60 minutes. The Daqu (fermentation starter) is pulverized, with ≤50% passing through a 1.2mm sieve.

[0044] Step 2: Preparation of *Xanthoceras sorbifolium* fermentation broth. *Xanthoceras sorbifolium* cake was pulverized through a 120-mesh sieve, and distilled water was added at a solid-liquid ratio of 1:5. The pH was adjusted to 6.0–6.5, and sterilized at 121℃ for 30 minutes. After cooling, the broth was inoculated with Y5 seed culture at a volume ratio of 5%, and cultured at 35℃ and 120 rpm for 72 hours.

[0045] Step 3: Steaming the raw materials. After soaking the sorghum, corn, and wheat, add rice and glutinous rice, and add rice husks at 5% of the total weight of the raw grains. Mix well and then sprinkle them into the steamer. After the steam rises, add 10% to 20% hot water (50-60℃) and steam for 60 minutes.

[0046] Step 4: Cooling. Remove the steamed grains from the steamer while they are still hot and spread them into a rectangle. Pour in hot water (55-60℃) equal to 30% of the weight of the raw materials to allow the starch to further absorb water and swell. Then stir and toss the mixture to allow it to cool and ventilate.

[0047] Step 5: Add the starter culture. Add 2% to 5% of the total grains of the crushed starter culture, stir well, and mix thoroughly.

[0048] Step 6: Stacking. Add the fermented *Sapindus mukorossi* syrup prepared in Step 2 at 5%–10% of the total grain, and add 2%–5% of the steamed rice husks. The moisture content should be controlled at 70%–80% before stacking. Stack the raw materials in a trapezoidal shape with a height of 20–40 cm. During the stacking process, control the temperature at the center of the pile to not exceed 35°C. Turn the material over immediately after it reaches 35°C.

[0049] Step 7: Fermentation. The experiment was conducted at the Baijiu Brewing Training Center of the School of Biological and Food Engineering, Huanghuai University. Stainless steel fermentation tanks were used as fermentation containers. Three fermentation tanks were designed as replicates, with each tank containing 200 kg of dry grain. The fermentation period was 30 days.

[0050] Step 8: Distillation. After 30 days of fermentation, remove the mash from the vat and mix it with auxiliary materials, adding 2%–5% steamed rice husks in proportion, and stirring evenly. Before loading the mash into the still, sprinkle a layer of rice husks at the bottom of the still grate, and add more material when steam is seen. After loading, cover the still and begin distillation; collect 1 kg of the first distillate from each still; collect the distillate in stages: high-proof (alcohol content below 60% V / V), medium-proof (alcohol content above 50% V / V), and low-proof (alcohol content below 35% V / V). When the alcohol content drops below 35% V / V, stop collecting the distillate and discard the mash.

[0051] Step 9: Storage and Aging. The high, medium, and low grades of distilled spirits are stored separately in ceramic jars. The sealed jars are then placed in a cellar for aging. The cellar temperature is controlled at 15℃-20℃, and the humidity is controlled at 60%-70%. The aging time is no less than 12 months.

[0052] Step 10: Blending. Based on the quality standards and taste requirements of the finished product, select base spirits from different years and batches for blending. The blending ratio is determined according to the spirit design plan, generally using the main spirit from the middle section as a base, adding appropriate amounts of high-proof and low-proof spirits. For finished spirits with a volume of 50% or higher, the proportion of the middle section spirit should not be less than 50%.

[0053] Three additional fermentation boxes were set up as a control group, in which no *Xanthoceras sorbifolium* fermentation liquid was added during step 6, but 2%–5% of steamed rice husks were added, and the moisture content was controlled at 70%–80% before piling. The rest of the operation was the same.

[0054] 1.5 Microbial community analysis of fermented mash after 30 days of fermentation

[0055] DNA was extracted from fermented mash samples after 30 days of fermentation. Bacterial PCR amplification of the 16S rRNA gene V3-V4 variable region was performed using 341F (5'-CCTAYGGGRBGCASCAG-3') and 806R (5'-GGACTACHVG GGTWTCTAAT-3'), respectively. PCR amplification of the 18S rRNA gene was performed using 547F (5'-CAGCASCYGCGGTAATTCC-3') and 2043R (5'-ACTTTCGTTCTTGATYRA-3'). The PCR products were then quantified and homogenized. Library construction was performed using the NEXTFLEX Rapid DNA-Seq Kit, and sequencing was performed using the Illumina Miseq PE300 platform. Raw data were uploaded to the SRA database for alignment analysis (https: / / www.arb-silva.de / ).

[0056] 1.6 Analysis of wine sample components

[0057] A 52% vol sample of the finished wine was selected for component analysis. Fourteen components, including ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl lactate, n-propanol, β-phenylethanol, acetic acid, hexanoic acid, and ethyl propionate, were determined according to the method in GB / T 10345-2022.

[0058] 1.7 Flavor component analysis (GC-MS)

[0059] Flavor component analysis of *Xanthoceras sorbifolium* cake. *Xanthoceras sorbifolium* cake was pre-frozen at -80℃ for 48 h, then ground with liquid nitrogen and vortexed until homogeneous. Approximately 500 mg was placed in a headspace vial. Saturated NaCl solution and 20 μL of 10 μg / mL internal standard solution were added. Finally, sample extraction was performed using automated headspace solid-phase microextraction (HS-SPME). Under HS-SPME conditions, the sample was shaken at 60℃ for 5 min, followed by extraction in the headspace for 15 min using a 120 μm DVB / CWR / PDMS extraction head, and finally eluented at 250℃ for 5 min. Chromatographic conditions: A DB-5MS capillary column was used with high-purity helium as the carrier gas at a flow rate of 1.2 mL / min and an injection port temperature of 250℃. Temperature program: 40℃ was held for 3.5 min, then increased to 100℃ at 10℃ / min, then to 180℃ at 7℃ / min, and finally to 280℃ at 25℃ / min and held for 5 min. Mass spectrometry conditions: Electron impact ionization (EI) source, ion source temperature 230℃, ion detection mode (SIM) selected for precise scanning of qualitative and quantitative ions. Data analysis was performed by Jiangsu Sanshu Biotechnology Co., Ltd.

[0060] Flavor component analysis of control group baijiu (Chinese white liquor) and *Vernicia fordii* wine samples. 1 mL of each sample was placed in a headspace vial, and saturated NaCl and 20 μL of internal standard solution were added. Sample extraction was performed using fully automated headspace solid-phase microextraction (HS-SPME) for GS-MS analysis, following the same procedure as above.

[0061] 1.8 Correlation Analysis

[0062] Based on flavor component analysis, four flavor components with the greatest significant differences and that have been labeled were selected, and Pearson correlation analysis was performed on the differentially expressed microorganisms and flavor components.

[0063] 1.9 Data Processing

[0064] All experiments were designed with three parallel samples. Experimental data are expressed as mean ± standard deviation. Analysis of variance (ANOVA) was performed using SPSS 20.0, and Duncan's multiple range test was used to compare differences in means. A p-value < 0.05 was considered statistically significant.

[0065] 2. Experimental Results and Analysis

[0066] 2.1 Effects of six yeast strains on the pH and viable cell count of the fermentation broth of *Sapindus mukorossi* cake

[0067] Table 1 shows the data from the fermentation of *Xanthoceras sorbifolium* cake by six yeasts. As can be seen from Table 1, yeast Y5 had the lowest pH value after fermentation, reaching 5.56, significantly lower than the pH values ​​of the other yeasts (P < 0.05). Simultaneously, the viable cell count reached 7.01 g CFU / mL, significantly higher than the other groups (P < 0.05).

[0068] Table 1. pH and viable cell counts after fermentation of six yeasts

[0069] strains pH viable bacteria count (1g CFU / mL) Y1 <![CDATA[7.08±0.03 b ]]> <![CDATA[5.5±0.2 b ]]> Y2 <![CDATA[7.03±0.10 b ]]> <![CDATA[6.0±0.7 a ]]> Y3 <![CDATA[7.12±0.22 b ]]> <![CDATA[1.5±0.04 c ]]> Y4 <![CDATA[5.69±0.14 c ]]> <![CDATA[0.05±0.03 d ]]> Y5 <![CDATA[5.56±0.09 c ]]> <![CDATA[7.0±0.6 a ]]> Y6 <![CDATA[8.04±0.05 a ]]> <![CDATA[0.1±0.04 d ]]>

[0070] Note: Identical lowercase superscript letters in the same column indicate no significant difference (P>0.05), while different lowercase superscript letters indicate significant difference (P<0.05). The same applies below.

[0071] 2.2 Changes in pH and viable cell count during the fermentation of three solid residues by yeast Y5

[0072] Table 2 shows the data analysis of yeast Y5 fermentation of Xanthoceras sorbifolium cake. As can be seen from the table, the pH value of the fermentation broth decreased with the extension of time during the fermentation process, reaching the lowest value of 5.59 at 72h; the viable cell count was the highest at 72h, reaching 7.101g CFU / mL.

[0073] Table 2. Effects of three types of solid residues on the pH and viable cell count of yeast Y5 fermentation broth.

[0074] Time h pH value viable bacteria count (1g CFU / mL) 24 <![CDATA[6.04±0.09 a ]]> <![CDATA[1.40±012 c ]]> 48 <![CDATA[5.88±0.12 a ]]> <![CDATA[3.70±0.03 b ]]> 72 <![CDATA[5.59±0.06 b ]]> <![CDATA[7.10±0.05 a ]]>

[0075] 2.3 Effect of yeast Y5 fermentation solid residue supernatant on antioxidant index

[0076] Table 3 shows the results of the in vitro antioxidant index evaluation of yeast Y5 before and after fermentation. As can be seen from Table 3, the antioxidant indexes in the fermentation broth increased significantly after fermentation. The results suggest that the structural components of *Xanthoceras sorbifolium* cake were destroyed after fermentation, and the antioxidant components (flavonoids, polyphenols, and terpenes) in *Xanthoceras sorbifolium* cake were released into the solution, resulting in a significant increase in antioxidant activity.

[0077] Table 3 Evaluation of antioxidant in vitro in fermentation broth before and after yeast Y5 fermentation.

[0078]

[0079] 2.4 Nutritional composition analysis of *Sapindus mukorossi* cake before and after fermentation

[0080] Table 4 shows the nutritional composition analysis of *Xanthoceras sorbifolium* cake before and after fermentation. As can be seen from Table 4, the crude protein, fat and starch content of *Xanthoceras sorbifolium* cake decreased significantly after fermentation.

[0081] Table 4. Nutritional composition analysis of *Xanthoceras sorbifolium* cake before and after fermentation (%)

[0082] crude protein Crude fat Coarse ash crude fiber starch Before fermentation 51.70±0.23 12.02±0.49 5.05±0.81 12.02±0.41 4.51±0.11 After fermentation 34.56±0.18 3.10±0.55 3.50±0.76 12.70±0.21 1.20±0.11

[0083] 2.5 Yeast Y5 sequencing

[0084] The sequence results of yeast strain Y5 are shown in the nucleotide sequence listing SEQ ID NO: 1, and the phylogenetic tree is shown in [link to phylogenetic tree]. Figure 1 The sequence of strain Y5 was compared with the NCBI database. This strain belongs to Saccharomycetes (family Saccharomycetes); Phaffomycetaceae (subfamily Phaffomycetes); Cyberlindnera (genus Cyberlindnera). It showed the highest similarity (99.64%) to Cyberlindnera jadinii CBS 621 (GenBank: EU568909.1). Based on the comparison results, yeast Y5 was identified as Cyberlindnera jadinii (Taxonomy ID: 4903, Cyberlindnera jadinii).

[0085] 2.6 Analysis of the microbial community of fermented mash

[0086] The relative abundance of bacteria and fungi in the mash after 30 days of fermentation are shown in the figure below. Figure 2 A and Figure 2B. The specific composition is shown in Table 5. *Lactobacillus acetotolerans* had the highest relative abundance in both groups, reaching 99.54% and 84.83% respectively, significantly higher than the relative abundance of other bacteria (P < 0.05). *Saccharomyces* had the highest relative abundance among fungi in both groups, reaching 99.45% and 97.26% respectively, significantly higher than the relative abundance of other fungi (P < 0.05). Table 6 shows the abundance of *Weissella cibaria* among bacteria and *Cyberlindnera jadinii* among fungi after adding *Xanthoceras sorbifolium* fermentation broth (P < 0.05). The results indicate that exogenously added HM-02 can survive in the fermented mash and alters the composition of bacteria and fungi in the microbial community of the mash.

[0087] Table 5. Abundance composition of bacteria and fungi in fermented mash after 30 days of fermentation.

[0088] control group Sapindus mukorossi group Top 10 Bacterial RRNAs (16S rRNA) Lactobacillus acetotolerans acid-resistant lactobacillus <![CDATA[59538.00±130.50 a ]]> <![CDATA[50400.00±1670.43 b ]]> Weissella cibaria, a bacterium that feeds on Weissella cibaria <![CDATA[205.67±97.55 b ]]> <![CDATA[7540.30±1721.37 a ]]> Lactobacillus sakei_g__Latilactobacillus (Lactobacillus salc) <![CDATA[3.33±2.52 b ]]> <![CDATA[543.33±79.43 a ]]> Lactobacillus hilgardii <![CDATA[9.00±6.56 b ]]> <![CDATA[352.00±18.33 a ]]> Leuconostoc citreum (a type of bacteria) <![CDATA[15.67±8.08 b ]]> <![CDATA[184.67±125.58 a ]]> unclassified_g_Lactococcus lactis <![CDATA[3.33±0.58 b ]]> <![CDATA[128.33±34.5 a ]]> Pediococcus penosaceus g__Pediococcus pentosaceus <![CDATA[26.33±11.72 b ]]> <![CDATA[67.33±16.86 a ]]> unclassified_g__Lacticaseibacillus paracasei Lactobacillus 0.0 82.33±21.46 Weissella ghanensis (Ghanaian Weissella) 0.0 82.00±19.52 Acinetobacter_johnsonii Acinetobacter johnsonii <![CDATA[13.00±3.0 b ]]> <![CDATA[30.67±9.07 a ]]> Top 10 fungal 18S rRNAs Saccharomyces sp. brewer's yeast <![CDATA[45757.0±40.75 a ]]> <![CDATA[44740.00±210.27 b ]]> Cyberlindnera jadinii <![CDATA[206.67±53.00 b ]]> <![CDATA[1158.00±219.06 a ]]> Wickerhamomyces_anomalus Abnormal Wickerhamomyces 11.00±6.25 <![CDATA[44.33±1.15 a ]]> Suhomyces_xylopsoci xyloside yeast 13.67±9.50 12.67±6.43 Saccharomycopsis_fibuligera Capsule-forming yeast 7.67±4.73 12.33±5.13 unclassified_g__Aspergillus genus Aspergillus 1.67±2.08 1.33±1.15 Fungi_sp is an undefined fungal genus. 9.00±7.21 2.67±3.06 unclassified_k__Fungi (Undefined genus of fungi) 13.67±7.64 11.67±4.62 Blumeria graminis (Fungiella graminis) 0.00±0.00 5.33±3.06 Geotrichum_candidum 2.67±0.58 0.33±0.58

[0089] Note: The *Xanthoceras sorbifolium* group was treated with *Xanthoceras sorbifolium* fermentation liquid in step 6 of the *Xanthoceras sorbifolium* wine production process, while the control group was not treated with this liquid. All other procedures were the same.

[0090] 2.7 Analysis of Wine Components

[0091] The composition of the control group and the *Xanthoceras sorbifolium* wine is shown in Table 6. Table 6 shows that ethyl lactate content was the highest in both wines, reaching 1059.02 and 820.96 mg / L, respectively. Compared with the control group, the addition of *Xanthoceras sorbifolium* cake significantly reduced the content of sec-butanol, ethyl butyrate, n-butanol, and ethyl lactate in the wine, while significantly increasing the content of β-phenylethanol and furfural (P < 0.05).

[0092] Table 6. Analysis of wine components (mg / L)

[0093] Element control group wine Sinensis wine sec-butanol 0.55±0.01 0 Ethyl butyrate 7.71±0.04 0 allyl alcohol 2.74±1.01 2.84±1.02 n-Butanol <![CDATA[10.77±0.83 a ]]> <![CDATA[3.88±0.95 b ]]> Ethyl hexanoate 3.11±0.86 5.31±0.82 β-Phenylenol <![CDATA[23.62±1.11 b ]]> <![CDATA[34.97±1.88 a ]]> methanol 68.29±2.31 64.7±2.01 furfural <![CDATA[16.52±0.03 b ]]> <![CDATA[67.43±0.01 a ]]> n-Propanol 83.94±1.1 85.24±3.3 Isoamyl alcohol 136.71±6.03 132.3±1.31 Isobutanol <![CDATA[326.75±3.8 a ]]> <![CDATA[280.57±2.1 b ]]> Ethyl acetate 570.73±5.6 572.4±4.3 Active pentanol 810.95±5.64 819.41±5.13 Ethyl lactate <![CDATA[1059.02±0.57 a ]]> <![CDATA[820.96±0.56 b ]]>

[0094] Note: Identical lowercase letters in the same row indicate no significant difference (P > 0.05); different lowercase letters in the same row indicate significant difference (P < 0.05); the same applies below.

[0095] 2.8 Flavor Component Analysis

[0096] 2.8.1 Analysis of flavor components in *Sapindus mukorossi* cake

[0097] The flavor analysis of *Sapindus mukorossi* fruit cake is shown in Table 7. As can be seen from Table 7, it contains 15 components, including terpenes, esters, and ketones. Among them, terpenes, esters, and ketones have the most types, with 324, 274, and 180 types, respectively. The content of D-limonene among terpenes is 24.19 mg / kg, which is significantly higher than other groups (P < 0.05); the content of ethyl 2-furanocarboxylate among esters is 12.07 mg / kg, which is significantly higher than other groups (P < 0.05); and the content of 1-(2-thienyl)acetone among ketones is 10.76 mg / kg, which is significantly higher than other groups (P < 0.05).

[0098] Table 7. Flavor Component Analysis of *Vernicia fordii* Cake

[0099]

[0100]

[0101]

[0102] 2.7.2 Analysis of Flavor Components in Sapindus mukorossi Wine

[0103] The flavor components of *Xanthoceras sorbifolium* wine are shown in Table 8. The wines were ranked according to their FC (*Xanthoceras sorbifolium* wine group / control group wine) values ​​greater than 1 and less than 1, and the top 10 aroma components were selected for analysis. Table 8 shows that 1-methoxy-2-propylacetate, an ester component, was significantly enriched in the *Xanthoceras sorbifolium* wine, with a 410-fold increase compared to the control group. The heterocyclic compound 2-methylbenzothiazole was significantly reduced in the *Xanthoceras sorbifolium* wine compared to the control group, with a 5917-fold decrease in content.

[0104] Table 8. Analysis of Flavor Components in Sapindus mukorossi Wine

[0105]

[0106]

[0107] 2.9 Correlation Analysis

[0108] Based on the flavor component analysis results, four flavor components with significant differences were selected and their correlation with differentially expressed microorganisms was analyzed. The results are shown below. Figure 3 .Depend on Figure 3 It was found that the abundance of Lactobacillus acetotolerans was positively correlated with the content of 2-vinyl-6-methylpyrazine (CAS:13925-09-2) (P<0.05); the abundance of Weissella cibaria was significantly negatively correlated with 2-vinyl-6-methylpyrazine (P<0.01) and negatively correlated with methyl heptanate (CAS:106-73-0) (P<0.05).

[0109] 3. Conclusion

[0110] This invention utilizes *Cyberlindnera jadinii* (HM-02) to ferment *Xanthoceras sorbifolium* cake, significantly increasing the viable cell count and antioxidant activity of HM-02 in the fermentation broth. By adding *Xanthoceras sorbifolium* cake fermentation broth during the stacking stage of the brewing process, *Cyberlindnera jadinii* HM-02 is added to the mash via the fermentation broth, significantly increasing the *Cyberlindnera* content in the post-fermentation mash. The abundance of *Jadinii* and *Weissella* was reduced, while the abundance of *Lactobacillus acetotolerans* and *Saccharomyces* was decreased. This further increased the content of aroma components such as β-phenylethanol in *Xanthoceras sorbifolium*-flavored baijiu, and increased pleasant aroma components such as 2,3-butanediol (CAS: 513-85-9, fruity and creamy aroma) and methyl heptanoate (CAS: 106-73-0, grassy and honey aroma); while reducing the content of undesirable flavors such as 2-vinyl-6-methylpyrazine (CAS: 13925-09-2, smoky flavor) and 1H-pyrrole-2-carboxaldehyde (CAS: 1003-29-8, musty flavor).

Claims

1. A type of *J. seldomochia* (J. seldomochia) Cyberlindnera jadinii HM-02, characterized in that: It is isolated from the fermentation pit with the flavor of malt, which is preserved in China Center for Type Culture Collection (CCTCC) in Wuhan, China on May 30, 2025, and the preservation number is CCTCCM20251229, and the nucleotide sequence of its 18S rDNA is shown in SEQ ID NO:

1.

2. The use of the Zeylnosaccharomyces cerevisiae HM-02 according to claim 1, characterized in that: It is used for preparing the fermentation liquor of Xueguanggu of Cyberlindnera jadinii HM-02, and the preparation method is as follows: Xueguanggu cake is added with distilled water according to the solid-liquid ratio of 1:5, sterilized at 121℃ for 30 min, and then inoculated with the seed liquid of Cyberlindnera jadinii HM-02 according to the inoculation ratio of 1%, the viable cell count in the seed liquid is 8.0 lg CFU / mL, after fermentation at 35℃ and 120 r / min for 72 h, the content of fatty acid in the Xueguanggu cake decreases from 12.02% to 3.1%, the viable cell count reaches 7.10 lg CFU / mL, and the in vitro antioxidant indexes PTIO, ABTS, DPPH and FRAP values in the fermentation liquor increase.

3. The use of the Zeylnosaccharomyces cerevisiae HM-02 according to claim 2, characterized in that: It is used for preparing Xueguanggu liquor with the flavor of malt, and the preparation method is that the fermentation liquor of Xueguanggu is added into the stacking step of liquor preparation according to the proportion of 5% to 10%, and then fermented, distilled, stored and blended to obtain the final product.

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