Yeast S19k and application thereof

Abstract

The invention belongs to the technical field of yeast strains and fermentation, and discloses a yeast S19k and application thereof. The strain is hansenula polymorpha S19K. According to the method, the exclusive strain S19k of the red wine is screened, and a sequential inoculation process is combined, so that the limitation of the prior art in the aspects of aroma complexity, color stability and environmental tolerance is broken through, and a differentiated solution is provided for producing area personalization of the domestic red wine. According to the invention, a traditional single enzyme activity screening mode is broken through, a comprehensive screening system of the bacterial strain and the enzyme produced by the bacterial strain in a gradient wine brewing stress environment is constructed, and the functional stability of the bacterial strain in a real brewing environment is ensured. Compared with existing patents, the strain specificity, the metabolism regulation and control mechanism and the industrial adaptability are remarkably innovated, and higher market competitiveness and technical barriers are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of yeast strains and fermentation technology, and particularly relates to a Western Hanseniaspora uvarum S19k with high β-glucosidase production and its application in improving the sensory quality of wine. Background Art

[0002] The domestic wine industry is undergoing a strategic transformation from scale expansion to quality improvement. Under the dual pressures of consumption upgrading and intensified competition from imported wines, enhancing the expression of regional terroir and flavor uniqueness has become the core demand for industrial upgrading. The core of wine sensory quality - aroma complexity and color stability - is essentially dynamically regulated by processes such as enzymatic conversion, microbial metabolism, and aging modification of glycoside-bound precursor substances in grape raw materials. Research shows that 60%-90% of the key aroma compounds such as terpenoids, norisoprenoids, and phenolic derivatives in grape fruits exist in the form of non-volatile glycosides, and the release of their sensory activities highly depends on the hydrolysis of β-glucosidase. However, the industrial fermentation system dominated by traditional Saccharomyces cerevisiae has significant limitations: its inherent β-glucosidase activity is low, and its tolerance to wine stress environments (such as high ethanol, low pH, etc.) is poor, resulting in insufficient conversion rate of aroma precursors, which is an important technical bottleneck causing the flavor homogenization of domestic wines.

[0003] In recent years, non-Saccharomyces yeasts (NS) have provided new biological solutions for breaking through this dilemma due to their high β-glucosidase production characteristics and metabolic diversity. However, existing research has mainly focused on the enzyme activity screening of single strains, lacking systematic analysis of the functional stability of strains in real brewing environments (such as high SO2 tolerance), the enzyme-substrate interaction mechanism, and the multi-strain co-metabolism network, severely restricting their industrial application.

[0004] The prior art solution discloses a rare Hanseniaspora occidentalis HO1 and its application in the brewing of highly acidic fruit wines. The preservation number of this strain is CGMCC No. 27007, and it has the following characteristics:

[0005] 1) Outstanding acid reduction ability: It can efficiently degrade citric acid, with a degradation rate as high as 20.39%, significantly reducing the acidity of the wine body.

[0006] 2) Excellent aroma production ability: It can increase the content of ester compounds in plum wine by 15.08%, among which the contents of isoamyl acetate and ethyl hexanoate increase by more than 60%, enhancing the fruity aroma characteristics.

[0007] 3) Strong environmental tolerance: It can grow normally within the ranges of pH = 3, ethanol concentration 12% v / v, glucose concentration 100 - 300 g / L, and temperature 15 - 30°C.

[0008] The existing technical solution uses the combined fermentation process of HO1 and Saccharomyces cerevisiae BV818 to brew prune wine. Through the acid-lowering and aroma-producing characteristics of HO1, combined with the efficient alcohol fermentation ability of Saccharomyces cerevisiae, the aroma complexity and flavor quality of high-acid fruit wine have been significantly improved.

[0009] However, although HO1 in the existing technology can increase the ester content, its β-glucosidase activity level is not clear; the growth of existing strains is limited when the glucose concentration > 300 g / L or the ethanol concentration > 12% v / v; although HO1 in the existing technology can enhance the fruity aroma, it may produce a small amount of off-flavor substances (such as hydrogen sulfide). Summary of the Invention

[0010] In order to overcome the deficiencies of the existing technology, the present invention provides a yeast S19k and its application. With the construction of local functional strain resources and the analysis of the co-fermentation regulation mechanism as the core, high-yield β-glucosidase strains are directionally isolated from grape skins and natural fermentation mash through p-NPG colorimetric screening technology, and a polyphasic classification and identification system is established by combining ITS-rDNA sequence analysis and physiological metabolomics characteristics; further, the wine stress environment is simulated to systematically evaluate the extracellular enzyme stability and self-tolerance of the screened strains; finally, a sequential inoculation fermentation scheme of non-Saccharomyces cerevisiae - Saccharomyces cerevisiae is constructed, and the fermentation kinetic parameters are monitored in real time to analyze the dynamic law of β-glucosidase, the conversion efficiency of aroma precursors and the correlation with sensory quality.

[0011] The above object of the present invention is achieved by the following technical solution: A yeast S19k, the strain is Hanseniaspora occidentalis S19K, and its Latin name is Hanseniaspora occidentalis S19k; it is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC M 2025424, and the preservation time is: March 10, 2025.

[0012] Another object of the present invention is to protect the application of the above yeast S19K.

[0013] Furthermore, the yeast S19K is specifically applied to improving the sensory quality of fruit wine, especially wine.

[0014] Furthermore, the improvement of the sensory quality of wine is specifically achieved by increasing the terpene compounds and ester compounds in the wine, and reducing C6 compounds, forming a "high floral aroma - low herbaceous flavor" differential flavor, improving color stability, delaying color degradation, and thus improving the sensory quality of wine.

[0015] Another object of the present invention is to protect the screening method of the above yeast S19K, and the steps are as follows:

[0016] S1. Screening strains: Under aseptic conditions, coat the grape epidermis on the YEPD solid medium. Invert the solid medium plate for incubation to form visible single colonies with the naked eye, and then streak and inoculate on the surface of the WL solid medium. Invert the inoculated medium plate and incubate it in a constant temperature incubator at 28 °C. After visible single colonies appear with the naked eye, perform streak purification until a pure culture with uniform morphology and stable color is obtained on the WLN medium.

[0017] S2. Preliminary screening of the enzyme-producing ability of strains: Under aseptic environment, inoculate the strains into test tubes containing YEPD liquid medium and place them in a shaker for oscillating culture to obtain bacterial solutions. Then, add the bacterial solutions of each strain into the 96-well standard enzyme-labeled plates containing aesculin-Fe 3+ . Invert the inoculated medium plate and incubate it in a constant temperature incubator at 28 °C. Since the strains with β-glucosidase activity can hydrolyze aesculin and the produced esculetin will react with Fe 3+ in the medium to form a dark brown complex, the ability of the strains to produce β-glucosidase can be preliminarily judged by observing the depth of the colony color. Select the strains with dark colors as the potentially high enzyme-producing strains obtained by preliminary screening.

[0018] S3. Re-screening of the enzyme-producing ability of strains: For the potentially high enzyme-producing strains obtained by preliminary screening, further re-screening is carried out to accurately determine their enzyme-producing ability. First, activate the strains obtained by preliminary screening. Similarly, under aseptic conditions, inoculate the preliminarily screened strains into YEPD liquid medium and culture them in a shaker to obtain the bacterial solutions for re-screening. Then, inoculate the obtained bacterial solutions into Erlenmeyer flasks containing enzyme-producing medium and place the Erlenmeyer flasks in a shaker for culture. During the culture process, sample every 24 h. When sampling, centrifuge the bacterial solution and take the supernatant as the crude enzyme solution. Use the p-NPG colorimetric method to measure the activity of β-glucosidase in the crude enzyme solution, and compare the enzyme-producing ability of the strains based on the enzyme activity. Finally, a strain of Hanseniaspora occidentalis is screened out and named strain S19k.

[0019] The beneficial effects of the present invention compared with the prior art are as follows:

[0020] Through directional screening in the natural environment, the present invention isolates strains (such as S19k) with higher β-glucosidase activity from the grape epidermis or natural fermentation mash in red wine production areas, further improving the hydrolysis efficiency of aroma precursor substances (such as terpenoids and norisoprenoids) and enhancing the floral and fruity characteristics of red wine.

[0021] The present invention can construct strains that are tolerant to higher sugar concentrations (such as 350 g / L) and alcohol contents (such as 15% v / v) through mutagenesis or genetic engineering means, broadening their application scope in industrial production.

[0022] Through the regulation of mixed bacteria fermentation, S19k significantly enhances terpenes (such as linalool and nerol) and esters (such as ethyl laurate), while inhibiting C6 compounds (the smell of grass), forming a differentiated flavor of "high floral fragrance - low herbaceous flavor".

[0023] The present invention focuses on the aroma complexity and color stability of red wine, and realizes the quality upgrade through the multi-metabolic regulation driven by β-glucosidase.

[0024] The present invention can apply the strain S19k or the process to the brewing of wine in a high-sugar and low-pH environment, covering a wider market demand.

[0025] The present invention combines the fermentation of strain S19k with technologies such as cold maceration to synergistically enhance the aroma complexity, color stability and aging potential of red wine.

[0026] By screening the exclusive strain S19k of red wine and combining the sequential inoculation process, the present invention breaks through the limitations of the existing technology in terms of aroma complexity, color stability and environmental tolerance, providing a differentiated solution for the "regional personalization" of domestic red wine. Compared with the existing patents, the present invention has significant innovations in strain specificity, metabolic regulation mechanism and industrial adaptability, and has stronger market competitiveness and technical barriers. Brief Description of the Drawings

[0027] The following further illustrates the present invention in conjunction with the drawings and specific embodiments

[0028] Figure 1 It is a schematic diagram of the morphological identification of strain S19k on WL medium;

[0029] Figure 2 It is a schematic diagram of the enzyme production ability of strain S19k on aesculin screening medium;

[0030] Figure 3 It is a schematic diagram of the tolerance analysis of strain S19k to different glucose concentrations;

[0031] Figure 4 It is a schematic diagram of the tolerance analysis of strain S19k to different alcohol contents;

[0032] Figure 5 It is a schematic diagram of the tolerance analysis of strain S19k to different SO2 concentrations;

[0033] Figure 6 It is a schematic diagram of the tolerance analysis of strain S19k to different pH values;

[0034] Figure 7 It is a schematic diagram of the effect of different glucose concentrations on the relative enzyme activity of β-glucosidase produced by strain S19k;

[0035] Figure 8 It is a schematic diagram showing the effect of different alcohol contents on the relative enzyme activity of β-glucosidase produced by strain S19k;

[0036] Figure 9 It is a schematic diagram showing the effect of different pH values on the relative enzyme activity of β-glucosidase produced by strain S19k;

[0037] Figure 10 It is a schematic diagram showing the effect of different temperatures on the relative enzyme activity of β-glucosidase produced by strain S19k;

[0038] Figure 11 It is a comparative diagram of the dynamic β-glucosidase enzyme activity during the fermentation of S19k-seq and D254 wine samples;

[0039] Figure 12 It is a VIP score chart of aroma compounds in the S19k-seq and D254 groups based on OPLS-DA analysis;

[0040] Figure 13 It is a chart of color characterization and absorption spectrum characteristics of sequential inoculation of S19k (S19k-seq) and single inoculation fermentation of commercial Saccharomyces cerevisiae D254. Detailed implementation manners

[0041] The present invention will be described in detail below by means of specific embodiments, but the protection scope of the present invention is not limited. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be obtained from commercial channels.

[0042] Example 1

[0043] 1. Strain screening and identification

[0044] The isolation method of the high-yield β-glucosidase non-Saccharomyces cerevisiae S19k described in the present invention is as follows: Taking the epidermis of Cabernet Sauvignon grapes in Huailai production area, Hebei, China as a sample, the sample is coated on YEPD solid medium under sterile conditions, and the solid medium plate is inverted and cultured at 28 °C for 24 h to form visible single colonies with the naked eye. Streak inoculate on the surface of WL solid medium. The inoculated medium plate is inverted and cultured in a constant temperature incubator at 28 °C for 24 hours. After visible single colonies appear with the naked eye, perform 2-3 times of streak purification until a pure culture with uniform morphology and stable color is obtained on the WLN medium. The results are shown in Figure 1 . It shows a relatively large diameter with a central dark green to green color, a narrow, neat, smooth and non-notch white edge, and a delicate and smooth surface.

[0045] The purified strain was preserved by glycerol freezing method: The sterilized 50% (v / v) glycerol solution was mixed with the strain culture solution at a volume ratio of 1:1, aliquoted into cryotubes, and stored frozen in an -80°C refrigerator. This preservation method can ensure the genetic stability of the strain and maintain the enzyme activity for at least 2 years.

[0046] 2. Screening method for enzyme-producing ability

[0047] 1) Primary screening: First, the selected non-Saccharomyces cerevisiae strains were separately activated. Under aseptic conditions, an appropriate amount of the strain was taken from the preservation tube and inoculated into a test tube containing 5 mL of YEPD liquid medium, and cultured in a shaker at 28°C and 180 r / min for 24 h to obtain a bacterial solution. Then, the bacterial solution of each strain was added dropwise into a 96-well standard enzyme-linked immunosorbent assay (ELISA) plate containing aesculin-Fe 3+ . The inoculated culture medium plate was placed in an incubator at 28°C and incubated upside down for 48 h. Since the strains with β-glucosidase activity can hydrolyze aesculin, and the produced esculetin will react with Fe in the medium 3+ to form a dark brown complex, the ability of the strain to produce β-glucosidase was preliminarily judged by observing the depth of the colony color. The strains with dark colors were selected as the potential high enzyme-producing strains obtained by primary screening, and the results are shown in Figure 2 .

[0048] 2) Secondary screening: For the potential high enzyme-producing strains obtained by primary screening, further secondary screening was carried out to accurately determine their enzyme-producing ability. First, the strains obtained by primary screening were activated. Similarly, under aseptic conditions, the strains obtained by primary screening were inoculated into 5 mL of YEPD liquid medium and cultured in a shaker at 28°C and 180 r / min for 24 h to obtain the bacterial solution for secondary screening. Then, the obtained bacterial solution was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of enzyme-producing medium at an inoculation amount of 2% (v / v). The Erlenmeyer flask was placed in a shaker at 28°C and 180 r / min and cultured for 72 h. During the culture process, samples were taken every 24 h. When sampling, the bacterial solution was centrifuged at 4°C and 8000 r / min for 10 min, and the supernatant was taken as the crude enzyme solution. The p-NPG (p-nitrophenyl-β-D-glucopyranoside) colorimetric method was used to determine the activity of β-glucosidase in the crude enzyme solution. The specific operation is as follows: Take 0.025 mL of the crude enzyme solution, add 0.1 mL of a p-NPG solution with a concentration of 5 mmol / L, react in a water bath at 50°C for 30 min, and then add 0.125 mL of 1 mol / L Na2CO3 solution to terminate the reaction. The absorbance value was measured at a wavelength of 405 nm, and the enzyme activity was calculated according to the standard curve.

[0049] Where U is the enzyme activity unit (U / mL); C is the pNP concentration (μmol / L); V1 is the total volume of the reaction system (250 μL); D is the dilution factor, if the crude enzyme solution is not diluted, D = 1; T is the reaction time (30 minutes); V2 is the volume of the enzyme solution added (25 μL).

[0050] The enzyme-producing ability of the strains was compared based on the level of enzyme activity, and finally a strain of Hanseniaspora occidentalis was screened out. The highest β-glucosidase activity of this strain was measured to be 167.66 ± 1.98 U / mL, and it was named strain S19k.

[0051] 3. Identification of strain S19k

[0052] 1) Genomic DNA extraction: Inoculate strain S19k into a test tube containing 5 mL of YEPD liquid medium and culture it in a shaker at 28 °C and 180 r / min for 24 hours to allow the strain to enter the logarithmic growth phase. Then, take 1.5 mL of the bacterial solution and place it in a centrifuge tube, add quartz sand and yeast lysis solution for cell disruption operations (such as grinding, sonication, etc.) to fully lyse the cells. After the disruption is completed, immediately place the sample in an ice bath to reduce the activity of nucleases and avoid DNA degradation. Add sodium acetate and isopropanol to the disrupted bacterial solution, mix well and then place it in an ice bath again. Sodium acetate adjusts the pH of the solution, and isopropanol promotes the precipitation of DNA. The precipitation effect is enhanced by ice bath, and then the DNA precipitate is collected. Add 1×TE buffer to dissolve the substance, and then add chloroform:isopropanol (24:1) for extraction. Chloroform removes impurities such as proteins, and isopropanol further concentrates DNA. The supernatant containing pure DNA is separated by centrifugation. Wash the DNA precipitate with the washing solution to remove residual salt ions and organic reagents, and finally dissolve the washed DNA in sterile double-distilled water (ddH2O) and store it at an appropriate temperature (such as -20 °C) to complete the extraction of genomic DNA. The extracted DNA can be stored at -20 °C for subsequent experiments.

[0053] 2) ITS sequence PCR amplification: Use the universal primers ITS1 (5’-CTTGGTCATTTAGACGAAGTAA-3’) and ITS4 (5’-GCATATCAATAAGCGGAGGA-3’) to amplify the ITS sequence of strain S19k. The PCR reaction system is as follows: 2.0 μL of genomic DNA, 4.0 μL of 10×Buffer, 0.2 μL of Taq DNA polymerase, 1.0 μL of dNTP (10 mM), 1.0 μL of each upstream and downstream primer, and make up to 25 μL with ddH2O. The PCR amplification program is set as follows: pre-denaturation at 95 °C for 5 minutes; denaturation at 95 °C for 30 seconds, annealing at 55 °C for 30 seconds, extension at 72 °C for 1 minute, for a total of 30 cycles; finally, extension at 72 °C for 10 minutes.

[0054] 3) Detection and Sequencing of PCR Products

[0055] After the completion of PCR amplification, 5 μL of the amplified product was taken for detection by 1% agarose gel electrophoresis. The gel was placed in a gel imaging system for observation. If a bright band appeared at approximately 500 - 700 bp, it indicated successful amplification. The remaining PCR amplified product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to obtain the ITS sequence of strain S19k.

[0056] 4) Sequence Homology Analysis and Species Identification

[0057] The ITS sequence of strain S19k was searched for homologous sequences in the GenBank nucleotide sequence database (https: / / www.ncbi.nlm.nih.gov / blast) using the BLAST software. The species of the tested strain was identified by comparing the similarity degree of the ITS sequence of the tested strain with that of known yeasts. Generally, a similarity degree above 99% could be regarded as the same species. The results showed that strain S19k was identified as Hanseniaspora occidentalis, that is, Hanseniaspora occidentalis S19k.

[0058] 5) Strain Preservation

[0059] Strain S19k was preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number was CCTCC M2025424.

[0060] 4. Tolerance Study of Non - Saccharomyces Strain S19k with High β - Glucosidase Yield

[0061] 1) Evaluation Method for Sugar Tolerance

[0062] Treatment 1: The activated seed liquid of strain S19k was inoculated into 20 mL of YEPD medium for cultivation. The effective viable count of strain S19k in the YEPD medium was 10 6 CFU / mL. The glucose concentrations in the YEPD medium were 100 g / L respectively. It was inoculated into the liquid medium at an inoculation amount of 10 6 CFU / mL and cultured at 28 °C and 200 r / min for 48 h. Three parallel experiments were set for each treatment.

[0063] Treatment 2: The same as Treatment 1, the only difference was that the glucose concentration in the YEPD medium was 150 g / L.

[0064] Treatment 3: The same as Treatment 1, the only difference was that the glucose concentration in the YEPD medium was 200 g / L.

[0065] Treatment 4: Identical to Treatment 1, the only difference being that the glucose concentration in the YEPD medium is 250 g / L.

[0066] Treatment 5: Identical to Treatment 1, the only difference being that the glucose concentration in the YEPD medium is 300 g / L.

[0067] After the experiment, the growth of the strains on the solid medium was observed to evaluate their growth performance under different glucose concentration stress conditions; the growth of the strains in the liquid medium was analyzed by measuring the OD value of the culture solution at 600 nm using a UV-spectrophotometer. The results are shown in Figure 3 .

[0068] According to the experimental results, when the glucose concentration was 100 g / L, the OD600 value of strain S19k was the highest (about 2.0), indicating that the strain grew most vigorously and had the strongest metabolic activity at this concentration, which was the optimal growth concentration of strain S19k under glucose concentration stress conditions. Strain S19k could maintain a relatively good growth state (OD600 values were all close to or higher than 1.9) at glucose concentrations of 100 - 200 g / L, indicating its tolerance to glucose stress in this concentration range.

[0069] (2) Evaluation method for alcohol tolerance

[0070] Treatment 1: The activated seed liquid of strain S19k was inoculated into 20 mL of YEPD medium for cultivation. The effective viable count of strain S19k in the YEPD medium was 10 6 CFU / mL. The ethanol concentrations in the YEPD medium were 3% v / v, and it was inoculated into the liquid medium at an inoculation amount of 10 6 CFU / mL and cultured at 28 °C and 200 r / min for 48 h. Three parallel experiments were set for each treatment.

[0071] Treatment 2: Identical to Treatment 1, the only difference being that the ethanol concentration in the YEPD medium was 6% (v / v).

[0072] Treatment 3: Identical to Treatment 1, the only difference being that the ethanol concentration in the YEPD medium was 9% (v / v).

[0073] Treatment 4: Identical to Treatment 1, the only difference being that the ethanol concentration in the YEPD medium was 12% (v / v).

[0074] Treatment 5: Identical to Treatment 1, the only difference being that the ethanol concentration in the YEPD medium was 15% (v / v).

[0075] After the experiment, observe the growth of the strains on the solid medium and evaluate their growth performance under different alcohol concentration stress conditions. For the strains in the liquid medium, analyze their growth by measuring the OD value of the culture solution at 600 nm using a UV-spectrophotometer. The results are shown in Figure 4 .

[0076] According to the experimental results, when the alcohol concentration is 3.0% v / v, the OD600 value of strain S19k is the highest (about 1.3), indicating that the strain grows most vigorously at this alcohol concentration. The low-alcohol environment promotes its growth and the cell metabolic activity is relatively strong. At an alcohol concentration of 6.0% v / v, the OD600 value still remains at a relatively high level (about 1.1), indicating that strain S19k has good tolerance to alcohol concentrations of 6.0% v / v and below. When the alcohol concentration rises to 9.0% v / v, the OD600 value drops to 1.0, showing that its tolerance to alcohol concentrations of 9.0% v / v and above decreases, but it can still grow.

[0077] (3) Evaluation method for SO2 tolerance

[0078] Inoculate the activated seed solution of strain S19k into 20 mL of YEPD medium for culture. The effective viable count of strain S19k in the YEPD medium is 10 6 CFU / mL. The SO2 concentrations in the YEPD medium are 60 mg / L respectively. Inoculate into the liquid medium at an inoculation amount of 10 6 CFU / mL and culture at 28 °C and 200 r / min for 48 h. Set three parallel experiments for each treatment.

[0079] Treatment 2: The same as Treatment 1, the only difference is that the SO2 concentration in the YEPD medium is 120 mg / L.

[0080] Treatment 3: The same as Treatment 1, the only difference is that the SO2 concentration in the YEPD medium is 180 mg / L.

[0081] Treatment 4: The same as Treatment 1, the only difference is that the SO2 concentration in the YEPD medium is 240 mg / L.

[0082] Treatment 5: The same as Treatment 1, the only difference is that the SO2 concentration in the YEPD medium is 300 mg / L.

[0083] After the experiment, observe the growth of the strains on the solid medium and evaluate their growth performance under different SO2 stress conditions. For the strains in the liquid medium, analyze their growth by measuring the OD value of the culture solution at 600 nm using a UV-spectrophotometer. The results are shown in Figure 5 .

[0084] According to the experimental results, when the SO2 concentration was 60 mg / L, the OD600 value of strain S19k was the highest (about 1.58), indicating that the growth state of the strain was the best at this concentration, the cell metabolic activity was the strongest, and it was the optimal growth concentration of strain S19k under SO2 stress conditions. Strain S19k could grow within the SO2 concentration range of 60 - 300 mg / L, among which 60 mg / L was the optimal concentration, and the growth performance at the concentration of 120 - 300 mg / L further proved its strong tolerance to SO2 stress, and this characteristic had important value in practical application scenarios such as actual brewing (which often involves SO2 addition).

[0085] (4) Evaluation method of pH tolerance

[0086] Treatment 1: Inoculate the activated seed liquid of strain S19k into 20 mL YEPD medium for cultivation. The effective viable count of strain S19k in the YEPD medium was 10 6 CFU / mL. The pH values in the YEPD medium were 2.8 respectively, and inoculate them into the liquid medium at an inoculation amount of 10 6 CFU / mL, and cultivate them at 28 °C and 200 r / min for 48 h. Set three parallel experiments for each treatment.

[0087] Treatment 2: Same as Treatment 1, the only difference was that the pH value in the YEPD medium was 3.2.

[0088] Treatment 3: Same as Treatment 1, the only difference was that the pH value in the YEPD medium was 3.6.

[0089] Treatment 4: Same as Treatment 1, the only difference was that the pH value in the YEPD medium was 3.8.

[0090] After the experiment, observe the growth of the strain on the solid medium and evaluate its growth performance under different pH stress conditions; the growth of the strain in the liquid medium was analyzed by measuring the OD value of the culture solution at 600 nm with an ultraviolet - spectrophotometer, and the results are shown in Figure 6 .

[0091] According to the experimental results, when the pH value is 3.6, the OD600 value of strain S19k is the highest (about 1.38), indicating that this pH condition is the most suitable for the growth of the strain, with the strongest cell metabolic activity, and it is the optimal growth environment of strain S19k under pH stress conditions. In the pH range of 2.8 - 3.8 tested, the OD600 values of strain S19k all remained above 1.2, showing its good tolerance to acidic environments. Even under the strong acidic condition of pH 2.8, the OD600 value still reached about 1.25, indicating that the strain can maintain growth within a relatively wide acidic pH range, reflecting its adaptive advantage in acidic environments (such as the wine and grape wine brewing environments). This result shows that strain S19k can grow stably in common acidic fermentation systems (such as the fruit wine and grape wine brewing environments with pH 2.8 - 3.8), providing an important basis for its tolerance in the fermentation processes of related acidic beverages.

[0092] 5. Tolerance analysis of β-glucosidase produced by strain S19k in the grape wine brewing environment

[0093] 1) Evaluation method of sugar tolerance

[0094] Treatment 1: Take 25 μL of the crude enzyme solution of β-glucosidase produced by strain S19k and add it to a 100 μL reaction system containing the substrate p-NPG (p-nitrophenyl-β-D-glucopyranoside). The temperature of the reaction system is 50 °C, the pH is 5.0, the glucose concentration in the reaction system is 0 g / L, react for 30 min, and set three parallel experiments. After the reaction ends, add 125 μL of 1 mol / L Na2CO3 solution to terminate the reaction, measure the absorbance at a wavelength of 405 nm, and calculate the enzyme activity according to the standard curve. Take the highest point of the enzyme activity obtained when the glucose content is 0 g / L as 100%, and compare other enzyme activities with it to obtain the relative enzyme activity.

[0095] Treatment 2: The same as Treatment 1, the only difference is that the glucose concentration in the reaction system is 50 g / L.

[0096] Treatment 3: The same as Treatment 1, the only difference is that the glucose concentration in the reaction system is 100 g / L.

[0097] Treatment 4: The same as Treatment 1, the only difference is that the glucose concentration in the reaction system is 150 g / L.

[0098] Treatment 5: The same as Treatment 1, the only difference is that the glucose concentration in the reaction system is 200 g / L.

[0099] Treatment 6: The same as Treatment 1, the only difference is that the glucose concentration in the reaction system is 250 g / L.

[0100] Treatment 7: Same as Treatment 1, the only difference being that the glucose concentration in the reaction system is 250 g / L.

[0101] According to the experimental results shown in Figure 7 it can be seen that as the glucose concentration increases, the enzyme activity decreases significantly. The relative enzyme activity drops to approximately 63% at 150 g / L, approximately 55% at 200 g / L, approximately 49% at 250 g / L, and approximately 45% at 300 g / L. High-concentration glucose may inhibit the enzyme activity by competing for the enzyme active site, changing the enzyme spatial conformation, etc. At a glucose concentration of 50 - 100 g / L, the relative enzyme activity is maintained at 95% - 98%, indicating that the β-glucosidase produced by strain S19k has a certain tolerance to glucose in this concentration range and can still maintain a relatively high catalytic activity. Taking the highest enzyme activity obtained at an alcohol concentration of 0.0% as 100%, the other enzyme activities are compared with it to obtain the relative enzyme activity.

[0102] 2) Evaluation method for alcohol tolerance

[0103] Treatment 1: Take 25 μL of the crude enzyme solution of β-glucosidase produced by strain S19k and add it to a 100 μL reaction system containing the substrate p-NPG. The temperature of the reaction system is 50 °C, the pH is 5.0, the ethanol concentration in the reaction system is 0.0% (v / v), react for 30 min, and set three parallel experiments. After the reaction is completed, add 125 μL of 1 mol / L Na2CO3 solution to terminate the reaction, measure the absorbance at a wavelength of 405 nm, and calculate the enzyme activity according to the standard curve.

[0104] Treatment 2: Same as Treatment 1, the only difference being that the ethanol concentration in the reaction system is 2.5% (v / v).

[0105] Treatment 3: Same as Treatment 1, the only difference being that the ethanol concentration in the reaction system is 5.0% (v / v).

[0106] Treatment 4: Same as Treatment 1, the only difference being that the ethanol concentration in the reaction system is 7.5% (v / v).

[0107] Treatment 5: Same as Treatment 1, the only difference being that the ethanol concentration in the reaction system is 10.0% (v / v).

[0108] Treatment 5: Same as Treatment 1, the only difference being that the ethanol concentration in the reaction system is 12.5% (v / v).

[0109] Treatment 6: Same as Treatment 1, the only difference being that the ethanol concentration in the reaction system is 15.0% (v / v).

[0110] According to the experimental results shown in Figure 8It can be seen that when the alcohol content is 0.0%, 2.5%, and 5.0%, the relative enzyme activity of β-glucosidase produced by strain S19k is close to 100%, indicating that the low-alcohol environment has no significant inhibition on enzyme activity, and the enzyme can maintain high activity under this condition. As the alcohol content increases, the enzyme activity gradually decreases. When the alcohol content is 7.5%-12.5%, the relative enzyme activity remains at 85%-94%, indicating that the enzyme has a certain tolerance to the alcohol content in this range, but the activity is lower than that in the alcohol-free environment.

[0111] 3) Evaluation method for pH tolerance

[0112] Treatment 1: Take 25 μL of the crude enzyme solution of β-glucosidase produced by strain S19k and add it to a 100 μL reaction system containing the substrate p-NPG. The temperature of the reaction system is 50 °C, the pH of the reaction system is 3.0, and the reaction lasts for 30 min. Set three parallel experiments. After the reaction ends, add 125 μL of 1 mol / L Na2CO3 solution to terminate the reaction, measure the absorbance at a wavelength of 405 nm, and calculate the enzyme activity according to the standard curve. Take the highest point of the enzyme activity obtained at pH 5.0 as 100%, and compare the other enzyme activities with it to obtain the relative enzyme activity.

[0113] Treatment 2: Same as Treatment 1, the only difference is that the pH of the reaction system is 4.0.

[0114] Treatment 3: Same as Treatment 1, the only difference is that the pH of the reaction system is 5.0.

[0115] Treatment 4: Same as Treatment 1, the only difference is that the pH of the reaction system is 6.0.

[0116] Treatment 5: Same as Treatment 1, the only difference is that the pH of the reaction system is 7.0.

[0117] Treatment 6: Same as Treatment 1, the only difference is that the pH of the reaction system is 8.0.

[0118] According to the experimental results, see Figure 9 It can be seen that when the pH value is 6.0, the relative enzyme activity of the enzyme produced by strain S19k is the highest (exceeding 100%), indicating that this is the optimal pH environment for the enzyme to exert its catalytic activity. In the range of pH 5.0-7.0, the relative enzyme activity remains at a relatively high level (above 90%), indicating that the enzyme has good tolerance to neutral and weak acid-base environments; while under the conditions of too low or too high pH values, the enzyme activity deviates significantly.

[0119] 4) Evaluation method for temperature tolerance

[0120] Treatment 1: Take 25 μL of the crude β-glucosidase solution produced by strain S19k and add it to a reaction system of 100 μL containing the substrate p-NPG. The pH of the reaction system is 5.0, and the reaction is carried out in a water bath at 10 °C for 30 min. Set three parallel experiments. After the reaction, add 125 μL of 1 mol / L Na2CO3 solution to terminate the reaction, measure the absorbance at a wavelength of 405 nm, and calculate the enzyme activity according to the standard curve. Take the highest enzyme activity obtained at 50 °C as 100%, and compare the other enzyme activities with it to obtain the relative enzyme activity.

[0121] Treatment 2: Same as Treatment 1, the only difference is that the reaction temperature is 20 °C.

[0122] Treatment 3: Same as Treatment 1, the only difference is that the reaction temperature is 30 °C.

[0123] Treatment 4: Same as Treatment 1, the only difference is that the reaction temperature is 40 °C.

[0124] Treatment 5: Same as Treatment 1, the only difference is that the reaction temperature is 50 °C.

[0125] Treatment 6: Same as Treatment 1, the only difference is that the reaction temperature is 60 °C.

[0126] According to the experimental results shown in Figure 10 It can be seen that when the temperature is 30 °C, 40 °C and 50 °C, the relative enzyme activity of the enzyme produced by strain S19k is the highest (exceeding 100%), indicating that this temperature range is the optimal temperature for the enzyme to exert its catalytic activity. And at the wine fermentation temperature of 20-30 °C, the relative enzyme activity of this β-glucosidase still reaches more than 96%, indicating that β-glucosidase has good adaptability to the temperature environment of wine brewing.

[0127] 6. Fermentation and determination of basic physical and chemical indexes

[0128] Treatment group 1 (sequential fermentation of S19k + D254)

[0129] ① Grape juice preparation

[0130] Using fresh Cabernet Sauvignon grapes as raw materials, select grape fruits with consistent maturity and no pests and diseases. Remove the stems and crush the grapes, separating the grape fruits from the stems and crushing them into a pulp. Fill the crushed grape pulp into the fermentation container at 80% filling volume, and add potassium metabisulfite with a mass fraction of 60 mg / L to inhibit the growth of miscellaneous bacteria and prevent oxidation. After allowing it to react fully for 1 hour, add 20 mg / L of pectinase to further promote the extraction of pigments, polyphenols, and aromatic substances from the grape berries. After the addition, stir well, then seal the glass bottle and place it in a low-temperature environment at 4°C for 24 hours of maceration. This allows the pigments, aroma substances, etc. in the grape skins to fully dissolve into the grape juice. After the maceration is completed, press through a pressing device and filter through two layers of gauze to remove solid impurities such as grape skins and seeds, obtaining clarified grape juice.

[0131] ② Fermentation process

[0132] Under aseptic conditions, inoculate the non-Saccharomyces cerevisiae strain S19k with high β-glucosidase activity into the above grape juice for continued fermentation. The effective viable count of S19k in the grape juice is 10 5 CFU / mL. After 2 days of fermentation, inoculate commercial Saccharomyces cerevisiae D254 into the grape fermentation juice for continued fermentation. The effective viable count of the inoculated commercial Saccharomyces cerevisiae D254 is 10 4 CFU / mL. The temperature throughout the fermentation process is controlled at 25 ± 2°C.

[0133] After the start of fermentation, samples are collected every 24 hours to measure the β-glucosidase activity, and the CO2 weight loss is used as an observation index. When the change in CO2 weight loss < 0.1 g / day and lasts for 3 days, it is regarded as the end of fermentation. After the fermentation is completed, measure the physicochemical indexes such as residual sugar content, total acid, pH, and alcohol content. Add 60 mg / L K2S2O5 to terminate the fermentation. Age for one month at 15°C, then filter for subsequent detection and analysis of color and aroma components. Set up 2 parallel experimental groups for each scheme, and measure each parameter 3 times for each group to ensure the accuracy and reliability of the experimental data.

[0134] Treatment group 2 (single fermentation with commercial Saccharomyces cerevisiae D254): In step ②, only inoculate Saccharomyces cerevisiae D254, and the remaining steps are the same as those in treatment group 1.

[0135] This study compared the key physicochemical indexes ( Figure 11 , Table 1, Figure 13 ) of treatment group 1 (sequential inoculation of S19k + D254) and treatment group 2 (single fermentation with D254) during the red wine fermentation process to deeply analyze the synergistic effect of the non-Saccharomyces cerevisiae S19k with high β-glucosidase activity.

[0136] Treatment group 1 (sequential fermentation of S19k + D254) had significantly higher enzyme activity throughout the fermentation cycle than the Sc group (pure fermentation with commercial yeast) because high-yield β-glucosidase screening strains were inoculated in the early stage. Especially in the initial stage of fermentation (such as 1 - 4 days), the screening strains rapidly expressed β-glucosidase, laying a foundation for the hydrolysis of subsequent aroma precursors; the enzyme activity maintained a relatively high level in the middle stage of fermentation (such as 6 - 7 days) and continuously acted on the substrate. Treatment 2 (single fermentation group with commercial Saccharomyces cerevisiae D254) relied on commercial Saccharomyces cerevisiae, which had weak β-glucosidase-producing ability, and the enzyme activity remained at a relatively low level throughout the fermentation cycle, making a limited contribution to the hydrolysis of aroma precursors. The enzyme activity curve of treatment group 2 was flat without obvious fluctuations, indicating that under the single fermentation mode of commercial yeast, there was a lack of a mechanism to enhance β-glucosidase activity and it was unable to effectively activate the hydrolysis metabolic pathway of aroma precursors.

[0137] After 7 days of fermentation in treatment group 2 and 10 days of fermentation in S19k-seq, the residual sugar content of both stabilized at 3.38 g / L. Moreover, the residual sugar and alcohol content data showed that there was no significant difference between the two groups in terms of the degree of residual sugar consumption and the final alcohol content (12.79% v / v and 12.49% v / v). This result indicated that although the process of treatment group 1 extended the fermentation cycle, its glycolysis efficiency was basically the same as that of treatment group 2. It was speculated that the non-Saccharomyces cerevisiae S19k inoculated in the initial stage of fermentation did not show a significant difference in alcohol conversion efficiency compared with commercial Saccharomyces cerevisiae D254. In terms of the total acid index, it was 5.29 ± 0.19 g / L in treatment group 2 and 5.28 ± 0.64 g / L in treatment group 1, and there was a significant difference in the total acid content between the two groups, which might be related to the acid-producing characteristics of the fermentation strains or process differences. It was worth noting that the difference in volatile acid content was particularly prominent: it was 0.23 ± 0.01 g / L in treatment group 2 and only 0.14 ± 0.00 g / L in treatment group 1 (p < 0.05), indicating that the process of treatment group 1 had a significant advantage in inhibiting the formation of volatile acids such as acetic acid, effectively improving the purity of the wine body. Given that excessive volatile acids would impart a spoilage odor to the wine, the positive effect of the process of treatment group 1 on controlling the content of volatile acids directly contributed to the optimization and upgrading of wine quality.

[0138] Analysis of the absorbance curves of the fermented wine samples of Treatment 1 and Treatment 2 revealed significant differences in absorbance within the wavelength range of 380 - 780 nm, indicating different light absorption characteristics and reflecting differences in color composition and optical properties. From the color characterization data, the L value of the wine sample of Treatment 1 (33.44 ± 0.59) was significantly lower than that of the wine sample of Treatment 2 (43.74 ± 0.43), suggesting that the color of the wine sample of Treatment 1 was deeper. In red wine, a deeper color is usually associated with a higher degree of polymerization of phenolic substances (such as anthocyanins and tannins). Such polymerized substances have a stable structure and can effectively resist the destruction of color by external factors such as oxidation, thereby delaying color degradation. In contrast, the higher L value and lighter color of the wine sample of Treatment 2 may indicate insufficient polymerization of its phenolic substances and are more likely to cause color degradation due to oxidation reactions during storage. In addition, the yellowness value b* of the wine sample of Treatment 1 (29.74 ± 0.87) was significantly higher than that of the wine sample of Treatment 2 (24.27 ± 1.57), further indicating that Treatment 1 was more prominent in inhibiting oxidative browning. If further analyzed in combination with the absorption spectrum, if Treatment 1 showed more stable absorption characteristics at key wavelengths (such as the wavelength band reflecting the absorption of pigment substances), it would better confirm its color stability advantage. In summary, the wine sample under Treatment 1 showed better color stability, while the wine sample of Treatment 2 had a higher risk of color degradation due to its lighter color and insufficient polymerization of phenolic substances.

[0139] Table 1 Comparison of Physicochemical Indexes during the Fermentation of Different Wine Samples

[0140]

[0141] During the entire fermentation process, a total of 71 volatile compounds were detected in the red wine sample treatment group 1 (sequential fermentation of S19k + D254), including 3 C6 compounds, 1 C13-norisoprenoid, 12 terpenoids, 4 benzene derivatives, 1 phenolic acid ester, 13 higher alcohols, 7 fatty acids, 25 esters, 3 aldehydes and ketones, and 2 phenols.

[0142] Through the VIP score plot of OPLSDA analysis ( Figure 12) It was found that there were 15 major differential metabolites between treatment group 1 and treatment group 2 (single fermentation of commercial Saccharomyces cerevisiae D254). The higher the VIP score, the more significant the difference in the compound between the two treatment groups. Among them, compounds such as ethyl acetate and (+)-limonene had relatively high VIP scores and were the core aroma substances differentiating the two wine samples. Some compounds such as ethyl acetate and geraniol showed specific high expression in treatment group 1 or treatment group 2, indicating that the S19k sequential inoculation process significantly affected the synthesis and accumulation of these aroma substances. The compounds with high VIP values mainly included esters (such as decyl acetate and benzyl acetate), alcohols (such as geraniol and nerol), and terpenes (such as (+)-limonene), and these substances made important contributions to the aroma characteristics of red wine. The research results showed that the S19SEQ sequential inoculation process directed the generation of characteristic aroma components by changing the yeast metabolic pathway and ultimately shaped an aroma profile significantly different from that of treatment group 2.

[0143] The present invention discloses a Hanseniaspora occidentalis S19k with high β-glucosidase activity and its application in improving the sensory quality of wine. This strain was directionally screened from the skin of Cabernet Sauvignon grapes in Huailai production area, Hebei, China, and its species was confirmed by ITS sequence identification and polyphasic taxonomic analysis, and it was deposited in the China Center for Type Culture Collection (CCTCC M 2025424).

[0144] 1. Efficient enzyme activity and metabolism coordination: The β-glucosidase activity of S19k was as high as 167.66 U / mL, significantly higher than that of traditional non-Saccharomyces cerevisiae. Sequential inoculation with commercial Saccharomyces cerevisiae D254 (S19k was inoculated 48 hours first) achieved a two-stage optimization of "release of aroma precursors - synthesis of flavor substances", with significant enrichment of terpenes (such as linalool +42%) and esters (such as ethyl laurate +35%), and at the same time inhibiting C6 compounds (green grass flavor -28%), forming a differential flavor of "high floral fragrance - low herbaceous flavor".

[0145] 2. Strong environmental tolerance: In the typical stress environment of red wine (pH 2.8 - 3.8, ethanol 15% v / v, SO2 300 mg / L), S19k had a relatively high survival rate and could ensure stable enzyme production throughout the fermentation process, breaking through the growth limitations of existing strains under high sugar and high alcohol content.

[0146] 3. Fermentation process optimization: Compared with the pure fermentation of commercial wine yeast by the sequential inoculation process, although the fermentation time is extended, there are no significant differences in sugar utilization rate and alcohol content. However, the sequential inoculation process significantly reduces the volatile acid content to 0.12 g / L, which is significantly better than 0.18 g / L of single yeast fermentation. In addition, the alcohol content of the sequential inoculation process reaches 12.0% v / v, which is the same as that of single yeast fermentation. These results indicate that the sequential inoculation process effectively reduces the volatile acid content while maintaining the alcohol production, thus significantly improving the sensory quality of the wine.

[0147] 4. Enhancement of color stability: The S19k-seq process maintains the color stability through the action of β-glucosidase, effectively inhibits oxidative browning, and the overall color performance is better than that of the D254 group, providing reliable technical support for the color quality of red wine.

[0148] 5. Application value: Through strain-specific screening, metabolic pathway regulation and process parameter optimization, the present invention systematically solves the core problems of aroma homogenization, color instability and low fermentation efficiency in red wine brewing, provides a differential technical solution for the "regional individuation" of domestic wines, and is applicable to the industrial production of various types of wines such as dry red and sweet red wines.

[0149] The above-described embodiments are only the preferred embodiments of the present invention, and not all the feasible embodiments of the present invention. Any obvious changes made by those of ordinary skill in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of the claims of the present invention.

Claims

1. A yeast S19k, characterized in that, The strain is Hanseniaspora occidentalis S19K, with the Latin name Hanseniaspora occidentalis S19k; it is deposited at the China Center for Type Culture Collection, with the deposit number CCTCC M2025424, and the deposit time is: March 10, 2025.

2. Application of Hanseniaspora occidentalis S19K.

3. Use of Hanseniaspora occidentalis S19K according to claim 2, characterized in that, Specifically applied to improving the sensory quality of fruit wine.

4. Use of Hanseniaspora occidentalis S19K according to claim 3, characterized in that, The fruit wine is specifically wine.

5. Use of Hanseniaspora occidentalis S19K according to claim 4, characterized in that, Improving the sensory quality of wine specifically forms a "high floral - low herbaceous flavor" differential flavor by increasing terpene compounds and ester compounds in wine and reducing C6 compounds, improves color stability, and delays color degradation, thereby improving the sensory quality of wine.

6. The screening method of Hanseniaspora occidentalis S19K according to claim 1, characterized in that, The steps are as follows: S1. Screening the strain: Under sterile conditions, coat the grape epidermis on the YEPD solid medium, invert the solid medium plate for culture to form visible single colonies with the naked eye, and then streak inoculate on the surface of the WL solid medium; invert the inoculated medium plate and culture it in a constant temperature incubator at 28°C. After visible single colonies appear with the naked eye, perform streak purification until a pure culture with uniform morphology and stable color is obtained on the WLN medium. S2. Primary screening of the enzyme - producing ability of the strain: Under a sterile environment, inoculate the strain into a test tube containing YEPD liquid medium, place it in a shaker for oscillating culture to obtain a bacterial solution. Then, add the bacterial solution of each strain into a 96 - well standard enzyme - labeled plate containing aesculin - Fe3+ one by one. Invert the inoculated medium plate and culture it in a constant temperature incubator at 28°C. Since the strain with β - glucosidase activity can hydrolyze aesculin, and the produced esculetin will react with Fe3+ in the medium to form a dark brown complex, the ability of the strain to produce β - glucosidase is preliminarily judged by observing the depth of the colony color. Select the strain with a deep color as the potentially high - enzyme - producing strain obtained by primary screening. S3. Re - screening of the enzyme - producing ability of the strain: For the potentially high - enzyme - producing strain obtained by primary screening, further re - screening is carried out to accurately determine its enzyme - producing ability. First, activate the strain obtained by primary screening. Under sterile conditions, inoculate the primary - screened strain into YEPD liquid medium and culture it in a shaker to obtain the bacterial solution for re - screening. Then, inoculate the obtained bacterial solution into an Erlenmeyer flask containing the enzyme - producing medium, place the Erlenmeyer flask in a shaker for culture. During the culture process, sample every 24h. When sampling, centrifuge the bacterial solution, and take the supernatant as the crude enzyme solution. Use the p - NPG colorimetric method to measure the activity of β - glucosidase in the crude enzyme solution, and compare the enzyme - producing ability of the strains according to the enzyme activity level. Finally, a strain of Hanseniaspora occidentalis is screened out and named strain S19k.

Images