Grape juice hansenula polymorpha YC-C-16 for producing lactic acid and application of grape juice hansenula polymorpha YC-C-16
By using Hansenula polysaccharide YC-C-16 from grape juice to co-ferment wine with Saccharomyces cerevisiae, the problem of the wine having a single aroma when fermented with Saccharomyces cerevisiae was solved, the complexity and acidity of the wine's flavor were enhanced, and the diversity and layering of the wine's flavor were achieved.
Patent Information
- Application Number
- CN202511789661.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-01
- Publication Date
- 2026-02-06
AI Technical Summary
In current winemaking practices, wines fermented solely using brewer's yeast lack complexity in aroma and unique style. Furthermore, the growth capacity of Hansenula polysaccharide strains in grape juice systems is relatively weak, and their lactic acid production capacity is scarce, limiting their potential application in regulating wine acidity and enhancing flavor.
A single grape juice culture of Hanseniaspora uvarum (C-C-16), preservation number CTCC M 20252302, was used to prepare a bacterial broth through fermentation. This broth was then co-fermented with Saccharomyces cerevisiae to produce wine, and the fermentation conditions were optimized to increase lactic acid content and the generation of flavor compounds.
It increases the lactic acid content of wine and adds flavor compounds such as ethyl butyrate, α-terpineol, ethyl salicylate, ethyl myristate, and trans-2-heptenal, enhancing the complexity and layering of the wine's flavor and improving the balance of acidity and aroma complexity.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of microorganisms and wine brewing, and particularly relates to a lactic acid-producing grape juice Hanseniaspora occidentalis YC-C-16 and application thereof. BACKGROUND
[0002] Wine brewing is a complex microbial-driven process, in which yeast as the key fermenting agent converts the sugar in grape juice into alcohol and a variety of flavor substances. In traditional wine brewing, Saccharomyces cerevisiae has long dominated due to its strong alcohol fermentation ability and reliable fermentation, and has become the preferred strain for commercial wine fermentation. However, the wine fermented by pure Saccharomyces cerevisiae is often lacking in aroma complexity and style uniqueness, which is difficult to meet the needs of consumers for diversified wine styles.
[0003] With the in-depth research on wine fermentation microorganisms, the potential of non-Saccharomyces cerevisiae in improving the flavor characteristics of wine has been increasingly concerned. Non-Saccharomyces cerevisiae is diverse, including Hanseniaspora, Kluyveromyces, Metschnikovia, etc., which naturally exist in the surface of grape skin, orchard soil and brewing environment. Among these non-Saccharomyces cerevisiae, grape juice Hanseniaspora occidentalis shows significant application value due to its unique metabolic characteristics. The yeast belongs to the kingdom of eukaryotes, the fungal kingdom, the ascomycota phylum, the yeast subphylum, the yeast class, the yeast order, the yeast family and the Hanseniaspora genus in taxonomy.
[0004] Grape juice Hanseniaspora occidentalis can synthesize a variety of ester substances with sugar, aldehyde, organic acid, salt, etc. as substrates under the catalysis of esterase, which has an advantage over Saccharomyces cerevisiae in wine aroma. Studies have shown that grape juice Hanseniaspora occidentalis can significantly increase the aroma of warm-temperate fruits in mixed fermentation of grape wine, especially in the aspect of rose wine aroma, which can effectively increase the content of 2-phenylethanol, monoterpene alcohol and other rose wine characteristic aroma substances. In addition, the yeast also shows high β-glucosidase activity, which can promote the hydrolysis of bound aroma substances and release free volatile compounds, further enhancing the aroma complexity of grape wine.
[0005] Although grape juice Hanseniaspora occidentalis shows obvious advantages in improving the aroma of grape wine, most natural strains have weak growth ability in juice systems and cannot complete fermentation alone. At the same time, the yeast produces alcohol during fermentation, and alcohol accumulation inhibits its own growth, resulting in low fermentation efficiency under single culture conditions. In addition, there are significant differences in metabolic characteristics among grape juice Hanseniaspora occidentalis strains from different sources, especially the relatively scarce resources of lactic acid-producing strains, which limits the application potential of the yeast in the adjustment of grape wine acidity. SUMMARY
[0006] In view of this, the present invention provides a lactic acid-producing grape juice Hansenula polysaccharide strain YC-C-16 and its application to solve the above problems.
[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a strain of grape juice spore-bearing Hanseniasporauvarum YC-C-16, taxonomically named Hanseniasporauvarum, deposited at the China Center for Type Culture Collection (CTCC) with accession number CTCC M 20252302, deposited on October 22, 2025, at Wuhan University.
[0008] The present invention also provides a method for preparing the bacterial culture of *Hansenula polymorpha* YC-C-16 from grape juice, characterized in that *Hansenula polymorpha* YC-C-16 from grape juice is inoculated into a culture medium for fermentation culture to obtain the bacterial culture.
[0009] Preferably, the fermentation culture temperature is 22~28℃ and the rotation speed is 180~220rpm.
[0010] The present invention also provides the application of the aforementioned grape juice containing Hansenula polysaccharide YC-C-16 in winemaking and / or in enhancing the aroma of wine.
[0011] The present invention also provides a method for increasing the aroma of winemaking by using the grape juice containing Hansenula polymorpha YC-C-16 in winemaking.
[0012] Preferably, brewing yeast is added during the winemaking process, including brewing yeast XR, FTH, STR, or TXL.
[0013] Preferably, the concentration of *Hansenula polymorpha* YC-C-16 added to the grape juice fermentation substrate is 1~9×10⁻⁶. 7 CFU / mL.
[0014] Preferably, the ratio of viable Hansenula polymorpha YC-C-16 to Saccharomyces cerevisiae in the grape juice is 1:0.5~2, and the sum of viable Hansenula polymorpha YC-C-16 and Saccharomyces cerevisiae in the grape juice is 1×10⁻⁶. 6 ~1×10 7 CFU / mL.
[0015] Preferably, the wine is brewed at a temperature of 18-25°C, the pH value is 3.0-3.5, and the dissolved oxygen content is in the range of trace to anaerobic.
[0016] Preferably, the fermentation substrate is grape juice and / or grapes.
[0017] By adopting the above technical solution, the present invention has the following beneficial effects: the grape juice contained *Hansenula polymorpha* YC-C-16, taxonomically named... Hanseniaspora uvarum The *Hansenula polysaccharide* strain YC-C-16 from grape juice is deposited at the China Center for Type Culture Collection (CTCC M 20252302) on October 22, 2025, at Wuhan University. This invention can ferment wine alone or co-ferment with *Saccharomyces cerevisiae*, increasing the lactic acid content and enhancing flavor compounds such as ethyl butyrate, α-terpineol, ethyl salicylate, ethyl myristate, and trans-2-heptenal, thus enriching the flavor profile of the wine. This *Hansenula polysaccharide* strain YC-C-16 from grape juice has wide applications in wine fermentation.
[0018] Biological Preservation Instructions The taxonomic name of the grape juice containing *Hansenula polymorpha* strain YC-C-16 of this invention is... Hanseniaspora uvarum It is deposited at the China Center for Type Culture Collection, accession number CTCC M 20252302, on October 22, 2025, at Wuhan University. Detailed Implementation
[0019] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0020] WL medium (g / L): yeast extract: 4.0g, casein: 5.0g, glucose: 50.0g, potassium dihydrogen phosphate: 0.55g, potassium chloride: 0.425g, calcium chloride: 0.125g, magnesium sulfate: 0.125g, ferric chloride: 0.0025g, manganese sulfate: 0.0025g, bromocresol green: 0.022g, pH 5.5±0.2.
[0021] YPD liquid culture medium (g / L): glucose 20.0g, peptone 20.0g, yeast extract 10.0g.
[0022] YPD medium plates (g / L): glucose 20.0g, peptone 20.0g, yeast extract 10.0g, agar powder 20.0g.
[0023] In the embodiments of this invention, the brewing yeast FTH and pectinase (10,000 U / g) were purchased from LAMOTHE-ABTET, France.
[0024] Example 1 1. Isolation and identification of strain YC-C-16 The *Hansenula polymorpha* strain YC-C-16 used in this invention was isolated from the surface of naturally fermented grapes from the vineyard of Hejinzun Winery in Yinchuan City, Ningxia. The strain was isolated using a serial dilution method, and the specific steps are as follows: In a clean bench, naturally fermented grape samples were collected. 1g of sample was added to 9mL of sterile phosphate-buffered saline (PBS, 0.1M, pH 7.4), and the mixture was shaken at 180rpm and 22℃ for 20min to prepare a sample suspension. Subsequently, a 10-fold serial dilution was performed, with six dilutions in total. The 10-fold dilution was then selected from the selected dilutions. -4 10 -5 and 10 -6 Three serially diluted solutions were evenly spread (200 μL each) onto WL solid medium. After 5 days of incubation at 22°C, the strains were preliminarily classified based on the color and morphology of the colonies on the WL solid medium. Yeast strains with typical colony characteristics were selected and inoculated onto WL solid medium for further purification, and their growth status was observed. The strains isolated and purified from WL solid medium were then subjected to lactic acid bacteria assays to screen for yeast strains with lactic acid-producing characteristics.
[0025] 2. Identification of strain YC-C-16 (1) Morphological identification Strain YC-C-16 was inoculated onto WL agar plates and its colony morphology was observed after 2 days of incubation.
[0026] The colonies are approximately 1-3 mm in diameter, round, with neat edges and a slightly raised center. The colony surface is smooth, moist, and glossy, with a creamy texture, exhibiting typical characteristics of *Hansenula polysaccharide*.
[0027] (2) Molecular biological identification The 26S rDNA gene sequence of strain YC-C-16 was identified, and the results showed that it was similar to *Hansenula polymorpha* found in grape juice. Hanseniaspora uvarum If the similarity reaches 100%, then strain YC-C-16 can be identified as a strain of *Hansenula polymorpha* from grape juice, and it will be named *Hansenula polymorpha* from grape juice. Hanseniaspora uvarum YC-C-16 is deposited at the China Center for Type Culture Collection, accession number CTCC M 20252302.
[0028] 26S rDNA fragment sequence: (SEQ ID NO.1). Example 2. Preparation of a suspension of *Hansenula polymorpha* YC-C-16 and *Saccharomyces cerevisiae* FTH from grape juice Under aseptic conditions, *Hansenula polymorpha* strain YC-C-16 and *Saccharomyces cerevisiae* FTH were inoculated onto plates containing YPD solid medium (streaked). The plates were then inverted and incubated at 28°C for 48 h to activate the strains. The activated target strains were then scraped into sterile water, shaken to mix thoroughly, and suspensions of *Hansenula polymorpha* strain NF26 and *Saccharomyces cerevisiae* FTH were obtained. The viable cell count in each suspension was adjusted to 1 × 10⁻⁶. 9 CFU / mL.
[0029] Example 3. Wine fermentation using *Hansenula polymorpha* YC-C-16 from grape juice. Grape variety: Cabernet Sauvignon Grape juice preparation: After harvesting, the grapes are pressed using a press, and 40 mg / L pectinase is added for enzymatic hydrolysis in a constant temperature water bath at 45℃ for 4 hours. The juice is then filtered through three layers of gauze to remove the residue. The sugar content is adjusted to 25.0°Bx with granulated sugar, and the pH is adjusted to 3.2 with anhydrous citric acid.
[0030] Grape juice fermentation: Grape juice is fermented at an initial concentration of 1×10⁻⁶. 7 CFU / mL of a suspension of *Hansenula polymorpha* strain YC-C-16 (prepared according to the method in Example 2) was inoculated into grape juice and fermented at 18°C. Fermentation was terminated when the weight loss of the grape juice remained unchanged for 3 consecutive days, and the fermentation broth stopped producing gas and the sugar content stopped decreasing. All wine samples were centrifuged at 5000 rpm for 10 minutes, and the supernatant was collected and stored at 4°C.
[0031] Example 4. Fermentation of grape juice with *Hansenula polymorpha* YC-C-16 and *Saccharomyces cerevisiae* FTH. Grape variety: Cabernet Sauvignon Grape juice preparation: After harvesting, the grapes are pressed using a press, and 40 mg / L pectinase is added for enzymatic hydrolysis in a constant temperature water bath at 45°C for 4 hours. The juice is then filtered through three layers of gauze to remove the residue. The sugar content is adjusted to 25.0°Bx with granulated sugar, and the pH is adjusted to 3.5 with anhydrous citric acid.
[0032] Preparation of mixed bacterial suspension: A suspension of Saccharomyces hansenulatus YC-C-16 and Saccharomyces cerevisiae FTH was prepared in grape juice according to the method in Example 2. The suspension of Saccharomyces hansenulatus YC-C-16 and the suspension of Saccharomyces cerevisiae FTH were mixed at a volume ratio of 1:1 to obtain a mixed bacterial suspension.
[0033] Grape juice fermentation: Grape juice is fermented at an initial concentration of 1×10⁻⁶. 7 Inoculate the above mixed bacterial suspension with CFU / mL and ferment at 18℃. Terminate fermentation when the grape juice weight loss remains constant for 3 consecutive days, and the fermentation broth stops producing gas and the sugar content no longer decreases. Centrifuge all wine samples at 5000 rpm for 10 minutes, and store the supernatant at 4℃.
[0034] Comparative Example 1 Unlike Example 3, Saccharomyces cerevisiae FTH was used instead of Saccharomyces cerevisiae YC-C-16 in the grape juice for wine fermentation.
[0035] Experimental Example The wines prepared in Examples 3, 4 and Comparative Example 1 were subjected to physicochemical index testing, volatile flavor component analysis and lactic acid content identification.
[0036] The alcohol content, total acid, total sugar, and pH value of the wine were determined according to the national standard GB / T 15038—2006 "General Analytical Methods for Wine and Fruit Wine", with three replicates for each group.
[0037] The volatile flavor components in the above-mentioned wine samples were detected using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). HS-SPME conditions: 5 mL of wine was added to a 20 mL headspace vial containing 1.5 g of NaCl, followed by the addition of an internal standard (10 μL of 4-methyl-2-pentanol, with a mass concentration of 1008.3 mg / L). The vial was placed in a headspace heater and heated to equilibrate for 30 min. An SPME fiber head aged at 250 °C for 10 min was inserted into the headspace vial and allowed to adsorb for 30 min. Thermal desorption of the analytes from the SPME fiber was achieved via non-branched injection, followed by thermal desorption at 240 °C for 10 min.
[0038] GC conditions: DB-WAX column (60 m × 25 mm × 0.25 μm), injection port temperature 250 ℃, held for 1 min; carrier gas: high-purity helium (He), flow rate 1.0 mL / min. Temperature program conditions: initial column oven temperature 40 ℃, increased to 150 ℃ at a rate of 10 ℃ / min, held for 30 min; increased to 240 ℃ at a rate of 10 ℃ / min.
[0039] MS conditions: electronic ionization (EI) source, electron energy 70 eV; transfer temperature 230℃; mass scan range: 40~450 m / z, three replicates per group. The results of the physicochemical indicators of the wine are shown in Table 1, and the results of the determination of volatile flavor compounds and lactic acid are shown in Table 2.
[0040] Table 1. Results of determination of basic physicochemical properties of wolfberry wine fermented with different commercial brewing yeasts
[0041] Table 1 shows that the alcohol contents of the wines fermented in Examples 3, 4, and Comparative Example 1 were 1.35% vol, 14.3% vol, and 12.8% vol, respectively. The wolfberry wine produced in Example 4 had the highest alcohol content at 14.3% vol, indicating the most thorough fermentation. The total sugar content was 13.01 g / mL, 13.32 g / mL, and 13.16 g / mL, respectively; the total acid content was 4.32 g / mL, 4.38 g / mL, and 3.81 g / mL, respectively; and the pH values were 3.94, 3.92, and 3.97, respectively. Based on the physicochemical results, the wolfberry wine produced in Example 4 was the best.
[0042] Table 2. Results of determination of volatile flavor compounds and lactic acid in fermented wines of each group.
[0043] As shown in Table 1, the wines fermented in Examples 3 and 4 showed the addition of several flavor compounds compared to Comparative Example 1. These compounds significantly enhanced the aroma of the wine, including ethyl butyrate (pineapple aroma), α-terpineol (lavender aroma), ethyl salicylate (wintergreen and mint aroma), ethyl myristate (iris oil aroma), and trans-2-heptenal (grassy aroma). Simultaneously, the contents of ethyl acetate, hexyl hexanoate, isoamyl acetate, ethyl octanoate, ethyl decanoate, phenylethyl acetate, 1-octen-3-ol, decanal, β-ionone, and citronellol were all significantly increased. These aromas enriched the flavor profile of the wine, contributing to its characteristic flavor profile and playing a crucial role in enhancing the balance, complexity, and typicality of the aroma. Compared to Comparative Example 1, the wines fermented in Examples 3 and 4 showed a more than twofold increase in lactic acid content, effectively increasing the acidity of the wine and improving its acidity balance and flavor complexity.
[0044] As can be seen from the above embodiments, the present invention provides a lactic acid-producing grape juice Hansenula polysaccharide YC-C-16 and its application. The grape juice Hansenula polysaccharide YC-C-16 of the present invention has the ability to produce lactic acid bacteria, which can increase the acidity of wine. It can effectively enhance the complexity and layering of wine flavor when fermented alone or co-fermented with wine yeast.
[0045] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A grape stalk containing *Hansenula polymorpha* YC-C-16, characterized in that, Taxonomic naming Hanseniaspora uvarum It is deposited at the China Center for Type Culture Collection, accession number CTCC M 20252302, on October 22, 2025, at Wuhan University.
2. The method for preparing the bacterial suspension of *Hansenula polymorpha* YC-C-16 in grape juice according to claim 1, characterized in that, Grape juice was inoculated with Saccharomyces hansenulatus YC-C-16 into a culture medium for fermentation to obtain the bacterial solution.
3. The preparation method according to claim 2, characterized in that, The fermentation culture temperature is 22~28℃, and the rotation speed is 180~220rpm.
4. The use of the grape juice containing Hansenula polysaccharide YC-C-16 as described in claim 1 in winemaking and / or in enhancing the aroma of winemaking.
5. A method for enhancing the aroma of wine, characterized in that, The grape juice containing Hansenula polysaccharide YC-C-16 as described in claim 1 is used in winemaking.
6. The method according to claim 5, characterized in that, Winemaking also involves the addition of brewing yeast, including brewing yeast XR, FTH, STR, or TXL.
7. The method according to claim 5, characterized in that, The concentration of Saccharomyces hannosa yeast YC-C-16 added to the grape juice fermentation substrate was 1~9×10⁻⁶. 7 CFU / mL.
8. The method according to claim 6, characterized in that, The ratio of viable Hansenula polymorpha YC-C-16 to Saccharomyces cerevisiae in the grape juice is 1:0.5~2, and the sum of the viable Hansenula polymorpha YC-C-16 and Saccharomyces cerevisiae in the grape juice is 1×10⁻⁶. 6 ~1×10 7 CFU / mL.
9. The method according to any one of claims 5 to 8, characterized in that, The wine is brewed at a temperature of 18-25°C, with a pH value of 3.0-3.5 and dissolved oxygen levels ranging from trace to anaerobic.
10. The method according to claim 9, characterized in that, The fermentation substrate is grape juice and / or grapes.