Novel hansenula polymorpha strain with grape juice spores and application of novel hansenula polymorpha strain

Abstract

The invention provides a novel hansenula polymorpha strain, which is classified and named as Hanseniaspora uvarium NF26, is preserved in the China Center for Type Culture Collection (CCTCC), and has the preservation number of CCTCC NO: M 2025860. The invention also provides an activation culture method of the strain. In addition, the invention also provides application of the strain in fruit wine fermentation preparation and a fruit wine fermentation preparation method. The hansenula polymorpha NF26 has the advantages that the hansenula polymorpha NF26 is screened out, the hansenula polymorpha NF26 is adopted to be independently inoculated into the fruit wine, or different types of fruit wine are sequentially inoculated into saccharomyces cerevisiae at an interval of 48 hours, so that the content of titratable acid in the fruit wine can be remarkably reduced, and meanwhile, the content of total phenols and anthocyanin substances is increased; in addition, the strain can also promote biosynthesis of terpenes and esters; in the aspect of sensory quality, the fruit wine fermented by the strain is brighter in color, and the fragrance of flowers and fruits is also remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of microbial fermentation, in particular to a novel Hansenula sporangiophora strain and application thereof. Background Art

[0002] Fresh berries, such as blueberries and grapes, are prone to rotting and spoiling after harvest due to their thin skins and high water content, making them difficult to store and transport. However, processing fresh fruit into wine preserves the fruit's rich nutrients and unique flavor.

[0003] Flavor is a key factor in determining the quality of fruit wine. Yeasts are the core microorganisms in fruit wine fermentation. They not only convert reducing sugars in juice into alcohol but also produce flavor compounds such as esters, higher alcohols, organic acids, and aldehydes and ketones, significantly impacting the taste and aroma of fruit wine.

[0004] Yeasts primarily include Saccharomyces cerevisiae and non-Saccharomyces. Saccharomyces cerevisiae possesses strong fermentation capacity and high tolerance to alcohol and temperature, generally capable of completing alcoholic fermentation on its own. Non-Saccharomyces is a general term for a large group of yeasts distinct from Saccharomyces cerevisiae used in winemaking. Common non-Saccharomyces include genera such as Torulaspora, Hanseniaspora, Pichia, Metschnikowia, and Kluyveromyces. In-depth research on non-Saccharomyces has revealed that they secrete a variety of hydrolytic enzymes that degrade bound aroma compounds into free forms and produce numerous secondary metabolites, enhancing the aroma quality of wine. However, non-Saccharomyces cerevisiae exhibit weak fermentation capacity and are generally unable to complete alcoholic fermentation on their own. Saccharomyces cerevisiae is often used in combination with non-Saccharomyces cerevisiae to complete the entire fermentation process.

[0005] Currently, most fruit wines on the market are produced industrially using commercial brewing yeast, resulting in a homogeneous flavor profile and limiting the product's market competitiveness. Therefore, screening for high-quality non-brewer yeast strains with regional characteristics and applying them to fruit wine brewing is crucial for improving the flavor quality of fruit wine and achieving high-value utilization of fruit resources.

[0006] According to existing reports, Hanseniaspora uvarum is believed to have the ability to enhance the floral and fruity aroma of fruit wine. Furthermore, according to relevant prior art, several patents also disclose different types of Hanseniaspora uvarum strains and their applications in enhancing aroma in winemaking:

[0007] For example, the patent "A Grape Juice Spore-bearing Hanseniaspora" (CN 202410454325.0) provides grape juice spore-bearing Hanseniaspora (Hanseniaspora vineae) FLSY-HV1 (strain deposit number CGMCC NO: 29536) and a brewing method for enhancing the fruity aroma of wolfberry hops;

[0008] The patent "A strain of Hanseniaspora vineae and its use in preparing blueberry wine" (CN202311139077.2) provides Hanseniaspora vineae YIM Y02353 (strain deposit number CGMCC No. 27960) and a brewing method for enhancing the fruity aroma and wine flavor of blueberry wine;

[0009] The patent "A Strain of Hanseniaspora Uvarum HX17 and Its Application" (CN 202210429351.9) provides Hanseniaspora uvarum HX17 (strain accession number CGMCC No. 24625) and a brewing method for enhancing the floral and fruity aroma of wine;

[0010] The patent "Hanseniaspora uvarum QTX-C10 and its screening method and application" (CN 202311034482.8) provides Hanseniaspora uvarum QTX-C10 (strain deposit number CCTCC NO: M20231084) and a brewing method for enhancing the black fruit flavor, tropical fruit flavor, jam flavor, and preserved fruit flavor of wine;

[0011] The patent "A strain of Hanseniaspora uvarum that can increase the aroma of wine brewing" (CN202110054639.8) provides Hanseniaspora uvarum JNB-HB-66 (strain collection number CGMCC No. 21227) and a brewing method for enhancing the temperate fruit aroma of hawthorn wine.

[0012] However, existing research has mostly focused on screening high-aroma strains based on the activity of a single flavor enzyme, and these strains are often applied to a single type of fruit wine. Currently, there are no reports in this field of screening for Hansenula sporogenes strains that produce high terpenes and esters by comprehensively evaluating their glycosidase and alcohol acyltransferase activities. These strains can then be applied to the production of different types of fruit wines, significantly improving the flavor quality of the products. Summary of the Invention

[0013] The technical problem to be solved by this invention is to provide a novel strain of Hansenula vitis vinifera and its application. This strain, when inoculated alone or mixed with Saccharomyces cerevisiae into various types of fruit wine (fermented blueberry wine, grape wine), significantly reduces the titratable acid content while increasing the levels of total phenols and anthocyanins. Furthermore, this strain promotes the biosynthesis of terpenes and esters (particularly acetates). In terms of sensory quality, wines fermented with this strain exhibit a brighter color (with a significantly enhanced red hue) and a significantly enhanced floral and fruity aroma.

[0014] The present invention adopts the following technical solutions to solve the above technical problems:

[0015] A new Hanseniaspora uvarum strain NF26, classified and named Hanseniaspora uvarum NF26, was deposited in the China Center for Type Culture Collection on April 23, 2025 and showed survival. The deposit number is CCTCC NO: M 2025860. The deposit address is: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0016] A method for activating and culturing the above-mentioned Hansenula sporangiophora strain NF26 comprises the following steps:

[0017] (1) Prepare YPD solid medium;

[0018] (2) Under sterile conditions, the target strain was inoculated onto a plate containing YPD solid medium and then activated;

[0019] (3) In a sterile operating environment, scrape the activated target strain into sterile water, shake and mix evenly to obtain the corresponding bacterial suspension.

[0020] As one of the preferred embodiments of the present invention, in step (2), the activation culture conditions are: temperature 25° C., time 5 days.

[0021] The invention relates to an application of the Hansenula sporogenes strain NF26 in grape juice in the fermentation preparation of fruit wine.

[0022] A method for preparing fruit wine by fermentation, using the above-mentioned Hansenula sporangiophora strain NF26 as a target fermentation strain, comprises the following steps:

[0023] (1) Sterilization

[0024] After crushing the fruit raw material, hydrolyzing the pectin, squeezing and filtering, adding sucrose, then sterilizing at high temperature, and adding diammonium hydrogen phosphate to obtain a sterilized juice raw material;

[0025] (2) Fermentation

[0026] Fermentation is carried out by single-bacteria fermentation or mixed-bacteria fermentation;

[0027] Single-bacteria fermentation method: a suspension of the target fermentation strain (Hansenula sporogenes NF26) is inoculated into the sterilized juice raw material, and the mixture is placed in an incubator for fermentation until the reducing sugar content does not decrease for three consecutive days, at which point the fermentation is considered complete, and the fruit wine is obtained;

[0028] The mixed fermentation method comprises the following steps: inoculating a bacterial suspension of the target fermentation strain (a bacterial suspension of Hansenula sporogenes strain NF26) and a commercial cerevisiae yeast suspension into the obtained sterilized juice raw material, placing the mixture in an incubator for mixed fermentation until the reducing sugar content does not decrease for three consecutive days, which is considered to be the end of the fermentation, thereby obtaining the fruit wine.

[0029] As one of the preferred embodiments of the present invention, in step (1), the fruit raw material is blueberry or wine grape fruit.

[0030] As one of the preferred embodiments of the present invention, in step (1), after the fruit raw material is crushed, pectin is hydrolyzed, squeezed and filtered, sucrose is added to adjust the total soluble solids of the juice to 20°Brix, and then sterilized at 90°C for 1 min, and 200 mg / L of diammonium hydrogen phosphate is added to obtain a sterilized juice raw material.

[0031] As one of the preferred embodiments of the present invention, in the single-bacteria fermentation process of step (2), the concentration of the inoculated target bacterial strain suspension for fermentation is 1.0×10 7 CFU / mL, culture and fermentation conditions: temperature 25℃.

[0032] As one of the preferred embodiments of the present invention, in the mixed fermentation process of step (2), the commercial Saccharomyces cerevisiae strain is Saccharomyces cerevisiae Lalvin ICV D254 (Raman Company, Bordeaux, France).

[0033] As one of the preferred embodiments of the present invention, during the mixed fermentation process of step (2), the target fermentation strain and the commercial brewing yeast are inoculated in the order of 48 hours apart; and the bacterial suspension concentrations of the target fermentation strain and the commercial brewing yeast are both 1.0×10 7 CFU / mL, the inoculation ratio was 1:1; the culture and fermentation conditions were: temperature 25℃.

[0034] The advantages of the present invention over the prior art are:

[0035] (1) The present invention screened and isolated a strain of Hanseniaspora uvarum NF26 from grape juice, and used this strain as a strain for fruit wine fermentation, which has the following advantages: ① The titratable acid content of the fruit wine product fermented by this strain is significantly lower than that of products fermented by other strains, and the total phenol and anthocyanin contents are significantly higher than those of products fermented by other strains, and the wine color is brighter (the red hue is enhanced); ② The inoculation of this strain into the fermented fruit wine can increase the content of terpenes and acetates, and enhance the intensity of sweet aroma and floral and fruity aroma;

[0036] (2) The Hansenula sporogenes NF26 of the present invention can be used to prepare high-anthocyanin and high-floral and fruity aroma fruit wine products, thereby increasing the economic value of the fruit wine;

[0037] (3) The production process of the fruit wine product of the present invention is simple, the fermentation is fast and the cost is low, and it is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a colony morphology diagram of the Hansenula sporogenes NF26 strain of the present invention on a culture medium;

[0039] Figure 2 This is a graph showing the color development results of the Hansenula sporangiophora strain NF26 of the present invention and reference strains 1 and 2 in aesculin culture medium;

[0040] Figure 3 Graph showing the results of alcohol acyltransferase activity assays of the Hansenula sporogenes strain NF26 of the present invention and reference strains 1 and 2;

[0041] Figure 4 Figure 1 is a graph showing the tolerance of the Hansenula sporangiophora strain NF26 of the present invention and reference strains 1 and 2 (Figure A shows the growth results of each strain at different SO2 concentrations, Figure B shows the growth results of each strain at different fermentation temperatures, Figure C shows the growth results of each strain at different mass concentrations of glucose, and Figure D shows the growth results of each strain at different volume fractions of anhydrous ethanol);

[0042] Figure 5 The present invention is a graph showing the glycosidase brewing adaptability of the Hansenula sporangiophora strain NF26 and reference strains 1 and 2 (Figure A shows the glycosidase activity results of each strain at different SO2 concentrations, Figure B shows the glycosidase activity results of each strain at different fermentation temperatures, Figure C shows the glycosidase activity results of each strain at different mass concentrations of glucose, Figure D shows the glycosidase activity results of each strain at different volume fractions of anhydrous ethanol, and Figure E shows the glycosidase activity results of each strain at different pH values);

[0043] Figure 6is a 26S rDNA phylogenetic tree of the Hansenula sporangiophora vitis NF26 strain of the present invention;

[0044] Figure 7 This is a simulated color palette showing the colors of blueberry wine and grape wine of the present invention;

[0045] Figure 8 Graphs showing the total terpenes, total esters, and total acetate contents in blueberry wine and grape wine of the present invention (graphs A to C show the total terpenes, total esters, and total acetate contents of blueberry wine, respectively, and graphs D to F show the total terpenes, total esters, and total acetate contents of grape wine, respectively);

[0046] Figure 9 The sensory evaluation diagrams of blueberry wine and wine of the present invention are shown (Figure A shows the sensory evaluation results of blueberry wine, and Figure B shows the sensory evaluation results of wine). DETAILED DESCRIPTION

[0047] The following embodiments of the present invention are described in detail. These embodiments are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operating procedures are provided. However, the scope of protection of the present invention is not limited to the following embodiments. Meanwhile, unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0048] The culture medium formula and preparation method involved in the following examples are as follows:

[0049] WL solid medium: 5 g / L yeast extract powder, 5 g / L acid hydrolyzed casein, 50 g / L glucose, 0.55 g / L potassium dihydrogen phosphate, 0.425 g / L potassium chloride, 0.125 g / L calcium chloride, 0.125 g / L magnesium sulfate, 0.0025 g / L ferric chloride, 0.0025 g / L manganese sulfate, 0.022 g / L bromocresol green, 17 g / L agar; weigh each ingredient, dissolve in 1000 mL of distilled water, wrap, sterilize at 121°C for 15 min, cool to 50°C, and pour into a plate.

[0050] YPD solid medium: 10.0 g / L peptone, 5.0 g / L yeast extract powder, 20.0 g / L glucose, 14.0 g / L agar; weigh each raw material, dissolve in 1000 mL distilled water, wrap, sterilize at 121°C for 15 min, cool to 50°C, and pour into a plate.

[0051] Esculin biochemical screening medium: 0.5 g / L tryptone, 0.3 g / L beef extract, 0.1 g / L esculin, 0.05 g / L ammonium ferric citrate; weigh each raw material, dissolve in 1000 mL distilled water, wrap, sterilize at 121°C for 15 min, cool to 50°C, and pour into a plate.

[0052] Example 1: Isolation and identification of Hansenula sporogenes strain NF26 from grape juice:

[0053] 1. Screening and isolation of strains

[0054] In the clean bench, take appropriate amount of Cabernet Sauvignon grape fermentation mash at different fermentation stages and dilute it in 6 gradients. -4 , 10 -5 and 10 -6 From each of the three gradient dilutions, 200 μL was evenly spread onto WL solid medium. After 5 days of growth in a 25°C incubator, the strains were preliminarily classified based on the color and morphology of the colonies on the WL solid medium. Yeasts with typical colony characteristics were selected and inoculated onto WL solid medium for further purification (two to three purifications depending on the isolation efficiency), and the yeast growth was observed. The strains isolated and purified on WL solid medium were initially screened for glycosidases to identify strains with high glycosidase activity. These strains were then rescreened for alcohol acyltransferase activity, resulting in the identification of a strain with high production of both glycosidases and alcohol acyltransferases.

[0055] A single colony of this strain was inoculated into YPD solid culture medium for enrichment, cultured at a constant temperature of 25°C for 5 days, and then stored for future use. It was subjected to molecular biological identification and named as Hansenula sporangiophora vitis vinifera strain NF26.

[0056] 2. Identification of strains

[0057] (1) Morphological identification

[0058] The strain "Hansenula vitis spores NF26" was inoculated on WL plate culture medium and its colony morphology was observed.

[0059] like Figure 1 As shown in the figure, the colony morphology is characterized by: the colonies are between 0 and 2 mm, round green colonies, complete edges, and smooth and moist surfaces.

[0060] (2) Initial screening of glycosidases from yeast strains

[0061] The glycosidase activity of yeast was preliminarily screened using the aesculin colorimetric method.

[0062] After the preserved strains were activated, 1×10 7CFU / mL bacterial suspension, add 200 μL of esculin to 96-well culture plates, inoculate 20 μL of the activated bacterial suspension respectively, and culture at 28°C for 48 hours. The glycosidase activity of the test strain was determined according to the color development grade (dark black indicates high enzyme activity, dark gray indicates medium enzyme activity, and gray indicates low enzyme activity), and the commercial Saccharomyces cerevisiae D254 was used as a reference 1, and the commercial non-Saccharomyces cerevisiae yeast Tourlaspora delbrueckii (Td) was used as a reference. Alpha is reference 2.

[0063] The color development results of the Hansenula spore-bearing yeast strain NF26 of the present invention and the reference strains 1 and 2 in the aesculin culture medium are as follows: Figure 2 shown.

[0064] (3) Rescreening of yeast strain alcohol acyltransferase

[0065] The yeast alcohol acyltransferase activity assay reaction system consists of: 2.5 mL of 5 mM MgCl₂ solution (5 mM MgCl₂ in 0.5 M Tris-HCl, pH 8.0), 150 μL of acetyl-CoA solution (5 mM acetyl-CoA in 0.5 M Tris-HCl, pH 8.0), 50 μL of butanol solution (200 mM butanol in 0.5 M Tris-HCl, pH 8.0), and 150 μL of enzyme extract. After mixing, the mixture was placed in a 35°C water bath for 15 minutes. 100 μL of 10 mM DTNB was added, and the reaction was allowed to stand at room temperature for 10 minutes. The absorbance of the reaction solution was then measured at 412 nm. A reaction solution without enzyme extract was used as a blank. Each sample was assayed in triplicate. Commercial Saccharomyces cerevisiae D254 was used as a reference, and the commercial non-Saccharomyces cerevisiae yeast Tourlaspora delbrueckii (Td) was used as a reference. Alpha is reference 2.

[0066] Every 1×10 7 One unit of enzyme activity (U) is defined as the production of 1 nmol of coenzyme A per minute by one bacterium or cell.

[0067] The alcohol acyltransferase activity of the Hansenula sporogenes strain NF26 of the present invention and the reference strains 1 and 2 is as follows: Figure 3 shown.

[0068] (4) Yeast strain tolerance study

[0069] The tolerance of isolated yeast strains was studied (commercial Saccharomyces cerevisiae D254 was used as reference 1, and commercial non-Saccharomyces cerevisiae Tourlaspora delbrueckii (Td) Alpha is reference 2), including SO2 addition amount, fermentation temperature, glucose content and ethanol concentration tolerance. The specific steps are as follows:

[0070] Blueberry juice was pasteurized (90°C, 1 min) and clarified. The supernatant was used, and sucrose was added to adjust the total soluble solids (TSS) of the blueberry juice to 20°Brix. A multifactorial experimental system was constructed by adjusting the SO2 concentration in the blueberry juice (0, 25, 50, 75, and 100 mg / L) by adding sodium metabisulfite. Different fermentation temperatures (10°C, 15°C, 20°C, and 25°C) were set, and different concentrations of glucose (100, 200, 300, and 400 g / L) and different volume fractions of anhydrous ethanol (6%, 8%, 10%, 12%, and 14%) were added. After activation, the yeast strain was added to the different blueberry juices for fermentation, with an inoculum size of 1×10 7 CFU / mL, the fermentation scale was 50mL, and the fermentation temperature was 25℃. 600 The values are used to reflect the growth of each yeast strain. 600 The strain was considered to be tolerant under the corresponding conditions if the value change was greater than 1. Three biological replicates were performed for each yeast strain.

[0071] The tolerance results of the Hansenula sporangiophora strain NF26 of the present invention and the reference strains 1 and 2 are as follows: Figure 4 Among them, Hansenula sporogenes NF26 showed the best tolerance at an initial sugar concentration of 200 g / L, a SO2 concentration of 0-50 mg / L, and a fermentation temperature of 25°C.

[0072] (5) Study on the adaptability of glycosidase of Hansenula spore-forming yeast NF26 in different fermentation environments

[0073] The brewing characteristics of the isolated yeast strains were evaluated under different SO2 addition amounts, fermentation temperatures, glucose concentrations, ethanol concentrations and pH conditions (commercial Saccharomyces cerevisiae D254 was used as reference 1, and commercial non-Saccharomyces cerevisiae Tourlaspora delbrueckii (Td) Alpha is reference 2). The specific method is as follows:

[0074] First, the selected strains stored at 4°C were activated and inoculated into blueberry juice for fermentation for 3 days. Glycosidase activity was then determined under various conditions: ① Using a citric acid-phosphate buffer system, the effects of varying SO₂ addition levels (0, 10, 20, 30, 40, 50, and 60 mg / L, calculated as Na₂S₂O₅), fermentation temperature (10, 20, 30, 40, 50, 60, and 70°C), glucose content (0, 50, 100, 150, 200, 250, and 300 g / L), and ethanol volume fraction (0%, 5%, 10%, 12%, 14%, and 15%) were investigated. ② A pH gradient (2.5, 3.0, 3.5, 4.0, 4.5, 5.0, and 6.0) was established to determine the effects of glycosidase activity. All assays were performed using the same method.

[0075] The results of the glycosidase adaptability of the Hansenula spore-bearing yeast strain NF26 of the present invention and the reference strains 1 and 2 under different fermentation environments are as follows: Figure 5 Among them, the β-D-glucosidase of Hansenula sporogenes strain NF26 in blueberry fermentation broth showed good stability under blueberry wine brewing conditions: 60% enzyme activity was retained at 50 mg / L SO2; 85% activity was retained at a fermentation temperature of 25°C; 79% enzyme activity was retained at a sugar concentration of 200 g / L; more than 75% activity was retained at an ethanol concentration of 5% to 15%; and more than 70% enzyme activity was retained in the pH range of 3.5 to 6.

[0076] The above results show that under common fruit wine fermentation conditions (initial sugar content 200 g / L, pH 3-4, 0-50 mg / L SO2, fermentation temperature 25°C, ethanol volume fraction 5%-15%), the β-D-glucosidase of the Hansenula sporogenes NF26 strain of the present invention exhibits excellent adaptability.

[0077] (6) 26S rDNA sequence identification

[0078] The 26S rDNA of the Hansenula sporogenes strain NF26 of the present invention was sequenced, and the sequencing result (shown in SEQ ID NO. 1) was compared with the sequences of known yeast species in the NCBI (National Center for Biotechnology Information) database to construct a phylogenetic tree as shown in FIG. Figure 6 As shown; combined with the morphology and 26S rRNA gene sequence analysis of the strain Hanseniaspora uvarum NF26, it was determined to be Hanseniaspora uvarum.

[0079] (7) Strain preservation and molecular biological identification in the collection center

[0080] The strain Hanseniaspora vitis NF26 screened by the present invention was sent to the China Center for Type Culture Collection for preservation, and was identified and named Hanseniaspora uvarum, with a preservation number of CCTCC NO: M 2025860.

[0081] Example 2: Activation culture and bacterial suspension preparation of Hansenula sporeifera strain NF26

[0082] Under sterile conditions, the Hansenula sporogenes strain NF26 was inoculated onto a plate containing YPD solid medium (streaked); then, the plate was inverted and activated in a constant temperature incubator at 25°C for 5 days; finally, the activated target strain was scraped into sterile water and shaken to mix evenly to obtain a bacterial suspension of the Hansenula sporogenes strain NF26.

[0083] Example 3: Preparation of blueberry wine by single-bacteria fermentation:

[0084] (1) Sterilization

[0085] After blueberries are crushed, pectin is hydrolyzed, squeezed and filtered, sucrose is added to adjust the total soluble solids of the blueberry juice to 20°Brix, and then the blueberry juice is sterilized at 90°C for 1 minute, and 200 mg / L diammonium hydrogen phosphate is added to obtain a sterilized blueberry juice raw material.

[0086] (2) Fermentation

[0087] Under aseptic conditions, a single bacterial suspension of Hansenula viticola NF26 (1.0×10 7 CFU / mL), and placed in an incubator for fermentation at 25°C. Fermentation was considered complete when the reducing sugar content no longer decreased, and blueberry wine was obtained.

[0088] Example 4: Preparation of wine by single-bacteria fermentation:

[0089] (1) Sterilization

[0090] After the grapes were crushed, pectin was hydrolyzed, pressed and filtered, sucrose was added to adjust the total soluble solids of the grape juice to 20°Brix, and then sterilized at 90°C for 1 minute to obtain a sterilized grape juice raw material.

[0091] (2) Fermentation

[0092] Under aseptic conditions, a single suspension of Hansenula viticola NF26 (1.0×10 7 CFU / mL) and placed in an incubator for fermentation at 25°C. Fermentation was considered complete when the reducing sugar content no longer decreased, and the wine was obtained.

[0093] Example 5: Preparation of blueberry wine by mixed fermentation:

[0094] (1) Sterilization

[0095] After blueberries are crushed, pectin is hydrolyzed, squeezed and filtered, sucrose is added to adjust the total soluble solids of the blueberry juice to 20°Brix, and then the blueberry juice is sterilized at 90°C for 1 minute, and 200 mg / L diammonium hydrogen phosphate is added to obtain a sterilized blueberry juice raw material.

[0096] (2) Fermentation

[0097] Under aseptic conditions, the sterilized blueberry juice was inoculated with a suspension of Hansenula spore-forming yeast strain NF26 (1.0×10 7 CFU / mL) and commercial Saccharomyces cerevisiae D254 bacterial suspension (1.0×10 7 CFU / mL) were inoculated in a 1:1 ratio, with a 48-h interval (NF26 first, then D254), and placed in an incubator for mixed fermentation at 25°C. Fermentation was considered complete when the reducing sugar content of the fermentation broth no longer decreased, and blueberry wine was obtained.

[0098] Example 6: Preparation of wine by mixed fermentation:

[0099] (1) Sterilization

[0100] After the grapes were crushed, pectin was hydrolyzed, pressed and filtered, sucrose was added to adjust the total soluble solids of the grape juice to 20°Brix, and then sterilized at 90°C for 1 minute to obtain a sterilized grape juice raw material.

[0101] (2) Fermentation

[0102] Under aseptic conditions, the sterilized grape juice raw material was inoculated with a suspension of Hansenula spore-forming yeast strain NF26 (1.0×10 7 CFU / mL) and commercial Saccharomyces cerevisiae D254 bacterial suspension (1.0×10 7 CFU / mL) were inoculated in a 1:1 ratio, with 48-h intervals between the order (NF26 first, then D254), and placed in an incubator for mixed fermentation at 25°C. Fermentation was considered complete and the wine was obtained when the reducing sugar content of the fermentation broth no longer decreased.

[0103] Comparative Example 1

[0104] The preparation of a blueberry wine by single-bacterial fermentation in this comparative example is basically the same as that in Example 3, with the main difference being that commercial Saccharomyces cescerevisiae D254 is used instead of Hansenula sporangiophora vitis NF26 as the target fermentation strain.

[0105] Comparative Example 2

[0106] The preparation of a blueberry wine by single-bacterial fermentation in this comparative example is basically the same as that in Example 3, except that the commercial non-Saccharomyces cerevisiae yeast Tourlaspora delbrueckii (Td) is used. Alpha replaced Hansenula sporangiophora NF26 as the target strain for fermentation.

[0107] Comparative Example 3

[0108] The single-bacterial fermentation preparation of a wine in this comparative example is basically the same as that in Example 4, with the main difference being that commercial Saccharomyces cescerevisiae D254 is used instead of Hansenula sporangiophora vitis NF26 as the target fermentation strain.

[0109] Comparative Example 4

[0110] The single-bacterial fermentation preparation of a wine in this comparative example is basically the same as that in Example 4, except that the commercial non-Saccharomyces cerevisiae yeast Tourlaspora delbrueckii (Td) is used. Alpha replaced Hansenula sporangiophora NF26 as the target strain for fermentation.

[0111] Comparative Example 5

[0112] The mixed fermentation preparation of a blueberry wine in this comparative example is basically the same as that in Example 5, except that the commercial non-Saccharomyces cerevisiae yeast Tourlaspora delbrueckii (Td) is used. Alpha replaces Hansenula vitis NF26.

[0113] Comparative Example 6

[0114] The mixed fermentation preparation of a wine in this comparative example is basically the same as that in Example 6, except that the commercial non-Saccharomyces cerevisiae yeast Tourlaspora delbrueckii (Td) is used. Alpha replaces Hansenula vitis NF26.

[0115] Test Example 1: Basic physical and chemical testing of fruit wine:

[0116] Basic physical and chemical properties of the blueberry wines fermented in Examples 3 and 5 of the present invention and Comparative Examples 1, 2, and 5, as well as the wines fermented in Examples 4 and 6 of the present invention and Comparative Examples 3, 4, and 6, were tested. These properties included reducing sugar content, alcohol content, pH, titratable acid content, yeast-assimilable nitrogen, total anthocyanin content, total phenolic content, and color characteristics.

[0117] 1. Detection method

[0118] (1) Use a pH meter to measure the pH value of blueberry wine. The reducing sugar content, titratable acid content, and alcohol content of fruit wine were determined according to GB / T15038-2006 "General Methods for Analysis of Grape and Fruit Wine".

[0119] (2) The total anthocyanin content was determined by the pH differential method: 2 mL of wine sample was taken and centrifuged at 4000 r / min for 10 min. The supernatant was diluted with pH 1.0 and pH 4.5 buffer solutions by the same multiple, mixed by vortex, and allowed to stand in the dark for 15 min. The absorbance of each sample was measured at 520 nm and 700 nm, respectively. The absorbance of the solution was recorded, and the anthocyanin content in the finished wine was calculated according to the following formula:

[0120] A=(A 520 -A 700 )pH 1.0-(A 520 -A 700 )pH 4.5;

[0121] TAC (mg / g)=A×MW×DF×1000 / (ε×L);

[0122] Where:

[0123] A 520 is the absorbance of the sample at 520 nm, A 700 is the absorbance of the sample at 700 nm;

[0124] pH 1.0 refers to the sample to be tested after dilution with pH 1.0 buffer, and pH 4.5 refers to the sample to be tested after dilution with pH 4.5 buffer;

[0125] MW represents the relative molecular mass of anthocyanins in the sample, calculated based on the relative molecular mass of cyanidin-3-glucoside, MW = 449.2; DF represents the dilution factor;

[0126] ε represents the molar absorptivity of anthocyanins in the sample, calculated based on the molar absorptivity of cyanidin-3-glucoside, ε = 26900;

[0127] L represents the optical path length.

[0128] (3) Determination of total phenols: Pipette 0.25 mL of a finished wine sample diluted a certain number of times into a test tube, add 1.25 mL of a 10-fold diluted fulvestrant reagent, vortex mix and let stand for 5 minutes, then add 1.25 mL of 7.5% (v / v) sodium carbonate solution and 2.5 mL of deionized water, vortex mix and react in the dark for 60 minutes. Immediately measure the absorbance of the solution at 765 nm. Express it as equivalent gallic acid (mg / L). Plot a standard curve with the gallic acid concentration as the horizontal axis and the absorbance as the vertical axis. Calculate the total phenol content based on the standard curve (y = 2.9031x + 0.0673, R2 = 0.99932).

[0129] (4) The colorimetric characteristics of the wine were characterized using CIELab parameters under a 10° standard observer and D65 standard illuminant. The absorbance values of the blueberry wine samples were recorded at wavelengths of 450, 520, 570, and 630 nm with an optical path length of 1 cm. The CIELab parameters, including lightness (L), red-green value (a*), and yellow-blue value (b*), were calculated according to the method described by Gordillo et al.

[0130] 2. Test Results

[0131] The test results are shown in Table 1.

[0132] The results in Table 1 (Basic Physical and Chemical Properties of Blueberry and Grape Wine) demonstrate that both the fermentation of wine using the present invention's Hansenula sporogenes NF26 alone (Example 4) and the sequential fermentation using Hansenula sporogenes NF26 and commercial Saccharomyces cerevisiae D254 (Example 6) were able to complete fermentation. The mixed blueberry wine system (Example 5) not only overcomes the inability of NF26 alone (Example 3) to complete fermentation independently, but also maintains comparable fermentation efficiency (similar to the initial reducing sugar consumption rate) to high-quality commercial strains (other comparative examples). In terms of wine quality, Example 3 significantly reduced the pH (0.04) compared with Comparative Example 1, increased the titratable acid by 0.22 g / L, and significantly increased the total phenol (65 mg / L) and anthocyanin (20.2 mg / L) contents; Example 4 significantly reduced the pH (0.06) compared with Comparative Example 3, increased the titratable acid by 0.25 g / L, and significantly increased the total phenol (51.4 mg / L) and anthocyanin (19.2 mg / L) contents. Figure 7 It is shown that, compared with Comparative Examples 1, 2, and 5, respectively, and compared with Comparative Examples 3, 4, and 6, respectively, Examples 3 and 5 increase the a* value, making the wine present a brighter red color.

[0133] In summary, mixed bacteria fermentation treatment can reduce the titratable acidity of fruit wine, and both single bacteria fermentation and mixed bacteria fermentation can significantly increase the total phenol and total anthocyanin contents in the produced blueberry wine and grape wine, making the wine appear more vivid red.

[0134] Table 1 Basic physical and chemical indicators of blueberry wine and wine

[0135]

[0136]

[0137] Test Example 2: Detection of aroma substances in fermented fruit wine:

[0138] The blueberry wines prepared by fermentation in Examples 3 and 5 of the present invention and Comparative Examples 1, 2, and 5, as well as the wines prepared by fermentation in Examples 4 and 6 of the present invention and Comparative Examples 3, 4, and 6, were respectively tested for volatile compounds in the wines.

[0139] 1. Detection method

[0140] Headspace solid phase microextraction (HS-SPME) was used to extract volatile compounds from fruit wine.

[0141] (1) HS-SPME extraction process

[0142] 9 mL of citrate buffer (0.2 M, pH 5.0) and 1 mL of the alcohol sample to be tested were added to a 20 mL headspace vial. 3 g of sodium chloride and 20 μL of a 20.71 μg / mL 3-nonanone solution (internal standard) were added. A rotor was placed in the vial, sealed with a cap, and the headspace vial was placed on a magnetic stirrer and equilibrated at 50°C for 15 minutes. A conditioned 50 / 30 μm DVB / CAR / PDMS extraction tip was inserted into the headspace vial, which had been equilibrated at 50°C for 15 minutes, maintaining a distance of 2 cm from the liquid surface. The extraction was performed at a fixed speed of approximately 500 rpm, a temperature of 50°C, and a time of 45 minutes. After extraction, the tip was inserted into the GC inlet and desorbed for 5 minutes before analysis by gas chromatography-mass spectrometry (GC-MS).

[0143] (2) GC-MS detection conditions

[0144] Gas phase conditions: HP-5MS capillary column (5% phenylmethylsiloxane, 30 m × 0.25 mm × 0.25 μm); high-purity helium carrier gas; carrier gas flow rate: 1.2 mL / min; splitless injection; inlet temperature: 250°C; mass spectrometer interface temperature: 250°C. Temperature program: initial temperature: 40°C, hold for 5 min, ramp to 180°C at 3°C / min, hold for 1 min, then ramp to 300°C at 30°C / min, hold for 2 min. Solvent delay: 0 min. Mass spectrometer conditions: electron impact ionization (EI); electron energy: 70 eV; ion source temperature: 250°C; quadrupole temperature: 150°C; mass scan range: 25–300 m / z; full scan.

[0145] 2. Test Results

[0146] The results are as follows Figure 8 shown.

[0147] Depend on Figure 8 (Total terpenes, total esters, and total acetate content in blueberry wine and wine) The results show that the use of the present invention's Hansenula sporangiophora NF26 single-strain fermentation of blueberry wine (Example 3) and wine (Example 4) significantly increased the total terpene, total ester, and total acetate contents compared to Comparative Example 1 and Comparative Example 3, respectively. The sequential fermentation of blueberry wine (Example 5) and wine (Example 6) by inoculating Hansenula sporangiophora NF26 with commercial cerevisiae D254 significantly increased the total terpene content compared to Comparative Example 1 and Comparative Example 3, respectively, and also significantly increased the total ester and total acetate contents compared to the other comparative examples.

[0148] In summary, the contents of terpenes and ester compounds, especially acetate, were significantly increased in the fruit wines fermented by single and mixed strains of Hansenula sporangiophora NF26 in grape juice of the present invention.

[0149] In addition, to identify the characteristic volatile compounds that contribute to the differences between different fermentation treatment groups of blueberry wine and wine, compounds with variable weight values (VIP values) greater than 1.0 and relative odor activity values (rOAVs) greater than 0.1 in the PLS-DA model were screened, and the results are shown in Table 2.

[0150] Table 2 Contents of key aroma compounds in blueberry wine and wine (μg / L)

[0151]

[0152]

[0153]

[0154] Table 2 (Contents of Key Aroma Compounds in Blueberry and Grape Wine) shows that, compared with Comparative Examples 1 and 2, the fermentation of blueberry wine with Hansenula sporangiophora NF26 alone (Example 3) increased the contents of α-terpinene, isoamyl acetate, isobutyl acetate, 2-methylbutyl acetate, phenylethyl acetate, isooctyl alcohol, phenylethyl alcohol, phenylacetaldehyde, and p-cymene; and compared with Comparative Example 2, the content of ethyl decanoate was increased. The fermentation of wine with Hansenula sporangiophora NF26 alone (Example 4) increased the contents of 4-terpene alcohols, isoamyl acetate, 2-methylbutyl acetate, phenylethyl acetate, phenylethyl alcohol, phenylacetaldehyde, and β-damascone; compared with Comparative Examples 3 and 4, the content of isooctyl alcohol was increased; and compared with Comparative Example 4, the content of methyl hexanoate and 1-octanol was increased. In conclusion, the fermentation of grape juice with Hansenula sporogenes NF26 alone can increase the contents of isoamyl acetate, 2-methylbutyl acetate, phenylethyl acetate, phenylethanol and phenylacetaldehyde.

[0155] Compared with other comparative examples, the mixed fermentation of blueberry wine with Hansenula sporangiophora NF26 in grape juice (Example 5) significantly increased the contents of isoamyl acetate, isobutyl acetate, 2-methylbutyl acetate, isooctyl alcohol, and phenylethyl alcohol; compared with comparative example 2, the content of isovaleric acid was increased; compared with comparative example 5, the content of phenylacetaldehyde was increased; and compared with comparative examples 2 and 5, the content of ethyl decanoate was increased. Compared with other comparative examples, the mixed fermentation of wine with Hansenula sporangiophora NF26 in grape juice (Example 6) significantly increased the contents of isoamyl acetate, 2-methylbutyl acetate, and phenylethyl alcohol; compared with comparative example 3, the contents of phenylacetaldehyde and isooctyl alcohol were increased; compared with comparative examples 3 and 6, the contents of 4-terpenol and phenylethyl acetate were increased; and compared with comparative examples 4 and 6, the contents of methyl hexanoate and 1-octanol were increased.

[0156] In conclusion, sequential inoculation of grape juice with Hansenula sporogenes NF26 and Saccharomyces cerevisiae can increase the contents of isoamyl acetate, 2-methylbutyl acetate, and phenylethanol in wine fermentation.

[0157] Test Example 3: Sensory evaluation of fermented fruit wine:

[0158] The blueberry wines prepared by fermentation according to Examples 3 and 5 of the present invention and Comparative Examples 1, 2, and 5, as well as the wines prepared by fermentation according to Examples 4 and 6 of the present invention and Comparative Examples 3, 4, and 6, were respectively subjected to sensory evaluation.

[0159] 1. Detection method

[0160] The evaluation panel consisted of ten evaluators (age range: 22-25 years old). Before the sensory evaluation of the fruit wines, each panel member received approximately 30 hours of training in aroma olfactory recognition and aroma intensity identification. Blueberry wine and wine samples were randomly coded with three-digit numbers, and 15mL of wine samples were dispensed into 125mL professional fruit wine tasting cups and randomly presented to the evaluators. First, an aroma attribute description experiment was conducted. The descriptive words of all evaluators were summarized. Through group discussion, descriptive words with the same meaning were merged, and the top eight descriptors with the highest frequency were selected. Subsequently, the evaluators ranked the blueberry wine samples from each treatment group according to the intensity of each aroma attribute. Each sample was evaluated three times.

[0161] 2. Test Results

[0162] The results are as follows Figure 9 shown.

[0163] Depend on Figure 9 (Sensory evaluation of blueberry wine and wine) The results show that: the blueberry wine fermented with the grape juice Hansenula NF26 single bacteria of the present invention (Example 3) significantly increased the banana, citrus and blueberry aromas compared with Comparative Example 1, and the wine fermented with NF26 single bacteria (Example 4) significantly increased the banana, citrus and honey, rose and fruit aromas compared with Comparative Example 3; the blueberry wine (Example 5) fermented with grape juice Hansenula NF26 and commercial brewer's yeast D254 significantly increased the banana, citrus, blueberry and fruit aromas compared with Comparative Example 1, and the sequential fermentation of wine (Example 6) significantly increased the banana, citrus and honey and fruit aromas compared with Comparative Example 3.

[0164] In summary, the floral and fruity aroma of the fruit wines fermented by the grape juice fermentation of the present invention using the single strain and mixed strains of Hansenula sporangiophora NF26 was significantly enhanced.

[0165] In summary, the Hansenula sporogenes NF26 strain of the present invention, when inoculated alone or sequentially with Saccharomyces cerevisiae 48 hours apart in different types of fruit wine, significantly reduced the titratable acid content in the wine while increasing the levels of total phenols and anthocyanins. Furthermore, the strain also promoted the biosynthesis of terpenes and esters (particularly acetates). In terms of sensory quality, wines fermented with this strain exhibited a brighter color (with a significantly enhanced red hue) and a significantly enhanced floral and fruity aroma.

[0166] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A novel strain of Hansenula vitis vinifera, characterized in that: The taxonomic name is Hanseniasporauvarum NF26 and it is deposited in China Center for Type Culture Collection with the deposit number CCTCCNO: M 2025860.

2. A method for activating and culturing the novel Hansenula vitis spore strain according to claim 1, characterized in that: Under sterile conditions, the target strain was inoculated on a plate containing YPD solid medium for activation culture.

3. The activation culture method according to claim 2, characterized in that The activation culture conditions are: temperature 25° C., time 5 days.

4. Use of the novel Hansenula sporogenes strain of claim 1 in the fermentation preparation of fruit wine.

5. A fermentation method for preparing fruit wine, characterized in that: The novel Hansenula sporogenes strain of claim 1 is used as a target strain for fermentation, and the steps are as follows: (1) Sterilization After crushing the fruit raw material, hydrolyzing the pectin, squeezing and filtering, adding sucrose, then sterilizing at high temperature, and adding diammonium hydrogen phosphate to obtain a sterilized juice raw material; (2) Fermentation Fermentation is carried out by single-bacteria fermentation or mixed-bacteria fermentation; Single-bacteria fermentation method: a single bacterial suspension of the target fermentation strain is inoculated into the sterilized juice raw material, and the mixture is placed in an incubator for fermentation until the reducing sugar content does not decrease for three consecutive days, at which point the fermentation is considered complete and the fruit wine is obtained; The mixed fermentation method comprises inoculating a bacterial suspension of the target fermentation strain and a commercial brewing yeast suspension into the obtained sterilized juice raw material, placing the mixture in an incubator for mixed fermentation until the reducing sugar content does not decrease for three consecutive days, which is considered to be the end of the fermentation, and obtaining the fruit wine.

6. The fermentation method for preparing fruit wine according to claim 5, characterized in that: In the step (1), the fruit raw material is blueberry or wine grape fruit.

7. The fermentation method for preparing fruit wine according to claim 5, characterized in that: In the step (1), after the fruit raw material is crushed, pectin is hydrolyzed, squeezed and filtered, sucrose is added to adjust the total soluble solids of the juice to 20°Brix, and then sterilized at 90°C for 1 minute, and 200 mg / L diammonium hydrogen phosphate is added to obtain a sterilized juice raw material.

8. The fermentation method for preparing fruit wine according to claim 5, characterized in that: In the single-bacteria fermentation process of step (2), the concentration of the inoculated target bacterial strain suspension for fermentation is 1.0×10 7 CFU / mL, culture and fermentation conditions: temperature 25℃.

9. The method for preparing fruit wine by fermentation according to claim 5, characterized in that: In the mixed fermentation process of step (2), the commercial brewer's yeast is brewer's yeast Lalvin ICV D254.

10. The fermentation method for preparing fruit wine according to claim 5, characterized in that: During the mixed fermentation process of step (2), the target strain for fermentation and the commercial brewing yeast were inoculated in the order of 48 hours apart; and the bacterial suspension concentrations of the target strain for fermentation and the commercial brewing yeast were both 1.0×10 7 CFU / mL, the inoculation ratio was 1:1; the culture and fermentation conditions were: temperature 25℃.

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