Novel hansenula polymorpha strain and application thereof in blueberry wine fermentation

Abstract

The invention provides a novel hansenula polymorpha strain, which is classified and named as Hanseniaspora thailandica CL49, is preserved in the China Center for Type Culture Collection (CCTCC), and has the preservation number of CCTCC NO: M 2025862. The invention also provides an activation culture method of the strain, an application of the strain in fermentation preparation of blueberry wine, and a fermentation preparation method of the blueberry wine. The hanseniaspora thailandica CL49 has the advantages that the hanseniaspora thailandica CL49 is screened and separated, and the hanseniaspora thailandica CL49 is inoculated to ferment the blueberry wine independently or in a manner of being mixed with saccharomyces cerevisiae, so that the content of total phenols and anthocyanin can be remarkably increased, and the red color of a wine body is enhanced; and terpene and acetate substances can be increased, particularly, accumulation of sweet and fragrant characteristic substances such as beta-damascenone, isoamyl acetate and phenethyl acetate is promoted, the flower and fruit fragrance and the sweet fragrance are enhanced, and the flavor bottleneck of existing blueberry wine is broken through.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation, and in particular to a novel strain of *Hansenula polymorpha* and its application in blueberry wine fermentation. Background Technology

[0002] Blueberries, due to their thin skin and high water content, are highly susceptible to spoilage after harvesting, exhibiting poor storage and transportability. Processing blueberries into wine is an effective way to preserve their rich nutrients and unique flavor, achieving high-value utilization of resources. Flavor plays a crucial role in the quality of blueberry wine. Yeast, as the core functional microorganism in the fermentation process, not only converts the reducing sugars in blueberry juice into alcohol but also produces diverse flavor compounds such as esters, higher alcohols, organic acids, aldehydes, and ketones through metabolism. These metabolites have a decisive influence on the final taste and aroma characteristics of blueberry wine.

[0003] In fruit wine brewing, yeasts are mainly divided into brewer's yeast (Saccharomyces cerevisiae). Saccharomyces cerevisiae ) and non-brewing yeast (Non- Saccharomyces There are two main categories: *Saccharomyces cerevisiae* and *Saccharomyces rubrum*. *Saccharomyces cerevisiae* has strong fermentation ability and high alcohol tolerance and temperature adaptability, and can usually complete alcoholic fermentation independently. *Non-Saccharomyces cerevisiae* is a collective term for various yeasts used in fruit wine brewing besides *Saccharomyces cerevisiae*, with common genera including *Cytomyces* (…). Torulaspora ), Hansenula sporeans ( Hanseniaspora ), Pichia pastoris ( Pichia ), genus *Megkistrodon* ( Metschnikowia ) and Kluyveromyces ( Kluyveromyces Studies have shown that non-sacchariculture yeasts can secrete a variety of hydrolytic enzymes that degrade bound aroma substances into volatile free forms, while producing abundant secondary metabolites, significantly improving and enhancing the aroma complexity and quality of fruit wines. However, non-sacchariculture yeasts generally have a weaker fermentation capacity and usually cannot complete alcoholic fermentation independently. Therefore, in practice, they are often used in combination with sacchariculture yeasts for fermentation.

[0004] Currently, the production of commercially available blueberry wine largely relies on industrial fermentation using commercial brewing yeasts. While this method ensures fermentation efficiency, it also leads to homogenized flavors and a lack of distinctive characteristics, thus limiting the market competitiveness of blueberry wine. Therefore, screening superior non-brewing yeast strains with regional characteristics from specific environments and applying them to blueberry wine production is of great significance for breaking through existing flavor bottlenecks, improving the overall flavor quality of blueberry wine, and promoting the high-value-added transformation of fruit resources.

[0005] According to existing reports, Hanseniaspora thailandicaStrain isolation and culture cases are relatively rare, and their application in fruit wine fermentation is even more scarce. Only a few patents involve related applications, such as the patent “Fruit wine and its fermentation method” (CN202211616248.1) provides a fruit wine fermentation method using a mixed inoculum of Saccharomyces cerevisiae and Hansenula polymorpha. Hanseniaspora guilliermondii NF6 (strain accession number CCTCC NO: M 20221615) and Hanseniaspora thailandica YLL16 (strain accession number CCTCC NO: M2018140) mixed inoculation to increase the content of aroma compounds and reduce the acidity of citrus fruit wine.

[0006] Currently, by comprehensively evaluating the glycosidase and alcohol acyltransferase activities of yeast strains, strains that can simultaneously produce terpenes and ester flavor substances are screened out Hanseniaspora thailandica and applied to blueberry wine fermentation to significantly improve the flavor quality of the product. This research strategy and application direction has not been reported in the field. SUMMARY

[0007] The technical problem to be solved by the present application is to provide a new Hansenula sp. yeast strain and its application in blueberry wine fermentation. Using the strain alone or mixed with Saccharomyces cerevisiae 24 hours apart to prepare blueberry wine can not only significantly increase the content of total phenols and anthocyanins, promote the biological accumulation of terpenes and acetate esters (especially β-damascenone, isoamyl acetate, phenethyl acetate, etc. Characteristic substances related to sweet aroma), but also make the wine body more vibrant (red tone significantly enhanced), and specifically enhance the fruit aroma and sweet aroma, thereby breaking through the existing blueberry wine flavor bottleneck and improving its exclusive quality and economic value.

[0008] The present application solves the above technical problems by using the following technical solutions: A new Hansenula sp. yeast strain, classified and named as Hanseniaspora thailandica CL49, was sent to the “China Typical Culture Collection Center” on April 23, 2025 and showed survival, with the accession number CCTCC NO: M2025862 and the address of the China Typical Culture Collection Center at Wuhan University, Biaoyi Road, Wuchang District, Wuhan City, Hubei Province, China.

[0009] As one of the preferred modes of the present application, the strain is used for blueberry wine fermentation, which specifically promotes the biosynthesis of sweet aroma characteristic substances during fermentation.

[0010] As one of the preferred modes of the present application, the sweet aroma characteristic substances include β-damascenone, isoamyl acetate and phenethyl acetate.

[0011] A method for activating the culture of the above-mentioned new Hansenula sp. yeast strain. Under sterile conditions, the target strain is inoculated into a plate containing YPD solid medium and incubated at 25°C for 5 days.

[0012] As one of the preferred modes of the present application, the concentration of the obtained bacterial suspension after activation culture is 1.0×10 7 CFU / mL.

[0013] The application of the above-mentioned new Hansenula yeast strain in the fermentation preparation of blueberry wine.

[0014] A fermentation preparation method of blueberry wine, which uses the above-mentioned new Hansenula yeast strain as a target strain for fermentation, and the steps are as follows: (1) Raw material treatment and sterilization: after crushing and pectinolysis of blueberry raw materials, sucrose is added, then high-temperature sterilization is performed, and diammonium hydrogen phosphate is added to obtain sterilized blueberry juice raw materials; (2) Fermentation: single-strain fermentation or mixed-strain fermentation is used for fermentation, and the fermentation is continued until the content of reducing sugar does not decrease for three consecutive days, and then the blueberry wine is obtained.

[0015] As one of the preferred modes of the present application, in the step (1), the fruit raw material is blueberry fruit; after crushing, pectinolysis, pressing and filtration of the blueberry fruit, sucrose is added to adjust the total soluble solids of the juice to 20 °Brix, then sterilization is performed at 80 ℃ for 3 min, and 200 mg / L of diammonium hydrogen phosphate is added to obtain sterilized blueberry juice raw materials.

[0016] As one of the preferred modes of the present application, in the step (2), the single-strain fermentation is: inoculating the sterilized blueberry juice raw materials with the bacterial suspension of the target strain for fermentation, and fermenting at 25 ℃ until the content of reducing sugar does not decrease for three consecutive days, and then considering the fermentation as finished.

[0017] As one of the preferred modes of the present application, in the single-strain fermented blueberry wine, the total phenol content is ≥448 mg / L, the anthocyanin content is ≥157 mg / L, and the red tone (a* value) is ≥47.15.

[0018] As one of the preferred modes of the present application, in the step (2), the mixed-strain fermentation is: first inoculating the sterilized blueberry juice raw materials with the bacterial suspension of the target strain for fermentation, then inoculating the commercial Saccharomyces cerevisiae bacterial suspension with the same concentration after 24 h, the inoculation ratio is 1:1, and the fermentation is carried out at 25 ℃ until the content of reducing sugar does not decrease for three consecutive days, and then considering the fermentation as finished.

[0019] As one of the preferred modes of the present application, the commercial Saccharomyces cerevisiae uses Saccharomyces cerevisiae strain Lalvin ICVD254 (Lallemand, Bordeaux, France).

[0020] As one of the preferred modes of the present application, in the mixed-strain fermented blueberry wine, the total phenol content is ≥543 mg / L, the anthocyanin content is ≥161 mg / L, and the red tone (a* value) is ≥30.81.

[0021] The advantages of this invention compared to the prior art are: (1) This invention screened and isolated a strain Hanseniaspora thailandica CL49, this strain can simultaneously and efficiently produce terpenes and esters, making it a significant advantage as a fermentation strain for blueberry wine: Blueberry wine prepared using this strain alone or mixed with *Saccharomyces cerevisiae* at 24-hour intervals exhibits significantly higher total phenol and anthocyanin content than products from other strains, and a more vibrant color (enhanced red hue); fermentation with this strain not only increases the content of terpenes and acetates, but also promotes the bioaccumulation of characteristic sweet-aroma compounds such as β-damascene, isoamyl acetate, and phenethyl acetate, enhancing the intensity of floral, fruity, and sweet aromas, thus breaking through the current flavor limitations of blueberry wine. (2) The present invention contains the Hansenula polymorpha yeast strain CL49, which can be used to prepare blueberry wine products with high anthocyanin, high floral and fruity aroma and sweet aroma. Its application in blueberry wine fermentation can significantly improve the flavor and quality of the product, specifically enhance the unique quality of blueberry wine, and thus improve the economic value of blueberry wine. (3) The production process of the blueberry wine product of the present invention is simple, the fermentation is fast and the cost is low, making it suitable for industrial production. Attached Figure Description

[0022] Figure 1 The image shows the colorimetric results of the present invention's *Hansenula polymorpha* strain CL49 and reference strains 1 and 2 in esculin medium. Figure 2 The figure shows the results of the alcohol acyltransferase activity assay of the present invention's yeast strain CL49 and reference strains 1 and 2; Figure 3 The diagram shows the β-D-glucosidase activity results of the *Hansenula polymorpha* strain CL49 and reference strains 1 and 2 of this invention. Figure 4 The tolerance results of the *Hansenula polymorpha* strain CL49 and reference strains 1 and 2 are shown in the figure (Figure A shows the growth results of each strain under different SO2 concentrations, Figure B shows the growth results of each strain under different fermentation temperatures, Figure C shows the growth results of each strain under different mass concentrations of glucose, and Figure D shows the growth results of each strain under different proportions of anhydrous ethanol). Figure 5 This is a 26S rDNA phylogenetic tree of the *Hansenula polymorpha* strain CL49 of the present invention. Figure 6 The figures show the results of the total terpenoid, total ester, and total acetate content in the blueberry wine of this invention (Figure A shows the total terpenoid results, Figure B shows the total ester results, and Figure C shows the total acetate results). Figure 7 The sensory evaluation chart of the blueberry wine of the present application (in the chart, A is the sensory evaluation result of the single strain fermented blueberry wine, and B is the sensory evaluation result of the mixed strain fermented blueberry wine). DETAILED DESCRIPTION

[0023] The following detailed description of the embodiments of the present application is based on the technical solution of the present application, and detailed implementation and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments. Meanwhile, unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the technical field.

[0024] The medium formula and preparation method involved in the following embodiments are as follows: WL solid medium: yeast extract powder 5 g / L, acid hydrolyzed casein 5 g / L, glucose 50 g / L, potassium dihydrogen phosphate 0.55 g / L, potassium chloride 0.425 g / L, calcium chloride 0.125 g / L, magnesium sulfate 0.125 g / L, ferric chloride 0.0025 g / L, manganese sulfate 0.0025 g / L, bromocresol green 0.022 g / L, agar 17 g / L; weigh each raw material, dissolve in 1000 mL of distilled water, seal, sterilize at 121℃ for 15 min, cool to 50℃, and then pour into a flat plate.

[0025] YPD solid medium: peptone 10.0 g / L, yeast extract powder 5.0 g / L, glucose 20.0 g / L, agar 14.0 g / L; weigh each raw material, dissolve in 1000 mL of distilled water, seal, sterilize at 121℃ for 15 min, cool to 50℃, and then pour into a flat plate.

[0026] Escin biochemical screening medium: tryptone 0.5 g / L, beef extract 0.3 g / L, escin 0.1 g / L, ferric ammonium citrate 0.05 g / L; weigh each raw material, dissolve in 1000 mL of distilled water, seal, sterilize at 121℃ for 15 min, cool to 50℃, and then pour into a flat plate.

[0027] YEPD expansion medium: peptone 20.0 g / L, yeast extract powder 10.0 g / L, glucose 20.0 g / L; weigh each raw material, dissolve in 1000 mL of distilled water, seal, sterilize at 121℃ for 15 min, cool to 28℃, and then use. Example 1, isolation and identification of strain CL49: 1. Screening and isolation of strains In the clean bench, take an appropriate amount of fermented mash of 'Canshan' blueberry of Anhui Huaining at different fermentation periods for dilution, dilute in 6 gradients in turn, select 10 -4 , 10 -5 and 10-6 Three serially diluted solutions were evenly spread (200 μL each) onto WL solid medium and incubated at 25°C for 5 days. Based on the color and morphology of the colonies on the WL medium, the strains were preliminarily classified. Yeast strains with typical colony characteristics were selected and inoculated onto WL solid medium for further purification (2-3 purification cycles depending on the isolation effect), and the growth status of the yeast was observed. The strains isolated and purified from WL solid medium were first screened for glycosidases and alcohol acyltransferases. Yeast strains with high glycosidase and alcohol acyltransferase activities were selected. Further screening was performed based on alcohol acyltransferase activity, followed by β-D-glucosidase activity, resulting in a strain with high glycosidase and alcohol acyltransferase production.

[0028] A single colony of this strain was inoculated onto YPD solid medium for enrichment, incubated at 25°C for 5 days, and then stored for later use. Molecular biological identification was performed, and the strain was named... Hanseniaspora thailandica CL49.

[0029] 2. Screening and identification of strains (1) Initial screening of yeast strains for glycosidase The activity of glycosidases in yeast was initially screened using the aescin colorimetric method.

[0030] After activating the preserved bacterial strain, it was prepared into 1×10⁻⁶ samples. 7 A bacterial suspension of CFU / mL was prepared, and 200 μL of aescin was added to each well of a 96-well plate. 20 μL of the activated bacterial suspension was then inoculated into each well, and the plates were incubated at 28°C for 48 h. The glycosidase activity of the tested strains was determined according to the colorimetric grade (dark black indicates high activity, dark gray indicates medium activity, and gray indicates low activity). Commercial brewer's yeast (Saccharomyces cerevisiae) was used as a reference. Saccharomy cescerevisiae D254 (Laman, Bordeaux, France) is used as a reference 1 for commercial non-brewing yeasts such as *Saccharomyces cerevisiae*. Tourlaspora delbrueckii Td) Zymaflore ® Alpha is reference 2.

[0031] The present invention provides colorimetric results of *Hansenula polymorpha* strain CL49 and reference strains 1 and 2 in esculin medium, as shown below. Figure 1 As shown.

[0032] (2) Rescreening of yeast strains with alcohol acyltransferase The reaction system for determining yeast acyltransferase activity included: 2.5 mL of 5 mM MgCl2 solution (0.5 M pH 8.0 Tris-HCl containing 5 mM MgCl2), 150 μL of acetyl-CoA solution (0.5 M pH 8.0 Tris-HCl containing 5 mM Macetyl-CoA), 50 μL of butanol solution (0.5 M pH 8.0 Tris-HCl containing 200 mM butanol), and 150 μL of enzyme extract. After mixing, the mixture was incubated in a 35°C water bath for 15 min. Then, 100 μL of 10 mM DTNB was added, and the mixture was allowed to stand at room temperature for 10 min. The absorbance of the reaction solution was then measured at 412 nm. The reaction solution without enzyme extract was used as a blank. Each sample was tested three times. Simultaneously, commercial Saccharomyces cerevisiae D254 (Laman, Bordeaux, France) was used as a reference 1, and commercial non-Saccharomyces cerevisiae (Cyclocarya delbue) was used as a control. Tourlaspora delbrueckii Td) Zymaflore ® Alpha is reference 2.

[0033] Each 1×10 7 One unit of enzyme activity (U) is defined as the amount of coenzyme A produced by bacteria or cells per minute.

[0034] This invention contains the *Hansenula polymorpha* strain CL49 and the acyltransferase activity of reference strains 1 and 2. Figure 2 As shown.

[0035] (3) Quantitative rescreening of yeast strains with β-D-glucosidase Methods for determining yeast glycosidase activity. Preparation of crude glycosidase solution: Yeast strain activation solution was inoculated at a ratio of 5% into YEPD amplification medium and cultured in a constant temperature shaker at 28℃ and 180 rpm for 24 h. The amplified culture was then inoculated at a ratio of 10% into YPD fermentation medium, followed by further culture in a constant temperature shaker for 72 h. The fermentation broth was then collected. The supernatant was extracted using a benchtop high-speed refrigerated centrifuge at 8000 rpm, 4℃, and 15 min. The supernatant was filtered through a 0.45 μm membrane to remove cell debris and other macromolecules, and the yeast glycosidase extract was collected.

[0036] Determination of β-D-glucosidase activity: The reaction system consisted of 0.1 mL glycosidase extract, 0.75 mL citrate-phosphate buffer (pH 5.0), and 0.25 mL 1 mmol / L p-nitrobenzene-β-D-glucosidase. The reaction conditions were: water bath temperature 40℃, reaction time 30 min. After the reaction, 1 mL of 0.5 mol / L sodium carbonate was added to terminate the reaction. The absorbance of the reaction solution was measured at 400 nm. Distilled water was used as a control group instead of the reaction substrate. Each sample was repeated three times. Commercial Saccharomyces cerevisiae D254 (Laman, Bordeaux, France) was used as reference 1, and commercial non-Saccharomyces cerevisiae Zymaflore® Alpha was used as reference 2. The enzyme activity was calculated based on the standard curve.

[0037] The enzyme activity unit (IU) of β-D-glucosidase is defined as the amount of enzyme required to catalyze the production of 1 μmol of p-nitrophenol within 30 min under specified reaction conditions (40℃, pH 5.0).

[0038] Plot a p-NP standard curve. Plot the p-nitrophenol concentration on the x-axis and the absorbance on the y-axis. Based on the standard curve (y = 0.0121x + 0.0435, R0),... 2 The final enzyme activity was calculated using the formula (=0.9993).

[0039] This invention contains the β-D-glucosidase activity of the *Hansenula polymorpha* strain CL49 and reference strains 1 and 2. Figure 3 As shown.

[0040] (4) Study on the tolerance of yeast strains Tolerance studies were conducted on the isolated yeast strains (using commercial brewer's yeast D254 (Laman, Bordeaux, France) as a reference 1, and commercial non-brewing yeast *Saccharomyces cerevisiae* as a control). Tourlaspora delbrueckii Td) Zymaflore ® Alpha (reference 2) includes SO2 addition amount, fermentation temperature, C glucose content, and ethanol concentration tolerance. The specific steps are as follows: Blueberry juice was clarified after pasteurization (80℃, 3min) and the supernatant was used, and sucrose was added to adjust the total soluble solids of the blueberry juice to 20°Brix. The SO2 concentration in the blueberry juice was adjusted by adding sodium metabisulfite (0, 25, 50, 75 and 100 mg / L), different fermentation temperatures (10℃, 15℃, 20℃ and 25℃) were set, and different mass concentrations of glucose (100, 200, 300 and 400 g / L) and different proportions of anhydrous ethanol (6%, 8%, 10%, 12% and 14%) were added to construct a multi-factor experimental system. After the yeast strain was activated, it was added to different blueberry juices for fermentation, and the inoculation amount was 1×10 7 CFU / mL, the fermentation volume was 50 mL, and the fermentation temperature was 25℃. The growth of each yeast strain was reflected by measuring the OD 600 value. If the OD 600 value of the yeast strain changes by more than 1 in the first 3 days, it is considered that the strain has tolerance under the corresponding conditions. Each yeast strain is biologically repeated 3 times.

[0041] The tolerance results of the Hanseniaspora sp. yeast strain CL49 and reference 1, 2 strains of the application are shown in Table 1. Figure 4 Among them, Hanseniaspora thailandica CL49 has the best tolerance under the conditions of initial sugar adjustment 200 g / L, SO2 concentration 0~50 mg / L, fermentation temperature 25℃, and ethanol concentration less than 12%.

[0042] (5) 26S rDNA sequence identification The 26S rDNA of the Hanseniaspora sp. yeast strain CL49 of the application was sequenced, and the sequencing results (shown in SEQ ID NO. 1) were compared with the known yeast species sequences in the NCBI (National Center for Biotechnology Information, American National Biotechnology Information Center) database, and the constructed phylogenetic tree is shown in Figure 1; combined with the morphology and 26S rRNA gene sequence analysis of the strain Figure 5 CL49, it is determined that it is Hanseniaspora thailandica CL49. Hanseniaspora thailandica

[0043] (6) Preservation of the strain in the preservation center and molecular biology identification The strain CL49 screened in the application was sent to the China Center for Type Culture Collection for preservation, and its classification and naming was identified as Hanseniaspora thailandica CL49, and the preservation number is CCTCC NO: M 2025862. Hanseniaspora thailandica

[0044] ​​Example 2, Activation culture and preparation of spore suspension of Hanseniaspora sp. strain CL49 Under sterile conditions, Hanseniaspora sp. strain CL49 was inoculated on a plate containing YPD solid medium (streaking); then, the plate was inverted in a constant temperature incubator at 25°C for 5 days of activation; finally, the activated target strain was scraped into sterile water, mixed uniformly by shaking, and a spore suspension of Hanseniaspora sp. strain CL49 was obtained.

[0045] Example 3, Single-strain fermentation preparation of blueberry wine (1) Sterilization After crushing, pectinolysis, pressing and filtering the blueberry fruits, sucrose was added to adjust the total soluble solids of the blueberry juice to 20°Brix, and then sterilized at 80°C for 3 min, and 200 mg / L of diammonium hydrogen phosphate was added to obtain the sterilized blueberry juice raw material.

[0046] (2) Fermentation Under sterile conditions, the sterilized blueberry juice raw material was inoculated with the spore suspension (1.0×10 7 CFU / mL) of the Hanseniaspora sp. strain CL49 of the present application, and placed in a culture incubator for fermentation. The fermentation conditions were: temperature 25°C. After fermentation, the blueberry wine was obtained when the reducing sugar content did not decrease for three consecutive days.

[0047] Example 4, Mixed-strain fermentation preparation of blueberry wine (1) Sterilization After crushing, pectinolysis, pressing and filtering the blueberry fruits, sucrose was added to adjust the total soluble solids of the blueberry juice to 20°Brix, and then sterilized at 80°C for 3 min, and 200 mg / L of diammonium hydrogen phosphate was added to obtain the sterilized blueberry juice raw material.

[0048] (2) Fermentation Under sterile conditions, the sterilized blueberry juice raw material was inoculated with the spore suspension (1.0×10 7 CFU / mL) of the Hanseniaspora sp. strain CL49 of the present application and the spore suspension (1.0×10 7 CFU / mL) of the commercial Saccharomyces cerevisiae strain D254 (Lallemand, Bordeaux, France) in a ratio of 1:1, sequentially (CL49 first, then D254) with an interval of 24 h, and placed in a culture incubator for mixed-strain fermentation. The fermentation conditions were: temperature 25°C. After fermentation, the blueberry wine was obtained when the reducing sugar content did not decrease for three consecutive days.

[0049] Comparative Example 1 The single-strain fermentation preparation of a blueberry wine in this comparative example was basically the same as in Example 3, with the main difference being that the commercial Saccharomyces cerevisiae (S. cerevisiae) Saccharomy cescerevisiae ) D254 (Lallemand, Bordeaux, France) was used instead of Hanseniaspora thailandicaCL49, as the target strain for fermentation.

[0050] Comparative Example 2 A single-strain fermentation of a blueberry wine was prepared according to the present comparative example, which was substantially the same as Example 3, except that a commercial non-Saccharomyces yeast, ZyMaflore® Delbus (Lachancea delbrueckii) (Zymaflore®) was used instead of CL49. Tourlaspora delbrueckii , Td) Zymaflore ® Alpha instead Hanseniaspora thailandica CL49, as the target strain for fermentation.

[0051] Comparative Example 3 A mixed-strain fermentation of a blueberry wine was prepared according to the present comparative example, which was substantially the same as Example 4, except that a commercial non-Saccharomyces yeast, ZyMaflore® Delbus (Lachancea delbrueckii) (Zymaflore®) was used instead of CL49. Tourlaspora delbrueckii , Td) Zymaflore ® Alpha instead Hanseniaspora thailandica CL49.

[0052] Test Example 1, Basic physicochemical detection of blueberry wine: The blueberry wines prepared by fermentation according to Examples 3 and 4 and Comparative Examples 1, 2 and 3 of the present application were subjected to physicochemical detection. The determination of basic physicochemical properties included: reducing sugar content, alcohol content, pH value, titratable acid content, yeast assimilable nitrogen, total anthocyanin content, total phenol content and colorimetric characteristics.

[0053] I. Detection method (1) The pH value of the blueberry wine was measured using a pH meter. The determination of the reducing sugar content, titratable acid content and alcohol content of the blueberry wine was in accordance with GB / T 15038-2006 “General analysis method for grape wine and fruit wine”.

[0054] (2) The total anthocyanin content was determined by pH differential method: 2 mL of the wine sample was centrifuged at 4000 r / min for 10 min, and the supernatant was diluted by the same multiple with pH 1.0 and pH 4.5 buffer solutions, respectively, and vortexed, and then placed in the dark for 15 min. The absorbance of each sample at 520 nm and 700 nm was measured, and the absorbance of the solution was recorded. The anthocyanin content in the finished product was calculated according to the following formula: A= (A 520 -A 700 ) pH 1.0 - (A 520 -A 700 ) pH 4.5; TAC (mg / g) = A x MW x DF x 1000 / (ε x L); In the formula: A 520 is the absorbance of the sample at 520 nm, and A 700The absorbance value of the sample at 700 nm; pH 1.0 is the sample to be tested diluted with pH 1.0 buffer, and pH 4.5 is the sample to be tested diluted with pH 4.5 buffer; MW represents the relative molecular mass of anthocyanin in the sample, calculated based on the relative molecular mass of cyanidin-3-glucoside, MW = 449.2; DF represents the dilution factor; ε represents the molar absorption of anthocyanin in the sample, calculated based on the molar absorption of cyanidin-3-glucoside, ε = 26900; L represents the optical path.

[0055] (3) Determination of total phenol: 0.25 mL of the product wine sample diluted by a certain multiple was taken into a test tube, 1.25 mL of Folin phenol reagent diluted by 10 times was added, vortex mixed and allowed to stand for 5 min, then 1.25 mL of 7.5% (v / v) sodium carbonate solution and 2.5 mL of deionized water were added, vortex mixed and allowed to react in the dark for 60 min, and then the absorbance value of the solution at 765 nm was immediately determined. It is expressed by equivalent gallic acid (mg / L). The gallic acid concentration is the abscissa, and the absorbance value is the ordinate. The standard curve is drawn, and the total phenol content is calculated according to the standard curve (y = 2.9031x + 0.0673, R 2 = 0.99932).

[0056] (4) The colorimetric characteristics of the fruit wine were characterized by CIELab parameters under a 10° standard observer and a D65 standard light source. The absorbance values of the blueberry wine sample at an optical path of 1 cm and wavelengths of 450, 520, 570 and 630 nm were recorded, and the CIE lab parameters including lightness (L), red-green color value (a a* ), and yellow-blue color value (b b* ) were calculated according to the method described by Gordillo et al.

[0057] II. Test results The test results are shown in Table 1.

[0058] Table 1 Basic physicochemical indexes of blueberry wine

[0059] From the results of Table 1 (basic physicochemical indexes of blueberry wine), it can be seen that the use of the present application Hanseniaspora thailandica CL49 single strain fermented blueberry wine (Example 3), and inoculation Hanseniaspora thailandicaBoth CL49 and commercial S. cerevisiae D254 could complete the fermentation (Example 4). In terms of wine quality, Example 3 significantly reduced the pH (0.08) and titratable acid (0.11 g / L) and significantly increased the total phenol (29.9 mg / L) and anthocyanin (25.5 mg / L) contents compared with Comparative Example 1; Example 4 significantly increased the pH (0.14) and titratable acid (1.95 g / L) and greatly increased the total phenol (125 mg / L) and anthocyanin (30.3 mg / L) contents compared with Comparative Example 1; Example 4 significantly reduced the pH (0.06) and increased the titratable acid (0.54 g / L), total phenol (1.8 mg / L) and anthocyanin (5.2 mg / L) contents compared with Comparative Example 3. Table 1 shows that Examples 3 and 4 increased the a* value, making the wine body present a more vibrant red color compared with Comparative Examples 1 and 2 and Comparative Example 3, respectively.

[0060] In summary, the present application Hanseniaspora thailandica CL49 can independently complete the alcoholic fermentation of blueberry wine. Hanseniaspora thailandica Both CL49 single-strain fermentation and mixed-strain fermentation can significantly increase the total phenol and total anthocyanin contents in the prepared blueberry wine, making the wine body present a more vibrant red color.

[0061] Test Example 2: Detection of aroma substances in fermented blueberry wine The blueberry wine prepared by fermentation according to Examples 3 and 4 and Comparative Examples 1, 2 and 3 of the present application was subjected to volatile compound detection.

[0062] I. Detection method The headspace solid-phase microextraction method (HS-SPME) was used to extract the volatile compounds in the fruit wine.

[0063] (1) HS-SPME extraction process 9 mL of citrate buffer (0.2 M, pH 5.0) and 1 mL of the wine sample to be tested were added to a 20 mL headspace bottle, 3 g of sodium chloride and 20 µL of 20.71 µg / mL 3-nonanone solution (internal standard) were added, a rotor was placed, sealed with a cover, and the headspace bottle was placed in a magnetic stirrer, 50°C for 15 min. The aged 50 / 30 µm DVB / CAR / PDMS extraction head was inserted into the headspace bottle which had been balanced at 50°C for 15 min, and the distance between the liquid surface and the extraction head was kept at 2 cm, the rotation speed was about 500 r / min, the extraction temperature was 50°C, and the extraction time was 45 min. After extraction, the extraction head was inserted into the GC injection port end, desorbed for 5 min, and then subjected to gas chromatography-mass spectrometry (GC-MS) analysis.

[0064] (2) GC-MS detection conditions Gas phase conditions: HP-5MS capillary column (5% phenylmethylsiloxane, 30 m x 0.25 mm x 0.25 µm); carrier gas: high purity helium; carrier gas flow rate: 1.2 mL / min; splitless injection; injection port temperature: 250℃; mass spectrometry interface temperature: 250℃. Temperature program: initial temperature 40℃, hold for 5 min, increase to 180℃ at 3℃ / min, hold for 1 min, then increase to 300℃ at 30℃ / min, hold for 2 min. Solvent delay: 0 min. Mass spectrometry conditions: electron impact ion source (EI); electron energy: 70 eV; ion source temperature: 250℃; quadrupole temperature: 150℃; mass scan range: 25~300 m / z; full scan.

[0065] II. Results of detection The results are shown in Figure 6 The results are shown in Figure 6 The results show that: the total terpenes, total esters and total acetate content of the blueberry wine fermented by CL49 single strain (Example 3) are significantly increased compared with those of Comparative Example 1 and Comparative Example 2. Hanseniaspora thailandica The results show that: the total terpenes, total esters and total acetate content of the blueberry wine fermented by CL49 single strain (Example 3) are significantly increased compared with those of Comparative Example 1 and Comparative Example 2. Hanseniaspora thailandica The results show that: the total terpenes, total esters and total acetate content of the blueberry wine fermented by CL49 single strain (Example 3) are significantly increased compared with those of Comparative Example 1 and Comparative Example 2.

[0066] In summary, the blueberry wine fermented by CL49 single strain and mixed strain of the present application Hanseniaspora thailandica The results show that: the total terpenes, total esters and total acetate content of the blueberry wine fermented by CL49 single strain (Example 3) are significantly increased compared with those of Comparative Example 1 and Comparative Example 2.

[0067] In addition, in order to determine the characteristic volatile compounds that contribute to the differences between different fermentation treatment groups of blueberry wine, the compounds with variable importance in the projection (VIP value) greater than 0.8 and relative odor activity value (rOAVs) greater than 0.1 in the PLS-DA model were screened, and the results are shown in Table 2.

[0068] Table 2. Contents of key aroma compounds in blueberry wine (µg / L)

[0069] From the results in Table 2, it can be seen that: Hanseniaspora thailandicaCompared with Comparative Example 1 and Comparative Example 2, the CL49 single-strain fermentation blueberry wine (Example 3) increases the content of isoamyl acetate, ethyl 9-decenoate, phenethyl acetate, linalool, alpha-terpineol and beta-damascenone; compared with Comparative Example 1, the content of octanoic acid and n-decanoic acid is increased; compared with Comparative Example 2, the content of ethyl hexanoic acid is increased. Among them, beta-damascenone is a marker substance that gives the wine a sweet aroma, and its content in Example 3 reaches 5.01 μg / L, which is significantly higher than that in Comparative Example 1 (3.19 μg / L) and Comparative Example 2 (4.00 μg / L); isoamyl acetate has sweet and fruity aroma, and its content in Example 3 reaches 1018.5 μg / L (2.3 times higher than that in Comparative Example 1), which is the core substance for improving the sweet intensity; phenethyl acetate has a sweet rose aroma, and forms a synergy with the sweet aroma, and its content in Example 3 reaches 223.7 μg / L (1.9 times higher than that in Comparative Example 1). The three substances together constitute the specific substance basis of the sweet aroma characteristics of the blueberry wine fermented by the strain.

[0070] Compared with other comparative examples, Hanseniaspora thailandica Compared with other comparative examples,

[0071] In summary, the present application Hanseniaspora thailandica The CL49 single-strain and mixed-strain fermented blueberry wine can specifically promote the biosynthesis of sweet aroma characteristic substances such as beta-damascenone, isoamyl acetate and phenethyl acetate, and increase the content of other flavor substances such as ethyl 9-decenoate and n-decanoic acid.

[0072] Test Example 3, sensory evaluation of fermented blueberry wine: The blueberry wine prepared by fermentation of Example 3 and Example 4 and Comparative Examples 1, 2 and 3 of the present application was subjected to sensory evaluation.

[0073] I. Detection method The judging panel consisted of ten evaluators (aged 22-25). Prior to the sensory evaluation of the blueberry wines, each panel member received approximately 30 hours of training in aroma identification and intensity recognition. Blueberry wine samples were randomly coded with three-digit numbers, dispensed in 15mL portions into 125mL professional fruit wine tasting glasses, and randomly presented to the evaluators. First, an aroma attribute description experiment was conducted. All evaluators' descriptive terms were compiled, and through group discussion, descriptive terms with the same meaning were merged, selecting the top eight most frequent terms. 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.

[0074] II. Test Results The results are as follows Figure 7 As shown.

[0075] Depend on Figure 7 The sensory evaluation results of the blueberry wine show that: using this invention... Hanseniaspora thailandica The CL49 single-strain fermented blueberry wine (Example 3) showed a significant increase in rose, apple, and sweet aromas compared to Comparative Examples 1 and 2. This is directly related to the high content of β-damascene (the core sweet aroma compound), isoamyl acetate (a synergistic compound of sweet aroma and floral-fruity aroma), and phenethyl acetate (a sweet aroma compound) in Example 2. Hanseniaspora thailandica The sequential fermentation of blueberry wine with CL49 and commercial brewing yeast D254 (Example 4) significantly increased the aromas of rose, honey, and pineapple compared to Comparative Example 3. The enhancement of the sweet aroma mainly depended on the specific accumulation of β-damascene, isoamyl acetate, and phenethyl acetate, rather than the increase of broad-spectrum aroma substances.

[0076] In summary, this invention Hanseniaspora thailandica In fruit wines fermented with CL49 single and mixed cultures, floral and fruity aromas and sweet aromas are significantly enhanced, and the sweet aroma characteristics are clearly associated with the accumulation of specific substances.

[0077] In summary, the present invention... Hanseniaspora thailandica When CL49 strain is inoculated alone or sequentially with Saccharomyces cerevisiae at 24-hour intervals into blueberry wine, it significantly increases the content of titratable acids, total phenols, and anthocyanins in the wine. Furthermore, this strain promotes the biosynthesis of terpenes and esters (especially acetates). From a sensory perspective, blueberry wine fermented with this strain has a more vibrant color (a significantly enhanced red hue), and the intensity of floral, fruity, and sweet aromas is also significantly improved.

[0078] 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 within the protection scope of the present invention.

Claims

1. A novel strain of the genus Hanseniaspora, characterized in that, Classification name is Hanseniaspora thailandica CL49, deposited with China Center for Type Culture Collection, with the accession number CCTCC NO: M 2025862.

2. The strain according to claim 1, characterized in that, The strain is used for blueberry wine fermentation, and specifically promotes biosynthesis of sweet-smelling characteristic substances during fermentation.

3. The strain according to claim 2, characterized in that, The sweet-smelling characteristic substances include β-damascenone, isoamyl acetate, and phenethyl acetate.

4. A method for activating the culture of a novel Hansenula sp. strain according to any one of claims 1 to 3, characterized in that, Under sterile conditions, the target strain is inoculated on a plate containing YPD solid medium, and activated culture is performed at 25°C for 5 days.

5. The activation culture method according to claim 4, characterized by, The concentration of the bacterial suspension obtained after activation of the culture was 1.0 x 10 7 CFU / mL.

6. Use of the novel Hanseniaspora yeast strain according to any one of claims 1-3 in the preparation of blueberry wine.

7. A method for the fermentation preparation of a blueberry wine, characterized in that, The novel Hanseniaspora yeast strain according to any one of claims 1-3 is used as a target strain for fermentation, and the steps are as follows: (1) Raw material treatment and sterilization: after crushing and pectinolysis of blueberry raw materials, sucrose is added, followed by high-temperature sterilization, and addition of diammonium hydrogen phosphate to obtain sterilized blueberry juice raw materials; (2) Fermentation: single-strain fermentation or mixed-strain fermentation is used for fermentation, and the fermentation is continued until the reducing sugar content does not decrease for three consecutive days, to obtain blueberry wine.

8. The fermentation production process of claim 7, wherein, In the step (1), the fruit raw material is blueberry fruit; after crushing, pectinolysis, pressing, and filtration of the blueberry fruit, sucrose is added to adjust the total soluble solids of the juice to 20°Brix, followed by sterilization at 80°C for 3 min, and addition of 200 mg / L diammonium hydrogen phosphate to obtain sterilized blueberry juice raw materials.

9. The fermentation production method according to claim 7, characterized by, In the step (2), single-strain fermentation is performed by inoculating the sterilized blueberry juice raw materials with a bacterial suspension of the target strain for fermentation, and fermentation is performed at 25°C until the reducing sugar content does not decrease for three consecutive days, which is considered as the end of fermentation.

10. The fermentation production method according to claim 7, wherein, In the step (2), mixed-strain fermentation is performed by first inoculating the sterilized blueberry juice raw materials with a bacterial suspension of the target strain for fermentation, and then inoculating the same concentration of commercial Saccharomyces cerevisiae Lalvin ICV D254 bacterial suspension after 24 h, at a ratio of 1:1, and fermentation is performed at 25°C until the reducing sugar content does not decrease for three consecutive days, which is considered as the end of fermentation.

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