A strain of Metschnikowia pulcherrima producing hydroxycinnamate decarboxylase

Abstract

The present invention discloses a Meyerozyma guilliermondii strain producing hydroxycinnamic acid decarboxylase, belonging to the technical field of microbiology. The present invention provides a Meyerozyma guilliermondii strain Y4 producing hydroxycinnamic acid decarboxylase, which was deposited at the China Center for Type Culture Collection on August 29, 2022. By using Meyerozyma guilliermondii Y4 with hydroxycinnamic acid activity to ferment blueberry juice, the hydroxycinnamic acid in the juice can be catalyzed to decarboxylate into the corresponding vinylphenol. During the 8-day fermentation process, the vinylphenol further reacts with anthocyanins to form three more stable pyranoanthocyanins. According to the detection and analysis results of anthocyanins in blueberry juice in our previous study, more pyranoanthocyanins will be formed by changing the fermentation conditions, such as extending the fermentation time, increasing the inoculum size, changing the fermentation temperature, and adjusting the juice concentration. Therefore, this technical solution can effectively improve the color stability of blueberry fruit wine.

Description

Technical Field

[0001] The present invention relates to a strain of Metschnikowia pulcherrima producing hydroxycinnamic acid decarboxylase, belonging to the field of microbial technology. Background Art

[0002] Blueberries have high nutritional and health care value. The cultivation of blueberries across the country has reached a considerable scale, and the output is also increasing day by day. However, fresh blueberries are not easy to store. Therefore, the significance of deep processing of blueberries has become increasingly prominent, and fermented fruit wine is an effective way for deep processing of blueberries. Anthocyanins play a decisive role in the color of blueberry fruit wine. However, the stability of anthocyanins after blueberry juice fermentation is poor, which affects the color and quality of the product, and greatly affects the storage and shelf life of blueberry fruit wine.

[0003] At present, using non-Saccharomyces yeasts with high hydroxycinnamic acid decarboxylase (HCDC) activity to improve the stability of fruit wine is a research hotspot among scholars at home and abroad. It has been proven that some non-Saccharomyces yeast strains have positive HCDC activity and can produce higher concentrations of vinylphenol pyranoanthocyanins compared with Saccharomyces cerevisiae. HCDC activity is common in yeast and has also been reported in non-yeast strains. However, it is strain-dependent, and the activity may vary greatly among different species. Other pyranoanthocyanins detected by Jelena et al. after experiments include delphinidin-3-O-glucoside-4-vinylphenol, petunidin-3-O-glucoside-4-vinylphenol, and peonidin-3-O-glucoside-4-vinylphenol.

[0004] Pyranoanthocyanins have interesting color characteristics, can display a variety of colors (from orange to blue), are more resistant to water and sulfur dioxide, and have a wider stability to pH changes compared with anthocyanins. Vinylphenol pyranoanthocyanins are one of the most well-known anthocyanin derivatives in fruit wine and are formed by the spontaneous addition reaction of anthocyanins and vinylphenols after the decarboxylation of pyruvic acid or hydroxycinnamic acid. In order to improve the stability of anthocyanins in blueberry fruit wine during fermentation and aging, it is necessary to study more appropriate methods to accelerate the formation of pyranoanthocyanins in blueberry fruit wine. Using yeasts with hydroxycinnamic acid decarboxylase (HCDC) activity can promote the decarboxylation of hydroxycinnamic acids (caffeic acid, ferulic acid, p-coumaric acid) to generate the corresponding 4-vinylcatechol, 4-vinylguaiacol, and 4-vinylphenol, which can spontaneously add to different anthocyanins to form stable vinylphenol pyranoanthocyanins, thereby improving the color of blueberry fruit wine and enhancing the stability of blueberry fruit wine.

[0005] In recent years, some of the simplest pyranoanthocyanins, such as A-type vitisins, have been identified as the starting point for the formation of more complex pyranoanthocyanins. However, only a few pyranoanthocyanins have been detected and quantified. Due to the complex mixture of anthocyanins present in blueberry fruit wine, a large number of different pyranoanthocyanins are expected to form. Monomeric anthocyanins are highly unstable and are susceptible to the effects of light, oxygen, temperature, pH, structure, and their own concentration, resulting in degradation. Pyranoanthocyanins, as a class of derivatives of anthocyanins, have higher stability than their precursors. Due to the important contribution of anthocyanins to the color and biological activity of fruit wine, it is very important to improve the understanding of the stability of pyranoanthocyanins.

[0006] Therefore, by screening yeast strains with HCDC activity, the stability of blueberry fruit wine can be enhanced without the additional addition of cofactors. By promoting the decarboxylation of phenolic acids contained in blueberries themselves, vinylphenols are generated, accelerating the adduct reaction with various anthocyanins to form more stable pyranoanthocyanins, providing a new way to solve the problem of unstable color in blueberry fruit wine. Summary of the Invention

[0007] The first object of the present invention is to provide a strain of Metschnikowia pulcherrima Y4 that produces hydroxycinnamic acid decarboxylase, which was deposited at the China Center for Type Culture Collection on August 29, 2022. The deposit address is Wuhan University, China, and the deposit number is CCTCC NO: M 20221344.

[0008] The Metschnikowia pulcherrima Y4 of the present invention was isolated from grape skins. The DNA fragment encoding the 26S ribosomal RNA of the strain was amplified by PCR using yeast ITS universal primers. The PCR product was sent to Sangon Biotech for sequencing. The sequencing results were submitted to the GenBank database, subjected to BLAST and compared with other reported sequences. A phylogenetic tree was constructed by the neighbor-joining method using MEGA11.0 software. By constructing a phylogenetic tree through characteristic sequences, Y4 has a high similarity with Metschnikowia pulcherrima SN-25. Therefore, the strain of the present invention is Metschnikowia pulcherrima, named Metschnikowia pulcherrima Y4.

[0009] The 26S rDNA sequence of this strain was amplified and sequenced (SEQ ID NO.1). A phylogenetic tree was constructed through characteristic sequences. The sequencing results and the phylogenetic tree are as Figure 4 shown.

[0010] The second object of the present invention is to provide a method for screening yeast with HCDC activity in YPD medium containing three different phenolic acids (caffeic acid, p-coumaric acid, ferulic acid) respectively.

[0011] In one embodiment of the present invention, caffeic acid standard, p-coumaric acid standard, ferulic acid standard and bromocresol purple are all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Yeast extract, peptone, anhydrous glucose, agar, potassium chloride, sodium acetate, 98% formic acid, methanol (HPLC), acetonitrile (HPLC) are purchased from Sinopharm Chemical Reagent Co., Ltd. All chemicals and reagents are of analytical grade if not otherwise stated.

[0012] In one embodiment of the present invention, AgilentSB-C 18 Liquid chromatography column (4.6×250mm, 5μm) is used to detect the contents of caffeic acid, ferulic acid and p-coumaric acid. The detection wavelength is 290nm, the flow rate is 1mL / min, the injection volume is 10μL, and the column temperature is 40℃. Mobile phase A is 0.1% formic acid-water, B is 0.1% formic acid-acetonitrile, A is 90%, B is 10%, isocratic elution, and the time is 35min.

[0013] In one embodiment of the present invention, the components of the YPD liquid medium include: 1% yeast extract, 2% peptone, 2% anhydrous glucose.

[0014] In one embodiment of the present invention, the components of the YPD solid screening medium include: 1% yeast extract, 2% peptone, 2% anhydrous glucose, 0.145% of p-coumaric acid, ferulic acid and caffeic acid are added respectively, and 2% agar.

[0015] In one embodiment of the present invention, the components of the YPD liquid screening medium include: 1% yeast extract, 2% peptone, 2% anhydrous glucose, 0.145% of p-coumaric acid, ferulic acid and caffeic acid are added respectively.

[0016] In one embodiment of the present invention, the inoculated yeast pre-culture is obtained by inoculating a single colony into YPD liquid medium and culturing at 28-30℃ and 200-220rpm for 24h.

[0017] In one embodiment of the present invention, the inoculated yeast is inoculated with its single colony into YPD solid screening medium and cultured in an incubator at 28-30℃ for 10d.

[0018] In one embodiment of the present invention, the hydroxycinnamic acid decarboxylase (HCDC) activity of the inoculated yeast is obtained by inoculating a preculture of the yeast into a YPD liquid screening medium and culturing it at 28 - 30 °C and 200 - 220 rpm for 72 h, with an inoculation amount of 1% - 5%.

[0019] The third object of the present invention is to provide a microbial inoculant, which contains the Metschnikowia pulcherrima Y4 or its fermentation broth, or contains the freeze-dried powder of the above-mentioned Metschnikowia pulcherrima Y4, or contains its lysate.

[0020] In one embodiment of the present invention, the microbial inoculant is a solid inoculant or a liquid inoculant.

[0021] In one embodiment of the present invention, in the microbial inoculant, the addition amount of the Metschnikowia pulcherrima Y4 is at least: 1.0×10 6 cells / mL.

[0022] The fourth object of the present invention is to provide a composition containing the above-mentioned Metschnikowia pulcherrima Y4.

[0023] The fifth object of the present invention is to provide the application of Metschnikowia pulcherrima Y4, or the above-mentioned microbial inoculant, or the above-mentioned composition in the production of fermented products.

[0024] In one embodiment of the present invention, the fermented product is a fermented food or a fermented beverage; the fermented beverage is a fermented alcoholic beverage.

[0025] In one embodiment of the present invention, the fermented alcoholic beverage includes but is not limited to: wine, blueberry fruit wine, mulberry fruit wine.

[0026] The sixth object of the present invention is to provide the application of Metschnikowia pulcherrima Y4 in the brewing of blueberry fruit wine. The application is to use a fermentation raw material containing blueberry juice and ferment to prepare fruit wine by using the above-mentioned Metschnikowia pulcherrima Y4 and Saccharomyces cerevisiae.

[0027] In one embodiment of the present invention, the experiment was carried out in a 500 ml fermentation flask containing 300 ml of pasteurized blueberry juice, with a pH of 2.70 and an initial sugar content of 200 g / L. After activating the Y4 yeast strain in YPD medium in a shaker at 30 °C at 200 rpm for 24 h, it was inoculated into the blueberry juice at a concentration of 1.0×10 6 cells / mL, and placed in a static fermentation in an incubator at 25 °C. The fermentation flask was sealed with a fermentation stopper to ferment under the condition of no interference from external microorganisms and release CO 2 . The weight of the fermentation flask was weighed every 24 h, and the weight loss was calculated by subtracting the weight of the fermentation flask on the previous day from the weight on the current day to monitor the fruit wine fermentation. When the weight of the fermentation flask remained constant, the fermentation was considered to be completed. After the fermentation was completed, the blueberry fermentation broth was separated by a refrigerated centrifuge at a speed of 8000×g for 10 min at a temperature of 4 °C. The separated samples were filtered through a 0.22 μm aqueous needle filter and stored in a -20 °C refrigerator until analysis. After the fermentation was completed, the reducing sugar was determined by the 3,5-dinitrosalicylic acid method, and the alcohol content was detected using an Anton Paar alcohol analyzer.

[0028] In one embodiment of the present invention, the inoculation method is: directly inoculating the yeast into the blueberry juice; the specific inoculated strain is:

[0029] Control group: only inoculating a single Saccharomyces cerevisiae (K1); mixed fermentation group: Y4 and K1 were inoculated simultaneously at a cell number ratio of 4:1. The initial inoculation amount of both yeasts was 1.0×10 6 cells / mL.

[0030] The pyran anthocyanins were analyzed using a WATERS ACQUITY UPLC system with a PDA detector and a BEHC 18 (2.1×150 mm, 1.7 μm) liquid chromatography column.

[0031] In one embodiment of the present invention, the pH differential method was used to determine the total monomer anthocyanin content in the juice and the samples after fermentation. Two blueberry fruit wine samples fermented by different fermentation methods were dissolved in 0.01 M KCl aqueous solution (pH = 1.0) at a ratio of sample: diluent 1:9 for stability analysis. 10 mL of the diluted samples of the two different fruit wines were each placed in a simulated storage environment. The total anthocyanin content was measured every day for 6 consecutive days. All the analyses in this study were repeated three times.

[0032] The seventh object of the present invention is to provide the application of Metschnikowia pulcherrima Y4 in improving the flavor of fruit wine.

[0033] In one embodiment of the present invention, flavor substances are detected by GC-MS, and volatile substances in blueberry fermentation broth are detected by gas chromatograph Trace1310 and mass spectrometer ISQLT. The column model is TG-WAX A 6mm×0.25mm×0.25μm. 2-octanol is used as the internal standard. The ion source type is EI source, the temperature is 260°C, the transfer line temperature is 230°C, the injection port temperature is 250°C, the helium flow rate is 1 mL / min, the electron energy is 70 eV, and the emission current is 25 μA.

[0034] In one embodiment of the present invention, the fruit wine includes but is not limited to: blueberry fruit wine, mulberry fruit wine, grape wine, strawberry fruit wine, hawthorn fruit wine, and bayberry fruit wine.

[0035] The eighth object of the present invention is to provide a method for improving the stability of anthocyanins in fruit wine, by adding the above-mentioned Metschnikowia pulcherrima Y4 during the fermentation process of fruit wine.

[0036] Beneficial effects

[0037] (1) In the present invention, fermenting blueberry juice with Metschnikowia pulcherrima Y4 having hydroxycinnamic acid activity can catalyze the decarboxylation of hydroxycinnamic acid in the juice into the corresponding vinylphenol. During the 8-day fermentation process, vinylphenol further reacts with anthocyanins to form three pyranoanthocyanins with better stability (delphinidin-3-O-arabinoside-4-vinylcatechol, cyanidin-3-O-arabinoside-4-vinylcatechol, malvidin-3-O-galactoside / glucoside-4-vinylcatechol). According to the previous detection and analysis results of anthocyanins in blueberry juice, by changing the fermentation conditions, such as extending the fermentation time, increasing the inoculum amount, changing the fermentation temperature, and adjusting the juice concentration, more pyranoanthocyanins will be formed. Therefore, this technical solution can effectively improve the color stability of blueberry fruit wine.

[0038] (2) The alcoholic fermentation of blueberries will produce relatively high levels of organic acids, which affect the taste of the finished blueberry fruit wine. However, adding Metschnikowia pulcherrima Y4 for mixed fermentation can appropriately reduce the content of five main organic acids in blueberry fruit wine. This technical solution helps to improve the palatability of blueberry fruit wine and makes the blueberry wine body more refreshing.

[0039] (3) Pyranoanthocyanins have higher stability compared to anthocyanins, but they usually form slowly during the aging process of wine (generally more than 1 year). However, this technical solution can form pyranoanthocyanins just by fermenting with Y4 for only 8 days, which shortens the period for improving the color stability of blueberry fruit wine.

[0040] (4) The aroma components of blueberry juice are not prominent. This technical solution can significantly increase the concentration of esters such as ethyl acetate in blueberry fruit wine, generating fruity and floral aromas. In particular, the increase in ethyl lactate will produce the aroma of coffee or strawberry and raspberry. This technology can increase the volatile substances in blueberry fruit wine, especially the formation of ester substances, making the taste of the finished blueberry fruit wine more rich. And this strain of Metschnikowia pulcherrima Y4 does not produce vinylphenol reductase, reducing the production of ethylphenol-like off-flavor substances, thus improving the flavor of blueberry fruit wine.

[0041] (5) Since the alcohol content of traditional blueberry fruit wine is relatively high and not suitable for the general public to drink, this technical solution can reduce the alcohol content of blueberry fruit wine, making the taste of blueberry wine softer and more suitable for the general public to drink.

[0042] Biological material preservation

[0043] A strain of Metschnikowia pulcherrima Y4, taxonomically named: Metschnikowia pulcherrima Y4; has been deposited at the China Center for Type Culture Collection on August 29, 2022. The deposit address is Wuhan University, China, and the deposit number is CCTCC NO: M 20221344. Description of the drawings

[0044] Figure 1 It is a diagram of the color-changing circle of Metschnikowia pulcherrima growing on YPD solid screening medium for 1, 3, and 10 days.

[0045] Figure 2 It is a diagram of the colony morphology of Metschnikowia pulcherrima.

[0046] Figure 3 It is a microscopic examination diagram of Metschnikowia pulcherrima Y4

[0047] Figure 4 It is a phylogenetic tree of Metschnikowia pulcherrima Y4

[0048] Figure 5 It is a speculative diagram of the newly formed pyran anthocyanin after fermentation with Metschnikowia pulcherrima.

[0049] Figure 6 It is a diagram of the research results on the stability of blueberry fruit wine after fermentation with Metschnikowia pulcherrima by different fermentation methods. Detailed implementation manners

[0050] The pectinase involved in the following examples was purchased from Shanghai Macklin Biochemical Co., Ltd. The Metschnikowia pulcherrima involved in the following examples was isolated from grape skins, and the commercial yeast K1 involved in the following examples was purchased from: Lallemand Group, France.

[0051] The culture media involved in the following examples are as follows:

[0052] The components of each liter of YPD liquid medium include: 1% yeast extract, 2% peptone, and 2% anhydrous glucose.

[0053] The components of each liter of YPD solid screening medium include: 1% yeast extract, 2% peptone, 2% anhydrous glucose, 0.145% of p-coumaric acid, ferulic acid, and caffeic acid respectively, 2% agar, and 0.01% bromocresol purple.

[0054] The components of each liter of YPD liquid screening medium include: 1% yeast extract, 2% peptone, 2% anhydrous glucose, 0.145% of p-coumaric acid, ferulic acid, and caffeic acid respectively, and 0.01% bromocresol purple.

[0055] The detection method of hydroxycinnamic acid decarboxylase activity involved in the following examples

[0056] Inoculate a single colony into a 250 mL conical flask containing 50 mL of YPD liquid medium to prepare a yeast preculture, and incubate at 30 °C at 200 rpm for 24 h. Subsequently, inoculate 200 μL (OD 600 = 1) of the yeast preculture into the YPD liquid screening medium, place it on a shaker at 30 °C at 200 rpm, incubate until it turns purple and stops growing. Use a refrigerated centrifuge to centrifuge 1 mL of the product after fermentation at 5000×g at 4 °C for 5 min, filter it through a 0.22 μm aqueous syringe filter, and analyze it by HPLC to quantify the metabolized caffeic acid, ferulic acid, and p-coumaric acid. The detection of HCDC activity is obtained according to the following formula, and the activity is calculated according to the following formula.

[0057]

[0058] In the formula, HA represents hydroxycinnamic acid (Hydroxycinnamic Acid, HA). In the following examples, the hydroxycinnamic acid decarboxylase activity of Y4 was measured using caffeic acid, ferulic acid, and p-coumaric acid as substrates respectively. HA initial represents the initial amount of hydroxycinnamic acid in the screening medium, HA remained represents the content of hydroxycinnamic acid that has not been decarboxylated after fermentation by Y4, HA control represents the content of hydroxycinnamic acid in the blank sample incubated under the same culture conditions without inoculating Y4.

[0059] Use with SB-C 18High-performance liquid chromatography (HPLC) with a chromatographic column (4.6×250 mm, 5 μm) (Agilent, USA) and a diode array detector (DAD) was used to determine the concentrations of caffeic acid and ferulic acid. The injection volume was 10 μL, the column temperature was 40 °C, and the detection wavelength was 320 nm. The mobile phase consisted of phase A: 0.1% formic acid-water and phase B: 0.1% formic acid-acetonitrile, with A:B being 90:10, and the flow rate was 1 mL / min.

[0060] Detection method for vinyl phenol reductase activity (VPR) (the ability to reduce vinyl phenol to ethyl phenol substances)

[0061] Solid-phase microextraction gas chromatography-mass spectrometry (SPME / GC / MS) combined with single-ion monitoring was used for quantitative analysis of ethyl phenols in the fermentation broth. Gas chromatography conditions: Agilent Technologies 6890N instrument and MSD-5973N mass spectrometry detector were used. The chromatographic column was a DB-WAX column (30 m × 0.25 mm × 0.25 μm), and splitless injection was used. The fiber had a total desorption time of 3 minutes, following the following temperature program: 60 °C for 1 minute, then rising at a gradient of 10 °C / min to 150 °C, and then rising at a gradient of 3 °C / min to 210 °C, holding for 20 minutes. The helium flow rate was 1 ml / min. Detection was performed using SIM mode mass spectrometry.

[0062] Detection of pyran anthocyanin content

[0063] The pyran anthocyanins were detected using a WATERS ACQUITY UPLC system. Gradient elution was performed using mobile phase A (0.1% formic acid-water) and mobile phase B (acetonitrile). The gradient elution rate was 0.3 mL / min, as follows. 0 - 40 minutes, 0 - 30% B; 40 - 45 minutes, 30 - 80% B; 45 - 50 minutes, 80% - 100% B; 50 - 55 minutes, 100% - 0 B. The column used was BEH C 18 (2.1×150 mm, 1.7 μm). The column temperature was set at 45 °C. 5 μL of the sample was injected into the HPLC. The ESI parameters were: heater temperature 400 °C, sheath gas flow rate 70 arb, auxiliary gas flow rate 10 arb, sweep gas flow rate 2 arb, spray voltage 3 kV, transfer capillary temperature 350 °C. Mass spectrometry was performed in the positive scan mode (scan range m / z 20 - 2000). Fragmentation of the precursor ions was performed at a normalized collision energy of 30%. The data was processed by MassLynx 4.1 software.

[0064] Detection of flavor substances in blueberry fruit wine

[0065] The volatile substances in blueberry fermentation broth were detected using a gas chromatograph and a mass spectrometer. The column model was TG-WAXA (6 mm × 0.25 mm × 0.25 μm). 2-Octanol was used as the internal standard. The ion source type was the EI source, the temperature was 260 °C, the transfer line temperature was 230 °C, the injection port temperature was 250 °C, the helium flow rate was 1 mL / min, the electron energy was 70 eV, and the emission current was 25 μA.

[0066] Detection of Organic Acids in Blueberry Fruit Wine

[0067] HPLC detection method for organic acids: Chromatographic column: ACQΜITY BEH C 18 Chromatographic column (2.1×100 mm, 1.7 μm, Waters, USA), injection volume 5 μL, column temperature 40 °C, mobile phase A - water (containing 0.1% formic acid), B - methanol (containing 0.1% formic acid). The gradient elution conditions were 0 - 3 min, 10 - 30% B; 3 - 5 min, 30 - 50% B; 5 - 7 min, 50 - 90% B; 7 - 9 min, 90% B; 9 - 12 min, 90 - 30% B; 12 - 13 min, 30 - 10% B. The flow rate was 0.4 ml / min.

[0068] Example 1: Obtaining Metschnikowia pulcherrima Y4 and Its Characteristics Using the Primary Screening and Re - screening Methods

[0069] 1. Screening and Identification of Strains

[0070] The screening medium used was YPD medium (containing 1% yeast extract, 2% peptone, 2% anhydrous glucose per liter) supplemented with 0.01% bromocresol purple and 0.145% ferulic acid, caffeic acid, and p - coumaric acid respectively. Forty - five yeast strains isolated from grape skins were preserved in 40% glycerol tubes at - 20 °C after multiple purifications. The activated bacterial solution was cultured on the YPD screening medium by the streak plate method for 10 d, and the growth of yeast colonies and the color change of the plate on the screening medium were observed. And it was cultured in the corresponding liquid screening medium for 10 d, and the activities of hydroxycinnamic acid decarboxylase (HCDC) and vinylphenol reductase of the strains were calculated.

[0071] A strain of Metschnikowia pulcherrima Y4 that was both highly productive in hydroxycinnamic acid decarboxylase (HCDC) and did not produce vinylphenol reductase (VPR) was screened. While providing more precursors for the formation of pyranoanthocyanins, it would not produce off - odors of ethylphenol - like substances. It was the first reported strain of Metschnikowia pulcherrima Y4 with HCDC activity in China.

[0072] 2. Strain Identification

[0073] The genomic DNA of the strain Metschnikowia pulcherrima Y4 was extracted using a kit. The DNA fragment encoding the 26S ribosomal RNA of the strain was amplified by PCR using universal yeast ITS primers (SEQ ID NO.1). The PCR product was sent to Sangon Biotech for sequencing. The sequencing results were submitted to the GenBank database, subjected to BLAST and compared with other reported sequences, and a phylogenetic tree was constructed by the neighbor-joining method using MEGA 11.0 software. By constructing a phylogenetic tree with characteristic sequences, Metschnikowia pulcherrima Y4 was highly similar to Metschnikowia pulcherrima SN-25, and Metschnikowia pulcherrima Y4 was identified as Metschnikowia pulcherrima.

[0074] The colony morphology of Metschnikowia pulcherrima Y4 is shown in Figure 2 the figure below, and the microscopic examination image is shown in Figure 3 the figure below. The sequencing results and the phylogenetic tree are shown in Figure 4 the figure below.

[0075] 3. Result characterization of Metschnikowia pulcherrima Y4 during the screening process

[0076] (1) The color change of Metschnikowia pulcherrima Y4 on the YPD screening medium is shown in Figure 1 the figure below;

[0077] The results showed that on the 1st day of cultivation, the diameter of the purple color change zone was 0.1 cm; on the 3rd day of cultivation, the diameter of the yeast colony increased to 1 cm, and a light purple color change zone with a diameter of 1.5 cm appeared, and the range of the color change zone increased by 140% compared with the first day; on the 10th day of cultivation, the diameter of the yeast colony increased to 5 cm, and the purple color change zone covered the entire plate, which was 90 times larger than the first day.

[0078] (2) The conversion rates of ferulic acid, caffeic acid, and p-coumaric acid by Metschnikowia pulcherrima Y4 and the VPR activity of this strain are shown in Table 1 below.

[0079] Table 1: Conversion rates of ferulic acid, caffeic acid, and p-coumaric acid by Metschnikowia pulcherrima Y4 and VPR activity

[0080]

[0081] The results showed that Metschnikowia pulcherrima Y4 was highly productive in hydroxycinnamic acid decarboxylase (HCDC) and did not produce vinylphenol reductase (VPR). The conversion rate of Metschnikowia pulcherrima Y4 for ferulic acid was as high as 76.8 ± 0.1%, and the conversion rate of Metschnikowia pulcherrima Y4 for ferulic acid was as high as 88.6 ± 0.1%; the conversion rate of Metschnikowia pulcherrima Y4 for p-coumaric acid was as high as 76.8 ± 0.1%.

[0082] Example 2: Preparation of Blueberry Fruit Wine

[0083] The specific steps are as follows:

[0084] 1. Preparation of blueberry juice

[0085] (1) Screen and then crush and pulp the fresh blueberries to obtain a mixed liquid;

[0086] (2) Add 20 mg / kg of pectinase to the mixed liquid and carry out enzymatic hydrolysis at pH 2.5 and 30 °C for 2 h, with an enzyme activity of 30000 U / g, to prepare an enzymolyzed liquid;

[0087] (3) Adjust the total sugar content of the enzymolyzed liquid to 200 g / L and the pH to 2.5, and centrifuge at 4 °C with a refrigerated centrifuge at a speed of 8000×g for 10 min, and take the supernatant.

[0088] (4) Add 60 - 80 mg / kg of potassium metabisulfite to the supernatant for sterilization for 12 - 24 hours, and then prepare blueberry juice.

[0089] 2. Preparation of blueberry wine

[0090] (1) Pretreatment of blueberry juice

[0091] Place 300 ml of the blueberry juice obtained in step (1) into a 500 ml fermentation flask, and adjust the initial pH to 2.70 and the initial sugar content to 200 g·L -1 ;

[0092] (2) Preparation of Metschnikowia pulcherrima Y4 seed liquid

[0093] Inoculate the Metschnikowia pulcherrima Y4 yeast strain in YPD medium and activate it in a shaker at 30 °C at 200 rpm for 24 h to prepare Metschnikowia pulcherrima Y4 seed liquid;

[0094] (3) Preparation of blueberry wine

[0095] Inoculation method 1: Prepare commercial yeast K1 seed liquid according to the method in step (2), and inoculate the prepared seed liquid at 1.0×10 6 cells / mL into the blueberry juice treated in step (1);

[0096] Inoculation method 2: Inoculate the Metschnikowia pulcherrima Y4 seed liquid obtained in step (2) at 1.0×10 6 cells / mL into the blueberry juice treated in step (1);

[0097] Inoculation method 3: Prepare a commercial yeast K1 seed solution according to the method in step (2). Mix the commercial yeast K1 seed solution with the Metschnikowia pulcherrima Y4 seed solution in a ratio of viable bacteria of 1:4, and then inoculate the mixture into the blueberry juice treated in step (1); the total viable bacteria count for inoculation is 1.0×10 6 cells / mL;

[0098] Using the above three inoculation methods respectively, inoculate into blueberry juice, and place them in a constant temperature incubator at 25°C for static fermentation for 10 days. Seal the fermentation bottle mouth with a fermentation plug. Ferment for a total of 8 days to obtain blueberry wine 1 (corresponding to inoculation method 1), blueberry wine 2 (corresponding to inoculation method 2), and blueberry wine 3 (corresponding to inoculation method 3) respectively.

[0099] Example 3: Influence of Metschnikowia pulcherrima Y4 on blueberry fruit wine

[0100] Detect the alcohol content, formation of anthocyanins, stability of the blueberry fruit wine body, and flavor substances in the blueberry fruit wine after fermentation in Example 2 respectively, as follows:

[0101] 1. Detect the alcohol content of blueberry wine 1, blueberry wine 2, and blueberry wine 3 after fermentation in Example 2 respectively. The results are shown in Table 2 below.

[0102] Table 2: Alcohol content of fruit wines after fermentation with different inoculation methods

[0103] Inoculation method Alcohol content Single inoculation of commercial yeast K1 (blueberry wine one) 10±1%v / v Single inoculation of Y4 (blueberry wine two) 6.8±1%v / v Commercial yeast K1:Y4 = 1:4 (blueberry wine three) 8.1±1%v / v

[0104] Using this strain of Metschnikowia pulcherrima Y4 for mixed fermentation with commercial yeast K1 can reduce the alcohol content of blueberry wine, making the taste of blueberry wine softer and more suitable for the general public to drink.

[0105] 2. Influence of Metschnikowia pulcherrima Y4 on anthocyanins during blueberry fermentation

[0106] Detect and analyze the pyran anthocyanins in blueberry juice (obtained in step 1 of Example 2) and blueberry wine 2 after fermentation in Example 2 respectively. The results are as Figure 5 shown.

[0107] From Figure 5 it can be seen that through HPLC analysis of blueberry juice and blueberry wine 2 fermented with Metschnikowia pulcherrima Y4, three newly generated pyran anthocyanins were detected in the fermented blueberry fruit wine. That is, Metschnikowia pulcherrima Y4 can quickly convert caffeic acid and ferulic acid, and combine with three anthocyanins to form pyran anthocyanins with more stable structures.

[0108] The three pyran anthocyanins are (a) delphinidin-3-O-arabinoside-4-vinylcatechol, (b) cyanidin-3-O-arabinoside-4-vinylcatechol, and (c) malvidin-3-O-galactoside / glucoside-4-vinylcatechol. The specific addition process and content are shown in Table 3 below:

[0109] Table 3: Catalyzing the decarboxylation of caffeic acid to add anthocyanins to form corresponding pyran anthocyanins during the preparation of fruit wine from Y4-fermented blueberry juice

[0110]

[0111] 3. Influence of Metschnikowia pulcherrima Y4 on the stability of blueberry fruit wine

[0112] The pH differential method was used to determine the total monomeric anthocyanin content in the samples (Blueberry Wine II and Blueberry Wine III) after blueberry juice fermentation. Blueberry Wine II and Blueberry Wine III were respectively dissolved in 0.01 M KCl aqueous solution (pH = 1.0) at a ratio of sample: diluent 1:9 for stability analysis.

[0113] Take 10 mL each and place them under natural indoor light at 20 °C (simulating the daily storage environment of blueberry fruit wine). Measure the total anthocyanin content every other day for 6 consecutive days. The results are as Figure 6 shown.

[0114] As Figure 6 can be seen, the stability of Blueberry Wine II fermented by K1 alone is poor, while after using Metschnikowia pulcherrima Y4 and K1 for mixed fermentation to prepare Blueberry Wine III, the stability of blueberry fruit wine can be greatly increased. Therefore, adding Metschnikowia pulcherrima to ferment blueberry fruit wine can improve the stability of blueberry fruit wine.

[0115] 4. Influence of Metschnikowia pulcherrima Y4 on the flavor substances of blueberry fruit wine

[0116] The volatile substances in Blueberry Wine II were detected using a gas chromatograph and a mass spectrometer. The results are shown in Table 4.

[0117] Table 4: Changes in flavor substances in blueberry fruit wine after single fermentation by Metschnikowia pulcherrima

[0118]

[0119] The results showed that, as can be seen from Table 4, after 8 days of fermentation with Y4, the ethyl acetate concentration in the blueberry fermentation broth increased significantly, reaching 1332.18 μg / L. Moreover, the concentration of total esters in the fermented blueberry fruit wine had the same trend as the ethyl acetate concentration. Generally speaking, all esters may produce fruity and floral aromas. In particular, the increase in ethyl lactate will produce the aroma of coffee or strawberries and raspberries. The fermentation of Y4 can increase volatile substances, especially the formation of ester substances, making the taste of the finished blueberry fruit wine richer.

[0120] 5. Ability of Metschnikowia pulcherrima Y4 to reduce organic acids in blueberry fruit wine

[0121] The contents of organic acids in blueberry wine I (commercial yeast K1), blueberry wine II (Y4), and blueberry wine III (mixture of K1 and Y4) were detected by HPLC, and the results are shown in Table 5.

[0122] Table 5: Changes in the contents of organic acids in blueberry fruit wine after fermentation by Metschnikowia pulcherrima in different fermentation methods

[0123]

[0124] As can be seen from Table 5, the contents of the five main organic acids in the blueberry wine fermented by mixing Y4 and commercial yeast K1 during the fermentation process decreased. During the participation of Metschnikowia pulcherrima, the fermentation process slowed down, resulting in a decrease in microbial metabolites, a slowdown in substrate consumption, and a reduction in appropriate organic acids, which helped to improve the palatability of the fruit wine and make the blueberry wine body more refreshing.

[0125] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A strain of Metschnikowia pulcherrima ( Metschnikowia pulcherrima Metschnikowia pulcherrima ) Y4 was deposited at the China Center for Type Culture Collection on August 29, 2022, with the deposit number CCTCC NO: M 20221344.

2. A microbial inoculant, characterized in that, the inoculant contains the Metschnikowia pulcherrima Y4 described in claim 1 or its fermentation broth.

3. The microbial inoculant according to claim 2, characterized in that, the microbial inoculant is a solid inoculant or a liquid inoculant.

4. A composition containing the Metschnikowia pulcherrima Y4 described in claim 1.

5. Application of the Metschnikowia pulcherrima Y4 described in claim 1, or the microbial inoculant described in claim 2 or 3, or the composition described in claim 4 in the production of blueberry fruit wine.

6. A method for preparing blueberry fruit wine, characterized in that, the method uses a fermentation raw material containing blueberry juice and ferments to prepare fruit wine by using the Metschnikowia pulcherrima described in claim 1 and Saccharomyces cerevisiae.

7. A method for improving the stability of anthocyanins in fruit wine, characterized in that, the Metschnikowia pulcherrima Y4 described in claim 1 is added during the fermentation process of fruit wine.

8. A method for improving the flavor of fruit wine, characterized in that, the Metschnikowia pulcherrima Y4 described in claim 1 is added during the fermentation process of fruit wine.

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