Independently-fermented broad-spectrum color-protecting saccharomycetes and application thereof

Abstract

The invention discloses a single-fermentation broad-spectrum color-protecting saccharomycetes capable of protecting anthocyanin and reducing degradation of the anthocyanin. The strain is named as hanseniaspora Opuntiae DYG1, the strain is preserved in the Guangdong Microbial Culture Collection Center, the preservation number is GDMCC No: 65703, and the preservation date is December 31, 2024. The invention further discloses a preparation method of the strain. The saccharomyces cerevisiae is non-saccharomyces cerevisiae, is resistant to acid, wide in growth temperature range, resistant to ethanol concentration of 9%, resistant to high glucose and high SO2, capable of remarkably improving the anthocyanin retention rate after fermentation of anthocyanin-rich fruits is finished, and capable of protecting the broad spectrum of anthocyanin. The invention further provides application of the saccharomycetes DYG1 in fruit wine brewing, and further provides a method for brewing fruit wine by adopting the saccharomycetes DYG1, and the saccharomycetes DYG1 has important guiding significance in strain screening and process optimization for brewing the fruit wine rich in anthocyanin.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and relates to a yeast, in particular to a yeast that is fermented alone and has broad-spectrum color protection, i.e., anthocyanin protection and anthocyanin degradation reduction, and application of the yeast. Background Art

[0002] Anthocyanins are water-soluble pigments widely found in plants, typically stored as glycosides within the vacuoles of epidermal cells. They are the primary colorant in a variety of fruits and vegetables, including grapes, blueberries, mulberries, strawberries, red cabbage, and purple sweet potatoes, as well as in juices, wines, and other products. However, anthocyanins are easily degraded during fermentation due to temperature, pH, oxygen, enzymes, metal ions, ascorbic acid, sulfur dioxide, yeast fermentation, and adsorption to cell walls.

[0003] Saccharomyces cerevisiae is the dominant yeast species in fruit wine fermentation. To regulate the winemaking process and prevent spoilage, numerous strains of S. cerevisiae are used as commercial starter yeasts. The impact of S. cerevisiae on wine color during fermentation primarily focuses on yeast metabolism, its metabolites, and adsorption to yeast cell walls. During the winemaking process, S. cerevisiae cell walls have been shown to adsorb compounds in wine, significantly impacting wine quality. Yeast also produces specific enzymes (such as glucosidase and phenoloxidase) that hydrolyze the glycosidic bonds of anthocyanins, leading to their degradation. After hydrolysis, anthocyanins may be converted to their non-pigmented form, reducing their stability and color. Oxidative reactions initiated by yeast during metabolism can also lead to oxidative degradation of anthocyanins. Oxidation disrupts the molecular structure of anthocyanins, resulting in color changes and reduced activity. Therefore, the metabolic characteristics of the yeast strain itself will also have a certain negative impact on the stability of anthocyanins in fruit wine. However, there is currently a lack of relevant systematic research on whether its negative impact is greater or smaller than that caused by cell wall adsorption.

[0004] In the past, non-Saccharomyces yeasts were often considered insignificant or strictly considered contaminants. Currently, an increasing number of non-Saccharomyces yeasts are being used in the winemaking process, significantly impacting the fermentation quality, flavor, and color of wine. However, while existing research focuses on the regulation of flavor components by non-Saccharomyces yeasts, systematic screening for their color-protecting properties remains lacking. While a significant amount of research has focused on the regulation of flavor by non-Saccharomyces yeasts in wine fermentation, color research has primarily focused on the production of anthocyanin derivatives. However, there are few reports on strains that significantly enhance anthocyanin retention or reduce anthocyanin degradation.

[0005] The types and structures of anthocyanins vary significantly among different fruits. Grapes primarily contain malvidin 3-O-glucoside (M3G), which is chemically stable. Blueberries are primarily M3G, but other derivatives (such as malvidin 3-O-galactoside) also coexist. Mulberries primarily contain cyanidin 3-O-glucoside (C3G), which is less stable than M3G. Strawberries, on the other hand, primarily contain pelargonidin 3-O-glucoside (Pe3G), which is more sensitive to pH changes. This structural diversity leads to significant differences in the stability of anthocyanins from different sources during fermentation. For example, wine retains a high anthocyanin rate after fermentation (reaching 60%-80%), while blueberry and mulberry wines often retain less than 50%. Currently, fermentation process research for non-grape wines (such as blueberries, mulberries, and strawberries) cannot directly replicate the strain selection and fermentation processes used for wine. Currently, there is a lack of fermentation strains with broad-spectrum color-protecting properties that can be used in fermentations of different fruits. Summary of the Invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a single fermentation yeast that has broad-spectrum color protection, i.e., protects anthocyanins and reduces anthocyanin degradation.

[0007] The broad-spectrum color-protecting yeast described in the present invention is named Hanseniaspora Opuntiae DYG1 and is deposited with the Guangdong Provincial Microbial Culture Collection Center under the deposit number GDMCC No: 65703 and the deposit date of December 31, 2024. It is hereinafter referred to as DYG1 or D1.

[0008] The second object of the present invention is to provide an application of the yeast DYG1 of the present invention, specifically an application of the yeast DYG1 in fruit wine brewing.

[0009] Preferably, the raw material of the fruit wine is a fruit selected from the following: grapes, mulberries, strawberries, blueberries, and pomegranates.

[0010] The third object of the present invention is to provide a method for brewing fruit wine using the yeast of the present invention.

[0011] The method for brewing fruit wine using the yeast of the present invention comprises the following steps:

[0012] A. Wash and juice the fruit to be fermented, add pectinase for enzymatic hydrolysis, filter, adjust sulfur and sugar to obtain juice;

[0013] B. pasteurizing the juice obtained in step A, cooling it to room temperature, adding the yeast of the present invention, and mixing them uniformly to obtain a juice to be fermented;

[0014] C. The juice to be fermented obtained in step B is placed in a fermentation tank with a one-way valve, with a liquid volume of 50% by volume, a fermentation temperature of 30° C., and an anaerobic fermentation for 18 days.

[0015] Preferably, in step A, the enzymatic hydrolysis temperature of the pectinase is 45 to 50° C., and the enzymatic hydrolysis time is 3 to 4 hours.

[0016] Preferably, in step B, the amount of yeast added is 1.0×10 9 The above yeast strains were calculated.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The present invention discloses for the first time a color-protecting yeast strain DYG1 that can significantly improve the retention rate of anthocyanins in fruit wine during the fermentation process of fruit wine, wherein the retention rate of anthocyanins such as cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, pelargonidin-3-O-glucoside, paeoniflorin-3-O-glucoside, and petunidin-3-O-glucoside is improved by more than 200%, and the strain has a broad spectrum of anthocyanin protection, which has important guiding significance for the subsequent strain screening and process optimization of anthocyanin-rich fruit wine brewing.

[0019] (2) Compared with the commercial brewing yeast mostly used to brew fruit wine, the yeast DYG1 provided by the present invention is a non-brewing yeast and can also be used in a separate fermentation process. It also has advantages that brewing yeast does not have, such as low alcohol content and enhanced flavor complexity.

[0020] (3) The yeast DYG1 described in the present invention has a better anthocyanin retention rate during the fermentation of anthocyanin-rich fruits than commercial brewer's yeast additives on the market, and effectively improves the anthocyanin retention rate of various anthocyanin-rich fruits during and after fermentation.

[0021] (4) The yeast DYG1 described in the present invention has good physiological and biochemical characteristics: acid resistance, wide growth temperature range, ethanol concentration tolerance of up to 9%, and tolerance to high sugar and high SO2, so it has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the phylogenetic tree of Hanseniaspora Opuntiae DYG1 and SY.

[0023] Figure 2Figure 1: The effects of commercial yeast SY and DYG1 (abbreviated as D1) on the color (A); total anthocyanin content (B) and retention (C); C3G content (D) and retention (E); and C3R content (F) and retention (G) of simulated mulberry anthocyanin juice using different fermentation methods. C3G is cyanidin-3-O-glucoside, and C3R is cyanidin-3-O-rutinoside. D1S refers to simultaneous inoculation of DYG1 and SY for mixed fermentation, while 4D1S refers to inoculation of DYG1 for 4 days followed by SY.

[0024] Figure 3 Figure 5. The effects of commercial yeast SY and DYG1 (abbreviated as D1) on the viable yeast count (A), total anthocyanin degradation rate (B), C3G degradation rate (C), C3R degradation rate (D), sludge color (E), L* (F), a* (G), b* (H), and ΔE (I) in a simulated mulberry anthocyanin juice fermented using different fermentation methods. C3G is cyanidin-3-O-glucoside, and C3R is cyanidin-3-O-rutinoside. D1S refers to simultaneous inoculation of DYG1 and SY for mixed fermentation, while 4D1S refers to inoculation of DYG1 for 4 days followed by SY.

[0025] Figure 4 Figure 3. The pH (A), acidity (B), residual sugar (C), and ethanol (D) of mulberry anthocyanin-containing simulated juice after 18 days of fermentation using commercial yeast SY and DYG1 (abbreviated as D1) in different fermentation modes. D1S refers to the simultaneous inoculation of DYG1 and SY for mixed fermentation, while 4D1S refers to the inoculation of DYG1 for 4 days followed by SY.

[0026] Figure 5 Figure 1 shows the effects of different fermentation methods on the color (A) of C3G simulated juice; C3G content (B) and retention rate (C); viable yeast count (D), total anthocyanin degradation rate (E), and sludge color (F). C3G is cyanidin-3-O-glucoside; D1S is a mixed fermentation method in which DYG1 and SY are inoculated simultaneously.

[0027] Figure 6 Figure 1 shows the color (A) of C3G simulated juice after one day of fermentation using commercial yeast SY and DYG1 (abbreviated as D1); C3G content (B) and retention rate (C); viable yeast count (D), total anthocyanin degradation rate (E), and sludge color (F). C3G is cyanidin-3-O-glucoside. Min is the minimum value, NS is not significant, and ** indicates extremely significant (P < 0.001). D1S represents a mixed fermentation using DYG1 and SY.

[0028] Figure 7Color change (A); anthocyanin retention rate (B); yeast viable count (C); and glucose content (D) changes after one day of fermentation of different anthocyanins by commercial yeast SY and DYG1 (abbreviated as D1).

[0029] Figure 8 Effects of commercial yeast SY and DYG1 (abbreviated as D1) fermentation for different times on the total anthocyanin retention rate and wine color (F) of mulberry (A), blueberry (B), grape (C), strawberry (D), and pomegranate (E)

[0030] Figure 9 Effects of commercial yeast SY and DYG1 (abbreviated as D1) fermentation on the retention of different anthocyanins in mulberry wine (A and B), blueberry wine (C and D), strawberry wine (E and F), grape wine (G and H and I and J), and pomegranate wine (K and L)

[0031] Figure 10 The results show the effects of commercial yeast SY and DYG1 (abbreviated as D1) fermented separately for different times on the number of viable fruit wine yeasts (A); total anthocyanin retention rate (B); C3G (C), M3G (D), M3Gt (E), C3R (F), C35G (G), Dp3G (H), Pn3G (I), Pe3G (J), Pe3R (K) and sludge color (L). Among them, C3G is cyanidin-3-O-glucoside, C3R is cyanidin-3-O-rutinoside, M3G is malvidin-3-O-glucoside, M3Gt is malvidin-3-O-galactoside, Pe3G is pelargonidin-3-O-glucoside, Pe3R is pelargonidin-3-O-rutinoside, Dp3G is delphinidin-3-O-glucoside, Pn3G is peonydin-3-O-glucoside, and C35G is cyanidin-3,5-O-glucoside.

[0032] Figure 11 Effects of commercial yeast SY and DYG1 (abbreviated as D1) fermentation time on mulberry wine (A), blueberry wine (B), wine (C), strawberry wine (D), and pomegranate wine (E); L*(a), a*(c), b*(c), and ΔE(d).

[0033] Figure 12 Effects of commercial yeast SY and DYG1 (abbreviated as D1) fermentation for different times on mulberry wine (A), blueberry wine (B), wine (C), strawberry wine (D); pomegranate wine (E); pH (a), total acid (c), residual sugar (d), and ethanol after 18 days of fermentation (d). DETAILED DESCRIPTION

[0034] The present invention will be described in further detail below with reference to the embodiments and accompanying drawings, but the embodiments do not limit the present invention in any form.

[0035] Example 1: Isolation, screening, physiological properties and identification of yeast

[0036] A total of 292 yeast strains were isolated from 30 materials including grapes, bananas, strawberries, doyi fruit, mangoes, watermelons, mulberries, blueberries, and lemons. Crystal violet staining and cell morphology observation were performed. Based on tolerance tests (pH tolerance reached 2.5, ethanol concentration tolerance reached 6%, and glucose concentration tolerance reached 20%) and microporous fermentation of mulberry juice medium, the yeast with the highest total anthocyanin retention rate was screened out. This strain was named DYG1, hereinafter referred to as D1.

[0037] The specific steps for yeast isolation and screening are as follows:

[0038] Sterilize a conical flask containing 50 mL of YPD liquid medium, a test tube containing 9 mL of distilled water, a WL solid medium, and a plate. After sterilization, pour the WL medium into the plate. Aseptically, take 10 g of sample into a conical flask containing 50 mL of malt extract liquid medium and shake it on a shaker at 180 rpm for 30 minutes. Then, take 1 mL of the diluted sample solution and add it to a test tube containing 9 mL of sterile distilled water and shake it evenly to dilute it by 10 -1 Concentration, now take 0.01mL of the liquid and spread it on the plate, incubate it anaerobically at 30℃ for 24-48h, then use an inoculation needle to pick up a single colony, streak it onto the WL plate, incubate it anaerobically at 30℃ for 24-48h, repeat the plate streaking separation until a single colony is obtained, insert the single colony into the center of the slope of the test tube containing YPD solid culture medium with an inoculation needle, and store it in a refrigerator at 4℃.

[0039] The 292 purified yeast strains were then inoculated into 48-well cell culture plates with 0.9 mL of mulberry juice medium (mulberry juice: YPD liquid medium at a ratio of 1:1, pasteurized) in each well, with three replicates for each strain. After anaerobic fermentation at 30°C for 3 days, the retention rate of anthocyanins was measured, and the strain with the highest retention rate and good tolerance was screened out, namely the DYG1 described in the present invention.

[0040] The main physiological and biochemical characteristics of the strain DYG1 are shown in Table 1.

[0041] Table 1: Physiological and biochemical characteristics of strain DYG1

[0042] index DYG1 index DYG1 index DYG1 shape Spindle Growth temperature Ethanol concentration <![CDATA[SO2 concentration]]> 15℃ + 3％ ++++ 100mg / L ++++ 20℃ +++ 6％ ++ 200mg / L ++++ 25℃ +++ 9％ ± 300mg / L ++++ 30℃ ± 12％ - 400mg / L +++ 35℃ 15％ - 500mg / L +++ 40℃ 18％ - 600mg / L +++ Growth pH Glucose concentration 2.00 + 10％ ++++ 2.50 +++ 15％ ++++ 3.00 +++ 20％ ++++ 3.50 +++ 25％ ++++ 4.0 ++++ 30％ +++ 4.50 +++ 40％ +++

[0043] +++: vigorous growth; ++: good growth; +: growth; ±: weak growth; -: no growth.

[0044] As shown in Table 1, DYG1 is acid-resistant, has a wide growth temperature range, can tolerate ethanol concentrations up to 9%, and is resistant to high sugar and high SO2.

[0045] The ingredients and preparation method of the above-mentioned YPD medium are as follows: 20.0 g of peptone, 10.0 g of yeast extract, 20.0 g of glucose, 20 g / L of agar (Agar) added to the solid medium, and distilled water (H2O) is added to 1000 mL. The mixture is sterilized at 121°C for 20 min.

[0046] The WL medium contains 5.0 g of yeast extract, 5.0 g of acid hydrolyzed casein, 50.0 g of glucose, 0.55 g of potassium dihydrogen phosphate (KH2PO4), 0.425 g of potassium chloride (KCl), 0.125 g of calcium chloride (CaCl2), 0.125 g of magnesium sulfate (MgSO4), 0.0025 g of ferric chloride (FeCl3), 0.0025 g of manganese sulfate (MnSO4), and 0.0025 g of bromocresol green (C 21 H 14 Br4O5S) 0.022g, agar 17.0g.

[0047] Identification of yeast species

[0048] First, the purity of the screened yeast was confirmed by multiple plate streaking and microscopic examination. Molecular identification used ITS1 (5'-TCCGTAGGTGAACCTGCG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') primers, and DNA extraction and analysis were performed according to the method described in the yeast DNA extraction kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.). PCR amplification was performed according to the method described in the kit, and the amplified products were sequenced by Shanghai Paisonno Biotechnology Co., Ltd. The obtained sequences were compared with the sequences of described species in the GenBank database and analyzed using the BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST / ) tool. The D1 / D2 region of the 26S rDNA was aligned using the MEGA6 software package, and a phylogenetic tree was constructed by the neighbor-joining method ( Figure 1 ), thus confirming that DYG1 is Hanseniaspora Opuntiae.

[0049] The inventors deposited Hanseniaspora Opuntiae DYG1 in the Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No: 65703 and the deposit date of December 31, 2024.

[0050] Example 2: Fermentation experiment of DYG1 and commercial yeast SY on mulberry anthocyanin simulated juice

[0051] The ingredients of mulberry anthocyanin simulated juice are: glucose 240g / L, yeast extract 1.5g / L, citric acid 0.3g / L, tartaric acid 5g / L, L-malic acid 5g / L, ammonium sulfate 2g / L, potassium dihydrogen phosphate 5g / L, magnesium sulfate 0.4g / L, sodium chloride 0.2g / L, and manganese sulfate 0.05g / L.

[0052] To prepare the simulated mulberry anthocyanin juice, adjust the pH of the solution to 4.0 with 0.5 mol / L sodium hydroxide and sterilize at 121°C for 20 minutes. After cooling to room temperature, add mulberry anthocyanins at a concentration of 1200 mg / L (similar to the total anthocyanin content of mulberry juice) with a purity of ≥95%. The mulberry anthocyanins were first dissolved in water, and the solution was sterilized by passing through a 0.22 μm filter before addition.

[0053] The group treatments included: SY (commercial yeast SY inoculated alone), D1 (DYG1 inoculated alone), D1S (DYG1 and SY co-inoculated), and 4D1S (DYG1 inoculated alone and fermented for 4 days before inoculation with SY).

[0054] The yeast strain (DYG1) and commercial yeast (SY, purchased from Angel Yeast Co., Ltd.) were activated twice in YPD liquid medium. 6 An inoculum of 100 cfu / ml was inoculated into a 100 ml fermentation vessel equipped with a one-way valve. The simulated juice was filled to 50% of its original volume, and the fermentation temperature was set at 30°C under anaerobic conditions. Three replicates were set for each treatment. Sampling was performed at 3, 6, 9, 12, and 18 days. Parameters measured included anthocyanin retention, CIE-LAB value, viable yeast cell count, anthocyanin degradation rate, yeast cell color, and fermentation quality indicators (pH, total titratable acidity, residual sugar, and ethanol content on day 18).

[0055] Here are the results:

[0056] like Figure 1 As shown in Figure 2, the degradation of anthocyanins in the simulated juice by SY mainly occurred in the first three days of fermentation, and nearly 90% of C3G was degraded during this period. At the same time, the mixed fermentation of DYG1 and SY and the sequential fermentation did not have a significant inhibitory effect on the degradation of C3G ( Figure 1F and G). The degradation of C3R is also mainly concentrated in the first 3 days ( Figure 1 D and E), and then the degradation rate slowed down, and the overall degradation rate was much lower than that of C3G. DYG1 fermentation alone had the best protection effect on C3G and C3R.

[0057] like Figure 2 As shown, during the first three days of fermentation, the number of viable bacteria in each treatment was in the hundreds of millions ( Figure 2 A), and there was no significant difference, indicating that DYG1 did not significantly degrade anthocyanins and it was not caused by the difference in viable bacteria count. Total anthocyanins ( Figure 2 B) C3G Figure 2 C) and C3R( Figure 2 The peak degradation rate of C3G occurred in the first 3 days and then decreased rapidly. Although sequential fermentation delayed the time when the degradation rate of C3G reached its peak, it had little effect on the final result. It is worth noting that in the first 3 days of fermentation, all yeast sludges were pink and white and the number of live bacteria did not decrease significantly ( Figure 2 A and E), indicating that there was no significant death of yeast cells and a decrease in cell viability, but significant adsorption occurred, indicating that the large-scale degradation of anthocyanins at this time was mainly due to metabolic degradation of the yeast itself. After 6 days of fermentation, the number of viable yeast cells decreased significantly and the color of the sludge began to turn black, indicating that the yeast cell wall began to adsorb anthocyanins on a large scale, which may be the reason for the loss of anthocyanins in the later stage. The degradation of color by SY mainly occurred in the first 3 days of fermentation, and the red color faded visibly with the naked eye ( Figure 1 A), L* value increased significantly ( Figure 2 F), the a* value decreased significantly ( Figure 2 G), ΔE value increased significantly ( Figure 2 K). The color of the fermentation with D1 alone was very stable, and the ΔE value tended to change slowly after the third day, indicating that D1 alone could effectively maintain the stability of mulberry anthocyanins, and the total anthocyanin retention rate could be increased by 252% compared with commercial yeast SY ( Figure 1 B), C3G's retention rate increased by 390% ( Figure 1 G), C3R retention rate increased by 32% ( Figure 1 E). Figure 3 The fermentation quality-related indicators shown indicate that the fermentation quality of SY and D1 meets industry standards. The alcohol content of DYG1 can reach about 5%, which can be used for low-alcohol fruit wine brewing.

[0058] Example 3: Fermentation experiment of DYG1 and commercial yeast SY on C3G simulated juice

[0059] The ingredients of C3G simulated juice are: glucose 240g / L, yeast extract 1.5g / L, citric acid 0.3g / L, tartaric acid 5g / L, L-malic acid 5g / L, ammonium sulfate 2g / L, potassium dihydrogen phosphate 5g / L, magnesium sulfate 0.4g / L, sodium chloride 0.2g / L, and manganese sulfate 0.05g / L.

[0060] To prepare simulated C3G juice, adjust the pH of the solution to 4.0 with 0.5 mol / L sodium hydroxide and sterilize at 121°C for 20 minutes. After cooling to room temperature, add C3G at a concentration of 700 mg / L (similar to the C3G content in mulberry juice) with a purity of ≥95%. Dissolve the C3G in water, then sterilize the solution by filtering it through a 0.22 μm filter before adding.

[0061] The group treatments included: SY (commercial yeast SY inoculated alone), D1 (DYG1 inoculated alone), and D1S (DYG1 and SY co-inoculated).

[0062] The yeast strain (DYG1) and commercial yeast (SY, purchased from Angel Yeast Co., Ltd.) were activated twice in YPD liquid medium. 6 An inoculum of 100 cfu / ml was inoculated into a 100 ml fermentation vessel equipped with a one-way valve. The simulated juice was filled to 50% of its original volume, and the fermentation temperature was set at 30°C under anaerobic conditions. Three replicates were used for each treatment. Sampling was performed at 0.5, 1, 2, 3, 6, 9, 12, and 18 days. Parameters measured included C3G retention, viable yeast cell count, C3G degradation rate, and yeast cell color.

[0063] Here are the results:

[0064] like Figure 3 As shown, the degradation of C3G by SY mainly occurred in the first day of fermentation, with obvious color fading ( Figure 3 A), the degradation was close to 90% within the first day of fermentation. The mixed fermentation of DYG1 and SY failed to inhibit the degradation of C3G by SY ( Figure 3 B and C), while the C3G retention rate of DYG1 alone increased by 338% compared with SY, which is the same trend as the results of mulberry anthocyanin simulated juice fermentation in Example 2 ( Figure 1 F and G). On the first day of fermentation, the number of viable bacteria among the treatments was in the hundreds of millions and there was no significant difference ( Figure 3 G, P>0.05), indicating that the degradation of C3G is not related to the difference in viable cell counts between strains. The mixed fermentation of DYG1 and SY can reduce the degradation rate of C3G on the first day of fermentation, but has little effect on the final results. The peak degradation rate of SY for C3G occurs on day 0.5, while the peak degradation rate of DYG1 occurs between day 0.5 and day 1 ( Figure 3H). The bacterial sludge at this time is obviously white, and the number of live bacteria is also the highest value, indicating that the bacterial activity is good and no anthocyanins are adsorbed. This shows that the degradation of C3G at this time is mainly due to the metabolism of yeast, and has little to do with bacterial adsorption ( Figure 3 I), further demonstrating the high efficiency and specificity of DYG1 fermentation alone in reducing anthocyanin degradation.

[0065] Example 4: Fermentation experiment of DYG1 and various commercial yeasts on C3G simulated juice

[0066] Objective: To investigate whether the rapid degradation of C3G by commercial Saccharomyces cerevisiae is common.

[0067] The inventors collected brewer's yeast for anthocyanin-rich fruit wine fermentation sold by world-renowned brewer's yeast manufacturers for verification testing. The relevant strain numbers and origins are shown in Table 2.

[0068] Table 2: Yeast species and sources used in this example

[0069]

[0070]

[0071] The ingredients of the simulated fruit juice are as follows: glucose 240 g / L, yeast extract 1.5 g / L, citric acid 0.3 g / L, tartaric acid 5 g / L, L-malic acid 5 g / L, ammonium sulfate 2 g / L, potassium dihydrogen phosphate 5 g / L, magnesium sulfate 0.4 g / L, sodium chloride 0.2 g / L, and manganese sulfate 0.05 g / L.

[0072] To prepare the simulated juice, adjust the pH of the solution to 4.0 with 0.5 mol / L sodium hydroxide and sterilize at 121°C for 20 minutes. After cooling to room temperature, add C3G at a concentration of 700 mg / L (similar to the C3G content in mulberry juice) with a purity of ≥95%. Dissolve the C3G in water and sterilize the solution by filtering it through a 0.22 μm filter before adding.

[0073] The group treatments included: SY (commercial yeast SY inoculated alone) and 18 other commercial yeasts available globally (see Table 2 ), and D1 (DYG1 inoculated alone).

[0074] The yeast strain (DYG1), commercial yeast (SY, purchased from Angel Yeast Co., Ltd.) and 18 other commercial yeast strains listed in Table 2 were activated twice in YPD liquid medium. 6An inoculum of 100 cfu / ml was inoculated into a 100 ml fermentation vessel equipped with a one-way valve. The simulated juice was filled to 50% of its original volume, and the fermentation temperature was set at 30°C under anaerobic conditions. Three replicates were used for each treatment, and sampling was performed every day. Parameters measured included C3G content and retention rate, viable yeast cell count, glucose content, and yeast cell color.

[0075] Here are the results:

[0076] like Figure 6 As shown in the figure, after 1 day of fermentation, the color of all C3G simulated juices fermented by commercial yeasts faded rapidly, and the color of the bacterial mud was lighter ( Figure 6 A), different commercial yeasts have very obvious commonalities in the degradation of C3G ( Figure 6 B and C), and the degradation rate is very fast, although the number of viable bacteria ( Figure 6 D) and glucose consumption ( Figure 6 E) Although there are differences among strains, the degradation of C3G can reach about 90% within 1 day. This may be the main reason why anthocyanins are degraded in large quantities and the color fades rapidly during the brewing process of fruits rich in C3G.

[0077] Example 5: Fermentation experiment of DYG1 and commercial yeast SY on simulated juices with different anthocyanins

[0078] Purpose of the experiment: To explore the types and key structures of anthocyanins that can resist degradation by Saccharomyces cerevisiae.

[0079] The inventors prepared simulated fruit juices containing 12 different types and structures of anthocyanins commonly found in nature. The classification and basic information of these anthocyanins are shown in Table 3.

[0080] Table 3: Classification and basic information of different anthocyanins

[0081]

[0082] The ingredients of the simulated fruit juice are as follows: glucose 240 g / L, yeast extract 1.5 g / L, citric acid 0.3 g / L, tartaric acid 5 g / L, L-malic acid 5 g / L, ammonium sulfate 2 g / L, potassium dihydrogen phosphate 5 g / L, magnesium sulfate 0.4 g / L, sodium chloride 0.2 g / L, and manganese sulfate 0.05 g / L.

[0083] To prepare simulated juice, adjust the pH of the solution to 4.0 with 0.5 mol / L sodium hydroxide and sterilize at 121°C for 20 minutes. After cooling to room temperature, add 500 mg / L of various anthocyanins with a purity of ≥95% (see Table 3). Dissolve the various anthocyanins in water, then sterilize the solution by passing it through a 0.22 μm filter membrane before adding.

[0084] The group treatments included: SY (commercial yeast SY inoculated alone) and D1 (DYG1 inoculated alone).

[0085] The yeast strain (DYG1) and commercial yeast (SY, purchased from Angel Yeast Co., Ltd.) were activated twice in YPD liquid medium. 6 An inoculum of 100 cfu / ml was inoculated into a 100 ml fermentation vessel equipped with a one-way valve. The simulated juice was filled to 50% of its original volume, and the fermentation temperature was set at 30°C under anaerobic conditions. Three replicates were used for each treatment, and the fermentation time was set to 1 day. Parameters measured included anthocyanin retention, viable yeast cell count, glucose content, and yeast cell color.

[0086] Here are the results:

[0087] From the perspective of wine color, different anthocyanins have obvious differences in color ( Figure 7 A) Combined Figure 7 B. The data on the retention rates of different anthocyanins showed that after 1 day of fermentation with Saccharomyces cerevisiae SY, anthocyanins such as Dp3G, Pe3G and C3G without methoxy groups on the mother nucleus and with glucose as glycosides were very unstable. At the same time, Pn3G with a methoxy group on the mother nucleus was also very unstable. M3G with two methoxy groups on the mother nucleus was very stable in color, and acetylated anthocyanins were also relatively stable. C35G and C37G, whose hydroxyl groups on the mother nucleus were replaced by two glucoses, were relatively more stable than C3G, among which C35G was more stable than C37G. Cyanidin 3-O-rhamnoside (C3Gt), whose mother nucleus was also cyanidin, was relatively more stable than C3G, and C3R and Pe3R, which had rutinose as glycosides, were both very stable. The retention rates of different anthocyanins before and after DYG1 fermentation were all above 90%, and there was no significant difference, indicating that it may have obvious broad-spectrum properties in protecting anthocyanins ( Figure 7 B).

[0088] Example 6: Fermentation experiment of DYG1 and commercial yeast SY on juices of different fruits

[0089] In order to verify the results of the above simulation system, the inventors selected 5 common anthocyanin-rich fruits used for fruit wine production for verification. The types of fruits and the main types of anthocyanins are shown in Table 4. All fruits were purchased from the Jiangnan Fruit Wholesale Market in Guangzhou.

[0090] Table 4: Different fruit types, their abbreviations and their main anthocyanin contents

[0091]

[0092] Wash and juice the five fruits separately, then add pectinase (0.04% by volume) for enzymatic hydrolysis. For example, the hydrolysis temperature is 45°C and the hydrolysis time is 4 hours, or the hydrolysis temperature is increased to 50°C and the hydrolysis time is adjusted to 3 hours. The juices are then filtered and the sulfur and sugar levels are adjusted. In this example, sucrose is used to adjust the sugar content to 240g / L and the sulfur content to 0.09g / L. The different juices are then pasteurized, cooled to room temperature, and set aside.

[0093] The group treatments included: SY (commercial yeast SY inoculated alone) and D1 (DYG1 inoculated alone).

[0094] The yeast strain (DYG1) and commercial yeast (SY, purchased from Angel Yeast Co., Ltd.) were activated twice in YPD liquid medium. 9 An inoculum of 100 cfu / L was inoculated into a 100 ml fermentation vessel equipped with a one-way valve. The volume of the different fruit juices was 50% (volume percentage), and the fermentation temperature was set at 30°C, with anaerobic fermentation. Three replicates were set for each treatment. Sampling was performed at 1, 3, 6, 9, 12, and 18 days. Measurement parameters included fermentation kinetics, retention of total anthocyanins and different anthocyanins, CIE-LAB value, viable yeast cell count, and fermentation quality indicators (pH, total titratable acidity, residual sugar content, and ethanol content on day 18).

[0095] Here are the results:

[0096] like Figure 8 As shown, mulberry ( Figure 8 A) Grapes Figure 8 C) Strawberry Figure 8 D) and pomegranate (Fig.8E) total anthocyanins showed a rapid decrease in the first day of fermentation, while the decline rate of blueberry was significantly slower ( Figure 8 B), the color degradation of mulberry juice mainly composed of C3G and C3R was visible to the naked eye after 1 day of fermentation, and it was more obvious after 3 days ( Figure 8 F), the main anthocyanins of blueberry are M3G and M3Gt, which contain two methoxy groups on the mother core and are relatively more stable. The color degradation will not be obvious until 3 days after fermentation ( Figure 8 F); Grape juice containing mainly M3G showed obvious color degradation after 9 days of fermentation; Strawberry juice containing mainly Pe3G showed significant color degradation after 1 day of fermentation; Pomegranate juice containing mainly C3G and C35G showed obvious color degradation after 3 days of fermentation, and the degradation was more obvious after 6 days. Figure 9 As shown in C3G( Figure 9 A and I and L), Dp3G ( Figure 9 G), Pe3G( Figure 9 E) and Pn3G( Figure 9H) were rapidly degraded within the first day of SY fermentation, with C3G and Pe3G both degrading by more than 90%, which was the same as the simulation system in Example 5 and had nothing to do with the type of fruit raw materials. Dp3G exceeded 60%, and Pn3G exceeded 70%, with a trend very close to that of the simulation system in Example 5. M3G ( Figure 9 C and J), C3R( Figure 9 B) C35G( Figure 9 K) and Pe3R( Figure 9 F) are relatively stable, and the trend is very close to that of the simulation system in Example 5 ( Figure 7 B), M3Gt( Figure 9 D) is also relatively stable, similar to the M3G results ( Figure 9 C and J). The viable bacterial counts of SY and DYG1 decreased rapidly and significantly after 6 days ( Figure 10 A), while the peak of total anthocyanin degradation is mainly concentrated in the first 1 day ( Figure 10 B) and the bacterial sludge is less and the color is obviously white 1d ( Figure 10 L) indicates that no obvious anthocyanin adsorption occurs. The peak degradation rate of different anthocyanin monomers is concentrated in the first 3 days, and the degradation rate after 9 days of fermentation is much lower than that of the first 3 days ( Figure 10 CK), and DYG1 began to turn black after 3 days of fermentation of mulberry juice and blueberry juice, while SY did not change color significantly until 9 days after fermentation of mulberry juice, grape juice, strawberry juice and pomegranate juice, and did not start to turn black until 12 days after fermentation of blueberry juice, indicating that the degradation and adsorption of anthocyanins in the early stage of fermentation were not closely related ( Figure 10 L). Figure 11 The color index changes shown in the figure further verify the degradation results of the above-mentioned different anthocyanins. The color-related indicators of anthocyanin-rich fruits fermented by DYG1 are more stable than those of SY after fermentation, especially for a* value and ΔE value ( Figure 10 B and F), further indicating that the color protection effect of DYG1 is broad-spectrum across fruit varieties. In addition, fermentation quality indicators showed that the ethanol yield of DYG1 was low, but the fermentation indicators of different fruit wines all met the relevant industry standards ( Figure 12 ), can be used for the processing and utilization of low-alcohol fruit wine or low-alcohol related fruit fermented beverages.

[0097] In Examples 2 to 6 above, the determination methods for anthocyanin retention rate, fermentation quality, and viable yeast count are as follows:

[0098] (1) Determination of total anthocyanin content:

[0099] The total anthocyanin content (TA) of the samples was determined as follows: the samples were diluted with appropriate amounts of hydrochloric acid-potassium chloride buffer (pH 1.0) and hydrochloric acid-sodium acetate buffer (pH 4.5), incubated in the dark for 30 minutes, and the absorbance at 520 nm and 700 nm was recorded. TA was expressed as milligram equivalents of cyanidin-3-O-glucoside using the following formula: ((A520-A700)pH1.0 - (A520-A700)pH4.5 × MW × DF × 10 3 ) / (ε×L). In this formula, "A" represents absorbance; "MW" is the molecular weight (449.2); DF is the dilution factor; ε is the molar extinction coefficient (26800); and "L" is the diameter of the test tube in centimeters.

[0100] (2) Determination of the content of different anthocyanin monomers:

[0101] Shimadzu high-performance liquid chromatography (HPLC) was used for analysis, equipped with a UV-vis diode array detector. For sample preparation, samples were filtered through a 0.22 μm filter membrane before HPLC analysis. The operating conditions for HPLC were as follows: mobile phase A was a 2% (volume fraction, the same below) formic acid solution, and mobile phase B was a 100% methanol solution; the flow rate was set at 1 mL / min, and the column temperature was 30°C. The gradient elution conditions were as follows: 0-2 min, mobile phase B was 6%; 2-30 min, mobile phase B increased from 6% to 90%; 30-31 min, mobile phase B increased from 90% to 95%; 31-34 min, mobile phase B was maintained at 95%; 34-37 min, mobile phase B was restored from 95% to 6%; 37-40 min, mobile phase B was maintained at 6%.

[0102] (3) CIELAB color parameter determination

[0103] Color indices are measured using a spectrophotometer, where the CIELAB color parameters L*, a*, b*, C*, h*, and ΔE reflect lightness, redness-greenness, yellowness-blueness, chroma, hue, and color difference, respectively.

[0104] (4) Fermentation quality determination:

[0105] Alcohol content is determined by gas chromatography, while total acidity and pH are determined using official OIV analytical methods. Total sugar content is measured using the anthrone-sulfuric acid method and is expressed in grams of glucose equivalent per liter.

[0106] (5) Determination of viable yeast count:

[0107] The number of viable yeast cells was determined by the dilution spread plate method. Briefly, the sample was diluted with sterile saline at different gradients, 100 μl was evenly spread on a YPD solid culture plate, and then placed in a 30°C constant temperature incubator for 2-3 days for determination. The number of viable yeast cells per unit volume (mL) of the sample was calculated according to the dilution multiple.

[0108] According to the experimental results of the above examples, the yeast DYG1 described in the present invention can be used to brew fruit wine.

[0109] The method for brewing fruit wine using the yeast DYG1 may comprise the following steps:

[0110] A. Wash and juice the fruit to be fermented, add pectinase for enzymatic hydrolysis, filter, adjust sulfur and sugar to obtain juice;

[0111] B. pasteurize the juice obtained in step A, cool it to room temperature, add yeast DYG1, and mix well to obtain a juice to be fermented;

[0112] C. The juice to be fermented obtained in step B is placed in a fermentation tank with a one-way valve, with a liquid volume of 50% by volume, a fermentation temperature of 30° C., and an anaerobic fermentation for 18 days.

[0113] In the step A, the enzymatic hydrolysis temperature of the pectinase can be 45 to 50° C., and the enzymatic hydrolysis time can be 3 to 4 hours.

[0114] In step B, the amount of yeast added is 1.0×10 9 The above yeast strains were calculated.

[0115] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A single fermentation broad-spectrum color-protecting yeast, characterized by: The yeast was named Hanseniaspora Opuntiae DYG1 and was deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 65703.

2. Application of the yeast as claimed in claim 1 in fruit wine brewing.

3. The use according to claim 2, characterized in that: The raw material of the fruit wine is one of the following fruits: grape, mulberry, strawberry, blueberry and pomegranate.

4. A method for brewing fruit wine using the yeast according to claim 1, characterized in that: The following steps are involved: A. Wash the fruit to be fermented, squeeze the juice, add pectinase for enzymatic hydrolysis, filter, adjust the sulfur and sugar content to obtain juice; B. pasteurizing the juice obtained in step A, cooling it to room temperature, adding the yeast according to claim 1, and mixing them uniformly to obtain a juice to be fermented; C. The juice to be fermented obtained in step B is placed in a fermentation tank with a one-way valve, with a liquid volume of 50% by volume, a fermentation temperature of 30° C., and an anaerobic fermentation for 18 days.

5. The method according to claim 4, characterized in that: In the step A, the enzymatic hydrolysis temperature of the pectinase is 45 to 50° C., and the enzymatic hydrolysis time is 3 to 4 hours.

6. The method according to claim 4, characterized in that: In step B, the amount of yeast added is 1.0×10 9 The above yeast strains were calculated.

Images