Color-protecting saccharomycetes mixed with commercial yeast or sequentially fermented with commercial yeast and application of color-protecting saccharomycetes
By mixing or fermenting yeast MG6 with commercial yeast, the problem of anthocyanins degradation of yeast strains is solved, and the efficient retention of anthocyanins in mulberry wine and the improvement of fruit wine quality is achieved.
Patent Information
- Application Number
- CN202510418157.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-03
- Publication Date
- 2025-08-08
AI Technical Summary
Existing yeast strains are prone to degradation of anthocyanins during the fermentation of fruit wine, lack effective color protection functions, and affect the color and quality of fruit wine.
The retention rate and biological activity of anthocyanins and biological activity were significantly improved by using Hanseniaspora Opuntiae MG6 and commercial yeast.
It significantly improves the anthocyanin retention rate during the fermentation of mulberry wine, maintains the stability and color of anthocyanins, and improves the quality and antioxidant ability of fruit wine.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of microbial fermentation, and relates to a yeast, in particular to a yeast that is mixed with commercial yeast or fermented sequentially, and can protect color, i.e., protect anthocyanins and reduce anthocyanin degradation, and the 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. The color depth of plants is positively correlated with their anthocyanin content. Anthocyanins are the primary colorant in most fruits and their processed products, such as juice and wine. Yeasts, the dominant players in wine fermentation, possess extensive genetic diversity. They metabolize sugars and other nutrients into ethanol, carbon dioxide, and various secondary metabolites, influencing the chemical composition, color, taste, and sensory quality of the wine. However, anthocyanins are easily degraded during fermentation by factors such as temperature, pH, oxygen, enzymes, metal ions, ascorbic acid, sulfur dioxide, yeast fermentation, and cell wall adsorption. Yeast metabolism and cell wall adsorption particularly influence anthocyanin stability.
[0003] To regulate the winemaking process and prevent spoilage, numerous strains of Saccharomyces cerevisiae are used as commercial yeasts to initiate fermentation. During the winemaking process, the cell walls of S. cerevisiae have been shown to adsorb compounds in wine, significantly impacting wine quality. Yeast also produces specific enzymes (such as glucosidases and phenoloxidases) that hydrolyze the glycosidic bonds of anthocyanins, leading to their degradation. This hydrolysis may convert anthocyanins to their non-pigmented forms, reducing their stability and color. Oxidative reactions initiated by yeast during metabolism can also lead to the oxidative degradation of anthocyanins. This oxidation process disrupts the molecular structure of anthocyanins, resulting in color changes and reduced activity. Therefore, the metabolic properties of the yeast strain itself can negatively impact the stability of anthocyanins in wine. Whether this negative impact is greater or lesser than that caused by cell wall adsorption remains a lack of systematic research.
[0004] Non-brewery yeasts can also have a significant impact on the fermentation quality, flavor, and color of fruit wine. In the past, non-brewery yeasts were often considered insignificant or strictly regarded as contaminants. Currently, more and more non-brewery yeasts are being used in the brewing process of fruit wine, especially the research on the combined use of non-brewery yeasts and brewer's yeasts has attracted much attention. However, a large number of studies have focused on the positive effects of non-brewery yeasts on the formation of fruit wine fermentation flavor, while research on color has mainly focused on the production of anthocyanin derivatives, while there is a lack of exploration of the "color protection function" of protecting anthocyanins and reducing degradation. Summary of the Invention
[0005] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a yeast strain that can be mixed with commercial yeast or fermented sequentially to protect the color, that is, to protect anthocyanins and reduce anthocyanin degradation.
[0006] The color-protecting yeast strain described herein, which is used for mixed or sequential fermentation with commercial yeast, is named Hanseniaspora opuntiae MG6 and is deposited with the Guangdong Provincial Microbial Culture Collection, located at Building 59, 5th Floor, No. 100 Xianlie Middle Road, Guangzhou. The deposit number is GDMCC No. 65702, and the deposit date is December 31, 2024. It is hereinafter referred to as MG6.
[0007] The second object of the present invention is to provide an application of the yeast MG6 described in the present invention, specifically an application of the yeast MG6 in the brewing of mulberry wine.
[0008] The third object of the present invention is to provide a method for brewing mulberry wine using the yeast of the present invention.
[0009] The method for brewing mulberry wine using the yeast of the present invention comprises the following steps:
[0010] A. Wash the mulberries, squeeze the juice, add pectinase for enzymatic hydrolysis, filter, and adjust the sulfur and sugar content to obtain mulberry juice;
[0011] B. pasteurizing the mulberry juice obtained in step A, cooling it to room temperature, and adding the yeast of the present invention and commercial yeast, mixing them evenly to obtain mulberry juice to be fermented;
[0012] C. The mulberry juice to be fermented obtained in step B is placed in a fermentation tank with a one-way valve, with a liquid filling volume of 50% by volume, a fermentation temperature of 30° C., and anaerobic fermentation for 18 days.
[0013] 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.
[0014] Preferably, in step B, the amount of yeast added is 1.0×10 9 The above yeast strains were calculated.
[0015] Another optional method for brewing mulberry wine using the yeast of the present invention comprises the following steps:
[0016] A. Wash the mulberries, squeeze the juice, add pectinase for enzymatic hydrolysis, filter, and adjust the sulfur and sugar content to obtain mulberry juice;
[0017] B. pasteurize the mulberry juice obtained in step A, cool it to room temperature, add the yeast of the present invention, mix well, place it in a fermentation tank equipped with a one-way valve, fill it with 50% by volume, ferment it at 30° C., and ferment it anaerobically for 4 days;
[0018] C. Inoculate commercial yeast in the fermentation tank, ferment at 30℃, and perform anaerobic fermentation for 14 days.
[0019] 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.
[0020] Preferably, in step B and step C, the amount of yeast added is 1.0×10 9 The above yeast strains were calculated.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] (1) The present invention provides a cactus spore-bearing Hanseniaspora (Hanseniaspora Opuntiae) MG6, which is a non-brewery yeast and can be mixed with commercial yeast or fermented with mulberry wine under sequential fermentation conditions.
[0023] (2) The yeast MG6 described in the present invention can significantly improve the retention rate of anthocyanins in mulberry wine during the winemaking process, and has a color-protecting effect of protecting anthocyanins and reducing anthocyanin degradation.
[0024] (3) The yeast MG6 described in the present invention can significantly improve the anthocyanin retention rate during and after the fermentation of mulberry wine, and effectively maintain the biological activity of anthocyanins, by mixing or sequential fermentation with commercial brewing yeast, compared with using commercial brewing yeast alone. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is the phylogenetic tree of the 7 screened yeast strains, which are PTP1, PTP4, PTP5, DYG1, DYG5, MG2, and MG6.
[0026] Figure 2 Figure 3. Changes in total anthocyanin content (A), retention rate (B), total phenolic content (C), total flavonoid content (D), pH (E), and total acidity (F) in mulberry wine fermented with seven screened yeast strains and commercial yeast SY. The fermentation treatments for the seven screened yeast strains and SY were PTP1SY, PTP4SY, PTP5SY, DYG1SY, DYG5SY, MG2SY, and MG6SY, respectively. **, P < 0.01, ***, P < 0.001.
[0027] Figure 3Figure 5. Comparison of pH (A), total acidity (B), residual sugar (C), ethanol (D), antioxidant capacity (DPPH scavenging rate (E), ABTS scavenging rate (F), and FRAP activity (G) in the supernatant of mulberry wine fermented with seven screened yeast strains and commercial yeast SY. The seven screened yeast strains mixed with SY were PTP1SY, PTP4SY, PTP5SY, DYG1SY, DYG5SY, MG2SY, and MG6SY, respectively. Different lowercase letters indicate significant differences among treatment groups (P < 0.05). **, P < 0.01.
[0028] Figure 4 Figure 3. Changes in total anthocyanin content (A), retention rate (B), total phenolic content (C), total flavonoid content (D), pH (E), and total acidity (F) of mulberry wine produced by sequential fermentation of seven screened yeast strains with commercial yeast SY. The seven strains screened with SY were 4-PTP1SY, 4-PTP4SY, 4-PTP5SY, 4-DYG1SY, 4-DYG5SY, 4-MG2SY, and 4-MG6SY, respectively. **, P < 0.01, ***, P < 0.001.
[0029] Figure 5 Figure 5. Comparison of pH (A), total acidity (B), residual sugar (C), ethanol (D), antioxidant capacity (DPPH scavenging rate (E), ABTS scavenging rate (F), and FRAP activity (G) in the supernatant of mulberry wine fermented with seven screened yeast strains and commercial yeast SY. The treatments used in the sequential fermentation of mulberry wine with the seven screened yeast strains and SY were 4-PTP1SY, 4-PTP4SY, 4-PTP5SY, 4-DYG1SY, 4-DYG5SY, 4-MG2SY, and 4-MG6SY, respectively. Different lowercase letters indicate significant differences among the treatment groups (P < 0.05). **, P < 0.01. DETAILED DESCRIPTION
[0030] The present invention will be further described in detail below with reference to the embodiments and the accompanying drawings, but the embodiments do not limit the present invention in any form.
[0031] Example 1: Isolation, screening, physiological properties and identification of yeast
[0032] A total of 292 yeast strains were isolated from 30 materials including grapes, bananas, strawberries, doyi fruits, mangoes, watermelons, mulberries, blueberries, and lemons. Crystal violet staining and cell shape observation were performed, and the yeast strain with the highest total anthocyanin retention rate was screened out based on tolerance tests (pH tolerance reached 2.5, ethanol concentration tolerance reached 6%, and glucose concentration tolerance reached 20%) and micropore fermentation of mulberry juice medium.
[0033] The specific steps for yeast isolation and screening are as follows:
[0034] 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℃.
[0035] YPD medium consists of: 20.0 g of peptone, 10.0 g of yeast extract, 20.0 g of glucose, 20 g / L of agar, and distilled water (H2O) to 1000 mL. Sterilize at 121°C for 20 min.
[0036] WL medium consists of: yeast extract 5.0 g, acid hydrolyzed casein 5.0 g, glucose 50.0 g, potassium dihydrogen phosphate (KH2PO4) 0.55 g, potassium chloride (KCl) 0.425 g, calcium chloride (CaCl2) 0.125 g, magnesium sulfate (MgSO4) 0.125 g, ferric chloride (FeCl3) 0.0025 g, manganese sulfate (MnSO4) 0.0025 g, bromocresol green (C 21 H 14 Br4O5S) 0.022g, agar 17.0g.
[0037] The 292 purified yeast strains were inoculated into 48-well cell culture plates with 0.9 mL of mulberry juice medium (mulberry juice: YPD liquid medium, pasteurized, 1:1) in each well. Each strain was replicated three times. After anaerobic fermentation at 30°C for 3 days, the retention rate of anthocyanins was measured. Seven yeast strains with high retention rate and good tolerance were screened out and named PTP1, PTP4, PTP5, DYG1, DYG5, MG2 and MG6. The main physiological and biochemical characteristics of MG6 are shown in Table 1.
[0038] Table 1: Physiological and biochemical characteristics of strain MG6
[0039]
[0040] +++: vigorous growth; ++: good growth; +: growth; ±: weak growth; -: no growth.
[0041] As shown in Table 1, yeast MG6 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.
[0042] Yeast species identification
[0043] 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 MG6 and the other 6 screened yeast strains were all Hanseniaspora Opuntiae.
[0044] The inventors deposited Hanseniaspora Opuntiae MG6 in the Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No: 65702 and the deposit date of December 31, 2024.
[0045] Example 2: Fermentation experiment of adding screened yeast strains (PTP1, PTP4, PTP5, DYG1, DYG5, MG2, MG6) and commercial yeast SY to mulberry juice
[0046] The mulberry variety selected was "Yuezhen Da 10", which was purchased from the Jiangnan Fruit Wholesale Market in Guangzhou. The fresh fruit was washed and juiced, and then pectinase (added in an amount of 0.04% by mass to volume) was added for enzymatic hydrolysis. For example, the enzymatic hydrolysis temperature is 45°C and the enzymatic hydrolysis time is 4 hours, or the enzymatic hydrolysis temperature is increased to 50°C and the enzymatic hydrolysis time is adjusted to 3 hours accordingly. Then filter and adjust the sulfur and sugar. In this example, sucrose is used to adjust the sugar to 240g / L and the sulfur to 0.09g / L. Subsequently, the sulfur and sugar adjusted products are pasteurized, cooled to room temperature, and set aside. Subsequently, the juice is placed in a 500mL fermentation container with a one-way valve, the liquid volume is 50% by volume, and the fermentation temperature is set to 30°C.
[0047] The group treatments include: SY (commercial yeast SY inoculated alone), PTP1SY (PTP1 and SY inoculated at the same time), PTP4SY (PTP4 and SY inoculated at the same time), PTP5SY (PTP5 and SY inoculated at the same time), DYG1SY (DYG1 and SY inoculated at the same time), DYG5SY (DYG5 and SY inoculated at the same time), MG2SY (MG2 and SY inoculated at the same time), and MG6SY (MG6 and SY inoculated at the same time).
[0048] The screening yeast strains (PTP1, PTP4, PTP5, DYG1, DYG5, MG2, MG6) and commercial yeast (SY, purchased from Angel Yeast Co., Ltd.) were activated twice in YPD liquid medium. The screening yeast strains PTP1, PTP4, PTP5, DYG1, DYG5, MG2, MG6 and SY were activated at 10 6 An inoculum of 100 cfu / mL was inoculated into a 100 ml fermentation vessel equipped with a one-way valve. A control was a single SY inoculation. The fruit juice was loaded at 50% by volume, and the fermentation temperature was set at 30°C. Three replicates were used for each treatment. Sampling was performed every 18 days. Parameters measured included total anthocyanin and anthocyanin retention rates, total phenolic and flavonoid content, fermentation quality indicators (pH, total titratable acidity, residual sugar, and ethanol content at day 18), and antioxidant activity at day 18.
[0049] Here are the results:
[0050] like Figure 2 As shown in Figure 2, although the total anthocyanin content and retention rate of all screened treatments were significantly higher than those of SY fermentation alone, the best treatment was MG6 ( Figure 2 A), the anthocyanin retention rate can reach 36% VS.26.8% (SY), the improvement rate is 34%, while the protection effects of PTP4, PTP5, DYG1 and DYG5 are significantly lower than those of other screening strains ( Figure 2 A). Total phenol content of MG6 ( Figure 2B) is at the highest level, the total flavonoid content ( Figure 2 C) was also significantly higher than other screened yeasts.
[0051] like Figure 3 As shown in the figure, the fermentation quality of mulberry wine from mixed fermentation of MG6 and SY is as follows: pH( Figure 3 A), total acid ( Figure 3 B) ethanol content ( Figure 3 C), residual sugar content ( Figure 3 D) are in line with industry standards, antioxidant capacity DPPH clearance rate ( Figure 3 E) and FRAP values ( Figure 3 G) were significantly higher than those in the other screened yeast treatments, indicating that the mixed fermentation of MG6 and commercial yeast SY had the best color protection effect compared with other screened strains.
[0052] Example 3: Fermentation experiment of screening yeast strains (PTP1, PTP4, PTP5, DYG1, DYG5, MG2, MG6) and commercial yeast SY sequentially added to mulberry juice
[0053] The mulberry variety selected was "Yuezhen Da 10", which was purchased from the Jiangnan Fruit Wholesale Market in Guangzhou. The fresh fruit was washed and juiced, and then pectinase (added in an amount of 0.04% by mass to volume) was added for enzymatic hydrolysis. For example, the enzymatic hydrolysis temperature is 45°C and the enzymatic hydrolysis time is 4 hours, or the enzymatic hydrolysis temperature is increased to 50°C and the enzymatic hydrolysis time is adjusted to 3 hours accordingly. Then filter and adjust the sulfur and sugar. In this case, sucrose is used to adjust the sugar to 240g / L and the sulfur to 0.09g / L. Subsequently, the sulfur- and sugar-adjusted juice is pasteurized, cooled to room temperature, and set aside. Subsequently, the juice is placed in a 500mL fermentation container with a one-way valve, the liquid volume is 50%, and the fermentation temperature is set to 30°C.
[0054] The group treatments included: SY (commercial yeast SY inoculated alone), 4-PTP1SY (PTP1 was fermented for 4 days before inoculation with SY), 4-PTP4SY (PTP4 was fermented for 4 days before inoculation with SY), 4-PTP5SY (PTP5 was fermented for 4 days before inoculation with SY), 4-DYG1SY (DYG1 was fermented for 4 days before inoculation with SY), 4-DYG5SY (DYG5 was fermented for 4 days before inoculation with SY), 4-MG2SY (MG2 was fermented for 4 days before inoculation with SY), and 4-MG6SY (MG6 was fermented for 4 days before inoculation with SY).
[0055] The screening yeast strains (PTP1, PTP4, PTP5, DYG1, DYG5, MG2, MG6) 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. After 4 days of fermentation, an equal amount of SY was inoculated. SY alone was used as a control. The fruit juice was loaded at 50% by volume, and the fermentation temperature was set at 30°C. Three replicates were used for each treatment. Sampling was performed every 18 days. Parameters measured included total anthocyanin and retention rates of different anthocyanins, total phenolic and total flavonoid contents, fermentation quality indicators (pH, total titratable acidity, residual sugar, and ethanol content at day 18), and antioxidant activity at day 18.
[0056] Here are the results:
[0057] like Figure 4 As shown in Figure 2, the total anthocyanin retention rates of all screened yeast treatments were significantly higher than those of commercial yeast SY alone. The best treatment was MG6, with an anthocyanin retention rate of 32% vs. 26.0% (SY), an increase of 23% ( Figure 4 A). Total phenol content of MG6 ( Figure 4 B) was also at the highest level, and the total flavonoid content was also significantly higher than that of other screened yeasts ( Figure 4 C).
[0058] like Figure 5 As shown in the figure, the fermentation quality of mulberry wine produced by sequential fermentation of MG6 and SY is shown in the figure. Figure 5 A), total acid ( Figure 5 B) ethanol content ( Figure 5 C), residual sugar content ( Figure 5 D) are in line with industry standards, antioxidant capacity DPPH clearance rate ( Figure 5 E) and ABTS clearance ( Figure 5 F) were significantly higher than those in other screened yeast treatments, indicating that the sequential fermentation of MG6 and commercial yeast SY had the best color protection effect compared with other screened strains.
[0059] Regarding the determination method in above-mentioned Example 2 and Example 3:
[0060] (1) Determination of total anthocyanin content:
[0061] 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.
[0062] (2) Determination of total phenol content
[0063] The total phenolic content (TP) of fruit wine samples was determined using the following simple steps: After appropriately diluting the sample, take 0.1 mL of the sample, add 0.1 mL of distilled water and 1.0 mL of Folin's phenol reagent, and react at 25°C for 5 minutes. Subsequently, add 3.0 mL of 7.5% Na2CO3, let it stand for 30 minutes, and finally measure and record the absorbance at 760 nm. The results were then calculated using a standard curve.
[0064] (3) Determination of total flavonoid content
[0065] The total flavonoid content (TF) of a sample was determined as follows: 0.5 mL of the diluted sample was added to 0.5 mL of distilled water and 0.3 mL of 5.0% NaNO₃ and incubated for 6 minutes. Next, 0.3 mL of 7.5% AlCl₃ was added, the mixture was allowed to stand for 30 minutes, and 2 mL of 4% NaOH was added to terminate the reaction. Finally, the absorbance at 510 nm was measured and recorded, and the results were calculated using a standard curve.
[0066] (4) Fermentation quality determination:
[0067] 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.
[0068] (5) Determination of antioxidant index:
[0069] The DPPH clearance rate was determined as follows:
[0070] Take 2 mL of DPPH solution with a concentration of 0.02 mg / mL (prepared with anhydrous ethanol), add 2 mL of fruit wine sample, mix well, and place in a constant temperature water bath at 25°C for 30 minutes. Then, measure the absorbance at a wavelength of 517 nm. For the blank group, 2 mL of anhydrous ethanol was used instead of the fermentation broth sample.
[0071] The calculation formula of DPPH free radical scavenging rate (%) is: [1-(A1-A2) / A0]×100.
[0072] Wherein, A1 is the absorbance of the sample solution; A2 is the background absorbance value of the corresponding concentration measured by replacing the DPPH solution with anhydrous ethanol; A0 is the absorbance of the blank group.
[0073] ABTS clearance was determined as follows ] :
[0074] Prepare a 7 mmol / L ABTS stock solution and a 2.6 mmol / L K2S2O8 stock solution. Mix 100 mL of ABTS stock solution with 1.75 mL of K2S2O8 stock solution, homogenize by sonication, and incubate in the dark for 12 hours to prepare the ABTS·+ radical working solution. Dilute the solution with 0.05 mmol / L pH 7.4 phosphate buffer to an absorbance of 0.70 ± 0.02 at 734 nm. Let stand at room temperature for 30 minutes before use.
[0075] After mixing the ABTS·+ free radical working solution with the sample, incubate in the dark for 30 minutes and read the absorbance at 734 nm using a UV spectrophotometer. Calculate the ABTS·+ free radical scavenging rate using the following formula:
[0076]
[0077] A1 is the sample group: 50 μL of samples of different concentrations + 950 μL of ABTS assay solution
[0078] A2 is the sample blank group: 50 μL samples of different concentrations + 950 μL anhydrous ethanol solution
[0079] A0 is the control group: 50 μL deionized water + 950 μL ABTS solution
[0080] A3 is the background blank group: 50 μL deionized water + 950 μL anhydrous ethanol solution
[0081] The FRAP value was determined as follows:
[0082] Preparation of FRAP working solution:
[0083] Accurately weigh 0.0312 g of TPTZ and dissolve it in 10 mL of 40 mM hydrochloric acid to obtain a TPTZ stock solution. Accurately weigh 0.0541 g of FeCl3·6H2O and dissolve it in 10 mL of distilled water to obtain a FeCl3 stock solution. Accurately weigh 3.1 g of C2H3O2Na·3H2O and dissolve it in 16 mL of glacial acetic acid, and finally dilute to 1 L with distilled water to obtain an acetate buffer solution. Separately, take 2.5 mL of TPTZ solution, 2.5 mL of FeCl3 solution, and 25 mL of acetate buffer and mix them evenly to obtain a FRAP working solution.
[0084] Construction of FeSO4 standard curve:
[0085] Prepare gradient FeSO4 solutions of 0.4mM, 0.8mM, 1.2mM, 1.6mM, and 2mM respectively, mix 3mL of FRAP working solution with 100μL of gradient concentration FeSO4 solution respectively, incubate in the dark for 30min, and measure the absorbance at 593nm. Establish a standard curve with the concentration of FeSO4 as the horizontal axis and the absorbance of the reactant at 593nm as the vertical axis.
[0086] Test of antioxidant activity of sample solution:
[0087] Sample solutions of varying concentrations were diluted 3-fold and then mixed with FRAP working solution at a volume ratio of 1:300. The mixture was incubated in the dark for 30 minutes, and the absorbance of the mixed solution was measured at 593 nm. The measured absorbance values of the samples were substituted into the standard curve to calculate the FeSO₄ equivalent. FRAP was expressed as FeSO₄ concentration. Higher FRAP values indicate stronger antioxidant activity.
[0088] According to the experimental results of the above examples, the yeast MG6 described in the present invention can be used to brew mulberry wine.
[0089] The method for brewing mulberry wine using yeast MG6 may comprise the following steps:
[0090] A. Wash the mulberries, squeeze the juice, add pectinase for enzymatic hydrolysis, filter, and adjust the sulfur and sugar content to obtain mulberry juice;
[0091] B. pasteurizing the mulberry juice obtained in step A, cooling it to room temperature, adding yeast MG6 and commercial yeast, and mixing them evenly to obtain mulberry juice to be fermented;
[0092] C. The mulberry juice to be fermented obtained in step B is placed in a fermentation tank with a one-way valve, with a liquid filling volume of 50% by volume, a fermentation temperature of 30° C., and anaerobic fermentation for 18 days.
[0093] 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.
[0094] In step B, the amount of yeast added is 1.0×10 9 The above yeast strains were calculated.
[0095] Another optional method for brewing mulberry wine using yeast MG6 comprises the following steps:
[0096] A. Wash the mulberries, squeeze the juice, add pectinase for enzymatic hydrolysis, filter, and adjust the sulfur and sugar content to obtain mulberry juice;
[0097] B. pasteurize the mulberry juice obtained in step A, cool it to room temperature, add yeast MG6, mix well, place it in a fermentation tank equipped with a one-way valve, fill it with 50% by volume, ferment it at 30° C., and ferment it anaerobically for 4 days;
[0098] C. Inoculate commercial yeast in the fermentation tank, ferment at 30℃, and perform anaerobic fermentation for 14 days.
[0099] 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.
[0100] Preferably, in step B and step C, the amount of yeast added is 1.0×10 9 The above yeast strains were calculated.
[0101] The above embodiments are preferred implementation modes of the present invention, but the implementation modes 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 as equivalent replacement methods and are included in the scope of protection of the present invention.
Claims
1. A color-protecting yeast strain for mixed or sequential fermentation with commercial yeast, characterized by: The yeast was named Hanseniaspora Opuntiae MG6 and was deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 65702.
2. Application of the yeast as claimed in claim 1 in mulberry wine brewing.
3. A method for brewing mulberry wine using the yeast according to claim 1, characterized in that: The steps include: A. Wash the mulberries, squeeze the juice, add pectinase for enzymatic hydrolysis, filter, and adjust the sulfur and sugar content to obtain mulberry juice; B. pasteurizing the mulberry juice obtained in step A, cooling it to room temperature, and adding the yeast according to claim 1 and commercial yeast, mixing them evenly to obtain mulberry juice to be fermented; C. The mulberry juice to be fermented obtained in step B is placed in a fermentation tank with a one-way valve, with a liquid filling volume of 50% by volume, a fermentation temperature of 30° C., and anaerobic fermentation for 18 days.
4. The method according to claim 3, wherein: 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.
5. The method according to claim 3, wherein: In step B, the amount of yeast added is 1.0×10 9 The above yeast strains were calculated.
6. A method for brewing mulberry wine using the yeast according to claim 1, characterized in that: The steps include: A. Wash the mulberries, squeeze the juice, add pectinase for enzymatic hydrolysis, filter, and adjust the sulfur and sugar content to obtain mulberry juice; B. pasteurizing the mulberry juice obtained in step A, cooling it to room temperature, adding the yeast of claim 1, mixing them evenly, and placing them in a fermentation tank equipped with a one-way valve, filling it with 50% by volume, fermenting it at a temperature of 30° C., and performing anaerobically fermentation for 4 days; C. Inoculate commercial yeast in the fermentation tank, mix well, and ferment anaerobically at 30°C for 14 days.
7. The method according to claim 6, 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.
8. The method according to claim 6, wherein: In step B and step C, the amount of yeast added is 1.0×10 9 The above yeast strains were calculated.