A method for improving the flavor of mulberry wine by screening aroma-producing yeast from mulberry fruits

Abstract

The invention discloses a method for improving the flavor of mulberry wine by screening aroma-producing yeast from mulberries. The method relates to the technical field of microorganisms and solves the problem that the existing mulberry wine preparation process cannot fully reflect the nutritional value and unique flavor of mulberries. Low-temperature treatment is used to increase the relative abundance of target bacterial genera in a sample, and a sample with a high relative abundance of desired bacterial species and a low fungal count is selected. Then, a specific screening method is adopted to separate and molecularly identify the aroma-producing yeast. Yeast with good tolerance and the ability to produce a strong aroma is selected from the sample through tolerance experiments and electronic nose analysis, and the yeast is compounded into a starter. The alcohol content of the obtained mulberry wine is increased by more than 2.2% vol to 13.5% vol, the total ester is increased by 25%, and the relative content of aromatic compounds such as aldehydes and ketones is increased. The aroma-producing yeast species are obtained through directional separation, which can avoid blindness, improve screening efficiency, and enhance the flavor of the fruit wine.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and more particularly to the technical field of a method for improving the flavor of mulberry wine by utilizing aroma-producing yeast. Background Art

[0002] Mulberries are rich in protein, essential amino acids, and easily absorbed fructose and glucose. They also contain vitamins such as VB1, VB2, VB3, VB5, VB6, VC, and VE, as well as minerals like Fe, Ca, and Zn, trace elements like selenium, carotene, and cellulose. Mulberries also contain active polysaccharides, flavonoids, and rutin, which have the potential to quench thirst, nourish yin and blood, tonify the liver and kidneys, strengthen essence and stabilize pregnancy, promote black hair and beard, improve hearing and eyesight, slow aging, soothe the mind and nourish the heart, moisten the intestines and promote bowel movements, improve gait, calm wind, clear internal heat, benefit joints, and combat rheumatism. Regular consumption of mulberries also offers beauty and anti-aging benefits.

[0003] With the deepening of scientific research, the advantages of fruit wine have been further recognized, and consumption is increasing. The development of fruit wine production is a key direction for my country's current alcoholic beverage industry. Mulberry juice is rich in glucose, organic acids, minerals, vitamins, and has a purple-red color, making it ideal for fermentation and winemaking. Mulberry wine produced from mulberry juice not only retains the vast majority of the nutrients found in mulberries, but also boasts a vibrant color, a rich and profound aroma, a full-bodied, mellow body, a pleasantly sweet and sour taste, a lingering flavor, and a unique style. It is considered a top-quality fruit wine. Using mulberry juice to ferment mulberry wine can transform waste into valuable resources, significantly reducing mulberry pollution and significantly improving the overall economic benefits of mulberry orchards. This has positive social significance for the stability and development of sericulture production. For example, patent number 201410619977.1 discloses a method for brewing mulberry wine using a fermentation process, which comprises the following steps: pre-filtering the squeezed mulberry juice with a 200-mesh filter cloth, measuring the sugar content of the pre-filtered juice, and adding white sugar to adjust the sugar content of the fermentation liquid to 20-22%, adjusting the acidity to a pH value of 3.3-3.5, and adding SO2 to adjust the SO2 content of the juice to 50 mg / L; adding 5% active dry yeast of wine to the juice and mixing evenly, placing the mixture in a water bath at a temperature of 30-40°C, activating the mixture after 10 minutes, fermenting the mixture for 4 hours, and sealing the mixture with a cover, at a fermentation temperature of 25-28°C; and fermenting the mixture for 9 days, replacing the container for post-fermentation; filtering the post-fermented wine once with 3% diatomaceous earth to obtain clarified wine.

[0004] The current preparation adopts the traditional wine processing technology and adds some other auxiliary materials. However, the nutritional components of mulberries and grapes are quite different. At the same time, brewing yeast ferments quickly, which seriously inhibits the activity of a small amount of non-brewing yeast on the surface of mulberries during fermentation, resulting in the homogenization of different types of fruit wine products. The nutritional value and unique flavor of mulberries cannot be fully reflected, and the commercial value is low. Summary of the Invention

[0005] The purpose of the present invention is to solve the problem that mulberry wine prepared by the existing mulberry wine preparation process cannot fully reflect the nutritional value and unique flavor and taste of mulberry. In order to solve the above technical problems, the present invention provides a method for improving the flavor of mulberry wine by screening aroma-producing yeast from mulberry.

[0006] In order to achieve the above-mentioned purpose, the present invention specifically adopts the following technical solutions:

[0007] A method for improving the flavor of mulberry wine by screening aroma-producing yeast from mulberries comprises the following steps:

[0008] Step 1: Storing fresh mulberries at low temperatures to obtain mulberry samples;

[0009] Step 2: Under sterile conditions, extract the original bacterial suspension of the strains from the surface of the mulberry samples. A portion of the original bacterial suspension is subjected to high-throughput sequencing to obtain the relative abundance of each yeast genus in different samples. A portion of the original bacterial suspension is subjected to fungal plate counts, and samples with high relative abundance of target genus and low fungal counts are selected as screening strain samples.

[0010] Step 3: Cultivate the screened strain sample in sterilized mulberry juice under sterile conditions, select the culture medium at different stages and the fermentation mud at the final stage for solid culture, and isolate and purify to obtain single colonies;

[0011] Step 4: Perform microscopic comparison and molecular biological identification on the single colony liquid to obtain the target bacterial genus;

[0012] Step 5: Perform tolerance analysis on single colonies and 10 6 After inoculation and culture of CFU / mL liquid, electronic nose analysis was performed to screen out strains with good tolerance and high alcohol and aromatic compound content;

[0013] Step 6: The strains screened in step 5 are divided into 10 groups according to the formula: Saccharomyces cerevisiae: Hansenula vitis spores: Pichia kluyveri = 10 7 :10 5 :10 5 The number of CFU / mL was inoculated into mulberry fermentation broth for mixed fermentation to obtain mulberry wine.

[0014] Furthermore, the low-temperature storage temperature of the mulberry fruit sample in step 1 includes 4°C and -1°C, and the storage time includes 0d, 4d, 8d, and 12d.

[0015] Furthermore, the method for extracting the original bacterial suspension of the strain on the surface of the mulberry sample in step 2 is as follows: the mulberry sample is cut with sterile scissors under a clean workbench and added to sterile physiological saline, and then the dilution is obtained after shaking and standing, the dilution is taken into a sterile centrifuge tube and centrifuged to remove the supernatant, and the original bacterial suspension is obtained after resuspending with sterile water; the original bacterial suspension is then used to extract total bacterial DNA using a D6005 fungal / bacterial DNA extraction kit, the extracted DNA is subjected to electrophoresis detection and analysis, and then high-throughput sequencing is performed, and the relative abundance of the target bacterial genus in different samples is displayed by a bar graph of the relative abundance of the fungal community at the genus level in the mulberry sample.

[0016] Furthermore, in step 2, the original bacterial suspension was gradiently diluted and spread on PDA solid culture medium, and the number of fungi was counted after inverted culture at 28° C. for 48 hours.

[0017] Furthermore, the strain sample screened in step 3 is placed in sterilized mulberry juice and fermented at 24° C. under aseptic conditions, and the juice of the mulberry fermentation liquid on the 0th and 2nd day of fermentation and the fermentation mud of the mulberry fermentation liquid on the 6th day of fermentation are respectively taken.

[0018] Furthermore, in step 3, based on the results of high-throughput sequencing and fungal plate counts, the samples for screening strains were finally selected as mulberry samples stored at -1°C for 4 days and mulberry samples stored at room temperature for 4 days; the mulberry samples stored at -1°C for 4 days were subjected to a gradient of 10 4 , 10 5 , 10 6 , 10 7 The mulberry samples stored at room temperature for 4 days were diluted by gradient 10 8 , 10 9 , 10 10 , 10 11 The solution was diluted, spread on YPD solid plates, cultured at 28°C for 48 h, and then single colonies were picked and streaked on YPD medium until single colonies were purified.

[0019] Furthermore, in step 4, the strains are examined microscopically, and the screened strains are examined by ordinary preparation and crystal violet staining, and compared with the results of microscopic examination of Saccharomyces cerevisiae. Single colonies with cell morphology and reproductive methods different from those of Saccharomyces cerevisiae are isolated, and molecular biological identification and preservation are performed.

[0020] Furthermore, in step 5, each single colony was subjected to tolerance analysis, and each single colony was subjected to 10 6 The number of CFU / mL was inoculated into sterilized mulberry juice and cultured at 24°C for 6 days before analysis using an electronic nose. The strains with good tolerance and high response values ​​for alcohol and aromatic compounds were selected.

[0021] Furthermore, in step 6, the strains screened in step 5 are divided into the following groups: Saccharomyces cerevisiae: Hansenula vitis spores: Pichia kluyveri = 10 7 :10 5 :10 5 The number of CFU / mL was inoculated into mulberry fermentation broth and fermented with mixed strains at 24°C for 6 days. The lees were separated and left to stand at 20°C for 1 month. The supernatant was filtered with a 0.22 μm plate and frame filter to obtain mulberry wine.

[0022] Furthermore, the enrichment culture in steps 3 and 5 uses sterilized mulberry juice, which is obtained by crushing fresh mulberries, removing the residue and retaining the juice, and then sterilizing with high-pressure steam at 121°C for 15 minutes; the mulberry fruit fermentation liquid in step 6 is directly inoculated by directly crushing fresh mulberries, removing the residue and retaining the juice.

[0023] The beneficial effects of the present invention are as follows:

[0024] (1) The present invention utilizes low-temperature treatment to increase the relative abundance of target bacterial genera in samples, selects samples with high relative abundance of desired bacterial species and low fungal count (in the event of a conflict between the two, the criterion of high relative abundance is prioritized), then employs a specific screening method to isolate and molecularly identify aroma-producing yeasts, and then utilizes tolerance experiments and electronic nose analysis to select yeasts with good tolerance and the ability to produce strong aroma, and compounding them into a fermentation agent to improve the flavor of mulberry wine. The alcohol content of the resulting mulberry wine is increased by more than 2.2% vol (relative alcohol content increased by 0.48%) to 13.5% vol, and the total ester content is increased by 25% (relative ester content increased by 0.76%). At the same time, the relative content of aromatic compounds such as aldehydes and ketones is also increased; aroma-producing yeast species are obtained by targeted separation, and this method can avoid blindness, improve screening efficiency, and enhance the flavor of fruit wine;

[0025] (2) The present invention uses sterilized mulberry juice as the culture medium for yeast. During the natural fermentation of mulberry juice, on the second day of fermentation, yeasts multiply in large quantities and are widely distributed in the mulberry culture medium, consuming sugars. By the sixth day of fermentation, most of the sugars have been consumed, and the yeasts will settle at the bottom of the culture bottle. Therefore, we take juice from the mulberry culture medium on the 0th and second days of fermentation, and fermentation mud from the mulberry culture medium on the sixth day of fermentation. Based on the results of high-throughput sequencing and fungal plate counts, we select the appropriate gradient for plate screening, thereby reducing the workload.

[0026] (3) The present invention adopts different temperature treatments, and the growth of some temperature-sensitive microorganisms is inhibited, while the growth of low-temperature-resistant microorganisms remains unchanged, thereby increasing the relative abundance of low-temperature-resistant microorganisms; while under normal temperature conditions, as the nutrients of mulberry fruits are consumed, the microorganisms form a situation of growth and decline, so the relative abundance of some microorganisms will also increase significantly, while the relative abundance of some microorganisms will decrease significantly. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a histogram of the relative abundance of fungal communities at the genus level in different mulberry samples;

[0028] Figure 2 is a bar graph of the relative abundance of fungal communities at the species level in different mulberry samples;

[0029] Figure 3 is the change in the number of culturable fungi in mulberry samples;

[0030] Figure 4 This is a comparison of the microscopic examination results of commercial Saccharomyces cerevisiae and the isolated strain (20x objective lens);

[0031] Figure 5 This is a comparison of the cell morphology of commercial Saccharomyces cerevisiae and the isolated strain (100x objective lens);

[0032] Figure 6 It is the phylogenetic tree constructed based on the complete 18S rDNA sequence of the screening strains;

[0033] Figure 7 This is a tolerance analysis chart for screening yeast strains. DETAILED DESCRIPTION

[0034] To make the objectives, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0035] Example 1

[0036] A method for improving the flavor of mulberry wine by screening aroma-producing yeast from mulberries comprises the following steps:

[0037] Step 1: Fresh mulberries (mulberry variety: Jialing 30) were stored at 4°C and -1°C respectively. Mulberries were collected as samples after 0, 4, 8, and 12 days of storage under sterile conditions.

[0038] Step 2: 10 g of the mulberry fruit sample was cut with sterile scissors under a clean bench and added to 90 mL of sterile 0.85% saline (containing 5 g of glass beads). The mixture was shaken at 120 rpm and 28°C for 30 min and allowed to stand for 5 min to obtain a 1:10 dilution. 10 mL of the dilution was centrifuged in a sterile centrifuge tube, the supernatant was removed, and the sample was resuspended in 1 mL of sterile water to obtain the original bacterial suspension. The original bacterial suspension was then used to extract total bacterial DNA using a D6005 fungal / bacterial DNA extraction kit. The extracted DNA was subjected to electrophoresis and analysis, followed by high-throughput sequencing. The relative abundance of the target genus in different samples was displayed by a bar graph of the relative abundance of the fungal community at the genus level in the mulberry fruit sample. At the same time, the original bacterial suspension was diluted in a gradient manner and spread on PDA solid culture medium. The fungi were counted after inverted culture at 28°C for 48 h.

[0039] Step 3: Screening strain samples, put them into sterilized mulberry juice (fresh mulberries are crushed, the residue is removed, and the juice is sterilized at 121℃ with high pressure steam for 15min) under aseptic conditions and fermented at 24℃, and take the juice of the mulberry fermentation liquid on the 0th and 2nd day of fermentation, and the fermentation mud of the mulberry fermentation liquid on the 6th day of fermentation; according to the results of high-throughput sequencing and fungal plate counts, the screening strain samples were finally selected as mulberry samples stored at -1℃ for 4 days and mulberry samples stored at room temperature for 4 days; the mulberry samples stored at -1℃ for 4 days were heated according to a gradient of 10 4 , 10 5 , 10 6 , 10 7 The mulberry samples stored at room temperature for 4 days were diluted by gradient 10 8 , 10 9 , 10 10 , 10 11 Dilute the solution, spread on YPD solid plates, culture at 28°C for 48 hours, then pick a single colony and streak it on YPD medium until a single colony is obtained;

[0040] Step 4: Perform conventional microscopic examination and crystal violet staining on the selected strains, and compare the results with those of Saccharomyces cerevisiae. Isolate single colonies with cell morphology and reproductive methods that are different from Saccharomyces cerevisiae, and perform molecular biological identification and preservation to obtain the target bacterial genus;

[0041] Step 5: Perform tolerance analysis on single colonies and 6 The number of CFU / mL was inoculated into sterilized mulberry juice (prepared by crushing fresh mulberries, removing the residue and retaining the juice, and then sterilizing with high-pressure steam at 121°C for 15 minutes). After incubation at 24°C for 6 days, the strains were analyzed using an electronic nose, and strains with good tolerance and high response values ​​for alcohol and aromatic compounds were selected.

[0042] Step 6: The strains screened in step 5 are divided into 10 groups according to the formula: Saccharomyces cerevisiae: Hansenula vitis spores: Pichia kluyveri = 10 7 :10 5 :10 5 The number of CFU / mL was inoculated into mulberry fermentation liquid (the fresh mulberries were directly crushed, the residues were removed and the juice was retained and then directly inoculated) and fermented with mixed strains at 24°C for 6 days to separate the lees. After standing at 20°C for 1 month, the supernatant was taken and filtered through a 0.22 μm plate and frame filter to obtain mulberry wine.

[0043] Experimental data and result analysis

[0044] 1. High-throughput sequencing and plate counting results

[0045] Table 1 Relative abundance of each genera in the yeast community

[0046]

[0047] Figure 1 As shown in Table 1, there were two non-Saccharomyces yeasts with relative abundances greater than 1% on the surface of fresh mulberries, namely Hanseniaspora and Pichia. The relative abundance of Hanseniaspora (15.51%) was the highest on the surface of mulberries stored at -1°C for 4 days. The relative abundance of Hanseniaspora was higher in all samples stored at -1°C than at 4°C. With the extension of storage time, the relative abundance of Hanseniaspora on the surface of mulberries stored at -1°C first increased, then decreased, and then tended to equilibrium. Low-temperature storage decreased the relative abundance of Pichia on the surface of mulberries, while room-temperature storage increased the relative abundance of Pichia. The relative abundance of Pichia (16.15%) was the highest on the surface of mulberries stored at room temperature for 4 days.

[0048] Table 2 Relative abundance of each species in the yeast community

[0049]

[0050] Figure 2As shown in Table 2, the genus Hansenia sporauvarum is mainly concentrated in Hansenia sporauvarum, while the genus Pichia is mainly concentrated in Pichia kluyveri. Among them, Hanseniaspora uvarum, the strain deposit number is CCTCC M 20221245S006-02, the deposit date is August 4, 2022, the deposit name is China Center for Type Culture Collection (CCTCC), and the deposit address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province; Pichia kluyveri, the strain deposit number is CCTCCM20221246S007-03, the deposit date is August 4, 2022, the deposit name is China Center for Type Culture Collection (CCTCC), and the deposit address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0051] Depend on Figure 3 It can be seen that under low temperature storage conditions, the number of fungi in each sample was 1.05×10 7 -4.90×10 9 The CFU / g of mulberry fruit was the lowest when stored at -1℃ for 4 days. However, the number of culturable fungi on the surface of mulberry fruit increased sharply under room temperature. After 4 days of storage, the surface of mulberry fruit was seriously moldy and a large amount of water seepage was observed. The maximum number was 3.39×10 11 CFU / g.

[0052] The comprehensive results showed that in order to increase the number of target bacteria in the samples on the plates, mulberry samples with relatively high abundances of Hansenula and Pichia were selected as screening samples for their respective species, and the results of the number of culturable fungi were used as a reference (because the number of culturable fungi on the surface of mulberry samples includes yeasts, which can indirectly reflect the number of each yeast genera. Therefore, selecting samples with high relative abundances of each yeast genera and low fungal counts can increase the number of target bacteria on the screening plates). Therefore, based on the high-throughput sequencing results and plate count results, mulberry samples stored at -1°C for 4 days were selected for Hansenula, and mulberry samples stored at room temperature for 4 days were selected for Pichia.

[0053] 2. Strain Screening Results

[0054] Reference Figure 4 、 Figure 5 、 Figure 6 ,in Figure 4 Note: A1: K, D254 Saccharomyces cerevisiae; A2: isolated strain S006-01, suspected to be Hansenula sporangiophora; A3: isolated strain S007-01, suspected to be Pichia pastoris. Figure 5Among them, B1: K, D254 cerevisiae; B2: isolated strain S006-01, suspected spore-forming Hansenula; B3: isolated strain S007-01, suspected Pichia; Figure 6 Note: K is D254 Saccharomyces cerevisiae, S006-01, S006-02, S007-01, S007-02, S007-03 are the screening strain numbers.

[0055] Two suspected Hansenula sporogenes strains were isolated from mulberry samples grown at -1°C for four days and were numbered S006-01 and S006-02. Three suspected Pichia strains were isolated from mulberry samples grown at room temperature for four days and were numbered S007-01, S007-02, and S007-03. These strains were identified using morphological observation, olfactory recognition, and molecular biological methods.

[0056] The strains S006-01 and S006-02 produced aroma in the culture medium, but the aroma and wine taste were both weak; the colonies on the YPD solid medium were milky white, shiny, with neat edges, raised edges, and moist surfaces; the staining results showed that the cells were lemon-shaped, reproduced asexually, and budded at both poles (see Figure 5 B2). The phylogenetic tree showed that strains S006-01 and S006-02 were on the same branch as Hanseniaspora uvarum (see Figure 6 ), and combined with the morphological identification results, strains S006-01 and S006-02 were identified as Hansenula sporangiophora vitis, which was consistent with the results of non-Saccharomyces species identification in the -1℃4d mulberry sample in Table 2.

[0057] Strain S007-01, S007-02, and S007-03 all have a white floating film on the surface of the culture medium and produce a fragrance that varies in intensity and has a wine-like flavor. On YPD solid medium, the colonies are white, with a dull, rounded surface and irregular, toothed edges. The cells are about the same size as Saccharomyces cerevisiae (see Figure 4 A3); staining results showed that the cells were oval or elliptical in shape, reproduced sexually, and produced asci, each of which contained 1-4 ascospores of different shapes (see Figure 5 B3). The phylogenetic tree showed that strains S007-01, S007-02, and S007-03 were on the same branch as Pichia kluyveri (see Figure 6 ), and combined with the morphological identification results, strains S007-01, S007-02, and S007-03 were identified as Pichia kluyveri, which was consistent with the results of non-Saccharomyces species identification in the mulberry samples grown at room temperature for 4 days in Table 2.

[0058] 3. Screening of yeast strains for tolerance analysis

[0059] Reference Figure 7 Note: A, yeast tolerance to temperature; B, yeast tolerance to high sugar; C, yeast tolerance to sulfur dioxide; D, yeast tolerance to low pH; E, yeast tolerance to alcohol content; S006-01, S006-02, S007-01, S007-02, S007-03, represent different yeast strains.

[0060] The six yeast strains screened above were subjected to temperature, sugar content, sulfur dioxide, acidity, and alcohol tolerance tests. The larger the yeast OD value, the more vigorous the yeast growth, that is, the stronger the tolerance to the conditions. The results are shown in Figure 7 .

[0061] Figure 7 The results showed that temperature had a greater impact on Hansenula sporangiophora S006-01 and S006-02, and both high and low temperatures would inhibit their growth. They grew better at 30℃. Pichia kluyveri S007-01 and S007-02 could withstand high temperatures of 40℃ but not low temperatures of 10℃, while S007-03 could withstand high and low temperatures. All three strains grew vigorously at 20-30℃.

[0062] All strains grew well at sugar concentrations of 150-300 g / L. As sugar concentration increased, the OD values ​​of non-brewer's yeast showed a negative correlation. High sugar concentration had a greater impact on Hansenula sporogenes S006-01 and S006-02.

[0063] The screened strains can all grow in culture medium containing 50-200 mg / L SO2. Among them, strains S007-01, S007-02, and S007-03 have stronger tolerance to SO2, while Hansenula sporangiophora S006-01 and S006-02 have weaker tolerance to SO2, and strain S006-01 has the weakest tolerance.

[0064] The results showed that all the screened strains could grow in low-acid environments. Among them, strains S007-01, S007-02, and S007-03 had stronger tolerance to low pH, while Hansenula sporangiophora S006-01 and S006-02 were not tolerant to low acid, and strain S006-01 had the weakest growth in low-acid environments.

[0065] from Figure 7It can be seen that Pichia kluyveri S007-03 has the strongest alcohol tolerance and can grow vigorously in culture medium with an alcohol content of 5%. The other strains are intolerant to alcohol. This fully demonstrates that most non-brewers' yeasts are sensitive to alcohol and only exist in the early stages of fermentation. Therefore, if brewer's yeast is used to quickly increase the alcohol content in the fermentation liquid during the fermentation process of mulberry wine, the growth of non-brewers' yeasts in mulberry resources will be greatly inhibited.

[0066] In summary, among the non-Saccharomyces yeast strains, Pichia kluyveri S007-03 thrived in the early stages of mulberry wine fermentation. However, the growth of strains S007-02, S007-03, and S006-02 was significantly inhibited as alcohol content increased during fermentation. Furthermore, strain S006-01 was unsuitable for mulberry wine fermentation. Therefore, S007-03 and S006-02 were selected as superior yeast strains for further testing.

[0067] 4. Analysis of the fermentation performance of mulberry wine by screening strains

[0068] Table 3 Analysis of components of mulberry wine fermented by yeast strains

[0069]

[0070] Note: Mulberry fermentation liquid 1, fermentation with Saccharomyces cerevisiae; Mulberry fermentation liquid 2, mixed fermentation with Saccharomyces cerevisiae and Hansenula sporangiophora vitis S006-02; Mulberry fermentation liquid 3, mixed fermentation with Saccharomyces cerevisiae and Pichia kluyveri S007-03; Mulberry fermentation liquid 4, mixed fermentation with Saccharomyces cerevisiae, Hansenula sporangiophora vitis S006-02 and Pichia kluyveri S007-03.

[0071] Table 4 Analysis of components of mixed-bacteria fermented mulberry wine

[0072]

[0073]

[0074] Table 5 Volatile aroma components and relative contents of mulberry wine fermented by screened yeast strains

[0075]

[0076]

[0077]

[0078] Table 3 shows that the screened strains can grow in a mulberry fermentation liquid with a sugar content of 330 g / L, and the alcohol content of the mulberry wine reaches above 11% vol, with the highest alcohol content reaching 13% vol. When brewing yeast and a single non-brewer's yeast, the lactic acid content increases, the volatile acid content decreases, and the alcohol content of the mulberry wine reaches above 13% vol. When brewing yeast and multiple flavor non-brewer's yeasts, the lactic acid content decreases, the volatile acid content also decreases slightly, and the alcohol content of the mulberry wine reaches above 13% vol. The mixed fermentation produces a higher alcohol content than fermentation with brewer's yeast alone, and the total ester content of the resulting mulberry wine is increased by 25%.

[0079] Tables 4 and 5 show that mixed fermentation, with the sugar content of the fermentation broth adjusted to achieve a final alcohol content of 12% vol, resulted in superior ethanol production and volatile acid content compared to single yeast fermentation. The volatile aroma components of the resulting wine increased by 0.76% for esters, 0.48% for alcohols, 0.05% for aldehydes, and 0.06% for ketones, while acids decreased by 0.54%. Furthermore, the volatile components in wine fermented with multiple strains were more abundant, with a total of 61 detected volatile aroma components, including 31 esters, 6 alcohols, 2 acids, 19 hydrocarbons, 1 aldehyde, 1 ketone, and 1 other. In contrast, 53 volatile aroma components were detected in wine fermented with Saccharomyces cerevisiae, including 23 esters, 3 alcohols, 3 acids, 21 hydrocarbons, 1 aldehyde, and 2 other.

Claims

1. A method for improving the flavor of mulberry wine using aroma-producing yeast screened from mulberries, characterized by: The aroma-producing yeast is Hansenula vitis spores ( Hanseniaspora uvarum ) and Pichia kluyveri ( Pichia kluyveri ), the deposit number of Hansenula vitis spores is CCTCC NO: M 20221245, the deposit number of Pichia kluyveri is CCTCC NO: M 20221246, and the method comprises the following steps: According to brewer's yeast: Hansenula vitis vinifera: Pichia kluyveri = 10 7 :10 5 :10 5 The number of CFU / mL was inoculated into mulberry fermentation broth and fermented with mixed strains at 24 ℃ for 6 days. The lees were separated and placed at 20 ℃ for 1 month. The supernatant was filtered through a 0.22 μm plate and frame filter to obtain mulberry wine.

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