Yeast and application thereof in wine fermentation
The mutant strain of Saccharomyces cerevisiae D3-1 (AE-43) obtained through laboratory adaptive evolution solved the problem of lack of wine aroma due to climate change, significantly increased the yield of 2-phenylethanol and phenethyl acetate, and enhanced the flower and fruit aroma of the wine.
Patent Information
- Application Number
- CN202510028466.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-08
- Publication Date
- 2025-05-02
AI Technical Summary
Global climate warming affects the aromatic form of wine aroma, especially the reduction of floral properties, mainly due to the inhibition of L-phenylalanine utilization by yeasts, resulting in a decrease in the yield of 2-phenylethanol and phenylethyl acetate.
Through laboratory adaptive evolutionary methods, a mutant strain of Saccharomyces cerevisiae D3-1 (AE-43) was obtained. This strain was able to utilize L-phenylalanine more efficiently and increase the yield of 2-phenylethanol and phenylethyl acetate.
It significantly increases the content of 2-phenylethanol and phenylethyl acetate in the wine, imparting more flower and fruit aroma to the wine and enhancing the aroma complexity of the wine.
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Figure CN119913053A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of yeast and wine fermentation, and particularly relates to a yeast and application thereof in wine fermentation. Background Art
[0002] Aroma is an important sensory characteristic of wine and is highly valued by experts and consumers in the wine field. Affected by many factors such as global warming, the secondary metabolism of grapes is affected during the growth process, resulting in a thin aroma of wine and a lack of aromatic aroma. Therefore, it has become a top priority to carry out targeted research and development breeding based on the huge Chinese native wine yeast germplasm resources to solve the negative impact of global warming on wine. Fruity ester compounds are the most abundant volatile components in the aroma of wine, which leads to the lack of typical "floral" attributes, mainly presented by 2-phenylethanol and its derivatives. Directed breeding of aroma-enhancing yeast to compensate for the lower aromatic complexity of wine caused by the secondary metabolism of grapes during growth due to global warming is the current research focus. L-phenylalanine is an important nitrogen-containing compound aroma in grape juice and a precursor of aroma compounds phenylethanol and phenylethyl acetate. However, L-phenylalanine is not the preferred nitrogen source for yeast during grape juice fermentation. When other preferred nitrogen sources exist in the grape juice, the utilization of L-phenylalanine by Saccharomyces cerevisiae is inhibited, thereby hindering the production of aroma compounds 2-phenylethanol and phenylethyl acetate.
[0003] Laboratory adaptive evolution refers to a strategy of changing the growth environment of microorganisms under laboratory conditions, causing spontaneous mutations in the process of environmental adaptation to achieve the purpose of evolution, thereby achieving the breeding goal. It has the characteristics of low cost, short screening time, direct breeding of mutant strains, and high safety of the obtained strains, and has received more and more attention and application. The present invention aims to improve the utilization of L-phenylalanine by brewer's yeast by using the method of laboratory adaptive evolution, increase the yield of 2-phenylethanol and phenylethyl acetate, and thus give wine more floral and fruity aroma. Summary of the invention
[0004] One of the purposes of the present invention is to provide a method for improving the production of 2-phenylethanol and phenylethyl acetate by brewer's yeast, and to obtain a flavor-enhancing mutant strain through laboratory adaptive evolution; the second purpose is to provide an application of the yeast in wine flavor enhancement, with clear and significant effects.
[0005] After research, the present invention provides the following technical solutions.
[0006] The present invention discloses a yeast Saccharomyces cerevisiae D3-1 (AE-43), which has a deposit number of CGMCCNO.31678 at the General Microbiological Center of China National Microbiological Culture Collection Administration on August 19, 2024.
[0007] The invention also discloses the application of yeast S.cerevisiae D3-1 (AE-43) in the preparation of wine.
[0008] The invention also discloses a method for preparing wine. The method uses grapes as raw materials, pre-treats the raw materials, and then adds yeast S.cerevisiae D3-1 (AE-43) to ferment and prepare the wine.
[0009] Furthermore, the grapes are Sauvignon Blanc grapes.
[0010] Beneficial effects:
[0011] The present invention provides a method for preparing wine, and obtains a flavor-enhancing mutant strain (S.cerevisiae AE-43) with high yield of phenylethanol and phenylethyl acetate through laboratory adaptive evolution, and the obtained mutant strain (S.cerevisiaeAE-43) can be used for flavor enhancement of Sauvignon Blanc wine. The present invention not only obtains a yeast strain that can significantly enhance the flavor, enriches the resource library of brewing yeast strains, broadens the application scope of yeast in the field of wine flavor enhancement, but also provides a new solution for wine flavor enhancement.
[0012] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 Schematic diagram of the selection and experimental method of laboratory adaptive evolution pressure, including the growth of the starting strain S. cescerevisiae D3-1 in CDJGM with different concentrations of L-Phe (A), the residual amount of L-Phe and the yield of 2-PE in the fermentation broth (B), the laboratory adaptive evolution process (C), and the growth curve of the starting strain S. cerevisiae D3-1 during the laboratory adaptive evolution process (D);
[0014] Figure 2The effects of different generations of populations on fermentation rate and phenylalanine utilization, among which the effects of S. cescerevisiae D3-1 evolutionary population on sugar metabolism (A) and L-Phe (B) metabolism;
[0015] Figure 3 The effect of different generation groups on the production of phenylethyl alcohol and phenylethyl acetate, where A is phenylethyl alcohol and B is phenylethyl acetate;
[0016] Figure 4 Screening of excellent aroma-enhancing mutant strains of native brewer's yeast S.cerevisiae D3-1, including the maximum biomass of a single clone isolate of the evolutionary population (A) and the maximum biomass of four excellent strains screened in 100 generations (B);
[0017] Figure 5 To study the effect of the aroma-enhancing S.cerevisiae D3-1 mutant strain on the content of phenylethanol and phenylethyl acetate in Sauvignon Blanc wine, where A is phenylethanol and B is phenylethyl acetate;
[0018] Figure 6 The effect of the mutant strain on other aroma substances in Sauvignon Blanc wine, where A is the concentration of different types of aroma compounds, B is the heat map of aroma compounds in Sauvignon Blanc wine, and C is the visualization of aroma compounds of different yeasts. DETAILED DESCRIPTION
[0019] In order to make the technical solutions, advantages and purposes of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of this application.
[0020] The invention discloses a yeast S.cerevisiae D3-1 (AE-43), whose preservation number in the General Microbiological Center of China Microbiological Culture Collection Administration is CGMCCNO.31678.
[0021] The invention also discloses the application of yeast S.cerevisiae D3-1 (AE-43) in the preparation of wine.
[0022] The invention also discloses a method for preparing wine. The method uses grapes as raw materials, pre-treats the raw materials, and then adds yeast S.cerevisiae D3-1 (AE-43) to ferment and prepare the wine.
[0023] 1. Experimental Methods
[0024] 1. Purification and activation of native yeast strain S.cerevisiae D3-1
[0025] Take out the frozen strains in glycerol tubes from the -80℃ freezer and purify them by streaking on YPD solid medium. Cultivate at 30℃ for 24-48h, then select single colonies with regular morphology and no overlap with other colonies. Pick a single colony and expand it in YEPD liquid medium, and cultivate it at 30℃ and 200r / min until the logarithmic phase to obtain seed liquid.
[0026] 2. Laboratory adaptive evolution of the native Saccharomyces cerevisiae D3-1
[0027] Saccharomyces cerevisiae D3-1 seed culture was carried out with 1×10 6 cell mL -1 The inoculum size was inoculated into sterile simulated grape juice (CDGJM) with L-phenylalanine as the sole nitrogen source. The fermentation system was 100 mL and the concentrations of L-phenylalanine were 1, 2, 3, 4, and 5 g L -1 , incubate at 25°C, measure OD every 12 h 600nm To evaluate the growth of yeast in different concentrations of phenylalanine, when it grows to the stable phase, the concentrations of L-phenylalanine and 2-phenylethanol in the fermentation broth are measured.
[0028] 1x10 cells were inoculated in CDGJM containing 2 g / L phenylalanine as the sole nitrogen source (CDGJM-phenylalanine). 6 S.cerevisiae D3-1 with a concentration of 1x10 cells / mL was cultured in a 25°C incubator. The yeast count was counted daily using a hemocytometer. When the yeast reached a stable growth period, the acclimated bacterial solution was inoculated into fresh CDGJM-phenylalanine medium and cultured under the same conditions. The inoculation volume was 1x10 6 cells / mL, and the acclimation culture of S.cerevisiaeD3-1 was terminated when the acclimation was carried out to the 200th generation.
[0029] The Mix strains domesticated to the 50th, 100th, 150th, and 200th generations were fermented at 1×10 6 cellmL -1 Inoculate into a conical flask containing 100 mL of LCDJM for fermentation, and ferment in a 25°C incubator. Use the starting strain S. cerevisiae D3-1 as the control. Each treatment has 3 replicates. Pipette 1 mL of sample under sterile operation every 24 hours, centrifuge, take a certain amount of supernatant to detect residual sugar, and place the remaining supernatant in a -40°C refrigerator.
[0030] 3. Screening of excellent mutant strains of local Saccharomyces cerevisiae D3-1
[0031] The strains of 50, 100, 150, and 200 generations of domestication were screened. The bacterial suspensions of different generations were diluted in gradients, spread on WLN solid medium, and cultured in a 30°C constant temperature incubator for 2 to 5 days. The dilution gradient plates with yeast counts of about 100 were selected, and the single colonies were dipped and stored in glycerol tubes and numbered. The single colony suspension (1x10 6 cells mL -1 ) were grown in 8 mL simulated juice with 2 g / L phenylalanine as the sole nitrogen source. The starting strain and commercial yeast were used as controls, with three replicates per group. The culture was placed in a 25°C incubator for static culture, and the absorbance of the bacterial solution was detected at a wavelength of 600 nm every 12 hours to characterize the growth of the strain.
[0032] 4. Fermentation of grape juice with aroma-enhancing mutant strains
[0033] Sauvignon Blanc grape juice was sterilized using DMDC and allowed to stand at 4°C for 24 hours. The yeast strains in this study were inoculated into YPD liquid medium and activated and cultured at 30°C and 180 r / min for 1-2 days. After centrifugation (3000 r / min, 5 min), the cells were washed with sterile water, counted with a hemocytometer, and diluted with sterile distilled water to the required test concentration. The cells were divided into 1x10 5 cells mL -1 Grape juice was added for fermentation. 600 mL of grape juice was placed in a 1 L wide-mouth bottle at 20°C for static fermentation. Three biological replicates were performed for each group.
[0034] 5. HPLC determination of phenylalanine content in fermentation broth
[0035] Use the VenusilAA amino acid analysis column to determine the phenylalanine content. Accurately measure 200μL of amino acid standard solution and sample and place them in 1.5mL centrifuge tubes. Then accurately add 20μL of norleucine internal standard solution to each centrifuge tube, add 100μL of triethylamine acetonitrile solution and 100μL of phenyl isothiocyanate acetonitrile solution to each centrifuge tube, mix well, and place at room temperature for 1 hour. Then add 400μL of n-hexane to each centrifuge tube, shake and place for 10min. Take the lower layer solution (PTC-AA) and filter it with a 0.45μm needle filter. Take 200μL of the filtrate, dilute it with 800μL of water, shake well, and inject 10μL.
[0036] VenusilAA amino acid analysis column 4.6×250mm, 5μm; column temperature: 40℃; injection volume: 10μL; wavelength: 254nm; flow rate: 1.0mL / min; Nle is the internal standard of norleucine. Mobile phase A: weigh 15.2g of anhydrous sodium acetate, add 1850mL of water, dissolve it, adjust the pH to 6.5 with glacial acetic acid, then add 140mL of acetonitrile, mix well, and filter with a 0.45μm filter membrane. Mobile phase B: 80% acetonitrile aqueous solution, gradient elution is as follows.
[0037]
[0038]
[0039] 6. Wine aroma measurement
[0040] The volatile compounds of wine were analyzed by gas chromatography and mass spectrometry (HS-SPME-GC-MS) with some modifications to a previously reported method. The method was as follows: 5 mL of wine, 1.3 g of NaCl, and 10 μL of internal standard (4-methyl-2-pentanol, 1.034 g / L) were placed in a 20 mL glass bottle, which was located in the thermostatic autosampler tray for HS-SPME sampling. Then, it was equilibrated at 40 °C with a shaking speed of 400 rpm for 30 min, and then the extraction head was inserted into the sample and adsorbed at 40 °C and 250 rpm for 30 min before injection. The carrier gas was high-purity helium with a flow rate of 1 mL / min and the injection port temperature was 250 °C for 25 min. The temperature program was 50 °C for 1 min, then increased to 220 °C at a rate of 3 °C / min and maintained for 5 min. The mass spectrometer interface temperature was 280°C, the ion source temperature was 230°C, the EI ionization mode, the ion energy was 70 eV, and the mass spectrometer scanning range was 29-350 u. Each sample was measured three times.
[0041] 2. Experimental results
[0042] 1. Laboratory adaptive evolution of the indigenous Saccharomyces cerevisiae D3-1
[0043] like Figure 1 As shown in the figure, the laboratory adaptive evolution pressure was selected. Different concentrations of L-phenylalanine inhibited the growth of S.cerevisiae D3-1. By measuring the residual L-phenylalanine, it was found that in 2g / L L-phenylalanine, 20mg of L-phenylalanine was not completely used when the growth of S.cerevisiae D3-1 reached the stable period. Therefore, in the subsequent experiments, 2g / L L-phenylalanine was used as the only nitrogen source as pressure for laboratory adaptive evolution. Figure 1C. The native brewer's yeast S.cerevisiae D3-1 was domesticated in CDGJM containing 2g / L phenylalanine for 101 days, and a total of 200 generations of S.cerevisiae D3-1 strains were obtained. As the domestication progressed, the generation time of S.cerevisiae D3-1 strain in CDGJM became shorter, and the adaptability of S.cerevisiae D3-1 to the culture conditions gradually increased ( Figure 1 D).
[0044] 2. Effect of different generations of population fermentation rate on phenylalanine utilization
[0045] The strains were screened for excellent strains based on their growth and metabolic capacity for sugar and phenylalanine. The study found that the evolved strain population was able to complete alcohol fermentation, and the fermentation rate of the G100 population was the fastest, significantly higher than that of the starting strain S.cerevisiae D3-1. Further analysis of the utilization of L-phenylalanine by the evolved population showed that the G100 population was able to utilize L-phenylalanine faster and more completely.
[0046] 3. Effects of different generation groups on the content of 2-phenylethanol and phenylethyl acetate
[0047] Inoculate 1x10 6 The fermentation verification test was carried out with 10 cells / mL bacterial liquid to detect its ability to produce phenylethanol and phenylethyl acetate. The study found that the evolved population was able to produce more 2-phenylethanol in normal simulated juice, among which the G100 generation population had the highest yield. Phenylethanol produces the aroma compound phenylethyl acetate under the action of acetyl coenzyme A. The study found that the ability of the G100 generation population to produce phenylethyl acetate was significantly higher than that of the starting strain.
[0048] 4. Screening of excellent aroma-enhancing mutant strains of local brewer's yeast S.cerevisiae D3-1
[0049] When the domestication was carried out to the 200th generation, the adaptive domestication of S.cerevisiae D3-1 was terminated, and the bacterial liquid at the 50th, 100th, 150th, and 200th generations was taken for YPD liquid culture expansion, and 500 μL of bacterial liquid was mixed with 40% glycerol and stored in a -40°C refrigerator. 50-60 strains were isolated from each of the G50, 100th, 150th, and 200th generation populations, and tolerance experiments were carried out in CDGJM with 2g / L L-phenylalanine as the only nitrogen source. The experimental results showed that the growth of most of the evolved populations in the simulated juice of 2g / L L-phenylalanine was better than that of the starting strain (Max.OD 600nm ), and the strains isolated in the 100th generation performed more prominently. Figure 2 , Figure 3Based on the results, we selected the 100th generation strains that showed obvious advantages as the next research object, and selected the four strains AE-1, AE-31, AE-43, and AE-45 that grew best in 2g / L L-phenylalanine simulated juice for the next grape juice fermentation verification study. Figure 4 shown.
[0050] 5. Effect of aroma-enhancing S.cerevisiae D3-1 mutant on the contents of phenylethanol and phenylethyl acetate in Sauvignon Blanc wine
[0051] The best performing strains AE-1, AE-31, AE-43, and AE-45 in the rescreening experiment were selected for grape juice fermentation verification test. 6 cells / mL were inoculated into a 1L glass bottle containing 600mL Sauvignon Blanc grape juice, with the starting strains S.cerevisiae D3-1, G100 and commercial yeast F15 as controls, with 3 parallels in each group. The cells were placed in a 20℃ constant temperature incubator for static culture, and the ability of yeast to produce phenylethanol and phenylethyl acetate was tested after the fermentation was completed.
[0052] like Figure 5 As shown in A, in terms of the ability to produce phenylethanol, the mutant strains are better than the starting strain S.cerevisiae D3-1 in terms of phenylethanol production. The AE-43 mutant strain performed outstandingly, with the content of phenylethanol produced being significantly higher than the starting strains S.cerevisiae D3-1 and G100, and even outstanding compared to the commercial yeast F15; the AE-1 mutant strain performed second, but was significantly higher than the starting strains S.cerevisiae D3-1 and G100 population.
[0053] like Figure 5 As shown in B, in terms of the ability to produce phenylethyl acetate, the AE-1, AE-31 and AE-43 mutants performed outstandingly, with the content of phenylethyl acetate produced being higher than that of the starting strains S.cerevisiae D3-1 and G100, and having no significant difference with the commercial yeast F15; while the ability of the AE-45 mutant to produce phenylethyl acetate was basically the same as that of the starting strain S.cerevisiae D3-1.
[0054] 6. Effects of mutant strains on other aroma substances in wine
[0055] In order to visually examine the relationship between wine samples and aroma substances, principal component analysis was performed on the aroma substances of each strain. Figure 6 .like Figure 6As shown in the figure, the evolved strain can significantly increase the content of higher alcohols, aromatic compounds, terpene compounds, fatty acid ethyl esters and ethyl acetate in Sauvignon Blanc wine. Further analysis of aroma compounds showed that the content of terpenes, ethyl acetate and other ethyl ester compounds in Sauvignon Blanc wine brewed by the evolved strain was significantly higher than that of the starting strain. From the PCA-Biplot diagram, it can be found that phenylethanol and phenylethyl acetate contribute more to the aroma of the mutant strain than the starting strain, especially the mutant strain AE-43. Further analysis of differential compounds showed that the content of phenylethanol (rose fragrance), phenylethyl acetate (floral fragrance), 1-hexanol (fruity fragrance), isoamyl acetate (banana flavor), geraniol (rose), ethyl acetate (pineapple) and ethyl propionate (pineapple) in the wine brewed by the evolved strain was significantly higher than that of the starting strain S.cerevisiae D3-1, so the evolved strain gave Sauvignon Blanc wine a richer floral and fruity aroma.
[0056] It is hereby declared that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions, which should be included in the scope of the claims of the present invention.
Claims
1. A yeast S.cerevisiae D3-1 (AE-43), whose deposit number in the General Microbiological Center of China Microorganism Culture Collection Administration is CGMCC NO.31678.
2. Use of the yeast S.cerevisiae D3-1 (AE-43) according to claim 1 in preparing wine.
3. A method for preparing wine, characterized in that: The method uses grapes as raw materials, pre-treats the raw materials, and adds the yeast S.cerevisiae D3-1 (AE-43) described in claim 1 to ferment and prepare wine.
4. The method for preparing wine according to claim 3, characterized in that: The grapes are selected from Sauvignon Blanc grapes.