Saccharomycetes, fungicide and application of saccharomycetes and fungicide in improvement of flavor and / or quality stability of wine

Abstract

The invention relates to the field of microorganisms, and discloses saccharomycetes, a microbial agent and application of the saccharomycetes and the microbial agent in improving the flavor and / or quality stability of wine. The saccharomycetes are Saccharomyces cerevisiae with the preservation number of CGMCC (China General Microbiological Culture Collection Center) NO.30497, and the saccharomycetes are saccharomyces cerevisiae with the preservation number of CGMCC NO.30497; the saccharomycetes are Hanseniaspora uvarium with the preservation number of CGMCC (China General Microbiological Culture Collection Center) NO.30499, or the saccharomycetes are Hanseniaspora uvarium with the preservation number of CGMCC NO.30499. The yeast provided by the invention can effectively improve the flavor and quality stability of wine when applied to wine fermentation.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a yeast and a bacterial agent and applications thereof in improving the flavor and / or quality stability of wine. Background Art

[0002] Adding Saccharomyces cerevisiae during the wine fermentation process can not only inhibit the growth of other harmful microorganisms, but also convert sugar into alcohol to the greatest extent and produce byproducts such as esters and higher alcohols that have a positive impact on the aroma of wine. In addition to Saccharomyces cerevisiae, non-Saccharomyces cerevisiae will affect the final quality of wine through the interaction of various factors such as synergy, inhibition, and accumulation. However, since non-Saccharomyces cerevisiae cannot complete alcohol fermentation independently, it is necessary to use a mixed fermentation method with Saccharomyces cerevisiae to complete alcohol fermentation in order to improve the flavor of the wine and enhance its sensory properties.

[0003] During the wine fermentation process, in order to avoid the stagnation phenomenon where the yeast stops being active before the alcohol content reaches the expected standard, the winery will optimize the fermentation temperature, oxygen supply, sulfur dioxide dosage and pH value. However, the sulfur dioxide and pH conditions set to inhibit the growth of bacteria may inhibit the activity of yeast or even inactivate it, so there is an urgent need for excellent yeast strains to match the appropriate grape varieties to avoid stagnation during the fermentation process, ensure the smooth fermentation of the wine, and improve the taste and flavor of the wine. The wine industry in the Changli production area mostly uses commercial yeast for fermentation, lacks local characteristics, and the flavor and taste tend to be consistent. Therefore, there is an urgent need for excellent yeast strains to ferment with high-quality grapes, so as to improve fermentation stability, shorten wine fermentation time, save production costs, and brew wines that meet the characteristics of this production area. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems existing in the prior art and provide a yeast and a bacterial agent and their use in improving the flavor and / or quality stability of wine. The yeast is applied to the fermentation of native grapes to improve the flavor and quality of wine while avoiding stagnation.

[0005] In order to achieve the above object, the present invention provides a yeast on one hand, wherein the yeast is Saccharomyces cerevisiae with a preservation number of CGMCC NO.30497; or, the yeast is Hanseniaspora uvarum with a preservation number of CGMCC NO.30499.

[0006] The second aspect of the present invention provides a bacterial agent, wherein the bacterial agent contains Saccharomyces cerevisiae with a preservation number of CGMCC NO.30497 and / or Hansenula sporogenes with a preservation number of CGMCC NO.30499.

[0007] A third aspect of the present invention provides a method for preparing wine.

[0008] A fourth aspect of the present invention provides a wine produced by the method provided by the present invention.

[0009] A fifth aspect of the present invention provides a use of the yeast or bacterial agent provided by the present invention in improving the flavor and / or quality stability of wine.

[0010] Through the above technical solution, the beneficial effects of the present invention at least include:

[0011] The present invention isolates and screens two strains from vineyard soil, wine grapes and wine fermentation in Changli area, and identifies them as Saccharomyces cerevisiae and Hansenula sporei in grape juice. Through fermentation performance and tolerance tests, it is found that the pH value tolerance of Saccharomyces cerevisiae is up to 2.5, and it can be grown in SO 2 The yeast can metabolize normally in an environment with a pH of 200 mg / L, a glucose content of 10-40 wt% and an ethanol content of 12 vol.%; Hansenula vitis spores can tolerate a pH value of 3.0 and can 2 It can survive and ferment normally in an environment with an aqueous solution of 150 mg / L, a glucose content of 10-30 wt% and an ethanol content of 18 vol.%.

[0012] The physical and chemical indices of the wine prepared by the invention all meet the requirements of the national standard GB / T 15037-2006. When the two strains of the present invention are used to ferment wine alone, the wine brewed by the brewer's yeast with the accession number of CGMCC NO.30497 has a relatively strong and coordinated aroma, good coordination, good palatability and the best taste; the wine brewed by the grape juice Hansen yeast with the accession number of CGMCC NO.30499 has a relatively obvious fruity aroma and chemical taste as a whole. When the above two strains are compounded in a ratio of 1:1, the fermentation rate is greater than that of commercial yeast or single brewer's yeast strain fermentation, the number of yeast reaches a peak on the first day of fermentation, and the fermentation can be completed in only six days, which greatly shortens the time required for fermentation, and the sugar content of the wine is the smallest after the fermentation is completed, and the utilization rate of the sugar in the wine is the highest. By using it in combination with grape varieties with high sugar content, a higher alcohol content and a faster fermentation speed can be obtained under the premise of minimizing the amount of grapes and not changing the fermentation process and fermentation conditions; and the sensory quality is more excellent: first, the aroma is more strong and complex, especially the floral aroma, honey aroma and fruit aroma, and these strong aromas express the flavor characteristics of local wine; the wine presents a bright purple-red color; the taste is coordinated and has good typicality. In addition, the content of higher alcohols in the wine of the present invention is significantly lower than that of wine brewed with commercial yeast, which can reduce discomfort such as a feeling of being in a state of dizziness after drinking.

[0013] Biological Deposit

[0014] The brewer's yeast (Saccharomyces cerevisiae) of the present invention was deposited on May 6, 2024 at the General Microbiology Center of China Culture Collection Administration (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101) (the abbreviation of the depository unit is CGMCC), with the deposit number CGMCC No. 30497, abbreviated as Sc28.

[0015] The grape juice spore Hanseniaspora uvarum of the present invention was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101) on May 6, 2024 (the abbreviation of the depository unit is CGMCC), and the deposit number is CGMCC No. 30499, abbreviated as Hu19. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 are soil (SO), fermentation tank equipment (FT), wine grapes (G), cold maceration stage (MC), and the separation of yeasts in the early, middle and late fermentation stages;

[0017] Figure 2 It is the different levels of yeast’s ability to produce alcohol;

[0018] Figure 3 Yeast produces H 2 Different levels of S ability;

[0019] Figure 4 It is the ability of yeast to produce β-glucosidase;

[0020] Figure 5 is a heat map summarizing the basic brewing characteristics, where (a) is brewing yeast and (b) is non-brewing yeast;

[0021] Figure 6 is a heat map of Saccharomyces cerevisiae tolerance;

[0022] Figure 7 is a heat map of non-Saccharomyces cerevisiae tolerance;

[0023] Figure 8 is the aroma intensity score of the wine, where (a) is a single strain and (b) is a composite yeast;

[0024] Fig. 9 The following are the conditions of yeasts under different fermentation modes, where (a) shows the dynamic changes in the number of yeasts under different fermentation modes, and (b) shows the differentiation of different yeasts in the culture medium;

[0025] Fig.10 It is the content of volatile flavor substances in wine under different fermentation methods;

[0026] Fig.11 is the PLS-DA of volatile flavor substances under different fermentation methods, where (a) is the score graph, (b) is the loading graph, and (c) is the VIP score graph;

[0027] Fig.12 The dynamic changes of organic acid content in wine under different fermentation methods, among which (a) is tartaric acid, (b) is malic acid, (c) is lactic acid, (d) is citric acid, and (e) is succinic acid;

[0028] Fig.13 It is a radar chart of sensory evaluation of wine under different fermentation methods. DETAILED DESCRIPTION

[0029] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0030] A first aspect of the present invention provides a yeast, wherein the yeast is Saccharomyces cerevisiae with a preservation number of CGMCC NO.30497; or the yeast is Hansenula sporogenes with a preservation number of CGMCC NO.30499.

[0031] In the present invention, the brewer's yeast with a preservation number of CGMCC NO.30497 and the Hansenula vitis spores with a preservation number of CGMCC NO.30499 are both separated and screened from the vineyard soil of Fenghuang Manor in Changli, Hebei Province, wine grapes, equipment samples and wine fermentation process under laboratory conditions.

[0032] The morphology of the Saccharomyces cerevisiae colony with the accession number of CGMCC NO.30497 on the WL medium is: round, cream-colored, smooth on the surface, and light green in the center.

[0033] The morphology of the Hansenula sporogenes colonies on the WL medium with the accession number of CGMCC NO.30499 is: round, dark green in color, with a layer of translucent lines on the outermost layer, and a smooth surface.

[0034] The second aspect of the present invention provides a bacterial agent, wherein the bacterial agent contains Saccharomyces cerevisiae with a preservation number of CGMCC NO.30497 and / or Hansenula sporogenes with a preservation number of CGMCC NO.30499.

[0035] In the present invention, in order to further improve the flavor and quality stability of wine, preferably, the bacterial agent contains Saccharomyces cerevisiae with a preservation number of CGMCC NO.30497 and Hansenula sporogenes with a preservation number of CGMCC NO.30499.

[0036] In the bacterial agent, the ratio of the number of live bacteria of Saccharomyces cerevisiae with a preservation number of CGMCC NO.30497 to that of Hansenula vitis spores with a preservation number of CGMCC NO.30499 is (0.1-6):1, preferably (0.9-1.1):1.

[0037] A third aspect of the present invention provides a method for preparing wine, the method comprising: inoculating the bacterial agent provided by the present invention into grape juice for fermentation.

[0038] In the present invention, the inoculation amount of the bacterial agent is such that the total number of viable bacteria in the grape slurry is 10 5 -10 7 CFU / mL.

[0039] In the present invention, the fermentation temperature is 15-30°C, preferably 19-24°C.

[0040] In the present invention, the fermentation time is 100-400 hours, preferably 100-200 hours.

[0041] In the present invention, the method for preparing the grape slurry comprises: mixing wine grapes with SO 2 Mix with pectinase.

[0042] In the present invention, the grape slurry can be obtained by using wine grapes as raw materials and using conventional processing methods in the art. In a preferred embodiment, the wine grapes can be crushed and destemmed first, and then the crushed and destemmed wine grapes and SO 2 and pectinase. 2 The function of cellulose is to inhibit bacteria and resist oxidation, and the function of pectinase is to clarify the brewed wine.

[0043] In the present invention, the wine grapes may be Marselan grapes.

[0044] The inventor of the present invention has found that when brewing wine using Saccharomyces cerevisiae with a preservation number of CGMCC NO.30497 and Hansenia sporei with a preservation number of CGMCC NO.30499, the utilization rate and conversion efficiency of sugar are significantly improved compared with patent CN117736890A. In one embodiment of the present invention, when the two yeasts are compounded in a ratio of 1:1, the sugar conversion rate reaches 70wt% after six days of fermentation. In addition, the two yeasts of the present invention have good sugar tolerance, can survive and stably ferment in high-sugar grape juice, are particularly suitable for use in combination with grape varieties with high sugar content, can maintain sweetness and alcohol content while shortening the fermentation time to the greatest extent, thereby reducing fermentation costs and increasing profits.

[0045] The "Marselan" grape used in the present invention is a wine grape commonly used in wineries in recent years. Compared with other wine grapes, such as Cabernet Sauvignon and Grenache, it has strong adaptability to the environment and strong resistance to common diseases such as aphids, mites, gray mold and fall; the ears are large and the grains are small, and the grape skin accounts for a larger proportion when making wine, and the wine brewed has a rich and rich flavor, a darker color and more high-quality peel tannins. However, the tannins in its peel are relatively high, which will affect the quality and style of the wine, and its sweetness and acidity are also relatively high. Therefore, when using "Marselan" grapes for winemaking, it is required that the brewing yeast must be resistant to sugar, acid and tannin at the same time. The inventors of the present invention have found that the brewing yeast provided by the present invention is very suitable for high-sugar, high-tannin and high-acid wine grapes, such as "Marselan" grapes, so the brewing yeast provided by the present invention is also applicable to other wine grapes with lower sugar content, acidity and tannin content and low requirements for yeast.

[0046] In the present invention, SO 2The dosage of pectinase is 60-150 mg / L grape slurry and 15-50 mg / kg wine grapes respectively.

[0047] In a preferred embodiment of the present invention, SO is first added to the crushed and destemmed wine grapes. 2 , then add pectinase.

[0048] The present invention is for SO 2 There is no particular limitation on the addition method of pectinase. According to a preferred embodiment of the present invention, SO 2 It is added to the crushed and destemmed wine grapes in the form of potassium metabisulfite, and then pectinase is added to the crushed and destemmed wine grapes in the form of solids.

[0049] In the present invention, the form of the bacterial agent can be a conventional bacterial agent form in the art, such as solid, liquid or semi-solid form.

[0050] The number of live bacteria in the bacterial agent can be selected within a wide range, as long as it meets the requirements of relevant standards. Taking liquid bacterial agent as an example, the content of live bacteria in the bacterial agent is preferably 10 6 CFU / mL and above.

[0051] In the present invention, the method for preparing wine further comprises: separating the skin and residue of the fermentation liquid obtained by the fermentation.

[0052] A fourth aspect of the present invention provides wine produced by the preparation method provided by the present invention.

[0053] A fifth aspect of the present invention provides a use of the yeast or bacterial agent provided by the present invention in improving the flavor and / or quality stability of wine.

[0054] The present invention will be described in detail below through examples and comparative examples. In the following examples, unless otherwise specified, all methods are conventional methods; and the reagents and materials used, unless otherwise specified, can be obtained from commercial sources.

[0055] Commercial yeast ES488 was purchased from Enartis; anhydrous ethanol was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; glycerol, TTC, potassium metabisulfite and citric acid were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; microplate reader was purchased from Thermo Company, USA; handheld saccharimeter (AK002B) was purchased from Shenzhen Ceyou Technology Co., Ltd.; pH meter (PHSJ-3F) was purchased from Shanghai Yidian Scientific Instrument Co., Ltd.; gas chromatography-mass spectrometry (Agilent 5975C-7890A) and high performance liquid chromatography (Agilent1200Agilent) were purchased from Agilent, USA; digital ultrasonic cleaner (KQ-600DE) was purchased from Kunshan Ultrasonic Instrument Co., Ltd.; BIGGY medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.; yeast extract powder, peptone, glucose and agar medium were purchased from Beijing Aoboxing Biotechnology Co., Ltd.

[0056] Example 1

[0057] This example is used to illustrate the method and results of yeast screening and identification.

[0058] YPD liquid medium: 1 wt% yeast extract powder, 2 wt% peptone and 2 wt% glucose, sterilized at 121°C for 20 min. In addition, 2 wt% agar powder was added to prepare YPD solid medium.

[0059] TTC medium: TTC 0.05wt%, glucose 0.5wt% and agar 1.5wt%, sterilized at 121°C and high temperature and high pressure for 20 min.

[0060] Esculin culture medium: esculin 0.3wt%, ferric citrate 0.05wt%, NaCl 0.2wt%, MgSO 4 7H 2 O0.05wt% and KH 2 PO 4 0.1wt%, sterilized at 121℃ for 20min.

[0061] 1. Isolation, purification and identification of yeast

[0062] (1) Methods for isolation, purification, screening and identification of yeast strains

[0063] The yeast samples in this experiment were derived from the vineyard soil, wine grapes, equipment samples and wine fermented under laboratory conditions in Fenghuang Manor in Changli, Hebei Province. Pre-culture was performed on Bengal red agar plates, and then yeasts of different colors and forms on the plates were streaked on YPD solid medium with an inoculation loop. The culture was carried out at 28°C for 2 days, and single colonies were streaked and cultured for 2 generations to obtain pure cultures. The single colony was inoculated in a test tube containing 10mL YPD liquid medium, and cultured at 28°C and 180r / min shaking for 12 hours. The cultured yeast solution and 50wt% glycerol were transferred into a cryopreservation tube at a ratio of 1:1 and stored in a -80°C refrigerator. The bacterial solution to be identified was sent to Beijing Deaoping Biological Company to extract DNA and sequence it. The obtained sequence was compared by BLAST at NCBI to obtain the identification results.

[0064] (2) Yeast strain rescreening method

[0065] The top three yeast species isolated with the highest abundance were analyzed for their ethanol production capacity, fermentation rate, activity of hydrolyzing β-glucosidase, and SO 2 , pH and glucose tolerance testing to identify the best performing strains.

[0066] a. Determination of alcohol production capacity: Inoculate the activated strain on the YPD solid medium by streaking, and after the colonies grow, pour into the TTC medium at about 45°C, and culture at 28°C in a dark environment for 3 hours, and observe the color of the colonies on the culture dish. The stronger the alcohol production capacity of the strain, the darker the red color of the colonies.

[0067] b. H production 2 Method for determining H production capacity: Spread the activated yeast strain on BIGGY medium at a 2 vol.% inoculation rate, incubate at 28°C for 3-5 days, and observe the color change of the strain on the culture dish. The darker the color, the higher the H production capacity. 2 The higher the S ability.

[0068] c. Fermentation rate determination method: The activated strain was inoculated into YPD liquid culture medium containing inverted Duroc tubules at 2 vol.%, and cultured at 28°C. The gas production in the Duroc tubules was observed every 12 hours, and the gas production content and the time for the Duroc tubules to be filled with gas were recorded to screen out strains with fast fermentation speed and strong gas production ability.

[0069] d. Determination of β-glucosidase activity: Yeast can hydrolyze the β-glycosidic bond of β-glucosidase and act on β-glucosidase to release glucose and aromatic glycosides, so β-glucosidase is an important factor in the release of aromatic substances. β-glucosidase can hydrolyze aesculin to aesculin, which reacts with Fe in the culture medium. 3+The effect is brown, so the activity of β-glucosidase produced by the strain is determined by the color depth of the aesculin culture medium. The 96-well ELISA plate was sterilized with UV light in advance, and 180 μL of aesculin culture medium at room temperature was added to the wells of the ELISA plate in the clean bench. After cooling for 1 hour, 20 μL of the bacterial solution to be screened was drawn into the well plate, and cultured in a constant temperature incubator at 28°C for 3 days to observe the color change.

[0070] e. Basic characteristics screening method: Based on the test results of the above four basic characteristics of wine, select wines with strong alcohol production and H production. 2 Draw a heat map of yeast strains with weak S, fast fermentation rate and strong β-glucosidase activity. Select excellent yeast based on the characteristics of the heat map.

[0071] f. Tolerance determination method: The activated strain was inoculated with 2 vol.% in YPD liquid medium with different tolerance conditions. After culturing at 28°C for 48 hours, 200 μL of the bacterial solution was placed in a 96-well ELISA plate and its absorbance was measured at 600 nm as OD 600实际 .

[0072] Different tolerance conditions: SO 2 The concentrations are 50, 100, 150 and 200 mg / L; the concentrations of citric acid (used to test pH tolerance) are 1, 5, 10 and 20 g / L; the volume fractions of anhydrous ethanol are 9, 12, 15, 18 vol.%; and the concentrations of glucose are 200, 300, 400 and 500 g / L.

[0073] In the experiment, the culture medium without inoculation was used as the blank group, and the absorbance was recorded as OD 600空白 Yeast was cultured synchronously in YPD liquid medium without tolerance conditions for 48 h as the control group, and the absorbance was recorded as OD 600对照 . Calculate the tolerance growth rate of each strain. The greater the growth rate, the stronger the tolerance.

[0074] Tolerance growth rate = (OD 600实际 -OD 600空白 ) / (OD 600对照 -OD 600空白 )

[0075] g. Method for testing the aroma of wine fermented by inoculation with a single strain: After the Marselan wine grapes are destemmed and crushed, they are added to a 250mL triangular flask, with 100mL added to each bottle. Potassium metabisulfite 0.1g / L is added and cold-soaked for one day. After the excellent yeast strains selected by the above indicators are activated, they are inoculated into a triangular flask containing 100mL of Marselan grape juice, with an inoculation volume of 10 6CFU / mL, cultured in a constant temperature incubator at 20℃ for 7 days, and grape juice inoculated with commercial yeast ES488 was used as the control group to evaluate the aroma intensity of wines inoculated with different strains.

[0076] h. Method for determining the compounding ratio of excellent yeast strains: The fermentation process is consistent with the aroma test of wine fermented by a single strain, except that the ratio of yeasts is different: the excellent brewing yeast screened out by the aroma test of a single strain is mixed with non-brewing yeast and inoculated, and the inoculation ratios are brewing yeast: non-brewing yeast = 1:5, 1:1 and 5:1 respectively. Brewing yeast, non-brewing yeast and commercial yeast ES488 are inoculated separately as the control group to evaluate the aroma intensity of wines fermented by different strains.

[0077] 2. Results of screening and identification

[0078] (1) Results of yeast strain isolation, purification, initial screening and identification

[0079] A total of 172 yeast strains were isolated, purified and identified. The specific strain numbers and species identification results are shown in Table 1 . A total of 19 species were found, including 66 strains of Saccharomyces cerevisiae, 30 strains of Starmerella bacillaris and 25 strains of Hansenula vitis var.

[0080] Table 1

[0081] Species name Number of isolated strains Strain number Saccharomyces cerevisiae 66 Sc1-Sc66 Starmerella bacillaris 30 Stb1-Stb30 Hanseniaspora uvarum 25 Hu1-Hu25 Saccharomyces boulardii 13 Sab1-Sab13 Torulaspora delbrueckii 7 Td1-Td7 Papiliotrema flavescens 5 Pf1-Pf5 Wickerhamomyces anomalus 4 Wa1-Wa4 Meyerozyma smithsonii 5 Ms1-Ms5 Rhodotorula mucilaginosa 3 Rm1-Rm3 Rhodotorula glutinis 3 Rg1-Rg3 Candida californica 2 Cc1-Cc2 Candida oleophila 1 Co1 Candida railenensis 1 Cr1 Sporidiobolus pararoseus 1 Sp1 Candida sorbosivorans 1 Cs1 Filobasidium magnum 1 Fm1 Kurtzmaniella quercitrusa 1 Kq Suhomyces kilbournensis 1 Sk1 Papiliotrema laurentii 2 Pl1-2

[0082] pass Figure 1 It can be seen that three species of bacteria, namely Saccharomyces cerevisiae, Rhodotorula glutinosus, and Papiliotremalaurentii, were mainly isolated from the soil; Saccharomyces cerevisiae and Starmerella bacillaris were isolated from the equipment and grape samples, respectively. For the MC samples, many strains of different species were isolated, among which Starmerella bacillaris and Hansenella spores in grape juice had higher abundance. A total of 9 species were isolated in the early stage of wine fermentation, among which Saccharomyces cerevisiae, Hansenella spores in grape juice, Starmerella bacillaris and Saccharomyces boulardii had higher abundance. As the fermentation progressed, the abundance of Saccharomyces cerevisiae gradually increased, and became the dominant genus in the middle and late stages of fermentation.

[0083] (2) Results of yeast strain rescreening

[0084] 121 yeast strains from the top three most abundant yeast species (Saccharomyces cerevisiae, Starmerella bacillaris and Hansenula vitis spores) were selected for research and screening.

[0085] a. Alcohol production capacity: According to the depth of red color of wine-related yeast cells on TTC medium, their alcohol production capacity can be divided into different levels. The classification standards and results of yeast alcohol production capacity are as follows: Figure 2 and as shown in Table 2. The more alcohol produced, the deeper the TTC medium turns red.

[0086] Table 2

[0087]

[0088] According to Table 2, there are a total of 28 TTC culture media containing yeast that are dark red and have strong ethanol production capabilities. These 28 strains are all brewer's yeast; there are 34 strains with average ethanol production capabilities. In subsequent brewing experiments, priority will be given to the two types of yeast with average and strong ethanol production capabilities.

[0089] b. H production 2 S ability: Wine yeast is divided into 5 levels according to the degree of browning of the yeast cells on BIGGY medium. The darker the browning color of the medium, the higher the H production of the wine yeast. 2 The stronger the S ability, the more specific the yeast is in producing H 2 S classification standard reference Figure 3 .

[0090] Table 3

[0091]

[0092]

[0093] During the wine fermentation process, sulfur compounds with bad flavors are usually produced, which affect the quality and taste of the wine. Among them, hydrogen sulfide is the most volatile sulfur compound. If it is not removed in time, it will further combine with alcohol substances to form mercaptans. In order to ensure the quality of wine fermentation and avoid odor, it is necessary to screen out hydrogen sulfide that does not produce hydrogen sulfide. 2 S or H 2 From Table 3, we can see that there are 21 yeast strains that do not produce H 2 S, including 3 strains of Saccharomyces cerevisiae, 7 strains of Hansenula vitis spores, and 11 strains of Starmerella bacillaris; 32 strains of yeast produced H 2 S is weaker.

[0094] Table 4

[0095]

[0096] As shown in Table 4, the fermentation rate of Saccharomyces cerevisiae was all full of gas within 24 hours, which shows that the fermentation rate of Saccharomyces cerevisiae is relatively fast. Among them, Sc2, Sc3, Sc13, Sc14, Sc17, Sc23, Sc27, and Sc36 produced gas and filled the Dulbecco's tubules within 12 hours. This result shows that these strains have the fastest fermentation rate and the most vigorous fermentation. The fermentation rate of non-Saccharomyces cerevisiae is not as good as that of Saccharomyces cerevisiae, but most of them are fermented within 48 hours.

[0097] d. Activity of β-glucosidase: The darker the color, the stronger the activity of β-glucosidase. Figure 4 It can be found that there are 38 strains with high β-glucosidase production, among which there are 10 strains of Saccharomyces cerevisiae with high β-glucosidase production, namely Sc2, Sc3, Sc7, Sc8, Sc18, Sc25, Sc28, Sc39, Sc40 and Sc52; all Hansenula sporogenes of grape juice have high β-glucosidase production, while only three strains of Starmerella bacillaris, Stb1, Stb5 and Stb6, have high β-glucosidase production.

[0098] e. Screening results of basic characteristics: Based on the test results of the above four types of wine basic characteristics, we selected wines with strong alcohol production and H production. 2 Draw a heat map of yeast strains with weak S ability, fast fermentation rate, and strong β-glucosidase activity. Figure 5 The excellent brewer's yeasts screened out were Sc2, Sc3, Sc7, Sc8, Sc12, Sc25, Sc26, Sc28, and Sc52-54, and the non-brewer's yeasts were Hu1, Hu2, Hu4-6, Hu11-13, Hu15, Hu19, Hu21, Hu23-25, Stb1, Stb5, and Stb6.

[0099] f. Tolerance test results: The strains were tested at different SO 2 The tolerance under concentration, pH, ethanol concentration and glucose concentration is plotted as heat map. Figure 6 As shown, the vertical axis represents the tolerance growth rate of yeast under different environments.

[0100] according to Figure 6 It can be seen that SO 2 The increase in concentration will lead to an increase in the inhibitory effect on yeast. Overall, the SO 2 The tolerance is good. Except for some strains that do not grow at 200 mg / L concentration, the strains can grow at other different concentrations. Among them, Sc5, Sc6, Sc16, Sc18, Sc21-25, Sc27-36, Sc41-47, Sc50-53, Sc55, and Sc66 have strong tolerance. The wine quality standard requires that the total SO in fermented wine2 The content should be less than 250mg / L. 2 The addition amount is usually 50-150 mg / L, so the brewer's yeast screened basically meets the requirements; Figure 6 It can also be found that the screened brewer's yeast has good tolerance to pH: when the pH is 3-4, the strains can grow in culture, and when the pH is 2.5, the yeast survival rate is basically below 40%, among which Sc6, Sc22-Sc24, Sc27-Sc32, and Sc41-44 are relatively tolerant; the test results of ethanol tolerance show that at 9 vol.% ethanol content, except for Sc3, the other strains can grow normally, and with the increase of alcohol concentration, the inhibitory effect on yeast increases. The strains with strong ethanol tolerance include Sc21, Sc24, Sc30, Sc38, Sc40, Sc42, Sc44-52, Sc56, Sc59, Sc63, and Sc65-66, all of which can tolerate 18 vol.% ethanol; the results of glucose tolerance test show that glucose has an inhibitory effect on yeast, and with the increase of concentration, the inhibitory effect on yeast increases. When the concentration reaches 500 g / L, the strain basically does not grow. Generally, the highest glucose concentration of wine grape juice is around 300g / L. The strains with stronger tolerance include Sc6, Sc15-16, Sc18, Sc23, Sc28, Sc36, Sc43, Sc56, Sc57-59, Sc62-63, and Sc66.

[0101] Combining the tolerance to different concentrations of the four factors, the strains of Saccharomyces cerevisiae with stronger tolerance are Sc4-7, Sc9, Sc12, Sc15-16, Sc21-25, Sc28, Sc33, Sc35-36, Sc40, Sc43, Sc46-47, Sc51-52, Sc55, and Sc66.

[0102] Unlike Saccharomyces cerevisiae, non-Saccharomyces cerevisiae are less tolerant. Figure 7 The tolerance of non-brew yeasts can be seen. Hansenula vitis spores and Starmerella bacillaris are generally weaker in tolerance, among which Hu3, Hu5-9, Hu11, Hu12, Hu14, Hu19, Hu20, Hu22, Stb5, Stb8, Stb11, Stb13, Stb11, Stb16, Stb19, Stb20, Stb23-25, Stb28 and Stb30 are more tolerant to SO 2 Hu21, Hu23-25, Stb4, Stb17, Stb18, Stb21, Stb22 and Stb27 have strong tolerance to SO 2The tolerance of non-brewer's yeast is poor. At pH 2.5, the growth rate of the strain is low, among which Hu5-6, Hu10-12, Hu14, Hu17, Hu19, Stb1-10, Stb14, Stb18, Stb19, Stb22-25 and Stb28 have strong pH tolerance. The overall sugar tolerance of non-brewer's yeast is average, among which Hu5, Hu17-21, Stb1-9, Stb14-19 and Stb28 have relatively strong tolerance.

[0103] Taking into account different tolerances, the strains with stronger tolerance to Hansenula sporangiophora are Hu2, Hu5, Hu10, Hu11, Hu14, Hu16, Hu17, and Hu19; the strains with stronger tolerance to Starmerella bacillaris are Stb5-10, Stb23, and Stb28.

[0104] By comprehensively comparing the basic characteristics and tolerance of yeasts, 10 excellent yeast strains were screened out, including Saccharomyces cerevisiae Sc7, Sc12, Sc25 and Sc28; Hansenula vitis spores Hu2, Hu5, Hu11 and Hu19; Starmerella bacillaris Stb5 and Stb6.

[0105] g. Aroma results of a single strain: Figure 8 (a) shows that among the fermenting yeasts, Sc28 has a relatively strong and harmonious aroma and the highest relative score. Among the non-fermenting yeasts, the grape juice Hansen group has a relatively obvious fruity and chemical smell as a whole. Although the fermentation effect of grape juice Hansen yeast alone is worse than Stb, it has been found in previous literature that mixed fermentation with brewer's yeast can improve the aroma of wine, so Hu19 with a higher score is temporarily retained for subsequent compounding. Starmerella bacillaris can give wine a watermelon-like, cool aroma, and Stb5 is selected for subsequent compounding.

[0106] h. Compound ratio of excellent yeast strains: Figure 8 (b) It can be seen that among all the blending groups, the wine aroma intensity score obtained by blending Sc28 and Hu19 in a 1:1 ratio (SH11 group) was the highest. It has a strong fruity aroma, moderate sourness and sweetness, and a more coordinated and balanced aroma and taste. Therefore, the SH11 group was finally selected for subsequent research.

[0107] Example 2

[0108] 1. Preparation method of Marselan wine:

[0109] Marselan grapes are sorted, destemmed and crushed, and then packaged into sterilized fermentation tanks. 6L of each tank is added with SO 20.12 g / L grape slurry, cold soaked for 3 days, pectinase 0.03 g / kg wine grapes were added, and commercial yeast (ES488 group), brewer's yeast Sc28 (Sc28 group) and Sc28 and Hu19 inoculated at a ratio of 1:1 (SH11 group) were inoculated into three tanks, and the number of live yeast cells in each tank after inoculation was 10 6 CFU / mL, fermentation was carried out at 22±2℃, sampling was carried out at 0d, 1d, 2d, 3d and 6d, three parallels in each group, and various parameters were measured.

[0110] 2. Parameter determination method:

[0111] (1) Yeast growth during fermentation: The number of yeasts in wine samples brewed under different inoculation methods was detected by gradient dilution and spreading method, with three parallels in each group. The live bacteria counts of wine samples fermented by ES488 and Sc28 groups were counted using YPD solid medium, and the SH11 group was streaked using WL medium. The live bacteria counts of Saccharomyces cerevisiae and Hansenula sporei were counted and the colony color and morphology were observed.

[0112] (2) Physical and chemical indicators of wine: Use a pH meter to test the pH of the wine; use a handheld sugar meter to test the soluble solids (°Brix, unit: °Bx) of the wine; refer to the acid-base titration method and density bottle method in the national standard GB / T15038-2006 "General Analytical Method for Grape and Fruit Wine" to test the total acid content and alcohol content of the wine after fermentation.

[0113] (3) Detection of volatile aroma components in wine:

[0114] Sample pretreatment method: 2.5 g NaCl was placed in a 20 mL headspace bottle, stirred using a magnetic stirring rotor, 10 mL of wine sample after fermentation and 50 μL of 2-octanol (400 mg / L) internal standard were added, sealed, placed in a magnetic stirrer and kept warm at 45 °C for 30 min, headspace extraction for 30 min, and GC-MS analysis for 5 min.

[0115] GC-MS detection conditions: DB-WAX column (30m×0.53mm, 1μM) was used, the injection port temperature was 250℃, the initial temperature was 50℃ and maintained for 2min, then the temperature was increased to 85℃ at a rate of 2℃ / min and maintained for 0.1min, and then the temperature was increased to 230℃ at a rate of 5℃ / min and maintained for 2min. The ion source temperature was 230℃, the ionization mode was EI, the ion energy was 70eV, the quadrupole temperature was 150℃, and the mass scanning range was 20-350u.

[0116] Quantitative and qualitative analysis of volatile flavor substances: The detected flavor substances were searched and qualitatively analyzed using the US NIST08.L database through mass spectrometry full ion scanning spectra.

[0117] Quantitative analysis of aroma substances: calculated by the following formula (unit: mg / L):

[0118]

[0119] Odor activity value (OAV): evaluates the actual odor contribution of an aroma substance. When it is greater than 1, the substance is considered to have an aroma contribution. It is calculated using the following formula:

[0120]

[0121] (4) Dynamic changes of organic acids in wine:

[0122] Chromatographic conditions: C18 column (6 mm × 250 mm, 5 μM), mobile phase is methanol-0.02 mol / L potassium dihydrogen sulfate (pH = 2.8) (3:97) solution, flow rate is 1 mL / min, injection volume is 10 μL, detection is at λ = 210 nm. The mobile phase is filtered and sonicated before use.

[0123] Draw the standard curve of organic acids: Weigh appropriate amounts of tartaric acid, malic acid, lactic acid, succinic acid and citric acid standards, dissolve and dilute with mobile phase to obtain the mother solution of organic acid standards, and prepare mixed standard solutions of different concentrations. Dilute each standard to an appropriate concentration, inject into HPLC respectively, and perform qualitative analysis of the standard according to its retention time; inject 5 standard solutions with different concentration gradients respectively to obtain the corresponding peak areas. Draw the standard curve with the peak area (mAU) as the horizontal coordinate and the organic acid concentration (g / L) as the vertical coordinate, and establish the regression equation.

[0124] Sample preparation and detection: Take 2 mL of wine in a 10 mL centrifuge tube, add mobile phase solution to make up the volume, centrifuge at 10000 r / min for 15 min at 4°C, take the supernatant and filter it with a 0.45 μM filter membrane, and place the filtrate in a 2 mL chromatographic injection vial for determination.

[0125] (5) Sensory evaluation: The wine scoring standard was formulated according to the national standard GB / T15038-2006. A team of 10 students who had received professional wine sensory evaluation training was selected to evaluate the wine after fermentation. The total score was set to 100 points.

[0126] 3. Results:

[0127] (1) Yeast growth during fermentation

[0128] The number of yeasts in the three groups of wine during fermentation Fig. 9 (a) As shown. The initial yeast count was 5.69LogCFU / mL. The yeast count in the ES488 group rose to 7.22LogCFU / mL on day 1, reached a peak of 7.57LogCFU / mL on day 2 of fermentation, and then dropped to 6.22LogCFU / mL. The overall yeast count in the Sc28 group was slightly larger than that in the ES488 group. It also reached a peak of 7.77LogCFU / mL on day 2 of fermentation, and then dropped to 6.81LogCFU / mL. In the mixed fermentation SH11 group, the number of brewer's yeast increased rapidly in the early stage of fermentation, reaching a peak of 7.64LogCFU / mL within 1 day. The number of Sc28 was smaller than that of single fermentation, but the overall trend was still rising first and then falling. The yeast count in the Hu19 group also reached a peak of 7.36LogCFU / mL on day 1 of fermentation, and then began to decrease significantly, eventually dropping to 5.24LogCFU / mL.

[0129] Fig. 9 (b) The two yeast strains Sc28 and Hu19 can be clearly distinguished. The colony morphology of Sc28 is: cream-colored, round, smooth on the surface, and light green in the center; the strain morphology of Hu19 is: round, smooth on the surface, dark green throughout, and has a layer of translucent lines on the outermost layer.

[0130] (2) Physical and chemical indicators of wine

[0131] The sugar content (°Brix), pH, total acidity and alcohol content of the grape juice before fermentation and the wine after fermentation are shown in Table 5.

[0132] Table 5

[0133]

[0134] Note: The data in the table represent mean ± standard deviation. Different letters in the same column indicate significant differences among samples (p<0.05, Duncan).

[0135] The results showed that at the end of fermentation, the residual sugar content of the Sc28 group and the SH11 group was lower than that of the ES488 group, indicating that the sugar utilization rate of the two groups was higher. After the fermentation, the pH of the SH11 group was the lowest. The total acid content of the SH11 group was significantly higher than that of the other two groups of single-bacteria fermentation. The results of this study are in line with the national standard for fermentation with a total acid content of 5.5-7.5g / L. There was no significant difference in the alcohol content of wine under different fermentation methods.

[0136] (3) Volatile aroma components in wine

[0137] The contents of volatile flavor substances in wine in each experimental group are as follows Fig.10 As shown in Table 6, a total of 52 volatile flavor substances were detected. SH11 is the group with the highest total concentration of ester substances among the three treatment groups, and the content of some ester substances is significantly higher than that of wine fermented by commercial yeast, such as ethyl acetate, ethyl hexanoate, ethyl octanoate, ethyl heptanoate, etc., which gives the wine a strong fruity aroma; the content of damasconone in the SH11 group is significantly higher than that of the ES488 group and the Sc28 group, which gives the wine a strong floral and honey aroma, making the aroma of the wine more intense and rich. The wine has a unique floral, honey and fruity aroma, which is conducive to expressing the flavor characteristics of local wine. The content of alcohol substances in the SH11 group is lower than that of single strain fermentation, and the content of acid substances is slightly higher than that of ES488. The content and types of other substances are relatively high.

[0138] Table 6

[0139]

[0140]

[0141]

[0142] Note: The data in the table represent mean ± standard deviation. Different letters in the same row indicate significant differences among samples (p<0.05, Duncan).

[0143] Ester compounds are the main flavor substances in wine, most of which have a fruity aroma. A total of 25 ester substances were detected in this experiment. Among them, ethyl octanoate is the fatty acid ester with the highest content in wine, and the content of SH11 group and Sc28 group is significantly greater than that of ES488 group. Ethyl acetate is a volatile flavor substance with a high content in wine. The concentration of SH11 group reached 2153.68μg / L, which is the highest content among the three treatment groups, but if the content is too high, it may produce a chemical smell similar to nail polish. The content of ethyl hexanoate, ethyl heptanoate, and ethyl decanoate in the SH11 group is significantly greater than that of the other two treatment groups, proving that mixed fermentation can increase the content of ester substances in wine. Among all the wine samples that have completed alcoholic fermentation, the OAV values ​​of isoamyl acetate, ethyl hexanoate, ethyl octanoate, and ethyl heptanoate are greater than 1, which are the main contributing compounds to the floral and fruity aromas in wine. The OAV values ​​of ethyl acetate, phenylethyl acetate, ethyl butyrate, ethyl decanoate and ethyl undecanoate were all greater than 0.1, playing a synergistic role in enhancing the aroma of the wine samples.

[0144] A total of 12 alcohol substances were detected, mainly higher alcohols, which are mainly formed by decarboxylation reduction of α-keto acids, intermediate products of glycolysis or amino acid biosynthesis and catabolism. The total alcohol content of the ES488 group was higher, among which the isobutanol and isopentanol of the SH11 group were lower than those of the other two treatment groups, and isopentanol accounted for the highest proportion of higher alcohols. Therefore, the content of higher alcohols in mixed fermentation wine samples is significantly lower than that of commercial yeast, which is conducive to reducing discomfort such as headiness after drinking.

[0145] A total of 7 acids were identified in the three groups of samples. Most of these volatile acids have an unpleasant smell, but they can also adjust the flavor of wine at low concentrations. Compared with the ES488 group, the total acid concentration of the Sc28 group is higher, while the content of acid substances in the SH11 group is less, and the flavor of the wine is better.

[0146] Aldehydes can give wine an unpleasant green taste. In this test, only one aldehyde was detected, 3,5-dimethylbenzaldehyde.

[0147] Terpenes are mostly found in grape skins and pulps. Although the content of terpenes is relatively small, they can give wine a rich floral and fruity aroma. As shown in Table 6, a total of three terpenes were detected, namely damascone, D-citronellol and nerol. The OAV value of damascone is greater than 1, which gives the wine a floral and honey aroma. The SH11 group has the highest damascone content, and the wine fermented with commercial yeast has the lowest damascone content, that is, mixed fermentation can increase the concentration of terpenes in wine.

[0148] To further distinguish the aroma of volatile flavor substances between different treatment groups, PLS-DA analysis was performed on volatile compounds with OAV>0.1 using SIMCA software. Fig.11 As shown, the explanatory variables of the model (R 2 x) and predictive power (R 2 y) are 0.675 and 0.951 respectively, indicating that the model classification is good, Q 2The value of VIP>1 was 0.881, which had a good predictive ability for volatile flavor compounds in wine. The larger the VIP value, the more significant the difference in flavor substances among the three treatments. There were 11 aroma substances with VIP>1, namely ethyl undecanoate, isoamyl acetate, phenylethyl acetate, ethyl hexanoate, damascone, ethyl octanoate, ethyl acetate, D-citronellol, ethyl heptanoate, ethyl butyrate and decanoic acid. Most of the characteristic aromas of the ES488 group were located in the fourth quadrant. Ethyl butyrate, D-citronellol, ethyl undecanoate, etc. were closer to the Sc28 group, indicating that these substances contributed greatly to the aroma of the Sc28 group wine. Compared with wine fermented by a single strain, the SH11 group was mainly located in the first and second quadrants, and was associated with more ester aroma compounds. Among them, the typical aroma substance damascone was also closer to the SH11 group, indicating that mixed bacterial fermentation had better retention of its variety characteristic aroma substances.

[0149] (4) Dynamic changes of organic acids in wine

[0150] The results of organic acid content in wines brewed by three different fermentation methods are as follows Fig.12 shown.

[0151] Tartaric acid is the main organic acid in grapes and wine. It has a weak acidity, a harsh and strong sour taste, and is relatively stable during the fermentation process and not easily metabolized. Fig.12 (a) shows that the tartaric acid content shows a downward trend with the increase of fermentation time, among which the tartaric acid content of SH11 group is slightly higher than that of ES488 group at 6 days of fermentation. Malic acid is an important organic acid naturally produced by grape fruit. When the content is high, it can give the wine a sour and astringent feeling. Fig.12 (b) shows that the overall malic acid content first increased in a small range and then decreased. The malic acid content of the SH11 group was slightly higher than that of the single strain fermentation at the beginning of fermentation, but after six days of fermentation, the content was lower than that of the other two groups. Lactic acid is mainly produced during the malic acid-lactic acid fermentation process, which makes the wine taste soft. Fig.12 (c) shows that the lactic acid content of the SH11 group was the highest, reaching 1.19 g / L on day 6. Citric acid is produced during alcohol fermentation and then consumed as a substrate in malic acid-lactic acid fermentation. Fig.12 (d) shows that the citric acid content in the three treatment groups was small and the changes were not obvious.

[0152] Succinic acid has a complex flavor, stable properties, and a certain inhibitory effect on bacteria. Fig.12 (e) shows that the initial succinic acid content is 0.21 g / L. As the fermentation progresses, the content increases rapidly on the first day and then increases slowly. At the end of the fermentation, the succinic acid content of the ES488 group, the Sc28 group, and the SH11 group is 1.54, 1.29, and 1.16 g / L, respectively.

[0153] (5) Sensory evaluation

[0154] The full score is 100 points, and the radar chart is as follows Fig.13 As shown, the total score of ES488 group is 76.05 points, characterized by clear wine body, prominent wine aroma, floral and fruity aroma, but thin taste and slightly bitter feeling; the total score of Sc28 group is 84.85 points, with relatively strong aroma, good coordination and good palatability; the total score of SH11 group is 85.75 points, the wine body is clear and free of impurities, with aromas of yellow fruits such as banana, apple, pineapple and pear, as well as aromas of black fruits and red fruits such as blackberry, raspberry, hawthorn and strawberry, rich and coordinated aroma, and good typicality. It is the darkest and most fragrant of the three groups of wines, with overall coordination and good typicality.

[0155] In summary, the mixed fermentation of different non-brew yeasts and brewer's yeast can improve the sensory quality of wine, make the wine have unique floral, honey and fruity aromas, increase the complexity and diversity of wine aroma, and help express the flavor characteristics of local wines.

[0156] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A yeast, characterized in that: The yeast is Saccharomyces cerevisiae with a preservation number of CGMCC NO.30497; or, the yeast is Hanseniaspora uvarum with a preservation number of CGMCC NO.30499.

2. A bacterial agent, characterized in that The bacterial agent contains saccharomyces cerevisiae with a preservation number of CGMCC NO.30497 and / or Hansenula sporogenes with a preservation number of CGMCC NO.30499.

3. The bacterial agent according to claim 2, wherein The bacterial agent contains brewer's yeast with a preservation number of CGMCC NO.30497 and Hansenula sporogenes with a preservation number of CGMCC NO.30499; in the bacterial agent, the ratio of the number of live bacteria of brewer's yeast with a preservation number of CGMCC NO.30497 to Hansenula sporogenes with a preservation number of CGMCC NO.30499 is (0.1-6):1, preferably (0.9-1.1):

1.

4. A method for preparing wine, characterized in that: The method comprises: inoculating the bacterial agent according to claim 2 or 3 into grape slurry for fermentation.

5. The method according to claim 4, wherein: The inoculation amount of the bacterial agent is such that the total number of viable bacteria in the grape slurry is 10 5 -10 7 CFU / mL.

6. The method according to claim 4, wherein: The fermentation conditions include: a temperature of 15-30°C, preferably 19-24°C; and / or, The time is 100-400h, preferably 100-200h.

7. The method according to claim 4, wherein: The method for preparing the grape slurry comprises: mixing wine grapes with SO2 and pectinase.

8. The method according to claim 7, wherein: The dosage of the SO2 and the pectinase is 60-150 mg / L grape slurry and 15-50 mg / kg wine grapes respectively.

9. Wine obtained by the method according to any one of claims 4 to 8.

10. Use of the yeast according to claim 1 or the bacterial agent according to claim 2 or 3 in improving the flavor and / or quality stability of wine.

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