Yeast and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to saccharomycetes and application thereof, and the preservation number of the saccharomycetes is CCTCC NO: M 20241483. The saccharomyces cerevisiae is high in fermentation speed, high in alcohol production capacity and relatively high in ethanol tolerance, sulfur dioxide tolerance and glucose tolerance. Compared with K yeast, the tangerine fruit wine obtained by fermentation based on the yeast provided by the invention has more unique fragrance, and specifically, in volatile components of the tangerine fruit wine obtained by fermentation based on the yeast provided by the invention, the relative contents of 2-methyl-1-butanol, heptanal, valeraldehyde and 3-methyl-2-butenal are higher; wherein the 2-methyl-1-butanol can enhance the roasted onion flavor, fruit flavor, flower flavor and wine flavor of the fruit wine; heptanal can endow the fruit wine with characteristic vanilla flavor; the valeraldehyde and the 3-methyl-2-butenal can provide certain grass smell, weak banana smell and fruit fragrance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and particularly relates to a yeast strain and application thereof. BACKGROUND

[0002] Fruit wine is a low-alcohol wine brewed by fermenting fresh fruits with yeast, which retains the natural flavor and nutrients of fruits. It has become an important category in the global beverage market due to its refreshing taste, moderate alcohol content, and high content of polyphenolic antioxidants. As the core microorganism in fruit wine fermentation, the strain characteristics of yeast directly affect the fermentation efficiency, flavor composition, and wine quality. Therefore, screening yeast strains with excellent fermentation performance is a key direction for innovation in fruit wine brewing technology.

[0003] With the upgrading of consumers' demand for natural and high-quality beverages, the fruit wine industry has higher requirements for the performance of fermentation strains. On the one hand, the strain's ability to biotransform flavor precursors (such as terpenes and ester precursors) in fruit raw materials needs to be strengthened to highlight the typical aroma characteristics of fruits. On the other hand, the strain's metabolic pathway needs to be optimized to efficiently convert sugar while reducing the generation of excessive higher alcohols, aldehydes, and other substances to improve the softness and palatability of the wine body. In addition, the adaptability of specific strains to different fruit raw materials (such as berries, stone fruits, and pome fruits) also plays an important role in influencing the flavor diversity of fruit wine.

[0004] To meet the demand for high-quality fermentation strains in the fruit wine industry, it is necessary to develop a yeast strain. SUMMARY

[0005] Therefore, the present application isolates a yeast strain (also referred to as yeast SXZY-GJ-Y2401 or GJ-45) from naturally fermented juice of mature fresh red oranges, with a preservation number of CCTCC NO: M 20241483. The yeast CCTCC NO: M 20241483 has a fast fermentation speed, strong ethanol-producing capacity, and strong tolerance to ethanol, sulfur dioxide, and glucose, which can be used in the brewing of fruit wine.

[0006] To achieve the above purpose, the present application can adopt the following technical solutions: The present application provides a yeast strain with a preservation number of CCTCC NO: M 20241483.

[0007] In another aspect, the present application provides a yeast agent, which includes the above-mentioned yeast strain.

[0008] Preferably, the dosage form of the above-mentioned yeast agent includes freeze-dried bacteria, solid bacteria, bacterial suspension, or fermentation broth.

[0009] In still another aspect of the present application, a fruit wine is provided, which is prepared by fermenting fruits using the yeast or the yeast agent described above.

[0010] Preferably, in the fruit wine, the volatile components of the fruit wine include one or more of ethyl acetate, methyl acetate, 3-methyl-2-butene acetate, 2-methyl-1-butanol, 3-methyl-2-butenal, propyl acetate, butyl acetate, isobutyl acetate, isoamyl acetate, pentanal or heptanal.

[0011] Preferably, in the fruit wine, the total sugar content is ≤5 g / L.

[0012] Preferably, in the fruit wine, the fruits are red oranges.

[0013] In still another aspect of the present application, a preparation method of the fruit wine described above is provided, which includes: (1) enzymatically hydrolyzing fruits to obtain an enzymatic hydrolysate; (2) inoculating the yeast and / or the yeast agent described above into the enzymatic hydrolysate to ferment and obtain the fruit wine.

[0014] Preferably, in the preparation method described above, the preparation method includes: (a) enzymatically hydrolyzing using pectinase, and the addition amount of the pectinase is 0.2% to 0.4%; and / or (b) on the basis of (a), the enzymatic hydrolysis temperature is 18°C to 22°C, and the time is 6h to 8h.

[0015] Preferably, in the preparation method described above, the fermentation temperature is 18°C to 22°C.

[0016] In still another aspect of the present application, the yeast and / or the yeast agent described above are used in the preparation of ethanol.

[0017] The preservation information of the yeast in the present application is as follows: preservation agency: China Center for Type Culture Collection (CCTCC); preservation address: Wuhan, China, Wuhan University; preservation date: July 4, 2024; preservation number: CCTCC NO: M 20241483; classification and naming: yeast SXZY-GJ-Y2401 Saccharomyces sp. SXZY-GJ-Y2401 ).

[0018] The beneficial effects of the present application include: (1) The yeast CCTCC NO: M 20241483 provided in the present application has a fast fermentation speed, a strong alcohol production capacity, and strong ethanol tolerance, sulfur dioxide tolerance and glucose tolerance.

[0019] (2) The red orange fruit wine obtained by fermentation of the yeast (CCTCC NO: M 20241483) provided in the present application has low methanol content, which is equivalent to the red orange fruit wine obtained by fermentation of the existing K yeast under the same conditions.

[0020] (3) Compared with the existing K yeast, the red orange fruit wine obtained by fermentation of the yeast (CCTCC NO: M 20241483) provided in the present application has a more unique aroma, specifically, the relative content of 2-methyl-1-butanol, heptanal, pentanal and 3-methyl-2-butenal in the volatile components of the red orange fruit wine obtained by fermentation of the yeast provided in the present application is higher; wherein 2-methyl-1-butanol can enhance the roasted onion flavor, fruit aroma, floral aroma and wine aroma of the fruit wine; heptanal can impart the fruit wine with a unique green grassy medicinal aroma; pentanal and 3-methyl-2-butenal can provide a certain green grassy aroma, weak banana flavor and fruit aroma. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Colony characteristics of different yeast strains on WLN medium; Figure 2 Colony morphology and cell morphology of the 7 screened yeast strains; Figure 3 Phylogenetic tree of the 7 screened yeast strains based on 26S rDNA gene sequences; Figure 4 Analysis of ethanol production capacity of the 7 screened yeast strains; Figure 5A Cultivation of the 7 screened yeast strains and K yeast in medium containing different alcohol contents; Figure 5B Cultivation of the 7 screened yeast strains and K yeast in medium containing different sulfur dioxide contents; Figure 5C Cultivation of the 7 screened yeast strains and K yeast in medium containing different glucose contents; Figure 5D Cultivation of the 7 screened yeast strains and K yeast in medium with different pH values; Figure 6 Schematic diagram of the preparation process of red orange fruit wine; Figure 7 Gas chromatography-ion mobility spectrometry 3D graph of six fruit wine samples fermented by the 5 screened yeast strains and K yeast; Figure 8 Gas chromatography-ion mobility spectrometry 2D top view of six fruit wine samples fermented by the 5 screened yeast strains and K yeast; Figure 9The gas chromatography-ion mobility spectrometry difference map of the six fruit wine samples prepared by fermenting the five screened yeast strains and K yeast; Figure 10 The volatile organic compound fingerprint map in the six fruit wine samples prepared by fermenting the five screened yeast strains and K yeast; Figure 11 The PCA analysis situation of the GC-IMS analysis data of the six fruit wine samples prepared by fermenting the five screened yeast strains and K yeast; Figure 12 The Euclidean distance situation of the GC-IMS analysis data of the six fruit wine samples prepared by fermenting the four screened yeast strains and K yeast. DETAILED DESCRIPTION

[0022] The embodiments are used to better illustrate the present application, but are not the only embodiments of the present application. Therefore, the skilled in the art can make non-essential improvements and adjustments to the embodiments according to the above description, which still belong to the protection scope of the present application.

[0023] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless there is a clear different meaning in the context, the singular form includes the plural form. As used herein, it should be understood that terms such as "include", "have", "contain", etc. are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present application are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations can exist or can be added. As used herein, " / " can be interpreted as "and" or "or" depending on the circumstances.

[0024] In a first aspect, the present application provides a yeast strain, which has a preservation number of CCTCC NO: M 20241483.

[0025] It should be noted that the yeast strain in the present application is isolated from red oranges, and the preservation information is as follows: preservation agency: China Center for Type Culture Collection (CCTCC); preservation address: Wuhan, China, Wuhan University; preservation date: July 4, 2024; preservation number: CCTCC NO: M 20241483; classification and naming: yeast strain SXZY-GJ-Y2401 Saccharomyces sp. SXZY-GJ-Y2401 Saccharomyces sp. In addition, the yeast strain CCTCC NO: M 20241483 provided by the present application has a fast fermentation speed, a strong alcohol production capacity, and strong ethanol tolerance, sulfur dioxide tolerance and glucose tolerance.

[0026] In a second aspect, the embodiments of the present application provide a yeast agent, which comprises the yeast bacteria as described above.

[0027] In some specific examples, the dosage form of the yeast agent as described above comprises a freeze-dried bacteria agent, a solid bacteria agent, a bacteria suspension or a fermentation liquid.

[0028] It should be noted that the preparation methods of the freeze-dried bacteria agent, the solid bacteria agent, the bacteria suspension or the fermentation liquid in the present application are all known in the art. For example, for the freeze-dried bacteria agent, the raw materials for the preparation of the freeze-dried bacteria agent can include bacteria mud and freeze-dried protectants, and the freeze-dried protectants include skimmed milk powder and malt dextrin. Specifically, the bacteria mud can be obtained by centrifugation of the bacteria liquid of the yeast bacteria in the present application, and the centrifugation parameters can be set according to specific conditions. In addition, the freeze-dried protectants can be selected from those known in the art, and the freeze-dried protectants in the present application are preferably skimmed milk powder and malt dextrin, and the freeze-dried bacteria agent prepared by the freeze-dried protectants has good stability and a large number of viable bacteria. In addition, the freeze-dried bacteria agent can be in different shapes, such as unbroken blocks or broken powders, and generally the powder form can be selected.

[0029] In some specific examples, the bacteria agent as described above can be a solid bacteria agent. The solid bacteria agent is prepared by carrying the yeast bacteria on a carrier. The carrier is a key medium for fixing the bacteria and maintaining the activity, and must have high adsorption, air permeability and chemical stability, which are known in the art. For example, natural organic carriers (corn cob, bran, sawdust, grass carbon, vermiculite, etc. provide carbon source and pore structure required for microbial growth), inorganic or composite carriers (ultra-high molecular material (for slow-release bacteriostatic agent), corn flour and wheat mixture, enhance mechanical strength or prolong the life of the bacteria agent) or functionalized carriers (add malt (contains natural enzyme system), marigold extract (synergistically degrades pollutants), improves the comprehensive performance of the bacteria agent) and the like.

[0030] In some specific examples, the bacteria agent as described above can be a bacteria suspension. The bacteria suspension refers to a homogeneous suspension liquid formed by dispersing microbial cells (viable bacteria or inactivated bacteria) in sterile liquid (such as physiological saline, buffer, culture medium) by physical or chemical methods. Those skilled in the art can prepare the bacteria suspension of Streptococcus salivarius subsp. thermophilus according to the conventional preparation method.

[0031] In some specific examples, the bacteria agent as described above can be a fermentation liquid. The fermentation liquid refers to a mixed liquid containing bacteria, metabolites and culture medium components produced after specific microbial strains are expanded in a liquid culture medium. Its core value lies in the active substances (such as enzymes, antibiotics, hormones, etc.) secreted by microorganisms during fermentation. Those skilled in the art can obtain it by culturing according to the conventional liquid culture medium.

[0032] In a third aspect, the embodiments of the present application provide a fruit wine, which is prepared by fermenting fruits using the yeast bacteria or the yeast agent as described above.

[0033] It should be noted that the red orange fruit wine obtained by fermentation of the yeast (CCTCC NO: M 20241483) provided in the present application has low methanol content, is equivalent to the red orange fruit wine obtained by fermentation of the existing K yeast under the same conditions, and is suitable for fruit wine brewing.

[0034] In some specific examples, the volatile components of the fruit wine include one or more of ethyl acetate, methyl acetate, 3-methyl-2-butene acetate, 2-methyl-1-butanol, 3-methyl-2-butenal, propyl acetate, butyl acetate, isobutyl acetate, isoamyl acetate, pentanal or heptanal.

[0035] It should be noted that compared with the existing K yeast, the red orange fruit wine obtained by fermentation of the yeast (CCTCC NO: M 20241483) provided in the present application has a more unique aroma, specifically, the relative content of 2-methyl-1-butanol, heptanal, pentanal and 3-methyl-2-butenal in the volatile components of the red orange fruit wine obtained by fermentation of the yeast provided in the present application is higher; wherein 2-methyl-1-butanol can enhance the roasted onion flavor, fruit aroma, floral aroma and wine aroma of the fruit wine; heptanal can impart a unique green grassy aroma to the fruit wine; pentanal and 3-methyl-2-butenal can provide a certain green grassy odor, weak banana flavor and fruit aroma.

[0036] In some specific examples, the total sugar content of the fruit wine is ≤5g / L.

[0037] It should be noted that the total sugar content of the fruit wine in the present application is ≤5g / L, i.e. dry fruit wine.

[0038] In some specific examples, the fruit of the fruit wine is red orange.

[0039] It should be noted that the fermentation bacteria in the present application are suitable for brewing of all fruits, especially citrus fruits (pH value of fruit juice is 3-4), preferably red oranges.

[0040] In a fourth aspect, the present application provides a preparation method of the fruit wine, the preparation method comprising: (1) enzymolysis of the fruit to obtain an enzymolysis solution; (2) inoculation of the yeast and / or the yeast agent in the enzymolysis solution for fermentation to obtain the fruit wine.

[0041] It should be noted that the fruit wine in the present application is preferably prepared according to the above preparation method, and in the above preparation method, the purpose of enzymolysis to obtain the enzymolysis solution is to make the fermentation more sufficient after enzymolysis.

[0042] In some specific examples, the preparation method comprises: (a) enzymolysis using pectinase, the addition amount of the pectinase being 0.2%-0.4%; and / or (b) on the basis of (a), the temperature of the enzymolysis being 18℃-22℃, and the time being 6h-8h.

[0043] It should be noted that the addition amount of the pectinase can be 0.2%-0.4%, 0.25%, 0.3% or 0.35%, etc.; in addition, the temperature of the enzymolysis can be 18℃-22℃, for example, 19℃, 20℃ or 21℃, etc., and the time of the enzymolysis can be 6h-8h, 6.5h, 7h or 7.5h, etc.

[0044] In some specific examples, in the preparation method, the fermentation temperature is 18℃-22℃.

[0045] It should be noted that the fermentation temperature can be 18℃-22℃, for example, 19℃, 20℃ or 21℃, etc.

[0046] In a fifth aspect, the embodiments of the present application provide a use of the above-mentioned yeast and / or the above-mentioned yeast agent in preparation of ethanol.

[0047] It should be noted that the above-mentioned yeast and / or the above-mentioned yeast agent have strong ethanol production capacity, and can be applied in preparation of ethanol.

[0048] In order to better understand the present application, the content of the present application will be further illustrated below in combination with specific examples, but the content of the present application is not limited to the following examples only.

[0049] In the following examples, the red orange fruit, red orange leaf and soil are respectively collected from the orchard in the red orange production area of Taolong Town, Wanzhou, Chongqing (east longitude 108°31'17", north latitude 30°53'42").

[0050] In the following examples, the La-Ma active fruit wine dry yeast and pectinase are purchased from Yantai Diboze Brewing Co., Ltd. (Yantai, China). The normal ketones include 2-butanone, 2-pentanone, 2-hexanone, 2-heptanone, 2-octanone and 2-nonanone, which are purchased from Aladdin Holding Group Co., Ltd. (Beijing, China). The primers are purchased from Shenguo Bioengineering (Shanghai) Co., Ltd. (Shanghai, China). The 2xPCR Mix is purchased from Beijing Quansijin Biotechnology Co., Ltd. (Beijing, China); the high-purity low-electrolytic agarose is purchased from Beijing Quansijin Biotechnology Co., Ltd. (Beijing, China). The DL5000 Marker is purchased from Nanjing Nuozan Biotechnology Co., Ltd. (Nanjing, China).

[0051] The following examples, the use of equipment and instrument information as follows: sequencer (Applied Biosystems, 3730XL); centrifuge (Thermo, Legend Micro17); PCR instrument (Applied Biosystems, 2720 thermal cycler); electrophoresis instrument (Junyi Oriental, JY300C); electrophoresis tank (Junyi Oriental, JYDF); water bath (Beijing Zhongxingweiye, DFD-700); plate centrifuge (Xiangyi, L550); FlavourSpec gas ion mobility spectrometry, G.A.S. Company (Germany, Dortmund); CTC-PAL 3 static headspace automatic sampling device, CTC Analytics AG Company (Switzerland, Zwingen); VOCal data processing software (0.4.03), G.A.S. Company (Germany, Dortmund); headspace bottle (Shandong Hai Neng Scientific Instrument Co., Ltd., 20mL); MXT-WAX capillary chromatographic column (Restek Corporation, USA, 30m x 0.53mm, 1.0μm).

[0052] Example 1 The present application provides a yeast strain SXZY-GJ-Y240 Figure 1 SXZY-GJ-Y2401) CCTCC NO: M 20241483 is screened and identified.

[0053] (1) Sample collection Fruit: 10 samples of fresh and 10 samples of rotten red oranges were collected from orchards in Tailong Town, Wanzhou District, Chongqing.

[0054] Orchard soil: 10 samples of soil attached to the surface of the orchard were collected into sterile self-sealing bags, placed in a cold storage box, and immediately taken back to a 4°C refrigerator for standby.

[0055] (2) Enrichment of strains Fresh red orange fermentation broth: fresh fruits were squeezed to obtain juice, and the juice was placed in a 500mL sterile blue cap bottle and naturally fermented at 28°C for 5 days to obtain fresh red orange fermentation broth.

[0056] (3) Isolation, purification and preliminary identification of strains Each 1mL of the above fresh red orange fermentation broth, rotten red orange fermentation broth and soil enrichment culture was diluted to 10 -6 , 10 -4 -10 -6Gradient dilution 150 μL was coated on WLN plate (WLN medium) (repeated 3 times in parallel), cultured at 28°C for 7d; different morphology, color of colonies were selected by streak dilution method on WLN plate, and cultured at 28°C for 3d, and single colonies were obtained after multiple purification; meanwhile, the purified strains were inoculated on slant culture for 3d, and stored in 4°C refrigerator.

[0057] The yeast colony on WLN plate is shown in Figure 1 Figure 2 The four culture media are WLN medium for separating fresh juice natural fermentation broth, and the left 1 and left 2 in the figure are the growth of bacteria cultured by post-coating culture of post-fermentation broth separated from different sampling points, and left 3 and left 4 are the growth of bacteria after streak separation and purification of typical characteristic strains; in addition, there are 65 strains of typical colony characteristics for preliminary screening, and the WLN culture types of each colony are preliminarily classified according to the yeast colony characteristics (color and morphology of the colony, surface and edge characteristics) on WLN plate. The results show that most of the yeast bacteria on WLN medium are metabolized and react with the components in WLN medium, and the colors of different types of yeast bacteria are different, with white, green and green-white interlaced colors; the colony morphology is spherical, round-roofed, flat, etc.

[0058] The purified colonies were divided into 7 groups (i.e. 7 strains) according to the shape and color, namely GJ-2, GJ-35, GJ-36, GJ-37, GJ-40, GJ-44 and GJ-45, and crystal violet staining was performed respectively; then the microscope was used to observe the purified single colonies and the stained yeast single colonies, and the colony color, morphology, reproduction mode and cell morphology of the yeast were recorded, and the results are shown in Figure 2 and Table 1.

[0059] Table 1 Morphological description of yeast colonies on WLN medium (cultured for 7d), cell morphology

[0060] Based on the colony color and morphology in Torulaspora delbrueckii and Table 1, reference is made to the colony morphology description of different yeast bacteria on WLN medium by Wang Xi et al. (Wang Xi, 2012) in "Wang Xi. Screening and identification of wild sea-buckthorn yeast and diversity analysis [D]. Inner Mongolia Agricultural University, 2012.", and the isolated and screened yeast bacteria are preliminarily classified; among them, GJ-35 is butter color with a little green color, the morphology is spherical protrusion, the surface is smooth, and it is preliminarily determined as Dekayomyces drosoplii. Pichia kluyveri Candida parapsilosis ​) ; GJ-40 was white with green, the shape of spherical protrusions, surface wrinkles, in the middle of the volcano, preliminary identified as Pichia kluyveri ( Meyerozyma caribbica ), and other fermentation strains no classification results.

[0061] (4) Molecular biology of the strain identification The 26S ribosomal ribonucleic acid (rRNA) gene D1 / D2 region of the above purified 7 strains were sequenced and identified, as follows: 1) The 26S rRNA D1 / D2 region gene sequence was amplified using universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), and the amplification product was obtained; the PCR amplification conditions were as follows: 98°C pre-denaturation for 3 min, 98°C denaturation for 10 s, 53°C annealing for 10 s, 72°C extension for 10 s, 37 cycles, and 72°C final extension for 5 min; 2) After PCR amplification, 5 μL of the amplification product was detected by 1% agarose gel electrophoresis (150 V, 100 mA for 20 min), and the desired DNA band was cut and purified from the PCR product. The primer sequencing was used, and the ITS sequence of GJ-45 was shown in SEQ ID NO: 1; 3) The purified PCR product was sent to Shengjiang Bioengineering (Shanghai) Co., Ltd. for sequencing, and the sequencing results were searched and aligned by basic local alignment search tool (BLAST) in the national center for biotechnology information (NCBI).

[0062] According to the above Table 1 and the above sequencing results, the identification of the above 7 strains can be obtained, as shown in Table 2.

[0063] Table 2 Comparison of different yeast sequences

[0064] From the above Table 2, it can be seen that strain GJ-2 is Candida parapsilosis ( Candida quercitrusa ), strain GJ-35 is Meyerozyma caribbica ( Meyerozyma guilliermondii ), strain GJ-36 is Candida aurantia ( Pichia fermentans Pichia kudriavzevii ), and strain GJ-37 is Pichia guilliermondii ( Pichia kluyveri), strain GJ-40 is Pichia fermentans (P. fermentans) Figure 3 ), strain GJ-44 is Pichia kudriavzevii (P. kudriavzevii) Figure 4 Figure 5A ), strain GJ-45 is Pichia kloeckera (P. kloeckera) Figure 5B .

[0065] In addition, a phylogenetic tree was constructed by the Neighbor-Joing method in MEGA11 software, and 1000 times of Bootstrap test was performed, and the results are shown in Figure 5C .

[0066] Example 2 The present application provides performance tests of different yeast strains (GJ-2, GJ-35, GJ-36, GJ-37, GJ-40, GJ-44 and GJ-45).

[0067] (1) Strain activation Take 7 strains of purified yeast strains preserved on a slant, streak on YPD solid medium (purchased from Qingdao Haibo Shengke Technology Co., Ltd., the same below), and culture for 48 h; pick single colonies and inoculate in sterilized YPD liquid medium (purchased from Qingdao Haibo Shengke Technology Co., Ltd., the same below), and culture in a shaking incubator at 180 r / min and 28℃ for 48 h to obtain strain activation liquid; the bottom of different strain activation liquids generally produces sediment and bubbles, the surface of the culture medium has a bacterial web, and most of the liquid medium is turbid, as shown in Table 3 below.

[0068] Table 3 Culture of single colonies of purified strains using YPD liquid medium

[0069] (2) Fermentation performance test of yeast strains The strain activation liquid obtained in the above (1) was inoculated into YPD liquid medium test tubes with inverted Durham tubes at 2%, and cultured at 28℃; the gas production was observed every 12 h, and the volume of gas in the Durham tube was recorded; the specific fermentation conditions are shown in Table 4 below.

[0070] Table 4 Analysis of fermentation capacity of different yeast strains

[0071] Note: "+", "++", "+++" respectively represent that the gas production reaches 1 / 3, 2 / 3 and the whole volume of the Durham tube, and "-" represents no gas production.

[0072] As shown in Table 4, the yeast strains GJ-2, GJ-36, GJ-40, GJ-44 and GJ-45 formed white and compact precipitates after 72 hours of fermentation, and the fermentation broth was slightly turbid, indicating that the five strains had good coagulation performance; GJ-44 produced gas in 24 hours to fill the du tube with gas, and GJ-2 and GJ-45 produced gas in 36 hours to fill the du tube with gas, indicating that the three yeast strains (GJ-44, GJ-2 and GJ-45) had relatively fast fermentation speed.

[0073] (3) Determination of ethanol production capacity of strains The strain activation liquid of the activated different yeast strains in (1) above was inoculated (using a inoculation ring to coat a 1 cm diameter bacterial plaque) on TTC solid medium (purchased from Hangzhou Microorganism City-level Co., Ltd.), and the color of the yeast was observed after 48 h of static culture in a 28°C incubator. Specifically, the color depth can be used to judge the respiratory enzyme activity of the yeast, and thus the ethanol production capacity of the yeast can be determined. The yeast with strong ethanol production capacity will show deep red color, followed by red color, light red color or no color.

[0074] The color of the fermentation broth of different strains is shown in Table 4. Figure 5D As shown in Table 4, the yeast strains GJ-2, GJ-36, GJ-40, GJ-44 and GJ-45 formed white and compact precipitates after 72 hours of fermentation, and the fermentation broth was slightly turbid, indicating that the five strains had good coagulation performance; GJ-44 produced gas in 24 hours to fill the du tube with gas, and GJ-2 and GJ-45 produced gas in 36 hours to fill the du tube with gas, indicating that the three yeast strains (GJ-44, GJ-2 and GJ-45) had relatively fast fermentation speed.

[0075] In addition, the yeast strains with darker color were used as primary screening strains, and were inoculated on YPD slant medium, cultured at 28°C for 48 h, and then stored in a refrigerator (4°C) for subsequent related tests of yeast strains.

[0076] (4) Tolerance of strains The fermentation process of yeast strains has certain requirements for alcohol content, SO2, sugar content and pH value, etc. Therefore, the tolerance of the 7 screened yeast strains was further tested. Specifically, the 7 screened strains were subjected to ethanol and SO2 tolerance tests, and the strains with strong tolerance were screened, and K yeast (La-Ma active fruit wine dry yeast, purchased from Yantai Dibo Self-brewing Co., Ltd.) was used as a comparison. The specific contents include: YPD liquid culture medium was dispensed into test tubes, sterilized at 121℃ for 20 min, and cooled to room temperature (25℃). Seven yeast strains were inoculated into the YPD liquid culture medium at a 2% (v / v) inoculum and cultured at 28℃ for 48 h. The absorbance (OD600nm) of the cell suspension was measured using a UV-Vis spectrophotometer (T6 New Century-UV-Vis spectrophotometer-Beijing Purkinje). The culture medium was adjusted according to conditions (a to d), and the absorbance was measured in triplicate for each treatment. (a) Different ethanol concentrations (volume percentage) (0% (blank control), 8%, 10%, 12%, 14%, 16%). (b) Different SO2 concentrations (0 (blank control), 80 mg / L, 130 mg / L, 180 mg / L, 230 mg / L, 280 mg / L); (c) Different sugar contents (mass percentage) (0 (blank control), 20%, 25%, 30%, 35%, 40%); (d) Different pH values ​​(2, 3, 4, 5 and 6), of which treatments (a) and (b) were sterilized first and then adjusted, and treatments (c) and (d) were adjusted first and then sterilized. The sterilization conditions were 121℃ for 15 min.

[0077] Under the above-mentioned environmental conditions of different ethanol concentrations, different SO2 concentrations, different sugar contents, and different pH values, the OD of the fermentation broth after yeast inoculation was measured. 600nm Value. The test results are as follows: Figure 5A , Figure 5B , Figure 5C and Figure 5D As shown, the results indicated that, compared with the seven screened yeast strains, the K yeast fermentation broth showed lower alcohol content (0%–12% vol), sulfur dioxide content (30 mg / L–280 mg / L), glucose content (0%–35%), and OD200 of the K yeast fermentation broth. 600nm The values ​​are all relatively large; details are as follows: Depend on Figure 6 It can be seen that the OD values ​​of GJ-2 and GJ-35 are 16% vol. 600nm The value was lower than that of other screened yeast strains and K yeast, indicating that at an alcohol content of ≤16% vol, the alcohol tolerance of other screened yeast strains, except for GJ-2 and GJ-35, was similar to that of K yeast. Depend on Figure 7 It can be seen that, with sulfur dioxide content (30m / L-280m / L), K yeast has stronger sulfur dioxide tolerance than the 7 selected fermentation strains. Among the 7 selected fermentation strains, GJ-36, GJ-44, and GJ-45 have stronger sulfur dioxide tolerance than the other selected yeast strains. Depend on Figure 7It can be seen that the glucose content (0%~25%) is close to the high sugar tolerance of K yeast, followed by GJ-2, GJ-35, GJ-40 and GJ-44; By Figure 8 It can be seen that the OD 600nm values of the fermentation broth of K yeast and the screened yeast strains are quite different in the fermentation environment pH (3~5). Generally, the pH value of mature citrus natural juice is 3~4, and the screened yeast strains can better propagate and metabolize in the pH value environment.

[0078] Example 2 The embodiment of the present application provides a preparation step (the preparation process can refer to the flow diagram shown in the figure) of red orange fruit wine and VOCs determination. Figure 9

[0079] (I) Preparation of red orange fruit wine (1) Fresh red oranges (not treated by preservation), select and remove bad fruits, add pectinase for enzymolysis (the addition amount of pectinase is 0.3%, and the enzymolysis is carried out at 20℃ for 8h), add sucrose to adjust the initial fermentation sugar degree to 18.0%, and the pH value is natural (pH 3.5-4.0), and the inoculation amount of K yeast is 0.12g / L (K yeast is added in 5% sucrose solution, activated at 35℃ for 30min, and used) to obtain the enzymolysis liquid; (2) Then, the above-mentioned five kinds of screened yeast strains (GJ-2, GJ-36, GJ-40, GJ-44 and GJ-45) are purified, and the purified colonies are inoculated into YPD liquid medium at a volume fraction of 2% and cultured at 28℃ for 48h to obtain the fermentation broth; (3) The fermentation broth is inoculated into the enzymolysis liquid in the above (1) at an inoculation amount of 2% for fermentation, and the fermentation is carried out at 20℃ until the dry fruit wine (the total sugar content is <5.0g / L) is obtained, and the fermentation is ended.

[0080] (II) VOCs determination of red orange fruit wine In the following determination, the method for analyzing by using GC-IMS is as follows: (a) Sample treatment method: 1mL of the above-mentioned fruit wine sample is taken and placed in a 20mL headspace bottle, incubated at 60℃ for 20min, and then sampled, and each sample is determined in three groups in parallel; (b) Headspace sampling: the upper volatile gas in the headspace bottle is analyzed by using gas chromatography-ion mobility spectrometry (GC-IMS); Among them, the sampling volume is 100µL; non-split sampling; incubation rotation speed: 500r / min; sampling needle temperature: 85℃; ​The GC conditions include: column temperature: 60°C; carrier gas: high-purity nitrogen (purity ≥ 99.999%); programmed pressure: initial flow rate 2.0 mL / min for 2 min, linearly increased to 10.0 mL / min in 8 min, linearly increased to 100.0 mL / min in 10 min, and maintained for 10 min; chromatographic run time: 30 min; injection port temperature: 80°C; The IMS conditions include: ionization source: tritium source (3H); migration tube length: 53 mm; electric field strength: 500 V / cm; migration tube temperature: 45°C; drift gas: high-purity nitrogen (purity ≥ 99.999%); flow rate: 75.0 mL / min; positive ion mode.

[0081] In the following determination, the mixed standard of 6 ketones was detected by the above GC-IMS to establish the calibration curve of retention time and retention index, and then the retention index of the target substance was calculated by the retention time of the target substance. The VOCal software built-in GC retention index (NIST2020) database and IMS migration time database were used for retrieval and comparison for qualitative analysis of the target substance.

[0082] In the following determination, the Reporter, GalleryPlot and DynamicPCA plug-ins in the VOCal data processing software were used to generate three-dimensional spectrum, two-dimensional spectrum, difference spectrum, fingerprint spectrum and PCA graph of volatile components, respectively, for comparison of VOCs between samples; OriginPro2024 was used for drawing.

[0083] (1) GC-IMS spectrum analysis determination The three-dimensional spectrum (see Figure 9 ) of volatile components (analyzed by the above GC-IMS method) was generated by the Reporter plug-in of VOCal data processing software, and the results are shown in Figure 9 , which showed that there were differences in VOCs in different samples, and there was also a certain degree of similarity; In addition, in order to more intuitively compare the differences in VOCs between different samples, dimensionality reduction processing was performed, and the two-dimensional spectrum (see Figure 10 ) was obtained by the Reporter plug-in of VOCal data processing software, and the results showed that there were differences in the positions of signal points and the depth of color in the above-mentioned selected yeast and K yeast fermented Hongge fruit wine volatile organic compounds in the overhead plane graph, and most of the VOCs signals were retained between 250-600 s, and a few VOCs signals were between 750-1000 s.

[0084] In addition, in order to further intuitively compare the VOCs difference in the samples, the Reporter plug-in of VOCal data processing software was used, taking the spectrum of sample GJ-2 as the reference, and the spectrum of other samples was subtracted from the reference to obtain the difference comparison chart of different samples (see Figure 10 ); Figure 10 In the figure, white represents that the VOCs content in the target sample and the reference is the same, red represents that the concentration of the substance is higher than the reference, blue represents that the concentration of the substance is lower than the reference, and the ion peak intensity is represented by the color depth, and the deeper the color, the greater the difference; according to Figure 10 In the figure, white represents that the VOCs content in the target sample and the reference is the same, red represents that the concentration of the substance is higher than the reference, blue represents that the concentration of the substance is lower than the reference, and the ion peak intensity is represented by the color depth, and the deeper the color, the greater the difference; according to

[0085] (2) GC-IMS qualitative analysis of VOCs in red orange wine The VOCs of red orange wine fermented by the above-mentioned screened yeasts and K yeast were analyzed by GC-IMS Library Search, and 42 VOCs and their dimers were identified (among them, -M represents monomer, -D represents dimer), see Table 5, and the fingerprint picture was drawn by using the GalleryPlot plug-in of VOCal data processing software, see Figure 10 , which includes 19 esters, 8 alcohols, 5 aldehydes, 3 ketones, 2 acids and 5 others.

[0086] Table 5 Qualitative results of volatile components in navel orange wine fermented by different yeasts

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[0090]

[0091] Specifically, esters are the most abundant VOCs in red orange wine. Since the wine was detected after fermentation was completed and before aging, the source of esters is mainly produced during fermentation. The literature “Kang Mingli [Research on the changes of volatile flavor compounds in citrus wine brewing process]” shows that ethyl octanoate, ethyl decanoate, and ethyl n-hexanoate are the main ester compounds in the fermented wine of clarified juice and residue-containing juice of Dayetanzhong mandarin oranges. The literature “Zhang Xinyuan. Application potential of optimized Pichia pastoris in acid reduction fermentation of citrus wine [D]. Central China Agricultural University, 2023. DOI: 10.27158 / d.cnki.ghznu.2023.000654.” screened Pichia pastoris for fermentation of citrus wine, and 3-phenylpropyl ethyl ester, ethyl decanoate, and phenyl ethyl acetate were the main ester aroma components, giving citrus wine a unique floral and fruity aroma. The literature “Gao Tianian. Effects of mixed fermentation on the flavor of citrus wine [D]. Hunan University, 2022. DOI: 10.27135 / d.cnki.ghudu.2022.003945.” selected non-Saccharomyces yeasts and Saccharomyces cerevisiae for mixed fermentation of citrus wine, and ethyl acetate, ethyl propionate, isobutyl acetate, propyl acetate, and propyl hexanoate were the esters with higher content in JW14+DV10 fermented wine, giving the wine a soft fruity aroma such as banana and pineapple. The literature “Fu Y, Gao Y, Yang M, et al. Effects of Non-Saccharomyces Yeasts and Their Pairwise Combinations in Co-Fermentation with Saccharomyces cerevisiae on the Quality of Chunjian Citrus Wine [J]. Molecules, 2024, 29(5): 14. DOI: 10.3390 / molecules29051028. https: / / doi.org / 10.3390 / molecules29051028.” used non-Saccharomyces yeasts and Saccharomyces cerevisiae for mixed inoculation fermentation of Chunjian citrus wine, increasing the content of ester VOCs such as ethyl octanoate, ethyl decanoate, and phenyl ethyl acetate.

[0092] From Table 5 above and Figure 11It can be known that in addition to the above VOCs such as octanoic acid, acetic acid, ethyl hexanoate and ethyl acetate, the fermentation sample of GJ-45 detected a relatively high content of esters such as propyl acetate, butyl acetate, isobutyl acetate, isoamyl acetate and 3-methyl-2-butene ester, which endows the fruit wine with unique pear and raspberry fruit aroma; the fruit wine fermented by K yeast mainly contains a plurality of esters such as ethyl lactate, 3-methyl butyric acid methyl ester, 3-methyl butyric acid ethyl ester, 2-methyl butyric acid ethyl ester, ethyl butyrate, ethyl isobutyrate, propyl 2-methyl butyrate and ethyl formate, which can endow the fruit wine with complex fruit aroma and wine aroma such as apple, banana, pineapple, fat, whiskey and rum; these aroma components are detected in grape wine, and the variety of esters and other VOCs may be an important factor leading to the flavor homogenization of the fruit wine fermented by K yeast.

[0093] In addition, alcohol substances are also important VOCs in fruit wine, and the variety is only second to esters, which is consistent with the research results of different non-Saccharomyces cerevisiae on the VOCs of mixed fermentation of navel orange wine in the literature “Liu X, Gao T, Liu W, et al. Analysis of volatile aroma components of navel orange wine fermented by non-Saccharomyces cerevisiae and Saccharomyces cerevisiae based on GC-IMS [J]. Food Science and Technology, 2023, 44(17): 139-148. DOI: 10.13386 / j.issn1002-0306.2022100310.”, which are mainly produced during fermentation. The relative content of 2-methyl-1-butanol in GJ-45 is higher than that of the screened yeast and K yeast, which can endow the fruit wine with roasted onion flavor, fruit aroma, floral aroma and wine aroma; the 3-methyl-1-butanol, 1-hexanol and 1-butanol in the fruit wine fermented by K yeast endow the fruit wine with banana, fruit aroma, green aroma, red wine flavor and whiskey wine aroma, and the overall aroma is diverse and complex.

[0094] In addition, aldehydes and ketones also contribute to the flavor of fruit wine. The content of heptanal in GJ-45 is higher than that of other screened yeast strains and K yeast, which endows the fruit wine with unique green grassy aroma, followed by the high content of pentanal and 3-methyl-2-butenal, which provides a certain green grassy aroma, weak banana flavor and fruit aroma; and the relatively high content of 3-hydroxy-2-butanone and acetone in the fermentation sample of K yeast makes the fruit wine have butter flavor, cream flavor, apple flavor and pear flavor.

[0095] Specifically, Figure 12In the table, each row represents all the signal peaks selected from a sample, and each column represents the signal peaks of the same VOC in different samples. The color intensity indicates the level of substance content. In the red mandarin orange wine sample, a total of 42 VOCs (including monomers and dimers) were identified by GC-IMS. Among them, there were 19 esters, 8 alcohols, 5 aldehydes, 3 ketones, 2 acids, and 5 other organic compounds. See Table 4 for details.

[0096] Depend on Figure 12 It can be seen that the VOCs formed by the fruit wine samples fermented by the five yeasts screened above and K yeast are quite different in terms of type and content. Among the five yeast-fermented fruit wine samples, GJ-2 and GJ-36 have similar fingerprint spectra and similar flavors, while GJ-40, GJ-44 and GJ-45 have significant differences in fingerprint spectra, indicating that the flavors of the samples are quite different. ​ In the middle, region C represents the main VOCs produced by yeast fermentation in sample K. In addition to heptanal (highest content in GJ-45), 3-methyl-1-butanol-M, and 3-methyl-1-butanol-D, other VOCs such as acetic acid-M, acetic acid-D, 3-hydroxy-2-butanone, ethyl lactate-M, ethyl lactate-D, methyl 3-methylbutyrate, ethyl 3-methylbutyrate, ethyl 2-methylbutyrate, ethyl butyrate, ethyl isobutyrate, propyl 2-methylbutyrate, ethyl formate, 1-hexanol, 1-butanol, acetone, and dimethyl sulfide are present in relatively high or unique amounts in sample K. Region B mainly contains VOCs that are unique to or present in high amounts in the GJ-45 sample, including 2-methyl-1-butanol, pentanal, 3-methyl-2-butenal, propyl acetate, butyl acetate, isobutyl acetate, isoamyl acetate, and 3-methyl-2-butenyl acetate.

[0097] Area A mainly contains VOCs specific to the five yeast strains screened above and fruit wine samples fermented by K yeast. Among them, 1-propanol, ethyl acetate, methyl acetate, and 2-methyl-1-propanol are VOCs commonly found in all six samples, while 2-methylpropanal, propane-M, and propane-D are VOCs commonly found in fruit wines fermented by the five yeast strains screened above. Methanol content is relatively high in GJ-2 and GJ-36.

[0098] From the above, it can be seen that the VOCs of the fruit wine sample fermented by the GJ-45 yeast strain are relatively typical, and the content of methanol produced is relatively low, which is comparable to that of K yeast, among which the ion peak intensity of ethyl acetate, methyl acetate, 3-methyl-2-butene acetate, 2-methyl-1-butanol, 3-methyl-2-butenal, propyl acetate, butyl acetate, isobutyl acetate, isoamyl acetate, valeraldehyde, heptanal and other VOCs are at a relatively high level compared with the fruit wine samples fermented by other screened yeast strains and K yeast, which contributes unique roasted onion, pear-like aroma, raw pear and raspberry aroma, banana fruit flavor and sweetness to the red orange wine, and can be used as a characteristic strain for developing characteristic flavor fermented citrus fruit wine.

[0099] (3) Sample cluster analysis (dynamic principal component PCA analysis) The data obtained by GC-IMS analysis was subjected to PCA analysis, and the Dynamic PCA plug-in of VOCal data processing software was used for dynamic principal component PCA analysis, and the results are shown in ​ The results show that the dispersion of each sample in the six samples of the fruit wine fermented by the above-mentioned five yeast strains (GJ-2, GJ-36, GJ-40, GJ-44 and GJ-45) and K yeast is small. Among them, GJ-2 is located on the positive axis of PC1, and the other four yeast strains are located on the negative axis of PC1 and far away from GJ-2, indicating that there is a large difference in the flavor of the fruit wine fermented by K yeast and the above-mentioned screened yeast strains. GJ-44 and GJ-45 are on the positive axis of PC2, GJ-2, GJ-36 and GI-40 are on the negative axis of PC2, indicating that there are differences in the flavor of the fruit wine fermented by the above-mentioned screened yeast strains. The flavor similarity of GJ-2 and GJ-36 is also close, and GJ-40, GJ-44 and GJ-45 are relatively dispersed, and there are differences in flavor. The analysis results are basically consistent with the comparison results of the above fingerprint. And the cumulative contribution rate of PC1 and PC2 is 81%, indicating that PCA analysis can better reflect most of the information of the samples, and can also better distinguish the fermentation fruit wine samples of K yeast and the above-mentioned screened yeast strains, and different screened yeast strains.

[0100] (4) Sample similarity analysis The data obtained by GC-IMS analysis was subjected to PCA analysis, and the Dynamic PCA plug-in of VOCal data processing software was used for dynamic principal component PCA analysis, and the results are shown in ​ , and the results are shown in ​It can be seen that the red orange fruit wines fermented by the above 5 screened yeast strains (GJ-2, GJ-36, GJ-40, GJ-44 and GJ-45) are far away from the red orange fruit wine fermented by K yeast, indicating that the volatile organic compounds of the red orange fruit wines fermented by the above 5 screened yeast strains are quite different from K yeast. Among them, the red orange fruit wine fermented by GJ-45 is farthest away from the red orange fruit wines fermented by the other 4 yeasts, and the similarity is the lowest; GJ-2 and GJ-36 are closest, and the similarity is higher. The results are basically consistent with the results of the fingerprint and the dynamic principal component PCA analysis.

[0101] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A yeast, with a preservation number of CCTCC NO: M 20241483.

2. A yeast preparation, characterized in that, The yeast of claim 1.

3. The yeast inoculant of claim 2, wherein, The dosage form of the yeast agent includes a freeze-dried agent, a solid agent, a bacterial suspension or a fermentation liquid.

4. A fruit wine, characterized by, The fruit is fermented using the yeast of claim 1 and / or the yeast agent of claim 2 or 3.

5. The fruit wine according to claim 4, characterized by, The volatile components of the fruit wine include one or more of ethyl acetate, methyl acetate, 3-methyl-2-butenyl acetate, 2-methyl-1-butanol, 3-methyl-2-butenal, propyl acetate, butyl acetate, isobutyl acetate, isoamyl acetate, pentanal or heptanal.

6. The fruit wine according to claim 4 or 5, characterized by, The total sugar content is ≤5 g / L.

7. The fruit wine according to claim 4 or 5, characterized by, The fruit is red orange.

8. The method of producing fruit wine according to any one of claims 4 to 7, characterized in that, The preparation method includes: (1) enzymolysis of the fruit to obtain an enzymolysis liquid; (2) inoculation of the yeast of claim 1 or the yeast agent of claim 2 or 3 in the enzymolysis liquid to obtain the fruit wine.

9. The production method according to claim 8, characterized by, The preparation method includes: (a) enzymolysis using pectinase, the addition amount of the pectinase being 0.2%-0.4%; and / or (b) on the basis of (a), the temperature of the enzymolysis being 18-22 ℃, and the time being 6-8 h; and / or The fermentation temperature is 18-22 ℃. 10.Use of the yeast of claim 1 and / or the yeast agent of claim 2 or 3 in the preparation of ethanol.

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