CN111484941B - Saccharomyces cerevisiae with low fusel oil yield and application thereof - Google Patents
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Abstract
The invention discloses a Saccharomyces cerevisiae Y276 with a preservation date of 2019, 12 and 27 months and a preservation number of CCTCC NO: m2020080. The saccharomyces cerevisiae strain is obtained by separating and purifying soy sauce flavor type fermented grains of Jinpaimaotaizzhen liquor industry Limited, can reduce the fusel oil in the faint scent Xiaoqu liquor by 25.7 percent, can be matched with rhizopus and other strains to prepare mixed yeast for brewing the Xiaoqu faint scent liquor, and plays roles in reducing the fusel oil of the Xiaoqu raw liquor and improving the quality of the Xiaoqu raw liquor.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to saccharomyces cerevisiae and application thereof.
Background
The metabolism rate of fusel oil (higher alcohols represented by isobutanol and isoamyl alcohol) in the human body is slower than that of ethanol, and the uncomfortable reactions such as headache, anesthesia and the like after drinking are generally considered to be caused by the fusel oil. Therefore, the content of fusel oil must be less than or equal to 0.2g/100mL according to the hygienic Standard GB2757-1981 for distilled liquor and wine prepared from the distilled liquor. However, after China enters the world trade agreement, as the foreign wine enters the China market, the fusel oil content of the foreign wine such as brandy and the like is always overproof, the index of the fusel oil content of the distilled wine is forced to be cancelled in China in 2006. The current distilled liquor and the liquor preparation standard GB2757-2012 have no fusel oil content index. However, the general feeling of Chinese consumers is that the foreign wine has large reaction after drinking and is easy to get up. The reduction of the fusel oil content in the distilled liquor is an important project for improving the quality of the distilled liquor and improving the reaction of consumers after drinking.
The contents of fusel oil in the liquor base liquor with different flavors in the Chinese inherent distilled liquor are greatly different, the fusel oil content of the liquor with higher flavor such as Xiaoqu fen-flavor is usually more than 0.2g/100mL, and the fusel oil content of the liquor with lower flavor such as Maotai-flavor is usually less than 0.05g/100 mL. The reasons for the relatively low content of the aromatic fusel oil such as Maotai-flavor type, Luzhou-flavor type and the like are various, such as grain varieties, brewing processes, distiller's yeast varieties and the like. Besides optimizing the grain variety, the brewing process and the like, the method for fermenting the low-yield fusel oil by adopting the yeast strain is also an idea for solving the problem of higher fusel oil. Because the low-yield fusel oil strain bred by genetic modification such as mutagenesis and the like is easy to have back mutation in production, the screening of the naturally existing yeast strains with low-yield fusel oil and high-yield ethanol is a realistic and reliable method.
Disclosure of Invention
The invention aims to solve the problems and provides a Saccharomyces cerevisiae Y276 CCTCC NO with low fusel oil yield: m2020080, the mixed yeast prepared by the yeast strain is used for brewing and producing Xiaoqu fen-flavor liquor, solves the problem that the fusel oil content of the Xiaoqu fen-flavor liquor is higher than that of other flavor type raw liquor, and achieves the purposes of improving the quality of the Xiaoqu fen-flavor raw liquor and improving the reaction of consumers after drinking. The invention is innovative in that excellent strains are found in a Daqu Maotai-flavor liquor microorganism resource library and are applied to brewing Xiaoqu fen-flavor liquor.
The Saccharomyces cerevisiae (Saccharomyces cerevisiae) Y276 with low fusel oil yield is obtained by separating fermented grains of Jinpaitai Zhenjiu, Inc., and the 18s rDNA is shown in Table 8. The strain has the following form: the bacterial colony is white, dull, flat and round, has smooth surface and edge, neat edge, moist and sticky property and is easy to pick up; the physiological and biochemical characteristics are that carbon sources such as glucose, sucrose, maltose, molasses and the like can be utilized, the glucose is metabolized to produce alcohol, and the yield of fusel oil in the metabolic products is low.
The low-yield fusel oil Saccharomyces cerevisiae (Saccharomyces cerevisiae) Y276 related by the invention is obtained by the following method:
the special process of the Maotai-flavor liquor with three highs and two changs, the open production environment and the long-term continuous production practice screening accumulate abundant brewing microorganism strains. In the test, the Maotai-flavor liquor Daqu and fermented grains are used as microorganism separation sources, yeast strains with high liquor production capacity and low fusel oil production are screened, and the yeast strains and Y1 and Y2 in the new process yeast are used as control strains. The method aims to screen yeast strains which are lower than fusel oil produced by Y1 and Y2 and have the wine production capacity not lower than or higher than that of Y1 and Y2.
Because the Maotai-flavor liquor is obtained by seven times, each turn has a unique style, and the style of the turn can be derived from the difference of the types and the contents of microorganisms in fermented grains of each turn. Therefore, the microorganisms in each turn of fermented grains of Maotai-flavor liquor are different and need to be researched and screened respectively. The production of the Maotai-flavor liquor in seven rounds is determined by the fermentation condition of the previous round, so the fermented grains and the yeast used in the round are used as the objects for screening and separating the microorganisms from the fermentation of the accumulated fermentation from the next round to the six rounds of the Maotai Zhenjiu GmbH.
In the test, a yeast strain with high ethanol yield is obtained by screening a TCC (cross-section resistance) culture medium, and then the yeast strain with low fusel oil yield is further screened. Transferring the high-yield ethanol strain to a culture medium which takes lactic acid as a unique carbon source for screening the low-yield fusel oil. And selecting the colony growing larger on the lactic acid culture medium, and further performing secondary screening. The fermentation termination is judged according to the weight loss condition through sorghum juice fermentation, and the contents of ethanol, ethyl acetate and fusel oil in the fermentation liquor are measured after the fermentation is finished. Screening out yeast strains superior to Y1 and Y2, mixing with Rhizopus to obtain mixed yeast with new formula, fermenting sorghum with the mixed yeast containing Y1 and Y2 as control yeast, distilling to obtain wine, and comparing wine yield, fusel oil content and other main quality indexes.
The invention also discloses application of the saccharomyces cerevisiae with low fusel oil yield in reducing fusel oil of the Xiaoqu fen-flavor liquor. And mixing the saccharomyces cerevisiae with low fusel oil yield with the rhizopus G1 three-level bacteria, and then fermenting and brewing.
The invention also discloses a microbial agent, which comprises the following components in percentage by weight: m2020080 Saccharomyces cerevisiae with low fusel oil yield. The microbial agent is a solid microbial agent or a liquid microbial agent.
The invention also discloses a Xiaoqu fen-flavor liquor which is prepared from the following components in percentage by weight, wherein the preservation number is CCTCC NO: m2020080 and is prepared by brewing Saccharomyces cerevisiae microbial preparation with low fusel oil yield.
Compared with the prior art, the invention has the following beneficial effects: the saccharomyces cerevisiae with low fusel oil yield can reduce the fusel oil content of the Xiaoqu fen-flavor liquor by 25.7 percent, and effectively improve the liquor quality. The saccharomyces cerevisiae is separated from fermented grains, can be used for producing white spirit, reduces the content of harmful substance fusel oil in the Xiaoqu fen-flavor raw wine, and provides a new way for improving the quality of the white spirit and improving the reaction after drinking by consumers.
The preservation date of the Saccharomyces cerevisiae Saccharomyces cerevisiae Y276 is 2020, 04 and 27 days, and the preservation number is CCTCC NO: m2020080. The name of the preservation unit is China typical culture Collection (CCTCC) and the address is eight-way Lopa mountain China typical culture Collection in Wuchang district, Wuhan city, Hubei province, the postal code: 430072.
drawings
FIG. 1 is a graph showing the dyeing effect of Saccharomyces cerevisiae Y267 Melan of the present invention (40X 10).
Detailed Description
The present invention is described in detail below with reference to the following embodiments and the attached drawings, it should be understood that the embodiments are only for illustrating the present invention and are not to be construed as limiting the present invention, and any modifications, equivalents and the like based on the present invention are within the scope of the present invention.
Example 1 screening of Low-yield fusel oil Saccharomyces cerevisiae strains
1. Separating and screening high-yield wine yeast of Jinpaitai Zhenjiu wine industry
Materials: piling fermented grains from sand to six times in Jinpaitai Zhenjiu industry
YPD medium: 10g of yeast powder; 20g of glucose; 20g of peptone; 20g of sodium propionate; 1g of penicillin; 1000ml of water; if a solid YPD medium is to be prepared, 20g of agar is also added. Bottling, sterilizing in a sterilizing pot at 121 deg.C for 30min, and cooling.
TTC upper medium: solution A: 5g of glucose; 15g of agar; 900mL of water. And B, liquid B: 100mL of 0.5% TTC aqueous solution. Sterilizing solution A and solution B in a sterilization pot at 121 deg.C for 30min, cooling to 60 deg.C, mixing, and immediately using.
Separating and purifying the yeast: adding 5g of fermented grains sample into 95ml of sterilized distilled water, placing into a 150r/min shaking table for oscillating for 30min, placing the oscillated bacterial suspension into a gradient dilution apparatus, and taking 200 microliters of 10 microliters-1、10-2And 10-3And adding the three gradient bacterial suspensions to a YPD solid culture medium for coating, putting the YPD solid culture medium into an incubator at 30 ℃ for culture and observation, and growing yeast colonies on a flat plate after 2-3 d. The YPD solid medium plate on which yeast colonies grew was poured as TTC upper medium. Culturing in dark 30 deg.C incubator for 4 hr. The color development of the colonies was observed, and dark red colonies were picked up and transferred onto YPD solid medium. And simultaneously, two flat plates are switched, one flat plate is used for repeated verification of color development, and the other flat plate is used for the next experiment. And (3) placing the transferred plate in an incubator at 30 ℃ for culture, and growing yeast colonies on the plate after 2-3 days. The TTC color test was repeated once for the transfer plate to confirm that the selected yeast colonies had higher dehydrogenase activity. The colonies of the transfer plate confirmed by repeated TTC color development were streaked out by YPD solid plates and cultured in a medium at 30 ℃ for 2-3d to obtain purified single colonies.
2. Confirmation of fermentation Performance of Low-yield Heterohydrozyma Strain
Materials: yeast strains screened for TTC
Sorghum juice culture medium: pulverizing 1000g sorghum into 40 mesh powder with a high speed pulverizer, adding 5L water, steaming at 105 deg.C for 30min, cooling to 60 deg.C, mashing cooked grain, adding 2g diastase and 2g amylase, stirring, and saccharifying at 60 deg.C for 24 hr. The mash is centrifuged to obtain a supernatant. Making filter paper into filter cake, filtering the supernatant with filter cake, vacuum filtering with suction filter to obtain sorghum juice, measuring sugar degree, and storing at-20 deg.C.
YPD medium: 10g of yeast powder; 20g of glucose; 20g of peptone; 20g of sodium propionate; 1g of penicillin; 1000ml of water; if a solid YPD medium is to be prepared, 20g of agar is also added. Bottling, sterilizing in a sterilizing pot at 121 deg.C for 30min, and cooling.
Isolated and purified TTC-screened yeast colonies were inoculated into 10mLYPD liquid medium and shaken overnight at 140rpm at 30 ℃ in a test tube. Adding water into sorghum juice culture medium to adjust sugar degree to Bx9, subpackaging 50-250 mL of triangular flask with fermentation plug, and sterilizing at 121 ℃ for 30 minutes in a sterilization pot. The YPD bacterial liquid cultured overnight was transferred to 100uL to 50mL of sterilized sorghum juice. The fermentation plug is sleeved on the triangular bottle mouth and is sealed by injecting clear water. The inoculated flask was weighed to the nearest 0.01 g. The weight loss was measured every 24 h. And after 5-6 days, stopping weightlessness and finishing fermentation. 50mL of the fermentation broth was shaken up and then introduced into a 50mL centrifuge tube. Centrifuge at 8000rpm for 5 minutes. 5mL of the supernatant was aspirated through a syringe, passed through a 0.45um filter and into a 5mL centrifuge tube. The filtered fermentation supernatant was analyzed by gas chromatography. Comparing the fermentation performance and fusel oil yield of the screened strains. And selecting the strains with higher weight loss and lower fusel oil content in the small liquid fermentation test, and carrying out the next solid fermentation verification.
3. Fermentation verification of low-yield fusel oil strain
The method comprises the following steps:
(1) preparing a koji: and mixing the YPD culture solution of the low-yield fusel-oil-producing yeast strain to be verified with the G1 rhizopus for the third-level clock according to a specific proportion, wherein 5G of the G1 third-level and 10ml of the YPD culture solution of the yeast strain to be verified are required for verifying the faint scent solid state fermentation effect of the Xiaoqu.
(2) Soaking grains: weighing the required grain according to the amount of 500g of Australian sorghum of each sample, adding warm water at 60 ℃ (the water surface is higher than the grain surface), and soaking the grain in an oven at 60 ℃ for 24 hours.
(3) Steaming grains: firstly, putting the soaked grains into a sterilizing steamer for primary steaming at the temperature of 121 ℃ for 30 minutes. And after primary steaming is finished, adding 100 ℃ water to stew grains for 40 minutes, filtering the water, and putting the water into a sterilizing steamer to re-steam the grains for 30 minutes at 115 ℃.
(4) Mixing the grains and the yeast: cooling the grains after re-steaming to 30 ℃, subpackaging in No. 12 self-sealing bags, each self-sealing bag is 1040g, and fully mixing with the yeast.
(5) Saccharification: and (3) putting the mixed grain yeast into an incubator at 30 ℃ for saccharification, keeping the seal of the self-sealing bag open for about 24 hours, and sealing the opening with a wet towel to ensure the supply of oxygen and moisture in the saccharification process.
(6) And (3) sterilizing vinasse: weighing the vinasse according to the amount of 500g of a single sample, putting the vinasse into a sterilization steamer, and sterilizing at 121 ℃ for 30 minutes.
(7) Fermenting in a tank: mixing saccharified grain and sterilized distiller's grains according to the ratio of 1: 1, placing the mixture into a gas sampling bag, sealing, vacuumizing, placing the bag into an incubator at 30 ℃ for fermentation, and continuing for 7 days.
(8) Distilling wine: and 7 days later, filling fermented grains after fermentation into a special triangular flask for open fire distillation, taking 100ml of distilled wine sample, and taking samples for measuring related gas phase index parameters and fusel oil indexes of the wine body. The results of gas chromatography for solid state fermentation for each run are shown in tables 1-7.
TABLE 1 gas chromatography indexes for strains screened in rounds of sand
TABLE 2 Brown sand gas chromatography indexes for round screening of strains
TABLE 3 gas chromatography indexes of strains screened in one round
TABLE 4 gas chromatography indexes of strains screened in two rounds
TABLE 5 gas chromatography indexes for three rounds of strain screening
TABLE 6 gas chromatography indexes for four rounds of strain screening
TABLE 7 gas chromatography indexes of five rounds of strain screening
The gas phase data of wine samples in each round show that the fermentation performance, namely the alcohol production capacity of the screened yeast strains is similar to that of the yeast No. 1 and the yeast No. 2 strains used in the faint scent starter at present, but the fusel oil content of the screened yeast strains is generally reduced by 25.7 percent compared with that of the yeast No. 1 and the yeast No. 2. The screened sauce No. 17 with high alcohol yield and low fusel oil content and prominent performance is recorded in a strain resource library of a microbial research laboratory and is named as Y267.
Example 2 identification of Saccharomyces cerevisiae strain Y267
1.18S rDNA sequence analysis
(1) DNA extraction
Collecting the thallus for useAnd (3) carrying out DNA extraction by using the soil genome DNA extraction kit.
(2) Amplification of 18S rDNA fragment of Saccharomyces cerevisiae Y267
The 18S rDNA fragment amplification is carried out by taking the extracted saccharomyces cerevisiae DNA as a template and adopting 18S rDNA universal primers 18S-P1 and 18S-P2 of fungi. The amplification reaction system is 30 μ l, in which Premix TaqTM(TaKaRa TaqTMVersion 2.0plus dye) 15. mu.l, ddH2O 13. mu.l, primers 18S-P1 and 18S-P2 each 0.5. mu.l, DNA template 1. mu.l. And (3) amplification reaction conditions: 5min at 94 ℃; 30s at 94 ℃; at 55 ℃ for 50s and at 72 ℃ for 30s, and circulating for 32 s; extension at 72 ℃ for 10 mm and storage at 4 ℃.
(3)18S rDNA sequence analysis
The amplified 18S rDNA fragment was sent to the Wuhanhua large gene for sequencing to obtain the corresponding 18S rDNA amplified sequence results (as shown in Table 8). And its species information (as shown in table 8) was obtained by comparison with the Blast search program in the national biotechnology information (NCBI). The gene difference of the strain is larger than that of the typical strain of the existing saccharomyces cerevisiae, but the strain can still be confirmed to be the saccharomyces cerevisiae by integrating the colony morphology, the cell morphology, the physiological and biochemical characteristics and the like of the strain.
TABLE 818S rDNA amplified sequence results and BLAST alignment results
2. Morphological characterization of Saccharomyces cerevisiae strain Y267
Saccharomyces cerevisiae strain Y267 is inoculated and activated by YPD solid culture medium, and cultured for 2-3 days at 30 ℃.
(1) Microscopic observation of bacterial colony and thallus
Observing the colony morphology of the saccharomyces cerevisiae on the YPD culture medium under the culture condition of 30 ℃; a small amount of the cells were picked up with an inoculating needle or a sterilized toothpick and placed on a slide glass, and microscopic observation of the cells was carried out under a microscope of 40X 10.
The observations were as follows: the thallus is elliptical or nearly circular, the length is 2.5-4 μm, and the width is 2-3 μm; the bacterial colony is white, dull, flat and round, has smooth surface and edge, neat edge, moist and sticky property and is easy to pick up;
(2) melan staining
The strain culture was mixed in a drop of physiological saline, applied to a pellicle having a diameter of about 1cm, and subjected to aseptic processing. Drying or airing on an alcohol lamp, and fixing on a lamp flame back and forth for three times. Staining with 0.1% methylene blue staining solution for 1min, decolorizing with hydrochloric acid alcohol for 30s, washing, drying, and performing microscopic examination. The results are shown in FIG. 1.
Claims (1)
1. Saccharomyces cerevisiae (saccharomyces cerevisiae) with low fusel oil yieldSaccharomyces cerevisiae) The application of Y276 in reducing fusel oil of Maotai-flavor liquor is characterized in that the saccharomyces cerevisiae with low fusel oil yield is obtained by separating Maotai-flavor liquor Daqu and fermented grains, and the preservation number is CCTCC NO: m2020080.
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