Application of Pichia kudriavzevii with high tolerance and low isoamyl alcohol production in liquor brewing and blending

Abstract

The present invention provides an application of a high-tolerance, low-yield isoamyl alcohol-producing Pichia kudriavzevii yeast in liquor brewing and blending. The strain is Pichia kudriavzevii ( Picea Kudryavzevi ) M3, the deposit number is: CCTCC NO: M 2022254. The yeast has high tolerance, high ethanol production and low fusel oil production. Its colony is round with serrated edges and large in shape. It is slightly raised, milky white in appearance, dry, and the cell morphology of the strain is elliptical or flat under a 400x microscope. The reproduction method is budding. The yeast is used in blending light-flavor liquor to improve the purity and quality rate of the liquor.

Description

Technical Field

[0001] The present invention is a divisional application of 2022105907768, and the parent application date is May 27, 2022. The present invention relates to the field of brewing microorganisms, and specifically to a Pichia yeast strain with good tolerance, high ethanol production, and low fusel oil production, and its application. Background Art

[0002] Higher alcohols refer to the general term for monohydric alcohols containing more than three carbon atoms, including n-propanol, isobutanol, isopentanol, etc. Higher alcohols are also one of the three major aromatic substances. Yeast is the main microorganism that produces higher alcohols in the winemaking process. In the early stage of fermentation, the substrate is sufficient, the yeast proliferates in large quantities, and higher alcohols are primary metabolites, so they are mainly produced in this stage; and in the later stage of fermentation, part of these higher alcohols will react with acids, mainly acetic acid, to generate corresponding ester compounds under the catalysis of enzymes, and part of them will continue to exist in the wine. In the wine, a moderate amount of higher alcohols has the effect of balancing the taste and aroma, but studies have shown that too much higher alcohol in the wine will not only bring bitterness and unpleasant "fusel oil taste", but also cause serious post-drinking stress effects such as dizziness, nausea, and palpitations after drinking. Due to the above-mentioned problems with excessive higher alcohols, reducing the content of higher alcohols in wine has become an important means to control and improve the quality of wine. The methods for removing higher alcohols mainly include process control, distillation separation, material adsorption, and breeding of low-yield higher alcohol microorganisms. Compared with the previous methods, the latter can solve the problem of excessive higher alcohol content in fermented wine and distilled wine from the source. Breeding excellent strains can also directly improve the quality of wine and increase the rate of high-quality wine. Summary of the invention

[0003] The invention provides a high-tolerance, low-yield isoamyl alcohol Pichia kudriavzevii strain and application thereof. The strain has the characteristics of sugar tolerance, ethanol tolerance, acid tolerance, high ethanol yield and low fusel oil yield.

[0004] The technical scheme of the invention is a strain of Pichia kudriavzevii with high tolerance and low isoamyl alcohol production. The strain is Pichia kudriavzevii M3, which is preserved in China Center for Type Culture Collection with a preservation number of CCTCC NO: M 2022254.

[0005] Furthermore, the DNA splicing sequence of the strain is shown in SEQ ID NO.1.

[0006] Furthermore, the colony morphology of this strain is round with serrated edges and a milky white and dry appearance; it appears oval or flat under a microscope, and its reproduction method is budding, with the cell buds in reproduction clearly visible.

[0007] The present invention also relates to the application of the Pichia yeast in the field of winemaking and / or yeast preparation.

[0008] Furthermore, the winemaking is white wine brewing or yellow wine brewing.

[0009] Furthermore, when applied in the field of winemaking, Pichia yeast is applied to any step of the winemaking process, including but not limited to mixing with koji-making raw materials during the koji-making process; or mixing with saccharified and gelatinized winemaking raw materials; or mixing with winemaking mash in the middle and late stages of fermentation.

[0010] The invention also relates to a fortified bran koji, which contains the Pichia yeast.

[0011] Furthermore, wheat bran is used as raw material, water is added to mix and sterilize, and then Pichia yeast is dispersed with physiological saline until the cell number is about 10 7 -10 8 and then inoculated into bran at a ratio of 6-8wt% for cultivation and drying to obtain Pichia yeast-fortified bran.

[0012] The present invention also relates to a wine for blending, wherein the Pichia yeast or the fortified bran koji is used for solid-state fermentation during the preparation of the wine. Furthermore, the wine for blending is mixed with a base wine to prepare a light-flavor liquor.

[0013] The present invention has the following beneficial effects

[0014] The present invention provides a Pichia kudriavzevii strain M3, which has excellent physiological tolerance and unique ability of high ethanol production and low fusel oil production. It can continuously adapt to changes in the physical and chemical environment during the fermentation process. It can be widely used in the field of winemaking, especially in the field of white wine and yellow wine. At the same time, the fortified bran koji made by the Pichia kudriavzevii is mixed with ordinary wine koji to significantly improve the wine quality and reduce the content of fusel oil in the original wine.

[0015] The present invention also provides a wine fermented by pure strain M3 of Pichia kudriavzevii for blending in light-flavor liquor. The wine stored for a suitable period of time is blended with a light-flavor liquor base wine, which can significantly enhance the ester aroma of the base wine, make the ester aroma and grain aroma harmonious, and reduce the harsh and spicy feeling and bitter feeling, so as to achieve the purpose of enhancing the purity and quality rate of the wine, and obtain a high-quality wine that is pure, sweet, and does not make you drunk. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 , tolerance analysis diagram of multiple low-fusel oil-producing yeast strains.

[0017] Figure 2, Colony morphology of Pichia kudriavzevii M3 (Pichia kudriavzevii) on Yeast Extract Peptone Dextrose Medium (YEPD).

[0018] Figure 3 , Pichia kudriavzevii M3 bacterial morphology under a 400x microscope (methylene blue staining).

[0019] Figure 4 , genetic development tree diagram of Pichia kudriavzevii M3.

[0020] Figure 5 , GC-MS total ion chromatogram of volatile components in the fermentation broth of Pichia kudriavzevii M3. DETAILED DESCRIPTION

[0021] The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0022] Embodiment 1:

[0023] Screening of strains

[0024] The present invention relates to the separation and screening of Pichia yeast with high ethanol production and low fusel oil production from high-quality fermented grains. The specific screening method is as follows:

[0025] Firstly, more than 60 yeast strains with excellent performance were screened from high-quality mash, and targeted screening was carried out for ethanol production and low fusel oil production. The prepared yeast extract peptone dextrose medium (YEPD) plate was evenly divided into six areas. Multiple plates were prepared in advance, and then all the strains were spotted on the plates and cultured at 30°C for two days. Then, TTC medium cooled to 45°C was poured into the upper layer of the yeast extract peptone dextrose medium (YEPD) plate and kept in a dark place for two hours to observe the color change, and strains with darker red were selected as candidate strains (specific numbers are Shao, FenⅡ, PD1, PT11, PT17, Y-3, JY26, PF4, M3, M4, 4-16, 2300, 1312, 10#). Then the alternative strain was placed in yeast extract peptone glucose medium (YEPD) liquid culture medium at 30℃, 180r / min for 18h as seed liquid, and inoculated into sorghum juice medium with a sugar content of 12Brix according to 5% inoculation amount, and cultured at 30℃, 180r / min for 12h, then switched to static culture. The CO2 weight loss was recorded every 12h and the fermentation was terminated when the weight loss was less than 0.2g. After the fermentation was terminated, gas chromatography was used to detect fusel oil, and distillation was used to measure ethanol. Strains with high ethanol yield and low fusel oil yield were selected and compared with the production strain SC (brewer's yeast of a certain company, commercially available). Compared with the production strain SC of a certain factory, the total amounts of three higher alcohols of strains FenⅡ, PT11, JY26, 2300, 1312 and M3 decreased by 22.32%, 22.05%, 23.29%, 10.41%, 11.13% and 15.93%, respectively. At the same time, these strains have the ability to produce high ethanol.

[0026] In this example, the yeast extract powder peptone glucose medium (YEPD) is composed of 10 g yeast extract powder, 25 g glucose, 25 g peptone, 1200 g water, and a natural pH value; 15 g agar powder is additionally added to the solid medium. The sorghum juice medium is composed of 2000 g sorghum powder, 3 g amylase, 75 g saccharifying enzyme, 8000 g water, and a natural pH value.

[0027] Table 1 Comparison of fermentation parameters between screening strains and production strains

[0028]

[0029]

[0030] Example 2: Physiological characteristics of Pichia pastoris with high ethanol production and low fusel oil production:

[0031] Scrape FenⅡ, PT11, JY26, 2300, 1312, and M3 from a ring of the slant and culture them in liquid yeast extract peptone glucose medium (YEPD) at 30°C, 180r / min for 16h, then inoculate them into YEPD liquid medium at 2% inoculum and culture them at 30°C, 180r / min for 24h. Use an ultraviolet spectrophotometer with OD600 as an indicator to detect the strain at ethanol concentrations of 1%, 3%, 5%, 7%, 9%, 11%, and 13%; pH values ​​of 2, 3, 4, 5, 6, 7, and 8; sugar contents of 5Brix, 10Brix, 15Brix, 20Brix, 25Brix, 30Brix, and 35Brix; culture temperatures of 18°C, 22°C, 26°C, 30°C, 34°C, 38°C, and 42°C; lactic acid concentrations of 0%, 1%, 2%, 3%, 4%,

[0032] 5%, caproic acid concentration: 0‰, 0.2‰, 0.4‰, 0.6‰, 0.8‰, 1.0‰; acetic acid concentration: 0‰,

[0033] The tolerance under 1‰, 2‰, 3‰, 4‰ and 5‰ conditions is shown in the attached Figure 1 It can be seen that the M3 strain has the best physiological tolerance.

[0034] The selected M3 strain has been deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan City, Hubei Province, with the deposit number being CCTCC NO: M 2022254, and the deposit date being 2022-03-14. After identification, the strain was identified as Pichia kudriavzevii M3.

[0035] (1) Microbiological morphological characteristics:

[0036] Take the cultured slant, scrape a loop with an inoculation loop and disperse it in sterile physiological saline to make a cell suspension, then dilute the cell suspension in a 10-fold gradient, and take 100 μL to spread on Yeast Extract Peptone Dextrose Medium (YEPD) medium, invert the plate, and culture it in a 30°C incubator for 48 hours. Observe the morphological characteristics of the colonies: on the YEPD plate, the colonies are round, with jagged edges, large in shape, slightly raised, milky white in appearance, and dry. For details, see Figure 2 .

[0037] The cell morphology of the strain was observed under a 400x microscope. The characteristics were: oval or flat, the reproduction method was budding, and the cell buds in reproduction were clearly visible. Figure 3 .

[0038] Example 3: Molecular Biological Identification of M3 Strain

[0039] The screened strains were sent to BGI for 18S rRNA gene sequencing. The sequencing results were placed in the NCBI (National Center for Biotechnology Information) database for comparison. The strain sequences with higher similarity were selected and the phylogenetic tree was constructed using the Maximum Likelihood Estimation (MLE) method in MEGA 11.0 software as shown in the attached figure. Figure 4 shown.

[0040] Example 4: Application of strain M3 in liquor brewing and blending

[0041] Strengthening wheat bran koji: weigh 20 parts of wheat bran into a 250 mL conical flask, add 15 parts of water and mix well, sterilize with high pressure steam at 121°C for 20 min, take out and cool to room temperature; centrifuge, wash and precipitate the M3 strain seed liquid cultured in liquid yeast extract peptone dextrose medium (YEPD), add physiological saline to disperse the cell number to 10 7 -10 8 Then, 8 wt% of the bran was inoculated into the bran, mixed well and placed in a 30°C constant temperature box for 3 days. During the culture period, the bottle was closed every 12 hours. After the culture was completed, the bran was placed in a 38-40°C constant temperature drying oven and dried until the moisture content was less than 10%, thereby obtaining a fortified wheat bran.

[0042] Liquor brewing: Take 3000g of high-quality sorghum and immerse it in 70-80℃ hot water for 20cm, soak it for 24h, wash it seven to eight times, and then steam it at 0.25MPa for 30min. Soak the sorghum that has been steamed for the first time in 80℃ hot water for 30min, then steam it at 0.16MPa for 20min until the skin is broken and blooms, add 0.7wt% mixed fortified bran koji and brewing koji in a ratio of 1:1, stir evenly, saccharify and pile for 24h, and then put it into small jars, seal it and ferment it at room temperature for 7d. After the fermentation is over, distill it in the upper steamer, and the two sides are slightly higher than the middle by 1-2cm when it is in the upper steamer. The distillate is collected by the method of "cutting the head and tail". Alternatively, after sorghum is steamed until it blooms and breaks its skin, 0.1% amylase, 0.2% saccharifying enzyme, and 0.01% acid protease may be added, followed by saccharification in a stacked saccharification box for 24 hours, and 2-5% of pure bran koji made from Pichia yeast M3 may be added, and the mixture may be sealed and fermented for 7-14 days. The mixture may be distilled in a retort, and the fractions may be collected to obtain a wine for blending.

[0043] Liquor blending: One of the above-mentioned liquors for blending can be blended with other raw liquor samples to replace flavors and spices to improve the quality of the liquor and meet consumers' pursuit of taste. The specific characteristics before and after blending are shown in Table 2 below.

[0044] Table 2 Scoring table after adjustment

[0045]

[0046] Example 5: Analysis of metabolites of strain M3

[0047] Scrape a ring of Pichia from the slant and place it in liquid yeast extract peptone dextrose medium (YEPD) and culture it at 30°C, 180 rpm for 16 h. Then inoculate it into sorghum juice liquid medium to ensure that the number of cells after inoculation is within 10 6 -10 7 The mixture was cultured at 30°C and 180 r / min for 18 h until the stable phase, and then statically cultured at the same temperature for 4 days. After the culture was completed, the flavor compounds were extracted by headspace-solid phase microextraction combined with GC-MS detection.

[0048] Table 3 Determination of volatile components by HS-SPME method (partial)

[0049]

[0050]

[0051] Note: When using HS-SPME extraction, issues such as extraction rate should be considered. The above does not include all volatile products of the M3 strain.

[0052] As shown in Table 3, the metabolites of strain M3 include esters, acids and alcohols commonly found in wine. It is suitable for purebred brewing of wine for blending to improve the quality of wine, or for making purebred koji for fermentation with traditional koji to improve the quality of wine.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. The application of Pichia kudriavzevii with high tolerance and low isoamyl alcohol production in liquor brewing is characterized by: The Pichia yeast is Pichia kudriavzevii ( Pichia kudriavzevii ) M3, deposited in China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 2022254; Specifically, when brewing liquor, 3000g of sorghum is immersed in 70-80℃ hot water for 20cm, soaked for 24h, washed seven to eight times, and then steamed at 0.25MPa for 30min. The sorghum steamed for the first time is soaked in 80℃ hot water for 30min, and then steamed at 0.16MPa for 20min until the skin is broken and blooms, and 0.7wt% of mixed fortified bran koji and brewing koji with a mixing ratio of 1:1 is added, stirred evenly, saccharified and piled for 24h, and then respectively loaded into small jars, sealed and fermented at room temperature for 7d; after the fermentation is completed, the sorghum is distilled in a retort, and the two sides are slightly higher than the middle by 1-2cm when the sorghum is distilled in the retort, and the distillate is collected by the method of "cutting the head and tail" to obtain brewed liquor; The fortified bran koji is made of wheat bran, which is mixed with water and sterilized. Then, Pichia yeast is beaten with physiological saline until the cell count is about 10 7 -10 8 Then, it is inoculated into bran at a ratio of 6-8wt% for cultivation and dried to obtain the product.

2. The application of Pichia kudriavzevii with high tolerance and low isoamyl alcohol production in liquor blending is characterized by: The Pichia yeast is Pichia kudriavzevii ( Pichia kudriavzevii ) M3, deposited in China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 2022254; When brewing liquor for blending, take 3000g sorghum and immerse it in 70-80℃ hot water for 20cm, soak it for 24h, wash it seven to eight times, and then steam it at 0.25MPa for 30min. The sorghum steamed for the first time is soaked in 80℃ hot water for 30min, and then steamed at 0.16MPa for 20min until the skin is broken and blooms, add 0.1% amylase, 0.2% saccharifying enzyme, and 0.01% acid protease, saccharify it in a stacking saccharification box for 24h, add 2-5% pure bran koji made from Pichia yeast M3, seal and ferment it for 7-14d, distill it in a retort, collect the distillate, and the liquor for blending is obtained; During the specific blending process, the wine to be used for blending is blended with other original wine samples.

3. The use according to claim 1 or 2, characterized in that: The Pichia yeast colony is round in shape with serrated edges, milky white in appearance and dry; it appears oval or flat under a microscope and reproduces by budding.

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