CN114836332B - Pichia kudriavzevii with high tolerance and low isoamyl alcohol yield and application thereof - Google Patents

Abstract

The invention provides a high-tolerance and low-isoamyl alcohol-producing Pichia kudriavzevii and application thereof, wherein the strain is the Pichia kudriavzevii @ kudriavzeviiPichia kudriavzevii) M3, deposit number is: cctccc NO: m2022254. The yeast has high tolerance, high ethanol yield and low fusel oil yield, and the colony is circular with sawtooth edge and large shape. The micro-bulge is milky white in appearance, dried, and observed under a 400-time microscope, the cell morphological characteristics of the strain are elliptic or prolate, and the propagation mode is bud. The invention also relates to application of the pichia pastoris in the field of brewing and/or in yeast preparations; the wine is blended with the base wine, so that the purposes of improving the purity and the high-quality rate of the wine can be achieved, and the high-quality wine which is pure, sweet and free of top-quality can be obtained.

Description

Pichia pastoris kudliaziwei with high tolerance and low isoamyl alcohol production and its application

Technical field

The invention relates to the field of brewing microorganisms, and specifically relates to a Pichia pastoris strain with good tolerance, high yield of ethanol and low yield of fusel oil and its application.

Background technique

Higher alcohols refer to the general term for those monohydric alcohols containing more than three carbon atoms, including n-propanol, isobutanol, isoamyl alcohol, etc.; higher alcohols are also one of the three major aromatic substances. Yeast is the key ingredient in the brewing process. The main microorganisms that produce higher alcohols in the fermentation process have sufficient substrates in the early stage of fermentation, and yeast proliferates in large quantities. Higher alcohols are primary metabolites, so they are mainly produced at this stage; in the later stages of fermentation, some of these higher alcohols will interact with acetic acid, which is the main product. The acidic substances generate corresponding ester compounds under the catalysis of enzymes, and part of them continues to exist in the wine body. In the wine body, an appropriate amount of higher alcohols can balance the taste and aroma. However, studies have shown that too high higher alcohols in the wine body will not only bring bitterness and unpleasant "fusel oil taste", but also cause a lingering after-drinking effect. It can cause severe drunken stress effects such as headache, nausea, and heart palpitations. Due to the above-mentioned problems of 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 and separation, material adsorption, and breeding. Microorganisms with low production of higher alcohols, etc. 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 content of wine. The rate of excellent products.

Contents of the invention

The invention provides a strain of Pichia pastoris kudliaziwei with high tolerance and low production of isoamyl alcohol and its application. The strain has the characteristics of sugar resistance, ethanol resistance, acid resistance, high ethanol production and low fusel oil production.

The technical solution of the present invention is a strain of Pichia kudriavzevii with high tolerance and low production of isoamyl alcohol. This strain is Pichia kudriavzevii M3, which is preserved in China Typical Culture Collection Center, the collection number is: CCTCC NO: M 2022254.

Further, the DNA splicing sequence of this strain is shown in SEQ ID NO. 1.

Furthermore, the colony shape of this strain is round, with jagged edges, milky white, and dry appearance; under the microscope, it is oval or oblong in shape, and the reproduction method is budding, and the cell buds in reproduction are clearly visible.

The present invention also relates to the use of the Pichia pastoris in the field of brewing and/or yeast preparations.

Further, the brewing is white wine brewing or rice wine brewing.

Further, when specifically applied in the field of brewing, Pichia pastoris can be used in any step of the brewing process, including but not limited to mixing with koji-making raw materials during the koji-making process; or mixing with saccharified and gelatinized brewing raw materials; Or mixed with brewing grains during the middle and late stages of fermentation.

The present invention also relates to a strengthened bran koji, which contains the Pichia pastoris.

Further, wheat bran is used as raw material, water is added, mixed and sterilized, Pichia pastoris is dispersed with physiological saline until the number of cells is between 10 ⁷ and 10 ⁸ , and then the bran is inoculated at a ratio of 6 to 8 wt%. The skin is cultured and dried to obtain Pichia pastoris-fortified bran koji.

The invention also relates to a wine for blending. In the preparation process of the wine, the Pichia pastoris or the enhanced bran koji are used for solid-state fermentation. Further, the wine used for blending is mixed with the base liquor to prepare a light-flavor liquor.

The present invention has the following beneficial effects

The Pichia kudriavzevii strain M3 provided by the invention has excellent physiological tolerance and unique ability to produce high ethanol and low fusel oil. 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 wine making, especially the field of liquor and rice wine. At the same time, the fortified bran koji produced by the Pichia pastoris is mixed with ordinary distiller's yeast, which can significantly improve the quality of wine and reduce the content of fusel oil in the original wine.

The invention also provides a kind of wine fermented by the pure strain M3 of Pichia kudriavzevii and used for blending in light-flavor liquor, using the wine stored for an appropriate time and the base liquor of light-flavor liquor Blending can significantly improve the ester aroma of the base wine, harmonize the ester aroma and grain aroma, and reduce the harsh, spicy, and bitter feeling. In order to achieve the purpose of improving the purity and quality rate of wine, and obtain high-quality wine that is pure, sweet and not overlying.

Description of the drawings

Figure 1. Tolerance analysis chart of multiple low-fusel oil-producing yeast strains.

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

Figure 3. Pichia kudriavzevii M3 (Pichia kudriavzevii) cell morphology under a 400x microscope (methylene blue staining).

Figure 4. Genetic development tree diagram of Pichia kudriavzevii M3.

Figure 5. GC-MS total ion chromatogram of volatile components in Pichia kudriavzevii M3 (Pichia kudriavzevii) fermentation broth.

Detailed ways

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

Example 1:

Screening of strains

The Pichia pastoris with high ethanol yield and low fusel oil yield involved in the present invention is separated and screened from high-quality wine grains. The specific screening method is as follows:

First, more than 60 yeast strains with excellent performance were screened out from high-quality fermented grains, and directional screening was conducted for ethanol production and low fusel oil production. The prepared Yeast Extract Peptone Glucose Medium (YEPD) plate was evenly divided into six areas. , prepare multiple plates in advance, and then inoculate all the strains on the plates and cultivate them at 30°C for two days. Pour the TTC medium cooled to 45°C into the upper layer of the Yeast Extract Peptone Dextrose Medium (YEPD) plate and store it in the dark for two days. Observe the color change after an hour, and select the strain with darker red as the candidate strain (specific numbers are Shao, FenⅡ, PD1, PT11, PT17, Y-3, JY26, PF4, M3, M4, 4-16, 2300, 1312 ,10#). Then place the candidate strain in a yeast extract peptone glucose medium (YEPD) liquid medium at 30°C and culture it at 180 r/min for 18 hours as a seed liquid, and inoculate it into a sorghum juice medium with a sugar content of 12 Brix according to an inoculation volume of 5%. After culturing for 12 hours at 30°C and 180 r/min, switch to static culture. Weigh every 12 hours to record _CO2 weight loss. When the weight loss is less than 0.2g, fermentation is terminated. After fermentation, gas chromatography is used to detect fusel oil and ethanol is measured by distillation. A strain with high ethanol yield and low fusel oil yield was selected and compared with the production strain SC (Saccharomyces cerevisiae of a certain company, commercially available). Compared with the production strain SC of a certain factory, strains FenⅡ, PT11, JY26, 2300, 1312, The total amounts of the three higher alcohols of 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.

In this embodiment, the composition of the yeast extract peptone glucose medium (YEPD) is: 10 g of yeast extract powder, 25 g of glucose, 25 g of peptone, 1200 g of water, and a natural pH value; an additional 15 g of agar powder is added to the solid medium. The composition of sorghum juice culture medium is: 2000g sorghum powder, 3g amylase, 75g glucoamylase, 8000g water, and natural pH value.

Table 1 Comparison of fermentation parameters between screening strains and production strains

Example 2: Physiological characteristics of Pichia pastoris that produces high ethanol and low fusel oil production:

Scrape FenⅡ, PT11, JY26, 2300, 1312, and M3 from a ring of slopes into liquid yeast extract peptone glucose medium (YEPD) at 30°C and culture at 180 r/min for 16 hours, and then inoculate into the YEPD liquid according to the 2% inoculation volume Cultivate the culture medium at 30°C and 180r/min for 24 hours. Use a UV spectrophotometer to use OD600 as an indicator to detect the ethanol concentration of the strain at 1%, 3%, 5%, 7%, 9%, 11%, and 13%; The pH value is 2, 3, 4, 5, 6, 7, 8; the sugar content is 5Brix, 10Brix, 15Brix, 20Brix, 25Brix, 30Brix, 35Brix; the culture temperature is 18℃, 22℃, 26℃, 30℃, 34 ℃, 38℃, 42℃; lactic acid concentration is: 0%, 1%, 2%, 3%, 4%, 5%, caproic acid concentration is: 0‰, 0.2‰, 0.4‰, 0.6‰, 0.8‰, 1.0‰; acetic acid concentration is: tolerance under conditions of 0‰, 1‰, 2‰, 3‰, 4‰, and 5‰. The results are shown in Figure 1. It can be seen that the M3 strain has the best physiological tolerance.

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

(1) Microbiological morphological characteristics:

Take the cultured slant, use an inoculation loop to scrape a ring and disperse it in sterile saline to make a cell suspension. Then dilute the cell suspension 10 times in a gradient, and take 100 μL and apply it to the yeast extract peptone glucose medium ( In YeastExtract Peptone Dextrose Medium (YEPD) medium, invert the plate and place it in a 30°C incubator for 48 hours. Observe the morphological characteristics of the colonies: In the YEPD plate, the colonies are round, with jagged edges, larger shapes, and slightly raised. , the appearance is milky white and dry, see Figure 2 for details.

Observe the cell morphology of the strain under a 400x microscope. The characteristics are: oval or oblong shape, the reproduction method is budding, and the cell buds in the process of reproduction are clearly visible. See Figure 3 for details.

Example 3: Molecular biological identification of M3 strain

The screened strains were sent to BGI Technology Co., Ltd. for 18S rRNA gene sequencing. The sequencing results were placed in the NCBI (National Center for Biotechnology Information) database for comparison, and strains with higher similarity were selected using MEGA 11.0 software. Maximum Likelihood Estimation (MLE) was used to construct a phylogenetic tree as shown in Figure 4.

Example 4. Strain M3 is used in liquor brewing and blending.

Strengthened wheat bran koji: Weigh 20 parts of wheat bran into a 250mL triangular flask, add 15 parts of water and mix well, sterilize with high-pressure steam at 121°C for 20 minutes, then take out and cool to room temperature; add it to the liquid yeast leaching peptone glucose medium (YEPD ). The M3 strain seed liquid cultured in ) was centrifuged, washed, and precipitated. Added physiological saline to disperse the cells until the number of cells was between 10 ⁷ and 10 ^8. Then inoculated into the bran at 8 wt%, mixed well, and placed in a 30°C incubator. Medium culture 3d. During the culture period, bottles were buckled every 12 hours. After the cultivation is completed, the wheat bran koji is placed in a constant temperature drying oven at 38-40°C and baked until the moisture content is less than 10% to obtain strengthened wheat bran koji.

Liquor brewing: immerse 3000g of high-quality sorghum in 70-80℃ hot water for 20cm, soak for 24 hours, wash it seven to eight times, then steam it at 0.25MPa high temperature and high pressure for 30 minutes. The first steamed sorghum is soaked in 80℃ hot water for 30 minutes, and then Cook at 0.16MPa high temperature and pressure for 20 minutes until the skin breaks and blooms. Add 0.7wt% mixed fortified bran koji and brewing koji with a mixing ratio of 1:1. Stir evenly and saccharify and accumulate for 24 hours. Then put them into small jars and seal them. Ferment at room temperature for 7 days. After the fermentation is completed, the steamer is placed on the steamer for distillation. When the steamer is placed, the sides are slightly higher than the middle by 1-2cm, and the distillate is collected using the method of "pinch off the head and remove the tail". You can also add 0.1% amylase, 0.2% glucoamylase, and 0.01% acid protease after the sorghum is cooked until it blossoms and breaks. It is saccharified in a stacking saccharification box for 24 hours, and 2-5% pure bran made from Pichia pastoris M3 can be added. The koji is fermented in a sealed manner for 7-14 days, distilled in a steamer, and the fractions are collected to obtain a wine for blending.

Liquor blending: The above-mentioned blending wine can be blended with other original liquor samples to replace flavors and fragrances to improve the quality of the wine and satisfy consumers' pursuit of taste. The specific characteristics before and after blending are shown in Table 2 below.

Table 2 Rating form after adjustment

Example 5. Analysis of metabolites of strain M3

Scrape a ring of Pichia pastoris from the slant into liquid yeast extract peptone glucose medium (YEPD) at 30°C and culture at 180r/min for 16 hours, and then inoculate it into sorghum juice liquid medium to ensure that the number of cells after inoculation is 10 ⁶ Between -10 ^{and 7} , incubate at 30°C and 180r/min for 18h until the stable phase, and then incubate at the same temperature for 4d. After the incubation, the headspace-solid-phase microextraction method is used to extract flavor compounds and combined with GC-MS detection.

Table 3 Application of HS-SPME method to determine volatile component content (part)

Note: When applying the HS-SPME method for extraction, issues such as extraction rate should be taken into consideration. The above does not include all volatile products of the M3 strain.

It can be seen from Table 3 that the metabolites of strain M3 include esters, acids, and alcohols commonly found in wine, and are suitable for pure brewing of wine, for blending and improving the quality rate of wine, or for making pure koji. It is used to ferment with traditional koji to improve the quality of the wine.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

SEQUENCE LISTING

SEQ ID NO.1

<110> Hubei Daohuaxiang Liquor Co., Ltd.

<120> A Pichia pastoris strain of Kudriazwei with low fusel oil production and its application

<130> 1

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 501

<212> DNA

<213> Pichia kudriavzevii M3

<400> 1

cttccgtagg gtgaacctgc ggaaggatca ttactgtgat ttactactac actgcgtgag 60

cggaacgaaa acaacaacac ctaaaatgtg gaatatagca tatagtcgac aagagaaatc 120

tacgaaaaaa caaacaaaac tttcaacaac ggatctcttg gttctcgcat cgatgaagag 180

cgcagcgaaa tgcgatacct agtgtgaatt gcagccatcg tgaatcatcg agttcttgaa 240

cgcacattgc gcccctcggc attccggggg gcatgcctgt ttgagcgtcg tttccatctt 300

gcgcgtgcgc agagttgggg gagcggagcg gacgacgtgt aaagagcgtc ggagctgcga 360

ctcgcctgaa agggagcgaa gctggccgag cgaactagac tttttttcag ggacgcttgg 420

cggccgagag cgagtgttgc gagacaacaa aaagctcgac ctcaaatcag gtaggaatac 480

ccgctgaact taagcatatc a 501

Claims (7)

1. A strain of Pichia kudriavzevii with high tolerance and low production of isoamyl alcohol, characterized in that: the Pichia pastoris is Pichia kudriavzevii M3, preserved in China Typical Culture Collection Center, the collection number is: CCTCC NO: M 2022254. 2. The Pichia pastoris Kudriazivi according to claim 1, characterized in that: the Pichia pastoris colony shape is round, with jagged edges, milky white appearance, dry; oval or oblong under the microscope. The form and the mode of reproduction are budding. 3. Application of Pichia pastoris according to any one of claims 1 to 2 in the field of brewing and/or yeast preparations. 4. The application according to claim 3, characterized in that: the brewing is white wine brewing or rice wine brewing. 5. Application according to claim 3, characterized in that: when used specifically in the field of brewing, Pichia pastoris is used in any step of the brewing process, including mixing with koji-making raw materials in the koji-making process; or with the koji-making raw materials. Mixing of saccharified and gelatinized brewing raw materials; or mixing with brewing grains in the middle and late stages of fermentation. 6. A kind of strengthened bran koji, characterized in that: the bran koji contains the Pichia pastoris described in any one of claims 1 to 2. 7. The fortified bran koji according to claim 6, characterized in that: it uses wheat bran as raw material, adds water, mixes and sterilizes, and then disperses Pichia pastoris with physiological saline until the number of cells is 10 ⁷ -10 ⁸ between them, and then inoculate it into the bran at a ratio of 6-8wt% for culture and drying to obtain Pichia pastoris-enhanced bran koji.