CN112831425A - Pichia kudriavzevii and application thereof in XIN-flavor type white spirit fermentation - Google Patents
Pichia kudriavzevii and application thereof in XIN-flavor type white spirit fermentation Download PDFInfo
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Abstract
The invention relates to the technical field of microbial white spirit fermentation, in particular to pichia kudriavzevii and application thereof in XIN flavor type white spirit fermentation. The applicant screens out a Pichia kudriavzevii yeast (A.kudriavzevii) from fermented grainsPichia kudriavzevii) DHX 19; the preservation number is: CCTCC NO: m2021172. Mixing the obtained extract with other ingredientsThe Pichia schwangsi fermentation liquor is added into the fermented grains according to the proportion of 0.01-0.1% (w/w), the total ester content in the white spirit, including ethyl acetate, ethyl caproate and the like, can be obviously improved, trace flavor components such as tetramethylpyrazine, 2-ethyl phenylacetate, methyl phenylacetate and the like can be generated, and the product has the characteristic of rich ester fragrance.
Description
Technical Field
The invention relates to the technical field of microbial white spirit fermentation, in particular to pichia kudriavzevii and application thereof in XIN flavor type white spirit fermentation.
Background
The XIN flavor type white spirit is an innovative wine body developed by Hubei Daohuaxiang wine industry limited company and integrates the production process of three flavors of 'clear sauce thick'. The brewing process comprises the processes of saccharification culture, high-temperature accumulation, cellar fermentation and the like, and effectively combines the process characteristics of saccharification culture of Xiaoqu fen-flavor liquor, high-temperature accumulation of Maotai-flavor liquor and mud cellar fermentation of Luzhou-flavor liquor, so that a unique production process of the Xin-flavor liquor is formed, and specifically comprises the following steps: adding the Xiaoqu into the cooked sorghum for cultivating and saccharifying, adding the high-temperature Daqu into the saccharified fermented grains for high-temperature stacking, adding the medium-high temperature Daqu into the stacked fermented grains, and allowing the fermented grains to enter a mud pit for fermentation. The produced original XIN-type wine is clear and transparent in color, mellow and elegant, and has harmonious fragrance, sweet and clean taste, long aftertaste and unique flavor.
The XIN flavor type Chinese liquor uses three types of distiller's yeast including high temperature Daqu, middle and high temperature Daqu and Xiaoqu. The Daqu is prepared from barley, wheat, pea, etc. by pulverizing, kneading with water, pressing to obtain fermented grains, and forming blocks with different sizes under certain temperature and humidity conditions. As a saccharification leaven, the yeast contains rich microbial communities, functional enzyme systems and flavor precursors formed in the yeast preparation process, and is an important guarantee for the quality and the yield of white spirit. The temperature is an important index in the process of making the yeast for making hard liquor, and the yeast for making hard liquor can be divided into low-temperature yeast for making hard liquor, medium-high temperature yeast for making hard liquor and high-temperature yeast for making hard liquor according to different product temperatures. The Xiaoqu is prepared from rice flour, rice bran or wheat bran, and optionally Chinese herbal medicines or kaolin, adding quantitative mother yeast, and making under certain temperature and humidity conditions. The Xiaoqu is used as the same important saccharification leavening agent and is the main source of the faint scent aroma of the white spirit. In the saccharification stage, the Xiaoqu is mixed with the raw materials after steaming, soaking and spreading for cooling according to a certain proportion and added, so that the saccharification is promoted to be carried out smoothly, and the advantages of high liquor yield and stable liquor quality are achieved.
For example, Chinese patent document CN202010877716.5 discloses a yeast which is solid-state fermented and has flower and fruit flavor and application thereof, the invention screens a Geotrichum yeast applied to the production process of strong-flavor liquor, the quality of the produced liquor is improved, and the fruit flavor of the liquor is more prominent and lasting. CN202010879643.3 discloses an application of Kazachstaniaturensis in white spirit production. The yeast has good adaptation to the solid-state fermentation environment of grains and vigorous metabolism, and the main volatile flavor substance of the yeast is isoamyl acetate. The application of CN201610414698.0 also discloses a Pichia kudriavzevii strain, which has the characteristics of low urea yield, flavor production, alcohol resistance and acid resistance, can reduce the content of harmful component ethyl carbamate in fermented food, is applied to the fields of brewed wine, distilled wine and other foods, and ensures the food safety.
The research shows that the functional microorganisms separated from the brewing environment can improve the content of flavor substances in the white spirit and improve the quality of the white spirit after being cultured and applied to the production process of the white spirit. Although one or more aroma-producing microorganisms are applied to all flavor substances which can not produce white spirit in the white spirit production process, the aroma-producing microorganisms play an important role in improving the quality of the white spirit. In actual production, due to the difference of natural conditions in different areas and brewing processes of enterprises, the types and the number of the bacteria for producing the flavor substances of the white spirit also can be different.
The invention screens out a Pichia kudriavzevii yeast strain by separation and purification, adds the Pichia kudriavzevii yeast strain into the fermented grains of the Xin flavor type wine after amplification culture to participate in microbial fermentation, and has rapid growth and produces a large amount of growth metabolites including esters, alcohols, organic acids, sulfides and the like, and the substances are used as key factors influencing the flavor and the aroma of finished products of the white wine, thereby improving the quality of the white wine.
Disclosure of Invention
The invention aims to provide a Pichia kudriavzevii DHX19 strain, which has the preservation number as follows: CCTCC NO: m2021172, the strain has stronger capacity of producing ethyl acetate and ethyl caproate.
The invention also aims to provide the application of the Pichia kudriavzevii in the fermentation of the Xin flavor type white spirit. In order to achieve the purpose, the invention adopts the following technical measures:
the pichia kudriavzevii DHX19 is obtained by screening through the following steps:
taking a saccharified fermented grain sample from a Xin-flavor liquor brewing workshop of the limited company of Hubei rice flower fragrance liquor industry, diluting by 10 times, treating in a shaking table at 30 ℃ for 15min to activate saccharomycetes, properly diluting, coating a flat plate, adding ampicillin into the flat plate to inhibit bacterial growth, and then selecting a typical single colony according to colony morphology. Through physiological and biochemical and 26SrDNA sequence identification, a strain of Pichia kudriavzevii (Pichia kudriavzevii) is finally screened, and the strain is sent to the China center for type culture collection for collection at 2021 and 02 months, and is classified and named: pichia kudriavzevii DHX 19; the preservation number is: CCTCC NO: m2021172, address: wuhan university in Wuhan, China.
The DHX19 strain has typical physiological and biochemical characteristics of pichia pastoris: the bacterial colony formed by the propagation and growth of the bacterial strain on a yeast extract peptone glucose medium (YPD) is nearly circular, white and dull, has a protruding center, and has blunt edges and grey white color of 3.5-5.0 mm. Chemoheterotrophic culture; d, temperature addiction; acid-tending; facultative anaerobic. The cell thallus is rod-shaped, closely arranged, single, double or string, 3.0-4.5 × 5.5-8.0 μm. Candida type hyphae; the vegetative propagation mode is multilateral budding. Producing beta-glucanase.
The application of the Pichia kudriavzevii yeast in the XIN-flavor liquor fermentation comprises the fermentation of the XIN-flavor liquor and is used for preparing liquor fermentation distiller's yeast; or directly used for producing ethyl acetate, ethyl caproate, acetal, isobutanol or acetic acid by fermentation.
Compared with the prior art, the invention has the following advantages:
the Pichia kudriavzevii DHX19 has the biological characteristics of lactic acid resistance, salt resistance, strong ester production capacity, multiple acid production types, wide growth temperature range and the like, and particularly has the advantages that the Pichia kudriavzevii DHX19 has strong ethyl acetate and ethyl caproate production capacity, and the contents of two esters in sorghum juice fermentation liquor can reach 6771.0 +/-23.5 and 288.1 +/-19.15 mu g/mL respectively.
The Pichia kudriavzevii DHX19 also has the capacity of producing tetramethylpyrazine and can endow a white spirit product with a health-care function.
Drawings
FIG. 1 is a photograph of a colony (left) and a photomicrograph of a strain (right) of Pichia kudriavzevii DHX 19.
Detailed Description
Unless otherwise specified, the experimental methods and conditions used in the examples of the present invention are conventional methods, and the reagents or materials, unless otherwise specified, are commercially available.
Example 1:
acquisition of pichia kudriavzevii DHX 19:
taking a saccharified fermented grain sample from Hubei Daohuaxiang wine industry GmbH, diluting by 10 times, treating in a shaking table at 30 ℃ for 15min to activate saccharomycetes, properly diluting, coating a flat plate, adding ampicillin into the flat plate to inhibit bacterial growth, and then selecting a typical single colony (a milky flat colony, with the increase of the age of the colony, the surface of the colony becomes rough, the edge extends in a zigzag manner, the thallus is from oval to long oval, and the thallus forms a lotus root-shaped connected Candida after budding and reproduction).
The strain has typical physiological and biochemical characteristics of pichia pastoris: the bacterial colony formed by the propagation and growth of the bacterial strain on a yeast extract peptone glucose medium (YPD) is nearly circular, white and dull, has a protruding central part, and has a blunt edge tooth shape and a gray color of 3.5-5.0mm (figure 1). Chemoheterotrophic culture; d, temperature addiction; acid-tending; facultative anaerobic. The cell thallus is rod-shaped, closely arranged, single, double or string, 3.0-4.5 × 5.5-8.0 μm. Candida type hyphae; the vegetative propagation mode is multilateral budding. Producing beta-glucanase. Physiological and biochemical characteristics: aerobic and resistant to 8% NaCl. The optimum temperature is 28-32 ℃, and the maximum temperature is 50 ℃.
Through the physiological and biochemical processes and the identification of 26SrDNA sequence, the strain is determined to be Pichia kudriavzevii, the strain is sent to the China center for type culture collection for collection at 2021, 02/01, and the classification and the naming are as follows: pichia kudriavzevii DHX 19; the preservation number is: CCTCC NO: m2021172, address: wuhan university in Wuhan, China.
Recipe (1L) of YPD medium: tryptone 20g, yeast extract 10g, glucose 20g, pH natural; agar 15g was added to the solid medium.
Example 2:
scale-up culture of Pichia kudriavzevii (Pichia kudriavzevii) DHX 19:
according to the weight percentage of 20g/L glucose, 10g/L yeast extract and 10g/L, NaCl 5g/L, KH peptone2PO43g/L (natural pH), preparing fermentation liquor by 65% (V/V) liquid loading, sterilizing for 30min by high-temperature steam at 121 ℃, cooling to 30 ℃, inoculating 1% (V/V) of Pichia kudriavzevii seed liquid with the age of 10h, culturing at 30 ℃, and controlling the stirring speed to maintain dissolved oxygen (DO value is 30% -90%). Fermenting for 48h, and placing in a tank, wherein the amount of the plate count Pichia kudriavzevii DHX19 live bacteria is 3.8 × 108cfu/ml, and obtaining a Pichia kudriavzevii (Pichia kudriavzevii) DHX19 fermentation broth.
Example 3:
determination of optimal growth temperature for Pichia kudriavzevii (Pichia kudriavzevii) DHX 19:
inoculating test strain into 10ml sterilized YPD liquid culture medium at 2% inoculation amount, culturing at 25 deg.C, 30 deg.C, 37 deg.C, 40 deg.C, 45 deg.C and 50 deg.C for 24 hr with no inoculation test tube as blank control, measuring OD600 value, and calculating relative thallus concentration with OD600 value at 30 deg.C as 100% to obtain the optimum growth temperature of the strain. The optimal growth temperature of the strain is 25-37 ℃, but the bacterial count is still 11.1% at 50 ℃, and the strain has good temperature resistance.
Survival (%). percent OD600 at different culture temperatures/OD 600 at 30 ℃ culture temperature
TABLE 1 growth of the strains at different temperatures
| Temperature (. degree.C.) | 25 | 30 | 37 | 45 | 50 |
| Survival rate (%) | 91.3 | 100 | 73.7 | 19.6 | 11.1 |
Example 4:
pichia kudriavzevii (Pichia kudriavzevii) DHX19 ethanol tolerance property assay:
inoculating a test strain into 10ml of sterilized YPD liquid culture medium according to the inoculation amount of 2%, respectively adjusting the ethanol concentration of the culture medium to 0% (control group), 2% (volume ratio, the rest is the same), 5%, 8%, 9%, 10%, 12%, 16%, culturing at 30 ℃ for 24h, measuring OD600 values, and analyzing and comparing the growth conditions of each group to obtain the ethanol tolerance conditions of the strain as follows:
the survival rate (%) — OD600 value after culture in media containing different ethanol concentrations/OD 600 value after culture in media containing no ethanol.
TABLE 2 growth of the strains at different ethanol concentrations
| Ethanol (%) | 2 | 5 | 8 | 9 | 10 | 12 | 16 |
| Survival rate (%) | 94.1 | 88.2 | 56.9 | 15.5 | 1.7 | 1.1 | 1.0 |
Example 5:
tolerance assay for lactic acid in Pichia kudriavzevii DHX 19:
the test strains were inoculated in 10ml of sterilized YPD medium at an inoculum size of 2%, and the pH of the YPD medium was adjusted to: 3.0, 4.0, 5.0, 6.0 and 7.0, culturing at the optimum temperature of 30 ℃ for 24h, measuring the OD600 value, and comparing the growth conditions of each group to obtain the acid tolerance condition of the strain. The strain shows good growth performance in the pH range of 3.0-7.0.
The survival rate (%) — OD600 value after culture at different pH of the medium/OD 600 value after culture at pH 5.0 of the medium.
TABLE 3 growth of the strains with lactic acid adjustment at different pH values
| pH | 3.0 | 4.0 | 5.0 | 6.0 | 7.0 |
| Survival rate (%) | 79.1 | 88 | 100 | 93.9 | 90.8 |
Example 6:
determination of organic acid production by Pichia Kuderliazevii (Pichia kudriavzevii) DHX19
Taking sorghum flour: water 1: 4 (mass ratio), steaming at 100 ℃ for 2h, cooling to 65 ℃, adding glucoamylase (5U/mL), liquefying for 4h, adding glucoamylase (5U/mL), saccharifying for 3h, filtering, centrifuging at 5000r/min for 15min, taking supernatant, diluting to 10 ° Brix, subpackaging, sterilizing at 121 ℃ for 30min, and thus obtaining the sorghum juice culture medium.
Inoculating 1% (v/v) of the Pichia kudriavzevii seed liquid into the sorghum juice culture medium, standing and fermenting for 5d, centrifuging at 5000r/min for 15min, and taking the fermentation supernatant for subsequent experiments.
Collecting the fermented supernatant, centrifuging at 12000 r/min for 5min, filtering with 0.22 μm microporous membrane, and collecting supernatant by use of UItimate 3000 of Agilent liquid phase system; setting: column C184.6X 150mm, 5 μm; mobile phase 20 mmol/L KH2PO4The pH value is 2.7; the sample volume is 10 mu L; the flow speed is 1.00 mL/min; detector UV210, nm; column temperature: measuring the content of organic acid in the fermented supernatant by using a series of parameters at 25 DEG C
TABLE 4 organic acid content of the strains (. mu.g/mL)
Example 7:
determination of related volatile substances in white spirit produced by bacterial strains
50mL of the fermentation supernatant obtained in example 6 was taken, distilled with a distiller, and 5mL of the distillate was put into a 10mL cuvette, and 200. mu.L of three internal standard mixed solutions (t-amyl alcohol, n-amyl acetate, diethyl n-butyric acid) were added thereto, shaken well, and quantitatively analyzed with an Agilent 7890A gas chromatograph.
The type of the chromatographic column: agilent CP-Wax 57 CB; specification: 50 mm. times.0.25 mm. times.0.20. mu.m. The instrument parameters are as follows: the chromatographic conditions are that the column temperature is 40 ℃ for 6 minutes, the temperature is 5 ℃ for 5 minutes, the temperature is increased to 200 ℃ for 5 minutes, the sample injection amount is 1 microliter, and the split ratio is 1: 10, the sample injection temperature is 220 ℃, and the detector is 200 ℃. The mobile phase is nitrogen: 30ml/min hydrogen: 30 ml/min; air: 300 ml/min. The results are shown in Table 5.
TABLE 5 content of volatile substances produced by the strains (. mu.g/mL)
The results show that: in the fermentation process of the strain, a large amount of ethyl acetate is produced to reach 6771 mu g/mL, and accounts for the main components of ester substances in the white spirit, and in addition, some aldehydes, alcohols and other types of ester substances are produced, have higher specific gravity and are main components influencing the flavor of the white spirit.
Example 8:
detecting the content of volatile aroma substances of the strain by headspace solid-phase microextraction-gas chromatography-mass spectrometry:
the fermentation supernatant of example 6 (6 mL) was accurately pipetted into a 15mL headspace bottle together with 20. mu.L of a diallyl disulfide diluent (20. mu.g/mL), the bottle was sealed with a lid, the bottle was equilibrated for 5min in a 60 ℃ water bath, and the aged extraction head was placed in the headspace of the bottle and extracted for 30 min.
GC conditions were as follows: separating with DB-5 MS elastic quartz capillary chromatographic column (30m × 0.25mm × 0.25 μm); the temperature raising procedure is that the initial temperature is 40 ℃, the temperature is raised to 120 ℃ at the speed of 3 ℃/min, and the temperature is kept for 2 min; heating to 250 deg.C at a speed of 10 deg.C/min, and maintaining for 3 min; heating to 300 ℃ at the speed of 15 ℃/min; and kept for 2 min. The carrier gas is high-purity helium (1.0mL/min), and the split-flow sample injection is not carried out.
MS conditions: the mass spectrum adopts an electron bombardment ionization source: electron energy 70 eV; the ion source temperature is 230 ℃ and the scanning range is 35-350 m/z.
TABLE 6 headspace solid phase microextraction-GC-MS detection of volatile aroma substances (μ g/mL) of the strains
Example 9:
the application of pichia kudriavzevii DHX19 in the shell flavor type white spirit fermentation is as follows:
after saccharification and stacking fermentation of the fermented grains are completed, spreading and cooling to 18-21 ℃, adding the pichia kudriavzevii DHX19 fermentation liquor prepared in example 2 according to different proportions, wherein the adding amount is 0.01%, 0.05% and 0.1% (mass ratio) of the fermented grains respectively, uniformly stirring, and then putting into a pool for fermentation, and comparing with 3 cellars without adding strains. After fermenting for 90 days, opening the cellar to detect indexes of the fermented grains such as moisture, acidity, reducing sugar, starch, alcohol content, ethyl acetate and the like, and the results are shown in table 7, wherein the percentages in table 7 are mass fractions.
TABLE 7 application of Pichia kudriavzevii DHX19 in XIN-flavor Chinese liquor
The results show that: and a large amount of esters such as ethyl acetate, ethyl caproate and the like in the fermented grains of the cellar of the Pichia Azrvitae DHX19 are added, so that the product has good ester fragrance.
Claims (10)
1. An isolated pichia kudriavzevii: (Pichia kudriavzevii) The preservation number of the Pichia kudriavzevii is CCTCC NO: m2021172.
2. The use of pichia kudriavzevii, according to claim 1, in the fermentation of an XIN-flavor liquor.
3. The use of pichia kudriavzevii of claim 1 for the preparation of white spirit distiller's yeast.
4. The use of pichia kudriavzevii (r) as claimed in claim 1 for increasing the total ester content in white spirit.
5. The use of pichia kudriavzevii of claim 1 for enhancing flavor components in white spirit.
6. The use of pichia kudriavzevii of claim 1 for the fermentative production of ethyl acetate.
7. The use of pichia kudriavzevii of claim 1 in the fermentative production of ethyl hexanoate.
8. The use of pichia kudriavzevii, as claimed in claim 1, for the fermentative production of acetal.
9. Use of pichia kudriavzevii as claimed in claim 1 for the fermentative production of isobutanol.
10. The use of pichia kudriavzevii of claim 1 in the fermentative production of acetic acid.
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| CN116083263A (en) * | 2022-07-06 | 2023-05-09 | 贵州大学 | Ester-producing type Pichia kudriavzevii strain and application thereof |
| CN117247850A (en) * | 2023-10-18 | 2023-12-19 | 甘肃省农业科学院农产品贮藏加工研究所 | Pichia pastoris GAAS-JG-1 strain resistant to acid and application thereof in preparation of high-acidity fruit fermented wine |
| CN117247850B (en) * | 2023-10-18 | 2024-05-17 | 甘肃省农业科学院农产品贮藏加工研究所 | Pichia pastoris GAAS-JG-1 strain resistant to acid and application thereof in preparation of high-acidity fruit fermented wine |
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