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Lactic acid-tolerant ester-producing pichia pastoris

A Pichia pastoris and lactic acid technology, which is applied in the fields of bioengineering and brewing, can solve the problems of being unsuitable for the growth and reproduction of ester-producing yeast, affecting the metabolism and production of esters, and not describing the tolerance status, and achieving rich ester-producing species. , strong lactic acid tolerance, good adaptability

Active Publication Date: 2017-10-24
KWEICHOW MOUTAI COMPANY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The brewing environment is an acidic environment, and the fermented grains contain a large amount of lactic acid, which is generally not suitable for the growth and reproduction of ester-producing yeast, thus affecting the metabolism of esters.
For example, in the patent "A Highly Tolerant Ester-Producing Yeast Strain and Its Application" (Patent No.: 201310191489.0), a strain of yeast with strong environmental tolerance and strong ability to produce ethyl acetate was screened, but the patent did not elaborate The tolerance of this yeast to lactic acid, the main acid in the fermented grains. At the same time, the main metabolite of this yeast is only ethyl acetate, and there are few types of esters.

Method used

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  • Lactic acid-tolerant ester-producing pichia pastoris
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  • Lactic acid-tolerant ester-producing pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Screening of Lactic Acid Resistance Pichia Estoris

[0021] Weigh 10g of Daqu or fermented grains sample into 90mL sterile water with glass beads, oscillate fully and evenly, draw 0.1mL supernatant into the enrichment medium liquid, and culture it statically at 30°C for 2-4 days, observe whether the culture medium is If the culture medium is obviously turbid and smells a pleasant fruity aroma, absorb 0.1mL of the enrichment solution and culture it again in a new screening medium. After 3-4 cultures, the culture medium is gradually Dilute it on a plate, and after culturing for 3-4 days, pick a single colony for fermentation. After the fermentation, the flavor substances of the fermentation broth were measured, and the strain with the highest ester-producing ability was selected as the target strain.

[0022] The composition of the screening medium is: lactic acid 20g / L, glucose 20g / L, peptone 20g / L, yeast extract 10g / L, sodium chloride 1g / L, pH 3.2 (natural pH...

Embodiment 2

[0024] Example 2 Identification of Lactic Acid-tolerant Pichia Estoris

[0025] Observation of the colony morphology of the obtained highly efficient lactic acid-degrading strain ( figure 1 ), it was found that the colony was white, the surface was rough, easy to pick, and the colony was larger. Utilize microscope to observe the cell shape of this bacterial strain, under 15 * 40 times, this bacterial strain cell is ellipse, and has budding, can preliminarily determine as yeast. Use yeast-specific classification identification primers to amplify the 26srDNA fragment of the strain, connect the amplified product to the pMD18-T vector, transform it into Escherichia coli competent cells, extract the plasmid for sequencing and comparison, and determine the species of the screened yeast. Its classification is named: Pichiakudriavzevii (Pichiakudriavzevii). It has been preserved in the General Microorganism Center of China Committee for the Collection of Microorganisms on April 24, ...

Embodiment 3

[0026] Strain growth ability under different tolerance conditions of embodiment 3

[0027] The brewing environment has a very important influence on the growth and metabolism of microorganisms. In the liquor brewing process, due to the constant changes in fermentation temperature, ethanol concentration, and substrate acidity, the environment in which the microorganisms are located is also constantly changing. growth situation.

[0028] Temperature tolerance test: the activated Pichia pastoris 2% (the volume of Pichia seed liquid / the volume of YPD liquid medium) seed liquid is inoculated in the Erlenmeyer flask that 100mL YPD liquid medium is housed, place respectively After static culture at 25°C, 30°C, 37°C, 42°C, and 45°C for 4 days, measure the OD value at a wavelength of 600nm. The bacterial solution with a large concentration can be diluted appropriately. The OD value measured after dilution is multiplied by The dilution factor is the actual OD value of the culture medi...

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Abstract

The invention discloses a lactic acid-tolerant ester-producing pichia pastoris, and belongs to the technical field of a bioengineering technology and a brewing biotechnology. The strain is preserved at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on April 24, 2017, the classification is named pichia kudriavzevii (Pichia kudriavzevii) and the preservation number is CGMCC No.14068. The pichia pastoris is high in environmental tolerance, is capable of tolerating 0-15% lactic acid, is capable of metabolizing to generate multiple ethyl ester volatile flavor substances, such as ethyl acetate, ethyl propionate, ethyl caprylate, phenethyl acetate and ethyl phenylacetate in the environment of 0-12% lactic acid, and is a brewing functional strain with excellent performance.

Description

technical field [0001] The invention belongs to the fields of bioengineering technology and brewing. It involves a lactic acid-tolerant ester-producing yeast. Background technique [0002] The main components of Chinese liquor are ethanol and water (accounting for 98%-99% of the total), and the trace components accounting for 1%-2% of the total (referring to many kinds of esters, acids, alcohols, aldehydes, etc. Trace organic compounds) as the aroma and taste substances of liquor determine the style and quality of liquor. Among them, the esters in the trace components have aromatic flavor substances, which are the main factors for the strong aroma of the wine body. In the national standards of various flavor-type liquors, the total ester content is an important index for classifying liquor grades. For example, the total ester content of Maotai-flavor premium liquor should be higher than 2.2g / L, and the total ester content of Luzhou-flavor premium liquor should be higher th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/16C12G3/02C12R1/84
CPCC12G3/02C12N1/165C12R2001/84
Inventor 陈良强林琳杨帆王和玉汪地强王莉陈宗校
Owner KWEICHOW MOUTAI COMPANY
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