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Pichia anomala for producing ethyl acetate and application of pichia anomala

A kind of abnormal Pichia pastoris, ethyl ester technology, applied in the direction of fermentation, alcoholic beverage preparation, microorganism-based methods, etc., can solve the problems of unprocessed ester production, strain restriction, etc., and achieve the effect of high tolerance

Active Publication Date: 2017-10-17
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the obtained ester-producing yeast mainly adopts pure liquid fermentation method to test its ester production ability, while the current liquor mainly adopts traditional solid-state fermentation, and the ester-producing effect of the strain in pure liquid fermentation has not been verified by multi-strain mixed solid-state fermentation , and the strains constructed by molecular biology methods are subject to certain limitations in practical application

Method used

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  • Pichia anomala for producing ethyl acetate and application of pichia anomala
  • Pichia anomala for producing ethyl acetate and application of pichia anomala
  • Pichia anomala for producing ethyl acetate and application of pichia anomala

Examples

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Effect test

Embodiment 1

[0030] Example 1, Screening and identification of Pichia anomaly P6

[0031] The inventors of the present invention used sorghum mash medium to screen and obtain a high ethyl acetate-producing yeast strain with yeast morphological characteristics, and used 26S rRNA primers to perform PCR amplification on the yeast genome to detect its 26S rRNA sequence. The sequence of the product is sequence 1 in the sequence table, and the sequence is compared with BLAST in GenBank. The highest consistency with sequence 1 is the 26S rRNA of Pichia anomala (Pichia anomala), and the consistency reaches 99%. Therefore, the The yeast was identified as Pichia anomala, and the yeast was named Pichia anomala P6.

[0032] Pichia anomaly P6 was deposited on July 4, 2017 in the General Microbiology Center of China Committee for Culture Collection of Microorganisms (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing), and the preservation number is CGMCCNo .1438...

Embodiment 2

[0033] Example 2, Detection of Pichia abnormal P6 ester production ability

[0034] Preparation of sorghum mash medium: prepare sorghum starch slurry with a material-to-liquid ratio of 0.25 (that is, the mass ratio of sorghum starch to water is 0.25:1), gelatinize it at 65°C for 30 minutes, and add 15 U / g of initial starch Glucoamylase, then liquefy at 95°C for 100min, adjust the pH to about 5.0 with 0.1M hydrochloric acid solution after cooling, then add glucoamylase at 150U / g initial starch, then saccharify at 55°C for 4h, filter with gauze, collect the filtered juice and separate packed, and autoclaved at 115°C for 20 minutes to obtain sorghum mash medium.

[0035] Pichia anomaly P6 was inoculated into the prepared sorghum mash medium at an inoculum size of 1%, and left to ferment in a constant temperature incubator at 28°C for 7 days, and three parallel experiments were set up. Shake and mix the fermented liquid obtained after 7 days of fermentation, absorb 2 mL, centrifu...

Embodiment 3

[0039] Example 3, Detection of tolerance of Pichia abnormal Pichia P6

[0040] 1. Determination of ethanol tolerance

[0041] YPD liquid medium (YPD liquid medium contains yeast extract powder 10g / L, peptone 20g / L, glucose 20g / L, autoclaved at 115°C for 20min). Absolute ethanol was added to the sterilized YPD liquid medium to obtain mediums with ethanol volume percentages of 0%, 4%, 8%, 12%, and 16%, respectively. Pichia anomaly P6 was inoculated into each of the above-mentioned mediums at an inoculation amount of 0.1%, and cultured on a shaking table at 150 r / min at 28°C for 20 hours to obtain fermentation broths treated with different ethanol concentrations. Absorbance at OD600.

[0042] The result is as figure 1 As shown, when the volume percentage of ethanol in the medium is 4% and 8%, Pichia anomaly P6 can grow well; when the volume percentage of ethanol is 12%, Pichia anomaly P6 is not completely inhibited, and can still grow slowly growth; when the volume percentag...

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Abstract

The invention discloses pichia anomala for producing ethyl acetate and an application of the pichia anomala. The pichia anomala for producing the ethyl acetate is the pichia anomala (Pichia anomala)P6, and the preservation number of the pichia anomala at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms is CGMCC No.14381. The pichia anomala (Pichia anomala)P6 has relatively high tolerance on ethanol and low pH, and high ethyl acetate producing capacity; the content of the ethyl acetate in strengthened fermented grains obtained through adding the pichia anomala P6 can be improved by 32.69% in comparison with a contrast, the content of total ester in the fermented grains can be improved by 28.22% and the content of the total ester in a base wine of a distilled liquor can be improved by 53.5%. It is shown that the pichia anomala (Pichia anomala)P6 can be wiely applied to fermented food industry, such as Chinese white spirit with requirements on ester flavor substances.

Description

technical field [0001] The invention relates to a Pichia anomaly producing ethyl acetate and its application in the field of biotechnology. Background technique [0002] The trace flavor components in liquor are the main substances that make up the aroma and style of different liquors, mainly including alcohols, aldehydes, acids, esters, lactones, ketones, etc. Among them, esters are important aroma components in liquor. Components such as ethyl acetate are the main aroma components of Fen-flavor liquor. Esterogenic yeast, as an important strain of liquor aroma production, can convert acetic acid and ethanol into ethyl acetate and other esters under the action of esterase under long-term cultivation and domestication. Esterogenic yeast is also called aroma yeast, which is a general term for yeasts that can produce more esters, not a taxonomic term. Some yeasts of the genus Toruceromyces and Brettanomyces are ester-producing yeasts. During the fermentation process, ester-p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/16C12G3/02C12P7/62C12P7/64C12R1/84
CPCC12G3/02C12N1/16C12P7/62C12P7/6436C12N1/165C12R2001/84
Inventor 韩北忠庞晓娜陈晶瑜吕辰侯林凤
Owner CHINA AGRI UNIV
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