CN102876592A - Pichia anomala - Google Patents
Pichia anomala Download PDFInfo
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- CN102876592A CN102876592A CN2012103585751A CN201210358575A CN102876592A CN 102876592 A CN102876592 A CN 102876592A CN 2012103585751 A CN2012103585751 A CN 2012103585751A CN 201210358575 A CN201210358575 A CN 201210358575A CN 102876592 A CN102876592 A CN 102876592A
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Abstract
The invention relates to pichia anomala, and belongs to the field of microbes. The invention provides a pichia anomala strain for producing ethyl acetate in high yield. According to the pichia anomala JNC-EA002, the conservation number is CGMCC No. 5955. The invention also provides application of the strain to the production of seasoning spirits. The pichia anomala strain is high in ester producing capacity and is suitable for the large-scale production of the seasoning spirits.
Description
Technical field
The present invention relates to unusual pichia spp, belong to microorganism field.
Background technology
High-grade liquor is very popular, but the matter a large amount is few, and its price also goes up year by year.A large amount of high-grade following liquor need to become the higher flavouring wine of flavor substances content to collude accent with one-tenth perfume, thereby reach higher quality level, meet corresponding quality standard.
Traditional flavouring wine production is solid-state with pure grain and technology pattern semi-solid ferment carries out, the normal operation Daqu is as saccharifying ferment, and utilizes various bacteria, yeast, mould and various enzyme (enzymes such as amylase, polygalacturonase, cellulase, the aspartic protease) acting in conjunction in the Daqu and obtain the purpose product.And modern flavouring wine production trends towards the pure strain fermentation gradually, according to the difference that local flavor requires, uses the specific functionality bacterial classification to carry out the production of corresponding local flavor flavouring wine.Wherein, the production of the fragrant flavouring wine of ester mainly utilizes ester-producing yeast to ferment.At biological field, yeast is commonly used to the Host Strains as expression alien gene as model animals.Pichia spp is as a kind of methanol using type yeast, and since the exploitation of the year eighties 20th century, by continuous research, existing a plurality of kinds are developed so far, mainly comprise two kinds of Candida and Pichia, have more than 30 and plant.Wherein especially with fastest developing speed with pichia spp, study the clearlyest, also be to produce the fragrant flavouring wine of ester in the present industry to use one of more bacterial classification.But, consider from the food safety angle that at present domestic also do not have drinks enterprise externally to declare to utilize the transgenosis bacterial strain directly to carry out liquor production because liquor industry is traditional industries.Thereby by natural seed selection, and the mode of use Traditional Man mutagenesis is the approach commonly used that present alcohol industry is obtained the specific functionality bacterial strain.Although some enterprises and research institution carried out research to the generation of EA flavour substances, also obtained the specific functionality bacterial strain of some high yield ethyl acetate, it compares traditional Daqu, although producing to some extent lifting on the ester ability, produce in general the ester amount and still can not satisfy production requirement.
Summary of the invention
The technical problem to be solved in the present invention provides the unusual pichia spp that a plant height produces ethyl acetate.
Unusual pichia spp Pichia anomala JNC-EA002 provided by the invention, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on April 6th, 2012, preserving number is CGMCC No.5955, preservation address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode 100101.
These bacterial strain characteristics are as follows: on the wort agar substratum, 30 ℃, behind the cultivation 24h, bacterium colony is projection slightly, oyster white, surface folding, edge-smoothing.By microscopic examination, this strain cell form be spherical, oval to Long Circle, the teething reproduction.
The present invention also provides the purposes of unusual pichia spp in the production ethyl acetate.
The present invention also provides the purposes of unusual pichia spp in wine brewing.
The present invention also provides the purposes of unusual pichia spp in the production flavouring wine.
The unusual pichia spp Pichia of bacterial strain of the present invention anomala JNC-EA002, from the Koji of limited liability company of Jian Nan Chun group and poor unstrained spirits, to separate to obtain, first by the production performance seed selection, pass through again the plant height that further ultraviolet ray (UV) mutagenesis obtains and produce the bacterial strain of ethyl acetate.
Bacterial strain of the present invention adopts following flow process to carry out seed selection:
Sample collecting → multiplication culture → Pure strain separation → production performance is tested → is prepared the separation screening of cultivations → mutagenesis before bacteria suspension → mutagenesis → dissociant → genetic stability and tests
Beneficial effect of the present invention: unusual pichia spp product ester ability of the present invention is strong, and going down to posterity property is good, is fit to long-term large-scale production and uses.The product ester ability of unusual pichia spp of the present invention can reach 8.75g/L, for the production of ethyl acetate provides new selection; Also can be used for making wine and produce and the production of flavouring wine.
Unusual pichia spp Pichia anomala JNC-EA002 provided by the invention, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on April 6th, 2012, and preserving number is CGMCC No.5955.
Description of drawings
The content of ethyl acetate in Fig. 1, the multi-strain bacteria strain meta-bolites, ordinate zou are the content (g/L of unit) of ethyl acetate.
Fig. 2, set out strain and mutagenic fungi product ester capability analysis, ordinate zou is the content (g/L of unit) of ethyl acetate.
Embodiment
Unusual pichia spp Pichia anomala JNC-EA002 of the present invention has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on April 6th, 2012, preserving number is CGMCC No.5955, preservation address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode 100101.
These bacterial strain characteristics are as follows: on the wort agar substratum, 30 ℃, behind the cultivation 24h, bacterium colony is projection slightly, oyster white, surface folding, edge-smoothing.By microscopic examination, this strain cell form be spherical, oval to Long Circle, the teething reproduction.
The present invention also provides the purposes of unusual pichia spp in the production ethyl acetate.
The present invention also provides the purposes of unusual pichia spp in wine brewing.
The present invention also provides the purposes of unusual pichia spp in the production flavouring wine.
Unusual pichia spp product ethyl acetate ability of the present invention is strong, can be used for the production ethyl acetate; This bacterial strain separates from fragrant yeast and poor unstrained spirits and obtains, and is applicable to wine brewing and produces; Ethyl acetate is the main fragrance matter in the liquor, so available unusual pichia spp production flavouring wine of the present invention.
Used diastatic fermentation equipment in the preparation of saccharified liquid among the following embodiment (saccharifying tank, Jet liquefier, vacuum are dodged but system etc. of chilling) manufactures and designs by Ming River machine works of Yibin Ming River group.
Gas-chromatography among the following embodiment is Agilent (Agilent) 6890GC of company, chromatographic column is HP-INNOWAX, 280 ℃ of detector temperatures, 260 ℃ of injector temperatures, splitting ratio is 20 ︰ 1, and the temperature programming condition is 40 ℃ and keeps 8min that the speed with 5 ℃/min rises to 100 ℃ again, speed with 15 ℃/min rises to 220 ℃ again, keeps 8min.
Employed substratum all belongs to the synthetic medium of purchase among the following embodiment, and malt extract medium and potato culture are Beijing biological company limited of extensive and profound in meaning star product, the article No. 02-183 of malt extract medium, the article No. 02-023 of potato culture; Yeast extract medium is Beijing bispin substratum Manufacturing Co., Ltd product, and article No. is 02-22.
Following bacterial classification is the higher functional bacterial classification of product ethyl acetate that separation and purification obtains in the Koji of Jian Nan Chun group company and the poor unstrained spirits.Detailed process is as follows:
1. the collection of sample:
Koji sample collecting storage period is at 2 to 3 months fragrant yeast, chooses the bent brick of half block (different storage vault) more than 10 through pulverizing, mixing, and obtains the comprehensive sample of Koji.Smashing fineness is can (be sieve 20 holes/cm by 20 mesh sieves
3) fine powder content account for 80% of Koji total amount.
The grain unstrained spirits is selected at the middle and upper levels the female grain of high-quality (going out the cellar for storing things grain), gets 5 points, and namely the poor unstrained spirits in four jiaos and centre is got the 200g mixing at every, as 1 poor unstrained spirits sub-sample.That gets at random 10 Kou Jiao ponds (different teams and groups) goes out the cellar for storing things grain, obtains 10 each poor unstrained spirits sub-sample.In each poor unstrained spirits sub-sample, respectively get the 200g mixing, i.e. the comprehensive sample of poor unstrained spirits that seed selection is required.
2. strain culturing:
The comprehensive sample of Koji is packed into by the standard of 20g/ bottle in the 250mL triangular flask that fills the 100mL sterilized water; The comprehensive sample of grain unstrained spirits is packed into by the standard of 100g/ bottle in the 250mL triangular flask that fills the 100mL sterilized water.Soaked 2 hours, per concussion half an hour once.Get the supernatant liquor 10mL of each soak solution, join 100mL sterilize (115 ℃, in malt juice liquid medium 20min), mixing, 30 ℃ of constant temperature, aerobic cultivation 24h.Then respectively get the 0.5mL nutrient solution and be coated on potato (PDA) culture medium flat plate, 30 ℃, cultivate 24h.The yeast of choosing different colonial morphologies carries out purifying at the wort agar substratum and cultivates.
The test of embodiment 2 production performances
The preparation of bacteria suspension: dip in a little distilled water with aseptic cotton carrier, allow cotton swab infiltrate.On the bacterium colony of purifying, rotate gently cotton swab, allow cotton head be stained with bacterial classification.The cotton swab that is stained with bacterial classification is inserted rapidly in the opacity tube of splendid attire 20mL sterilized water.The rotation cotton swab is dispersed in the sterilized water by bacterial classification, and from inside to outside, from slow to fast, cotton swab is extracted in rotation out.With bacteria suspension be placed on shake up on the vortex vibrator after, insert in the cell concn instrument.Continue the method by this method or interpolation sterilized water, the bacterial concentration of adjusting respectively various bacteria suspensions is 10
6Individual/mL.
Fermentation: draw 2mL bacteria suspension and 3mL dehydrated alcohol, join (115 ℃, sterilization 20min) in the 100mL malt extract medium that has prepared, 30 ℃ of constant temperature, aerobic, leave standstill and cultivate 72h.After cultivating end, all fermented liquids are poured in the 2000mL round bottom distilling flask, and adding 60mL massfraction is 96% alcohol (analytical pure) and 340mL pure water.Round-bottomed flask is put into the 2000mL electric mantle, by experiment chamber distillation (distillation voltage 220V), carry out the equal-volume distillation, ethyl acetate in the fermented liquid is all distilled, get distillate 100mL, do gas chromatographic analysis, understand the product ester ability of every strain bacterial strain, pick out the high dominant strain of production performance.Analytical results sees Table 1 and Fig. 1.
Table 1 separation and purification gained produces the main metabolites of the stronger bacterial strain of ester ability
Annotate: symbol "-" expression does not detect in the table.
Can find out that from table 1 data the content with ethyl acetate in the meta-bolites of each bacterial strain is the highest, also contain a certain amount of aldehyde material, fusel wet goods by product simultaneously.Wherein, the content of acetal and potato spirit is higher than other bacterial strain in the meta-bolites of strains A D11 and AD22.As shown in Figure 1, the content of ethyl acetate is minimum in the meta-bolites of strains A D22 and X2; The content of ethyl acetate is slightly high in the meta-bolites of strains A D11 and S1, and the content of ethyl acetate is the highest in the meta-bolites of bacterial strain JNC1.This shows that bacterial strain JNC1 has the advantage that product ester ability is strong, fermentation byproduct is few, be chosen as the starting strain of selection by mutation.
The bacterial strain JNC1 that the contriver obtains embodiment 2 uses the breeding mode of ultraviolet ray (UV) mutagenesis to carry out seed selection as starting strain.
1) cultivates before the mutagenesis
Before the mutagenic treatment, prepare the bacteria suspension (preparation method and embodiment 2 are together) of the JNC1 bacterial strain of choosing through embodiment 2, draw the 10mL bacteria suspension and add in the 100mL yeast extract paste liquid nutrient medium, it is for subsequent use that 30min is cultivated in the aerobic concussion of 130r/min.Continue to cultivate, get nutrient solution one time every 1h, in 4 ℃ of refrigerators, get 2mL at every turn, take a sample altogether 10 times.The complete rear spectrophotometric determination light absorption value of using of taking a sample, the adjustment wavelength is 560nm, take the yeast extract paste liquid nutrient medium as blank, draws out the production curve of bacterial classification according to data, determines that its logarithmic phase is about 8 ~ 12 hours.
2) mutation rate and lethality rate are measured
The bacteria suspension that will be in logarithmic phase is diluted to 10
6Individual/mL.Open 20W ultraviolet lamp preheating 30min, make its power reach stable.The culture dish that to get 7 diameters be 9cm is drawn respectively 4mL dilution bacteria suspension in culture dish, and liquid layer thickness is 2mm, and 7 culture dish are placed apart from ultraviolet lamp 20cm place, takes 1 culture dish away every 30S.Getting 1 culture dish without uv irradiating compares.With the culture dish after 7 mutagenesis and 1 not the nutrient solution in the culture dish of mutagenesis carry out 10 times of dilutions and be coated with flat board, dull and stereotyped potato (PDA) substratum that uses, Duplicate Samples is 3,28 ℃ of constant temperature culture 48h, and calculate its mutation rate and lethality rate.
3) ultraviolet mutagenesis
The selection lethality rate is 85% the mutagenic exposure time of UV irradiation time for testing, and the starting strain JNC1 that is in logarithmic phase coats on potato (PDA) culture medium flat plate after UV irradiation mutagenesis, and lucifuge is cultivated 48h.Each 7 bacterium colonies of inoculating needle picking and numbering used done respectively production performance test (with embodiment 2) with these 7 kinds of mutant strains and the strain of setting out, and detects the ability of its product ethyl acetate.The contriver picks out 9 plant heights and produces bacterial strain through the too much screening of the some samples of wheel, and with its difference called after: JNC-EA001～009, each bacterial strain produces ester ability such as Fig. 2.
By upper figure as can be known, the ability that this 9 strain bacterial strain produces ethyl acetate all is higher than starting strain, and wherein the product ester ability with JNC-EA002 is the strongest again, reaches 8.75g/L.Because this bacterial strain belongs to the production application bacterial strain, carries out immediately the inheritance stability property testing.Through identifying that bacterial strain JNC-EA002 is unusual pichia spp Pichia anomala.
Subsequently, to this unusual pichia spp Pichia anomala JNC-EA002 inheritance stability test, the results are shown in Table 2 again.
The content of main metabolites after the table 2 bacterial strain JNC-EA002 continuous passage
As can be seen from Table 2, through continuous passage nine times, the metabolic end product ethyl acetate content not have the obviously trend of reduction, and in respectively going down to posterity, the variable quantity of ethyl acetate content (comparing with 1st generation) when all being no more than 1st generation ethyl acetate content 4%.Simultaneously, other metabolic by-prods content is extremely low and stable, satisfies production requirement fully.This shows that unusual pichia spp Pichia anomala JNC-EA002 heredity of the present invention is good, production performance is stable.
The application of embodiment 4 bacterial strains of the present invention in flavouring wine is produced
1. the preparation of saccharified liquid
Fructus Hordei Germinatus is pulverized, and the pulverizing standard is can (be sieve 20 holes/cm by 20 mesh sieves
3) fine powder content account for 75% of Fructus Hordei Germinatus total amount.Add water by grain quality 1:5 and mix, 60 ℃ of simultaneously liquefaction, saccharification of constant temperature 6 hours.Drip behind rare allusion quotation liquid constant indigo plant take saccharified liquid and filter as the terminal point of liquefaction, saccharification, for subsequent use.The saccharified liquid that above method is made adds water, and to regulate pol be that (Brix, symbol be a ° Bx to 12 degree, namely are converted in 20 ° of C situations, and per 100 restrain the sucrose grams of dissolving in the aqueous solution.); 4 times of concentrated hydrochloric acid (food grade) dilutions, regulating pH is 3.4, and sterilising temp is 115 ℃, and sterilization time is 20min, cools off for subsequent use.
2. inoculation fermentation
The present invention adopts the technology pattern of the aerobic static fermentation of intermittent type, and with the saccharified liquid injection fermentor tank of preparation in early stage, inoculating cell concentration is the bacteria suspension of 106/mL, and the bacterial suspension inoculation volume is 3% of saccharified liquid volume.Add the dehydrated alcohol of saccharified liquid volume 2.5%.Mixing, standing for fermentation.Fermentation period is 72h, and leavening temperature is 28 ℃.
3. distillation and concentration
By tray column rectifying tower (buying the Ming River machinery from Yibin Ming River group) ethyl acetate and alcohol are separated from fermented liquid, with the ethyl acetate simmer down to 3872mg/100mL in the flavouring wine, alcoholic strength is 60%voL.
4. quality evalution
The flavouring wine that distillation and concentration obtains is carried out the analysis of aesthetic quality's evaluation and physical and chemical parameter.
A, aesthetic quality identify
In 24 ℃ of environment, the flavouring wine that distillation and concentration is obtained with the alcohol of 60%voL is diluted to 200mg/100m L.The personnel that taste and appraise carry out aesthetic quality's evaluation to color, 4 of styles respectively by 7 sole duties, the results are shown in Table 3.
Table 3 utilizes the sensory test of flavouring wine that the unusual pichia spp of the present invention produces
Sensory test result from table 3, flavouring wine water white transparency, the delicate fragrance of utilizing the unusual pichia spp of the present invention to produce is pure, the ethyl acetate smell outstanding, and the wine body is pure and mild, continuous gentle, and the clean flavor characteristic such as refreshing of smell coordination, tail flavor is fit to seasoning and uses very much.
B, physicochemical data analyzing and testing
Utilize gas-chromatography that aroma component in the product is detected, test sample is through the parallel sample after sensory test.
According to concentrated solution concentration, ethyl acetate concentration is as calculated in the fermented liquid: 702.5mg/100mL.
Claims (4)
1. unusual pichia spp, preserving number is CGMCC No.5955.
2. the purposes of the described unusual pichia spp of claim 1 in the production ethyl acetate.
3. the purposes of the described unusual pichia spp of claim 1 in wine brewing.
4. the purposes of the described unusual pichia spp of claim 1 in the production flavouring wine.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103184167A (en) * | 2013-03-27 | 2013-07-03 | 河北衡水老白干酒业股份有限公司 | Wickerhamomyces anomalus strain and application thereof |
| CN103740603A (en) * | 2014-01-26 | 2014-04-23 | 北京绿环国际科技有限公司 | Abnormal pichia anomala and application thereof |
| CN105349444A (en) * | 2015-11-27 | 2016-02-24 | 四川理工学院 | Saccharomycete for high-yield production of ethyl acetate under low temperature and application thereof |
| CN107254415A (en) * | 2017-08-08 | 2017-10-17 | 中国农业大学 | One plant production ethyl acetate Pichia anomala and its application |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107699503B (en) * | 2017-11-22 | 2020-06-09 | 内蒙古农业大学 | Abnormal yeast Weikehan yeast BT-1-1 and application thereof |
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2012
- 2012-09-24 CN CN201210358575.1A patent/CN102876592B/en active Active
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103184167A (en) * | 2013-03-27 | 2013-07-03 | 河北衡水老白干酒业股份有限公司 | Wickerhamomyces anomalus strain and application thereof |
| CN103184167B (en) * | 2013-03-27 | 2015-05-06 | 河北衡水老白干酒业股份有限公司 | Wickerhamomyces anomalus strain and application thereof |
| CN103740603A (en) * | 2014-01-26 | 2014-04-23 | 北京绿环国际科技有限公司 | Abnormal pichia anomala and application thereof |
| CN103740603B (en) * | 2014-01-26 | 2015-08-05 | 北京绿环国际科技有限公司 | A kind of Abnormal pichia anomala and application thereof |
| CN105349444A (en) * | 2015-11-27 | 2016-02-24 | 四川理工学院 | Saccharomycete for high-yield production of ethyl acetate under low temperature and application thereof |
| CN105349444B (en) * | 2015-11-27 | 2018-07-13 | 四川理工学院 | The saccharomycete of high yield ethyl acetate and its application under one plant of cryogenic conditions |
| CN107254415A (en) * | 2017-08-08 | 2017-10-17 | 中国农业大学 | One plant production ethyl acetate Pichia anomala and its application |
| CN107254415B (en) * | 2017-08-08 | 2020-12-22 | 中国农业大学 | Pichia anomala for producing ethyl acetate and application thereof |
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