CN102876592B - Pichia anomala - Google Patents
Pichia anomala Download PDFInfo
- Publication number
- CN102876592B CN102876592B CN201210358575.1A CN201210358575A CN102876592B CN 102876592 B CN102876592 B CN 102876592B CN 201210358575 A CN201210358575 A CN 201210358575A CN 102876592 B CN102876592 B CN 102876592B
- Authority
- CN
- China
- Prior art keywords
- strain
- ethyl acetate
- production
- pichia spp
- bacterial strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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 abnormal pichia spp, belong to microorganism field.
Background technology
High-grade white wine is very popular, but matter a large amount is few, and its price also goes up year by year.A large amount of high-grade following white wine need to become the higher flavouring wine of taste substances content to hook tune with one-tenth perfume, thereby reaches higher quality level, meets corresponding quality standard.
It is solid-state with pure grain that traditional flavouring wine is produced and technology pattern semi-solid ferment carries out, the general Daqu that uses is as saccharifying ferment, and utilizes various bacteria, yeast, mould and various enzyme in Daqu enzymes such as () amylase, polygalacturonase, cellulase, aspartic proteases acting in conjunction and obtain object product.And modern flavouring wine production trends towards pure strain fermentation gradually, the difference requiring according to local flavor, is used 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, as model animals, is commonly used to the Host Strains as expression alien gene.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, be also to produce the fragrant flavouring wine of ester in current industry to use one of more bacterial classification.But because liquor industry is traditional industries, from food safety angle, consider, current domestic Hai Weiyou drinks enterprise externally declares to utilize transgenosis bacterial strain directly to carry out white wine production.Thereby by natural seed selection, and to use the mode of Traditional Man mutagenesis be the conventional approach that current alcohol industry is obtained 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 lifting to some extent in ester ability, produce in general ester amount and still can not meet production requirement.
Summary of the invention
The technical problem to be solved in the present invention is to provide the abnormal pichia spp that a plant height produces ethyl acetate.
Abnormal pichia spp Pichia anomala JNC-EA002 provided by the invention, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on April 6th, 2012, preserving number is CGMCC No.5955, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode 100101.
This bacterial strain feature is as follows: on wort agar substratum, and 30 ℃, to cultivate after 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, teething reproduction.
The present invention also provides the purposes of abnormal pichia spp in production ethyl acetate.
The present invention also provides the purposes of abnormal pichia spp in wine brewing.
The present invention also provides the purposes of abnormal pichia spp in production flavouring wine.
The abnormal pichia spp Pichia of bacterial strain of the present invention anomala JNC-EA002, the Koji of limited liability company of Shi Cong Jian Nan Chun group and separated obtaining in poor unstrained spirits, first by production performance seed selection, then pass through 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:
Separation screening → genetic stability test of the front cultivation → mutagenesis → dissociant of bacteria suspension → mutagenesis is tested → prepared to sample collecting → multiplication culture → Pure strain separation → production performance
Beneficial effect of the present invention: abnormal pichia spp product ester ability of the present invention is strong, and going down to posterity property is good, is applicable to long-term large-scale production and uses.The product ester ability of abnormal 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 the production of wine brewing production and flavouring wine.
Abnormal pichia spp Pichia anomala JNC-EA002 provided by the invention, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on April 6th, 2012, and preserving number is CGMCC No.5955.
Accompanying drawing explanation
The content of ethyl acetate in Fig. 1, multi-strain bacteria strain meta-bolites, the content that ordinate zou is ethyl acetate (g/L of unit).
Fig. 2, set out strain and mutagenic fungi product ester capability analysis, the content that ordinate zou is ethyl acetate (g/L of unit).
Embodiment
Abnormal pichia spp Pichia anomala JNC-EA002 of the present invention has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on April 6th, 2012, preserving number is CGMCC No.5955, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode 100101.
This bacterial strain feature is as follows: on wort agar substratum, and 30 ℃, to cultivate after 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, teething reproduction.
The present invention also provides the purposes of abnormal pichia spp in production ethyl acetate.
The present invention also provides the purposes of abnormal pichia spp in wine brewing.
The present invention also provides the purposes of abnormal pichia spp in production flavouring wine.
Abnormal pichia spp product ethyl acetate ability of the present invention is strong, can be used for production ethyl acetate; This bacterial strain, from fragrant yeast and separated obtaining poor unstrained spirits, is applicable to wine brewing and produces; Ethyl acetate is the main fragrance matter in white wine, therefore available abnormal pichia spp production flavouring wine of the present invention.
In following embodiment, in the preparation of saccharified liquid, diastatic fermentation equipment used (saccharifying tank, Jet liquefier, vacuum are dodged anxious cooling system etc.) manufactures and designs by Yibin Ming River machine works of Ming River group.
Gas-chromatography in 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 temperature programming condition is 40 ℃ and keeps 8min, then rises to 100 ℃ with the speed of 5 ℃/min, speed with 15 ℃/min rises to 220 ℃ again, keeps 8min.
The substratum using in following embodiment all belongs to the synthetic medium of purchase, 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.
The natural seed selection of embodiment 1
Following bacterial classification is the higher functional bacterial classification of product ethyl acetate that in the Koji of Jian Nan Chun group company and poor unstrained spirits, separation and purification obtains.Detailed process is as follows:
1. the collection of sample:
Koji sample collecting storage period, at the fragrant yeast of 2 to 3 months, is chosen 10 bent bricks of above half block (different storage vault) 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.
Grain unstrained spirits is selected at the middle and upper levels the female grain of high-quality (going out cellar for storing things grain), gets 5 points, and the poor unstrained spirits in four jiaos and centre, gets 200g at every and mix, as 1 poor unstrained spirits sub-sample.That gets at random 10 Kou Jiao ponds (different teams and groups) goes out cellar for storing things grain, obtains 10 each poor unstrained spirits sub-samples.In each poor unstrained spirits sub-sample, respectively get 200g and mix, be i.e. the comprehensive sample of poor unstrained spirits that seed selection is required.
2. strain culturing:
The comprehensive sample of Koji packs in the 250mL triangular flask that fills 100mL sterilized water by the standard of 20g/ bottle; The comprehensive sample of grain unstrained spirits packs in the 250mL triangular flask that fills 100mL sterilized water by the standard of 100g/ bottle.Soak 2 hours, per concussion half an hour once.Get the supernatant liquor 10mL of each soak solution, join 100mL sterilizing (115 ℃, in malt juice liquid medium 20min), mix 30 ℃ of constant temperature, aerobic cultivation 24h.Then respectively get 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 cultivation on wort agar substratum.
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.Rotation cotton swab is dispersed in sterilized water by bacterial classification, and from inside to outside, from slow to fast, cotton swab is extracted in rotation out.After bacteria suspension is placed on and is shaken up on vortex vibrator, insert in cell concn instrument.Continue by the method for 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 ℃, sterilizing 20min) in the 100mL malt extract medium having prepared, 30 ℃ of constant temperature, aerobic, standing cultivation 72h.After cultivation finishes, all fermented liquids are poured in 2000mL round bottom distilling flask, and to add 60mL massfraction be 96% alcohol (analytical pure) and 340mL pure water.Round-bottomed flask is put into 2000mL electric mantle, chamber distillation by experiment (distillation voltage 220V), carry out equal-volume distillation, ethyl acetate in fermented liquid is all distilled, obtain distillate 100mL, do gas chromatographic analysis, understand the product ester ability of every strain bacterial strain, pick out the dominant strain that production performance is high.Analytical results is in Table 1 and Fig. 1.
Table 1 separation and purification gained produces the main metabolites of the stronger bacterial strain of ester ability
Note: in table, symbol "-" represents not detect.
From table 1 data, can find out, the content with ethyl acetate in the meta-bolites of each bacterial strain is the highest, also contains a certain amount of aldehyde material, fusel wet goods by product simultaneously.Wherein, in the meta-bolites of strains A D11 and AD22 the content of acetal and potato spirit higher than other bacterial strain.As shown in Figure 1, in the meta-bolites of strains A D22 and X2, the content of ethyl acetate is minimum; In the meta-bolites of strains A D11 and S1, the content of ethyl acetate is slightly high, and in the meta-bolites of bacterial strain JNC1, the content of ethyl acetate is the highest.As can be seen here, bacterial strain JNC1 has the advantage that product ester ability is strong, fermentation byproduct is few, is chosen as the starting strain of selection by mutation.
The bacterial strain JNC1 that contriver obtains embodiment 2, as starting strain, is used the breeding mode of ultraviolet ray (UV) mutagenesis to carry out seed selection.
1) before mutagenesis, cultivate
Before mutagenic treatment, the bacteria suspension of the JNC1 bacterial strain that preparation is chosen through embodiment 2 (preparation method and embodiment 2 are same), draws 10mL bacteria suspension and adds in 100mL yeast extract paste liquid nutrient medium, and it is standby that 30min is cultivated in the aerobic concussion of 130r/min.Continue to cultivate, every 1h, get nutrient solution one time, in 4 ℃ of refrigerators, get 2mL at every turn, sample altogether 10 times.After sampling, use spectrophotometric determination light absorption value, adjustment wavelength is 560nm, take 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
Bacteria suspension in logarithmic phase is diluted to 10
6individual/mL.Open 20W ultraviolet lamp preheating 30min, make its power reach stable.Get the culture dish that 7 diameters are 9cm and draw respectively 4mL dilution bacteria suspension in culture dish, liquid layer thickness is 2mm, and 7 culture dish are placed in apart from ultraviolet lamp 20cm place, every 30S, takes 1 culture dish away.Getting 1 culture dish without uv irradiating compares.By the culture dish after 7 mutagenesis and 1 not the nutrient solution in the culture dish of mutagenesis carry out 10 times of dilutions be coated with dull and stereotyped, 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
Selecting the UV irradiation time that lethality rate is 85% is the mutagenic exposure time of test, and the starting strain JNC1 in logarithmic phase coats on potato (PDA) culture medium flat plate after UV irradiates mutagenesis, and lucifuge is cultivated 48h.Each 7 bacterium colonies of inoculating needle picking numbering used, does respectively production performance test (with embodiment 2) by these 7 kinds of mutant strains and the strain of setting out, and detects the ability of its product ethyl acetate.Contriver, through the too much screening of the some samples of wheel, picks out 9 strain superior strains, is distinguished called after: JNC-EA001～009, each bacterial strain produces ester ability as Fig. 2.
From upper figure, this 9 strain bacterial strain produces the ability of ethyl acetate all higher than starting strain, wherein the strongest with the product ester ability of JNC-EA002 again, reaches 8.75g/L.Because this bacterial strain belongs to production application bacterial strain, carry out immediately genetic stability test.Through identifying that bacterial strain JNC-EA002 is abnormal pichia spp Pichia anomala.
Subsequently, to this abnormal pichia spp Pichia anomala JNC-EA002 inheritance stability test, the results are shown in Table 2 again.
The content of main metabolites after table 2 bacterial strain JNC-EA002 continuous passage
As can be seen from Table 2, through continuous passage nine times, metabolic end product ethyl acetate content not have the obviously trend of reduction, and in respectively going down to posterity, when the variable quantity of ethyl acetate content (comparing with 1st generation) is all no more than 1st generation 4% of ethyl acetate content.Meanwhile, other metabolic by-prods content is extremely low and stable, meets production requirement completely.As can be seen here, abnormal pichia spp Pichia anomala JNC-EA002 heredity of the present invention is good, and production performance is stable.
The application of embodiment 4 bacterial strain of the present invention in flavouring wine is produced
1. the preparation of saccharified liquid
Fructus Hordei Germinatus is pulverized, and 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.By grain quality 1:5, add water and mix, 60 ℃ of liquefaction, saccharification simultaneously of constant temperature 6 hours.It is the terminal of liquefaction, saccharification that the saccharified liquid of take drips constant indigo plant after rare allusion quotation liquid, filters, standby.It is that (Brix, symbol is a ° Bx to 12 degree, is converted in 20 ° of C situations the sucrose grams of dissolving in every 100 grams of aqueous solution that the saccharified liquid that above method is made adds water adjusting pol.); Concentrated hydrochloric acid (food grade) dilutes 4 times, and regulating pH is 3.4, and sterilising temp is 115 ℃, and sterilization time is 20min, cooling standby.
2. inoculation fermentation
The present invention adopts the technology pattern of the aerobic static fermentation of intermittent type, and the saccharified liquid of preparation in early stage is injected to fermentor tank, and inoculating cell concentration is the bacteria suspension of 106/mL, and bacterial suspension inoculation volume is 3% of saccharified liquid volume.Add the dehydrated alcohol of saccharified liquid volume 2.5%.Mix 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, by the ethyl acetate simmer down to 3872mg/100mL in flavouring wine, alcoholic strength is 60%voL.
4. quality evalution
The flavouring wine that distillation and concentration is obtained is carried out the analysis of aesthetic quality's evaluation and physical and chemical parameter.
A, aesthetic quality identify
In 24 ℃ of environment, by the flavouring wine that the alcohol of 60%voL obtains distillation and concentration, be diluted to 200mg/100m L.By 7 sole duties, the personnel that taste and appraise carry out aesthetic quality's evaluation to color, 4 of styles respectively, the results are shown in Table 3.
Table 3 utilizes the sensory test of flavouring wine that the abnormal pichia spp of the present invention produces
Sensory test result from table 3, flavouring wine water white transparency, the delicate fragrance of utilizing the abnormal pichia spp of the present invention to produce are pure, ethyl acetate smell outstanding, wine body alcohol and, continuous soft, smell coordination, tail taste be the flavor characteristic such as refreshing only, is applicable to very much seasoning and uses.
B, physicochemical data analyzing and testing
Utilize gas-chromatography to detect aroma component in product, detecting sample is the parallel sample after sensory test.
According to concentrated solution concentration, in fermented liquid, ethyl acetate concentration is as calculated: 702.5mg/100mL.
Claims (4)
1. abnormal pichia spp (Pichia anomala), preserving number is CGMCC No.5955.
2. the purposes of abnormal pichia spp in production ethyl acetate described in claim 1.
3. the purposes of abnormal pichia spp in wine brewing described in claim 1.
4. the purposes of abnormal pichia spp in production flavouring wine described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210358575.1A CN102876592B (en) | 2012-09-24 | 2012-09-24 | Pichia anomala |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210358575.1A CN102876592B (en) | 2012-09-24 | 2012-09-24 | Pichia anomala |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102876592A CN102876592A (en) | 2013-01-16 |
| CN102876592B true CN102876592B (en) | 2014-01-22 |
Family
ID=47478114
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210358575.1A Active CN102876592B (en) | 2012-09-24 | 2012-09-24 | Pichia anomala |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102876592B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107699503A (en) * | 2017-11-22 | 2018-02-16 | 内蒙古农业大学 | A kind of abnormal Brunswick Durham yeast BT 11 and application thereof |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| 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 |
| CN107254415B (en) * | 2017-08-08 | 2020-12-22 | 中国农业大学 | Pichia anomala for producing ethyl acetate and application thereof |
-
2012
- 2012-09-24 CN CN201210358575.1A patent/CN102876592B/en active Active
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107699503A (en) * | 2017-11-22 | 2018-02-16 | 内蒙古农业大学 | A kind of abnormal Brunswick Durham yeast BT 11 and application thereof |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102876592A (en) | 2013-01-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102876592B (en) | Pichia anomala | |
| CN106916758A (en) | A kind of Hansenula yeast and its application in wine production | |
| CN106635832B (en) | One plant of abnormal Brunswick Durham saccharomycete and its application | |
| CN109266562A (en) | One plant height produces ethyl acetate exception Brunswick Durham yeast and its cultural method and application | |
| CN107177519A (en) | Schizosaccharomyces pombe bacterium, its composition and application | |
| CN108018218A (en) | One plant height produces ethyl acetate yeast strain and its cultural method and application | |
| CN102796716B (en) | Method for preparing tannase | |
| CN107287127A (en) | The production ester Pichia pastoris of one plant of resistance to lactic acid | |
| CN103013737B (en) | Production method of EA (Ester Aroma) flavoring liquor | |
| CN109370929A (en) | A kind of application of S. cervisiae in wine brewing | |
| CN106753994B (en) | Method for improving alcohol content of alcohol fermentation liquor and reducing isoamyl alcohol content by using high-ester-yield indigenous aroma-producing yeast enhanced yeast | |
| CN109182156B (en) | Saccharomyces cerevisiae suitable for brewing red-core pitaya wine and application thereof | |
| CN108251318A (en) | A kind of Dell is furnished with spore torula and its application in Wine Aroma quality is improved | |
| CN109988720A (en) | One saccharomycete ZB412 and its application | |
| CN109971661B (en) | Ester-producing yeast ZB406 and application thereof | |
| Yang et al. | Characteristics of traditional Chinese shanlan wine fermentation | |
| CN107164251A (en) | One Accharomyces cerevisiae and its purposes for improving grape wine Ester content | |
| CN102242072A (en) | Strain produced by screening beta-damascenone in Chinese liquor brewing by utilizing flavor directional technology and application thereof | |
| CN108315271A (en) | One primary yeast and its application | |
| CN105802865A (en) | Ice wine yeast with high fermentation activity and outstanding aroma-producing characteristics and application thereof | |
| CN110305804A (en) | A kind of Non-Saccharomyces bacterial strain and its application | |
| CN103045495A (en) | Method for screening citrus fruit wine brewing saccharomycetes and method for producing citrus fruit wine | |
| CN103865841B (en) | A kind of bacillus aceticus and the fruit vinegar utilizing apricot skin slag solid state fermentation to prepare thereof | |
| CN108865910A (en) | A kind of application of wine waste mash, screening technique and the saccharomycete in blueberry red wine fermentation | |
| CN110218662A (en) | A kind of thermophilic low temperature produces fragrant characteristic U.S. pole plum surprise yeast outstanding and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |