CN104531536A - High-kojic-acid-yield aspergillus oryzae bacterial strain and application thereof - Google Patents

High-kojic-acid-yield aspergillus oryzae bacterial strain and application thereof Download PDF

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Publication number
CN104531536A
CN104531536A CN201410564556.3A CN201410564556A CN104531536A CN 104531536 A CN104531536 A CN 104531536A CN 201410564556 A CN201410564556 A CN 201410564556A CN 104531536 A CN104531536 A CN 104531536A
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kojic acid
aspergillus oryzae
bacterial strain
kojic
acid
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杨海泉
华珊
宋江宁
王一恬
沈微
陈献忠
樊游
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Jiangnan University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/69—Aspergillus oryzae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein

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Abstract

The invention discloses a high-kojic-acid-yield aspergillus oryzae bacterial strain (Aspergillus oryzae QS). The preservation serial number is CCTCC M 2014436. The bacterial strain is screened from soil; kojic acid starts to be produced after fermentation cultivation is carried out in a 30 DEG C shake flask for 2 d; after fermentation is carried out for 7 d, the yield of the kojic acid can reach 25 g/L; and the bacterial strain is an excellent wild bacterial strain with the high kojic acid yield. The kojic acid has a wide application prospect, and is mainly used for the industries of food anticorrosion and fresh keeping, cosmetics, medicines, agriculture, chemical engineering and the like.

Description

One plant height produces aspergillus oryzae strain and the application thereof of kojic acid
Technical field
The present invention relates to Aspergillus oryzae bacterial strain and an application thereof, particularly a plant height produces aspergillus oryzae strain and the application thereof of kojic acid.
Background technology
In human body, tyrosine oxidase energy catalytic tyrosine and oxyradical through oxidase complex, be polymerized, last synthesis of melanin.Check melanin is formed by the activity of restraint of tyrosinase for kojic acid and derivative thereof, and safety non-toxic, thus at makeup with food antiseptic is fresh-keeping etc. that field tool has been widely used.Pharmaceutically, kojic acid can as the intermediate producing cephalosporin analog antibiotic.Meanwhile, can be used as synthetic perfume intermediate, sterilant raw material etc., have wide practical use in industries such as food, makeup, medicine, agricultural, chemical industry.Much aspergillus is as aspergillus oryzae, flavus, aspergillus effusus, Aspergillus nidulans, Aspergillus parasiticus, Aspergillus albicans, Aspergillus fumigatus, huge aspergillus, Aspergillus amstelodami etc. and minority mould, and as produced kojic acid mould etc., energy metabolism produces kojic acid.At present, generally use aspergillus oryzae in production and do not produce the flavus of flavacin.1998, the people such as Zhou Weidong report to produce after aspergillus oryzae 2336 cultivates 6.5d kojic acid 7.45g/L (Zhou Weidong, Shen Yufen, Chen Yiliang, etc. the mutagenic and breeding [J] of Kojic Acid-producing Mold. subtropical plant communication, 1998,02:12-6).Before Shen Weirong equals report in 2003 mutagenesis the kojic acid output of flavus C-527 be 9.26g/L (Shen Weirong, Shen Jian, Han Liping, etc. the complex mutation breeding [J] of Kojic Acid-producing Mold. microbiology is circulated a notice of, 2003,04:60-64).In 2006, the people such as Lee one Jing in 2006 report that aspergillus oryzae QA96.8 kojic acid output before mutagenesis is 4.79g/L, even if after repeatedly ultraviolet mutagenesis, kojic acid output only rises to 18.13g/L (Li Yijing, Ma Weichao. the screening of Kojic Acid-producing Mold kind and the optimization [J] of technological condition for fermentation. China brewages, 2006,06:42-44).Shen Yufen etc. screen a plant mutant strain with aspergillus oryzae 2336 through ultraviolet mutagenesis in calendar year 2001, kojic acid output reaches 26.36g/L (Shen Yufen, Chen Yiliang, Zhou Weidong, Deng. the seed selection of kojic acid producing strains F31012 mutant and the research [J] of fermentation condition. Fujian Normal University's journal (natural science edition), 2001,02:73-77).Therefore, kojic acid high yield strain excellent is screened significant.
Summary of the invention
The invention provides an Aspergillus oryzae bacterial strain (Aspergillus oryzae QS), be preserved in China typical culture collection center on September 20th, 2014, deposit number CCTCC M 2014436, preservation address is China, Wuhan, Wuhan University's China typical culture collection center.
Described product kojic acid aspergillus oryzae screens and obtains from soil, and its storage conditions is as follows: washed out by the spore on inclined-plane with sterile distilled water, and granulated glass sphere is broken up the filtration of rear band absorbent cotton funnel and prepared spore suspension.Add 0.5mL spore suspension and 0.5mL 30% glycerine inside each glycerine pipe, put into-70 DEG C of cryogenic refrigerators and preserve.
Solid state flat panel substratum is (w/v): 5% glucose, 1% W-Gum, 0.5% yeast extract paste, 0.5%KH 2pO 4, 0.25%MgSO 47H 2o, agar powder 2%, natural pH, sterilizing.
Liquid seed culture medium is (w/v): 10% glucose, 2% W-Gum, 1% yeast extract paste, 0.5%KH 2pO 4, 0.25%MgSO47H 2o, adjusts pH to 4.0 with rare HCl, sterilizing.
Liquid fermentation medium is (w/v): 12% glucose, 1.0% soybean cake powder, 0.3% yeast extract paste, 0.2%KH 2pO 4, 0.02%MgSO 47H 2o, natural pH, sterilizing.
The culture condition of aspergillus oryzae is: rule on solid plate substratum by the aspergillus oryzae spore of preservation glycerine pipe, 30 DEG C cultivate 5d after spore is accessed liquid seed culture medium, 30 DEG C, 200rpm, 27h cultivated by shaking table.Be inoculated in fermentation and acid substratum with the inoculum size of 10% (v/v) by cultured liquid seeds, 30 DEG C, 200rpm, shaking table is cultivated.
By producing the fermented liquid after the fermentation of kojic acid aspergillus oryzae, the centrifugal 2min of 10000rpm, adopt the kojic acid output of the centrifugal secondary fermentation supernatant liquor of iron trichloride colorimetric method for determining.Start to produce kojic acid after fermentation 2d, after 7d, kojic acid output can reach 25g/L.
The present invention filters out a strain and produces kojic acid aspergillus oryzae in soil, is the wild strain that a strain kojic acid output is higher.The kojic acid that this bacterial strain is produced can be widely used in the industries such as food antiseptic is fresh-keeping, makeup, medicine, agricultural, chemical industry, has important production application and is worth.
Accompanying drawing illustrates:
Fig. 1: kojic acid bioassay standard curve
Fig. 2: aspergillus oryzae screening color reaction and colonial morphology
Fig. 3: kojic acid fermentative production
Embodiment:
Embodiment 1: iron trichloride measures kojic acid content method
The making of kojic acid bioassay standard curve:
1) preparation of 0.1% kojic acid standardized solution: accurately take kojic acid standard substance 0.1000g in 100mL volumetric flask, adds appropriate distilled water and makes it to dissolve completely, and be settled to 100mL with distilled water, obtain the kojic acid standardized solution of 1mg/mL.
2) 1.0%FeCl 3prepared by-HCl nitrite ion: accurately take FeCl 36H 2o 1.0000g, in 100mL volumetric flask, adds appropriate distilled water and makes it to dissolve completely, add 2mL concentrated hydrochloric acid, and be settled to 100mL with distilled water, obtain 1.0%FeCl 3-HCl nitrite ion, for subsequent use.
3) with transfer pipet draw respectively accurately 0.1% kojic acid standardized solution 0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0mL in 100 mL volumetric flasks, respectively add 2.0mL 1.0%FeCl 3-HCl nitrite ion, is settled to 100mL with distilled water, after fully shaking up, adds nitrite ion as blank using distilled water, the light absorption value (A under 500nm place measures different concns 500nm).With kojic acid concentration be X-coordinate, A 500nmvalue is ordinate zou drawing standard curve (Fig. 1).
In fermented liquid, kojic acid measures:
After fermented liquid is centrifugal, get supernatant liquor and suitably dilute, accurately draw 1.0mL diluent and measure A by standard curve making method 500nm, and the content of kojic acid in fermented liquid is calculated according to kojic acid typical curve.
Embodiment 2: produce kojic acid aspergillus oryzae screening method
Take a certain amount of soil, put into the 250mL triangular flask that 100mL sterilized water and granulated glass sphere are housed, vibration, dilution different concns gradient, sample good for different concns gradient dilution is applied on screening culture medium flat board respectively, in 30 DEG C of constant incubators, cultivates 3d.Observe dull and stereotyped discoloration, the large single bacterium colony spore inoculating of picking color dark variable color circle is in screening culture medium, be inoculated on czapek agar medium plate with bikini method during inoculation, spore must be avoided during inoculation to be scattering into other position of plate, under being placed on mold incubator Plays culture condition afterwards, to cultivate 7d.Isolation and purification culture, until without living contaminants, is inoculated in inclined-plane and preserves, for next step qualification.
Described solid screening culture medium (w/v) is: 10% glucose, 2% W-Gum, 1% yeast extract paste, 0.5%KH 2pO 4, 0.25%MgSO 47H 2o, 0.1%FeCl 36H 2o, 0.08% sodium deoxycholate, agar 2%, natural pH, sterilizing.
Identify mainly through: 1) colony characteristics is observed: the later stage be brown or pale green brown, and the back side is colourless, and conidium is more in white early stage in bacterium colony front, and bacterium colony central authorities are slightly in loose column, and bacterium colony presents concentric annular line, and bacterium colony is surperficial without transudate.2) colour developing circle is observed: single bacterium colony (Fig. 2) that color dark variable color circle is large.
The single bacterium colony selecting color dark variable color circle large carries out shake flask fermentation cultivation, get the centrifugal 2min of fermented liquid 10000rpm after cultivation, after centrifugal end, get supernatant liquor, measured kojic acid content in fermented liquid by iron trichloride colorimetry after suitable dilution, filter out the strain excellent that a strain kojic acid output is higher, kojic acid output is about 25g/L, China typical culture collection center is preserved in, deposit number CCTCCM 2014436 on September 1st, 2014.
Embodiment 3: kojic acid fermentative production
The aspergillus oryzae spore of preservation glycerine pipe is rule on solid plate substratum, 30 DEG C cultivate 5d after spore is accessed liquid seed culture medium, 30 DEG C, 200rpm, 27h cultivated by shaking table.Be inoculated in fermentation and acid substratum with the inoculum size of 10% (v/v) by cultured liquid seeds, 30 DEG C, 200rpm, shaking table is cultivated.Take out 1mL fermented liquid every 24h, by centrifugal 2min under fermented liquid 10000rpm, 4 DEG C of conditions, collect supernatant liquor, and the volume of Measurement accuracy supernatant liquor.After suitably being diluted by the centrifugal supernatant liquor obtained, accurately draw 1.0mL diluent and measure A by standard curve making method 500nm, and calculate the content of kojic acid in fermented liquid.This strain fermentation is cultivated after 2d and is started to produce kojic acid, and after cultivating 7d, kojic acid output reaches 25g/L (Fig. 3), because of but the higher excellent wild strain of a strain kojic acid output.

Claims (7)

1. an Aspergillus oryzae bacterial strain (Aspergillus oryzae QS), be preserved in China typical culture collection center on September 20th, 2014, deposit number is: CCTCC M 2014436.
2. aspergillus oryzae according to claim 1, is characterized in that this bacterium high yield kojic acid.
3. aspergillus oryzae according to claim 2, starts to produce kojic acid after it is characterized in that cultivating 2d.
4. aspergillus oryzae according to claim 3, after it is characterized in that 30 DEG C of fermentation 7d, kojic acid output can reach 25g/L.
5. the arbitrary described aspergillus oryzae of claim 1-4 is applied to production kojic acid.
6. application according to claim 5, is characterized in that producing the fermention medium component of kojic acid for (w/v): 12% glucose, 1.0% soybean cake powder, 0.3% yeast extract paste, 0.2%KH 2pO 4, 0.02%MgSO 47H 2o.
7. the industry such as, makeup, medicine, agricultural, chemical industry fresh-keeping at food antiseptic according to the arbitrary described aspergillus oryzae of claim 1-4 has and applies more widely.
CN201410564556.3A 2014-10-21 2014-10-21 High-kojic-acid-yield aspergillus oryzae bacterial strain and application thereof Pending CN104531536A (en)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105018352A (en) * 2015-07-23 2015-11-04 云南大学 Fungal strain capable of producing kojic acid and preparation method of fungal strain
CN105647815A (en) * 2015-12-16 2016-06-08 江南大学 Method for increasing kojic acid yield of Aspergillus oryzae
CN106755164A (en) * 2016-12-23 2017-05-31 江南大学 A kind of method for improving aspergillus oryzae kojic acid yield
CN106893681A (en) * 2017-03-10 2017-06-27 云南中烟工业有限责任公司 A kind of method for preparing tobacco aromaticss with aspergillus oryzae degraded bata-carotene
CN108866119A (en) * 2015-10-10 2018-11-23 吴联凯 A kind of whitening cosmetics kojic acid producing process
CN115197933A (en) * 2022-04-29 2022-10-18 成都金开生物工程有限公司 Breeding method of high-yield kojic acid strain

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105018352A (en) * 2015-07-23 2015-11-04 云南大学 Fungal strain capable of producing kojic acid and preparation method of fungal strain
CN108866119A (en) * 2015-10-10 2018-11-23 吴联凯 A kind of whitening cosmetics kojic acid producing process
CN105647815A (en) * 2015-12-16 2016-06-08 江南大学 Method for increasing kojic acid yield of Aspergillus oryzae
CN105647815B (en) * 2015-12-16 2019-08-06 江南大学 A method of improving aspergillus oryzae kojic acid yield
CN106755164A (en) * 2016-12-23 2017-05-31 江南大学 A kind of method for improving aspergillus oryzae kojic acid yield
CN106893681A (en) * 2017-03-10 2017-06-27 云南中烟工业有限责任公司 A kind of method for preparing tobacco aromaticss with aspergillus oryzae degraded bata-carotene
CN115197933A (en) * 2022-04-29 2022-10-18 成都金开生物工程有限公司 Breeding method of high-yield kojic acid strain

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Application publication date: 20150422