CN107201359A - A kind of selection of high yield kojic acid aspergillus oryzae - Google Patents

A kind of selection of high yield kojic acid aspergillus oryzae Download PDF

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CN107201359A
CN107201359A CN201710411496.5A CN201710411496A CN107201359A CN 107201359 A CN107201359 A CN 107201359A CN 201710411496 A CN201710411496 A CN 201710411496A CN 107201359 A CN107201359 A CN 107201359A
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kojic acid
aspergillus oryzae
selection
high yield
strain
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李镇江
杨志刚
谢彪
唐小芳
王宏
杨晓琴
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CHENGDU JINKAI BIOLOGICAL ENGINEERING Co Ltd
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    • C12R2001/69—Aspergillus oryzae

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Abstract

The invention discloses a kind of selection of high yield kojic acid aspergillus oryzae, with microwave/ultraviolet complex mutation breeding, the high strain of kojic acid level is produced in screening.Method is related to the complex mutation of a microwave radiation and ultraviolet irradiation twice, includes screening, induction mutation of bacterium and the seed culture of initial strain.The excellent species obtained using this compound mutation breeding technology, yield height, high conversion rate, fermentation period are short, kojic acid zymotic fluid is of light color, and genetic stability is good.

Description

A kind of selection of high yield kojic acid aspergillus oryzae
Technical field
The invention belongs to bioengineering field, more particularly to a kind of selection of high yield kojic acid aspergillus oryzae.
Background technology
Kojic acid is a kind of important organic acid, is widely used in the fields such as cosmetics, food processing, medicine, agricultural.It is special It is not, in food processing field, due to its safety non-toxic, to be used as preferable polyphenol oxidase enzyme inhibitor, most application prospect.It is micro- Biofermentation is the main path of kojic acid industrialized production, because kojic acid is the secondary species of microbial metabolism, its building-up process More complicated, synthesis mechanism is still unclear so far, therefore, improves kojic acid yield using the method for genetic engineering and still has any problem.At present, Production kojic acid bacterial strain focuses primarily upon aspergillus oryzae and aspergillus flavus, wherein, aspergillus flavus is conditionity pathogenic bacteria.Manabe etc. is to 149 plants Aspergillus oryzae and 46 plants of aspergillus flavus find that aspergillus flavus all produces kojic acid and has 34 plants while producing aflatoxin when being studied；And Aspergillus oryzae only has 105 plants of production kojic acids and does not all produce aflatoxin.In order to improve kojic acid yield, domestic and foreign scholars have been carried out greatly Quantity research works, mainly including strain breeding thereof, the aspect of fermentation process optimization two.
For many years, by carrying out the seed selection that kojic acid produces bacterial strain with a variety of different method of mutagenesis, some are obtained bent Sour superior strain.Method of mutagenesis is still in the majority with traditional physical mutagenesis and mutagenesis, there is that method of mutagenesis is random, direction is difficult Grasp, preferable individual screens the limitations such as difficulty.Some new mutagenesis means, such as cobalt, N~+ implantation are occurred in that in recent years, from Sub- injection technique is to integrate mutagenesis, the synthesis method of mutagenesis of physical mutagenesis, inhereditary material can be made in gene level or Change or lack on molecular level, significantly improve the aberration rate of biology, obtain light damage, mutation rate height, the spectrum of mutation is wide, lose Stable mutagenesis is passed, but this kind of method pervasive device is expensive, it is not easy to operate, so that its application is restricted.Also there is document （Chen Xijian etc.）Report, atmospheric pressure at room plasma（ARTP）Biological induced-mutation breeding technique is a kind of new mutagenesis means, tool The plasma for have the advantages that jet temperature is low, producing is uniform, obvious with large biological molecule and cytosis.
Microwave refers to the electromagnetic wave that frequency is 300MHz-300GHz, and microwave frequency is higher than general radio wave frequency, leads to Often also referred to as " uhf electromagnetic wave ".Microwave also has duality principle as a kind of electromagnetic wave, and the fundamental property of microwave is usual It is rendered as penetrating, reflects, absorbs three characteristics.Microwave radiation process be frequency electromagnetic waves penetrate medium reach inside microtubule fasolculus With the process of glandular cell's system, many covibrations that molecular atoms core is presented under the cyclic force effect of additional electromagnetic field are all sent out Life is in microwave range, thus microwave provides effective research meanses to explore the internal structure and fundamental characteristics of material.
The content of the invention
It is an object of the invention to：A kind of selection of high yield kojic acid aspergillus oryzae is provided.
To achieve the above object, the technical scheme is that：With microwave/ultraviolet complex mutation breeding, song is produced in screening Sour water puts down high strain.
Specific technology path is such：A kind of selection of high yield kojic acid aspergillus oryzae, is related to microwave/ultraviolet compound Screening, induction mutation of bacterium and the seed culture of mutagenesis, repeated screening, including initial strain.The screening of the initial strain is by song After mould species activation, obtained bacterial strain is screened, the sour highest bacterial strain of choosing production is mutagenesis starting strain；The induction mutation of bacterium bag Include complex mutation, high flux screening, shaking flask secondary screening and the passage checking of a microwave radiation and ultraviolet irradiation twice；Described Seed culture is cultivated in seeding tank, and condition of culture is 28 DEG C of temperature, air quantity 1.8M3/ h, rotating speed 150rpm, tank pressure 0.05MPa；
It is preferred that, microwave frequency is 1500MHz to 2450MHz, and radiated time is 10-80s (second)；
It is preferred that, ultraviolet is that 2 ~ 6min is irradiated at 20W uviol lamps 20cm；
Further, by strain passage assays, the genetic stability of kojic acid superior strain is investigated,
As a result show, pass on and verify through 6 generations, the strain production acid of institute's seed selection is 6.3 ~ 6.7%.
Present invention process flow：
1st, bacterial screening
After complex mutation, with 96 orifice plate flat board high flux primary dcreening operations, shaking flask is verified after secondary screening, the superior strain filtered out, Slant medium culture is accessed, 4 DEG C of preservations are put into.
2nd, the culture of seed
Cultivated in seeding tank, condition of culture:28 DEG C of temperature, air quantity 1.8M3/ h, rotating speed 150rpm, tank pressure 0.05MPa.
3rd, fermentation cylinder for fermentation culture
Access in fermentation tank and cultivate, fermentation condition:30 DEG C of temperature, rotating speed 200rpm, air quantity 40M3/ h, tank pressure 0.05MPa.Fermentation During every 4h sampling detected, the residual sugar in zymotic fluid is monitored, production fermentation period be 60 ~ 70 hours, production Sour ability is 63 ~ 67g/L.
Formula：Glucose 14-15%, yeast extract 0.6-1%, potassium dihydrogen phosphate 0.2-0.5%, magnesium sulfate 0.06-0.1%, second Alcohol 1-3%.
As a result of such scheme, the beneficial effects of the present invention are：Using microwave/ultraviolet compound mutation breeding technology The excellent species of acquisition, yield height, high conversion rate, fermentation period are short, zymotic fluid is of light color.High yield kojic acid is obtained by complex mutation Aspergillus oryzae CK2147 strains, acid producing ability are 63-65g/L, and fermentation is steady in a long-term, is very beneficial for building for optimal production technology It is vertical.
Obviously, according to the above of the present invention, according to the ordinary technical knowledge and customary means of this area, do not departing from Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification of other diversified forms can also be made, replaces or changes.
The embodiment of form, remakes further specifically to the above of the invention by the following examples It is bright.But this should not be interpreted as to the scope of above-mentioned theme of the invention and be only limitted to following example, it is all to be based on the above of the present invention The technology realized belongs to the scope of the present invention.
Embodiment
The present invention is described in further detail below in conjunction with specific embodiment.
Embodiment 1：Microwave/ultraviolet mutagenesis bacterial strain shaking flask screening test
First, experiment purpose
Microwave/ultraviolet mutagenesis（Microwave 1 time, ultraviolet 2 times）Bacterial strain by after the simple primary dcreening operation of high-throughput flat, it is necessary to resulting Bacterial strain carries out quantitative screening, is screened by shaking flask, directly perceived, quantitative selects superior strain.
2nd, experiment material
1. kojic acid bacterial strain
2.1% liquor ferri trichloridi：It is accurate to weigh 10g iron chloride, 22.5mL concentrated hydrochloric acids are added, are arrived with distilled water constant volume 1000mL。
3. kojic acid：Standard items, purity >=99.5%.
4. fermentation medium：Glucose 14 ~ 15%, yeast extract 0.6 ~ 1%, potassium dihydrogen phosphate 0.2 ~ 0.5%, magnesium sulfate 0.06 ~ 0.1%th, ethanol 1 ~ 3%.
3rd, laboratory operating procedures
1st, by the fermentation medium prepared, it is filled in 500mL triangular flasks, liquid amount is 60mL, is sterilized at 120 DEG C respectively 20min, is cooled down standby.
2nd, strain obtained by microwave/ultraviolet mutagenesis is seeded in fermentation medium respectively, at 28 DEG C, cultivated under 180rm During 120h, analysis detection is carried out to the zymotic fluid that culture is obtained.
3rd, kojic acid equation of linear regression is determined：Kojic acid standard items 0.1000g accurately is weighed, is dissolved in distilled water, is settled to 100mL, accurately draws 0mL with pipette, 1mL, 2mL, 3mL, 4mL, 5mL totally 6 standard sample mother liquors, and 6 are moved into respectively In 100mL volumetric flasks, developer 2mL being separately added into, then being settled to 100mL, its concentration is respectively 0mg/L, 10mg/L, 20mg/L, 30mg/L, 40mg/L, 50mg/L, determine its light absorption value in the case where wavelength is 500nm, try to achieve equation of linear regression respectively.
4th, zymotic fluid kojic acid assay：Take zymotic fluid to centrifuge 10min at 3,000 rpm, take fermented supernatant fluid, by one Fixed dilution, it is standby.Take dilution after fermentation supernatant 2mL in 100mL volumetric flasks, add 2mL1% liquor ferri trichloridis, then it is fixed Hold to 100mL.Its light absorption value is surveyed at wavelength 500nm, the linear equation tried to achieve above is substituted into and produces kojic acid content.
4th, result
As a result as shown in table 1, draw one plant of bacterial strain M/UV-5 than original strain output increased 5.1% by shaking flask screening.
Embodiment 2：Mutant strain mitotic stability is tested
First, experiment purpose
Resulting superior strain is subjected to mitotic stability experiment, the genetic stability whether mutant strain has had is determined.
2nd, experiment material
5. kojic acid mutant strain
6.1% liquor ferri trichloridi：It is accurate to weigh 10g iron chloride, 22.5mL concentrated hydrochloric acids are added, are arrived with distilled water constant volume 1000mL。
7. kojic acid：Standard items, purity >=99.5%.
8. slant medium：Glucose 14 ~ 15%, yeast extract 0.6 ~ 1%, potassium dihydrogen phosphate 0.2 ~ 0.5%, magnesium sulfate 0.06 ~ 0.1% 。
9. fermentation medium：Glucose 14 ~ 15%, yeast extract 0.6 ~ 1%, potassium dihydrogen phosphate 0.2 ~ 0.5%, magnesium sulfate 0.06 ~ 0.1%th, ethanol 1 ~ 3%.
3rd, laboratory operating procedures
1st, by the slant medium prepared, dispense into test tube, sterilize 15min at 120 DEG C, standby after cooling.
2nd, the inclined-plane after cooling is entered into line blank culture 24h in incubator at 28 DEG C.
3rd, mutant strain inoculation is placed in 4 DEG C of refrigerators and preserved with inclined-plane, carrying out Secondary Culture, cultured bacterial strain, It is stand-by.
4th, by cultured inclined-plane, it will be eluted with sterilized water, certain density spore suspension is made, is connect by 5% inoculum concentration Into triangular flask fermentation medium, terminated at 28 DEG C, 180rpm culture 120h, analysis inspection is carried out to the zymotic fluid that culture is obtained Survey.
5th, kojic acid equation of linear regression is determined：Kojic acid standard items 0.1000g accurately is weighed, is dissolved in distilled water, is settled to 100mL, accurately draws 0mL with pipette, 1mL, 2mL, 3mL, 4mL, 5mL totally 6 standard sample mother liquors, and 6 are moved into respectively In 100mL volumetric flasks, developer 2mL being separately added into, then being settled to 100mL, its concentration is respectively 0mg/L, 10mg/L, 20mg/L, 30mg/L, 40mg/L, 50mg/L, determine its light absorption value in the case where wavelength is 500nm, try to achieve equation of linear regression respectively.
6th, zymotic fluid kojic acid assay：Take zymotic fluid to centrifuge 10min at 3,000 rpm, take fermented supernatant fluid, by one Fixed dilution, it is standby.Take dilution after fermentation supernatant 2mL in 100mL volumetric flasks, add 2mL1% liquor ferri trichloridis, then it is fixed Hold to 100mL.Its light absorption value is surveyed at wavelength 500nm, the linear equation tried to achieve above is substituted into and produces kojic acid content.
4th, result
As it can be seen from table 1 mutant strain can have preferable genetic stability within 6 generations.
Conclusion：
Using aspergillus oryzae CK0113 as starting strain, mutagenesis is carried out using new microwave/ultraviolet mutagenesis means, one plant of kojic acid is obtained Yield is 63 ~ 67g/L superior strain, and acid producing ability improves 6.1 ~ 6.5% than starting strain CK0113 and the bacterial strain is produced Acidity can be stable, and it is a kind of breeding technique of effective and feasible raising kojic acid yield to illustrate microwave/UV mutation.

Claims (8)

1. a kind of selection of high yield kojic acid aspergillus oryzae, is related to mutation breeding, it is characterised in that：Microwave/ultraviolet complex mutation, Repeated screening, includes screening, induction mutation of bacterium and the seed culture of initial strain.
2. a kind of selection of high yield kojic acid aspergillus oryzae according to claim 1, it is characterised in that：The sieve of the initial strain Choosing is, by after aspergillus actication of culture, obtained strain bacterium to be screened, and the sour highest bacterial strain of choosing production is mutagenesis starting strain.
3. a kind of selection of high yield kojic acid aspergillus oryzae according to claim 1, it is characterised in that：The induction mutation of bacterium includes 1 microwave radiation and the complex mutation of 2 ultraviolet irradiations, high flux screening, shaking flask secondary screening and passage checking.
4. a kind of selection of high yield kojic acid aspergillus oryzae according to claim 1, it is characterised in that：Described seed culture is Cultivated in seeding tank, condition of culture is 28 DEG C of temperature, air quantity 1.8M3/ h, rotating speed 150rpm, tank pressure 0.05MPa.
5. a kind of selection of high yield kojic acid aspergillus oryzae according to claim 3, it is characterised in that：Described microwave frequency is 1500MHz to 2450MHz, radiated time is 10 ~ 80s.
6. a kind of selection of high yield kojic acid aspergillus oryzae according to claim 3, it is characterised in that：Described ultraviolet is 2 ~ 6 min are irradiated at 20W uviol lamps 20cm.
7. a kind of selection of high yield kojic acid aspergillus oryzae according to claim 3, it is characterised in that：Passage checking refer to through 6 generations production acid is passed 6.3 ~ 6.7%.
8. according to a kind of high yield kojic acid aspergillus oryzae of claim 1 ~ 7, it is characterised in that：Fermentation medium is：Glucose 14 ~ 15%th, yeast extract 0.6 ~ 1%, potassium dihydrogen phosphate 0.2 ~ 0.5%, magnesium sulfate 0.06 ~ 0.1%, ethanol 1 ~ 3%, production fermentation period are 60 ~ 70 hours, acid producing ability was 63 ~ 67g/L.
CN201710411496.5A 2017-06-05 2017-06-05 A kind of selection of high yield kojic acid aspergillus oryzae Pending CN107201359A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114045293A (en) * 2021-10-28 2022-02-15 江西省农业科学院园艺研究所 Gene Aokap1 for improving aspergillus oryzae kojic acid yield, method and application
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

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114045293A (en) * 2021-10-28 2022-02-15 江西省农业科学院园艺研究所 Gene Aokap1 for improving aspergillus oryzae kojic acid yield, method and application
CN114045293B (en) * 2021-10-28 2023-06-30 江西省农业科学院园艺研究所 Gene Aokap1 for improving kojic acid yield of aspergillus oryzae, method and application
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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