CN105087511A - Culture medium and method for preparing polyphenol oxidase through fungus fermentation process - Google Patents

Culture medium and method for preparing polyphenol oxidase through fungus fermentation process Download PDF

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Publication number
CN105087511A
CN105087511A CN201510647605.4A CN201510647605A CN105087511A CN 105087511 A CN105087511 A CN 105087511A CN 201510647605 A CN201510647605 A CN 201510647605A CN 105087511 A CN105087511 A CN 105087511A
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polyphenoloxidase
substratum
legal system
formula
spore suspension
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杨民和
张婉蓉
巫婷玉
黄鹭强
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Fujian Normal University
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0059—Catechol oxidase (1.10.3.1), i.e. tyrosinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
    • C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
    • C12Y110/03001—Catechol oxidase (1.10.3.1), i.e. tyrosinase

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Abstract

The invention relates to a method for producing polyphenol oxidase through fungus fermentation process. The method comprises the following steps: mixing wheat bran, tea leaves, a nitrogen source additive, KH2PO4, MgSO4.7H2O, MnSO4.5H2O and CuSO4.5H2O, and diluting to 1L; under the aseptic operating condition, adding fungi spore suspending liquid, performing shaking-flask culture under the temperature of 26-28 DEG C and at the rotating speed of 120 r/min for 3-5 days, and performing centrifugation, so as to obtain an aqueous solution of the polyphenol oxidase. According to the invention, the tea leaves are used as main components of a culture medium, and polyphenol compounds contained in the tea leaves are used as induction ingredients to facilitate the increase of yield of the polyphenol oxidase; besides, the wheat bran and tea leaves as the raw materials are rich in resources and low in price; through the adoption of the culture medium in the method, the enzyme activity can reach 241-256.6 U/mL.min.

Description

For the substratum and preparation method thereof of fungi fermentation legal system for polyphenoloxidase
Technical field
The invention belongs to technical field of microbial fermentation, be specifically related to a kind of for the substratum and preparation method thereof of fungi fermentation legal system for polyphenoloxidase.
Background technology
Polyphenoloxidase (Polyphenoloxidase, PPO) be the class cuproprotein all extensively existed in bacterium, fungi, plant and mammalian body, comprise laccase (Laccase), tyrosine oxidase (Tyrosinase) and catechol-oxydase (Catecholase).Polyphenoloxidase the oxidation of catalysis polyphenolic compound can form corresponding quinones substance effectively, is the key enzyme of melanochrome metabolism and catecholamine synthesis.Polyphenoloxidase is the Main Function enzyme causing plant leaf, fruit and vegetables brown stain; Meanwhile, owing to participating in the degradation process of polyphenolic compound, obtain good investigation and application in phenolic wastewater treatment, lignin degradation and the field such as dye decolored.
Being rich in polyphenoloxidase in fresh tea leaf in its cell, catechins can being impelled to be oxidized and form theoflavin, theabrownin and other oxypolymers etc., produce volatile compound, is the biochemical basis forming black tea local flavor and speciality.Therefore, polyphenoloxidase is the crucial enzyme during black tea manufactures, and is formed with vital effect to black tea quality.External when preparing instant black tea, add polyphenoloxidase and can improve black tea preparation rate, increase the content of soluble substance, alleviate the bitter taste of black tea, significantly improve the quality of instant black tea.
The polyphenoloxidase used in Tea Processing investigation and application field at present derives from plant mostly, due to the restriction by season and extraction cost, is difficult to reach large-scale production.Meanwhile, derive from the polyphenoloxidase of different plant, because the difference of substrate specificity, be also difficult to give full play to its catalysis efficiency in Tea Processing.Many units, attempting the polyphenoloxidase of being expressed different plant origin by genetic engineering technique, are also difficult to broadened application because expression activity is low.
Microorganism has that source is wide, kind is many, breeding is fast and the advantage of easy cultivation, by fermentable production polyphenoloxidase by technical solution bottleneck.In recent years, produce in the screening of bacterial strain at polyphenoloxidase, some bacterium producing multi enzyme preparations are all obtained from tea garden soil, tealeaves, black tea fermenting process and black tea finished product, as fungi has the ability of certain production polyphenoloxidase, but content is lower, in order to improve the enzymatic production ability of fungi, optimizing and formulating the substratum producing polyphenoloxidase, improving technological condition for fermentation and just seem particularly important.
Summary of the invention
An object of the present invention is to provide a kind of for the culture medium prescription of fungi fermentation legal system for polyphenoloxidase.This culture medium prescription main component is wheat bran and tealeaves, and submember comprises peptone and inorganic salt, without the need to adjust ph after having prepared, can use after carrying out sterilizing according to a conventional method.Wheat bran in substratum and tea component, all can promote the generation of polyphenoloxidase significantly.
Two of object of the present invention is that method provides a kind of for fungi, and particularly coronoid process falls apart the method preparing polyphenoloxidase of capsule bacterium fermentation method.
As follows for realizing the technical scheme that above-mentioned purpose of the present invention adopts:
1, culture medium prescription
Take final volume as the hydration mixed solution of the fermention medium of 1L, the formula of its each component is:
Wheat bran 25 ~ 45g
Tealeaves 3.5 ~ 10g
Peptone 1.5 ~ 4g
KH 2PO 42.0g
MgSO 4.7H 2O0.2～0.5g
MnSO 4.5H 2O0.5g
CuSO 4.5H 2O0.01～0.02g
Described wheat bran, both can as carbon source, also can as nitrogenous source.
Described tealeaves is commercially available low price Tie Guanyin tea.Tealeaves, after abundant drying, suitably grinds or directly adds in substratum.
After each for substratum component being weighed by described formula, join successively in the container of distilled water, be settled to 1L after stirring, divide and be filled to after in triangular flask, sterilizing can use according to a conventional method.
2, eurotium cristatum strain and cultivation
Under aseptic technique, coronoid process is fallen apart capsule bacterium ( eurotiumcristatum) bacterial strain move and receive on fresh potato dextrose agar, under 26 ~ 28 DEG C of conditions, dark culturing is after 5 ~ 7 days, and with conidium ripe under aseptic washing, preparation spore suspension, the concentration of spore suspension is 1 ~ 2 × 10 8individual spore/mL.
The formula of described potato dextrose agar slant substratum, take final volume as the substratum aqueous solution of 1L, the formula of its each component is:
Potato 200g
Glucose 18 ~ 20g
Agar 15 ~ 20g
The preparation method of described potato dextrose agar slant substratum is:
After the potato wash clean that market is bought, about 1cm is cut in peeling 3fritter, takes 200g and puts into 1L beaker, adds water boil 30 minutes, filtered through gauze.Add the glucose and agar that weigh up, heat and stir, melt completely to agar, after 2 layers of filtered through gauze, supply water to 1L.Packing, wrap up, label, in high-pressure steam sterilizing pan, 121 DEG C of sterilizings 20 ~ 30 minutes, take out pendulum inclined-plane, are potato dextrose agar slant substratum.
Described coronoid process fall apart capsule bacterium ( eurotiumcristatum), purchased from China General Microbiological culture presevation administrative center (CGMCC), it is numbered: 3.07934.
3, the preparation of polyphenoloxidase
By above formulated fermention medium, in triangular flask, load the fermention medium of triangular flask capacity 1/4th 'shydration mixed solution, according to a conventional method sterilizing.In each triangular flask, access coronoid process after sterilizing to fall apart capsule bacterium spore suspension, temperature 26 ~ 28 DEG C, under the condition of rotating speed 120r/min, shake-flask culture is after 3 ~ 4 days, get fermentation liquor filtered through gauze, through 4 DEG C, centrifugal 10 minutes of 10000r/min, the supernatant liquor of acquisition is the aqueous solution of polyphenoloxidase.
Described access coronoid process falls apart capsule bacterium spore suspension, and access amount is fermention medium 'sone of 1/30 of hydration mixing liquid accumulated amount.
Beneficial effect of the present invention is the main ingredient utilizing tealeaves as substratum, and polyphenolic compound contained in tealeaves, as inducing component, is conducive to the output improving polyphenoloxidase.Meanwhile, the main ingredient of the fermention medium of the present invention's preparation is as wheat bran and tea raw material abundance, cheap, is conducive to reducing production cost.Utilize substratum described in the inventive method, coronoid process fall apart capsule bacterium ( eurotiumcristatum) after spore suspension in 28 DEG C, 120 rmin -1constant temperature culture, ferment to the 5th day and reach product enzyme peak, enzyme work can reach 241 ~ 256.6 U/mLmin.
Embodiment
In following examples, the substratum be not particularly illustrated, is conventional disclosed substratum.
embodiment 1
1, culture medium prescription
Take final volume as the fermentation of 1L substratumhydration mixed solution meter, the formula of its each component is:
Wheat bran 30g
Tealeaves 6g
Peptone 2.5g
KH 2PO 42.0g
MgSO 4.7H 2O0.2g
MnSO 4.5H 2O0.5g
CuSO 4.5H 2O0.02g。
2, eurotium cristatum strain and cultivation
Use front coronoid process fall apart capsule bacterium ( eurotiumcristatum) be kept on potato dextrose agar slant substratum under 4 DEG C of conditions.Under aseptic technique, moved by bacterial strain and receive on fresh potato dextrose agar, under 28 DEG C of conditions, dark culturing is after 5 days, and with conidium ripe under aseptic washing, preparation spore suspension, the concentration of spore suspension is 1 ~ 2 × 10 8individual spore/mL.
The formula of described potato dextrose agar slant substratum, take final volume as the substratum aqueous solution of 1L, the formula of its each component is:
Potato 200g
Glucose 18 ~ 20g
Agar 15 ~ 20g
The preparation method of described potato dextrose agar slant substratum is:
After the potato wash clean that market is bought, about 1cm is cut in peeling 3fritter, takes 200g and puts into 1L beaker, adds water boil 30 minutes, filtered through gauze.Add the glucose and agar that weigh up, heat and stir, melt completely to agar, after 2 layers of filtered through gauze, supply water to 1L.Packing, wrap up, label, in high-pressure steam sterilizing pan, 121 DEG C of sterilizings 20 ~ 30 minutes, take out pendulum inclined-plane, are potato dextrose agar slant substratum.
3. the preparation of polyphenoloxidase
By above formulated fermention medium, in each 250mL triangular flask, load 60mL fermention medium 'shydration mixed solution, according to a conventional method sterilizing.In each triangular flask, access coronoid process to fall apart capsule bacterium spore suspension 2mL, described coronoid process falls apart capsule bacterium spore suspension, and its concentration is 1-2 × 10 8individual spore/mL.Temperature 26 DEG C, under the condition of rotating speed 120r/min, shake-flask culture is after 3 ~ 4 days, gets fermentation liquor filtered through gauze, and through 4 DEG C, centrifugal 10 minutes of 10000r/min, the supernatant liquor of acquisition is the aqueous solution of polyphenoloxidase.
4. the detection of polyphenoloxidase
1) flat board of phenol oxidase detects
On the differential medium adding methyl catechol, the Laccase Catalyzed methyl catechol in polyphenoloxidase reacts and produces purple variable color circle;
After interpolation naphthyl alcohol, the Laccase Catalyzed naphthyl alcohol in polyphenoloxidase reacts and generates purple zone of oxidation;
After interpolation tyrosine, the tyrosinase catalysis tyrosine in polyphenoloxidase reacts and generates yellowish chromosphere;
After interpolation Weibull, polyphenol oxidase catalyzed Weibull reaction generates tawny endless belt;
After interpolation gallic acid, polyphenol oxidase catalyzed gallic acid reaction generates tawny endless belt.Choose the bacterial strain that bacterium colony periphery has color reaction, repeat to be confirmed.
The polyphenoloxidase differential medium of described interpolation methyl catechol and naphthyl alcohol, take final volume as the substratum aqueous solution of 1L, the formula of its each component is: add methyl catechol 0.4 g, naphthyl alcohol 5 × 10 in potato dextrose agar respectively -5mol/L.
The polyphenoloxidase differential medium of described interpolation gallic acid and Weibull, take final volume as the substratum aqueous solution of 1L, the formula of its each component is: the volumetric molar concentration of adding Weibull and gallic acid in minimum medium 1 is 4 × 10 respectively -4mol/L and 0.4 mol/L.
The polyphenoloxidase differential medium of described interpolation TYR, take final volume as the substratum aqueous solution of 1L, the formula of its each component is: glucose 2 g, TYR 10 g, KH 2pO 41.52 g, KCl0.52 g, MgSO 47H 2o0.52 g, Cu (NO 3) 23H 2o0.001 g, ZnSO 47H 2o0.001 g, FeSO 47H 2o0.001 g, agar 20g.
Above substratum is sterilizing 20 ~ 30min under 121 DEG C of temperature condition all.
2) enzyme activity determination
The aqueous solution getting 0.1 mL polyphenoloxidase adds cuvette, adds pH5.5 phosphate buffered saline buffer 3 mL, after to add concentration be 0.1 molL -1pyrocatechol solution l mL, rapid test 410 nm place absorbancy change.With phosphate buffered saline buffer (pH5.5) 3 mL+0.1 molL -1in contrast, successive reaction 30 min, every 3 min record 1 absorbancy to pyrocatechol solution l mL.L mL enzyme liquid makes absorbance change 0.001 and is defined as 1 enzyme activity unit (UmL in 1 min -1min -1).
Enzyme work=1/V × Δ OD/ Δ T
Or middle V: the volume (mL) of crude enzyme liquid, Δ OD: absorbancy changing value, Δ T: reaction times (min).
Coronoid process is fallen apart capsule bacterium ( eurotiumcristatum) inoculate in described fermention medium, in 28 DEG C, 120 rmin -1constant temperature culture, measures polyphenol oxidase enzyme activity every day.Fermentation reached to the 5th day produces enzyme peak, and enzyme work is 241 U/mLmin.
Described coronoid process is fallen apart capsule bacterium ( eurotiumcristatum) aqueous solution of polyphenoloxidase that obtains for 5 days of the fermentation culture flat board that carries out polyphenoloxidase detects, this crude enzyme liquid presents color reaction respectively on the differential medium flat board adding methyl catechol, gallic acid and naphthyl alcohol.
embodiment 2
1. be the substratum aqueous solution of 1L with final volume, the formula of its each component is:
Wheat bran 48.9g
Tealeaves 3.5g
Ammonium nitrate 10.5g
KH 2PO 42.0g
MgSO 4.7H 2O0.5g
Anhydrous CaCl 20.075g
CuSO 4.5H 2O0.01g
2, eurotium cristatum strain and cultivation
Use front coronoid process fall apart capsule bacterium ( eurotiumcristatum) be kept on potato dextrose agar slant substratum under 4 DEG C of conditions.Under aseptic technique, moved by bacterial strain and receive on fresh potato dextrose agar, under 26 DEG C of conditions, dark culturing is after 7 days, and with conidium ripe under aseptic washing, preparation spore suspension, the concentration of spore suspension is 1 ~ 2 × 10 8individual spore/mL.
Formula of described potato dextrose agar slant substratum and preparation method thereof is with embodiment 1.
3. the preparation of polyphenoloxidase
By above formulated fermention medium, in each 250mL triangular flask, load 60mL fermention medium 'shydration mixed solution, according to a conventional method sterilizing.In each triangular flask, access coronoid process to fall apart capsule bacterium spore suspension 2mL, described coronoid process falls apart capsule bacterium spore suspension, and its concentration is 1-2 × 10 8individual spore/mL.Temperature 28 DEG C, under the condition of rotating speed 120r/min, shake-flask culture is after 3 ~ 4 days, gets fermentation liquor filtered through gauze, and through 4 DEG C, centrifugal 10 minutes of 10000r/min, the supernatant liquor of acquisition is the aqueous solution of polyphenoloxidase.
The formula of described polyphenoloxidase differential medium and preparation side are with embodiment 1.
The dull and stereotyped detection method of described polyphenoloxidase is with embodiment 1.
After testing, the enzyme activity of the polyphenoloxidase produced afterwards for 4 days in fermentation culture reaches 246.7U/mL.min, and the enzyme activity of the polyphenoloxidase produced afterwards for 5 days reaches 255.6U/mL.min.

Claims (8)

1., for the substratum of fungi fermentation legal system for polyphenoloxidase, it is characterized in that the formula of substratum is in the hydration mixed solution of 1L in final volume, the formula of its each component is:
Wheat bran 25 ~ 45g
Tealeaves 3.5 ~ 10g
Peptone 2.5 ~ 4g
KH 2PO 42.0g
MgSO 4.7H 2O0.2～0.5g
MnSO 4.5H 2O0.5g
CuSO 4.5H 2O0.01～0.02g。
2. according to claim 1ly a kind ofly it is characterized in that described tealeaves for the substratum of fungi fermentation legal system for polyphenoloxidase, after abundant drying, suitably grind or directly add.
3., for the method for fungi fermentation legal system for polyphenoloxidase, it is characterized in that:
1) take final volume as the hydration mixed solution of substratum of 1L, the formula of its each component is:
Wheat bran 25 ~ 45g
Tealeaves 3.5 ~ 10g
Peptone 2.5 ~ 4g
KH 2PO 42.0g
MgSO 4.7H 2O0.2～0.5g
MnSO 4.5H 2O0.5g
CuSO 4.5H 2O0.01～0.02g；
2) preparation of polyphenoloxidase
By above formulated substratum, in triangular flask, load the substratum of triangular flask capacity 1/4th 'shydration mixed solution, sterilizing according to a conventional method, in each triangular flask, access coronoid process after sterilizing to fall apart capsule bacterium spore suspension, temperature 26 ~ 28 DEG C, under the condition of rotating speed 120r/min, shake-flask culture is after 3 ~ 5 days, get fermentation liquor filtered through gauze, through 4 DEG C, centrifugal 10 minutes of 10000r/min, the supernatant liquor of acquisition is the aqueous solution of polyphenoloxidase.
4. according to claim 3 a kind of for the method for fungi fermentation legal system for polyphenoloxidase, it is characterized in that described access coronoid process falls apart capsule bacterium spore suspension, access amount is fermention medium 'sone of 1/30 of hydration mixing liquid accumulated amount.
5. according to claim 3 a kind of for the method for fungi fermentation legal system for polyphenoloxidase, it is characterized in that described access coronoid process falls apart capsule bacterium spore suspension, refers under aseptic technique, capsule bacterium that coronoid process is fallen apart ( eurotiumcristatum) bacterial strain move and receive on fresh potato dextrose agar, under 26 ~ 28 DEG C of conditions, dark culturing is after 5 ~ 7 days, and with conidium ripe under aseptic washing, preparation spore suspension, the concentration of spore suspension is 1 ~ 2 × 10 8individual spore/mL.
6. according to claim 5 a kind of for the method for fungi fermentation legal system for polyphenoloxidase, it is characterized in that the formula of described potato dextrose agar slant substratum, take final volume as the substratum aqueous solution of 1L, the formula of its each component is:
Potato 200g
Glucose 18 ~ 20g
Agar 15 ~ 20g.
7. according to claim 6 a kind of for the method for fungi fermentation legal system for polyphenoloxidase, it is characterized in that the preparation method of described potato dextrose agar slant substratum is: after potato wash clean, 1cm is cut in peeling 3fritter, takes 200g and puts into 1L beaker, adds water boil 30 minutes, filtered through gauze; Add the glucose and agar that weigh up, heat and stir, melt completely to agar, after 2 layers of filtered through gauze, supply water to 1L; Packing, wrap up, label, in high-pressure steam sterilizing pan, 121 DEG C of sterilizings 20 ~ 30 minutes, take out pendulum inclined-plane, are potato dextrose agar slant substratum.
8. according to claim 4 a kind of for the method for fungi fermentation legal system for polyphenoloxidase, it is characterized in that described coronoid process fall apart capsule bacterium ( eurotiumcristatum), purchased from China General Microbiological culture presevation administrative center (CGMCC), it is numbered: 3.07934.
CN201510647605.4A 2015-10-09 2015-10-09 Culture medium and method for preparing polyphenol oxidase through fungus fermentation process Pending CN105087511A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117801967A (en) * 2024-03-01 2024-04-02 烟台市供销社茶业有限公司 Method for culturing Eurotium cristatum and application

Citations (2)

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Publication number Priority date Publication date Assignee Title
CN102120964A (en) * 2010-12-21 2011-07-13 湖南城市学院 Preparation method of eurotium cristatum spore suspension
CN103275876A (en) * 2013-05-23 2013-09-04 湖南农业大学 Tea source eurotium cristatum strain and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102120964A (en) * 2010-12-21 2011-07-13 湖南城市学院 Preparation method of eurotium cristatum spore suspension
CN103275876A (en) * 2013-05-23 2013-09-04 湖南农业大学 Tea source eurotium cristatum strain and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117801967A (en) * 2024-03-01 2024-04-02 烟台市供销社茶业有限公司 Method for culturing Eurotium cristatum and application
CN117801967B (en) * 2024-03-01 2024-04-30 烟台市供销社茶业有限公司 Method for culturing Eurotium cristatum and application

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Application publication date: 20151125