CN103436505B - Preparation method of tannase - Google Patents

Preparation method of tannase Download PDF

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CN103436505B
CN103436505B CN201310350225.5A CN201310350225A CN103436505B CN 103436505 B CN103436505 B CN 103436505B CN 201310350225 A CN201310350225 A CN 201310350225A CN 103436505 B CN103436505 B CN 103436505B
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tannase
citric acid
sodium citrate
damping fluid
crude enzyme
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CN103436505A (en
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杨洋
严东
候杰
马万良
胡振兴
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Guangxi University
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Guangxi University
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Abstract

The invention discloses a preparation method of tannase. The method comprises the following steps: enlarging cultivation is performed on aspergillus ficuum slant strains through a shake flask; the mixture of corn flour and soybean meal or the mixture of bagasse and bran are taken as fermentation medium substrates; a salt solution is added to prepare into fermentation media; aspergillus ficuum suspension is added into the fermentation media; the fermentation media are fermented for 65 h at the constant temperature of 35 DEG C, and a citric acid-sodium citrate buffer solution with the pH value of 5.5 is added; concussion extraction is performed for 90 minutes, and crude enzyme is collected after the fermentation media are filtered by filter paper, tannase freeze-dried powder is prepared after the crude enzyme is purified and freeze-dried.

Description

A kind of method of preparing tannase
Technical field
The present invention relates to a kind of method of preparing tannase, belong to biological fermentation field.
Background technology
Tannase (Tannase EC3.1.1.20), claims again Tannase, is a kind of inducible enzyme, can in the time that the inductors such as Weibull exist, be induced by certain micro-organisms synthetic.Tannase can be hydrolyzed ester bond and the contracting phenol carboxylic key in Nutgalls tannin, generates gallic acid and other compounds.Tannase is widely used in the fields such as beverage, wine brewing, medicine, process hides, makeup, particularly preparing medicinal intermediate gallic acid and Food Antioxidant Propyl Gallate and processing the aspects such as tealeaves " cream down " and beer precipitation, thering is important using value.
Summary of the invention
The object of the present invention is to provide a kind of method of preparing tannase.
Object of the present invention is achieved through the following technical solutions:
A method of preparing tannase, comprises the following steps:
(1) seed enlarged culturing: by Fructus Fici aspergillus slant strains access shaking flask enlarged culturing;
(2) configuration fermention medium: make fermentation culture substrate with Semen Maydis powder, dregs of beans mixture or bagasse, bran mixture, add salts solution, sterilizing, cooling;
(3) tannase fermentation: after substratum is cooling, access 1mL Fructus Fici aspergillus suspension, ferment at constant temperature;
(4) crude enzyme liquid preparation: the fermention medium after finishing toward ferment at constant temperature adds the citric acid-sodium citrate damping fluid of pH5.5, is placed in shaking table concussion and extracts 90min, filters, and collects crude enzyme liquid;
(5) tannase purifying.
Semen Maydis powder described in step (2), dregs of beans mixture are evenly mixed with the mass ratio of 2 ︰ 1 by Semen Maydis powder, dregs of beans, bagasse, bran mixture are to be evenly mixed with 1 ︰ 1 mass ratio by bagasse, wheat bran, substratum substrate becomes 6 ︰ 7 mass volume ratio ratios with the salts solution adding, every liter of salts solution contains 10gNH 4cl, 1gKCl, 2gK 2hPO 4, 1gMgSO 47H 2o.
Fructus Fici aspergillus suspension described in step (3) is Fructus Fici aspergillus Gim3.6 (being purchased from DSMZ of Guangdong Province) suspension, and concentration is 1 × 10 8individual spore/mL, tannase fermentation condition is 35 DEG C of ferment at constant temperature 65h.
Fermention medium described in step (4) becomes 1 ︰ 10 mass volume ratios with pH5.5 citric acid-sodium citrate damping fluid, shaking table shakes speed for 160r/min.
Tannase purifying described in step (5) comprises three kinds of methods, tannase purifying comprises three kinds of methods, method one is ammonium sulfate graded precipitation, be 30%～70% ammonium sulfate toward adding saturation ratio in crude enzyme liquid, collecting precipitation, add the citric acid-sodium citrate damping fluid dissolution precipitation of 5 DEG C that becomes 1 ︰ 1 volume ratio with pH5.5 citric acid-sodium citrate damping fluid in step (4), taking dextrane gel Sephacryl200-HR as chromatography column chromatography, obtain tannase refined solution, lyophilize makes tannase lyophilized powder, method two is acetone precipitation, in crude enzyme liquid, add-20 DEG C of acetone, 4 DEG C of standing 3h, 6000r/min centrifugation 20min, collecting precipitation, adds the citric acid-sodium citrate damping fluid dissolution precipitation of 5 DEG C that becomes 1 ︰ 1 volume ratio with pH5.5 citric acid-sodium citrate damping fluid in step (4), taking dextrane gel Sephacryl200-HR as chromatography column chromatography, obtain tannase refined solution, lyophilize makes tannase lyophilized powder, method three is reverse micelle extraction methods, using the crude enzyme liquid of collecting as water, add organic phase 75mmol/LCTAB-isooctane Reversed Micelles solution with 1 ︰ 1 volume ratio, on 200r/min shaking table, concussion mixes 13min, 4000r/min centrifugation 5min, get organic phase, add strip aqueous 0.5mol/L NaCl solution by 1:1 volume ratio, on 200r/min shaking table, concussion mixes 30min, 4000r/min centrifugation 5min, obtain water, concentrated with polyethylene glycol 6000 embedding, taking dextrane gel Sephacryl200-HR as chromatography column chromatography, obtain tannase refined solution, lyophilize makes tannase lyophilized powder.
With Fructus Fici aspergillus fermentative production tannase, fermentation period is short, and zymotechnique is simple, and culturing process is easy to control.Reverse micelle extraction technology is carried out purifying to crude enzyme liquid, economic and practical, easily carries out suitability for industrialized production.
Brief description of the drawings
Fig. 1 is tannase preparation technology flow process of the present invention.
Embodiment
Below in conjunction with specific embodiment, the present invention is done to further detailed elaboration, but embodiments of the present invention are not limited to the scope that embodiment represents.These embodiment are only for the present invention is described, but not for limiting the scope of the invention.In addition, reading after content of the present invention, those skilled in the art can do various amendments to the present invention, and these equivalent variations fall within appended claims limited range of the present invention equally.
Embodiment 1
Fructus Fici aspergillus Gim3.6 (being purchased from DSMZ of Guangdong Province) slant strains is through shaking flask enlarged culturing.Semen Maydis powder, dregs of beans evenly mix as fermentation culture substrate by the mass ratio of 2 ︰ 1, and (every liter of salts solution contains 10gNH to add salts solution by 6 ︰ 7 mass volume ratios 4cl, 1gKCl, 2gK 2hPO 4, 1gMgSO 47H 2o.), sterilizing, cooling.Access concentration is 1 × 10 8the Fructus Fici aspergillus suspension 1mL of individual spore/mL, 35 DEG C of ferment at constant temperature 65h.After ferment at constant temperature finishes, by 1 ︰ 10 mass volume ratios, the citric acid-sodium citrate damping fluid of pH5.5 is added in fermention medium, be placed in and shake concussion extraction 90min on the shaking table that speed is 160r/min, filter, collect crude enzyme liquid.Be 30% ammonium sulfate toward adding saturation ratio in crude enzyme liquid, collecting precipitation, add the citric acid-sodium citrate damping fluid dissolution precipitation of 5 DEG C that becomes 1 ︰ 1 volume ratio with pH5.5 citric acid-sodium citrate damping fluid, taking dextrane gel Sephacryl200-HR as chromatography column chromatography, obtain tannase refined solution, lyophilize makes tannase lyophilized powder.
Embodiment 2
Fructus Fici aspergillus Gim3.6 (being purchased from DSMZ of Guangdong Province) slant strains is through shaking flask enlarged culturing.Semen Maydis powder, dregs of beans evenly mix as fermentation culture substrate by the mass ratio of 2 ︰ 1, and (every liter of salts solution contains 10gNH to add salts solution by 6 ︰ 7 mass volume ratios 4cl, 1gKCl, 2gK 2hPO 4, 1gMgSO 47H 2o.), sterilizing, cooling.Access concentration is 1 × 10 8the Fructus Fici aspergillus suspension 1mL of individual spore/mL, 35 DEG C of ferment at constant temperature 65h.After ferment at constant temperature finishes, by 1 ︰ 10 mass volume ratios, the citric acid-sodium citrate damping fluid of pH5.5 is added in fermention medium, be placed in and shake concussion extraction 90min on the shaking table that speed is 160r/min, filter, collect crude enzyme liquid.Be 50% ammonium sulfate toward adding saturation ratio in crude enzyme liquid, collecting precipitation, add the citric acid-sodium citrate damping fluid dissolution precipitation of 5 DEG C that becomes 1 ︰ 1 volume ratio with pH5.5 citric acid-sodium citrate damping fluid, taking dextrane gel Sephacryl200-HR as chromatography column chromatography, obtain tannase refined solution, lyophilize makes tannase lyophilized powder.
Embodiment 3
Fructus Fici aspergillus Gim3.6 (being purchased from DSMZ of Guangdong Province) slant strains is through shaking flask enlarged culturing.Bagasse, wheat bran evenly mix as fermentation culture substrate by 1 ︰ 1 mass ratio, and (every liter of salts solution contains 10gNH to add salts solution by 6 ︰ 7 mass volume ratios 4cl, 1gKCl, 2gK 2hPO 4, 1gMgSO 47H 2o.), sterilizing, cooling.Access concentration is 1 × 10 8the Fructus Fici aspergillus suspension 1mL of individual spore/mL, 35 DEG C of ferment at constant temperature 65h.After ferment at constant temperature finishes, by 1 ︰ 10 mass volume ratios, the citric acid-sodium citrate damping fluid of pH5.5 is added in fermention medium, be placed in and shake concussion extraction 90min on the shaking table that speed is 160r/min, filter, collect crude enzyme liquid.Be 70% ammonium sulfate toward adding saturation ratio in crude enzyme liquid, collecting precipitation, add the citric acid-sodium citrate damping fluid dissolution precipitation of 5 DEG C that becomes 1 ︰ 1 volume ratio with pH5.5 citric acid-sodium citrate damping fluid, taking dextrane gel Sephacryl200-HR as chromatography column chromatography, obtain tannase refined solution, lyophilize makes tannase lyophilized powder.
Embodiment 4
Fructus Fici aspergillus Gim3.6 (being purchased from DSMZ of Guangdong Province) slant strains is through shaking flask enlarged culturing.Bagasse, wheat bran evenly mix as fermentation culture substrate by 1 ︰ 1 mass ratio, and (every liter of salts solution contains 10gNH to add salts solution by 6 ︰ 7 mass volume ratios 4cl, 1gKCl, 2gK 2hPO 4, 1gMgSO 47H 2o.), sterilizing, cooling.Access concentration is 1 × 10 8the Fructus Fici aspergillus suspension 1mL of individual spore/mL, 35 DEG C of ferment at constant temperature 65h.After ferment at constant temperature finishes, by 1 ︰ 10 mass volume ratios, the citric acid-sodium citrate damping fluid of pH5.5 is added in fermention medium, be placed in and shake concussion extraction 90min on the shaking table that speed is 160r/min, with double-deck qualitative filter paper filtration, collect crude enzyme liquid, add-20 DEG C of acetone, 4 DEG C of standing 3h, 6000r/min centrifugation 20min, collecting precipitation, add the citric acid-sodium citrate damping fluid dissolution precipitation of 5 DEG C that becomes 1 ︰ 1 volume ratio with pH5.5 citric acid-sodium citrate damping fluid, taking dextrane gel Sephacryl200-HR as chromatography column chromatography, obtain tannase refined solution, lyophilize makes tannase lyophilized powder.
Embodiment 5
Fructus Fici aspergillus Gim3.6 (being purchased from DSMZ of Guangdong Province) slant strains is through shaking flask enlarged culturing.Semen Maydis powder, dregs of beans evenly mix as fermentation culture substrate by the mass ratio of 2 ︰ 1, and (every liter of salts solution contains 10gNH to add salts solution by 6 ︰ 7 mass volume ratios 4cl, 1gKCl, 2gK 2hPO 4, 1gMgSO 47H 2o.), sterilizing, cooling.Access concentration is 1 × 10 8the Fructus Fici aspergillus suspension 1mL of individual spore/mL, 35 DEG C of ferment at constant temperature 65h.After ferment at constant temperature finishes, by 1 ︰ 10 mass volume ratios, the citric acid-sodium citrate damping fluid of pH5.5 is added in fermention medium, be placed in and shake concussion extraction 90min on the shaking table that speed is 160r/min, filter, collect crude enzyme liquid.Using the crude enzyme liquid of collecting as water, be the CTAB-isooctane Reversed Micelles solution that adds organic phase 75mmol/L under the condition of 30 DEG C by the volume ratio of 1 ︰ 1 in temperature, on the shaking table that is 200r/min at rotating speed, concussion mixes 13min, 4000r/min rotating speed centrifugation 5min, get organic phase, add the NaCl solution of strip aqueous 0.5mol/L by the volume ratio of 1 ︰ 1, on the shaking table that is 200r/min at rotating speed, concussion mixes 30min, with 4000r/min rotating speed centrifugation 5min, obtain water; With polyethylene glycol 6000, the water embedding of obtaining is concentrated, taking dextrane gel Sephacryl200-HR as chromatography column, loading, wash-out, obtain tannase refined solution, and lyophilize makes tannase lyophilized powder.
With Fructus Fici aspergillus fermentative production tannase, fermentation period is short, and zymotechnique is simple, and culturing process is easy to control.Reverse micelle extraction technology is carried out purifying to crude enzyme liquid, economical and practical, easily carries out suitability for industrialized production.

Claims (6)

1. a method of preparing tannase, comprises the following steps:
(1) seed enlarged culturing: by Fructus Fici aspergillus slant strains access shaking flask enlarged culturing;
(2) configuration fermention medium: make fermentation culture substrate with Semen Maydis powder, dregs of beans mixture or bagasse, bran mixture, add salts solution, sterilizing, cooling, described Semen Maydis powder, dregs of beans mixture are evenly mixed with the mass ratio of 2 ︰ 1 by Semen Maydis powder, dregs of beans, bagasse, bran mixture are to be evenly mixed with 1 ︰ 1 mass ratio by bagasse, wheat bran, substratum substrate becomes 6 ︰ 7 mass volume ratio ratios with the salts solution adding, every liter of salts solution contains 10gNH 4cl, 1gKCl, 2gK 2hPO 4, 1gMgSO 47H 2o;
(3) tannase fermentation: after substratum is cooling, access 1mL Fructus Fici aspergillus tubigensis Gim3.6 suspension, concentration is 1 × 10 8individual spore/mL, 35 DEG C of ferment at constant temperature 65h;
(4) crude enzyme liquid preparation: the fermention medium after finishing toward ferment at constant temperature adds the citric acid-sodium citrate damping fluid of pH5.5, is placed in shaking table concussion and extracts 90min, filters, and collects crude enzyme liquid;
(5) tannase purifying.
2. according to a kind of method of preparing tannase described in right 1, it is characterized in that: the fermention medium described in step (4) becomes 1 ︰ 10 mass volume ratios with pH5.5 citric acid-sodium citrate damping fluid, shaking table shakes speed for 160r/min.
3. according to the method for preparing tannase described in right 1, it is characterized in that: described tannase purifying can adopt any purification process in ammonium sulfate graded precipitation, acetone precipitation, three methods of reverse micelle extraction method.
4. according to the method for preparing tannase described in right 3, it is characterized in that: described ammonium sulfate graded precipitation concrete operations are, be 30%～70% ammonium sulfate toward adding saturation ratio in crude enzyme liquid, collecting precipitation, add the citric acid-sodium citrate damping fluid dissolution precipitation of 5 DEG C that becomes 1 ︰ 1 volume ratio with pH5.5 citric acid-sodium citrate damping fluid in step (4), taking dextrane gel Sephacryl200-HR as chromatography column chromatography, obtain tannase refined solution, lyophilize makes tannase lyophilized powder.
5. according to the method for preparing tannase described in right 3, it is characterized in that: described acetone precipitation concrete operations are, in crude enzyme liquid, add-20 DEG C of acetone, 4 DEG C of standing 3h, 6000r/min centrifugation 20min, collecting precipitation, add the citric acid-sodium citrate damping fluid dissolution precipitation of 5 DEG C that becomes 1 ︰ 1 volume ratio with pH5.5 citric acid-sodium citrate damping fluid in step (4), taking dextrane gel Sephacryl200-HR as chromatography column chromatography, obtain tannase refined solution, lyophilize makes tannase lyophilized powder.
6. according to the method for preparing tannase described in right 3, it is characterized in that: the concrete operations of described reverse micelle extraction method are, using the crude enzyme liquid of collecting as water, add organic phase 75mmol/L CTAB-isooctane Reversed Micelles solution with 1 ︰ 1 volume ratio, on 200r/min shaking table, concussion mixes 13min, 4000r/min centrifugation 5min, get organic phase, add strip aqueous 0.5mol/L NaCl solution by 1:1 volume ratio, on 200r/min shaking table, concussion mixes 30min, 4000r/min centrifugation 5min, obtain water, concentrated with polyethylene glycol 6000 embedding, taking dextrane gel Sephacryl200-HR as chromatography column chromatography, obtain tannase refined solution, lyophilize makes tannase lyophilized powder.
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CN104195121A (en) * 2014-09-05 2014-12-10 广西大学 Solid culture medium for cassava leaves as well as preparation method and application of solid culture medium
CN104263706A (en) * 2014-10-15 2015-01-07 广西大学 Purification method for tannase
CN104962544B (en) * 2015-06-17 2017-11-03 集美大学 A kind of method of the immobilized tannase directly from zymotic fluid
CN107904219B (en) * 2017-12-29 2020-04-03 集美大学 Preparation method and application of tannase solid-state fermentation medium

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