CN103436505A - Preparation method of tannase - Google Patents

Preparation method of tannase Download PDF

Info

Publication number
CN103436505A
CN103436505A CN2013103502255A CN201310350225A CN103436505A CN 103436505 A CN103436505 A CN 103436505A CN 2013103502255 A CN2013103502255 A CN 2013103502255A CN 201310350225 A CN201310350225 A CN 201310350225A CN 103436505 A CN103436505 A CN 103436505A
Authority
CN
China
Prior art keywords
tannase
citric acid
sodium citrate
damping fluid
add
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.)
Granted
Application number
CN2013103502255A
Other languages
Chinese (zh)
Other versions
CN103436505B (en
Inventor
杨洋
严东
候杰
马万良
胡振兴
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangxi University
Original Assignee
Guangxi University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Guangxi University filed Critical Guangxi University
Priority to CN201310350225.5A priority Critical patent/CN103436505B/en
Publication of CN103436505A publication Critical patent/CN103436505A/en
Application granted granted Critical
Publication of CN103436505B publication Critical patent/CN103436505B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Landscapes

  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

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 for preparing tannase
Technical field
The present invention relates to a kind of method for preparing tannase, belong to the biological fermentation field.
Background technology
Tannase (Tannase EC3.1.1.20), claim again Tannase, is a kind of inducible enzyme, can when the inductors such as Weibull exist, induce synthetic by certain micro-organisms.Tannase can be hydrolyzed ester bond and the contracting phenol carboxylic key in the 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 for preparing tannase.
Purpose of the present invention is achieved through the following technical solutions:
A kind of method for 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 the 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 the 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 evenly to be mixed with 1 ︰ 1 mass ratio by bagasse, wheat bran, the substratum substrate becomes 6 ︰ 7 mass volume ratio ratios with the salts solution added, 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 Guangdong Province DSMZ) suspension, and concentration is 1 * 10 8individual spore/mL, the tannase fermentation condition is 35 ℃ 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, the tannase purifying comprises three kinds of methods, method one is ammonium sulfate graded precipitation, toward adding saturation ratio in crude enzyme liquid, be 30%～70% ammonium sulfate, collecting precipitation, add the citric acid-sodium citrate damping fluid dissolution precipitation of 5 ℃ that becomes 1 ︰ 1 volume ratio with pH5.5 citric acid-sodium citrate damping fluid in step (4), take dextrane gel Sephacryl200-HR as the chromatography column chromatography, obtain the tannase refined solution, lyophilize makes the tannase lyophilized powder, method two is acetone precipitation, add-20 ℃ of acetone in crude enzyme liquid, 4 ℃ of standing 3h, 6000r/min centrifugation 20min, collecting precipitation, add the citric acid-sodium citrate damping fluid dissolution precipitation of 5 ℃ that becomes 1 ︰ 1 volume ratio with pH5.5 citric acid-sodium citrate damping fluid in step (4), take dextrane gel Sephacryl200-HR as the chromatography column chromatography, obtain the tannase refined solution, lyophilize makes the tannase lyophilized powder, method three is reverse micelle extraction methods, using the crude enzyme liquid collected as water, add organic phase 75mmol/LCTAB-isooctane Reversed Micelles solution with 1 ︰ 1 volume ratio, on the 200r/min shaking table, concussion mixes 13min, 4000r/min centrifugation 5min, get organic phase, add strip aqueous 0.5mol/L NaCl solution by the 1:1 volume ratio, on the 200r/min shaking table, concussion mixes 30min, 4000r/min centrifugation 5min, obtain water, concentrated with the polyethylene glycol 6000 embedding, take dextrane gel Sephacryl200-HR as the chromatography column chromatography, obtain the tannase refined solution, lyophilize makes the 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.The reverse micelle extraction technology is carried out purifying to crude enzyme liquid, economic and practical, easily carries out suitability for industrialized production.
The accompanying drawing explanation
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 means.These embodiment are only for the present invention is described, but not for limiting the scope of the invention.In addition, after reading content of the present invention, those skilled in the art can do various modifications 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 Guangdong Province DSMZ) slant strains is through the shaking flask enlarged culturing.Semen Maydis powder, dregs of beans evenly mix as the 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 ℃ of ferment at constant temperature 65h.Ferment at constant temperature adds the citric acid-sodium citrate damping fluid of pH5.5 in fermention medium by 1 ︰ 10 mass volume ratios after finishing, and is placed in and shakes concussion extraction 90min on the shaking table that speed is 160r/min, filters, and collects crude enzyme liquid.Toward adding saturation ratio in crude enzyme liquid, be 30% ammonium sulfate, collecting precipitation, add the citric acid-sodium citrate damping fluid dissolution precipitation of 5 ℃ that becomes 1 ︰ 1 volume ratio with pH5.5 citric acid-sodium citrate damping fluid, take dextrane gel Sephacryl200-HR as the chromatography column chromatography, obtain the tannase refined solution, lyophilize makes the tannase lyophilized powder.
Embodiment 2
Fructus Fici aspergillus Gim3.6 (being purchased from Guangdong Province DSMZ) slant strains is through the shaking flask enlarged culturing.Semen Maydis powder, dregs of beans evenly mix as the 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 ℃ of ferment at constant temperature 65h.Ferment at constant temperature adds the citric acid-sodium citrate damping fluid of pH5.5 in fermention medium by 1 ︰ 10 mass volume ratios after finishing, and is placed in and shakes concussion extraction 90min on the shaking table that speed is 160r/min, filters, and collects crude enzyme liquid.Toward adding saturation ratio in crude enzyme liquid, be 50% ammonium sulfate, collecting precipitation, add the citric acid-sodium citrate damping fluid dissolution precipitation of 5 ℃ that becomes 1 ︰ 1 volume ratio with pH5.5 citric acid-sodium citrate damping fluid, take dextrane gel Sephacryl200-HR as the chromatography column chromatography, obtain the tannase refined solution, lyophilize makes the tannase lyophilized powder.
Embodiment 3
Fructus Fici aspergillus Gim3.6 (being purchased from Guangdong Province DSMZ) slant strains is through the shaking flask enlarged culturing.Bagasse, wheat bran evenly mix as the 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 ℃ of ferment at constant temperature 65h.Ferment at constant temperature adds the citric acid-sodium citrate damping fluid of pH5.5 in fermention medium by 1 ︰ 10 mass volume ratios after finishing, and is placed in and shakes concussion extraction 90min on the shaking table that speed is 160r/min, filters, and collects crude enzyme liquid.Toward adding saturation ratio in crude enzyme liquid, be 70% ammonium sulfate, collecting precipitation, add the citric acid-sodium citrate damping fluid dissolution precipitation of 5 ℃ that becomes 1 ︰ 1 volume ratio with pH5.5 citric acid-sodium citrate damping fluid, take dextrane gel Sephacryl200-HR as the chromatography column chromatography, obtain the tannase refined solution, lyophilize makes the tannase lyophilized powder.
Embodiment 4
Fructus Fici aspergillus Gim3.6 (being purchased from Guangdong Province DSMZ) slant strains is through the shaking flask enlarged culturing.Bagasse, wheat bran evenly mix as the 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 ℃ 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, filter, collect crude enzyme liquid, add-20 ℃ of acetone, 4 ℃ of standing 3h, 6000r/min centrifugation 20min, collecting precipitation, add the citric acid-sodium citrate damping fluid dissolution precipitation of 5 ℃ that becomes 1 ︰ 1 volume ratio with pH5.5 citric acid-sodium citrate damping fluid, take dextrane gel Sephacryl200-HR as the chromatography column chromatography, obtain the tannase refined solution, lyophilize makes the tannase lyophilized powder.
Embodiment 5
Fructus Fici aspergillus Gim3.6 (being purchased from Guangdong Province DSMZ) slant strains is through the shaking flask enlarged culturing.Semen Maydis powder, dregs of beans evenly mix as the 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 ℃ of ferment at constant temperature 65h.Ferment at constant temperature adds the citric acid-sodium citrate damping fluid of pH5.5 in fermention medium by 1 ︰ 10 mass volume ratios after finishing, and is placed in and shakes concussion extraction 90min on the shaking table that speed is 160r/min, filters, and collects crude enzyme liquid.Using the crude enzyme liquid collected as water, add the CTAB-isooctane Reversed Micelles solution of organic phase 75mmol/L under the condition that is 30 ℃ in temperature by the volume ratio of 1 ︰ 1, 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, that the water embedding of obtaining is concentrated, take dextrane gel Sephacryl200-HR as chromatography column, loading, wash-out, obtain the tannase refined solution, and lyophilize makes the 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.The reverse micelle extraction technology is carried out purifying to crude enzyme liquid, economical and practical, easily carries out suitability for industrialized production.

Claims (8)

1. a method for 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 the 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 the shaking table concussion and extracts 90min, filters, and collects crude enzyme liquid;
(5) tannase purifying.
2. according to the described method for preparing tannase of right 1, it is characterized in that: the 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 evenly to be mixed with 1 ︰ 1 mass ratio by bagasse, wheat bran, the substratum substrate becomes 6 ︰ 7 mass volume ratio ratios with the salts solution added, every liter of salts solution contains 10gNH 4cl, 1gKCl, 2gK 2hPO 4, 1gMgSO 47H 2o.
3. according to the described method for preparing tannase of right 1, it is characterized in that: the Fructus Fici aspergillus suspension described in step (3) is Fructus Fici aspergillus Gim3.6 (being purchased from Guangdong Province DSMZ) suspension, and concentration is 1 * 10 8individual spore/mL, the tannase fermentation condition is 35 ℃ of ferment at constant temperature 65h.
4. according to a kind of described method for preparing tannase of 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, and shaking table shakes speed for 160r/min.
5. according to the described method for preparing tannase of right 1, it is characterized in that: the tannase purifying comprises three kinds of methods.
6. according to the described tannase purifying of right 1 or 5, it is characterized in that: first method is ammonium sulfate graded precipitation, toward adding saturation ratio in crude enzyme liquid, be 30%～70% ammonium sulfate, collecting precipitation, add the citric acid-sodium citrate damping fluid dissolution precipitation of 5 ℃ that becomes 1 ︰ 1 volume ratio with pH5.5 citric acid-sodium citrate damping fluid in step (4), take dextrane gel Sephacryl200-HR as the chromatography column chromatography, obtain the tannase refined solution, lyophilize makes the tannase lyophilized powder.
7. according to the described tannase purifying of right 1 or 5, it is characterized in that: second acetone precipitation of tannase purifying, add-20 ℃ of acetone in crude enzyme liquid, 4 ℃ of standing 3h, 6000r/min centrifugation 20min, collecting precipitation, add the citric acid-sodium citrate damping fluid dissolution precipitation of 5 ℃ that becomes 1 ︰ 1 volume ratio with pH5.5 citric acid-sodium citrate damping fluid in step (4), take dextrane gel Sephacryl200-HR as the chromatography column chromatography, obtain the tannase refined solution, lyophilize makes the tannase lyophilized powder.
8. according to the described tannase purifying of right 1 or 5, it is characterized in that: the 3rd method of tannase purifying is the reverse micelle extraction method, using the crude enzyme liquid collected as water, add organic phase 75mmol/L CTAB-isooctane Reversed Micelles solution with 1 ︰ 1 volume ratio, on the 200r/min shaking table, concussion mixes 13min, 4000r/min centrifugation 5min, get organic phase, add strip aqueous 0.5mol/L NaCl solution by the 1:1 volume ratio, on the 200r/min shaking table, concussion mixes 30min, 4000r/min centrifugation 5min, obtain water, concentrated with the polyethylene glycol 6000 embedding, take dextrane gel Sephacryl200-HR as the chromatography column chromatography, obtain the tannase refined solution, lyophilize makes the tannase lyophilized powder.
CN201310350225.5A 2013-08-13 2013-08-13 Preparation method of tannase Active CN103436505B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201310350225.5A CN103436505B (en) 2013-08-13 2013-08-13 Preparation method of tannase

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201310350225.5A CN103436505B (en) 2013-08-13 2013-08-13 Preparation method of tannase

Publications (2)

Publication Number Publication Date
CN103436505A true CN103436505A (en) 2013-12-11
CN103436505B CN103436505B (en) 2014-09-17

Family

ID=49690223

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201310350225.5A Active CN103436505B (en) 2013-08-13 2013-08-13 Preparation method of tannase

Country Status (1)

Country Link
CN (1) CN103436505B (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN104962544A (en) * 2015-06-17 2015-10-07 集美大学 Method for directly immobilizing tannase in fermentation liquor
CN107904219A (en) * 2017-12-29 2018-04-13 集美大学 A kind of tannase solid-state fermentation culture medium preparation method and applications

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1289665C (en) * 2004-06-09 2006-12-13 中山大学 Recombined aspergillus oryzae tannase and its expression and purification
CN102203248A (en) * 2008-10-24 2011-09-28 天野酶株式会社 Tannase, gene encoding same, and process for producing same
CN102796716A (en) * 2012-08-10 2012-11-28 集美大学 Method for preparing tannase
CN102260656B (en) * 2011-05-31 2012-12-12 天津市畜牧兽医研究所 Recombinant aspergillus niger tannase and expression and purification method thereof
WO2013020189A1 (en) * 2011-08-05 2013-02-14 Universidade Estadual De Campinas - Unicamp Media for the culture of waste castor-oil-plant cake for simultaneous production of the phytase and thanase enzymes, and detoxification of the castor-oil-plant cake, using the microorganism paecilomyces variotii and using solid fermentation, the enzymes obtained and the uses thereof
CN103215193A (en) * 2013-04-28 2013-07-24 曹庸 Tannase high-producing strain and preparation method thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1289665C (en) * 2004-06-09 2006-12-13 中山大学 Recombined aspergillus oryzae tannase and its expression and purification
CN102203248A (en) * 2008-10-24 2011-09-28 天野酶株式会社 Tannase, gene encoding same, and process for producing same
CN102260656B (en) * 2011-05-31 2012-12-12 天津市畜牧兽医研究所 Recombinant aspergillus niger tannase and expression and purification method thereof
WO2013020189A1 (en) * 2011-08-05 2013-02-14 Universidade Estadual De Campinas - Unicamp Media for the culture of waste castor-oil-plant cake for simultaneous production of the phytase and thanase enzymes, and detoxification of the castor-oil-plant cake, using the microorganism paecilomyces variotii and using solid fermentation, the enzymes obtained and the uses thereof
CN102796716A (en) * 2012-08-10 2012-11-28 集美大学 Method for preparing tannase
CN103215193A (en) * 2013-04-28 2013-07-24 曹庸 Tannase high-producing strain and preparation method thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
H.V.DIFO等: "Isolation of Aspergillus niger from three varieties of Bambara nuts for simultaneous production of phytase and tannase", 《JOURNAL OF YEAST AND FUNGAL RESEARCH》 *
余钧池等: "米曲霉单宁酶产酶条件及其应用的研究", 《广西轻工业》 *
杨全明等: "饲料酶制剂的研究与应用", 《云南畜牧兽医》 *
郭鲁宏等: "单宁酶产生菌的筛选和发酵条件研究", 《应用与环境生物学报》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN104962544A (en) * 2015-06-17 2015-10-07 集美大学 Method for directly immobilizing tannase in fermentation liquor
CN104962544B (en) * 2015-06-17 2017-11-03 集美大学 A kind of method of the immobilized tannase directly from zymotic fluid
CN107904219A (en) * 2017-12-29 2018-04-13 集美大学 A kind of tannase solid-state fermentation culture medium preparation method and applications

Also Published As

Publication number Publication date
CN103436505B (en) 2014-09-17

Similar Documents

Publication Publication Date Title
CN102696811B (en) Preparation method of instant black tea
CN101491326B (en) Perilla sauce and preparation technique thereof
CN103305396B (en) Method for producing cordyceps vinegar by use of cordyceps taishanensis fermentation liquor
CN103436505B (en) Preparation method of tannase
CN103131590B (en) Brewage method of Chinese caterpillar fungus health-care yellow rice wine
CN103421645A (en) Walnut polypeptide liquor and production method thereof
CN105368646A (en) Method for preparing maple-flavor Baijiu by sugar maple
CN104031816B (en) A kind of working method of fermented type Paeonia suffruticosa vinegar
CN104726250B (en) The brewage process of chestnut flower boy's wine
CN105441271A (en) Brewing method of similar soybean-flavor baijiu
CN103923900A (en) Preparation method and application of cross-linked enzyme aggregate of bifunctional enzyme for rice wine
CN102628020B (en) Walnut-red date wine and brewing method thereof
CN104489134A (en) Method for making fermented tea beverage
CN104711143A (en) Dioscorea polystachya wine preparation method
CN102388996A (en) Method for producing instant fermented tea powder through liquid fermentation of microorganisms
CN104171155A (en) Preparation method of oolong tea drink
CN107625018A (en) A kind of preparation method of fermented ginger leaf beverage
CN106106910B (en) Preparation method of tea concentrated solution
CN107034095A (en) The manufacture method of full juice Lichee sweet wine
CN103462119B (en) A kind of fermented type passiflora edulis vinegar beverage and preparation technology thereof
CN103421637A (en) Mixed fruit wine and preparation method thereof
CN105112263A (en) Method for producing flavouring lotion by using saccharomycopsis fibuligera
CN105400858A (en) Preparing method for cordyceps militaris polypeptide
CN102154078B (en) Preparation method of nutrient solution required by cultivating strong aromatic Chinese spirits pit mud
CN102805171B (en) Process for utilizing tea comprehensively

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