CN102604902B - Method for preparing laccase by liquid fermentation of Pleurotus ferulae - Google Patents
Method for preparing laccase by liquid fermentation of Pleurotus ferulae Download PDFInfo
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- CN102604902B CN102604902B CN 201210094741 CN201210094741A CN102604902B CN 102604902 B CN102604902 B CN 102604902B CN 201210094741 CN201210094741 CN 201210094741 CN 201210094741 A CN201210094741 A CN 201210094741A CN 102604902 B CN102604902 B CN 102604902B
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Abstract
The invention discloses a method for preparing laccase by liquid fermentation of Pleurotus ferulae, belonging to the technical field of microbial fermentation. The method provided by the invention takes Pleurotus ferulae as a production strain, generates laccase by liquid fermentation, and is realized by the following main steps: (1) slant culture; (2) seed culture; and (3) liquid fermentation culture. Cultivated seed culture is inoculated into a liquid fermentation medium at an inoculum size of 4%, and cultivated at 22-26 DEG C for 7 days under the shaker rotation speed of 150rpm, wherein the fermentation medium comprises the following components: 15-20g/L of glucose, 10-20g/L of corn flour, 10-15g/L of wheat bran, 3g/L of KH2PO4 and 2g/L of MgSO4.7H2O, and the initial pH value is 7.0-9.0. The method realizes a goal of generating laccase with Pleurotus ferulae, and the fermentation level of Pleurotus ferulae reaches or exceeds the fermentation level of other Pleurotus fungi. The new method for generating laccase with the microbial fermentation is provided, and in addition, a new direction for application of Pleurotus ferulae in fermentation industry is developed.
Description
Technical field
A kind of method of utilizing the Resina Ferulae mushroom liquid fermenting to produce laccase belongs to the microbial fermentation field.
Background technology
Laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) is a kind of polyphenoloxidase of cupric, is distributed widely in plant, bacterium and fungi, in addition, has also found the existence of laccase in some insects.Laccase is the one-electron oxidation reductase enzyme, and its catalytic substrate comprises phenol and derivative, arylamine and derivative thereof, carboxylic acid and derivative thereof and some metal ions etc.Because the effect substrate is extensive, laccase has huge value for fields such as, novel environment friendly material developments dye decolored in foodstuffs industry, pulping and paper-making, synthetic.
In foodstuffs industry, laccase is widely used in controlling clarification and the color and luster of beverage.Traditional method is that beverage is carried out adsorption treatment through materials such as gel, bentonite and silicon-dioxide, thereby increases its stability.Compare with traditional method, utilize laccase to stablize fruit juice and have plurality of advantages, stablize, need not adopt finings or flocculating aids and be beneficial to automatization as clarifying effect and process.There is result of study to show, utilizes laccase treatment fruit juice can obtain fruit juice stable and clarification, and do not need too much investment.
In paper-making technique, pulping process mainly needs the fiber substance in wood material, and in wood material, the tight glue of fibrous matter and xylogen connects together, therefore need to adopt the method for chemistry or physics, degraded or removal xylogen just can reach the purpose of separation.Laccase has the ability of good lignin degrading, and the shortcomings such as, pollution high without energy consumption in chemical method and Physical is large, therefore have broad application prospects in paper-making pulping, and the ability of the good lignin degrading of laccase, well solved the existence of xylogen to the impact of intensity and the color of paper.
In textile industry, 10%-20% dyestuff is arranged along with waste water is discharged in environment every year.These dyestuff great majority are for having anti-illumination, oxidation resistant difficult degradation arene compounds, and this class material has not only caused harm to environment, also owing to himself having poisonous, carcinogenic substance, HUMAN HEALTH has also been caused threat.To the substrates such as single phenol, pyrocatechol and Resorcinol oxidative degradation preferably, make it be widely used in the decolored degradation of dyestuff due to laccase.Laccase not only can be applied to the processing of textile waste, can also participate directly in the production link of denim dyeing, have the loss of strength that reduces denim, reduce the treatment time, laccase can reuse, reduce water loss and avoid the plurality of advantages such as pungency chemical substance bleaching.
Resina Ferulae mushroom (Pleurotus ferulae Lenzi), the formal name used at school Pleurotus ferulae Lanzi, being the distinctive mushroom class in Xinjiang in China, mainly being distributed in the areas such as Yi Li, Tacheng, Altay and wooden base in Xinjiang, is a kind of important edible and medicinal fungi that colonizes on the corrupt rhizome of medicinal plant " asafoetide ".
At present, have been found that the multiple class fungi of picking up the ears with laccase production ability, as oyster cap fungus (flat mushroom), eryngo pick up the ears (Pleurotus eryngii), handle pick up the ears (Pleurotus sajor-caju) etc., but not relevant for correlative study and the report of Pleurotus ferulae Lanzi (Resina Ferulae mushroom) fermentation lacquer producing enzyme.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing Resina Ferulae mushroom to produce laccase.
Technical scheme of the present invention: concrete, realize by following steps.(1) slant culture is inoculated into the Resina Ferulae mushroom bacterial classification that obtains through separate tissue on slant medium, cultivates under 25 ℃ of conditions, covers with the inclined-plane to mycelium; (2) seed culture is inoculated into slant strains in seed culture medium, at 25 ℃, cultivates 7 days under the condition that shaking speed is 150 rev/mins; (3) fermentation culture is inoculated into cultured seed in fermention medium, inoculum size 4%(volume percent), at 22-26 ℃, cultivated 7 days under the condition that shaking speed is 150 rev/mins.
The Resina Ferulae mushroom bacterial classification that the present invention uses is to obtain through separate tissue by the wild Resina Ferulae mushroom that picks up from Xinjiang.The method of separate tissue all has statement in multiple textbook and paper, list no longer in detail here (for example compile the Academy of Agricultural Sciences, Shanghai City, " the 12nd-13 page of edible fungus culturing handbook,, press of editorial office of edible mushrooms periodical publisher in 1981).
Slant medium of the present invention is potato substratum (PDA substratum), forms (unit grams per liter): potato 200, and glucose 20, agar 20 with tap water preparation, pH nature, was sterilized 20 minutes for 121 ℃.
Seed culture medium of the present invention forms (unit grams per liter): glucose 20, Semen Maydis powder 20, wheat bran 10, KH
2PO
43, MgSO
47H
2O 2, with tap water preparation, pH nature, sterilize 20 minutes for 121 ℃.
The applied fermention medium of the present invention forms (unit grams per liter): glucose 15-20, Semen Maydis powder 10 – 20, wheat bran 10-15, KH
2PO
43, MgSO
47H
2O 2, and with the tap water preparation, initial pH is 7.0-9.0,121 ℃ of sterilizations 20 minutes.
The measuring method of Laccase activity sees embodiment for details.
Beneficial effect of the present invention: the realization of success of the present invention utilize Resina Ferulae mushroom to produce the technique of laccase, its fermentation level meets or exceeds the fermentation level of other class fungies of picking up the ears.This provides a kind of new method for microbial fermentation produces laccase, also goes out a kind of new direction for the application and development of Resina Ferulae mushroom on fermentation industry simultaneously.
Embodiment
In order further to set forth related material and the technique of technical scheme of the present invention, following examples have been provided.But the scope that these embodiment do not limit the present invention in any way.
The separate tissue of embodiment 1, Resina Ferulae mushroom bacterial classification
Get the sporophore of the fresh not parachute-opening of wild Resina Ferulae mushroom of picking up from In Altay, xinjiang, prune stem base portion and stem exterior skin after carrying out the sporophore surface sterilization with 75% ethanol, move into Bechtop ultra violet lamp 20 minutes.With the cooled scalper of the bacterium of going out, sporophore is divided into two, in stem or in stem, stem and cap intersection, cap and lamella place's cutting epidermis inner tissue, tissue is cut into the fritter of soya bean size, can not cut sporophore face tissue during cutting.With aseptic nipper access test tube slant substratum (being the PDA substratum), each tissue part respectively inoculates 10 test tube slants, 25 ℃ of cultivations, and every day, observation at interval mycelial growth pollution condition, when growing to 7 days, used the test tube slant switching of PDA substratum.
Embodiment 2, Resina Ferulae mushroom liquid fermenting produce laccase
(1) slant culture: the Resina Ferulae mushroom bacterial classification that embodiment 1 is obtained is inoculated on slant medium (being the PDA substratum), cultivates about 7 day time under 25 ℃ of environment, covers with the inclined-plane to mycelium;
(2) seed culture: slant strains is inoculated in the triangular flask of 250 milliliters that 60 milliliters of seed culture mediums are housed, seed culture medium forms (unit grams per liter): glucose 20, Semen Maydis powder 20, wheat bran 10, KH
2PO
43, MgSO
47H
2O
2, pH nature was sterilized 20 minutes for 121 ℃; 150 rev/mins of shaking speed, cultivated 7 days under 25 ℃ of conditions;
(3) liquid fermentation and culture: get 6 milliliters, cultured seed and be inoculated in the triangular flask of 500 milliliters that 150 milliliters of fermention mediums are housed.Fermention medium forms (unit grams per liter): glucose 15, Semen Maydis powder 10, wheat bran 10, KH
2PO
43, MgSO
47H
2O 2, and initial pH is 7.0,121 ℃ of sterilizations 20 minutes; 150 rev/mins of shaking speed, cultivated 7 days under 22 ℃ of conditions, finally obtain containing the Resina Ferulae mushroom fermented liquid of laccase, record Laccase activity 7156.3 U/L in fermented liquid.
The concrete mensuration of laccase activity is carried out by the following method:
Get 1 milliliter of fermented liquid, under the condition of 8000 rev/mins centrifugal 10 minutes; Get supernatant liquor, measure laccase activity.Be substrate with 2,2-connection nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS) when measuring Laccase activity, the per minute oxidation 1 needed enzyme amount of micromole ABTS substrate is defined as 1 enzyme work unit (U).In 1 milliliter of reaction system, substrate A BTS final concentration is 1 mM/l, wherein contain 100 mM/ls of pH values and be 4.5 sodium acetate buffer 880 microlitres, 100 microlitre ABTS and 20 microlitre fermented supernatant fluids, whole reaction system was reacted 5 minutes under 30 ℃, measured the variation of its absorbancy under 420 nm.Enzyme activity calculates as follows:
A wherein
1Be the blank light absorption value;
A
2For reacting rear light absorption value;
T is the reaction times;
ε is the molar absorptivity of substrate A BTS, is 3.6 * 10
4M
-1cm
-1
Embodiment 3, Resina Ferulae mushroom liquid fermenting produce laccase
Cultured seed is inoculated in fermention medium, and fermention medium forms (unit grams per liter): glucose 20, Semen Maydis powder 20, wheat bran 15, KH
2PO
43, MgSO
47H
2O 2, and initial pH is 9.0, cultivate 7 days under 26 ℃ of conditions, and other conditions are with embodiment 2.Fermentation ends records that in fermented liquid, Laccase activity reaches 8629.5 U/L.
Embodiment 4, Resina Ferulae mushroom liquid fermenting produce laccase
Cultured seed is inoculated in fermention medium, and fermention medium forms (unit grams per liter): glucose 15, Semen Maydis powder 10, wheat bran 10, KH
2PO
43, MgSO
47H
2O 2, and initial pH is 9.0, cultivate 7 days under 26 ℃ of conditions, and other conditions are with embodiment 2.Fermentation ends records that in fermented liquid, Laccase activity reaches 8327.8 U/L.
Embodiment 5, Resina Ferulae mushroom liquid fermenting produce laccase
Cultured seed is inoculated in fermention medium, and fermention medium forms (unit grams per liter): glucose 20, Semen Maydis powder 20, wheat bran 15, KH
2PO
43, MgSO
47H
2O 2, and initial pH is 7.0, cultivate 7 days under 22 ℃ of conditions, and other conditions are with embodiment 2.Fermentation ends records that in fermented liquid, Laccase activity reaches 7843.4 U/L.
Embodiment 6, Resina Ferulae mushroom liquid fermenting produce laccase
Cultured seed is inoculated in fermention medium, and fermention medium forms (unit grams per liter): glucose 15, Semen Maydis powder 20, wheat bran 10, KH
2PO
43, MgSO
47H
2O 2, and initial pH is 9.0, cultivate 7 days under 22 ℃ of conditions, and other conditions are with embodiment 2.Fermentation ends records that in fermented liquid, Laccase activity reaches 7895.8 U/L.
Embodiment 7, Resina Ferulae mushroom liquid fermenting produce laccase
Cultured seed is inoculated in fermention medium, and fermention medium forms (unit grams per liter): glucose 20, Semen Maydis powder 10, wheat bran 15, KH
2PO
43, MgSO
47H
2O 2, and initial pH is 7.0, cultivate 7 days under 26 ℃ of conditions, and other conditions are with embodiment 2.Fermentation ends records that in fermented liquid, Laccase activity reaches 9017.3 U/L.
Embodiment 8, Resina Ferulae mushroom liquid fermenting produce laccase
Cultured seed is inoculated in fermention medium, and fermention medium forms (unit grams per liter): glucose 18, Semen Maydis powder 15, wheat bran 12, KH
2PO
43, MgSO
47H
2O 2, and initial pH is 8.0, cultivate 7 days under 24 ℃ of conditions, and other conditions are with embodiment 2.Fermentation ends records that in fermented liquid, Laccase activity reaches 12018.6 U/L.
Embodiment 9, Resina Ferulae mushroom liquid fermenting produce laccase
Cultured seed is inoculated in fermention medium, and fermention medium forms (unit grams per liter): glucose 18, Semen Maydis powder 20, wheat bran 10, KH
2PO
43, MgSO
47H
2O 2, and initial pH is 7.0, cultivate 7 days under 24 ℃ of conditions, and other conditions are with embodiment 2.Fermentation ends records that in fermented liquid, Laccase activity reaches 9655.1 U/L.
Embodiment 10, Resina Ferulae mushroom liquid fermenting produce laccase
Cultured seed is inoculated in fermention medium, and fermention medium forms (unit grams per liter): glucose 17, Semen Maydis powder 15, wheat bran 12, KH
2PO
43, MgSO
47H
2O 2, and initial pH is 7.5, cultivate 7 days under 25 ℃ of conditions, and other conditions are with embodiment 2.Fermentation ends records that in fermented liquid, Laccase activity reaches 11385.3 U/L.
Embodiment 11, Resina Ferulae mushroom liquid fermenting produce laccase
Cultured seed is inoculated in fermention medium, and fermention medium forms (unit grams per liter): glucose 18, Semen Maydis powder 18, wheat bran 12, KH
2PO
43, MgSO
47H
2O 2, and initial pH is 8.5, cultivate 7 days under 26 ℃ of conditions, and other conditions are with embodiment 2.Fermentation ends records that in fermented liquid, Laccase activity reaches 10493.9 U/L.
Embodiment 12, Resina Ferulae mushroom liquid fermenting produce laccase
Cultured seed is inoculated in fermention medium, and fermention medium forms (unit grams per liter): glucose 18, Semen Maydis powder 12, wheat bran 12, KH
2PO
43, MgSO
47H
2O 2, and initial pH is 7.2, cultivate 7 days under 23 ℃ of conditions, and other conditions are with embodiment 2.Fermentation ends records that in fermented liquid, Laccase activity reaches 8713.4 U/L.
Embodiment 13, Resina Ferulae mushroom liquid fermenting produce laccase
Cultured seed is inoculated in fermention medium, and fermention medium forms (unit grams per liter): glucose 20, Semen Maydis powder 20, wheat bran 15, KH
2PO
43, MgSO
47H
2O 2, and initial pH is 8.5, cultivate 7 days under 23 ℃ of conditions, and other conditions are with embodiment 2.Finally obtain containing the Resina Ferulae mushroom fermented liquid of laccase, record Laccase activity 9481.2 U/L in fermented liquid.
Below described embodiment of the present invention in detail, can do a lot of improvement and variation obviously for a person skilled in the art and can not deviate from essence spirit of the present invention.All these changes and improvements are all within protection scope of the present invention.
Claims (1)
1. a method of producing laccase by the Resina Ferulae mushroom liquid fermenting, is characterized in that take Resina Ferulae mushroom as starting strain, through slant culture, seed culture and liquid fermentation and culture, realizes utilizing the purpose of Resina Ferulae mushroom liquid fermenting high yield laccase;
(1) slant culture
Slant medium is the potato substratum, i.e. PDA substratum forms in grams per liter: potato 200, and glucose 20, agar 20, with tap water preparation, pH nature, 121 ℃ of sterilizations 20 minutes;
The Resina Ferulae mushroom bacterial classification that obtains through separate tissue is inoculated on slant medium, cultivates under 25 ℃ of conditions, cover with the inclined-plane to mycelium;
(2) seed culture
Seed culture medium forms in grams per liter: glucose 20, Semen Maydis powder 20, wheat bran 10, KH
2PO
43, MgSO
47H
2O 2, with tap water preparation, pH value nature, sterilize 20 minutes for 121 ℃;
Slant strains is inoculated in seed culture medium, at 25 ℃, cultivated 7 days under the condition that shaking speed is 150 rev/mins;
(3) fermentation culture
Fermention medium forms in grams per liter: glucose 15-20, Semen Maydis powder 10-20, wheat bran 10-15, KH
2PO
43, MgSO
47H
2O 2, and with the tap water preparation, initial pH is 7.0-9.0,121 ℃ of sterilizations 20 minutes;
Cultured seed is inoculated in fermention medium, and inoculum size 4% at 22-26 ℃, was cultivated 7 days under the condition that shaking speed is 150 rev/mins.
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| CN103734022B (en) * | 2012-10-16 | 2017-09-05 | 中国科学院天津工业生物技术研究所 | The method for producing the bacterial strain of erythrothioneine and preparing erythrothioneine |
| CN103409379B (en) * | 2013-06-09 | 2015-09-09 | 江南大学 | A kind of Resina Ferulae mushroom and rhodotorula mucilaginosa are total to the method for fermentative production of laccase |
| CN105993587B (en) * | 2016-05-13 | 2019-04-09 | 新疆农业科学院微生物应用研究所 | The method that research Pleurotus ferulae fungi bacterium solution influences ermophyte asafoetide growth of seedling |
| CN105907731B (en) * | 2016-06-30 | 2019-08-20 | 江南大学 | A method of promoting microbial laccase production |
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| CN101812491A (en) * | 2010-01-31 | 2010-08-25 | 陈恒雷 | Method for producing pleurotus ferulae polysaccharide and oral liquid thereof through fermentation |
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| CN101812491A (en) * | 2010-01-31 | 2010-08-25 | 陈恒雷 | Method for producing pleurotus ferulae polysaccharide and oral liquid thereof through fermentation |
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