CN101591620A - A kind of method of propagation culture and solid fermentation of white-rot fungi - Google Patents

Abstract

A kind of method of propagation culture and solid fermentation of white-rot fungi, the method present method that belongs to the fungus solids fermentation comprises actication of culture: fluid enlargement culture: fermentor tank fluid enlargement culture; Cans are trained domestication admittedly, fermentation cylinder for fermentation: the inoculum size that Cans are trained domestication and fermentation cylinder for fermentation stage admittedly is respectively 10% and 5%.It is simple that the present invention has technical process, can directly apply to factory's large fermentation tank; With strong points, be applied to mix in sewage disposal and the paper industry whiterot fungi fermentation, practical value height; The fermentation efficiency height, advantage such as fermentation costs is low.

Description

A kind of method of propagation culture and solid fermentation of white-rot fungi

Technical field

The present invention is the method that belongs to a kind of fungus solids fermentation, particularly a kind of method of propagation culture and solid fermentation of white-rot fungi.

Background technology

The uniqueness of whiterot fungi and effective degradation capability; make it become powerful fresh combatants of field of environment protection; the active potential that this class is had spectrum offensiveness filamentous fungus to xenobiontics; being converted into the reality control to the pollutent in the different states medium in the environment, is that various countries scientist and related industries circle common pursue one's goal and the unremitting effort direction.Although than sophisticated bacterium system, the application of white-rot fungi in environment protection also is in and explores and establishment stage; Although be close to harsh requirement, become and realize industrialized serious hindrance owing to the singularity of this class bacterium physiological metabolism with to environmental factor; But everything does not stop that people utilize all research, exploitation and the trial of white-rot fungi solid fermentation in the environment protection practice.

The whiterot fungi solid fermentation is applied to pollute initial and maximum practice of processing, be in bio-pulping and water pollution control field, bio-pulping is to utilize the ability of the decomposition lignin that Institute of Micro-biology has to remove lignin in the pulping raw material, makes plant tissue and the fiber process of pulp of making separated from one another.Generally be before plant fiber material is carried out chemical treatment or mechanical jordaning it to be carried out Biological Pretreatment, bio-pulping has entered the batch production stage at present, and has obtained good effect.

The bio-pulping technology is a kind of pulping process of high-efficiency cleaning.Utilize the whiterot fungi solid fermentation that the Degradation of lignin is removed lignin, can alleviate the problem of environmental pollution of paper industry.Whiterot fungi is the most effective quasi-microorganism of present nature xylogen degradation, it can produce the organized enzyme system of xylogen degradation or halse lignin, (laccase, Laccase) system has constituted the main organized enzyme system of whiterot fungi xylogen degradation for lignin peroxidase (LiP), manganese peroxidase (MnP) and phenol oxidase.This characteristic of whiterot fungi has been utilized in research fields such as bio-pulping, bio-bleachings, and some has entered the pilot scale stage, has shown its good prospects for application.

But the liquid or solid culture condition of the whiterot fungi of mentioning in the bibliographical information all only is applicable to fundamental research and the non-system in the laboratory at present, be not suitable for the application in the large scale industry production, do not have explanation in the disclosed patent yet at the propagation culture and solid fermentation of white-rot fungi aspect.As: Chinese invention patent CN1415736 discloses the solid phase fermentation process of a kind of low value old rice as Hyphomycetes spore powder main raw material.After this method is soaked the low value old rice,, in fermenting case or flat chassis, cultivate, will ferment again and expect heating, drying, sieve and make spore powder, do not relate to the method for fungi enlarged culturing solid fermentation through sterilization slaking and fermented hypha liquid, additive combined inoculation.Chinese invention patent CN88101266 discloses a kind of microbial fermentation processes that contains culture medium of carbohydrate.This method is carried out at least in two steps, and the first step mainly is the cultured continuously microorganism, and second step mainly was to generate product by continuous or batch charging.In the processing of second step, microorganism is fixed on the solid support material, can make fermenting process keep stability for a long time thus, and obtain high yield, the utilization of increase sugar.But the working method such as medium component, condition control that does not relate to microorganism enlarged culturing and solid fermentation.Chinese invention patent CN1032956 discloses a kind of strain expanded culture method.This method replaces glassware and inoculating needle, ring with culture vessel and inoculation spoon, need not plant bent three generations cultivates, make song with enlarged culturing two pickup kinds, it is characterized in that with eggplant formula or the former bacterium enlarged culturing of a triangular flask generation be second-generation bacterial, be inoculated in the treated fermented product raw material and be made into song.But do not relate to the method for fungi enlarged culturing to solid fermentation.Chinese invention patent CN86100658 discloses the solid culture method of a kind of muscardine.This method is characterized in that in cultivating raw material based on soil (and adding a small amount of urea and fused(calcium magnesium)phosphate), wherein Tu ratio is 50～85% except part wheat bran or fine rice bran or flour are arranged in cultivating raw material.But the method that does not relate to the enlarged culturing solid fermentation.Chinese invention patent CN1057483 discloses a kind of method for enlarging yeast culture.The characteristics of this method are the saccharification wort made first, except that using for kind of a jar, rest part refrigerates respectively in enlarging jar and fermentor tank, when treating that expansion jar and fermentation need to import yeast juice, again the wheat juice temperature in it is increased to 12～13～15 ℃, this method is applicable to that seasonal beer producer strong or the maintenance duration uses.But do not relate to the method for fungi enlarged culturing to solid fermentation.Japanese Patent JP11269019 discloses a kind of shake flask fermentation and has produced the method for muscardine as the fungus insecticide material, does not relate to enlarged culturing and is the solid-fermented technique of matrix with bamboo timber.Japanese Patent JP200083471 discloses a kind of method of cultivating Paecilomyces varioti with the mixed-matrix of sawdust, rice bran, dish pupa and water, does not relate to the enlarged culturing solid fermentation method of fungi.Chinese invention patent CN1394232 discloses a kind of improved fermentation process, and this method comprises a kind of fermention medium, and it contains: (a) the metabolizable carbon source and the energy; (b) inorganic nitrogen-sourced; (c) phosphate source; (d) at least a metal that is selected from the group of basic metal, alkaline-earth metal, transition metal and composition thereof composition; (e) be substantially free of the vitamin H of particulate matter and bacterium.But the method that does not relate to the fermentation of enlarged culturing and fungus solids.Chinese invention patent CN1757614 discloses the method for a kind of plant cellulose, xylogen producing organic compound fertilizer by using fungi fermentation, this method solution is that feed composition and weight percent are: Mierocrystalline cellulose: 12～55%, xylogen: 43～85%, zymophyte: 1～10%, technological process is: raw material processing-Jia fermented bacterium-Jia water stirring-fermentation-stirring-screening-drying, taste removal-auxiliary material and the compound preparation of raw material-detection technique index-packing, the auxiliary material of compound preparation is a urea, and its nitrogen content is 46.6%; Diammonium phosphate, its nitrogen content are 21%, and phosphorus content is 23%; Vitriolate of tartar, its potassium content are 44%.But the method that does not relate to the fermentation of whiterot fungi enlarged culturing and fungus solids.

New patent searching is the result show, do not see the method record of propagation culture and solid fermentation of white-rot fungi both at home and abroad.Though whiterot fungi has a lot of fundamental researchs aspect lignin degrading, also shown extraordinary effect, aspect practical application, still there are a lot of problems.At first, because whiterot fungi is subjected to influences such as the nutritional needs of substrate pH value, redox potential, thalline and temperature and humidity at wood chip or paper pulp surface growth breeding and enzyme secretion, the lignin degradation enzyme enzyme activity that makes whiterot fungi produce is lower, and fermentation efficiency is low; Secondly, can't satisfy in the industrial production in a large number demand efficiently at the cultural method that only pursues fundamental studies of laboratory using at indivedual single bacterial classifications; At last, whiterot fungi also is attended by cellulosic degraded in xylogen degradation, and this will influence the strength of paper performance.In a word, the greatest difficulty of whiterot fungi bio-pulping process is the next effective lignin degrading of generation lignin degradation enzyme that is difficult to rapidly, ferments in large quantities, lacks the effective fermentation process of system now and solves these problems.

Summary of the invention

The objective of the invention is in order to overcome above-mentioned the deficiencies in the prior art, and proposed a kind of method of propagation culture and solid fermentation of white-rot fungi, problem such as solve that efficient is low, complicated operation, cost height, specific aim are not strong.

Involved in the present invention is a kind of method of propagation culture and solid fermentation of white-rot fungi, and its scheme is as follows:

(1) actication of culture: the whiterot fungi bacterial classification with preserving, be positioned in the PDA substratum that is inoculated in sterilization in 35 ℃ of growth cabinets after 25 minutes, activate 3～5 days;

(2) fluid enlargement culture: will activate mycelium inoculation and cultivate 3～5 days in the liquid nutrient medium of sterilization, every liter of liquid nutrient medium contains: bean sprouts juice 200mL, glucose 10.0g, potassium primary phosphate (KH ₂PO ₄) 3.0g, VB ₁0.001g, polysorbas20 2.0g, sal epsom (MgSO ₄7H ₂O) 1.5g, ferrous sulfate (FeSO ₄7H ₂O) 0.2g, manganous sulfate (MnSO ₄H ₂O) 0.2g, copper sulfate (CuSO ₄5H ₂O) 0.015g;

(3) fermentor tank fluid enlargement culture: above-mentioned gained bacterial classification inoculation was cultivated 3～5 days in liquid fermentation tank, and liquid nutrient medium is formed the same (2);

(4) Cans are trained domestication admittedly: liquid-spawn inoculation is carried out solid fermentation in Cans, inoculum size is 5%～15%, through fermentation domestication in 12 days, solid culture based formulas: draft or woody fibrous material are mixed (according to the water-retaining capacity of different material with an amount of synthetic nutrient solution, need blended nutrient solution volume difference), synthetic nutrient solution prescription (every liter): glucose 10.0g, ammonium tartrate (C ₄H ₁₂N ₂O ₆) 22.0g, potassium primary phosphate (KH ₂PO ₄) 10g, VB ₁0.1g, methyl catechol 1g, polysorbas20 2.0g, H ₂O ₂5g, sal epsom (MgSO ₄7H ₂O) 3.5g, ferrous sulfate (FeSO ₄7H ₂O) 0.4g, manganous sulfate (MnSO ₄H ₂O) 0.35g, copper sulfate (CuSO ₄5H ₂O) 0.065g, Calcium Chloride Powder Anhydrous 0.4g, dissolving back trim is to 1L;

(5) fermentation cylinder for fermentation: with the white rot bacterial classification layer-specific access fermentor tank behind the solid fermentation domestication 12d in the vial, inoculum size is 3%～7%, culture medium prescription the same (4).

The inoculum size that described Cans are trained domestication and fermentation cylinder for fermentation stage admittedly is respectively 10% and 5%.

Advantage of the present invention

(1) technical process is simple, and handled easily can directly apply to factory's large fermentation tank.

(2) with strong points, be applied to mix in sewage disposal and the paper industry whiterot fungi fermentation, practical value height.

(3) fermentation efficiency height, enzyme is lived: in the time of 10 days, the work of lips enzyme can reach 1100U/L, and the work of lacs enzyme can reach 450U/L, and in the time of 15 days, the work of mnps enzyme can reach 360U/L.The lignin degradation rate can reach 50.2% in 30 days.

(4) fermentation costs is low.

Embodiment

Below in conjunction with embodiment, further specify as follows to technical scheme of the present invention:

From refrigerator, take out the whiterot fungi bacterial classification of preserving down in 4 ℃ (Phanerochaete chrysosporium, Trametes cinnabarina (Jacg) Franeh (buying Institute of Micro-biology), flat mushroom (purchasing the institute of agricultural sciences)) in the Chongqing City in the Chinese Academy of Sciences, placed in 35 ℃ of thermostat containers 25 minutes, the PDA substratum is through 0.5MPa, under 121 ℃, sterilization 30min, divide to be filled to cooling back inoculation in the culture dish, put into 32 ℃ of biochemical incubators 3～5 days with insurance film parcel then.(every liter contains bean sprouts juice 200mL, glucose 10.0g, potassium primary phosphate (KH to the bottled liquid 90ml of 250ml triangle ₂PO ₄) 3.0g, VB ₁0.001g, polysorbas20 2.0g, sal epsom (MgSO ₄7H ₂O) 1.5g, ferrous sulfate (FeSO ₄7H ₂O) 0.2g, manganous sulfate (MnSO ₄H ₂O) 0.2g, copper sulfate (CuSO ₄5H ₂O) 0.015g), through 0.5MPa, under 121 ℃, sterilization 30min, the bacterial classification after the activation is inserted in cooling back, is 32 ℃ in temperature, humidity be 80 growth cabinet leave standstill cultivate 24h after, transfer to and cultivate 3～5 days in the constant temperature oscillator, culture temperature is 32 ℃, and rotating speed is 160r/min.Above-mentioned gained liquid spawn and bacterium ball inserted in the 7L liquid fermentation tank behind calibrated, sterilization, cooling and the steadiness parameter cultivated 3～5 days, the liquid culture based formulas is the same.To carry out solid fermentation domestication 12 days in the Cans (every bottled 20g solid medium) of gained liquid-spawn inoculation after the sterilization then, solid culture based formulas: the mixture 900g of bamboo wood and wheat bran (mass ratio is 8: 2), mix (water 1000mL with synthetic nutrient solution, glucose 10.0g, ammonium tartrate (C ₄H ₁₂N ₂O ₆) 22.0g, potassium primary phosphate (KH ₂PO ₄) 10g, VB ₁0.1g, methyl catechol 1g, polysorbas20 2.0g, H ₂O ₂5g, sal epsom (MgSO ₄7H ₂O) 3.5g, ferrous sulfate (FeSO ₄7H ₂O) 0.4g, manganous sulfate (MnSO ₄H ₂O) 0.35g, copper sulfate (CuSO ₄5H ₂O) 0.065g, Calcium Chloride Powder Anhydrous 0.4g).Then with (the autonomous invention: publication number CN201241154) of the white rot bacterial classification layer-specific access 20L fermentor tank after domestication in the Cans, culture medium prescription is the same, water ratio about 60%, be divided in the gauze sack, in vertical high-pressure sterilizing pot, 0.5MPa, under 121 ℃, sterilization 30min puts into fermentor tank after the cooling, the layering inoculation.Spontaneous fermentation begins to get material after 5 days, extrude enzyme liquid, measures the enzyme of lignin peroxidase (LiP), manganese peroxidase (MnP), laccase (Laccase) and lives, and measures an enzyme and lives per 10 days plain and lignin degradation rates of mensuration primary fiber in later per 5 days.

The changing conditions of bamboo wood lignin degradation rate 30 days time the in the table 1 different vaccination amount condition bottom fermentation jar

Below be the contrast experiment:

Table 2 changes the activity change situation of three kinds of main enzymes in time

And the one group of enzyme live data that draws when carrying out whiterot fungi fermentative degradation bamboo wood with additive method is:

Table 3 changes the activity change situation of three kinds of main enzymes in time

The changing conditions of bamboo wood main ingredient degradation rate under the table 4 different treatment time

And the one group of degradation rate data that draws when carrying out whiterot fungi fermentative degradation bamboo wood with additive method are:

The changing conditions of bamboo wood main ingredient degradation rate under the table 5 different treatment time

Contrast from table 2 to table 4 as can be seen, adopting said method ferments to paper making raw material, the high-efficiency lignin degrading aspect is significantly improved, enzyme lives that peak value occurs in advance, the lignin degradation rate is apparently higher than the treatment effect that utilizes additive method, can shorten the processing cycle, and cellulose degradation rate is lower, the ideal effect that this needs most in paper just, be high-efficiency lignin degrading, lower degraded cellulose is with the saving of guaranteeing paper making raw material with make full use of.Can alleviate the efficient degradation of xylogen and to utilize highly basic in the paper industry the chemical pollution of lignin degradation and then the contaminated wastewater that causes.This effect to the environmental protection aspect is very obvious.

Claims (2)

1, a kind of method of propagation culture and solid fermentation of white-rot fungi is characterized in that, step is as follows:

(1) actication of culture: the whiterot fungi bacterial classification with preserving, be positioned in the PDA substratum that is inoculated in sterilization in 35 ℃ of growth cabinets after 25 minutes, activate 3～5 days;

(2) fluid enlargement culture: will activate mycelium inoculation and cultivate 3～5 days in the liquid nutrient medium of sterilization, every liter of liquid nutrient medium contains: bean sprouts juice 200mL, glucose 10.0g, potassium primary phosphate (KH ₂PO ₄) 3.0g, VB ₁0.001g, polysorbas20 2.0g, sal epsom (MgSO ₄7H ₂O) 1.5g, ferrous sulfate FeSO ₄7H ₂O) 0.2g, manganous sulfate (MnSO ₄H ₂O) 0.2g, copper sulfate (CuSO ₄5H ₂O) 0.015g;

(3) fermentor tank fluid enlargement culture: above-mentioned gained bacterial classification inoculation was cultivated 3～5 days in liquid fermentation tank, and liquid nutrient medium is formed the same (2);

(4) Cans are trained domestication admittedly: liquid-spawn inoculation is carried out solid fermentation in Cans, inoculum size is 5%～15%, through fermentation domestication in 12 days, solid culture based formulas: draft or woody fibrous material are mixed (according to the water-retaining capacity of different material with an amount of synthetic nutrient solution, need blended nutrient solution volume difference), synthetic nutrient solution prescription (every liter): glucose 10.0g, ammonium tartrate (C ₄H ₁₂N ₂O ₆) 22.0g, potassium primary phosphate (KH ₂PO ₄) 10g, VB ₁0.1g, methyl catechol 1g, polysorbas20 2.0g, H ₂O ₂5g, sal epsom (MgSO ₄7H ₂O) 3.5g, ferrous sulfate (FeSO ₄7H ₂O) 0.4g, manganous sulfate (MnSO ₄H ₂O) 0.35g, copper sulfate (CuSO ₄5H ₂O) 0.065g, Calcium Chloride Powder Anhydrous 0.4g, dissolving back trim is to 1L;

(5) fermentation cylinder for fermentation: with the white rot bacterial classification layer-specific access fermentor tank behind the solid fermentation domestication 12d in the vial, inoculum size is 3%～7%, culture medium prescription the same (4).

2, the method for a kind of propagation culture and solid fermentation of white-rot fungi according to claim 1 is characterized in that: the inoculum size that described Cans are trained domestication and fermentation cylinder for fermentation stage admittedly is respectively 10% and 5%.