CN105255848A - Efficient production method for cellulase - Google Patents

Abstract

The invention relates to an efficient production method for cellulase, and particularly discloses a method for controlling cellulase fermentation through the oxygen uptake rate. Through optimal screening of culture media and optimal controlling over oxygen uptake rate conditions, the stable fermentation method is determined, and the enzyme activity of a produced cellulase preparation is improved by more than 50 percent; the technology can be widely applied to cellulase production through filamentous fungi such as penicillium and aspergillus.

Description

A kind of cellulase high-efficiency method for producing

Technical field

The present invention relates to fermentation engineering field, be specifically related to a kind of Cellulase Producing Methods.

Background technology

Cellulase wide material sources, complicated component, extensively be present in the meta-bolites of the microorganisms such as insect body, mollusk body and fungi, bacterium and actinomycetes, main production ways is fermentable, wherein be most widely used with filamentous fungus and bacteria cellulose enzyme, mainly still utilize fungi to the cellulase-producing that ferments at present.

In bacterium, most of aerobic bacteria and anaerobic bacterium are all rich in abundant cellulase system, particularly cud the anerobe of being rich in multiple cellulose degrading enzyme system.Cellulase in world's cellulose market has 20% from Trichoderma and Aspergillus.Mainly because filamentous fungus has the plurality of advantages of producing enzyme: the cellulase 1. produced is extracellular enzyme, is convenient to the separation and extraction of enzyme; 2. produce enzyme efficiency high, and the enzyme architecture producing cellulase is comparatively reasonable; 3. can produce many hemicellulases, polygalacturonase, amylase etc. simultaneously.Cellulase is a multiply anchor-pile, and by three class functions, the different but enzyme of complementation forms in enzyme system.This three fermentoid is endoglucanase (EG, Cx enzyme, also known as CMC enzyme), exoglucanase (cellobiohydrolase, CBH, C1 enzyme) and beta-glucosidase (BGL, cellobiase) respectively; The gene of most of encoding cellulase is chromogene.Cellulase system degradation principles there is no final conclusion at present, mainly contain three kinds of mechanisms of action: Synergy, fragment hypothesis and primary reaction hypothesis, wherein with Synergy by most of researcher and expert approve, its main points of view is the noncrystalline domain that first this fermentoid of endo-glucanase acts on cellulosic molecule inside, random identification is also hydrolyzed β-1,4-glycosidic link, by long chain cellulose molecular cut, produces a large amount of non reducing end.Then exoglucanase cuts the hydrolysis of cellulosic non-reducing end one by one and produces cellobiose; Beta-glucosidase then hydrolysis fiber disaccharides generates glucose.Three is indispensable, synergy, also only under suitable ratio and composition, acting in conjunction could realize cellulosic complete hydrolysis fast.

Cellulase fermentations process is easily subject to the impact that carbon water reptation behavior (CCR) synthesizes enzymic fermentation, namely, in hemicellulose and/or cellulase building-up process, often inhibit the synthesis of enzyme by carbon water reptation behavior (CCR) owing to there is the monose such as glucose, wood sugar in substratum.Conventional selection eliminates part CCR effect at present, such as, the people such as Zhou Guanglin successively have studied ubiquitin protein enzyme gene creB and to lack and transcriptional modulatory gene bglR lacks impact on producing strain growth state and form, cellulase activity, cellulase character.CreB gene deletion mutants cellulase expression secretes anti-glucose metabolism reptation behavior, the filter paper enzyme activity of bacterial strain, endo cellulase are lived, xylanase activity and exocellulase are lived improves 1.8 times, 1.71 times, 2.06 times and 2.04 times respectively, and its exoprotein content improves 2.68 times; And bglR gene is the disappearance of transcriptional regulator, β glucuroide enzyme activity is greatly improved, and improves 40%, but filter paper enzyme activity, endoglucanase and xylanase activity obviously reduce.Although prior art achieves certain effect, how to solve cellulase fermentations process carbon water reptation behavior (CCR), improve Mierocrystalline cellulose throughput, be still the subject matter that those skilled in the art face.

Summary of the invention

For solving above-mentioned problems of the prior art, contriver is found by research, in the process of penicillium oxalicum fermentative production cellulase, the fermentation condition such as feed supplement, DO, pH and cellulase expression amount closely related, achieved increasing considerably of the expression amount of cellulosic expression amount and extracellular protein by the technique such as feed supplement, DO, pH; Further, found by research, in feed supplement process, the metabolism of bacterial strain is very relevant to oxygen uptake rate, the carbon reptation behavior can removing feed supplement process is controlled by OUR, control OUR maintains certain level and maintains the metabolism of bacterial strain normal growth to strengthen the generation of secondary species cellulase, can significantly improve FPA, β glucoside enzyme activity and the protein content of production of cellulose enzyme.

Concrete, the invention provides following technical scheme:

A kind of method of penicillium oxalicum liquid state fermentation production of cellulose enzyme, it is characterized in that, controlling pH between whole yeast phase maintains between 3.5 ~ 6.0, carry out feed-batch culture during the fermentation middle and later periods to fermentation ends, period controls oxygen uptake rate (OUR) and maintains 12 (± 5) ~ 35 (± 5) mmol/L/h.

Those skilled in the art should understand in penicillium oxalicum fermentative production cellulase the time entering the fermentation middle and later periods, now glucose consumption, and carbon reptation behavior is removed, and cellulase starts synthesis, preferably, carry out feed-batch culture during fermentation 24h to fermentation ends.

Preferably, in feed-batch culture process, OUR and dissolved oxygen, feed supplement three variable relation, dissolved oxygen and feed supplement are cooperative compensating relation, and OUR and dissolved oxygen have certain proportionlity, by changing blade, regulate mixing speed to keep dissolved oxygen more than 15%.

Preferably, control oxygen uptake rate (OUR) during feed-batch culture and maintain 15 (± 5) ~ 30 (± 5) mmol/L/h.

Preferably, fermenting process pH regulator is: fermentation beginning pH is adjusted to 5.5-6.0,0-20hpH and declines gradually, and after controlling more than fermention medium pH3.5,50-60h with ammoniacal liquor, pH gos up to 5.5-6.0, starts with below phosphorus acid for adjusting pH to 6.0;

Preferably, fermenting process carries out air flow control, per minute air flow: fermentation volume >=1:1;

Preferably, fermenting process regulates dissolved oxygen, and the initial dissolved oxygen that ferments is adjusted to 100%, by regulating mixing speed to keep dissolved oxygen more than 15% in fermenting process;

Preferably, whole fermenting process temperature keeps 28 ~ 33 DEG C;

More preferred, by penicillium oxalicum streak inoculation 28 ~ 32 DEG C of cultivation 3-5 days in bran mass, after growing peach spore, be forwarded to fresh bran mass cultivate 3 days as bacterial classification.Meet 0.2-20cm ²strain inoculation is in seed culture medium, 28 ~ 32 DEG C of 200rpm fermentation 24 ~ 48h, be prepared into first order seed, inoculum size inoculation first order seed according to 5% ~ 10% is in seed culture medium, 28 ~ 32 DEG C of 200rpm cultivate 30 ~ 42h and make secondary seed, then secondary seed are inoculated in culture medium and carry out product enzymic fermentation.Producing enzymic fermentation design parameter controls as follows:

(1) pH regulator: fermentation beginning pH is adjusted to 5.5-6.0,0-20hpH and declines gradually, after controlling more than fermention medium pH3.5,50-60h with ammoniacal liquor, pH gos up to 5.5-6.0, starts with below phosphorus acid for adjusting pH to 6.0;

(2) air flow controls: per minute air flow: fermentation volume >=1:1;

(3) dissolved oxygen regulates: the initial dissolved oxygen that ferments is adjusted to 100%, by regulating mixing speed to keep dissolved oxygen more than 15% in fermenting process;

(4) temperature adjusting: whole fermenting process temperature keeps 28 ~ 33 DEG C;

(5) OUR regulates:

Fermentation starts, OUR increases with cell concentration and raises gradually, under this culture medium condition, maximum is reached at about 40h, about 23-27mmol/L/h, after maintaining about 10 ~ 15h, along with exhausting of Carbon and nitrogen sources in substratum, fermentation starts to occur that carbon nitrogen source limits, speed of reaction starts to decline, OUR starts to reduce, after there is obviously decline in OUR, about decline 4h (fermentation time is about about 50h), start to add glucose, wood sugar or Microcrystalline Cellulose, 100h is maintained between control OUR to 15 (± 5)-30 (± 5) mmol/L/h, there is most high-cellulose enzyme filter paper vigor, extracellular protein content is maximum, now stop fermentation.

Culturing process substratum used is as follows:

Bran mass: in 7 ~ 10% wheat brans and appropriate tap water, boil 30min, 8 layers of filtered through gauze, then with tap water constant volume to required volume, add the agar of 1.5%, be configured to wheat bran slant medium, for actication of culture and screening.

Seed culture medium: 1% xylose residue, 1% wheat bran, 1% glucose, 1% peptone, 0.2% ammonium sulfate, 0.3% potassium primary phosphate, 0.05% magnesium sulfate, 50mL in 500mL triangular flask, 121 DEG C of sterilizing 20min.

Culture medium: 2 ~ 7% xylose residues, 1 ~ 4% wheat bran, 0.2 ~ 1% Microcrystalline Cellulose, 0.5% soybean cake powder, 0.2% ammonium sulfate, 0.1% urea, 0.3% potassium primary phosphate, 0.05% magnesium sulfate, 0.3% tween 80,121 DEG C of sterilizing 30min.

Adopt mass spectrograph or exhaust analyzer to carry out real-time online collection analysis to the air inlet in fermenting process and tail gas, this mass spectrograph can hydrogen in Accurate Measurement fermenting process tail gas, nitrogen, volatile gas within carbon dioxide and oxygen equimolecular quantity 300.Demarcate by the responsive of standard gas to instrument before use.

Oxygen uptake rate (OUR) measures:

The calculating of OUR is by calculating the analytical data of fermentation tail gas.Maintain constant equilibrium establishment equation with rare gas element N2 in air inlet and tail gas, the calculation formula of trying to achieve OUR is as follows:

$f=\frac{273}{273+t_{in}}\·P_{in}.\frac{1}{1+h}\×{10}^{-5}$

F _in: charge flow rate L/min; V: fermentating liquid volume L; C _{lazy in}cO _2incCO _2in: the mass fraction being respectively rare gas element in air inlet, oxygen and carbonic acid gas; CO _2outcCO _2out: the mass fraction being respectively oxygen and carbonic acid gas in exhaust; P _in: the pounds per square inch absolute (psia) Pa of air inlet; t _in: the temperature DEG C of air inlet; H: the relative humidity (%) of air inlet.

The invention has the advantages that,

(1) the best OUR value by maintaining bacterial strain realizes the carbon-nitrogen ratio of strain growth the best, prevents thalline from occurring nutrition hunger phenomenon during the fermentation and causing autophagy phenomenon, and causes protein content on the low side, the phenomenon that enzyme activity is not high.

(2) control the whole physiological status of thalline and growth conditions by OUR, define thalli growth, cellulase produces and the core controlling factor of scale operation, can amplify, accurately control large scale fermentation by decades of times.

(3) react thalli growth demand by OUR, partial solution de-carbon reptation behavior, increases substantially enzyme activity level and protein level.

Accompanying drawing explanation

Fig. 1 embodiment 1 fermentation results

Fig. 2 embodiment 2 fermentation results

Fig. 3 embodiment 3 fermentation results

Fig. 4 comparative example 1 fermentation results

Embodiment

Embodiment 1:

Seed culture medium: 1% xylose residue, 1% wheat bran, 1% glucose, 1% peptone, 0.2% ammonium sulfate, 0.3% potassium primary phosphate, 0.05% magnesium sulfate, 50mL in 500mL triangular flask, 121 DEG C of sterilizing 20min.

Culture medium: 3% xylose residue, 3% wheat bran, 0.2% Microcrystalline Cellulose, 0.5% soybean cake powder, 0.2% ammonium sulfate, 0.1% urea, 0.3% potassium primary phosphate, 0.05% magnesium sulfate, 0.3% tween 80,121 DEG C of sterilizing 30min.

Filter paperlyase (FPA) enzyme activity determination method: the centrifugal 10min of fermented liquid 8000rpm, getting supernatant is crude enzyme liquid, for enzyme activity and determining the protein quantity.Measure with reference to light industry industry standard (QB2583-2003), get the crude enzyme liquid 0.5mL of suitably dilution, with 50mg (1cm × 6cm) Xinhua filter paper bar for substrate, add pH4.8 acetate buffer solution 1.5mL and 0.5mL, not add substrate for contrast, 50 DEG C of reaction 60min, take out, add 3mlDNS solution, keep 10min in boiling water bath, water-bath cools, and is settled to 25ml, mixing, in 540nm place colorimetric.

Bradford method measures protein content: with bovine serum albumin production standard curve, calculate the protein content in crude enzyme liquid according to typical curve.

By penicillium oxalicum JUA10-1 streak inoculation in bran mass 28 ~ 32 DEG C cultivate 7 days, after growing peach spore, be forwarded to fresh bran mass cultivate 5 days as bacterial classification.Meet 1cm ²strain inoculation is in seed culture medium, 28 ~ 32 DEG C of 200rpm fermentation 24 ~ 48h, be prepared into first order seed, inoculum size inoculation first order seed according to 5% ~ 10% is in seed culture medium, 28 ~ 32 DEG C of 200rpm cultivate 30 ~ 42h and make secondary seed, then secondary seed is inoculated in 4L culture medium, protects in emerging fermentor tank at 5L and carry out product enzymic fermentation, adopt gas chromatograph-mass spectrometer (model GC-MS6800) to carry out tails assay OUR, CER index.(OUR and CER index is basically identical, characterizes with OUR).

(1) pH regulator: fermentation starts pH and is adjusted to 5.5-6.0, and earlier fermentation pH declines gradually, and control slag more than 4.0 with ammoniacal liquor, middle and later periods pH rise is with below phosphorus acid for adjusting pH to 5.5; (2) air flow controls: per minute air flow: fermentation volume=1:1; (3) dissolved oxygen regulates: the initial dissolved oxygen that ferments is adjusted to 100%, by regulating mixing speed to keep dissolved oxygen more than 30% in fermenting process; (4) temperature adjusting: whole fermenting process temperature keeps 30 DEG C; (5) fermentation starts, and OUR increases with cell concentration and raises gradually, under this culture medium condition, maximum is reached at about 40h, about 23 (± 5)-27 (± 5) mmol/L/h, after maintaining about 10 ~ 15h, along with exhausting of Carbon and nitrogen sources in substratum, fermentation starts to occur that carbon nitrogen source limits, speed of reaction starts to decline, and OUR starts to reduce, and starts to add glucose, maintain OUR to 20 ± 5mmol/L/h, the later stage controls 17 ± 5mmol/L/h and maintains 100h.Fermentation results: cellulase activity is when fermenting 96h, and enzyme activity is the highest, and FPA is about 9.3U/mL, and β glucoside enzyme activity is 10.1U/mL, and protein content is 8.8mg/mL.

Embodiment 2

Seed culture medium: 1% xylose residue, 1% wheat bran, 1% glucose, 1% peptone, 0.2% ammonium sulfate, 0.3% potassium primary phosphate, 0.05% magnesium sulfate, 50mL in 500mL triangular flask, 121 DEG C of sterilizing 20min.

Culture medium: 6% xylose residue, 4% wheat bran, 0.2% Microcrystalline Cellulose, 0.5% soybean cake powder, 0.2% ammonium sulfate, 0.1% urea, 0.3% potassium primary phosphate, 0.05% magnesium sulfate, 0.3% tween 80,121 DEG C of sterilizing 30min.

It is described above that Filter paperlyase (FPA) enzyme activity determination method and Bradford method measure protein content.

By penicillium oxalicum JUA10-1 streak inoculation in bran mass 30 DEG C cultivate 5 days, after growing peach spore, be forwarded to fresh bran mass cultivate 5 days as bacterial classification.Meet 2cm ²strain inoculation is in seed culture medium, 28 ~ 32 DEG C of 200rpm fermentation 24 ~ 48h, be prepared into first order seed, inoculum size inoculation first order seed according to 5% ~ 10% is in seed culture medium, 28 ~ 32 DEG C of 200rpm cultivate 30 ~ 42h and make secondary seed, then secondary seed is inoculated in 40L culture medium, protects in emerging fermentor tank at 50L and carry out product enzymic fermentation, adopt gas chromatograph-mass spectrometer (model GC-MS6800) to carry out tails assay OUR, CER index.

(1) pH regulator: fermentation starts pH and is adjusted to 5.5-6.0, and earlier fermentation pH declines gradually, and control slag more than 4.0 with ammoniacal liquor, middle and later periods pH rise is with below phosphorus acid for adjusting pH to 5.5; (2) air flow controls: per minute air flow: fermentation volume=1:1; (3) dissolved oxygen regulates: the initial dissolved oxygen that ferments is adjusted to 100%, by regulating mixing speed to keep dissolved oxygen more than 30% in fermenting process; (4) temperature adjusting: whole fermenting process temperature keeps 30 DEG C; (5) fermentation starts, and OUR increases with cell concentration and raises gradually, under this culture medium condition, maximum is reached at about 60h, about 24 ± 5mmol/L/h, after maintaining about 10 ~ 15h, along with exhausting of Carbon and nitrogen sources in substratum, fermentation starts to occur that carbon nitrogen source limits, speed of reaction starts to decline, and OUR starts to reduce, and starts to add glucose mother liquid, maintain OUR to 24 ± 5mmol/L/h, the later stage controls 27 ± 5mmol/L/h and maintains 100h.Fermentation results: cellulase activity is when fermenting 96h, and enzyme activity is the highest, and FPA is about 22.1U/mL, and β glucoside enzyme activity is 24.3U/mL protein content is 24.7mg/mL.

Embodiment 3

Seed culture medium: 1% xylose residue, 1% wheat bran, 1% glucose, 1% peptone, 0.2% ammonium sulfate, 0.3% potassium primary phosphate, 0.05% magnesium sulfate, 50mL in 500mL triangular flask, 121 DEG C of sterilizing 20min.

Culture medium: 6% xylose residue, 4% wheat bran, 0.2% Microcrystalline Cellulose, 0.5% soybean cake powder, 0.2% ammonium sulfate, 0.1% urea, 0.3% potassium primary phosphate, 0.05% magnesium sulfate, 0.3% tween 80,121 DEG C of sterilizing 30min.

It is described above that Filter paperlyase (FPA) enzyme activity determination method and Bradford method measure protein content.

By penicillium oxalicum JUA10-1 streak inoculation in bran mass 30 DEG C cultivate 5 days, after growing peach spore, be forwarded to fresh bran mass cultivate 5 days as bacterial classification.Meet 10cm ²strain inoculation is in seed culture medium, 28 ~ 32 DEG C of 200rpm fermentation 24 ~ 48h, be prepared into first order seed, inoculum size inoculation first order seed according to 5% is in seed culture medium, 28 ~ 32 DEG C of 200rpm cultivate 30 ~ 42h and make secondary seed, then by secondary seed by 5% inoculum size be inoculated in 1.6t culture medium, in 2t fermentor tank, carry out product enzymic fermentation, adopt gas chromatograph-mass spectrometer (model GC-MS6800) carry out tails assay OUR, CER index.

(1) pH regulator: fermentation starts pH and is adjusted to 5.5, and earlier fermentation pH declines gradually, with ammoniacal liquor control pH more than 4.0, middle and later periods pH rise phosphoric acid maintains between pH5.0-6.6; (2) air flow controls: per minute air flow: fermentation volume=1:1; (3) dissolved oxygen regulates: the initial dissolved oxygen that ferments is adjusted to 100%, by regulating mixing speed to keep dissolved oxygen more than 20% in fermenting process; (4) temperature adjusting: whole fermenting process temperature keeps 30 DEG C; (5) fermentation starts, and OUR increases with cell concentration and raises gradually, under this culture medium condition, maximum is reached at about 60h, about 18 ± 5mmol/L/h, after maintaining about 10 ~ 15h, along with exhausting of Carbon and nitrogen sources in substratum, fermentation starts to occur that carbon nitrogen source limits, speed of reaction starts to decline, and OUR starts to reduce, and starts to add glucose mother liquid, maintain OUR to 20 ± 5mmol/L/h, the later stage controls 20 ± 5mmol/L/h and maintains 100h.Fermentation results: cellulase activity is when fermenting 96h, and enzyme activity is the highest, and FPA is about 17.5U/mL, and β glucoside enzyme activity is 16.1U/mL protein content is 20.2mg/mL, sees Fig. 3.

Comparative example 1:

Filter paperlyase (FPA) enzyme activity determination method: the centrifugal 10min of fermented liquid 8000rpm, getting supernatant is crude enzyme liquid, for enzyme activity and determining the protein quantity.Measure with reference to light industry industry standard (QB2583-2003), get the crude enzyme liquid 0.5mL of suitably dilution, with 50mg (1cm × 6cm) Xinhua filter paper bar for substrate, add pH4.8 acetate buffer solution 1.5mL and 0.5mL, not add substrate for contrast, 50 DEG C of reaction 60min, take out, add 3mlDNS solution, keep 10min in boiling water bath, water-bath cools, and is settled to 25ml, mixing, in 540nm place colorimetric.

Bradford method measures protein content: with bovine serum albumin production standard curve, calculate the protein content in crude enzyme liquid according to typical curve.

Seed culture medium: 1% xylose residue, 1% wheat bran, 1% glucose, 1% peptone, 0.2% ammonium sulfate, 0.3% potassium primary phosphate, 0.05% magnesium sulfate, 50mL in 500mL triangular flask, 121 DEG C of sterilizing 20min.

Culture medium: 3% xylose residue, 3% wheat bran, 0.2% Microcrystalline Cellulose, 0.5% soybean cake powder, 0.2% ammonium sulfate, 0.1% urea, 0.3% potassium primary phosphate, 0.05% magnesium sulfate, 0.3% tween 80,121 DEG C of sterilizing 30min.

By penicillium oxalicum JUA10-1 streak inoculation in bran mass 28 ~ 32 DEG C cultivate 6 days, after growing peach spore, be forwarded to fresh bran mass cultivate 3 days as bacterial classification.Meet 1cm ²strain inoculation is in 50mL seed culture medium, 28 ~ 32 DEG C of 200rpm fermentation 24 ~ 48h, be prepared into first order seed, inoculum size inoculation first order seed according to 5% is in 400mL seed culture medium, 28 ~ 32 DEG C of 200rpm cultivate 42h and make secondary seed, then by secondary seed according to 10% inoculum size be inoculated in 4L culture medium, protect in emerging fermentor tank at 5L and carry out product enzymic fermentation.

Producing enzymic fermentation design parameter controls as follows: pH fermentation starts pH and is adjusted to 5.5, and earlier fermentation pH declines gradually, and with ammoniacal liquor control pH more than 4.0, middle and later periods pH rise phosphoric acid maintains between pH5.0-6.6; Air flow is per minute air flow: fermentation volume is 1:1, and initial for fermentation dissolved oxygen is adjusted to 100%, and rotating speed controls at more than 200rpm, feed supplement is carried out by 2g/L/h speed, by regulating mixing speed to keep dissolved oxygen more than 20% in fermenting process, OUR does not control, and naturally falls after rise.Whole fermenting process temperature keeps 30 DEG C, ferment 96h reach the highest enzyme live reach 6FPA/mL, β glucoside enzyme activity is 3.1U/mL, protein content 3.6mg/mL.Between dissolved oxygen, OUR and FPA, relation as shown in Figure 4.

The present invention, by other production of cellulose enzyme penicillium oxalicum bacterial strains of test, has similar cellulase metabolic condition.Although above-mentioned, the specific embodiment of the present invention is described; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various amendment or distortion that creative work can make still within protection scope of the present invention.

Claims (10)

1. the method for a penicillium oxalicum liquid state fermentation production of cellulose enzyme, it is characterized in that, whole fermenting process pH regulator is between 3.5 ~ 6.0, carry out feed-batch culture during the fermentation middle and later periods to fermentation ends, period controls oxygen uptake rate (OUR) and maintains 15 (± 5) ~ 35 (± 5) mmol/L/h.

2. method according to claim 1, is characterized in that, carries out feed-batch culture during fermentation 24h to fermentation ends.

3. method according to claim 1 or 2, is characterized in that, in feed-batch culture process, by changing blade, regulates mixing speed to keep dissolved oxygen more than 15%, by control of additive raw material oxygen uptake rate (OUR).

4. method according to claim 1, is characterized in that, control oxygen uptake rate (OUR) and maintain 18 (± 5) ~ 27 (± 5) mmol/L/h during feed-batch culture.

5. method according to claim 1, it is characterized in that, fermenting process pH regulator is: fermentation starts pH and is adjusted to 5.5-6.0,0-20hpH declines gradually, after controlling more than fermention medium pH3.5,50-60h with ammoniacal liquor, pH gos up to 5.5-6.0, starts with below phosphorus acid for adjusting pH to 6.0.

6. method according to claim 1, it is characterized in that, fermenting process carries out air flow control, air flow per minute air flow: fermentation volume >=1:1.

7. method according to claim 1, it is characterized in that, the initial dissolved oxygen that ferments is adjusted to 100%, by regulating mixing speed to keep dissolved oxygen more than 15% in fermenting process.

8. method according to claim 1, is characterized in that, whole fermenting process temperature keeps 28 ~ 33 DEG C.

9. method according to claim 1, is characterized in that, fermentation design parameter controls as follows:

(1) pH regulator: fermentation starts pH and is adjusted to 5.5-6.0, and 0-20hpH declines gradually, after controlling more than fermention medium pH3.5,50-60h with ammoniacal liquor, pH gos up to 5.5-6.0, starts with below phosphorus acid for adjusting pH to 6.0;

(2) air flow controls: per minute air flow: fermentation volume >=1:1;

(3) dissolved oxygen regulates: the initial dissolved oxygen that ferments is adjusted to 100%, by regulating mixing speed to keep dissolved oxygen more than 15% in fermenting process;

(4) temperature adjusting: whole fermenting process temperature keeps 28 ~ 33 DEG C;

(5) OUR regulates: fermentation starts, OUR increases with cell concentration and raises gradually, maximum is reached at about 40-60h, along with exhausting of Carbon and nitrogen sources in substratum, fermentation starts to occur that carbon nitrogen source limits, and speed of reaction starts to decline, OUR starts to reduce, after obviously decline appears in OUR, start to add glucose, wood sugar or Microcrystalline Cellulose, ammonium sulfate, between control OUR to 18-27mmol/L/h, maintain 100h.

10. method according to claim 9, is characterized in that, before fermentation by penicillium oxalicum streak inoculation in bran mass 28 ~ 32 DEG C cultivate 3-5 days, after growing peach spore, be forwarded to fresh bran mass cultivate 3 days as bacterial classification; Connect bacterial classification in seed culture medium, 28 ~ 32 DEG C of 200rpm fermentation 24 ~ 48h, be prepared into first order seed, inoculation first order seed is in seed culture medium, 28 ~ 32 DEG C of 200rpm cultivate 30 ~ 42h and make secondary seed, then secondary seed are inoculated in culture medium and carry out product enzymic fermentation;

Culturing process substratum used is:

Bran mass: in 7 ~ 10% wheat brans and appropriate tap water, boil 30min, 8 layers of filtered through gauze, then with tap water constant volume to required volume, add the agar of 1.5%, be configured to wheat bran slant medium, for actication of culture and screening;

Seed culture medium: 1% xylose residue, 1% wheat bran, 1% glucose, 1% peptone, 0.2% ammonium sulfate, 0.3% potassium primary phosphate, 0.05% magnesium sulfate, 50mL in 500mL triangular flask, 121 DEG C of sterilizing 20min;

Culture medium: 2 ~ 7% xylose residues, 1 ~ 4% wheat bran, 0.2 ~ 1% Microcrystalline Cellulose, 0.5% soybean cake powder, 0.2% ammonium sulfate, 0.1% urea, 0.3% potassium primary phosphate, 0.05% magnesium sulfate, 0.3% tween 80,121 DEG C of sterilizing 30min.