CN107586765A - A kind of industrial fermentation process of phytase - Google Patents

Abstract

The invention discloses a kind of industrial fermentation process of phytase, methods described includes：Step 1：One grade fermemtation tank ferments；Step 2：Second order fermentation tank ferments；Step 3：High density fermentation；Step 4：Fed-batch fermentation, it is hungry；Step 5：Induction, mixed feeding.The industrial fermentation process of phytase of the present invention, have and put the characteristics of tank enzyme activity is high, fermentation efficiency is high.

Description

A kind of industrial fermentation process of phytase

Technical field

The present invention relates to field of fermentation engineering, specifically, is related to a kind of industrial fermentation process of phytase.

Background technology

Phytic acid (Phytate, Phytic, IP6) is also known as phytic acid, is phosphorus in feed containing 6 phosphate groups Important storage form.The molecular formula of phytic acid is C₆H₁₈O₂₄P₆, molecular weight is 660.09.Phytase (Phytase) can be catalyzed Phytic acid and phytate hydrolysis are into inositol and phosphoric acid (or phosphate).

Phytic acid is mainly widely present in vegetable seeds in the form of phytin sylvite, and existing in animal has core red thin Intracellular, the presence of phytase is had been found that into extracellular enzyme in soil, plant and microorganism secretion.Therefore, phytase pair The metabolic cycles of phosphorus play the role of important in natural environment.In addition, phytic acid has extremely strong network to most metal ions Conjunction ability, complexing power are similar to EDTA.Under normal circumstances, the phosphoric acid ester bond in phytic acid is highly stable, for the drop of phytic acid Solution typically has two methods, when chemical method, but be extremely difficult come phytic acid of degrading using chemical method；Another side Method is enzymatic isolation method, and the enzyme for the phytic acid that can effectively degrade being currently known only has phytase.

Lack the phytase for decomposing phytic acid in nonruminant body, phosphorus existing in the form of phytic acid is difficult to be absorbed by animal And bring following problem：First, phytic acid exists in feed and has an anti-oxidant action, phytate phosphorus be difficult in itself by pig fowl with The alimentary canal hydrolysis of aquatic livestock itself utilizes；Phytic acid can be with various metals ion and egg in the digestion process of animal gastrointestinal tract White matter combines, and forms insoluble compound, is suppressed the effect of some digestive ferments so that mineral element in feed and The digestibility of other nutriments substantially reduces.Second, in order to meet needs of the animal to phosphorus, it is necessary to added in feed inorganic Phosphate.The cost of Feed Manufacturing is so not only considerably increased, and is not expelled directly out in vitro by the phosphorus of animal use largely, Cause the waste of phosphorus source and serious environmental problem.Therefore, phytase is added in feed, can not only improve animal in feed The utilization rate of phytate phosphorus, pollution of the phosphorus to environment is reduced, the anti-oxidant action of phytate phosphorus can also be reduced.

Phytase gene high efficient expression in recombinant bacterial strain is set to be that phytase is extensive honest and clean by genetic engineering means The effective way of valency production.At present, the production of phytase relies primarily on the microbial fermentation of fungi, bacterium.The group that they are produced Histidine acidic phosphatases (HAP) phytase, because of its optimal pH meta-acid and than the features such as high of living, it can significantly improve chicken and pig Growth performance.The industrial fermentation of traditional phytase, frequently with fermentation, fed-batch fermentation, methanol induction, it is old to there is zymotechnique Old, cost is high and the problems such as benefit is low.

The content of the invention

The purpose of the present invention is in view of the shortcomings of the prior art, there is provided a kind of industrial fermentation process of phytase.

The technical solution adopted by the present invention is as follows.

A kind of industrial fermentation process of phytase, it is characterised in that methods described comprises the following steps.

Step 1：One grade fermemtation tank ferments

Load one grade fermemtation culture medium in one grade fermemtation tank and polyether-modified silicon defoaming agent, fermentation medium and polyethers change The volume ratio of property silicon defoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121- 123 DEG C, tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in one grade fermemtation tank is entered Row cooling, using volume ratio it is 5-10% inoculum concentration culture transferring to one grade fermemtation by fermentation seed liquid when temperature is down to 30-35 DEG C Expand culture in tank, trace element solution is added into fermentation tank, and be 4.8-5.0 with the pH value of ammoniacal liquor regulation zymotic fluid, it is micro- The volume ratio of secondary element solution and fermentation medium is 1：20-25；Fermentation seed liquid inoculum concentration is 1.5-2%；One grade fermemtation tank Rotating speed is 180-200rpm；Cultivation temperature is 29-32 DEG C；When the zymotic fluid in one grade fermemtation tank residual sugar less than 3-4g/L or During weight in wet base >=60-65g/L, stop；Ventilation, 0-6h 40-45m³/ h, 6h- terminate as 60-70m³/h；One grade fermemtation tank tank pressure Control is in 0.03-0.08Mpa.The trace element solution is the aqueous solution for the PTM1 that volume ratio is 0.8-1.2%.

Step 2：Second order fermentation tank ferments

Load second order fermentation culture medium in second order fermentation tank and polyether-modified silicon defoaming agent, fermentation medium and polyethers change The volume ratio of property silicon defoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121- 123 DEG C, tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in second order fermentation tank is entered Row cooling, when temperature is down to 30-35 DEG C, inoculum concentration of the thalline of one grade fermemtation tank culture using volume ratio as 10-15% is moved Kind expands culture into second order fermentation tank, and the culture of second order fermentation tank is incubated, and cultivation temperature is 29-32 DEG C, second order fermentation Tank rotating speed is 180-200rpm, is stopped as weight in wet base >=80g/L of the zymotic fluid in second order fermentation tank；Ventilation, 0-6h are 500-600m³/ h, 6h- terminate as 800-1000m³/h；The pH of zymotic fluid is 4.5-4.7 in the second order fermentation tank incubation； The voltage-controlled system of second order fermentation tank tank is in 0.03-0.08Mpa.

Step 3：High density fermentation

Load fermentation medium and polyether-modified silicon defoaming agent in high density fermentation tank, fermentation medium and polyether-modified The volume ratio of silicon defoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121-123 DEG C, tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in high density fermentation tank is entered Row cooling, when temperature is down to 30-35 DEG C, inoculum concentration of the thalline of second order fermentation tank culture using volume ratio as 10-13% is moved Kind expands culture into high density fermentation tank, and the culture of high density fermentation tank is incubated, and high density fermentation tank rotating speed is 180- 200rpm；Originated from fermentation in 24 hours and control the pH value of zymotic fluid in 4.5-4.7 with ammoniacal liquor, until dissolving quick rebound extremely Stop when 80%；Cultivation temperature is controlled at 29-32 DEG C；Ventilation：0h-4h, 1500-1600m³/h；More than 4h：2200- 2400m³/h；The voltage-controlled system of high density fermentation tank is in 0.03-0.08Mpa；

Step 4：Fed-batch fermentation

Feed liquid is added into high density fermentation tank；

Within 0-2h hours, with 300-350L/h speed stream charging liquid；

Within 2-4h hours, with 500-550L/h speed stream sugaring liquid；After 4 hours, with 650-700L/h speed Degree stream charging liquid；In fed-batch fermentation whole process, when DO is less than 25%, feed velocity 20% in day part is reduced；DO is less than When 20%, then stop material flow and add, until dissolved oxygen gos up；When the thalline weight in wet base of zymotic fluid reaches 190G/L, stop stream and add；

Glycerine 15-20kg, NH containing glucose 55-70kg, 50% concentration in feed liquid per ton₄H₂PO₄ 5-7kg、K₂SO₄ 10-15kg、MgSO₄ 4-9kg、CaSO₄0.8-1.5kg, KOH 0.3-0.6kg, remaining is water.

Step 5：It is hungry

After stopping feed supplement, any carbon source is not added, observation DO gos up to 80%, stops during pH value rise 0.5-0.6.

Step 6：Induction, mixed feeding

0-1h is flowed by 40L/h flow velocity into high density fermentation tank plus the glucose solution and first of mass percentage concentration 50% Alcohol presses 8:The mixed liquor of 1 volume ratio；1-2h is flowed by 45L/h flow velocity into high density fermentation tank adds mass percentage concentration 50% Glucose solution and methanol press 8:The mixed liquor of 1 volume ratio；2-3h is flowed by 50L/h flow velocity into high density fermentation tank plus quality The glucose solution and methanol of percentage concentration 50% press 8:The mixed liquor of 1 volume ratio；3-5h is sent out by 65L/h flow velocity to high density The glucose solution and methanol of stream plus mass percentage concentration 50% press 7 in fermentation tank:The mixed liquor of 1 volume ratio；5-7h is by 80L/h's Flow velocity flows the glucose solution for adding mass percentage concentration 50% into high density fermentation tank and methanol presses 6:The mixing of 1 volume ratio Liquid；7-9h is flowed the glucose solution for adding mass percentage concentration 50% by 95L/h flow velocity into high density fermentation tank and methanol is pressed 5:The mixed liquor of 1 volume ratio；9-11h is flowed by 110L/h flow velocity into high density fermentation tank plus the Portugal of mass percentage concentration 50% Grape sugar juice and methanol press 4:The mixed liquor of 1 volume ratio；11-13h is flowed by 120L/h flow velocity into high density fermentation tank plus matter The glucose solution and methanol for measuring percentage concentration 50% press 3:The mixed liquor of 1 volume ratio；13-19h is by 130L/h flow velocity to height The glucose solution and methanol of stream plus mass percentage concentration 50% press 2 in density fermentation tank:The mixed liquor of 1 volume ratio；19-22h The glucose solution for adding mass percentage concentration 50% is flowed into high density fermentation tank by 140L/h flow velocity and methanol presses 1:1 volume The mixed liquor of ratio；

After 22h, flowed by 145L/h flow velocity into high density fermentation tank and add salting liquid and methanol to press 1:The mixing of 4 volume ratios Liquid simultaneously adjusts rotating speed, air mass flow and mixing flow velocity according to the weight in wet base and dissolved oxygen amount of zymotic fluid, is specially：Control dissolved oxygen exists More than 20%, when dissolved oxygen rises to 60%, mixing flow velocity is increased 20%, can not such as keep dissolved oxygen then to stop more than 20% Only mixing liquid stream adds, until dissolved oxygen gos up；

The formula of wherein salting liquid is：

CuSO₄·5H₂O 3-3.8g、NaI0.03-0.08g、MnSO₄·H₂O1.5-2g、H₃BO₃ 0.01-0.03g、 Na₂MoO₄.2H₂O 0.1-0.3g、CoCl₂0.2-0.5g、ZnCl₂ 10-16g、FeSO₄·7H₂O30-45g、H₂SO₄ 2.5-3g、 Biotin 0.2-0.3g；

When until wet bacterium weighs to 180g/L in zymotic fluid, stream glycerol adding carries out mixed feeding, and mixed feeding flow velocity is 40-60L/h； Induction starts rear 90-110h and stops mixed feeding；The concentration of glycerine is 30-40%；

After induction is not less than 160-170h, terminate fermentation.

Further, in step 1, the preparation of the seed liquor includes：Pichia yeast is grown through slant medium culture Go out Pichia yeast single bacterium colony, be then inoculated in shaking flask and cultivated, control 180-220rpm, 30-32 DEG C of perseverances of shaking flask rotating speed Warm shaking table culture 24-62h or so, obtain shake-flask seed, weight in wet base 45-70g/L.

Further, in step 1, contain in first cell culture medium per ton：Dusty yeast 2-4kg, peptone 6-15kg, glucose 6-10kg, surplus are water.

Further, in step 2, contain in secondary medium per ton：Glucose 160-240kg, NH4H2PO4 30- 50kg, K2SO460-80kg, MgSO425-40kg, CaSO43-6kg%, KOH3-5kg, surplus are water.

Further, in step 3, glucose 55-70kg, NH4H are contained in fermentation medium per ton₂PO₄ 5-7kg、 K₂SO₄10-15kg MgSO₄ 4-9kg、CaSO₄0.8-1.5kg, KOH0.3-0.6kg, peptone 1.0-1.5kg, dusty yeast 0.3-0.6kg、CuSO₄·5H₂O 300-380g、NaI:3-8g、MnSO₄·H₂O 150-200g、H₃BO₃ 1-3g、 Na₂MoO₄.2H₂O:10-20g、CoCl₂:20-35g、ZnCl₂ 1-1.6kg、FeSO₄·7H₂O₃-4.5kg、H₂SO₄ 250- 300g, biotin 20-30g.

Further, in steps of 5, hungry phase duration is not less than 4h.

Further, the industrial fermentation process of the phytase also includes the step for isolating and purifying the zymotic fluid obtained from step 6 Suddenly, it is specially：Crude enzyme liquid is centrifuged to obtain, crude enzyme liquid is purified with acetone precipitation and ion-exchange chromatography, acetone concentration 60- 63%, ion exchange column pH are 5.2-5.4.

Further, the industrial fermentation process of the phytase is additionally included in the zymotic fluid that step 6 obtains and adds phytase Synergist, through adsorbing, dry after the step of obtaining phytase.

Further, the phytase synergist is mainly made up of formic acid, cyclodextrin and starch；The quality of wherein described formic acid It is 0.1-0.12% with fermentating liquid volume ratio；Cyclodextrin and fermentating liquid volume ratio are 1.1-1.3%, starch and fermented liquid Long-pending ratio is 1.1-1.3%.

Further, dry process is no more than 100 DEG C using spray drying, dry temperature；The phytase finally given Enzyme product moisture is no more than 10%.

The beneficial effects of the invention are as follows：The complete dissolved oxygen of sugar will be mended on original Process ba- sis to go up to mend methanol induction immediately, more New is that processing time is 4-5 hour, wherein requiring that dissolved oxygen gos up to more than 80%, pH rises 0.5- using Nature enemy 0.6, it can completely be consumed based on sugar using this processing, beneficial to what cell more optimized induce.On original Process ba- sis Mixed feeding is carried out using suitable concentration glycerine (30%-40%) and methanol during induction.Because glycerine as carbon source cell biochemistry It is more beneficial for absorbing in reaction cycle, while the molecular structure of glycerine and permeability are more beneficial for keeping Premeabilisation of cells pressure, so as to Promote the metabolism producing enzyme of cell.After taking the new cultural method of the above, it puts tank enzyme activity by the 32000u/ml that is averaged originally, improves extremely Averagely enzyme activity 35000u/ml at present, improves fermentation level about 8.5%.

Brief description of the drawings

Fig. 1 shows the weight in wet base and enzyme activity test chart of the phytase of art methods production.

Fig. 2 illustrates the weight in wet base and enzyme activity test chart of the phytase of the production of method shown in the embodiment of the present invention 2.

Wherein：Series 1 is the curves of enzyme activity * 100, and series 2 is weight in wet base curve.

Embodiment

Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment In the conventional meanses that are well known to those skilled in the art of used technological means, raw materials used is commercial goods.

Embodiment 1.A kind of industrial fermentation process of phytase, it is characterised in that methods described comprises the following steps.

Step 1：One grade fermemtation tank ferments

Load one grade fermemtation culture medium in one grade fermemtation tank and polyether-modified silicon defoaming agent, fermentation medium and polyethers change The volume ratio of property silicon defoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121- 123 DEG C, tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in one grade fermemtation tank is entered Row cooling, using volume ratio it is 5-10% inoculum concentration culture transferring to one grade fermemtation by fermentation seed liquid when temperature is down to 30-35 DEG C Expand culture in tank, trace element solution is added into fermentation tank, and be 4.8-5.0 with the pH value of ammoniacal liquor regulation zymotic fluid, it is micro- The volume ratio of secondary element solution and fermentation medium is 1：20-25；Fermentation seed liquid inoculum concentration is 1.5-2%；One grade fermemtation tank Rotating speed is 180-200rpm；Cultivation temperature is 29-32 DEG C；When the zymotic fluid in one grade fermemtation tank residual sugar less than 3-4g/L or During weight in wet base >=60-65g/L, stop；Ventilation, 0-6h 40-45m³/ h, 6h- terminate as 60-70m³/h；One grade fermemtation tank tank pressure Control is in 0.03-0.08Mpa.

Step 2：Second order fermentation tank ferments

Load second order fermentation culture medium in second order fermentation tank and polyether-modified silicon defoaming agent, fermentation medium and polyethers change The volume ratio of property silicon defoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121- 123 DEG C, tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in second order fermentation tank is entered Row cooling, when temperature is down to 30-35 DEG C, inoculum concentration of the thalline of one grade fermemtation tank culture using volume ratio as 10-15% is moved Kind expands culture into second order fermentation tank, and the culture of second order fermentation tank is incubated, and cultivation temperature is 29-32 DEG C, second order fermentation Tank rotating speed is 180-200rpm, is stopped as weight in wet base >=80g/L of the zymotic fluid in second order fermentation tank；Ventilation, 0-6h are 500-600m³/ h, 6h- terminate as 800-1000m³/h；The pH of zymotic fluid is 4.5-4.7 in the second order fermentation tank incubation； The voltage-controlled system of second order fermentation tank tank is in 0.03-0.08Mpa.

Step 3：High density fermentation

Load fermentation medium and polyether-modified silicon defoaming agent in high density fermentation tank, fermentation medium and polyether-modified The volume ratio of silicon defoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121-123 DEG C, tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in high density fermentation tank is entered Row cooling, when temperature is down to 30-35 DEG C, inoculum concentration of the thalline of second order fermentation tank culture using volume ratio as 10-13% is moved Kind expands culture into high density fermentation tank, and the culture of high density fermentation tank is incubated, and high density fermentation tank rotating speed is 180- 200rpm；Originated from fermentation in 24 hours and control the pH value of zymotic fluid in 4.5-4.7 with ammoniacal liquor, until dissolving quick rebound extremely Stop when 80%；Cultivation temperature is controlled at 29-32 DEG C；Ventilation：0h-4h, 1500-1600m³/h；More than 4h：2200- 2400m³/h；The voltage-controlled system of high density fermentation tank is in 0.03-0.08Mpa.

Step 4：Fed-batch fermentation

Feed liquid is added into high density fermentation tank；

Within 0-2h hours, with 300-350L/h speed stream charging liquid；In feed liquid per ton containing glucose 55-70kg, Glycerine 15-20kg, NH4H2PO4 5-7kg, K2SO4 10-15kg, MgSO4 4-9kg, the CaSO4 0.8- of 50% concentration 1.5kg, KOH 0.3-0.6kg, remaining is water.Glycerine absorbs fast, and initial stage, glycerine was advantageous to absorb, and long-living sugared will not tire out Product.

Within 2-4h hours, with 500-550L/h speed stream sugaring liquid；After 4 hours, with 650-700L/h speed Degree stream charging liquid；In fed-batch fermentation whole process, when DO is less than 25%, feed velocity 20% in day part is reduced；DO is less than When 20%, then stop material flow and add, until dissolved oxygen gos up；When the thalline weight in wet base of zymotic fluid reaches 190G/L, stop stream and add.

Step 5：It is hungry

After stopping feed supplement, any carbon source is not added, observation DO gos up to 80%, stops during pH value rise 0.5-0.6.

Step 6：Induction, mixed feeding

0-1h is flowed by 40L/h flow velocity into high density fermentation tank plus the glucose solution and first of mass percentage concentration 50% Alcohol presses 8:The mixed liquor of 1 volume ratio；1-2h is flowed by 45L/h flow velocity into high density fermentation tank adds mass percentage concentration 50% Glucose solution and methanol press 8:The mixed liquor of 1 volume ratio；2-3h is flowed by 50L/h flow velocity into high density fermentation tank plus quality The glucose solution and methanol of percentage concentration 50% press 8:The mixed liquor of 1 volume ratio；3-5h is sent out by 65L/h flow velocity to high density The glucose solution and methanol of stream plus mass percentage concentration 50% press 7 in fermentation tank:The mixed liquor of 1 volume ratio；5-7h is by 80L/h's Flow velocity flows the glucose solution for adding mass percentage concentration 50% into high density fermentation tank and methanol presses 6:The mixing of 1 volume ratio Liquid；7-9h is flowed the glucose solution for adding mass percentage concentration 50% by 95L/h flow velocity into high density fermentation tank and methanol is pressed 5:The mixed liquor of 1 volume ratio；9-11h is flowed by 110L/h flow velocity into high density fermentation tank plus the Portugal of mass percentage concentration 50% Grape sugar juice and methanol press 4:The mixed liquor of 1 volume ratio；11-13h is flowed by 120L/h flow velocity into high density fermentation tank plus matter The glucose solution and methanol for measuring percentage concentration 50% press 3:The mixed liquor of 1 volume ratio；13-19h is by 130L/h flow velocity to height The glucose solution and methanol of stream plus mass percentage concentration 50% press 2 in density fermentation tank:The mixed liquor of 1 volume ratio；19-22h The glucose solution for adding mass percentage concentration 50% is flowed into high density fermentation tank by 140L/h flow velocity and methanol presses 1:1 volume The mixed liquor of ratio.In this way, sugar accumulation will not be produced, is advantageous to improve product quality.

After 22h, flowed by 145L/h flow velocity into high density fermentation tank and add salting liquid and methanol to press 1:The mixing of 4 volume ratios Liquid simultaneously adjusts rotating speed, air mass flow and mixing flow velocity according to the weight in wet base and dissolved oxygen amount of zymotic fluid, is specially：Control dissolved oxygen exists More than 20%, when dissolved oxygen rises to 60%, mixing flow velocity is increased 20%, can not such as keep dissolved oxygen then to stop more than 20% Only mixing liquid stream adds, until dissolved oxygen gos up；

The formula of wherein salting liquid is：

CuSO₄·5H₂O 3-3.8g、NaI0.03-0.08g、MnSO₄·H₂O1.5-2g、H₃BO₃ 0.01-0.03g、 Na₂MoO₄.2H₂O 0.1-0.3g、CoCl₂0.2-0.5g、ZnCl₂ 10-16g、FeSO₄·7H₂O30-45g、H₂SO₄ 2.5-3g、 Biotin 0.2-0.3g.

When until wet bacterium weighs to 180g/L in zymotic fluid, stream glycerol adding carries out mixed feeding, and mixed feeding flow velocity is 40L/h；Lure 90h stops mixed feeding after leading beginning；

After induction is not less than 160h, terminate fermentation.

The preparation of the seed liquor includes：Pichia yeast is grown into Pichia yeast single bacterium through slant medium culture Fall, be then inoculated in shaking flask and cultivated, 180-220rpm, 30-32 DEG C of constant-temperature table culture 24-62h of control shaking flask rotating speed Left and right, obtain shake-flask seed, weight in wet base 45-70g/L.

Used yeast is pichia pastoris phaff, is that one kind in methanotrophic yeast can be by the use of methanol as uniquely The saccharomycete of carbon source and the energy.As other yeast, mainly exist in the asexual growth phase in the form of monoploid, work as environmental nutrient During limitation, the maqting type haploid cell mating that 2 physiological-types are different is often induced, is fused into amphiploid.Koichi Ogata Et al. to be found that some yeast can utilize methanol first in 1969 be that sole carbon source and the energy grow (Ogata, et A1.1969), hereafter, the potentiality by the use of methanol using type yeast production single cell protein as animal feed just cause extensive pass Note.1987, Cregg et al. reported that (HbsAg is subsequent with methanotrophic Yeast expression hepatitis B surface antibody first Philip Petroleum companies and Salk Institute Biotechnology/Industrial Associates (SIBIA) cooperative development of pichia yeast expression system has been begun to.SIBIA. researcher has separated the startup of AOX genes Son and host strain, construct carrier, and have developed corresponding Pichia pastoris gene manipulation techniques, with reference to Philip Petroleum companies produce the zymotechnique of single cell protein, realize the high efficient expression of foreign protein.1993, Philip The patent of pichia yeast expression system is sold to Research Corporation Technologies public affairs by Petroleum companies Department, and entrust Invitrogea companies to carry out article sale.Its advantage has：(1) there is alcohol oxidase AOX1 gene promoters Son, this is most strong at present, one of most stringent of promoter of Regulation Mechanism；(2) expression efficiency is high, and its foreign protein expressed can account for More than the 90% of total expressing protein, is advantageous to isolating and purifying for destination protein；(3) can be achieved in simple synthetic media highly dense Degree culture；(4) expression plasmid can be integrated in the specific site of genome with the form stable of single copy or multicopy；(5) due to The yeast can be using methanol as sole carbon source and the energy, and most microorganisms be able to not can be reduced using methanol as carbon source Pollution.Pichia pastoris Pichia pastoris have been the most frequently used protein expression system for being only second to Escherichia coli at present, extensively should Protein preparation, sign and structure elucidation for laboratory scale etc., existing thousands of kinds of albumen are in Pichia pastoris system Express with being succeeded in system.In recent years, Pichia pastoris regarded as GRAS (Generally recognized as by U.S. FDA Safe) microorganism, road has been paved for its application on food and medicine.In medical albumen field, existing insulin, hepatitis B The multiple proteins such as surface antigen, human serum albumins, EGF realize prepared by commercialization using Pichia anomala expression. Industrial enzyme preparation field, also there are many enzyme preparations to include phytase, lipase, mannonase zytase etc. red using finishing Yeast realizes the production of industrialized scale.

In step 1, contain in first cell culture medium per ton：Dusty yeast 2-4kg, peptone 6-15kg, glucose 6-10kg, Surplus is water.

In step 2, contain in secondary medium per ton：Glucose 160-240kg, NH₄H₂PO₄ 30-50kg、 K₂SO₄60-80kg、MgSO₄25-40kg、CaSO₄3-6kg%, KOH3-5kg, surplus are water.

In step 3, glucose 55-70kg, NH4H are contained in fermentation medium per ton₂PO₄ 5-7kg、K₂SO₄10- 15kg MgSO₄ 4-9kg、CaSO₄0.8-1.5kg, KOH0.3-0.6kg, peptone 1.0-1.5kg, dusty yeast 0.3-0.6kg, CuSO₄·5H₂O 300-380g、NaI:3-8g、MnSO₄·H₂O 150-200g、H₃BO₃ 1-3g、Na₂MoO₄.2H₂O:10- 20g、CoCl₂:20-35g、ZnCl₂ 1-1.6kg、FeSO₄·7H₂O₃-4.5kg、H₂SO₄250-300g, biotin 20-30g.

In steps of 5, hungry phase duration is not less than 4h；In step 6, the concentration of glycerine is 30-40%.

The industrial fermentation process of the phytase also include isolate and purify from the zymotic fluid that step 6 obtains the step of, specifically For：Crude enzyme liquid is centrifuged to obtain, crude enzyme liquid is purified with acetone precipitation and ion-exchange chromatography, acetone concentration 60-63%, from Sub- displacement chromatography post pH is 5.2-5.4.

Embodiment 2.A kind of industrial fermentation process of phytase, it is characterised in that methods described comprises the following steps.

Step 1：One grade fermemtation tank ferments

Load one grade fermemtation culture medium in one grade fermemtation tank and polyether-modified silicon defoaming agent, fermentation medium and polyethers change The volume ratio of property silicon defoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121- 123 DEG C, tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in one grade fermemtation tank is entered Row cooling, using volume ratio it is 5-10% inoculum concentration culture transferring to one grade fermemtation by fermentation seed liquid when temperature is down to 30-35 DEG C Expand culture in tank, trace element solution is added into fermentation tank, and be 4.8-5.0 with the pH value of ammoniacal liquor regulation zymotic fluid, it is micro- The volume ratio of secondary element solution and fermentation medium is 1：20-25；Fermentation seed liquid inoculum concentration is 1.5-2%；One grade fermemtation tank Rotating speed is 180-200rpm；Cultivation temperature is 29-32 DEG C；When the zymotic fluid in one grade fermemtation tank residual sugar less than 3-4g/L or During weight in wet base >=60-65g/L, stop；Ventilation, 0-6h 40-45m³/ h, 6h- terminate as 60-70m³/h；One grade fermemtation tank tank pressure Control is in 0.03-0.08Mpa.

Step 2：Second order fermentation tank ferments

Load second order fermentation culture medium in second order fermentation tank and polyether-modified silicon defoaming agent, fermentation medium and polyethers change The volume ratio of property silicon defoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121- 123 DEG C, tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in second order fermentation tank is entered Row cooling, when temperature is down to 30-35 DEG C, inoculum concentration of the thalline of one grade fermemtation tank culture using volume ratio as 10-15% is moved Kind expands culture into second order fermentation tank, and the culture of second order fermentation tank is incubated, and cultivation temperature is 29-32 DEG C, second order fermentation Tank rotating speed is 180-200rpm, is stopped as weight in wet base >=80g/L of the zymotic fluid in second order fermentation tank；Ventilation, 0-6h are 500-600m³/ h, 6h- terminate as 800-1000m³/h；The pH of zymotic fluid is 4.5-4.7 in the second order fermentation tank incubation； The voltage-controlled system of second order fermentation tank tank is in 0.03-0.08Mpa.

Step 3：High density fermentation

Load fermentation medium and polyether-modified silicon defoaming agent in high density fermentation tank, fermentation medium and polyether-modified The volume ratio of silicon defoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121-123 DEG C, tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in high density fermentation tank is entered Row cooling, when temperature is down to 30-35 DEG C, inoculum concentration of the thalline of second order fermentation tank culture using volume ratio as 10-13% is moved Kind expands culture into high density fermentation tank, and the culture of high density fermentation tank is incubated, and high density fermentation tank rotating speed is 180- 200rpm；Originated from fermentation in 24 hours and control the pH value of zymotic fluid in 4.5-4.7 with ammoniacal liquor, until dissolving quick rebound extremely Stop when 80%；Cultivation temperature is controlled at 29-32 DEG C；Ventilation：0h-4h, 1500-1600m³/h；More than 4h：2200- 2400m³/h；The voltage-controlled system of high density fermentation tank is in 0.03-0.08Mpa.

Step 4：Fed-batch fermentation

Feed liquid is added into high density fermentation tank；

Within 0-2h hours, with 300-350L/h speed stream charging liquid；

Within 2-4h hours, with 500-550L/h speed stream sugaring liquid；After 4 hours, with 650-700L/h speed Degree stream charging liquid；In fed-batch fermentation whole process, when DO is less than 25%, feed velocity 20% in day part is reduced；DO is less than When 20%, then stop material flow and add, until dissolved oxygen gos up；When the thalline weight in wet base of zymotic fluid reaches 190G/L, stop stream and add；

Glycerine 15-20kg, NH containing glucose 55-70kg, 50% concentration in feed liquid per ton₄H₂PO₄ 5-7kg、K₂SO₄ 10-15kg、MgSO₄ 4-9kg、CaSO₄0.8-1.5kg, KOH 0.3-0.6kg, remaining is water.

Step 5：It is hungry

After stopping feed supplement, any carbon source is not added, observation DO gos up to 80%, stops during pH value rise 0.5-0.6.

Step 6：Induction, mixed feeding

0-1h is flowed by 40L/h flow velocity into high density fermentation tank plus the glucose solution and first of mass percentage concentration 50% Alcohol presses 8:The mixed liquor of 1 volume ratio；1-2h is flowed by 45L/h flow velocity into high density fermentation tank adds mass percentage concentration 50% Glucose solution and methanol press 8:The mixed liquor of 1 volume ratio；2-3h is flowed by 50L/h flow velocity into high density fermentation tank plus quality The glucose solution and methanol of percentage concentration 50% press 8:The mixed liquor of 1 volume ratio；3-5h is sent out by 65L/h flow velocity to high density The glucose solution and methanol of stream plus mass percentage concentration 50% press 7 in fermentation tank:The mixed liquor of 1 volume ratio；5-7h is by 80L/h's Flow velocity flows the glucose solution for adding mass percentage concentration 50% into high density fermentation tank and methanol presses 6:The mixing of 1 volume ratio Liquid；7-9h is flowed the glucose solution for adding mass percentage concentration 50% by 95L/h flow velocity into high density fermentation tank and methanol is pressed 5:The mixed liquor of 1 volume ratio；9-11h is flowed by 110L/h flow velocity into high density fermentation tank plus the Portugal of mass percentage concentration 50% Grape sugar juice and methanol press 4:The mixed liquor of 1 volume ratio；11-13h is flowed by 120L/h flow velocity into high density fermentation tank plus matter The glucose solution and methanol for measuring percentage concentration 50% press 3:The mixed liquor of 1 volume ratio；13-19h is by 130L/h flow velocity to height The glucose solution and methanol of stream plus mass percentage concentration 50% press 2 in density fermentation tank:The mixed liquor of 1 volume ratio；19-22h The glucose solution for adding mass percentage concentration 50% is flowed into high density fermentation tank by 140L/h flow velocity and methanol presses 1:1 volume The mixed liquor of ratio.

After 22h, flowed by 145L/h flow velocity into high density fermentation tank and add salting liquid and methanol to press 1:The mixing of 4 volume ratios Liquid simultaneously adjusts rotating speed, air mass flow and mixing flow velocity according to the weight in wet base and dissolved oxygen amount of zymotic fluid, is specially：Control dissolved oxygen exists More than 20%, when dissolved oxygen rises to 60%, mixing flow velocity is increased 20%, can not such as keep dissolved oxygen then to stop more than 20% Only mixing liquid stream adds, until dissolved oxygen gos up；

The formula of wherein salting liquid is：

CuSO₄·5H₂O 3-3.8g、NaI0.03-0.08g、MnSO₄·H₂O1.5-2g、H₃BO₃ 0.01-0.03g、 Na₂MoO₄.2H₂O 0.1-0.3g、CoCl₂0.2-0.5g、ZnCl₂ 10-16g、FeSO₄·7H₂O30-45g、H₂SO₄ 2.5-3g、 Biotin 0.2-0.3g.

When until wet bacterium weighs to 180g/L in zymotic fluid, stream glycerol adding carries out mixed feeding, and mixed feeding flow velocity is 60L/h；Lure 110h stops mixed feeding after leading beginning；

After induction is not less than 170h, terminate fermentation.

The preparation of the seed liquor includes：Pichia yeast is grown into Pichia yeast single bacterium through slant medium culture Fall, be then inoculated in shaking flask and cultivated, 180-220rpm, 30-32 DEG C of constant-temperature table culture 24-62h of control shaking flask rotating speed Left and right, obtain shake-flask seed, weight in wet base 45-70g/L.

In step 1, contain in first cell culture medium per ton：Dusty yeast 2-4kg, peptone 6-15kg, glucose 6-10kg, Surplus is water.

In step 2, contain in secondary medium per ton：Glucose 160-240kg, NH₄H₂PO₄ 30-50kg、 K₂SO₄60-80kg、MgSO₄25-40kg、CaSO₄3-6kg%, KOH3-5kg, surplus are water.

In step 3, glucose 55-70kg, NH4H are contained in fermentation medium per ton₂PO₄ 5-7kg、K₂SO₄10- 15kgMgSO₄ 4-9kg、CaSO₄0.8-1.5kg, KOH0.3-0.6kg, peptone 1.0-1.5kg, dusty yeast 0.3-0.6kg, CuSO₄·5H₂O 300-380g、NaI:3-8g、MnSO₄·H₂O 150-200g、H₃BO₃ 1-3g、Na₂MoO₄.2H₂O:10- 20g、CoCl₂:20-35g、ZnCl₂ 1-1.6kg、FeSO₄·7H₂O₃-4.5kg、H₂SO₄250-300g, biotin 20-30g.

In steps of 5, hungry phase duration is not less than 4h；In step 6, the concentration of glycerine is 30-40%.

The industrial fermentation process of the phytase is additionally included in addition phytase synergist in the zymotic fluid that step 6 obtains, Through adsorbing, dry after the step of obtaining phytase.

The phytase synergist is mainly made up of formic acid, cyclodextrin and starch；The quality of wherein described formic acid and fermentation Liquid volume ratio is 0.1-0.12%；Cyclodextrin and the ratio that fermentating liquid volume ratio is 1.1-1.3%, starch and fermentating liquid volume It is worth for 1.1-1.3%.

Dry process is no more than 100 DEG C using spray drying, dry temperature；The phytase enzyme product water finally given Divide and be no more than 10%.

Embodiment 3.A kind of industrial fermentation process of phytase, it is characterised in that methods described comprises the following steps.

Step 1：One grade fermemtation tank ferments

Load one grade fermemtation culture medium in one grade fermemtation tank and polyether-modified silicon defoaming agent, fermentation medium and polyethers change The volume ratio of property silicon defoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121- 123 DEG C, tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in one grade fermemtation tank is entered Row cooling, using volume ratio it is 5-10% inoculum concentration culture transferring to one grade fermemtation by fermentation seed liquid when temperature is down to 30-35 DEG C Expand culture in tank, trace element solution is added into fermentation tank, and be 4.8-5.0 with the pH value of ammoniacal liquor regulation zymotic fluid, it is micro- The volume ratio of secondary element solution and fermentation medium is 1：20-25；Fermentation seed liquid inoculum concentration is 1.5-2%；One grade fermemtation tank Rotating speed is 180-200rpm；Cultivation temperature is 29-32 DEG C；When the zymotic fluid in one grade fermemtation tank residual sugar less than 3-4g/L or During weight in wet base >=60-65g/L, stop；Ventilation, 0-6h 40-45m³/ h, 6h- terminate as 60-70m³/h；One grade fermemtation tank tank pressure Control is in 0.03-0.08Mpa.

Step 2：Second order fermentation tank ferments

Load second order fermentation culture medium in second order fermentation tank and polyether-modified silicon defoaming agent, fermentation medium and polyethers change The volume ratio of property silicon defoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121- 123 DEG C, tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in second order fermentation tank is entered Row cooling, when temperature is down to 30-35 DEG C, inoculum concentration of the thalline of one grade fermemtation tank culture using volume ratio as 10-15% is moved Kind expands culture into second order fermentation tank, and the culture of second order fermentation tank is incubated, and cultivation temperature is 29-32 DEG C, second order fermentation Tank rotating speed is 180-200rpm, is stopped as weight in wet base >=80g/L of the zymotic fluid in second order fermentation tank；Ventilation, 0-6h are 500-600m³/ h, 6h- terminate as 800-1000m³/h；The pH of zymotic fluid is 4.5-4.7 in the second order fermentation tank incubation； The voltage-controlled system of second order fermentation tank tank is in 0.03-0.08Mpa.

Step 3：High density fermentation

Load fermentation medium and polyether-modified silicon defoaming agent in high density fermentation tank, fermentation medium and polyether-modified The volume ratio of silicon defoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121-123 DEG C, tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in high density fermentation tank is entered Row cooling, when temperature is down to 30-35 DEG C, inoculum concentration of the thalline of second order fermentation tank culture using volume ratio as 10-13% is moved Kind expands culture into high density fermentation tank, and the culture of high density fermentation tank is incubated, and high density fermentation tank rotating speed is 180- 200rpm；Originated from fermentation in 24 hours and control the pH value of zymotic fluid in 4.5-4.7 with ammoniacal liquor, until dissolving quick rebound extremely Stop when 80%；Cultivation temperature is controlled at 29-32 DEG C；Ventilation：0h-4h, 1500-1600m³/h；More than 4h：2200- 2400m³/h；The voltage-controlled system of high density fermentation tank is in 0.03-0.08Mpa.

Step 4：Fed-batch fermentation

Feed liquid is added into high density fermentation tank；

Within 0-2h hours, with 300-350L/h speed stream charging liquid；

Within 2-4h hours, with 500-550L/h speed stream sugaring liquid；After 4 hours, with 650-700L/h speed Degree stream charging liquid；In fed-batch fermentation whole process, when DO is less than 25%, feed velocity 20% in day part is reduced；DO is less than When 20%, then stop material flow and add, until dissolved oxygen gos up；When the thalline weight in wet base of zymotic fluid reaches 190G/L, stop stream and add；

Glycerine 15-20kg, NH containing glucose 55-70kg, 50% concentration in feed liquid per ton₄H₂PO₄ 5-7kg、K₂SO₄ 10-15kg、MgSO₄ 4-9kg、CaSO₄0.8-1.5kg, KOH 0.3-0.6kg, remaining is water.

Step 5：It is hungry

After stopping feed supplement, any carbon source is not added, observation DO gos up to 80%, stops during pH value rise 0.5-0.6.

Step 6：Induction, mixed feeding

0-1h is flowed by 40L/h flow velocity into high density fermentation tank plus the glucose solution and first of mass percentage concentration 50% Alcohol presses 8:The mixed liquor of 1 volume ratio；1-2h is flowed by 45L/h flow velocity into high density fermentation tank adds mass percentage concentration 50% Glucose solution and methanol press 8:The mixed liquor of 1 volume ratio；2-3h is flowed by 50L/h flow velocity into high density fermentation tank plus quality The glucose solution and methanol of percentage concentration 50% press 8:The mixed liquor of 1 volume ratio；3-5h is sent out by 65L/h flow velocity to high density The glucose solution and methanol of stream plus mass percentage concentration 50% press 7 in fermentation tank:The mixed liquor of 1 volume ratio；5-7h is by 80L/h's Flow velocity flows the glucose solution for adding mass percentage concentration 50% into high density fermentation tank and methanol presses 6:The mixing of 1 volume ratio Liquid；7-9h is flowed the glucose solution for adding mass percentage concentration 50% by 95L/h flow velocity into high density fermentation tank and methanol is pressed 5:The mixed liquor of 1 volume ratio；9-11h is flowed by 110L/h flow velocity into high density fermentation tank plus the Portugal of mass percentage concentration 50% Grape sugar juice and methanol press 4:The mixed liquor of 1 volume ratio；11-13h is flowed by 120L/h flow velocity into high density fermentation tank plus matter The glucose solution and methanol for measuring percentage concentration 50% press 3:The mixed liquor of 1 volume ratio；13-19h is by 130L/h flow velocity to height The glucose solution and methanol of stream plus mass percentage concentration 50% press 2 in density fermentation tank:The mixed liquor of 1 volume ratio；19-22h The glucose solution for adding mass percentage concentration 50% is flowed into high density fermentation tank by 140L/h flow velocity and methanol presses 1:1 volume The mixed liquor of ratio.

After 22h, flowed by 145L/h flow velocity into high density fermentation tank and add salting liquid and methanol to press 1:The mixing of 4 volume ratios Liquid simultaneously adjusts rotating speed, air mass flow and mixing flow velocity according to the weight in wet base and dissolved oxygen amount of zymotic fluid, is specially：Control dissolved oxygen exists More than 20%, when dissolved oxygen rises to 60%, mixing flow velocity is increased 20%, can not such as keep dissolved oxygen then to stop more than 20% Only mixing liquid stream adds, until dissolved oxygen gos up；

The formula of wherein salting liquid is：

CuSO₄·5H₂O 3-3.8g、NaI0.03-0.08g、MnSO₄·H₂O1.5-2g、H₃BO₃ 0.01-0.03g、 Na₂MoO₄.2H₂O 0.1-0.3g、CoCl₂0.2-0.5g、ZnCl₂ 10-16g、FeSO₄·7H₂O30-45g、H₂SO₄ 2.5-3g、 Biotin 0.2-0.3g；

When until wet bacterium weighs to 180g/L in zymotic fluid, stream glycerol adding carries out mixed feeding, and mixed feeding flow velocity is 40-60L/h； Induction starts rear 100h and stops mixed feeding；

After induction is not less than 160-170h, terminate fermentation.

The preparation of the seed liquor includes：Pichia yeast is grown into Pichia yeast single bacterium through slant medium culture Fall, be then inoculated in shaking flask and cultivated, 180-220rpm, 30-32 DEG C of constant-temperature table culture 24-62h of control shaking flask rotating speed Left and right, obtain shake-flask seed, weight in wet base 45-70g/L.

In step 1, contain in first cell culture medium per ton：Dusty yeast 2-4kg, peptone 6-15kg, glucose 6-10kg, Surplus is water.

In step 2, contain in secondary medium per ton：Glucose 160-240kg, NH₄H₂PO₄ 30-50kg、 K₂SO₄60-80kg、MgSO₄25-40kg、CaSO₄3-6kg%, KOH3-5kg, surplus are water.

In step 3, glucose 55-70kg, NH4H are contained in fermentation medium per ton₂PO₄ 5-7kg、K₂SO₄10- 15kg MgSO₄ 4-9kg、CaSO₄0.8-1.5kg, KOH0.3-0.6kg, peptone 1.0-1.5kg, dusty yeast 0.3-0.6kg, CuSO₄·5H₂O 300-380g、NaI:3-8g、MnSO₄·H₂O 150-200g、H₃BO₃ 1-3g、Na₂MoO₄.2H₂O:10- 20g、CoCl₂:20-35g、ZnCl₂ 1-1.6kg、FeSO₄·7H₂O₃-4.5kg、H₂SO₄250-300g, biotin 20-30g.

In steps of 5, hungry phase duration is not less than 4h；In step 6, the concentration of glycerine is 30-40%.

The industrial fermentation process of the phytase also include isolate and purify from the zymotic fluid that step 6 obtains the step of, specifically For：Crude enzyme liquid is centrifuged to obtain, crude enzyme liquid is purified with acetone precipitation and ion-exchange chromatography, acetone concentration 60-63%, from Sub- displacement chromatography post pH is 5.2-5.4.Pichia pastoris not producing enzyme in itself, it is by different producing enzyme genes by genetic engineering means It is transferred in yeast genes, therefore same yeast can produce different enzymes.

Embodiment 4.A kind of industrial fermentation process of phytase, it is characterised in that methods described comprises the following steps.

Step 1：One grade fermemtation tank ferments

Load one grade fermemtation culture medium in one grade fermemtation tank and polyether-modified silicon defoaming agent, fermentation medium and polyethers change The volume ratio of property silicon defoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121- 123 DEG C, tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in one grade fermemtation tank is entered Row cooling, using volume ratio it is 5-10% inoculum concentration culture transferring to one grade fermemtation by fermentation seed liquid when temperature is down to 30-35 DEG C Expand culture in tank, trace element solution is added into fermentation tank, and be 4.8-5.0 with the pH value of ammoniacal liquor regulation zymotic fluid, it is micro- The volume ratio of secondary element solution and fermentation medium is 1：20-25；Fermentation seed liquid inoculum concentration is 1.5-2%；One grade fermemtation tank Rotating speed is 180-200rpm；Cultivation temperature is 29-32 DEG C；When the zymotic fluid in one grade fermemtation tank residual sugar less than 3-4g/L or During weight in wet base >=60-65g/L, stop；Ventilation, 0-6h 40-45m³/ h, 6h- terminate as 60-70m³/h；One grade fermemtation tank tank pressure Control is in 0.03-0.08Mpa.

Step 2：Second order fermentation tank ferments

Load second order fermentation culture medium in second order fermentation tank and polyether-modified silicon defoaming agent, fermentation medium and polyethers change The volume ratio of property silicon defoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121- 123 DEG C, tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in second order fermentation tank is entered Row cooling, when temperature is down to 30-35 DEG C, inoculum concentration of the thalline of one grade fermemtation tank culture using volume ratio as 10-15% is moved Kind expands culture into second order fermentation tank, and the culture of second order fermentation tank is incubated, and cultivation temperature is 29-32 DEG C, second order fermentation Tank rotating speed is 180-200rpm, is stopped as weight in wet base >=80g/L of the zymotic fluid in second order fermentation tank；Ventilation, 0-6h are 500-600m³/ h, 6h- terminate as 800-1000m³/h；The pH of zymotic fluid is 4.5-4.7 in the second order fermentation tank incubation； The voltage-controlled system of second order fermentation tank tank is in 0.03-0.08Mpa.

Step 3：High density fermentation

Load fermentation medium and polyether-modified silicon defoaming agent in high density fermentation tank, fermentation medium and polyether-modified The volume ratio of silicon defoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121-123 DEG C, tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in high density fermentation tank is entered Row cooling, when temperature is down to 30-35 DEG C, inoculum concentration of the thalline of second order fermentation tank culture using volume ratio as 10-13% is moved Kind expands culture into high density fermentation tank, and the culture of high density fermentation tank is incubated, and high density fermentation tank rotating speed is 180- 200rpm；Originated from fermentation in 24 hours and control the pH value of zymotic fluid in 4.5-4.7 with ammoniacal liquor, until dissolving quick rebound extremely Stop when 80%；Cultivation temperature is controlled at 29-32 DEG C；Ventilation：0h-4h, 1500-1600m³/h；More than 4h：2200- 2400m³/h；The voltage-controlled system of high density fermentation tank is in 0.03-0.08Mpa.

Step 4：Fed-batch fermentation

Feed liquid is added into high density fermentation tank；

Within 0-2h hours, with 300-350L/h speed stream charging liquid；

Within 2-4h hours, with 500-550L/h speed stream sugaring liquid；After 4 hours, with 650-700L/h speed Degree stream charging liquid；In fed-batch fermentation whole process, when DO is less than 25%, feed velocity 20% in day part is reduced；DO is less than When 20%, then stop material flow and add, until dissolved oxygen gos up；When the thalline weight in wet base of zymotic fluid reaches 190G/L, stop stream and add；

Glycerine 15-20kg, NH containing glucose 55-70kg, 50% concentration in feed liquid per ton₄H₂PO₄ 5-7kg、K₂SO₄ 10-15kg、MgSO₄ 4-9kg、CaSO₄0.8-1.5kg, KOH 0.3-0.6kg, remaining is water.

Step 5：It is hungry

After stopping feed supplement, any carbon source is not added, observation DO gos up to 80%, stops during pH value rise 0.5-0.6.

Step 6：Induction, mixed feeding

0-1h is flowed by 40L/h flow velocity into high density fermentation tank plus the glucose solution and first of mass percentage concentration 50% Alcohol presses 8:The mixed liquor of 1 volume ratio；1-2h is flowed by 45L/h flow velocity into high density fermentation tank adds mass percentage concentration 50% Glucose solution and methanol press 8:The mixed liquor of 1 volume ratio；2-3h is flowed by 50L/h flow velocity into high density fermentation tank plus quality The glucose solution and methanol of percentage concentration 50% press 8:The mixed liquor of 1 volume ratio；3-5h is sent out by 65L/h flow velocity to high density The glucose solution and methanol of stream plus mass percentage concentration 50% press 7 in fermentation tank:The mixed liquor of 1 volume ratio；5-7h is by 80L/h's Flow velocity flows the glucose solution for adding mass percentage concentration 50% into high density fermentation tank and methanol presses 6:The mixing of 1 volume ratio Liquid；7-9h is flowed the glucose solution for adding mass percentage concentration 50% by 95L/h flow velocity into high density fermentation tank and methanol is pressed 5:The mixed liquor of 1 volume ratio；9-11h is flowed by 110L/h flow velocity into high density fermentation tank plus the Portugal of mass percentage concentration 50% Grape sugar juice and methanol press 4:The mixed liquor of 1 volume ratio；11-13h is flowed by 120L/h flow velocity into high density fermentation tank plus matter The glucose solution and methanol for measuring percentage concentration 50% press 3:The mixed liquor of 1 volume ratio；13-19h is by 130L/h flow velocity to height The glucose solution and methanol of stream plus mass percentage concentration 50% press 2 in density fermentation tank:The mixed liquor of 1 volume ratio；19-22h The glucose solution for adding mass percentage concentration 50% is flowed into high density fermentation tank by 140L/h flow velocity and methanol presses 1:1 volume The mixed liquor of ratio.

After 22h, flowed by 145L/h flow velocity into high density fermentation tank and add salting liquid and methanol to press 1:The mixing of 4 volume ratios Liquid simultaneously adjusts rotating speed, air mass flow and mixing flow velocity according to the weight in wet base and dissolved oxygen amount of zymotic fluid, is specially：Control dissolved oxygen exists More than 20%, when dissolved oxygen rises to 60%, mixing flow velocity is increased 20%, can not such as keep dissolved oxygen then to stop more than 20% Only mixing liquid stream adds, until dissolved oxygen rise

The formula of wherein salting liquid is：

CuSO₄·5H₂O 3-3.8g、NaI0.03-0.08g、MnSO₄·H₂O1.5-2g、H₃BO₃ 0.01-0.03g、 Na₂MoO₄.2H₂O 0.1-0.3g、CoCl₂0.2-0.5g、ZnCl₂ 10-16g、FeSO₄·7H₂O30-45g、H₂SO₄ 2.5-3g、 Biotin 0.2-0.3g；

When until wet bacterium weighs to 180g/L in zymotic fluid, stream glycerol adding carries out mixed feeding, and mixed feeding flow velocity is 40-60L/h； Induction starts rear 90-110h and stops mixed feeding；

After induction is not less than 160-170h, terminate fermentation.

The preparation of the seed liquor includes：Pichia yeast is grown into Pichia yeast single bacterium through slant medium culture Fall, be then inoculated in shaking flask and cultivated, 180-220rpm, 30-32 DEG C of constant-temperature table culture 24-62h of control shaking flask rotating speed Left and right, obtain shake-flask seed, weight in wet base 45-70g/L.

In step 1, contain in first cell culture medium per ton：Dusty yeast 2-4kg, peptone 6-15kg, glucose 6-10kg, Surplus is water.

In step 2, contain in secondary medium per ton：Glucose 160-240kg, NH₄H₂PO₄ 30-50kg、 K₂SO₄60-80kg、MgSO₄25-40kg、CaSO₄3-6kg%, KOH3-5kg, surplus are water.

In step 3, glucose 55-70kg, NH4H are contained in fermentation medium per ton₂PO₄ 5-7kg、K₂SO₄10- 15kg MgSO₄ 4-9kg、CaSO₄0.8-1.5kg, KOH0.3-0.6kg, peptone 1.0-1.5kg, dusty yeast 0.3-0.6kg, CuSO₄·5H₂O 300-380g、NaI:3-8g、MnSO₄·H₂O 150-200g、H₃BO₃ 1-3g、Na₂MoO₄.2H₂O:10- 20g、CoCl₂:20-35g、ZnCl₂ 1-1.6kg、FeSO₄·7H₂O₃-4.5kg、H₂SO₄250-300g, biotin 20-30g.

In steps of 5, hungry phase duration is not less than 4h；In step 6, the concentration of glycerine is 30-40%.

The industrial fermentation process of the phytase is additionally included in addition phytase synergist in the zymotic fluid that step 6 obtains, Through adsorbing, dry after the step of obtaining phytase.

The phytase synergist is mainly made up of formic acid, acetic acid, cyclodextrin and starch；The quality of wherein described formic acid with Fermentating liquid volume ratio is 0.05-0.0.06%；The quality of wherein described formic acid and fermentating liquid volume ratio are 0.05- 0.06%；Cyclodextrin and fermentating liquid volume ratio are 1.1-1.3%, and the ratio of starch and fermentating liquid volume is 1.1-1.3%.

Dry process is no more than 100 DEG C using spray drying, dry temperature；The phytase enzyme product water finally given Divide and be no more than 10%.

Embodiment 5.A kind of industrial fermentation process of phytase, it is characterised in that methods described comprises the following steps.

Step 1：One grade fermemtation tank ferments

Load one grade fermemtation culture medium in one grade fermemtation tank and polyether-modified silicon defoaming agent, fermentation medium and polyethers change The volume ratio of property silicon defoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121- 123 DEG C, tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in one grade fermemtation tank is entered Row cooling, using volume ratio it is 5-10% inoculum concentration culture transferring to one grade fermemtation by fermentation seed liquid when temperature is down to 30-35 DEG C Expand culture in tank, trace element solution is added into fermentation tank, and be 4.8-5.0 with the pH value of ammoniacal liquor regulation zymotic fluid, it is micro- The volume ratio of secondary element solution and fermentation medium is 1：20-25；Fermentation seed liquid inoculum concentration is 1.5-2%；One grade fermemtation tank Rotating speed is 180-200rpm；Cultivation temperature is 29-32 DEG C；When the zymotic fluid in one grade fermemtation tank residual sugar less than 3-4g/L or During weight in wet base >=60-65g/L, stop；Ventilation, 0-6h 40-45m³/ h, 6h- terminate as 60-70m³/h；One grade fermemtation tank tank pressure Control is in 0.03-0.08Mpa.

Step 2：Second order fermentation tank ferments

Load second order fermentation culture medium in second order fermentation tank and polyether-modified silicon defoaming agent, fermentation medium and polyethers change The volume ratio of property silicon defoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121- 123 DEG C, tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in second order fermentation tank is entered Row cooling, when temperature is down to 30-35 DEG C, inoculum concentration of the thalline of one grade fermemtation tank culture using volume ratio as 10-15% is moved Kind expands culture into second order fermentation tank, and the culture of second order fermentation tank is incubated, and cultivation temperature is 29-32 DEG C, second order fermentation Tank rotating speed is 180-200rpm, is stopped as weight in wet base >=80g/L of the zymotic fluid in second order fermentation tank；Ventilation, 0-6h are 500-600m³/ h, 6h- terminate as 800-1000m³/h；The pH of zymotic fluid is 4.5-4.7 in the second order fermentation tank incubation； The voltage-controlled system of second order fermentation tank tank is in 0.03-0.08Mpa.

Step 3：High density fermentation

Load fermentation medium and polyether-modified silicon defoaming agent in high density fermentation tank, fermentation medium and polyether-modified The volume ratio of silicon defoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121-123 DEG C, tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in high density fermentation tank is entered Row cooling, when temperature is down to 30-35 DEG C, inoculum concentration of the thalline of second order fermentation tank culture using volume ratio as 10-13% is moved Kind expands culture into high density fermentation tank, and the culture of high density fermentation tank is incubated, and high density fermentation tank rotating speed is 180- 200rpm；Originated from fermentation in 24 hours and control the pH value of zymotic fluid in 4.5-4.7 with ammoniacal liquor, until dissolving quick rebound extremely Stop when 80%；Cultivation temperature is controlled at 29-32 DEG C；Ventilation：0h-4h, 1500-1600m³/h；More than 4h：2200- 2400m³/h；The voltage-controlled system of high density fermentation tank is in 0.03-0.08Mpa.

Step 4：Fed-batch fermentation

Feed liquid is added into high density fermentation tank；

Within 0-2h hours, with 300-350L/h speed stream charging liquid；

Within 2-4h hours, with 500-550L/h speed stream sugaring liquid；After 4 hours, with 650-700L/h speed Degree stream charging liquid；In fed-batch fermentation whole process, when DO is less than 25%, feed velocity 20% in day part is reduced；DO is less than When 20%, then stop material flow and add, until dissolved oxygen gos up；When the thalline weight in wet base of zymotic fluid reaches 190G/L, stop stream and add；Ton Position is directly proportional with oxygen amount is melted, and dissolved oxygen amount is directly proportional to rotating speed, air mass flow and mixing flow velocity.

Glycerine 15-20kg, NH containing glucose 55-70kg, 50% concentration in feed liquid per ton₄H₂PO₄ 5-7kg、K₂SO₄ 10-15kg、MgSO₄ 4-9kg、CaSO₄0.8-1.5kg, KOH 0.3-0.6kg, remaining is water.

Step 5：It is hungry

After stopping feed supplement, any carbon source is not added, observation DO gos up to 80%, stops during pH value rise 0.5-0.6.

Step 6：Induction, mixed feeding

0-1h is flowed by 40L/h flow velocity into high density fermentation tank plus the glucose solution and first of mass percentage concentration 50% Alcohol presses 8:The mixed liquor of 1 volume ratio；1-2h is flowed by 45L/h flow velocity into high density fermentation tank adds mass percentage concentration 50% Glucose solution and methanol press 8:The mixed liquor of 1 volume ratio；2-3h is flowed by 50L/h flow velocity into high density fermentation tank plus quality The glucose solution and methanol of percentage concentration 50% press 8:The mixed liquor of 1 volume ratio；3-5h is sent out by 65L/h flow velocity to high density The glucose solution and methanol of stream plus mass percentage concentration 50% press 7 in fermentation tank:The mixed liquor of 1 volume ratio；5-7h is by 80L/h's Flow velocity flows the glucose solution for adding mass percentage concentration 50% into high density fermentation tank and methanol presses 6:The mixing of 1 volume ratio Liquid；7-9h is flowed the glucose solution for adding mass percentage concentration 50% by 95L/h flow velocity into high density fermentation tank and methanol is pressed 5:The mixed liquor of 1 volume ratio；9-11h is flowed by 110L/h flow velocity into high density fermentation tank plus the Portugal of mass percentage concentration 50% Grape sugar juice and methanol press 4:The mixed liquor of 1 volume ratio；11-13h is flowed by 120L/h flow velocity into high density fermentation tank plus matter The glucose solution and methanol for measuring percentage concentration 50% press 3:The mixed liquor of 1 volume ratio；13-19h is by 130L/h flow velocity to height The glucose solution and methanol of stream plus mass percentage concentration 50% press 2 in density fermentation tank:The mixed liquor of 1 volume ratio；19-22h The glucose solution for adding mass percentage concentration 50% is flowed into high density fermentation tank by 140L/h flow velocity and methanol presses 1:1 volume The mixed liquor of ratio；

After 22h, flowed by 145L/h flow velocity into high density fermentation tank and add salting liquid and methanol to press 1:The mixing of 4 volume ratios Liquid simultaneously adjusts rotating speed, air mass flow and mixing flow velocity according to the weight in wet base and dissolved oxygen amount of zymotic fluid, is specially：Control dissolved oxygen exists More than 20%, when dissolved oxygen rises to 60%, mixing flow velocity is increased 20%, can not such as keep dissolved oxygen then to stop more than 20% Only mixing liquid stream adds, until dissolved oxygen gos up；

The formula of wherein salting liquid is：

CuSO₄·5H₂O 3-3.8g、NaI0.03-0.08g、MnSO₄·H₂O1.5-2g、H₃BO₃ 0.01-0.03g、 Na₂MoO₄.2H₂O 0.1-0.3g、CoCl₂0.2-0.5g、ZnCl₂ 10-16g、FeSO₄·7H₂O30-45g、H₂SO₄ 2.5-3g、 Biotin 0.2-0.3g；

When until wet bacterium weighs to 180g/L in zymotic fluid, stream glycerol adding carries out mixed feeding, and mixed feeding flow velocity is 60L/h；Lure 110h stops mixed feeding after leading beginning；

After induction is not less than 160-170h, terminate fermentation.

The preparation of the seed liquor includes：Pichia yeast is grown into Pichia yeast single bacterium through slant medium culture Fall, be then inoculated in shaking flask and cultivated, 180-220rpm, 30-32 DEG C of constant-temperature table culture 24-62h of control shaking flask rotating speed Left and right, obtain shake-flask seed, weight in wet base 45-70g/L.

In step 1, contain in first cell culture medium per ton：Dusty yeast 2-4kg, peptone 6-15kg, glucose 6-10kg, Surplus is water.

In step 2, contain in secondary medium per ton：Glucose 160-240kg, NH₄H₂PO₄ 30-50kg、 K₂SO₄60-80kg、MgSO₄25-40kg、CaSO₄3-6kg%, KOH3-5kg, surplus are water.

In step 3, glucose 55-70kg, NH4H are contained in fermentation medium per ton₂PO₄ 5-7kg、K₂SO₄10- 15kg MgSO₄ 4-9kg、CaSO₄0.8-1.5kg, KOH0.3-0.6kg, peptone 1.0-1.5kg, dusty yeast 0.3-0.6kg, CuSO₄·5H₂O 300-380g、NaI:3-8g、MnSO₄·H₂O 150-200g、H₃BO₃ 1-3g、Na₂MoO₄.2H₂O:10- 20g、CoCl₂:20-35g、ZnCl₂ 1-1.6kg、FeSO₄·7H₂O₃-4.5kg、H₂SO₄250-300g, biotin 20-30g.

In steps of 5, hungry phase duration is not less than 4h；In step 6, the concentration of glycerine is 30-40%.

The industrial fermentation process of the phytase also include isolate and purify from the zymotic fluid that step 6 obtains the step of, specifically For：Crude enzyme liquid is centrifuged to obtain, crude enzyme liquid is purified with acetone precipitation and ion-exchange chromatography, acetone concentration 60-63%, from Sub- displacement chromatography post pH is 5.2-5.4.

Test example 1.To verify fed-batch fermentation effect of the present invention, it is (simple with mending sugar in the prior art that sugar is mended by embodiment 2 Apply glucose and mend sugared speed increase block) contrasted, as a result such as following table.

It can be seen that the embodiment of the present invention 2 mends sugar amount and the sugared speed of benefit by changing, hence it is evident that improves weight in wet base.

Methanol of the present invention, glycerine mixed feeding inducing effect are verified, passes through the methanol of embodiment 2, the induction of glycerine mixed feeding and existing skill Methanol induction is contrasted in art, as a result such as following table.

Methanol, the induction of glycerine mixed feeding and the parameter comparison of the simple methanol induction of prior art

Note：Mixed feeding technique uses 25% glycerine, and mixed feeding process flow is 20~25L/h.

It can be seen that the embodiment of the present invention 2 significantly improves induction weight in wet base.

Test example 2.Averagely put tank Enzyme activity assay.

In fermentation process, zymotic fluid measure OD600nm and thalline weight in wet base are taken every 24h, takes supernatant to carry out phytase Activity determination.Put tank centrifugation and Enzyme activity assay：Tank is put in induction, mixed feeding after terminating, received under the conditions of 4 DEG C with 5000rpm centrifugations 30min Collect supernatant.

According to National Standard of the People's Republic of China《GB/T 18634-2009》Carry out.Phytase activity definition refers to Under conditions of 37 DEG C of pH value 5.50, every min is that 1 μm of ol Phos is discharged in 5.0mmol/L sodium phytate solutions from concentration, as one Individual phytase activity unit, is represented with U.

X=ym × t × n]]>

The activity of phytase in X-sample, unit are every gram of unit of enzyme activity (U/g) or unit of enzyme activity's units per ml (U/mL)；The amount of the Phos for y-calculated according to the light absorption value of actual sample liquid by linear regression equation, unit is micromole； T-enzyme digestion reaction time, unit min；The extension rate of n-sample；The amount of m-sample, unit for gram or milliliter.

50 μ L dilutions enzyme liquid is taken to add the μ L of substrate 4mmol/L sodium phytates 950 (to be matched somebody with somebody with 0.1mol/L pH5.5 acetate buffer solution System), 37 DEG C reaction 30min, add 1mL 10%TCA terminating reactions, add 2mL nitrite ions (10g Ammonium Molybdate Tetrahydrate+32mL sulfuric acid+ 73.2g ferrous sulfate, adds water to be settled to 1L), reaction solution is developed the color.First add TCA to mix after compareing then enzyme-added liquid, then add bottom Thing.Develop the color after 10min, survey its OD value at 700 nm, calculate enzyme activity.

To verify experiment effect of the present invention, using the second order fermentation tank fermentation of prior art, stream plus glucose, starvation, first Alcohol-induced technique productions phytase, it is as follows with comparing result of the present invention.

Fig. 1 shows weight in wet base of the prior art using the phytase after two-stage fermentation, feeding glucose fermentation, starvation and methanol induction With enzyme activity test chart.Fig. 2 illustrates the weight in wet base and enzyme activity test chart of the phytase of the production of method shown in the embodiment of the present invention 2.Can To find out, under the conditions of same period, the enzyme activity rate of rise being adjusted is very fast, and final enzyme activity can reach 3.57 ten thousand activity Unit, improve about 8.5%.Tank enzyme activity is minimum to reach 35000U/mL to final the putting of embodiment 1- embodiments 5, than using tradition side Method prepares and improves a class.

Test example 3.Experiment selection average weight is 8.48kg healthy sucking pig 360, and statistical analysis weight differences are not Significantly.It is randomly divided into 6 groups (5 test groups and 1 control group), every group of 6 repetitions, it is each to repeat 10.Control group daily ration is Basal diet, adds phytase made from embodiment 1,3,5 in the daily ration of test group 1,3,5 respectively, test group, in 2,4 daily rations point Not Tian Jia phytase made from embodiment 2,4, experimental group addition phytase total amount it is identical, piglet free choice feeding and drinking-water, press Routine carries out immune and expelling parasite, is observed daily in experimental period and records the diarrhoea situation of piglet, totally 30 days experimental period.Off-test Sucking pig daily gain and feedstuff-meat ratio are calculated afterwards.

Result of the test shows, compared with control group, product of the present invention can effectively improve the growth performance of sucking pig, has very Obvious advantage, illustrate that product of the present invention has very great help to improving sucking pig cultivation benefit tool.

Test example 4.The property measure of phytase

1st, the optimal pH assay method of recombinant phytase is as follows：

Zymotic fluid is subjected to enzymatic reaction to determine its optimal pH under different pH.The different pH of substrate sodium phytate buffering Phytase activity measure is carried out at 37 DEG C of liquid.As a result show, the optimal pH of phytase is 6.0, should under the conditions of pH4.0~7.5 Enzyme has preferable catalytic activity.

2nd, the optimum temperature of phytase and thermal stability determination method are as follows：

Being determined as under pH5.5 buffer solution systems and different temperatures for the optimum temperature of phytase carries out enzymatic reaction.Not Frozen water cooling is put into rapidly after heating 5min in synthermal water-bath.Separately setting one does not heat control group.Routinely side Method carries out enzyme activity determination, not heat sample result as 100%, detects remaining enzyme activity after each temperature heat treatment 5min, then Enzyme assay is carried out at 37 DEG C.Enzyme reaction optimum temperature measurement result shows that its optimum temperature is 45 DEG C.

Claims (10)

1. a kind of industrial fermentation process of phytase, it is characterised in that methods described comprises the following steps：

Step 1：One grade fermemtation tank ferments

Load one grade fermemtation culture medium and polyether-modified silicon defoaming agent, fermentation medium and polyether-modified silicon in one grade fermemtation tank The volume ratio of defoamer is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121-123 DEG C, Tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in one grade fermemtation tank is carried out cold But, using volume ratio it is 5-10% inoculum concentration culture transferring to one-level by Pichia pastoris fermentation seed liquid when temperature is down to 30-35 DEG C Expand culture in fermentation tank, trace element solution is added into fermentation tank, and be 4.8- with the pH value of ammoniacal liquor regulation zymotic fluid 5.0, the volume ratio of trace element solution and fermentation medium is 1：20-25；Fermentation seed liquid inoculum concentration is 1.5-2%；One-level Fermentation tank rotating speed is 180-200rpm；Cultivation temperature is 29-32 DEG C；When the residual sugar of the zymotic fluid in one grade fermemtation tank is less than 3- During 4g/L or weight in wet base >=60-65g/L, stop；Ventilation, 0-6h 40-45m³/ h, 6h- terminate as 60-70m³/h；One-level is sent out The voltage-controlled system of fermentation tank tank is in 0.03-0.08Mpa；

Step 2：Second order fermentation tank ferments

Load second order fermentation culture medium and polyether-modified silicon defoaming agent, fermentation medium and polyether-modified silicon in second order fermentation tank The volume ratio of defoamer is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121-123 DEG C, Tank pressure 1.1-1.4MPa is kept, sterilize 25-40min；After sterilizing terminates, the fermentation medium in second order fermentation tank is carried out cold But, when temperature is down to 30-35 DEG C, inoculum concentration culture transferring of the thalline of one grade fermemtation tank culture using volume ratio as 10-15% is arrived Expand culture in second order fermentation tank, the culture of second order fermentation tank is incubated, and cultivation temperature is 29-32 DEG C, and second order fermentation tank turns Speed is 180-200rpm, is stopped as weight in wet base >=80g/L of the zymotic fluid in second order fermentation tank；Ventilation, 0-6h 500- 600m³/ h, 6h- terminate as 800-1000m³/h；The pH of zymotic fluid is 4.5-4.7 in the second order fermentation tank incubation；Two level The voltage-controlled system of fermentation tank tank is in 0.03-0.08Mpa；

Step 3：High density fermentation

Load fermentation medium in high density fermentation tank and polyether-modified silicon defoaming agent, fermentation medium and polyether-modified silicon disappear The volume ratio of infusion is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is risen to 121-123 DEG C, is protected Tank pressure 1.1-1.4MPa is held, sterilize 25-40min；After sterilizing terminates, the fermentation medium in high density fermentation tank is carried out cold But, when temperature is down to 30-35 DEG C, inoculum concentration culture transferring of the thalline of second order fermentation tank culture using volume ratio as 10-13% is arrived Expand culture in high density fermentation tank, the culture of high density fermentation tank is incubated, and high density fermentation tank rotating speed is 180- 200rpm；Originated from fermentation in 24 hours and control the pH value of zymotic fluid in 4.5-4.7 with ammoniacal liquor, until dissolving quick rebound extremely Stop when 80%；Cultivation temperature is controlled at 29-32 DEG C；Ventilation：0h-4h, 1500-1600m³/h；More than 4h：2200- 2400m³/h；The voltage-controlled system of high density fermentation tank is in 0.03-0.08Mpa；

Step 4：Fed-batch fermentation

Feed liquid is added into high density fermentation tank；

Within 0-2h hours, with 300-350L/h speed stream charging liquid；

Within 2-4h hours, with 500-550L/h speed stream sugaring liquid；After 4 hours, with 650-700L/h speed stream Feed liquid；In fed-batch fermentation whole process, when DO is less than 25%, feed velocity 20% in day part is reduced；DO is less than 20% When, then stop material flow and add, until dissolved oxygen gos up；When the thalline weight in wet base of zymotic fluid reaches 190G/L, stop stream and add；

Glycerine 15-20kg, NH containing glucose 55-70kg, 50% concentration in feed liquid per ton₄H₂PO₄ 5-7kg、K₂SO₄ 10- 15kg、MgSO₄ 4-9kg、CaSO₄0.8-1.5kg, KOH 0.3-0.6kg, remaining is water；

Step 5：It is hungry

After stopping feed supplement, any carbon source is not added, observation DO gos up to 80%, stops during pH value rise 0.5-0.6；

Step 6：Induction, mixed feeding

0-1h is flowed the glucose solution for adding mass percentage concentration 50% by 40L/h flow velocity into high density fermentation tank and methanol is pressed 8:The mixed liquor of 1 volume ratio；1-2h is flowed by 45L/h flow velocity into high density fermentation tank plus the grape of mass percentage concentration 50% Sugar juice and methanol press 8:The mixed liquor of 1 volume ratio；2-3h is flowed by 50L/h flow velocity into high density fermentation tank plus quality percentage The glucose solution and methanol of concentration 50% press 8:The mixed liquor of 1 volume ratio；3-5h is by 65L/h flow velocity to high density fermentation tank Middle stream plus the glucose solution and methanol of mass percentage concentration 50% press 7:The mixed liquor of 1 volume ratio；5-7h presses 80L/h flow velocity Into high density fermentation tank, the glucose solution and methanol of stream plus mass percentage concentration 50% press 6:The mixed liquor of 1 volume ratio；7- 9h is flowed the glucose solution for adding mass percentage concentration 50% by 95L/h flow velocity into high density fermentation tank and methanol presses 5:1 body The mixed liquor of product ratio；9-11h is flowed by 110L/h flow velocity into high density fermentation tank plus the glucose of mass percentage concentration 50% Solution and methanol press 4:The mixed liquor of 1 volume ratio；11-13h is flowed by 120L/h flow velocity into high density fermentation tank plus quality hundred The glucose solution and methanol for dividing concentration 50% press 3:The mixed liquor of 1 volume ratio；13-19h is by 130L/h flow velocity to high density The glucose solution and methanol of stream plus mass percentage concentration 50% press 2 in fermentation tank:The mixed liquor of 1 volume ratio；19-22h is pressed 140L/h flow velocity flows the glucose solution for adding mass percentage concentration 50% into high density fermentation tank and methanol presses 1:1 volume ratio Mixed liquor；

After 22h, flowed by 145L/h flow velocity into high density fermentation tank and add salting liquid and methanol to press 1:The mixed liquor of 4 volume ratios is simultaneously According to the weight in wet base of zymotic fluid and dissolved oxygen amount regulation rotating speed, air mass flow and mixing flow velocity, it is specially：Dissolved oxygen is controlled 20% More than, when dissolved oxygen rises to 60%, mixing flow velocity is increased 20%, can not such as keep dissolved oxygen more than 20%, then is stopped mixed Close liquid stream to add, until dissolved oxygen gos up；

The formula of wherein salting liquid is：

CuSO₄·5H₂O 3-3.8g、NaI0.03-0.08g、MnSO₄·H₂O1.5-2g、H₃BO₃ 0.01-0.03g、 Na₂MoO₄.2H₂O 0.1-0.3g、CoCl₂0.2-0.5g、ZnCl₂ 10-16g、FeSO₄·7H₂O30-45g、H₂SO₄ 2.5-3g、 Biotin 0.2-0.3g；

When until wet bacterium weighs to 180g/L in zymotic fluid, stream glycerol adding carries out mixed feeding, and mixed feeding flow velocity is 40-60L/h；Induction 90-110h stops mixed feeding after beginning；The concentration of glycerine is 30-40%；

After induction is not less than 160-170h, terminate fermentation.

A kind of 2. industrial fermentation process of phytase as claimed in claim 1, it is characterised in that：In step 1, the seed The preparation of liquid includes：Pichia yeast is grown into Pichia yeast single bacterium colony through slant medium culture, is then inoculated in shaking flask In cultivated, control shaking flask rotating speed 180-220rpm, 30-32 DEG C of constant-temperature table culture 24-62h or so, obtain shake-flask seed, Weight in wet base 45-70g/L.

A kind of 3. industrial fermentation process of phytase as claimed in claim 1, it is characterised in that：In step 1, one-level per ton Contain in culture medium：Dusty yeast 2-4kg, peptone 6-15kg, glucose 6-10kg, surplus are water.

A kind of 4. industrial fermentation process of phytase as claimed in claim 1, it is characterised in that：In step 2, two level per ton Contain in culture medium：Glucose 160-240kg, NH₄H₂PO₄ 30-50kg、K₂SO₄60-80kg、MgSO₄25-40kg、CaSO₄3- 6kg%, KOH3-5kg, surplus are water.

A kind of 5. industrial fermentation process of phytase as claimed in claim 1, it is characterised in that：In step 3, fermentation per ton Contain glucose 55-70kg, NH4H in culture medium₂PO₄ 5-7kg、K₂SO₄10-15kg MgSO₄ 4-9kg、CaSO₄ 0.8- 1.5kg, KOH0.3-0.6kg, peptone 1.0-1.5kg, dusty yeast 0.3-0.6kg, CuSO₄·5H₂O 300-380g、NaI3- 8g、MnSO₄·H₂O 150-200g、H₃BO₃ 1-3g、Na₂MoO₄.2H₂O10-20g、CoCl₂20-35g、ZnCl₂ 1-1.6kg、 FeSO₄·7H₂O₃-4.5kg、H₂SO₄250-300g, biotin 20-30g.

A kind of 6. industrial fermentation process of phytase as claimed in claim 1, it is characterised in that：In steps of 5, the hungry stage Duration is not less than 4h.

A kind of 7. industrial fermentation process of phytase as claimed in claim 1, it is characterised in that：The industry hair of the phytase Fermenting process also include isolate and purify from the zymotic fluid that step 6 obtains the step of, be specially：Crude enzyme liquid is centrifuged to obtain, uses acetone precipitation Crude enzyme liquid is purified with ion-exchange chromatography, acetone concentration 60-63%, ion exchange column pH is 5.2-5.4.

A kind of 8. industrial fermentation process of phytase as claimed in claim 1, it is characterised in that：The industry hair of the phytase Fermenting process is additionally included in addition phytase synergist in the zymotic fluid that step 6 obtains, and the step of phytase is obtained through adsorbing, after drying Suddenly.

A kind of 9. industrial fermentation process of phytase as claimed in claim 8, it is characterised in that：The phytase synergist master To be made up of formic acid, cyclodextrin and starch；The quality of wherein described formic acid and fermentating liquid volume ratio are 0.1-0.12%；Ring is pasted Essence and fermentating liquid volume ratio are 1.1-1.3%, and the ratio of starch and fermentating liquid volume is 1.1-1.3%.

A kind of 10. industrial fermentation process of phytase as claimed in claim 8, it is characterised in that：Dry process is using spray Mist is dried, and dry temperature is no more than 100 DEG C；The phytase enzyme product moisture finally given is no more than 10%.