CN108396017A

CN108396017A - A kind of industrial fermentation process of mannase

Info

Publication number: CN108396017A
Application number: CN201710989548.7A
Authority: CN
Inventors: 付振山; 赵士辉; 黄兴义; 王淑华
Original assignee: Shandong Aubio Biological Technology Co Ltd
Current assignee: Shandong Aubio Biological Technology Co Ltd
Priority date: 2017-10-20
Filing date: 2017-10-20
Publication date: 2018-08-14

Abstract

The invention discloses a kind of industrial fermentation process of mannase, the method 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 mannase of the present invention, has the characteristics that put that tank enzyme activity is high, fermentation efficiency is high.

Description

A kind of industrial fermentation process of mannase

Technical field

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

Background technology

Plant cell wall is mainly made of substances such as cellulose, hemicellulose and lignin.Mannase, i.e. β-sweet dew Dextranase is a kind of multi-functional growth accelerator, can promote the secretion of quasi-insulin growthing factor I GF-I, promote protein Synthesis improves lean meat percentage, promotes growth.Mannosan is the important component of plant hemicellulose, is by β-Isosorbide-5-Nitrae-D-MANNOSE The linear polymer being formed by connecting, mainly there is the substituent groups such as glucosyl group, acetyl group and galactosyl on the side chain of polysaccharide. 'beta '-mannase (β-mannanase EC3.2.1.78) is a kind of endo hydrolysis enzyme of hydrolyzing mannan, by endo-cleavage Degrade mannose backbone β-l, and 4 glycosidic bonds release short β-Isosorbide-5-Nitrae manna oligosacchride.

In recent years, with the discovery of manna oligosacchride physiological function, the rise of green feed and the increasing of people's environmental consciousness By force, the regeneration research of the energy, people have had been enter into a new stage to the research and utilization of 'beta '-mannase.β-is sweet Dew dextranase has been widely used in food, medicine, feed, papermaking, textile printing and dyeing, oil exploitation, fine chemistry industry and biological skill The numerous areas such as art are a kind of novel industrial enzymes, have prodigious potential using value.

'beta '-mannase is a kind of multi-functional growth accelerator, can promote point of quasi-insulin growthing factor I GF-I It secretes, promotes the synthesis of protein, improve lean meat percentage, growth, main function is promoted to have：1, eliminate feed in mannosan to Portugal The interference that grape sugar absorbs, is greatly improved the energy digestibility of dregs of beans, corn bean pulp type daily ration can be given to improve 100-150kcal/kg Metabolic energy.2, mannosan decomposes the manna oligosacchride generated, can be absorbed by the beneficial bacterium in animal intestinal tract, improves flora group At the infection of reduction Escherichia coli, salmonella.The harm of broiler chicken globidiosis is reduced, the broiler chicken uniformity is improved.3, enteron aisle is reduced Viscosity promotes the digestion and absorption of energy, albumen, cellulose.

'beta '-mannase is widely present in the biology such as bacterium, actinomyces, fungi, plant, animal.Bacterial origin it is sweet Reveal the mannase that dextranase is mainly sour partial neutral.Its molecular weight is mostly between 35kDa~55kDa, optimal reaction effect Temperature is 50 DEG C~70 DEG C.Most study is bacillus at present, except the most suitable action pH of Bacillus alcalophilus reaches PH9.0 or more, most optimal reaction pH is between 5.5~8.0.For the 'beta '-mannase of fungi generally in acidity, molecular weight is big About in 45kDa~55kDa, most suitable action pH is 4.0~6.0, and optimum temperature is 55 DEG C~75 DEG C.For opposite bacterium, 'beta '-mannase optimal reaction pH value, the pH stability of originated from fungus are all relatively low, and heat resistance is poorer than bacterium.At present both at home and abroad, Although many 'beta '-mannases are cloned separation and property measures, there is some defects, example in the nature and characteristic of these enzymes Such as, pH sphere of actions are improper, and thermal stability is poor, and expression quantity is low etc., cannot meet the needs of practical application.Therefore people are uncommon A kind of new 'beta '-mannase that disclosure satisfy that practical application request can be found by hoping, so as to further genralrlization, the β-is sweet Dew dextranase is applied in the industries such as feed, food, medicine.

Mannase gene high efficient expression in recombinant bacterial strain is set to be mannase by genetic engineering means The extensive effective way inexpensively produced.Currently, the production of mannase relies primarily on the microbial fermentation of fungi, bacterium. The problems such as that there are zymotechniques is outmoded for the industrial fermentation of traditional mannase, and of high cost and benefit is low.

Invention content

In view of the deficiencies of the prior art, it is an object of the present invention to provide a kind of industrial fermentation process of mannase.

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

A kind of industrial fermentation process of mannase, it is characterised in that described method includes following steps.

Step 1：One grade fermemtation tank ferments

It is packed into one grade fermemtation culture medium in one grade fermemtation tank and polyether-modified silicon defoaming agent, fermentation medium and polyethers change Property silicon defoaming agent volume ratio be 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is made to rise to 121- 123 DEG C, tank is kept to press 1.1-1.4MPa, sterilize 25-40min；After sterilizing, to the fermentation medium in one grade fermemtation tank into Row cooling, when temperature is down to 29-32 DEG C, by fermentation seed liquid with volume ratio for 5-10% inoculum concentration culture transferring to one grade fermemtation Expand culture in tank, add trace element solution into fermentation tank, the volume ratio of trace 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 residual sugar of the zymotic fluid in one grade fermemtation tank is less than 3-3.5g/L or weight in wet base >=55-60g/L, stop；Ventilation quantity, 0-6h For 45-50m³/ h, 6h- terminate as 65-70m³/h；The voltage-controlled system of one grade fermemtation tank tank is in 0.03-0.08Mpa.The trace element is molten Liquid is the aqueous solution for the PTM1 that volume ratio is 0.8-1.2%.

Step 2：Second order fermentation tank ferments

It is packed into second order fermentation culture medium in second order fermentation tank and polyether-modified silicon defoaming agent, fermentation medium and polyethers change Property silicon defoaming agent volume ratio be 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is made to rise to 121- 123 DEG C, tank is kept to press 1.1-1.4MPa, sterilize 25-40min；After sterilizing, to the fermentation medium in second order fermentation tank into Row cooling is moved the thalline of one grade fermemtation tank culture with volume ratio when temperature is down to 29-32 DEG C for the inoculum concentration of 10-15% Expand culture in kind to second order fermentation tank, second order fermentation tank culture is constant temperature incubation, 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 quantity, 0-6h are 550-650m³/ h, 6h- terminate as 850-950m³/h；The pH of zymotic fluid is 4.5-4.6 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

It is packed into 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 made to rise to 121-123 DEG C, keep tank to press 1.1-1.4MPa, sterilize 25-40min；After sterilizing, to the fermentation medium in high density fermentation tank into Row cooling is moved the thalline of second order fermentation tank culture with volume ratio when temperature is down to 29-32 DEG C for the inoculum concentration of 10-13% Expand culture in kind to high density fermentation tank, high density fermentation tank culture is constant temperature incubation, and high density fermentation tank rotating speed is 120- 140rpm；The pH value for controlling zymotic fluid with ammonium hydroxide in 24 hours is originated in 4.5-4.7 from fermentation, is quickly rebounded extremely until dissolving Stop when 80%；Cultivation temperature is controlled at 29-32 DEG C；Ventilation quantity：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；

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

In 2-4h hours, with the speed stream sugaring liquid of 500-550L/h；After 4 hours, with the speed of 650-700L/h 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, stops stream and add；

Glycerine 15-20kg, NH containing glucose 55-70kg, 30% concentration in feed liquid per ton₄H₂PO₄ 5-7kg、 K₂SO₄10-15kg、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%, and when pH value rise 0.2-0.3 stops.

Step 6：Induction, mixed feeding

0-1h is flowed into high density fermentation tank by the flow velocity of 40L/h plus the glucose solution and first of mass percentage concentration 50% Alcohol presses 8:The mixed liquor of 1 volume ratio；1-2h, which is flowed by the flow velocity of 45L/h 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 into high density fermentation tank by the flow velocity of 50L/h 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 the flow velocity of 65L/h 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, which is flowed by the flow velocity of 95L/h into high density fermentation tank, adds the glucose solution of mass percentage concentration 50% and methanol to press 5:The mixed liquor of 1 volume ratio；9-11h is flowed into high density fermentation tank by the flow velocity of 110L/h 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 into high density fermentation tank by the flow velocity of 120L/h 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 the flow velocity of 130L/h 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 Being flowed into high density fermentation tank by the flow velocity of 140L/h adds the glucose solution of mass percentage concentration 50% and methanol to press 1:1 volume The mixed liquor of ratio；

After 22h, is flowed into high density fermentation tank by the flow velocity of 145L/h and salting liquid and methanol is added to press 1:The mixing of 4 volume ratios Liquid simultaneously adjusts rotating speed, air mass flow and mixing flow velocity according to the tonnage of zymotic fluid and dissolved oxygen amount, specially：Control dissolved oxygen exists 20% or more, when dissolved oxygen rises to 60%, mixing flow velocity is increased 20%, such as dissolved oxygen cannot be kept 20% or more, then stopped 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、 Na₂MoO₄.2H₂O0.1-0.3g、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；A concentration of 10-15% of glycerine.

Induction terminates fermentation not less than after 160-170h.

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

Further, in step 1, contain in first cell culture medium per ton：Yeast powder 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, yeast powder 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 mannase further includes isolating and purifying the zymotic fluid obtained from step 6 The step of, specially：Crude enzyme liquid is centrifuged to obtain, is purified to crude enzyme liquid with acetone precipitation and ion-exchange chromatography, acetone concentration It is 5.2-5.4 for 60-63%, ion exchange column pH.

Further, the industrial fermentation process of the mannase further include be added in the zymotic fluid that step 6 obtains it is sweet The step of revealing dextranase synergist, mannase obtained after adsorbing, drying.

Further, the mannase synergist is mainly made of formic acid, cyclodextrin and starch；The wherein described formic acid Quality is 0.1-0.12% with fermentating liquid volume ratio；Cyclodextrin is 1.1-1.3%, starch and fermentation with fermentating liquid volume ratio The ratio of liquid product is 1.1-1.3%.

Further, dry process is no more than 100 DEG C using spray drying, dry temperature；Finally obtained sweet dew is poly- Carbohydrase enzyme product moisture is no more than 10%.

The beneficial effects of the invention are as follows：The complete dissolved oxygen rise of sugar will be mended on original Process ba- sis and mends methanol induction immediately, more New is using Nature enemy, and processing time is 4-5 hour, wherein dissolved oxygen is required to go up to 80% or more, pH rises 0.5- 0.6, it can be completely consumed based on sugar using this processing, be induced conducive to cell is more optimized.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 Absorption is more advantageous in reaction cycle, while the molecular structure of glycerine and permeability are more advantageous to holding Premeabilisation of cells pressure, to Promote the metabolism producing enzyme of cell.After taking the above new cultural method, tank enzyme activity is put by the 11000u/ml that is averaged originally, is improved extremely Averagely enzyme activity 13000u/ml at present, improves fermentation level about 18%.

Description of the drawings

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

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

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

Specific implementation mode

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.

Embodiment 1.A kind of industrial fermentation process of mannase, it is characterised in that described method includes following steps.

Step 1：One grade fermemtation tank ferments

It is packed into one grade fermemtation culture medium in one grade fermemtation tank and polyether-modified silicon defoaming agent, fermentation medium and polyethers change Property silicon defoaming agent volume ratio be 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is made to rise to 121- 123 DEG C, tank is kept to press 1.1-1.4MPa, sterilize 25-40min；After sterilizing, to the fermentation medium in one grade fermemtation tank into Row cooling, when temperature is down to 29-32 DEG C, by fermentation seed liquid with volume ratio for 5-10% inoculum concentration culture transferring to one grade fermemtation Expand culture in tank, add trace element solution into fermentation tank, the volume ratio of trace 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 residual sugar of the zymotic fluid in one grade fermemtation tank is less than 3-3.5g/L or weight in wet base >=55-60g/L, stop；Ventilation quantity, 0-6h For 45-50m³/ h, 6h- terminate as 65-70m³/h；The voltage-controlled system of one grade fermemtation tank tank is in 0.03-0.08Mpa.

Step 2：Second order fermentation tank ferments

Step 3：High density fermentation

It is packed into 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 made to rise to 121-123 DEG C, keep tank to press 1.1-1.4MPa, sterilize 25-40min；After sterilizing, to the fermentation medium in high density fermentation tank into Row cooling is moved the thalline of second order fermentation tank culture with volume ratio when temperature is down to 29-32 DEG C for the inoculum concentration of 10-13% Expand culture in kind to high density fermentation tank, high density fermentation tank culture is constant temperature incubation, and high density fermentation tank rotating speed is 120- 140rpm；The pH value for controlling zymotic fluid with ammonium hydroxide in 24 hours is originated in 4.5-4.7 from fermentation, is quickly rebounded extremely until dissolving Stop when 80%；Cultivation temperature is controlled at 29-32 DEG C；Ventilation quantity：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；

In 0-2h hours, with the speed stream charging liquid of 300-350L/h；In feed liquid per ton containing glucose 55-70kg, Glycerine 15-20kg, NH4H2PO4 5-7kg, K2SO4 10-15kg, MgSO4 4-9kg, the CaSO40.8- of 30% concentration 1.5kg, KOH 0.3-0.6kg, remaining is water.Glycerine is absorbed faster, and initial stage glycerine is conducive to absorb, and long-living sugared will not be tired out Product.

In 2-4h hours, with the speed stream sugaring liquid of 500-550L/h；After 4 hours, with the speed of 650-700L/h 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, stops stream and add.

Step 5：It is hungry

Step 6：Induction, mixed feeding

0-1h is flowed into high density fermentation tank by the flow velocity of 40L/h plus the glucose solution and first of mass percentage concentration 50% Alcohol presses 8:The mixed liquor of 1 volume ratio；1-2h, which is flowed by the flow velocity of 45L/h 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 into high density fermentation tank by the flow velocity of 50L/h 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 the flow velocity of 65L/h 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, which is flowed by the flow velocity of 95L/h into high density fermentation tank, adds the glucose solution of mass percentage concentration 50% and methanol to press 5:The mixed liquor of 1 volume ratio；9-11h is flowed into high density fermentation tank by the flow velocity of 110L/h 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 into high density fermentation tank by the flow velocity of 120L/h 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 the flow velocity of 130L/h 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 Being flowed into high density fermentation tank by the flow velocity of 140L/h adds the glucose solution of mass percentage concentration 50% and methanol to press 1:1 volume The mixed liquor of ratio.In this way, it not will produce sugar accumulation, be conducive to improve product quality.

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、 Na₂MoO₄.2H₂O0.1-0.3g、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；It lures 90h stops mixed feeding after leading beginning；

Induction terminates fermentation not less than after 160h.

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

Used yeast is pichia pastoris yeast, is that one kind in methanotrophic yeast can be using methanol as unique The saccharomycete of carbon source and the energy.As other yeast, mainly exists in the form of monoploid in the asexual growth phase, work as environmental nutrient When limitation, the maqting type haploid cell mating that 2 physiological-types are different is often induced, amphiploid is fused into.Koichi Ogata Et al. to be found that certain yeast can utilize methanol for the first time in 1969 be that sole carbon source and the energy grow (Ogata, et A1.1969), hereafter, the potentiality for using 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 for the first time Philip Petroleum companies and Salk Institute Biotechnology/Industrial Associates (SIBIA) cooperative development of pichia yeast expression system has been begun to.SIBIA. researcher has detached the startup of AOX genes Son and host strain, construct carrier, and have developed corresponding Pichia pastoris gene manipulation techniques, in conjunction with 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 Invitrogea companies is entrusted 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 the foreign protein of expression can account for Summary table reaches 90% or more of albumen, is conducive to isolating and purifying for destination protein；(3) it 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 can 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, are answered extensively Protein preparation, characterization and structure elucidation etc. for laboratory scale, have thousands of kinds of albumen in Pichia pastoris system It expresses with succeeding in system.In recent years, Pichia pastoris regarded as GRAS (Generally recognized as by U.S. FDA Safe) microorganism has paved road for its application on food and medicine.In medical albumen field, have insulin, hepatitis B The multiple proteins such as surface antigen, human serum albumins, epidermal growth factor realize prepared by commercialization using Pichia anomala expression. Industrial enzyme preparation field also includes the utilizations such as mannonase lipase, mannonase zytase there are many enzyme preparation Pichia pastoris realizes the production of industrialized scale.

In step 1, contain in first cell culture medium per ton：Yeast powder 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, yeast powder 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, a concentration of 10-15% of glycerine.

The industrial fermentation process of the mannase further includes the steps that isolating and purifying the zymotic fluid obtained from step 6, Specially：Crude enzyme liquid is centrifuged to obtain, is purified to crude enzyme liquid with acetone precipitation and ion-exchange chromatography, acetone concentration 60- 63%, ion exchange column pH are 5.2-5.4.

Embodiment 2.A kind of industrial fermentation process of mannase, it is characterised in that described method includes following steps.

Step 1：One grade fermemtation tank ferments

It is packed into one grade fermemtation culture medium in one grade fermemtation tank and polyether-modified silicon defoaming agent, fermentation medium and polyethers change Property silicon defoaming agent volume ratio be 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is made to rise to 121- 123 DEG C, tank is kept to press 1.1-1.4MPa, sterilize 25-40min；After sterilizing, to the fermentation medium in one grade fermemtation tank into Row cooling, when temperature is down to 29-32 DEG C, by fermentation seed liquid with volume ratio for 5-10% inoculum concentration culture transferring to one grade fermemtation Expand culture in tank, add trace element solution into fermentation tank, the volume ratio of trace 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 residual sugar of the zymotic fluid in one grade fermemtation tank is less than 3-3.5g/L or weight in wet base >=55-60g/L, stop；Ventilation quantity, 0-6h For 45-50m³/ h, 6h- terminate as 65-70m³/h；The voltage-controlled system of one grade fermemtation tank tank is in 0.03-0.08Mpa.Pichia pastoris itself is simultaneously Not producing enzyme is by genetic engineering means by different producing enzyme gene transfers to yeast genes, therefore same yeast can produce not Same enzyme.

Step 2：Second order fermentation tank ferments

Step 3：High density fermentation

Step 4：Fed-batch fermentation

Feed liquid is added into high density fermentation tank；

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

Step 5：It is hungry

Step 6：Induction, mixed feeding

0-1h is flowed into high density fermentation tank by the flow velocity of 40L/h plus the glucose solution and first of mass percentage concentration 50% Alcohol presses 8:The mixed liquor of 1 volume ratio；1-2h, which is flowed by the flow velocity of 45L/h 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 into high density fermentation tank by the flow velocity of 50L/h 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 the flow velocity of 65L/h 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, which is flowed by the flow velocity of 95L/h into high density fermentation tank, adds the glucose solution of mass percentage concentration 50% and methanol to press 5:The mixed liquor of 1 volume ratio；9-11h is flowed into high density fermentation tank by the flow velocity of 110L/h 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 into high density fermentation tank by the flow velocity of 120L/h 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 the flow velocity of 130L/h 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 Being flowed into high density fermentation tank by the flow velocity of 140L/h adds the glucose solution of mass percentage concentration 50% and methanol to press 1:1 volume The mixed liquor of ratio.

After 22h, is flowed into high density fermentation tank by the flow velocity of 145L/h and salting liquid and methanol is added to press 1:The mixing of 4 volume ratios Liquid simultaneously adjusts rotating speed, air mass flow and mixing flow velocity according to the tonnage of zymotic fluid and dissolved oxygen amount, specially：Control dissolved oxygen exists 20% or more, when dissolved oxygen rises to 60%, mixing flow velocity is increased 20%, such as dissolved oxygen cannot be kept 20% or more, then stopped Only mixing liquid stream adds, until dissolved oxygen gos up；Zymotic fluid tonnage and methanol additive amount are the relationships of proportional example, can pass through practice Groped to fix.

The formula of wherein salting liquid is：

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；It lures 110h stops mixed feeding after leading beginning；

Induction terminates fermentation not less than after 170h.

The preparation of the seed liquor includes：It willPichia pastorisBacterium grows through slant medium culturePichia pastorisBacterium single bacterium It falls, is then inoculated in shaking flask and is cultivated, 180-220rpm, 30-32 DEG C of constant-temperature table culture 24-62h of control shaking flask rotating speed Left and right, obtains shake-flask seed, weight in wet base 45-70g/L.

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, yeast powder 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.

The industrial fermentation process of the mannase further includes that mannase is added in the zymotic fluid that step 6 obtains Synergist, the step of obtaining mannase after adsorbing, drying.

The mannase synergist is mainly made of formic acid, cyclodextrin and starch；The quality of the wherein described formic acid with Fermentating liquid volume ratio is 0.1-0.12%；Cyclodextrin is 1.1-1.3%, starch and fermentating liquid volume with fermentating liquid volume ratio Ratio be 1.1-1.3%.

Dry process is no more than 100 DEG C using spray drying, dry temperature；Finally obtained mannase enzyme production Product moisture is no more than 10%.

Embodiment 3.A kind of industrial fermentation process of mannase, it is characterised in that described method includes following steps.

Step 1：One grade fermemtation tank ferments

Step 2：Second order fermentation tank ferments

Step 3：High density fermentation

Step 4：Fed-batch fermentation

Feed liquid is added into high density fermentation tank；

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

Step 5：It is hungry

Step 6：Induction, mixed feeding

After 22h, is flowed into high density fermentation tank by the flow velocity of 145L/h and salting liquid and methanol is added to press 1:The mixing of 4 volume ratios Liquid simultaneously adjusts rotating speed, air mass flow and mixing flow velocity according to the tonnage of zymotic fluid and dissolved oxygen amount, specially：Control dissolved oxygen exists 20% or more, when dissolved oxygen rises to 60%, mixing flow velocity is increased 20%, such as dissolved oxygen cannot be kept 20% or more, then stopped Only mixing liquid stream adds, until dissolved oxygen gos up；Tonnage is directly proportional with oxygen amount is melted, dissolved oxygen amount and rotating speed, air mass flow and mixed liquor Flow velocity is directly proportional.

The formula of wherein salting liquid is：

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；

Induction terminates fermentation not less than after 160-170h.

The industrial fermentation process of the mannase further includes the steps that isolating and purifying the zymotic fluid obtained from step 6, Specially：Crude enzyme liquid is centrifuged to obtain, is purified to crude enzyme liquid with acetone precipitation and ion-exchange chromatography, acetone concentration 60- 63%, ion exchange column pH are 5.2-5.4.Pichia pastoris not producing enzyme itself, being will be different by genetic engineering means In producing enzyme gene transfer to yeast genes, therefore same yeast can produce different enzymes.

Embodiment 4.A kind of industrial fermentation process of mannase, it is characterised in that described method includes following steps.

Step 1：One grade fermemtation tank ferments

Step 2：Second order fermentation tank ferments

Step 3：High density fermentation

Step 4：Fed-batch fermentation

Feed liquid is added into high density fermentation tank；

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

Step 5：It is hungry

Step 6：Induction, mixed feeding

After 22h, is flowed into high density fermentation tank by the flow velocity of 145L/h and salting liquid and methanol is added to press 1:The mixing of 4 volume ratios Liquid simultaneously adjusts rotating speed, air mass flow and mixing flow velocity according to the tonnage of zymotic fluid and dissolved oxygen amount, specially：Control dissolved oxygen exists 20% or more, when dissolved oxygen rises to 60%, mixing flow velocity is increased 20%, such as dissolved oxygen cannot be kept 20% or more, then stopped Only mixing liquid stream adds, until dissolved oxygen rise

The formula of wherein salting liquid is：

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；

Induction terminates fermentation not less than after 160-170h.

The mannase synergist is mainly made of formic acid, acetic acid, cyclodextrin and starch；The matter of the wherein described formic acid Amount is 0.05-0.0.06% with fermentating liquid volume ratio；The quality of the wherein described formic acid is 0.05- with fermentating liquid volume ratio 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%.

Embodiment 5.A kind of industrial fermentation process of mannase, it is characterised in that described method includes following steps.

Step 1：One grade fermemtation tank ferments

Step 2：Second order fermentation tank ferments

Step 3：High density fermentation

Step 4：Fed-batch fermentation

Feed liquid is added into high density fermentation tank；

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

Step 5：It is hungry

Step 6：Induction, mixed feeding

The formula of wherein salting liquid is：

Induction terminates fermentation not less than after 160-170h.

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

It can be seen that the embodiment of the present invention 2 mends sugar amount by change and mends sugared rate, hence it is evident that improve weight in wet base.

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

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

Note：It is 20~25L/h that mixed feeding technique, which uses 25% glycerine, mixed feeding process flow,.

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, every taking zymotic fluid to measure OD600nm and thalline weight in wet base for 24 hours, supernatant is taken to carry out sweet dew poly- Anase activity detects.Put tank centrifugation and Enzyme activity assay：Tank is put after induction, mixed feeding, is centrifuged with 5000rpm under the conditions of 4 DEG C 30min collects supernatant.

According to National Standard of the People's Republic of China《GB/T 18634-2009》It carries out.Mannanase Activity defines Refer under conditions of 37 DEG C of pH value 5.50, discharges 1 μm of ol Phos from a concentration of 5.0mmol/L sodium phytate solutions per min, i.e., For a Mannanase Activity unit, indicated with U.

X=ym × t × n]]>

The activity of mannase in X-sample, unit are that every gram of unit of enzyme activity (U/g) or unit of enzyme activity's unit are every Milliliter (U/mL)；For y-according to the light absorption value of practical sample liquid by the amount of the calculated Phos of linear regression equation, unit is micro- rubs You；T-enzyme digestion reaction time, unit min；The extension rate of n-sample；The amount of m-sample, unit be gram or milliliter.

Take 50 μ L dilution enzyme solutions that 950 μ L of substrate 4mmol/L sodium phytates is added (to be matched with the acetate buffer solution of 0.1mol/L pH5.5 System), 37 DEG C reaction 30min, add 1mL 10%TCA terminate reaction, add 2mL developing solutions (10g Ammonium Molybdate Tetrahydrate+32mL sulfuric acid+ 73.2g ferrous sulfate, adds water to be settled to 1L), it develops the color to reaction solution.Control then plus after enzyme solution first adds TCA mixings, then adds bottom Object.It develops the color after 10min, surveys 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 the prior art, stream plus glucose, starvation, first Alcohol-induced technique productions mannase, it is as follows with comparing result of the present invention.

	Put tank enzyme activity
		Embodiment 1	12900U/mL
Embodiment 2	13000U/mL
		Embodiment 3	13500U/mL
Embodiment 4	13200U/mL
		Embodiment 5	12950U/mL
Conventional method	11000U/mL

Fig. 1 shows the prior art using the mannase after two-stage fermentation, feeding glucose fermentation, starvation and methanol induction Weight in wet base and enzyme activity test chart.Fig. 2 illustrates the weight in wet base of the mannase of the production of method shown in the embodiment of the present invention 2 and enzyme activity is surveyed Attempt.As can be seen that under the conditions of same period, the enzyme activity rate of rise being adjusted is very fast, after taking the above new cultural method, It puts tank enzyme activity by the 11000u/ml that is averaged originally, improves to averagely enzyme activity 13000u/ml at present, improves fermentation level about 18%.

Test example 3.Experiment selects average weight for the healthy sucking pig 360 of 8.48kg, 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 mannase made from embodiment 1,3,5, test group, 2,4 daily rations in 1,3,5 daily ration of test group respectively Middle to add mannase made from embodiment 2,4 respectively, the mannase total amount of experimental group addition is identical, and piglet is freely adopted Food and drinking-water, routinely carry out immune and expelling parasite, observe and record the diarrhea situation of piglet in experimental period daily, experimental period totally 30 It.Sucking pig daily gain and feedstuff-meat ratio are calculated after the test.

Test result shows that compared with the control group product of the present invention can effectively improve the growth performance of sucking pig, has especially non- Normal apparent advantage illustrates that product of the present invention has very great help to improving sucking pig cultivation benefit tool.

Claims

1. a kind of industrial fermentation process of mannase, which is characterized in that described method includes following steps：

Step 1：One grade fermemtation tank ferments

One grade fermemtation culture medium and polyether-modified silicon defoaming agent, fermentation medium and polyether-modified silicon are packed into one grade fermemtation tank The volume ratio of antifoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is made to rise to 121-123 DEG C, Tank is kept to press 1.1-1.4MPa, sterilize 25-40min；After sterilizing, the fermentation medium in one grade fermemtation tank is carried out cold But, when temperature is down to 29-32 DEG C, by Pichia pastoris fermentation seed liquid with volume ratio for 5-10% inoculum concentration culture transferring to level-one Expand culture in fermentation tank, trace element solution, the volume ratio of trace element solution and fermentation medium are added into fermentation tank It 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℃；When the residual sugar of the zymotic fluid in one grade fermemtation tank is less than 3-3.5g/L or weight in wet base >=55-60g/L, stop；Ventilation Amount, 0-6h 45-50m³/ h, 6h- terminate as 65-70m³/h；The voltage-controlled system of one grade fermemtation tank tank is in 0.03-0.08Mpa；

Step 2：Second order fermentation tank ferments

Second order fermentation culture medium and polyether-modified silicon defoaming agent, fermentation medium and polyether-modified silicon are packed into second order fermentation tank The volume ratio of antifoaming agent is 6000-8500：1；Fermentation medium is heated, fermentation medium temperature is made to rise to 121-123 DEG C, Tank is kept to press 1.1-1.4MPa, sterilize 25-40min；After sterilizing, the fermentation medium in second order fermentation tank is carried out cold But, when temperature is down to 29-32 DEG C, the thalline of one grade fermemtation tank culture is arrived with volume ratio for the inoculum concentration culture transferring of 10-15% Expand culture in second order fermentation tank, second order fermentation tank culture is constant temperature incubation, 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 quantity, 0-6h 550- 650m³/ h, 6h- terminate as 850-950m³/h；The pH of zymotic fluid is 4.5-4.6 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

It is packed into 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 made to rise to 121-123 DEG C, is protected Tank pressure 1.1-1.4MPa is held, sterilize 25-40min；After sterilizing, the fermentation medium in high density fermentation tank is carried out cold But, when temperature is down to 29-32 DEG C, the thalline of second order fermentation tank culture is arrived with volume ratio for the inoculum concentration culture transferring of 10-13% Expand culture in high density fermentation tank, high density fermentation tank culture is constant temperature incubation, and high density fermentation tank rotating speed is 120- 140rpm；The pH value for controlling zymotic fluid with ammonium hydroxide in 24 hours is originated in 4.5-4.7 from fermentation, is quickly rebounded extremely until dissolving Stop when 80%；Cultivation temperature is controlled at 29-32 DEG C；Ventilation quantity：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；

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

In 2-4h hours, with the speed stream sugaring liquid of 500-550L/h；After 4 hours, with the speed stream of 650-700L/h 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, stops stream and add；

Glycerine 15-20kg, NH containing glucose 55-70kg, 30% 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%, and when pH value rise 0.2-0.3 stops；

Step 6：Induction, mixed feeding

0-1h, which is flowed by the flow velocity of 40L/h into high density fermentation tank, adds the glucose solution of mass percentage concentration 50% and methanol to press 8:The mixed liquor of 1 volume ratio；1-2h is flowed into high density fermentation tank by the flow velocity of 45L/h 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 into high density fermentation tank by the flow velocity of 50L/h plus quality percentage The glucose solution and methanol of concentration 50% press 8:The mixed liquor of 1 volume ratio；3-5h is by the flow velocity of 65L/h 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 the flow velocity of 80L/h 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, which is flowed by the flow velocity of 95L/h into high density fermentation tank, adds the glucose solution of mass percentage concentration 50% and methanol to press 5:1 body The mixed liquor of product ratio；9-11h is flowed into high density fermentation tank by the flow velocity of 110L/h plus the glucose of mass percentage concentration 50% Solution and methanol press 4:The mixed liquor of 1 volume ratio；11-13h is flowed into high density fermentation tank by the flow velocity of 120L/h 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 the flow velocity of 130L/h 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 The flow velocity of 140L/h 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, is flowed into high density fermentation tank by the flow velocity of 145L/h and salting liquid and methanol is added to press 1:The mixed liquor of 4 volume ratios is simultaneously Rotating speed, air mass flow and mixing flow velocity are adjusted according to the tonnage of zymotic fluid and dissolved oxygen amount, specially：Dissolved oxygen is controlled 20% More than, when dissolved oxygen rises to 60%, mixing flow velocity is increased 20%, cannot such as keep dissolved oxygen 20% or more, then is stopped mixed It closes 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 starts rear 90-110h and stops mixed feeding；A concentration of 10-15% of glycerine；

Induction terminates fermentation not less than after 160-170h.

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

3. a kind of industrial fermentation process of mannase as described in claim 1, it is characterised in that：In step 1, per ton Contain in first cell culture medium：Yeast powder 2-4kg, peptone 6-15kg, glucose 6-10kg, surplus are water.

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

5. a kind of industrial fermentation process of mannase as described in claim 1, it is characterised in that：In step 3, per ton Contain glucose 55-70kg, NH4H in fermentation medium₂PO₄ 5-7kg、K₂SO₄10-15kg MgSO₄ 4-9kg、CaSO₄ 0.8-1.5kg, KOH0.3-0.6kg, peptone 1.0-1.5kg, yeast powder 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.

6. a kind of industrial fermentation process of mannase as described in claim 1, it is characterised in that：In steps of 5, hungry Phase duration is not less than 4h.

7. a kind of industrial fermentation process of mannase as described in claim 1, it is characterised in that：The mannase Industrial fermentation process further include the steps that isolating and purifying the zymotic fluid obtained from step 6, specially：Crude enzyme liquid is centrifuged to obtain, is used Acetone precipitation and ion-exchange chromatography purify crude enzyme liquid, acetone concentration 60-63%, and ion exchange column pH is 5.2-5.4。

8. a kind of industrial fermentation process of mannase as described in claim 1, it is characterised in that：The mannase Industrial fermentation process further include the addition mannase synergist in the zymotic fluid that step 6 obtains, after adsorb, dry The step of to mannase.

9. a kind of industrial fermentation process of mannase as claimed in claim 8, it is characterised in that：The mannase Synergist is mainly made of formic acid, cyclodextrin and starch；The quality of the wherein described formic acid is 0.1- with fermentating liquid volume ratio 0.12%；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%.

10. a kind of industrial fermentation process of mannase as claimed in claim 8, it is characterised in that：Dry process is adopted With spray drying, dry temperature is no more than 100 DEG C；Finally obtained mannase enzyme product moisture is no more than 10%.