CN104450815A - Fermentation method for improving yield of isoleucine - Google Patents

Abstract

The invention discloses a fermentation method for improving the yield of isoleucine. The fermentation method comprises the following steps: culturing microorganisms, fermenting, separating fermentation liquor, carrying out ion exchange on fermentation supernatant, deaminizing with a deamination tower, decolorizing with activated carbon and crystallizing by evaporating. Because the corn steep liquor which serves as a hydrolysis liquor in the culture medium and accounts for 40% of the total weight of the culture medium replaces the hydrochloric acid adopted by the traditional method to regulate the pH value, the corrosion of the hydrochloric acid to the stainless steel tank is avoided. In addition, because biotin and VB1 are used as growth factors in the process, the viscosity of the fermentation liquor can be reduced, the respiratory metabolism of the microorganisms can be promoted, and the down-stream finished product can be extracted easily. By adopting the fermentation method disclosed by the invention, a good fermentation index can be achieved, the yield of the isoleucine which is a metabolite can be improved significantly, the average yield of the isoleucine can be up to 36.9g/L and can be increased by 30% compared with that of the metabolite isoleucine obtained by adopting the original method, the conversion rate of the metabolite isoleucine can be increased by 2-4%, and the fermentation cycle can be shortened by 10 hours.

Description

A kind of fermentation process improving Isoleucine output

Technical field

the invention belongs to technical field of amino acid production, be specifically related to a kind of fermentation process improving Isoleucine output.

Background technology

ILE, as one of branched-chain amino acid, is essential amino acid, has different physiological roles.Because of the structure and function that it is special, account for the status of particularly important in human life's metabolism, mainly in order to prepare amino acid transfusion, synthetic polypeptide medicaments and food antioxidant etc., the effect especially in medical research and treatment comes into one's own day by day.It the dietary therapy of the treatment of hemato encephalic barrier, hepatic coma, chronic cirrhosis and renal failure, Organic acidemia, table mass formed by blood stasis and postoperative diabetic subject treatment, accelerate surgical wound healing, tumour patient nutritional support treatment in be widely used.And China's branched-chain amino acid cost can fall, and poor product quality, price does not increase, and most branched-chain amino acid all can only be used in the production of feed, and quality does not reach pharmaceutical grade.In addition, domestic acid production rate is up to 28-33 g/L, and Isoleucine has realized large-scale industrial production abroad, and acid yield has reached more than 30-35 g/L; From productive rate, the peak rate of conversion of current fermentative Production Isoleucine is only 18-20%.Domestic many Amino Acid Factories are all in the urgent need to the production technology of high-quality, low cost.Therefore, carry out technological innovation to zymotechnique, the production and technical indication of Isoleucine is improved, particularly the raising of glucose acid invert ratio makes production cost be declined, and promotes that the healthy and rapid development of China's branched-chain amino acid industry is necessary very much.

Summary of the invention

The object of the invention is the technical problem for the yielding poorly of fermentative Production Isoleucine in prior art, low conversion rate, a kind of fermentation process improving Isoleucine output is provided, the fermentation index of the method is all better, Isoleucine meta-bolites output significantly improves, and the acid of average product can reach 36.9g/L.

The technical scheme that the present invention realizes above-mentioned purpose employing is: a kind of fermentation process improving Isoleucine output, and step is as follows:

Step one, cultivation bacterial classification

Thalline streak inoculation in activated inclined plane, 31 DEG C of constant temperature culture 36-40h, first order seed is produced in bacterial classification room enlarged culturing, then enlarged culturing is continued by the first order seed obtained access secondary seed tank, in secondary seed medium, pass into sterile air, air flow is 0.5V/V/M-0.8V/V/M, and secondary seed process in secondary seed tank disappears in fact, lowers the temperature, cultivates, obtain secondary seed nutrient solution, for subsequent use;

Step 2, fermentation

Being linked into by secondary seed nutrient solution installs in the fermentor tank of fermention medium, and the volume of secondary seed nutrient solution is the 15%-20% of fermention medium volume; Pass into sterile air, continuously ferment 50-60h; Temperature from fermentation control as 31-32 DEG C in the process of fermentation 25h, control after fermentation 25h as 32.5-33 DEG C; Dissolved oxygen from fermentation control as being not less than 18.75mol/L in the process of fermentation 12h, control as 11.25-15.62mol/L after fermentation 12h; PH from fermentation control as 6.8-7.0 in the process of fermentation 36h, control after fermentation 36h as 7.0-7.2; The mass percent controlling residual sugar in fermenting process is 0.005-0.05%;

Step 3, separation of fermentative broth

After fermentation ends, first fermented liquid is warming up to 60-80 DEG C and carries out sterilizing, then 40 DEG C are cooled to, adopt membrane filtration treatment process, carry out being separated under 0.2-0.5Mpa pressure and obtain mycelium in fermented liquid and albumen and fermented liquid clear liquid, in isolated mycelium and albumen, add flocculation agent flocculation, the add-on of flocculation agent is the 5-20% of isolated mycelium and protein mass, then must to wet protein fodder through Plate Filtration at 0.02-0.04Mpa, 20-40 DEG C, for subsequent use;

Step 4, the ion-exchange of fermented liquid clear liquid

The pH regulator of the fermented liquid clear liquid first step 3 obtained is 3.0-4.0, then join in ion exchange column and adsorb Isoleucine, stop when resin absorption is saturated adding fermented liquid clear liquid, with the ammonia soln of the 0.5-1.0mol/L Isoleucine as elution resin absorption, what first flow out during wash-out is clear liquid, be recycled to fermentation clear liquid tank, for subsequent use; Secondly flow out for pH value be the Isoleucine elutriant of 2-10, for subsequent use; The elutriant finally flowed out is tail washings, and back scrubbing takes off tank and reuses, and ion exchange process is normal temperature, and pressure is 0.01-0.03Mpa;

Step 5, deammoniation tower deamination

The Isoleucine elutriant that step 4 is collected is entered deammoniation tower deamination, obtains deamination liquid, for subsequent use;

Step 6, activated carbon decolorizing

The deamination liquid vitriol oil first step 5 obtained regulates pH to be 6.0-7.0, then add the gac of Isoleucine total amount 30% in deamination liquid, under the condition of 60-70 DEG C, leave standstill 1h, then filter gac at the pressure of 0.01-0.02Mpa, obtain the Isoleucine clear liquid decoloured, for subsequent use;

Step 7, evaporative crystallization

Isoleucine clear liquid step 6 obtained joins in vaporizer, vacuum tightness be 0.09Mpa, under temperature is less than 70 DEG C of conditions, pass into 0.5Mpa saturation steam, carry out evaporation concentration, after being concentrated into 10-12 times of Isoleucine clear liquid density, educate crystalline substance, centrifugation removing mother liquor, obtain Isoleucine crystal and enter packaging process after expansion drying, namely complete the fermentative production of Isoleucine.

Secondary seed medium described in step one is made up of the raw material of following mass concentration: glucose 25-35g/L, magnesium sulfate 0.3-0.8g/L, potassium primary phosphate 1.0-2.0g/L, ammonium sulfate 3-8g/L, yeast extract powder 1.5-3.0g/L, corn syrup hydrolyzate 25-40g/L, vitamin H 0.3-0.8mg/L, Vb1 1.5-3.0mg/L, defoamer 0.3mg/L, and surplus is water;

Described corn syrup hydrolyzate is the corn steep liquor magma of the soak solution evaporation concentration gained of corn after sulfurous acid soaks, or is that corn steep liquor magma is again through vitriol oil heating hydrolysis gained slurries.

Fermention medium described in step 2 is made up of the raw material of following mass concentration: glucose 100-200g/L, magnesium sulfate 0.3-0.8g/L, potassium primary phosphate 1.0-2.0g/L, ammonium sulfate 5-10g/L, yeast extract powder 1.5-3.0g/L, corn syrup hydrolyzate 15-35g/L, vitamin H 0.05-0.25mg/L, Vb1 0.05-0.2mg/L, Trisodium Citrate 2-6g/L, defoamer 0.1-0.5mg/L, and surplus is water;

Described corn syrup hydrolyzate is the corn steep liquor magma of the soak solution evaporation concentration gained of corn after sulfurous acid soaks, or is that corn steep liquor magma is again through vitriol oil heating hydrolysis gained slurries.

60% of corn syrup hydrolyzate weight in described secondary seed medium is directly prepared in the medium, and remaining 40% auto-feeding is to regulate the pH of fermenting process.

beneficial effect of the present invention

The fermentation process of Isoleucine provided by the invention, fermentation index is better; Isoleucine meta-bolites output significantly improves, and the acid of average product can reach 36.9g/L, improves acid production rate 30% than average before improvement; Isoleucine meta-bolites transformation efficiency improves 2-4%; Producing strains bulk-growth and metabolism are rapid, and fermentation period shortens 10h, and equipment turnover rate is improved, and correspondingly improve output; Decrease the generation of metabolic by-prods, make the corresponding raising of the purity of meta-bolites in fermented liquid, to the extraction operation of product and the purity of product favourable, reduce fermentation costs and extraction cost simultaneously.

In fermentation process substratum of the present invention, 40% of corn syrup hydrolyzate gross weight replaces the way with salt acid for adjusting pH in traditional technology, avoids the corrosion of hydrochloric acid to the tank body of stainless steel.Vitamin H and VB is used in addition in this technique ₁as somatomedin, reduce the viscosity of feed liquid, advantageously in the respiratory metabolism of thalline, be conducive to the extraction of downstream takeaway simultaneously.

Embodiment

Improve a fermentation process for Isoleucine output, step is as follows:

Step one, cultivation bacterial classification

Thalline streak inoculation in activated inclined plane, 31 DEG C of constant temperature culture 36-40h, first order seed is produced in bacterial classification room enlarged culturing, then enlarged culturing is continued by the first order seed obtained access secondary seed tank, in secondary seed medium, pass into sterile air, air flow is 0.5V/V/M-0.8V/V/M, and secondary seed process in secondary seed tank disappears in fact, lowers the temperature, cultivates, obtain secondary seed nutrient solution, for subsequent use;

Step 2, fermentation

Being linked into by secondary seed nutrient solution installs in the fermentor tank of fermention medium, and the volume of secondary seed nutrient solution is the 15%-20% of fermention medium volume; Pass into sterile air, continuously ferment 50-60h; Temperature from fermentation control as 31-32 DEG C in the process of fermentation 25h, control after fermentation 25h as 32.5-33 DEG C; Dissolved oxygen from fermentation control as being not less than 18.75mol/L in the process of fermentation 12h, control as 11.25-15.62mol/L after fermentation 12h; PH from fermentation control as 6.8-7.0 in the process of fermentation 36h, control after fermentation 36h as 7.0-7.2; The mass percent controlling residual sugar in fermenting process is 0.005-0.05%;

Step 3, separation of fermentative broth

After fermentation ends, first fermented liquid is warming up to 60-80 DEG C and carries out sterilizing, then 40 DEG C are cooled to, adopt membrane filtration treatment process, carry out being separated under 0.2-0.5Mpa pressure and obtain mycelium in fermented liquid and albumen and fermented liquid clear liquid, in isolated mycelium and albumen, add flocculation agent flocculation, the add-on of flocculation agent is the 5-20% of isolated mycelium and protein mass, then must to wet protein fodder through Plate Filtration at 0.02-0.04Mpa, 20-40 DEG C, for subsequent use;

Step 4, the ion-exchange of fermented liquid clear liquid

The pH regulator of the fermented liquid clear liquid first step 3 obtained is 3.0-4.0, then join in ion exchange column and adsorb Isoleucine, stop when resin absorption is saturated adding fermented liquid clear liquid, with the ammonia soln of the 0.5-1.0mol/L Isoleucine as elution resin absorption, what first flow out during wash-out is clear liquid, be recycled to fermentation clear liquid tank, for subsequent use; Secondly flow out for pH value be the Isoleucine elutriant of 2-10, for subsequent use; The elutriant finally flowed out is tail washings, and back scrubbing takes off tank and reuses, and ion exchange process is normal temperature, and pressure is 0.01-0.03Mpa;

Step 5, deammoniation tower deamination

The Isoleucine elutriant that step 4 is collected is entered deammoniation tower deamination, obtains deamination liquid, for subsequent use;

Step 6, activated carbon decolorizing

The deamination liquid vitriol oil first step 5 obtained regulates pH to be 6.0-7.0, then add the gac of Isoleucine total amount 30% in deamination liquid, under the condition of 60-70 DEG C, leave standstill 1h, then filter gac at the pressure of 0.01-0.02Mpa, obtain the Isoleucine clear liquid decoloured, for subsequent use;

Step 7, evaporative crystallization

Isoleucine clear liquid step 6 obtained joins in vaporizer, vacuum tightness be 0.09Mpa, under temperature is less than 70 DEG C of conditions, pass into 0.5Mpa saturation steam, carry out evaporation concentration, after being concentrated into 10-12 times of Isoleucine clear liquid density, educate crystalline substance, centrifugation removing mother liquor, obtain Isoleucine crystal and enter packaging process after expansion drying, namely complete the fermentative production of Isoleucine.

Secondary seed medium described in step one is made up of the raw material of following mass concentration: glucose 25-35g/L, magnesium sulfate 0.3-0.8g/L, potassium primary phosphate 1.0-2.0g/L, ammonium sulfate 3-8g/L, yeast extract powder 1.5-3.0g/L, corn syrup hydrolyzate 25-40g/L, vitamin H 0.3-0.8mg/L, Vb1 1.5-3.0mg/L, defoamer 0.3mg/L, and surplus is water; Described corn syrup hydrolyzate is the corn steep liquor magma of the soak solution evaporation concentration gained of corn after sulfurous acid soaks, or is that corn steep liquor magma is again through vitriol oil heating hydrolysis gained slurries.

Fermention medium described in step 2 is made up of the raw material of following mass concentration: glucose 100-200g/L, magnesium sulfate 0.3-0.8g/L, potassium primary phosphate 1.0-2.0g/L, ammonium sulfate 5-10g/L, yeast extract powder 1.5-3.0g/L, corn syrup hydrolyzate 15-35g/L, vitamin H 0.05-0.25mg/L, Vb1 0.05-0.2mg/L, Trisodium Citrate 2-6g/L, defoamer 0.1-0.5mg/L, and surplus is water; Described corn syrup hydrolyzate is the corn steep liquor magma of the soak solution evaporation concentration gained of corn after sulfurous acid soaks, or is that corn steep liquor magma is again through vitriol oil heating hydrolysis gained slurries.

60% of corn syrup hydrolyzate weight in described secondary seed medium is directly prepared in the medium, and remaining 40% auto-feeding is to regulate the pH of fermenting process.

below in conjunction with specific embodiment, the present invention will be further described:

embodiment 1:

Improve a fermentation process for Isoleucine output, step is as follows:

Step one, cultivation bacterial classification

Thalline streak inoculation in activated inclined plane, 31 DEG C of constant temperature culture 36-40h, first order seed is produced in bacterial classification room enlarged culturing, then enlarged culturing is continued by the first order seed obtained access secondary seed tank, in secondary seed medium, pass into sterile air, air flow is 0.5V/V/M, and secondary seed process in secondary seed tank disappears in fact, lowers the temperature, cultivates, obtain secondary seed nutrient solution, for subsequent use; Described secondary seed medium is made up of the raw material of following mass concentration: glucose 25g/L, magnesium sulfate 0.5g/L, potassium primary phosphate 1.3g/L, ammonium sulfate 5g/L, yeast extract powder 2.2g/L, corn syrup hydrolyzate 33.75g/L, vitamin H 0.3-0.8mg/L, Vb1 1.5-3.0mg/L, defoamer 0.3mg/L, and surplus is water; Described corn syrup hydrolyzate is the corn steep liquor magma of the soak solution evaporation concentration gained of corn after sulfurous acid soaks, or is that corn steep liquor magma is again through vitriol oil heating hydrolysis gained slurries.In described corn syrup hydrolyzate, 20.25g/L allocates substratum into as bed material, and the independent sterilizing of 13.5g/L auto-feeding when seed culture reduces pH value as acid solution.

Step 2, fermentation

Being linked into by secondary seed nutrient solution installs in the fermentor tank of fermention medium, and the volume of secondary seed nutrient solution is 15% of fermention medium volume; Pass into sterile air, continuously ferment 50-60h; Temperature from fermentation control as 31-32 DEG C in the process of fermentation 25h, control after fermentation 25h as 32.5-33 DEG C; Dissolved oxygen from fermentation control as being not less than 18.75mol/L in the process of fermentation 12h, control as 11.25-15.62mol/L after fermentation 12h; PH just from control as 6.8-7.0 in fermentation to the process of fermentation 36h, control after fermentation 36h as 7.0-7.2; The mass percent controlling residual sugar in fermenting process is 0.005-0.05%;

Described fermention medium is made up of the raw material of following mass concentration: glucose 120g/L, magnesium sulfate 0.6g/L, potassium primary phosphate 1.2g/L, ammonium sulfate 8.4g/L, yeast extract powder 2.4g/L, corn syrup hydrolyzate 28.5g/L, vitamin H 0.14mg/L, Vb1 0.1mg/L, Trisodium Citrate 4g/L, defoamer 0.3mg/L, and surplus is water; Described corn syrup hydrolyzate is the corn steep liquor magma of the soak solution evaporation concentration gained of corn after sulfurous acid soaks, or is that corn steep liquor magma is again through vitriol oil heating hydrolysis gained slurries.

Step 3, separation of fermentative broth

After fermentation ends, first fermented liquid is warming up to 60-80 DEG C and carries out sterilizing, then 40 DEG C are cooled to, adopt membrane filtration treatment process, carry out being separated under 0.2-0.5Mpa pressure and obtain mycelium in fermented liquid and albumen and fermented liquid clear liquid, in isolated mycelium and albumen, add flocculation agent flocculation, the add-on of flocculation agent is 5% of isolated mycelium and protein mass, then must to wet protein fodder through Plate Filtration at 0.02-0.04Mpa, 20-40 DEG C, for subsequent use;

Step 4, the ion-exchange of fermented liquid clear liquid

The pH regulator of the fermented liquid clear liquid first step 3 obtained is 3.0, then join in ion exchange column and adsorb Isoleucine, stop when resin absorption is saturated adding fermented liquid clear liquid, with the ammonia soln of the 0.5mol/L Isoleucine as elution resin absorption, what first flow out during wash-out is clear liquid, be recycled to fermentation clear liquid tank, for subsequent use; Secondly flow out for pH value be the Isoleucine elutriant of 2-10, for subsequent use; The elutriant finally flowed out is tail washings, and back scrubbing takes off tank and reuses, and ion exchange process is normal temperature, and pressure is 0.01-0.03Mpa;

Step 5, deammoniation tower deamination

The Isoleucine elutriant that step 4 is collected is entered deammoniation tower deamination, obtains deamination liquid, for subsequent use;

Step 6, activated carbon decolorizing

The deamination liquid vitriol oil first step 5 obtained regulates pH to be 6.5, then add the gac of Isoleucine total amount 30% in deamination liquid, under the condition of 60-70 DEG C, leave standstill 1h, then filter gac at the pressure of 0.01-0.02Mpa, obtain the Isoleucine clear liquid decoloured, for subsequent use;

Step 7, evaporative crystallization

Isoleucine clear liquid step 6 obtained joins in vaporizer, vacuum tightness be 0.09Mpa, under temperature is less than 70 DEG C of conditions, pass into 0.5Mpa saturation steam, carry out evaporation concentration, after being concentrated into 10-12 times of Isoleucine clear liquid density, educate crystalline substance, centrifugation removing mother liquor, obtain Isoleucine crystal and enter packaging process after expansion drying, namely complete the fermentative production of Isoleucine.

Fermentation index is as shown in table 1:

Table 1: the fermentation index of embodiment 1

embodiment 2:

Improve a fermentation process for Isoleucine output, step is as follows:

Step one, cultivation bacterial classification

Thalline streak inoculation in activated inclined plane, 31 DEG C of constant temperature culture 36-40h, first order seed is produced in bacterial classification room enlarged culturing, then enlarged culturing is continued by the first order seed obtained access secondary seed tank, in secondary seed medium, pass into sterile air, air flow is 0.8V/V/M, and secondary seed process in secondary seed tank disappears in fact, lowers the temperature, cultivates, obtain secondary seed nutrient solution, for subsequent use; Described secondary seed medium is made up of the raw material of following mass concentration: glucose 35g/L, magnesium sulfate 0.3g/L, potassium primary phosphate 1.3g/L, ammonium sulfate 5g/L, yeast extract powder 2.2g/L, corn syrup hydrolyzate 33.75g/L(wherein 20.25g/L allocate substratum into as bed material, the independent sterilizing of 13.5g/L reduces pH value when seed culture as acid solution), vitamin H 0.5mg/L, Vb1 2.5mg/L, defoamer 0.3mg/L, surplus is water; Described corn syrup hydrolyzate is the corn steep liquor magma of the soak solution evaporation concentration gained of corn after sulfurous acid soaks, or is that corn steep liquor magma is again through vitriol oil heating hydrolysis gained slurries.

Step 2, fermentation

Being linked into by secondary seed nutrient solution installs in the fermentor tank of fermention medium, and the volume of secondary seed nutrient solution is the 15%-20% of fermention medium volume; Pass into sterile air, continuously ferment 50-60h; Temperature from fermentation control as 31-32 DEG C in the process of fermentation 25h, control after fermentation 25h as 32.5-33 DEG C; Dissolved oxygen from fermentation control as being not less than 18.75mol/L in the process of fermentation 12h, control as 11.25-15.62mol/L after fermentation 12h; PH from fermentation control as 6.8-7.0 in the process of fermentation 36h, control after fermentation 36h as 7.0-7.2; The mass percent controlling residual sugar in fermenting process is 0.005-0.05%;

Described fermention medium is made up of the raw material of following mass concentration: glucose 120g/L, magnesium sulfate 0.6g/L, potassium primary phosphate 1.2g/L, ammonium sulfate 7.8g/L, yeast extract powder 1.5g/L, corn syrup hydrolyzate 25g/L, vitamin H 0.12mg/L, Vb1 0.08mg/L, Trisodium Citrate 4g/L, defoamer 0.3mg/L, and surplus is water; Described corn syrup hydrolyzate is the corn steep liquor magma of the soak solution evaporation concentration gained of corn after sulfurous acid soaks, or is that corn steep liquor magma is again through vitriol oil heating hydrolysis gained slurries.

Step 3, separation of fermentative broth

After fermentation ends, first fermented liquid is warming up to 60-80 DEG C and carries out sterilizing, then 40 DEG C are cooled to, adopt membrane filtration treatment process, carry out being separated under 0.2-0.5Mpa pressure and obtain mycelium in fermented liquid and albumen and fermented liquid clear liquid, in isolated mycelium and albumen, add flocculation agent flocculation, the add-on of flocculation agent is 20% of isolated mycelium and protein mass, then must to wet protein fodder through Plate Filtration at 0.02-0.04Mpa, 20-40 DEG C, for subsequent use;

Step 4, the ion-exchange of fermented liquid clear liquid

The pH regulator of the fermented liquid clear liquid first step 3 obtained is 3.5, then join in ion exchange column and adsorb Isoleucine, stop when resin absorption is saturated adding fermented liquid clear liquid, with the ammonia soln of the 1.0mol/L Isoleucine as elution resin absorption, what first flow out during wash-out is clear liquid, be recycled to fermentation clear liquid tank, for subsequent use; Secondly flow out for pH value be the Isoleucine elutriant of 2-10, for subsequent use; The elutriant finally flowed out is tail washings, and back scrubbing takes off tank and reuses, and ion exchange process is normal temperature, and pressure is 0.01-0.03Mpa;

Step 5, deammoniation tower deamination

The Isoleucine elutriant that step 4 is collected is entered deammoniation tower deamination, obtains deamination liquid, for subsequent use;

Step 6, activated carbon decolorizing

The deamination liquid vitriol oil first step 5 obtained regulates pH to be 7.0, then add the gac of Isoleucine total amount 30% in deamination liquid, under the condition of 60-70 DEG C, leave standstill 1h, then filter gac at the pressure of 0.01-0.02Mpa, obtain the Isoleucine clear liquid decoloured, for subsequent use;

Step 7, evaporative crystallization

Isoleucine clear liquid step 6 obtained joins in vaporizer, vacuum tightness be 0.09Mpa, under temperature is less than 70 DEG C of conditions, pass into 0.5Mpa saturation steam, carry out evaporation concentration, after being concentrated into 10-12 times of Isoleucine clear liquid density, educate crystalline substance, centrifugation removing mother liquor, obtain Isoleucine crystal and enter packaging process after expansion drying, namely complete the fermentative production of Isoleucine.

Fermentation index is as shown in table 2:

Table 2: the fermentation index of embodiment 2

comparative experimental example

The secondary seed medium formula of simultaneous test is: glucose 28g/L, magnesium sulfate 0.5g/L, potassium primary phosphate 1.3g/L, ammonium sulfate 5g/L, yeast extract powder 2.2g/L, corn steep liquor 22.5g/L, Semen Maydis oil 1.56ml/L, defoamer 0.3ml/L; Secondary seed medium is 0.5-0.8 with the volume ratio of the air flow passing into sterile air, and the bacterial classification index that obtain of secondary seed through disappearing in fact, lowering the temperature, cultivate in secondary seed tank is: have a net increase of OD >=0.8, Gram-positive;

Secondary seed nutrient solution is linked into the fermentor tank installing fermention medium, the volume ratio of fermention medium and secondary seed solution is 1:0.15, and this fermentative medium formula is: glucose 120g/L, magnesium sulfate 0.6g/L, potassium primary phosphate 1.2g/L, ammonium sulfate 8.4g/L, yeast extract powder 2.4g/L, corn steep liquor 19g/L, Semen Maydis oil 0.44ml/L, Trisodium Citrate 4g/L, defoamer 0.3ml/L, surplus is water; Pass into the volume ratio of sterile air ventilation and secondary seed solution and fermention medium: 0.3V/V/M, continuously ferment 65-70 hour under this technique, and make thalline utilize glucose metabolism to accumulate Isoleucine, other processes are similar with 2 to embodiment 1.

The fermentation index of simultaneous test is as shown in table 3:

Table 3: the fermentation index of simultaneous test

By table 1,2 and table 3 contrast known, every fermentation index of method provided by the invention is all higher, and acid production rate improves 30% than average before improving; Isoleucine meta-bolites transformation efficiency improves 2-4%; Producing strains bulk-growth and metabolism are rapid, and fermentation period shortens 10h, and equipment turnover rate is improved, and correspondingly improve output; Decrease the generation of metabolic by-prods, make the corresponding raising of the purity of meta-bolites in fermented liquid, to the extraction operation of product and the purity of product favourable, reduce fermentation costs and extraction cost simultaneously.

Claims (4)

1. improve a fermentation process for Isoleucine output, it is characterized in that: step is as follows:

Step one, cultivation bacterial classification

Thalline streak inoculation in activated inclined plane, 31 DEG C of constant temperature culture 36-40h, first order seed is produced in bacterial classification room enlarged culturing, then enlarged culturing is continued by the first order seed obtained access secondary seed tank, in secondary seed medium, pass into sterile air, air flow is 0.5V/V/M-0.8V/V/M, and secondary seed process in secondary seed tank disappears in fact, lowers the temperature, cultivates, obtain secondary seed nutrient solution, for subsequent use;

Step 2, fermentation

Being linked into by secondary seed nutrient solution installs in the fermentor tank of fermention medium, and the volume of secondary seed nutrient solution is the 15%-20% of fermention medium volume; Pass into sterile air, continuously ferment 50-60h; Temperature from fermentation control as 31-32 DEG C in the process of fermentation 25h, control after fermentation 25h as 32.5-33 DEG C; Dissolved oxygen from fermentation control as being not less than 18.75mol/L in the process of fermentation 12h, control as 11.25-15.62mol/L after fermentation 12h; PH from fermentation control as 6.8-7.0 in the process of fermentation 36h, control after fermentation 36h as 7.0-7.2; The mass percent controlling residual sugar in fermenting process is 0.005-0.05%;

Step 3, separation of fermentative broth

After fermentation ends, first fermented liquid is warming up to 60-80 DEG C and carries out sterilizing, then 40 DEG C are cooled to, adopt membrane filtration treatment process, carry out being separated under 0.2-0.5Mpa pressure and obtain mycelium in fermented liquid and albumen and fermented liquid clear liquid, in isolated mycelium and albumen, add flocculation agent flocculation, the add-on of flocculation agent is the 5-20% of isolated mycelium and protein mass, then must to wet protein fodder through Plate Filtration at 0.02-0.04Mpa, 20-40 DEG C, for subsequent use;

Step 4, the ion-exchange of fermented liquid clear liquid

The pH regulator of the fermented liquid clear liquid first step 3 obtained is 3.0-4.0, then join in ion exchange column and adsorb Isoleucine, stop when resin absorption is saturated adding fermented liquid clear liquid, with the ammonia soln of the 0.5-1.0mol/L Isoleucine as elution resin absorption, what first flow out during wash-out is clear liquid, be recycled to fermentation clear liquid tank, for subsequent use; Secondly flow out for pH value be the Isoleucine elutriant of 2-10, for subsequent use; The elutriant finally flowed out is tail washings, and back scrubbing takes off tank and reuses, and ion exchange process is normal temperature, and pressure is 0.01-0.03Mpa;

Step 5, deammoniation tower deamination

The Isoleucine elutriant that step 4 is collected is entered deammoniation tower deamination, obtains deamination liquid, for subsequent use;

Step 6, activated carbon decolorizing

The deamination liquid vitriol oil first step 5 obtained regulates pH to be 6.0-7.0, then add the gac of Isoleucine total amount 30% in deamination liquid, under the condition of 60-70 DEG C, leave standstill 1h, then filter gac at the pressure of 0.01-0.02Mpa, obtain the Isoleucine clear liquid decoloured, for subsequent use;

Step 7, evaporative crystallization

Isoleucine clear liquid step 6 obtained joins in vaporizer, vacuum tightness be 0.09Mpa, under temperature is less than 70 DEG C of conditions, pass into 0.5Mpa saturation steam, carry out evaporation concentration, after being concentrated into 10-12 times of Isoleucine clear liquid density, educate crystalline substance, centrifugation removing mother liquor, obtain Isoleucine crystal and enter packaging process after expansion drying, namely complete the fermentative production of Isoleucine.

2. a kind of fermentation process improving Isoleucine output as claimed in claim 1, it is characterized in that: the secondary seed medium described in step one is made up of the raw material of following mass concentration: glucose 25-35g/L, magnesium sulfate 0.3-0.8g/L, potassium primary phosphate 1.0-2.0g/L, ammonium sulfate 3-8g/L, yeast extract powder 1.5-3.0g/L, corn syrup hydrolyzate 25-40g/L, vitamin H 0.3-0.8mg/L, Vb1 1.5-3.0mg/L, defoamer 0.3mg/L, surplus is water;

Described corn syrup hydrolyzate is the corn steep liquor magma of the soak solution evaporation concentration gained of corn after sulfurous acid soaks, or is that corn steep liquor magma is again through vitriol oil heating hydrolysis gained slurries.

3. a kind of fermentation process improving Isoleucine output as claimed in claim 1, it is characterized in that: the fermention medium described in step 2 is made up of the raw material of following mass concentration: glucose 100-200g/L, magnesium sulfate 0.3-0.8g/L, potassium primary phosphate 1.0-2.0g/L, ammonium sulfate 5-10g/L, yeast extract powder 1.5-3.0g/L, corn syrup hydrolyzate 15-35g/L, vitamin H 0.05-0.25mg/L, Vb1 0.05-0.2mg/L, Trisodium Citrate 2-6g/L, defoamer 0.1-0.5mg/L, surplus is water;

Described corn syrup hydrolyzate is the corn steep liquor magma of the soak solution evaporation concentration gained of corn after sulfurous acid soaks, or is that corn steep liquor magma is again through vitriol oil heating hydrolysis gained slurries.

4. a kind of fermentation process improving Isoleucine output as claimed in claim 2, it is characterized in that: 60% of the corn syrup hydrolyzate weight in described secondary seed medium is directly prepared in the medium, and remaining 40% auto-feeding is to regulate the pH of fermenting process.