CN112080533B - Full-nutrition fed-batch fermentation control process for improving yield of L-isoleucine - Google Patents

Full-nutrition fed-batch fermentation control process for improving yield of L-isoleucine Download PDF

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CN112080533B
CN112080533B CN202010918523.XA CN202010918523A CN112080533B CN 112080533 B CN112080533 B CN 112080533B CN 202010918523 A CN202010918523 A CN 202010918523A CN 112080533 B CN112080533 B CN 112080533B
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CN112080533A (en
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徐庆阳
熊海波
张成林
李燕军
范晓光
马倩
谢希贤
陈宁
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Tianjin University of Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q3/00—Condition responsive control processes

Abstract

The invention provides a full-nutrition fed-batch fermentation control process for improving the yield of L-isoleucine, which improves the fermentation yield and the conversion rate and solves the defects of easy decay and weak vitality of thalli in the middle and later stages of fermentation by adopting a low-concentration full-nutrition fed-batch strategy in the middle and later stages of fermentation; the low-concentration initial fermentation medium is adopted for fermentation by reducing the nutrition components of the initial fermentation medium, so that full nutrition is fed in the fermentation process, the high nutrition inhibition in the initial stage of fermentation is reduced, and the problem of insufficient nutrition in the later stage is solved by a fed-batch full nutrition strategy. The consumption of corn steep liquor is reduced, and a part of corn steep liquor is replaced by using a clean organic nitrogen source, so that the problems of easy bacteria contamination, insufficient dissolved oxygen, high ash content, high impurity content of separated and extracted products and the like caused by excessive consumption of corn steep liquor in the fermentation process are solved.

Description

Full-nutrition fed-batch fermentation control process for improving yield of L-isoleucine
Technical Field
The invention relates to a production process of amino acid, in particular to a full-nutrition fed-batch fermentation control process for improving the yield of L-isoleucine.
Background
L-isoleucine belongs to branched chain amino acid, is one of essential amino acid of human body, can be used as an important food additive, can regulate the amino acid balance in food, strengthen the nutritive value of food, can not be degraded in liver, can directly enter blood, can directly influence the blood sugar level in liver, and is used for preparing special therapeutic transfusion and medicines, so that the L-isoleucine is widely applied to industries such as food, medicine and the like and has huge commercial value.
In recent years, research on isoleucine has been greatly advanced in China. In patent CN 104878051A applied by Tianjin science and technology university 2015, choline chloride is added to a fermentation medium; in 2019 application patent CN 109609565A of inner Mongolian Bayer biological Co-Ltd, the ammonium nitrogen content in fermentation broth is obviously reduced by controlling the proportion of glucose and ammonium sulfate in the fermentation medium. In the patent application CN 110396493A filed in 2019 by the company of the gallery plum blossom biotechnology development limited, the mixed solution of corn steep liquor and hydrolysate (soybean meal hydrolysate or corn steep liquor) is used to improve the formation rate and yield of isoleucine. In the production process of amino acid, one-time feeding fermentation is often adopted, but high-concentration nutrition in fermentation liquor during initial fermentation can cause nutrition poisoning to thalli, inhibit the growth of thalli, and cause abnormality of thallus metabolic pathways, so that certain nutrient substances in a fermentation tank are consumed too fast, and the phenomenon of premature decay of thalli is very easy to occur.
Disclosure of Invention
The invention aims to provide a full-nutrition fed-batch fermentation control process for improving the yield of L-isoleucine.
A full-nutrition fed-batch fermentation control process for improving the yield of L-isoleucine comprises the following specific steps:
(1) And (3) activating and culturing: taking out a corynebacterium glutamicum bacteria-retaining tube from a refrigerator at the temperature of-80 ℃, transferring two generations on an inclined plane, performing activation culture at the culture temperature of 32 ℃ for 16 hours per generation to obtain an activated strain, wherein the adopted inclined plane culture medium is as follows: 5g/L peptone, 10g/L beef extract, 4g/L yeast powder, 20g/L corn steep liquor dry powder and KH 2 PO 4 1 g/L，MgSO 4 0.2 g1g/L NaCl, 25g/L agar powder, 1g/L lysine, and pH=6.8-7.0;
(2) Seed culture: inoculating all the activated strains into a seed tank to obtain seed liquid, wherein the seed culture medium is as follows: glucose 20g/L, mgSO 4 0.8 g/L，KH 2 PO 4 1.2 g/L，(NH 4 ) 2 SO 4 0.8g/L, 6g/L yeast powder, 30g/L corn steep liquor dry powder, V B1 0.3mg/L, 0.2g/L of dichlord, regulating the pH value to 4.5-5 by NaOH, regulating the pH value to 6.7-7.0 by ammonia water for seed fermentation tank culture;
(3) Fermentation culture: inoculating 15% of seed liquid into a fermentation tank, continuously culturing, and supplementing materials in the middle to obtain fermentation liquid, wherein the adopted fermentation medium is as follows: a first medium and a second medium, wherein,
the first culture medium is: 70g/L glucose, 8g/L peptone, 2g/L yeast powder, (NH) 4 ) 2 SO 4 ·7H 2 0 3g/L，KH 2 PO 4 2.2g/L，VB 1 0.2mg/L, 20ml/L of bean cake hydrolysate, 1g/L of lysine, 3g/L of glutamic acid, 0.2g/L of methionine and 0-50g/L of corn steep liquor dry powder;
the second culture medium is: 70g/L glucose, 8g/L peptone, 2g/L yeast powder, (NH) 4 ) 2 SO 4 ·7H 2 0 3g/L，KH 2 PO 4 2.2g/L，VB 1 0.2mg/L, 20ml/L of bean cake hydrolysate, 1g/L of lysine, 3g/L of glutamic acid, 0.2g/L of methionine and 0-50g/L of silk peptide powder;
the total amount of the corn steep liquor dry powder and the silk peptide powder in the first culture medium and the second culture medium is 50g/L.
On the basis of the first culture medium, the second culture medium only replaces 50g/L of corn steep liquor dry powder with 50g/L of silk peptide powder, and the rest culture medium components are unchanged.
Preferably, in the above full-nutrition fed-batch fermentation control process for improving the yield of L-isoleucine, the two media in the step (3) are synergistic, and the low-concentration full-nutrition fed-batch, low-initial-nutrition coupled fermentation full-nutrition fed-batch, silk peptide powder and the like replace corn steep liquor full-nutrition fed-batch to improve the yield of L-isoleucine in three stages, specifically:
the high-concentration bottom material and low-concentration total nutrient feeding method comprises the following steps: preparing 3L of a first culture medium to 2.7L as an initial bottom material in a fermentation tank, adding 300ml of a supplementing liquid into the fermentation tank at the beginning of fermentation for 14h, wherein the preparation method of the 300ml of the supplementing liquid is that the components of the 3L of the first culture medium are fixed to 300ml (if the components are less than 300ml and can be supplemented with sterile water to 300 ml), and the supplementing liquid is fed for 30h, and the fermentation is finished for 35 h;
low initial nutrient coupled fermentation total nutrient feeding strategy: preparing 10-70% of 3L culture medium I to 2.7L as an initial base material in a fermentation tank, adding the initial base material into the fermentation tank at one time at the beginning of fermentation, and simultaneously adding 300ml of supplementing liquid (the preparation method of the supplementing liquid 300ml is that the volume of the culture medium I is 30-90% of 3L, the concentration is 300ml, if the concentration is less than 300ml, sterile water can be added to supplement 300 ml) in a flowing way when the fermentation is carried out for 0h, and adding the supplementing liquid in a flowing way in the whole process; meanwhile, the initial bottom material and the supplementary material are ensured to be 3L of culture medium I, for example, the initial bottom material is 10 percent of culture medium I with 3L of culture medium I, and the supplementary material is 90 percent of culture medium I with 3L of culture medium I; if the initial substrate is 70% of the No. 3L medium, the supplement is 30% of the No. 3L medium.
The full-nutrition feeding method for replacing corn steep liquor by silk peptide powder in equal proportion comprises the following steps: the method comprises the steps of replacing 0% -100% of a first culture medium with a second culture medium in an equal proportion, wherein the essence is that only 0% -100% of corn steep liquor dry powder in the first culture medium is replaced with 0% -100% of silk peptide powder in an equal proportion (if 3L of the first culture medium is prepared originally, 2L of the first culture medium and 1L of the second culture medium are optimized or 1L of the first culture medium and 2L of the second culture medium are optimized), and the components of the rest culture mediums are unchanged; the fermentation tank is prepared to 30% of No. 3L culture medium, the volume of which is fixed, and the concentration is carried out until 2.7L is used as an initial bottom material, 300ml of No. 70% of No. 3L culture medium replenishing solution is added from 14h, and the replenishing solution is Cheng Liujia.
Preferably, in the full-nutrition fed-batch fermentation control process for improving the yield of the L-isoleucine, the corn steep liquor dry powder in the steps (1), (2) and (3) has complex components, high protein content, large ash content and easy foaming and bacteria contamination during fermentation; the silk peptide powder belongs to a clean nitrogen source, the biotin content is low, the nitrogen content is high, the corn steep liquor dry powder and the silk peptide powder are substances difficult to sterilize, the water electromagnetic oven needs to be added to be pre-boiled for 40-60min, and then the sterilization pot is used for sterilizing for 20-40min at 121 ℃.
Preferably, the whole nutrition fed-batch fermentation control process for improving the yield of the L-isoleucine is characterized in that the corn steep liquor dry powder in the steps (1), (2) and (3) is diluted to 100-400g/L in a high-power manner in the sterilization process.
Preferably, the total nutrient fed-batch fermentation control process for improving the yield of the L-isoleucine comprises the following fermentation conditions in the step (3): the inoculation is carried out in a 5L fermentation tank, the inoculation amount is 600-800ml, and the fermentation conditions are as follows: culturing at constant temperature of 32deg.C, and maintaining pH at 6.7-7.0; the initial ventilation is 2.0L/min, the tank pressure is less than 0.05MPa, the initial rotating speed is 200r/min, the dissolved oxygen and the rotating speed are linked, 20% -30% of the dissolved oxygen is kept, the rotating speed is gradually regulated to 900r/min, the ventilation is gradually increased to 6.0L/min, and the fermentation time is 40h.
The beneficial effects are that:
the full-nutrition fed-batch fermentation control process for improving the yield of the L-isoleucine effectively solves the problem of activity reduction of the strain in the middle and later stages of the L-isoleucine fermentation, reduces the generation of the mixed acid, and greatly reduces the content of the mixed acid on the premise of not reducing the biomass and the acid yield.
The full-nutrition feeding control process for improving the yield of the L-isoleucine is characterized in that the optimal concentration of a substrate culture medium and feeding materials is explored through the full-nutrition feeding process, so that thalli are always in an optimal fermentation environment, an experiment is divided into three stages of low-concentration full-nutrition feeding, low-initial-nutrition coupling fermentation full-nutrition feeding, corn steep liquor full-nutrition feeding is replaced by silk peptide powder in equal proportion, the optimal concentration of the substrate culture medium and feeding materials is obtained, so that thalli are always in the optimal fermentation environment, and feeding conditions with the highest yield of the L-isoleucine and the lowest byproduct Val, leu, ala are found; meanwhile, the influence of corn steep liquor and silk peptide powder on fermentation is explored by utilizing a nitrogen source replacement experiment, so that the generation of L-isoleucine is obviously improved, the byproducts are minimized, the fed-batch is finished after 30 hours, the fermentation is finished after 35 hours, the biomass and the L-isoleucine yield respectively reach 44.2g/L, 36.4g/L, and the byproducts Val, leu, ala are respectively 1.4g/L, 0.8g/L and 0.5g/L.
The full-nutrition feeding control process for improving the yield of the L-isoleucine improves the utilization rate and the acid production capacity of glucose through a low-concentration full-nutrition feeding strategy in the middle and later stages of fermentation, and overcomes the defect of rapid decay and weak vitality of thalli in the middle and later stages of fermentation; the method adopts low-concentration initial fermentation medium to ferment by reducing the nutrition components of the initial fermentation medium, feeds full nutrition during the fermentation process, reduces the high nutrition inhibition at the initial stage of fermentation, and solves the problem of insufficient nutrition at the later stage by feeding. The consumption of corn steep liquor is reduced, and the corn steep liquor is replaced by a clean organic nitrogen source, so that the problems that the corn steep liquor is excessively large in consumption, easy to dye bacteria and insufficient in dissolved oxygen caused by excessive foam in the fermentation process are solved; high ash content, high impurity content of the separated and extracted products, and the like.
Detailed Description
Example 1
(1) And (3) activating and culturing: and taking out the bacteria-retaining tube (Corynebacterium glutamicum (Corynebacterium glutamate) YILW, supplied by Tianjin university metabolic engineering laboratory) from the refrigerator at-80 ℃ and transferring the bacteria-retaining tube on an inclined plane for two generations to perform activation culture to obtain an activated strain.
The slant culture medium comprises: peptone 5g/L, mgSO 4 0.2g/L, beef extract 10g/L, yeast powder 4g/L, KH 2 PO 4 1g/L g/L, 20g/L of corn steep liquor dry powder, 1g/L of NaCl, 25g/L of agar powder and 1g/L of lysine, and the pH=6.8-7.0;
(2) Seed culture: inoculating all the activated strain into a seed tank to obtain seed liquid.
The seed medium comprises: glucose 20g/L, mgSO 4 0.8 g/L，KH 2 PO 4 1.2 g/L，V B1 0.3 mg/L，(NH 4 ) 2 SO 4 0.8g/L, yeast extract powder 6g/L, corn steep liquor dry powder 30g/L, enemy soaking 0.2g/L, adjusting pH to 4.5-5 with NaOH, and adjusting and maintaining pH to 6.7-7.0 with ammonia water for seed fermenter culture.
(3) Fermentation culture: the inoculation is carried out in a 5L fermentation tank, the inoculation amount is 600-800ml, and the fermentation conditions are as follows: culturing at constant temperature of 32deg.C, and maintaining pH at 6.7-7.0. The initial ventilation is 2.0L/min, the tank pressure is less than 0.05MPa, the initial rotating speed is 200r/min, the dissolved oxygen and the rotating speed are linked, 20% -30% of the dissolved oxygen is kept, the rotating speed is gradually regulated to 900r/min, and the ventilation is gradually increased to 6.0L/min. Preparing a No. 3L culture medium as an initial bottom material, and adding the initial bottom material into a fermentation tank without feeding.
Medium No. one: 70g/L glucose, 8g/L peptone, 2g/L yeast powder, (NH) 4 ) 2 SO 4 ·7H 2 0 3g/L，KH 2 PO 4 2.2g/L，VB 1 0.2mg/L, 20ml/L of bean cake hydrolysate, 1g/L of lysine, 3g/L of glutamic acid, 0.2g/L of methionine and 50g/L of corn steep liquor dry powder;
example 2
Referring to embodiment 1, the difference is that: 3L of first culture medium is prepared in a fermentation tank to a volume of 2.7L and is added into the fermentation tank as an initial bottom material at one time, 300ml of supplementary liquid (300 ml of 5%3L of first culture medium is concentrated to a volume of 300 ml) is fed in a flow manner from 14h, the supplementary liquid is fed in a flow manner for 30h, and the fermentation is finished for 35 h.
Example 3
Referring to embodiment 1, the difference is that: 3L of first culture medium is prepared in a fermentation tank, the first culture medium is concentrated to 2.7L in a constant volume manner, the first culture medium is added into the fermentation tank as an initial bottom material at one time, 300ml of supplementary solution (10% of the first culture medium is concentrated to 300ml in a constant volume manner) is fed into the fermentation tank from 14h, the supplementary solution is fed into the fermentation tank for 30h, and the fermentation is finished for 35 h.
Example 4
Referring to embodiment 1, the difference is that: 3L of first culture medium is prepared in a fermentation tank to a volume of 2.7L and is added into the fermentation tank as an initial bottom material at one time, 300ml of supplementary liquid (the volume of 15% of the first culture medium is concentrated to 300 ml) is fed in a flow manner from 14h, the supplementary liquid is fed in a flow manner for 30h, and the fermentation is finished for 35 h.
Example 5
Referring to embodiment 1, the difference is that: preparing 10%3L of first culture medium to a volume of 2.7L in a fermentation tank, adding the first culture medium as an initial base material into the fermentation tank at one time, adding 300ml of supplementary liquid (90% 3L of first culture medium is concentrated to 300 ml) from 0h, adding the supplementary liquid in a whole process, and ending the fermentation for 35 h.
Example 6
Referring to embodiment 1, the difference is that: preparing 30% of a No. 3L culture medium to a volume of 2.7L in a fermentation tank, adding the culture medium as an initial base material into the fermentation tank at one time, adding 300ml of a supplementing liquid (70% of the No. 3L culture medium to a volume of 300 ml) from 0h, adding the supplementing liquid in a whole process, and ending the fermentation for 35 h.
Example 7
Referring to embodiment 1, the difference is that: the fermentation tank is prepared into a volume of 2.7L of 50%3L of first culture medium, the volume of the first culture medium is added into the fermentation tank as an initial bottom material at one time, 300ml of supplementary liquid (300 ml of 50%3L of first culture medium is concentrated into 300ml of first culture medium) is fed in a flow manner from 0h, and the whole flow of the supplementary liquid is fed in a flow manner, so that the fermentation is finished for 35 h.
Example 8
Referring to embodiment 1, the difference is that: the fermentation tank is prepared with 70 percent of No. 3L culture medium to 2.7L, the culture medium is added into the fermentation tank as a bottom material at one time, 300ml of supplementary liquid (30 percent of No. 3L culture medium is concentrated to 300 ml) is fed into the fermentation tank from 0h, the whole process of the supplementary liquid is fed into the fermentation tank, and the fermentation is finished for 35 h.
Example 9
The difference from example 6 is only that the initial base material of 30%3L of the medium was replaced with 21%3L of the medium I and 9%3L of the medium II, and the medium I of 50%3L of the medium II was replaced with 35%3L of the medium I and 15%3L of the medium II.
The first medium and the second medium differ only in the mutual substitution of the corn steep liquor dry powder and the silk peptide powder, for example:
medium No. one in the replenishment solution: 70g/L glucose, 8g/L peptone, 2g/L yeast powder, (NH) 4 ) 2 SO 4 ·7H 2 0 3g/L，KH 2 PO 4 2.2g/L，VB 1 0.2mg/L, 20ml/L of bean cake hydrolysate, 1g/L of lysine, 3g/L of glutamic acid, 0.2g/L of methionine and 35g/L of corn steep liquor dry powder;
the second culture medium in the supplementing solution is: 70g/L glucose, 8g/L peptone, 2g/L yeast powder, (NH) 4 ) 2 SO 4 ·7H 2 0 3g/L，KH 2 PO 4 2.2g/L，VB 1 0.2mg/L, 20ml/L of bean cake hydrolysate, 1g/L of lysine, 3g/L of glutamic acid, 0.2g/L of methionine and 15g/L of silk peptide powder.
Example 10
The difference from example 6 is only that 30% of the initial base material of the 3L medium is replaced by 9% of the 3L medium I and 21% of the 3L medium II, and 50% of the 3L medium I in the supplementing solution is replaced by 15% of the 3L medium I and 35% of the 3L medium II.
Example 11
The difference from example 6 is only that 30% of the initial substrate of the 3L medium was replaced with 30% of the 3L medium II, and 50% of the medium 3L was replaced with 50% of the medium 3L II in the supplemented solution.
Table 1 comparative acid production and biomass index (g/L)
As is clear from Table 1, in example 6, the acid production effect was best, in a 5L fermenter, 2.7L of 30%3L medium No. 1 was prepared in the fermenter, 300ml of a supplementary solution (70% 3L medium No. 1 was concentrated to 300ml in a constant volume) was fed from 0h, the whole procedure of the supplementary solution was fed, and finally, 36.8g/L of L-isoleucine was obtained, and valine, leucine and alanine were 5.6g/L, 2.3g/L and 2.1g/L, respectively. The byproduct in example 11 is the lowest, the corn steep liquor in the first culture medium is replaced by silk peptide powder to form a second culture medium, 30%3L of the second culture medium is prepared in a fermentation tank to reach a constant volume of 2.7L, 300ml of a supplementing liquid (70% 3L of the second culture medium is added to reach a constant volume and concentrated to 300 ml) is fed in a flowing manner from 0h, the supplementing liquid is fed in a flowing manner in the whole process, and finally, the L-isoleucine reaches 33.1g/L, and valine, leucine and alanine are respectively 1.0g/L, 0.5g/L and 0.3g/L. In the comprehensive view, the effect of the embodiment 9 is optimal, the fermentation tank is prepared to be fixed with 0.63L of a first culture medium and 0.27L of a second culture medium to be 2.7L, 300ml of a supplementing liquid (the fixed volume of the first culture medium and the second culture medium of 1.47L and 0.63L of the second culture medium are concentrated to be 300 ml) is fed from 0h, the supplementing liquid is fed in the whole process, and finally the L-isoleucine reaches 36.1g/L, and valine, leucine and alanine are respectively 1.4g/L, 0.8g/L and 0.5g/L.
In conclusion, three lifting strategies are provided for producing L-isoleucine by fermenting corynebacterium glutamicum, and the three lifting strategies are divided into a low-concentration full-nutrition feeding strategy and a low-initial-nutrition coupling fermentation full-nutrition feeding strategy, and the corn steep liquor full-nutrition feeding is replaced by silk peptide powder in equal proportion, so that the optimal concentration of a substrate culture medium and feeding is obtained, and therefore, thalli are always in an optimal fermentation environment. The low-concentration full-nutrition feeding strategy is adopted, so that the yield of L-isoleucine can be effectively improved, the problem that the high nutrition in the early stage causes nutritional toxicity to thalli is solved, meanwhile, the full-nutrition feeding of corn steep liquor is replaced by clean nitrogen source silk peptide powder in equal proportion, the glucose utilization rate is optimized, the accumulation of byproducts is reduced, and the metabolic flux is more reasonable. And then, the influence of the corn steep liquor dry powder and the silk peptide powder on fermentation is explored through nitrogen source replacement, so that the generation of L-isoleucine is obviously improved, the byproducts are minimized, the fed-batch is finished after 30 hours, the fermentation is finished after 35 hours, the biomass and the L-isoleucine yield respectively reach 44.2g/L, 36.4g/L, and the byproducts Val, leu, ala are respectively 1.4g/L, 0.8g/L and 0.5g/L.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (3)

1. A full-nutrition fed-batch fermentation control method for improving the yield of L-isoleucine is characterized by comprising the following steps: the method comprises the following specific steps:
(1) And (3) activating and culturing: taking out a corynebacterium glutamicum bacteria-retaining tube from a refrigerator at the temperature of-80 ℃, transferring two generations on an inclined plane, performing activation culture at the culture temperature of 32 ℃ for 16 hours per generation to obtain an activated strain, wherein the adopted inclined plane culture medium is as follows: 5g/L peptone, 10g/L beef extract, 4g/L yeast powder, 20g/L corn steep liquor dry powder and KH 2 PO 4 1g/L，MgSO 4 0.2g/L of NaCl 1g/L, agar powder 25g/L, lysine 1g/L, and pH=6.8-7.0;
(2) Seed culture: inoculating all the activated strains into a seed tank to obtain seed liquid, wherein the seed culture medium is as follows: glucose 20g/L, mgSO 4 0.8g/L，KH 2 PO 4 1.2g/L，(NH 4 ) 2 SO 4 0.8g/L, 6g/L yeast powder, jade30g/L of rice milk dry powder, VB 1 0.3mg/L, 0.2g/L of dichlord, regulating the pH value to 4.5-5 by NaOH, regulating the pH value to 6.7-7.0 by ammonia water for seed fermentation tank culture;
(3) Fermentation culture: inoculating 15% of seed liquid into a fermentation tank, continuously culturing, and supplementing materials in the middle to obtain fermentation liquid, wherein the adopted fermentation medium is as follows: a first culture medium and a second culture medium, wherein the first culture medium is: 70g/L glucose, 8g/L peptone, 2g/L yeast powder, (NH) 4 ) 2 SO 4 ·7H 2 0 3g/L，KH 2 PO 4 2.2g/L，VB 1 0.2mg/L, 20ml/L of bean cake hydrolysate, 1g/L of lysine, 3g/L of glutamic acid, 0.2g/L of methionine and 35g/L of corn steep liquor dry powder;
the second culture medium is: 70g/L glucose, 8g/L peptone, 2g/L yeast powder, (NH) 4 ) 2 SO 4 ·7H 2 0 3g/L，KH 2 PO 4 2.2g/L，VB 1 0.2mg/L, 20ml/L of bean cake hydrolysate, 1g/L of lysine, 3g/L of glutamic acid, 0.2g/L of methionine and 15g/L of silk peptide powder; the total amount of the corn steep liquor dry powder and the silk peptide powder in the first culture medium and the second culture medium is 50g/L;
the initial bottom materials are 0.63L of first culture medium and 0.27L of second culture medium, the volume is fixed to 2.7L, the supplementary solution is 1.47L of first culture medium and 0.63L of second culture medium, the volume is fixed, the concentration is fixed to 300ml, 300ml of supplementary solution is added from 0h, and the supplementary solution is Cheng Liujia;
the fermentation conditions are as follows: the inoculation is carried out in a 5L fermentation tank, the inoculation amount is 600-800ml, and the fermentation conditions are as follows: culturing at constant temperature of 32deg.C, and maintaining pH at 6.7-7.0; the initial ventilation is 2.0L/min, the tank pressure is 0.05MPa, the initial rotating speed is 200r/min, the dissolved oxygen is linked with the rotating speed, 20% -30% of the dissolved oxygen is kept, the rotating speed is gradually regulated to 900r/min, the ventilation is gradually increased to 6.0L/min, and the fermentation time is 40h.
2. The full-nutrient fed-batch fermentation control method for improving the yield of L-isoleucine as claimed in claim 1, wherein: the corn steep liquor dry powder and the silk peptide powder are pre-boiled for 40-60min by adding a water electromagnetic oven, and then sterilized for 20-40min at 121 ℃.
3. The full-nutrient fed-batch fermentation control method for improving the yield of L-isoleucine as claimed in claim 2, wherein: the corn steep liquor dry powder is diluted to 100-400g/L in high power in the sterilization process.
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