CN112094873B - Method for improving fermentation yield of L-isoleucine - Google Patents

Abstract

The invention provides a method for improving L-isoleucine fermentation yield, which adopts a method of adding amino acid chelated microelements into a fermentation medium to improve the saccharic acid conversion rate and the yield of an isoleucine producing strain in the L-isoleucine fermentation process, the amino acid chelated microelements comprise glutamic acid chelated iron, alanine chelated zinc, arginine chelated calcium, glutamic acid chelated copper and glutamic acid chelated manganese, and the metal elements are chelated with the amino acid, not only increases the nutrient content of effective amino acid, but also enhances the absorption effect of trace elements, improves the growth rate and the cell activity of cells, further improves the yield of isoleucine and the conversion efficiency of saccharic acid, under the condition of not increasing additional equipment and manpower input, the method realizes the shortening of the whole fermentation period and the great improvement of the yield and the conversion rate of the L-isoleucine, and is suitable for industrial production.

Description

Method for improving fermentation yield of L-isoleucine

Technical Field

The invention relates to the technical field of amino acid production by a fermentation method, in particular to a method for improving fermentation yield of L-isoleucine.

Background

In recent years, with the application and research of L-isoleucine in medicine and health care, food processing and feed industries, the market demand for L-isoleucine is increasing. The domestic branch chain market demand in 2019 is about 5 ten thousand tons, but isoleucine domestic production capacity is only 0.4 ten thousand tons, the gap between supply and demand is large, and the annual demand increases at a rate of 10%. The production method of L-isoleucine mainly comprises the following steps: protein hydrolysis method, chemical synthesis method, fermentation method and enzyme method. Currently, the only way to industrially produce L-isoleucine on a large scale is microbial fermentation. Therefore, the further optimization of the production process of the L-isoleucine is of great significance.

At present, the L-isoleucine production in China has the problems of low fermentation acid yield, low conversion rate, many byproducts and the like. The acid production level of L-isoleucine in China is 35-40 g/L, and the extraction rate is 78-80%. The recent rise in raw material prices and the rise in labor costs have led to challenges in the fermentative production of L-isoleucine, which in turn has limited the use of L-isoleucine. When the glutamic acid helper bacillus is used for fermenting isoleucine, the saccharic acid conversion rate is 12-15%, and the promotion space is larger than the theoretical value. Therefore, how to further improve the yield and the sugar-acid conversion rate of the L-isoleucine produced by the microbial fermentation method becomes a key problem for promoting the sustainable development of the L-isoleucine industry.

Disclosure of Invention

The invention aims to provide a method for improving the fermentation yield of L-isoleucine.

In order to solve the technical problems, the technical scheme of the invention is as follows:

a method for improving L-isoleucine fermentation yield is characterized in that L-isoleucine is obtained by fermenting isoleucine producing bacteria, and amino acid chelated trace elements are added into a culture medium to improve the saccharic acid conversion rate and the yield of the isoleucine producing bacteria in the L-isoleucine fermentation process, wherein the amino acid chelated trace elements are composed of glutamic acid chelated iron, alanine chelated zinc, arginine chelated calcium, glutamic acid chelated copper and glutamic acid chelated manganese, wherein the weight ratio of the glutamic acid chelated iron to the alanine chelated zinc to the arginine chelated calcium to the glutamic acid chelated copper to the glutamic acid chelated manganese is as follows: 200-300: 50-100: 100-150: 20-40: 150-250.

Preferably, in the method for improving the fermentation yield of the L-isoleucine, amino acid chelated trace elements are added along with glucose flow in the fermentation culture process: the contents of glutamic acid chelated iron, alanine chelated zinc, arginine chelated calcium, glutamic acid chelated copper and glutamic acid chelated manganese in the glucose solution are respectively 200-300mg/L, 50-100mg/L, 100-150mg/L, 20-40mg/L and 150-250mg/L, and the dosage of the glucose is as follows: the initial fermentation medium is 30g/L, the glucose concentration is maintained to be 3-5g/L in the fermentation process, and the fed-batch sugar concentration is 600-700 g/L.

Preferably, in the method for improving the fermentation yield of L-isoleucine, the weight ratio of the iron glutamate chelate, the zinc alanine chelate, the calcium arginine chelate, the copper glutamate chelate and the manganese glutamate chelate is as follows: 240-260: 70-80: 120-140: 25-30: 180-210.

Preferably, the method for improving the fermentation yield of L-isoleucine comprises the following specific steps:

(1) the corynebacterium glutamicum is activated from a glycerol tube to a culture slant, and the medium components: 1.0g/L glucose, 10.0g/L yeast extract powder, 5.0g/L peptone, 1.0g/L potassium dihydrogen phosphate, 0.2g/L magnesium sulfate and 20.0g/L agar powder, culturing at 32 ℃ for 20-24h, culturing at 32 ℃ on a secondary transfer culture inclined plane for 16-20h, and inoculating into a seed culture medium for seed culture to obtain a seed solution;

(2) inoculating the seed liquid into a fermentation culture medium for culture, and controlling dissolved oxygen by adopting a staged oxygen supply mode: 30-50% in 0-20 h and 20-40% in 20-45h, controlling the pH value at 7.0-7.2, and controlling the residual sugar at 0.3-0.5% by feeding glucose solution with the concentration of 600-700g/L, wherein the glucose solution contains 300mg/L of glutamic acid chelated iron, 50-100mg/L of alanine chelated zinc, 50-100mg/L of arginine chelated calcium, 150mg/L of 100-150mg/L of glutamic acid chelated manganese, and 250mg/L of 150-L of glutamic acid chelated manganese.

Preferably, in the method for improving the fermentation yield of L-isoleucine, the Corynebacterium glutamicum is Corynebacterium glutamicum CGMCC.12153 (deposited in China general microbiological culture Collection center).

Has the advantages that:

the method for improving the fermentation yield of the L-isoleucine adopts a method of adding amino acid chelated microelements into a fermentation medium to improve the saccharic acid conversion rate and the yield of the L-isoleucine fermentation process of the isoleucine producing bacteria, the amino acid chelated microelements comprise glutamic acid chelated iron, alanine chelated zinc, arginine chelated calcium, glutamic acid chelated copper and glutamic acid chelated manganese, and the metal elements are chelated with the amino acid, not only increases the nutrient content of effective amino acid, but also enhances the absorption effect of trace elements, improves the growth rate and the cell activity of cells, further improves the yield of isoleucine and the conversion efficiency of saccharic acid, under the condition of not increasing additional equipment and manpower input, the method realizes the shortening of the whole fermentation period and the great improvement of the yield and the conversion rate of the L-isoleucine, and is suitable for industrial production.

Detailed Description

Example 1

The strain adopts Corynebacterium glutamicum CGMCC.12153;

the culture medium is prepared from the existing commonly used fermentation medium [ glucose 30g/L (Xiaoxiao, 0.075 MPa moist heat sterilization for 15 min), (NH)₄)₂SO₄ 3 g/L，MgSO₄·7H₂O 0.6 g/L，KH₂PO₄ 2.5 g/L，V_B10.2 mg/L, 20g/L (0.01 MPa moist heat sterilization for 50 min) of corn steep liquor dry powder, and adjusting the pH value to 7.0 by using NaOH and hydrochloric acid. And (3) sterilization conditions: sterilizing with 0.1 MPa for 20 min]；

Iron glutamate chelate, zinc alanine chelate, calcium arginine chelate, copper glutamate chelate and manganese glutamate chelate all purchased from the national medicine group;

the culture method comprises the following steps: activating a strain (Corynebacterium glutamicum CGMCC.12153) from a glycerol tube to a culture slant, wherein the culture medium comprises the following components: 1.0g/L glucose, 10.0g/L yeast extract powder, 5.0g/L peptone, 1.0g/L potassium dihydrogen phosphate, 0.2g/L magnesium sulfate, 20.0g/L agar powder, culturing at 32 deg.C for 20-24h, culturing at 32 deg.C on secondary transfer culture slant for 16-20h, inoculating seed culture medium [ 30g/L glucose, 5g/L yeast powder, 5g/L ammonium sulfate, 15 g/L, KH g/L corn steep powder dry powder₂PO₄ 2 g/L、MgSO₄ ·7H₂O 0.8 g/L、V_B1 0.2 mg/L]The inoculation amount is 15 percent; culturing for 15 h to the middle and later logarithmic phase in a 5L automatic control fermentation tank under the conditions of 32 ℃, pH 7.0 and dissolved oxygen of 20-50%, inoculating the culture medium into the 5L automatic control fermentation tank containing a fermentation culture medium according to the inoculation amount of 15%, introducing air, controlling the initial ventilation ratio to be 0.6, adjusting the stirring speed according to the dissolved oxygen requirement and the highest ventilation ratio to be 1.5, and controlling the dissolved oxygen by adopting a staged oxygen supply mode: 30-50% in 0-20 h and 20-40% in 20-45h, controlling pH at 7.0-7.2 by automatically feeding 25% ammonia water,defoaming by feeding 0.02-0.05% of foam enemy, controlling the residual sugar at 0.3-0.5% by feeding glucose solution (containing iron glutamate chelate, zinc alanine chelate, calcium arginine chelate, copper glutamate chelate and manganese glutamate chelate, respectively 250mg/L, 80mg/L, 130mg/L, 30mg/L and 200 mg/L) with the concentration of 700g/L, and finishing the fermentation for 45 h. The components of the fermentation medium of the control group are the same as those of the experimental group, glucose solution with fed-batch concentration of 700g/L (containing equimolar amounts of glutamic acid, alanine, arginine, ferrous chloride, zinc chloride, calcium chloride, copper chloride and manganese chloride, the contents of which are 417.3 mg/L, 58.6 mg/L, 116.6 mg/L, 90.7 mg/L, 44.8 mg/L, 37.2mg/L, 7.6mg/L and 43.8 mg/L respectively, and various amino acids and metal ions are added according to the equimolar amounts fed-batch in the experimental group), and the fermentation is finished for 45 h. When the strain is placed in a pot, the dry weight of the strain reaches 73.9g/L, the yield of L-isoleucine is 58.2g/L, and the saccharic acid conversion rate is 18.6%, which are respectively improved by 18.1%, 38.2% and 10.7% compared with a control experiment (the dry weight of the strain is 62.6g/L, the yield of L-isoleucine is 42.1 g/L, and the saccharic acid conversion rate is 16.8%).

To sum up, the embodiment 1 adopts the fed-batch fermentation technology of amino acid chelated trace elements along with glucose, fed-batch chelates include glutamic acid chelated iron, alanine chelated zinc, arginine chelated calcium, glutamic acid chelated copper and glutamic acid chelated manganese, and metal elements are subjected to a chelating reaction with amino acids, so that the nutrient content of effective amino acids is increased, the absorption effect of trace elements is enhanced, the cell growth rate and the cell activity are improved, and the isoleucine yield and the sugar acid conversion efficiency are further improved. After fermentation for 45 hours, the dry weight of the thalli reaches the highest 73.9g/L, the yield of L-isoleucine is 58.2g/L, the saccharic acid conversion rate is 18.6%, and the saccharic acid conversion rate is respectively improved by 18.1%, 38.2% and 10.7% compared with a control experiment.

Example 2

Adopting corynebacterium glutamicum;

the culture medium is a fermentation medium commonly used in the prior art (same as example 1);

the cultivation method was the same as in example 1.

During the fermentation process, glucose solution with the concentration of 600 g/L (containing 200mg/L, 50mg/L, 100mg/L, 20mg/L and 150mg/L of glutamic acid chelated iron, alanine chelated zinc, arginine chelated calcium, glutamic acid chelated copper and glutamic acid chelated manganese respectively) is fed, and glucose solution with the concentration of 600 g/L (containing equimolar amounts of glutamic acid, alanine, arginine, ferrous chloride, zinc chloride, calcium chloride, copper chloride and manganese chloride respectively, with the contents of 321.2mg/L, 36.6 mg/L, 89.7 mg/L, 72.6 mg/L, 28.0mg/L, 28.6mg/L, 5.1mg/L and 32.9 mg/L) is fed in a control group. Controlling the residual sugar at 0.3-0.5%, and finishing fermentation for 45 h. When the strain is placed in a pot, the dry weight of the strain reaches 67.2g/L, the yield of L-isoleucine is 49.7g/L, and the saccharic acid conversion rate is 17.2%, which are respectively improved by 13.1%, 23.9% and 5.5% compared with a control experiment (the dry weight of the strain is 59.4g/L, the yield of L-isoleucine is 40.1 g/L, and the saccharic acid conversion rate is 16.3%).

Example 3

Adopting corynebacterium glutamicum;

the culture medium is a fermentation medium commonly used in the prior art (same as example 1);

the cultivation method was the same as in example 1.

During the fermentation process, glucose solution with the concentration of 600 g/L (containing 260mg/L, 75mg/L, 130mg/L, 30mg/L and 200mg/L of glutamic acid iron chelate, alanine zinc chelate, arginine calcium chelate, glutamic acid copper chelate and glutamic acid manganese chelate respectively) is fed, and glucose solution with the concentration of 600 g/L (containing equal molar amounts of glutamic acid, alanine, arginine, ferrous chloride, zinc chloride, calcium chloride, copper chloride and manganese chloride respectively and containing 425.7mg/L, 54.9 mg/L, 116.6 mg/L, 94.3 mg/L, 42.1mg/L, 37.2mg/L, 7.6mg/L and 43.8 mg/L) is fed in a control group. Controlling the residual sugar at 0.3-0.5%, and finishing fermentation for 45 h. When the strain is placed in a pot, the dry weight of the strain reaches 76.2g/L, the yield of L-isoleucine is 56.7g/L, and the saccharic acid conversion rate is 17.9%, which are respectively improved by 24.1%, 32.2% and 7.8% compared with a control experiment (the dry weight of the strain is 61.4g/L, the yield of L-isoleucine is 42.9 g/L, and the saccharic acid conversion rate is 16.6%).

Example 4

Adopting corynebacterium glutamicum;

the culture medium is a fermentation medium commonly used in the prior art (same as example 1);

the cultivation method was the same as in example 1.

In the fermentation process, glucose solution with the concentration of 650 g/L (containing 240mg/L, 100mg/L, 140mg/L, 25mg/L and 180mg/L of glutamic acid chelated iron, alanine chelated zinc, arginine chelated calcium, glutamic acid chelated copper and glutamic acid chelated manganese respectively) is fed, and glucose solution with the concentration of 650 g/L (containing equimolar amounts of glutamic acid, alanine, arginine, ferrous chloride, zinc chloride, calcium chloride, copper chloride and manganese chloride, and the contents of 386.4mg/L, 73.3mg/L, 125.6 mg/L, 87.1 mg/L, 55.9mg/L, 40.1mg/L, 6.3mg/L and 39.4 mg/L respectively) is fed in a control group. Controlling the residual sugar at 0.3-0.5%, and finishing fermentation for 45 h. When the strain is placed in a pot, the dry weight of the strain reaches 72.7g/L, the yield of L-isoleucine is 53.1g/L, and the saccharic acid conversion rate is 17.7%, which are respectively improved by 20.6%, 29.2% and 6.6% compared with a control experiment (the dry weight of the strain is 60.3g/L, the yield of L-isoleucine is 41.1 g/L, and the saccharic acid conversion rate is 16.6%).

Example 5

Adopting corynebacterium glutamicum;

the culture medium is a fermentation medium commonly used in the prior art (same as example 1);

the cultivation method was the same as in example 1.

During the fermentation process, glucose solution with the concentration of 700g/L (containing 260mg/L, 70mg/L, 120mg/L, 28mg/L and 210mg/L of glutamic acid chelated iron, alanine chelated zinc, arginine chelated calcium, glutamic acid chelated copper and glutamic acid chelated manganese respectively) is fed, and glucose solution with the concentration of 700g/L (containing equimolar amounts of glutamic acid, alanine, arginine, ferrous chloride, zinc chloride, calcium chloride, copper chloride and manganese chloride, and the contents of 433.0mg/L, 51.3mg/L, 107.6 mg/L, 94.3 mg/L, 39.1mg/L, 34.4 mg/L, 7.2mg/L and 45.9 mg/L respectively) is fed in a control group. Controlling the residual sugar at 0.3-0.5%, and finishing fermentation for 45 h. When the strain is placed in a pot, the dry weight of the strain reaches 69.9g/L, the yield of L-isoleucine is 54.2g/L, and the saccharic acid conversion rate is 17.3%, which are respectively 22.4%, 28.4% and 5.5% higher than that of a control experiment (the dry weight of the strain is 57.1g/L, the yield of L-isoleucine is 42.2 g/L, and the saccharic acid conversion rate is 16.4%).

Example 6

Adopting corynebacterium glutamicum;

the culture medium is a fermentation medium commonly used in the prior art (same as example 1);

the cultivation method was the same as in example 1.

During the fermentation process, glucose solution with the concentration of 700g/L (containing 300mg/L, 100mg/L, 150mg/L, 40mg/L and 250mg/L of glutamic acid chelated iron, alanine chelated zinc, arginine chelated calcium, glutamic acid chelated copper and glutamic acid chelated manganese respectively) is fed, and glucose solution with the concentration of 700g/L (containing equimolar amounts of glutamic acid, alanine, arginine, ferrous chloride, zinc chloride, calcium chloride, copper chloride and manganese chloride, and the contents of 513.3mg/L, 73.3mg/L, 134.5 mg/L, 108.8mg/L, 55.9mg/L, 32.4 mg/L, 10.2 mg/L and 54.8 mg/L respectively) is fed in a control group. Controlling the residual sugar at 0.3-0.5%, and finishing fermentation for 45 h. When the strain is placed in a pot, the dry weight of the strain reaches 77.4g/L, the yield of L-isoleucine is 56.2g/L, and the saccharic acid conversion rate is 17.4%, which are respectively 21.9%, 27.4% and 4.8% higher than those of a control experiment (the dry weight of the strain is 63.5g/L, the yield of L-isoleucine is 44.1 g/L, and the saccharic acid conversion rate is 16.6%).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (4)

1. A method for improving fermentation yield of L-isoleucine, which is characterized by comprising the following steps: adopting isoleucine-producing bacteria to obtain L-isoleucine by adopting an amino acid chelated trace element and glucose fed-batch fermentation technology, wherein the concentration of glucose is maintained to be 3-5g/L in the fermentation process, the residual sugar is controlled to be 0.3-0.5%, and the amino acid chelated trace element is added into a culture medium and consists of glutamic acid chelated iron, alanine chelated zinc, arginine chelated calcium, glutamic acid chelated copper and glutamic acid chelated manganese, wherein the weight ratio of the glutamic acid chelated iron, the alanine chelated zinc, the arginine chelated calcium, the glutamic acid chelated copper and the glutamic acid chelated manganese is as follows: 200-300: 50-100: 100-150: 20-40: 150- > 250; the isoleucine producing strain is Corynebacterium glutamicum CGMCC.12153.

2. The method for improving fermentation yield of L-isoleucine according to claim 1, wherein: in the fermentation culture process, amino acid chelated trace elements are added along with glucose: the contents of glutamic acid chelated iron, alanine chelated zinc, arginine chelated calcium, glutamic acid chelated copper and glutamic acid chelated manganese in the glucose solution are respectively 200-300mg/L, 50-100mg/L, 100-150mg/L, 20-40mg/L and 150-250mg/L, and the dosage of the glucose is as follows: the initial fermentation medium is 30g/L, the glucose concentration is maintained to be 3-5g/L in the fermentation process, and the fed-batch sugar concentration is 600-700 g/L.

3. The method for improving fermentation yield of L-isoleucine according to claim 1, wherein: the weight ratio of the glutamic acid chelated iron to the alanine chelated zinc to the arginine chelated calcium to the glutamic acid chelated copper to the glutamic acid chelated manganese is as follows: 240-260: 70-80: 120-140: 25-30: 180-210.

4. The method for improving fermentation yield of L-isoleucine according to claim 2, wherein: the method comprises the following specific steps:

(1) activating corynebacterium glutamicum from a glycerol tube to a culture inclined plane, and then inoculating the corynebacterium glutamicum into a seed culture medium for seed culture to obtain a seed solution;

(2) inoculating the seed liquid into a fermentation culture medium for culture, and controlling dissolved oxygen by adopting a staged oxygen supply mode: 30-50% in 0-20 h and 20-40% in 20-45h, controlling the pH value at 7.0-7.2, and controlling the residual sugar at 0.3-0.5% by feeding glucose solution with the concentration of 600-700g/L, wherein the glucose solution contains 300mg/L of glutamic acid chelated iron, 50-100mg/L of alanine chelated zinc, 50-100mg/L of arginine chelated calcium, 150mg/L of 100-150mg/L of glutamic acid chelated manganese, and 250mg/L of 150-L of glutamic acid chelated manganese.