CN107022583B - Method for producing L-alanine by feeding fermentation - Google Patents
Method for producing L-alanine by feeding fermentation Download PDFInfo
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Abstract
The invention relates to a method for producing L-alanine by fed-batch fermentation, which is characterized in that in the process of culturing a mutant strain Lds.0108 of lactobacillus delbrueckii by using a glucose culture medium with the glucose concentration of 95-105g/L, when the residual sugar content in fermentation liquor is lower than 45g/L, a glucose solution with the concentration of 50-75% is fed into the fermentation liquor at one time until the total concentration of glucose added into the fermentation liquor is 125-170 g/L, and the fermentation is continued until the fermentation is finished. The method improves the yield of the L-alanine by supplementing the glucose solution on the basis of the basic fermentation medium, is simple and easy to implement, has obvious effect, improves the concentration of the L-alanine in the fermentation tank from 80-100g/L to 120g/L of 110-90 percent, and has the fermentation conversion rate of more than 90 percent.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a method for producing L-alanine by fed-batch fermentation.
Background
L-alanine is colorless to white crystalline powder, soluble in water, ethanol, and insoluble in diethyl ether and acetone. L-alanine is a medical intermediate, and is a main raw material for producing vitamin B6 and L-aminopropanol. In the field of food, L-alanine has unique flavor improving effect, and can obviously improve the utilization rate of protein in food and beverage and improve the sour taste of organic acid.
At present, L-aspartic acid- β -decarboxylase is mainly used for converting L-aspartic acid to produce L-alanine industrially, the production equipment investment is small, the reaction is simple, but the price fluctuation of the main raw material L-aspartic acid is too large, and the stable production throughout the year is difficult to ensure.
Disclosure of Invention
The invention aims to provide a method for producing L-alanine by fed-batch fermentation, which is characterized in that in the process of culturing a Lactobacillus delbrueckii mutant strain Lds.0108 by using a glucose culture medium with the initial concentration of glucose of 95-105g/L, when the content of residual sugar in fermentation liquor is lower than 45g/L, a glucose solution with the concentration of 50-75% is fed into the fermentation liquor at one time until the total concentration of glucose added into the fermentation liquor is 125-170 g/L, and the fermentation is continued until the end of the fermentation.
On the other hand, the method of the present invention does not add all glucose at the time of initially preparing the culture medium, and does not cause damage to the cells due to high osmotic pressure caused by adding high concentration glucose at once. On the other hand, the production of L-alanine can be further increased by adding glucose during the culture. In the fermentation process of the lactobacillus delbrueckii mutant strain Lds.0108, a metabolite L-alanine can be generated, and can generate a certain osmotic pressure for the bacteria, the osmotic pressure of the bacteria can be increased by continuously supplementing glucose, a glucose grain solution with the concentration of 50-75% is added into the culture medium when the concentration of the glucose in the culture medium is controlled to be less than 45g/L, and the osmotic pressure generated by the L-alanine and the glucose in the culture solution can not bring adverse effects to the growth of the bacteria due to the dilution effect of water in the supplemented glucose solution. Moreover, the glucose with the concentration does not generate pigment substances due to overlarge concentration in the culture process, does not influence the extraction of effective substances, and does not influence the growth of microorganisms.
Preferably, when the residual sugar content in the fermentation liquor is 30-41g/L, the glucose solution is supplemented to the fermentation liquor. When the concentration of residual sugar is too high, the concentration of sugar is too high after high-concentration glucose is supplemented, high osmotic pressure is formed, and the damage to thalli is large; the residual sugar is too low, the thalli are aged, the activity of an enzyme system is small, and the integral conversion speed after glucose is supplemented is influenced.
As a preferable supplementing scheme, when the content of residual sugar in the fermentation liquor is lower than 33-35 g/L, glucose solution with the concentration of 68-72% is supplemented into the fermentation liquor at one time until the total concentration of glucose added into the fermentation liquor is 160-168 g/L, and the culture is continued until the fermentation is finished.
Preferably, the glucose medium comprises, per liter: 95-105g of glucose and Na2HPO4·12H2O 20g，KH2PO42.0g，NaCl 0.5g，MgSO4·7H20.2g of O, 10mL/L of trace inorganic salt solution and the balance of purified water;
in the trace inorganic salt solution: FeCl3·6H2O 2.5g/L，CoCl2·6H2O 0.2g/L，CuCl2·2H2O0.1g/L，ZnCl20.2g/L，Na2MoO4·2H2O 0.3g/L，H3BO30.1g/L，MnCl2·4H20.5g/L of O and the balance of purified water.
Preferably, the conditions for culturing the lactobacillus delbrueckii mutant strain lds.0108 using a glucose medium are: the temperature is 30 +/-2 ℃; air volume of 0m3H, the pressure is 0.04MPa to 0.06 MPa; pH: 6.5 +/-0.1.
Preferably, in the method of the present invention, the pH in the fermentation broth is adjusted by liquid ammonia. L-alanine was synthesized using ammonia while supplying amino groups to the glucose decomposition products.
Preferably, the method of the present invention comprises the following steps:
1) preparing seed solution of mutant strain LDS.0108 of Lactobacillus delbrueckii with OD value of 3.5-7.0;
2) the seed liquid with the seed volume fraction of 5-10% is added into the glucose culture medium according to the seed volume fraction to carry out fermentation culture;
3) and when the concentration of the glucose in the fermentation liquor is less than 45g/L, adding the glucose with the concentration of 50-75% into the fermentation liquor until the concentration of the glucose in the fermentation liquor is higher than that of the glucose in the glucose culture medium, and continuously culturing until the fermentation is finished.
Preferably, the OD value of the seed liquid of the lactobacillus delbrueckii mutant strain LDS.0108 is 3.5-7.0.
Preferably, the preparation of seed solution of the lactobacillus delbrueckii mutant strain lds.0108 comprises the following steps:
(1) under the aseptic condition, selecting a Lactobacillus delbrueckii mutant strain by using an inoculating loop, inoculating the strain on the slope of a solid LB culture medium eggplant-shaped bottle, placing the solid LB culture medium eggplant-shaped bottle in a constant-temperature incubator at 28-30 ℃ and culturing for 16-18 h;
(2) under the aseptic condition, washing the thalli on the inclined plane by using aseptic normal saline to obtain a bacterial suspension;
(3) inoculating the bacterial suspension liquid into a liquid LB culture medium, and culturing under the conditions that the temperature is 30 ℃, the air flow is 2L/min, and the tank pressure is 0.04-0.06MPa until the OD value of the fermentation liquid is 3.5-7.0.
In the process of large-scale fermentation, secondary fermentation is required on the basis of the method, namely, the step (3) is repeated twice to obtain the seed liquid with the OD value of 5-7.0.
The lactobacillus delbrueckii mutant strain Lds.0108 used in the invention is preserved in China center for type culture Collection with the preservation number: CCTCC NO: m2013361.
Further preferably, the method of the present invention comprises the steps of:
1) preparing a seed solution with OD value of 3.5-7.0;
2) inoculating the seed liquid into a glucose culture medium according to the seed volume fraction of 5-10%, and performing fermentation culture;
the formula of the glucose culture medium is that each liter of the culture medium contains 95-105g of glucose and Na2HPO4·12H2O 20g，KH2PO42.0g，NaCl 0.5g，MgSO4·7H20.2g of O0.2g, 10mL/L of trace inorganic salt solution and the balance of purified water;
in the trace inorganic salt solution: FeCl3·6H2O 2.5g/L，CoCl2·6H2O 0.2g/L，CuCl2·2H2O0.1g/L，ZnCl20.2g/L，Na2MoO4·2H2O 0.3g/L，H3BO30.1g/L，MnCl2·4H2O0.5 g/L and the balance of purified water;
the culture conditions were: the temperature is 30 +/-2 ℃; air volume of 0m3H, the pressure is 0.04MPa to 0.06 MPa; pH: 6.5 plus or minus 0.1;
3) supplemented culture
And when the concentration of glucose in the fermentation liquor is 30-41g/L, adding 50-75% of glucose into the fermentation liquor until the total concentration of the glucose added into the fermentation liquor is 125-170 g/L, and continuing culturing until the concentration of residual sugar in the culture solution is less than or equal to 2g/L, thereby finishing the fermentation.
Most preferably, the method of the present invention comprises the steps of:
1) preparing a seed solution with OD value of 3.5-7.0;
2) inoculating the seed liquid into a glucose culture medium according to the seed volume fraction of 5-10%, and performing fermentation culture;
the formula of the glucose culture medium is 95-105g of glucose and Na2HPO4·12H2O20g，KH2PO42.0g，NaCl 0.5g，MgSO4·7H20.2g of O, 10mL/L of trace inorganic salt solution and the balance of purified water;
in the trace inorganic salt solution: FeCl3·6H2O 2.5g/L，CoCl2·6H2O 0.2g/L，CuCl2·2H2O0.1g/L，ZnCl20.2g/L，Na2MoO4·2H2O 0.3g/L，H3BO30.1g/L，MnCl2·4H2O0.5 g/L and the balance of purified water;
the culture conditions were: the temperature is 30 +/-2 ℃; air volume of 0m3H, the pressure is 0.04MPa to 0.06 MPa; pH: 6.5 plus or minus 0.1;
3) supplemented culture
When the content of residual sugar in the fermentation liquor is lower than 33-35 g/L, adding 68-72% glucose solution into the fermentation liquor at one time until the total concentration of the added glucose in the fermentation liquor is 160-168 g/L.
The method has the following beneficial effects:
the invention uses the Lactobacillus delbrueckii mutant strain LDS.0108 as the starting strain, and the yield of L-alanine is improved by supplementing glucose solution on the basis of the basic fermentation medium, the method is simple and easy to implement, the concentration of L-alanine in the fermentation tank is improved from 80-100g/L to 110-123g/L, the fermentation conversion rate reaches over 90 percent, the concentration of residual fermentation sugar is kept unchanged, and the unit product cost is greatly reduced although the fermentation period is increased.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The glucose medium referred to in the examples was prepared by the following method:
preparing a fermentation medium according to the following component contents: glucose 100g/L, Na2HPO4·12H2O 20g/L，KH2PO42.0g/L，NaCl 0.5g/L，MgSO4·7H2O0.2g/L, trace inorganic salt 10ml/L and the balance of purified water;
wherein the trace inorganic salts include: FeCl3·6H2O 2.5g/L，CoCl2·6H2O 0.2g/L，CuCl2·2H2O0.1g/L，ZnCl20.2g/L，Na2MoO4·2H2O 0.3g/L，H3BO30.1g/L，MnCl2·4H2O0.5 g/L, and the balance of purified water.
Sterilizing the culture medium at 121 deg.C, maintaining the pressure for 30min, and cooling to 30 deg.C;
the glucose solution was prepared as follows in the following examples: preparing 50-75% glucose solution according to required volume, diluting to desired volume, sterilizing at 115 deg.C, maintaining pressure for 20min, and cooling.
In the following examples, examples 1 to 4 were all carried out in a 10L fermentor, and example 5 was carried out in a 10T fermentor, so that a secondary seed solution was prepared during the preparation of the seed solution, which had a much higher OD value than in examples 1 to 4.
Example 1
The embodiment relates to a preparation method of L-alanine, which specifically comprises the following steps:
1. preparation of Lactobacillus delbrueckii mutant strain Lds.0108 seed liquid
1) Under the aseptic condition, the lactobacillus delbrueckii mutant strain is selected by an inoculating loop and inoculated on the inclined plane of a solid LB culture medium eggplant-shaped bottle, and the solid LB culture medium eggplant-shaped bottle is placed in a constant temperature incubator at the temperature of 28-30 ℃ for culture for 17 hours.
2) Under the aseptic condition, the thalli is eluted by sterilized 100ml of normal saline to obtain bacterial suspension.
3) Inoculating the bacterial suspension into a liquid LB culture medium at the temperature of 30 ℃; the air flow is 2L/min, and the tank pressure is 0.04-0.06 MPa; incubate 5.5h to OD (600 nm): 4.05, obtaining the seed liquid.
2. Inoculating the seed liquid into the glucose culture medium according to the volume fraction of 7%, and controlling the temperature to be 30 ℃; air flow rate of 2m3Fermenting and culturing under the condition of/h, and keeping the tank pressure at 0.04-0.06MPa by feeding air in the culture process; the pH was controlled to 6.5 with liquid ammonia.
3. When the residual glucose concentration of the fermentation liquid is 32.8g/L, adding 50% glucose solution at one time until the total glucose addition concentration in the fermentation liquid is 158.4g/L, and continuing until the glucose concentration in the fermentation liquid is 1.85g/L, stopping fermentation.
After the fermentation is finished, sampling and carrying out HPLC (high performance liquid chromatography) determination, wherein the content of L-alanine in the fermentation liquid is 110g/L, the concentration of residual glucose is 1.85g/L, and the fermentation conversion rate is 90%.
Example 2
The embodiment relates to a preparation method of L-alanine, which specifically comprises the following steps:
1. a seed solution was prepared in the same manner as in example 1 to obtain a seed solution having an OD value of 3.75.
2. Inoculating the seed liquid into the glucose culture medium according to the volume fraction of 7%, and controlling the temperature to be 30 ℃; air flow rate of 2m3Fermenting and culturing under the condition of/h, and keeping the tank pressure at 0.04-0.06MPa by feeding air in the culture process; the pH was controlled to 6.50 with liquid ammonia.
3. When the residual glucose concentration in the fermentation liquid is 40.6g/L, 60% glucose solution is supplemented once until the total glucose concentration in the fermentation liquid is 159.2g/L, and the fermentation is stopped when the glucose concentration in the fermentation liquid is 1.77 g/L.
After the fermentation is finished, sampling and carrying out HPLC (high performance liquid chromatography) determination, wherein the content of L-alanine in the fermentation liquid is 115g/L, the concentration of residual glucose is changed to 1.77g/L, and the fermentation conversion rate is 90.5%.
Example 3
The embodiment relates to a preparation method of L-alanine, which specifically comprises the following steps:
1. a seed solution was prepared in the same manner as in example 1 to obtain a seed solution having an OD value of 3.86.
2. Inoculating the seed liquid into the glucose culture medium according to the volume fraction of 7%, and controlling the temperature to be 30 ℃; air flow rate of 2m3Fermenting and culturing under the condition of/h, and keeping the tank pressure at 0.04-0.06MPa by feeding air in the culture process; the pH was controlled to 6.50 with liquid ammonia.
3. When the residual glucose concentration in the fermentation liquid is 33.5g/L, 70% glucose solution is supplemented once until the total glucose concentration in the fermentation liquid is 166.5g/L, and the fermentation is stopped when the glucose concentration in the fermentation liquid is 1.80 g/L.
After the fermentation is finished, sampling and carrying out HPLC determination, wherein the L-alanine content in the fermentation liquid is 123g/L, the residual glucose concentration is 1.80g/L, and the fermentation conversion rate is 91%.
Example 4
The embodiment relates to a preparation method of L-alanine, which specifically comprises the following steps:
1. a seed solution was prepared in the same manner as in example 1 to obtain a seed solution having an OD value of 3.9.
2. Inoculating the seed liquid into the glucose culture medium according to the volume fraction of 7%, and controlling the temperature to be 30 ℃; air flow rate of 2m3Fermenting and culturing under the condition of/h, and keeping the tank pressure at 0.04-0.06MPa by feeding air in the culture process; the pH was controlled to 6.50 with liquid ammonia.
3. When the residual glucose concentration in the fermentation liquid is 31.0g/L, 75% glucose solution is supplemented once until the total glucose concentration in the fermentation liquid is 127.2g/L, and the fermentation is stopped when the residual glucose concentration in the fermentation liquid is 1.62 g/L.
After the fermentation is finished, sampling and carrying out HPLC (high performance liquid chromatography) determination, wherein the content of L-alanine in the fermentation liquid is 113g/L, the concentration of residual glucose is 1.62g/L, and the fermentation conversion rate is 90.2%.
Example 5
The embodiment relates to a preparation method of L-alanine, which specifically comprises the following steps:
1. seed liquid was prepared by two-stage seed treatment in the same manner as in example 1 to obtain a seed liquid having an OD of 6.85.
2. Inoculating the seed liquid into the glucose culture medium according to the volume fraction of 7%, and controlling the temperature to be 30 ℃; air flow rate of 2m3Fermenting and culturing under the condition of/h, and keeping the tank pressure at 0.04-0.06MPa by feeding air in the culture process; the pH was controlled to 6.50 with liquid ammonia.
3. When the residual glucose concentration of the fermentation liquid is 31.0g/L, 75% glucose is supplemented once until the total addition concentration of the glucose in the fermentation liquid is 152.54g/L, and the fermentation is stopped when the glucose concentration in the fermentation liquid is 1.70 g/L.
After the fermentation is finished, sampling and carrying out HPLC (high performance liquid chromatography) determination, wherein the content of L-alanine in the fermentation liquid is 125g/L, the concentration of residual glucose is 1.70g/L, and the fermentation conversion rate is 90.2%.
Comparative example 1
The difference between this example and example 3 is that step 3 is omitted, i.e. no additional glucose is added and only the initial medium is used for the cultivation until the end of the fermentation.
After the fermentation is finished, sampling and carrying out HPLC (high performance liquid chromatography) determination, wherein the content of L-alanine in the fermentation liquid is 82g/L, the concentration of residual glucose is 1.86g/L, and the fermentation conversion rate is 90%.
Comparative example 2
Compared with example 3, this example is different in that in step 3, glucose is added when the glucose concentration in the fermentation broth is 60 g/L.
After the fermentation is finished, sampling and carrying out HPLC (high performance liquid chromatography) determination, wherein the content of L-alanine in the fermentation liquid is 115g/L, the concentration of residual glucose is 2.86g/L, and the fermentation conversion rate is 85%. It can be seen that when the concentration of residual glucose is too high, the addition of glucose will significantly reduce the conversion rate of fermentation, which is detrimental to the conversion and utilization of glucose in the culture medium, and that when the glucose concentration is 2.86, the microorganism will stop fermentation due to too high osmotic pressure.
Comparative example 3
Compared with example 3, this example is distinguished in that, in step 3, glucose at a concentration of 80% is added to the fermentation broth at a concentration of 166.5 g/L.
After the fermentation is finished, sampling and carrying out HPLC (high performance liquid chromatography) determination, wherein the content of L-alanine in the fermentation liquid is 120g/L, the concentration of residual glucose is 1.89g/L, and the fermentation conversion rate is 88%.
Comparative example 4
The difference between this example and example 3 is that in step 3, the glucose concentration in the fermentation broth after glucose supplementation was 140 g/L.
After the fermentation is finished, sampling and carrying out HPLC (high performance liquid chromatography) determination, wherein the content of L-alanine in the fermentation liquid is 110g/L, the concentration of residual glucose is 1.58g, and the fermentation conversion rate is 90.1%.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (15)
1. A method for producing L-alanine by fed-batch fermentation is characterized in that in the process of culturing a Lactobacillus delbrueckii mutant strain Lds.0108 by using a glucose culture medium with an initial glucose concentration of 95-105g/L, when the residual sugar content in fermentation liquor is lower than 45g/L, a glucose solution with a concentration of 50-75% is supplemented to the fermentation liquor at one time until the total concentration of glucose added to the fermentation liquor is 125-170 g/L, and the fermentation is continued until the fermentation is finished;
the lactobacillus delbrueckii mutant strain Lds.0108 is deposited in China center for type culture Collection with the deposition number: CCTCC NO: m2013361.
2. The method of claim 1, wherein the glucose solution is supplemented to the fermentation broth when the residual sugar content of the fermentation broth is 30-41 g/L.
3. The method according to claim 1 or 2, characterized in that when the residual sugar content in the fermentation liquor is less than 33-35 g/L, 68-72% glucose solution is added to the fermentation liquor at one time until the total concentration of glucose added to the fermentation liquor is 160-168 g/L.
4. The method of claim 1, wherein each liter of the glucose medium comprises: 95-105g of glucose and Na2HPO4·12H2O 20g，KH2PO42.0g，NaCl 0.5g，MgSO4·7H20.2g of O, 10mL/L of trace inorganic salt solution and the balance of purified water;
in the trace inorganic salt solution: FeCl3·6H2O 2.5g/L，CoCl2·6H2O 0.2g/L，CuCl2·2H2O0.1g/L，ZnCl20.2g/L，Na2MoO4·2H2O 0.3g/L，H3BO30.1g/L，MnCl2·4H20.5g/L of O and the balance of purified water.
5. The method of claim 3, wherein each liter of the glucose medium comprises: 95-105g of glucose and Na2HPO4·12H2O 20g，KH2PO42.0g，NaCl 0.5g，MgSO4·7H20.2g of O, 10mL/L of trace inorganic salt solution and the balance of purified water;
in the trace inorganic salt solution: FeCl3·6H2O 2.5g/L，CoCl2·6H2O 0.2g/L，CuCl2·2H2O0.1g/L，ZnCl20.2g/L，Na2MoO4·2H2O 0.3g/L，H3BO30.1g/L，MnCl2·4H20.5g/L of O and the balance of purified water.
6. The method according to claim 1, 4 or 5, wherein the Lactobacillus delbrueckii mutant strain Lds.0108 is cultured on a glucose medium under the following conditions: temperature 30
2 ℃; air volume of 0m3H, the pressure is 0.04MPa to 0.06 MPa; pH6.5
0.1。
7. The method according to claim 1 or 2 or 4 or 5, characterized by comprising the steps of:
1) preparing a lactobacillus delbrueckii mutant strain Lds.0108 seed solution;
2) the seed liquid with the seed volume fraction of 5-10% is added into the glucose culture medium according to the seed volume fraction to carry out fermentation culture;
3) and when the concentration of the glucose in the fermentation liquor is less than 45g/L, adding 50-75% of glucose into the fermentation liquor until the total concentration of the glucose added into the fermentation liquor is 125-170 g/L, continuing culturing until the concentration of the glucose is less than or equal to 2g/L, and finishing fermentation.
8. A method according to claim 3, characterized by the steps of:
1) preparing a lactobacillus delbrueckii mutant strain Lds.0108 seed solution;
2) the seed liquid with the seed volume fraction of 5-10% is added into the glucose culture medium according to the seed volume fraction to carry out fermentation culture;
3) and when the concentration of the glucose in the fermentation liquor is less than 45g/L, adding 50-75% of glucose into the fermentation liquor until the total concentration of the glucose added into the fermentation liquor is 125-170 g/L, continuing culturing until the concentration of the glucose is less than or equal to 2g/L, and finishing fermentation.
9. The method of claim 6, comprising the steps of:
1) preparing a lactobacillus delbrueckii mutant strain Lds.0108 seed solution;
2) the seed liquid with the seed volume fraction of 5-10% is added into the glucose culture medium according to the seed volume fraction to carry out fermentation culture;
3) and when the concentration of the glucose in the fermentation liquor is less than 45g/L, adding 50-75% of glucose into the fermentation liquor until the total concentration of the glucose added into the fermentation liquor is 125-170 g/L, continuing culturing until the concentration of the glucose is less than or equal to 2g/L, and finishing fermentation.
10. The method of claim 7, comprising the steps of:
(1) preparing seed solution of mutant strain Lds.0108 of Lactobacillus delbrueckii with OD value of 3.5-7.0;
(2) inoculating the seed liquid into a glucose culture medium according to the seed volume fraction of 5-10%, and performing fermentation culture;
the formula of the glucose culture medium is that each liter of the glucose culture medium comprises 95-105g of glucose and Na2HPO4·12H2O 20g，KH2PO42.0g，NaCl 0.5g，MgSO4·7H20.2g of O, 10mL/L of trace inorganic salt solution and the balance of purified water;
in the trace inorganic salt solution: FeCl3·6H2O 2.5g/L，CoCl2·6H2O 0.2g/L，CuCl2·2H2O0.1g/L，ZnCl20.2g/L，Na2MoO4·2H2O 0.3g/L，H3BO30.1g/L，MnCl2·4H2O0.5 g/L and the balance of purified water;
the culture conditions were: temperature 30
2 ℃; air volume of 0m3H, the pressure is 0.04MPa to 0.06 MPa; pH: 6.5
0.1；
(3) Supplemented culture
And when the concentration of glucose in the fermentation liquor is 30-41g/L, adding 50-75% of glucose into the fermentation liquor until the total concentration of the glucose added into the fermentation liquor is 125-170 g/L, continuing culturing until the concentration of residual sugar in the culture solution is less than or equal to 2g/L, and finishing fermentation.
11. Method according to claim 8 or 9, characterized in that it comprises the following steps:
(1) preparing seed solution of mutant strain Lds.0108 of Lactobacillus delbrueckii with OD value of 3.5-7.0;
(2) inoculating the seed liquid into a glucose culture medium according to the seed volume fraction of 5-10%, and performing fermentation culture;
the formula of the glucose culture medium is that each liter of the glucose culture medium comprises 95-105g of glucose and Na2HPO4·12H2O 20g，KH2PO42.0g，NaCl 0.5g，MgSO4·7H20.2g of O, 10mL/L of trace inorganic salt solution and the balance of purified water;
in the trace inorganic salt solution: FeCl3·6H2O 2.5g/L，CoCl2·6H2O 0.2g/L，CuCl2·2H2O0.1g/L，ZnCl20.2g/L，Na2MoO4·2H2O 0.3g/L，H3BO30.1g/L，MnCl2·4H2O0.5 g/L and the balance of purified water;
the culture conditions were: temperature 30
2 ℃; air volume of 0m3H, the pressure is 0.04MPa to 0.06 MPa; pH: 6.5
0.1；
(3) Supplemented culture
And when the concentration of glucose in the fermentation liquor is 30-41g/L, adding 50-75% of glucose into the fermentation liquor until the total concentration of the glucose added into the fermentation liquor is 125-170 g/L, continuing culturing until the concentration of residual sugar in the culture solution is less than or equal to 2g/L, and finishing fermentation.
12. The method as claimed in claim 10, wherein in the step 3), when the residual sugar content in the fermentation liquor is less than 33-35 g/L, 68-72% glucose solution is added to the fermentation liquor at one time, until the total concentration of the added glucose in the fermentation liquor is 160-168 g/L.
13. The method as claimed in claim 11, wherein in the step 3), when the residual sugar content in the fermentation liquor is less than 33-35 g/L, 68-72% glucose solution is added to the fermentation liquor at one time, until the total concentration of the added glucose in the fermentation liquor is 160-168 g/L.
14. The method according to claim 10 or 12 or 13, wherein the preparation of seed fluid of said mutant strain of lactobacillus delbrueckii lds.0108 comprises the steps of:
(1) under the aseptic condition, selecting a lactobacillus delbrueckii mutant strain Lds.0108 by an inoculating loop, inoculating the lactobacillus delbrueckii mutant strain on the surface of a solid LB solid culture medium, and culturing for 16-18h at the constant temperature of 28-30 ℃;
(2) under the aseptic condition, washing the thalli on the inclined plane by using aseptic normal saline to obtain a bacterial suspension;
(3) inoculating the bacterial suspension liquid into a liquid LB culture medium, and culturing at the temperature of 30 ℃, the air flow of 2L/min and the tank pressure of 0.04-0.06MPa until the OD value of the fermentation liquid is 3.5-7.0.
15. The method according to claim 11, wherein the preparation of seed fluid of lactobacillus delbrueckii mutant strain lds.0108 comprises the steps of:
(1) under the aseptic condition, selecting a lactobacillus delbrueckii mutant strain Lds.0108 by an inoculating loop, inoculating the lactobacillus delbrueckii mutant strain on the surface of a solid LB solid culture medium, and culturing for 16-18h at the constant temperature of 28-30 ℃;
(2) under the aseptic condition, washing the thalli on the inclined plane by using aseptic normal saline to obtain a bacterial suspension;
(3) inoculating the bacterial suspension liquid into a liquid LB culture medium, and culturing at the temperature of 30 ℃, the air flow of 2L/min and the tank pressure of 0.04-0.06MPa until the OD value of the fermentation liquid is 3.5-7.0.
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| CN103602609A (en) * | 2013-09-05 | 2014-02-26 | 淮北新旗氨基酸有限公司 | High-yield strain for producing L-alanine by fermentation and preparation method thereof |
| CN106222309A (en) * | 2016-07-28 | 2016-12-14 | 山东金朗生物科技有限公司 | A kind of fermentable produces the control of additive raw material method improving L alanine yield |
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| CN103602609A (en) * | 2013-09-05 | 2014-02-26 | 淮北新旗氨基酸有限公司 | High-yield strain for producing L-alanine by fermentation and preparation method thereof |
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