CN112251474A - Method for improving fermentation yield and saccharic acid conversion rate of L-glutamic acid - Google Patents
Method for improving fermentation yield and saccharic acid conversion rate of L-glutamic acid Download PDFInfo
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Abstract
The invention provides a method for improving fermentation yield and saccharic acid conversion rate of L-glutamic acid, which comprises the following steps of adding one part of amino acid chelated trace elements into a fermentation culture medium, feeding the other part of the amino acid chelated trace elements along with glucose for fermentation, wherein the amino acid chelated trace elements consist of methionine chelated iron, methionine chelated manganese and alanine chelated zinc, and the weight ratio of the methionine chelated iron, the methionine chelated manganese and the alanine chelated zinc is as follows: 10-20: 10-15: 2-6; according to the method, the amino acid chelated trace elements are adopted, so that the nutrient components of effective amino acid in a fermentation medium are supplemented, the absorption effect of the thalli on the trace elements is increased, the growth activity and the production performance of the thalli are improved, the yield of the L-glutamic acid and the saccharic acid conversion rate are improved, the fermentation lasts for 38 hours, the maximum bacterial body quantity (OD 600) reaches 40, the yield of the L-glutamic acid is 181g/L, the saccharic acid conversion rate is 68.1%, and the yield is respectively improved by 17.6%, 13.8% and 3.1% compared with the conventional fermentation.
Description
Technical Field
The invention relates to the field of L-glutamic acid fermentation, in particular to a method for improving the fermentation yield and the saccharic acid conversion rate of L-glutamic acid.
Background
In recent years, with the wide application of L-glutamic acid in the industries of food, medicine, chemical industry, feed and the like, the L-glutamic acid has huge demand on yield, the annual yield reaches more than 170 million tons only by using monosodium glutamate, more than 50 countries are exported, the sales income is nearly 180 million yuan, and the market prospect is huge. There are various methods for producing L-glutamic acid, including: chemical synthesis, protein hydrolysis, enzymatic methods and microbial fermentation, but the only methods that can be used for large-scale industrial production of L-glutamic acid are microbial fermentation.
However, the existing L-glutamic acid fermentation industry in China still has the problems of low fermentation acid yield, low sugar-acid conversion rate, more byproducts, low extraction yield and the like. Meanwhile, along with the rising of raw material cost, the improvement of labor cost and environmental problems in recent years, the difficulty of the L-glutamic acid industry is further aggravated, and the economic benefit of enterprises is limited. Therefore, how to further improve the capacity of producing L-glutamic acid by microbial fermentation and the sugar-acid conversion rate becomes important.
Disclosure of Invention
The invention aims to provide a method for improving the fermentation yield and the sugar-acid conversion rate of L-glutamic acid.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a method for improving fermentation yield and saccharic acid conversion rate of L-glutamic acid comprises the steps of adding one part of amino acid chelated trace elements into a fermentation culture medium, feeding the other part of the amino acid chelated trace elements along with glucose for fermentation, wherein the amino acid chelated trace elements are composed of methionine chelated iron, methionine chelated manganese and alanine chelated zinc, and the weight ratio of the methionine chelated iron to the methionine chelated manganese to the alanine chelated zinc is as follows: 10-20: 10-15: 2-6.
After the amino acid is chelated with the trace elements, on one hand, the nutrient components of the effective amino acid in the fermentation medium are supplemented, on the other hand, the absorption effect of the thalli on the trace elements is increased, the growth activity and the production performance of the thalli are improved, and therefore the yield of the L-glutamic acid and the conversion rate of the saccharic acid are improved.
Preferably, the method for improving the fermentation yield and the sugar-acid conversion rate of L-glutamic acid comprises the following steps:
(1) and (3) activation of thalli: inoculating a sub-appropriate amount of corynebacterium glutamicum preserved in a refrigerator at the temperature of-80 ℃ to a slant, carrying out passage activation and carrying out passage twice; the slant culture medium is peptone 5g/L, beef extract 10g/L, yeast powder 4g/L, corn steep liquor dry powder 25mL/L, KH2PO41g/L,MgSO40.2g/L, NaCl1g/L, agar powder 25g/L, methionine 0.2g/L, pH = 6.8-7.0;
(2) seed culture: eluting the activated strain with sterile water, and completely inoculating the eluted strain to a prepared seed culture medium for seed culture, wherein the seed culture medium comprises: 40g/L glucose, 10g/L corn steep liquor dry powder and KH2PO4 2g/L,MgSO4·7H20.8g/L of O, 80mg/L of methionine chelated iron, 60mg/L of methionine chelated manganese and 20mg/L of alanine chelated zinc; the seed culture conditions are as follows: the temperature is maintained at 32 +/-2 ℃, the dissolved oxygen is controlled at 20-40%, and the pH is controlled to be approximately 7.0 by ammonia water;
(3) fermentation culture: when the amount of bacteria (OD) in the seed culture medium600) When the content of the amino acid chelated trace elements reaches more than 15 percent, the amino acid chelated trace elements are inoculated to a fermentation medium according to the inoculation amount of 20 percent, the amino acid chelated trace elements are fed along with a glucose solution in the fermentation process, the amino acid chelated trace elements comprise methionine chelated iron, methionine chelated manganese and alanine chelated zinc, the content of each component in the glucose solution is respectively 100-200mg/L, 100-150mg/L and 20-60mg/L, the fermentation medium is 60g/L of glucose, 30g/L of corn steep liquor, 20 mL/L of soybean concentrate, and KH2PO4 2.5g/L,MgSO4·7H2O1.2g/L, KCL1g/L, iron methionine chelate 80mg/L, manganese methionine chelate 60mg/L, zinc alanine chelate 20mg/L, betaine 1g/L, VB10.5 mg/L; the fermentation culture conditions are as follows: the temperature is controlled at 34 +/-2 ℃, the dissolved oxygen is maintained at 30-50%, and the pH is controlled to be approximately 7.0 by ammonia water.
Preferably, in the method for improving the fermentation yield and the sugar acid conversion rate of the L-glutamic acid, the contents of the methionine chelated iron, the methionine chelated manganese and the alanine chelated zinc in the glucose solution are 150-170mg/L, 110-130mg/L and 30-50mg/L respectively.
Preferably, in the method for improving the fermentation yield and the sugar-acid conversion rate of the L-glutamic acid, the contents of the iron methionine chelate, the manganese methionine chelate and the zinc alanine chelate in the glucose solution are 160mg/L, 120mg/L and 40mg/L respectively.
Preferably, in the method for improving the fermentation yield and the sugar-acid conversion rate of the L-glutamic acid, the mass concentration of the fed-in glucose solution is 80%, and the residual sugar content is maintained to be 3-5g/L in the fermentation process.
Has the advantages that:
according to the method for improving the fermentation yield and the saccharic acid conversion rate of the L-glutamic acid, the amino acid is chelated with the trace elements, so that on one hand, the nutrient components of effective amino acid in a fermentation culture medium are supplemented, on the other hand, the absorption effect of the thalli on the trace elements is increased, the growth activity and the production performance of the thalli are improved, the yield and the saccharic acid conversion rate of the L-glutamic acid are improved, the fermentation time is 38 hours, the maximum bacterial body mass (OD 600) reaches 40, the yield of the L-glutamic acid is 181g/L, and the saccharic acid conversion rate is 68.1%, which are respectively improved by 17.6%, 13.8% and 3.1% compared with a.
Detailed Description
Example 1
A method for improving L-glutamic acid fermentation yield and saccharic acid conversion rate comprises the following specific steps:
and (3) activation of thalli: inoculating appropriate amount of biotin sub-type Corynebacterium glutamicum GKG-047 (CGMCC No.5481 strain, China Committee for culture Collection of microorganisms) stored in a refrigerator at-80 deg.C to the inclined plane for successive generation and activation,inoculating to slant for successive activation and twice passages. The slant culture medium is peptone 5g/L, beef extract 10g/L, yeast powder 4g/L, corn steep liquor dry powder 25mL/L, KH2PO41g/L,MgSO40.2g/L, NaCl1g/L, agar powder 25g/L, methionine 0.2g/L, pH = 6.8-7.0;
seed culture: and (3) eluting the activated bacterial strains by using sterile water, and completely inoculating the eluted bacterial strains into a prepared seed culture medium for seed culture. The seed culture medium is as follows: 40g/L glucose, 10g/L corn steep liquor dry powder and KH2PO4 2g/L,MgSO4·7H20.8g/L of O, 80mg/L of methionine chelated iron, 60mg/L of methionine chelated manganese and 20mg/L of alanine chelated zinc. The seed culture conditions are as follows: the temperature is maintained at about 32 ℃, the dissolved oxygen is controlled at 20-40%, and the pH is controlled at about 7.0 by ammonia water.
Fermentation culture: when the amount of bacteria (OD) in the seed culture medium600) When the inoculation amount reaches more than 15 percent, the mixture is inoculated to a fermentation culture medium according to the inoculation amount of 20 percent, and amino acid chelated trace elements are fed along with a glucose solution in the fermentation process. The fermentation medium is glucose 60g/L, corn steep liquor 30g/L, soybean concentrate 20 mL/L, KH2PO4 2.5g/L,MgSO4·7H2O1.2g/L, KCL1g/L, iron methionine chelate 80mg/L, manganese methionine chelate 60mg/L, zinc alanine chelate 20mg/L, betaine 1g/L, VB1 0.5 mg/L. The fermentation culture conditions are as follows: the temperature is controlled at 34 ℃, the dissolved oxygen is maintained at 30-50%, the pH is controlled at about 7.0 by ammonia water, the residual sugar in the fermentation liquor is controlled at 3-5g/L, and the mass concentration of the fed-batch glucose is 80%, wherein the contents of methionine chelated iron, methionine chelated manganese and alanine chelated zinc are respectively 150mg/L, 110mg/L and 30 mg/L.
Example 2
The contents of methionine chelated iron, methionine chelated manganese and alanine chelated zinc in the fed-batch glucose are respectively 160mg/L, 120mg/L and 40mg/L, and the other steps refer to example 1.
Example 3
The contents of methionine chelated iron, methionine chelated manganese and alanine chelated zinc in the fed-batch glucose are respectively 170mg/L, 130mg/L and 50mg/L, and the other steps refer to example 1.
Example 4
The contents of methionine chelated iron, methionine chelated manganese and alanine chelated zinc in the fed-batch glucose are respectively 100mg/L, 100mg/L and 20mg/L, and the other steps refer to example 1.
Example 5
The contents of methionine chelated iron, methionine chelated manganese and alanine chelated zinc in the fed-batch glucose are respectively 200mg/L, 150mg/L and 60mg/L, and the other steps refer to example 1.
Example 6
Adopting conventional fermentation, adding 0.45mmol/L FeSO instead of adding iron methionine chelate, manganese methionine chelate and zinc alanine chelate to the fermentation medium and fed-batch glucose4、0.34mmol/L MnSO4And 0.18mmol/L ZnSO4(equimolar concentration of the corresponding inorganic salt ion) and the other steps are as in example 1.
Example 7
In the conventional fermentation, 0.79mmol/L methionine and 0.18mmol/L alanine (equivalent molar concentration of corresponding amino acids) were selectively added to the fermentation medium and fed-batch glucose without adding iron methionine chelate, manganese methionine chelate and zinc alanine chelate, and the other steps were performed as in example 1.
Example 8
Adopting conventional fermentation, adding 0.45mmol/L FeSO instead of adding iron methionine chelate, manganese methionine chelate and zinc alanine chelate to the fermentation medium and fed-batch glucose4、0.34mmol/L MnSO4、0.18mmol/L ZnSO40.79mmol/L methionine and 0.18mmol/L alanine (equimolar concentrations of corresponding inorganic salt ion and amino acid), other steps were referred to example 1.
Example 9
The conventional fermentation is adopted, the iron methionine chelate, the manganese methionine chelate and the zinc alanine chelate are not added into a fermentation medium and fed-batch glucose, and other steps refer to example 1.
TABLE 1 comparative indices for biomass, L-glutamic acid production and saccharic acid conversion
Maximum amount of bacteria | L-glutamic acid yield (g/L) | Conversion ratio of sugar to acid (%) | |
Example 1 | 38 | 167 | 66.7 |
Example 2 | 40 | 181 | 68.1 |
Example 3 | 38.9 | 175 | 67.2 |
Example 4 | 36.6 | 163 | 66.4 |
Example 5 | 38.1 | 165 | 67.3 |
Example 6 | 34 | 159 | 65 |
Example 7 | 35.5 | 158 | 64.6 |
Example 8 | 37.8 | 173 | 66.7 |
Example 9 | 32 | 155 | 64.5 |
The comparative analysis of the examples 1, 2, 3, 4 and 5 shows that the growth performance and production performance of the cells tend to increase and decrease with the increase of the addition amount of the iron methionine chelate, the manganese methionine chelate and the zinc alanine chelate, this indicates that when the addition amount is too low, the lack of nutrients leads to a decrease in the viability of the cells, whereas when the addition amount is too high, the negative effects of high substrate concentration and high osmotic pressure also reduce the growth and productivity of the cells, wherein example 2, namely when the contents of methionine chelated iron, methionine chelated manganese and alanine chelated zinc in the fed-batch glucose are respectively 160mg/L, 120mg/L and 40mg/L, the maximum bacterial amount reaches 40, the yield of the L-glutamic acid is 181g/L, the saccharic acid conversion rate is 68.1%, and the effect is best.
Analysis of examples 2, 6, 7, 8 and 9 shows that the addition of an amino acid chelating agent, the addition of an equivalent amount of a corresponding inorganic salt ion alone, the addition of an equivalent amount of a corresponding amino acid alone, the addition of a corresponding inorganic salt ion and an amino acid in combination, and the addition of no corresponding inorganic salt ion and an amino acid in combination showed that the addition of an inorganic salt ion and an amino acid to a fermentation broth actually facilitated the growth of cells and the production of acid by fermentation, wherein the addition of an amino acid chelating agent was most effective, and the addition of an amino acid chelating agent was estimated by increasing the maximum cell mass, the L-glutamic acid production, and the sugar acid conversion by 20%, 16.7%, and 3.6%, respectively, as compared with the conventional fermentation (maximum cell mass 32, L-glutamic acid production 155 g/L, and sugar acid conversion 64.5%) in which no corresponding inorganic salt ion and amino acid were added, accelerating the absorption effect of the thalli on inorganic salt ions and amino acid, thereby showing positive effects on improving the activity of the thalli, increasing the yield of the L-glutamic acid and increasing the conversion rate of saccharic acid.
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 (5)
1. A method for improving L-glutamic acid fermentation yield and saccharic acid conversion rate is characterized in that: adding one part of amino acid chelated trace elements into a fermentation medium, feeding the other part of the amino acid chelated trace elements along with glucose for fermentation, wherein the amino acid chelated trace elements are composed of methionine chelated iron, methionine chelated manganese and alanine chelated zinc, and the weight ratio of the methionine chelated iron, the methionine chelated manganese and the alanine chelated zinc is as follows: 10-20: 10-15: 2-6.
2. The method for improving the fermentation yield and sugar-acid conversion rate of L-glutamic acid according to claim 1, wherein: comprises the following steps:
(1) and (3) activation of thalli: inoculating a sub-appropriate amount of corynebacterium glutamicum preserved in a refrigerator at the temperature of-80 ℃ to a slant, carrying out passage activation and carrying out passage twice; the slant culture medium is protein5g/L of peptone, 10g/L of beef extract, 4g/L of yeast powder, 25mL/L of corn steep liquor dry powder and KH2PO41g/L,MgSO4 0.2g/L, NaCl1g/L, agar powder 25g/L, methionine 0.2g/L, pH = 6.8-7.0;
(2) seed culture: eluting the activated strain with sterile water, and completely inoculating the eluted strain to a prepared seed culture medium for seed culture, wherein the seed culture medium comprises: 40g/L glucose, 10g/L corn steep liquor dry powder and KH2PO4 2g/L,MgSO4·7H20.8g/L of O, 80mg/L of methionine chelated iron, 60mg/L of methionine chelated manganese and 20mg/L of alanine chelated zinc; the seed culture conditions are as follows: the temperature is maintained at 32 +/-2 ℃, the dissolved oxygen is controlled at 20-40%, and the pH is controlled to be approximately 7.0 by ammonia water;
(3) fermentation culture: when the amount of bacteria (OD) in the seed culture medium600) When the content of the amino acid chelated trace elements reaches more than 15 percent, the amino acid chelated trace elements are inoculated to a fermentation medium according to the inoculation amount of 20 percent, the amino acid chelated trace elements are fed along with a glucose solution in the fermentation process, the amino acid chelated trace elements comprise methionine chelated iron, methionine chelated manganese and alanine chelated zinc, the content of each component in the glucose solution is respectively 100-200mg/L, 100-150mg/L and 20-60mg/L, the fermentation medium is 60g/L of glucose, 30g/L of corn steep liquor, 20 mL/L of soybean concentrate, and KH2PO42.5g/L,MgSO4·7H2O1.2g/L, KCL1g/L, iron methionine chelate 80mg/L, manganese methionine chelate 60mg/L, zinc alanine chelate 20mg/L, betaine 1g/L, VB10.5 mg/L; the fermentation culture conditions are as follows: the temperature is controlled at 34 +/-2 ℃, the dissolved oxygen is maintained at 30-50%, and the pH is controlled to be approximately 7.0 by ammonia water.
3. The method for improving the fermentation yield and sugar-acid conversion rate of L-glutamic acid according to claim 2, wherein: the contents of the methionine chelated iron, the methionine chelated manganese and the alanine chelated zinc in the glucose solution are respectively 150-170mg/L, 110-130mg/L and 30-50 mg/L.
4. The method for improving the fermentation yield and sugar-acid conversion rate of L-glutamic acid according to claim 3, wherein: the contents of the methionine chelated iron, the methionine chelated manganese and the alanine chelated zinc in the glucose solution are respectively 160mg/L, 120mg/L and 40 mg/L.
5. The method for improving the fermentation yield and sugar-acid conversion rate of L-glutamic acid according to claim 2, wherein: the mass concentration of the glucose solution fed back is 80%, and the residual sugar content is maintained to be 3-5g/L in the fermentation process.
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