CN112695061A - L-glutamic acid total nutrient fed-batch high-density fermentation method - Google Patents
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- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 title claims abstract description 80
- 238000000855 fermentation Methods 0.000 title claims abstract description 66
- 230000004151 fermentation Effects 0.000 title claims abstract description 66
- 235000015097 nutrients Nutrition 0.000 title claims abstract description 48
- 229960002989 glutamic acid Drugs 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims description 24
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- 230000012010 growth Effects 0.000 claims abstract description 15
- 239000002609 medium Substances 0.000 claims description 24
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 21
- 238000011218 seed culture Methods 0.000 claims description 16
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- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 12
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 12
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- 239000004455 soybean meal Substances 0.000 claims description 8
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
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- 239000004473 Threonine Substances 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
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- 239000001888 Peptone Substances 0.000 claims description 3
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
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Abstract
The invention belongs to the technical field of amino acid fermentation, and discloses a total nutrient fed-batch high-density fermentation method for producing L-glutamic acid by fermenting corynebacterium glutamicum, which comprises the following steps: inoculating the temperature-sensitive corynebacterium glutamicum into a fermentation broth, and continuously feeding a total nutrient medium into the fermentation broth when the temperature-sensitive corynebacterium glutamicum grows to a logarithmic phase, wherein the total nutrient medium comprises inorganic salt ions such as potassium salt and magnesium salt and various other growth factors required by the growth of the temperature-sensitive corynebacterium glutamicum. The full nutrient medium is fed in at the beginning of the logarithmic growth phase of the thalli, and the fed-in full nutrient medium can compensate nutrient substances consumed by the thalli due to growth and production in time, so that the thalli are always in the most suitable fermentation environment, and the density of the thalli in the fermentation liquor is improved; meanwhile, in the later stage of fermentation, the activity of the thalli can be improved by feeding the total nutrient medium, and the production performance of the thalli is ensured, so that the yield of the L-glutamic acid is increased.
Description
Technical Field
The invention belongs to the technical field of amino acid fermentation, relates to a total nutrient fed-batch high-density fermentation method of L-glutamic acid, and particularly relates to a total nutrient fed-batch high-density fermentation method for producing L-glutamic acid by fermenting corynebacterium glutamicum with temperature sensitivity.
Background
At present, the monosodium glutamate industry in China is rapidly developed, the yield is at the top of the world, the annual demand reaches 119 ten thousand tons, meanwhile, with the continuous increase of various small foods, refrigerated salted foods in the food processing industry and convenient foods, the monosodium glutamate products are prosperous in market potential, the development of the glutamic acid industry is further driven, according to statistics, the national glutamic acid yield is nearly 300 ten thousand tons, and the production scale tends to be further increased.
The temperature-sensitive corynebacterium glutamicum is a new strain for producing L-glutamic acid, and has the characteristics that the structure of a cell membrane of a strain can be changed at high temperature, so that the permeability of the cell membrane is increased, and L-glutamic acid is secreted, so that the temperature-sensitive corynebacterium glutamicum can still perform fermentation production of the L-glutamic acid even under the condition of high biotin. At present, the method for fermenting L-glutamic acid by utilizing temperature-sensitive corynebacterium glutamicum mainly controls the temperature to be 28-33 ℃ in the early stage of fermentation so that thalli grow rapidly; when the thallus OD in the fermentation liquor600And when the temperature reaches 20-24 ℃, the temperature is increased to 37-40 ℃ within 30 min, and then the thalli grow moderately and accumulate a large amount of L-glutamic acid in the fermentation liquor. However, when the production is carried out according to the existing production method, the growth rate of the initial temperature-sensitive Corynebacterium glutamicum is slower OD600The time for reaching 20-24 is longer, and the subsequent residual growth is lessThe amount of the bacteria is small, the yield of the L-glutamic acid is influenced, meanwhile, the activity of the bacteria is obviously reduced in the later fermentation period, and the performance of the bacteria for producing the L-glutamic acid is further influenced.
Disclosure of Invention
Aiming at the technical problems mentioned above, the invention provides a method for L-glutamic acid complete-nutrition fed-batch high-density fermentation.
A method for L-glutamic acid complete-nutrition fed-batch high-density fermentation, which comprises the following steps:
(1) and (3) activation of thalli: inoculating temperature sensitive corynebacterium glutamicum preserved in a refrigerator at-80 ℃ to a slant, activating for passage, and carrying out passage twice. The slant culture medium is peptone 5 g/L, beef extract 10 g/L, yeast powder 4 g/L, corn steep liquor dry powder 25 mL/L, KH2PO41 g/L，MgSO40.2 g/L, 1g/L NaCl, 25 g/L agar powder, 0.2 g/L methionine and pH = 6.8-7.0;
(2) seed culture: and (3) eluting the activated bacterial strains by using sterile water, and completely inoculating the eluted bacterial strains into prepared seed fermentation liquor for seed culture. The seed culture medium is as follows: 40 g/L glucose, 10 g/L corn steep liquor dry powder and KH2PO42. g/L，MgSO4·7H2O0.8 g/L，MnSO45 mg/L，FeSO45 mg/L, threonine 1g/L, VH10 mg/L. 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.
(3) Fermentation culture: when the amount of bacteria (OD) in the seed culture medium600) And when the inoculation amount reaches more than 40%, inoculating the strain into a fermentation culture medium according to the inoculation amount of 20%, and feeding a full-nutrient culture medium with the volume fraction of 40-80% when the thalli reach the logarithmic phase. The fermentation medium comprises 60 g/L glucose, 30 g/L corn steep liquor, 5 g/L yeast extract, 20 mL/L soybean meal hydrolysate and KH2PO42.5 g/L，MgSO4·7H2O1.2 g/L，KCL1 g/L，MnSO410 mg/L，FeSO410 mg/L, threonine 1g/L, betaine 1g/L, VH3 mg/L，VB10.5 mg/L。
Preferably, the above-mentioned oneThe total nutrient medium of the L-glutamic acid fed-batch high-density fermentation method comprises the following steps: 30 g/L of corn steep liquor dry powder, 30 mL/L of soybean meal hydrolysate and KH2PO42.5 g/L，MgSO4·7H2O1.2 g/L，KCL1 g/L，MnSO410 mg/L，FeSO410 mg/L, methionine 0.5 g/L, VH3 mg/L，VB10.5 mg/L。
Preferably, the method for L-glutamic acid total nutrient fed-batch high-density fermentation is implemented by using bacterial biomass (OD) in logarithmic growth phase600) The value is about 12 to 18.
Preferably, the total nutrient medium feeding mode is constant-speed feeding.
Preferably, the L-glutamic acid total nutrient feeding high-density fermentation method is characterized in that feeding of the total nutrient medium is stopped 4-8h before the fermentation is finished.
Preferably, the method for fermenting the L-glutamic acid by total nutrient feeding and high density comprises the following fermentation conditions: the initial fermentation temperature is 32 ℃, and when the bacterial load (OD) in the fermentation liquid is600) When the temperature reaches 28 ℃, the temperature is increased to 37.5 ℃ within 30 min, and the temperature is increased to 38 ℃ after 1 h; controlling the dissolved oxygen to be 20-40% by controlling the rotating speed and ventilation; the pH was controlled to around 7.0 by ammonia.
Preferably, the strain is Corynebacterium glutamicum sensitive to temperature
Has the advantages that:
according to the method for the L-glutamic acid total nutrient fed-batch high-density fermentation, nutrients consumed by the growth of the thalli are timely compensated through total nutrient fed-batch at the early stage of fermentation, so that the thalli are always in the optimal fermentation environment, the growth speed of the thalli is accelerated, the thalli amount is increased, meanwhile, due to temperature change, the thalli need to grow moderately to a certain extent, and the fed-batch total nutrient medium can provide enough nutrients for the thalli, so that the thalli amount is further increased and enters an acid production stage more quickly, and high-density fermentation is realized; along with the fermentation, the activity of the thalli is gradually reduced in the later stage of fermentation, the metabolism is abnormal, and then the production of the heteroacid is caused, the full-nutrition feeding can relieve the aging speed of the thalli, stabilize the metabolic flux and improve the yield of the L-glutamic acid. In conclusion, the method for the total nutrient fed-batch high-density fermentation of the L-glutamic acid effectively solves the problems that when the temperature-sensitive corynebacterium glutamicum is subjected to L-glutamic acid fermentation, the early-stage thallus grows slowly, the transformation time is long, the residual growth is small, and the activity of the thallus is reduced in the later stage of fermentation, so that the yield of the L-glutamic acid is reduced and the content of the heteropolyacid is increased.
Detailed Description
The present invention will be described in further detail below with reference to specific embodiments.
Example 1
(1) And (3) activation of thalli: inoculating temperature-sensitive Corynebacterium glutamicum (GKG-047, preservation number CGMCC No. 5481) preserved in a refrigerator at-80 ℃ to a slant for successive generation and activation, and carrying out passage twice.
The slant culture medium is peptone 5 g/L, beef extract 10 g/L, yeast powder 4 g/L, corn steep liquor dry powder 25 mL/L, KH2PO41 g/L，MgSO40.2 g/L, 1g/L NaCl, 25 g/L agar powder, 0.2 g/L methionine and pH = 6.8-7.0;
(2) seed culture: and (3) eluting the activated bacterial strains by using sterile water, and completely inoculating the eluted bacterial strains into prepared seed fermentation liquor for seed culture.
The seed culture medium is as follows: 40 g/L glucose, 10 g/L corn steep liquor dry powder and KH2PO42. g/L，MgSO4·7H2O0.8 g/L，MnSO45 mg/L，FeSO45 mg/L, threonine 1g/L, VH10 mg/L。
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.
(3) Fermentation culture: when the amount of bacteria (OD) in the seed culture medium600) Inoculating into 5L fermentation medium in an inoculum size of 20% when the inoculum size reaches above 40, feeding 60% total nutrient medium when the thallus reaches 15, and feeding to fermentation nodeStopping 4-8h before bundling.
The fermentation medium comprises 60 g/L glucose, 30 g/L corn steep liquor, 5 g/L yeast extract, 20 mL/L soybean meal hydrolysate and KH2PO42.5 g/L，MgSO4·7H2O1.2 g/L，KCL1 g/L，MnSO410 mg/L，FeSO410 mg/L, threonine 1g/L, betaine 1g/L, VH3 mg/L，VB10.5 mg/L。
The total nutrient medium comprises: 30 g/L of corn steep liquor dry powder, 30 mL/L of soybean meal hydrolysate and KH2PO42.5 g/L，MgSO4·7H2O1.2 g/L，KCL1 g/L，MnSO410 mg/L，FeSO410 mg/L, methionine 0.5 g/L, VH3 mg/L，VB10.5 mg/L。
The fermentation conditions are as follows: the initial fermentation temperature is 32 ℃, and when the bacterial load (OD) in the fermentation liquid is600) When the temperature reaches 28 ℃, the temperature is increased to 37.5 ℃ within 30 min, and the temperature is increased to 38 ℃ after 1 h; controlling the dissolved oxygen to be 20-40% by controlling the rotating speed and ventilation; the pH was controlled to around 7.0 by ammonia.
The preparation method of the soybean meal hydrolysate comprises the following steps: preheating 20% soybean meal solution at 90 deg.C for 10min, hydrolyzing at 40 deg.C, pH3, adding 20000U/g acid protease (calculated on dry soybean meal), and inactivating enzyme when the hydrolysis time is 3 hr.
Example 2
Reference is made to example 1, except that when the cells reached 12, a total nutrient medium was fed in an amount of 60% by volume.
Example 3
Referring to example 1, the difference is that when the bacterial cells reached 18, the feeding of the complete nutrient medium was started at a volume fraction of 60%.
Example 4
Reference is made to example 1, except that the total nutrient medium is fed in a volume fraction of 40% of the total nutrient medium.
Example 5
Reference is made to example 1, except that the total nutrient medium is fed in at a volume fraction of 80% of the total nutrient medium.
Example 6
Reference is made to example 1, except that the total nutrient fed-batch strategy is not employed.
TABLE 1 comparative indices for biomass, L-glutamic acid production and saccharic acid conversion
| Maximum bacterial body volume (OD)600) | Final bacterial Mass (OD)600） | L-glutamic acid yield (g/L) | Sugar consumption (g/L) | High saccharic acid conversion rate (%) | |
| Example 1 | 36.2 | 34 | 261 | 360.3 | 72.5 |
| Example 2 | 32.1 | 29.5 | 225 | 316 | 71.2 |
| Example 3 | 34.3 | 32.1 | 239 | 333.8 | 71.6 |
| Example 4 | 34.1 | 30.8 | 237 | 328.7 | 72.1 |
| Example 5 | 35.6 | 32.9 | 249 | 344.4 | 72.3 |
| Example 6 | 30 | 26 | 209 | 282.4 | 71 |
As can be seen from Table 1, when the optimum feeding time was investigated by performing a one-way test on example 1, example 2 and example 3, it was found that example 1 is the amount of bacteria (OD)600) When the total nutrient medium reaches 15 ℃, the feeding of the total nutrient medium is started to achieve the best effect. The feeding time is too early, the nutrient substances in the fermentation liquor are not consumed much, and the nutrient substances supplemented subsequently can cause high osmotic pressure and a nutrient-rich environment, so that the growth of thalli is not facilitated; the time of feeding is too late, the subsequent growth potential of the thalli is insufficient, the transformation time is prolonged, and the thalli produce acid. In order to examine the optimum volume percentage of the total nutrient medium fed, it was found by comparing examples 1, 4 and 5 that the cell growth and L-glutamic acid production were the best when the total nutrient medium was fed at 60% by volume. When the volume percentage of the fed-batch is too large, the excessively abundant nutrient substances can bring about substrate inhibition and a high osmotic pressure environment, and are not beneficial to the growth and metabolism of thalli; when the volume percentage of fed-batch is too low, the fed-batch total nutrient medium is not enough to make up the nutrient substances consumed by the growth and production of the thalli, thereby influencing the growth and acid production of the thalli. Through comparison between example 1 and example 6, the maximum bacterial amount of the total nutrient fed-batch strategy is 36.2, the final bacterial amount is 34, the reduction range is 6.1%, the yield of L-glutamic acid reaches 261 g/L, the conversion rate of saccharic acid is 72.5%, and the method is greatly improved compared with the conventional fermentation method without the total nutrient fed-batch. Therefore, the feeding of the total nutrient medium has obvious positive effects on accelerating the growth of thalli, delaying the senescence of thalli and increasing the yield of L-glutamic acid.
Although the present invention has been described in detail with respect to the general description and the specific embodiments, it will be apparent to those skilled in the art that modifications and improvements can be made based on the present invention. Accordingly, it is intended that all such modifications and variations as fall within the true spirit of this invention be included within the scope thereof.
Claims (10)
1. A method for L-glutamic acid total nutrient fed-batch high-density fermentation, which is characterized by comprising the following steps:
and (3) activation of thalli: inoculating temperature-sensitive corynebacterium glutamicum preserved in a refrigerator at the temperature of-80 ℃ to a slant culture medium for passage activation and passage twice;
seed culture: eluting the activated strains by using sterile water, and completely inoculating the eluted strains into a prepared seed culture medium for seed culture; 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 the bacteria in the seed culture medium is OD600And when the inoculation amount reaches more than 40%, inoculating the strain into a fermentation culture medium according to the inoculation amount of 20%, and feeding a full-nutrient culture medium with the volume fraction of 40-80% when the thalli reach the logarithmic phase.
2. The method for complete-nutrition fed-batch high-density fermentation of L-glutamic acid according to claim 1, which is characterized in that: the total nutrient medium comprises: 30 g/L of corn steep liquor dry powder, 20 mL/L of soybean meal hydrolysate and KH2PO42.5 g/L，MgSO4·7H2O1.2 g/L，KCl 1 g/L，MnSO410 mg/L，FeSO410 mg/L, methionine 0.5 g/L, VH3 mg/L，VB10.5 mg/L。
3. The method for complete-nutrition fed-batch high-density fermentation of L-glutamic acid according to claim 1, which is characterized in that: the bacterial body amount OD of the logarithmic growth phase600The value is about 12 to 18.
4. The method for complete-nutrition fed-batch high-density fermentation of L-glutamic acid according to claim 1, which is characterized in that: the feeding mode of the total nutrient medium is constant-speed feeding.
5. The method for complete-nutrition fed-batch high-density fermentation of L-glutamic acid according to claim 1, which is characterized in that: the feeding time is 4-8h before the fermentation is finished.
6. The method for complete-nutrition fed-batch high-density fermentation of L-glutamic acid according to claim 1, which is characterized in that: the fermentation conditions are as follows: the initial fermentation temperature is 32 ℃, and when the bacterial body amount OD in the fermentation liquid600When the temperature reaches 28 ℃, the temperature is increased to 37.5 ℃ within 30 min, and the temperature is increased to 38 ℃ after 1 h; controlling the dissolved oxygen to be 20-40% by controlling the rotating speed and ventilation; the pH was controlled to around 7.0 by ammonia.
7. The method for complete-nutrition fed-batch high-density fermentation of L-glutamic acid according to claim 1, which is characterized in that: the strain is temperature-sensitive corynebacterium glutamicum.
8. The method for complete-nutrition fed-batch high-density fermentation of L-glutamic acid according to claim 1, which is characterized in that: the slant culture medium is peptone 5 g/L, beef extract 10 g/L, yeast powder 4 g/L, corn steep liquor dry powder 25 mL/L, KH2PO41 g/L，MgSO40.2 g/L, NaCl 1g/L, agar powder 25 g/L, methionine 0.2 g/L, and pH = 6.8-7.0.
9. The method for complete-nutrition fed-batch high-density fermentation of L-glutamic acid according to claim 1, which is characterized in that: the seed culture medium is as follows: 40 g/L glucose, 10 g/L corn steep liquor dry powder and KH2PO42. g/L，MgSO4·7H2O0.8 g/L，MnSO45 mg/L，FeSO45 mg/L, threonine 1g/L, VH10 mg/L。
10. The method for complete-nutrition fed-batch high-density fermentation of L-glutamic acid according to claim 1, which is characterized in that: the fermentation medium is as follows: 60 g/L glucose, 30 g/L corn steep liquor, 5 g/L yeast extract and soybean mealHydrolysate of 20 mL/L, KH2PO42.5 g/L，MgSO4·7H2O1.2 g/L，KCl 1 g/L，MnSO410 mg/L，FeSO410 mg/L, threonine 1g/L, betaine 1g/L, VH3 mg/L，VB10.5 mg/L。
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| CN113981019A (en) * | 2021-10-14 | 2022-01-28 | 呼伦贝尔东北阜丰生物科技有限公司 | Process for improving glutamic acid fermentation rate |
| CN115386529A (en) * | 2022-06-15 | 2022-11-25 | 呼伦贝尔东北阜丰生物科技有限公司 | Method for improving glutamic acid fermentation bacteria amount and acid production efficiency |
| CN115399466A (en) * | 2022-06-29 | 2022-11-29 | 呼伦贝尔东北阜丰生物科技有限公司 | Concentration and crystallization process of sodium glutamate |
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| CN115386529A (en) * | 2022-06-15 | 2022-11-25 | 呼伦贝尔东北阜丰生物科技有限公司 | Method for improving glutamic acid fermentation bacteria amount and acid production efficiency |
| CN115399466A (en) * | 2022-06-29 | 2022-11-29 | 呼伦贝尔东北阜丰生物科技有限公司 | Concentration and crystallization process of sodium glutamate |
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