CN116987743A - Technology for producing gamma-aminobutyric acid based on silkworm chrysalis powder coupled glucose acid control - Google Patents
Technology for producing gamma-aminobutyric acid based on silkworm chrysalis powder coupled glucose acid control Download PDFInfo
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- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 title claims abstract description 138
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- 229960003692 gamma aminobutyric acid Drugs 0.000 title claims abstract description 69
- 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 title claims abstract description 59
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- 238000004519 manufacturing process Methods 0.000 abstract description 16
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Abstract
The invention discloses a technology for producing gamma-aminobutyric acid based on silkworm chrysalis powder coupled glucose acid control. The method for efficiently producing the gamma-aminobutyric acid by using the gamma-aminobutyric acid producing strain-lactobacillus johnsonii as an initial strain, using low-cost silkworm chrysalis powder to replace high-cost peptone, and simultaneously controlling acidity by feeding low-concentration glucose. According to the invention, through optimizing the addition proportion of silkworm chrysalis powder, the concentration of glucose and the concentration of sodium glutamate, and adopting fed-batch glucose to maintain the growth of cells, the production of lactic acid is increased, the pH value of fermentation is maintained, and the capability of continuously maintaining high-yield gamma-aminobutyric acid of the strain is realized, wherein the accumulated concentration of gamma-aminobutyric acid is 229.28 g/L. According to the invention, the silkworm chrysalis is used for replacing peptone and is coupled with glucose to control acid, so that the capability of producing gamma-aminobutyric acid by lactobacillus fermentation is obviously improved, the production cost of the gamma-aminobutyric acid is greatly reduced, and the prepared gamma-aminobutyric acid can be used for food development and has no potential safety hazard.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a technology for producing gamma-aminobutyric acid based on silkworm chrysalis powder coupled glucose acid control.
Background
Gamma-aminobutyric acid is an important central nervous system inhibitory neurotransmitter widely existing in vertebrates, plants and microorganisms, has the effects of reducing blood pressure, treating epilepsy, treating alcohol dependence, improving brain functions and lipid metabolism, promoting neuroleptic, promoting secretion of memory and growth hormone, activating kidney functions and liver functions, preventing skin aging, and removing body odor, and is widely used in the fields of medicine, food, chemical industry and the like.
The microbial fermentation method utilizes safe, nontoxic, harmless and stable strains, takes sodium glutamate or glutamic acid as a substrate, and produces gamma-aminobutyric acid under the action of glutamate decarboxylase. The producing strain of gamma-aminobutyric acid comprises Escherichia coli, lactobacillus, yeast, monascus, etc. Wherein, the escherichia coli has potential safety hazard and can not be directly used in medicines or foods. The yield of the gamma-aminobutyric acid of the yeast and monascus is low, and the method cannot be suitable for wide application of the gamma-aminobutyric acid. Thus, lactic acid bacteria are currently recognized as safe and high-yielding strains of gamma-aminobutyric acid. Although the yield of the gamma-aminobutyric acid can be obviously improved through culture medium and fermentation process optimization, in the actual production process, the laboratory-scale efficient production process cannot meet the requirements of low cost and high profit of industrial production, and compared with the low cost of the fermentation culture medium, the method has important industrialized effect on the production of the gamma-aminobutyric acid.
Lactic acid bacteria are bacteria which take sugar as a carbon source and ferment to produce lactic acid, most amino acid auxotrophs cannot synthesize amino acid and are mainly obtained by hydrolyzing protein, so a fermentation culture medium of gamma-aminobutyric acid mainly comprises a composite nitrogen source, in particular yeast powder, peptone, beef extract and the like. The cost of yeast powder, peptone and yeast powder is high, so that the production cost of gamma-aminobutyric acid is obviously increased. Therefore, searching for an alternative nitrogen source for gamma-aminobutyric acid is a key to reducing the production cost of gamma-aminobutyric acid. The silkworm chrysalis is a byproduct of silkworm cocoon after spinning, has high nutritive value, contains a large amount of complete protein, and is ideal pure natural high-quality protein. In China, the national mulberry field is used for producing more than 80 ten thousand tons of silkworm chrysalis annual, and silkworm chrysalis comprehensive products including silkworm chrysalis powder, silkworm chrysalis compound amino acid, chitosan, silkworm chrysalis protein powder and the like have been developed. Because of high decoloring and deodorizing cost, the method can not be widely applied, thereby causing resource waste and environmental pollution.
The production of gamma-aminobutyric acid by lactobacillus fermentation is based on the transport of glutamate or sodium glutamate substrate into cells, and the catalytic decarboxylation of glutamate decarboxylase to produce gamma-aminobutyric acid while consuming intracellular H + The intracellular pH is maintained stable, and the cells are protected from acid stress. In order to maintain efficient catalysis of the enzyme, researchers often adopt to control the pH of the fermentation process so that the enzyme can be continuously and efficiently catalyzed. The process control method of fermentation pH mainly comprises the step of adding sulfuric acid, and in industrial production, particularly in the field of food and medicine, the safety hazard of sulfuric acid residues is large, and meanwhile, the purification and separation cost of gamma-aminobutyric acid is increased.
In summary, how to use the dry silkworm chrysalis powder as a substitute nitrogen source of the gamma-aminobutyric acid producing bacteria reduces the industrial production cost of the gamma-aminobutyric acid, and meanwhile, by adjusting the addition of glucose, the advantage of lactic acid production by the metabolism of lactic acid bacteria is relied on, and by the production of the gamma-aminobutyric acid, the intracellular acid stress is relieved, the control of fermentation pH is realized, the pH value of the fermentation process is maintained, and the method has important significance for the industrial production and application of the gamma-aminobutyric acid and the reutilization of silkworm chrysalis resources.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a technology for producing gamma-aminobutyric acid based on coupling of silkworm chrysalis powder with glucose for controlling acid, which uses low-cost silkworm chrysalis powder to replace high-cost peptone and coupling of glucose for controlling acid, maintains the activity of glutamate decarboxylase, remarkably improves the capability of producing gamma-aminobutyric acid by lactobacillus fermentation, greatly reduces the production cost of gamma-aminobutyric acid, and can be used for food development without potential safety hazard.
In order to solve the problems in the prior art, the invention adopts the following technical scheme:
a technology for preparing gamma-aminobutyric acid based on coupling of silkworm chrysalis powder and glucose by controlling acid includes such steps as using gamma-aminobutyric acid generating bacteria as initial strain, replacing high-cost peptone with low-cost silkworm chrysalis powder, and adding low-concentration glucose to control fermentation pH.
As an improvement, the starting strain is lactobacillus johnsoniiLactobacillus hilgardii)。
The technology for producing gamma-aminobutyric acid based on silkworm chrysalis powder coupled glucose acid control comprises the following specific steps:
step 1, lactobacillus johnsonii is selected and inoculated in a GYP liquid culture medium, activated and cultured at 37 ℃ for 24 h, the lactobacillus johnsonii is a primary seed culture solution, inoculated in the GYP liquid culture medium according to 10 percent of inoculum size, and cultured at 37 ℃ for 15 h, and the lactobacillus johnsonii is a secondary seed culture solution;
step 2, inoculating the second-level seed culture solution into a GYP liquid culture medium of which the silkworm chrysalis meal replaces peptone according to 10% of inoculation amount, adding 10-50 g/L sodium glutamate and 10-50 g/L glucose, and culturing 24 h in a 5L bioreactor at 37 ℃ and 200 rpm;
step 3, after 24-h fermentation, the thalli reach the logarithmic growth phase, glucose with the flow rate of 0.01-0.05 g/L is fed, the flow rate is 1 mL/min, sodium glutamate with the flow rate of 0.1-0.5 g/L is fed, the flow rate is 1 mL/min, the feeding of glucose and sodium glutamate is stopped after the fermentation is carried out for 54 hours, the fermentation is continued until the fermentation is carried out until the fermentation time reaches 72 h, and the fermentation is stopped;
and 4, part of the culture solution in the fermentation process is centrifuged at 6000 rpm at 4 ℃ to remove thalli and silkworm chrysalis meal, and the pH value and the gamma-aminobutyric acid content of the fermentation supernatant are detected.
As an improvement, in the step 2, 50/g/L of sodium glutamate and 50/g/L of glucose are adopted.
As an improvement, glucose in step 3 was 0.02/g/L and sodium glutamate was 0.2/g/L.
Advantageous effects
Compared with the prior art, the technology for producing the gamma-aminobutyric acid based on the coupling glucose acid control of the silkworm chrysalis powder has the advantages that lactic acid bacteria capable of producing the gamma-aminobutyric acid at high yield are adopted, the production cost of the gamma-aminobutyric acid is reduced by adopting the silkworm chrysalis powder with low cost to replace peptone, the addition proportion of the silkworm chrysalis powder, the concentration of glucose and the concentration of sodium glutamate are optimized, the food-grade fermentation liquor containing the high gamma-aminobutyric acid product is prepared through the coupling glucose acid control technology, the cumulative concentration of the gamma-aminobutyric acid is 229.28 g/L, and the prepared gamma-aminobutyric acid can be applied to food and medicine industrialization and has no potential safety hazard.
Drawings
FIG. 1 shows the effect of silkworm chrysalis meal replacement peptone on gamma-aminobutyric acid production by Lactobacillus johnsonii GZ 2;
FIG. 2 shows the effect of different silkworm chrysalis meal concentrations on gamma-aminobutyric acid production by Lactobacillus his GZ2 instead of peptone;
FIG. 3 (a) is the effect of glucose concentration on fermentation pH after substitution of silkworm chrysalis meal for peptone;
FIG. 3 (b) is a graph showing the effect of glucose concentration on gamma-aminobutyric acid production by Lactobacillus johnsonii GZ2 after substitution of peptone with silkworm chrysalis meal;
FIG. 4 shows the efficient synthesis of gamma-aminobutyric acid by continuous feeding of glucose-controlled acid with silkworm chrysalis meal instead of peptone.
Detailed Description
GYP liquid medium: glucose 1%, yeast powder 1%, peptone 0.5%, sodium acetate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.01%, ferrous sulfate 0.01% and sodium chloride 0.01%. Adding 2% of agar powder into the solid culture medium; the GYP fermentation medium is GYP liquid medium added with sodium glutamate with different concentrations.
The GYP solid slant culture medium is GYP liquid culture medium added with 2% (W/V) agar, and the solid slant is prepared.
GYP1 liquid Medium: glucose 1%, silkworm chrysalis meal 1%, peptone 0.5%, sodium acetate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.01%, ferrous sulfate 0.01%, sodium chloride 0.01%.
GYP2 liquid Medium: glucose 1%, yeast powder 1%, silkworm chrysalis powder 0.5%, sodium acetate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.01%, ferrous sulfate 0.01% and sodium chloride 0.01%.
GYP3 liquid Medium: glucose 1%, silkworm chrysalis meal 1.5%, sodium acetate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.01%, ferrous sulfate 0.01%, and sodium chloride 0.01%.
GYP4 liquid Medium: glucose 1%, yeast powder 1%, silkworm chrysalis powder 0.1%, sodium acetate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.01%, ferrous sulfate 0.01% and sodium chloride 0.01%.
GYP5 liquid Medium: glucose 1%, yeast powder 1%, silkworm chrysalis powder 1.5%, sodium acetate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.01%, ferrous sulfate 0.01% and sodium chloride 0.01%.
GYP6 liquid Medium: glucose 1%, yeast powder 1%, silkworm chrysalis powder 2%, sodium acetate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.01%, ferrous sulfate 0.01% and sodium chloride 0.01%.
GYP7 liquid Medium: glucose 1%, yeast powder 1%, silkworm chrysalis powder 1%, sodium acetate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.01%, ferrous sulfate 0.01% and sodium chloride 0.01%.
The starting strain used in the following examples was Lactobacillus johnsonii GZ2, designated as Lactobacillus johnsonii in the classificationLactobacillus hilgardiiCMCC No.26755, stored in the China general microbiological culture Collection center, at 2023, 03, 06, address of storage: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
Example 1: silkworm chrysalis powder for replacing nitrogen to culture lactobacillus johnsonii to produce gamma-aminobutyric acid
Preparing liquid culture media of GYP1, GYP2 and GYP 3. Selecting a proper amount of lactobacillus johnsonii GZ2 from the activated GYP solid slant culture medium, inoculating the lactobacillus johnsonii GZ2 into a GYP liquid culture medium, and culturing at 37 ℃ and 200 rpm for 24 h; inoculating seed culture solution of 24 h into GYP liquid culture medium according to 10% of inoculum size, and culturing at 37deg.C and 200 rpm for 15 h to obtain fermentation seed; the fermented seeds were inoculated into GYP1, GYP2 and GYP3 liquid medium at a rate of 10% and cultured at 37℃and 200 rpm for 72 h, and each 12: 12 h samples were taken. After fermentation, centrifuging the fermentation broth at 6000 rpm at 4deg.C for 5 min to remove thallus, and detecting gamma-aminobutyric acid content in fermentation supernatant by high performance liquid chromatography. As shown in FIG. 1, the Silkworm chrysalis meal is adopted to replace peptone in GYP liquid culture medium, and the gamma-aminobutyric acid yield is highest and is 14.49 g/L, wherein Yeast extract/Tryptone is 10 g/L Yeast powder/5 g/L peptone, yeast extract/Silkworm is 10 g/L Yeast powder/5 g/L Silkworm chrysalis meal, silkworm/Tryptone is 10 g/L Silkworm chrysalis meal/5 g/L peptone, and Silkworm is 15 g/L Silkworm chrysalis meal.
Example 2: lactobacillus johnsonii GZ2 is used for replacing peptone with glucose to control acid and efficiently fermenting to produce gamma-aminobutyric acid
1. The concentration of silkworm chrysalis powder influences fermentation of lactobacillus johnsonii GZ2 to produce gamma-aminobutyric acid
Selecting a proper amount of lactobacillus johnsonii GZ2 from the activated GYP solid slant culture medium, inoculating the lactobacillus johnsonii GZ2 into a GYP liquid culture medium, and culturing at 37 ℃ and 200 rpm for 24 h; inoculating 24 h seed culture solution according to 10% of inoculum size, and culturing at 37deg.C and 200 rpm for 15 h to obtain fermentation seed; inoculating to liquid culture mediums of GYP2, GYP4, GYP5, GYP6 and GYP7 respectively according to 10% inoculum size, wherein each culture medium contains 50 g/L sodium glutamate, and culturing at 37 ℃ and 200 rpm for 72 h, and sampling every fermentation 12 h. After fermentation, the fermentation broth is centrifuged at 6000 rpm at 4 ℃ to remove thalli, and the gamma-aminobutyric acid content of the fermentation supernatant is detected by high performance liquid chromatography. As shown in FIG. 2, when 10 g/L silkworm chrysalis meal is used for replacing peptone, the yield of gamma-aminobutyric acid is the highest and is 18.81 g/L, and the GYP7 liquid culture medium is used as a fermentation medium for subsequent coupled glucose acid control fermentation.
2. Glucose acid-controlling fermentation production of gamma-aminobutyric acid after silkworm chrysalis meal replaces peptone
Selecting proper amount of lactobacillus johnsonii from the activated GYP solid slant culture medium, inoculating the lactobacillus johnsonii into GYP liquid culture medium, and culturing at 37 ℃ and 200 rpm for 24 h; taking seed culture solution of 24 h, inoculating the seed culture solution into GYP liquid culture medium according to 10% of inoculum size, culturing 15 h as fermentation seeds at 37 ℃ and 200 rpm, and respectively inoculating the seed culture solution into GYP7 liquid culture medium containing 10 g/L-50 g/L glucose according to 10% of inoculum size, culturing 72 h at 37 ℃ and 200 rpm. During the fermentation process, samples were taken every 12 th h, and the pH and gamma-aminobutyric acid content of the fermentation supernatant were measured.
As a result, as shown in FIG. 3 (a), the pH of the fermentation process was continuously increased by 10 to 20 g/L of glucose, the fermentation pH was started to decrease by maintaining 8 to 9, 30 g/L of glucose was added, and finally 6 was maintained, while as 50 g/L of glucose was added, the fermentation pH was started to decrease from 24 h, and finally 5 was maintained, indicating that control of fermentation pH was achieved by adding glucose, and as shown in FIG. 3 (b), the yield of gamma-aminobutyric acid was increased by increasing with increasing glucose, and as 50 g/L of glucose was added, 50 g/L of sodium glutamate was added, and the yield of gamma-aminobutyric acid was the highest and was 31.22 g/L.
3. Lactobacillus johnsonii GZ2 is used for synthesizing gamma-aminobutyric acid by using silkworm chrysalis powder to replace peptone coupled glucose to control acid and fed-batch fermentation
Inoculating activated Lactobacillus shigella GZ2 on an inclined plane into a GYP liquid culture medium, culturing at 37 ℃ for 24 h to prepare a first seed culture solution, culturing the first seed culture solution in the GYP seed culture medium at 37 ℃ and 200 rpm for 15 h according to 10% of inoculation amount to prepare a second seed solution, inoculating the second seed solution in the GYP7 liquid culture medium containing 50 g/L sodium glutamate and 50 g/L glucose according to 10% of inoculation amount, controlling the temperature to 37 ℃ by using a 5L bioreactor, and stirring at 200 rpm. After 24-h is fermented, the strain grows stably, glucose is continuously added at a flow rate of 1 mL/min for 24-54 h by adopting 0.02-g/mL glucose, so as to control the fermentation pH, and meanwhile, a sodium glutamate substrate is continuously added at a flow rate of 1-mL/min for 24-54 h by adopting 0.2-g/mL, so that 72-h is fermented. In the fermentation process, sampling is carried out every 6 h, and the concentration OD of the thalli is detected 600 pH and gamma-aminobutyric acid content of fermentation supernatant.
As shown in FIG. 4, after glucose is fed in, the growth activity of the strain is maintained, and meanwhile, the fermentation pH is maintained at about 5 all the time, so that the activity of glutamate decarboxylase is maintained high, the fermentation of lactobacillus johnsonii GZ2 is enhanced to produce gamma-aminobutyric acid, the fermentation is carried out for 72 h, and the accumulated concentration of the gamma-aminobutyric acid is 229.27 g/L.
Claims (5)
1. A technology for producing gamma-aminobutyric acid based on coupling of silkworm chrysalis powder and glucose for acid control is characterized in that gamma-aminobutyric acid producing bacteria are used as starting strains, low-cost silkworm chrysalis powder is used for replacing high-cost peptone, and fermentation pH is controlled by feeding low-concentration glucose.
2. The process for producing gamma-aminobutyric acid based on silkworm chrysalis powder coupled glucose acid control as claimed in claim 1, wherein the starting strain is lactobacillus johnsonii @Lactobacillus hilgardii)。
3. The process for producing gamma-aminobutyric acid based on silkworm chrysalis meal coupled glucose acid control as claimed in claim 2, comprising the following steps:
step 1, lactobacillus johnsonii is selected and inoculated in a GYP liquid culture medium, activated and cultured at 37 ℃ for 24 h, the lactobacillus johnsonii is a primary seed culture solution, inoculated in the GYP liquid culture medium according to 10 percent of inoculum size, and cultured at 37 ℃ for 15 h, and the lactobacillus johnsonii is a secondary seed culture solution;
step 2, inoculating the second-level seed culture solution into a GYP liquid culture medium of which the silkworm chrysalis meal replaces peptone according to 10% of inoculation amount, adding 10-50 g/L sodium glutamate and 10-50 g/L glucose, and culturing 24 h in a 5L bioreactor at 37 ℃ and 200 rpm;
step 3, after 24-h fermentation, the thalli reach the logarithmic growth phase, glucose with the flow rate of 0.01-0.05 g/L is fed, the flow rate is 1 mL/min, sodium glutamate with the flow rate of 0.1-0.5 g/L is fed, the flow rate is 1 mL/min, the feeding of glucose and sodium glutamate is stopped after the fermentation is carried out for 54 hours, the fermentation is continued until the fermentation is carried out until the fermentation time reaches 72 h, and the fermentation is stopped;
and 4, part of the culture solution in the fermentation process is centrifuged at 6000 rpm at 4 ℃ to remove thalli and silkworm chrysalis meal, and the pH value and the gamma-aminobutyric acid content of the fermentation supernatant are detected.
4. The process for producing gamma-aminobutyric acid based on silkworm chrysalis meal coupled glucose acid control as claimed in claim 3, wherein in the step 2, sodium glutamate is 50 g/L and glucose is 50 g/L.
5. The process for producing gamma-aminobutyric acid based on silkworm chrysalis meal coupled glucose acid control as claimed in claim 3, wherein glucose is 0.02 g/L and sodium glutamate is 0.2 g/L in step 3.
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