CN113832053B - Fermentation method of polyglutamic acid - Google Patents
Fermentation method of polyglutamic acid Download PDFInfo
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- CN113832053B CN113832053B CN202111062120.0A CN202111062120A CN113832053B CN 113832053 B CN113832053 B CN 113832053B CN 202111062120 A CN202111062120 A CN 202111062120A CN 113832053 B CN113832053 B CN 113832053B
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- 238000000855 fermentation Methods 0.000 title claims abstract description 121
- 230000004151 fermentation Effects 0.000 title claims abstract description 121
- 229920002643 polyglutamic acid Polymers 0.000 title claims abstract description 31
- 108010020346 Polyglutamic Acid Proteins 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 25
- 230000001954 sterilising effect Effects 0.000 claims abstract description 20
- 239000007788 liquid Substances 0.000 claims abstract description 13
- 238000001035 drying Methods 0.000 claims abstract description 4
- 238000000605 extraction Methods 0.000 claims abstract description 4
- 239000002609 medium Substances 0.000 claims description 41
- 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 claims description 28
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 28
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims description 28
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims description 28
- 238000004659 sterilization and disinfection Methods 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 claims description 14
- 229960002685 biotin Drugs 0.000 claims description 14
- 235000020958 biotin Nutrition 0.000 claims description 14
- 239000011616 biotin Substances 0.000 claims description 14
- 229960000304 folic acid Drugs 0.000 claims description 14
- 235000019152 folic acid Nutrition 0.000 claims description 14
- 239000011724 folic acid Substances 0.000 claims description 14
- 229960003966 nicotinamide Drugs 0.000 claims description 14
- 235000005152 nicotinamide Nutrition 0.000 claims description 14
- 239000011570 nicotinamide Substances 0.000 claims description 14
- 235000001968 nicotinic acid Nutrition 0.000 claims description 14
- 239000011664 nicotinic acid Substances 0.000 claims description 14
- 229960003512 nicotinic acid Drugs 0.000 claims description 14
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 13
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 13
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 claims description 12
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 12
- 235000019743 Choline chloride Nutrition 0.000 claims description 12
- 240000008042 Zea mays Species 0.000 claims description 12
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 12
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 12
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 12
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 12
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 12
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 claims description 12
- 229960002079 calcium pantothenate Drugs 0.000 claims description 12
- 229960003178 choline chloride Drugs 0.000 claims description 12
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 claims description 12
- 235000005822 corn Nutrition 0.000 claims description 12
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims description 12
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 12
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 12
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 12
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 claims description 12
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 12
- 150000003839 salts Chemical class 0.000 claims description 12
- 229940073490 sodium glutamate Drugs 0.000 claims description 12
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 10
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 10
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 9
- YRMLFORXOOIJDR-UHFFFAOYSA-N Dichlormid Chemical compound ClC(Cl)C(=O)N(CC=C)CC=C YRMLFORXOOIJDR-UHFFFAOYSA-N 0.000 claims description 8
- 238000004090 dissolution Methods 0.000 claims description 8
- 239000011574 phosphorus Substances 0.000 claims description 8
- 229910052698 phosphorus Inorganic materials 0.000 claims description 8
- 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 7
- 239000008103 glucose Substances 0.000 claims description 7
- 230000001502 supplementing effect Effects 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims 1
- 239000003337 fertilizer Substances 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 238000007630 basic procedure Methods 0.000 description 2
- 235000010633 broth Nutrition 0.000 description 2
- FAPWYRCQGJNNSJ-CTWWJBIBSA-L calcium;3-[[(2s)-2,4-dihydroxy-3,3-dimethylbutanoyl]amino]propanoate Chemical compound [Ca+2].OCC(C)(C)[C@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-CTWWJBIBSA-L 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000009920 chelation Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000013557 nattō Nutrition 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000003175 pesticide synergist Substances 0.000 description 1
- 108700022290 poly(gamma-glutamic acid) Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
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- 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
- 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
- C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
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- Life Sciences & Earth Sciences (AREA)
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- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a fermentation method of polyglutamic acid, which comprises the following steps: preparing a basic fermentation medium; sterilizing the basic fermentation medium; inoculating the seed liquid into a basic fermentation medium for fermentation culture to obtain fermentation liquid; performing filter pressing extraction on the fermentation liquor, and drying to obtain polyglutamic acid; wherein, the fermentation conditions are as follows: the pH is 6.5 plus or minus 0.05, the temperature is 34 plus or minus 0.5 ℃, and the fermentation time is 80-120 hours. The fermentation liquor obtained by the fermentation method has high polyglutamic acid content, and the specific content can reach 58-70g/L.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a fermentation method of polyglutamic acid.
Background
Polyglutamic acid (gamma-PGA), also known as natto gum and polyglutamic acid, is a water-soluble, biodegradable, non-toxic biopolymer produced by microbial fermentation. A large number of free carboxyl groups on the main chain of the polyglutamic acid molecule can carry out crosslinking, chelation, derivatization and other reactions. According to research reports, polyglutamic acid can be used as fertilizer, pesticide synergist, water-retaining agent, heavy metal ion adsorbent, flocculant, slow release agent, drug carrier and the like. Polyglutamic acid has been widely used in recent years due to its special effect on agricultural fertilizers. Mainly has the advantages of water and fertilizer retention, fertilizer utilization rate improvement, crop growth promotion, stress resistance and yield increase, fruit quality improvement, soil pH value adjustment and the like.
However, in terms of the current industry of the fermentation level of polyglutamic acid, the content of the fermented product is relatively low, and therefore, it is very important to provide a fermentation method of polyglutamic acid with high content.
Disclosure of Invention
In view of the above problems, an object of the present invention is to: a fermentation method of polyglutamic acid with high content is provided.
In order to achieve the above object, the present invention provides the following technical solutions:
A fermentation method of polyglutamic acid, comprising the steps of:
preparing a basic fermentation medium;
sterilizing the basic fermentation medium;
inoculating the seed liquid into a basic fermentation medium for fermentation culture to obtain fermentation liquid;
Performing filter pressing extraction on the fermentation liquor, and drying to obtain polyglutamic acid;
wherein, the fermentation conditions are as follows: the pH is 6.5 plus or minus 0.05, the temperature is 34 plus or minus 0.5 ℃, and the fermentation time is 80-120 hours.
Preferably, the basal fermentation medium comprises the following components:
13-14g/L glucose, 4-5g/L corn steep liquor dry powder, 3-4g/L ammonium sulfate, 3-4g/L sodium glutamate, 0.5-0.6g/L monopotassium phosphate, 1.5-1.6g/L ferrous sulfate heptahydrate, 14-15g/L magnesium sulfate heptahydrate, 2-3g/L industrial salt, 0.06-0.08g/L manganese sulfate monohydrate, 0.08-0.10g/L anhydrous calcium chloride and 0.003-0.004g/L choline chloride.
Preferably, the basal fermentation medium comprises the following components:
13.5g/L glucose, 4.44g/L corn steep liquor dry powder, 3.33g/L ammonium sulfate, 3.33g/L sodium glutamate, 0.56g/L potassium dihydrogen phosphate, 1.56g/L ferrous sulfate heptahydrate, 14.44g/L magnesium sulfate heptahydrate, 2.89g/L industrial salt, 0.07g/L manganese sulfate monohydrate, 0.09g/L anhydrous calcium chloride and 0.0033g/L choline chloride.
Preferably, the basal fermentation medium further comprises the following components:
VB 1.008-0.009 g/L, VB 2.008-0.009 g/L, VB 6.0.0014-0.0015 g/L, nicotinic acid 0.009-0.010g/L, nicotinamide 0.009-0.010g/L, folic acid 0.0004-0.0005g/L, calcium pantothenate 0.0001-0.0002g/L, biotin 0.0002-0.0003g/L and dichlormid 0.17-0.019mL/L.
Preferably, the basal fermentation medium further comprises the following components:
VB1 0.00807g/L, VB2 0.00807g/L, VB6 0.00148g/L, nicotinic acid 0.00917g/L, nicotinamide 0.00917g/L, folic acid 0.00044g/L, calcium pantothenate 0.00015g/L, biotin 0.00029g/L and dichlormid 0.18mL/L.
Preferably, when the basal fermentation medium is sterilized, the sterilization conditions are as follows:
the sterilization temperature is 121-125deg.C, the sterilization time is 25-35min, and the sterilization pressure is 0.5-0.8bar.
Preferably, after 8 hours of fermentation culture, adding sugar water; the specific additional process is as follows:
fermenting for 8-20 hours: the sugar concentration in the fermentation liquor is controlled to be 10-15g/L; after 20 hours of fermentation: the sugar concentration in the fermentation liquor is controlled to be 4-8g/L.
Preferably, after 2 hours of fermentation culture, adding phosphorus supplementing water, and controlling the phosphorus dissolution in the fermentation liquor to be 100-150mg/L.
Preferably, a feed fermentation medium is also added in the fermentation process, and the feed fermentation medium comprises the following components:
19-21g/L of corn steep liquor dry powder, 4.9-5.1g/L of ammonium sulfate, 4.9-5.1g/L of sodium glutamate, 1.9-2.1g/L of monopotassium phosphate, 1.4-1.6g/L of ferrous sulfate heptahydrate, 17.4-17.6g/L of magnesium sulfate heptahydrate, 3.9-4.1g/L of industrial salt, 0.074-0.076g/L of manganese sulfate monohydrate, 0.1-0.2g/L of anhydrous calcium chloride, 0.0037-0.0038g/L of choline chloride and 0.9-1.1mL/L of dichlord;
VB 1.24-0.25 g/L, VB 2.0009-0.0010 g/L, VB 6.0.073-0.074 g/L, nicotinic acid 0.29-0.30g/L, nicotinamide 0.29-0.30g/L, folic acid 0.0004-0.0006g/L, calcium pantothenate 0.0001-0.0002g/L and biotin 0.0069-0.0070g/L.
Preferably, a feed fermentation medium is also added in the fermentation process, and the feed fermentation medium comprises the following components:
Corn steep liquor dry powder 20g/L, ammonium sulfate 5.0g/L, sodium glutamate 5.0g/L, monopotassium phosphate 2.0g/L, ferrous sulfate heptahydrate 1.5g/L, magnesium sulfate heptahydrate 17.5g/L, industrial salt 4.0g/L, manganese sulfate monohydrate 0.075g/L, anhydrous calcium chloride 0.1g/L, choline chloride 0.00375g/L, and dichlormid 1mL/L;
VB 1.243 g/L, VB 2.00098 g/L, VB 6. 6 0.0735g/L, nicotinic acid 0.295g/L, nicotinamide 0.295g/L, folic acid 0.0005g/L, calcium pantothenate 0.00019g/L and biotin 0.00698g/L.
The invention has the beneficial effects that:
the fermentation liquor obtained by the fermentation method has high polyglutamic acid content, and the specific content can reach 58-70g/L.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A fermentation method of polyglutamic acid, comprising the steps of:
s1: preparing basic fermentation culture medium
The amounts and ratios of the basal fermentation medium are shown in tables 1 and 2 below:
TABLE 1 addition amount and ratio of main materials in basic fermentation Medium
TABLE 2 addition and formulation of auxiliary liquid in basic fermentation Medium
| Name of the name | Standard quantity | Proportioning of |
| VB1 | 322.8g | 0.00807g/L |
| VB2 | 32.28g | 0.000807g/L |
| VB6 | 59.2g | 0.00148g/L |
| Nicotinic acid | 366.8g | 0.00917g/L |
| Nicotinamide | 366.8g | 0.00917g/L |
| Folic acid | 17.6g | 0.00044g/L |
| Calcium pantothenate | 6.0g | 0.00015g/L |
| Biotin | 11.6g | 0.00029g/L |
S2: sterilizing the basic fermentation medium
The sterilization conditions were as follows: the sterilization temperature is 121-125 ℃, the sterilization time is 25-35min, the sterilization pressure is 0.5-0.8bar, and preferably, the following sterilization conditions are selected: the sterilization temperature is 123 ℃, the sterilization time is 30min, and the sterilization pressure is 0.7bar.
S3: inoculating seed liquid (here, bacillus subtilis secondary seed liquid capable of producing polyglutamic acid) into a basic fermentation medium for fermentation culture to obtain fermentation liquid, wherein the specific process is as follows:
(1) Raising the pressure of the seed tank to about 1.5-2bar, exhausting steam in the seed transfer pipe, opening various valves of the seed transfer pipe, introducing seed liquid into the fermentation tank, and recording time.
(2) Closing a seed transferring valve of the fermentation tank, closing a bottom valve of the seed tank and an air inlet valve, opening an air outlet valve of the seed tank, reducing the pressure to zero, opening an air outlet valve of a seed transferring pipeline, and flushing the pipeline for 5 minutes by steam.
(3) The cover of the seed tank is opened, the tank body is flushed by drinking water, the bottom valve and the sampling valve are checked by compressed air to see whether leakage exists or not, the seed tank is soaked by water for standby after normal use, and abnormal conditions are found to be treated in time and recorded.
(4) The air flow rate of the fermentation tank is regulated to 1300-1700 Nm 3/h, the temperature is 34+/-0.5 ℃, the tank pressure is 0.3+/-0.05 bar, and the rotating speed opening degree is: 78, the pH value is 6.5 plus or minus 0.05, and the culture is started for 80 to 120 hours;
Fermentation termination conditions: the bacterial body becomes light in dyeing, part of hyphae is autolyzed, the titer is slowly increased, and the fermentation can be stopped.
Specifically, the material is required to be fed in the fermentation process, and the specific feeding mode is as follows:
Sugar supplement
After fermentation culture for 8 hours, adding sugar supplementing water, and fermenting for 8-20 hours: the sugar concentration in the fermentation liquor is controlled to be 10-15g/L; after 20 hours of fermentation: the sugar concentration in the fermentation liquor is controlled to be 4-8g/L.
The sugar water is prepared by the following steps:
1) Feed amount and ratio
TABLE 3 dosage and formulation of sugar water
2) Operation method and procedure
The lid of the dissolution tank was opened, 7260kg glucose was added to the dissolution tank, and water was added for dissolution, the total volume was 13.2T, and the sugar water concentration was 55%.
Phosphorus supplementing water
After 2 hours of fermentation culture, adding phosphorus supplementing water, and controlling the phosphorus dissolution in the fermentation liquor at 100-150mg/L.
Wherein, the configuration process of the phosphor water is as follows:
1) Feed amount and ratio
TABLE 4 phosphorus Water dosage and formulation
2) Operation method and procedure
The lid of the dissolution tank was opened, 1333.360kg of potassium dihydrogen phosphate was added to the dissolution tank, and 0.4kg of VB1 was dissolved in water to give a total volume of 7200L.
Complement material
The fermentation process also needs to add a feed-supplementing fermentation medium, and the feed-supplementing medium can be supplemented for three times after 55 hours of fermentation according to actual needs.
The main components of the feed fermentation medium are shown in tables 5 and 6 below:
TABLE 5 feeding amount and ratio of the main materials of the fed-batch fermentation medium
TABLE 6 addition and formulation of the supplemented fermentation Medium auxiliary solution
| Name of the name | Standard quantity | Proportioning of |
| VB1 | 388.8g | 0.243g/L |
| VB2 | 1.568g | 0.00098g/L |
| VB6 | 117.6g | 0.0735g/L |
| Nicotinic acid | 472g | 0.295g/L |
| Nicotinamide | 472g | 0.295g/L |
| Folic acid | 0.8g | 0.0005g/L |
| Calcium pantothenate | 0.304g | 0.00019g/L |
| Biotin | 11.168g | 0.00698g/L |
S4: performing filter pressing extraction on the fermentation liquor, and drying to obtain polyglutamic acid;
After fermentation is finished, the fermentation tank stops ventilation, the pH value is regulated to 4.0+/-0.5 by hydrochloric acid, the temperature is raised to 76-80 ℃, the temperature is not kept, the fermentation liquid is immediately cooled to about 70 ℃, the fermentation liquid is put into a plate-frame press filter for press filtration, mycelium is collected, and the mycelium is dried and powdered.
Example 2
Example 2 is identical to the basic procedure of example 1, except that:
The basic fermentation medium and the feed fermentation medium are different in component ratio, and the specific component ratio of the basic fermentation medium and the feed fermentation medium in the embodiment is as follows:
The basic fermentation medium comprises the following components in percentage by weight:
13g/L of oral glucose, 4g/L of corn steep liquor dry powder, 3g/L of ammonium sulfate, 3g/L of sodium glutamate, 0.5g/L of monopotassium phosphate, 1.5g/L of ferrous sulfate heptahydrate, 14g/L of magnesium sulfate heptahydrate, 2g/L of industrial salt, 0.06g/L of manganese sulfate monohydrate, 0.08g/L of anhydrous calcium chloride and 0.003g/L of choline chloride;
VB 1.008 g/L, VB 2.008 g/L, VB 6.0.0014 g/L, nicotinic acid 0.009g/L, nicotinamide 0.009g/L, folic acid 0.0004g/L, calcium pantothenate 0.0001g/L, biotin 0.0002g/L and bufomide 0.17mL/L.
The feed fermentation medium comprises the following components in percentage by weight:
corn steep liquor dry powder 19g/L, ammonium sulfate 4.9g/L, sodium glutamate 4.9g/L, monopotassium phosphate 1.9g/L, ferrous sulfate heptahydrate 1.4g/L, magnesium sulfate heptahydrate 17.4g/L, industrial salt 3.9g/L, manganese sulfate monohydrate 0.074g/L, anhydrous calcium chloride 0.1g/L, choline chloride 0.0037g/L and dichlormid 0.9mL/L;
VB 1.24 g/L, VB g/L, VB 6.0009 g/3924.0.073 g/L, nicotinic acid 0.29g/L, nicotinamide 0.29g/L, folic acid 0.0004g/L, calcium pantothenate 0.0001g/L and biotin 0.0069g/L.
Example 3
Example 3 is identical to the basic procedure of example 1, except that:
The basic fermentation medium and the feed fermentation medium are different in component ratio, and the specific component ratio of the basic fermentation medium and the feed fermentation medium in the embodiment is as follows:
The basic fermentation medium comprises the following components in percentage by weight:
14g/L of oral glucose, 5g/L of corn steep liquor dry powder, 4g/L of ammonium sulfate, 4g/L of sodium glutamate, 0.6g/L of monopotassium phosphate, 1.6g/L of ferrous sulfate heptahydrate, 15g/L of magnesium sulfate heptahydrate, 3g/L of industrial salt, 0.08g/L of manganese sulfate monohydrate, 0.10g/L of anhydrous calcium chloride and 0.004g/L of choline chloride;
VB 1.009 g/L, VB g/L, VB 6.0.009 g/3924.0.0015 g/L, nicotinic acid 0.010g/L, nicotinamide 0.010g/L, folic acid 0.0005g/L, calcium pantothenate 0.0002g/L, biotin 0.0003g/L and bufomesangial 0.019mL/L.
The feed fermentation medium comprises the following components in percentage by weight:
Corn steep liquor dry powder 21g/L, ammonium sulfate 5.1g/L, sodium glutamate 5.1g/L, monopotassium phosphate 2.1g/L, ferrous sulfate heptahydrate 1.6g/L, magnesium sulfate heptahydrate 17.6g/L, industrial salt 4.1g/L, manganese sulfate monohydrate 0.076g/L, anhydrous calcium chloride 0.2g/L, choline chloride 0.0038g/L and dichlormid 1.1mL/L;
VB 1.25 g/L, VB g/L, VB 6.0.0010 g/3924.0.074 g/L, nicotinic acid 0.30g/L, nicotinamide 0.30g/L, folic acid 0.0006g/L, calcium pantothenate 0.0002g/L and biotin 0.0070g/L.
The polyglutamic acid content of the fermentation broths of examples 1-3 was measured and the results are shown in Table 7 below:
TABLE 7 polyglutamic acid content in fermentation broths
| Project | Polyglutamic acid content |
| Example 1 | 62.8g/L |
| Example 2 | 70.2g/L |
| Example 3 | 65.4g/L |
As can be seen from table 7: the content of polyglutamic acid in the fermentation liquor obtained by the fermentation method is high and can reach 70.2g/L at most.
Although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (5)
1. A fermentation method of polyglutamic acid is characterized in that: the method comprises the following steps:
Preparing a basic fermentation medium; the basic fermentation medium comprises the following components: 13-14g/L glucose, 4-5g/L corn steep liquor dry powder, 3-4g/L ammonium sulfate, 3-4g/L sodium glutamate, 0.5-0.6g/L monopotassium phosphate, 1.5-1.6g/L ferrous sulfate heptahydrate, 14-15g/L magnesium sulfate heptahydrate, 2-3g/L industrial salt, 0.06-0.08g/L manganese sulfate monohydrate, 0.08-0.10g/L anhydrous calcium chloride and 0.003-0.004g/L choline chloride; VB 1.008-0.009 g/L, VB 2.008-0.009 g/L, VB 6.0.0014-0.0015 g/L, nicotinic acid 0.009-0.010g/L, nicotinamide 0.009-0.010g/L, folic acid 0.0004-0.0005g/L, calcium pantothenate 0.0001-0.0002g/L, biotin 0.0002-0.0003g/L and dichlormid 0.17-0.019mL/L;
sterilizing the basic fermentation medium;
Inoculating the seed liquid into a basic fermentation medium for fermentation culture to obtain fermentation liquid; after 2 hours of fermentation culture, adding phosphorus supplementing water, wherein the phosphorus dissolution in the fermentation liquor is controlled at 100-150mg/L; after fermenting and culturing for 8 hours, adding sugar water; the specific additional process is as follows: fermenting for 8-20 hours: the sugar concentration in the fermentation liquor is controlled to be 10-15g/L; after 20 hours of fermentation: the sugar concentration in the fermentation liquor is controlled to be 4-8g/L; and a feed fermentation medium is also added in the fermentation process, and comprises the following components: 19-21g/L of corn steep liquor dry powder, 4.9-5.1g/L of ammonium sulfate, 4.9-5.1g/L of sodium glutamate, 1.9-2.1g/L of monopotassium phosphate, 1.4-1.6g/L of ferrous sulfate heptahydrate, 17.4-17.6g/L of magnesium sulfate heptahydrate, 3.9-4.1g/L of industrial salt, 0.074-0.076g/L of manganese sulfate monohydrate, 0.1-0.2g/L of anhydrous calcium chloride, 0.0037-0.0038g/L of choline chloride and 0.9-1.1mL/L of dichlord; VB 1.24-0.25 g/L, VB 2.0009-0.0010 g/L, VB 6.0.073-0.074 g/L, nicotinic acid 0.29-0.30g/L, nicotinamide 0.29-0.30g/L, folic acid 0.0004-0.0006g/L, calcium pantothenate 0.0001-0.0002g/L and biotin 0.0069-0.0070g/L;
Performing filter pressing extraction on the fermentation liquor, and drying to obtain polyglutamic acid;
wherein, the fermentation conditions are as follows: the pH is 6.5 plus or minus 0.05, the temperature is 34 plus or minus 0.5 ℃, and the fermentation time is 80-120 hours.
2. The fermentation method of polyglutamic acid according to claim 1, wherein: the basic fermentation culture medium is divided into the following parts:
13.5g/L glucose, 4.44g/L corn steep liquor dry powder, 3.33g/L ammonium sulfate, 3.33g/L sodium glutamate, 0.56g/L potassium dihydrogen phosphate, 1.56g/L ferrous sulfate heptahydrate, 14.44g/L magnesium sulfate heptahydrate, 2.89g/L industrial salt, 0.07g/L manganese sulfate monohydrate, 0.09g/L anhydrous calcium chloride and 0.0033g/L choline chloride.
3. The fermentation method of polyglutamic acid according to claim 1, wherein: the basic fermentation culture medium is divided into the following parts:
VB1 0.00807g/L, VB2 0.00807g/L, VB6 0.00148g/L, nicotinic acid 0.00917g/L, nicotinamide 0.00917g/L, folic acid 0.00044g/L, calcium pantothenate 0.00015g/L, biotin 0.00029g/L and dichlormid 0.18mL/L.
4. The fermentation method of polyglutamic acid according to claim 1, wherein: when the basic fermentation medium is sterilized, the sterilization conditions are as follows:
the sterilization temperature is 121-125deg.C, the sterilization time is 25-35min, and the sterilization pressure is 0.5-0.8bar.
5. The fermentation method of polyglutamic acid according to claim 1, wherein: and a feed fermentation medium is also added in the fermentation process, and comprises the following components:
Corn steep liquor dry powder 20g/L, ammonium sulfate 5.0g/L, sodium glutamate 5.0g/L, monopotassium phosphate 2.0g/L, ferrous sulfate heptahydrate 1.5g/L, magnesium sulfate heptahydrate 17.5g/L, industrial salt 4.0g/L, manganese sulfate monohydrate 0.075g/L, anhydrous calcium chloride 0.1g/L, choline chloride 0.00375g/L, and dichlormid 1mL/L;
VB 1.243 g/L, VB 2.00098 g/L, VB 6. 6 0.0735g/L, nicotinic acid 0.295g/L, nicotinamide 0.295g/L, folic acid 0.0005g/L, calcium pantothenate 0.00019g/L and biotin 0.00698g/L.
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