CN116855550A - Method for producing sodium gluconate by enzyme method - Google Patents
Method for producing sodium gluconate by enzyme method Download PDFInfo
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 54
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 54
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 32
- AEQDJSLRWYMAQI-UHFFFAOYSA-N 2,3,9,10-tetramethoxy-6,8,13,13a-tetrahydro-5H-isoquinolino[2,1-b]isoquinoline Chemical compound C1CN2CC(C(=C(OC)C=C3)OC)=C3CC2C2=C1C=C(OC)C(OC)=C2 AEQDJSLRWYMAQI-UHFFFAOYSA-N 0.000 title claims abstract description 29
- 239000000176 sodium gluconate Substances 0.000 title claims abstract description 29
- 235000012207 sodium gluconate Nutrition 0.000 title claims abstract description 29
- 229940005574 sodium gluconate Drugs 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000000855 fermentation Methods 0.000 claims abstract description 105
- 230000004151 fermentation Effects 0.000 claims abstract description 105
- 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 abstract description 86
- 239000008103 glucose Substances 0.000 claims abstract description 86
- 229940088598 enzyme Drugs 0.000 claims abstract description 58
- 235000019420 glucose oxidase Nutrition 0.000 claims abstract description 45
- 102000016938 Catalase Human genes 0.000 claims abstract description 44
- 108010053835 Catalase Proteins 0.000 claims abstract description 44
- 108010015776 Glucose oxidase Proteins 0.000 claims abstract description 36
- 239000004366 Glucose oxidase Substances 0.000 claims abstract description 36
- 229940116332 glucose oxidase Drugs 0.000 claims abstract description 36
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 24
- 238000006911 enzymatic reaction Methods 0.000 claims abstract description 9
- 230000000694 effects Effects 0.000 claims description 39
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 36
- 240000008042 Zea mays Species 0.000 claims description 28
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 28
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 28
- 235000005822 corn Nutrition 0.000 claims description 28
- 230000001580 bacterial effect Effects 0.000 claims description 27
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 22
- 239000002002 slurry Substances 0.000 claims description 20
- -1 iron ion Chemical class 0.000 claims description 15
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 14
- 229910052742 iron Inorganic materials 0.000 claims description 14
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 13
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 13
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 13
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 13
- 230000004763 spore germination Effects 0.000 claims description 13
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 11
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 11
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 11
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 11
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 11
- 235000015097 nutrients Nutrition 0.000 claims description 5
- 150000003839 salts Chemical class 0.000 claims description 5
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 claims description 3
- 229910001447 ferric ion Inorganic materials 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 2
- 230000002255 enzymatic effect Effects 0.000 claims 3
- 239000000126 substance Substances 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 25
- 241001052560 Thallis Species 0.000 abstract description 2
- 229910021645 metal ion Inorganic materials 0.000 abstract description 2
- 230000001954 sterilising effect Effects 0.000 description 44
- 230000001276 controlling effect Effects 0.000 description 27
- 230000000052 comparative effect Effects 0.000 description 20
- 238000001816 cooling Methods 0.000 description 15
- 239000007788 liquid Substances 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 239000012526 feed medium Substances 0.000 description 9
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 3
- 229910001448 ferrous ion Inorganic materials 0.000 description 3
- 229910001414 potassium ion Inorganic materials 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000012459 cleaning agent Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- BITYAPCSNKJESK-UHFFFAOYSA-N potassiosodium Chemical compound [Na].[K] BITYAPCSNKJESK-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000006052 feed supplement Substances 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000004381 surface treatment Methods 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/58—Aldonic, ketoaldonic or saccharic acids
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- 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
- C12N1/14—Fungi; Culture media therefor
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- 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
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
- C12R2001/685—Aspergillus niger
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Abstract
The invention discloses a method for producing sodium gluconate by an enzymatic method, which relates to the technical field of biochemical industry, and is characterized in that catalase, glucose oxidase and aspergillus niger strains are added into glucose fermentation liquor for fermentation, glucose is converted into sodium gluconate by means of two enzymes in the early stage of fermentation, and meanwhile, feeding and feeding are carried out to promote the rapid growth of aspergillus niger thalli, and the metal ion feeding rate is controlled by detecting the glucose consumption rate so as to realize the mass production of enzymes by hyphae; and adding a small amount of catalase and glucose oxidase again at the end of fermentation to realize efficient completion of conversion. The invention utilizes the advantages of the double-enzyme method and the strain fermentation to realize the rapid conversion at the initial stage and the final stage of the fermentation, controls the fermentation time within 15 hours, and improves the production efficiency; meanwhile, the addition amount of glucose oxidase and catalase is reduced, and the production cost is greatly reduced.
Description
Technical Field
The invention relates to the technical field of biochemical engineering, in particular to a method for producing sodium gluconate by an enzymatic method.
Background
Sodium gluconate is polyhydroxy sodium carboxylate, has very wide industrial application, can be applied to industries such as building, textile printing and dyeing, metal surface treatment, water treatment and the like, is used as a high-efficiency chelating agent, a steel surface cleaning agent, a glass cleaning agent and the like, and can be used as a high-efficiency retarder in the concrete industry.
At present, the production method of sodium gluconate mainly comprises a microbial fermentation method and a double-enzyme method. The microbial fermentation method mainly adopts Aspergillus niger for fermentation, and has mild reaction conditions and lower cost; however, the Aspergillus niger strain is transferred from the flat plate to the fermentation tank and needs to be cultured by the seed tank, the process is complicated, the required time is about 40 hours, and the production efficiency of sodium gluconate is seriously influenced; in the initial stage of fermentation, the conversion efficiency of glucose into sodium gluconate is lower due to the low enzyme activity produced by the aspergillus niger; in the fermentation ending stage, as the glucose content in the fermentation tank is low, the combination ability of enzyme and glucose is reduced, and the fermentation ending time is prolonged; compared with the fermentation method, the method for producing the sodium gluconate by the enzyme method does not need seed culture, and the fermentation time is about 20 hours, but the cost of the enzyme is higher, so that the large-scale utilization of the enzyme is restricted.
Therefore, it is a problem to be solved by those skilled in the art to provide a method for producing sodium gluconate which saves the addition amount of enzyme, reduces the production cost and improves the production rate.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a method for producing sodium gluconate by an enzyme method, which utilizes the advantages of a double-enzyme method and strain fermentation to realize rapid conversion at the initial stage and the final stage of fermentation, controls the fermentation time within 15 hours and improves the production efficiency; meanwhile, the addition amount of glucose oxidase and catalase is reduced, and the production cost is greatly reduced.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for producing sodium gluconate by enzyme method comprises adding catalase, glucose oxidase and Aspergillus niger strain into glucose fermentation liquid for fermentation;
wherein the addition amount of the catalase is 0.8g/kg of dry matter, the addition amount of the glucose oxidase is 0.6g/kg, the inoculation amount of the Aspergillus niger strain is 3-5% of the volume of the glucose fermentation liquor, and the total spore number is more than or equal to 2 x 10 8 And each.
In the early fermentation stage, glucose is converted into sodium gluconate by means of two enzymes, the feeding flow acceleration rate is adjusted by detecting the bacterial concentration increase rate in the period to promote the rapid growth of aspergillus niger, and the metal ion feeding rate is controlled by detecting the glucose consumption rate to realize the mass enzyme production of hyphae; and adding a small amount of catalase and glucose oxidase again at the end of fermentation to realize efficient completion of conversion.
Preferably, the dry matter is the mass of glucose in the fermentation broth.
Preferably, the glucose fermentation broth comprises glucose solution and nutrient salt;
wherein the concentration of glucose in the glucose solution is 300-320g/L;
the nutrient salt comprises potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate and ammonium sulfate, the addition amounts are 3-5g/L, 9-15g/L, 4g/L and 12g/L respectively, and the mass ratio of the potassium dihydrogen phosphate to the disodium hydrogen phosphate is 1:2-3;
the pH value of the glucose fermentation liquor is 6.0-6.2.
Preferably, the fermentation conditions are:
stage before spore germination: the temperature is 39 ℃, the rotating speed is 50-100rpm, the air quantity is 0.03-0.05wwm, the pressure is 0.1-0.12MPa, and the PH is 5.2-5.4;
spore germination and strain growth stage: the temperature is 39 ℃, the rotating speed is 200-300rpm, the air quantity is 0.09-0.1vvm, the pressure is 0.1-0.12MPa, and the PH is 5.2-5.4.
Preferably, the fermentation process further comprises a feeding step, wherein the feeding step comprises corn slurry and ferric ion solution.
Preferably, the corn steep liquor has a Baume of 17-20 DEG Be, a pH of 4.5-5.0 and a flow rate of 0.6-0.8mL/min.
Preferably, the concentration of the iron ion solution is 0.1mol/L, and the flow rate is 0.2-0.4mL/min.
Sodium ions and potassium ions with proper proportion are added in the fermentation of the aspergillus niger, so that the growth of thalli can be promoted, and iron ions and magnesium ions have a promoting effect on the hypha development and enzyme production of the aspergillus niger.
Preferably, the iron ion is FeCl 2 ·4H 2 O and/or FeSO 4 ·7H 2 O。
Preferably, the specific steps of the feed supplement are as follows:
performing microscopic examination on the fermentation liquor every 1h, and starting to feed the corn slurry and the ferric ion solution when the microscopic examination finds that the hypha length is higher than the spore diameter;
feeding for 7h from the beginning of fermentation: the bacterial concentration increasing rate is less than 0.1g/L, the corn slurry flow adding rate is controlled to be 0.8ml/min, the bacterial concentration increasing rate is more than or equal to 0.1g/L, and the corn slurry flow adding rate is controlled to be 0.6ml/min; the glucose consumption rate is more than 20g/L/h, the iron ion solution feeding rate is controlled to be 0.2ml/min, the glucose consumption rate is less than or equal to 20g/L/h, and the iron ion solution feeding rate is controlled to be 0.4ml/min;
fermenting for 7-13 h: the bacterial concentration increasing rate is less than 0.1g/L, the feeding rate of the corn slurry is controlled to be 0.6ml/min, the bacterial concentration increasing rate is more than or equal to 0.1g/L, and the feeding of the corn slurry is stopped; the glucose consumption rate is more than 40g/L/h, the iron ion solution feeding rate is controlled to be 0.2ml/min, the glucose consumption rate is less than or equal to 40g/L/h, and the iron ion solution feeding rate is controlled to be 0.4ml/min; stopping feeding the feed when the glucose content is lower than 30g/L, and stopping fermenting when the glucose content is less than or equal to 2g/L by adding catalase and glucose oxidase.
Preferably, the catalase is added in a ratio of 0.08g/kg of initial dry matter and the glucose oxidase is added in a ratio of 0.06g/kg of initial dry matter.
The enzyme with the addition amount can meet the conversion of residual glucose to sodium gluconate and does not cause waste.
Preferably, the pH is adjusted during the fermentation to sodium hydroxide solution.
Preferably, the enzyme activity of the catalase is more than or equal to 10 mu/ml, and the enzyme activity of the glucose oxidase is more than or equal to 1.2 mu/ml.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention utilizes Aspergillus niger strains and a double-enzyme method to jointly produce sodium gluconate, controls the fermentation time within 15 hours, greatly shortens the fermentation time and improves the production efficiency;
(2) Compared with the double-enzyme method for producing sodium gluconate, the method reduces the addition of the two enzymes by more than 50%, and realizes cost reduction;
(3) According to the invention, through controlling the nutrient salt composition and feeding, the rapid growth and mass enzyme production of aspergillus niger spores are realized, and the technical innovation is realized.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described, and the drawings in the description are only embodiments of the present invention.
FIG. 1 is a microscopic view of the initial germination of spores of example 1 of the present invention;
FIG. 2 is a microscopic image of the hyphae length higher than the spore diameter in example 1 of the present invention.
Detailed Description
The following describes embodiments of the invention, examples of which are illustrated in the accompanying drawings, and the embodiments described with reference to the drawings are illustrative and intended to be in the way of explanation of the invention and not to be construed as limiting the invention.
Raw materials and sources:
catalase: the enzyme activity of catalase purchased from Shandong Long Kete enzyme preparation limited company is more than or equal to 10 mu/ml;
glucose oxidase: the enzyme activity of glucose oxidase purchased from Shandong Long Kete enzyme preparation limited company is more than or equal to 1.2 mu/ml;
corn slurry: the Shandong Fuyang biotechnology Co., ltd, the preparation method is as follows: soaking corn in 0.2-0.3% sulfurous acid solution for about 50 hr, concentrating the soaked water until Baume degree=17-20 deg. Baume to obtain corn steep liquor, wherein Baume degree is 17-20 deg. Baume and pH is 4.5-5.0;
aspergillus niger: purchased from university of south of the Yangtze river;
example 1:
(1) Preparing a fermentation medium and sterilizing: adding 320g/L glucose solution 10L, potassium dihydrogen phosphate 30g, disodium hydrogen phosphate 90g, magnesium sulfate 40g, and ammonium sulfate 120g into 15L fermentation tank, adjusting pH=6.0 with 32% (wt) sodium hydroxide solution, and sterilizing at 105deg.C for 20min;
(2) Preparing a feed medium and sterilizing: adding 3L of corn slurry with Baume=17° Be and PH=4.5 into a 5L feeding bottle, sterilizing at 121 ℃ for 20min, and cooling to room temperature; feed B was a 2L feed bottle charged with 39.76g FeCl 2 ·4H 2 O, sterilizing at 121 ℃ for 20min, and cooling to room temperature;
(3) Adding 2.56g of catalase and 1.92g of glucose oxidase (original enzyme liquid enzyme activity: catalase enzyme activity=10.1 mu/mL and glucose oxidase enzyme activity=1.21 mu/mL) into a fermentation tank, inoculating 400mL of aspergillus niger strain, wherein the total number of spores is 2.2 x 10 8 And (3) controlling fermentation conditions: temperature 39 ℃, rotating speed 50-100rpm, air quantity 0.03-0.05wwm, pressure 0.1-0.12MPa, PH=5.2-5.4;
(4) Fermenting for 2h, microscopic examination shows spore germination (as shown in figure 1), and controlling fermentation conditions: the temperature is 39 ℃, the rotating speed is 200-300rpm, the air quantity is 0.09-0.1vvm, the pressure is 0.1-0.12MPa, and the PH=5.2-5.4; fermenting for 3h, microscopic examination shows that the hypha length is higher than the spore diameter (shown in figure 2), feeding the feed A and the feed B, fermenting for 3h to 7h, wherein the average bacterial concentration growth rate is 1.12g/L, the glucose consumption rate=19.3 g/L/h, controlling the feeding rate of the feed A to be 0.6ml/min, and the feeding rate of the feed B to be 0.4ml/min; fermenting for 7-13h, wherein the average bacterial concentration increasing rate is 0.88g/L, the glucose consumption rate=43.3 g/L/h, the feeding A feeding rate is 0ml/min, and the feeding B feeding rate is 0.2ml/min;
(5) Fermenting for 14h, wherein the glucose content=26.3 g/L, stopping feeding, and feeding 0.256g of catalase and 0.192g of glucose oxidase; fermentation was stopped until 15h, glucose content = 1.2g/L, conversion was 109.8%.
Example 2:
(1) Preparing a fermentation medium and sterilizing: adding 10L of 300g/L glucose solution, 40g of potassium dihydrogen phosphate, 100g of disodium hydrogen phosphate, 40g of magnesium sulfate and 120g of ammonium sulfate into a 15L fermentation tank, adjusting pH=6.0 by using 32% (wt) sodium hydroxide solution, and sterilizing at 105 ℃ for 20min;
(2) Preparing a feed medium and sterilizing: adding 3L of corn slurry with Baume=18° Be and PH=4.6 into a 5L feeding bottle, sterilizing at 121 ℃ for 20min, and cooling to room temperature; the feed B is a 2L feed bottle filled with 55.6g FeSO 4 ·7H 2 O, sterilizing at 121 ℃ for 20min, and cooling to room temperature;
(3) Adding 2.4g of catalase and 1.8g of glucose oxidase (original enzyme liquid enzyme activity: catalase enzyme activity=10.1 mu/mL and glucose oxidase enzyme activity=1.21 mu/mL) into a fermentation tank, inoculating 300mL of Aspergillus niger strain, wherein the total number of spores is 2.5 x 10 8 And (3) controlling fermentation conditions: temperature 39 ℃, rotating speed 50-100rpm, air quantity 0.03-0.05wwm, pressure 0.1-0.12MPa, PH=5.2-5.4;
(4) Fermenting for 2h, detecting spore germination by microscopic examination, and controlling fermentation conditions: the temperature is 39 ℃, the rotating speed is 200-300rpm, the air quantity is 0.09-0.1vvm, the pressure is 0.1-0.12MPa, and the PH=5.2-5.4; fermenting for 3h, wherein the length of hypha is higher than the diameter of spores by microscopic examination, feeding the feed A and the feed B, fermenting for 3h to 7h, wherein the average growth rate of the bacterial concentration is 1.09g/L, the glucose consumption rate=19.4 g/L/h, controlling the feeding rate of the feed A to be 0.6ml/min and the feeding rate of the feed B to be 0.4ml/min; fermenting for 7-13h, wherein the average bacterial concentration increasing rate is 0.82g/L, the glucose consumption rate=41.6 g/L/h, the feeding A feeding rate is 0ml/min, and the feeding B feeding rate is 0.2ml/min;
(5) Fermenting for 14h, wherein the glucose content=27.4 g/L, stopping feeding, and feeding 0.24g of catalase and 0.18g of glucose oxidase; fermentation was stopped until 15h, glucose content = 1.5g/L, conversion 110.2%.
Example 3:
(1) Preparing a fermentation medium and sterilizing: 10L of 310g/L glucose solution, 50g of potassium dihydrogen phosphate, 100g of disodium hydrogen phosphate, 40g of magnesium sulfate and 120g of ammonium sulfate are added into a 15L fermentation tank, the PH=6.0 is regulated by 32% (wt) sodium hydroxide solution, and the fermentation tank is sterilized for 20min at 105 ℃;
(2) Preparing a feed medium and sterilizing: adding 3L of corn slurry with Baume=19° Be and PH=4.8 into a 5L feeding bottle, sterilizing at 121 ℃ for 20min, and cooling to room temperature; feed B was a 2L feed bottle charged with 19.88g FeCl 2 ·4H 2 O、27.8g FeSO 4 ·7H 2 O, sterilizing at 121 ℃ for 20min, and cooling to room temperature;
(3) 2.48g of catalase and 1.86g of glucose oxidase (raw enzyme liquid enzyme activity: catalase enzyme activity=10.1 mu/ml, glucose oxidase enzyme activity=1.21 mu/ml) are added into a fermentation tank; inoculating 500mL of Aspergillus niger strain with total spore count of 2.3×10 8 And (3) controlling fermentation conditions: temperature 39 ℃, rotating speed 50-100rpm, air quantity 0.03-0.05wwm, pressure 0.1-0.12MPa, PH=5.2-5.4;
(4) Fermenting for 2h, detecting spore germination by microscopic examination, and controlling fermentation conditions: the temperature is 39 ℃, the rotating speed is 200-300rpm, the air quantity is 0.09-0.1vvm, the pressure is 0.1-0.12MPa, and the PH=5.2-5.4; fermenting for 3h, wherein the length of hypha is higher than the diameter of spores by microscopic examination, feeding the feed A and the feed B, fermenting for 3h to 7h, wherein the average growth rate of the bacterial concentration is 0.98g/L, the glucose consumption rate=20.2 g/L/h, controlling the feeding rate of the feed A to be 0.6ml/min and the feeding rate of the feed B to be 0.2ml/min; fermenting for 7-13h, wherein the average bacterial concentration increasing rate is 0.85g/L, the glucose consumption rate=42.7 g/L/h, the feeding A feeding rate is 0ml/min, and the feeding B feeding rate is 0.2ml/min;
(5) Fermenting for 14h, wherein the glucose content=29.1 g/L, stopping feeding, and feeding 0.248g of catalase and 0.186g of glucose oxidase; fermentation was stopped until 15h, glucose content=1.1 g/L, conversion was 109.6%.
Example 4:
(1) Preparing a fermentation medium and sterilizing: adding 315g/L glucose solution 10L, potassium dihydrogen phosphate 50g, disodium hydrogen phosphate 150g, magnesium sulfate 40g, and ammonium sulfate 120g into 15L fermentation tank, adjusting pH=6.0 with 32% (wt) sodium hydroxide solution, and sterilizing at 105deg.C for 20min;
(2) Preparing a feed medium and sterilizing: adding 3L of corn slurry with Baume=20° Be and PH=5.0 into a 5L feeding bottle, sterilizing at 121 ℃ for 20min, and cooling to room temperature; the feed B is a 2L feed bottle filled with 15.904 FeCl 2 ·4H 2 O、33.36g FeSO 4 ·7H 2 O, sterilizing at 121 ℃ for 20min, and cooling to room temperature;
(3) 2.52g of catalase and 1.89g of glucose oxidase (raw enzyme liquid enzyme activity: catalase enzyme activity=10.1 mu/ml, glucose oxidase enzyme activity=1.21 mu/ml) were added to the fermenter; inoculating 500mL of Aspergillus niger strain with total spore count of 2.3×10 8 And (3) controlling fermentation conditions: temperature 39 ℃, rotating speed 50-100rpm, air quantity 0.03-0.05wwm, pressure 0.1-0.12MPa, PH=5.2-5.4;
(4) Fermenting for 2h, detecting spore germination by microscopic examination, and controlling fermentation conditions: the temperature is 39 ℃, the rotating speed is 200-300rpm, the air quantity is 0.09-0.1vvm, the pressure is 0.1-0.12MPa, and the PH=5.2-5.4; fermenting for 3h, wherein the length of hypha is higher than the diameter of spores by microscopic examination, feeding the feed A and the feed B, fermenting for 3h to 7h, wherein the average growth rate of the bacterial concentration is 0.98g/L, the glucose consumption rate=20.7 g/L/h, controlling the feeding rate of the feed A to be 0.6ml/min and the feeding rate of the feed B to be 0.2ml/min; fermenting for 7-13h, wherein the average bacterial concentration increasing rate is 0.85g/L, the glucose consumption rate=44.3 g/L/h, the feeding A feeding rate is 0ml/min, and the feeding B feeding rate is 0.2ml/min;
(5) Fermenting for 14h, wherein the glucose content=29.6 g/L, stopping feeding, and feeding 0.252g of catalase and 0.189g of glucose oxidase; fermentation was stopped until 15h, glucose content=0.9 g/L, conversion was 109.3%.
Comparative example 1 (initial no enzyme added):
(1) Preparing a fermentation medium and sterilizing: adding 320g/L glucose solution 10L, potassium dihydrogen phosphate 30g, disodium hydrogen phosphate 90g, magnesium sulfate 40g, and ammonium sulfate 120g into 15L fermentation tank, adjusting pH=6.0 with 32% (wt) sodium hydroxide solution, and sterilizing at 105deg.C for 20min;
(2) Preparing a feed medium and sterilizing: the feed A is prepared by adding 3L corn slurry with Baume=17° Be and PH=4.6 into a 5L feed bottle, sterilizing at 121deg.C for 20minCooling to room temperature; feed B was a 2L feed bottle charged with 39.76g FeCl 2 ·4H 2 O, sterilizing at 121 ℃ for 20min, and cooling to room temperature;
(3) Inoculating Aspergillus niger strain 400mL into fermentation tank, wherein the total spore count is 2.2X10 8 And (3) controlling fermentation conditions: temperature 39 ℃, rotating speed 50-100rpm, air quantity 0.03-0.05wwm, pressure 0.1-0.12MPa, PH=5.2-5.4;
(4) Fermenting for 1h, detecting spore germination by microscopic examination, and controlling fermentation conditions: the temperature is 39 ℃, the rotating speed is 200-300rpm, the air quantity is 0.09-0.1vvm, the pressure is 0.1-0.12MPa, and the PH=5.2-5.4; fermenting for 2h, microscopic examination shows that the hypha length is higher than the spore diameter, feeding the feed A and the feed B, fermenting for 2h to 7h, wherein the average bacterial concentration growth rate is 1.03g/L, the glucose consumption rate=10.3 g/L/h, controlling the feeding rate of the feed A to be 0.6ml/min and the feeding rate of the feed B to be 0.4ml/min; fermenting for 7-13h, wherein the average bacterial concentration increasing rate is 1.22g/L, the glucose consumption rate=21.3 g/L/h, the feeding A feeding rate is 0ml/min, and the feeding B feeding rate is 0.4ml/min;
(5) Fermenting for 14h, wherein the glucose content=187.6 g/L; fermenting for 22h, wherein the glucose content is=27.6 g/L, and stopping feeding; adding 0.256g of catalase and 0.192g of glucose oxidase (enzyme activity of original enzyme liquid: catalase activity=10.1 mu/ml, glucose oxidase activity=1.21 mu/ml); fermentation was stopped until 23h, glucose content=1.1 g/L, conversion was 107.5%.
Comparative example 2 (no enzyme added at end):
(1) Preparing a fermentation medium and sterilizing: adding 320g/L glucose solution 10L, potassium dihydrogen phosphate 30g, disodium hydrogen phosphate 90g, magnesium sulfate 40g, and ammonium sulfate 120g into 15L fermentation tank, adjusting pH=6.0 with 32% (wt) sodium hydroxide solution, and sterilizing at 105deg.C for 20min;
(2) Preparing a feed medium and sterilizing: adding 3L of corn slurry with Baume=18° Be and PH=4.8 into a 5L feeding bottle, sterilizing at 121 ℃ for 20min, and cooling to room temperature; feed B was a 2L feed bottle charged with 39.76g FeCl 2 ·4H 2 O, sterilizing at 121 ℃ for 20min, and cooling to room temperature;
(3) Adding 2.56g of catalase and grape into a fermentation tank1.92g of sugar oxidase (stock enzyme activity: catalase enzyme activity=10.1 mu/ml, glucose oxidase enzyme activity=1.21 mu/ml); 400mL of Aspergillus niger strain is inoculated, and the total spore count is 2.2-10 8 And (3) controlling fermentation conditions: temperature 39 ℃, rotating speed 50-100rpm, air quantity 0.03-0.05wwm, pressure 0.1-0.12MPa, PH=5.2-5.4;
(4) Fermenting for 2h, detecting spore germination by microscopic examination, and controlling fermentation conditions: the temperature is 39 ℃, the rotating speed is 200-300rpm, the air quantity is 0.09-0.1vvm, the pressure is 0.1-0.12MPa, and the PH=5.2-5.4; fermenting for 3h, wherein the length of hypha is higher than the diameter of spores by microscopic examination, feeding the feed A and the feed B, fermenting for 3h to 7h, wherein the average growth rate of the bacterial concentration is 1.12g/L, the glucose consumption rate=19.3 g/L/h, controlling the feeding rate of the feed A to be 0.6ml/min and the feeding rate of the feed B to be 0.4ml/min; fermenting for 7-13h, wherein the average bacterial concentration increasing rate is 0.88g/L, the glucose consumption rate=43.3 g/L/h, the feeding A feeding rate is 0ml/min, and the feeding B feeding rate is 0.2ml/min;
(5) Fermenting for 14h, wherein the glucose content is=26.3 g/L, and stopping feeding; fermentation was stopped until 25h, glucose content = 1.2g/L, conversion 106.4%.
Comparative example 3 (no added seed):
(1) Preparing a fermentation medium and sterilizing: adding 320g/L glucose solution 10L into 15L fermentation tank, adjusting pH to 6.0 with 32% (wt) sodium hydroxide solution, and sterilizing at 105deg.C for 20min;
(2) 2.56g of catalase and 1.92g of glucose oxidase (raw enzyme liquid enzyme activity: catalase enzyme activity=10.1 mu/ml, glucose oxidase enzyme activity=1.21 mu/ml) are added into a fermentation tank; controlling fermentation conditions: temperature 39 ℃, rotating speed 50-100rpm, air quantity 0.03-0.05wwm, pressure 0.1-0.12MPa, PH=5.2-5.4;
(3) Fermenting for 10h, wherein the glucose content=126.8g/L, detecting that the enzyme activity of glucose oxidase is 0, and adding 0.256g of catalase and 0.192g of glucose oxidase; fermenting for 13h, wherein the glucose content=96.3 g/L, detecting the enzyme activity of glucose oxidase to be 0, stopping fermenting, and the conversion rate is 69.9%.
Comparative example 4 (no corn steep liquor fed):
(1) Preparing a fermentation medium and sterilizing: adding 320g/L glucose solution 10L, potassium dihydrogen phosphate 30g, disodium hydrogen phosphate 90g, magnesium sulfate 40g, and ammonium sulfate 120g into 15L fermentation tank, adjusting pH=6.0 with 32% (wt) sodium hydroxide solution, and sterilizing at 105deg.C for 20min;
(2) Preparing a feed medium and sterilizing: feed B was a 2L feed bottle charged with 39.76g FeCl 2 ·4H 2 O, sterilizing at 121 ℃ for 20min, and cooling to room temperature;
(3) 2.56g of catalase and 1.92g of glucose oxidase (raw enzyme liquid enzyme activity: catalase enzyme activity=10.1 mu/ml, glucose oxidase enzyme activity=1.21 mu/ml) are added into a fermentation tank; 400mL of Aspergillus niger strain is inoculated, and the total spore count is 2.2-10 8 And (3) controlling fermentation conditions: temperature 39 ℃, rotating speed 50-100rpm, air quantity 0.03-0.05wwm, pressure 0.1-0.12MPa, PH=5.2-5.4;
(4) Fermenting for 2h, detecting spore germination by microscopic examination, and controlling fermentation conditions: the temperature is 39 ℃, the rotating speed is 200-300rpm, the air quantity is 0.09-0.1vvm, the pressure is 0.1-0.12MPa, and the PH=5.2-5.4; fermenting for 3h, wherein microscopic examination shows that the hypha length is higher than the spore diameter, feeding a feed B, fermenting for 3h to 7h, wherein the average growth rate of the bacterial concentration is 0.62g/L, the glucose consumption rate is=14.3 g/L/h, and the feeding rate of the feed B is 0.4ml/min; fermenting for 7-13h, wherein the average bacterial concentration growth rate is 0.78g/L, the glucose consumption rate=23.3 g/L/h, and the feeding B flow rate is 0.4ml/min;
(5) Fermenting for 25h, wherein the glucose content=28.1 g/L, stopping feeding, and feeding 0.256g of catalase and 0.192g of glucose oxidase; fermentation was stopped until 26h, glucose content=1.2 g/L, conversion was 104.2%.
Comparative example 5 (no ferrous ion fed):
(1) Preparing a fermentation medium and sterilizing: adding 320g/L glucose solution 10L, potassium dihydrogen phosphate 30g, disodium hydrogen phosphate 90g, magnesium sulfate 40g, and ammonium sulfate 120g into 15L fermentation tank, adjusting pH=6.0 with 32% (wt) sodium hydroxide solution, and sterilizing at 105deg.C for 20min;
(2) Preparing a feed medium and sterilizing: adding 3L of corn slurry with Baume=19° Be and PH=4.9 into a 5L feeding bottle, sterilizing at 121 ℃ for 20min, and cooling to room temperature;
(3) 2.56g of catalase and 1.92g of glucose oxidase (raw enzyme liquid enzyme activity: catalase enzyme activity=10.1 mu/ml, glucose oxidase enzyme activity=1.21 mu/ml) are added into a fermentation tank; 400mL of Aspergillus niger strain is inoculated, and the total spore count is 2.2-10 8 And (3) controlling fermentation conditions: temperature 39 ℃, rotating speed 50-100rpm, air quantity 0.03-0.05wwm, pressure 0.1-0.12MPa, PH=5.2-5.4;
(4) Fermenting for 2h, detecting spore germination by microscopic examination, and controlling fermentation conditions: the temperature is 39 ℃, the rotating speed is 200-300rpm, the air quantity is 0.09-0.1vvm, the pressure is 0.1-0.12MPa, and the PH=5.2-5.4; fermenting for 3h, microscopic examination shows that the hypha length is higher than the spore diameter, feeding the feed A, fermenting for 3h to 7h, wherein the average bacterial concentration increasing rate is 1.12g/L, the glucose consumption rate=19.3 g/L/h, and controlling the feeding rate of the feed A to be 0.6ml/min; fermenting for 7-13h, wherein the average bacterial concentration growth rate is 0.88g/L, the glucose consumption rate=20.2 g/L/h, and the feeding A flow rate is 0ml/min;
(5) Fermenting for 22h, wherein the glucose content=28.8 g/L, and 0.256g of catalase and 0.192g of glucose oxidase are added; fermentation was stopped until 23h, glucose content = 1.1g/L, conversion was 103.9%.
Comparative example 6 (adjustment of sodium potassium ion ratio):
(1) Preparing a fermentation medium and sterilizing: adding 320g/L glucose solution 10L, potassium dihydrogen phosphate 30g, disodium hydrogen phosphate 50g, magnesium sulfate 40g, and ammonium sulfate 120g into 15L fermentation tank, adjusting pH=6.0 with 32% (wt) sodium hydroxide solution, and sterilizing at 105deg.C for 20min;
(2) Preparing a feed medium and sterilizing: adding 3L of corn slurry with Baume=20° Be and PH=5.0 into a 5L feeding bottle, sterilizing at 121 ℃ for 20min, and cooling to room temperature; feed B was a 2L feed bottle charged with 39.76g FeCl 2 ·4H 2 O, sterilizing at 121 ℃ for 20min, and cooling to room temperature;
(3) 2.56g of catalase and 1.92g of glucose oxidase (raw enzyme liquid enzyme activity: catalase enzyme activity=10.1 mu/ml, glucose oxidase enzyme activity=1.21 mu/ml) are added into a fermentation tank; 400mL of Aspergillus niger strain is inoculated, and the total spore count is 2.2-10 8 Number, controlFermentation conditions: temperature 39 ℃, rotating speed 50-100rpm, air quantity 0.03-0.05wwm, pressure 0.1-0.12MPa, PH=5.2-5.4;
(4) Fermenting for 2h, detecting spore germination by microscopic examination, and controlling fermentation conditions: the temperature is 39 ℃, the rotating speed is 200-300rpm, the air quantity is 0.09-0.1vvm, the pressure is 0.1-0.12MPa, and the PH=5.2-5.4; fermenting for 3h, wherein the length of hypha is higher than the diameter of spores by microscopic examination, feeding the feed A and the feed B, fermenting for 3h to 7h, wherein the average growth rate of the bacterial concentration is 1.12g/L, the glucose consumption rate=17.3 g/L/h, controlling the feeding rate of the feed A to be 0.6ml/min and the feeding rate of the feed B to be 0.4ml/min; fermenting for 7-13h, wherein the average bacterial concentration increasing rate is 0.88g/L, the glucose consumption rate=36.3 g/L/h, the feeding A feeding rate is 0ml/min, and the feeding B feeding rate is 0.4ml/min;
(5) Fermenting for 17h, wherein the glucose content=27.2 g/L, stopping feeding, and feeding 0.256g of catalase and 0.192g of glucose oxidase; fermentation was stopped until 22h, glucose content=1.2 g/L, conversion was 106.8%.
Comparative examples 1 to 6 are comparative examples of example 1, comparative example 1 is a comparative example in which two enzymes are not added initially, comparative example 2 is a comparative example in which two enzymes are not added at the end, and other conditions are the same as those of example 1, and as can be seen from comparative examples 1 and 2, the fermentation time period is 20 hours or more and the conversion rate is lower due to the absence of double enzymes at the initial and end; comparative example 3 was not added with strain, and as seen from comparative example 3, the addition of only double enzymes without strain resulted in incomplete conversion; comparative example 4 is no-fed corn steep liquor, comparative example 5 is no-fed ferrous ion, and as can be seen from comparative examples 4 and 5, no-fed corn steep liquor and ferrous ion result in fermentation time longer than 20h, and the conversion rate is lower; as is clear from comparative example 6, the adjustment of the sodium-potassium ion ratio at the 6-position also has the problems of long conversion time and low conversion rate.
As can be seen from examples 1-4 and comparative examples 1-6, the method for efficiently producing sodium gluconate of the invention can realize that the conversion of glucose to sodium gluconate is completed within 15 hours, improves the conversion efficiency and conversion rate, reduces the addition of two enzymes by more than 50%, and realizes cost reduction.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; 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 (10)
1. A method for producing sodium gluconate by an enzymatic method is characterized in that catalase, glucose oxidase and aspergillus niger strains are added into glucose fermentation liquor for fermentation;
wherein the addition amount of the catalase is 0.8g/kg of dry matter, the addition amount of the glucose oxidase is 0.6g/kg of dry matter, the inoculation amount of the Aspergillus niger strain is 3-5% of the volume of the glucose fermentation liquor, and the total spore number is more than or equal to 2 x 10 8 And each.
2. The method for producing sodium gluconate by an enzymatic method according to claim 1, wherein the glucose fermentation broth comprises a glucose solution and a nutrient salt;
wherein the concentration of glucose in the glucose solution is 300-320g/L;
the nutrient salt comprises potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate and ammonium sulfate, the addition amounts are 3-5g/L, 9-15g/L, 4g/L and 12g/L respectively, and the mass ratio of the potassium dihydrogen phosphate to the disodium hydrogen phosphate is 1:2-3;
the pH value of the glucose fermentation liquor is 6.0.
3. The method for producing sodium gluconate by using an enzymatic method according to claim 1, wherein the fermentation conditions are as follows:
stage before spore germination: the temperature is 39 ℃, the rotating speed is 50-100rpm, the air quantity is 0.03-0.05wwm, the pressure is 0.1-0.12MPa, and the PH is 5.2-5.4;
spore germination and strain growth stage: the temperature is 39 ℃, the rotating speed is 200-300rpm, the air quantity is 0.09-0.1vvm, the pressure is 0.1-0.12MPa, and the PH is 5.2-5.4.
4. The method for producing sodium gluconate by an enzymatic process according to claim 1, wherein the fermentation process further comprises a feeding step comprising corn steep liquor and an iron ion solution.
5. The method for producing sodium gluconate by an enzymatic process of claim 4, wherein the corn steep liquor has a Baume of 17-20 degrees Be, a pH of 4.5-5.0, and a flow rate of 0.6-0.8mL/min.
6. The method for producing sodium gluconate by an enzymatic process according to claim 4, wherein the concentration of the iron ion solution is 0.1mol/L and the flow rate is 0.2 to 0.4mL/min.
7. The method for producing sodium gluconate by using an enzymatic method according to claim 4, wherein the iron ion is FeCl 2 ·4H 2 O and/or FeSO 4 ·7H 2 O。
8. The method for producing sodium gluconate by using the enzymatic method according to claim 4, wherein the specific steps of the feed are as follows:
performing microscopic examination on the fermentation liquor every 1h, and starting to feed the corn slurry and the ferric ion solution when the microscopic examination finds that the hypha length is higher than the spore diameter;
feeding for 7h from the beginning of fermentation: the bacterial concentration increasing rate is less than 0.1g/L, the corn slurry flow adding rate is controlled to be 0.8ml/min, the bacterial concentration increasing rate is more than or equal to 0.1g/L, and the corn slurry flow adding rate is controlled to be 0.6ml/min; the glucose consumption rate is more than 20g/L/h, the iron ion solution feeding rate is controlled to be 0.2ml/min, the glucose consumption rate is less than or equal to 20g/L/h, and the iron ion solution feeding rate is controlled to be 0.4ml/min;
fermenting for 7-13 h: the bacterial concentration increasing rate is less than 0.1g/L, the feeding rate of the corn slurry is controlled to be 0.6ml/min, the bacterial concentration increasing rate is more than or equal to 0.1g/L, and the feeding of the corn slurry is stopped; the glucose consumption rate is more than 40g/L/h, the iron ion solution feeding rate is controlled to be 0.2ml/min, the glucose consumption rate is less than or equal to 40g/L/h, and the iron ion solution feeding rate is controlled to be 0.4ml/min; stopping feeding the feed when the glucose content is lower than 30g/L, and stopping fermenting when the glucose content is less than or equal to 2g/L by adding catalase and glucose oxidase.
9. The method for producing sodium gluconate by using the enzymatic method according to claim 1, wherein the PH-adjusting substance in the fermentation process is sodium hydroxide solution.
10. The method for producing sodium gluconate by using the enzymatic method according to claim 1, wherein the enzyme activity of the catalase is more than or equal to 10 mu/ml, and the enzyme activity of the glucose oxidase is more than or equal to 1.2 mu/ml.
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