CN110760622B - Feeding method for producing glucosamine by fermenting bacillus subtilis - Google Patents
Feeding method for producing glucosamine by fermenting bacillus subtilis Download PDFInfo
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- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 title claims abstract description 25
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 title claims abstract description 25
- 229960002442 glucosamine Drugs 0.000 title claims abstract description 25
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 24
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 24
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 18
- 238000000855 fermentation Methods 0.000 claims abstract description 69
- 230000004151 fermentation Effects 0.000 claims abstract description 69
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 59
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 59
- 239000001301 oxygen Substances 0.000 claims abstract description 59
- 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 43
- 239000008103 glucose Substances 0.000 claims abstract description 43
- 230000002572 peristaltic effect Effects 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 17
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 46
- 229960001031 glucose Drugs 0.000 claims description 46
- 230000001502 supplementing effect Effects 0.000 claims description 22
- 239000000463 material Substances 0.000 claims description 17
- 230000001105 regulatory effect Effects 0.000 claims description 14
- 238000012544 monitoring process Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 238000009423 ventilation Methods 0.000 claims description 6
- 230000001276 controlling effect Effects 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 5
- 239000013589 supplement Substances 0.000 claims description 5
- 239000007858 starting material Substances 0.000 claims 1
- 239000006052 feed supplement Substances 0.000 abstract description 17
- 238000000605 extraction Methods 0.000 abstract description 4
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 238000007086 side reaction Methods 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 12
- 230000004060 metabolic process Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 3
- 238000005265 energy consumption Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 244000005700 microbiome Species 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- SPFMQWBKVUQXJV-BTVCFUMJSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal;hydrate Chemical compound O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O SPFMQWBKVUQXJV-BTVCFUMJSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 229960000673 dextrose monohydrate Drugs 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q3/00—Condition responsive control processes
-
- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
Abstract
The invention relates to a feeding method for producing glucosamine by fermenting bacillus subtilis, belonging to the technical field of microbial fermentation production. The main steps include taking glucose solution with a certain concentration as feed supplement liquid; feeding by using a PLC control system and a peristaltic pump quantitative feeding mode; the starting time and the feeding amount are controlled according to the glucose content and the dissolved oxygen content in the fermentation tank, and the process parameters are simple to control, the glucosamine yield is high, the fermentation side reaction is less, and the extraction yield is high.
Description
Technical Field
The invention relates to the technical field of microbial fermentation production, in particular to a feeding method for producing glucosamine by bacillus subtilis.
Background
The existing glucosamine production method mainly comprises an enzyme conversion method, a shrimp and crab shell extraction method, a microorganism fermentation method and the like, wherein the microorganism fermentation method has the characteristics of high yield, low cost, no limitation of raw material sources and seasons, stable product quality and the like, and can replace the other two production methods. Compared with other microbial fermentation, the method for producing the glucosamine by fermenting the bacillus subtilis genetically engineered bacteria has great advantages in the aspects of strain, product safety and the like, and because the bacillus subtilis is a food safety-grade strain, the safety of the glucosamine produced by fermenting the bacillus subtilis at the gene level is ensured, and the method has very important significance in modern society which pay more attention to food and drug safety.
In the fermentation production process of the bacillus subtilis, the feeding is an important link for maintaining the high efficiency of the fermentation process, and the existing fermentation production process of the bacillus subtilis only simply uses the content of glucose as a basis which cannot be added, but affects the fermentation process of the bacillus subtilis, so that the method cannot be well adapted to the fermentation process of the bacillus subtilis.
Disclosure of Invention
In order to solve the problems in the prior art, the invention designs a feeding method for producing glucosamine by bacillus subtilis, which comprises the following steps,
(a) Feeding with 700-800 g/L food grade glucose monohydrate solution as feed liquid;
(b) Regulating the tank pressure of the material supplementing tank to 0.05-0.15 MPa, and keeping the tank pressure of the fermentation tank at 0.03-0.05 MPa;
(c) Feeding glucose feed liquid in a quantitative feed supplement mode by adopting a peristaltic pump, controlling the peristaltic pump by adopting a PLC control system to perform feed supplement, monitoring the content of glucose and the dissolved oxygen in the fermentation tank by the PLC control system at the same time, and starting to supplement after the dissolved oxygen in the fermentation tank rapidly rises to 90%;
(d) After the material supplementing is started, regulating the rotating speed of a peristaltic pump by a PLC control system according to the content of glucose and the dissolved oxygen amount in the fermentation tank, gradually increasing the material supplementing amount to control the glucose concentration in the tank to be 3-5 g/L, and reducing the dissolved oxygen in the tank when the material supplementing speed is increased, and regulating the dissolved oxygen in the tank by increasing the ventilation amount and the stirring rotating speed;
(e) In the fermentation process, dissolved oxygen is subjected to multistage control, the dissolved oxygen amount is monitored by a PLC control system, and the starting and stopping of the ventilation device and the stirring rotating speed are controlled to be adjusted.
The feeding method for producing the glucosamine by the bacillus subtilis is characterized in that the feeding starting time is judged according to the change condition of dissolved oxygen, and after the dissolved oxygen is rapidly increased to 90, feeding is started, so that the situation that the feeding is delayed due to the fact that the concentration of glucose in a tank cannot be rapidly detected, and the glucose is deficient in fermentation metabolism is avoided; meanwhile, a peristaltic pump is adopted for quantitative feeding, the feeding amount of the feeding flow is controlled more accurately, and the strain grows and metabolizes in a more stable environment, so that the strain conversion rate and the glucosamine conversion rate are high, the strain metabolism speed is high, the fermentation period is shortened, and the energy consumption is reduced.
Further, in the step (d), the glucose concentration in the tank is measured every 1 hour, the rotating speed of the peristaltic pump is regulated, the feeding amount is gradually increased, the feeding speed is not increased when the glucose concentration in the tank reaches 4.5g/L, and the feeding speed is reduced when the glucose concentration in the tank exceeds 5g/L.
Further, the control process of dissolved oxygen in the fermentation process in the step (e) is 0 to 7 hours of fermentation, and the dissolved oxygen is 30%; 7 to 15 hours, and dissolved oxygen is 50 percent; 15 to 50h, and 40 percent of dissolved oxygen; 50 to 65 hours and 30 percent of dissolved oxygen.
Furthermore, the feed supplement liquid needs to be subjected to steam wet heat sterilization for 20min under the condition of 0.1MPa and 121 ℃.
Further, the feed supplement liquid is 800g/L of food-grade dextrose monohydrate solution.
Further, the step (b) adjusts the tank pressure of the feeding tank to 0.1MPa, and maintains the tank pressure of the fermentation tank to 0.04MPa
Compared with the prior art, the feed supplementing method for producing the glucosamine by fermenting the bacillus subtilis is characterized in that firstly, the feed supplementing process is carried out by monitoring and adjusting a PLC control system, the feed supplementing starting time is judged according to the change condition of dissolved oxygen, and after the dissolved oxygen is rapidly increased to 90%, the feed supplementing is started, so that the situation that the glucose concentration in a tank cannot be rapidly detected to delay the feed supplementing, and the glucose deficiency in fermentation metabolism is avoided.
In addition, as the peristaltic pump is adopted for quantitative feeding, the feeding amount of the feeding flow is controlled more accurately, and the strain grows and metabolizes in a more stable environment, so that the strain conversion rate and the glucosamine conversion rate are high, the strain metabolism speed is high, the fermentation period is shortened, and the energy consumption is reduced.
In addition, the concentration of glucose in the fermentation tank is controlled to be 3-5 g/L, so that the fermentation side reaction can be greatly reduced, the quality of fermentation liquor is improved, the post-extraction difficulty is reduced, and the extraction yield is increased.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are provided for the purpose of illustrating the invention in detail and are not to be construed as limiting the scope of the invention in any way.
Example 1
According to the first embodiment of the feeding method for producing glucosamine by fermenting bacillus subtilis, the bacillus subtilis used in the embodiment is a commercial product, and the feeding method comprises the following steps:
(a) Feeding with 700g/L food-grade glucose monohydrate solution as feed supplement liquid, wherein the feed supplement liquid needs steam wet heat sterilization for 20min under the conditions of 0.1MPa and 121 ℃;
(b) Regulating the tank pressure of the material supplementing tank to 0.05MPa, and keeping the tank pressure of the fermentation tank at 0.03MPa;
(c) Feeding glucose feed liquid in a quantitative feed supplement mode by adopting a peristaltic pump, controlling the peristaltic pump by adopting a PLC control system to perform feed supplement, monitoring the content of glucose and the dissolved oxygen in the fermentation tank by the PLC control system at the same time, and starting to supplement after the dissolved oxygen in the fermentation tank rapidly rises to 90%;
(d) After the feeding is started, measuring the glucose concentration in the tank every 1 hour, regulating the rotation speed of the peristaltic pump by the PLC control system according to the glucose content and dissolved oxygen amount in the fermentation tank, gradually increasing the feeding amount, and when the glucose concentration in the tank reaches 4.5g/L, not increasing the feeding speed any more, and when the glucose concentration in the tank exceeds 5g/L, reducing the feeding speed. The glucose concentration in the tank is controlled to be more than 3g/L, when the feeding speed is increased, the dissolved oxygen in the tank can be reduced, and at the moment, the ventilation quantity and the stirring rotating speed are increased to regulate the dissolved oxygen in the tank;
(e) The dissolved oxygen is controlled in a multistage manner in the fermentation process, wherein the process is 0 to 7 hours of fermentation, and the dissolved oxygen is 30 percent; 7 to 15 hours, and dissolved oxygen is 50 percent; 15 to 50h, and 40 percent of dissolved oxygen; 50 to 65 hours and 30 percent of dissolved oxygen.
After fermentation, the detection shows that the strain conversion rate reaches 40% and the glucosamine content reaches 105g/L in the fermentation process of the bacillus subtilis by the feeding method of the embodiment after fermentation reaches 65 hours. For the traditional feeding technology, the fermentation period is 80-85 hours, the strain conversion rate is 28-30%, and the glucosamine content is 70-75 g/L. Compared with the prior art, the embodiment has obvious progress, improves the strain conversion rate, shortens the fermentation period and obviously improves the yield of the glucosamine. The peristaltic pump is adopted for quantitative feeding, feeding amount control is more accurate, glucose content and dissolved oxygen in the fermentation process are detected and controlled in time, so that strains grow and metabolize in a more stable environment, strain conversion rate and glucosamine conversion rate are high, strain metabolism speed is high, fermentation period is shortened, and energy consumption is reduced.
Example 2
The second embodiment of the feeding method for producing glucosamine by bacillus subtilis fermentation, which is designed by the invention, comprises the following steps:
(a) Feeding with 750g/L food-grade glucose monohydrate solution as feed supplement liquid, wherein the feed supplement liquid needs steam wet heat sterilization for 20min under the conditions of 0.1MPa and 121 ℃;
(b) Regulating the tank pressure of the material supplementing tank to 0.1MPa, and keeping the tank pressure of the fermentation tank at 0.03MPa;
(c) Feeding glucose feed liquid in a quantitative feed supplement mode by adopting a peristaltic pump, controlling the peristaltic pump by adopting a PLC control system to perform feed supplement, monitoring the content of glucose and the dissolved oxygen in the fermentation tank by the PLC control system at the same time, and starting to supplement after the dissolved oxygen in the fermentation tank rapidly rises to 90%;
(d) After the feeding is started, measuring the glucose concentration in the tank every 1 hour, regulating the rotating speed of the peristaltic pump by a PLC control system according to the glucose content and dissolved oxygen amount in the fermentation tank, gradually increasing the feeding amount, and when the glucose concentration in the tank reaches 4.5g/L, not increasing the feeding speed any more, and when the glucose concentration in the tank exceeds 5g/L, reducing the feeding speed. The glucose concentration in the tank is controlled to be more than 4g/L, when the feeding speed is increased, the dissolved oxygen in the tank can be reduced, and at the moment, the ventilation quantity and the stirring rotating speed are increased to regulate the dissolved oxygen in the tank;
(e) The dissolved oxygen is controlled in a multistage manner in the fermentation process, wherein the process is 0 to 7 hours of fermentation, and the dissolved oxygen is 30 percent; 7 to 15 hours, and dissolved oxygen is 50 percent; 15 to 50h, and 40 percent of dissolved oxygen; 50 to 65 hours and 30 percent of dissolved oxygen.
After fermentation, the detection shows that the strain conversion rate reaches 42% and the glucosamine content reaches 115g/L in the fermentation process of the bacillus subtilis by the feeding method of the embodiment after fermentation reaches 65 hours.
Example 3
The invention relates to a third embodiment of a feeding method for producing glucosamine by bacillus subtilis fermentation, which comprises the following steps:
(a) Feeding with 800g/L food-grade glucose monohydrate solution as feed supplement liquid, wherein the feed supplement liquid needs steam wet heat sterilization for 20min under 0.1MPa at 121 ℃;
(b) Regulating the tank pressure of the material supplementing tank to 0.15MPa, and keeping the tank pressure of the fermentation tank at 0.05MPa;
(c) Feeding glucose feed liquid in a quantitative feed supplement mode by adopting a peristaltic pump, controlling the peristaltic pump by adopting a PLC control system to perform feed supplement, monitoring the content of glucose and the dissolved oxygen in the fermentation tank by the PLC control system at the same time, and starting to supplement after the dissolved oxygen in the fermentation tank rapidly rises to 90%;
(d) After the feeding is started, measuring the glucose concentration in the tank every 1 hour, regulating the rotating speed of the peristaltic pump by a PLC control system according to the glucose content and dissolved oxygen amount in the fermentation tank, gradually increasing the feeding amount, and when the glucose concentration in the tank reaches 4.5g/L, not increasing the feeding speed any more, and when the glucose concentration in the tank exceeds 5g/L, reducing the feeding speed. The glucose concentration in the tank is controlled to be 5g/L, when the feeding speed is increased, the dissolved oxygen in the tank is reduced, and at the moment, the ventilation quantity and the stirring rotating speed are increased to regulate the dissolved oxygen in the tank;
(e) The dissolved oxygen is controlled in a multistage manner in the fermentation process, wherein the process is 0 to 7 hours of fermentation, and the dissolved oxygen is 30 percent; 7 to 15 hours, and dissolved oxygen is 50 percent; 15 to 50h, and 40 percent of dissolved oxygen; 50 to 72 hours and 30 percent of dissolved oxygen.
After fermentation, the detection shows that the strain conversion rate reaches 45% and the glucosamine content reaches 120g/L in the fermentation process of the bacillus subtilis by the feeding method of the embodiment after fermentation reaches 65 hours.
The foregoing is merely illustrative of the present invention and is not intended to limit the scope of the invention, i.e., all such modifications and variations are within the scope of the invention as defined in the appended claims and their equivalents.
Claims (3)
1. A method for producing glucosamine by fermenting bacillus subtilis is characterized by comprising the following steps of (a) taking a food-grade glucose monohydrate solution with the concentration of 800g/L as a feed liquid for feeding;
(b) Regulating the tank pressure of the material supplementing tank to 0.05-0.15 MPa, and keeping the tank pressure of the fermentation tank at 0.03-0.05 MPa;
(c) Feeding a material supplementing liquid in a peristaltic pump quantitative material supplementing mode, controlling the peristaltic pump to supplement materials by adopting a PLC control system, monitoring the content of glucose and the dissolved oxygen in the fermentation tank by the PLC control system at the same time, and starting material supplementing after the dissolved oxygen in the fermentation tank rapidly rises to 90%;
(d) After the material supplementing starts, regulating the rotating speed of a peristaltic pump by a PLC control system according to the content of glucose and the dissolved oxygen amount in the fermentation tank, gradually increasing the material supplementing amount to control the glucose concentration in the tank to 3-5 g/L, reducing the dissolved oxygen in the tank when the material supplementing speed is increased, regulating the dissolved oxygen in the tank by increasing the ventilation amount and the stirring rotating speed at the moment, measuring the glucose concentration in the tank every 1 hour, regulating the rotating speed of the peristaltic pump, gradually increasing the material supplementing amount, and not increasing the material supplementing speed when the glucose concentration in the tank reaches 4.5g/L, and reducing the material supplementing speed when the glucose concentration in the tank exceeds 5 g/L;
(e) The dissolved oxygen is controlled in a plurality of sections in the fermentation process, wherein the control process of the dissolved oxygen is 0 to 7 hours of fermentation, and the dissolved oxygen is 30 percent; 7 to 15 hours, and dissolved oxygen is 50 percent; 15 to 50h, and 40 percent of dissolved oxygen; 50 to 65 hours and 30 percent of dissolved oxygen.
2. The method for feeding glucosamine by bacillus subtilis fermentation according to claim 1, wherein the feeding liquid is subjected to steam wet heat sterilization for 20min under the condition of 0.1MPa and 121 ℃.
3. The method for feeding glucosamine by fermentation of Bacillus subtilis according to claim 1, wherein the step (b) adjusts the tank pressure of the feeding tank to 0.1MPa, and maintains the tank pressure of the fermentation tank at 0.04MPa.
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Denomination of invention: A feeding method for producing glucosamine through Bacillus subtilis fermentation Effective date of registration: 20231215 Granted publication date: 20230627 Pledgee: Bank of Rizhao Co.,Ltd. Juxian sub branch Pledgor: DAZIRAN BIOLOGICAL GROUP Co.,Ltd. Registration number: Y2023980071960 |