CN112592944B - Production method of glucosamine - Google Patents

Production method of glucosamine Download PDF

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CN112592944B
CN112592944B CN202011495086.1A CN202011495086A CN112592944B CN 112592944 B CN112592944 B CN 112592944B CN 202011495086 A CN202011495086 A CN 202011495086A CN 112592944 B CN112592944 B CN 112592944B
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朱志春
陈必钦
严必能
李丹
胡丽娜
刘子睿
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INNER MONGOLIA KINGDOMWAY PHARMACEUTICAL CO Ltd
Xiamen Kingdomway Group Co
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Abstract

The invention belongs to the field of glucosamine production, and relates to a production method of glucosamine, which comprises the steps of monitoring at least one of oxygen consumption rate, oxidation-reduction potential, acetic acid concentration and specific carbon dioxide release rate in fermentation liquor on line and controlling the numerical value thereof in a specific range in stages in the fermentation process of the glucosamine. The method provided by the invention feeds back and regulates the process parameters by monitoring and controlling at least one of the oxygen consumption rate, the oxidation-reduction potential, the acetic acid concentration and the specific carbon dioxide release rate in the fermentation liquid on line, can meet the growth requirement of thalli, effectively promote the metabolic synthesis of glucosamine and improve the conversion rate, and has higher content and conversion rate of the glucosamine in the obtained fermentation product.

Description

Production method of glucosamine
Technical Field
The invention belongs to the field of glucosamine production, and particularly relates to a production method of glucosamine.
Background
The glucosamine is a constituent unit of various polysaccharides in organisms, has wide application field, is a medicament for treating rheumatic and rheumatoid arthritis clinically, can be used as a food antioxidant, an infant food additive and a sweetener for diabetics, can be used for clinically enhancing the function of a human immune system and inhibiting overgrowth of cancer cells or fibroblasts, has inhibiting and treating effects on cancers and malignant tumors, can effectively treat various inflammations, and has treating effects on osteoarthritis and arthralgia.
The current methods for producing glucosamine mainly comprise an acid hydrolysis method, an enzymolysis method and a microbial fermentation method. The acid hydrolysis method and the enzymolysis method for producing the glucosamine have adverse effects on the environment, low production efficiency and allergic hidden trouble. In recent years, the microbial fermentation method for producing glucosamine has been paid more attention to, but the microbial fermentation method has the phenomena of low yield, low conversion rate, more byproducts and the like, and a fermentation process with high yield and high conversion rate needs to be developed to meet the industrial production.
When glucose is used as a carbon source for expression or synthesis of a product, a large part of the carbon source is used for synthesizing byproducts, such as acetic acid, and when the concentration of acetic acid is accumulated to a certain extent, the effect on the growth of thalli and the synthesis of the product is large, and particularly when escherichia coli is used as a production strain, high-concentration acetic acid can have a serious inhibition effect. In addition, the conversion rate of the product is obviously reduced due to the fact that more byproducts are generated. At present, the domestic glucosamine fermentation process mainly feeds back process feed-back and process regulation and control by limiting the sugar-supplementing or using the dissolved oxygen level, the process control has the problem of lag, the yield and conversion rate of products are unstable, and the feed-back or oxygen supply regulation and control according to the dissolved oxygen level easily causes the problems of insufficient sugar-supplementing or more and higher or lower oxygen supply regulation. Insufficient sugar supplement affects the growth of thalli, excessive sugar supplement leads to increased byproducts of thalli synthesis, and the product conversion rate is low. The lower oxygen supply affects the growth of thalli and the synthesis rate of products, and the higher oxygen supply leads to the faster growth of thalli and the lower conversion rate of products.
Disclosure of Invention
The invention aims to solve the problems of low yield and conversion rate of glucosamine produced by the existing method, and provides a production method of glucosamine capable of improving the yield and the conversion rate.
After intensive and extensive research, the inventor of the invention finds that in the glucosamine fermentation process, the oxygen consumption rate, the oxidation-reduction potential, the acetic acid concentration and the specific carbon dioxide release rate in the fermentation liquid can well feed back the glucosamine fermentation process, and at least one of the four parameters is monitored on line and the numerical value is controlled within a specific range, so that the consistency of the thallus growth and the metabolite in the complicated glucosamine fermentation process can be effectively ensured, the stable and high-yield fermentation level is achieved, the process reproducibility is good, and the production cost can be effectively reduced. Based on this, the present invention has been completed.
Specifically, the invention provides a production method of glucosamine, wherein the method comprises the steps of monitoring at least one of oxygen consumption rate, oxidation-reduction potential, acetic acid concentration and specific carbon dioxide release rate in fermentation liquid on line and controlling the value thereof in the following range in the fermentation process of the glucosamine: the oxygen consumption rate is 10-150 mmol/L.h, the oxidation-reduction potential is-300-50 mV, the acetic acid concentration is 0.1-20 g/L, and the specific carbon dioxide release rate is 0.05-0.8.
Preferably, the oxygen consumption rate is controlled in stages and in the following manner: controlling the oxygen consumption rate to be 10-90 mmol/L.h during the fermentation culture for 0-8 h; controlling the oxygen consumption rate to be 60-150 mmol/L.h during the fermentation culture for 8-24 h; controlling the oxygen consumption rate to be 70-120 mmol/L.h during the fermentation culture for 24-40 h; and controlling the oxygen consumption rate to be 50-100 mmol/L.h during the period from 40h of fermentation culture to the end of fermentation.
Preferably, the oxygen consumption rate is controlled in stages and in the following manner: controlling the oxygen consumption rate to be 10-90 mmol/L.h during the fermentation culture for 0-8 h; controlling the oxygen consumption rate to be 60-120 mmol/L.h during the fermentation culture for 8-16 h; controlling the oxygen consumption rate to be 80-150 mmol/L.h during the fermentation culture for 16-24 h; controlling the oxygen consumption rate to be 70-120 mmol/L.h during the fermentation culture for 24-32 h; controlling the oxygen consumption rate to be 70-100 mmol/L.h during the fermentation culture for 32-40 h; controlling the oxygen consumption rate to be 70-90 mmol/L.h during the fermentation culture for 40-48 h; and controlling the oxygen consumption rate to be 50-80 mmol/L.h during the period from 48h of fermentation culture to the end of fermentation.
Preferably, the oxidation-reduction potential is controlled in stages, and the control manner is as follows: during the fermentation culture for 0-8 h, the oxidation-reduction potential is controlled at-100-50 mV; during the fermentation culture for 8-24 h, the oxidation-reduction potential is controlled to be minus 300 mV to minus 50mV; during the fermentation culture for 24-40 h, the oxidation-reduction potential is controlled to be minus 250 mV to minus 100mV; and controlling the oxidation-reduction potential to be-200 mV to-50 mV in the period from 40h of fermentation culture to the end of fermentation.
Preferably, the oxidation-reduction potential is controlled in stages, and the control manner is as follows: during the fermentation culture for 0-8 h, the oxidation-reduction potential is controlled at-100-50 mV; during the fermentation culture for 8-16 h, the oxidation-reduction potential is controlled to be-200 mV to-50 mV; during the fermentation culture for 16-24 h, the oxidation-reduction potential is controlled to be minus 300 mV to minus 50mV; during the fermentation culture for 24-32 h, the oxidation-reduction potential is controlled to be minus 250 mV to minus 100mV; during the fermentation culture for 32-40 h, the oxidation-reduction potential is controlled to be-200 mV to-100 mV; during the fermentation culture for 40-48 h, the oxidation-reduction potential is controlled to be-150 mV to-100 mV; and controlling the oxidation-reduction potential to be-150 mV to-50 mV in the period from 48h of fermentation culture to the end of fermentation.
Preferably, the acetic acid concentration is controlled in stages, and the control mode is as follows: acetic acid concentration is controlled to be 0.1-20 g/L in the period of 0-8 h of fermentation culture; acetic acid concentration is controlled to be 0.5-10 g/L during fermentation culture for 8-24 h; acetic acid concentration is controlled to be 0.3-10 g/L in the fermentation culture period of 24-40 h; and controlling the acetic acid concentration to be 0.2-10 g/L during the period from 40h of fermentation culture to the end of fermentation.
Preferably, the acetic acid concentration is controlled in stages, and the control mode is as follows: acetic acid concentration is controlled to be 0.1-20 g/L in the period of 0-8 h of fermentation culture; acetic acid concentration is controlled to be 0.5-10 g/L during fermentation culture for 8-16 h; acetic acid concentration is controlled to be 0.5-6 g/L during the fermentation culture for 16-24 h; acetic acid concentration is controlled to be 0.3-10 g/L in the fermentation culture period of 24-32 h; acetic acid concentration is controlled to be 0.3-8 g/L in the fermentation culture period of 32-40 h; acetic acid concentration is controlled to be 0.2-10 g/L in the fermentation culture period of 40-48 h; and controlling the acetic acid concentration to be 0.2-8 g/L during the period from 48h of fermentation culture to the end of fermentation.
Preferably, the specific carbon dioxide release rate is controlled in stages, and the control manner is as follows: controlling the release rate of specific carbon dioxide to be 0.05-0.8 in the period of fermenting and culturing for 0-8 h; controlling the release rate of specific carbon dioxide to be 0.1-0.6 during the fermentation culture for 8-24 h; controlling the release rate of specific carbon dioxide to be 0.1-0.5 during the fermentation culture for 24-40 h; and controlling the release rate of the specific carbon dioxide to be 0.05-0.3 in the period from 40h of fermentation culture to the end of fermentation.
Preferably, the specific carbon dioxide release rate is controlled in stages, and the control manner is as follows: controlling the release rate of specific carbon dioxide to be 0.05-0.8 in the period of fermenting and culturing for 0-8 h; controlling the release rate of specific carbon dioxide to be 0.1-0.6 during the fermentation culture for 8-16 h; controlling the release rate of specific carbon dioxide to be 0.15-0.4 during the fermentation culture for 16-24 h; controlling the release rate of specific carbon dioxide to be 0.15-0.5 during the fermentation culture for 24-32 h; controlling the release rate of specific carbon dioxide to be 0.1-0.5 during the fermentation culture for 32-40 h; controlling the release rate of specific carbon dioxide to be 0.05-0.3 in the fermentation culture period of 40-48 hours; and controlling the release rate of the specific carbon dioxide to be 0.05-0.15 in the period from 48 hours of fermentation culture to the end of fermentation.
Preferably, the numerical ranges of the oxygen consumption rate, the oxidation-reduction potential, the acetic acid concentration, and the specific carbon dioxide release rate in the fermentation liquid are controlled by adjusting at least one of the air flow rate, the rotation speed, and the tank pressure.
Preferably, the strain used for the glucosamine fermentation is at least one selected from the group consisting of Escherichia coli (Escherichia coli), bacillus subtilis (Bacillus subtilis), saccharomyces cerevisiae (Saccharomyces cerevisiae), rhizopus oligosporus (Rhizopus oligosporus), aspergillus sp, and Mucor (Monascus pilosus).
Preferably, the production method of the glucosamine provided by the invention further comprises the steps of adding alkali in the fermentation process to control the pH value of a fermentation system to be 5.5-7.5, adding a carbon source and a nitrogen source to control the concentration of the carbon source in the fermentation system to be 0.1-15 g/L and the concentration of the nitrogen source to be 0.01-2 g/L; the carbon source is at least one selected from glucose, sucrose and glycerol, and the nitrogen source is ammonium sulfate and/or yeast extract powder.
Preferably, the glucosamine fermentation comprises the steps of:
(1) Seed activation: absorbing bacterial liquid from a seed retaining tube for gradient dilution, absorbing the diluted bacterial suspension into a flat plate culture medium, and culturing for 12-72 hours at 28-38 ℃ to obtain a mature single colony;
(2) Shake flask culture: 1 to 20 single colonies are picked from a mature flat plate culture medium and are connected into a shake flask culture medium, the shake flask culture conditions comprise a culture temperature of 28 to 38 ℃, a rotation speed of 150 to 250rpm and a culture period of 4 to 48 hours, when the wet weight of thalli reaches 1 to 20g/L, the thalli is transferred to a seed tank, and the inoculum size is controlled to be 0.1 to 5 percent;
(3) Seed expansion culture: the seed culture conditions comprise a culture temperature of 28-38 ℃, a pot pressure of 0.025-0.08 MPa, an aeration ratio of 0.2-2 VVM, a rotating speed of 100-500 rpm, a culture period of 4-48 hours, and when the wet weight of the thalli reaches 1-20 g/L, the thalli is transferred into a fermentation tank, and the inoculum size is controlled at 5-30%;
(4) Culturing in a fermentation tank: the fermentation culture conditions comprise culture temperature 28-38 ℃, tank pressure 0.02-0.08 MPa, aeration ratio 0.2-2 VVM, rotation speed 50-500 rpm, and fermentation process by adding alkali to control pH value of the fermentation system at 5.5-7.5, adding carbon source and nitrogen source to control carbon source concentration in the fermentation system at 0.1-15 g/L and nitrogen source concentration at 0.01-2 g/L, stopping fermentation when the product growth rate is obviously slowed down or the thallus is shallow; the carbon source is at least one selected from glucose, sucrose and glycerol, and the nitrogen source is ammonium sulfate and/or yeast extract powder.
The method provided by the invention feeds back and regulates the process parameters by monitoring and controlling at least one of the oxygen consumption rate, the oxidation-reduction potential, the acetic acid concentration and the specific carbon dioxide release rate in the fermentation liquid on line, can meet the growth requirement of thalli, effectively promote the metabolic synthesis of glucosamine and improve the conversion rate, and has higher yield and conversion rate of the glucosamine in the obtained fermentation product.
Detailed Description
In some embodiments of the invention, the oxygen consumption rate, oxidation-reduction potential, acetic acid concentration and specific carbon dioxide release rate in the fermentation broth are regulated in four stages (namely 0-8 h, 8-24 h, 24-40 h and 40h to the end of fermentation). 0 to 8h include 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h and 16h, 10h … … h and 24h and 25h and 26h … … h and 40h and 16h,40h and 40h include 40h and 41h … … fermentation end.
In some embodiments of the invention, the oxygen consumption rate, oxidation-reduction potential, acetic acid concentration and specific carbon dioxide release rate in the fermentation broth are regulated in seven stages (namely 0-8 h, 8-16 h, 16-24 h, 24-32 h, 32-40 h, 40-48 h and 48h to the end of fermentation). It should be noted that the number of the substrates, 0 to 8h including 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h and 8h,8 to 16h including 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h and 8h,16 to 24h including 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h and 8h,24 to 32h including 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h and 8h,32 h and 34h, 35h, 36h, 37h, 38h, 39h, 40h and 48h including 41h, 42h, 43h, 44h, 45h, 46h, 47h, 48h and finishing fermentation.
In some embodiments of the invention, the manner in which the rate of oxygen consumption is controlled in stages is as follows: during the fermentation culture for 0 to 8 hours, the oxygen consumption rate is controlled to 10 to 90 mmol/L.h, and for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 mmol/L.h, etc. can be used; during the fermentation culture for 8 to 24 hours, the oxygen consumption rate is controlled to 60 to 150 mmol/L.h, for example, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 mmol/L.h, etc.; the oxygen consumption rate is controlled to 70 to 120 mmol/L.h during the fermentation culture for 24 to 40 hours, and may be, for example, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 mmol/L.h, etc.; the oxygen consumption rate is controlled to 50 to 100 mmol/L.multidot.h from the fermentation culture time of 40 hours to the fermentation completion, and may be, for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mmol/L.multidot.h, etc.
In some embodiments of the invention, the manner in which the rate of oxygen consumption is controlled in stages is as follows: during the fermentation culture for 0 to 8 hours, the oxygen consumption rate is controlled to 10 to 90 mmol/L.h, and for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 mmol/L.h, etc. can be used; during the fermentation culture for 8 to 16 hours, the oxygen consumption rate is controlled to 60 to 120 mmol/L.h, and for example, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 mmol/L.h, etc. can be used; during the fermentation culture for 16 to 24 hours, the oxygen consumption rate is controlled to 80 to 150 mmol/L.h, for example, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 mmol/L.h, etc.; the oxygen consumption rate is controlled to 70 to 120 mmol/L.h during the fermentation culture for 24 to 32 hours, and may be, for example, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 mmol/L.h, etc.; the oxygen consumption rate is controlled to be 70 to 100 mmol/L.h during the fermentation culture for 32 to 40 hours, and may be, for example, 70, 75, 80, 85, 90, 95, 100 mmol/L.h, etc.; during the fermentation culture for 40 to 48 hours, the oxygen consumption rate is controlled to 70 to 90 mmol/L.h, for example, 70, 75, 80, 85, 90 mmol/L.h, etc.; the oxygen consumption rate is controlled to 50 to 80 mmol/L.multidot.h from 48 hours to the end of fermentation, and may be, for example, 50, 55, 60, 65, 70, 75, 80 mmol/L.multidot.h. The fermentation has different environmental conditions and different thallus metabolic demands in different periods, and the oxygen consumption rate can be controlled in stages so as to provide the most suitable culture conditions for thallus product synthesis.
In the present invention, the oxygen consumption rate (OUR) is calculated according to the following formula:
F in : ventilation (L/h or m) 3 /h)；
V: volume of fermentation broth (L or m) 3 )；
Co 2in : oxygen concentration (%) in intake air;
C idle in : concentration (%) of inert gas in the intake air;
Co 2out : oxygen concentration (%) in the exhaust gas of the fermentation broth;
Cco 2out : carbon dioxide concentration (%) in the effluent gas of the fermentation broth.
Wherein Co is 2in 、C Idle in 、Co 2out 、Cco 2 out is monitored in real time by a mass spectrometer.
In some embodiments of the invention, the manner in which the redox potential is controlled in stages is as follows: during the fermentation culture for 0-8 hours, the oxidation-reduction potential is controlled to be-100-50 mV, for example, -100, -90, -80, -70, -60, -50, -40, -30, -20, -10, 0, 10, 20, 30, 40, 50mV and the like; during the fermentation culture for 8-24 hours, the oxidation-reduction potential is controlled to be-300 to-50 mV, for example, -300, -290, -280, -270, -260, -250, -240, -230, -220, -210, -200, -190, -180, -170, -160, -150, -140, -130, -120, -110, -100, -90, -80, -70, -60, -50mV and the like; during the fermentation culture for 24-40 h, the oxidation-reduction potential is controlled to be-250 to-100 mV, for example, the oxidation-reduction potential can be-250, -240, -230, -220, -210, -200, -190, -180, -170, -160, -150, -140, -130, -120, -110, -100mV and the like; during the period from 40h to the end of fermentation, the oxidation-reduction potential is controlled to be-200 to-50 mV, and can be, for example, -200, -190, -180, -170, -160, -150, -140, -130, -120, -110, -100, -90, -80, -70, -60, -50mV, etc.
In some embodiments of the invention, the manner in which the redox potential is controlled in stages is as follows: during the fermentation culture for 0-8 hours, the oxidation-reduction potential is controlled to be-100-50 mV, for example, -100, -90, -80, -70, -60, -50, -40, -30, -20, -10, 0, 10, 20, 30, 40, 50mV and the like; during the fermentation culture for 8-16 h, the oxidation-reduction potential is controlled to be-200 to-50 mV, for example, -200, -190, -180, -170, -160, -150, -140, -130, -120, -110, -100, -90, -80, -70, -60, -50mV and the like; during the fermentation culture for 16-24 hours, the oxidation-reduction potential is controlled to be-300 to-50 mV, for example, -300, -290, -280, -270, -260, -250, -240, -230, -220, -210, -200, -190, -180, -170, -160, -150, -140, -130, -120, -110, -100, -90, -80, -70, -60, -50mV and the like; during the fermentation culture for 24-32 h, the oxidation-reduction potential is controlled to be-250 to-100 mV, for example, -250, -240, -230, -220, -210, -200, -190, -180, -170, -160, -150, -140, -130, -120, -110, -100mV and the like; during the fermentation culture for 32-40 h, the oxidation-reduction potential is controlled to be-200 to-100 mV, for example, -200, -190, -180, -170, -160, -150, -140, -130, -120, -110, -100mV and the like; during the fermentation culture for 40-48 h, the oxidation-reduction potential is controlled to be-150 to-100 mV, for example, -150, -140, -130, -120, -110, -100mV and the like; during the period from 48 hours of fermentation culture to the end of fermentation, the oxidation-reduction potential is controlled to be-150 to-50 mV, and can be, for example, -150, -140, -130, -120, -110, -100, -90, -80, -70, -60, -50mV, etc. The oxidation-reduction potential is controlled in stages according to different environmental conditions and different thallus metabolic demands of fermentation in different periods, so that the optimal culture conditions can be provided for thallus product synthesis.
In the present invention, the redox potential is measured by means of a redox potential electrode.
In some embodiments of the invention, the acetic acid concentration is controlled in stages in the following manner: during the fermentation culture for 0 to 8 hours, the acetic acid concentration is controlled to be 0.1 to 20g/L, for example, may be 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20g/L, etc.; during the fermentation culture for 8-24 hours, the acetic acid concentration is controlled to be 0.5-10 g/L, for example, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10g/L, etc.; during the fermentation culture for 24-40 hours, the acetic acid concentration is controlled to be 0.3-10 g/L, for example, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10g/L and the like; the acetic acid concentration is controlled to be 0.2 to 10g/L, for example, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10g/L, etc. in the period from 40 hours of fermentation culture to the end of fermentation.
In some embodiments of the invention, the acetic acid concentration is controlled in stages in the following manner: during the fermentation culture for 0 to 8 hours, the acetic acid concentration is controlled to be 0.1 to 20g/L, for example, may be 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20g/L, etc.; during the fermentation culture for 8-16 hours, the acetic acid concentration is controlled to be 0.5-10 g/L, for example, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10g/L, etc.; during the fermentation culture for 16-24 hours, the acetic acid concentration is controlled to be 0.5-6 g/L, for example, 0.5, 1, 2, 3, 4, 5, 6g/L and the like; during the fermentation culture for 24-32 hours, the acetic acid concentration is controlled to be 0.3-10 g/L, for example, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10g/L and the like; during the fermentation culture for 32 to 40 hours, the acetic acid concentration is controlled to be 0.3 to 8g/L, for example, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8g/L, etc.; during the fermentation culture for 40-48 hours, the acetic acid concentration is controlled to be 0.2-10 g/L, for example, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10g/L and the like; the acetic acid concentration is controlled to be 0.2 to 8g/L, for example, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8g/L, etc. in the period from 48 hours of fermentation culture to the end of fermentation. The fermentation has different environmental conditions and different thallus metabolic demands, and the acetic acid concentration can be controlled in stages to provide optimal culture conditions for thallus product synthesis.
In the present invention, the acetic acid concentration is measured in real time by an acetic acid on-line meter (e.g., a BRS-CH COOH fully automatic acetic acid concentration meter).
In some embodiments of the invention, the specific carbon dioxide release rate is controlled in stages as follows: during the fermentation culture for 0 to 8 hours, the specific carbon dioxide release rate is controlled to be 0.05 to 0.8, and for example, may be 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, etc.; the specific carbon dioxide release rate is controlled to be 0.1 to 0.6 during the fermentation culture for 8 to 24 hours, and may be, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, etc.; the specific carbon dioxide release rate is controlled to be 0.1 to 0.5 during the fermentation culture for 24 to 40 hours, and may be, for example, 0.1, 0.2, 0.3, 0.4, 0.5, etc.; the specific carbon dioxide release rate is controlled to be 0.05 to 0.3, for example, 0.05, 0.1, 0.2, 0.3, etc. in the period from 40 hours of fermentation culture to the end of fermentation.
In some embodiments of the invention, the specific carbon dioxide release rate is controlled in stages as follows: during the fermentation culture for 0-8 hours, the release rate of the fermentation process relative to the carbon dioxide is controlled to be 0.05-0.8, for example, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and the like; during the fermentation culture for 8-16 h, the release rate of the fermentation process to carbon dioxide is controlled to be 0.1-0.6, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and the like; during the fermentation culture for 16-24 hours, the release rate of the fermentation process to carbon dioxide is controlled to be 0.15-0.4, for example, 0.15, 0.2, 0.3, 0.4 and the like; during the fermentation culture for 24-32 h, the release rate of the fermentation process to carbon dioxide is controlled to be 0.15-0.5, for example, 0.15, 0.2, 0.3, 0.4, 0.5 and the like; during the fermentation culture for 32-40 hours, the release rate of the fermentation process to carbon dioxide is controlled to be 0.1-0.5, for example, 0.1, 0.2, 0.3, 0.4, 0.5 and the like; during the fermentation culture for 40-48 hours, the release rate of the fermentation process to carbon dioxide is controlled to be 0.05-0.3, for example, 0.05, 0.1, 0.2, 0.3 and the like; the fermentation process ratio carbon dioxide release rate is controlled to be 0.05 to 0.15, for example, 0.05, 0.1, 0.15, etc. in the period from 48 hours of fermentation culture to the end of fermentation. The environmental conditions and the bacterial metabolic demands of the fermentation are different in different periods, and the carbon dioxide release rate is controlled by stages, so that the optimal culture conditions can be provided for synthesizing bacterial products.
In the present invention, the specific carbon dioxide release rate is calculated by the following formula:
C CO2 : carbon dioxide concentration (%) in the waste gas discharged from the fermentation broth, which is monitored in real time by a mass spectrometer;
v: volume of fermentation broth (L or m) 3 )；
dt: unit fermentation time (h);
DCW: the thallus content (g/L) of the fermentation liquid is monitored in real time by a living cell sensor.
In the present invention, the strain used for the fermentation culture may be any of various existing strains suitable for producing glucosamine, and specific examples thereof include, but are not limited to: at least one of Escherichia coli (Escherichia coli ACCC 01548), bacillus subtilis (Bacillus subtilis ACCC 10242), saccharomyces cerevisiae (Saccharomyces cerevisiae ACCC 20037), rhizopus oligosporus (Rhizopus oligosporus ACCC 30496), aspergillus (Aspergillus sp ACCC 30005), mucor (Monascus pilosus ACCC 30504), etc. The fermentation process flow of the glucosamine comprises seed activation, shake flask culture, seed expansion culture and fermentation tank culture, wherein the formula of the culture medium adopted in each stage is not particularly limited, and the culture medium can be selected conventionally in the field, and a proper strain growth culture medium can be selected according to different strains, so that the culture medium can be known to those skilled in the art, and is not repeated herein.
In a specific embodiment, the strain is escherichia coli (Escherichia coli ACCC 01548), and the specific process flow of the fermentation culture is as follows:
(1) Seed activation: preparing a flat culture medium, sterilizing for 20-30 min at 121-123 ℃, and adjusting the pH value to 5.5-7.5 before sterilization; a small amount of bacterial liquid is absorbed from a seed retaining tube for gradient dilution, and the bacterial suspension after a small amount of dilution is absorbed into a flat plate culture medium, and is cultured for 12 to 72 hours at the temperature of 28 to 38 ℃ to obtain a mature single colony; the components of the plate culture medium are as follows: 1-10 g/L of sodium chloride, 1-15 g/L of peptone, 1-15 g/L of yeast extract powder and 10-20 g/L of agar powder;
(2) Shake flask culture: preparing a shake flask culture medium, sterilizing for 20-30 min at 121-123 ℃, and adjusting the pH value to 5.5-7.5 before sterilization; 1 to 20 single colonies are picked from a mature flat plate culture medium and are connected into a shake flask culture medium, the shake flask culture conditions comprise a culture temperature of 28 to 38 ℃, a rotation speed of 150 to 250rpm and a culture period of 4 to 48 hours, when the wet weight of thalli reaches 1 to 20g/L, the thalli is transferred to a seed tank, and the inoculum size is controlled to be 0.1 to 5 percent; the shake flask culture medium comprises the following components: 1-10 g/L of sodium chloride, 1-15 g/L of peptone and 1-15 g/L of yeast extract powder;
(3) Seed expansion culture: preparing a seed culture medium, sterilizing for 20-30 min at 121-123 ℃, and adjusting the pH value to 5.5-7.5 before sterilization; the seed culture condition is that the culture temperature is 28-38 ℃, the tank pressure is 0.025-0.08 MPa, the aeration ratio is 0.2-2 VVM, the rotating speed is 100-500 rpm, the culture period is 4-48 hours, when the wet weight of the thalli reaches 1-20 g/L, the thalli is transferred into the fermentation tank, and the inoculation amount is controlled at 5-30%; the seed culture medium comprises the following components: glucose 1-15 g/L, sodium chloride 1-10 g/L, peptone 1-15 g/L, yeast extract 1-15 g/L;
(4) Culturing in a fermentation tank: preparing a fermentation medium, sterilizing for 20-30 min at 121-123 ℃, and adjusting the pH value to 5.5-7.5 before sterilization; the fermentation culture conditions are that the culture temperature is 28-38 ℃, the tank pressure is 0.02-0.08 MPa, the aeration ratio is 0.2-2 VVM, the rotating speed is 50-500 rpm, the pH value of a fermentation system is controlled to be 5.5-7.5 by adding alkali in the fermentation process, the carbon source and the nitrogen source are added to control the concentration of the carbon source in the fermentation system to be 0.1-15 g/L, and the concentration of the nitrogen source is controlled to be 0.01-2 g/L; the components of the fermentation medium are as follows: glucose 1-20 g/L, sodium dihydrogen phosphate 1-20 g/L, magnesium chloride 0.5-10 g/L, sodium sulfate 0.5-10 g/L, yeast extract 1-15 g/L, calcium sulfate 0.01-5 g/L, zinc chloride 0.005-0.5 g/L, and manganese sulfate 0.0001-0.5 g/L. Wherein the carbon source may be selected from at least one of glucose, sucrose and glycerol. The nitrogen source may be ammonium sulfate and/or yeast extract. The carbon source and nitrogen source may be added in the form of a feed medium. In one embodiment, the feed medium comprises the following components in mass concentration: glucose, sucrose or glycerin 50-60%, ammonia water 20-28%, sodium hydroxide 10-40%, ammonium sulfate 0.1-5%, and yeast extract powder 0.1-5%.
The fermentation process monitors at least one of oxygen consumption rate, oxidation-reduction potential, acetic acid concentration and specific carbon dioxide release rate in fermentation liquid in real time to feed back and regulate the glucosamine fermentation process, and maintains the oxygen consumption rate, the oxidation-reduction potential, the acetic acid concentration and the specific carbon dioxide release rate in a preset range by increasing or decreasing at least one of rotating speed, air flow and tank pressure.
As a standard for terminating fermentation when the rate of product growth is significantly slowed or the bacterial stain is shallow, HPLC is used to measure the concentration of glucosamine in the fermentation broth, and Athena NH made of stainless steel can be used 2 The column measures the concentration of glucosamine. In one embodiment, the diameter of the column is 4.6mm, the height is 250mm, and the packing size in the column is 3 μm; the wavelength for measuring the concentration of the glucosamine is 195nm; the mobile phase comprises 80% chromatographic acetonitrile and 20% dipotassium hydrogen phosphate solution with the concentration of 2.68 g/L; the flow rate of the mobile phase is controlled to be about 13 minutes for glucosamine retention time, typically 0.8mL/min, and the measurement temperature is maintained at 35 ℃.
The following detailed description of embodiments of the invention is intended to be illustrative of the invention and is not to be taken as limiting the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The strain used in examples 1 to 18 and examples 1-1 to 5-1 was E.coli (Escherichia coli ACCC 01548).
Example 1:100L tank fermentation production process
(1) Seed activation
And (3) sucking a small amount of bacterial liquid from the seed holding tube for gradient dilution, sucking a small amount of bacterial suspension onto a flat culture medium, and culturing at 32 ℃ for 24 hours to obtain a mature single colony. The plate culture medium comprises the following components: 5g/L of sodium chloride, 10g/L of peptone, 10g/L of yeast extract powder and 20g/L of agar powder, adjusting the pH value to 7.5 before sterilization, and sterilizing at 121 ℃ for 25min.
(2) Shake flask culture
3 single colonies are picked from the mature flat plate culture medium and put into a shake flask filled with 100mL shake flask culture medium, the shake flask is a 1L triangular flask, the shake flask is placed on a shaking table for culture, the culture temperature is 32 ℃, the rotation speed is 220rpm, the culture is carried out for 20 hours, and when the wet weight of thalli reaches 8g/L, the thalli can be moved into a seed tank for culture. The shake flask culture medium comprises the following components: 5g/L of sodium chloride, 10g/L of peptone and 10g/L of yeast extract powder, adjusting the pH value to 7.5 before sterilization, and sterilizing at 121 ℃ for 25min.
(3) Seed expansion culture
Preparing a seed culture medium, sterilizing at 121 ℃ for 25min, adjusting the pH value to 7.5 before sterilization, inoculating the shake flask seed liquid to a 15L seed tank according to the inoculation amount of 1%, and filling the liquid amount to 9L. The seed culture medium comprises the following components: glucose 10g/L, sodium chloride 5g/L, peptone 10g/L, yeast extract 10g/L. The seed culture conditions comprise a culture temperature of 32 ℃, a pot pressure of 0.05MPa, an aeration ratio of 1.5VVM and a rotation speed of 500rpm, the pH value is controlled to be about 7.5 by adding ammonia water in the culture process, the culture period is 12 hours, and when the wet weight reaches 10g/L, the seed can be moved into a fermentation tank for culture.
(4) Fermentation culture
Preparing a fermentation medium, sterilizing at 121 ℃ for 25min, adjusting the pH value to 7.5 before sterilization, transferring the seed tank seed liquid to a 100L fermentation tank according to 15% of inoculation amount, and keeping the liquid loading amount to be 50L. Initial culture conditions: the culture temperature is 32 ℃, the rotating speed is 200rpm, the aeration ratio is 0.5VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 7.5 in the fermentation culture process by supplementing ammonia water, and the glucose solution with the concentration of 50% and the ammonium sulfate with the concentration of 0.2% are fed in the fermentation culture process so as to respectively control the content of a carbon source and a nitrogen source to be 0.6g/L and 0.8g/L.
The components of the fermentation medium are 15g/L glucose, 15g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 10g/L yeast extract powder, 2.5g/L calcium sulfate, 0.25g/L zinc chloride and 0.25g/L manganese sulfate.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the concentration of acetic acid in fermentation liquor, and controlling the concentration of acetic acid in the following range by increasing or decreasing at least one of rotating speed, air quantity and tank pressure:
acetic acid concentration is controlled to be 0.1-20 g/L in the period of 0-8 h of fermentation culture;
acetic acid concentration is controlled to be 0.5-10 g/L during fermentation culture for 8-24 h;
acetic acid concentration is controlled to be 0.3-10 g/L in the fermentation culture period of 24-40 h;
And controlling the acetic acid concentration to be 0.2-10 g/L in the period from 40h of fermentation culture to the end of fermentation.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the fermentation is stopped, the content of the glucosamine in the fermentation liquid is measured by using HPLC, the content of the glucosamine reaches 125g/L when the fermentation is stopped, and the conversion rate reaches 45 percent.
Example 2:5m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 7.2, and transferring seed solution of seed tank to 5m 3 Fermentation tank, liquid loading amount is 2.5m 3 . Initial culture conditions: the culture temperature is 32 ℃, the rotating speed is 150rpm, the aeration ratio is 0.5VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 7.2 in the fermentation culture process by supplementing ammonia water, and the glucose solution with the concentration of 55% and the ammonium sulfate with the concentration of 0.4% are fed in the fermentation culture process so as to respectively control the content of a carbon source and a nitrogen source to be 0.5g/L and 1.1g/L.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 10g/L magnesium chloride, 10g/L sodium sulfate, 15g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.3g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 7.2 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the concentration of acetic acid in fermentation liquor, and controlling the concentration of acetic acid in the following range by increasing or decreasing at least one of rotating speed, air quantity and tank pressure:
acetic acid concentration is controlled to be 0.1-20 g/L in the period of 0-8 h of fermentation culture;
acetic acid concentration is controlled to be 0.5-10 g/L during fermentation culture for 8-16 h;
acetic acid concentration is controlled to be 0.5-6 g/L during the fermentation culture for 16-24 h;
acetic acid concentration is controlled to be 0.3-10 g/L in the fermentation culture period of 24-32 h;
acetic acid concentration is controlled to be 0.3-8 g/L in the fermentation culture period of 32-40 h;
acetic acid concentration is controlled to be 0.2-10 g/L in the fermentation culture period of 40-48 h;
and controlling the acetic acid concentration to be 0.2-8 g/L in the period from 48h of fermentation culture to the end of fermentation.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the fermentation is stopped, the content of the glucosamine in the fermentation liquid is measured by using HPLC, the content of the glucosamine reaches 130g/L when the fermentation is stopped, and the conversion rate reaches 45 percent.
Example 1-1:5m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 7.2, and transferring seed solution of seed tank to 5m 3 Fermentation tank, liquid loading amount is 2.5m 3 . Initial culture conditions: the culture temperature is 32 ℃, the rotating speed is 150rpm, the aeration ratio is 0.5VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 7.2 by supplementing ammonia water in the fermentation culture process, and the glucose solution with the concentration of 55 percent and the ammonium sulfate with the concentration of 0.4 percent are fed in the fermentation culture process so as to respectively control the content of a carbon source and a nitrogen source to be 0.5g/L and 0.4 percent1.1g/L。
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 10g/L magnesium chloride, 10g/L sodium sulfate, 15g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.3g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 7.2 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the concentration of acetic acid in fermentation liquor, and controlling the concentration of acetic acid in the following range by increasing or decreasing at least one of rotating speed, air quantity and tank pressure:
acetic acid concentration is controlled to be 0.1-20 g/L in the period of 0-8 h of fermentation culture;
acetic acid concentration is controlled to be 0.5-10 g/L during fermentation culture for 8-16 h;
acetic acid concentration is controlled to be 0.5-6 g/L during the fermentation culture for 16-24 h;
acetic acid concentration is controlled to be 10-20 g/L in the fermentation culture period of 24-32 h;
Acetic acid concentration is controlled to be 0.1-0.2 g/L in the fermentation culture period of 32-40 h;
acetic acid concentration is controlled to be 0.2-10 g/L in the fermentation culture period of 40-48 h;
and controlling the acetic acid concentration to be 0.2-8 g/L in the period from 48h of fermentation culture to the end of fermentation.
When the product growth rate is obviously slowed down or the bacterial stain is shallow, the fermentation is stopped, the content of the glucosamine in the fermentation liquid is measured by using HPLC, the content of the glucosamine reaches 100g/L when the fermentation is ended, the conversion rate reaches 35 percent, and compared with the example 2, the partial interval is not controlled within the acetic acid concentration requirement range, and the fermentation level and the conversion rate of the glucosamine are lower than those of the example 2.
Example 3:60m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 60m 3 A fermentation tank with liquid loading amount of30m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 150rpm, the aeration ratio is 0.8VVM, the tank pressure is 0.04MPa, the pH value is controlled to be about 6.0 in the fermentation culture process by supplementing ammonia water, and the concentration of a glucose solution with concentration of 60% and ammonium sulfate with concentration of 1% are fed in the fermentation culture process so as to respectively control the content of a carbon source and a nitrogen source to be 0.25g/L and 0.7g/L.
The components of the fermentation medium are 15g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 10g/L yeast extract powder, 2g/L calcium sulfate, 0.2g/L zinc chloride and 0.2g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the oxygen consumption rate in the fermentation liquid, and controlling the oxygen consumption rate in the fermentation liquid within the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
controlling the oxygen consumption rate to be 10-90 mmol/L.h during the fermentation culture for 0-8 h;
controlling the oxygen consumption rate to be 60-150 mmol/L.h during the fermentation culture for 8-24 h;
controlling the oxygen consumption rate to be 70-120 mmol/L.h during the fermentation culture for 24-40 h;
and controlling the oxygen consumption rate to be 50-100 mmol/L.h in the period from 40h to the end of fermentation culture.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 140g/L when the fermentation is ended, and the conversion rate reaches 50%.
Example 4:60m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 60m 3 Fermentation tank, liquid loading amount is 30m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 150rpm, the aeration ratio is 0.8VVM, the tank pressure is 0.04MPa, and the fermentation culture is completedThe pH value is controlled to be about 6.0 by supplementing ammonia water, and the concentration of a glucose solution with 60 percent and the concentration of ammonium sulfate with 1 percent are fed in the fermentation culture process so as to respectively control the content of a carbon source and a nitrogen source to be 0.4g/L and 0.65g/L.
The components of the fermentation medium are 15g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 10g/L yeast extract powder, 2g/L calcium sulfate, 0.2g/L zinc chloride and 0.2g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the oxygen consumption rate in the fermentation liquid, and controlling the oxygen consumption rate in the fermentation liquid within the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
controlling the oxygen consumption rate to be 10-90 mmol/L.h during the fermentation culture for 0-8 h;
controlling the oxygen consumption rate to be 60-120 mmol/L.h during the fermentation culture for 8-16 h;
Controlling the oxygen consumption rate to be 80-150 mmol/L.h during the fermentation culture for 16-24 h;
controlling the oxygen consumption rate to be 70-120 mmol/L.h during the fermentation culture for 24-32 h;
controlling the oxygen consumption rate to be 70-100 mmol/L.h during the fermentation culture for 32-40 h;
controlling the oxygen consumption rate to be 70-90 mmol/L.h during the fermentation culture for 40-48 h;
and controlling the oxygen consumption rate to be 50-80 mmol/L.h during the period from 48h of fermentation culture to the end of fermentation.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 150g/L when the fermentation is ended, and the conversion rate reaches 50%.
Example 2-1:60m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, and sterilizingH value was adjusted to 6.0, and the seed tank seed solution was transferred to 60m at 20% inoculum size 3 Fermentation tank, liquid loading amount is 30m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 150rpm, the aeration ratio is 0.8VVM, the tank pressure is 0.04MPa, the pH value is controlled to be about 6.0 in the fermentation culture process by supplementing ammonia water, and the concentration of a glucose solution with concentration of 60% and ammonium sulfate with concentration of 1% are fed in the fermentation culture process so as to respectively control the content of a carbon source and a nitrogen source to be 0.4g/L and 0.65g/L.
The components of the fermentation medium are 15g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 10g/L yeast extract powder, 2g/L calcium sulfate, 0.2g/L zinc chloride and 0.2g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the oxygen consumption rate in the fermentation liquid, and controlling the oxygen consumption rate in the fermentation liquid within the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
controlling the oxygen consumption rate to be 10-90 mmol/L.h during the fermentation culture for 0-8 h;
controlling the oxygen consumption rate to be 60-120 mmol/L.h during the fermentation culture for 8-16 h;
controlling the oxygen consumption rate to be 60-70 mmol/L.h during the fermentation culture for 16-24 h;
controlling the oxygen consumption rate to be 120-150 mmol/L.h during the fermentation culture for 24-32 h;
controlling the oxygen consumption rate to be 50-60 mmol/L.h during the fermentation culture for 32-40 h;
controlling the oxygen consumption rate to be 70-100 mmol/L.h during the fermentation culture for 40-48 h;
and controlling the oxygen consumption rate to be 50-80 mmol/L.h during the period from 48h of fermentation culture to the end of fermentation.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 110g/L when the fermentation is ended, and the conversion rate reaches 35 percent. In contrast to example 4, the partial interval was not controlled within the oxygen consumption rate requirement, and both the glucosamine fermentation level and conversion were lower than in example 4.
Example 5:120m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 120m 3 Fermentation tank with liquid loading of 60m 3 . Initial culture conditions: the culture temperature is 30 ℃, the rotating speed is 60rpm, the aeration ratio is 0.5VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 in the fermentation culture process by supplementing ammonia water, and the glucose solution with the concentration of 60% and the yeast extract with the concentration of 0.3% are fed in the fermentation culture process so as to respectively control the content of a carbon source and a nitrogen source to be 0.25g/L and 0.8g/L.
The components of the fermentation medium are 12g/L glucose, 12g/L sodium dihydrogen phosphate, 8g/L magnesium chloride, 8g/L sodium sulfate, 12g/L yeast extract powder, 2.5g/L calcium sulfate, 0.3g/L zinc chloride and 0.3g/L manganese sulfate, and the pH value is adjusted to 6.0 before sterilization under 121 ℃ for 25 min.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the oxidation-reduction potential, and the oxidation-reduction potential is controlled in the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
during the fermentation culture for 0-8 h, the oxidation-reduction potential is controlled at-100-50 mV;
during the fermentation culture for 8-24 h, the oxidation-reduction potential is controlled to be minus 300 mV to minus 50mV;
during the fermentation culture for 24-40 h, the oxidation-reduction potential is controlled to be minus 250 mV to minus 100mV;
and controlling the oxidation-reduction potential to be-200 mV to-50 mV in the period from 40h of fermentation culture to the end of fermentation.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 135g/L when the fermentation is ended, and the conversion rate reaches 44%.
Example 6:120m 3 Tank fermentation productionProcess for producing a solid-state image sensor
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 120m 3 Fermentation tank with liquid loading of 60m 3 . Initial culture conditions: the culture temperature is 30 ℃, the rotating speed is 60rpm, the aeration ratio is 0.5VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 in the fermentation culture process by supplementing ammonia water, and the glucose solution with the concentration of 60% and the yeast extract with the concentration of 0.3% are fed in the fermentation culture process so as to respectively control the content of a carbon source and a nitrogen source to be 0.8g/L and 1.2g/L.
The components of the fermentation medium are 12g/L glucose, 12g/L sodium dihydrogen phosphate, 8g/L magnesium chloride, 8g/L sodium sulfate, 12g/L yeast extract powder, 2.5g/L calcium sulfate, 0.3g/L zinc chloride and 0.3g/L manganese sulfate, and the pH value is adjusted to 6.0 before sterilization under 121 ℃ for 25 min.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the oxidation-reduction potential, and the oxidation-reduction potential is controlled in the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
during the fermentation culture for 0-8 h, the oxidation-reduction potential is controlled at-100-50 mV;
during the fermentation culture for 8-16 h, the oxidation-reduction potential is controlled to be-200 mV to-50 mV;
during the fermentation culture for 16-24 h, the oxidation-reduction potential is controlled to be minus 300 mV to minus 50mV;
during the fermentation culture for 24-32 h, the oxidation-reduction potential is controlled to be minus 250 mV to minus 100mV.
And controlling the oxidation-reduction potential to be-200 to-100 mV during the fermentation culture period of 32-40 h.
And controlling the oxidation-reduction potential to be-150 to-100 mV in the fermentation culture period of 40-48 hours.
And controlling the oxidation-reduction potential to be-150 mV to-50 mV during the fermentation culture period of 48-56 h.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 145g/L when the fermentation is ended, and the conversion rate reaches 48 percent.
Example 3-1:120m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 120m 3 Fermentation tank with liquid loading of 60m 3 . Initial culture conditions: the culture temperature is 30 ℃, the rotating speed is 60rpm, the aeration ratio is 0.5VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 in the fermentation culture process by supplementing ammonia water, and the glucose solution with the concentration of 60% and the yeast extract with the concentration of 0.3% are fed in the fermentation culture process so as to respectively control the content of a carbon source and a nitrogen source to be 0.8g/L and 1.2g/L.
The components of the fermentation medium are 12g/L glucose, 12g/L sodium dihydrogen phosphate, 8g/L magnesium chloride, 8g/L sodium sulfate, 12g/L yeast extract powder, 2.5g/L calcium sulfate, 0.3g/L zinc chloride and 0.3g/L manganese sulfate, and the pH value is adjusted to 6.0 before sterilization under 121 ℃ for 25 min.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the oxidation-reduction potential, and the oxidation-reduction potential is controlled in the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
during the fermentation culture for 0-8 h, the oxidation-reduction potential is controlled at-100-50 mV;
During the fermentation culture for 8-16 h, the oxidation-reduction potential is controlled to be-200 mV to-50 mV;
during the fermentation culture for 16-24 h, the oxidation-reduction potential is controlled to be minus 40 mV to minus 10mV;
during the fermentation culture for 24-32 h, the oxidation-reduction potential is controlled to be minus 90 mV to minus 10mV.
And controlling the oxidation-reduction potential to be-200 to-100 mV during the fermentation culture period of 32-40 h.
And controlling the oxidation-reduction potential to be-300 to-210 mV during the fermentation culture period of 40-48 hours.
And controlling the oxidation-reduction potential to be-150 mV to-50 mV during the fermentation culture period of 48-56 h.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 125g/L when the fermentation is ended, and the conversion rate reaches 30 percent. Compared with example 6, the oxidation-reduction potential of a part of interval is not controlled within the process requirement range, and the fermentation level and the conversion rate of glucosamine are lower than those of example 6.
Example 7:160m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 160m 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 32 ℃, the rotating speed is 80rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 in the fermentation culture process by supplementing ammonia water, and the glucose solution with the concentration of 58% and the yeast extract with the concentration of 0.2% are fed in the fermentation culture process so as to respectively control the content of a carbon source and a nitrogen source to be 3.2g/L and 0.8g/L.
The components of the fermentation medium are 12g/L glucose, 15g/L sodium dihydrogen phosphate, 8g/L magnesium chloride, 8g/L sodium sulfate, 10g/L yeast extract powder, 0.6g/L calcium sulfate, 0.2g/L zinc chloride and 0.05g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the specific carbon dioxide release rate, and controlling the specific carbon dioxide release rate within the following range by increasing or decreasing at least one of rotating speed, air quantity and tank pressure:
during the fermentation culture for 0-8 h, controlling the release rate of the fermentation process to 0.05-0.8;
during the fermentation culture for 8-24 h, controlling the release rate of the fermentation process to 0.1-0.6;
during the fermentation culture for 24-40 h, controlling the release rate of the fermentation process to 0.1-0.5;
And controlling the release rate of the fermentation process to be 0.05-0.3 in the fermentation culture period of 40-56 h.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 150g/L when the fermentation is ended, and the conversion rate reaches 50%.
Example 8:160m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 160m 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 70rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 by supplementing ammonia water in the fermentation culture process, and the glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of the carbon source to be 3.5g/L.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 5g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.5g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the specific carbon dioxide release rate, and controlling the specific carbon dioxide release rate within the following range by increasing or decreasing at least one of rotating speed, air quantity and tank pressure:
During the fermentation culture for 0-8 h, controlling the release rate of the fermentation process to 0.05-0.8;
during the fermentation culture for 8-16 h, controlling the release rate of the fermentation process to 0.1-0.6;
during the fermentation culture for 16-24 h, controlling the release rate of the fermentation process to 0.15-0.4;
during the fermentation culture for 24-32 h, controlling the release rate of the fermentation process to 0.15-0.5;
during the fermentation culture for 32-40 h, controlling the release rate of the fermentation process to 0.1-0.5;
during the fermentation culture for 40-48 h, controlling the release rate of the fermentation process to 0.05-0.3;
and controlling the release rate of the fermentation process to be 0.05-0.15 in the period of 48-56 hours of fermentation culture.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 160g/L when the fermentation is ended, and the conversion rate reaches 55 percent.
Example 4-1:160m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 160m 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 70rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 by supplementing ammonia water in the fermentation culture process, and the glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of the carbon source to be 3.5g/L.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 5g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.5g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the specific carbon dioxide release rate, and controlling the specific carbon dioxide release rate within the following range by increasing or decreasing at least one of rotating speed, air quantity and tank pressure:
during the fermentation culture for 0-8 h, controlling the release rate of the fermentation process to 0.05-0.8;
during the fermentation culture for 8-16 h, controlling the release rate of the fermentation process to 0.05-0.09;
during the fermentation culture for 16-24 h, controlling the release rate of the fermentation process to 0.4-0.6;
during the fermentation culture for 24-32 h, controlling the release rate of the fermentation process to 0.15-0.5;
During the fermentation culture for 32-40 h, controlling the release rate of the fermentation process to 0.1-0.5;
during the fermentation culture for 40-48 h, controlling the release rate of the fermentation process to 0.3-0.5;
and controlling the release rate of the fermentation process to be 0.05-0.15 in the period of 48-56 hours of fermentation culture.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 130g/L when the fermentation is ended, and the conversion rate reaches 40%. Compared with example 8, the partial interval ratio carbon dioxide release rate is not controlled within the process requirement range, and the glucosamine fermentation level and the conversion rate are lower than those of example 8.
Example 9:160m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 160m 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 70rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 by supplementing ammonia water in the fermentation culture process, and the glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of the carbon source to be 10.8g/L.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 5g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.5g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the oxygen consumption rate and the oxidation-reduction potential in the fermentation liquid, and controlling the oxygen consumption rate and the oxidation-reduction potential in the fermentation liquid within the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
during the fermentation culture for 0-8 h, controlling the oxygen consumption rate to be 10-90 mmol/L.h and the oxidation-reduction potential to be-100-50 mV;
during the fermentation culture for 8-24 h, controlling the oxygen consumption rate at 60-150 mmol/L.h and the oxidation-reduction potential at-300 to-50 mV;
during the fermentation culture for 24-40 h, controlling the oxygen consumption rate at 70-120 mmol/L.h and the oxidation-reduction potential at-250 to-100 mV;
and controlling the oxygen consumption rate at 50-100 mmol/L.h and the oxidation-reduction potential at-200 to-50 mV during the period from 40h of fermentation culture to the end of fermentation.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 150g/L when the fermentation is ended, and the conversion rate reaches 50%.
Example 10:160m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 160m 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 70rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 by supplementing ammonia water in the fermentation culture process, and the glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of the carbon source to be 13.5g/L.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 5g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.5g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the oxygen consumption rate and the acetic acid concentration in the fermentation liquid, and controlling the oxygen consumption rate and the acetic acid concentration in the fermentation liquid within the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
during the fermentation culture for 0-8 h, controlling the oxygen consumption rate to be 10-90 mmol/L.h and the acetic acid concentration to be 0.1-20 g/L;
During the fermentation culture for 8-24 h, controlling the oxygen consumption rate to be 60-150 mmol/L.h and the acetic acid concentration to be 0.5-10 g/L;
during the fermentation culture for 24-40 h, controlling the oxygen consumption rate to be 70-120 mmol/L.h and the acetic acid concentration to be 0.3-10 g/L;
and controlling the oxygen consumption rate to be 50-100 mmol/L.h and the acetic acid concentration to be 0.2-10 g/L during the period from 40h of fermentation culture to the end of fermentation.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 145g/L when the fermentation is ended, and the conversion rate reaches 52 percent.
Example 11:160m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 160m 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 70rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 by supplementing ammonia water in the fermentation culture process, and the glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of the carbon source to be 12g/L.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 5g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.5g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the oxygen consumption rate and the specific carbon dioxide release rate in the fermentation liquid, and controlling the oxygen consumption rate and the specific carbon dioxide release rate in the fermentation liquid within the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
during the fermentation culture for 0-8 h, controlling the oxygen consumption rate to be 10-90 mmol/L.h and the specific carbon dioxide release rate to be 0.05-0.8;
during the fermentation culture for 8-24 h, controlling the oxygen consumption rate to be 60-150 mmol/L.h and the specific carbon dioxide release rate to be 0.1-0.6;
during the fermentation culture for 24-40 h, controlling the oxygen consumption rate to be 70-120 mmol/L.h and the specific carbon dioxide release rate to be 0.1-0.5;
and controlling the oxygen consumption rate to be 50-100 mmol/L.h and the specific carbon dioxide release rate to be 0.05-0.3 during the period from 40h of fermentation culture to the end of fermentation.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 148g/L when the fermentation is ended, and the conversion rate reaches 51 percent.
Example 12:160m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 160m 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 70rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 by supplementing ammonia water in the fermentation culture process, and the glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of the carbon source to be 0.15g/L.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 5g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.5g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the oxidation-reduction potential and the acetic acid concentration, and controlling the oxidation-reduction potential and the acetic acid concentration in the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
During the fermentation culture for 0-8 h, the oxidation-reduction potential is controlled at-100-50 mV, and the acetic acid concentration is controlled at 0.1-20 g/L;
during the fermentation culture for 8-24 h, the oxidation-reduction potential is controlled to be minus 300 mV to minus 50mV, and the acetic acid concentration is controlled to be 0.5-10 g/L;
during the fermentation culture for 24-40 h, the oxidation-reduction potential is controlled to be minus 250 mV to minus 100mV, and the acetic acid concentration is controlled to be 0.3-10 g/L;
and (3) controlling the oxidation-reduction potential to be-200 to-50 mV and controlling the acetic acid concentration to be 0.2-10 g/L in the period from 40h of fermentation culture to the end of fermentation.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 145g/L when the fermentation is ended, and the conversion rate reaches 52 percent.
Example 13:160m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 160m 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 70rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 by supplementing ammonia water in the fermentation culture process, and the glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of the carbon source to be 0.2g/L.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 5g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.5g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the oxidation-reduction potential and the specific carbon dioxide release rate, and controlling the oxidation-reduction potential and the specific carbon dioxide release rate within the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
during the fermentation culture for 0-8 h, the oxidation-reduction potential is controlled at-100-50 mV, and the specific carbon dioxide release rate is controlled at 0.05-0.8;
during the fermentation culture for 8-24 h, the oxidation-reduction potential is controlled to be minus 300 mV to minus 50mV, and the specific carbon dioxide release rate is controlled to be 0.1-0.6;
during the fermentation culture for 24-40 h, the oxidation-reduction potential is controlled to be minus 250 mV to minus 100mV, and the specific carbon dioxide release rate is controlled to be 0.1-0.5;
and controlling the oxidation-reduction potential to be-200 to-50 mV and controlling the specific carbon dioxide release rate to be 0.05 to 0.3 in the period from 40 hours of fermentation culture to the end of fermentation.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 148g/L when the fermentation is ended, and the conversion rate reaches 53 percent.
Example 14:160m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 160m 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 70rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 by supplementing ammonia water in the fermentation culture process, and the glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of the carbon source to be 3g/L.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 5g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.5g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the specific carbon dioxide release rate and the acetic acid concentration, and controlling the specific carbon dioxide release rate and the acetic acid concentration in the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
during the fermentation culture for 0-8 h, controlling the specific carbon dioxide release rate to be 0.05-0.8 and the acetic acid concentration to be 0.1-20 g/L;
During the fermentation culture for 8-24 h, the specific carbon dioxide release rate is controlled to be 0.1-0.6, and the acetic acid concentration is controlled to be 0.5-10 g/L;
during the fermentation culture for 24-40 h, the specific carbon dioxide release rate is controlled to be 0.1-0.5, and the acetic acid concentration is controlled to be 0.3-10 g/L;
and controlling the specific carbon dioxide release rate to be 0.05-0.3 and the acetic acid concentration to be 0.2-10 g/L during the period from 40h of fermentation culture to the end of fermentation.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 150g/L when the fermentation is ended, and the conversion rate reaches 53 percent.
Example 15:160m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 160m 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 70rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 by supplementing ammonia water in the fermentation culture process, and the glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of the carbon source to be 10.3g/L.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 5g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.5g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the oxygen consumption rate, the oxidation-reduction potential and the acetic acid concentration in the fermentation liquid, and controlling the oxygen consumption rate, the oxidation-reduction potential and the acetic acid concentration in the fermentation liquid in the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
during the fermentation culture for 0-8 h, controlling the oxygen consumption rate to be 10-90 mmol/L.h, the oxidation-reduction potential to be-100-50 mV, and the acetic acid concentration to be 0.1-20 g/L;
during the fermentation culture for 8-24 h, controlling the oxygen consumption rate at 60-150 mmol/L.h, the oxidation-reduction potential at-300 to-50 mV, and the acetic acid concentration at 0.5-10 g/L;
during the fermentation culture for 24-40 h, controlling the oxygen consumption rate at 70-120 mmol/L.h, the oxidation-reduction potential at-250 to-100 mV, and the acetic acid concentration at 0.3-10 g/L;
and controlling the oxygen consumption rate at 50-100 mmol/L.h, the oxidation-reduction potential at-200 to-50 mV and the acetic acid concentration at 0.2-10 g/L during the period from 40h of fermentation culture to the end of fermentation.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 154g/L when the fermentation is ended, and the conversion rate reaches 52 percent.
Example 16:160m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 160m 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotation speed is 70rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 by supplementing ammonia water in the fermentation culture process, and the fermentation culture is completedA60% glucose solution was fed in a stream to control the carbon source content to 9.6g/L.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 5g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.5g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the oxygen consumption rate, the oxidation-reduction potential and the specific carbon dioxide release rate in the fermentation liquid, and controlling the oxygen consumption rate, the oxidation-reduction potential and the specific carbon dioxide release rate in the fermentation liquid within the following ranges by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
During the fermentation culture for 0-8 h, controlling the oxygen consumption rate to be 10-90 mmol/L.h, the oxidation-reduction potential to be-100-50 mV, and the specific carbon dioxide release rate to be 0.05-0.8;
during the fermentation culture for 8-24 h, controlling the oxygen consumption rate at 60-150 mmol/L.h, the oxidation-reduction potential at-300 to-50 mV, and the specific carbon dioxide release rate at 0.1-0.6;
during the fermentation culture for 24-40 h, controlling the oxygen consumption rate at 70-120 mmol/L.h, the oxidation-reduction potential at-250 to-100 mV, and the specific carbon dioxide release rate at 0.1-0.5;
and controlling the oxygen consumption rate at 50-100 mmol/L.h, the oxidation-reduction potential at-200 to-50 mV and the specific carbon dioxide release rate at 0.05-0.3 during the period from 40h of fermentation culture to the end of fermentation.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 155g/L when the fermentation is ended, and the conversion rate reaches 53 percent.
Example 17:160m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25minThe pH value is adjusted to 6.0, and the seed solution of the seed tank is moved to 160m according to the inoculation amount of 20 percent 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 70rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 by supplementing ammonia water in the fermentation culture process, and the glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of the carbon source to be 8g/L.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 5g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.5g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the acetic acid concentration, the oxidation-reduction potential and the specific carbon dioxide release rate, and controlling the acetic acid concentration, the oxidation-reduction potential and the specific carbon dioxide release rate within the following ranges by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
during the fermentation culture for 0-8 h, controlling the acetic acid concentration to be 0.1-20 g/L, the oxidation-reduction potential to be-100-50 mV, and the specific carbon dioxide release rate to be 0.05-0.8;
during the fermentation culture for 8-24 h, controlling the acetic acid concentration to be 0.5-10 g/L, the oxidation-reduction potential to be-300-50 mV, and the specific carbon dioxide release rate to be 0.1-0.6;
During the fermentation culture for 24-40 h, controlling the acetic acid concentration to be 0.3-10 g/L, the oxidation-reduction potential to be-250-100 mV, and the specific carbon dioxide release rate to be 0.1-0.5;
and during the period from 40h of fermentation culture to the end of fermentation, controlling the concentration of acetic acid to be 0.2-10 g/L, the oxidation-reduction potential to be-200 to-50 mV, and the specific carbon dioxide release rate to be 0.05-0.3.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 149g/L when the fermentation is ended, and the conversion rate reaches 53%.
Example 18:160m 3 Tank fermentation productionProcess for producing a solid-state image sensor
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 160m 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 70rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 by supplementing ammonia water in the fermentation culture process, and the glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of the carbon source to be 3.6g/L.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 5g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.5g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the oxygen consumption rate, the acetic acid concentration, the oxidation-reduction potential and the specific carbon dioxide release rate in the fermentation liquid, and controlling the oxygen consumption rate, the acetic acid concentration, the oxidation-reduction potential and the specific carbon dioxide release rate in the fermentation liquid within the following ranges by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
during the fermentation culture for 0-8 h, controlling the oxygen consumption rate to 10-90 mmol/L.h, the acetic acid concentration to 0.1-20 g/L, the oxidation-reduction potential to-100-50 mV and the specific carbon dioxide release rate to 0.05-0.8;
during the fermentation culture for 8-24 h, controlling the oxygen consumption rate at 60-150 mmol/L.h, the acetic acid concentration at 0.5-10 g/L, the oxidation-reduction potential at-300 to-50 mV and the specific carbon dioxide release rate at 0.1-0.6;
during the fermentation culture for 24-40 h, controlling the oxygen consumption rate at 70-120 mmol/L.h, the acetic acid concentration at 0.3-10 g/L, the oxidation-reduction potential at-250 to-100 mV and the specific carbon dioxide release rate at 0.1-0.5;
and controlling the oxygen consumption rate at 50-100 mmol/L.h, the acetic acid concentration at 0.2-10 g/L, the oxidation-reduction potential at-200 to-50 mV and the specific carbon dioxide release rate at 0.05-0.3 during the period from 40h to the end of fermentation culture.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 160g/L when the fermentation is ended, and the conversion rate reaches 55 percent.
Example 5-1:160m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 160m 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 70rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 by supplementing ammonia water in the fermentation culture process, and the glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of the carbon source to be 3.6g/L.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 5g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.5g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The oxygen consumption rate, the oxidation-reduction potential, the acetic acid concentration and the specific carbon dioxide release rate are not monitored and regulated in stages in the fermentation culture process, the oxygen consumption rate control range is 10-150 mmol/L.h, the oxidation-reduction potential control range is-300-50 mV, the acetic acid concentration control range is 0.1-20 g/L, and the specific carbon dioxide release rate control range is 0.05-0.8. The fermentation process maintains the oxygen consumption rate, redox potential, acetic acid concentration, and specific carbon dioxide release rate within the above process ranges by increasing or decreasing one or more of the rotational speed or air volume or tank pressure.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the glucosamine content in the fermentation liquid, the glucosamine content reaches 120g/L when the fermentation is ended, the conversion rate reaches 42 percent, and compared with the staged control, the glucosamine fermentation level and the conversion rate are lower.
Example 19:160m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 160m 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 70rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 by supplementing ammonia water in the fermentation culture process, and the glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of the carbon source to be 10.3g/L.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 5g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.5g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the oxygen consumption rate, the acetic acid concentration and the oxidation-reduction potential in the fermentation liquid, and controlling the oxygen consumption rate, the acetic acid concentration and the oxidation-reduction potential in the fermentation liquid within the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
During the fermentation culture for 0-8 h, controlling the oxygen consumption rate to be 10-90 mmol/L.h, controlling the acetic acid concentration to be 0.1-20 g/L and controlling the oxidation-reduction potential to be-100-50 mV;
during the fermentation culture for 8-16 h, controlling the oxygen consumption rate to be 60-120 mmol/L.h, controlling the acetic acid concentration to be 0.5-10 g/L, and controlling the oxidation-reduction potential to be-200 to-50 mV;
during the fermentation culture for 16-24 h, controlling the oxygen consumption rate to 80-150 mmol/L.h, the acetic acid concentration to 0.5-6 g/L, and the oxidation-reduction potential to-300 to-50 mV;
during the fermentation culture for 24-32 h, controlling the oxygen consumption rate at 70-120 mmol/L.h, the acetic acid concentration at 0.3-10 g/L, and the oxidation-reduction potential at-250 to-100 mV;
during the fermentation culture for 32-40 h, controlling the oxygen consumption rate at 70-100 mmol/L.h, the acetic acid concentration at 0.3-8 g/L, and the oxidation-reduction potential at-200 to-100 mV;
during the fermentation culture for 40-48 h, controlling the oxygen consumption rate at 70-90 mmol/L.h, the acetic acid concentration at 0.2-10 g/L, and the oxidation-reduction potential at-150 to-100 mV;
and controlling the oxygen consumption rate at 50-80 mmol/L.h, the acetic acid concentration at 0.2-8 g/L and the oxidation-reduction potential at-150 to-50 mV during the period from 48h of fermentation culture to the end of fermentation.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 162g/L when the fermentation is ended, and the conversion rate reaches 56.2 percent.
Example 20:160m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 160m 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 70rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 by supplementing ammonia water in the fermentation culture process, and the glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of the carbon source to be 8g/L.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 5g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.5g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the concentration of acetic acid, the oxidation-reduction potential and the specific carbon dioxide release rate in the fermentation liquid, and controlling the concentration of acetic acid, the oxidation-reduction potential and the specific carbon dioxide release rate in the fermentation liquid within the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
During the fermentation culture for 0-8 h, controlling the acetic acid concentration to be 0.1-20 g/L, the oxidation-reduction potential to be-100-50 mV, and the specific carbon dioxide release rate to be 0.05-0.8;
during the fermentation culture for 8-16 h, controlling the acetic acid concentration to be 0.5-10 g/L, the oxidation-reduction potential to be-200 to-50 mV, and the specific carbon dioxide release rate to be 0.1-0.6;
during 16-24 h of fermentation culture, controlling the concentration of acetic acid to be 0.5-6 g/L, the oxidation-reduction potential to be-300-50 mV, and the release rate of specific carbon dioxide to be 0.15-0.4;
during the fermentation culture for 24-32 h, controlling the acetic acid concentration to be 0.3-10 g/L, the oxidation-reduction potential to be-250-100 mV, and the specific carbon dioxide release rate to be 0.15-0.5;
during the fermentation culture for 32-40 h, controlling the acetic acid concentration to be 0.3-8 g/L, the oxidation-reduction potential to be-200-100 mV, and the specific carbon dioxide release rate to be 0.1-0.5;
during the fermentation culture for 40-48 h, controlling the acetic acid concentration to be 0.2-10 g/L, the oxidation-reduction potential to be-150-100 mV, and the specific carbon dioxide release rate to be 0.05-0.3;
and during the period from 48h of fermentation culture to the end of fermentation, controlling the concentration of acetic acid to be 0.2-8 g/L, the oxidation-reduction potential to be-150 to-50 mV, and the specific carbon dioxide release rate to be 0.05-0.15.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 159g/L when the fermentation is ended, and the conversion rate reaches 54 percent.
Example 21:160m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, and sterilizing at 121deg.C25min, adjusting pH to 6.0 before sterilization, and transferring seed solution of seed tank to 160m according to 20% inoculum size 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 70rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 by supplementing ammonia water in the fermentation culture process, and the glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of the carbon source to be 9.6g/L.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 5g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.5g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the oxygen consumption rate, the oxidation-reduction potential and the specific carbon dioxide release rate in the fermentation liquid, and controlling the oxygen consumption rate, the oxidation-reduction potential and the specific carbon dioxide release rate in the fermentation liquid within the following ranges by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
During the fermentation culture for 0-8 h, controlling the oxygen consumption rate to be 10-90 mmol/L.h, the oxidation-reduction potential to be-100-50 mV, and the specific carbon dioxide release rate to be 0.05-0.8;
during the fermentation culture for 8-16 h, controlling the oxygen consumption rate at 60-120 mmol/L.h, the oxidation-reduction potential at-200 to-50 mV, and the specific carbon dioxide release rate at 0.1-0.6;
during the fermentation culture for 16-24 h, controlling the oxygen consumption rate to 80-150 mmol/L.h, the oxidation-reduction potential to-300-50 mV, and the specific carbon dioxide release rate to 0.15-0.4;
during the fermentation culture for 24-32 h, controlling the oxygen consumption rate at 70-120 mmol/L.h, the oxidation-reduction potential at-250 to-100 mV, and the specific carbon dioxide release rate at 0.15-0.5;
during the fermentation culture for 32-40 h, controlling the oxygen consumption rate at 70-100 mmol/L.h, the oxidation-reduction potential at-200 to-100 mV, and the specific carbon dioxide release rate at 0.1-0.5;
during the fermentation culture for 40-48 h, controlling the oxygen consumption rate at 70-90 mmol/L.h, the oxidation-reduction potential at-150-100 mV, and the specific carbon dioxide release rate at 0.05-0.3;
and controlling the oxygen consumption rate at 50-80 mmol/L.h, the oxidation-reduction potential at-150-50 mV and the specific carbon dioxide release rate at 0.05-0.15 during the period from 48h of fermentation culture to the end of fermentation.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 161g/L when the fermentation is ended, and the conversion rate reaches 55 percent.
Example 22:160m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 160m 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 70rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 by supplementing ammonia water in the fermentation culture process, and the glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of the carbon source to be 1.2g/L.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 5g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.5g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the oxygen consumption rate, the acetic acid concentration and the specific carbon dioxide release rate in the fermentation liquid, and controlling the oxygen consumption rate, the acetic acid concentration and the specific carbon dioxide release rate in the fermentation liquid within the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
During the fermentation culture for 0-8 h, controlling the oxygen consumption rate to be 10-90 mmol/L.h, controlling the acetic acid concentration to be 0.1-20 g/L and controlling the specific carbon dioxide release rate to be 0.05-0.8;
during the fermentation culture for 8-16 h, controlling the oxygen consumption rate to be 60-120 mmol/L.h, controlling the acetic acid concentration to be 0.5-10 g/L and controlling the specific carbon dioxide release rate to be 0.1-0.6;
during the fermentation culture for 16-24 h, controlling the oxygen consumption rate to 80-150 mmol/L.h, the acetic acid concentration to 0.5-6 g/L and the specific carbon dioxide release rate to 0.15-0.4;
during the fermentation culture for 24-32 h, controlling the oxygen consumption rate to be 70-120 mmol/L.h, controlling the acetic acid concentration to be 0.3-10 g/L and controlling the specific carbon dioxide release rate to be 0.15-0.5;
during the fermentation culture for 32-40 h, controlling the oxygen consumption rate to be 70-100 mmol/L.h, controlling the acetic acid concentration to be 0.3-8 g/L and controlling the specific carbon dioxide release rate to be 0.1-0.5;
during the fermentation culture for 40-48 h, controlling the oxygen consumption rate to 70-90 mmol/L.h, the acetic acid concentration to 0.2-10 g/L and the specific carbon dioxide release rate to 0.05-0.3;
and controlling the oxygen consumption rate to be 50-80 mmol/L.h, the acetic acid concentration to be 0.2-8 g/L and the specific carbon dioxide release rate to be 0.05-0.15 in the period from 48h of fermentation culture to the end of fermentation.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 162g/L when the fermentation is ended, and the conversion rate reaches 54.4 percent.
Example 23:160m 3 Tank fermentation production process
(1) - (3) as in example 1;
(4) Fermentation culture
Preparing fermentation medium, sterilizing at 121deg.C for 25min, adjusting pH to 6.0, and transferring seed solution of seed tank to 160m 3 Fermentation tank with liquid loading of 90m 3 . Initial culture conditions: the culture temperature is 33 ℃, the rotating speed is 70rpm, the aeration ratio is 0.6VVM, the tank pressure is 0.03MPa, the pH value is controlled to be about 6.0 by supplementing ammonia water in the fermentation culture process, and the fermentation culture process is fed-batch concentratedThe concentration of the carbon source was controlled to 3.6g/L with a 60% glucose solution.
The components of the fermentation medium are 10g/L glucose, 10g/L sodium dihydrogen phosphate, 5g/L magnesium chloride, 5g/L sodium sulfate, 5g/L yeast extract powder, 5g/L calcium sulfate, 0.5g/L zinc chloride and 0.5g/L manganese sulfate, and the fermentation medium is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process is used for feeding back and regulating the process technology in real time by monitoring the oxygen consumption rate, the acetic acid concentration, the oxidation-reduction potential and the specific carbon dioxide release rate in the fermentation liquid, and controlling the oxygen consumption rate, the acetic acid concentration, the oxidation-reduction potential and the specific carbon dioxide release rate in the fermentation liquid within the following ranges by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
During the fermentation culture for 0-8 h, controlling the oxygen consumption rate to 10-90 mmol/L.h, the acetic acid concentration to 0.1-20 g/L, the oxidation-reduction potential to-100-50 mV and the specific carbon dioxide release rate to 0.05-0.8;
during the fermentation culture for 8-16 h, controlling the oxygen consumption rate at 60-120 mmol/L.h, the acetic acid concentration at 0.5-10 g/L, the oxidation-reduction potential at-200 to-50 mV and the specific carbon dioxide release rate at 0.1-0.6;
during 16-24 h of fermentation culture, controlling the oxygen consumption rate to 80-150 mmol/L.h, the acetic acid concentration to 0.5-6 g/L, the oxidation-reduction potential to-300-50 mV and the specific carbon dioxide release rate to 0.15-0.4;
during the fermentation culture for 24-32 h, controlling the oxygen consumption rate at 70-120 mmol/L.h, the acetic acid concentration at 0.3-10 g/L, the oxidation-reduction potential at-250 to-100 mV and the specific carbon dioxide release rate at 0.15-0.5;
during the fermentation culture for 32-40 h, controlling the oxygen consumption rate at 70-100 mmol/L.h, the acetic acid concentration at 0.3-8 g/L, the oxidation-reduction potential at-200 to-100 mV and the specific carbon dioxide release rate at 0.1-0.5;
during the fermentation culture for 40-48 h, controlling the oxygen consumption rate at 70-90 mmol/L.h, the acetic acid concentration at 0.2-10 g/L, the oxidation-reduction potential at-150 to-100 mV and the specific carbon dioxide release rate at 0.05-0.3;
And controlling the oxygen consumption rate at 50-80 mmol/L.h, the acetic acid concentration at 0.2-8 g/L, the oxidation-reduction potential at-150 to-50 mV and the specific carbon dioxide release rate at 0.05-0.15 during the period from 48h to the end of fermentation culture.
When the growth rate of the product is obviously slowed down or the bacterial stain is shallow, the HPLC is used for measuring the content of the glucosamine in the fermentation liquid, the content of the glucosamine reaches 166g/L when the fermentation is ended, and the conversion rate reaches 56.5 percent.
Examples 24 to 43: the difference between the production method of glucosamine and examples 1 to 18 is that the strain is different, and the specific conditions and the glucosamine content and conversion rate in the fermentation broth are shown in Table 1.
TABLE 1 fermentation levels of different glucosamine-producing strains
Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives, and variations may be made in the above embodiments by those skilled in the art without departing from the spirit and principles of the invention.

Claims (7)

1. A method for producing glucosamine, comprising on-line monitoring at least one of oxygen consumption rate, oxidation-reduction potential, acetic acid concentration, specific carbon dioxide release rate in fermentation broth during fermentation of glucosamine and controlling the value thereof within the following range: the oxygen consumption rate is 10-150 mmol/L.h, the oxidation-reduction potential is-300-50 mV, the acetic acid concentration is 0.1-20 g/L, and the specific carbon dioxide release rate is 0.05-0.8; the strain adopted by the glucosamine fermentation is escherichia coli @ Escherichia coli) The method comprises the steps of carrying out a first treatment on the surface of the The fermentation ofThe numerical ranges of the oxygen consumption rate, the oxidation-reduction potential, the acetic acid concentration and the specific carbon dioxide release rate in the liquid are controlled by adjusting at least one of the air flow rate, the rotating speed and the tank pressure;
the oxygen consumption rate is controlled in stages and is controlled as follows: controlling the oxygen consumption rate to be 10-90 mmol/L.h in the fermentation culture period of 0-8 h; controlling the oxygen consumption rate to be 60-150 mmol/L.h in the fermentation culture period of 8-24 h; controlling the oxygen consumption rate to be 70-120 mmol/L.h in the fermentation culture period of 24-40 h; controlling the oxygen consumption rate to be 50-100 mmol/L.h in the period from 40h of fermentation culture to the end of fermentation;
the oxidation-reduction potential is controlled in stages, and the control mode is as follows: during the fermentation culture for 0-8 h, controlling the oxidation-reduction potential to be-100-50 mV; during the fermentation culture for 8-24 h, the oxidation-reduction potential is controlled at-300 to-50 mV; during the fermentation culture for 24-40 h, the oxidation-reduction potential is controlled at-250 to-100 mV; during the period from 40h of fermentation culture to the end of fermentation, the oxidation-reduction potential is controlled at-200 mV to-50 mV;
the acetic acid concentration is controlled in stages, and the control mode is as follows: acetic acid concentration is controlled to be 0.1-20 g/L in the fermentation culture period of 0-8 h; acetic acid concentration is controlled to be 0.5-10 g/L in the fermentation culture period of 8-24 hours; acetic acid concentration is controlled to be 0.3-10 g/L in the fermentation culture period of 24-40 h; controlling the concentration of acetic acid to be 0.2-10 g/L in the period from fermentation culture for 40h to fermentation completion;
The specific carbon dioxide release rate is controlled in stages, and the control mode is as follows: controlling the release rate of specific carbon dioxide to be 0.05-0.8 in the fermentation culture period of 0-8 h; controlling the release rate of specific carbon dioxide to be 0.1-0.6 in the fermentation culture period of 8-24 hours; controlling the release rate of specific carbon dioxide to be 0.1-0.5 in the fermentation culture period of 24-40 h; and controlling the release rate of the specific carbon dioxide to be 0.05-0.3 in the period from 40h of fermentation culture to the end of fermentation.
2. The method for producing glucosamine according to claim 1, wherein the oxygen consumption rate is controlled in stages in the following manner: controlling the oxygen consumption rate to be 10-90 mmol/L.h in the fermentation culture period of 0-8 h; controlling the oxygen consumption rate to be 60-120 mmol/L.h in the fermentation culture period of 8-16 h; controlling the oxygen consumption rate to be 80-150 mmol/L.h in the fermentation culture period of 16-24 h; controlling the oxygen consumption rate to be 70-120 mmol/L.h in the fermentation culture period of 24-32 h; controlling the oxygen consumption rate to be 70-100 mmol/L.h in the fermentation culture period of 32-40 h; controlling the oxygen consumption rate to be 70-90 mmol/L.h in the fermentation culture period of 40-48 h; and controlling the oxygen consumption rate to be 50-80 mmol/L.h in the period from 48h of fermentation culture to the end of fermentation.
3. The method for producing glucosamine according to claim 1, wherein the oxidation-reduction potential is controlled in stages in the following manner: during the fermentation culture for 0-8 h, controlling the oxidation-reduction potential to be-100-50 mV; during the fermentation culture for 8-16 h, the oxidation-reduction potential is controlled at-200 to-50 mV; during the fermentation culture for 16-24 h, the oxidation-reduction potential is controlled at-300 to-50 mV; during the fermentation culture for 24-32 h, controlling the oxidation-reduction potential at-250 to-100 mV; during the fermentation culture for 32-40 h, controlling the oxidation-reduction potential at-200 to-100 mV; during the fermentation culture for 40-48 h, controlling the oxidation-reduction potential at-150 to-100 mV; and controlling the oxidation-reduction potential to be-150 to-50 mV in the period from 48 hours of fermentation culture to the end of fermentation.
4. The method for producing glucosamine according to claim 1, wherein the acetic acid concentration is controlled in stages in the following manner: acetic acid concentration is controlled to be 0.1-20 g/L in the fermentation culture period of 0-8 h; acetic acid concentration is controlled to be 0.5-10 g/L in the fermentation culture period of 8-16 h; acetic acid concentration is controlled to be 0.5-6 g/L in the fermentation culture period of 16-24 hours; controlling the concentration of acetic acid to be 0.3-10 g/L in the fermentation culture period of 24-32 h; during the fermentation culture for 32-40 h, controlling the acetic acid concentration to be 0.3-8 g/L; controlling the concentration of acetic acid to be 0.2-10 g/L during the fermentation culture for 40-48 h; and controlling the concentration of acetic acid to be 0.2-8 g/L in the period from 48h of fermentation culture to the end of fermentation.
5. The method for producing glucosamine according to claim 1, wherein the specific carbon dioxide release rate is controlled in stages in the following manner: controlling the release rate of specific carbon dioxide to be 0.05-0.8 in the fermentation culture period of 0-8 h; controlling the release rate of specific carbon dioxide to be 0.1-0.6 in the fermentation culture period of 8-16 h; controlling the release rate of specific carbon dioxide to be 0.15-0.4 in the fermentation culture period of 16-24 hours; controlling the release rate of specific carbon dioxide to be 0.15-0.5 in the fermentation culture period of 24-32 h; controlling the release rate of specific carbon dioxide to be 0.1-0.5 in the fermentation culture period of 32-40 h; controlling the release rate of specific carbon dioxide to be 0.05-0.3 in the fermentation culture period of 40-48 h; and controlling the release rate of the specific carbon dioxide to be 0.05-0.15 in the period from 48 hours of fermentation culture to the end of fermentation.
6. The method for producing glucosamine according to claim 1, further comprising adding alkali during fermentation to control the pH of the fermentation system to 5.5-7.5, adding a carbon source and a nitrogen source to control the concentration of the carbon source in the fermentation system to 0.1-15 g/L, and controlling the concentration of the nitrogen source to 0.01-2 g/L; the carbon source is at least one selected from glucose, sucrose and glycerol, and the nitrogen source is ammonium sulfate and/or yeast extract powder.
7. The method for producing glucosamine according to claim 1, wherein the glucosamine fermentation comprises the steps of:
(1) Seed activation: sucking bacterial liquid from a seed retaining tube for gradient dilution, sucking the diluted bacterial suspension into a flat culture medium, and culturing for 12-72 hours at 28-38 ℃ to obtain a mature single colony;
(2) Shake flask culture: 1-20 single colonies are picked from a mature flat plate culture medium and are connected to a shake flask culture medium, shake flask culture conditions comprise a culture temperature of 28-38 ℃, a rotation speed of 150-250 rpm and a culture period of 4-48 hours, and when the wet weight of thalli reaches 1-20 g/L, the thalli is transferred to a seed tank, and the inoculum size is controlled to be 0.1-5%;
(3) Seed expansion culture: the seed culture conditions comprise a culture temperature of 28-38 ℃, a pot pressure of 0.025-0.08 MPa, an aeration ratio of 0.2-2 VVM, a rotating speed of 100-500 rpm, a culture period of 4-48 hours, and when the wet weight of the thalli reaches 1-20 g/L, the thalli is transferred into a fermentation tank, and the inoculum size is controlled to be 5% -30%;
(4) Culturing in a fermentation tank: the fermentation culture conditions comprise a culture temperature of 28-38 ℃, a tank pressure of 0.02-0.08 MPa, a ventilation ratio of 0.2-2 VVM and a rotation speed of 50-500 rpm, wherein in the fermentation process, alkali is added to control the pH value of a fermentation system to 5.5-7.5, a carbon source and a nitrogen source are added to control the concentration of the carbon source in the fermentation system to 0.1-15 g/L, the concentration of the nitrogen source is controlled to 0.01-2 g/L, and fermentation is stopped when the growth rate of a product is obviously slowed down or the bacterial stain is shallow; the carbon source is at least one selected from glucose, sucrose and glycerol, and the nitrogen source is ammonium sulfate and/or yeast extract powder.
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