CN112592944A - Production method of glucosamine - Google Patents
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Abstract
The invention belongs to the field of glucosamine production, and relates to a glucosamine production method, 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 in a specific range in stages in the glucosamine fermentation process. The method provided by the invention feeds back and controls process parameters by monitoring and controlling at least one of oxygen consumption rate, oxidation-reduction potential, acetic acid concentration and specific carbon dioxide release rate in the fermentation liquor on line, can meet the growth requirement of thalli, effectively promotes the metabolic synthesis of glucosamine and improves the conversion rate, and the glucosamine content and the conversion rate in the obtained fermentation product are high.
Description
Technical Field
The invention belongs to the field of glucosamine production, and particularly relates to a glucosamine production method.
Background
Glucosamine is a composition unit of various polysaccharides in organisms, has wide application field, is a medicament for clinically treating rheumatic and rheumatoid arthritis, can be used as a food antioxidant, an infant food additive and a sweetener for diabetics, can also be used for clinically enhancing the function of a human immune system and inhibiting the overgrowth of cancer cells or fiber cells, has the effects of inhibiting and treating cancers and malignant tumors, can effectively treat various inflammations, and also has the effect of treating osteoarthritis and arthralgia.
The current methods for producing glucosamine mainly comprise an acid hydrolysis method, an enzymolysis method and a microbial fermentation method. Among them, the production of glucosamine by acid hydrolysis and enzymatic hydrolysis has adverse effects on the environment, low production efficiency and allergic potential. In recent years, more and more researchers pay attention to the production of glucosamine by a microbial fermentation method, 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 requirement of industrial production.
When the product is expressed or synthesized by using glucose as a carbon source, a large part of the carbon source is used for synthesizing some byproducts, such as acetic acid, when the concentration of the acetic acid is accumulated to a certain degree, the influence on the growth of the thallus and the product synthesis is large, and particularly when escherichia coli is used as a production strain, the high-concentration acetic acid can generate a serious inhibition effect. In addition, the production of more by-products significantly reduces the conversion of the product. At present, the domestic glucosamine fermentation process mainly comprises sugar supplement by limitation or feedback of dissolved oxygen level during process supplement and process regulation, the process control has the problem of hysteresis, the yield and the conversion rate of products are unstable, and the problems of insufficient or excessive sugar supplement and high or low oxygen supply regulation are easily caused by the feedback of the supplement or the oxygen supply regulation according to the dissolved oxygen level. The growth of thalli is influenced by insufficient sugar supplement, the increase of thalli synthesis byproducts is caused by excessive sugar supplement, and the conversion rate of products is low. The low oxygen supply affects the growth of the thallus and the synthesis rate of the product, the high oxygen supply leads to the rapid growth of the thallus and the low conversion rate of the product.
Disclosure of Invention
The invention aims to overcome the problems of low yield and low conversion rate of glucosamine produced by adopting the existing method, and provides a glucosamine production method capable of improving the yield and the conversion rate.
The inventor of the invention finds that the oxygen consumption rate, the oxidation-reduction potential, the acetic acid concentration and the specific carbon dioxide release rate in the fermentation liquor can be well fed back to the glucosamine fermentation process in the glucosamine fermentation process after intensive and extensive research, and can effectively ensure the consistency of thallus growth and metabolites in the complex glucosamine fermentation process by monitoring at least one of the four parameters on line and controlling the numerical value of the parameters within a specific range, thereby achieving the stable and high-yield fermentation level, having good process reproducibility and effectively reducing the production cost. Based on this, the present invention has been completed.
Specifically, the invention provides a glucosamine production method, 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 liquor on line and controlling the value in the following ranges during the glucosamine fermentation process: the oxygen consumption rate is 10 to 150 mmol/L.h, the oxidation-reduction potential is-300 to 50mV, the acetic acid concentration is 0.1 to 20g/L, and the specific carbon dioxide release rate is 0.05 to 0.8.
Preferably, the oxygen consumption rate is controlled in stages in the following manner: controlling the oxygen consumption rate to be 10-90 mmol/L.h during the fermentation culture period of 0-8 h; controlling the oxygen consumption rate to be 60-150 mmol/L.h during the fermentation culture period of 8-24 h; controlling the oxygen consumption rate to be 70-120 mmol/L.h during the fermentation culture period of 24-40 h; controlling the oxygen consumption rate to be 50-100 mmol/L.h during the period from the fermentation culture for 40h to the end of the fermentation.
Preferably, the oxygen consumption rate is controlled in stages in the following manner: controlling the oxygen consumption rate to be 10-90 mmol/L.h during the fermentation culture period of 0-8 h; controlling the oxygen consumption rate to be 60-120 mmol/L.h during the fermentation culture period of 8-16 h; controlling the oxygen consumption rate to be 80-150 mmol/L.h during 16-24 h of fermentation culture; controlling the oxygen consumption rate to be 70-120 mmol/L.h during the fermentation culture period of 24-32 h; controlling the oxygen consumption rate to be 70-100 mmol/L.h during the fermentation culture period of 32-40 h; controlling the oxygen consumption rate to be 70-90 mmol/L.h during the fermentation culture period of 40-48 h; 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 in the following manner: controlling the oxidation-reduction potential to be-100-50 mV during the fermentation culture period of 0-8 h; controlling the oxidation-reduction potential to be-300 to-50 mV during the fermentation culture period of 8-24 h; controlling the oxidation-reduction potential to be-250 to-100 mV during the fermentation culture period of 24-40 h; during the fermentation culture for 40h to the end of fermentation, the oxidation-reduction potential is controlled to be-200 to-50 mV.
Preferably, the oxidation-reduction potential is controlled in stages in the following manner: controlling the oxidation-reduction potential to be-100-50 mV during the fermentation culture period of 0-8 h; controlling the oxidation-reduction potential to be-200 to-50 mV during the fermentation culture period of 8-16 h; controlling the oxidation-reduction potential to be-300 to-50 mV during 16-24 h of fermentation culture; controlling the oxidation-reduction potential to be-250 to-100 mV during the fermentation culture period of 24-32 h; controlling the oxidation-reduction potential to be-200 to-100 mV during the fermentation culture period of 32-40 h; controlling the oxidation-reduction potential to be-150 to-100 mV during the fermentation culture for 40-48 h; during the fermentation culture period from 48h to the end of fermentation, the oxidation-reduction potential is controlled to-150 to-50 mV.
Preferably, the acetic acid concentration is controlled in stages in the following manner: controlling the concentration of acetic acid to be 0.1-20 g/L during fermentation culture for 0-8 h; controlling the concentration of acetic acid to be 0.5-10 g/L during the fermentation culture period of 8-24 h; controlling the concentration of acetic acid to be 0.3-10 g/L during the fermentation culture period of 24-40 h; controlling the concentration of acetic acid to be 0.2-10 g/L during the period from fermentation culture for 40h to the end of fermentation.
Preferably, the acetic acid concentration is controlled in stages in the following manner: controlling the concentration of acetic acid to be 0.1-20 g/L during fermentation culture for 0-8 h; controlling the concentration of acetic acid to be 0.5-10 g/L during the fermentation culture period of 8-16 h; controlling the concentration of acetic acid to be 0.5-6 g/L during 16-24 h of fermentation culture; controlling the concentration of acetic acid to be 0.3-10 g/L during the fermentation culture period of 24-32 h; controlling the concentration of acetic acid to be 0.3-8 g/L during the fermentation culture period of 32-40 h; controlling the concentration of acetic acid to be 0.2-10 g/L during fermentation culture for 40-48 h; controlling the concentration of acetic acid to be 0.2-8 g/L during the period from fermentation culture for 48h to the end of fermentation.
Preferably, the specific carbon dioxide release rate is controlled in stages in the following manner: controlling the specific carbon dioxide release rate to be 0.05-0.8 during the fermentation culture period of 0-8 h; controlling the specific carbon dioxide release rate to be 0.1-0.6 during the fermentation culture period of 8-24 h; controlling the specific carbon dioxide release rate to be 0.1-0.5 during the fermentation culture period of 24-40 h; controlling the specific carbon dioxide release rate to be 0.05-0.3 during the period from fermentation culture for 40h to the end of fermentation.
Preferably, the specific carbon dioxide release rate is controlled in stages in the following manner: controlling the specific carbon dioxide release rate to be 0.05-0.8 during the fermentation culture period of 0-8 h; controlling the specific carbon dioxide release rate to be 0.1-0.6 during the fermentation culture period of 8-16 h; controlling the specific carbon dioxide release rate to be 0.15-0.4 during the fermentation culture period of 16-24 h; controlling the specific carbon dioxide release rate to be 0.15-0.5 during the fermentation culture period of 24-32 h; controlling the specific carbon dioxide release rate to be 0.1-0.5 during the fermentation culture period of 32-40 h; controlling the specific carbon dioxide release rate to be 0.05-0.3 during the fermentation culture for 40-48 h; controlling the specific carbon dioxide release rate to be 0.05-0.15 during the period from fermentation culture for 48h 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 rotating speed and the tank pressure.
Preferably, the strain employed for glucosamine fermentation is selected from at least one of Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Rhizopus oligosporus (Rhizopus oligosporus), Aspergillus sp (Aspergillus sp), and Mucor (Monascus pilosus).
Preferably, the method for producing glucosamine further comprises the steps of supplementing alkali in the fermentation process to control the pH value of the fermentation system to be 5.5-7.5, supplementing 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 of glucose, sucrose and glycerol, and the nitrogen source is ammonium sulfate and/or yeast extract powder.
Preferably, the glucosamine fermentation comprises the following steps:
(1) seed activation: absorbing bacterial liquid from a seed-preserving tube for gradient dilution, absorbing the diluted bacterial suspension into a plate culture medium, and culturing at 28-38 ℃ for 12-72 h to obtain mature single bacterial colonies;
(2) and (3) shake flask culture: selecting 1-20 single colonies from a mature plate culture medium, inoculating the single colonies into a shake flask culture medium, wherein the shake flask culture conditions comprise the culture temperature of 28-38 ℃, the rotation speed of 150-250 rpm, and the culture period of 4-48 h, and when the wet weight of thalli reaches 1-20 g/L, inoculating the thalli into a seeding tank, and controlling the inoculation amount to be 0.1-5%;
(3) seed amplification culture: the seed culture conditions comprise the culture temperature of 28-38 ℃, the tank pressure of 0.025-0.08 MPa, the aeration ratio of 0.2-2 VVM, the rotating speed of 100-500 rpm, the culture period of 4-48 h, when the wet weight of the thalli reaches 1-20 g/L, the thalli are planted into a fermentation tank, and the inoculation amount is controlled at 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 be 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 be 0.1-15 g/L, the concentration of the nitrogen source is controlled to be 0.01-2 g/L, and fermentation is stopped when the growth rate of a product is obviously slowed down or the staining of thalli is shallow; the carbon source is at least one of 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 controls process parameters by monitoring and controlling at least one of oxygen consumption rate, oxidation-reduction potential, acetic acid concentration and specific carbon dioxide release rate in the fermentation liquor on line, can meet the growth requirement of thalli, effectively promotes the metabolic synthesis of glucosamine and improves the conversion rate, and the yield and the conversion rate of the glucosamine in the obtained fermentation product are high.
Detailed Description
In some embodiments of the invention, the oxygen consumption rate, the oxidation-reduction potential, the acetic acid concentration and the specific carbon dioxide release rate in the fermentation broth are regulated in four stages (i.e., 0-8 h, 8-24 h, 24-40 h, 40h to the end of fermentation). It is to be noted that 0 to 8 hours include 8 hours including 1 st, 2 nd, 3 rd, 4 th, 5 th, 6 th, 7 th and 8 th, 8 to 24 hours include 16 hours including 24 hours including 9 th and 10 th … … th, 24 to 40 hours include 16 hours including 25 th and 26 th … … th and 40 th, and 40 hours include 40 th and 41 th … … after fermentation is finished.
In some embodiments of the invention, the oxygen consumption rate, the oxidation-reduction potential, the acetic acid concentration and the specific carbon dioxide release rate in the fermentation liquid are regulated and controlled in seven stages (i.e., 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 0 to 8 hours include 8 hours of 1 st, 2 nd, 3 rd, 4 th, 5 th, 6 th, 7 th and 8 th, 8 to 16 hours include 8 hours of 9 th, 10 th, 11 th, 12 th, 13 th, 14 th, 15 th and 16 th, 16 to 24 hours include 8 hours of 17 th, 18 th, 19 th, 20 th, 21 th, 22 th, 23 th and 24 th, 24 to 32 hours include 8 hours of 25 th, 26 th, 27 th, 28 th, 29 th, 30 th, 31 th and 32 th, 32 to 40 hours include 8 hours of 33 th, 34 th, 35 th, 36 th, 37 th, 38 th, 39 th and 40 th, 8 hours of 40 to 48 hours include 8 hours of 41 th, 42 th, 43 th, 44 th, 48 th and 48 th, 8 hours of fermentation, The fermentation was ended at 50h … ….
In some embodiments of the invention, the manner in which the oxygen consumption rate is controlled in stages is as follows: controlling the oxygen consumption rate to be 10-90 mmol/L.h, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 mmol/L.h, etc. during the fermentation culture for 0-8 h; controlling the oxygen consumption rate to be 60-150 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. during the fermentation culture for 8-24 h; controlling the oxygen consumption rate to 70-120 mmol/L.h, for example, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 mmol/L.h, etc., during the fermentation culture for 24-40 h; the oxygen consumption rate is controlled to be 50 to 100 mmol/L.h, for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mmol/L.h, or the like, during the period from the fermentation culture for 40h to the end of the fermentation.
In some embodiments of the invention, the manner in which the oxygen consumption rate is controlled in stages is as follows: controlling the oxygen consumption rate to be 10-90 mmol/L.h, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 mmol/L.h, etc. during the fermentation culture for 0-8 h; controlling the oxygen consumption rate to be 60-120 mmol/L.h, for example, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 mmol/L.h, etc., during the fermentation culture for 8-16 h; controlling the oxygen consumption rate to be 80-150 mmol/L.h, for example, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 mmol/L.h, etc., during 16-24 h of fermentation culture; controlling the oxygen consumption rate to 70-120 mmol/L.h, for example, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 mmol/L.h, etc., during the fermentation culture for 24-32 h; controlling the oxygen consumption rate to 70-100 mmol/L.h, for example, 70, 75, 80, 85, 90, 95, 100 mmol/L.h, etc. during the fermentation culture for 32-40 h; controlling the oxygen consumption rate to be 70-90 mmol/L.h, for example, 70, 75, 80, 85, 90 mmol/L.h, etc. during the fermentation culture for 40-48 h; the oxygen consumption rate is controlled to be 50 to 80 mmol/L.multidot.h, for example, 50, 55, 60, 65, 70, 75, 80 mmol/L.multidot.h, or the like, during the period from 48 hours of the fermentation culture to the completion of the fermentation. The environmental conditions and the thallus metabolism requirements are different at different stages of fermentation, and the optimal culture conditions can be provided for thallus product synthesis by controlling the oxygen consumption rate in stages.
In the present invention, the oxygen consumption rate (OUR) is calculated according to the following formula:
Fin: ventilation (L/h or m)3/h);
V: volume of fermentation broth (L or m)3);
Co2in: oxygen concentration (%) in intake air;
Cidle in: inert gas concentration (%) in intake air;
Co2out: the oxygen concentration (%) in the exhaust gas discharged from the fermentation broth;
Cco2out: carbon dioxide concentration (%) in the exhaust gas discharged from the fermentation broth.
Wherein Co2in、CIdle in、Co2out、Cco2out is monitored by the mass spectrometer in real time.
In some embodiments of the invention, the redox potential is controlled in stages as follows: during fermentation culture for 0-8 h, the oxidation-reduction potential is controlled at-100-50 mV, which may be-100, -90, -80, -70, -60, -50, -40, -30, -20, -10, 0, 10, 20, 30, 40, 50mV, etc.; during the fermentation culture for 8-24 h, the oxidation-reduction potential is controlled to-300 to-50 mV, which can be-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, etc.; during the fermentation culture for 24-40 h, the oxidation-reduction potential is controlled to-250-100 mV, which may be-250, -240, -230, -220, -210, -200, -190, -180, -170, -160, -150, -140, -130, -120, -110, -100mV, for example; the redox 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, etc., during the period from the fermentation culture for 40h to the end of the fermentation.
In some embodiments of the invention, the redox potential is controlled in stages as follows: during fermentation culture for 0-8 h, the oxidation-reduction potential is controlled at-100-50 mV, which may be-100, -90, -80, -70, -60, -50, -40, -30, -20, -10, 0, 10, 20, 30, 40, 50mV, etc.; during the fermentation culture for 8-16 h, the oxidation-reduction potential is controlled to-200 to-50 mV, which can be-200, -190, -180, -170, -160, -150, -140, -130, -120, -110, -100, -90, -80, -70, -60, -50mV, etc.; during the fermentation culture for 16-24 h, the oxidation-reduction potential is controlled to-300-50 mV, which can be-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, etc.; during the fermentation culture for 24-32 h, the oxidation-reduction potential is controlled to-250-100 mV, which can be-250, -240, -230, -220, -210, -200, -190, -180, -170, -160, -150, -140, -130, -120, -110, -100 mV; during the fermentation culture for 32-40 h, the oxidation-reduction potential is controlled to-200 to-100 mV, which can be-200, -190, -180, -170, -160, -150, -140, -130, -120, -110, -100mV, etc.; during the fermentation culture for 40-48 h, the oxidation-reduction potential is controlled to-150 to-100 mV, which can be-150, -140, -130, -120, -110, -100mV, etc.; the redox potential is controlled to be-150 to-50 mV, for example, -150, -140, -130, -120, -110, -100, -90, -80, -70, -60, -50mV, etc., during the period from 48h of fermentation culture to the end of fermentation. The environmental conditions and the thallus metabolism requirements are different in different fermentation periods, and the optimal culture conditions can be provided for thallus product synthesis by controlling the oxidation-reduction potential by stages.
In the present invention, the oxidation-reduction potential is measured by an oxidation-reduction potential electrode.
In some embodiments of the invention, the acetic acid concentration is controlled in stages as follows: controlling the concentration of acetic acid to 0.1-20 g/L, for example, 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 during the fermentation culture for 0-8 h; controlling the concentration of acetic acid to be 0.5-10 g/L, for example, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10g/L and the like during the fermentation culture for 8-24 h; controlling the concentration of acetic acid to be 0.3-10 g/L, for example, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10g/L, etc. during the fermentation culture for 24-40 h; during the fermentation culture for 40h to the end of fermentation, the concentration of acetic acid is controlled to be 0.2-10 g/L, for example, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 g/L.
In some embodiments of the invention, the acetic acid concentration is controlled in stages as follows: controlling the concentration of acetic acid to 0.1-20 g/L, for example, 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 during the fermentation culture for 0-8 h; controlling the concentration of acetic acid to be 0.5-10 g/L, for example, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10g/L and the like during the fermentation culture for 8-16 h; controlling the concentration of acetic acid to be 0.5-6 g/L, for example, 0.5, 1, 2, 3, 4, 5, 6g/L and the like during 16-24 h of fermentation culture; controlling the concentration of acetic acid to be 0.3-10 g/L, for example, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10g/L, etc. during the fermentation culture for 24-32 h; controlling the concentration of acetic acid to be 0.3-8 g/L, for example, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8g/L and the like during the fermentation culture for 32-40 h; controlling the concentration of acetic acid to be 0.2-10 g/L, for example, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10g/L, etc. during the fermentation culture for 40-48 h; during the period from 48 hours of fermentation culture to the end of fermentation, the concentration of acetic acid is controlled to be 0.2-8 g/L, for example, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8 g/L. The environmental conditions and the thallus metabolism requirements are different at different periods of fermentation, and the optimal culture conditions can be provided for thallus product synthesis by controlling the acetic acid concentration by stages.
In the present invention, the acetic acid concentration is measured in real time by an acetic acid on-line measuring instrument (e.g., a BRS-CH COOH full-automatic acetic acid concentration meter).
In some embodiments of the invention, the specific carbon dioxide release rate is controlled in stages as follows: controlling the specific carbon dioxide release rate 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, etc. during the fermentation culture for 0-8 h; controlling the specific carbon dioxide release rate to be 0.1-0.6, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, etc. during the fermentation culture for 8-24 h; controlling the specific carbon dioxide release rate to be 0.1-0.5, for example, 0.1, 0.2, 0.3, 0.4, 0.5, etc. during the fermentation culture for 24-40 h; 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, or the like, during the period from 40 hours of the fermentation culture to the completion of the fermentation.
In some embodiments of the invention, the specific carbon dioxide release rate is controlled in stages as follows: controlling the specific carbon dioxide release rate of the fermentation process 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, etc. during the fermentation culture for 0-8 h; controlling the specific carbon dioxide release rate of the fermentation process to be 0.1-0.6, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, etc. during the fermentation culture for 8-16 h; controlling the specific carbon dioxide release rate of the fermentation process to be 0.15-0.4, for example, 0.15, 0.2, 0.3, 0.4 and the like during the fermentation culture for 16-24 h; controlling the specific carbon dioxide release rate of the fermentation process 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 24-32 h; controlling the specific carbon dioxide release rate of the fermentation process to be 0.1-0.5, for example, 0.1, 0.2, 0.3, 0.4, 0.5, etc. during the fermentation culture for 32-40 h; controlling the specific carbon dioxide release rate of the fermentation process to be 0.05-0.3, for example, 0.05, 0.1, 0.2, 0.3 and the like during the fermentation culture for 40-48 h; the specific carbon dioxide release rate during the fermentation is controlled to be 0.05-0.15, for example, 0.05, 0.1, 0.15, etc., during the period from 48 hours to the end of the fermentation. The environmental conditions and the thallus metabolism requirements are different at different periods of fermentation, and the optimal culture conditions can be provided for thallus product synthesis by controlling the carbon dioxide release rate in stages.
In the present invention, the specific carbon dioxide release rate is calculated by the following formula:
CCO2: the concentration (%) of carbon dioxide in the waste gas discharged by the fermentation liquor is monitored in real time by a mass spectrometer;
v: volume of fermentation broth (L or m)3);
dt: unit fermentation time (h);
DCW: and (3) monitoring the thallus content (g/L) of the fermentation liquor in real time by a living cell sensor.
In the present invention, the strain used in the fermentation culture may be any of various strains suitable for producing glucosamine, and specific examples thereof include, but are not limited to: escherichia coli (Escherichia coli ACCC01548), Bacillus subtilis ACCC 10242, Saccharomyces cerevisiae (Saccharomyces cerevisiae ACCC 20037), Rhizopus oligosporus (Rhizopus oligosporus ACCC 30496), Aspergillus sp ACCC 30005, Mucor (Monascus pilosus ACCC 30504), etc. The fermentation process flow of glucosamine comprises seed activation, shake flask culture, seed expansion culture and fermenter culture, wherein the formula of the culture medium adopted in each stage is not particularly limited, and can be selected conventionally in the field, and the culture medium suitable for growth of the strain can be selected according to different strains, which can be known by those skilled in the art and is not described herein again.
In a specific embodiment, the strain is Escherichia coli (Escherichia coli ACCC01548), and the specific process flow of the fermentation culture is as follows:
(1) seed activation: preparing a plate culture medium, sterilizing at 121-123 ℃ for 20-30 min, and adjusting the pH value to 5.5-7.5 before sterilization; sucking a small amount of bacterial liquid from a seed-preserving pipe for gradient dilution, sucking a small amount of diluted bacterial suspension into a plate culture medium, and culturing at 28-38 ℃ for 12-72 h to obtain mature single bacterial colonies; 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) and (3) shake flask culture: preparing a shake flask culture medium, sterilizing at 121-123 ℃ for 20-30 min, and adjusting the pH value to 5.5-7.5 before sterilization; selecting 1-20 single colonies from a mature plate culture medium, inoculating the single colonies into a shake flask culture medium, wherein the shake flask culture conditions comprise the culture temperature of 28-38 ℃, the rotation speed of 150-250 rpm, and the culture period of 4-48 h, and when the wet weight of thalli reaches 1-20 g/L, inoculating the thalli into a seeding tank, and controlling the inoculation amount to be 0.1-5%; the shake flask culture medium had the following composition: 1-10 g/L of sodium chloride, 1-15 g/L of peptone and 1-15 g/L of yeast extract powder;
(3) seed amplification culture: preparing a seed culture medium, sterilizing at 121-123 ℃ for 20-30 min, and adjusting the pH value to 5.5-7.5 before sterilization; the seed culture conditions comprise the culture temperature of 28-38 ℃, the tank pressure of 0.025-0.08 MPa, the aeration ratio of 0.2-2 VVM, the rotation speed of 100-500 rpm, the culture period of 4-48 h, when the wet weight of the thalli reaches 1-20 g/L, the thalli are planted into a fermentation tank, and the inoculation amount is controlled at 5-30%; the components of the seed culture medium are as follows: 1-15 g/L of glucose, 1-10 g/L of sodium chloride, 1-15 g/L of peptone and 1-15 g/L of yeast extract powder;
(4) culturing in a fermentation tank: preparing a fermentation culture medium, sterilizing at 121-123 ℃ for 20-30 min, and adjusting the pH value to 5.5-7.5 before sterilization; 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 be 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 be 0.1-15 g/L, and the concentration of the nitrogen source is controlled to be 0.01-2 g/L; the fermentation medium comprises the following components: 1-20 g/L glucose, 1-20 g/L sodium dihydrogen phosphate, 0.5-10 g/L magnesium chloride, 0.5-10 g/L sodium sulfate, 1-15 g/L yeast extract powder, 0.01-5 g/L calcium sulfate, 0.005-0.5 g/L zinc chloride and 0.0001-0.5 g/L manganese sulfate. Wherein the carbon source may be at least one selected from glucose, sucrose and glycerol. The nitrogen source may be ammonium sulfate and/or yeast extract. The carbon source and the nitrogen source may be added in the form of a feed medium. In one embodiment, the feed medium comprises the following components in mass concentrations: 50-60% of glucose, sucrose or glycerol, 20-28% of ammonia water, 10-40% of sodium hydroxide, 0.1-5% of ammonium sulfate and 0.1-5% of yeast extract powder.
The fermentation process monitors at least one of oxygen consumption rate, oxidation-reduction potential, acetic acid concentration and specific carbon dioxide release rate in the fermentation liquor in real time to feed back and regulate the process of the fermentation process of the glucosamine, 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 reducing at least one of rotating speed, air flow and tank pressure.
Measuring the glucosamine concentration in the fermentation broth by HPLC when the product growth rate is significantly slowed or the staining of the bacteria is light, specifically Athena NH made of stainless steel2The column measures the glucosamine concentration. In one embodiment, the diameter of the column is 4.6mm, the height is 250mm, and the particle size of the packing in the column is 3 μm; the wavelength for measuring the glucosamine concentration is 195 nm; the mobile phase comprises 80% chromatographic acetonitrile and 20% dipotassium hydrogen phosphate solution with the concentration of 2.68 g/L; flow rate of mobile phase to control the retention time of glucosamineAt about 13min, the flow rate is 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 construed as limiting the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The strain used in examples 1 to 18 and 1-1 to 5-1 was Escherichia coli ACCC 01548.
Example 1: 100L tank fermentation production process
(1) Seed activation
And (3) sucking a little bacterial liquid from the seed-preserving tube for gradient dilution, sucking a little bacterial suspension to a plate culture medium, and culturing at 32 ℃ for 24h to obtain a mature single bacterial colony. The components of the plate culture medium are as follows: 5g/L sodium chloride, 10g/L peptone, 10g/L yeast extract powder and 20g/L agar powder, adjusting the pH value to 7.5 before sterilization, and then sterilizing at 121 ℃ for 25 min.
(2) Shaking culture
3 single colonies are picked from a mature 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 culture is carried out on a shaking table at the culture temperature of 32 ℃ and the rotation speed of 220rpm for 20h, and when the wet weight of the thalli reaches 8g/L, the thalli can be transferred into a seeding tank for culture. The shake flask culture medium comprises the following components: 5g/L sodium chloride, 10g/L peptone and 10g/L yeast extract powder, adjusting the pH value to 7.5 before sterilization, and then sterilizing at 121 ℃ for 25 min.
(3) Seed scale-up 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 solution to a 15L seed tank according to 1% inoculation amount, and filling the solution into 9L seed tanks. The seed culture medium comprises the following components: 10g/L of glucose, 5g/L of sodium chloride, 10g/L of peptone and 10g/L of yeast extract powder. The seed culture conditions comprise culture temperature of 32 deg.C, tank pressure of 0.05MPa, aeration ratio of 1.5VVM, rotation speed of 500rpm, pH value controlled at about 7.5 by adding ammonia water during culture, culture period of 12h, and culturing in a fermenter when wet weight reaches 10 g/L.
(4) Fermentation culture
Preparing a fermentation culture medium, sterilizing at 121 ℃ for 25min, adjusting the pH value to 7.5 before sterilization, transferring seed liquid in a seeding tank to a 100L fermentation tank according to 15% inoculation amount, and filling 50L of liquid. Initial culture conditions: the culture temperature is 32 ℃, the rotation 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 a glucose solution with the concentration of 50 percent and 0.2 percent of ammonium sulfate are fed in the fermentation culture process so as to respectively control the contents of a carbon source and a nitrogen source to be 0.6g/L and 0.8 g/L.
The fermentation medium comprises 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 feeds back and regulates the process technology in real time by monitoring the concentration of acetic acid in fermentation liquor, and controls the concentration of the acetic acid in the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
controlling the concentration of acetic acid to be 0.1-20 g/L during fermentation culture for 0-8 h;
controlling the concentration of acetic acid to be 0.5-10 g/L during the fermentation culture period of 8-24 h;
controlling the concentration of acetic acid to be 0.3-10 g/L during the fermentation culture period of 24-40 h;
controlling the concentration of acetic acid to be 0.2-10 g/L during the period from fermentation culture to fermentation completion.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 125g/L when the fermentation is ended, and the conversion rate reaches 45%.
Example 2: 5m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, and sterilizingAdjusting pH to 7.2, transferring seed solution to 5m at 15% inoculation amount3The liquid filling amount of the fermentation tank is 2.5m3. Initial culture conditions: the culture temperature is 32 ℃, the rotation 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 a glucose solution with the concentration of 55% and 0.4% of ammonium sulfate are fed in the fermentation culture process so as to respectively control the contents of a carbon source and a nitrogen source to be 0.5g/L and 1.1 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 7.2 before sterilization.
The fermentation culture process feeds back and regulates the process technology in real time by monitoring the concentration of acetic acid in fermentation liquor, and controls the concentration of the acetic acid in the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
controlling the concentration of acetic acid to be 0.1-20 g/L during fermentation culture for 0-8 h;
controlling the concentration of acetic acid to be 0.5-10 g/L during the fermentation culture period of 8-16 h;
controlling the concentration of acetic acid to be 0.5-6 g/L during 16-24 h of fermentation culture;
controlling the concentration of acetic acid to be 0.3-10 g/L during the fermentation culture period of 24-32 h;
controlling the concentration of acetic acid to be 0.3-8 g/L during the fermentation culture period of 32-40 h;
controlling the concentration of acetic acid to be 0.2-10 g/L during fermentation culture for 40-48 h;
controlling the concentration of acetic acid to be 0.2-8 g/L during the period from 48 hours of fermentation culture to the end of fermentation.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 130g/L when the fermentation is ended, and the conversion rate reaches 45%.
Example 1-1: 5m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 7.2 before sterilization, transferring seed solution to 5m according to 15% inoculum size3The liquid filling amount of the fermentation tank is 2.5m3. Initial culture conditions: the culture temperature is 32 ℃, the rotation 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 a glucose solution with the concentration of 55% and 0.4% of ammonium sulfate are fed in the fermentation culture process so as to respectively control the contents of a carbon source and a nitrogen source to be 0.5g/L and 1.1 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 7.2 before sterilization.
The fermentation culture process feeds back and regulates the process technology in real time by monitoring the concentration of acetic acid in fermentation liquor, and controls the concentration of the acetic acid in the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
controlling the concentration of acetic acid to be 0.1-20 g/L during fermentation culture for 0-8 h;
controlling the concentration of acetic acid to be 0.5-10 g/L during the fermentation culture period of 8-16 h;
controlling the concentration of acetic acid to be 0.5-6 g/L during 16-24 h of fermentation culture;
controlling the concentration of acetic acid to be 10-20 g/L during the fermentation culture period of 24-32 h;
controlling the concentration of acetic acid to be 0.1-0.2 g/L during the fermentation culture period of 32-40 h;
controlling the concentration of acetic acid to be 0.2-10 g/L during fermentation culture for 40-48 h;
controlling the concentration of acetic acid to be 0.2-8 g/L during the period from 48 hours of fermentation culture to the end of fermentation.
And (3) when the product growth rate is obviously slowed or the bacterial body is lightly stained, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by using HPLC, the glucosamine content reaches 100g/L when the fermentation is stopped, the conversion rate reaches 35 percent, compared with the example 2, part of the interval is not controlled within the acetic acid concentration required range, and the glucosamine fermentation level and the conversion rate are lower than those in the example 2.
Example 3: 60m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 60m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 30m3. Initial culture conditions: the culture temperature is 33 ℃, the rotation 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 a glucose solution with the concentration of 60 percent and ammonium sulfate with the concentration of 1 percent are fed in the fermentation culture process so as to respectively control the contents of a carbon source and a nitrogen source to be 0.25g/L and 0.7 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
In the fermentation culture process, the process technology is fed back and regulated in real time by monitoring the oxygen consumption rate in the fermentation liquor, and the oxygen consumption rate in the fermentation liquor is controlled within the following range by increasing or reducing 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 period of 0-8 h;
controlling the oxygen consumption rate to be 60-150 mmol/L.h during the fermentation culture period of 8-24 h;
controlling the oxygen consumption rate to be 70-120 mmol/L.h during the fermentation culture period of 24-40 h;
controlling the oxygen consumption rate to be 50-100 mmol/L.h during the period from fermentation culture to fermentation ending.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 140g/L when the fermentation is ended, and the conversion rate reaches 50%.
Example 4: 60m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 60m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 30m3. Initial culture conditions: the culture temperature is 33 ℃, the rotation 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 a glucose solution with the concentration of 60 percent and ammonium sulfate with the concentration of 1 percent are fed in the fermentation culture process so as to respectively control the contents of a carbon source and a nitrogen source to be 0.4g/L and 0.65 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
In the fermentation culture process, the process technology is fed back and regulated in real time by monitoring the oxygen consumption rate in the fermentation liquor, and the oxygen consumption rate in the fermentation liquor is controlled within the following range by increasing or reducing 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 period of 0-8 h;
controlling the oxygen consumption rate to be 60-120 mmol/L.h during the fermentation culture period of 8-16 h;
controlling the oxygen consumption rate to be 80-150 mmol/L.h during 16-24 h of fermentation culture;
controlling the oxygen consumption rate to be 70-120 mmol/L.h during the fermentation culture period of 24-32 h;
controlling the oxygen consumption rate to be 70-100 mmol/L.h during the fermentation culture period of 32-40 h;
controlling the oxygen consumption rate to be 70-90 mmol/L.h during the fermentation culture period of 40-48 h;
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 product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 150g/L when the fermentation is ended, and the conversion rate reaches 50%.
Example 2-1: 60m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 60m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 30m3. Initial culture conditions: the culture temperature is 33 ℃, the rotation 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 a glucose solution with the concentration of 60 percent and ammonium sulfate with the concentration of 1 percent are fed in the fermentation culture process so as to respectively control the contents of a carbon source and a nitrogen source to be 0.4g/L and 0.65 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
In the fermentation culture process, the process technology is fed back and regulated in real time by monitoring the oxygen consumption rate in the fermentation liquor, and the oxygen consumption rate in the fermentation liquor is controlled within the following range by increasing or reducing 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 period of 0-8 h;
controlling the oxygen consumption rate to be 60-120 mmol/L.h during the fermentation culture period of 8-16 h;
controlling the oxygen consumption rate to be 60-70 mmol/L.h during 16-24 h of fermentation culture;
controlling the oxygen consumption rate to be 120-150 mmol/L.h during the fermentation culture period of 24-32 h;
controlling the oxygen consumption rate to be 50-60 mmol/L.h during the fermentation culture period of 32-40 h;
controlling the oxygen consumption rate to be 70-100 mmol/L.h during the fermentation culture period of 40-48 h;
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 product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 110g/L when the fermentation is ended, and the conversion rate reaches 35%. Compared with example 4, part of the interval is not controlled in the required range of oxygen consumption rate, and the fermentation level and conversion rate of glucosamine are lower than those of example 4.
Example 5: 120m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 120m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 60m3. Initial culture conditions: the culture temperature is 30 ℃, the rotation 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 a 60% glucose solution and 0.3% yeast extract powder are fed in the fermentation culture process so as to respectively control the contents of a carbon source and a nitrogen source to be 0.25g/L and 0.8 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process feeds back a regulation process technology in real time by monitoring the oxidation-reduction potential, and controls the oxidation-reduction potential in the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
controlling the oxidation-reduction potential to be-100-50 mV during the fermentation culture period of 0-8 h;
controlling the oxidation-reduction potential to be-300 to-50 mV during the fermentation culture period of 8-24 h;
controlling the oxidation-reduction potential to be-250 to-100 mV during the fermentation culture period of 24-40 h;
during the fermentation culture for 40h to the end of fermentation, the oxidation-reduction potential is controlled to be-200 to-50 mV.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 135g/L when the fermentation is ended, and the conversion rate reaches 44%.
Example 6: 120m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 120m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 60m3. Initial culture conditions: the culture temperature is 30 ℃, the rotation 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 a 60% glucose solution and 0.3% yeast extract powder are fed in the fermentation culture process so as to respectively control the contents of a carbon source and a nitrogen source to be 0.8g/L and 1.2 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process feeds back a regulation process technology in real time by monitoring the oxidation-reduction potential, and controls the oxidation-reduction potential in the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
controlling the oxidation-reduction potential to be-100-50 mV during the fermentation culture period of 0-8 h;
controlling the oxidation-reduction potential to be-200 to-50 mV during the fermentation culture period of 8-16 h;
controlling the oxidation-reduction potential to be-300 to-50 mV during 16-24 h of fermentation culture;
controlling the oxidation-reduction potential to be-250 mV to-100 mV during the fermentation culture period of 24-32 h.
During the fermentation culture for 32-40 h, the oxidation-reduction potential is controlled to-200 to-100 mV.
During the fermentation culture for 40-48 h, the oxidation-reduction potential is controlled to-150 to-100 mV.
During the fermentation culture for 48-56 h, the oxidation-reduction potential is controlled to-150 to-50 mV.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 145g/L when the fermentation is ended, and the conversion rate reaches 48 percent.
Example 3-1: 120m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 120m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 60m3. Initial culture conditions: the culture temperature is 30 ℃, the rotation 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 a 60% glucose solution and 0.3% yeast extract powder are fed in the fermentation culture process so as to respectively control the contents of a carbon source and a nitrogen source to be 0.8g/L and 1.2 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process feeds back a regulation process technology in real time by monitoring the oxidation-reduction potential, and controls the oxidation-reduction potential in the following range by increasing or decreasing at least one of the rotating speed, the air quantity and the tank pressure:
controlling the oxidation-reduction potential to be-100-50 mV during the fermentation culture period of 0-8 h;
controlling the oxidation-reduction potential to be-200 to-50 mV during the fermentation culture period of 8-16 h;
controlling the oxidation-reduction potential to be-40 to-10 mV during 16-24 h of fermentation culture;
controlling the oxidation-reduction potential to be between 90 and 10mV below zero during the fermentation culture period of 24 to 32 hours.
During the fermentation culture for 32-40 h, the oxidation-reduction potential is controlled to-200 to-100 mV.
During the fermentation culture for 40-48 h, the oxidation-reduction potential is controlled to-300 to-210 mV.
During the fermentation culture for 48-56 h, the oxidation-reduction potential is controlled to-150 to-50 mV.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 125g/L when the fermentation is ended, and the conversion rate reaches 30%. Compared with example 6, the oxidation-reduction potential of part of the interval is not controlled in the process requirement range, and the fermentation level and the conversion rate of the glucosamine are lower than those of example 6.
Example 7: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 160m according to 15% inoculum size3The liquid filling amount of the fermentation tank is 90m3. Initial culture conditions: the culture temperature is 32 ℃, the rotation 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 a glucose solution with the concentration of 58 percent and yeast extract powder with the concentration of 0.2 percent are fed in the fermentation culture process so as to respectively control the contents of a carbon source and a nitrogen source to be 3.2g/L and 0.8 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process feeds back and regulates the process technology in real time by monitoring the specific carbon dioxide release rate, and controls the specific carbon dioxide release rate in the following range by increasing or reducing at least one of the rotating speed, the air quantity and the tank pressure:
controlling the specific carbon dioxide release rate in the fermentation process to be 0.05-0.8 during the fermentation culture period of 0-8 h;
controlling the specific carbon dioxide release rate in the fermentation process to be 0.1-0.6 during the fermentation culture period of 8-24 h;
controlling the specific carbon dioxide release rate in the fermentation process to be 0.1-0.5 during the fermentation culture period of 24-40 h;
during the fermentation culture for 40-56 h, the specific carbon dioxide release rate in the fermentation process is controlled to be 0.05-0.3.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 150g/L when the fermentation is ended, and the conversion rate reaches 50%.
Example 8: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 160m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 90m3. 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 in the fermentation culture process by supplementing ammonia water, and a glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of a carbon source to be 3.5 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process feeds back and regulates the process technology in real time by monitoring the specific carbon dioxide release rate, and controls the specific carbon dioxide release rate in the following range by increasing or reducing at least one of the rotating speed, the air quantity and the tank pressure:
controlling the specific carbon dioxide release rate in the fermentation process to be 0.05-0.8 during the fermentation culture period of 0-8 h;
controlling the specific carbon dioxide release rate in the fermentation process to be 0.1-0.6 during the fermentation culture period of 8-16 h;
controlling the specific carbon dioxide release rate in the fermentation process to be 0.15-0.4 during the fermentation culture period of 16-24 h;
controlling the specific carbon dioxide release rate in the fermentation process to be 0.15-0.5 during the fermentation culture period of 24-32 h;
controlling the specific carbon dioxide release rate in the fermentation process to be 0.1-0.5 during the fermentation culture period of 32-40 h;
controlling the specific carbon dioxide release rate in the fermentation process to be 0.05-0.3 during the fermentation culture period of 40-48 h;
and during the fermentation culture period of 48-56 h, controlling the specific carbon dioxide release rate in the fermentation process to be 0.05-0.15.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 160g/L when the fermentation is ended, and the conversion rate reaches 55%.
Example 4-1: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 160m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 90m3. 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 in the fermentation culture process by supplementing ammonia water, and a glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of a carbon source to be 3.5 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process feeds back and regulates the process technology in real time by monitoring the specific carbon dioxide release rate, and controls the specific carbon dioxide release rate in the following range by increasing or reducing at least one of the rotating speed, the air quantity and the tank pressure:
controlling the specific carbon dioxide release rate in the fermentation process to be 0.05-0.8 during the fermentation culture period of 0-8 h;
controlling the specific carbon dioxide release rate in the fermentation process to be 0.05-0.09 during the fermentation culture period of 8-16 h;
controlling the specific carbon dioxide release rate in the fermentation process to be 0.4-0.6 during the fermentation culture period of 16-24 h;
controlling the specific carbon dioxide release rate in the fermentation process to be 0.15-0.5 during the fermentation culture period of 24-32 h;
controlling the specific carbon dioxide release rate in the fermentation process to be 0.1-0.5 during the fermentation culture period of 32-40 h;
controlling the specific carbon dioxide release rate in the fermentation process to be 0.3-0.5 during the fermentation culture period of 40-48 h;
and during the fermentation culture period of 48-56 h, controlling the specific carbon dioxide release rate in the fermentation process to be 0.05-0.15.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 130g/L when the fermentation is ended, and the conversion rate reaches 40%. Compared with the example 8, the specific carbon dioxide release rate of the partial section is not controlled within the process requirement range, and the fermentation level and the conversion rate of the glucosamine are lower than those of the example 8.
Example 9: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 160m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 90m3. Initial cultureConditions are as follows: 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 in the fermentation culture process by supplementing ammonia water, and a glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of a carbon source to be 10.8 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
In the fermentation culture process, the process technology is fed back and regulated in real time by monitoring the oxygen consumption rate and the oxidation-reduction potential in the fermentation liquid, and the oxygen consumption rate and the oxidation-reduction potential in the fermentation liquid are controlled within the following ranges by increasing or reducing 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 and the oxidation-reduction potential to be-100-50 mV during the fermentation culture period of 0-8 h;
controlling the oxygen consumption rate to be 60-150 mmol/L.h and the oxidation-reduction potential to be-300 to-50 mV during the fermentation culture period of 8-24 h;
controlling the oxygen consumption rate at 70-120 mmol/L.h and the oxidation-reduction potential at-250 to-100 mV during the fermentation culture period of 24-40 h;
during the period from fermentation culture for 40h to the end of fermentation, the oxygen consumption rate is controlled to be 50-100 mmol/L.h, and the oxidation-reduction potential is controlled to be-200 to-50 mV.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 150g/L when the fermentation is ended, and the conversion rate reaches 50%.
Example 10: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 160m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 90m3. 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 in the fermentation culture process by supplementing ammonia water, and a glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of a carbon source to be 13.5 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
In the fermentation culture process, the process technology is fed back and regulated in real time by monitoring the oxygen consumption rate and the acetic acid concentration in the fermentation broth, and the oxygen consumption rate and the acetic acid concentration in the fermentation broth are controlled within the following ranges by increasing or reducing 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 and the acetic acid concentration to be 0.1-20 g/L during the fermentation culture period of 0-8 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 period of 8-24 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 during the fermentation culture period of 24-40 h;
during the period from fermentation culture for 40h to the end of fermentation, the oxygen consumption rate is controlled to be 50-100 mmol/L.h, and the acetic acid concentration is controlled to be 0.2-10 g/L.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 145g/L when the fermentation is ended, and the conversion rate reaches 52 percent.
Example 11: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 160m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 90m3. 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 in the fermentation culture process by supplementing ammonia water, and a glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of a carbon source to be 12 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
In the fermentation culture process, the process technology is fed back and regulated in real time by monitoring the oxygen consumption rate and the specific carbon dioxide release rate in the fermentation broth, and the oxygen consumption rate and the specific carbon dioxide release rate in the fermentation broth are controlled within the following ranges by increasing or reducing 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 and the specific carbon dioxide release rate to be 0.05-0.8 during the fermentation culture period of 0-8 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 period of 8-24 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 during the fermentation culture period of 24-40 h;
during the period from fermentation culture for 40h to the end of fermentation, the oxygen consumption rate is controlled to be 50-100 mmol/L.h, and the specific carbon dioxide release rate is controlled to be 0.05-0.3.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 148g/L when the fermentation is ended, and the conversion rate reaches 51%.
Example 12: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, and sterilizingAdjusting pH to 6.0, transferring seed solution to 160m with 20% inoculation amount3The liquid filling amount of the fermentation tank is 90m3. 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 in the fermentation culture process by supplementing ammonia water, and a glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of a carbon source to be 0.15 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process feeds back and regulates the process technology in real time by monitoring the oxidation-reduction potential and the acetic acid concentration, and controls the oxidation-reduction potential and the acetic acid concentration in the following ranges by increasing or reducing at least one of the rotating speed, the air quantity and the tank pressure:
controlling the oxidation-reduction potential to be-100-50 mV and controlling the acetic acid concentration to be 0.1-20 g/L during fermentation culture for 0-8 h;
controlling the oxidation-reduction potential to be-300 to-50 mV and controlling the acetic acid concentration to be 0.5 to 10g/L during the fermentation culture period of 8 to 24 hours;
controlling the oxidation-reduction potential to be-250 to-100 mV and controlling the acetic acid concentration to be 0.3 to 10g/L during the fermentation culture for 24 to 40 hours;
during the fermentation culture for 40h to the end of fermentation, the oxidation-reduction potential is controlled to be-200 to-50 mV, and the acetic acid concentration is controlled to be 0.2 to 10 g/L.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 145g/L when the fermentation is ended, and the conversion rate reaches 52 percent.
Example 13: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, and adjusting pH before sterilizationAdjusting to 6.0, transferring seed liquid to 160m according to 20% of inoculation amount3The liquid filling amount of the fermentation tank is 90m3. 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 in the fermentation culture process by supplementing ammonia water, and a glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of a carbon source to be 0.2 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process feeds back a regulation process technology in real time by monitoring the oxidation-reduction potential and the specific carbon dioxide release rate, and controls the oxidation-reduction potential and the specific carbon dioxide release rate within the following range by increasing or reducing at least one of the rotating speed, the air quantity and the tank pressure:
controlling 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 0-8 h;
controlling the oxidation-reduction potential to be-300 to-50 mV and the specific carbon dioxide release rate to be 0.1 to 0.6 during the fermentation culture for 8 to 24 hours;
controlling the oxidation-reduction potential to be-250 to-100 mV and the specific carbon dioxide release rate to be 0.1 to 0.5 during the fermentation culture for 24 to 40 hours;
during the fermentation culture for 40h to the end of fermentation, the oxidation-reduction potential is controlled to be-200 to-50 mV, and the specific carbon dioxide release rate is controlled to be 0.05 to 0.3.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 148g/L when the fermentation is ended, and the conversion rate reaches 53%.
Example 14: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing a fermentation culture medium,sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 160m according to 20% inoculation amount3The liquid filling amount of the fermentation tank is 90m3. 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 in the fermentation culture process by supplementing ammonia water, and a glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of a carbon source to be 3 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process feeds back and regulates the process technology in real time by monitoring the specific carbon dioxide release rate and the acetic acid concentration, and controls the specific carbon dioxide release rate and the acetic acid concentration in the following ranges by increasing or reducing at least one of the rotating speed, the air quantity and the tank pressure:
controlling the specific carbon dioxide release rate to be 0.05-0.8 and controlling the acetic acid concentration to be 0.1-20 g/L during the fermentation culture period of 0-8 h;
controlling the specific carbon dioxide release rate to be 0.1-0.6 and controlling the acetic acid concentration to be 0.5-10 g/L during the fermentation culture period of 8-24 h;
controlling the specific carbon dioxide release rate to be 0.1-0.5 and controlling the acetic acid concentration to be 0.3-10 g/L during the fermentation culture period of 24-40 h;
during the period from fermentation culture for 40h to the end of fermentation, the specific carbon dioxide release rate is controlled to be 0.05-0.3, and the acetic acid concentration is controlled to be 0.2-10 g/L.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 150g/L when the fermentation is ended, and the conversion rate reaches 53 percent.
Example 15: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparation of fermentation cultureCulturing, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 160m according to 20% inoculation amount3The liquid filling amount of the fermentation tank is 90m3. 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 in the fermentation culture process by supplementing ammonia water, and a glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of a carbon source to be 10.3 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
In the fermentation culture process, the process technology is fed back and regulated in real time by monitoring the oxygen consumption rate, the oxidation-reduction potential and the acetic acid concentration in the fermentation liquor, and the oxygen consumption rate, the oxidation-reduction potential and the acetic acid concentration in the fermentation liquor are controlled within the following ranges by increasing or reducing 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, 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 period of 0-8 h;
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;
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 during the fermentation culture period of 24-40 h;
during the period from fermentation culture for 40h to the end of fermentation, the oxygen consumption rate is controlled to be 50-100 mmol/L.h, the oxidation-reduction potential is controlled to be-200 to-50 mV, and the acetic acid concentration is controlled to be 0.2-10 g/L.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 154g/L when the fermentation is ended, and the conversion rate reaches 52 percent.
Example 16: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 160m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 90m3. 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 in the fermentation culture process by supplementing ammonia water, and a glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of a carbon source to be 9.6 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
In the fermentation culture process, the process technology is fed back and regulated in real time by monitoring the oxygen consumption rate, the oxidation-reduction potential and the specific carbon dioxide release rate in the fermentation broth, and the oxygen consumption rate, the oxidation-reduction potential and the specific carbon dioxide release rate in the fermentation broth are controlled within the following ranges by increasing or reducing 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, 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 period of 0-8 h;
controlling the oxygen consumption rate to be 60-150 mmol/L.h, 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 period of 8-24 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 during the fermentation culture period of 24-40 h;
during the period from fermentation culture for 40h to the end of fermentation, the oxygen consumption rate is controlled to be 50-100 mmol/L.h, the oxidation-reduction potential is controlled to be-200 to-50 mV, and the specific carbon dioxide release rate is controlled to be 0.05-0.3.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 155g/L when the fermentation is ended, and the conversion rate reaches 53 percent.
Example 17: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 160m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 90m3. 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 in the fermentation culture process by supplementing ammonia water, and a glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of a carbon source to be 8 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
The fermentation culture process feeds back and regulates the process technology in real time by monitoring the acetic acid concentration, the oxidation-reduction potential and the specific carbon dioxide release rate, and controls the acetic acid concentration, the oxidation-reduction potential and the specific carbon dioxide release rate within the following ranges by increasing or reducing at least one of the rotating speed, the air quantity and the tank pressure:
during fermentation culture for 0-8 h, controlling the concentration of acetic acid at 0.1-20 g/L, the oxidation-reduction potential at-100-50 mV, and the specific carbon dioxide release rate at 0.05-0.8;
during the fermentation culture for 8-24 h, controlling the concentration of acetic acid 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;
controlling the concentration of acetic acid 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 during the fermentation culture period of 24-40 h;
during the fermentation culture for 40h to the end of fermentation, the concentration of acetic acid is controlled to be 0.2-10 g/L, the oxidation-reduction potential is controlled to be-200 to-50 mV, and the specific carbon dioxide release rate is controlled to be 0.05-0.3.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 149g/L when the fermentation is ended, and the conversion rate reaches 53 percent.
Example 18: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 160m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 90m3. 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 in the fermentation culture process by supplementing ammonia water, and a glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of a carbon source to be 3.6 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
In the fermentation culture process, the process technology is fed back and regulated 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 liquor, and the oxygen consumption rate, the acetic acid concentration, the oxidation-reduction potential and the specific carbon dioxide release rate in the fermentation liquor are controlled in the following ranges by increasing or reducing at least one of the rotating speed, the air quantity and the tank pressure:
during fermentation culture for 0-8 h, controlling the oxygen consumption rate at 10-90 mmol/L.h, controlling the acetic acid concentration at 0.1-20 g/L, controlling the oxidation-reduction potential at-100-50 mV, and controlling the specific carbon dioxide release rate at 0.05-0.8;
during the fermentation culture for 8-24 h, controlling the oxygen consumption rate at 60-150 mmol/L.h, controlling the acetic acid concentration at 0.5-10 g/L, controlling the oxidation-reduction potential at-300 to-50 mV, and controlling the specific carbon dioxide release rate at 0.1-0.6;
controlling the oxygen consumption rate to be 70-120 mmol/L.h, controlling the acetic acid concentration to be 0.3-10 g/L, controlling the oxidation-reduction potential to be-250-100 mV, and controlling the specific carbon dioxide release rate to be 0.1-0.5 during the fermentation culture period of 24-40 h;
during the period from fermentation culture for 40h to the end of fermentation, the oxygen consumption rate is controlled to be 50-100 mmol/L.h, the acetic acid concentration is controlled to be 0.2-10 g/L, the oxidation-reduction potential is controlled to be-200 to-50 mV, and the specific carbon dioxide release rate is controlled to be 0.05-0.3.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 160g/L when the fermentation is ended, and the conversion rate reaches 55%.
Example 5-1: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 160m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 90m3. 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 in the fermentation culture process by supplementing ammonia water, and a glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of a carbon source to be 3.6 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
Monitoring and staged regulation of oxygen consumption rate, oxidation-reduction potential, acetic acid concentration and specific carbon dioxide release rate are not carried out in the fermentation culture process, the control range of the oxygen consumption rate in the fermentation process is 10-150 mmol/L.h, the control range of the oxidation-reduction potential is-300-50 mV, the control range of the acetic acid concentration is 0.1-20 g/L, and the control range of the specific carbon dioxide release rate is 0.05-0.8. The fermentation process maintains the oxygen consumption rate, oxidation-reduction potential, acetic acid concentration and specific carbon dioxide release rate within the process ranges described above by increasing or decreasing one or more of the rotation speed or air volume or tank pressure in combination.
And when the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the content of the glucosamine in the fermentation liquid is measured by using HPLC, the glucosamine content reaches 120g/L when the fermentation is ended, the conversion rate reaches 42 percent, and the fermentation level and the conversion rate of the glucosamine are lower than those of staged control.
Example 19: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 160m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 90m3. 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 in the fermentation culture process by supplementing ammonia water, and a glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of a carbon source to be 10.3 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
In the fermentation culture process, the process technology is fed back and regulated in real time by monitoring the oxygen consumption rate, the acetic acid concentration and the oxidation-reduction potential in the fermentation liquor, and the oxygen consumption rate, the acetic acid concentration and the oxidation-reduction potential in the fermentation liquor are controlled within the following ranges by increasing or reducing 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, 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 period of 0-8 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 period of 8-16 h;
controlling the oxygen consumption rate to be 80-150 mmol/L.h, controlling the acetic acid concentration to be 0.5-6 g/L and controlling the oxidation-reduction potential to be-300 to-50 mV during 16-24 h of fermentation culture;
controlling the oxygen consumption rate at 70-120 mmol/L.h, controlling the acetic acid concentration at 0.3-10 g/L and controlling the oxidation-reduction potential at-250 to-100 mV during the fermentation culture period of 24-32 h;
during fermentation culture for 32-40 h, controlling the oxygen consumption rate at 70-100 mmol/L.h, controlling the acetic acid concentration at 0.3-8 g/L, and controlling the oxidation-reduction potential at-200 to-100 mV;
controlling the oxygen consumption rate at 70-90 mmol/L.h, controlling the acetic acid concentration at 0.2-10 g/L and controlling the oxidation-reduction potential at-150 to-100 mV during the fermentation culture period of 40-48 h;
during the period from 48 hours of fermentation culture to the end of fermentation, the oxygen consumption rate is controlled to be 50-80 mmol/L.h, the acetic acid concentration is controlled to be 0.2-8 g/L, and the oxidation-reduction potential is controlled to be-150 to-50 mV.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 162g/L when the fermentation is ended, and the conversion rate reaches 56.2%.
Example 20: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 160m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 90m3. 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, and the fermentation culture process is controlled by supplementing ammonia waterThe pH value is controlled to be about 6.0, and a glucose solution with the concentration of 60 percent is fed in the fermentation culture process to control the content of the carbon source to be 8 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
In the fermentation culture process, the process technology is fed back and regulated in real time by monitoring the acetic acid concentration, the oxidation-reduction potential and the specific carbon dioxide release rate in the fermentation broth, and the acetic acid concentration, the oxidation-reduction potential and the specific carbon dioxide release rate in the fermentation broth are controlled within the following ranges by increasing or reducing at least one of the rotating speed, the air quantity and the tank pressure:
during fermentation culture for 0-8 h, controlling the concentration of acetic acid at 0.1-20 g/L, the oxidation-reduction potential at-100-50 mV, and the specific carbon dioxide release rate at 0.05-0.8;
during the fermentation culture for 8-16 h, controlling the concentration of acetic acid 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;
controlling the concentration of acetic acid to be 0.5-6 g/L, the oxidation-reduction potential to be-300-50 mV and the specific carbon dioxide release rate to be 0.15-0.4 during 16-24 h of fermentation culture;
controlling the concentration of acetic acid 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 period of 24-32 h;
during fermentation culture for 32-40 h, controlling the concentration of acetic acid 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;
controlling the concentration of acetic acid 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 during the fermentation culture for 40-48 h;
during the period from 48 hours of fermentation culture to the end of fermentation, the concentration of acetic acid is controlled to be 0.2-8 g/L, the oxidation-reduction potential is controlled to be-150 to-50 mV, and the specific carbon dioxide release rate is controlled to be 0.05-0.15.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 159g/L when the fermentation is ended, and the conversion rate reaches 54%.
Example 21: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 160m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 90m3. 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 in the fermentation culture process by supplementing ammonia water, and a glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of a carbon source to be 9.6 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
In the fermentation culture process, the process technology is fed back and regulated in real time by monitoring the oxygen consumption rate, the oxidation-reduction potential and the specific carbon dioxide release rate in the fermentation broth, and the oxygen consumption rate, the oxidation-reduction potential and the specific carbon dioxide release rate in the fermentation broth are controlled within the following ranges by increasing or reducing 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, 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 period of 0-8 h;
controlling the oxygen consumption rate to be 60-120 mmol/L.h, the oxidation-reduction potential to be-200-50 mV and the specific carbon dioxide release rate to be 0.1-0.6 during the fermentation culture period of 8-16 h;
controlling the oxygen consumption rate to be 80-150 mmol/L.h, the oxidation-reduction potential to be-300 to-50 mV and the specific carbon dioxide release rate to be 0.15-0.4 during 16-24 h of fermentation culture;
controlling the oxygen consumption rate to be 70-120 mmol/L.h, 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 period of 24-32 h;
during 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 to-100 mV, and the specific carbon dioxide release rate at 0.05-0.3;
during the period from 48 hours of fermentation culture to the end of fermentation, the oxygen consumption rate is controlled to be 50-80 mmol/L.h, the oxidation-reduction potential is controlled to be-150 to-50 mV, and the specific carbon dioxide release rate is controlled to be 0.05-0.15.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 161g/L when the fermentation is ended, and the conversion rate reaches 55%.
Example 22: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 160m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 90m3. 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 in the fermentation culture process by supplementing ammonia water, and a glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of a carbon source to be 1.2 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
In the fermentation culture process, the process technology is fed back and regulated in real time by monitoring the oxygen consumption rate, the acetic acid concentration and the specific carbon dioxide release rate in the fermentation broth, and the oxygen consumption rate, the acetic acid concentration and the specific carbon dioxide release rate in the fermentation broth are controlled within the following ranges by increasing or reducing at least one of the rotating speed, the air quantity and the tank pressure:
during fermentation culture for 0-8 h, controlling the oxygen consumption rate at 10-90 mmol/L.h, controlling the acetic acid concentration at 0.1-20 g/L, and controlling the specific carbon dioxide release rate at 0.05-0.8;
during the fermentation culture for 8-16 h, controlling the oxygen consumption rate at 60-120 mmol/L.h, controlling the acetic acid concentration at 0.5-10 g/L, and controlling the specific carbon dioxide release rate at 0.1-0.6;
controlling the oxygen consumption rate to be 80-150 mmol/L.h, controlling the acetic acid concentration to be 0.5-6 g/L and controlling the specific carbon dioxide release rate to be 0.15-0.4 during the fermentation culture period of 16-24 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 period of 24-32 h;
during fermentation culture for 32-40 h, controlling the oxygen consumption rate at 70-100 mmol/L.h, controlling the acetic acid concentration at 0.3-8 g/L, and controlling 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, controlling the acetic acid concentration at 0.2-10 g/L and controlling the specific carbon dioxide release rate at 0.05-0.3;
during the period from 48 hours of fermentation culture to the end of fermentation, the oxygen consumption rate is controlled to be 50-80 mmol/L.h, the acetic acid concentration is controlled to be 0.2-8 g/L, and the specific carbon dioxide release rate is controlled to be 0.05-0.15.
When the product growth rate is obviously slowed down or the bacterial staining is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 162g/L when the fermentation is ended, and the conversion rate reaches 54.4%.
Example 23: 160m3Tank fermentation production process
(1) - (3) same as in example 1;
(4) fermentation culture
Preparing fermentation culture medium, sterilizing at 121 deg.C for 25min, adjusting pH to 6.0 before sterilization, transferring seed solution to 160m according to 20% inoculum size3The liquid filling amount of the fermentation tank is 90m3. 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 in the fermentation culture process by supplementing ammonia water, and a glucose solution with the concentration of 60% is fed in the fermentation culture process so as to control the content of a carbon source to be 3.6 g/L.
The fermentation medium comprises 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 is sterilized at 121 ℃ for 25min, and the pH value is adjusted to 6.0 before sterilization.
In the fermentation culture process, the process technology is fed back and regulated 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 liquor, and the oxygen consumption rate, the acetic acid concentration, the oxidation-reduction potential and the specific carbon dioxide release rate in the fermentation liquor are controlled in the following ranges by increasing or reducing at least one of the rotating speed, the air quantity and the tank pressure:
during fermentation culture for 0-8 h, controlling the oxygen consumption rate at 10-90 mmol/L.h, controlling the acetic acid concentration at 0.1-20 g/L, controlling the oxidation-reduction potential at-100-50 mV, and controlling the specific carbon dioxide release rate at 0.05-0.8;
during the fermentation culture for 8-16 h, controlling the oxygen consumption rate at 60-120 mmol/L.h, controlling the acetic acid concentration at 0.5-10 g/L, controlling the oxidation-reduction potential at-200 to-50 mV, and controlling the specific carbon dioxide release rate at 0.1-0.6;
controlling the oxygen consumption rate to be 80-150 mmol/L.h, controlling the acetic acid concentration to be 0.5-6 g/L, controlling the oxidation-reduction potential to be-300-50 mV, and controlling the specific carbon dioxide release rate to be 0.15-0.4 during the 16-24 h period of fermentation culture;
controlling the oxygen consumption rate to be 70-120 mmol/L.h, controlling the acetic acid concentration to be 0.3-10 g/L, controlling the oxidation-reduction potential to be-250-100 mV, and controlling the specific carbon dioxide release rate to be 0.15-0.5 during the fermentation culture period of 24-32 h;
during fermentation culture for 32-40 h, controlling the oxygen consumption rate at 70-100 mmol/L.h, controlling the acetic acid concentration at 0.3-8 g/L, controlling the oxidation-reduction potential at-200 to-100 mV, and controlling the specific carbon dioxide release rate at 0.1-0.5;
during fermentation culture for 40-48 h, controlling the oxygen consumption rate at 70-90 mmol/L.h, controlling the acetic acid concentration at 0.2-10 g/L, controlling the oxidation-reduction potential at-150 to-100 mV, and controlling the specific carbon dioxide release rate at 0.05-0.3;
during the period from 48 hours of fermentation culture to the end of fermentation, the oxygen consumption rate is controlled to be 50-80 mmol/L.h, the acetic acid concentration is controlled to be 0.2-8 g/L, the oxidation-reduction potential is controlled to be-150 to-50 mV, and the specific carbon dioxide release rate is controlled to be 0.05-0.15.
When the product growth rate is obviously slowed down or the bacterial stain is light, the fermentation is stopped, the glucosamine content in the fermentation liquor is measured by HPLC, the glucosamine content reaches 166g/L when the fermentation is ended, and the conversion rate reaches 56.5 percent.
Examples 24 to 43: a glucosamine production method is different from the glucosamine production methods in the embodiments 1 to 18 in that the production strains are different, and the specific conditions, the glucosamine content in fermentation liquor and the conversion rate are shown in the table 1.
TABLE 1 fermentation levels of different glucosamine production strains
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.
Claims (9)
1. A method for producing glucosamine, comprising monitoring at least one of oxygen consumption rate, oxidation-reduction potential, acetic acid concentration, and specific carbon dioxide release rate in a fermentation broth on-line during fermentation of glucosamine, and controlling the values thereof within the following ranges: the oxygen consumption rate is 10 to 150 mmol/L.h, the oxidation-reduction potential is-300 to 50mV, the acetic acid concentration is 0.1 to 20g/L, and the specific carbon dioxide release rate is 0.05 to 0.8.
2. The method for producing glucosamine according to claim 1, wherein the glucosamine is produced by the step of,
the oxygen consumption rate is controlled in stages in the following way: controlling the oxygen consumption rate to be 10-90 mmol/L.h during the fermentation culture period of 0-8 h; controlling the oxygen consumption rate to be 60-150 mmol/L.h during the fermentation culture period of 8-24 h; controlling the oxygen consumption rate to be 70-120 mmol/L.h during the fermentation culture period of 24-40 h; controlling the oxygen consumption rate to be 50-100 mmol/L.h during the period from fermentation culture for 40h to the end of fermentation;
preferably, the oxygen consumption rate is controlled in stages in the following manner: controlling the oxygen consumption rate to be 10-90 mmol/L.h during the fermentation culture period of 0-8 h; controlling the oxygen consumption rate to be 60-120 mmol/L.h during the fermentation culture period of 8-16 h; controlling the oxygen consumption rate to be 80-150 mmol/L.h during 16-24 h of fermentation culture; controlling the oxygen consumption rate to be 70-120 mmol/L.h during the fermentation culture period of 24-32 h; controlling the oxygen consumption rate to be 70-100 mmol/L.h during the fermentation culture period of 32-40 h; controlling the oxygen consumption rate to be 70-90 mmol/L.h during the fermentation culture period of 40-48 h; 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.
3. The method for producing glucosamine according to claim 1, wherein the glucosamine is produced by the step of,
the oxidation-reduction potential is controlled in stages in the following manner: controlling the oxidation-reduction potential to be-100-50 mV during the fermentation culture period of 0-8 h; controlling the oxidation-reduction potential to be-300 to-50 mV during the fermentation culture period of 8-24 h; controlling the oxidation-reduction potential to be-250 to-100 mV during the fermentation culture period of 24-40 h; controlling the oxidation-reduction potential to be-200 to-50 mV during the fermentation culture period from 40h to the end of fermentation;
preferably, the oxidation-reduction potential is controlled in stages in the following manner: controlling the oxidation-reduction potential to be-100-50 mV during the fermentation culture period of 0-8 h; controlling the oxidation-reduction potential to be-200 to-50 mV during the fermentation culture period of 8-16 h; controlling the oxidation-reduction potential to be-300 to-50 mV during 16-24 h of fermentation culture; controlling the oxidation-reduction potential to be-250 to-100 mV during the fermentation culture period of 24-32 h; controlling the oxidation-reduction potential to be-200 to-100 mV during the fermentation culture period of 32-40 h; controlling the oxidation-reduction potential to be-150 to-100 mV during the fermentation culture for 40-48 h; during the fermentation culture period from 48h to the end of fermentation, the oxidation-reduction potential is controlled to-150 to-50 mV.
4. The method for producing glucosamine according to claim 1, wherein the glucosamine is produced by the step of,
the acetic acid concentration is controlled by stages in the following manner: controlling the concentration of acetic acid to be 0.1-20 g/L during fermentation culture for 0-8 h; controlling the concentration of acetic acid to be 0.5-10 g/L during the fermentation culture period of 8-24 h; controlling the concentration of acetic acid to be 0.3-10 g/L during the fermentation culture period of 24-40 h; controlling the concentration of acetic acid to be 0.2-10 g/L during the period from fermentation culture for 40h to the end of fermentation;
preferably, the acetic acid concentration is controlled in stages in the following manner: controlling the concentration of acetic acid to be 0.1-20 g/L during fermentation culture for 0-8 h; controlling the concentration of acetic acid to be 0.5-10 g/L during the fermentation culture period of 8-16 h; controlling the concentration of acetic acid to be 0.5-6 g/L during 16-24 h of fermentation culture; controlling the concentration of acetic acid to be 0.3-10 g/L during the fermentation culture period of 24-32 h; controlling the concentration of acetic acid to be 0.3-8 g/L during the fermentation culture period of 32-40 h; controlling the concentration of acetic acid to be 0.2-10 g/L during fermentation culture for 40-48 h; controlling the concentration of acetic acid to be 0.2-8 g/L during the period from fermentation culture for 48h to the end of fermentation.
5. The method for producing glucosamine according to claim 1, wherein the glucosamine is produced by the step of,
the specific carbon dioxide release rate is controlled in stages in the following manner: controlling the specific carbon dioxide release rate to be 0.05-0.8 during the fermentation culture period of 0-8 h; controlling the specific carbon dioxide release rate to be 0.1-0.6 during the fermentation culture period of 8-24 h; controlling the specific carbon dioxide release rate to be 0.1-0.5 during the fermentation culture period of 24-40 h; controlling the specific carbon dioxide release rate to be 0.05-0.3 during the period from fermentation culture for 40h to the end of fermentation;
preferably, the specific carbon dioxide release rate is controlled in stages in the following manner: controlling the specific carbon dioxide release rate to be 0.05-0.8 during the fermentation culture period of 0-8 h; controlling the specific carbon dioxide release rate to be 0.1-0.6 during the fermentation culture period of 8-16 h; controlling the specific carbon dioxide release rate to be 0.15-0.4 during the fermentation culture period of 16-24 h; controlling the specific carbon dioxide release rate to be 0.15-0.5 during the fermentation culture period of 24-32 h; controlling the specific carbon dioxide release rate to be 0.1-0.5 during the fermentation culture period of 32-40 h; controlling the specific carbon dioxide release rate to be 0.05-0.3 during the fermentation culture for 40-48 h; controlling the specific carbon dioxide release rate to be 0.05-0.15 during the period from fermentation culture for 48h to the end of fermentation.
6. The method for producing glucosamine according to any one of claims 1 to 5, wherein the ranges of the oxygen consumption rate, the oxidation-reduction potential, the acetic acid concentration, and the specific carbon dioxide release rate in the fermentation broth are controlled by adjusting at least one of the air flow rate, the rotation speed, and the tank pressure.
7. A method for producing glucosamine according to any one of claims 1 to 5, wherein the strain used for glucosamine fermentation is at least one strain selected from Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Rhizopus oligosporus (Rhizopus oligosporus), Aspergillus sp, and Mucor (Monascus pilosus).
8. The method for producing glucosamine according to any one of claims 1 to 5, wherein the method further comprises adding an alkali to control the pH of the fermentation system to 5.5 to 7.5 during the fermentation, adding a carbon source and a nitrogen source to control the concentration of the carbon source and the concentration of the nitrogen source in the fermentation system to 0.1 to 15g/L and 0.01 to 2 g/L; the carbon source is at least one of glucose, sucrose and glycerol, and the nitrogen source is ammonium sulfate and/or yeast extract powder.
9. The method for producing glucosamine according to any one of claims 1 to 5, wherein the fermentation of glucosamine comprises the steps of:
(1) seed activation: absorbing bacterial liquid from a seed-preserving tube for gradient dilution, absorbing the diluted bacterial suspension into a plate culture medium, and culturing at 28-38 ℃ for 12-72 h to obtain mature single bacterial colonies;
(2) and (3) shake flask culture: selecting 1-20 single colonies from a mature plate culture medium, inoculating the single colonies into a shake flask culture medium, wherein the shake flask culture conditions comprise the culture temperature of 28-38 ℃, the rotation speed of 150-250 rpm, and the culture period of 4-48 h, and when the wet weight of thalli reaches 1-20 g/L, inoculating the thalli into a seeding tank, and controlling the inoculation amount to be 0.1-5%;
(3) seed amplification culture: the seed culture conditions comprise the culture temperature of 28-38 ℃, the tank pressure of 0.025-0.08 MPa, the aeration ratio of 0.2-2 VVM, the rotating speed of 100-500 rpm, the culture period of 4-48 h, when the wet weight of the thalli reaches 1-20 g/L, the thalli are planted into a fermentation tank, and the inoculation amount is controlled at 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 be 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 be 0.1-15 g/L, the concentration of the nitrogen source is controlled to be 0.01-2 g/L, and fermentation is stopped when the growth rate of a product is obviously slowed down or the staining of thalli is shallow; the carbon source is at least one of glucose, sucrose and glycerol, and the nitrogen source is ammonium sulfate and/or yeast extract powder.
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CN114196790A (en) * | 2021-12-24 | 2022-03-18 | 内蒙古金达威药业有限公司 | Production control method of glyceric acid |
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CN116064489A (en) * | 2023-04-03 | 2023-05-05 | 内蒙古金达威药业有限公司 | Preparation method of nattokinase |
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