CN111073918A - Pleurotus ostreatus fermentation method for preparing pleuromutilin - Google Patents
Pleurotus ostreatus fermentation method for preparing pleuromutilin Download PDFInfo
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Abstract
The invention discloses a pleurotus ostreatus fermentation method for preparing pleuromutilin, which comprises the following specific steps: s1: preparing a culture medium: preparing a primary culture tank filled with a primary culture medium, a secondary seed culture tank filled with a secondary culture medium and a fermentation culture tank filled with fermentation liquor; s2: first-order seed culture: inoculating the seed liquid of the cultured mother bottle of the pleurotus ostreatus into a primary seed culture tank for primary seed culture, and transferring the mother bottle of the pleurotus ostreatus into a secondary seed culture tank when the concentration of thalli is 15-25% and the pH value is 6-8; s3: secondary seed culture: when the concentration of the bacterial liquid is 20-40% and the pH value is 6-8, transferring the bacterial liquid into a fermentation culture tank; s4: fermentation culture: culturing fermentation liquor in a fermentation culture tank, and stopping fermentation when the chemical titer reaches 18000-20000 u/ml; s5: and (5) feeding. Fructose is adopted to replace glucose as a carbon source, the yield is improved by 10 percent compared with that of a basic fermentation culture medium, and the addition of ferrous sulfate has a promoting effect on the synthesis of pleuromutilin and further improves the chemical potency.
Description
Technical Field
The invention relates to the technical field of fermentation, in particular to a fermentation method of pleurotus ostreatus for preparing pleuromutilin.
Background
Pleuromutilins (pleuromutilins) are a broad spectrum of diterpene antibiotics produced by fermentation of Pleurotus citrinopileus (Pleurotus mutilus) and are precursors to semisynthetic derivatives of pleuromutilins. Pleuromutilins are a large family of antibiotics with good antibacterial activity and can effectively inhibit most gram-positive bacteria and part of gram-negative bacteria.
At present, the basic raw materials of the seed culture medium of the pleurotus ostreatus of most manufacturers at home are yeast powder, glucose, dextrin, monopotassium phosphate and ammonium sulfate; the basic raw materials of the fermentation culture medium comprise soybean oil, glucose, hot-pressed soybean cake powder, yeast powder, potassium dihydrogen phosphate, magnesium sulfate, calcium nitrate, sodium chloride and ferric sulfate. At present, the cold-pressed soybean cake powder and corn oil are used for replacing the traditional soybean oil and hot-pressed soybean cake powder to reduce the cost and improve the fermentation titer, but the yield of pleuromutilin can be effectively improved by adopting glucose compared with sucrose, maltose, soluble starch and the like, but experiments prove that the yield of glucose and soluble starch is not very different, but the yield of fructose can be improved by about 10 percent relative to the yield of glucose, and the existing adopted culture medium does not contain Fe2+, and experiments prove that the addition of a proper amount of Fe2+ can clearly influence the yield of pleuromutilin.
Based on the above, the present invention designs a fermentation method of Pleurotus for preparing pleuromutilin, so as to solve the above mentioned problems.
Disclosure of Invention
The invention aims to provide a fermentation method of pleuromutilin by using pleurotus, which aims to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a fermentation method of Pleurotus for preparing pleuromutilin comprises the following steps:
s1: preparing a culture medium: preparing a primary culture tank filled with a primary culture medium, a secondary seed culture tank filled with a secondary culture medium and a fermentation culture tank filled with fermentation liquor;
s2: first-order seed culture: seed liquid of the cultured Pleurotus ostreatus mother bottle is 1-3L/m3Inoculating the inoculum size into a primary seed culture tank for primary seed culture, and completely transferring into a secondary seed culture tank when the thallus concentration is 15% -25% and the pH value is 6-8;
s3: secondary seed culture: performing secondary seed culture through a secondary seed culture tank until the concentration of the bacterial liquid is 20-40% and the pH value is 6-8, and completely transferring the bacterial liquid into a fermentation culture tank;
s4: fermentation culture: culturing the fermentation liquor in a fermentation culture tank until the concentration of thalli in the fermentation liquor is 30-40%, the concentration of amino nitrogen is 5-10 mg/100ml, the concentration of fat is 1-2%, the concentration of reducing sugar is 1-2%, and the chemical valence reaches 18000-20000 u/ml, and then terminating fermentation;
s5: feeding: feeding during the fermentation in the fermentation culture tank.
Preferably, the composition of the primary medium is: 5-20 g/L of fructose, 20-50 g/L of cold-pressed soybean cake powder, 1-10 g/L of dipotassium hydrogen phosphate, 0.1-0.4 g/L of magnesium sulfate, 0.5-1.0 g/L of calcium nitrate, 0.1-0.2 g/L of sodium chloride, 0.02-0.08 g/L of ferrous sulfate and 0.3-0.9 g/L of defoaming agent.
Preferably, the composition of the secondary medium is: 18-24 g/L of fructose, 10-15 g/L of cold-pressed soybean cake powder, 1-4 g/L of dipotassium hydrogen phosphate, 0.2-0.4 g/L of magnesium sulfate, 0.2-0.4 g/L of calcium nitrate, 0.1-0.2 g/L of sodium chloride, 0.04-0.08 g/L of ferrous sulfate and 0.6-0.9 g/L of defoaming agent.
Preferably, the composition of the fermentation medium is: 20-30 g/L of fructose, 15-20 g/L of cold-pressed soybean cake powder, 15-20 g/L of corn steep liquor, 1-4 g/L of dipotassium phosphate, 0.2-0.4 g/L of magnesium sulfate, 0.6-1.2 g/L of calcium nitrate, 2-5 g/L of yeast extract, 0.1-0.2 g/L of sodium chloride, 5-15 ml/L of corn oil, 0.04-0.08 g/L of ferrous sulfate and 0.6-0.9 g/L of defoaming agent.
Preferably, before the seed solution of the mother bottle of the lateral ear fungus is inoculated into the primary seed culture tank, the primary seed culture medium is sterilized and cooled, and sterile air is used for maintaining pressure, wherein the pressure is controlled to be 0.01-0.02 MPa, and the pH value of the sterilized seed solution is controlled to be 5-8; the content of amino nitrogen is controlled to be 10mg/100 ml-50 mg/100 ml; the content of reducing sugar is controlled to be 1-5%.
Preferably, before the primary seed liquid is transferred into a secondary seed culture tank, sterilizing and cooling a secondary seed culture medium, maintaining the pressure with sterile air, controlling the pressure at 0.01-0.02 MPa, and controlling the pH of the sterilized seed liquid at 5-8; the content of amino nitrogen is controlled to be 10mg/100 ml-50 mg/100 ml; the content of reducing sugar is controlled to be 1-5%.
Preferably, before the secondary seed liquid is transferred into a fermentation culture tank, sterilizing and cooling a fermentation culture medium, maintaining the pressure with sterile air, controlling the pressure to be 0.01-0.02 MPa, and controlling the pH value of the sterilized seed liquid to be 6-8; controlling the content of amino nitrogen to be 20-80 mg/100 ml; the content of reducing sugar is controlled to be 1-5%.
Preferably, the
The first-order seed culture conditions are as follows: and (3) tank pressure: controlling the pressure to be 0.02-0.06 MPa; the temperature of the tank is as follows: controlling the temperature of the tank to be 28-30 ℃; air flow rate: 0 to 20 hours, 1 to 5m3H; controlling the pH value to be 7-8; stirring speed: mechanical stirring is adopted, and the rotating speed is controlled to be 120-150 r/min; the primary seed culture time is 30-60 h,
the secondary seed culture conditions are as follows: and (3) tank pressure: controlling the pressure to be 0.04-0.06 MPa; the temperature of the tank is as follows: controlling the temperature of the tank to be 28-30 ℃; air flow rate: 0 to 20 hours, 1 to 5m3H; controlling the pH value to be 7-8; stirring speed: mechanical stirring is adopted, and the rotating speed is controlled to be 160-180 r/min; the primary seed culture time is 40-60 h.
Preferably, in the fermentation culture tank, the fermentation culture conditions are as follows:
a. amino nitrogen control: the fermentation period is controlled to be 100-10 mg/100ml within 50-180 h; the fermentation period is 181 h-fermentation is finished, and the concentration is controlled to be 50-10 mg/100 ml;
b. and (3) fermentation period: the culture time is 200-300 h;
c. and (3) pH control: the pH is natural and uncontrolled after 0-60 h; controlling the pH value to be 6-8 within 81-180 h; 181h to end of fermentation, and controlling the pH value to be 6.2 to 7.8;
d. air flow rate: 0-40 h: 10 to 20m3/h；41～180h：20～40m3H; 181 h-fermentation end: 15 to 20m3/h；
e. And (3) pressure control: the tank pressure is 0.01-0.05 MPa;
f. temperature control: controlling the temperature to be 20-28 ℃ in the whole fermentation process;
g. and (4) sterile inspection: performing bacteria detection in the fermentation process, wherein other mixed bacteria are required to be absent;
h. the thallus concentration: the thalli naturally grow and the concentration is not controlled within 0-90 h; controlling the thallus concentration to be 50-60% within 91-180 h; 181h to end of fermentation, and controlling the concentration of the thalli to be 30-40%;
I. stirring speed: the stirring speed is controlled to be 80-100 r/min for 0-80 h; stirring for 81-200 h, and controlling the stirring speed to be 120-150 r/min; 181h to the end of fermentation, and controlling the stirring speed at 100 to 120 r/min.
Preferably, the feeding during the fermentation in the fermentation culture tank is as follows:
(1) oil supplement: supplementing corn oil, wherein in the process of fermenting for 50-180 hours, when the fat content is less than 2%, the corn oil is supplemented in a fed-batch mode, and the fat content is controlled to be 2-3%; in the process from 181h fermentation to 181h fermentation, when the fat content is less than 1%, corn oil is supplemented in a fed-batch mode, and the fat content is controlled to be 1-2%;
(2) sugar supplement: fructose with the concentration of 29-30% is selected for sugar supplementation, in the process of fermentation for 60-180 h, when the concentration of reducing sugar is less than 2%, fruit solution is supplemented, the content of reducing sugar is controlled to be 2-3%, in the process of fermentation for 181 h-fermentation ending, when the concentration of reducing sugar is less than 1%, fruit solution is supplemented, and the content of reducing sugar is controlled to be 1-2%;
(3) water replenishing: in the process of a fermentation period of 91-180 h, when the concentration of the thalli is more than 60%, supplementing sterilized tap water, controlling the concentration of the thalli to be 50-60%, and when the concentration of the thalli is more than 40%, supplementing the sterilized tap water, and controlling the concentration of the thalli to be 30-40% after fermentation is 181h to be finished;
(4) supplement of Fe2+: in the process of fermentation period of 80-180 h, when Fe2+The concentration is less than 0.06 percent, and ferrous sulfate solution and Fe are added2+The content is controlled to be 0.06-0.08%, and in the process from 181h to end of fermentation, when Fe2+Concentration less than 0.03%, supplementAdding ferrous sulfate solution, Fe2+The content is controlled to be 0.03-0.06%.
(5) Acid or alkali supplementation: 0-80 h: the pH value is in a natural fermentation state; controlling the pH value to be 6-8 within 81-180 h; 181h to the end of fermentation, and the pH value is 6.2 to 7.8. And when the pH detection result is lower than the lower limit of the control range, adding 10-20% of sterilized sodium hydroxide solution for regulation.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, in the component proportion of the seed culture medium and the fermentation culture medium in the pleuromutilin fermentation production process, cold-pressed soybean cake powder and corn oil are used for replacing traditional soybean oil and hot-pressed soybean cake powder so as to reduce the cost and improve the fermentation titer, fructose is used for replacing glucose as a carbon source, the yield is improved by 10% compared with that of a basic fermentation culture medium, ferrous sulfate is added into the culture medium, and Fe is firstly added2+Can participate in electron transfer chain reaction in the form of ferritin by adding Fe2+—Fe3+The iron ions can be combined with the heme to participate in the transportation of oxygen and increase the oxygen intake, and the iron ions are 0.02 to 0.04 percent in proportion to Fe2+The concentration of the pleuromutilin is increased, the product is improved by 61.4 percent compared with the basic fermentation culture medium, the pleuromutilin has a promoting effect on the synthesis of the pleuromutilin, and the chemical titer is further improved.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The species of the following examples were selected from Pleurotus mutilus (Pleurotus mutilus): the strain source used for production is mother bottle fermentation liquor. Mother bottle fermentation liquor quality: the pH is 5-7; the concentration of the thalli is 10-30%; microscopic examination is carried out to ensure that no mixed bacteria exist; 13000-14000 u/ml of shake flask fermentation unit.
In the following examples the antifoam agent selection was made by the Weifang Fumigao Water resources chemical Co., Ltd.
Example 1
The invention provides a technical scheme that: a fermentation method of Pleurotus ostreatus for preparing pleuromutilin comprises the following steps
S1: preparing a culture medium: preparing a primary culture tank filled with a primary culture medium, a secondary seed culture tank filled with a secondary culture medium and a fermentation culture tank filled with fermentation liquor;
at 1m35kg of fructose, 20kg of cold-pressed soybean cake powder, 1kg of dipotassium phosphate, 0.1kg of magnesium sulfate, 0.5kg of calcium nitrate, 0.1kg of sodium chloride, 0.02kg of ferrous sulfate and 0.3kg of defoaming agent are added into the first-stage seed culture tank.
At 1m3The second-level seed culture tank is added with 18kg of fructose, 10kg of cold-pressed soybean cake powder, 1kg of dipotassium hydrogen phosphate, 0.2kg of magnesium sulfate, 0.2kg of calcium nitrate, 0.1kg of sodium chloride, 0.04kg of ferrous sulfate and 0.6kg of defoaming agent.
At 1m3The fermentation culture tank is added with 20kg of fructose, 15kg of cold-pressed soybean cake powder, 15kg of corn steep liquor, 1kg of dipotassium hydrogen phosphate, 0.2kg of magnesium sulfate, 0.6kg of calcium nitrate, 2kg of yeast extract, 0.1kg of sodium chloride, 5L of corn oil, 0.04kg of ferrous sulfate and 0.6kg of defoaming agent.
S2: first-order seed culture: before the seed liquid of the mother bottle of the lateral ear fungus is inoculated into a primary seed culture tank, firstly, sterilizing and cooling a primary seed culture medium, and maintaining the pressure by using sterile air, wherein the pressure is controlled to be 0.01-0.02 MPa, and the pH value of the sterilized seed liquid is required to be controlled to be 5-8; the content of amino nitrogen is controlled to be 10mg/100 ml-50 mg/100 ml; the content of reducing sugar is controlled to be 1-5%.
Seed liquid of the cultured Pleurotus ostreatus mother bottle is 1-3L/m3Inoculating the inoculum size into a primary seed culture tank for primary seed culture, and completely transferring into a secondary seed culture tank when the thallus concentration is 15% -25% and the pH value is 6-8;
the first-order seed culture conditions are as follows: and (3) tank pressure: controlling the pressure to be 0.02-0.06 MPa; the temperature of the tank is as follows: controlling the temperature of the tank to be 28-30 ℃; air flow rate: 0 to 20 hours, 1 to 5m3H; controlling the pH value to be 7-8; stirring speed: by machinesStirring, and controlling the rotating speed at 120-150 r/min; the primary seed culture time is 30-60 h.
S3: secondary seed culture: before the primary seed liquid is transferred into a secondary seed culture tank, firstly, sterilizing and cooling a secondary seed culture medium, maintaining the pressure with sterile air, controlling the pressure to be 0.01-0.02 MPa, and controlling the pH value of the sterilized seed liquid to be 5-8; the content of amino nitrogen is controlled to be 10mg/100 ml-50 mg/100 ml; the content of reducing sugar is controlled to be 1-5%.
Performing secondary seed culture through a secondary seed culture tank until the concentration of the bacterial liquid is 20-40% and the pH value is 6-8, and completely transferring the bacterial liquid into a fermentation culture tank;
the secondary seed culture conditions are as follows: and (3) tank pressure: controlling the pressure to be 0.04-0.06 MPa; the temperature of the tank is as follows: controlling the temperature of the tank to be 28-30 ℃; air flow rate: 0 to 20 hours, 1 to 5m3H; controlling the pH value to be 7-8; stirring speed: mechanical stirring is adopted, and the rotating speed is controlled to be 160-180 r/min; the primary seed culture time is 40-60 h.
S4: fermentation culture: before the secondary seed liquid is transferred into a fermentation culture tank, firstly, sterilizing and cooling a fermentation culture medium, and maintaining the pressure by using sterile air, wherein the pressure is controlled to be 0.01-0.02 MPa, and the pH value of the sterilized seed liquid is required to be controlled to be 6-8; the content of amino nitrogen is controlled to be 20mg/100 ml-80 mg/100 ml; the content of reducing sugar is controlled to be 1-5%. Culturing the fermentation liquor in a fermentation culture tank until the concentration of thalli in the fermentation liquor is 40-60 percent, the concentration of amino nitrogen is 5-10 mg/100ml, the concentration of fat is 1-2 percent, the concentration of reducing sugar is 1-2 percent, and the chemical valence reaches 18000-20000 u/ml;
the fermentation culture conditions are as follows:
a. amino nitrogen control: the fermentation period is controlled to be 100-10 mg/100ml within 50-180 h; the fermentation period is 181 h-fermentation is finished, and the concentration is controlled to be 50-10 mg/100 ml;
b. and (3) fermentation period: the culture time is 200-300 h;
c. and (3) pH control: the pH is natural and uncontrolled after 0-60 h; controlling the pH value to be 6-8 within 81-180 h; 181h to end of fermentation, and controlling the pH value to be 6.2 to 7.8;
d. air flow rate: 0-40 h: 10-20 m 3/h; 41-180 h: 20-40 m 3/h; 181 h-fermentation end: 15-20 m 3/h;
e. and (3) pressure control: the tank pressure is 0.01-0.05 MPa;
f. temperature control: controlling the temperature to be 20-28 ℃ in the whole fermentation process;
g. and (4) sterile inspection: performing bacteria detection in the fermentation process, wherein other mixed bacteria are required to be absent;
h. the thallus concentration: the thalli naturally grow and the concentration is not controlled within 0-90 h; controlling the thallus concentration to be 50-60% within 91-180 h; 181h to end of fermentation, and controlling the concentration of the thalli to be 30-40%;
I. stirring speed: the stirring speed is controlled to be 80-100 r/min for 0-80 h; stirring for 81-200 h, and controlling the stirring speed to be 120-150 r/min; 181h to the end of fermentation, and controlling the stirring speed at 100 to 120 r/min.
S5: feeding: feeding during the fermentation in the fermentation culture tank.
Corn oil 954L is supplemented in the fermentation process; 834L water supplement, 4275kg sugar supplement, and Fe supplement2+16.9kg。
After the fermentation is finished, the thallus concentration is 32%, the amino nitrogen is 5.3%, the fat is 0.03%, the reducing sugar is 0.10%, the fermentation titer is 18065u/ml, and the fermentation period is 246 h.
Example 2
The invention provides a technical scheme that: a fermentation method of Pleurotus ostreatus for preparing pleuromutilin comprises the following steps
S1: preparing a culture medium: preparing a primary culture tank filled with a primary culture medium, a secondary seed culture tank filled with a secondary culture medium and a fermentation culture tank filled with fermentation liquor;
the first-stage culture medium comprises the following components: 15kg of fructose, 35kg of cold-pressed soybean cake powder, 5kg of dipotassium phosphate, 0.3kg of magnesium sulfate, 0.8kg of calcium nitrate, 0.15kg of sodium chloride, 0.05kg of ferrous sulfate and 0.6kg of defoaming agent.
The composition of the secondary culture medium is as follows: 20kg of fructose, 12kg of cold-pressed soybean cake powder, 3kg of dipotassium phosphate, 0.3kg of magnesium sulfate, 0.3kg of calcium nitrate, 0.15kg of sodium chloride, 0.06kg of ferrous sulfate and 0.7kg of defoaming agent.
The fermentation medium comprises the following components: 25kg of fructose, 18kg of cold-pressed soybean cake powder, 18kg of corn steep liquor, 3kg of dipotassium phosphate, 0.3kg of magnesium sulfate, 0.9kg of calcium nitrate, 3kg of yeast extract, 0.15kg of sodium chloride, 10L of corn oil, 0.06kg of ferrous sulfate and 0.7kg of defoaming agent.
S2: first-order seed culture: before the seed liquid of the mother bottle of the lateral ear fungus is inoculated into a primary seed culture tank, firstly, sterilizing and cooling a primary seed culture medium, and maintaining the pressure by using sterile air, wherein the pressure is controlled to be 0.01-0.02 MPa, and the pH value of the sterilized seed liquid is required to be controlled to be 5-8; the content of amino nitrogen is controlled to be 10mg/100 ml-50 mg/100 ml; the content of reducing sugar is controlled to be 1-5%.
Seed liquid of the cultured Pleurotus ostreatus mother bottle is 1-3L/m3Inoculating the inoculum size into a primary seed culture tank for primary seed culture, and completely transferring into a secondary seed culture tank when the thallus concentration is 15% -25% and the pH value is 6-8;
the first-order seed culture conditions are as follows: and (3) tank pressure: controlling the pressure to be 0.02-0.06 MPa; the temperature of the tank is as follows: controlling the temperature of the tank to be 28-30 ℃; air flow rate: 0 to 20 hours, 1 to 5m3H; controlling the pH value to be 7-8; stirring speed: mechanical stirring is adopted, and the rotating speed is controlled to be 120-150 r/min; the primary seed culture time is 30-60 h.
S3: secondary seed culture: before the primary seed liquid is transferred into a secondary seed culture tank, firstly, sterilizing and cooling a secondary seed culture medium, maintaining the pressure with sterile air, controlling the pressure to be 0.01-0.02 MPa, and controlling the pH value of the sterilized seed liquid to be 5-8; the content of amino nitrogen is controlled to be 10mg/100 ml-50 mg/100 ml; the content of reducing sugar is controlled to be 1-5%.
Performing secondary seed culture through a secondary seed culture tank until the concentration of the bacterial liquid is 20-40% and the pH value is 6-8, and completely transferring the bacterial liquid into a fermentation culture tank;
the secondary seed culture conditions are as follows: and (3) tank pressure: controlling the pressure to be 0.04-0.06 MPa; the temperature of the tank is as follows: controlling the temperature of the tank to be 28-30 ℃; air flow rate: 0 to 20 hours, 1 to 5m3H; controlling the pH value to be 7-8; stirring speed: mechanical stirring is adopted, and the rotating speed is controlled to be 160-180 r/min; the primary seed culture time is 40-60 h.
S4: fermentation culture: before the secondary seed liquid is transferred into a fermentation culture tank, firstly, sterilizing and cooling a fermentation culture medium, and maintaining the pressure by using sterile air, wherein the pressure is controlled to be 0.01-0.02 MPa, and the pH value of the sterilized seed liquid is required to be controlled to be 6-8; the content of amino nitrogen is controlled to be 20mg/100 ml-80 mg/100 ml; the content of reducing sugar is controlled to be 1-5%. Culturing the fermentation liquor in a fermentation culture tank until the concentration of thalli in the fermentation liquor is 40-60 percent, the concentration of amino nitrogen is 5-10 mg/100ml, the concentration of fat is 1-2 percent, the concentration of reducing sugar is 1-2 percent, and the chemical valence reaches 18000-20000 u/ml;
the fermentation culture conditions are as follows:
a. amino nitrogen control: the fermentation period is controlled to be 100-10 mg/100ml within 50-180 h; the fermentation period is 181 h-fermentation is finished, and the concentration is controlled to be 50-10 mg/100 ml;
b. and (3) fermentation period: the culture time is 200-300 h;
c. and (3) pH control: the pH is natural and uncontrolled after 0-60 h; controlling the pH value to be 6-8 within 81-180 h; 181h to end of fermentation, and controlling the pH value to be 6.2 to 7.8;
d. air flow rate: 0-40 h: 10-20 m 3/h; 41-180 h: 20-40 m 3/h; 181 h-fermentation end: 15-20 m 3/h;
e. and (3) pressure control: the tank pressure is 0.01-0.05 MPa;
f. temperature control: controlling the temperature to be 20-28 ℃ in the whole fermentation process;
g. and (4) sterile inspection: performing bacteria detection in the fermentation process, wherein other mixed bacteria are required to be absent;
h. the thallus concentration: the thalli naturally grow and the concentration is not controlled within 0-90 h; controlling the thallus concentration to be 50-60% within 91-180 h; 181h to end of fermentation, and controlling the concentration of the thalli to be 30-40%;
I. stirring speed: the stirring speed is controlled to be 80-100 r/min for 0-80 h; stirring for 81-200 h, and controlling the stirring speed to be 120-150 r/min; 181h to the end of fermentation, and controlling the stirring speed at 100 to 120 r/min.
S5: feeding: feeding during the fermentation in the fermentation culture tank.
Corn oil supplement in fermentation process835L; 789L water supplement, 4086kg sugar supplement and Fe supplement2+16.2kg。
After the fermentation, the thallus concentration is 43.2%, the amino nitrogen is 6.1%, the fat is 1.4%, the reducing sugar is 1.3%, the fermentation titer is 19472u/ml, and the fermentation period is 234 h.
Example 3
The invention provides a technical scheme that: a fermentation method of Pleurotus ostreatus for preparing pleuromutilin comprises the following steps
S1: preparing a culture medium: preparing a primary culture tank filled with a primary culture medium, a secondary seed culture tank filled with a secondary culture medium and a fermentation culture tank filled with fermentation liquor;
the first-stage culture medium comprises the following components: 20kg of fructose, 50kg of cold-pressed soybean cake powder, 10kg of dipotassium phosphate, 0.4kg of magnesium sulfate, 1.0kg of calcium nitrate, 0.2kg of sodium chloride, 0.08kg of ferrous sulfate and 0.9kg of defoaming agent.
The composition of the secondary culture medium is as follows: 24kg of fructose, 15kg of cold-pressed soybean cake powder, 4kg of dipotassium phosphate, 0.4kg of magnesium sulfate, 0.4kg of calcium nitrate, 0.2kg of sodium chloride, 0.08kg of ferrous sulfate and 0.9kg of defoaming agent.
The fermentation medium comprises the following components: 30kg of fructose, 20kg of cold-pressed soybean cake powder, 20kg of corn steep liquor, 4kg of dipotassium phosphate, 0.4kg of magnesium sulfate, 1.2kg of calcium nitrate, 5kg of yeast extract, 0.2kg of sodium chloride, 15L of corn oil, 0.08kg of ferrous sulfate and 0.9kg of defoaming agent.
S2: first-order seed culture: before the seed liquid of the mother bottle of the lateral ear fungus is inoculated into a primary seed culture tank, firstly, sterilizing and cooling a primary seed culture medium, and maintaining the pressure by using sterile air, wherein the pressure is controlled to be 0.01-0.02 MPa, and the pH value of the sterilized seed liquid is required to be controlled to be 5-8; the content of amino nitrogen is controlled to be 10mg/100 ml-50 mg/100 ml; the content of reducing sugar is controlled to be 1-5%.
Seed liquid of the cultured Pleurotus ostreatus mother bottle is 1-3L/m3Inoculating the inoculum size into a primary seed culture tank for primary seed culture, and completely transferring into a secondary seed culture tank when the thallus concentration is 15% -25% and the pH value is 6-8;
first-order seed culture stripThe parts are as follows: and (3) tank pressure: controlling the pressure to be 0.02-0.06 MPa; the temperature of the tank is as follows: controlling the temperature of the tank to be 28-30 ℃; air flow rate: 0 to 20 hours, 1 to 5m3H; controlling the pH value to be 7-8; stirring speed: mechanical stirring is adopted, and the rotating speed is controlled to be 120-150 r/min; the primary seed culture time is 30-60 h.
S3: secondary seed culture: before the primary seed liquid is transferred into a secondary seed culture tank, firstly, sterilizing and cooling a secondary seed culture medium, maintaining the pressure with sterile air, controlling the pressure to be 0.01-0.02 MPa, and controlling the pH value of the sterilized seed liquid to be 5-8; the content of amino nitrogen is controlled to be 10mg/100 ml-50 mg/100 ml; the content of reducing sugar is controlled to be 1-5%.
Performing secondary seed culture through a secondary seed culture tank until the concentration of the bacterial liquid is 20-40% and the pH value is 6-8, and completely transferring the bacterial liquid into a fermentation culture tank;
the secondary seed culture conditions are as follows: and (3) tank pressure: controlling the pressure to be 0.04-0.06 MPa; the temperature of the tank is as follows: controlling the temperature of the tank to be 28-30 ℃; air flow rate: 0 to 20 hours, 1 to 5m3H; controlling the pH value to be 7-8; stirring speed: mechanical stirring is adopted, and the rotating speed is controlled to be 160-180 r/min; the primary seed culture time is 40-60 h.
S4: fermentation culture: before the secondary seed liquid is transferred into a fermentation culture tank, firstly, sterilizing and cooling a fermentation culture medium, and maintaining the pressure by using sterile air, wherein the pressure is controlled to be 0.01-0.02 MPa, and the pH value of the sterilized seed liquid is required to be controlled to be 6-8; the content of amino nitrogen is controlled to be 20mg/100 ml-80 mg/100 ml; the content of reducing sugar is controlled to be 1-5%. Culturing the fermentation liquor in a fermentation culture tank until the concentration of thalli in the fermentation liquor is 40-60 percent, the concentration of amino nitrogen is 5-10 mg/100ml, the concentration of fat is 1-2 percent, the concentration of reducing sugar is 1-2 percent, and the chemical valence reaches 18000-20000 u/ml;
the fermentation culture conditions are as follows:
a. amino nitrogen control: the fermentation period is controlled to be 100-10 mg/100ml within 50-180 h; the fermentation period is 181 h-fermentation is finished, and the concentration is controlled to be 50-10 mg/100 ml;
b. and (3) fermentation period: the culture time is 200-300 h;
c. and (3) pH control: the pH is natural and uncontrolled after 0-60 h; controlling the pH value to be 6-8 within 81-180 h; 181h to end of fermentation, and controlling the pH value to be 6.2 to 7.8;
d. air flow rate: 0-40 h: 10-20 m 3/h; 41-180 h: 20-40 m 3/h; 181 h-fermentation end: 15-20 m 3/h;
e. and (3) pressure control: the tank pressure is 0.01-0.05 MPa;
f. temperature control: controlling the temperature to be 20-28 ℃ in the whole fermentation process;
g. and (4) sterile inspection: performing bacteria detection in the fermentation process, wherein other mixed bacteria are required to be absent;
h. the thallus concentration: the thalli naturally grow and the concentration is not controlled within 0-90 h; controlling the thallus concentration to be 50-60% within 91-180 h; 181h to end of fermentation, and controlling the concentration of the thalli to be 30-40%;
I. stirring speed: the stirring speed is controlled to be 80-100 r/min for 0-80 h; stirring for 81-200 h, and controlling the stirring speed to be 120-150 r/min; 181h to the end of fermentation, and controlling the stirring speed at 100 to 120 r/min.
S5: feeding: feeding during the fermentation in the fermentation culture tank.
Corn oil 954L is supplemented in the fermentation process; 834L water supplement, 4275kg sugar supplement, and Fe supplement2+16.7 kg. After the fermentation is finished, the thallus concentration is 32%, the amino nitrogen is 5.3%, the fat is 0.03%, the reducing sugar is 0.10%, the fermentation titer is 18065u/ml, and the fermentation period is 242 h.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (10)
1. A fermentation method of Pleurotus ostreatus for preparing pleuromutilin is characterized in that: the method comprises the following specific steps:
s1: preparing a culture medium: preparing a primary culture tank filled with a primary culture medium, a secondary seed culture tank filled with a secondary culture medium and a fermentation culture tank filled with fermentation liquor;
s2: first-order seed culture: seed liquid of the cultured Pleurotus ostreatus mother bottle is 1-3L/m3Inoculating the inoculum size into a primary seed culture tank for primary seed culture, and completely transferring into a secondary seed culture tank when the thallus concentration is 15% -25% and the pH value is 6-8;
s3: secondary seed culture: performing secondary seed culture through a secondary seed culture tank until the concentration of the bacterial liquid is 20-40% and the pH value is 6-8, and completely transferring the bacterial liquid into a fermentation culture tank;
s4: fermentation culture: culturing the fermentation liquor in a fermentation culture tank until the concentration of thalli in the fermentation liquor is 40-60 percent, the concentration of amino nitrogen is 5-10 mg/100ml, the concentration of fat is 1-2 percent, the concentration of reducing sugar is 1-2 percent, and the chemical valence reaches 18000-20000 u/ml;
s5: feeding: feeding during the fermentation in the fermentation culture tank.
2. A fermentation process of pleuromutilin according to claim 1, characterized in that: the first-stage culture medium comprises the following components: 5-20 g/L of fructose, 20-50 g/L of cold-pressed soybean cake powder, 1-10 g/L of dipotassium hydrogen phosphate, 0.1-0.4 g/L of magnesium sulfate, 0.5-1.0 g/L of calcium nitrate, 0.1-0.2 g/L of sodium chloride, 0.02-0.08 g/L of ferrous sulfate and 0.3-0.9 g/L of defoaming agent.
3. A fermentation process of pleuromutilin according to claim 1, characterized in that: the composition of the secondary culture medium is as follows: 18-24 g/L of fructose, 10-15 g/L of cold-pressed soybean cake powder, 1-4 g/L of dipotassium hydrogen phosphate, 0.2-0.4 g/L of magnesium sulfate, 0.2-0.4 g/L of calcium nitrate, 0.1-0.2 g/L of sodium chloride, 0.04-0.08 g/L of ferrous sulfate and 0.6-0.9 g/L of defoaming agent.
4. A fermentation process of pleuromutilin according to claim 1, characterized in that: the fermentation medium comprises the following components: 20-30 g/L of fructose, 15-20 g/L of cold-pressed soybean cake powder, 15-20 g/L of corn steep liquor, 1-4 g/L of dipotassium phosphate, 0.2-0.4 g/L of magnesium sulfate, 0.6-1.2 g/L of calcium nitrate, 2-5 g/L of yeast extract, 0.1-0.2 g/L of sodium chloride, 5-15 ml/L of corn oil, 0.04-0.08 g/L of ferrous sulfate and 0.6-0.9 g/L of defoaming agent.
5. A fermentation process of pleuromutilin according to claim 1, characterized in that: before the seed liquid of the mother bottle of the lateral ear fungus is inoculated into a primary seed culture tank, firstly, sterilizing and cooling a primary seed culture medium, and maintaining the pressure by using sterile air, wherein the pressure is controlled to be 0.01-0.02 MPa, and the pH value of the sterilized seed liquid is required to be controlled to be 5-8; the content of amino nitrogen is controlled to be 10mg/100 ml-50 mg/100 ml; the content of reducing sugar is controlled to be 1-5%.
6. A fermentation process of pleuromutilin according to claim 1, characterized in that: before the primary seed liquid is transferred into a secondary seed culture tank, firstly, sterilizing and cooling a secondary seed culture medium, maintaining the pressure with sterile air, controlling the pressure to be 0.01-0.02 MPa, and controlling the pH value of the sterilized seed liquid to be 5-8; the content of amino nitrogen is controlled to be 10mg/100 ml-50 mg/100 ml; the content of reducing sugar is controlled to be 1-5%.
7. A fermentation process of pleuromutilin according to claim 1, characterized in that: before the secondary seed liquid is transferred into a fermentation culture tank, firstly, sterilizing and cooling a fermentation culture medium, and maintaining the pressure by using sterile air, wherein the pressure is controlled to be 0.01-0.02 MPa, and the pH value of the sterilized seed liquid is required to be controlled to be 6-8; the content of amino nitrogen is controlled to be 20mg/100 ml-80 mg/100 ml; the content of reducing sugar is controlled to be 1-5%.
8. A fermentation process of pleuromutilin according to claim 1, characterized in that: the above-mentioned
The first-order seed culture conditions are as follows: and (3) tank pressure: controlling the pressure to be 0.02-0.06 MPa; the temperature of the tank is as follows: controlling the temperature of the tank to be 28-30 ℃; air flow rate: 0 to 20 hours, 1 to 5m3H; controlling the pH value to be 7-8; stirring speed: mechanical stirring is adopted, and the rotating speed is controlled to be 120-150 r/min; the primary seed culture time is 30-60 h,
the secondary seed culture conditions are as follows: and (3) tank pressure: controlling the pressure to be 0.04-0.06 MPa; the temperature of the tank is as follows: controlling the temperature of the tank to be 28-30 ℃; air flow rate: 0 to 20 hours, 1 to 5m3H; controlling the pH value to be 7-8; stirring speed: mechanical stirring is adopted, and the rotating speed is controlled to be 160-180 r/min; the primary seed culture time is 40-60 h.
9. A fermentation process of pleuromutilin according to claim 1, characterized in that: in the fermentation culture tank, the fermentation culture conditions are as follows:
a. amino nitrogen control: the fermentation period is controlled to be 100-10 mg/100ml within 50-180 h; the fermentation period is 181 h-fermentation is finished, and the concentration is controlled to be 50-10 mg/100 ml;
b. and (3) fermentation period: the culture time is 200-300 h;
c. and (3) pH control: the pH is natural and uncontrolled after 0-60 h; controlling the pH value to be 6-8 within 81-180 h; 181h to end of fermentation, and controlling the pH value to be 6.2 to 7.8;
d. air flow rate: 0-40 h: 10 to 20m3/h；41～180h：20～40m3H; 181 h-fermentation end: 15 to 20m3/h；
e. And (3) pressure control: the tank pressure is 0.01-0.05 MPa;
f. temperature control: controlling the temperature to be 20-28 ℃ in the whole fermentation process;
g. and (4) sterile inspection: performing bacteria detection in the fermentation process, wherein other mixed bacteria are required to be absent;
h. the thallus concentration: the thalli naturally grow and the concentration is not controlled within 0-90 h; controlling the thallus concentration to be 50-60% within 91-180 h; 181h to end of fermentation, and controlling the concentration of the thalli to be 30-40%;
I. stirring speed: the stirring speed is controlled to be 80-100 r/min for 0-80 h; stirring for 81-200 h, and controlling the stirring speed to be 120-150 r/min; 181h to the end of fermentation, and controlling the stirring speed at 100 to 120 r/min.
10. A fermentation process of pleuromutilin according to claim 1, characterized in that: the feeding in the fermentation process in the fermentation culture tank comprises the following steps:
(1) oil supplement: supplementing corn oil, wherein in the process of fermenting for 50-180 hours, when the fat content is less than 2%, the corn oil is supplemented in a fed-batch mode, and the fat content is controlled to be 2-3%; in the process from 181h fermentation to 181h fermentation, when the fat content is less than 1%, corn oil is supplemented in a fed-batch mode, and the fat content is controlled to be 1-2%;
(2) sugar supplement: fructose with the concentration of 29-30% is selected for sugar supplementation, in the process of fermentation for 60-180 h, when the concentration of reducing sugar is less than 2%, fruit solution is supplemented, the content of reducing sugar is controlled to be 2-3%, in the process of fermentation for 181 h-fermentation ending, when the concentration of reducing sugar is less than 1%, fruit solution is supplemented, and the content of reducing sugar is controlled to be 1-2%;
(3) water replenishing: in the process of a fermentation period of 91-180 h, when the concentration of the thalli is more than 60%, supplementing sterilized tap water, controlling the concentration of the thalli to be 50-60%, and when the concentration of the thalli is more than 40%, supplementing the sterilized tap water, and controlling the concentration of the thalli to be 30-40% after fermentation is 181h to be finished;
(4) supplement of Fe2+: in the process of fermentation period of 80-180 h, when Fe2+The concentration is less than 0.06 percent, and ferrous sulfate solution and Fe are added2+The content is controlled to be 0.06-0.08%, and the fermentation is carried out for 181h to the end of fermentationIn the equation, when Fe2+The concentration is less than 0.03%, ferrous sulfate solution and Fe are added2+The content is controlled to be 0.03-0.06%.
(5) Acid or alkali supplementation: 0-80 h: the pH value is in a natural fermentation state; controlling the pH value to be 6-8 within 81-180 h; 181h to the end of fermentation, and the pH value is 6.2 to 7.8. And when the pH detection result is lower than the lower limit of the control range, adding 10-20% of sterilized sodium hydroxide solution for regulation.
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