CN112029750A - OUR-based fed-batch fermentation method for producing medium-temperature alpha-amylase by using bacillus subtilis - Google Patents
OUR-based fed-batch fermentation method for producing medium-temperature alpha-amylase by using bacillus subtilis Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01001—Alpha-amylase (3.2.1.1)
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Abstract
The invention discloses a OUR-based fed-batch fermentation method for producing medium-temperature alpha-amylase by bacillus subtilis, which is characterized in that during batch culture and fermentation of the bacillus subtilis, the change trend of OUR in tail gas is detected on line, fed-batch feeding is carried out at the time point when the OUR is reduced, so that the OUR is in a stable or slowly reduced state, and the alpha-amylase is continuously fermented and synthesized; the feed is glucose or a complete culture medium. According to the method, the change trend of the OUR in the tail gas is detected on line to flow and add the supplementary materials, the fermentation parameters are regulated and controlled, the bacillus subtilis is promoted to produce the medium-temperature alpha-amylase, and the enzyme activity yield of the alpha-amylase is greatly improved.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a OUR-based fed-batch fermentation method for producing medium-temperature alpha-amylase by using bacillus subtilis.
Background
With the development of brewing and fermentation industries, alpha-amylase is widely applied to industrial production of soy sauce, table vinegar, wine, monosodium glutamate, sugar color and the like, and replaces the traditional starter propagation technology. Compared with the traditional yeast making method, the enzyme preparation saves labor and equipment, improves the yield, reduces the cost and increases the benefit. Bacillus subtilis BF7658 alpha-amylase is a high-efficiency liquefying amylase with the largest yield and the widest application in China, is an enzyme preparation which is obtained by mutagenizing Bacillus subtilis with high heat resistance, performing amplification culture step by step, and purifying, and has a light grey brown powder odor, and the main function is to liquefy starchiness in raw materials into dextrin. In 1965, China began to apply Bacillus subtilis BF7658 to produce alpha-amylase, wherein the fermentation unit is about 200U/mL. The BF7658 variant 209 is subjected to UV and NTG compound mutagenesis in Hechichun and the like to obtain a mutagenic strain 8a5, the enzyme production activity is improved by 17-35% compared with that of the original strain, and the mutagenic strain has the advantages of fast growth and early enzyme production compared with the original strain. In the pilot scale production of a 2t fermentation tank and the scale production of a 16t fermentation tank, the activity of the alpha-amylase reaches 529U/mL and 505U/mL respectively, and the economic benefit is obvious. The medium-temperature alpha-amylase refers to alpha-amylase with the optimal reaction temperature of 50-70 ℃, is widely applied to industries such as starch sugar, baking industry, beer brewing, alcohol industry and the like, and is also applied to various fields such as feed, textile, papermaking, medicine and the like. At present, few studies on medium-temperature alpha-amylase are performed in China, and most scientific research institutions turn to the fields of high-temperature resistant alpha-amylase, acid alpha-amylase, alkaline alpha-amylase and the like.
The strain for producing the mesophilic alpha-amylase has wide sources, mainly comprises bacteria and fungi, such as Aspergillus kawachi, Aspergillus niger, Bacillus acidocaldarius, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus brevis and the like, and the Bacillus type bacteria are the main sources of the mesophilic alpha-amylase of microbial sources. Amylases are generally induction enzymes which induce a large amount of secretion of alpha-amylase in the presence of starch or its hydrolysates. The research on the optimization of the fermentation conditions of amylases at home and abroad has been long, Syu and the like research the influence of different carbon sources in a fermentation culture medium on the enzyme production of the medium-temperature alpha-amylase, and the research shows that the glucose can accelerate the growth of microorganisms when being used as a carbon source, and the starch can obviously improve the enzyme production when being used as a carbon source. Gangadharan D, Sharma A and the like effectively improve the enzyme yield by optimizing the fermentation conditions of the bacillus for producing the alpha-amylase through the response surface.
Tail gas in fermentationThe change of the component concentration reflects the change of the substances in the whole fermentation process. Especially CO in tail gas2And O2Contains very valuable process reaction information. CO 22Are the end products of cellular respiration and catabolism, and are also some of the anabolic substrates. Almost all fermentations produce large amounts of CO2。CO2Is an important growth indicator, and is particularly suitable for early growth stages. CO in logarithmic growth phase2Is proportional to the cell mass under certain conditions. Monitoring CO2Is an effective way to track growth activity. Oxygen is one of the constituents that make up the cell itself and metabolites. For aerobic fermentation, a large amount of oxygen is required for oxidation of substrates, growth of cells, or metabolism of products.
By fermentation of CO in the tail gas2And O2On-line detection and analysis can obtain important respiratory metabolism parameters of fermentation process, such as CO2Release rate (CER), Oxygen Uptake Rate (OUR), Respiratory Quotient (RQ), and the like. These parameters reflect the metabolic status of the microorganism and in particular provide an indication of the transition from growth to production or metabolic transition between the main substrates. CO 22The release Rate (CER) refers to the CO released by fermentation broth cells in unit time and unit volume2Amount of the compound (A). Oxygen Uptake Rate (OUR) refers to the Oxygen consumed by cells of a fermentation broth per unit time and per unit volume. OUR depends on the cell concentration and also on factors such as nutrient content of the fermentation broth, dissolved oxygen level, growth rate of the cells, and type and concentration of carbon source. The Quotient of CER divided by OUR is called Respiratory Quotient (RQ). Present fermentation tail gas CO2And O2Detection and analysis techniques are becoming mature. The performance is stable, the reliability is high, and continuous on-line detection can be realized. Because the gas is extracted from the tail gas for analysis, the fermentation is not influenced, and high-temperature sterilization is not needed, thereby creating favorable conditions for the application of the fermentation.
In recent years, the application research of tail gas analysis in fermentation is more and more extensive. The method has great significance for deeply researching the fermentation process mechanism, exploring and optimizing the fermentation process and comprehensively controlling the fermentation process. Particularly in the fields of gene engineering and biological pharmacy, can greatly accelerate the research and development and industrialization of new products, stabilize production and improve yield.
The domestic research of the feed supplement process for producing the medium-temperature alpha-amylase by using the bacillus subtilis is not many, related research is carried out for feed supplement by measuring the total sugar concentration in the fermentation process, and the growth metabolic process degree of the thalli cannot be quickly and accurately observed due to the fact that the total sugar concentration cannot be quickly measured and the fermentation process has certain fluctuation, so that labor is consumed, and the accuracy is not high. Moreover, the low concentration of total sugar can not completely indicate the bacterial decay, and the research of Sunjing et al shows that the low concentration of total sugar and the enzyme activity of amylase are still in the stage of rapid synthesis. In the fermentation liquor containing a large amount of solid compound nitrogen sources, the solid-liquid ratio of the fermentation system is high, so that the full absorption of carbon and nitrogen sources in surrounding solution by thalli is influenced to a certain extent, the sugar limit condition occurs already under the condition that the carbon source concentration in the fermentation liquor is not lower than 5g/L, and the optimal feeding time can be missed only by detecting the sugar concentration.
Therefore, it is urgently needed to provide a OUR-based fed-batch fermentation method for producing medium-temperature alpha-amylase by using Bacillus subtilis, which utilizes tail gas on-line detection, accurately observes the change of fermentation parameters in real time, and can reasonably control the fed-batch time and the fed-batch rate, thereby timely regulating and controlling the parameters to control the fermentation process and improve the production level.
Disclosure of Invention
The invention aims to provide a OUR-based fed-batch fermentation method for producing medium-temperature alpha-amylase by using bacillus subtilis, which can be used for detecting fermentation tail gas on line, accurately observing the change of fermentation parameters in real time and controlling fed-batch materials in time so as to improve the production level of the alpha-amylase.
In order to achieve the purpose, the invention adopts the following technical scheme.
The invention provides a OUR-based fed-batch fermentation method for producing medium-temperature alpha-amylase by bacillus subtilis, which is characterized in that during batch culture and fermentation of the bacillus subtilis, the change trend of OUR in tail gas is detected on line, fed-batch feeding is carried out at the time point when the OUR is reduced, so that the OUR is in a stable or slowly reduced state, and the alpha-amylase is continuously fermented and synthesized; the feed is glucose or a complete culture medium.
Further, the OUR-based fed-batch fermentation method for producing the mesophilic alpha-amylase by the bacillus subtilis comprises the following steps:
batch culture and fermentation: a fermentation tank culture medium is filled in the fermentation tank, the temperature is adjusted to 34 ℃, bacillus subtilis is inoculated, the inoculation amount is 10% (V/V), fermentation is carried out, and the pH is controlled to be 6.2-6.8 and the dissolved oxygen is more than 40% in the fermentation process;
feeding and fermenting: when the OUR in the batch culture fermentation tail gas is reduced, feeding the whole culture medium according to the total sugar amount or feeding the glucose according to the reducing sugar to ensure that the OUR keeps stable and does not reduce any more, and then fermenting until the fermentation is finished.
Further, the feeding rate of the feeding is the same as the sugar consumption in the stationary phase in the batch culture fermentation process. Feeding the whole culture medium according to the consumption rate of the total sugar; glucose was fed at the rate of reducing sugar consumption.
Further, the whole medium was fed at a feed rate of 4g/L/h in total sugar amount or glucose was fed at a feed rate of 3g/L/h in reducing sugar amount.
Further, batch culture fermentation: the fermentation tank is filled with a fermentation tank culture medium, the liquid loading amount is 3/5, the temperature is adjusted to 34 ℃, then bacillus subtilis is inoculated, the inoculation amount is 10% (V/V), the dissolved oxygen is 150% under the conditions of stirring speed of 300epm rpm, ventilation capacity of 6L/min and tank pressure of 0.05Kpa, and fermentation is carried out, wherein the pH is controlled to be 6.2-6.8 and the dissolved oxygen is more than 40% in the fermentation process.
Further, before the batch culture fermentation, the dissolved oxygen electrode was calibrated to 0% with a saturated sodium sulfite solution. And calibrating a pH electrode before batch culture and fermentation.
Further, during the whole batch culture fermentation process: feeding strong ammonia water to control the pH value to be 6.2-6.8; regulating the rotation speed and controlling the dissolved oxygen to be more than 40 percent.
Further, the fermentor medium comprises: 158g/L of corn starch, 37.5g/L of soybean meal, 40g/L of cottonseed meal, 1g/L of calcium chloride, 2.3g/L of ammonium chloride, 17.25g/L of corn steep liquor, 8.3g/L of disodium hydrogen phosphate and 0.01mL/100mL of high-temperature amylase; sterilizing at 121 deg.C for 60 min.
And further, finishing the fermentation when the pH value of the fermentation is increased back to the range of 7.3-7.7 after feeding is carried out.
Further, the fermentation is finished when the amylase activity in the fermentation process is not increased any more after fed-batch fermentation.
Further, the glucose concentration was 42.5g/100mL, and sterilized at 115 ℃ for 20 min.
Further, the whole medium comprises: 210.67g/L of corn starch, 50g/L of soybean meal, 53.33g/L of cottonseed meal, 1.33g/L of calcium chloride, 3.07g/L of ammonium chloride, 20g/L of corn steep liquor, 11.07g/L of disodium hydrogen phosphate and 0.01mL/100mL of high-temperature amylase; sterilizing at 121 deg.C for 30 min.
Further, the inoculated bacillus subtilis is obtained by seed shake flask culture, and the steps are as follows: inoculating the strain liquid in the glycerinum tube into a seed shake flask culture medium with the inoculation amount of 0.6% (V/V), and carrying out shake flask fermentation for 24h at the temperature of 34 ℃ and the rotation speed of 220 r/min.
Further, the seed shake flask culture medium comprises: 47.4g/L of corn starch, 11.25g/L of soybean meal, 12g/L of cotton meal, 0.5g/L of calcium chloride, 1.15g/L of ammonium chloride, 4.5g/L of corn steep liquor, 3g/L of disodium hydrogen phosphate and 0.01mL/100mL of high-temperature amylase; adjusting the pH value to 7.3-7.7, sterilizing at 121 ℃ for 30min, cooling, and adding kanamycin to a final concentration of 25 ug/mL.
In the present invention, the consumption rate of total sugars or the consumption rate of reducing sugars is measured by the DNS method. The sugar consumption in the rapid enzyme production period, namely the stabilization period, in the batch fermentation process is detected to determine the feed rate.
In the present invention, Bacillus subtilis is provided by limited Biotechnology from the company Shinshima, Schneider.
In the invention, samples are taken every 4-5 h in the whole process to determine related samples, and the fermentation star and the mass spectrometer are used for monitoring related parameters in the whole process.
In the present invention, the pH measurement can be performed by the following method: and (4) measuring the pH of the fermentation liquor stock solution off line by using a pH meter, comparing with the reading of a pH electrode, and calibrating the pH parameter in real time.
In the present invention, the biomass of cells can be measured by the following method: and (3) diluting the fermentation liquor stock solution to an appropriate multiple, and measuring OD600 on a spectrophotometer, wherein OD is the measured absorbance value multiplied by the dilution multiple.
In the present invention, the reducing sugar can be measured by the following method:
taking 1mL of diluted sample (diluted by 100 times) into a 10mL colorimetric tube, then adding 1.5mL of DNS reagent, uniformly mixing on a vortex oscillator, developing in a 100 ℃ boiling water bath for 5min, then rapidly cooling, cooling to room temperature, then fixing the volume to 10mL, uniformly mixing on the vortex oscillator, respectively absorbing 200 mu L of solution from the tube into a 96-hole enzyme label plate, and measuring the absorbance of each sample at 550nm by using an enzyme label instrument. And substituting the measured absorbance into a standard curve formula, calculating the content of the reducing sugar in the diluted sample, and multiplying the content by the dilution factor to obtain the content of the reducing sugar in the original sample.
In the present invention, the total sugar can be measured by the following method:
taking 1mL of diluted sample (diluted by 10 times) to a 10mL colorimetric tube, adding 1.5mL of 3M hydrochloric acid, uniformly mixing, hydrolyzing in a 100 ℃ boiling water bath for 20min, cooling to room temperature, adding 1.5mL of 3M NaOH solution, cooling to room temperature, and diluting to the constant volume of 10 mL. Taking 1mL of the solution with constant volume into a 10mL colorimetric tube, then adding 1.5mL of DNS reagent, uniformly mixing, developing in a boiling water bath at 100 ℃ for 5min, cooling to room temperature, then fixing the volume to 10mL, uniformly mixing on a vortex oscillation instrument, respectively absorbing 200 mu L of the solution from the tube into a 96-hole enzyme label plate, and measuring the absorbance of each sample at 550nm by using an enzyme label instrument. And substituting the measured absorbance into a standard curve formula, calculating the total sugar content in the diluted sample, and multiplying the total sugar content by the dilution multiple to obtain the total sugar content (the total sugar content is expressed by the content of reducing sugar) in the original sample.
In the present invention, the amylase activity can be measured by the following method:
taking out about 10mL of fermentation liquor in the fermentation tank, sucking 1mL of fermentation liquor, diluting with a phosphate buffer solution to an appropriate multiple for later use;
sucking 4mL of starch solution into a 10mL centrifuge tube, adding 1mL of phosphate buffer solution, uniformly mixing, and preheating in a 60 ℃ water bath for 8 min. Adding 0.2mL of fermentation liquid which is diluted to a proper amount, mixing uniformly, and then accurately reacting for 5min in a water bath kettle at 60 ℃. Immediately using a pipette to suck 1mL of the solution after the reaction, adding the solution into a test tube containing 5mL of dilute iodine solution and 0.5mL of hydrochloric acid in advance, mixing the solution uniformly, and measuring the absorbance at the wavelength of 660nm by taking 5mL of dilute iodine solution and 0.5mL of hydrochloric acid as blanks. And searching the relevant enzyme activity corresponding table according to the absorbance so as to obtain the enzyme activity concentration of the amylase in the zymophyte liquid.
In the present invention, the OUR can be measured by the following method:
and monitoring the tail gas in the fermentation process by adopting a process mass spectrometer, and calculating the Oxygen Uptake Rate (OUR) of the thallus in unit time by using biostar acquisition software according to a quasi-steady state measurement principle. The principle is based on the assumption that: the dissolved oxygen does not change in short time intervals and the gas composition change above the tank is ignored. In a closed fermenter, the DO (dissolved oxygen) in the fermentation broth is subjected to a mass balance:
calculating OTR (oxygen transmission rate) by the oxygen partial pressure ratio and the flow of the inlet gas and the outlet gas:
OTR＝(FinyO2,in-FoutyO2,out)/V (2-2)
calculation of F from Nitrogen balanceout：
Fin·yN2,in＝Fout·yN2,out (2-3)
OUR is obtained from equations (2-2) and (2-3):
wherein, FinAs intake air flow rate, FoutIs the exhaust gas flow rate, yO2,inIs the partial pressure ratio of intake oxygen, yO2,outAs tail gasThe oxygen partial pressure ratio and V is the volume of the fermentation liquor.
According to the invention, the change of the early bacterial concentration can be accurately observed in real time through the OUR in the tail gas, so that the early bacterial strain can be rapidly increased by timely regulating and controlling parameters.
The invention has the beneficial effects that:
according to the OUR-based fed-batch fermentation method for producing the medium-temperature alpha-amylase by the bacillus subtilis, the change trend of the OUR in the tail gas is detected on line to feed materials, fermentation parameters are regulated and controlled, the bacillus subtilis is promoted to produce the medium-temperature alpha-amylase, and the enzyme activity yield of the alpha-amylase is greatly improved.
The fed-batch fermentation method can maintain very low substrate concentration in the system, thereby avoiding the occurrence of repression effect, maintaining proper oxygen supply according to the equipment capacity, slowing down the adverse effect of metabolic harmful substances, further controlling the fermentation process and improving the production level of amylase. In the invention, the on-line OUR detection technology can identify different stages of the whole fermentation process on line, and the fed-batch time and the fed-batch rate can be reasonably and accurately controlled by utilizing the on-line detection and analysis of the tail gas, so that the feedback control of the fed-batch is realized, the fermentation parameters are effectively regulated and controlled, and the synthesis rate of the amylase and the enzyme activity of the medium-temperature alpha-amylase are improved.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph showing the variation of fermentation parameters in example 2 of the present invention.
FIG. 2 is a graph showing the variation of parameters of reducing sugars and total sugars in example 2 of the present invention.
FIG. 3 is a graph showing the variation of fermentation parameters in example 3 of the present invention.
FIG. 4 is a graph showing the variation of parameters of reducing sugars and total sugars in example 3 of the present invention.
FIG. 5 is a graph showing the variation of fermentation parameters in example 4 of the present invention.
FIG. 6 is a graph showing the change of reducing sugar and total sugar in example 4 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without inventive exercise, are within the scope of the present invention.
Example 1
The embodiment provides a OUR-based fed-batch fermentation method for producing medium-temperature alpha-amylase by using bacillus subtilis, which comprises the steps of detecting the change trend of OUR in tail gas on line in batch culture fermentation of the bacillus subtilis, feeding materials at the time point of OUR reduction to enable the OUR to be in a stable or slowly-reduced state, and continuing to ferment and synthesize the alpha-amylase; the feed is glucose or a complete culture medium.
In this embodiment, the OUR-based fed-batch fermentation method for producing mesophilic alpha-amylase by bacillus subtilis includes the following steps:
batch culture and fermentation: a fermentation tank culture medium is filled in the fermentation tank, the temperature is adjusted to 34 ℃, bacillus subtilis is inoculated, the inoculation amount is 10% (V/V), fermentation is carried out, and the pH is controlled to be 6.2-6.8 and the dissolved oxygen is more than 40% in the fermentation process;
feeding and fermenting: when the OUR in the batch culture fermentation tail gas is reduced, feeding the whole culture medium according to the total sugar amount or feeding the glucose according to the reducing sugar to ensure that the OUR keeps stable and does not reduce any more, and then fermenting until the fermentation is finished.
The feeding rate of the feeding is the same as the sugar consumption in the stationary phase in the batch culture fermentation process. Feeding the whole culture medium according to the consumption rate of the total sugar; glucose was fed at the rate of reducing sugar consumption.
Example 2
The embodiment provides a OUR-based fed-batch fermentation method for producing medium-temperature alpha-amylase by using bacillus subtilis, which comprises the steps of detecting the change trend of OUR in tail gas on line in batch culture fermentation of the bacillus subtilis, feeding materials at the time point of OUR reduction to enable the OUR to be in a stable or slowly-reduced state, and continuing to ferment and synthesize the alpha-amylase; the feed is glucose.
In this example, 170g of glucose was taken to a constant volume of 400mL, and sterilized at 115 ℃ for 20min to obtain supplemented glucose.
In this embodiment, the OUR-based fed-batch fermentation method for producing mesophilic alpha-amylase by bacillus subtilis includes the following steps:
inoculating 0.3mL of bacterial liquid in a glycerin pipe into a 500mL triangular shake flask, wherein the liquid loading amount of a seed shake flask culture medium in the shake flask is 50mL, wrapping with 8 layers of gauze, and shaking the flask for culture for 24h on a round shaking table at the temperature of 34 ℃ and the rotating speed of 220 r/min.
Adjusting the rotating speed and temperature of the fermentation tank, covering the wetted cleaning cloth on a motor and various pipelines of the fermentation tank after the tank is pressed, and reserving an inoculation port of the fermentation tank; the inoculation port sleeve is filled with a flame ring of alcohol cotton, a door and a window of a fermentation room are closed (so that the door and the window are prevented from being infected by bacteria caused by large-scale air flow in the inoculation process), the flame ring is ignited, and the seed liquid cultured for 24 hours is poured into the fermentation tank quickly.
The pH electrode was calibrated, and the dissolved oxygen electrode was calibrated to 0% with saturated sodium sulfite solution. The liquid loading amount of the fermentation tank culture medium in a 5L fermentation tank is 3.0L, sterilization is carried out for 60min at 121 ℃, the temperature is adjusted to 34 ℃ before inoculation, the stirring speed is 300epm rpm, the ventilation volume is 6L/min, and the dissolved oxygen is calibrated to be 150 percent under the condition that the tank pressure is 0.05 Kpa. The inoculation amount is 10% (V/V), the pH value is controlled to be about 6.5 by adding concentrated ammonia water in the whole process, and the rotating speed is adjusted to control the dissolved oxygen to be more than 40%.
When the OUR is in the descending trend, namely the period when the fermentation is started to enter the final stage, the glucose is fed at the feeding rate of 3g/L/h of reducing sugar, so that the OUR is kept stable and does not decline any more. And sampling every 4h in the whole process to determine related samples, and monitoring related parameters in the whole process by a fermentation star and a mass spectrometer. After feeding, the pH value of the fermentation is increased back to about 7.5 or the enzyme activity of the amylase is detected not to be increased any more, which represents that the fermentation is finished.
When OUR begins to decrease and dissolved oxygen begins to rise, it indicates that fermentation begins to enter the final stage.
In this embodiment, the fermenter medium comprises: 158g/L of corn starch, 37.5g/L of soybean meal, 40g/L of cottonseed meal, 1g/L of calcium chloride, 2.3g/L of ammonium chloride, 17.25g/L of corn steep liquor, 8.3g/L of disodium hydrogen phosphate and 0.01mL/100mL of high-temperature amylase, wherein the volume is determined to be 2.7L, and the sterilization is carried out for 60min at 121 ℃.
In this embodiment, the seed shake flask culture medium comprises: 47.4g/L of corn starch, 11.25g/L of soybean meal, 12g/L of cottonseed meal, 0.5g/L of calcium chloride, 1.15g/L of ammonium chloride, 4.5g/L of corn steep liquor, 3g/L of disodium hydrogen phosphate and 0.01mL/100mL of high-temperature amylase; adjusting the pH value to 7.3-7.7, and sterilizing at 121 ℃ for 30 min; after cooling, kanamycin was added to a final concentration of 25 ug/mL.
In this embodiment, during the whole fermentation process, the fermentation related parameters are monitored in the whole process, and fig. 1 and fig. 2 are obtained.
FIG. 1 is a graph showing the variation of fermentation parameters in example 2 of the present invention.
FIG. 2 is a graph showing the variation of parameters of reducing sugars and total sugars in example 2 of the present invention.
As can be seen from fig. 1:
the OUR of fermentation parameter shows a rapid descending trend when fermenting for 34h, because at this time, due to the limitation of nutrient substances, the strain can not utilize sufficient nutrition to maintain the growth of itself and carry out the synthesis of products, enters a decline death period, and then the OUR is rapidly descended. At the moment, glucose with a certain concentration is fed, and the feeding speed and the concentration of the glucose are determined by the consumption rate of reducing sugar when the enzyme is rapidly produced in the strain stabilization phase.
After the fed-batch culture medium is fed, the OUR does not decrease any more, the stable or slow decreasing state is presented, the DO value is continuously in a state close to zero, the enzyme activity is increased at a synthesis rate of 19.64U/mL.h, which indicates that the thalli start to utilize the supplemented nutrient substances to further synthesize the fermentation enzyme activity, the glucose feeding is stopped until the enzyme activity is not increased any more for 54h, the fermentation is finished, and finally the fermentation enzyme activity in the fermentation broth reaches about 1039U/mL.
As can be seen from the figure, the OD value is substantially consistent with the variation trend of OUR, the OD value represents the parameter of bacterial concentration, continuous sampling is needed for measuring the OD value, the accuracy of the OD value is greatly influenced because of the large amount of insoluble substances in the culture medium, and the feeding is performed by the variation trend of OD, so that the feeding is time-consuming and labor-consuming, and the accuracy of the parameter is not good.
Example 3
The embodiment provides a OUR-based fed-batch fermentation method for producing medium-temperature alpha-amylase by using bacillus subtilis, which comprises the steps of detecting the change trend of OUR in tail gas on line in batch culture fermentation of the bacillus subtilis, feeding materials at the time point of OUR reduction to enable the OUR to be in a stable or slowly-reduced state, and continuing to ferment and synthesize the alpha-amylase; the feed is a complete medium.
In this embodiment, the whole culture medium comprises: 210.67g/L of corn starch, 50g/L of soybean meal, 53.33g/L of cottonseed meal, 1.33g/L of calcium chloride, 3.07g/L of ammonium chloride, 20g/L of corn steep liquor, 11.07g/L of disodium hydrogen phosphate and 0.01mL/100mL of high-temperature amylase, wherein the volume is up to 1.5L, and the sterilization is carried out for 30min at 121 ℃.
In this embodiment, the OUR-based fed-batch fermentation method for producing mesophilic alpha-amylase by bacillus subtilis includes the following steps:
inoculating 0.3mL of bacterial liquid in a glycerin pipe into a 500mL triangular shake flask, wherein the liquid loading amount of a seed shake flask culture medium in the shake flask is 50mL, wrapping with 8 layers of gauze, and shaking the flask for culture for 24h on a round shaking table at the temperature of 34 ℃ and the rotating speed of 220 r/min.
Adjusting the rotating speed and temperature of the fermentation tank, covering the wetted cleaning cloth on a motor and various pipelines of the fermentation tank after the tank is pressed, and reserving an inoculation port of the fermentation tank; the inoculation port sleeve is filled with a flame ring of alcohol cotton, a door and a window of a fermentation room are closed (so that the door and the window are prevented from being infected by bacteria caused by large-scale air flow in the inoculation process), the flame ring is ignited, and the seed liquid cultured for 24 hours is poured into the fermentation tank quickly.
The pH electrode was calibrated, and the dissolved oxygen electrode was calibrated to 0% with saturated sodium sulfite solution. The liquid loading amount of the culture medium in the fermentation tank of 5L is 3.0L, sterilization is carried out for 60min at 121 ℃, the temperature is adjusted to 34 ℃ before inoculation, the stirring speed is 300epm, the ventilation volume is 6L/min, and the dissolved oxygen is 150% under the condition that the tank pressure is 0.05 Kpa. The inoculation amount is 10% (V/V), the pH value is controlled to be about 6.5 by adding concentrated ammonia water in the whole process, and the rotation speed is adjusted to control the dissolved oxygen to be more than 40%.
When the OUR is in the decline trend, namely the period when the fermentation is started to enter the final stage, the whole culture medium is fed at the feeding rate of 4g/L/h of total sugar, so that the OUR is kept stable and does not decline any more. Sampling every 4h in the whole process to determine related samples, and monitoring related parameters in a whole process by a fermentation star and a mass spectrometer. After feeding, the pH value of the fermentation rises back to about 7.5, which represents the end of the fermentation.
In this embodiment, the fermenter medium comprises: 158g/L of corn starch, 37.5g/L of soybean meal, 40g/L of cottonseed meal, 1g/L of calcium chloride, 2.3g/L of ammonium chloride, 17.25g/L of corn steep liquor, 8.3g/L of disodium hydrogen phosphate and 0.01mL/100mL of high-temperature amylase, wherein the volume is determined to be 2.7L, and the sterilization is carried out for 60min at 121 ℃.
In this embodiment, the seed shake flask culture medium comprises: 47.4g/L of corn starch, 11.25g/L of soybean meal, 12g/L of cottonseed meal, 0.5g/L of calcium chloride, 1.15g/L of ammonium chloride, 4.5g/L of corn steep liquor, 3g/L of disodium hydrogen phosphate and 0.01mL/100mL of high-temperature amylase; adjusting the pH value to 7.3-7.7, and sterilizing at 121 ℃ for 30 min; after cooling, kanamycin was added to a final concentration of 25 ug/mL.
In this embodiment, during the whole fermentation process, the fermentation related parameters are monitored in the whole process, and fig. 3 and 4 are obtained.
FIG. 3 is a graph showing the variation of fermentation parameters in example 3 of the present invention.
FIG. 4 is a graph showing the change in reducing sugar and total sugar in example 3 of the present invention.
As can be seen in fig. 3:
referring to FIG. 3, the initial stage of fermentation is shown when OUR begins to decrease and dissolved oxygen begins to rise again, and it is understood from FIG. 3 that the whole medium is fed in approximately 34 hours.
After the whole culture medium is fed in a flowing mode, the OUR basically maintains a relatively stable trend, which indicates that the strain does not continuously fade into empty spores, but the fed-in culture medium is used for continuously synthesizing the amylase. As can be seen from the variation trend of enzyme activity, the synthesis rate of amylase is faster than that of glucose fed-batch culture medium, reaches 35.5U/mL.h, is basically the same as that of amylase in batch fermentation process, and the final enzyme activity is much better than that of glucose fed-batch culture medium, reaches 1247U/mL, which indicates that the culture medium with richer fed-batch nutrient components has better effect than that of carbon source fed-batch culture medium. During the synthesis of the amylase, the Dissolved Oxygen (DO) value is basically kept in a state of falling to zero until the feeding of the culture medium is stopped for 46h, the dissolved oxygen begins to rise back, the OUR value begins to rapidly fall again until the fermentation time is 62h and falls below 20 mmol/L.h, at the moment, more than 90% of thalli are changed into empty spores through sampling microscopic examination, the fermentation is ended, and at the moment, the enzyme activity of the medium-temperature alpha-amylase reaches 1247U/mL.
Example 4
The embodiment provides a fermentation method for producing mesophilic alpha-amylase by bacillus subtilis, which comprises the following steps:
inoculating 0.3mL of bacterial liquid in a glycerin pipe into a 500mL triangular shake flask, wherein the liquid loading amount of a seed shake flask culture medium in the shake flask is 50mL, wrapping with 8 layers of gauze, and shaking the flask for culture for 24h on a round shaking table at the temperature of 34 ℃ and the rotating speed of 220 r/min.
Adjusting the rotating speed and temperature of the fermentation tank, covering the wetted cleaning cloth on a motor and various pipelines of the fermentation tank after the tank is pressed, and reserving an inoculation port of the fermentation tank; the inoculation port sleeve is filled with a flame ring of alcohol cotton, a door and a window of a fermentation room are closed (so that the door and the window are prevented from being infected by bacteria caused by large-scale air flow in the inoculation process), the flame ring is ignited, and the seed liquid cultured for 24 hours is poured into the fermentation tank quickly.
The pH electrode was calibrated, and the dissolved oxygen electrode was calibrated to 0% with saturated sodium sulfite solution. The liquid loading amount of a fermentation tank culture medium in a 5L fermentation tank is 3.0L, sterilization is carried out for 60min at 121 ℃, the temperature is adjusted to 34 ℃ before inoculation, the stirring speed is 300epm, the ventilation volume is 6L/min, and the dissolved oxygen is 150% under the condition that the tank pressure is 0.05 Kpa; the inoculation amount is 10% (V/V), the pH value is controlled to be about 6.5 by adding concentrated ammonia water in the whole process, and the rotating speed is adjusted to control the dissolved oxygen to be more than 40%; until the fermentation is finished.
In this embodiment, the seed shake flask culture medium comprises: 47.4g/L of corn starch, 11.25g/L of soybean meal, 12g/L of cottonseed meal, 0.5g/L of calcium chloride, 1.15g/L of ammonium chloride, 4.5g/L of corn steep liquor, 3g/L of disodium hydrogen phosphate and 0.01mL/100mL of high-temperature amylase; adjusting the pH value to 7.3-7.7, and sterilizing at 121 ℃ for 30 min; after cooling, kanamycin was added to a final concentration of 25 ug/mL.
In this embodiment, the fermenter medium comprises: 158g/L of corn starch, 37.5g/L of soybean meal, 40g/L of cottonseed meal, 1g/L of calcium chloride, 2.3g/L of ammonium chloride, 17.25g/L of corn steep liquor, 8.3g/L of disodium hydrogen phosphate and 0.01mL/100mL of high-temperature amylase, wherein the volume is determined to be 2.7L, and the sterilization is carried out for 60min at 121 ℃.
In this embodiment, during the whole fermentation process, the fermentation related parameters are monitored in the whole process, and fig. 5 and fig. 6 are obtained.
FIG. 5 is a graph showing the variation of fermentation parameters in example 4 of the present invention.
FIG. 6 is a graph showing the change of reducing sugar and total sugar in example 4 of the present invention.
As can be seen from fig. 5:
according to the change trend of Dissolved Oxygen (DO) in FIG. 5, it can be seen that the Bacillus consumes a large amount of oxygen at the early stage, and in order to increase the bacterial concentration, the OUR value is increased exponentially, and the rotation speed is continuously increased at the early stage to provide sufficient oxygen to enable the strain to rapidly propagate, so as to ensure that a large amount of strains synthesize amylase at the stationary stage.
According to the change trend of OUR, 1-6h is the lag phase of the bacillus subtilis, 6-20h is the exponential growth phase, the Dissolved Oxygen (DO) value is continuously reduced before the strain enters the stabilization phase, and the rotation speed is continuously regulated to ensure that sufficient oxygen is supplied in the early stage.
20-34h is a stable period, during which the strain mainly synthesizes amylase and the dissolved oxygen drops to zero.
The amylase can be detected after about 8 hours, but the rapid synthesis period of the amylase is the stable period of the strain growth, such as the trend that the enzyme activity of the amylase is sharply increased after 20 hours in the figure.
Dissolved oxygen begins to rise again within 32h, the OUR value begins to decrease rapidly, the strain enters a decline period, and the fermentation enters a final stage. And (3) until 42h, the dissolved oxygen is increased back to the initial saturation level, the OUR is reduced to be below 20 mmol/L.h, the enzyme activity is not increased any more, 90% of thalli are changed into empty spores through microscopic examination of fermentation liquor, the fermentation is completely finished, and the final fermentation enzyme activity is about 880U/mL.
In conclusion, compared with the conventional batch fermentation method, the fed-batch fermentation method has the advantages that the synthesis rate of the amylase is higher, and the enzyme activity of the final amylase after the fermentation is finished is also higher. The invention carries out fed-batch fermentation on the basis of stable batch fermentation, and respectively feeds glucose and a complete culture medium, so that the fermentation enzyme activity of the medium-temperature alpha-amylase reaches 1039U/mL and 1247U/mL respectively, the enzyme activity yield of the amylase is greatly improved, the effect is obvious, and the application value is higher.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A OUR-based fed-batch fermentation method for producing medium-temperature alpha-amylase by bacillus subtilis is characterized in that in batch culture fermentation of bacillus subtilis, the change trend of OUR in tail gas is detected on line, fed-batch feeding is carried out at the time point of OUR reduction, the OUR is in a stable or slowly-reduced state, and the fermentation is continued to synthesize the alpha-amylase; the feed is glucose or a complete culture medium.
2. The OUR-based Fed-feed fermentation process of Bacillus subtilis mesophilic alpha-amylase according to claim 1, characterized in that the complete medium comprises: 210.67g/L of corn starch, 50g/L of soybean meal, 53.33g/L of cottonseed meal, 1.33g/L of calcium chloride, 3.07g/L of ammonium chloride, 20g/L of corn steep liquor, 11.07g/L of disodium hydrogen phosphate and 0.01mL/100mL of high-temperature amylase.
3. The OUR-based Fed-batch fermentation method of Bacillus subtilis mesophilic alpha-amylase production according to claim 1 or 2, characterized in that in the Bacillus subtilis batch fermentation, the following steps are included:
batch culture and fermentation: a fermentation tank culture medium is filled in the fermentation tank, the temperature is adjusted to 34 ℃, then bacillus subtilis is inoculated, the inoculation amount is 10%, fermentation is carried out, and the pH is controlled to be 6.2-6.8 and the dissolved oxygen is controlled to be more than 40% in the fermentation process;
feeding and fermenting: when the OUR in the batch culture fermentation tail gas is reduced, feeding the whole culture medium according to the total sugar amount or feeding the glucose according to the reducing sugar to ensure that the OUR keeps stable and does not reduce any more, and then fermenting until the fermentation is finished.
4. The OUR-based fed-batch fermentation process for producing mesophilic alpha-amylase by Bacillus subtilis according to claim 3, characterized in that the feeding rate of the feed is the same as the sugar consumption during the stationary phase of the batch fermentation process.
5. The OUR-based Fed-batch fermentation process of mesophilic alpha-amylase production by Bacillus subtilis according to claim 3, characterized by batch fermentation: the fermentation tank is filled with a fermentation tank culture medium, the liquid loading amount is 3/5, the temperature is adjusted to 34 ℃, then bacillus subtilis is inoculated, the inoculation amount is 10%, the dissolved oxygen is determined to be 150% under the conditions of stirring speed of 300epm rpm, ventilation volume of 6L/min and tank pressure of 0.05Kpa, and fermentation is carried out, wherein the pH value is controlled to be 6.2-6.8 and the dissolved oxygen is controlled to be more than 40% in the fermentation process.
6. The OUR-based Fed-batch fermentation process of mesophilic alpha-amylase production by Bacillus subtilis of claim 3, characterized in that the dissolved oxygen electrode is calibrated to 0% with saturated sodium sulfite solution before the batch fermentation.
7. The OUR-based Fed-in fermentation process of a mesophilic alpha-amylase produced by Bacillus subtilis of claim 3, wherein the fermentor medium comprises: 158g/L of corn starch, 37.5g/L of soybean meal, 40g/L of cottonseed meal, 1g/L of calcium chloride, 2.3g/L of ammonium chloride, 17.25g/L of corn steep liquor, 8.3g/L of disodium hydrogen phosphate and 0.01mL/100mL of high-temperature amylase.
8. The OUR-based fed-batch fermentation method for producing the medium-temperature alpha-amylase by the Bacillus subtilis according to claim 3, wherein the fermentation is finished when the pH value is raised to 7.3-7.7 after fed-batch fermentation.
9. The OUR-based Fed-batch fermentation method of Bacillus subtilis for producing mesophilic alpha-amylase according to claim 3, characterized in that the inoculated Bacillus subtilis is obtained by seed shake flask culture, comprising the following steps: inoculating the strain liquid in the glycerinum pipe into a seed shake flask culture medium, wherein the inoculation amount is 0.6%, and carrying out shake flask fermentation for 24h under the conditions that the temperature is 34 ℃ and the rotating speed is 220 r/min.
10. The OUR-based Fed-in fermentation process of a mesophilic alpha-amylase produced by Bacillus subtilis according to claim 9, characterized in that the seed shake flask culture medium comprises: 47.4g/L of corn starch, 11.25g/L of soybean meal, 12g/L of cottonseed meal, 0.5g/L of calcium chloride, 1.15g/L of ammonium chloride, 4.5g/L of corn steep liquor, 3g/L of disodium hydrogen phosphate, 0.01mL/100mL of high-temperature amylase and 25ug/mL of kanamycin.
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