CN111592996A

CN111592996A - High-density fermentation culture method for bacillus CQN-2

Info

Publication number: CN111592996A
Application number: CN201911249696.0A
Authority: CN
Inventors: 杨求华; 周宸; 张燎原; 黄瑞芳; 林辉; 林琪; 陆振; 杨福元
Original assignee: Fisheries Research Institute Of Fujian (fujian Aquatic Disease Prevention Center)
Current assignee: Fisheries Research Institute Of Fujian (fujian Aquatic Disease Prevention Center)
Priority date: 2019-12-09
Filing date: 2019-12-09
Publication date: 2020-08-28

Abstract

The invention provides a high-density fermentation culture method of bacillus CQN-2, which is a high-density fermentation culture method of bacillus CQN-2 obtained through the steps of growth condition optimization, carbon source and nitrogen source optimization, single metal ion selection, carbon source, nitrogen source and molasses concentration optimization and the like.

Description

High-density fermentation culture method for bacillus CQN-2

Technical Field

The invention relates to the technical field of bacillus culture, in particular to a high-density fermentation culture method of bacillus CQN-2.

Background

The bacillus CQN-2 is bacillus amyloliquefaciens which is one of probiotics separated from intestinal tracts of healthy apostichopus japonicus, and after the apostichopus japonicus is fed by the bacillus CQN-2 added into feed, the weight gain rate and the specific growth rate of the apostichopus japonicus can be obviously improved, and meanwhile, the disease resistance of the apostichopus japonicus against pathogenic bacteria infection can be effectively improved. However, a pilot scale high density fermentation culture method for Bacillus CQN-2 has not been established.

Based on the above series of problems, the following technical solutions have been developed.

Disclosure of Invention

The invention aims to obtain a high-density fermentation culture method of bacillus CQN-2 by the steps of growth condition optimization, carbon source and nitrogen source optimization, single metal ion selection, carbon source, nitrogen source and molasses concentration optimization and the like.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides a high-density fermentation culture method of bacillus CQN-2, which comprises the following steps:

s1, preparing a PDA enriched medium, an LB solid medium, a fermentation medium, experimental equipment and a strain to be tested;

s2, firstly, carrying out enlarged culture and preservation on the strains, taking out the strains after preservation, starting activation, transferring the strains to fermentation, and measuring a growth curve in the fermentation process;

s3, optimizing conditions in the growth process, wherein the conditions to be optimized are the pH value and the liquid loading amount of the culture medium and the inoculation amount of the transfer during fermentation, and the culture medium is activated, transferred and fermented after being optimized and prepared, and data recording and analysis are carried out;

s4, optimizing a carbon source and a nitrogen source, selecting single metal ions and optimizing molasses concentration in the optimizing process, recording and analyzing data through orthogonal experiments, and then establishing a comparison effect of growth curves of an original culture condition and an optimal culture condition;

s5, performing pH regulation in a fermentation tank, and then drawing the OD of the fermentation liquor₆₀₀And (4) calculating the number of the thalli after fermentation is finished according to a standard curve of the value and the thalli weight.

Further, the PDA enriched medium of step S1 includes: 200g of potato, 20g of glucose, 3g of peptone and KH₂PO₄1g，MgSO₄0.5g, vitamin B₁0.1g, pH7.0, adding water to 1000 mL; the LB solid medium comprises: LB solid medium: 10g of peptone, 5g of yeast powder, 10g of NaCl, 20g of agar, pH7.0, and each 1000mL of the agar; the fermentation medium comprises: 20g of molasses, 20g of yeast powder and KH₂PO₄0.5g，MgSO₄0.5g, NaCl 0.3g, pH7.0, per 1000 mL.

Further, the experimental device comprises: beaker, graduated cylinder, erlenmeyer flask, liquid-transfering gun, rifle head box, coating rod, flat board, glass cell, centrifuge tube rack.

Further, the optimization of the carbon source and the nitrogen source in step S4 includes using sucrose, xylose, glucose, soluble starch, and dextrin, and the nitrogen source uses bacteriological peptone, yeast extract, fish meal peptone, beef extract, and NH₄Cl、 NaNO₂Preparing culture medium for each group of comparison tests, activating, transferring, fermenting, recording and analyzing data, and performing OD (optical density) on fermented liquid₆₀₀And (4) measuring values, recording data, and analyzing and comparing to obtain the optimal carbon source and nitrogen source.

Further, the selection of the single metal ion includes selecting Fe²⁺、Fe³⁺、Zn²⁺、Ca²⁺、 Mn²⁺、Ba²⁺、Al³ ⁺The activation, transfer and fermentation are carried out by the preparation of the culture medium, wherein the inoculation amount during the fermentation is inoculated according to the optimal conditions obtained by the previous experiment, and the metal ions which have the promotion effect on the growth conditions are obtained by recording data and analyzing.

Further, the optimization of the molasses concentration comprises the steps of preparing fermentation culture media with different molasses concentrations for activation, switching and fermentation, wherein the inoculation amount during fermentation is inoculated according to the optimal conditions obtained by the previous experiment, and then the optimal molasses concentration is obtained through data recording and analysis.

Further, the process of adjusting and controlling pH in step S5 includes preparing two fermentation media based on the optimal media formulation, performing activated fermentation, selecting one of the two fermentation media to control pH by using NaOH, and setting the fermentation temperature at 30 ℃, the rotation speed at 600rpm, and the pH of the fermentation media to be adjusted to 7.0. And inoculating the activated bacillus into a fermentation tank for fermentation at the optimal inoculation amount.

Further, the OD of the fermentation liquid is drawn₆₀₀The value and the standard curve of thallus weight include that a plurality of groups of fermentation liquids are taken and added into a centrifuge tube, the centrifuge tube is used for centrifugally precipitating the thallus at 10000rpm and 15 ℃, then sterile water is used for re-suspending a thallus cleaning culture medium, the thallus is centrifugally precipitated under the same conditions for 2-3 times, after washing is completed, residual liquid in the centrifuge tube is abandoned, the centrifuge tube is placed into a drying box for drying, after drying is completed, the weight of the centrifuge tube and the thallus is weighed, then the weight of the centrifuge tube is subtracted, the dry weight of the thallus can be obtained, and then the obtained data is used for drawing the standard curve.

Further, the step of calculating the number of the bacteria after the fermentation is completed comprises taking a proper amount of OD₆₀₀The 72.5 fermentation liquid is diluted to 10 degrees by using sterile water in a super clean bench in a gradient way^-11Diluting to 10^-9、10^-10、 10^-11And taking 100L of the three-gradient diluent, coating a flat plate, coating 2-3 flat plates on each gradient, taking the solid culture medium used here as LB solid culture medium, carrying out inverted culture on the coated flat plate in a 37 ℃ incubator for 48h, counting after the culture is finished, and calculating the average value to obtain the total number of the thalli.

Compared with the prior art, the invention has the following beneficial effects:

1. the high-density fermentation culture method of the bacillus CQN-2 is obtained by the steps of optimizing growth conditions, optimizing a carbon source and a nitrogen source, selecting single metal ions, optimizing the concentrations of the carbon source, the nitrogen source and molasses and the like;

2. according to the invention, through multiple groups of comparative experiments, a culture method suitable for growth of the bacillus CQN-2 is scientifically determined, and the survival rate of the bacillus CQN-2 is effectively improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without any creative effort.

FIG. 1 is a design table of orthogonal experiments performed using Minitab software according to the present invention;

FIG. 2 shows the OD of the fermentation broth of the present invention₆₀₀Standard curve of value versus cell weight;

FIG. 3 is a growth curve of Bacillus CQN-2;

FIG. 4 is a graph showing the pH optimization results in the growth optimization of the present invention;

FIG. 5 is a schematic diagram showing the result of the optimization of the liquid loading amount according to the present invention;

FIG. 6 is a graph showing the optimization results of the present invention with respect to the amount of seeds;

FIG. 7 is a graph comparing the results of carbon source optimization according to the present invention;

FIG. 8 is a graph comparing the results of the present invention for optimizing a nitrogen source;

FIG. 9 is a graph showing the results of optimization of the carbon source concentration according to the present invention;

FIG. 10 is a graph showing the results of optimizing the nitrogen source concentration according to the present invention;

FIG. 11 is a graph showing the results of optimizing the concentration of molasses according to the present invention;

FIG. 12 is a graph showing the results of the optimization of metal ions according to the present invention;

FIG. 13 is a graph showing the results of different sets of orthogonal experiments according to the present invention;

FIG. 14 is a graph of the mean effect of the quadrature experiment of the present invention;

FIG. 15 is a schematic diagram comparing growth curves of original culture conditions and optimal culture conditions;

FIG. 16 is a schematic comparison of pH versus fermentation process in a fermentor;

FIG. 17 is a schematic flow chart of the fermentation culture method of the present invention

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. 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 examples given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a high-density fermentation culture method of bacillus CQN-2. The technical solution provided by the present invention will be explained in more detail with reference to fig. 1 to 16.

As shown in figures 1 to 16, in order to improve the weight gain rate and specific growth rate of bacillus CQN-2 and effectively improve the anti-infection capacity, the invention provides a high-density fermentation culture method of bacillus CQN-2, which comprises the following steps:

The specific implementation process and the technical method comprise the following contents:

1 test materials and reagents

1.1 test strains

Bacillus CQN-2.

1.2 culture Medium

PDA enriched medium: 200g of potato, 20g of glucose, 3g of peptone and KH₂PO₄1g， MgSO₄0.5g, vitamin B₁0.1g, pH7.0, water to 1000 mL.

LB solid medium: 10g of peptone, 5g of yeast powder, 10g of NaCl, 20g of agar, pH7.0, and every 1000mL of the agar.

Fermentation medium: 20g of molasses, 20g of yeast powder and KH₂PO₄0.5g，MgSO₄0.5g, NaCl 0.3g, pH7.0, per 1000 mL.

1.3 Experimental equipment and consumables

Equipment: beaker (1000mL), graduated cylinder (1000mL, 50mL), Erlenmeyer flask (250mL), pipette gun (1mL, 200. mu.L, 25. mu.L, 10. mu.L), gun head box, coating rod, plate, glass cuvette, centrifuge tube rack.

Note: the equipment and consumables for strain operation are autoclaved in autoclave under 121 deg.C for 20min before use.

2 method of experiment

2.1 expanded culture and preservation of the Strain

1) Coating a plate: pouring the melted and cooled solid culture medium into a flat plate in an ultraclean workbench, taking out the preserved bacillus from a refrigerator at the temperature of-20 ℃ after the culture medium is solidified, taking 100L of bacterial liquid on the flat plate, uniformly coating the bacterial liquid on a coating rod, sealing the flat plate, and inverting the flat plate in an incubator at the temperature of 37 ℃ for overnight culture.

2) And (3) amplification culture: in the clean bench, the single colony with the largest colony size was picked from the plate after the culture was completed and cultured overnight in PDA liquid medium in a shaker at 37 ℃ and 200 rpm.

3) And (3) strain preservation: in an ultraclean workbench, a proper amount of bacterial liquid and glycerol are mixed in a 1.5mL centrifuge tube, the final concentration of the glycerol is 30%, the mixture is sealed by a sealing film and fully mixed by a vortex oscillator, and the mixture is stored in a refrigerator at the temperature of minus 20 ℃.

2.3 determination of growth Curve

1) And (3) activation: the bacillus preserved in a refrigerator at the temperature of-20 ℃ is taken out and inoculated in a prepared PDA liquid culture medium in a super clean bench and activated in a shaker at the temperature of 37 ℃ and the speed of 200rpm overnight.

2) Transferring: the activated bacteria solution was transferred to a new PDA liquid medium in a clean bench at 5% transfer amount, and cultured in a shaker at 37 ℃ and 200rpm for 24 hours.

3) Fermentation: inoculating 5% of the cultured bacterial liquid into the prepared fermentation medium in an ultraclean workbench, and culturing for 36h in a shaker at 30 ℃ and 200 rpm.

4) Measurement of growth curves: during the fermentation, samples were taken every 6h, and the OD was determined in a spectrophotometer by appropriately diluting the samples₆₀₀And fermenting for 36 h.

2.4 optimization of growth conditions

1) Determining an optimization condition: the conditions to be optimized are the pH of the medium, the liquid loading and the inoculum size transferred during fermentation.

2) Preparation of a culture medium: preparing a fermentation culture medium with the pH value of 7.0 and the liquid loading amount of 30mL for optimizing the inoculation amount, wherein the pH values are 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5 respectively; the fermentation medium with a liquid loading volume of 30mL was used for pH optimization, and the fermentation medium with a pH of 7.0 and liquid loading volumes of 20, 25, 30, 40, 50, 60, 75, 100, 125mL were prepared for liquid loading volume optimization.

3) The same procedures of activation, transfer and fermentation are carried out for 2.3, and the inoculation amounts are respectively 1%, 3%, 5%, 7% and 9% when the inoculation amounts are optimized.

4) Data were recorded and analyzed: performing OD (optical density) on the fermentation liquor after fermentation for 36h₆₀₀And (4) measuring values, recording data, and analyzing and comparing to obtain the optimal growth condition.

2.5 optimization of carbon and Nitrogen sources

1) Determining the types of carbon sources and nitrogen sources: the carbon source is sucrose, xylose, glucose, soluble starch and dextrin, and the nitrogen source is bacteriological peptone, yeast extract, fish meal peptone,Beef extract, NH₄Cl、NaNO₂。

2) Preparation of a culture medium: preparing a fermentation culture medium without the carbon source and the nitrogen source as a control group, then preparing the fermentation culture medium, and respectively adding the carbon source or the nitrogen source to the fermentation culture medium to obtain a final concentration of 20g/L, wherein the rest components are consistent with the control group.

3) The activation, transfer and fermentation are the same as 2.3

4) Data were recorded and analyzed: performing OD (optical density) on the fermentation liquor after fermentation for 36h₆₀₀And (4) measuring values, recording data, and analyzing and comparing to obtain the optimal carbon source and nitrogen source.

2.6 selection of Single Metal ions

1) Determining the type of the metal ions: selection of metal ions to Fe²⁺、Fe³⁺、Zn²⁺、Ca²⁺、Mn²⁺、 Ba²⁺、Al³⁺。

2) Preparation of a culture medium: the conditions of the prepared medium were prepared according to the optimum conditions obtained in the previous experiment, and then the above-mentioned metal ions were added to the medium to a final concentration of 1mM, 5mM, wherein any of the above-mentioned metal ions was not added as a control group.

3) The same procedure for activation, transfer and fermentation was followed as 2.3, wherein the inoculation amount during fermentation was carried out according to the optimal conditions obtained from the previous experiment.

4) Data were recorded and analyzed: performing OD (optical density) on the fermentation liquor after fermentation for 36h₆₀₀The values are measured and the data are recorded, and the analysis and comparison are carried out to obtain 3 metal ions which have promotion effect on the growth conditions.

2.7 optimization of the concentrations of carbon, Nitrogen and molasses

1) Determining the concentration range: the carbon source concentration was set at 5, 10, 15, 20, 25, 30, 35g/L, and the nitrogen source and molasses concentration were set at 20, 30, 40, 50, 60 g/L.

2) Preparation of a culture medium: preparing a fermentation medium with inconsistent concentrations of only the carbon source or the nitrogen source or the molasses according to the concentration of the step 1).

4) Data were recorded and analyzed: performing OD (optical density) on the fermentation liquor after fermentation for 36h₆₀₀And (4) measuring values, recording data, and analyzing and comparing to obtain optimal carbon source, nitrogen source and molasses concentration.

2.8 orthogonal experiments

1) Orthogonal design of orthogonal experiment was performed using Minitab software, the design table is shown in fig. 1;

2) preparation of the culture Medium the preparation of the fermentation medium was carried out according to the orthogonal experimental design table obtained, and the remaining ingredients were prepared according to the optimum conditions of the previous experiment.

3) Recording data and analyzing OD of fermentation liquor after fermentation for 36h₆₀₀Values were determined and data were recorded and analyzed using software to obtain optimal culture conditions.

2.9 comparison of growth curves for Primary and optimal culture conditions

1) Preparation of culture Medium fermentation culture Medium was prepared for OD according to the original and optimal conditions₆₀₀And (4) measuring the value.

2) The activation, transfer and fermentation are the same as 2.3.

3) Measurement of growth curves: during the fermentation, samples were taken every 6h, and the OD was determined in a spectrophotometer by appropriately diluting the samples₆₀₀The value is 48h in the whole fermentation process, and the measured data are recorded and the average value is taken to obtain the growth curve.

2.10 Effect of pH control in fermentors on fermentation Processes

1) Preparation of a culture medium: two 3L portions of fermentation medium were prepared according to the optimal medium formulation, and 4M NaOH solution was prepared for pH control in two fermentors, and sterilized in an autoclave at 115 deg.C for 30 min.

2) Activating, transferring the mixture for 2.3,

3) fermentation: the fermentors were properly connected to the apparatus, one of which was pH controlled using NaOH and the other was not used, the temperature of the fermentation was set at 30 ℃ and the rotation speed was 600rpm, and the fermenter requiring pH adjustment was set to pH 7.0. And inoculating the activated bacillus into a fermentation tank for fermentation at the optimal inoculation amount.

4) Measurement of growth curves: during the fermentation, samples were taken every 8h, and the OD was determined in a spectrophotometer by appropriately diluting the samples₆₀₀The wavelength is 600nm, the whole fermentation process is 36h, and two groups of data are respectively drawn into growth curves to be compared to obtain a conclusion.

2.11 plotting fermentation broth OD₆₀₀Standard curve of value versus cell weight

1) Taking a proper amount of fermentation broth after completion of fermentation, adding the fermentation broth into 10mL centrifuge tubes with known weights, wherein the amount of each tube is 0.5 mL, 1.0 mL, 1.5mL, 2.0 mL, 2.5 mL, 3.0 mL, 3.5 mL, 4.0 mL, 4.5 mL and 5.0mL respectively.

2) And (3) centrifugally precipitating the thalli in a centrifugal tube filled with the fermentation liquor at 10000rpm and 15 ℃, then resuspending the thalli cleaning culture medium with sterile water, centrifugally precipitating the thalli under the same conditions, and cleaning for 2-3 times.

3) And after cleaning, discarding residual liquid in the centrifugal tube, putting the centrifugal tube into a drying oven for drying, weighing the centrifugal tube and the thalli after drying, subtracting the weight of the centrifugal tube to obtain the dry weight of the thalli, and drawing a standard curve by using the obtained data.

The standard curve is plotted as shown in fig. 2.

2.12 calculate the number of cells after fermentation

1) Gradient dilution: taking appropriate amount of OD₆₀₀Diluting 72.5 fermentation broth in ultra-clean bench with sterile water to 10%^-11。

2) Coating a flat plate: diluting to 10^-9、10^-10、10^-11The dilution of the three gradients was plated at 100L, and 2-3 plates were plated per gradient, and it should be noted that the solid medium used here was LB solid medium.

3) Counting: and (3) inversely culturing the coated flat plate in an incubator at 37 ℃ for 48h, counting after the culture is finished, and calculating the average value to obtain the total number of the thalli.

Diluting to 10^-10The average number of the posterior cells was 30.3, and the OD was calculated₆₀₀The total thallus amount of the fermentation liquor is 72.53.03×10¹⁵Per L, each OD₆₀₀The amount of the strain is 4.18 × 10¹³。

Third, experimental verification

3.1 growth curves of Bacillus

As shown in FIG. 3, it can be seen that the bacterial concentration of Bacillus rapidly increases with the increase of culture time, the growth rate gradually decreases after 30h of culture, the hypha concentration is basically stable after 36h, and the bacterial concentration reaches 18.8 OD₆₀₀。

3.2 optimization of growth conditions

3.2.1 optimization of pH

As shown in FIG. 4, it is evident that the Bacillus bacteria have a cell concentration gradually increased from the increase of pH of the fermentation solution, and the differences of the cell concentrations between pH4.5 and pH 8.5 are not obvious and are all at 19 OD₆₀₀Above, the fermentation broth has the highest cell concentration and OD at the initial pH of 6.5₆₀₀The value reached 19.78.

3.2.2 optimization of liquid Loading

As shown in FIG. 5, it is evident that the liquid loading amount has a significant influence on the cell concentration of Bacillus, and the overall tendency of the cell concentration gradually decreases with the increase in the liquid loading amount, indicating that the cell is an aerobic type bacterium, in which the cell concentration is the highest at 25mL of the liquid loading amount and reaches 20.46 OD₆₀₀。

3.2.3 optimization of the amount of seed

As shown in FIG. 6, it can be clearly seen that the difference of the inoculation amounts has a certain influence on the cell concentration of Bacillus subtilis, the cell concentrations of 1% and 3% of the inoculation amounts are not significant, and the cell concentrations of 5%, 7% and 9% of the inoculation amounts are not significant, wherein the cell concentrations of 1% and 3% of the inoculation amounts are higher than those of 5%, 7% and 9%, so that the proper reduction of the inoculation amount is helpful for the growth of Bacillus subtilis.

3.3 optimization of carbon and Nitrogen sources

As shown in FIG. 7, it can be seen that the difference of the carbon source has a significant effect on the bacterial amount of the bacillus, the bacterial amount is significantly higher than that of sucrose, xylose and glucose when the soluble starch and dextrin are used as the carbon source, wherein the soluble starch is used as the carbon source and has the highest concentration and OD₆₀₀Up to 20.2.

As shown in FIG. 8, it can be seen that the growth of Bacillus under organic nitrogen source is significantly better than that of inorganic nitrogen source, wherein sodium nitrite is used as nitrogen source to cause no growth of thallus, and the thallus concentration difference among bacteriological peptone, yeast extract and fish meal peptone is not significant in organic nitrogen source, wherein bacteriological peptone is optimal, and the thallus amount can reach 17.88 OD₆₀₀。

3.4 optimization of the concentrations of carbon, Nitrogen and molasses

3.4.1 optimization of carbon Source concentration

As shown in FIG. 9, it is evident that the concentration of Bacillus gradually increases with the increase of the concentration of soluble starch, the specific concentration is the highest when the concentration of carbon source is 30g/L, and the amount of bacteria reaches 19.58 OD₆₀₀。

3.4.2 optimization of Nitrogen Source concentration

As shown in FIG. 10, the difference in the nitrogen source concentration significantly affects the cell concentration of Bacillus, as shown in FIG. 10, in which the increase in the cell amount was insignificant at a nitrogen source concentration of 30g/L, and was stabilized at 18 OD or more, and the cell concentration reached a maximum of 21.2 at a nitrogen source concentration of 60 g/L.

3.4.3 optimization of molasses concentration

As shown in FIG. 11, it is apparent that molasses as an additive has a remarkable promoting effect on the growth of Bacillus, the cell concentration starts to increase rapidly with the increase of the amount of molasses, the increase of cell amount starts to decrease after 30g/L, and the cell concentration reaches the maximum value of 21.03 at 50 g/L.

3.5 optimization of Metal ions

As shown in FIG. 12, it can be seen that the addition of different metals has different effects on the growth of Bacillus, where Fe²⁺、Mn²⁺、Al³⁺The three metal ions are beneficial to the growth of thalli, and Fe³⁺、Ca²⁺、Ba²⁺Addition of (2) has no significant influence on the cells, and Zn is added²⁺Growth of the cells is inhibited. Thus selecting Fe²⁺、Mn²⁺、 Al³⁺Orthogonal experiments were performed for these three metal ions.

3.6 orthogonal experiments

As shown in figure 13, 7-factor 3 horizontal orthogonal experiment design 27 groups of experiments are carried out according to software Minitab, and the obvious difference of the thallus concentration among different groups can be seen from the fermentation experiment result figure 13, wherein the thallus concentration of the group 6 is optimal and can reach 29.45 ODs₆₀₀。

As shown in FIG. 14, the mean effect of the orthogonal experiment using the software Minitab FIG. 14 can be obtained, and it can be seen that the optimal fermentation medium formulation for this strain is 20g of soluble starch, 30g of bacteriological peptone, 50g of molasses, KH₂PO₄0.5g，MgSO₄0.5g，NaCl 0.3g，Mn²⁺5mM，Al³⁺1mM per 1000 mL. The culture conditions were 15mL in a 250mL conical flask, pH7.0, and the inoculum size was 3%.

3.7 comparison of growth curves for Primary and optimal culture conditions

As shown in FIG. 15, the culture medium after orthogonal optimization and the initial culture medium are used for fermentation, the change of the thallus concentration in the process of stationary culture is observed, as can be seen from FIG. 15, the growth speed of the bacillus in the optimal culture medium is obviously faster than that of the initial culture medium, after the bacillus is cultured for 24h, the bacillus in the optimal culture medium continues to grow rapidly after the initial culture medium enters a stable period until 42h, the bacillus grows into the stable period, and the thallus concentration reaches 39 OD₆₀₀23.37 ODs significantly higher than the stationary phase of the original medium₆₀₀。

3.8 influence of pH control in fermenter on fermentation Process

As shown in FIG. 16, based on the orthogonal experiment results, fermentation conditions of the fermentation tank are designed, pH control and uncontrolled fermentation experiments are continued, the growth condition of the thalli under the fermentation conditions of uncontrolled pH is obviously better than that of the thalli under the fermentation conditions of uncontrolled pH, the concentration of the thalli is higher than that of the thalli at the beginning of fermentation, the concentration of the thalli is rapidly increased, the increase of the concentration of the thalli begins to slow down after 30 hours, and the concentration of the thalli reaches up to 36 hours.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the technical scope of the present invention.

Claims

1. A high-density fermentation culture method of bacillus CQN-2 is characterized in that: the culture method comprises the following steps:

2. The method for high-density fermentation culture of bacillus CQN-2 according to claim 1, wherein: step S1 the PDA enriched medium includes: 200g of potato, 20g of glucose, 3g of peptone and KH₂PO₄1g，MgSO₄0.5g, vitamin B₁0.1g, pH7.0, adding water to 1000 mL; the LB solid medium comprises: LB solid medium: 10g of peptone, 5g of yeast powder, 10g of NaCl, 20g of agar, pH7.0, and each 1000mL of the agar; the fermentation medium comprises: 20g of molasses, 20g of yeast powder and KH₂PO₄0.5g，MgSO₄0.5g, NaCl 0.3g, pH7.0, per 1000 mL.

3. The method for high-density fermentation culture of bacillus CQN-2 according to claim 1, wherein: the experimental device comprises: beaker, graduated cylinder, erlenmeyer flask, liquid-transfering gun, rifle head box, coating rod, flat board, glass cell, centrifuge tube rack.

4. The method for high-density fermentation culture of bacillus CQN-2 according to claim 1, wherein: the optimization of the carbon source and the nitrogen source in the step S4 comprises the use of sucrose, xylose, glucose, soluble starch and dextrin, and the nitrogen source uses bacteriological peptone, yeast extract, fish meal peptone, beef extract and NH₄Cl、NaNO₂Preparing culture medium for each group of comparison tests, activating, transferring, fermenting, recording and analyzing data, and performing OD (optical density) on fermented liquid₆₀₀And (4) measuring values, recording data, and analyzing and comparing to obtain the optimal carbon source and nitrogen source.

5. The method for high-density fermentation culture of bacillus CQN-2 according to claim 1, wherein: the selection of the single metal ion includes selecting Fe²⁺、Fe³⁺、Zn²⁺、Ca²⁺、Mn²⁺、Ba²⁺、Al³⁺The activation, transfer and fermentation are carried out by the preparation of the culture medium, wherein the inoculation amount during the fermentation is inoculated according to the optimal conditions obtained by the previous experiment, and the metal ions which have the promotion effect on the growth conditions are obtained by recording data and analyzing.

6. The method for high-density fermentation culture of bacillus CQN-2 according to claim 1, wherein: the optimization of the molasses concentration comprises the steps of preparing fermentation culture media with different molasses concentrations for activation, transfer and fermentation, wherein the inoculation amount during fermentation is inoculated according to the optimal conditions obtained by the previous experiment, and then the optimal molasses concentration is obtained by recording data and analyzing.

7. The method for high-density fermentation culture of bacillus CQN-2 according to claim 1, wherein: the process of adjusting and controlling the pH in the step S5 includes preparing two fermentation culture bases according to the formula of the optimal culture medium in the fermentation tanks, performing activated fermentation, selecting one of the two fermentation culture bases to control the pH by using NaOH, setting the fermentation temperature to be 30 ℃ and the rotation speed to be 600rpm, and setting the pH to be 7.0 by the fermentation tank needing to adjust the pH, wherein the other fermentation base is not used. And inoculating the activated bacillus into a fermentation tank for fermentation at the optimal inoculation amount.

8. The method for high-density fermentation culture of bacillus CQN-2 according to claim 1, wherein: the fermentation broth OD is drawn₆₀₀The standard curve of value and thallus weight includes that a plurality of groups of fermentation liquor are taken and added into a centrifuge tube, the centrifuge tube is centrifuged and precipitated at 10000rpm and 15 ℃, then sterile water is used for resuspending thallus cleaning culture medium, the thallus is centrifuged and precipitated under the same conditions for 2-3 times, after the thallus is washed, residual liquid in the centrifuge tube is discarded, the centrifuge tube is placed into a drying box for drying, after the drying is finished, the weight of the centrifuge tube and the thallus is weighed, then the self weight of the centrifuge tube is subtracted, the dry weight of the thallus can be obtained, and then the obtained data is used for drawing the standard curve.

9. The method for high-density fermentation culture of bacillus CQN-2 according to claim 1, wherein: the step of calculating the number of the thalli after fermentation is finished comprises the step of taking a proper amount of OD₆₀₀Diluting 72.5 fermentation broth in ultra-clean bench with sterile water to 10%^-11Diluting to 10^-9、10^-10、10^-11And taking 100L of the three-gradient dilution liquid, coating a flat plate, coating 2-3 flat plates on each gradient, taking the solid culture medium used here as LB solid culture medium, carrying out inverted culture on the coated flat plate in an incubator at 37 ℃ for 48h, counting after the culture is finished, and calculating the average value to obtain the total number of the thalli.