CN114081098B - Method for preparing composite immunopotentiator from Subtilosin A and dandelion root polysaccharide and application thereof - Google Patents
Method for preparing composite immunopotentiator from Subtilosin A and dandelion root polysaccharide and application thereof Download PDFInfo
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- 229930014626 natural product Natural products 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Biotechnology (AREA)
- Animal Husbandry (AREA)
- Molecular Biology (AREA)
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- Gastroenterology & Hepatology (AREA)
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- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention discloses a method for preparing a composite immunopotentiator from Subtilosin A and dandelion root polysaccharide and application thereof, wherein the preparation comprises the following steps: fermenting and culturing bacillus subtilis, centrifuging fermentation liquor to obtain supernatant, and regulating pH value to generate precipitate; leaching the precipitate to obtain Subtilosin A crude extract, and separating and purifying Subtilosin A crude extract; weighing dandelion root dry powder, leaching, centrifuging and collecting filtrate to obtain dandelion root polysaccharide crude extract; carrying out ion exchange column chromatography on the dandelion root polysaccharide crude extract, collecting effluent, and purifying the effluent by using a gel column to obtain a purified dandelion root polysaccharide extract; mixing Subtilosin A extractive solutions with herba Taraxaci. The composite immunopotentiator prepared by the invention has good acid resistance, can not reduce curative effect after entering intestinal tracts, has low production cost and can be produced in a large scale.
Description
Technical Field
The invention relates to a preparation method of a composite immunopotentiator, in particular to a method for preparing the composite immunopotentiator by Subtilosin A and dandelion root polysaccharide and application thereof.
Background
Subtilosin A is an antibacterial substance produced by bacillus subtilis, which can well inhibit the growth of gram-positive bacteria and partial gram-negative bacteria, wherein the antibacterial substance comprises a plurality of Nisin insensitive strains, can inhibit vagina-bound pathogenic bacteria without any toxic or side effect on normal lactic acid bacteria of a human body, has synergistic effect with glycerol monolaurate, lauric Arginate and the like, and can better inhibit the growth of pathogenic bacteria by combined use.
The bacillus subtilis and the metabolites thereof have remarkable antibacterial capability, and can achieve the effect of enhancing immunity by being combined with natural products. The dandelion polysaccharide is used as an important component in dandelion, can be used as an important nutritional component in organisms, and also participates in various biological activities, and has various physiological functions of resisting viruses, inhibiting bacteria, enhancing immunity, resisting radiation and the like. The polysaccharide can be used as a natural extract with almost no side effect, and has unique curative effects on prevention and treatment of various diseases.
At present, the existing composite immunopotentiator is prepared by using a live bacteria preparation, but the problems of poor synergism, poor compatibility and high cost of people in the intestinal environment exist, and the existing composite immunopotentiator is easy to be affected by gastric acid and bile when entering the intestinal tract, and the number of live bacteria can be greatly reduced before reaching the effective part of the intestinal tract, so that the curative effect of a probiotic product is affected.
Disclosure of Invention
The invention aims to: aiming at the problems existing in the prior art, the invention provides a method for preparing a composite immunopotentiator by extracting Subtilosin A from bacillus subtilis JCL16 and dandelion root polysaccharide, the composite immunopotentiator prepared by the invention adopts an antibacterial substance Subtilosin A generated by JCL16, has the advantages of good synergistic effect, good compatibility and low cost, and the two substances adopted by the invention have the characteristics of acid resistance, alkali resistance and the like, so that the problem that the traditional microbial inoculum cannot exert the due antibacterial effect under the action of gastric acid is solved.
The invention provides a prepared composite immunopotentiator and application thereof.
The technical scheme is as follows: in order to achieve the above object, the method for preparing the composite immunopotentiator from Subtilosin A and dandelion root polysaccharide is characterized by comprising the following steps:
(1) Fermenting and culturing bacillus subtilis, centrifuging fermentation liquor to remove thalli, taking supernatant, and regulating pH value to be strong acid to enable the supernatant to generate sediment;
(2) Leaching the precipitate, collecting leaching solution, adjusting pH to neutrality, and stirring;
(3) Centrifuging the leaching solution, removing precipitate, and collecting supernatant to obtain Subtilosin A crude extract;
(4) Separating and purifying the Subtilosin A crude extract in the step (3) through a solid phase extraction column to obtain Subtilosin A extract after separation and purification;
(5) Weighing dandelion root dry powder, adding water, stirring and mixing uniformly, then leaching by using ultrasonic waves, and cooling to room temperature after the leaching is finished; centrifuging, vacuum filtering, and collecting filtrate to obtain crude extract of dandelion root polysaccharide;
(6) Carrying out ion exchange column chromatography on the dandelion root polysaccharide crude extract, collecting effluent, and purifying the effluent by using a gel column to obtain a purified dandelion root polysaccharide extract;
(7) Evaporating the Subtilosin A extracting solution collected in the step (4) and the dandelion polysaccharide solution in the step (6) to dryness, and then mixing the two solutions according to a volume ratio of 1:1-10, and preparing the mixture into composite particles to obtain the final composite immune granules.
Wherein, the bacillus subtilis in the step (1) is bacillus subtilis JCL16.
The bacillus subtilis in the step (1) is subjected to fermentation culture, namely a single bacillus subtilis JCL16 colony is selected from a solid culture medium, inoculated into 30ml of seed culture medium and cultured for 16-18 hours at the rotating speed of 180r/min and the temperature of 37 ℃, and then seed liquid is inoculated into the fermentation culture medium according to the volume ratio of 5-10% and cultured for 24-48 hours at the temperature of 35-43 ℃ and the rotating speed of 160r/min-240r/min to obtain the fermentation culture medium.
Wherein the seed liquid culture time in the step (1) is 16-18h, the seed culture medium is beef extract 5g/L, peptone 10g/L, naCl 5g/L, and the pH value is 7.2. The fermentation medium is LB medium: 10g/L of tryptone, 10g/L of NaCl and 5g/L of yeast powder.
Wherein, in the step (1), HCl is used for adjusting the pH value to 2-3, and the mixture is left to stand overnight at the temperature of 4 ℃ to generate precipitate.
Preferably, the centrifugation conditions in the step (1) are 8000r/min,3min and HCl concentration of 5-7mol/L.
Wherein, in the step (2), the sediment is leached by methanol, the methanol leaching solution is collected, and the PH value of the methanol leaching solution is regulated to 7.0 by NaOH solution and stirred.
Preferably, in the step (2), the fermentation supernatant after standing overnight is centrifuged, the supernatant is removed by centrifugation, the sediment is reserved, the centrifugation condition is 10000r/min and 5min, the concentration of NaOH solution is 1-3mol/L, and the stirring is performed by a magnetic stirrer for 7-9h.
Preferably, the centrifugation in step (3) is performed under centrifugation conditions of 10000g and 20min.
In the step (4), the crude extract of Subtilosin A in the step (3) is separated and purified by a C18 solid phase extraction column, and then 3 times of column volume of 30%,50%,70% and 100% of methanol aqueous solution are used for gradient elution, and effluent liquid eluted by 100% of methanol aqueous solution is collected, so that Subtilosin A extract after separation and purification can be obtained.
Wherein, in the step (5), the dry powder of dandelion root and the water ratio are 1:10-50g/mL, wherein the ultrasonic time in the ultrasonic leaching is 0.5-2.5h, the temperature is 50-90 ℃, and the ultrasonic power is 60-140W.
Preferably, the centrifugation conditions in the step (7) are 8000r/min and 20min.
And (3) carrying out chromatography on the dandelion root polysaccharide crude extract in the step (6) by using a DEAE-52 ion exchange column, opening a constant flow pump to carry out sample loading at a flow rate of 0.6-1ml/min, eluting by using 0.1-0.5mol/LNaCl solution at an elution rate of 0.6-1ml/min, collecting effluent, purifying the effluent by using a glucose gel column SephadexG-100, wherein the loading amount of the effluent is 1.5ml each time, eluting by using 0.1-0.5mol/LNaCl solution at a flow rate of 0.6-1ml/min, and finally obtaining the purified dandelion root polysaccharide extract.
Further, the seed culture time in the step (1) is 17 hours, and the HCL concentration is 6mol/L. The concentration of the NaOH solution in the step (2) is 2mol/L, and the stirring time of a magnetic stirrer is 8 hours. The time in the ultrasonic leaching method in the step (5) is 1.5 hours, the temperature is 60 ℃, the ultrasonic power is 100W, and the feed-liquid ratio is 1:30g/ml. The concentration of the NaCl solution in the step (6) is 0.1mol/L. The volume ratio of the solution in the step (7) is 1:5.
The invention relates to an immunopotentiator prepared by a method for preparing a composite immunopotentiator by Subtilosin A and dandelion root polysaccharide.
The invention relates to an application of an immunopotentiator in preparing animal feed.
The method optimizes the culture conditions of the bacillus subtilis JCL 16; subtilosin A generated by extracting JCL 16; extracting polysaccharide in dandelion; the two materials were compounded by spray drying. Compared with the existing composite immunizing agent, the composite immunizing agent disclosed by the invention uses an antibacterial product, has the advantages of good synergistic effect, good compatibility and low cost, and has the advantages of acid resistance, alkali resistance and the like.
The invention utilizes antibacterial substances Subtilosin A generated by bacillus subtilis JCL16 and dandelion polysaccharide to compound to form the immunopotentiator, has good synergistic effect and immunopotentiation capability, and can tolerate acidity of gastric acid.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
The composite immunopotentiator prepared by the invention has good acid resistance, wherein the used dandelion has wide sources and low cost, subtilosin A and dandelion polysaccharide have good synergistic effect when being compounded under the acidic condition, and the composite immunopotentiator has good compatibility, has the effect of enhancing immunity and can not reduce the curative effect after entering intestinal tracts. And the production cost is low, and the large-scale production can be realized.
Drawings
FIG. 1 is a schematic diagram showing the change of the bacteriostasis condition of the composite immune granule along with the pH value.
Fig. 2 is a diagram showing the bacteriostasis of the composite immune granule in simulated intestinal fluid.
Detailed description of the preferred embodiments
The invention is further described below with reference to the drawings and examples.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The experimental methods for which specific conditions are not specified in the examples are generally conducted under conventional conditions or under conditions recommended by the manufacturer.
The strain JCL16 is identified as bacillus subtilis (Bacillus subtilis) and is preserved in China center for type culture Collection of microorganisms, wherein the preservation time is 2018, 06, 01 and the preservation number is CCTCC NO: M2018336, and the strain is preserved in the prior patent CN 111411053A of the applicant and belongs to the disclosed strain.
Example 1
(1) Activating the preserved strain JCL16, coating the strain JCL16 in a solid LB culture medium, picking a single colony from the solid culture medium, placing the single colony into 30mL of prepared seed culture solution for culture for 17 hours at 37 ℃ at the rotating speed of 180r/min, and inoculating the seed solution into a fermentation culture solution for culture for 24 hours according to the volume ratio of 5%, wherein the temperature is 37 ℃ and the rotating speed is 200r/min.
(2) And (3) collecting the fermented and cultured bacterial liquid, centrifuging at 8000r/min for 3min, removing bacterial cells, collecting supernatant, adjusting the pH value of the collected supernatant to 2.0 by using HCL solution with the concentration of 6mol, and standing overnight at 4 ℃ to generate precipitate.
(3) Centrifuging the supernatant after overnight at 10000r/min for 5min, centrifuging to remove supernatant, collecting precipitate, and adding methanol: supernatant = 1: the precipitate was leached at a volume ratio of 10, after which the pH of the methanol extract was adjusted to 7.0 with 2mol/L NaOH solution and stirred with a magnetic stirrer for 8 hours.
(4) Centrifuging the methanol leaching solution treated in the step (3) by using a centrifugal machine under the centrifugation condition of 10000g for 20min, removing sediment, and collecting a centrifugal supernatant to obtain Subtilosin A crude extract.
(5) Separating and purifying the collected Subtilosin A crude extract by using a C18 solid phase extraction column, respectively eluting with 30%,50%,70% and 100% methanol water solution of 3 times of column volume in a gradient way, and collecting effluent liquid eluted by 100% methanol solution to obtain Subtilosin A extract after separation and purification.
(6) Weighing 100g of dandelion root dry powder, and mixing the materials according to a feed liquid ratio of 1: adding 30g/mL distilled water, stirring, mixing, adjusting the ultrasonic power to 100W, the ultrasonic temperature to 60 ℃, performing ultrasonic treatment for 1.5h, taking out the solution, and cooling to room temperature.
(7) Centrifuging the solution in the step (6) by using a centrifugal machine under the conditions of 8000r/min and 20min, collecting the centrifuged solution, performing suction filtration by using a vacuum suction filter, and collecting filtrate to obtain dandelion root polysaccharide crude extract.
(8) And (3) carrying out DEAE-52 ion exchange column chromatography on the dandelion root polysaccharide crude extract, accurately measuring 5mL of sample, starting a constant flow pump to carry out sample loading at a flow rate of 1mL/min, eluting the polysaccharide solution with 0.1mol/L NaCl solution after the sample loading is finished, and collecting effluent liquid, wherein the elution speed is 1 mL/min. Further purifying by Sephadex G-100 glucose gel column under the following conditions: loading the collected effluent liquid by using an elution column with the volume of 2.6 x 60cm, wherein the loading amount is 1.5mL each time, the eluent is 0.1mol/L NaCl solution, eluting at the flow rate of 1mL/min, and collecting the effluent liquid, namely the dandelion root polysaccharide after purification.
(9) Because substances in the two solutions cannot be evaporated, the collected Subtilosin A extracting solution and dandelion polysaccharide solution are evaporated to dryness by using a rotary evaporator, wherein the temperature is adjusted to be 50 ℃, the rotating speed is properly adjusted, and then the two solutions are mixed according to the volume ratio of 1: and 5, preparing a mixed solution, and preparing the mixed solution into composite particles by a spray drying method to obtain the final composite immune particles.
Sterile water is used for regulating the pH value to 2.0,2.5,3.0 by using 6mol/L hydrochloric acid, then the granules are dissolved in the sterile water according to the mass ratio of 1:5, and the mixture is placed at 37 ℃ for treatment for 12 hours. After that, the pH of the solution was adjusted back to 7.0 with a 2mol/LNaOH solution, and an antibacterial (Staphylococcus aureus) experiment was performed, and the antibacterial diameter was measured as shown in FIG. 1.
The antibacterial activity is measured by adopting an oxford cup method, and the specific steps comprise: staphylococcus aureus cultured to logarithmic phase is used as indicator bacteria, a flat oxford cup is vertically placed on the flat plate, 0.2mL of composite immune granule solution is respectively taken and is injected into the oxford cup, experimental comparison is carried out, the mixture is cultured for 24 hours at 37 ℃, a bacteriostasis ring around the oxford cup is observed, the diameter/mm=total diameter/mm of the bacteriostasis ring is measured, and the bacteriostasis diameter is shown as figure 1.
Simulation of intestinal juice: weighing 6.8g of potassium dihydrogen phosphate, dissolving in sterile water, regulating the pH value to 6.8, weighing 10g of trypsin, dissolving in a proper amount of sterile water, mixing and diluting the two solutions to 1000mL, dissolving the granules in the sterile water according to the mass ratio of 1:5, and standing at 37 ℃ for 12h. After that, the solution was brought back to pH 7.0 with 2mol/LNaOH solution and an antibacterial (Staphylococcus aureus) experiment (the same method as above) was performed. The blank control group is free of granules, and shows no bacteriostasis.
Example 2
(1) Activating the preserved strain JCL16, coating the strain JCL16 in a solid LB culture medium, picking single colony from the solid culture medium, placing the single colony into 30ml of prepared seed culture solution for culture for 17 hours at 37 ℃ and the rotating speed of 180r/min, and inoculating the seed solution into a fermentation culture solution for culture for 36 hours according to the volume ratio of 6%, wherein the temperature is 35 ℃ and the rotating speed of 240r/min.
(2) And (3) collecting the fermented and cultured bacterial liquid, centrifuging at 8000r/min for 3min, removing bacterial cells, collecting supernatant, adjusting the pH value of the collected supernatant to 2.0 by using HCL solution with the concentration of 6mol, and standing overnight at 4 ℃ to generate precipitate.
(3) Centrifuging the supernatant after overnight at 10000r/min for 5min, centrifuging to remove supernatant, collecting precipitate, and adding methanol: supernatant = 1: the precipitate was leached at a volume ratio of 10, after which the pH of the methanol extract was adjusted to 7.0 with 2mol/L NaOH solution and stirred with a magnetic stirrer for 7 hours.
(4) Centrifuging the methanol leaching solution treated in the step (3) by using a centrifugal machine under the centrifugation condition of 10000g for 20min, removing sediment, and collecting a centrifugal supernatant to obtain Subtilosin A crude extract.
(5) Separating and purifying the collected Subtilosin A crude extract by using a C18 solid phase extraction column, respectively eluting with 30%,50%,70% and 100% methanol water solution of 3 times of column volume in a gradient way, and collecting effluent liquid eluted by 100% methanol solution to obtain Subtilosin A extract after separation and purification.
(6) Weighing 100g of dandelion root dry powder, and mixing the materials according to a feed liquid ratio of 1: adding 10g/ml distilled water, stirring, mixing, adjusting ultrasonic power to 60W, ultrasonic temperature to 50deg.C, ultrasonic for 0.5h, taking out the solution, and cooling to room temperature.
(7) Centrifuging the solution in the step (6) by using a centrifugal machine under the conditions of 8000r/min and 20min, collecting the centrifuged solution, performing suction filtration by using a vacuum suction filter, and collecting filtrate to obtain dandelion root polysaccharide crude extract.
(8) And (3) carrying out DEAE-52 ion exchange column chromatography on the dandelion root polysaccharide crude extract, accurately measuring 5ml of sample, starting a constant flow pump to carry out sample loading at a flow rate of 1ml/min, eluting the polysaccharide solution with 0.1mol/L NaCl solution after the sample loading is finished, and collecting effluent liquid, wherein the elution speed is 1 ml/min. Further purifying by Sephadex G-100 glucose gel column under the following conditions: loading the collected effluent liquid by using an elution column with the volume of 2.6 x 60cm, wherein the loading amount is 1.5ml each time, the eluent is 0.1mol/L NaCl solution, eluting at the flow rate of 1ml/min, and collecting the effluent liquid, namely the dandelion root polysaccharide after purification.
(9) Evaporating volatile solvents in the collected Subtilosin A extracting solution and dandelion polysaccharide solution by using a rotary evaporator, wherein the temperature is adjusted to 50 ℃, the rotating speed is properly adjusted, and then the two solutions are mixed according to the volume ratio of 1: and 5, preparing a mixed solution, and preparing the mixed solution into composite particles by a spray drying method to obtain the final composite immune particles. The bacteriostasis test was the same as in example 1.
Example 3
(1) Activating the preserved strain JCL16, coating the strain JCL16 in a solid LB culture medium, picking single colony from the solid culture medium, placing the single colony into 30ml of prepared seed culture solution for culture for 17 hours at 37 ℃ at the rotating speed of 180r/min, and inoculating the seed solution into a fermentation culture solution for culture for 48 hours according to the volume ratio of 7%, wherein the temperature is 39 ℃ and the rotating speed is 220r/min.
(2) And (3) collecting the fermented and cultured bacterial liquid, centrifuging at 8000r/min for 3min, removing bacterial cells, collecting supernatant, adjusting the pH value of the collected supernatant to 2.0 by using HCL solution with the concentration of 6mol, and standing overnight at 4 ℃ to generate precipitate.
(3) Centrifuging the supernatant after overnight at 10000r/min for 5min, centrifuging to remove supernatant, collecting precipitate, and adding methanol: supernatant = 1: the precipitate was leached at a volume ratio of 10, after which the pH of the methanol extract was adjusted to 7.0 with 2mol/L NaOH solution and stirred with a magnetic stirrer for 9 hours.
(4) Centrifuging the methanol leaching solution treated in the step (3) by using a centrifugal machine under the centrifugation condition of 10000g for 20min, removing sediment, and collecting a centrifugal supernatant to obtain Subtilosin A crude extract.
(5) Separating and purifying the collected Subtilosin A crude extract by using a C18 solid phase extraction column, respectively eluting with 30%,50%,70% and 100% methanol water solution of 3 times of column volume in a gradient way, and collecting effluent liquid eluted by 100% methanol solution to obtain Subtilosin A extract after separation and purification.
(6) Weighing 100g of dandelion root dry powder, and mixing the materials according to a feed liquid ratio of 1: adding distilled water into 20g/ml, stirring, mixing, adjusting ultrasonic power to 80W, ultrasonic temperature to 70deg.C, ultrasonic for 1 hr, taking out the solution, and cooling to room temperature.
(7) Centrifuging the solution in the step (6) by using a centrifugal machine under the conditions of 8000r/min and 20min, collecting the centrifuged solution, performing suction filtration by using a vacuum suction filter, and collecting filtrate to obtain dandelion root polysaccharide crude extract.
(8) And (3) carrying out DEAE-52 ion exchange column chromatography on the dandelion root polysaccharide crude extract, accurately measuring 5ml of sample, starting a constant flow pump to carry out sample loading at a flow rate of 1ml/min, eluting the polysaccharide solution with 0.1mol/L NaCl solution after the sample loading is finished, and collecting effluent liquid, wherein the elution speed is 1 ml/min. Further purifying by Sephadex G-100 glucose gel column under the following conditions: loading the collected effluent liquid by using an elution column with the volume of 2.6 x 60cm, wherein the loading amount is 1.5ml each time, the eluent is 0.1mol/L NaCl solution, eluting at the flow rate of 1ml/min, and collecting the effluent liquid, namely the dandelion root polysaccharide after purification.
(9) Evaporating volatile solvents in the collected Subtilosin A extracting solution and dandelion polysaccharide solution by using a rotary evaporator, wherein the temperature is adjusted to 50 ℃, the rotating speed is properly adjusted, and then the two solutions are mixed according to the volume ratio of 1: and 5, preparing a mixed solution, and preparing the mixed solution into composite particles by a spray drying method to obtain the final composite immune particles. The bacteriostasis test was the same as in example 1.
Example 4
(1) Activating the preserved strain JCL16, coating the strain JCL16 in a solid LB culture medium, picking single colony from the solid culture medium, placing the single colony into 30ml of prepared seed culture solution for culture for 17 hours at 37 ℃ at the rotating speed of 180r/min, and inoculating the seed solution into a fermentation culture solution for culture for 48 hours at the rotating speed of 180r/min according to the volume ratio of 8 percent, wherein the temperature is 41 ℃.
(2) And (3) collecting the fermented and cultured bacterial liquid, centrifuging at 8000r/min for 3min, removing bacterial cells, collecting supernatant, adjusting the pH value of the collected supernatant to 3.0 by using HCL solution with the concentration of 6mol, and standing overnight at 4 ℃ to generate precipitate.
(3) Centrifuging the supernatant after overnight at 10000r/min for 5min, centrifuging to remove supernatant, collecting precipitate, and adding methanol: supernatant = 1: the precipitate was leached at a volume ratio of 10, after which the pH of the methanol extract was adjusted to 7.0 with 2mol/L NaOH solution and stirred with a magnetic stirrer for 8 hours.
(4) Centrifuging the methanol leaching solution treated in the step (3) by using a centrifugal machine under the centrifugation condition of 10000g for 20min, removing sediment, and collecting a centrifugal supernatant to obtain Subtilosin A crude extract.
(5) Separating and purifying the collected Subtilosin A crude extract by using a C18 solid phase extraction column, respectively eluting with 30%,50%,70% and 100% methanol water solution of 3 times of column volume in a gradient way, and collecting effluent liquid eluted by 100% methanol solution to obtain Subtilosin A extract after separation and purification.
(6) Weighing 100g of dandelion root dry powder, and mixing the materials according to a feed liquid ratio of 1: adding 40g/ml distilled water, stirring, mixing, adjusting ultrasonic power to 120W, ultrasonic temperature to 80deg.C, ultrasonic for 2 hr, taking out the solution, and cooling to room temperature.
(7) Centrifuging the solution in the step (6) by using a centrifugal machine under the conditions of 8000r/min and 20min, collecting the centrifuged solution, performing suction filtration by using a vacuum suction filter, and collecting filtrate to obtain dandelion root polysaccharide crude extract.
(8) And (3) carrying out DEAE-52 ion exchange column chromatography on the dandelion root polysaccharide crude extract, accurately measuring 5ml of sample, starting a constant flow pump to carry out sample loading at a flow rate of 1ml/min, eluting the polysaccharide solution with 0.1mol/L NaCl solution after the sample loading is finished, and collecting effluent liquid, wherein the elution speed is 1 ml/min. Further purifying by Sephadex G-100 glucose gel column under the following conditions: loading the collected effluent liquid by using an elution column with the volume of 2.6 x 60cm, wherein the loading amount is 1.5ml each time, the eluent is 0.1mol/L NaCl solution, eluting at the flow rate of 1ml/min, and collecting the effluent liquid, namely the dandelion root polysaccharide after purification.
(9) Evaporating volatile solvents in the collected Subtilosin A extracting solution and dandelion polysaccharide solution by using a rotary evaporator, wherein the temperature is adjusted to 50 ℃, the rotating speed is properly adjusted, and then the two solutions are mixed according to the volume ratio of 1: and 5, preparing a mixed solution, and preparing the mixed solution into composite particles by a spray drying method to obtain the final composite immune particles. The bacteriostasis test was the same as in example 1.
Example 5
(1) Activating the preserved strain JCL16, coating the strain JCL16 in a solid LB culture medium, picking single colony from the solid culture medium, placing the single colony into 30ml of prepared seed culture solution for culture for 17 hours at the temperature of 37 ℃ and the rotating speed of 180r/min, and then inoculating the seed solution into a fermentation culture solution for culture for 48 hours according to the volume ratio of 9%, wherein the temperature is 43 ℃ and the rotating speed is 160r/min.
(2) And (3) collecting the fermented and cultured bacterial liquid, centrifuging at 8000r/min for 3min, removing bacterial cells, collecting supernatant, adjusting the pH value of the collected supernatant to 3.0 by using HCL solution with the concentration of 6mol, and standing overnight at 4 ℃ to generate precipitate.
(3) Centrifuging the supernatant after overnight at 10000r/min for 5min, centrifuging to remove supernatant, collecting precipitate, and adding methanol: supernatant = 1: the precipitate was leached at a volume ratio of 10, after which the pH of the methanol extract was adjusted to 7.0 with 2mol/L NaOH solution and stirred with a magnetic stirrer for 7 hours.
(4) Centrifuging the methanol leaching solution treated in the step (3) by using a centrifugal machine under the centrifugation condition of 10000g for 20min, removing sediment, and collecting a centrifugal supernatant to obtain Subtilosin A crude extract.
(5) Separating and purifying the collected Subtilosin A crude extract by using a C18 solid phase extraction column, respectively eluting with 30%,50%,70% and 100% methanol water solution of 3 times of column volume in a gradient way, and collecting effluent liquid eluted by 100% methanol solution to obtain Subtilosin A extract after separation and purification.
(6) Weighing 100g of dandelion root dry powder, and mixing the materials according to a feed liquid ratio of 1:50g/ml of distilled water is added, stirred and mixed uniformly, the ultrasonic power is adjusted to 140W, the ultrasonic temperature is 90 ℃, the ultrasonic is carried out for 2.5 hours, and the solution is taken out and cooled to the room temperature.
(7) Centrifuging the solution in the step (6) by using a centrifugal machine under the conditions of 8000r/min and 20min, collecting the centrifuged solution, performing suction filtration by using a vacuum suction filter, and collecting filtrate to obtain dandelion root polysaccharide crude extract.
(8) And (3) carrying out DEAE-52 ion exchange column chromatography on the dandelion root polysaccharide crude extract, accurately measuring 5ml of sample, starting a constant flow pump to carry out sample loading at a flow rate of 1ml/min, eluting the polysaccharide solution with 0.1mol/L NaCl solution after the sample loading is finished, and collecting effluent liquid, wherein the elution speed is 1 ml/min. Further purifying by Sephadex G-100 glucose gel column under the following conditions: loading the collected effluent liquid by using an elution column with the volume of 2.6 x 60cm, wherein the loading amount is 1.5ml each time, the eluent is 0.1mol/L NaCl solution, eluting at the flow rate of 1ml/min, and collecting the effluent liquid, namely the dandelion root polysaccharide after purification.
(9) Evaporating volatile solvents in the collected Subtilosin A extracting solution and dandelion polysaccharide solution by using a rotary evaporator, wherein the temperature is adjusted to 50 ℃, the rotating speed is properly adjusted, and then the two solutions are mixed according to the volume ratio of 1: and 5, preparing a mixed solution, and preparing the mixed solution into composite particles by a spray drying method to obtain the final composite immune particles. The bacteriostasis test was the same as in example 1.
Comparative example 1
(1) Activating the preserved strain JCL16, coating the strain JCL16 in a solid LB culture medium, picking single colony from the solid culture medium, placing the single colony into 30ml of prepared seed culture solution for culture for 17 hours at 37 ℃ at the rotating speed of 180r/min, and inoculating the seed solution into a fermentation culture solution for culture for 24 hours according to the volume ratio of 5%, wherein the temperature is 37 ℃ and the rotating speed is 200r/min.
(2) And (3) collecting the fermented and cultured bacterial liquid, centrifuging at 8000r/min for 3min, removing the supernatant, collecting bacterial cells, cleaning the bacterial cells with 0.9% physiological saline, centrifuging again at 6000r/min for 3min, repeating for three times, and collecting bacterial sludge. The final volume is 1/10 of the volume of the original fermentation liquor, and the bacterial suspension is prepared.
(3) The bacterial suspension and skimmed milk are mixed according to the ratio of 1:2, respectively packaging into freeze-drying bottles after uniformly mixing, wherein the sample loading height is 0.5-0.6cm, and pre-freezing in a refrigerator at-80 ℃ for 24 hours. And (3) after the quick freeze dryer is started and precooled for 1 hour, rapidly placing the pre-frozen sample into a material tray, after the freeze dryer is covered, starting vacuumizing, and performing vacuum freeze drying for 24 hours. And (3) obtaining freeze-dried bacterial powder, and then placing the freeze-dried sample in a drying place for storage.
(4) Subtilosin A in example 1 was replaced with lyophilized powder, and the rest was unchanged.
Comparative example 2 is the same as inventive example 1 except that the antibacterial substance Subtilosin A was removed and replaced with an equivalent amount of dandelion polysaccharide.
Comparative example 3 is the same as inventive example 1 except that dandelion polysaccharide was removed and replaced with an equivalent amount of antibacterial substance Subtilosin A.
The bacteriostasis experiments and simulated intestinal fluid of comparative examples 1-3 were identical to example 1.
As can be seen from FIG. 1, the effect of inhibition is slightly higher in example 1 than in examples 2-5, and much higher in acid conditions than in comparative examples 1,2 and 3. The results show that Subtilosin A or dandelion polysaccharide has good effect after being singly used or the dandelion polysaccharide has no combination of the Subtilosin A and dandelion polysaccharide, and the effect of using the fungus powder is not good under the acidic condition. The Subtilosin A and dandelion polysaccharide have good synergistic effect, can play a role in enhancing immunity, can resist gastric acid, and ensure curative effect because effective components are not easy to lose before reaching the intestinal tract of an animal effective part. As can be seen from fig. 2, in the intestinal juice with the simulated pH value of 6.8, the antibacterial condition of the granule is not changed, and the example is obviously higher than the comparative example, further illustrating that the composite immunopotentiator prepared by the invention does not reduce the curative effect after entering the intestinal tract.
Claims (3)
1. A method for preparing a composite immunopotentiator for inhibiting staphylococcus aureus from Subtilosin A and dandelion root polysaccharide, which is characterized by comprising the following steps:
(1) Fermenting and culturing bacillus subtilis, centrifuging fermentation liquor to remove thalli, taking supernatant, and regulating pH value to be strong acid to enable the supernatant to generate sediment;
(2) Leaching the precipitate, collecting leaching solution, adjusting pH to neutrality, and stirring;
(3) Centrifuging the leaching solution, removing precipitate, and collecting supernatant to obtain Subtilosin A crude extract;
(4) Separating and purifying the Subtilosin A crude extract in the step (3) through a solid phase extraction column to obtain Subtilosin A extract after separation and purification;
(5) Weighing dandelion root dry powder, adding water, stirring and mixing uniformly, then leaching by using ultrasonic waves, and cooling to room temperature after the leaching is finished; centrifuging, vacuum filtering, and collecting filtrate to obtain crude extract of dandelion root polysaccharide;
(6) Carrying out ion exchange column chromatography on the dandelion root polysaccharide crude extract, collecting effluent, and purifying the effluent by using a gel column to obtain a purified dandelion root polysaccharide extract;
(7) Evaporating the Subtilosin A extracting solution collected in the step (4) and the dandelion polysaccharide solution in the step (6) to dryness, and then mixing the two solutions according to a volume ratio of 1:1-10, and preparing the mixture into composite particles to obtain the final composite immune granules;
In the step (1), the bacillus subtilis is bacillus subtilis JCL16, and the preservation number is CCTCC NO: M2018336;
The bacillus subtilis in the step (1) is fermented and cultured to pick single bacillus subtilis JCL16 colony from a solid culture medium, inoculated into a seed culture medium and cultured for 16-18h at the rotating speed of 180-200r/min and the temperature of 35-37 ℃, and then inoculated into a fermentation culture medium according to the volume ratio of 5-10% and cultured for 24-48h at the temperature of 35-43 ℃ and the rotating speed of 160-240 r/min to obtain the fermentation culture medium;
In the step (1), HCl is used for regulating the pH value to 2-3, and the mixture is left to stand overnight to generate sediment;
Leaching the precipitate by using methanol in the step (2), collecting methanol leaching liquor, and regulating the pH value of the methanol leaching liquor to 7.0 by using NaOH solution to stir;
Separating and purifying the Subtilosin A crude extract in the step (3) through a C18 solid phase extraction column in the step (4), respectively carrying out gradient elution by using 30%,50%,70% and 100% methanol water solution by 3 times of column volume, and collecting 100% methanol water eluted effluent to obtain Subtilosin A extract after separation and purification;
In the step (5), the ratio of dandelion root dry powder to water is 1:10-50g/mL, wherein the ultrasonic time in the ultrasonic leaching is 0.5-2.5h, the temperature is 50-90 ℃, and the ultrasonic power is 60-140W;
And (6) carrying out chromatography on the dandelion root polysaccharide crude extract by using a DEAE-52 ion exchange column, opening a constant flow pump to carry out sample loading at a flow rate of 0.6-1ml/min, eluting by using 0.1-0.5mol/LNaCl solution at an elution rate of 0.6-1ml/min, collecting effluent, purifying the effluent by using a glucose gel column Sephadex G-100, wherein the sample loading amount of the effluent is 1.5ml each time, and eluting by using 0.1-0.5mol/LNaCl solution at a flow rate of 0.6-1ml/min to finally obtain the purified dandelion root polysaccharide extract.
2. An immunopotentiator prepared by the method for preparing a composite immunopotentiator for inhibiting staphylococcus aureus from Subtilosin A and dandelion root polysaccharide according to claim 1.
3. Use of the composite immunopotentiator for inhibiting staphylococcus aureus according to claim 2 in the preparation of animal feed.
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Application publication date: 20220225 Assignee: Huai'an Yongrun Instrument Equipment Co.,Ltd. Assignor: HUAIYIN INSTITUTE OF TECHNOLOGY Contract record no.: X2024980016637 Denomination of invention: A method for preparing a composite immune enhancer from Subtilosin A and dandelion root polysaccharides and its application Granted publication date: 20240419 License type: Common License Record date: 20240926 |