CN113812518A - Novel salmonella bacteriophage microencapsulated microsphere and preparation method thereof - Google Patents
Novel salmonella bacteriophage microencapsulated microsphere and preparation method thereof Download PDFInfo
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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
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- 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
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- 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
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/24—Compounds of alkaline earth metals, e.g. magnesium
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- 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
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/30—Oligoelements
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K40/00—Shaping or working-up of animal feeding-stuffs
- A23K40/30—Shaping or working-up of animal feeding-stuffs by encapsulating; by coating
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides a preparation method of a novel salmonella bacteriophage microencapsulated microsphere, which comprises the following steps: mixing the cationic etherified starch solution with the bacteriophage suspension, and uniformly stirring to obtain a first mixed solution; then the first mixed solution, the sodium alginate solution, the xanthan gum solution and the nano TiO are mixed2Stirring and uniformly mixing the solution to obtain a second mixed solution; dripping the second mixed solution into calcium chloride solution drop by drop to form calcium alginate gel, filtering and collecting phage microspheresPutting the phage microspheres into a cationic chitosan oligosaccharide solution for coating for 30min, and filtering and collecting the phage microencapsulated microspheres. When the phage microspheres enter intestinal juice, the material coated on the surfaces of the phage can react with the intestinal juice and is cracked, and the phage in the material can be released, so that the sterilization effect is achieved. The invention has the advantages that: the microencapsulation preparation is carried out at room temperature, the process is simple, the application range is wide, and the cost is low; the raw materials are wide in source, cheap and easy to obtain, the phage activity in the product is high, the encapsulation rate is high, and the product can be directly applied to feed addition.
Description
Technical Field
The invention belongs to the technical field of preparation of biological feed additives, and particularly relates to novel phage microencapsulated microspheres and a preparation method thereof.
Background
The bacteriophage is a kind of bacterial virus, and widely exists in nature, a bacteriophage with strong lytic property exists in the bacteriophage, and the bacteriophage can infect or lyse bacteria, can be rapidly replicated and proliferated, and finally achieves the antibacterial effect, and the current method for sterilizing by using the bacteriophage is widely applied. Antibiotics still occupy a large market as a traditional treatment method, but the phage sterilization method has many advantages which are not possessed by the antibiotic treatment method, and has the advantages of strong specificity, safety, no toxicity, self-replication, wide existence and the like of the phage, so the phage sterilization method is a very ideal method for replacing antibiotics. The bacteriophage has very remarkable effect on the aspect of animal bacterial diseases, but the resistance of the bacteriophage is not ideal, the bacteriophage is generally used for killing animal pathogenic bacteria by an oral method, and when the bacteriophage enters the stomach of an animal, the activity of the bacteriophage is easily damaged by gastric acid, digestive enzyme and the like, so that the bacteriophage is inactivated. Therefore, the problem of maintaining the viability of the phage during the sterilization process is an important problem to be solved.
The microencapsulation technology has a very broad prospect although it is not a long time to come out, mainly uses some special methods to make a certain special substance reach the desired density and be wrapped by another substance or several substances, thereby forming a special state to meet the special requirements of medicine, and the technology is widely applied to the field of controlled release of oral medicine at present. The application of the microencapsulation technology is more, but the microencapsulation phage has less reports, and the technology has a great development space. Although the microencapsulation technology has many advantages, it needs to select proper microencapsulation coating material, and if the selected coating material is not proper, not only the activity of the phage is reduced, but also the encapsulation efficiency is reduced, which affects the release in intestinal tract, and finally the sterilization effect is reduced.
Disclosure of Invention
In order to solve the problems of the prior art, the invention adopts cationic etherified starch/sodium alginate/xanthan gum/nano TiO2The chitosan oligosaccharide is used for coating the phage in a micro-encapsulation way so as to obtain an ideal method for improving the bactericidal effect of the phage.
The technical scheme adopted by the invention is as follows: a novel salmonella bacteriophage microencapsulated microsphere comprises a cationic etherified starch solution with a concentration of 2.2-2.6%, a sodium alginate solution with a concentration of 1-2%, a xanthan solution with a concentration of 0.5-1.5%, and a nano TiO core with a concentration of 0.5-1.0mmol/L2Solution, cationic chitosan oligosaccharide solution with the concentration of 0.4-0.8%, calcium chloride solution and salmonella phage suspension.
Further, the raw materials comprise a cationic etherified starch solution with the concentration of 2.4 percent, a sodium alginate solution with the concentration of 2 percent, a xanthan gum solution with the concentration of 1 percent and a xanthan gum solution with the concentration of 2 percent0.5mmol/L nano TiO2The preparation method comprises the following steps of solution, cationic chitosan oligosaccharide solution with the concentration of 0.6%, calcium chloride solution and salmonella phage suspension.
The method of preparing the novel microencapsulated microspheres of Salmonella bacteriophage according to claim 1 or 2, comprising the steps of: firstly, mixing a cationic etherified starch solution and a salmonella bacteriophage suspension, and uniformly stirring to obtain a first mixed solution; then the first mixed solution, the sodium alginate solution, the xanthan gum solution and the nano TiO are mixed2Putting the solution into the same beaker, uniformly stirring, and removing bubbles in the solution by vacuum degassing or standing at 4 ℃ to remove bubbles to obtain a second mixed solution; dropwise adding the second mixed solution into a calcium chloride solution at a constant speed by using a sterile syringe to form calcium alginate gel, standing at normal temperature for reaction for 30min, filtering and collecting phage microspheres, washing with deionized water, putting the phage microspheres into a cationic chitosan oligosaccharide solution for coating for 30min, filtering and collecting phage microencapsulated microspheres, and washing with deionized water to obtain the salmonella phage microencapsulated microspheres.
Further, the salmonella bacteriophage microencapsulated microspheres are stored in 4 ℃ physiological saline in a sealed manner or are stored in an electrothermal blowing drying oven in a sealed manner at the temperature of 30 ℃ for 24 hours and at the temperature of 4 ℃.
Further, the preparation method of the cationic etherified starch solution comprises the steps of dissolving the cationic etherified starch in deionized water, heating to 95 ℃, keeping the temperature for 20 minutes at the temperature, adding deionized water for dilution, and keeping the temperature at 60 ℃ for later use; the preparation method of the sodium alginate solution comprises the step of dissolving sodium alginate powder in a Tris-HCl solution. The beneficial effects obtained by the invention are as follows: the invention provides a novel salmonella bacteriophage microencapsulated microsphere, which can be used for preparing bacteriophage/cationic etherified starch/sodium alginate/xanthan gum/nano TiO prepared in advance in practical use2The cationic chitosan oligosaccharide phage microencapsulated microspheres are taken orally, when entering gastric juice, the material coated on the surfaces of the phage can effectively resist the erosion of gastric acid, reduce the inactivation speed of the phage and effectively improve the survival time of the phage, and when entering intestinal juice, the material coated on the surfaces of the phage can effectively resist the erosion of gastric acid, so thatThe material can react with intestinal juice and be cracked to release bacteriophage, thereby achieving the effect of sterilization.
The invention has the specific working principle that: 1. according to the invention, by using the Cationic Etherified Starch (CES), which has positive charges, the bacteriophage has anions under a neutral condition, the cationic etherified starch has affinity to the bacteriophage with anions, and after the cationic etherified starch is mixed with the bacteriophage, the cations and the anions are combined with each other, so that the components of the bacteriophage microsphere are combined more tightly, the stability of the bacteriophage microsphere can be improved, the maintenance of the bacteriophage activity is facilitated, the bacteriophage microsphere is slowly released, and the sterilization time of the bacteriophage in an intestinal tract is prolonged.
2. The invention uses Sodium Alginate (SA) as a natural polysaccharide substance, which has good sensitivity, biocompatibility and mild emulsion process, and has good characteristics of coating materials. The system of sodium alginate and calcium chloride can realize microencapsulation of bacteriophage mainly due to sodium alginate and Ca2+The ions react to form calcium alginate polymer, which has good biodegradability and biocompatibility and can effectively microencapsulate the phage.
3. The Xanthan Gum (XG) used in the invention is a monospore polysaccharide produced by fermentation of pseudoxanthomonas, and is an acidic extracellular heteropolysaccharide formed by cutting 1, 6-glycosidic bond and opening a branched chain by aerobic fermentation bioengineering technology by taking carbohydrate as a main raw material of xanthomonas campestris with black cabbage rot, and bonding 1, 4-into a straight chain. The microsphere has good macromolecule special structure and colloid characteristics, and can be used as an emulsifier, a stabilizer, a gel thickener, a wetting agent, a film forming agent and the like, so that the encapsulation rate of the phage microsphere can be effectively improved.
4. The invention is based on nano TiO2To improve the sterilization efficiency of the bacteriophage, the nano TiO2Has the advantages of no toxicity, good thermal stability, good chemical stability, and good optical, mechanical and electrical properties, and is made of nanometer TiO2With TiO2Compared with the prior art, the composite material has better adsorption capacity and biocompatibility. When nano TiO2After binding to the phage, it can be expressedThe method obviously promotes the infection efficiency of the phage to the receptor bacteria, and obviously increases the aggregation of the phage around the bacteria, thereby achieving the purpose of enhancing the sterilization of the phage.
5. The cationic Chitosan Oligosaccharide (COS) is only cationic basic amino oligosaccharide with positive charge in nature, has the advantages of low molecular weight, good water solubility, large functional action, easy absorption by small intestine of an organism, high biological activity and the like, is usually processed and generated by chitosan, has better compatibility, no toxicity and good film forming property, and can be used for coating the phage microencapsulated microspheres with the cationic chitosan oligosaccharide after the reaction of sodium alginate and calcium chloride, thereby improving the stability of the phage microcapsules, being beneficial to preservation and not influencing the release of the phage by the microcapsules.
Drawings
FIG. 1 is a graph showing the results of experiments on the concentration of cationic etherified starch according to the present invention;
FIG. 2 is a graph showing the results of the sodium alginate concentration test of the present invention;
FIG. 3 is a graph showing the results of a xanthan gum concentration test according to the present invention;
FIG. 4 shows the nano TiO of the present invention2A concentration experiment result graph;
FIG. 5 is a graph showing the results of the concentration experiment of chitosan oligosaccharide of the present invention;
FIG. 6 is a graph showing the effect of pH on the activity of phage microencapsulated microspheres in accordance with the present invention;
FIG. 7 is a graph showing the effect of different temperatures on the activity of phage microencapsulated microspheres in accordance with the present invention;
FIG. 8 is a graph showing the activity of the phage microencapsulated microspheres in simulated gastric fluid;
FIG. 9 is a graph showing the release profile of the phage microencapsulated microspheres of the invention in simulated intestinal fluid;
FIG. 10 is an activity diagram showing the storage stability of the phage microencapsulated microspheres of the present invention.
Detailed Description
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments or the prior art descriptions will be briefly described 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 to obtain other drawings based on these drawings without inventive exercise.
Example 1: a novel Salmonella bacteriophage microencapsulated microsphere comprises raw materials of 2.2% cationic etherified starch solution, 1% sodium alginate solution, 0.5% xanthan gum solution, and 0.5mmol/L nano TiO2The preparation method comprises the following steps of solution, cationic chitosan oligosaccharide solution with the concentration of 0.4%, calcium chloride solution and salmonella phage suspension.
Example 2: a novel Salmonella bacteriophage microencapsulated microsphere comprises raw materials of 2.6% cationic etherified starch solution, 2% sodium alginate solution, 1.5% xanthan gum solution, and 1.0mmol/L nano TiO2The kit comprises a solution, a cationic chitosan oligosaccharide solution with the concentration of 0.8%, a calcium chloride solution and a salmonella phage suspension.
Example 3: novel salmonella bacteriophage microencapsulated microspheres, which are characterized in that: the raw materials comprise a cationic etherified starch solution with the concentration of 2.4 percent, a sodium alginate solution with the concentration of 2 percent, a xanthan gum solution with the concentration of 1 percent and nano TiO with the concentration of 0.5mmol/L2Solution, cationic chitosan oligosaccharide solution with the concentration of 0.6%, calcium chloride solution and phage suspension.
The preparation method comprises the steps of dissolving required cationic etherified starch in deionized water, heating to 95 ℃, keeping the temperature for 20 minutes, adding deionized water to dilute to a certain concentration, and keeping the temperature at 60 ℃ for later use to obtain solution A. And (4) carrying out centrifugal filtration on the amplified and purified salmonella bacteriophage stock solution to obtain a solution B. And mixing the solution A and the solution B, and uniformly stirring to obtain solution C. And dissolving the required sodium alginate powder in a Tris-HCl solution to obtain a solution D. Dissolving required xanthan gum powder in deionized water, and uniformly stirring to obtain solution E. Adding a certain amount of TiO2Placing in deionized water to obtain mother liquor, ultrasonic treating for 1 hr, diluting, and ultrasonic treating for 15min to obtain solution F. Putting the C, D, E, F solutions into the same beaker, stirring and mixing uniformly, removing bubbles in the solution by vacuum degassing or standing at 4 ℃ for removing bubbles to obtain solution G. And (2) dropwise adding the solution G into a required calcium chloride solution, namely the solution H, at a constant speed by using a 5mL sterile syringe to form calcium alginate gel, standing at normal temperature for reaction for 30min, filtering and collecting phage microspheres, washing with deionized water, then placing into a required cationic chitosan oligosaccharide solution, namely the solution I, laminating for 30min, filtering and collecting phage microencapsulated microspheres, washing with deionized water, and then placing into physiological saline at 4 ℃ for sealed storage, or placing into an electrothermal blowing drying box for drying at 30 ℃ for 24H, and sealing at 4 ℃.
1. Measurement of encapsulation efficiency of phage microencapsulated microspheres
And (3) putting 1g of wet phage microspheres into the microsphere disruption solution, completely dissolving at room temperature, filtering by using a 0.22-micron filtering device, detecting the titer of phage in the microsphere disruption solution, and calculating the encapsulation rate of phage. The dried phage microspheres are firstly put into SM buffer solution to be hydrated for 6 hours and then put into the microsphere disruption solution to measure the titer.
The entrapment rate is x 100 percent of the medicine content in the phage microspheres/the initially input phage content
2 Single factor experiment
By changing the concentration ratio of different single factors, an analysis experiment is designed, and the most appropriate factor value of the phage microsphere is selected.
2.1CES concentration
The addition amount of fixed SA is 1.0g/100mL, the addition amount of XG is 1.0g/100mL, the addition amount of COS is 1.0g/100mL, the phage microspheres are prepared respectively at CES concentrations of 2.0, 2.2, 2.4 and 2.6g/100mL, the influence of different addition amounts of CES on the phage encapsulation efficiency is analyzed, and the design scheme of CES concentration is shown in Table 1:
TABLE 1 cationic etherified starch concentration design
Unit: is based on
2.2 SA concentration
The addition amount of a fixed CES concentration is 2.4g/100mL, the addition amount of XG is 1.0g/100mL, the addition amount of COS is 1.0g/100mL, phage microspheres are prepared respectively at SA concentrations of 1.0, 2.0, 3.0 and 4.0g/100mL, the influence of different addition amounts of SA on the phage encapsulation efficiency is analyzed, and the design scheme of the SA concentration is shown in Table 2:
TABLE 2 sodium alginate concentration design protocol
Unit: is based on
2.3 XG concentration
The fixed CES concentration addition amount is 2.4g/100mL, the SA addition amount is 2.0g/100mL, the COS addition amount is 1.0g/100mL, the phage microspheres are prepared at XG concentrations of 0.5, 1.0, 1.5 and 2.0g/100mL respectively, the influence of different addition amounts of XG on the phage encapsulation efficiency is analyzed, and the XG concentration design scheme is shown in Table 3:
table 3 xanthan gum concentration design
Unit: is based on
2.4 nanometer TiO2Concentration of
The addition amount of fixed CES concentration is 2.4g/100mL, the addition amount of SA is 2.0g/100mL, the addition amount of XG is 1.0g/100mL, the addition amount of COS is 1.0g/100mL, and the concentration of nano TiO is respectively 0.5, 1.0, 1.5 and 2.0mmol/L2Preparing bacteriophage microball in concentration and analyzing nanometer TiO2Influence of different addition amounts on phage encapsulation efficiency, nano TiO2The concentration design is shown in table 4:
TABLE 4 Nano TiO2Design scheme of concentration
Unit: is based on
2.5 COS concentration
The addition amount of fixed CES concentration is 2.4g/100mL, the addition amount of SA is 2.0g/100mL, the addition amount of XG is 1.0g/100mL, and nano TiO is added2The addition amount is 0.5mmol/L, phage microspheres are prepared with COS concentrations of 0.6, 0.8, 1.0 and 1.2g/100mL respectively, the influence of different addition amounts of COS on the phage encapsulation efficiency is analyzed, and the design scheme of the COS concentration is shown in Table 5:
TABLE 5 Chitosan oligosaccharide concentration design protocol
Unit: is based on
pH stability of 3 phage microencapsulated microspheres
NaCl solution is used as starting solution, HCl solution is added into the NaCl solution to adjust the pH value of the starting solution, the pH value of the starting solution is adjusted to be 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0, phage microencapsulation microspheres are respectively added into the adjusted starting solution, after a period of time, the phage microencapsulation microspheres are taken out and put into microsphere lysate for cracking, after complete cracking, the reaction is stopped by SM buffer solution, a 0.22 mu m filter device is used for filtering, and the titer is determined after proper gradient dilution.
Stability of 4 bacteriophage microencapsulated microspheres at different temperatures
In order to investigate the stability of the phage microencapsulated microspheres at different temperatures, the same amount of phage microencapsulated microspheres were placed in water baths at different temperatures, 10 ℃, 20 ℃, 37 ℃, 45 ℃, 55 ℃, 65 ℃ and 75 ℃ respectively, and after 1h, the phage microencapsulated microspheres were taken out and placed in a microsphere lysate for lysis, after complete lysis, the reaction was stopped with SM buffer, filtered by a 0.22 μm filter device, and the titer was determined after appropriate amount of gradient dilution.
Stability of 5 bacteriophage microencapsulated microspheres in simulated gastric fluid
Respectively putting prepared simulated gastric juice with pH 2 and pH 3 into 7 test tubes, wherein each test tube represents a reaction time, heating the simulated gastric juice to 37 ℃, respectively adding about 200mg of dry microspheres into each test tube, putting the test tubes into a constant-temperature shaking table, respectively taking out the test tubes at 30min, 60min, 90 min, 120 min, 150 min and 180min, filtering the simulated gastric juice, adding SM buffer solution into the dry microspheres to terminate the reaction, finally putting the dry microspheres soaked in the SM buffer solution into a microsphere lysate for complete lysis, filtering the dry microspheres by a 0.22 mu m filter device, and measuring the titer after appropriate gradient dilution. This experiment was repeated 3 times with SM buffer as a control.
Stability of 6 bacteriophage microencapsulated microspheres in simulated intestinal fluid
The prepared simulated intestinal juice is respectively put into 6 test tubes, each test tube represents a reaction time, the simulated intestinal juice is heated to 37 ℃, about 200mg of dry microspheres are respectively added into each test tube, the test tubes are put into a constant-temperature shaking table, 100 mu l of test solution is respectively taken out at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours and added into 900 mu l of SM buffer solution, a 0.22 mu m filtering device is used for filtering, and the titer is determined after proper amount of gradient dilution. This experiment was repeated 3 times with SM buffer as a control.
7 bacteriophage microencapsulated microsphere in simulated intestinal fluid release behavior
Placing prepared simulated intestinal juice into 6 test tubes respectively, wherein each test tube represents a reaction time, heating the simulated intestinal juice to 37 ℃, adding about 200mg of dried microspheres into each test tube respectively, placing the test tubes into a constant-temperature shaking table, taking 100 mu l of test solution at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours respectively, adding release medium with the same temperature and volume, filtering by a 0.22 mu m filter device, and measuring the titer after appropriate amount of gradient dilution. This experiment was repeated 3 times with SM buffer as a control. And (3) drawing a release curve of the phage microencapsulated microspheres in simulated intestinal fluid by taking the release time as a horizontal coordinate and the titer of the phage microencapsulated microspheres as a vertical coordinate.
8 storage stability study of phage microencapsulated microspheres
The prepared wet phage microencapsulated microspheres are stored in physiological saline at 4 ℃, the dry phage microencapsulated microspheres are stored at normal temperature and 4 ℃ respectively, and a small amount of wet microspheres and dry microspheres are extracted every week to measure the titer of the wet phage microencapsulated microspheres and the dry phage microencapsulated microspheres.
Results and analysis
9. Results of single factor experiments
9.1 CES concentration test results
As can be seen from FIG. 1, the encapsulation efficiency of the phage microencapsulated microspheres gradually increases with the increase of the CES concentration, gradually decreases after reaching the peak value, the peak value is 2.4g/100mL, and the encapsulation efficiency begins to decrease when the CES concentration exceeds 2.4g/100 mL. Therefore, the best concentration of the CES solution is selected to be 2.4g/100 mL.
9.2 SA concentration test results
As can be seen from FIG. 2, the encapsulation efficiency of the phage microencapsulated microspheres gradually increases with the increase of SA concentration, and gradually decreases after reaching the peak value, the peak value is 2g/100mL, when the SA concentration is less than 1g/100mL, the mixed solution is too diluted to form microspheres, and when the SA concentration exceeds 3g/100mL, the mixed solution is too viscous to form microspheres. Therefore, the best concentration of SA solution is chosen to be 2g/100 mL.
9.3 XG concentration test results
As shown in FIG. 3, XG is relatively adhesive, and an excessively high concentration of XG causes the microsphere system to be damaged and no microspheres are formed, and the encapsulation efficiency of the microencapsulated phage microspheres is gradually reduced with the increase of the concentration of XG, and when the peak value is 1g/100mL, the encapsulation efficiency of the microencapsulated phage microspheres is highest. Therefore, the best concentration for XG solution is 1g/100 mL.
9.4 nanometer TiO2Results of concentration experiments
As can be seen from FIG. 4, the addition of nano TiO2Compared with the method without adding nano TiO2The encapsulation rate of the phage microencapsulated microspheres is improved, and nano TiO is added2Then, the infection efficiency of the phage microencapsulated microspheres to host bacteria is obviously improved (P)<0.05) when nano TiO2When the amount of (B) was 0.5mmol/L, the peak was reached. Thus, the nano TiO is selected2The best concentration of the solution is 0.5 mmol/L.
9.5 COS concentration test results
As can be seen from FIG. 5, the encapsulation efficiency of the phage microencapsulated microspheres gradually increased with the increase of the COS concentration, gradually decreased after reaching the peak value of 0.6g/100mL, and began to decrease after the COS concentration exceeded 0.6g/100 mL. Therefore, the best concentration of COS solution is selected to be 0.6g/100 mL.
10 pH stability results of microencapsulated phage microspheres
The phage is not acid-resistant, the phage loses activity after being directly put into an initial solution with the pH value of 2.0, the phage still has no activity after the reaction of the phage losing activity and an SM buffer solution is stopped, and the inactivation of the phage in gastric juice is an irreversible reaction. The phage microencapsulated microsphere lysate is added into solutions with different pH values respectively, when the pH value is 2.0, the phage can retain most of activity, as shown in FIG. 6, the phage microencapsulated microsphere can effectively retain the activity of the phage under an acidic condition, but the activity is gradually reduced along with the increase of time, the activity of the phage in the phage microencapsulated microsphere is basically unchanged under a neutral condition, and the activity of the phage is gradually reduced along with the gradual increase of the pH value under an alkaline condition.
Stability results of 11 phage microencapsulated microspheres at different temperatures
The activity of the phage microencapsulated microspheres is different at different temperatures, as shown in fig. 7, the activity of the phage microencapsulated microspheres is basically unchanged at 10 ℃, 20 ℃ and 37 ℃, which indicates that at low temperature and normal temperature, the stability of the phage microencapsulated microspheres to temperature is good, the activity is basically unchanged, and the activity of the phage microencapsulated microspheres starts to gradually decrease with the increase of temperature, indicating that the activity of the phage microencapsulated microspheres can slowly decrease until inactivation under the condition of high temperature.
Stability results of 12 phage microencapsulated microspheres in simulated gastric fluid
The phage microencapsulated microspheres can effectively improve the acid resistance of the phage, and although the phage microencapsulated microspheres lose activity for a long time in simulated gastric juice with the pH of 2.0, the phage survival time is effectively prolonged, and the complete inactivation time is obviously prolonged when the pH is 3. The activity of the phage microencapsulated microspheres taken out when the reaction time is 30, 60, 90, 120, 150 and 180min is shown in FIG. 8, and compared with the uncoated phage, the phage retains a large amount of activity and the titer is not obviously reduced in a short time.
13 bacteriophage microencapsulated microsphere in simulated intestinal fluid release behavior
The bacteriophage microencapsulated microspheres are used for killing salmonella, so that the bacteriophage microencapsulated microspheres can not only be stored in gastric juice for a long time and be released in intestinal juice in time. Tests prove that the phage microencapsulated microspheres are not released in deionized water because the titer of the phage microencapsulated microspheres in deionized water is unchanged. The release characteristics of the microspheres in simulated intestinal fluid are shown in figure 9, the phage in the phage microencapsulated microspheres are gradually released in the simulated intestinal fluid along with the increase of time, and after 4 hours, the titer of the phage is basically unchanged, namely, all the phage in the phage microencapsulated microspheres are released.
14 storage stability of microencapsulated phage microspheres
The wet phage microencapsulated microspheres have good stability when stored at 4 ℃, and the activity of the wet phage microencapsulated microspheres is basically unchanged within 6 weeks as shown in figure 10 by the result of measuring the activity of the wet phage microencapsulated microspheres every week, which indicates that the cationic etherified starch/sodium alginate/xanthan gum/nano TiO2The chitosan oligosaccharide system has good compatibility with bacteriophage. The titer of the dried phage microencapsulated microspheres stored at 4 ℃ and normal temperature is respectively reduced by 3.51 log10 PFU/mL and 7.11log10 PFU/mL within 6 weeks, which indicates that the dried phage microencapsulated microspheres are more stable to be stored at 4 ℃ than normal temperature.
4 conclusion and discussion
The application mode of the phage is mainly oral administration, and the phage is inactivated after entering gastric juice through oral administration. Wherein the natural polysaccharides such as alginate, xanthan gum and the like have rich resources, simple microencapsulation process and wide application in the technical field of biological feed additive preparation, and the formed cation etherified starch/sodium alginate/xanthan gum/nano TiO2The chitosan oligosaccharide system can effectively improve the acid resistance of the bacteriophage and release the bacteriophage in intestinal tracts. The result shows that the bacteriophage has anions under neutral condition, and the bacteriophage is combined with positive and negative charges of cationic etherified starch to ensure that the bacteriophageThe components of the microsphere are combined more tightly, the stability of the phage microsphere can be improved, the activity of the phage can be maintained, the phage microsphere can slowly release the phage, and the sterilization time of the phage in intestinal tracts can be prolonged. The addition of xanthan gum in the system of sodium alginate and calcium chloride can improve the encapsulation efficiency. Adding nano TiO into microsphere system2The infection efficiency of the phage on the specific bacteria can be improved, and the sterilization effect of the phage is further improved. Finally, after the microspheres are formed, the microspheres are put into a cationic chitosan oligosaccharide solution for coating, so that the stability of the phage microcapsules can be improved, the preservation is facilitated, and the release of phage by the microcapsules is not influenced.
Cationic etherified starch/sodium alginate/xanthan gum/nano TiO2The system formed by the chitosan oligosaccharide can effectively coat the phage, and the activity difference of the coated phage is not obvious (p)>0.05), the encapsulation rate of the microencapsulated phage microspheres tends to increase and then decrease with the increase of the addition amount of the coating material, and the cationic etherified starch/sodium alginate/xanthan gum/nano TiO is proved2The chitosan oligosaccharide has obvious interaction on the encapsulation efficiency. Experiments show that the coated phage microencapsulated microspheres have high stability on pH, anions on the phage and cations in the cationic etherified starch are combined with each other to generate acting force, and the stability of the phage microspheres can be improved. The phage microencapsulated microspheres can survive in gastric juice for a long time, although phage in the phage microencapsulated microspheres is inactivated in 60min at a pH of 2.0, compared with uncoated phage suspension, the phage microencapsulated microspheres effectively prolong the survival time of phage in gastric juice, and the activity of phage after passing through gastric acid can be better preserved and smoothly reach intestinal tract for directional release and play a role in sterilization. The phage microencapsulated microspheres are released in intestinal juice, and are released less in a short time, probably because the phage microencapsulated microspheres do not fully react with a release medium, only a small amount of phage on the surfaces of the microspheres are separated from the microspheres and enter the release medium, and as the time increases, the microsphere coating material fully reacts with the intestinal juice, the microspheres are broken, and the phage is released rapidly. The activity of the wet or dry phage microencapsulated microspheres is relatively stable when the microspheres are stored at 4 ℃ (p)>0.05), drying the phageWhen the microencapsulated microspheres are stored at normal temperature, the activity of the microencapsulated microspheres is reduced obviously (p)<0.05). When the concentration of the cationic etherified starch is 2.4 percent, the concentration content of the sodium alginate is 2 percent, the concentration of the xanthan gum is 1 percent, the mass ratio of the xanthan gum to the sodium alginate is 1:2, and the nano TiO is2The concentration of the chitosan oligosaccharide is 0.5mmol/L, the concentration of the chitosan oligosaccharide is 0.6%, the prepared phage microencapsulated microspheres have regular shapes, the encapsulation rate is 97.5%, the survival time can be prolonged in simulated gastric juice, and the phage microencapsulated microspheres can be quickly released in an intestinal simulation system. Therefore, cationic etherified starch/sodium alginate/xanthan gum/nano TiO2The preparation method of the chitosan oligosaccharide system can effectively coat the phage, has high encapsulation efficiency and small influence on the titer of the phage (p)>0.05), the microsphere can be used as a slow-release carrier of the bacteriophage.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. A novel salmonella bacteriophage microencapsulated microsphere is characterized in that: the raw materials comprise a cationic etherified starch solution with the concentration of 2.2-2.6 percent, a sodium alginate solution with the concentration of 1-2 percent, a xanthan gum solution with the concentration of 0.5-1.5 percent and nano TiO with the concentration of 0.5-1.0mmol/L2Solution, cationic chitosan oligosaccharide solution with the concentration of 0.4-0.8%, calcium chloride solution and salmonella phage suspension.
2. The novel microencapsulated microsphere of a bacteriophage for Salmonella according to claim 1, wherein: the raw materials comprise a cationic etherified starch solution with the concentration of 2.4 percent, a sodium alginate solution with the concentration of 2 percent, a xanthan gum solution with the concentration of 1 percent and nano TiO with the concentration of 0.5mmol/L2The preparation method comprises the following steps of solution, cationic chitosan oligosaccharide solution with the concentration of 0.6%, calcium chloride solution and salmonella phage suspension.
3. The method of claim 1 or 2, wherein the method comprises the steps of: the method comprises the following steps: firstly, mixing a cationic etherified starch solution and a salmonella bacteriophage suspension, and uniformly stirring to obtain a first mixed solution; then the first mixed solution, the sodium alginate solution, the xanthan gum solution and the nano TiO are mixed2Putting the solution into the same beaker, uniformly stirring, and removing bubbles in the solution by vacuum degassing or standing at 4 ℃ to remove bubbles to obtain a second mixed solution; dropwise adding the second mixed solution into a calcium chloride solution at a constant speed by using a sterile syringe to form calcium alginate gel, standing at normal temperature for reaction for 30min, filtering and collecting phage microspheres, washing with deionized water, putting the phage microspheres into a cationic chitosan oligosaccharide solution for coating for 30min, filtering and collecting phage microencapsulated microspheres, and washing with deionized water to obtain the salmonella phage microencapsulated microspheres.
4. The method of claim 3, wherein the method comprises the steps of: the salmonella bacteriophage microencapsulated microspheres are placed in physiological saline at 4 ℃ for sealed storage or placed in an electrothermal blowing drying oven for drying for 24 hours at 30 ℃ and sealed storage at 4 ℃.
5. The method of claim 3, wherein the method comprises the steps of: dissolving cationic etherified starch in deionized water, heating to 95 ℃, preserving heat for 20 minutes at the temperature, adding deionized water for dilution, and preserving heat at 60 ℃ for later use; the preparation method of the sodium alginate solution comprises the step of dissolving sodium alginate powder in a Tris-HCl solution.
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