CN115300480B - Calcium alginate suspension microcapsule for encapsulating probiotics and preparation method and application thereof - Google Patents
Calcium alginate suspension microcapsule for encapsulating probiotics and preparation method and application thereof Download PDFInfo
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- 239000000725 suspension Substances 0.000 title claims abstract description 45
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- 235000018291 probiotics Nutrition 0.000 title claims abstract description 32
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5089—Processes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
- A61K9/0065—Forms with gastric retention, e.g. floating on gastric juice, adhering to gastric mucosa, expanding to prevent passage through the pylorus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/501—Inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/02—Antidotes
Abstract
The invention discloses a calcium alginate suspension microcapsule for encapsulating probiotics and a preparation method and application thereof, belonging to the field of biomedical engineering. The core of the suspension microcapsule is that calcium alginate microcapsule with pores is used for encapsulating acetobacter, the acetobacter can directly metabolize alcohol reaching the stomach after drinking, and the concentration of the alcohol in the stomach is reduced, so that the purpose of reducing alcohol absorption is realized; the porous structure reduces the density of the structure, so that the microcapsule can be suspended in gastric juice, the gastric retention time is prolonged, and the alcohol metabolism time is improved; the acid resistance is enhanced by the high crosslinking degree and regular structure, the survival rate of probiotics is improved, and the bioavailability is improved. Therefore, the invention provides the calcium alginate suspension microcapsule which is resistant to gastric acid, long in gastric retention time and high in alcohol dispelling efficiency and encapsulates the probiotics.
Description
Technical Field
The invention belongs to the field of biomedical engineering, and in particular relates to a calcium alginate suspension microcapsule for encapsulating probiotics, and a preparation method and application thereof.
Background
In recent years, alcoholism and alcoholism of China are increasing, and the physical health of people and the stability of families and society are seriously affected. Long-term drinking can cause mental disorder, gastric ulcer, fatty liver, alcoholic hepatitis, cirrhosis and other diseases, and investigation and research show that about 57.5% of patients with long-term drinking have fatty liver and 15% of patients have cirrhosis. The increased incidence of some digestive system tumors such as gastric cancer, esophageal cancer and liver cancer is also closely related to alcoholism.
Most of the alcohol-relieving medicines in the market focus on delaying alcohol absorption and accelerating alcohol metabolism, and can relieve headache and nausea symptoms. However, they cannot directly reduce alcohol intake and metabolism, and they still cause metabolic burden to the liver and kidneys to cause different degrees of injury. Oxidative metabolism of ethanol can often cause a decrease in the in vivo coenzyme NAD, which in turn affects the operation of the tricarboxylic acid cycle energy supply chain of liver cells, resulting in insufficient intracellular energy supply. Second, the ethanol metabolites generate a large amount of free radicals after oxidative metabolism by xanthine oxidase, thereby causing lipid peroxidation of liver cell membranes and cell damage.
In order to solve the problem that the prior anti-alcoholic drug cannot fundamentally reduce the alcohol content, how to innovate a novel anti-alcoholic drug which can directly convert alcohol in the stomach through the structure of a carrier is an important problem to be solved at present.
Disclosure of Invention
In order to solve the defects and shortcomings in the prior art, the primary object of the invention is to provide a preparation method of calcium alginate suspension microcapsules for encapsulating probiotics. Dispersing calcium carbonate into sodium alginate and acetobacter aceti solution in oil phase, and dripping acetic acid solution to generate calcium ions and cross-linking the sodium alginate to form calcium alginate microcapsule; the released carbon dioxide permeates into the calcium alginate matrix, leaving behind pores that provide buoyancy to the microcapsules, enabling them to suspend in gastric fluid. In the prior art, probiotics are directly orally taken, and pass through the stomach rapidly under the effect of gastric emptying, so that the emptying is fast, the acting time is short, and the absorption is irregular, thereby greatly influencing the acting effect. The gastric retention anti-alcoholic oral acetobacter probiotic microcapsule prepared by the invention can improve the retention time of probiotics in the stomach, concentrate on gastric administration and improve the bioavailability through a gastric retention suspension administration system.
A second object of the present invention is to provide a probiotic-encapsulated calcium alginate suspension microcapsule, which is a novel preparation for decomposing alcohol by oral probiotics. Acetobacter is a kind of short bacillus capable of oxidizing alcohol into acetic acid and other products, and has strong alcohol resistance and acetic acid resistance. The acetobacter is delivered to the stomach, alcohol is converted into acetic acid, the content of alcohol in the stomach is reduced, and the acetobacter has the advantages of convenience in administration, good patient tolerance, easiness in large-scale popularization and the like.
A third object of the present invention is to provide the use of the probiotic-encapsulated calcium alginate suspension microcapsules described above for anti-hangover.
The primary purpose of the invention is realized by the following technical scheme:
a method for preparing calcium alginate suspension microcapsules encapsulating probiotics, comprising the following steps:
centrifuging activated bacillus aceticus, removing supernatant and sodium alginate solution, suspending, adding calcium carbonate particles, placing in an oil phase, adding mixed solution of oil and acetic acid, stirring, removing supernatant, and washing to obtain the calcium alginate suspension microcapsule for encapsulating probiotics.
Preferably, the acetobacter is cultured to an OD of 0.4-0.6.
Preferably, the volume ratio of the acetobacter to the sodium alginate is 1: 10-20.
Preferably, the molecular weight of the sodium alginate is 1-20 ten thousand, and the mass concentration of the sodium alginate solution is 0.5-2.5%.
Preferably, the mass-volume ratio of the calcium carbonate particles to the sodium alginate is 1:4-4:1.
Preferably, the volume ratio of acetic acid to oil in the mixed solution of oil and acetic acid is 0.005-0.02.
Preferably, the stirring speed is 400-800rpm, and the stirring time is 10-40min.
The second object of the invention is achieved by the following technical scheme:
the calcium alginate suspension microcapsule for encapsulating probiotics is prepared by the preparation method.
The third object of the invention is achieved by the following technical scheme:
use of calcium alginate suspension microcapsule encapsulating probiotics in relieving hangover.
The principle of the invention is as follows:
dispersing calcium carbonate into sodium alginate and acetobacter aceti solution in oil phase, and dripping acetic acid solution to generate calcium ions and cross-linking the sodium alginate to form calcium alginate suspension microcapsule; the released carbon dioxide permeates into the calcium alginate matrix, leaving behind pores that provide buoyancy to the microcapsules, enabling them to suspend in gastric fluid. In the prior art, probiotics are directly taken orally, and pass through the stomach rapidly under the effect of gastric emptying, so that the emptying is fast, the acting time is short, and the absorption is irregular, thereby greatly influencing the acting effect. Firstly, the invention can improve the retention time of probiotics in stomach, concentrate the stomach for administration and concentrate the part with the highest alcohol concentration to play a role through the gastric retention suspension administration system, thereby prolonging the action time; secondly, the acetobacter can directly contact with alcohol through pores, and ethanol is converted into acetic acid through reaction, so that the alcohol decomposition efficiency is improved; meanwhile, the suspension microcapsule has higher crosslinking degree, and the result is more regular, so that the survival rate of acetobacter is improved, and the bioavailability is improved.
Compared with the prior art, the invention has the following advantages and effects:
(1) The invention directly converts alcohol into acetic acid by introducing oral acetobacter, solves the problem that the prior anti-alcoholic drug can not directly reduce the alcohol concentration, improves the anti-alcoholic efficiency, and avoids damage to organs such as kidneys, livers and the like.
(2) The invention uses the suspending microcapsule as carrier to encapsulate probiotics for treatment, so that the probiotics concentrate on stomach to play a role, thereby prolonging the acting time and improving the bioavailability. The pore structure of the suspension microcapsule enables the acetobacter to directly contact with alcohol to convert the alcohol into acetic acid through reaction, so that the efficiency of reducing the concentration of the alcohol is improved.
(3) The bacteria deposition is avoided by the embedding technology, the dispersibility is improved, and the suspension property of the material is improved, so that the contact probability of the bacteria and alcohol is greatly increased, and the alcohol metabolism rate is greatly improved.
(4) The suspension microcapsule has a more regular structure, and the survival rate of the acetobacter is improved.
(5) Compared with other probiotics oral delivery systems, the probiotics microcapsule does not depend on the effect of digestive enzymes in vivo, is less influenced by individual factors, and is beneficial to popularization.
(6) The invention adopts the emulsion method, the preparation process is simple, rapid and mild, no activity loss exists, the microcapsule particle size is uniform, and the invention is suitable for industrial production.
(7) The raw materials (sodium alginate, calcium chloride and calcium carbonate) used by the microcapsule have good biological safety and biocompatibility, are widely used in the field of food processing, and acetic acid generated by acetobacter does not cause adverse reaction on a human body because the stomach is an acidic environment, and meanwhile, the acetic acid can be further converted into carbon dioxide, so that the biological safety is ensured.
Drawings
FIG. 1 is a scanning electron microscope image of the microcapsule of Experimental example 1 after freeze-drying, wherein AC represents the suspension microcapsule prepared in example 1, and Control represents the normal microcapsule prepared in comparative example 1;
FIG. 2 is an image of the microcapsules of experimental example 2 after shaking and standing for 3min, AC represents the suspension microcapsules prepared in example 1, and Control represents the ordinary microcapsules prepared in comparative example 1.
FIG. 3 is a graph of an anatomic fluorescence image of the digestive tract of a Balb/c mouse after 24h of gastric lavage, where Plain represents unencapsulated, control represents the normal microcapsule of comparative example 1, and AC represents the suspension microcapsule prepared in example 1.
FIG. 4 shows the viability of the microcapsules of experimental example 4 in artificial gastric juice for different periods of time, wherein Plain represents the unencapsulated, control represents the normal microcapsules of comparative example 1, and AC represents the suspended microcapsules prepared in example 1.
FIG. 5 shows the blood alcohol concentrations of different groups of experimental example 5, wherein Plain represents unencapsulated, control represents the normal microcapsules of comparative example 1, and AC represents the suspension microcapsules prepared in example 1.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
Example 1
(1) Culturing activated Acetobacter (from the collection of microorganism strains of Guangdong province, model: GDMCC 1.152) to OD value of 0.4, centrifuging, discarding supernatant, and mixing at volume ratio of 1:10 was resuspended in 0.5% sodium alginate (molecular weight 20 ten thousand) solution and 0.01g of calcium carbonate particles were added.
(2) Uniformly dispersing the mixed solution into 3ml of oil, dropwise adding 1ml of acetic acid and oil mixed solution with volume ratio of 1:200, stirring at 800rpm for 10min, sucking the supernatant, and washing with deionized water for 2 times to obtain calcium alginate suspension microcapsule (AC) encapsulating probiotics.
Example 2
(1) Culturing activated Acetobacter (from the collection of microorganism strains of Guangdong province, model: GDMCC 1.152) to OD value of 0.5, centrifuging, discarding supernatant, and mixing at volume ratio of 1:20 are resuspended in 2.5% sodium alginate (molecular weight 1 ten thousand) solution and 0.03g of calcium carbonate particles are added.
(2) Uniformly dispersing the mixed solution into 3ml of oil, dropwise adding 1ml of acetic acid and oil mixed solution with volume ratio of 1:100, stirring at 600rpm for 20min, sucking the supernatant, and washing with deionized water for 2 times to obtain calcium alginate suspension microcapsule (AC) encapsulating probiotics. .
Example 3
(1) Culturing activated Acetobacter (from the collection of microorganism strains of Guangdong province, model: GDMCC 1.152) to OD value of 0.6, centrifuging, discarding supernatant, and mixing at volume ratio of 1:15 was resuspended in 2% sodium alginate (molecular weight 10 ten thousand) solution and 0.06g of calcium carbonate particles were added.
(2) Uniformly dispersing the mixed solution into 3ml of oil, dropwise adding 1ml of acetic acid and oil mixed solution with volume ratio of 1:50, stirring at 400rpm for 40min, sucking the supernatant, and washing with deionized water for 2 times to obtain calcium alginate suspension microcapsule (AC) encapsulating probiotics.
Comparative example 1
(1) Culturing activated Acetobacter (from the collection of microorganism strains of Guangdong province, model: GDMCC 1.152) to OD value of 0.4, centrifuging, discarding supernatant, and mixing at volume ratio of 1:10 was resuspended in 0.5% sodium alginate (molecular weight 20 ten thousand) solution.
(2) Uniformly dispersing the mixed solution into 4ml of oil, dropwise adding 500ml of 5% calcium chloride solution, stirring at 800rpm for 10min, sucking the supernatant, and washing with deionized water for 2 times to obtain calcium alginate ordinary microcapsule with Control group for encapsulating probiotics.
Comparative example 2
(1) Culturing activated Acetobacter (from the collection of microorganism strains of Guangdong province, model: GDMCC 1.152) to OD value of 0.5, centrifuging, discarding supernatant, and mixing at volume ratio of 1:20 are resuspended in 2.5% sodium alginate (molecular weight 1 ten thousand).
(2) Uniformly dispersing the mixed solution into 4ml of oil, dropwise adding 1000ml of 3% calcium chloride solution, stirring at 600rpm for 20min, sucking the supernatant, and washing with deionized water for 2 times to obtain calcium alginate ordinary microcapsule with Control group for encapsulating probiotics.
Comparative example 3
(1) Culturing activated Acetobacter (from the collection of microorganism strains of Guangdong province, model: GDMCC 1.152) to OD value of 0.6, centrifuging, discarding supernatant, and mixing at volume ratio of 1:15 was resuspended in 2% sodium alginate (molecular weight 10 ten thousand) solution.
(2) Uniformly dispersing the mixed solution into 4ml of oil, dropwise adding 1500ml of 1% calcium chloride solution, stirring at 400rpm for 40min, sucking the supernatant, and washing with deionized water for 2 times to obtain calcium alginate ordinary microcapsule with Control group for encapsulating probiotics.
Experimental example 1 surface morphology Observation
After freeze-drying the suspension microcapsules (AC) of example 1 and the ordinary microcapsules of comparative example 1, the surface morphology was observed with a scanning electron microscope.
As can be taken from fig. 1, AC has higher regularity and pores exist on the surface compared to the Control group normal microcapsules. This is because calcium ions obtained by the reaction of acetic acid and calcium carbonate in the preparation process are crosslinked with sodium alginate to form microcapsules, and the released carbon dioxide gas leaves pores on the surface.
Experimental example 2 suspension State test
The suspension microcapsules (AC) of example 1 and the ordinary microcapsules of comparative example 1 were resuspended in deionized water, and after shaking, left to stand for 3 minutes, and the suspended state of the microcapsules was observed with a high-resolution digital camera.
As shown in fig. 2, 3min after oscillation, the ordinary microcapsules had all settled to the bottom; the AC obtains larger buoyancy due to the pores on the surface and carbon dioxide gas retained in the microcapsules, and a large number of the microcapsules are in a suspension state after 3 min.
Experimental example 3 gastric retention detection
For ease of characterization, a characterization experiment was performed using E.coli MG1655 harboring a plasmid instead of Acetobacter. MG1655 and plasmid puc57-tac-mcherry were all from Wohan vast, biotech. MG1655 (150. Mu.L, 2 MG) was incubated with plasmid DNA (5 ng) on ice for 30 minutes, heat-shocked at 42℃for 45s, and then returned to ice for 2 minutes. Next, LB medium (800. Mu.L) was added to the tube and incubated at 37℃for 1 hour. The bacterial suspension (50. Mu.L) was plated on LB agar plates containing ampicillin and incubated overnight at 37 ℃. And picking single colonies for subsequent experiments. The specific operation is as follows: after culturing E.coli MG1655 to OD 0.5, the supernatant was centrifuged off for use. Referring to the methods of examples 1 and 4, suspension microcapsules and floating microcapsules were prepared and prepared for the relevant detection.
The isolated gastrointestinal tract was obtained by intragastric lavage of 12 week old Balb/c mice for 24 hours and dissecting the experimental group suspension microcapsules of example 1 and the control group normal microcapsules of comparative example 1, respectively, for fluorescence imaging analysis.
As shown in FIG. 3, the pure bacterial fluid of Plain group and the fluorescence signal of Control group are concentrated at the end of intestinal tract after 24 hours. The fluorescence of the AC group was closer to the stomach than that of Plain and Control groups, demonstrating that the microcapsules exhibited good gastric retention under suspension, prolonged the duration of action in the stomach, and was substantially consistent with the results of experimental example 2.
Experimental example 4 acid resistance test
The obtained suspension microcapsules of example 1 and ordinary microcapsules of comparative example 1 were resuspended in 1mL of sterile physiological saline, and added to 9mL of artificial gastric juice with ph=2.5, followed by shake culture for 1h, 2h, 6h, 12h, and 24h, gradient dilution, plating plate count, and viable bacteria ratio measurement.
As shown in FIG. 4, the survival rate of the probiotics in the AC group is higher than that in the Plain group and the Control group in the artificial gastric acid culture for 24 hours. Compared with Plain groups and Control groups, the AC group has good encapsulation and protection effects due to the regular structure, the survival rate of probiotics under gastric juice is improved, and the feasibility of long-time gastric retention is demonstrated.
Experimental example 5 in vivo anti-hangover test
The experimental group suspension microcapsules of example 1 and the control group common microcapsules of comparative example 1 were respectively subjected to gastric lavage on 12-week-old Balb/c mice, and 50% ethanol was added to the same amount to the stomach of all groups, and blood was taken at tail veins for 15, 30, 45 and 60min, respectively, and the alcohol content in the blood was measured by using an alcohol concentration meter.
As can be seen from fig. 5, after the mice were perfused with ethanol, the concentration of ethanol in the blood of Plain group gradually increased and reached a maximum value at 40min, followed by gradual decrease under the effect of in vivo metabolism. Within 2 hours, the alcohol content of the AC group was lower than that of both the Plain group and the Control group, indicating the ability of the AC group to rapidly metabolize gastric alcohol. The pores of the AC group suspension microcapsule enable the encapsulated acetobacter to be in direct contact with stomach alcohol, so that the metabolism efficiency is improved; meanwhile, the suspended microcapsule prolongs the gastric retention time, concentrates on the stomach and achieves the effects of accurate and long-acting alcohol dispelling. .
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (5)
1. A method for preparing a calcium alginate suspension microcapsule for encapsulating probiotics, comprising the following steps:
centrifuging activated bacillus aceticus, discarding supernatant and sodium alginate solution, adding calcium carbonate particles, placing in an oil phase, adding mixed solution of oil and acetic acid, stirring, removing supernatant, and washing to obtain calcium alginate suspension microcapsule encapsulating probiotics;
the volume ratio of the acetobacter to the sodium alginate is 1:10-20 configuration;
the mass volume ratio of the calcium carbonate particles to the sodium alginate is 1:4-4:1;
the molecular weight of the sodium alginate is 1-20 ten thousand, and the mass concentration of the sodium alginate solution is 0.5-2.5%;
the volume ratio of the acetic acid to the oil in the mixed solution of the oil and the acetic acid is 0.005-0.02.
2. The method of preparing a probiotic-encapsulated calcium alginate suspension microcapsule according to claim 1, wherein the acetobacter is cultured to an OD value of 0.4-0.6.
3. The method for preparing the probiotic-encapsulated calcium alginate suspension microcapsule according to claim 1, wherein the stirring speed is 400-800rpm and the stirring time is 10-40min.
4. A probiotic-encapsulated calcium alginate suspension microcapsule prepared according to the method of preparation of the probiotic-encapsulated calcium alginate suspension microcapsule of any one of claims 1 to 3.
5. Use of a calcium alginate suspension microcapsule encapsulating a probiotic according to claim 4 for the preparation of an anti-hangover medicament.
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