CN115300480A - Probiotics-encapsulated calcium alginate suspension microcapsule and preparation method and application thereof - Google Patents
Probiotics-encapsulated calcium alginate suspension microcapsule and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
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- 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
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- 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
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Abstract
The invention discloses a probiotic encapsulated calcium alginate suspension microcapsule, a preparation method and application thereof, and belongs to the field of biomedical engineering. The suspension microcapsule has the core that the calcium alginate microcapsule with pores is used for encapsulating the acetobacter aceti, the acetobacter aceti can directly metabolize alcohol which reaches the stomach after drinking, and the concentration of the alcohol in the stomach is reduced, so that the aim of reducing the absorption of the alcohol is fulfilled; 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 prolonged; the high crosslinking degree and regular structure enhances the acid resistance, improves the survival rate of probiotics and improves the bioavailability. Therefore, the invention provides the probiotic-encapsulated calcium alginate suspension microcapsule which is resistant to gastric acid, long in gastric retention time and high in alcoholism relieving efficiency.
Description
Technical Field
The invention belongs to the field of biomedical engineering, and particularly relates to a calcium alginate suspension microcapsule for encapsulating probiotics, and a preparation method and application thereof.
Background
In recent years, people suffering from alcoholism and alcoholism in China are increasing, and the 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, liver cirrhosis and other diseases, and investigation studies show that about 57.5% of patients who drink for a long time have fatty liver and 15% of patients have liver cirrhosis. The incidence of some digestive system tumors such as gastric cancer, esophageal cancer and liver cancer is also closely related to alcoholism.
Most of the anti-alcoholics in the market are focused on delaying alcohol absorption and accelerating alcohol metabolism, and can relieve headache and nausea symptoms. However, they cannot directly reduce the intake and metabolism of alcohol, and still cause metabolic burden to liver and kidney to cause various degrees of damage. The oxidative metabolism of ethanol can cause the reduction of coenzyme NAD in vivo, and further influences the running of the tricarboxylic acid cycle energy supply chain of liver cells, thereby causing the shortage of energy supply in the cells. Secondly, the ethanol metabolite generates a large amount of free radicals after undergoing 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 antialcoholic drug cannot radically reduce the content of alcohol, how to design a novel antialcoholic drug capable of directly converting alcohol in the stomach through the structural innovation of a carrier is an important problem to be solved urgently at present.
Disclosure of Invention
In order to solve the disadvantages and shortcomings of the above technology, the primary object of the present invention is to provide a method for preparing calcium alginate suspension microcapsule encapsulating probiotics. The preparation method comprises the steps of dispersing calcium carbonate into sodium alginate and acetic acid bacillus solution in an oil phase, and dripping acetic acid solution to generate calcium ions to be crosslinked with the sodium alginate to form calcium alginate microcapsules; the released carbon dioxide permeates into the calcium alginate matrix, leaving behind pores that provide buoyancy to the microcapsules, enabling them to be suspended in gastric fluid. In the prior art, probiotics are directly orally taken and can quickly pass through the stomach under the action of gastric emptying, so that the emptying is quick, the action time is short, the absorption is irregular, and the action effect is greatly influenced. The oral bacillus aceticus probiotics microcapsule for alleviating hangover by gastric retention can improve the retention time of probiotics in the stomach through a gastric retention suspension drug delivery system, and can be concentrated in the stomach for drug delivery, so that the bioavailability is improved.
The second purpose of the invention is to provide a probiotic bacteria-encapsulated calcium alginate suspension microcapsule, which is a novel preparation for decomposing alcohol by oral probiotic bacteria. Acetobacter is a kind of brevibacterium which can oxidize alcohol into acetic acid and other products, and has strong alcohol resistance and acetic acid resistance. The acetobacter is delivered to the stomach, so that alcohol is converted into acetic acid, the content of the alcohol in the stomach is reduced, and the method has the advantages of convenience in administration, good tolerance of patients, easiness in large-scale popularization and the like.
The third purpose of the invention is to provide the application of the calcium alginate suspension microcapsule for encapsulating probiotics in the aspect of relieving alcoholism.
The primary purpose of the invention is realized by the following technical scheme:
a preparation method of calcium alginate suspension microcapsules encapsulating probiotics comprises the following steps:
centrifuging activated bacillus aceticus, removing supernatant and suspending weight of sodium alginate solution, adding calcium carbonate particles, placing into an oil phase, adding a mixed solution of oil and acetic acid, stirring, removing supernatant, and washing to obtain the probiotic encapsulated calcium alginate suspended microcapsule.
Preferably, the acetobacter is cultured to OD value of 0.4-0.6.
Preferably, the acetobacter and sodium alginate are mixed according to a volume ratio of 1:10-20 configuration.
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.
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 purpose of the invention is realized by the following technical scheme:
a calcium alginate suspension microcapsule encapsulating probiotics is prepared by the preparation method.
The third purpose of the invention is realized by the following technical scheme:
application of calcium alginate suspension microcapsule for encapsulating probiotics in relieving hangover is provided.
The principle of the invention is as follows:
calcium carbonate is dispersed into sodium alginate and acetic acid bacillus solution in an oil phase, and the acetic acid solution is dripped into the calcium carbonate and the acetic acid solution to generate calcium ions which are crosslinked with the sodium alginate to form a 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 be suspended in gastric fluid. In the prior art, probiotics are directly orally taken and can quickly pass through the stomach under the action of gastric emptying, so that the emptying is quick, the action time is short, the absorption is irregular, and the action effect is greatly influenced. Firstly, the suspension drug delivery system for gastric retention can improve the retention time of probiotics in the stomach, and the drug delivery system concentrates on the stomach to play the role of the probiotics in the part with the highest alcohol concentration, thereby prolonging the acting time; secondly, the acetobacter can directly contact with alcohol through pores, and ethanol is converted into acetic acid through reaction, so that the efficiency of decomposing the alcohol is improved; meanwhile, the suspension microcapsule has higher crosslinking degree, the result is more regular, the survival rate of the acetobacter is improved, and the bioavailability is improved.
Compared with the prior art, the invention has the following advantages and effects:
(1) The invention introduces the mode of orally taking acetobacter to directly convert alcohol into acetic acid, solves the problem that the alcohol concentration cannot be directly reduced by the existing anti-alcoholism drug, improves the efficiency of understanding the alcohol, and avoids the damage to organs such as kidney, liver and the like.
(2) The invention uses the suspension microcapsule as a carrier to encapsulate probiotics for treatment, so that the probiotics can be concentrated in the stomach to play a role, the acting time is prolonged, and the bioavailability is improved. The pore structure of the suspension microcapsule enables the acetobacter aceti to directly contact with alcohol to convert the ethanol into the acetic acid through reaction, and the efficiency of reducing the concentration of the ethanol is improved.
(3) The embedding technology avoids bacterial deposition, improves dispersibility, and the suspension property of the material greatly increases the contact probability of bacteria and alcohol and greatly improves the alcohol metabolic rate.
(4) The suspension microcapsule has a more regular structure, and the survival rate of the acetobacter is improved.
(5) Compared with other probiotic oral delivery systems, the probiotic microcapsule disclosed by the invention is independent of the action of digestive enzymes in vivo, is less influenced by individual factors, and is beneficial to popularization.
(6) The invention adopts an emulsion method, has simple, rapid and mild preparation process, no activity loss and uniform microcapsule grain diameter, and 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, and are widely used in the field of food processing, the acetic acid generated by acetobacter does not cause adverse reaction to human bodies, and the biological safety is guaranteed because the stomach is in an acidic environment and the acetic acid can be further converted into carbon dioxide.
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 general microcapsule prepared in comparative example 1;
fig. 2 is an image of different groups of microcapsules of experimental example 2 after shaking and standing for 3min, AC represents the suspension microcapsules prepared in example 1, and Control represents the general microcapsules prepared in comparative example 1.
FIG. 3 is a photograph of fluorescence image of digestive tract anatomy 24h after gastric lavage of Balb/c mice in Experimental example 3, wherein Plain represents non-embedded, control represents the ordinary microcapsule of comparative example 1, and AC represents the suspension microcapsule prepared in example 1.
Fig. 4 is a graph showing the survival rate of different groups of microcapsules of experimental example 4 cultured in artificial gastric juice for different periods of time, wherein pain represents non-embedded, control represents the common microcapsules of comparative example 1, and AC represents the suspension microcapsules prepared in example 1.
FIG. 5 is a graph showing the blood alcohol concentrations of different groups of Experimental example 5, in which Plain represents non-embedded, control represents general microcapsules of comparative example 1, and AC represents suspension microcapsules prepared in example 1.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
(1) After culturing activated Acetobacter aceti (purchased from Guangdong province culture Collection of microorganisms, model: GDMCC 1.152) to OD value of 0.4, centrifuging and discarding the supernatant, and mixing the supernatant according to a volume ratio of 1:10 are resuspended in a 0.5% sodium alginate solution (molecular weight 20 ten thousand) and 0.01g of calcium carbonate particles are added.
(2) Uniformly dispersing the mixed solution into 3ml of oil, dropwise adding 1ml of mixed solution of acetic acid and oil with the volume ratio of 1.
Example 2
(1) After culturing activated Acetobacter aceti (purchased from Guangdong province culture Collection of microorganisms, model: GDMCC 1.152) to OD 0.5, centrifuging and discarding the supernatant, and mixing the supernatant according to a volume ratio of 1:20 was resuspended in a 2.5% sodium alginate solution (molecular weight 1 ten thousand) and 0.03g of calcium carbonate particles were added.
(2) Uniformly dispersing the mixed solution into 3ml of oil, dropwise adding 1ml of mixed solution of acetic acid and oil in a volume ratio of 1. .
Example 3
(1) After culturing activated acetobacter aceti (purchased from Guangdong province culture collection of microorganisms, type: GDMCC 1.152) to OD value of 0.6, centrifuging and discarding supernatant, and mixing the supernatant according to a volume ratio of 1:15 are resuspended in a 2% sodium alginate solution (molecular weight 10 ten thousand) and 0.06g of calcium carbonate particles are added.
(2) Uniformly dispersing the mixed solution into 3ml of oil, dropwise adding 1ml of mixed solution of acetic acid and oil with the volume ratio of 1.
Comparative example 1
(1) After culturing activated Acetobacter aceti (purchased from Guangdong province culture Collection of microorganisms, model: GDMCC 1.152) to OD value of 0.4, centrifuging and discarding the supernatant, and mixing the supernatant according to a volume ratio of 1:10 resuspended in a 0.5% sodium alginate solution (molecular weight 20 ten thousand).
(2) Uniformly dispersing the mixed solution into 4ml of oil, dropwise adding 500ml of 5% calcium chloride solution, stirring at 800rpm for 10min, sucking supernatant, and washing with deionized water for 2 times to obtain the Control group probiotic-encapsulated calcium alginate common microcapsule.
Comparative example 2
(1) After culturing activated Acetobacter aceti (purchased from Guangdong province culture Collection of microorganisms, model: GDMCC 1.152) to OD 0.5, centrifuging and discarding the supernatant, and mixing the supernatant according to a volume ratio of 1:20 was resuspended in a 2.5% sodium alginate (molecular weight 1 ten thousand) solution.
(2) Uniformly dispersing the mixed solution into 4ml of oil, dropwise adding 1000ml of 3% calcium chloride solution, stirring at 600rpm for 20min, sucking supernatant, and washing with deionized water for 2 times to obtain the Control group probiotic-encapsulated calcium alginate common microcapsule.
Comparative example 3
(1) After culturing activated Acetobacter aceti (purchased from Guangdong province culture Collection of microorganisms, model: GDMCC 1.152) to OD value of 0.6, centrifuging and discarding the supernatant, and mixing the supernatant according to a volume ratio of 1:15 were resuspended in a 2% sodium alginate solution (molecular weight 10 ten thousand).
(2) Uniformly dispersing the mixed solution into 4ml of oil, dropwise adding 1500ml of 1% calcium chloride solution, stirring at 400rpm for 40min, sucking supernatant, and washing with deionized water for 2 times to obtain the Control group probiotic-encapsulated calcium alginate common microcapsule.
Experimental example 1 surface morphology Observation
The suspension microcapsules (AC) of example 1 and the conventional microcapsules of comparative example 1 were freeze-dried, and then the surface morphology was observed by a scanning electron microscope.
As can be seen from fig. 1, AC has a higher regularity and pores exist on the surface compared to the Control group of ordinary microcapsules. The 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 pores are left on the surface of the released carbon dioxide gas.
Experimental example 2 suspension State test
The suspension microcapsules (AC) of example 1 and the common microcapsules of comparative example 1 were resuspended in deionized water, shaken and then allowed to stand for 3min, and the suspension state of the microcapsules was observed with a high-resolution digital camera.
As shown in fig. 2, 3min after oscillation, the common microcapsule has completely sunk to the bottom; the AC obtains larger buoyancy due to the pores on the surface and carbon dioxide gas retained in the microcapsule, and a large amount of microcapsules are still in a suspension state after 3 min.
Experimental example 3 gastric Retention assay
For ease of characterization, the characterization experiment was performed using the plasmid-carrying red fluorescent E.coli MG1655 instead of Acetobacter. MG1655 and plasmid puc57-tac-mcherry are both from Wuhan, inc. \\565656569. 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 placed back on ice for 2min. 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 to detect. The specific operation is as follows: escherichia coli MG1655 was cultured to OD 0.5, and centrifuged to discard the supernatant. Referring to the methods of examples 1 and 4, suspension microcapsules and floating microcapsules were prepared and prepared for the relevant detection.
And (3) respectively gavage the Balb/c mice aged 12 weeks with the suspension microcapsules of the experimental group in the example 1 and the common microcapsules of the control group in the comparative example 1, and dissecting for 24 hours to obtain isolated gastrointestinal tracts for fluorescence imaging analysis.
As shown in FIG. 3, after 24h, fluorescence signals of the Plain group bacterial suspension and the Control group were concentrated at the end of the intestinal tract. Compared with the Plain group and the Control group, the fluorescence of the AC group is closer to the stomach, which shows that the microcapsule shows good gastric retention effect under the action of suspension, prolongs the action time of the microcapsule on the stomach, and is basically consistent with the result of the experimental example 2.
Experimental example 4 detection of acid resistance
The obtained suspension microcapsule of example 1 and the common microcapsule of comparative example 1 are re-suspended in 1mL sterile physiological saline, added into 9mL artificial gastric juice with pH =2.5 respectively, and cultured by shaking for 1h, 2h, 6h, 12h and 24h, and then diluted in a gradient way, coated plates are counted, and the viable cell rate is measured.
As shown in FIG. 4, the survival rate of probiotics in the AC group was higher than that in the Plain group and the Control group within 24h of culturing in artificial gastric acid. Compared with the Plain group and the Control group, the AC group shows good encapsulation protection effect due to the self-regulated structure, improves the survival rate of probiotics in gastric juice, and shows the feasibility of long-time gastric retention effect.
Experimental example 5 in vivo hangover alleviation test
The suspension microcapsules of the experimental group of example 1 and the common microcapsules of the control group of comparative example 1 were used for intragastric administration to 12-week-old Balb/c mice, and then 50% ethanol with the same amount as the intragastric administration was administered to all groups, blood was taken from the tail vein at 15 min, 30 min, 45 min, and 60min, and the alcohol content in the blood was measured using an alcohol concentration meter.
As can be seen from FIG. 5, after the mice were gavaged with ethanol, the blood alcohol concentration in the blood of the Plain group gradually increased and reached a maximum at 40min, and then gradually decreased by the action of in vivo metabolism. Within 2h, the alcohol content of the AC group was lower than that of the Plain group and that of the Control group, indicating the ability of the AC group to rapidly metabolize alcohol in the stomach. The pores of the suspension microcapsule of the AC group enable the encapsulated acetobacter to be directly contacted with alcohol in the stomach, so that the metabolism efficiency is improved; meanwhile, the suspended microcapsule prolongs the gastric retention time, focuses on the stomach function and achieves the effect of accurately and long-acting sobering up. .
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (9)
1. A preparation method of a calcium alginate suspension microcapsule for encapsulating probiotics is characterized by comprising the following steps:
centrifuging activated bacillus aceticus, removing supernatant and suspending weight of sodium alginate solution, adding calcium carbonate particles, placing into an oil phase, adding a mixed solution of oil and acetic acid, stirring, removing supernatant, and washing to obtain the probiotic encapsulated calcium alginate suspended microcapsule.
2. The method for preparing probiotic bacteria-encapsulated calcium alginate suspension microcapsules according to claim 1, wherein the acetobacter is cultured to OD value of 0.4-0.6.
3. The preparation method of the probiotic bacteria-encapsulated calcium alginate suspension microcapsule according to claim 1, wherein the ratio of the acetobacter aceti to the sodium alginate is 1:10-20 configuration.
4. The method for preparing the probiotic bacteria-encapsulated calcium alginate suspension microcapsule as claimed in claim 1, wherein 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%.
5. The preparation method of the probiotic bacteria-encapsulated calcium alginate suspension microcapsule according to claim 1, wherein the mass volume ratio of the calcium carbonate particles to the sodium alginate is 1.
6. The method for preparing probiotic bacteria-encapsulated calcium alginate suspension microcapsules according to claim 1, wherein the volume ratio of acetic acid to oil in the mixed solution of oil and acetic acid is 0.005-0.02.
7. The method for preparing probiotic bacteria-encapsulated calcium alginate suspension microcapsules according to claim 1, wherein the stirring speed is 400-800rpm and the stirring time is 10-40min.
8. A probiotic bacteria-encapsulated calcium alginate suspension microcapsule, characterized in that it is prepared according to the method for preparing probiotic bacteria-encapsulated calcium alginate suspension microcapsule of any one of claims 1 to 7.
9. Use of probiotic bacteria encapsulated calcium alginate suspension microcapsules according to claim 8 for anti-hangover purposes.
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