CN115553469B - Stomach mucosa-adhered probiotic slow-release microsphere, preparation method and medicine for preparing helicobacter pylori resisting or stomach mucositis relieving medicine - Google Patents
Stomach mucosa-adhered probiotic slow-release microsphere, preparation method and medicine for preparing helicobacter pylori resisting or stomach mucositis relieving medicine Download PDFInfo
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- 235000018291 probiotics Nutrition 0.000 title claims abstract description 200
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- 229940037467 helicobacter pylori Drugs 0.000 title claims abstract description 10
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
- A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P10/00—Shaping or working of foodstuffs characterised by the products
- A23P10/30—Encapsulation of particles, e.g. foodstuff additives
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Mycology (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Organic Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Preparation (AREA)
Abstract
The application relates to the technical field of probiotic microspheres, and discloses a gastric mucosa-adhered probiotic sustained-release microsphere, which comprises a probiotic microsphere body, wherein the surface of the probiotic microsphere body is modified with hericium erinaceus polysaccharide or a compound of hericium erinaceus polysaccharide and bacterial outer membrane protein, and the probiotic is stored in the probiotic microsphere body and can be released from the probiotic microsphere body. The probiotic slow-release microsphere provided by the application has higher gastric mucosa adhesiveness, can stably and properly release probiotics in the stomach, keeps higher activity under the probiotic action of hericium erinaceus polysaccharide, better plays the stomach protection and repair roles, and has obvious effects on relieving and treating helicobacter pylori and gastric mucosa inflammation.
Description
Technical Field
The application relates to the technical field of probiotic microspheres, in particular to a gastric mucosa-adhered probiotic slow-release microsphere, a preparation method and a drug for preparing helicobacter pylori resistance or relieving gastric mucositis.
Background
Probiotics are the general term for bacteria beneficial to human body, and can play a unique role in restoring gastrointestinal motility, improving mucous membrane secretion and relieving symptoms. However, the acidic environment in the stomach is unfavorable for the growth of most bacteria, and the peristaltic rhythm of the stomach is high, so that the variety and quantity of the bacteria in the stomach are obviously reduced compared with the intestinal tract, and the externally-supplemented probiotics are weak in activity and easy to inactivate, and only bacteria with strong gastric acid resistance can stay in the stomach. In order to ensure the activity of shelf life, the probiotics preparations on the market at present are embedded and released in the body too slowly, so that the probiotics cannot be fully released and exert the effect in the stomach.
The gastric mucosa adhesion slow release microspheres can enable the functional components to be released slowly or at a constant speed according to a certain rule, promote the functional components to keep a certain concentration in the stomach for a long time, reduce the loss of the functional components and improve the effects of disease treatment or health improvement. For example, chinese patent CN201310108525.2 discloses a bioadhesive microsphere which is retained in stomach, and the effective components of the bioadhesive microsphere are jateorhizine hydrochloride, palmatine hydrochloride and the like. So the preparation of the probiotic slow release microsphere to replace the current common probiotic embedding preparation can be used as a beneficial attempt for improving the state of the probiotics in the stomach. However, most of the functional components cannot be completely released in the stomach or are released too quickly to stably act due to the influence of the problems of short and unstable gastric emptying in the stomach, heterogeneity of the gastrointestinal system, low bioavailability of gastrointestinal flora, dosage form, surface area, enzyme activity and the like, so that the bioavailability of the functional components is low, and the frequency and cost for oral supplement are increased. Meanwhile, the active ingredients of the existing adhesive microspheres are mainly compounds, benefit from the limitations of activity of probiotics and the like, and the preparation method of the adhesive microspheres is directly applied to probiotics and has a certain obstacle. Therefore, how to realize the effect that the microspheres adhere to the gastric mucosa and slowly release the embedded functional components at the mucosa is a technical problem that the gastric mucosa-adhering slow-release microspheres are required to face, and is a technical problem that the preparation of the gastric mucosa-adhering probiotic slow-release microspheres is required to face.
Disclosure of Invention
The application aims to provide the probiotic slow-release microsphere adhered to the gastric mucosa, which has higher adhesion rate and proper and stable release rate in the stomach, thereby playing the effect of probiotics and playing the role of gastrointestinal protection.
The application provides the following technical scheme:
a probiotic slow release microsphere adhered to gastric mucosa comprises a probiotic microsphere body, wherein the surface of the probiotic microsphere body is modified with Hericium erinaceus polysaccharide or a compound of Hericium erinaceus polysaccharide and bacterial outer membrane protein, and the probiotic is stored in the probiotic microsphere body and can be released from the probiotic microsphere body. According to the probiotic slow-release microsphere disclosed by the application, the hericium erinaceus polysaccharide or the compound of the hericium erinaceus polysaccharide and the bacterial outer membrane protein is modified on the surface of the probiotic microsphere body, so that the adhesion rate of the probiotic slow-release microsphere on gastric mucosa is improved under the condition of keeping a higher embedding rate of the probiotic microsphere body, the stable and proper release of the probiotic in the stomach is ensured, the corresponding effect of the probiotic in the stomach is promoted, and the adhesion rate can be further improved by adding the bacterial outer membrane protein. The hericium erinaceus polysaccharide has a probiotic effect, can improve the tolerance of probiotics in the stomach, has a gastrointestinal protection effect, and is in complement with the probiotics released by the microspheres.
As the preferential selection of the application, the probiotic microsphere body is obtained by reacting probiotic feed liquid containing probiotics and sodium alginate with a calcium ion-containing solution, and after carboxymethyl, chitosan is connected with the hericium erinaceus polysaccharide or the compound of the hericium erinaceus polysaccharide and the bacterial outer membrane protein, and then the chitosan is connected with the surface of the probiotic microsphere body. In the research, the hericium erinaceus polysaccharide has better performance compared with other polysaccharides under the condition of using sodium alginate as a wall material, and the performance is higher in both adhesion rate and embedding rate.
As the preferable mode of the application, the mass ratio of the hericium erinaceus polysaccharide to the bacterial outer membrane protein is 1:1-15. Too much bacterial outer membrane protein may rather reduce the adhesion rate relatively.
Preferably, the probiotic feed liquid also contains gelatin. Gelatin is a hydrophilic protein and has strong emulsifying and film forming properties. Preferably, the mass ratio of the sodium alginate to the gelatin is 1.5:1-1.5.
Preferably, the probiotic microsphere body is prepared by spraying probiotic feed liquid containing probiotics and sodium alginate to calcium ion-containing solution for reaction under high voltage static electricity. In the technical proposal of the application, the high-voltage electrostatic spray forming is adoptedThe adhesion rate of the microspheres is higher, probably due to the fact that the particle size and the surface of the microsphere body obtained by high-voltage electrostatic spraying are more uniform, and the microsphere body is more easily modified by hericium erinaceus polysaccharide. Preferably, the electrostatic spraying method comprises the following steps: filtering the composite solution of sodium alginate and gelatin by a microporous filtering membrane to obtain a wall material solution, mixing the wall material solution with a probiotic suspension to obtain a probiotic feed liquid, sucking the probiotic feed liquid into a syringe, spraying the mixed liquid into a sterilized calcium chloride solution which is magnetically stirred by a high-voltage electrostatic spraying device, and carrying out Ca 2+ And carrying out crosslinking reaction on the mixture and sodium alginate to form microspheres, and standing and solidifying the microspheres for 30 to 60 minutes. Wherein the mass concentration of the sodium alginate is 1.5-2%, the mass concentration of the calcium chloride is 2-3%, the mass ratio of the wall material solution to the probiotic suspension is 1-3:1, the voltage of the high-voltage electrostatic spray is 15-23 KV, and the receiving distance is 10-20 cm. Preferably, the probiotic concentration in the probiotic suspension is 1.0X10 8 ～1.0×10 9 cfu/mL。
The preparation method of the probiotic slow-release microsphere comprises the following steps:
(1) Carboxymethylation of Hep or Hep-OmpA:
dispersing Hericium erinaceus polysaccharide Hep or a compound Hep-ompA of Hericium erinaceus polysaccharide and bacterial outer membrane protein in an alkaline solution of isopropanol to obtain a suspension, adding chloroacetic acid into the alkaline solution of isopropanol to dissolve the suspension to obtain a carboxymethylated solution, dropwise adding the carboxymethylated solution into the suspension, stirring the suspension to react, heating the suspension to 60-70 ℃, continuously dropwise adding the carboxymethylated solution to stir the mixture to react, regulating pH to be neutral after the reaction, dialyzing the mixture, and drying the mixture to obtain carboxymethylated Hep or Hep-ompA;
(2) Connection chitosan
Dissolving chitosan in acetic acid aqueous solution, regulating pH to 4-6 to obtain reaction solution I, preparing carboxymethylated Hep or Hep-ompA into polysaccharide solution, adding N-hydroxy thiosuccinimide and EDC-HCL to react, regulating pH to be neutral to obtain reaction solution II, mixing the reaction solution I and the reaction solution II, stirring to react, dialyzing, and drying to obtain the Hep or Hep-ompA connected with chitosan, namely Hep-CS and Hep-ompA-CS respectively;
(3) Acquisition of probiotic slow-release microspheres
Washing the probiotic microsphere body Pro-MSs by using 1.5-2wt% sodium alginate solution, chitosan solution and 0.2-0.3wt% sodium alginate solution, immersing the probiotic microsphere body Pro-MSs into a polysaccharide solution of Hep or Hep-OmpA connected with chitosan, and separating and drying the probiotic slow-release microsphere Hep-CS-Pro-MSs or Hep-OmpA-CS-Pro-MSs.
Preferably, in step (1),
the alkaline solution of the isopropanol is obtained by dissolving the isopropanol in a sodium hydroxide aqueous solution with the mass concentration of 20-30%, and the volume ratio of the isopropanol to the sodium hydroxide aqueous solution is 1:0.5-1;
and/or the concentration of the alkaline solution of Hep or Hep-OmpA relative to isopropanol is 0.02-0.03 g/mL;
and/or the concentration of the chloroacetic acid relative to the alkaline solution of isopropanol is 0.1-0.2 g/mL;
and/or the volume ratio of the suspension to the carboxymethylation solution is 1:1-2;
and/or, the dialysis process is: dialyzing with tap water for 2-4 days, and dialyzing with distilled water for 4-6 days.
Preferably, in step (2),
the concentration of chitosan in the reaction solution I is 0.02-0.03 mmol/mL;
and/or, in the reaction solution II, the mass concentration of carboxymethylated Hep or Hep-ompA is 1-2%, the mass concentration of N-hydroxy thiosuccinimide is 1-2%, and the mass concentration of EDC and HCL is 1-2%;
and/or the volume ratio of the reaction liquid I to the reaction liquid II is 4:1-2;
and/or the reaction temperature of the reaction liquid I and the reaction liquid II is 20-30 ℃ and the reaction time is 8-12 h;
and/or, the dialysis process is: dialyzing with deionized water for 40-50 h, dialyzing with Tris-phosphate buffer solution for 6-10 h, and dialyzing with deionized water for 20-30 h. The dialysis effect is more stable.
Preferably, in step (3),
the chitosan solution is acetic acid solution of chitosan, the concentration of the chitosan is 1-2%, and the washing time is 1-2 h;
and/or washing the sodium alginate solution for 10-20 min;
and/or, the concentration of the polysaccharide solution is 0.4-1.6%, and the mass ratio of Pro-MSs to the polysaccharide solution is 1:3-5;
and/or, the immersion time of Pro-MSs in the polysaccharide solution is 1-2 h.
The probiotic slow release microsphere or the probiotic slow release microsphere obtained by the preparation method is used for preparing the medicine for resisting helicobacter pylori or relieving gastric mucositis. Proved by researches, when the probiotic slow-release microspheres are adopted to treat helicobacter pylori or gastric inflammation, a higher treatment effect can be obtained. The probiotics can be selected from common strains with effects of restoring gastrointestinal motility, improving mucous membrane secretion, relieving chronic gastritis, gastric ulcer, duodenal ulcer, etc., such as lactobacillus rhamnosus L.rhamnosus, lactobacillus acidophilus L.acidophilus, lactobacillus reuteri, lactobacillus plantarum, etc.
The beneficial effects of the application are as follows:
the probiotic slow-release microsphere provided by the application has higher gastric mucosa adhesiveness, can stably and properly release probiotics in the stomach, keeps higher activity under the probiotic action of hericium erinaceus polysaccharide, better plays the stomach protection and repair roles, and has obvious effects on relieving and treating helicobacter pylori and gastric mucosa inflammation.
Drawings
FIG. 1 is an electron microscope scan of the probiotic slow release microspheres prepared in example 1.
FIG. 2 shows the viable count of probiotics at different wall material contents.
Figure 3 shows the entrapment rate of the probiotic slow-release microspheres at different core wall material ratios.
Figure 4 is an effect of polysaccharide species on the adhesion rate and probiotic survival rate of probiotic sustained release microspheres.
FIG. 5 is a graph showing the effect of concentration of Hericium erinaceus polysaccharide and bacterial outer membrane protein complex on the adhesion rate of probiotic sustained release microspheres.
FIG. 6 is the effect of varying amounts of Hericium erinaceus polysaccharide and bacterial outer membrane protein on the adhesion rate of a probiotic sustained release microsphere.
Figure 7 is the storage stability of the probiotic slow release microspheres.
Figure 8 is a release profile of probiotic slow release microspheres in simulated gastric fluid.
Detailed Description
The following is a further description of embodiments of the application.
Unless otherwise indicated, all starting materials used in the present application are commercially available or are commonly used in the art, and unless otherwise indicated, the methods in the examples below are all conventional in the art.
The application provides a probiotic slow-release microsphere adhered to gastric mucosa, which comprises a probiotic microsphere body, wherein the surface of the probiotic microsphere body is modified with hericium erinaceus polysaccharide or a compound of hericium erinaceus polysaccharide and bacterial outer membrane protein, and the probiotic is stored in the probiotic microsphere body and can be released from the probiotic microsphere body.
In some embodiments provided by the application, the probiotic microsphere body is obtained by reacting a probiotic feed liquid containing probiotics and sodium alginate with a calcium ion-containing solution, and after carboxymethyl, chitosan is connected to the Hericium erinaceus polysaccharide or a compound of the Hericium erinaceus polysaccharide and a bacterial outer membrane protein, and then the probiotic feed liquid is connected to the surface of the probiotic microsphere body.
In some embodiments provided by the application, the mass ratio of hericium erinaceus polysaccharide to bacterial outer membrane protein is 1:1-15.
In some embodiments provided herein, the probiotic feed solution further comprises gelatin. Gelatin is a hydrophilic protein and has strong emulsifying and film forming properties. Preferably, the mass ratio of the sodium alginate to the gelatin is 1.5:1-1.5.
In some embodiments provided by the application, the probiotic microsphere body is obtained by high-voltage electrostatic spraying of a probiotic feed liquid containing probiotics and sodium alginate to a calcium ion-containing solution for reaction.
The electrostatic spraying method adopted is as follows:
filtering the composite solution composed of sodium alginate and gelatin by a microporous filter membrane to obtain a wall material solution, and mixing the wall material solution with a probiotic suspension to obtainThe probiotic feed liquid is sucked into a syringe and sprayed into the sterilized calcium chloride solution which is magnetically stirred by a high-voltage electrostatic spraying device, ca 2+ And carrying out crosslinking reaction on the mixture and sodium alginate to form microspheres, and standing and solidifying the microspheres for 30 to 60 minutes. Wherein the mass concentration of the sodium alginate is 1.5-2%, the mass concentration of the calcium chloride is 2-3%, the mass ratio of the wall material solution to the probiotic suspension is 1-3:1, the voltage of the high-voltage electrostatic spray is 15-23 KV, and the receiving distance is 10-20 cm. Preferably, the probiotic concentration in the probiotic suspension is 1.0X10 8 ～1.0×10 9 cfu/mL。
The application provides a preparation method of the probiotic slow release microsphere in some embodiments, which comprises the following steps:
(1) Carboxymethylation of Hep or Hep-OmpA:
dispersing Hericium erinaceus polysaccharide Hep or a compound Hep-ompA of Hericium erinaceus polysaccharide and bacterial outer membrane protein in an alkaline solution of isopropanol to obtain a suspension, adding chloroacetic acid into the alkaline solution of isopropanol to dissolve the suspension to obtain a carboxymethylated solution, dropwise adding the carboxymethylated solution into the suspension, stirring the suspension to react, heating the suspension to 60-70 ℃, continuously dropwise adding the carboxymethylated solution to stir the mixture to react, regulating pH to be neutral after the reaction, dialyzing the mixture, and drying the mixture to obtain carboxymethylated Hep or Hep-ompA.
In some embodiments provided by the application, the alkaline solution of the isopropyl alcohol is obtained by dissolving the isopropyl alcohol in a sodium hydroxide aqueous solution with the mass concentration of 20-30%, and the volume ratio of the isopropyl alcohol to the sodium hydroxide aqueous solution is 1:0.5-1.
In some embodiments provided herein, the concentration of Hep or Hep-OmpA relative to the alkaline solution of isopropanol is from 0.02 to 0.03g/mL.
In some embodiments provided herein, the concentration of chloroacetic acid to the alkaline solution of isopropanol is 0.1 to 0.2g/mL.
In some embodiments provided herein, the volume ratio of suspension to carboxymethylation solution is 1:1-2.
In some embodiments provided herein, the dialysis process is: tap water for 2-4 days and distilled water for 4-6 days.
(2) Connection chitosan
Dissolving chitosan in acetic acid aqueous solution, regulating pH to 4-6 to obtain reaction solution I, preparing carboxymethylated Hep or Hep-ompA into polysaccharide solution, adding N-hydroxy thiosuccinimide and EDC.HCL to react, regulating pH to neutrality to obtain reaction solution II, mixing the reaction solution I and the reaction solution II, stirring to react, dialyzing, and drying to obtain the Hep or Hep-ompA connected with chitosan, namely Hep-CS and Hep-ompA-CS respectively.
In some embodiments provided herein, the chitosan concentration in reaction solution I is 0.02-0.03 mmol/mL.
In some embodiments provided by the application, in the reaction solution II, the mass concentration of carboxymethylated Hep or Hep-ompA is 1-2%, the mass concentration of N-hydroxy thiosuccinimide is 1-2%, and the mass concentration of EDC & HCL is 1-2%.
In some embodiments provided herein, the volume ratio of reaction solution I to reaction solution II is 4:1-2.
In some embodiments provided by the application, the reaction temperature of the reaction liquid I and the reaction liquid II is 20-30 ℃ and the reaction time is 8-12 h.
In some embodiments provided herein, the dialysis process is: dialyzing with deionized water for 40-50 h, dialyzing with Tris-phosphate buffer solution with a buffer range of 5.0-9.0 for 6-10 h, and dialyzing with deionized water for 20-30 h.
(3) Acquisition of probiotic slow-release microspheres
Washing the probiotic microsphere body Pro-MSs by using 1.5-2wt% sodium alginate solution, chitosan solution and 0.2-0.3wt% sodium alginate solution, immersing the probiotic microsphere body Pro-MSs into a polysaccharide solution of Hep or Hep-OmpA connected with chitosan, and separating and drying the probiotic slow-release microsphere Hep-CS-Pro-MSs or Hep-OmpA-CS-Pro-MSs.
In some embodiments provided by the application, the chitosan solution is acetic acid solution of chitosan, the mass concentration of chitosan is 1-2%, and the washing time is 1-2 h.
In some embodiments provided herein, the sodium alginate solution is washed for a period of time ranging from 10 to 20 minutes.
In some embodiments provided herein, the polysaccharide solution has a mass concentration of 0.4 to 1.6% and a mass ratio of Pro-MSs to polysaccharide solution of 1:3 to 5.
In some embodiments provided herein, the Pro-MSs are immersed in the polysaccharide solution for a period of time ranging from 1 to 2 hours.
The application also provides a technical scheme of the probiotic slow-release microsphere or the application of the probiotic slow-release microsphere in preparing anti-helicobacter pylori medicines or medicines for relieving gastric mucosal inflammation. The probiotics can be selected from common strains with effects of restoring gastrointestinal motility, improving mucous membrane secretion, relieving chronic gastritis, gastric ulcer, duodenal ulcer, etc., such as lactobacillus rhamnosus L.rhamnosus, lactobacillus acidophilus L.acidophilus, lactobacillus reuteri, lactobacillus plantarum, etc.
The technical scheme of the application is described in more detail through specific examples.
Example 1 (preparation of probiotic microsphere body)
(1) Cultivation of probiotics
Inoculating 400mL of probiotic bacteria liquid (lactobacillus acidophilus L.acidophilus) into a test tube of 9mL of skim milk by using a liquid-transferring device, culturing at 37 ℃ for 24 hours, which is the first generation of probiotic bacteria, then sucking 1mL of probiotic bacteria liquid from the uniformly-oscillated first generation of bacteria liquid by using the liquid-transferring device, inoculating into the test tube containing 9mL of skim milk, and subculturing at 37 ℃ for 24 hours, which is the second generation; inoculating 1mL of the second generation well-shaken bacterial solution into MRS liquid culture medium, culturing for 18h, centrifuging for 10min at 4500r/min, collecting bacterial cells, washing bacterial cells with sterilized normal saline for 3 times, adding sterilized PBS on bacterial cell precipitate, and regulating bacterial count to 1.0X10 8 cfu/mL, shaking and mixing uniformly to obtain probiotic suspension;
(2) Probiotics microsphere bulk cross-linking
Filtering the composite solution of sodium alginate and gelatin by a microporous filtering membrane to obtain a wall material solution, wherein the mass concentration of the sodium alginate is 1.5wt% and the mass concentration of the gelatin is 1wt%, mixing the wall material solution with the probiotic suspension liquid obtained in the step (1) according to the mass ratio of 3:1 to obtain probiotic feed liquid, and sucking the probiotic feed liquid into a syringe and fixing the syringeSpraying the mixed solution into sterilized calcium chloride solution with mass concentration of 3wt% under magnetic stirring on injection pump by high-voltage electrostatic spraying device, wherein the voltage of high-voltage electrostatic spraying is 20KV, the receiving distance is 15cm, standing and solidifying for 30min to obtain Ca 2+ And (3) carrying out a crosslinking reaction with sodium alginate to form microspheres, thereby obtaining a probiotic microsphere body.
Example 2 (preparation of probiotic sustained release microspheres)
(1) Carboxymethylation of Hep-OmpA:
dispersing 0.9g of hericium erinaceus polysaccharide-bacterial outer membrane protein in 15mL of a mixed solution of 25% NaOH and 20mL of isopropanol under ice bath condition, continuously stirring to form uniform suspension, dissolving 5.25g of chloroacetic acid in the mixed solution of 15mL of 20% NaOH and 20mL of isopropanol, slowly dripping half of the mixed solution into a reaction system, stirring at room temperature for reaction for 4 hours, heating the reaction solution to 65 ℃, continuously stirring for 40 minutes, dripping the other half of the mixed solution, continuously reacting at 65 ℃ for 1.5 hours, cooling to room temperature after stopping the reaction, and regulating the pH of the reaction solution to be neutral. Pouring the obtained reaction liquid into a regenerated cellulose dialysis bag, sequentially dialyzing with tap water for 3 days, dialyzing with distilled water for 5 days, performing rotary evaporation concentration, and finally performing freeze drying to obtain a powdery compound of carboxymethylated Hericium erinaceus polysaccharide and bacterial outer membrane protein;
(2) Connection chitosan
Dissolving 4mmol of chitosan in 100mL of deionized water containing 1mL of acetic acid, stirring until the chitosan is fully dissolved, slowly adding 40mL of deionized water, and adjusting the pH of the mixed solution to 4.5 by using 1mol/L NaOH to obtain a reaction solution I; dissolving a complex of carboxymethylated hericium erinaceus polysaccharide and bacterial outer membrane protein in deionized water to prepare a polysaccharide solution with the mass fraction of 1%, adding EDC (EDC)/HCL (mass fraction of 1%) and NHS (mass fraction of 1%), regulating the pH to 5.0, reacting for 30min, and regulating the pH to be neutral to obtain a reaction solution II; mixing the reaction solution I and the reaction solution II according to a volume ratio of 2:1, and stirring at 25 ℃ for reaction for 8 hours; transferring the reacted mixed solution into a dialysis bag for dialysis, deionized water 48h, tris-phosphate buffer solution 8h, deionized water for 1 day, and then freeze-drying to obtain the hericium erinaceus polysaccharide-bacterial outer membrane protein modified chitosan polymer;
(3) Acquisition of probiotic slow-release microspheres
Washing the probiotic microsphere body Pro-MSs obtained in the example 1 by using sodium alginate solution (with the concentration of 1.5 wt%), chitosan solution (with the concentration of 1 wt%), sodium alginate solution (with the concentration of 0.2 wt%) respectively for 10min, 1h and 10min, immersing the Pro-MSs in the solution (with the concentration of 1.2 wt%) of the chitosan polymer modified by the hericium erinaceus polysaccharide-bacterial outer membrane protein for 1h, keeping the mass ratio of 1:5, and separating and freeze-drying the probiotic slow-release microsphere Hep-ompA-CS-Pro-MSs.
The scanning electron microscope image of the beneficial bacteria slow-release microsphere Hep-ompA-CS-Pro-MSs is shown in figure 1, and the microspheres are uniformly spherical, and the hericium erinaceus polysaccharide-bacterial outer membrane protein complex is adhered on the surfaces of the microspheres.
Example 3 (study of the Effect of wall Material on probiotics)
The wall material solutions obtained in example 1 were added to MRS liquid medium in different amounts (0, 2wt%, 4wt%, 6 wt%) respectively, and then the probiotic suspension in example 1 was inoculated into MRS liquid medium in an amount of 5wt% for anaerobic culture at 37℃for 48 hours, and the viable count was calculated as required by the plate dilution counting method in GB4789.2-94, and the results are shown in FIG. 2.
As can be seen from the results in FIG. 2, the presence or absence of the wall material solution has no obvious effect on the viable count of the lactic acid bacteria, and the selected sodium alginate and gelatin have good biocompatibility with the lactic acid bacteria.
Example 4 (influence of core wall Material ratio)
The procedure of example 1 was repeated, the mass ratio of the wall material solution to the probiotic suspension in the probiotic microsphere bodies Pro-MSs was adjusted to 1:1, 2:1, and the embedding rate of the microsphere bodies obtained in example 1 and example 4 was tested, and the results are shown in FIG. 3.
Embedding rate = number of viable bacteria embedded by microsphere body/number of viable bacteria in probiotic feed liquid x 100%.
Viable count was measured according to the plate dilution counting method in GB 4789.2-94.
Wherein, the microsphere body is disintegrated by 180r/min in a shaking table at 37 ℃ of artificial intestinal juice, and then the viable count is tested.
As can be seen from FIG. 3, the embedding rate is highest when the core-wall material ratio is 1:2.
Example 5 (Effect of polysaccharide species on the adhesion Rate of probiotic sustained release microspheres)
The procedure of example 2 was repeated, and the modified release microspheres were prepared by replacing the Hericium erinaceus polysaccharide bacterial outer membrane protein complexes with different polysaccharide bacterial outer membrane protein complexes, and the adhesion rate of the microspheres and the survival condition of probiotics in simulated gastric fluid were tested, and the results are shown in FIG. 4.
The testing method comprises the following steps:
(1) Preparing simulated gastric juice:
taking 16.4mL of dilute hydrochloric acid and adding about 800mL of water to prepare an acidic aqueous solution with the pH value of 1.2, specifically taking 10g of pepsin, placing the pepsin into the dilute hydrochloric acid solvent, shaking to dissolve the pepsin, and finally adding water to the volume of 1000mL to obtain simulated gastric fluid;
(2) Adhesion test:
the 6-week old Hangzhou mice were anesthetized with diethyl ether and sacrificed by cervical removal, immediately dissected and isolated from the stomach, and after removal of the stomach contents, rinsed clean with normal saline. Placing gastric mucosa in a closed container for holding simulated gastric juice, uniformly scattering the probiotic microsphere body and different slow release microspheres into the simulated gastric juice, and culturing in a shaking table at 37 ℃ in a dark place for 1h. After culture, the A of simulated gastric juice is respectively measured at 600nm of ultraviolet spectrophotometer 600 According to A 600 Calculation of light transmittance T by = -log T 2 And placing the same amount of probiotics or microspheres in simulated gastric fluid to test light transmittance T 1 As the initial transmittance, to simulate the gastric juice transmittance T 0 For blank, the adhesion rate was calculated from the following formula:
adhesion rate= (|t 0 -T 1 |-|T 0 -T 2 |)/|T 0 -T 1 The results are shown in fig. 4, |×100%;
(3) Survival calculation
The number of viable bacteria before and after gastric juice culture simulation of the gastric mucosa was tested, and the survival rate, i.e., viable bacteria rate was calculated, and the result is shown in fig. 4.
As can be seen from FIG. 4, the viable bacteria rates of algal polysaccharide and yeast polysaccharide are lower than those of the probiotic microsphere bodies. The pullulan has high viable bacteria rate, but the adhesion rate is lower than that of the probiotic microsphere body. In combination, hericium erinaceus polysaccharide performs better.
Example 6 (Effect of Hericium erinaceus polysaccharide bacterial outer membrane protein concentration on the adhesion Rate of probiotic sustained release microspheres)
The procedure of example 2 was repeated, and in step (3), the solution concentrations of the hericium erinaceus polysaccharide-bacterial outer membrane protein modified chitosan polymer were 0.4, 0.8, 1.6 and 2.0wt% respectively, different probiotic slow release microspheres were prepared, and the adhesion rates of the probiotic slow release microspheres obtained under different conditions were tested, and the results are shown in fig. 5.
From the figure, when the concentration of the chitosan polymer modified by the hericium erinaceus polysaccharide-bacterial outer membrane protein is lower than 2%, the adhesion rate of the probiotic slow-release microspheres gradually increases along with the increase of the concentration.
Example 7 (effect of the compounding ratio of hericium erinaceus polysaccharide and bacterial outer membrane protein on the adhesion rate of the probiotic sustained-release microspheres) the procedure of example 2 was repeated, and in step (1), the mass ratio of hericium erinaceus polysaccharide to bacterial outer membrane protein was controlled to be 1:0, 1:1, 1:5, 1:15 and 1:20 respectively, different probiotic sustained-release microspheres were prepared, and the adhesion rate was tested, and the results are shown in fig. 6.
From the figure, the adhesion rate of the probiotic slow-release microspheres on the gastric mucosa shows a decreasing trend after increasing along with the increase of the outer membrane proteins of bacteria, namely, the adhesion rate is improved in the range of 1:1-15 compared with the adhesion rate of the probiotic slow-release microspheres only using hericium erinaceus polysaccharide.
Example 8 (Effect of Probiotics microsphere Mass preparation method on Probiotics sustained Release microsphere adhesion)
Lactobacillus rhamnosus L.rhamnosus, lactobacillus acidophilus L.acidophilus, lactobacillus reuteri L.plantarum, lactobacillus plantarum L.plantarum and compound bacteria with the concentration ratio of 1:1:1:1 are respectively selected as probiotics to prepare two groups of probiotic slow-release microspheres, wherein the probiotic microsphere bodies used in the group I are prepared by dripping probiotic feed liquid into a calcium chloride solution through a syringe according to a conventional instillation method, and the probiotics without microspheres are used as a control, and the adhesion is tested, and the results are shown in the following table 1.
From the table, the probiotic slow release microspheres can obviously improve the adhesiveness of probiotics. Compared with different preparation methods of the probiotic microsphere body, the probiotic microsphere prepared by the electrostatic spraying method has higher adhesiveness compared with the instillation method.
Example 9 (influence of dialysis method)
The procedure of example 2 was repeated to prepare probiotic sustained-release microspheres except that the dialysis in step (2) was performed in deionized water for 80 hours, five groups of probiotic sustained-release microspheres were prepared 5 times in this manner, the adhesion rate was tested, and compared with five groups of probiotic sustained-release microspheres prepared 5 times in example 2, as a result, it was found that the average adhesion rate of the probiotic sustained-release microspheres prepared by dialysis with deionized water for 80 hours was 86.2% (85.9%, 87.1%, 86.2%, 85.3%, 86.5% each time) and the standard deviation was 0.0067, whereas the average adhesion rate of the five groups of probiotic sustained-release microspheres obtained by repeating example 2 was 86.3% (86.2%, 85.7%, 86.5%, 86.4%, 86.7% each time) and the standard deviation was 0.0038. When the phosphate buffer solution (pH 6-8) is used instead of Tris-phosphate buffer solution, the volatility is lower than that of the Tris-phosphate buffer solution, but the Tris-phosphate buffer solution is still not as good as that of the deionized water. It can be seen that the dialysis method of example 2 can improve the adhesion stability of the probiotic sustained release microspheres of different batches.
Example 10 (method of cleaning Probiotics microsphere body)
The procedure of example 2 was repeated to prepare probiotic slow release microspheres, except that:
microsphere 1: in the step (3), the probiotic microsphere body is not cleaned, and five groups are repeatedly prepared;
microsphere 2: in the step (3), the probiotic microsphere body is washed by deionized water, and five groups are repeatedly prepared;
microsphere 3: in the step (3), the probiotic microsphere body only uses chitosan solution clear liquid, and five groups of probiotic microsphere bodies are repeatedly prepared;
microsphere 4: in the step (3), the probiotic microsphere body is only 1.5 weight percent sodium alginate solution clear liquid, and five groups are repeatedly prepared;
microsphere 5: in the step (3), the probiotic microsphere body is cleaned by using 1.5 weight percent sodium alginate solution and 1 weight percent chitosan solution, and five groups are repeatedly prepared;
microsphere 6: and (3) repeatedly preparing five groups, wherein the concentration of the chitosan solution used in the step (3) of cleaning the probiotic microsphere body is 3%.
Microsphere 0 was then five sets of microspheres prepared in accordance with repeat example 2.
The adhesion rates of the different microspheres were measured, averaged, and the standard deviation of the adhesion rates was calculated, and the results are shown in table 2.
It can be seen from the above table that the washing of the probiotic microsphere body before use has a certain influence on the adhesion of the prepared microsphere, which may be mainly caused by the change of the connection capacity of the probiotic microsphere body with the polysaccharide solution in different washing modes. The washing mode of the application has better effect, wherein the proper concentration of the chitosan solution is 1-2 wt%, and the excessive concentration can cause relative negative effect.
Example 11 (storage stability of probiotic sustained release microspheres)
The probiotic microsphere body prepared in example 1 and the probiotic slow release microsphere prepared in example 2 were placed under normal temperature conditions, the viable count was measured every 10d, and the storage performance was observed, and the results are shown in fig. 7.
From the above graph, the viable count of the probiotic sustained-release microsphere prepared in example 2 is a trend of firstly decreasing and then gradually flattening, 40d is a demarcation point, probably because the viable count of the probiotic is decreased due to the influence of environmental heat in the early stage, and reaches a flat and gradual period due to the protection effect of wall materials in 40d, so that the viable count is decreased and slowed down, and the viable count can still approach 10 after 40d of storage at normal temperature 8 cfu/mL, viable count of 10 10 cfu/mL and 10 7 cfu/mL. The probiotic microsphere body prepared in example 1 is in a similar trend, and it is seen that the surface modification of the hericium erinaceus polysaccharide and the bacterial outer membrane protein does not affect the storage stability of the microsphere. Of course, the results should be distinguished from the testing of viable bacteria rate in simulated gastric fluid in example 5. The number of viable bacteria of the non-embedded lactobacillus plantarum freeze-dried powder is reduced with the passage of time, and no viable bacteria can be detected by 40 d. This demonstrates that Pro-MSs are effective against external disturbances and also have an effect on their storage properties, prolonging the shelf life of the probiotic.
Example 12 (Release Effect of microspheres in simulated gastric fluid)
The probiotic microsphere body prepared in the example 1 and the probiotic slow release microsphere prepared in the example 2 are subjected to no embedding treatment, and the controlled release effect is tested in simulated gastric juice:
(1) 1mL of aqueous solution, 3.6mg/mL of microsphere concentration, 4mL of simulated gastric fluid (pH=1.2) was added, 8 samples were prepared in parallel, and the samples were placed in a 5mL centrifuge tube and mixed vertically at constant temperature and 37℃with the rotation speed of a vertical mixer: 20rpm/min;
(2) One sample was taken at 0, 1, 2, 4, 8, 12, 18, 24 hours, centrifuged at 3000rpm for 5min, 200. Mu.L of supernatant was taken, the activity of the bacterial liquid was determined by ultraviolet spectrophotometry, the blank was 200. Mu.L of simulated gastric juice, after the blank was subtracted, the release effect of active probiotics in Hep-ompA-CS-Pro-MSs in the simulated gastric juice was obtained by calculation and arrangement, and the result is shown in FIG. 8.
From the graph, the accumulated release rate of the probiotic slow-release microspheres at 1h is 18.1%, the accumulated release rate at 8h is 54.8%, the accumulated release rate of the probiotics at 20h is 89.1%, the accumulated rate of the probiotics is not changed any more in the later time, and compared with the body of the probiotic slow-release microspheres, the release rate is stable, faster and regular.
Example 13 (anti-helicobacter pylori experiment)
(1) Construction of infected mouse model
40 clean BALB/c mice with 6-8 weeks of age, male and female halves, body weight of 18+ -2 g, animal feeding control conditions of clean level, and water and feed used by the mice were sterilized.
(2) Mice were pre-antibiotic treated and PPI plus Hp lavage modeled, and 30 BALB/c mice that were successfully modeled were randomly divided into 3 groups of 10:
group 1 (ppi+antibiotic treatment group): fasted for 12h from day 3-10, perfused with PPI solution 0.25ml and ampicillin 0.25ml and clarithromycin 0.1ml, fasted for 3-4 hours after the completion of the stomach, used for 7 days, and sacrificed at the 4 th week after the last time of the stomach;
group 2 (probiotic sustained release microsphere treatment group): the probiotic slow-release microsphere prepared in example 2 was administered in a fresh bacterial suspension of 0.5 ml/lavage with bacterial count of 1×10, after a daily fast of 12h on days 1-10 9 CFU, fasted for 3-4 hours after the last gastric lavage, once daily for 10 days, and sacrificed the mice on the 4 th week whole after the last gastric lavage;
group 3 (model control): from day 1-10, 0.5ml of physiological saline was infused per day, once a day for 10 days, and all mice were sacrificed at week 4 after the last infusion;
group 4 (blank): from day 1-10, 0.5ml of physiological saline was infused per day, once a day for 10 days, and all mice were sacrificed at week 4 after the last infusion.
Treatment of mice: the mice were sacrificed by cervical scission and immediately dissected, gastric cavity removed, anterior stomach removed, cut along the greater curvature side, and intact stomach tissue (including duodenum, antrum, body, etc.) was removed. Half of the gastric mucosa was fixed with 10% formalin and then submitted to the pathology department for HE staining and Giemsa staining, and the other half of the tissues were subjected to the rapid urease test (observation time 5 min), smear and bacterial culture, and the results are shown in table 3.
Table 3 eradication rate of mice Hp for each group (n=10)
Group a compared to group B (P > 0.05) compared to group C model control (P < 0.05).
The results showed that the positive rate of the gastric mucosa fast urease test was significantly reduced in the antibiotic treatment group and the treatment group compared with the model control group, with a significant difference (P < 0.05), while the treatment group was lower than the antibiotic group, with no significant difference (P > 0.05) between the two. The eradication rate of the model control group was 0%, and the blank control group did not find Hp infection.
Example 14 (alleviation of gastric mucositis criteria)
After HE staining, observing the stomach histopathological change of the mice under a high-power microscope, scoring the inflammation degree of the mice, wherein the grading integral of the gastric mucosa inflammation refers to the Rauws grading standard, and is respectively the density (0-2 points) of the infiltration of the mucosa lamina propria inflammatory cells (mononuclear cells), the density (0-3 points) of the infiltration of the mucosa lamina propria neutrophil granulocyte, the density (0-3 points) of the mucosa intraepithelial neutrophil granulocyte and the degree (0-2 points) of the superficial erosion; no wetting was 0 score, mild wetting was 1 score, moderate wetting was 2 score, and severe wetting was 3 score for each of the projects. The sum of the 3 item scores is recorded as the change of the pathology score to measure the degree of gastritis.
All data are analyzed by using statistical software GmbphPad Prism 6.0, the metering data are expressed by mean ± standard deviation (i ± s), the difference between groups is compared by using One-way ANOVA, the comparison between every two is by using t test, and P <0.05 is the difference, which has statistical significance. Gastric mucosal inflammation changes were observed by light microscopy, and gastric mucosal inflammation grading at the time of sacrifice for each group of mice is shown in table 4.
Table 4 grading results of the degree of inflammation after treatment for each group of mice
The results show that the gastric mucosa epithelium of the normal mice is complete, the glands are densely arranged in the intrinsic layer, and part of the mucosa bottoms of the gastric sinus parts of the mice can be seen with little scattered lymphocytes. The model control mice had a higher degree of antral inflammation, and more lymphocyte, eosinophil and neutrophil infiltration was seen in the lamina propria. One animal has dense inflammatory cell infiltration in the intrinsic membrane, but no change of erosion, hemorrhage, ulcer and the like of the mucous membrane is seen. The antral inflammation of animals in both treatment groups A and B was reduced compared to the model group, with significant differences (P < 0.05), and no significant differences (P > 0.05) between treatment groups A and B.
Claims (5)
1. The probiotic slow release microsphere is characterized by comprising a probiotic microsphere body, wherein the surface of the probiotic microsphere body is modified with hericium erinaceus polysaccharide or a compound of hericium erinaceus polysaccharide and bacterial outer membrane protein, and the probiotic is stored in the probiotic microsphere body and can be released from the probiotic microsphere body;
the probiotic microsphere body is prepared by spraying probiotic feed liquid containing probiotics and sodium alginate to calcium ion-containing solution for reaction under high pressure and static electricity, and is connected with chitosan after carboxymethyl of hericium erinaceus polysaccharide or a compound of hericium erinaceus polysaccharide and bacterial outer membrane protein, and then is connected to the surface of the probiotic microsphere body;
gelatin is also contained in the probiotic feed liquid;
in the compound of the hericium erinaceus polysaccharide and the bacterial outer membrane protein, the mass ratio of the hericium erinaceus polysaccharide to the bacterial outer membrane protein is 1:1-15;
the preparation method of the probiotic slow-release microsphere comprises the following steps:
(1) Carboxymethylation of Hep or Hep-OmpA:
dispersing Hericium erinaceus polysaccharide Hep or a compound Hep-ompA of Hericium erinaceus polysaccharide and bacterial outer membrane protein in an alkaline solution of isopropanol to obtain a suspension, adding chloroacetic acid into the alkaline solution of isopropanol to dissolve the suspension to obtain a carboxymethylated solution, dropwise adding the carboxymethylated solution into the suspension, stirring the suspension to react, heating to 60-70 ℃, continuously dropwise adding the carboxymethylated solution to stir the mixture to react, regulating pH to be neutral after the reaction, dialyzing and drying to obtain carboxymethylated Hep or Hep-ompA, namely Hep-CS and Hep-ompA-CS respectively;
(2) Connection chitosan
Dissolving chitosan in acetic acid aqueous solution, regulating pH to 4-6 to obtain reaction solution I, preparing carboxymethylated Hep or Hep-OmpA into polysaccharide solution, adding N-hydroxy thiosuccinimide and EDC.HCL for reaction, regulating pH to be neutral to obtain reaction solution II, mixing the reaction solution I and the reaction solution II, stirring for reaction, dialyzing, and drying to obtain the Hep or Hep-OmpA connected with chitosan;
(3) Acquisition of probiotic slow-release microspheres
Washing the probiotic microsphere body Pro-MSs by using 1.5-2wt% sodium alginate solution, chitosan solution and 0.2-0.3wt% sodium alginate solution, immersing the probiotic microsphere body Pro-MSs into a polysaccharide solution of Hep or Hep-OmpA connected with chitosan, and separating and drying the probiotic slow-release microsphere Hep-CS-Pro-MSs or Hep-OmpA-CS-Pro-MSs.
2. The probiotic delayed release microsphere according to claim 1, wherein in step (1),
the alkaline solution of the isopropanol is obtained by dissolving the isopropanol in a sodium hydroxide aqueous solution with the mass concentration of 20-30%, and the volume ratio of the isopropanol to the sodium hydroxide aqueous solution is 1:0.5-1;
and/or the concentration of the alkaline solution of Hep or Hep-OmpA relative to isopropanol is 0.02-0.03 g/mL;
and/or the concentration of the chloroacetic acid relative to the alkaline solution of isopropanol is 0.1-0.2 g/mL;
and/or the volume ratio of the suspension to the carboxymethylation solution is 1:1-2;
and/or, the dialysis process is: dialyzing with tap water for 2-4 days, and dialyzing with distilled water for 4-6 days.
3. The probiotic delayed release microsphere according to claim 1, wherein in step (2),
the concentration of chitosan in the reaction solution I is 0.02-0.03 mmol/mL;
and/or, in the reaction solution II, the mass concentration of carboxymethylated Hep or Hep-ompA is 1-2%, the mass concentration of N-hydroxy thiosuccinimide is 1-2%, and the mass concentration of EDC.HCL is 1-2%;
and/or the volume ratio of the reaction liquid I to the reaction liquid II is 4:1-2;
and/or the reaction temperature of the reaction liquid I and the reaction liquid II is 20-30 ℃ and the reaction time is 8-12 h;
and/or, the dialysis process is: dialyzing with deionized water for 40-50 h, dialyzing with Tris-phosphate buffer solution for 6-10 h, and dialyzing with deionized water for 20-30 h.
4. The probiotic delayed release microsphere according to claim 1, wherein in step (3),
the chitosan solution is acetic acid solution of chitosan, the concentration of the chitosan is 1-2%, and the washing time is 1-2 h;
and/or washing the sodium alginate solution for 10-20 min;
and/or, the concentration of the polysaccharide solution is 0.4-1.6%, and the mass ratio of Pro-MSs to the polysaccharide solution is 1:3-5;
and/or, the immersion time of Pro-MSs in the polysaccharide solution is 1-2 h.
5. The use of the probiotic sustained-release microsphere according to any one of claims 1 to 4 for the preparation of a medicament against helicobacter pylori or for alleviating gastric mucositis;
the probiotics are lactobacillus rhamnosus, lactobacillus acidophilus, lactobacillus reuteri or lactobacillus plantarum.
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