WO2022266840A1 - Microcapsule de probiotiques, son procédé de préparation et son utilisation - Google Patents

Microcapsule de probiotiques, son procédé de préparation et son utilisation Download PDF

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Publication number
WO2022266840A1
WO2022266840A1 PCT/CN2021/101531 CN2021101531W WO2022266840A1 WO 2022266840 A1 WO2022266840 A1 WO 2022266840A1 CN 2021101531 W CN2021101531 W CN 2021101531W WO 2022266840 A1 WO2022266840 A1 WO 2022266840A1
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probiotic
inner shell
polydopamine
kda
core
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PCT/CN2021/101531
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English (en)
Chinese (zh)
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王涛
郑斌
郭庆陆
王志云
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天津大学
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Priority to PCT/CN2021/101531 priority Critical patent/WO2022266840A1/fr
Publication of WO2022266840A1 publication Critical patent/WO2022266840A1/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • 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
    • 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
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the invention relates to the field of probiotic products, in particular to a probiotic microcapsule.
  • the invention also relates to the preparation method and application of the probiotic microcapsule.
  • Type 2 diabetes is the most common type of diabetes.
  • the main diagnostic criterion is elevated blood sugar.
  • the occurrence of diabetes can cause lesions in blood vessels, kidneys, retina and nerves, which have a great impact on the lives of patients.
  • the main mechanism of its pathogenesis is that long-term insulin resistance leads to damage to the function of pancreatic ⁇ -cells, which leads to insufficient insulin secretion and elevated blood sugar.
  • Short-chain fatty acids are the fermentation products of probiotics in the intestine, which can promote the production of insulin by ⁇ cells and exert the effect of lowering blood sugar.
  • SCFA can stimulate the expression of GPR41 and/or GPR43 in colonic epithelial cells, thereby activating the AMPK signaling pathway in skeletal muscle, adipose tissue, and liver, and accelerating the uptake of glucose in the blood. Therefore, SCFA is a potential drug candidate for lowering blood sugar.
  • SCFA-producing probiotics refer to a class of active microorganisms that are beneficial to the host. They are a general term for active beneficial microorganisms that colonize the human intestinal tract or reproductive system and can produce definite health effects, thereby improving the host's micro-ecological balance and exerting beneficial effects. Widely used in bioengineering, industry and agriculture, food safety and life and health fields. SCFA produced by the metabolism of probiotics can promote the secretion of insulin by pancreatic beta cells after reaching the intestinal tract and increase the activity of AMPK enzyme in the liver and muscle tissue, thereby accelerating the uptake of blood sugar by cells and achieving the purpose of lowering blood sugar.
  • probiotics examples include, for example, Lactobacillus plantarum (Lactobacillus plantarum, L.P.), Bifidobacterium adolescentis, B.A.), Clostridium butyricum (Clostridium butyricum, C.B.) et al.
  • probiotics have found that human supplementation with probiotics must ensure that a sufficient amount of viable bacteria colonize the intestines to function. Therefore, the US FDA recommends that the minimum amount of active probiotics added to food be 10 6 cfu/g or 10 6 cfu/ml.
  • the survival conditions of probiotics are extremely harsh. Oxygen, temperature, humidity, etc. have a great impact on the survival of probiotics, which can lead to a significant reduction in the number of viable bacteria during production, transportation, storage and sales. The stability of probiotic preparations limits its application.
  • probiotics are easily damaged by gastric acid, bile salts, and various digestive enzymes after entering the digestive tract, and it is difficult to maintain a sufficient number of viable bacteria to colonize the intestinal tract and play a role.
  • the probiotic preparations on the market focus on solving the problem that the number of viable bacteria contained in the shelf-life products meets the requirements, but how to improve the acid resistance and bile salt resistance of probiotics, and how to improve the intestinal activity and colonization effect of probiotics are still unclear. The lack of effective solutions severely limits the efficacy of probiotic formulations.
  • Microencapsulation (microencapsulation) technology is an effective means of embedding probiotics. It uses natural or synthetic polymer materials as capsule materials, and uses chemical, physical or physicochemical methods to coat active substances, that is, capsule cores, to form a Microcapsules with semipermeable or hermetic membranes. Microencapsulation of live probiotic bacteria can isolate them from the external environment to a certain extent and improve their tolerance to adverse environments.
  • the wall materials of probiotic microcapsules mainly include casein, isolated soybean egg, whey protein isolate, gelatin (GEL), xanthan gum, chitosan, sodium alginate or cellulose acetate phthalate. species or several.
  • the microencapsulation in the prior art does not have a significant protective effect on the survival rate of probiotics in the digestive fluid of the gastrointestinal tract.
  • the reason may be that the capsule wall skeleton of the microcapsules is too loose, the structure is porous, and the surface hardness of the microcapsules is small, so that the digestive juice can enter the capsule core and cause the inactivation of probiotics.
  • the purpose of the present invention is to provide a kind of probiotic microcapsule, this probiotic microcapsule has acid resistance, bile salt resistance, the performance of resistance to digestive enzymes, has improved probiotic stability and probiotic adhesion, has enhanced probiotic Intestinal colonization effect, so that SCFA can be efficiently produced in situ in the intestine, and the purpose of lowering blood sugar can be achieved.
  • the present invention provides the following technical solutions:
  • a probiotic microcapsule comprising a core and a shell covering the core, wherein the core comprises one or more probiotics, and the shell comprises a polydopamine (PDA) inner shell and a sodium carboxymethylcellulose-gelatin polymer (CMC-GEL) outer shell, wherein the PDA inner shell coats the core, and the CMC-GEL outer shell is located outside the PDA inner shell And cover the PDA inner shell.
  • PDA polydopamine
  • CMC-GEL sodium carboxymethylcellulose-gelatin polymer
  • Aspect 2 Probiotic microcapsules according to Aspect 1, wherein said probiotic bacteria are selected from L.P., B.A., C.B. or a combination of two or more thereof.
  • Scheme 7 A method for preparing probiotic microcapsules according to any one of schemes 1 to 6, the method comprising the following steps:
  • probiotics are preferably selected from L.P., B.A., C.B. or a combination of two or more thereof;
  • the suspension of the spores obtained in step (i) in an aqueous solution is mixed with DA to form a mixture, wherein for each milliliter of said suspension
  • the probiotic content is preferably about 0.1 ⁇ 10 8 cfu to about 0.5 ⁇ 10 8 cfu, more preferably about 0.2 ⁇ 10 8 cfu to about 0.4 ⁇ 10 8 cfu, most preferably about 0.3 ⁇ 10 8 cfu, so
  • the amount of said DA is preferably about 1 mg to about 3 mg, more preferably about 2 mg, and said aqueous solution is preferably tris-hydrochloride (Tris-HCl) buffer solution;
  • step (iii) placing the mixed solution obtained in step (ii) under conditions capable of self-polymerizing the DA, preferably at a temperature of about 20°C to about 40°C, preferably about 25°C, and The pH is adjusted to about 8.0 to about 9.0, preferably about 8.5, and the polymerization is continued for about 0.1 hour to about 3 hours, preferably about 1 hour to about 2 hours, thereby depositing and forming the PDA inner shell on the periphery of the core, the PDA inner shell
  • the layer preferably has a thickness of about 0.1 ⁇ m to about 0.5 ⁇ m, preferably about 0.2 ⁇ m to about 0.4 ⁇ m, most preferably about 0.3 ⁇ m;
  • step (iv) Mix the mixture obtained from step (iii) containing the particles having the core and the PDA inner shell coating the core with the CMC-GEL aqueous solution, preferably for about 1 minute to about 10 minutes, preferably About 5 minutes to about 7 minutes, more preferably about 6 minutes, to deposit the CMC-GEL shell layer on the outer periphery of the PDA inner shell layer, the CMC-GEL shell layer preferably has a thickness of about 0.7 ⁇ m to about 1.0 ⁇ m, preferably about 0.8 ⁇ m to about 0.9 ⁇ m, most preferably a thickness of about 0.85 ⁇ m, thereby obtaining the probiotic microcapsules, wherein in the CMC-GEL aqueous solution, the concentration of the CMC-GEL is preferably about 1 to about 3% by weight, preferably About 2% by weight, the CMC-GEL molecular weight is preferably from about 50 kDa to about 200 kDa, more preferably about 100 kDa to about 110
  • Scheme 8 Probiotic microcapsules according to any one of schemes 1 to 6 or probiotic microcapsules prepared according to the method of scheme 7 for the reduction of organisms, preferably animals, more preferably mammals, most preferably humans, by oral administration The use of blood sugar levels in the body.
  • the probiotic microcapsule of the present invention overcomes the deficiencies of the prior art. Through the double-layer modification of the PDA layer and the CMC-GEL layer, it has the properties of acid resistance, bile salt resistance, and digestive enzyme resistance, and has improved probiotic stability and probiotic Bacterial adhesion, enhanced intestinal colonization effect of probiotics, so that SCFA can be efficiently produced in situ in the intestinal tract, and the purpose of lowering blood sugar can be achieved.
  • the SCFA produced by the probiotics included in the probiotic microcapsules of the present invention exerts hypoglycemic effect through two pathways.
  • the probiotic microcapsule of the present invention has safety and high efficiency by producing SCFA in situ.
  • the synthesis method of the PDA layer included in the probiotic microcapsule of the present invention is simple.
  • DA can form a uniform coating on the surface of the core after simple self-polymerization in alkaline Tris-HCl solution.
  • PDA has super adhesive properties, can adhere to intestinal epithelial cells, and has good biological safety, and is widely used in multifunctional modification of materials.
  • the PDA endows the probiotics with good adhesion ability and can enhance the effect of intestinal colonization of the probiotics.
  • the CMC-GEL included in the probiotic microcapsule of the present invention is formed by polymerization of CMC-Na and GEL.
  • CMC-Na is usually made from natural cellulose. After being polymerized with GEL, it can remain stable in the environment of strong acid and active protease in the stomach. After reaching the intestinal tract, it can be fermented and decomposed by intestinal bacteria.
  • the coating of CMC-GEL endows probiotics with strong acid resistance, bile salt resistance, digestive enzyme resistance, and improves the stability of probiotics.
  • the coating of the double-layer film endows more functions to the probiotic live cell therapy.
  • the method of oral administration of the probiotic microcapsules of the present invention is simpler and safer than other administration methods such as injection.
  • Figure 1 schematically depicts the structure of the probiotic microcapsules of the present invention.
  • Figure 2 schematically depicts an aggregate formed by two probiotic microcapsules of the present invention.
  • Figure 3 schematically depicts another aggregate formed by two probiotic microcapsules of the present invention.
  • Figure 4 is a scanning electron micrograph of a core containing L.P. probiotics.
  • Fig. 5 is a scanning electron micrograph of a particle "L.P.@PDA" having an L.P. core and a PDA inner shell covering the L.P. core.
  • Fig. 6 is the L.P. probiotic microcapsule "L.P.@PDA@CMC-GEL" with the L.P. bacterium core, the PDA inner shell layer coating the L.P. bacterium core layer and the CMC-GEL outer shell layer coating the PDA inner shell layer scanning electron microscope image.
  • the present invention provides a probiotic microcapsule.
  • the probiotic microcapsules of the present invention comprise a bacterium core 1 and a shell covering the bacterium core 1, wherein the bacterium core 1 contains one or more probiotics, and the shell contains PDA inner shell 2 and CMC-GEL shell 3, wherein said PDA inner shell 2 coats said bacterium core 1, and said CMC-GEL shell 3 is positioned outside said PDA inner shell 2 and wraps Cover the inner shell layer 2 of the PDA.
  • the number of probiotics contained in the bacterial core 1 is not particularly limited, it may contain one or more probiotics, preferably 1, 2, 3, 4, 5 , 6, 7, 8, 9 or 10 probiotics, most preferably comprising 1 probiotic.
  • the selection of the probiotics is not particularly limited, and it may be able to colonize the human intestinal tract or reproductive system to achieve efficient production of SCFA in situ in the intestinal tract, thereby achieving Any active beneficial microorganism for the purpose of lowering blood sugar.
  • the probiotics may include, but not limited to, L.P., B.A., C.B. or a combination of two or more of them.
  • two or more of the probiotic microcapsules can be aggregated to form probiotic microcapsule aggregates, as shown in Figures 2 and 3 of the description (where only aggregates formed by two probiotic microcapsules are shown).
  • the inner shell layer 2 of PDA may exist between two or more cores 1
  • the outer shell layer 3 of CMC-GEL may not necessarily exist, as shown in Figure 3 of the specification.
  • the PDA inner shell 2 is preferably in direct contact with the outer periphery of the bacterium core 1 .
  • the PDA inner shell layer 2 can be formed by self-polymerization of DA. Synthesis of PDA layers is well known in the art. For example, DA can form a uniform coating on the surface of the core through simple self-polymerization in alkaline Tris-HCl solution. Such self-polymerization process and its mechanism can be found for example in the prior art literature: Chao Pan et al., Polymerization-Mediated Multifunctionalization of Living Cells for Enhanced Cell-Based Therapy, Advanced Material, 2021, 2007379, DOI: 10.1002/adma.202007379 and supporting information for this article.
  • the PDA inner shell layer 2 preferably has a thickness of about 0.1 ⁇ m to about 0.5 ⁇ m, preferably about 0.2 ⁇ m to about 0.4 ⁇ m, most preferably 0.3 ⁇ m.
  • the "thickness" of the PDA inner shell 2 means the shortest distance from a point on the outer surface of the PDA inner shell 2 to the outer surface of the bacterium core 1 covered by the PDA inner shell 2 .
  • the "thickness" of the PDA inner shell 2 may be the same or different.
  • the thickness of the reported PDA inner shell 2 is measured by using a particle size analyzer to measure the particle diameter of the particles having the bacterium 1 and the PDA inner shell 2 covering the bacterium 1 and measuring the bacterium 1, and subtract them to get the value.
  • the thickness of the inner shell layer 2 of the PDA is preferably an average value obtained after performing the above-mentioned measurements several times (preferably at least 3 times).
  • the thickness of the inner shell layer 2 of the PDA is too large, such as greater than about 0.5 ⁇ m, the activity of the probiotic microcapsules of the present invention in the intestinal environment will be reduced, and the effect of producing SCFA will be affected; on the contrary, If the thickness of the inner shell layer 2 of the PDA is too small, such as less than about 0.1 ⁇ m, the probiotic microcapsules of the present invention may not adhere well to the intestinal epithelial cells, and thus be excreted prematurely.
  • the CMC-GEL outer shell layer 3 is preferably in direct contact with the outer periphery of the PDA inner shell layer 2 .
  • the CMC-GEL shell layer 3 is obtained by polymerizing CMC-Na and GEL.
  • the synthesis method is well known in the art, for example, refer to prior art documents: Sara Esteghlal et al., Physical and mechanical properties of gelatin-CMC Composite films under the influence of electrostatic interactions, International Journal of Biological Macromolecules, Volume 114, Pages 1-9, July 2018, DOI: 10.1016/j.ijbiomac.2018.03.079.
  • the CMC-GEL outer shell layer 3 is mainly bonded to the surface of the PDA inner shell layer 2 through the reaction between the carboxyl groups of PDA and the amino groups of GEL.
  • the weight ratio of the CMC-Na and GEL used is not particularly limited, but preferably the weight of the CMC-Na is greater than the weight of the GEL, more preferably The weight ratio of CMC-Na adopted and GEL is about (50-99):(1-50), preferably about (70-97):(3-30), more preferably about (80-95):(5 -20), most preferably about 90:10.
  • the CMC-GEL shell layer 3 synthesized by using CMC-Na and GEL within the above weight ratio range can well maintain stability in the environment of strong stomach acid, active protease and the like.
  • the CMC-GEL preferably has about 50 kDa to about 200 kDa, more preferably about 100 to about 110 kDa, most preferably a molecular weight of about 107 kDa. If the molecular weight of the CMC-GEL is too large, such as greater than about 200 kDa, then when the probiotic microcapsules of the present invention reach the intestinal tract, the CMC-GEL shell layer 3 cannot be quickly destroyed by the intestinal flora of the intestinal juice , may cause the probiotics to fail to produce SCFA well.
  • the CMC-GEL shell layer 3 may be destroyed prematurely in the gastric juice, thereby exposing the probiotics to the gastric acid environment, reducing the The probiotic activity.
  • the CMC-GEL synthesis process in addition to the CMC-Na and GEL in the solution of the CMC-Na and GEL, other substances can also be included as required, and these other substances can be synthesized between the CMC-Na and GEL.
  • the GEL is incorporated into the CMC-GEL during the polymerization process, so that it is deposited on the surface of the inner shell layer 2 of the PDA together with the CMC-GEL to realize the multifunctional modification of the cell surface.
  • the CMC-GEL shell layer 3 preferably has a thickness of about 0.7 ⁇ m to about 1.0 ⁇ m, preferably about 0.8 ⁇ m to about 0.9 ⁇ m, most preferably about 0.85 ⁇ m.
  • the "thickness" of the CMC-GEL shell layer 3 means the distance between a point on the outer surface of the CMC-GEL shell layer 3 and the PDA inner shell layer 2 covered by the CMC-GEL shell layer 3. The shortest distance to the outer surface. At different positions of the CMC-GEL shell layer 3, the "thickness" of the CMC-GEL shell layer 3 may be the same or different.
  • the thickness of the reported CMC-GEL shell layer 3 is by measuring the particle diameter of the probiotic microcapsules of the present invention and measuring the PDA having the bacterium 1 and coating the bacterium 1 with a particle size analyzer The particle diameters of the particles in the inner shell layer 2, and the value obtained after subtracting them.
  • the thickness of the CMC-GEL shell layer 3 is preferably an average value obtained after performing the above measurements multiple times (preferably at least 3 times).
  • the thickness of the CMC-GEL shell layer 3 is too large, such as greater than about 1.0 ⁇ m, then when the probiotic microcapsules of the present invention reach the intestinal tract, the CMC-GEL shell layer 3 cannot be quickly absorbed by the intestinal fluid. The destruction of intestinal flora may cause the probiotics to fail to produce SCFA well. If the thickness of the CMC-GEL shell layer 3 is too small, such as less than about 0.7 ⁇ m, the CMC-GEL shell layer 3 may be destroyed prematurely in the gastric juice, thereby exposing the probiotics to the gastric acid environment , reducing the activity of the probiotics.
  • the particle size range and average particle size of the probiotic microcapsules are not particularly limited. According to various factors such as the kind and quantity of the probiotics included in the single probiotic microcapsule of the present invention, the thickness of the CMC-GEL shell layer 3 and the thickness of the PDA inner shell layer 2, the particle size of the probiotic microcapsule of the present invention And the average particle size can vary within a wide range.
  • the probiotic microcapsules of the present invention may have an average particle size of about 2.8 ⁇ m to about 3.5 ⁇ m, preferably about 3.0 ⁇ m to about 3.3 ⁇ m.
  • the particle size range and average particle size of the probiotic microcapsules of the present invention can be determined by various methods such as electron microscope and particle size analyzer.
  • a plurality of probiotic microcapsules of the present invention may further aggregate to form probiotic microcapsule aggregates (as shown in Figure 2 or 3 of the specification) after standing for a period of time, resulting in the measured particle size range and average
  • the particle size is greatly increased, which is also confirmed by the electron micrographs of the probiotic microcapsules of the present invention.
  • the outer shell may include other layers besides the PDA inner shell layer 2 and the CMC-GEL outer shell layer 3 .
  • the outer shell may include other layers besides the PDA inner shell layer 2 and the CMC-GEL outer shell layer 3 .
  • it can be a medicament protection layer commonly used in the art, as long as these layers do not affect the activity of the probiotics, do not affect the decomposition of the CMC-GEL shell layer 3 in the intestinal tract, The stability of the CMC-GEL outer shell layer 3 in gastric juice and the adhesion of the PDA inner shell layer 2 to intestinal epithelial cells are sufficient. It should be noted that those skilled in the art are fully capable of making appropriate selections for the other layers according to desired application conditions.
  • the present invention relates to a method for preparing the probiotic microcapsules of the present invention as described above, the method comprising the following steps i to iv.
  • Step i providing a core 1 comprising one or more probiotics.
  • the step i may, for example, include the following specific process: Obtain subcultured probiotics through a probiotic culture process known in the art, and centrifuge them to obtain the required core 1 containing one or more probiotics.
  • the probiotics are also preferably selected from L.P., B.A., C.B. or a combination of two or more of them.
  • the core 1 is obtained from the above step i, as an example, a core containing one or more L.P. probiotics is shown, and the scale bar of the figure is 500 nm.
  • Step ii mixing the suspension of the core 1 obtained in step i in the aqueous solution with DA at a temperature of about 20°C to about 40°C, preferably about 25°C, to form a mixture.
  • the content of probiotics in the suspension solution is not particularly limited, as long as it can match the amount of PDA and CMC-GEL used in the method of the present invention to form the microcapsules of the present invention.
  • the probiotic content is preferably from about 0.1 ⁇ 10 8 cfu to about 0.5 ⁇ 10 8 cfu per milliliter of the suspension, more preferably from about 0.2 ⁇ 10 8 cfu to about 0.4 ⁇ 10 8 cfu, most preferably about 0.3 ⁇ 10 8 cfu.
  • the amount of the DA is not particularly limited, as long as the amount of the DA can form PDA under the self-polymerization conditions of the following step iii and the PDA can be deposited on the core
  • the inner shell layer 2 of the PDA can be formed on the surface.
  • said DA is used in an amount of about 1 mg to about 3 mg, more preferably about 2 mg per ml of said suspension.
  • the aqueous solution is not particularly limited, and here even only deionized water may be used instead of the aqueous solution.
  • the aqueous solution is preferably a Tris-HCl buffer solution having a concentration of about 10 to about 1000 mM, preferably 100 mM.
  • Step iii placing the mixture obtained in step ii under conditions capable of self-polymerizing the DA.
  • the conditions preferably include adjusting the pH of the mixed solution to about 8.0 to about 9.0, preferably about 8.5 at a temperature of about 20°C to about 40°C, preferably about 25°C, and continuing the polymerization for about 0.1 hour to about 3 hours, preferably about 1 hour to about 2 hours.
  • the particles with the PDA inner shell 2 with the bacterium 1 and the coating of the bacterium 1 have been obtained from the above steps i to iii, as an example, it is shown that there is an L.P. bacterium and a coating Particles "L.P.@PDA" of the PDA inner shell covering the L.P. core, the scale bar in this figure is 500 nm.
  • Step iv Mix the mixture obtained in step iii, which contains the particles having the core 1 and the PDA inner shell 2 covering the core 1, with the CMC-GEL aqueous solution and mix evenly.
  • the composition and concentration of the CMC-GEL aqueous solution are not limited, as long as it can match the amount of the particles comprising the PDA inner shell 2 and the bacterium core 1, so as to form the microcapsules of the present invention, namely Can.
  • the concentration of the CMC-GEL is preferably about 1 to about 3% by weight, preferably about 2% by weight; the solvent is preferably deionized water, buffer solution, etc.
  • the molecular weight of the CMC-GEL is not particularly limited.
  • the CMC-GEL preferably has about 50 kDa to about 200 kDa, more preferably about 100 to about 110 kDa, most preferably a molecular weight of 107 kDa.
  • the molecular weight of the CMC-GEL is too large, such as greater than about 200 kDa, then when the probiotic microcapsules of the present invention reach the intestinal tract, the CMC-GEL shell layer 3 cannot be quickly absorbed by the intestinal tract of the intestinal juice. The destruction of the flora may cause the probiotics to fail to produce SCFA well.
  • the CMC-GEL shell layer 3 may be destroyed prematurely in the gastric juice, thereby exposing the probiotics to the gastric acid environment, reducing the The probiotic activity.
  • the amount of the CMC-GEL aqueous solution is not particularly limited, as long as it can match the amount of the particles comprising the PDA inner shell 2 and the bacterium core 1, so as to form the microcapsules of the present invention .
  • the volume ratio of the CMC-GEL aqueous solution used in step iv to the mixed solution obtained from step iii comprising particles with bacterium core 1 and polydopamine inner shell 2 coating the bacterium core 1 is preferably From about 300:1 to about 100:1, more preferably from about 250:1 to about 150:1, most preferably about 200:1.
  • the mixing process is not particularly limited.
  • the CMC-GEL aqueous solution and the polydopamine containing bacterium 1 and the polydopamine coated bacterium 1 obtained in step 3 can be mixed.
  • the mixture of particles in the inner shell 2 is mixed by various means such as stirring, shaking, vortexing, etc. for about 1 minute to about 10 minutes, preferably about 5 minutes to about 7 minutes, more preferably about 6 minutes.
  • the CMC-GEL can be bound to the surface of the PDA inner shell layer 2 through the reaction between the amino group of GEL and the carboxyl group of the above PDA.
  • the PDA inner shell 2 with the bacterium core 1, the PDA inner shell 2 coated with the bacterium core 1 and the CMC-GEL shell of the PDA inner shell 2 coated with the above steps i to iv are obtained
  • Probiotic microcapsules of layer 3 shown as an example are L.P. probiotics with L.P. bacterium core, PDA inner shell layer covering said L.P. bacterium core layer and CMC-GEL outer shell layer coating said PDA inner shell layer
  • Microcapsules "L.P.@PDA@CMC-GEL” the scale bar in this figure is 500 nm.
  • the present invention relates to the probiotic microcapsules as above in the first aspect or the probiotic microcapsules prepared according to the method as in the above second aspect for the reduction of organisms, preferably animals, more preferably lactating Use of blood glucose levels in an animal, most preferably a human.
  • the present invention relates to the use of the probiotic microcapsules of the first aspect above or the probiotic microcapsules prepared according to the method of the second aspect above for the preparation of oral preparations, which can be administered orally
  • the medicament lowers blood glucose levels in an organism, preferably an animal, more preferably a mammal, most preferably a human.
  • GEL glue strength 250g Bloom, purchased from Shanghai Dibo Biotechnology Co., Ltd.;
  • CMC-Na viscosity 300-800, commercially purchased from Shanghai Yuanye Biotechnology Co., Ltd.;
  • Tris-HCl commercially purchased from Beijing Suo Laibao Technology Co., Ltd.;
  • MRS liquid medium commercially purchased from Hangzhou Best Biotechnology Co., Ltd.;
  • UV spectrophotometer evolution 220, commercially available from Thermo Scientific.
  • the probiotic microcapsules of the present invention were prepared according to the following general preparation process, and the preparation examples 1 to 7 were prepared.
  • the general preparation method comprises the following steps:
  • the specific process includes inoculating the MRS liquid medium with probiotics (L.P., B.A. or C.B.) after autoclaving, and culturing at 37°C for about 12 hours to obtain Passaging a liquid medium of probiotics, centrifuging the liquid medium at about 3000 to about 4000 rpm to obtain the bacterium core comprising one or more probiotics;
  • probiotics L.P., B.A. or C.B.
  • step (ii) adding the bacterium core obtained in step (i) to an aqueous Tris-HCl buffer solution to form a suspension, and adding DA to the suspension under stirring to form a mixed solution;
  • step (iii) placing the mixed solution obtained in step (ii) under conditions capable of self-polymerizing the DA;
  • step (iv) Add CMC-GEL aqueous solution to the mixture obtained in step (iii) and vortex to mix well.
  • the unencapsulated L.P., B.A. and C.B. bacterium cores obtained from the above step (i) (as L.P. control, B.A. control and C.B. control) and the probiotic microcapsules of Preparation Examples 1 to 7 were respectively placed in simulated intestinal fluid, so that The initial probiotic OD600 values of the control and the probiotic microcapsules were substantially the same. The OD600 value of the probiotic bacteria in the medium was determined after each sample was incubated at 37°C for 1, 3, 5, 7 and 9 hours.
  • Table 2 Probiotic proliferation results of control samples and probiotic microcapsules of the present invention.
  • probiotics L.P., B.A. and C.B.
  • probiotics were coated before (L.P. control, B.A. control and C.B. control) and after coating (preparation examples 1 to 3, preparation Examples 4 to 5 and Preparation Examples 6 to 7) all proliferate in the simulated intestinal fluid, and the proliferation trends over time are basically the same.
  • preparation examples 4 to 5 and Preparation Examples 6 to 7 all proliferate in the simulated intestinal fluid, and the proliferation trends over time are basically the same.
  • the unencapsulated L.P., B.A. and C.B. cores obtained from the above step (i) (as L.P. control, B.A. control and C.B. control) and the probiotic microcapsules of Preparation Examples 1 to 7 were placed in a simulated In gastric juice, the initial probiotic OD600 values of the control and probiotic microcapsules were substantially the same. The OD600 value of the probiotics was determined after each sample was incubated at 37°C for 0.5, 1, 2, and 3 hours.
  • Table 3 Test results of tolerance of control samples and probiotic microcapsules of the present invention to simulated gastric juice.
  • the L.P. control, B.A. control and C.B. control samples in the simulated gastric juice the OD600 value of the probiotics gradually decreased over time, and the reduction rate was as high as 25.4% to 29.4% after 3 hours; while Example 1 of the present invention
  • the OD600 value of samples up to 7 in the simulated gastric juice gradually decreased over time, but the decrease rate did not exceed 9.5% after 3 hours. Therefore, the probiotic microcapsules of the present invention have good tolerance to simulated gastric juice.
  • the unencapsulated L.P., B.A. and C.B. cores (as L.P. control, B.A. control and C.B. control) obtained from the above step (i) and the probiotic microcapsules prepared in Examples 1 to 7 were placed in the Simulated gastric juice for 3 hours, and adjusted the initial probiotic OD600 values of the control and probiotic microcapsules to be substantially the same. Afterwards, each sample was placed in simulated intestinal fluid, and the OD600 value of probiotics was determined after incubation at 37°C for 1, 3, 5, 7 and 9 hours.
  • Table 4 Test results of proliferation of control samples and probiotic microcapsules of the present invention in simulated gastric fluid after 3 hours in simulated intestinal fluid.
  • L.P. control, B.A. control and C.B. control are basically inactivated after being treated in simulated gastric juice for 3 hours, and their vitality will not recover substantially in the intestinal environment, while the probiotic microcapsules of the present invention are treated in simulated gastric acid After environmental protection, it is not affected by the acidic environment of gastric juice, and can still significantly expand the number in the intestinal environment.
  • Table 5 Test results of hypoglycemic effect of control samples and probiotic microcapsules of the present invention.
  • the probiotic microcapsules prepared in Examples 1 to 7 of the present invention have a better effect of lowering blood sugar, and can more obviously improve the blood sugar level of diabetic patients role.
  • the terms “inner” and “outer” mean that in the probiotic microcapsules of the present invention, the probiotics are located in the most “inner” part, while the outer shell exists in the “outer” department.
  • the shell comprises a PDA inner shell and a CMC-Gel shell in order from inside to outside.
  • the term “about” used in the present invention has the meaning known to those skilled in the art, and preferably refers to the value modified by this term within its ⁇ 50%, ⁇ 40%, ⁇ 30%, ⁇ 20%, ⁇ 10%, ⁇ 5% % or ⁇ 1% range.

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Abstract

L'invention concerne une microcapsule de probiotiques contenant un noyau de probiotiques et une enveloppe externe recouvrant le noyau de probiotiques. Le noyau de probiotiques contient un ou plusieurs probiotiques, et l'enveloppe externe contient une couche d'enveloppe interne en polydopamine et une couche d'enveloppe externe en carboxyméthylcellulose sodique et en polymère de gélatine, la couche d'enveloppe interne en polydopamine recouvrant le noyau de probiotiques, et la couche d'enveloppe externe en carboxyméthylcellulose sodique et en polymère de gélatine étant disposée à l'extérieur de la couche d'enveloppe interne en polydopamine et recouvrant la couche d'enveloppe interne en polydopamine. La microcapsule de probiotiques de la présente invention est résistante aux acides, aux sels biliaires et aux enzymes digestives, présente une stabilité et une adhérence améliorées des probiotiques, présente des effets améliorés en matière de colonisation intestinale par les probiotiques, et peut assurer une production in situ efficace d'acides gras à chaîne courte dans les intestins, ce qui permet d'atteindre l'objectif de réduction de la glycémie.
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CN116622594B (zh) * 2023-07-21 2023-10-17 深圳市东荣生物科技有限责任公司 一种促消化的复合微生物制剂及其制备方法

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