WO2021088460A1 - 一种载益生菌的微胶囊的制备方法及由其制得产品和应用 - Google Patents

一种载益生菌的微胶囊的制备方法及由其制得产品和应用 Download PDF

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WO2021088460A1
WO2021088460A1 PCT/CN2020/109716 CN2020109716W WO2021088460A1 WO 2021088460 A1 WO2021088460 A1 WO 2021088460A1 CN 2020109716 W CN2020109716 W CN 2020109716W WO 2021088460 A1 WO2021088460 A1 WO 2021088460A1
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microcapsules
probiotic
probiotics
preparing
sodium alginate
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PCT/CN2020/109716
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French (fr)
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丁钢强
霍军生
王连艳
黄建
张贵锋
唐艳斌
曹科
崔迎彬
曹政
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中国疾病预防控制中心营养与健康所
中国科学院过程工程研究所
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Priority to US17/264,515 priority Critical patent/US20220142933A1/en
Publication of WO2021088460A1 publication Critical patent/WO2021088460A1/zh

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    • 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
    • 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
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • 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
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
    • A23L29/256—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin from seaweeds, e.g. alginates, agar or carrageenan
    • 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
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/16—Inorganic salts, minerals or trace elements
    • 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
    • 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
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • 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
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/11—Encapsulated compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/733—Alginic acid; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5089—Processes
    • 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

Definitions

  • This application belongs to the field of microbiology technology, and specifically relates to a preparation method of microcapsules containing probiotics, and products prepared therefrom and applications.
  • Probiotics are active microorganisms that can improve the host's intestinal microecological balance and are beneficial to the host. They are colonized in the human intestines and reproductive system and can produce definite health effects. When a certain amount of probiotics is ingested by the human body, it will produce many health-promoting effects, including controlling intestinal infections, enhancing immunity, regulating plasma and cholesterol levels, and fighting cancer. It can promote lactose intolerance for patients Their use of lactose. In recent years, more and more studies have confirmed that probiotics can inhibit the growth of pathogenic bacteria, eliminate carcinogenic factors, maintain normal intestinal flora, and have important physiological effects such as relieving lactose intolerance, lowering cholesterol, and improving immunity. When probiotics are lacking, it will lead to adverse effects such as imbalance of intestinal flora, growth of spoilage bacteria, weakened intestinal peristalsis, weakened immune function, and decreased function of synthesizing B vitamins.
  • probiotics are easily inactivated when they are taken orally through the strong acid environment of the stomach, resulting in weaker activity in the intestinal tract.
  • the microencapsulation of probiotics can provide a good isolation and protection effect, so that it can play a better role when it reaches the intestinal tract.
  • Lactobacillus acidophilus belonging to the genus Lactobacillus, is a gram-positive bacillus that is widely found in the intestines of humans and some animals.
  • CN104911171A discloses a method for preparing probiotic microcapsules with sodium alginate and gelatin composite attapulgite.
  • the method firstly purifies the attapulgite, and then mixes the attapulgite with sodium alginate and gelatin to form a composite wall material, and then combines the prepared attapulgite
  • the probiotic bacteria suspension is uniformly mixed with the above-mentioned composite wall material under certain conditions, and then slowly dropped into CaCl 2 to solidify and form, and finally dried by vacuum freezing to obtain probiotic microcapsules.
  • the probiotic microcapsules prepared by this method The buried material has low activity and low survival rate.
  • CN102228235A discloses a preparation method and application of probiotic microcapsules.
  • the sodium alginate solution and the modified starch solution are mixed in equal volume, and then mixed with probiotics, sprayed into the CaCl 2 solution through the sprayer nozzle, and stand for solidification. Washing and filtering with physiological saline.
  • this method enhances the mechanical strength of the capsule wall to a certain extent, the size of the obtained capsule is difficult to control, and it is difficult to apply to the industrial production of food and medicine.
  • the pH of the microcapsule After the addition of modified starch, the pH of the microcapsule The sensitive release characteristics are weakened, resulting in that after being immersed in the stomach, some of the microcapsules cannot become dense under the acidic environment of the stomach, and they cannot disintegrate quickly under the weak acid environment of the intestine to release the embedded probiotics. bacteria.
  • This application provides a method for preparing microcapsules containing probiotics, and products prepared therefrom and applications.
  • the probiotic-containing microcapsules prepared by the method have the advantages of good capsule spherical shape, controllable particle size, good dispersibility, good pH sensitivity, and easy release in the intestinal tract, which can meet the needs of subsequent animal evaluation.
  • this application provides a method for preparing microcapsules containing probiotics, the method comprising the following steps:
  • step (3) Spray a salt solution into the probiotic suspension obtained in step (2), stir and solidify to obtain the probiotic-carrying microcapsules.
  • This application uses sodium alginate as the wall material of probiotic microcapsules. Because sodium alginate has good biocompatibility and biodegradability, sodium alginate can absorb water in the presence of certain divalent cations such as calcium and barium. The resulting gel can be used for the preparation of microcapsules.
  • the alginate microcapsules have excellent pH-sensitive release characteristics. They have a denser surface in an acidic environment and protect the embedded probiotics. Under the pH environment of the intestinal tract, they can quickly disintegrate and release the package. The buried probiotics play a role in regulating the intestinal flora.
  • the spray drying method is the most widely used and has the advantages of simple operation and low cost.
  • the preparation method described in this application uses sodium alginate as the wall material of the microcapsules, and the spray method combined with the ion solidification method to prepare the microcapsules.
  • the two methods work together to achieve a good spherical shape, a controllable particle size, and good dispersion of the microcapsules. It can meet the needs of subsequent animal in vivo evaluation.
  • the solvent in step (1) is water and/or an aqueous sodium chloride solution, preferably an aqueous sodium chloride solution.
  • the mass percentage concentration of the sodium chloride aqueous solution is 0.85-0.9%, for example, it can be 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.9%.
  • the solvent in step (1) is physiological saline.
  • the mass percentage concentration of the sodium alginate solution in step (1) is 0.5-3%, for example, it can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2% , 1.4%, 1.6%, 1.8%, 1.9%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%.
  • the amount of probiotics added in step (2) is 10 8 -10 11 CFU/mL, for example, 1 ⁇ 10 8 CFU/mL, 2 ⁇ 10 8 CFU/mL, 3 ⁇ 10 8 CFU/mL, 4 ⁇ 10 8 CFU/mL, 5 ⁇ 10 8 CFU/mL, 6 ⁇ 10 8 CFU/mL, 7 ⁇ 10 8 CFU/mL, 8 ⁇ 10 8 CFU/mL, 9 ⁇ 10 8 CFU/ mL, 1 ⁇ 10 9 CFU/mL, 2 ⁇ 10 9 CFU/mL, 3 ⁇ 10 9 CFU/mL, 4 ⁇ 10 9 CFU/mL, 5 ⁇ 10 9 CFU/mL, 6 ⁇ 10 9 CFU/mL, 7 ⁇ 10 9 CFU/mL, 8 ⁇ 10 9 CFU/mL, 9 ⁇ 10 9 CFU/mL, 1 ⁇ 10 10 CFU/mL, 2 ⁇ 10 10 CFU/mL, 3 ⁇ 10 10 CFU/mL, 4 ⁇ 10 10 CFU/mL, 5 ⁇ 10 10 CFU/m
  • the probiotic in step (2) is selected from any one or a combination of at least two of Lactobacillus acidophilus, Bifidobacterium, or Gram-positive cocci.
  • the method of spraying the salt solution in step (3) is spraying through the nozzle of a spray dryer.
  • the salt solution is a calcium salt solution.
  • the concentration of the calcium salt solution is 0.5-1.5 mol/L, for example, it can be 0.5 mol/L, 0.6 mol/L, 0.7 mol/L, 0.8 mol/L, 0.9 mol/L, 1 mol /L, 1.1mol/L, 1.2mol/L, 1.3mol/L, 1.4mol/L, 1.5mol/L.
  • the calcium salt solution includes any one or a combination of at least two of calcium gluconate, calcium hydrogen phosphate, calcium lactate, calcium sulfate, or calcium chloride, preferably calcium chloride.
  • the gas velocity of the sprayed ionic solution is 450-550L/h, for example, it can be 450L/h, 460L/h, 470L/h, 480L/h, 490L/h, 500L/h, 510L /h, 520L/h, 530L/h, 540L/h, 550L/h.
  • the feed rate of the spray dryer is 1-3 mL/min, for example, it can be 1 mL/min, 1.2 mL/min, 1.4 mL/min, 1.6 mL/min, 1.8 mL/min, 2 mL/min. min, 2.2mL/min, 2.4mL/min, 2.6mL/min, 2.8mL/min, 3mL/min.
  • the present application provides a preparation process of calcium alginate microcapsules for improving the embedding rate of microcapsules and sensitive release from the gastrointestinal tract.
  • the preparation process controls the gas velocity of the wall material suspension containing the core material during the formation of the microcapsules.
  • the feed rate of the liquid feed of the jet device to further increase the microcapsule embedding rate, and the calcium alginate microcapsules formed at the same time can form a dense protective layer under the acidic environment of the stomach, without releasing the embedded material, and reach neutral to neutral.
  • the weakly alkaline post-crosslinked calcium ion valence bond in the intestinal tract can quickly dissociate and release the core material embedded in the microcapsule, making it have a good pH-sensitive release behavior.
  • the size of the capsule can also be controlled through nozzle size selection and injection process control, thereby improving the overall performance of the microcapsule.
  • step (3) further includes step (4): washing, collecting, and suspending the microcapsules carrying probiotics obtained in step (3) in a solvent.
  • the washing is a centrifugal washing.
  • the solvent in step (4) is physiological saline.
  • the method includes the following steps:
  • step (2) Slowly stir and mix the probiotics with the sodium alginate solution obtained in step (1), and the added amount of the probiotics is 10 8 -10 11 CFU/mL to obtain a probiotic suspension;
  • step (3) The probiotic suspension obtained in step (2) is sprayed into the calcium salt solution through the nozzle of the spray device, the concentration of the calcium salt solution is 0.5-1.5 mol/L, and the spraying gas velocity is 450-550L/ h, the feed rate of the spray dryer is 1-3 mL/min, stir and solidify to obtain microcapsules containing probiotics; and
  • step (3) The microcapsules carrying probiotics obtained in step (3) are centrifuged, washed, collected, and suspended and stored in physiological saline.
  • the present application provides a probiotic-containing microcapsule prepared by the method for preparing a probiotic-containing microcapsule as described in the first aspect.
  • the particle size of the microcapsules is 25-40 ⁇ m, for example, 25 ⁇ m, 26 ⁇ m, 27 ⁇ m, 28 ⁇ m, 29 ⁇ m, 30 ⁇ m, 31 ⁇ m, 32 ⁇ m, 34 ⁇ m, 35 ⁇ m, 36 ⁇ m, 37 ⁇ m, 38 ⁇ m, 39 ⁇ m, 40 ⁇ m.
  • the particle size of the microcapsules is 30-35 ⁇ m.
  • this application provides a use of the probiotic-containing microcapsules as described in the second aspect.
  • the microcapsules are used to prepare health foods, beverages and foods, clinical nutritional preparations, pharmaceutical microcapsule preparations or cosmetics.
  • the method for preparing probiotic-containing microcapsules described in the present application solves the problems of large particle size, irregular spherical shape, poor dispersion, high cost, and cumbersome preparation of probiotic microcapsules prepared by traditional methods.
  • the probiotic microcapsules of the present application have a particle size of 25-40 ⁇ m, good pH sensitivity, a denser surface in an acidic environment, and protect the embedded probiotics, while in the pH environment of the intestinal tract, they can quickly It disintegrates and releases the embedded probiotics, which can meet the needs of subsequent animal in vivo evaluation.
  • Figure 1 is an optical microscope characterization diagram of Lactobacillus acidophilus.
  • Figure 2 is a scanning electron microscope characterization diagram of Lactobacillus acidophilus.
  • FIG. 3 is an optical microscope characterization diagram of the microcapsules containing Lactobacillus acidophilus prepared in Example 1.
  • Fig. 4 is a scanning electron microscope characterization diagram of the microcapsules containing Lactobacillus acidophilus prepared in Example 1.
  • FIG. 5 is a diagram showing the particle size and particle size distribution of the microcapsules containing Lactobacillus acidophilus prepared in Example 1.
  • FIG. 5 is a diagram showing the particle size and particle size distribution of the microcapsules containing Lactobacillus acidophilus prepared in Example 1.
  • Fig. 7A is an immunohistochemical chart of intestinal cell proliferation in unadministered mice.
  • Fig. 7B is a partial enlarged view of an immunohistochemical image of intestinal cell proliferation of unadministered mice.
  • Figure 7C is an immunohistochemical image of mouse intestinal cell proliferation on the 7th day after administration of normal saline.
  • Figure 7D is an immunohistochemical image of mouse intestinal cell proliferation on the 7th day after intragastric administration of Lactobacillus acidophilus solution.
  • Figure 7E is an immunohistochemical image of mouse intestinal cell proliferation on the 7th day after low-dose water-filled Lactobacillus acidophilus microcapsules prepared in Example 1.
  • Figure 7F is an immunohistochemical image of mouse intestinal cell proliferation on the 7th day after intragastric administration of the water-filled Lactobacillus acidophilus microcapsules prepared in Example 1.
  • Figure 7G is an immunohistochemical image of mouse intestinal cell proliferation on the 7th day after high-dose water-filled Lactobacillus acidophilus microcapsules prepared in Example 1.
  • Figure 7H is an immunohistochemical image of mouse intestinal cell proliferation on the 14th day after administration of normal saline.
  • Figure 7I is an immunohistochemical image of mouse intestinal cell proliferation on the 14th day after intragastric administration of Lactobacillus acidophilus solution.
  • Figure 7J is an immunohistochemical image of mouse intestinal cell proliferation on the 14th day after low-dose water-filled Lactobacillus acidophilus microcapsules prepared in Example 1.
  • Figure 7K is an immunohistochemical image of mouse intestinal cell proliferation on the 14th day after intragastric administration of the water-filled Lactobacillus acidophilus microcapsules prepared in Example 1.
  • Figure 7L is an immunohistochemical image of mouse intestinal cell proliferation on the 14th day after high-dose water-filled Lactobacillus acidophilus microcapsules prepared in Example 1.
  • Fig. 7M is an immunohistochemical image of mouse intestinal cell proliferation on the 21st day after administration of normal saline.
  • Figure 7N is an immunohistochemical image of mouse intestinal cell proliferation on the 21st day after intragastric administration of Lactobacillus acidophilus solution.
  • Figure 70 is an immunohistochemical image of mouse intestinal cell proliferation on the 21st day after low-dose water-filled Lactobacillus acidophilus microcapsules prepared in Example 1.
  • Figure 7P is an immunohistochemical image of mouse intestinal cell proliferation on the 21st day after intragastric administration of the water-filled Lactobacillus acidophilus microcapsules prepared in Example 1.
  • Figure 7Q is an immunohistochemical image of mouse intestinal cell proliferation on the 21st day after high-dose water-filled Lactobacillus acidophilus microcapsules prepared in Example 1.
  • Fig. 8A is a statistical histogram of mouse intestinal cell proliferation after intragastric administration of the microcapsules containing Lactobacillus acidophilus prepared in Example 1. Figs.
  • FIG. 8B is a statistical curve diagram of the proliferation of mouse intestinal cells after intragastric administration of the microcapsules containing Lactobacillus acidophilus prepared in Example 1.
  • FIG. 8B is a statistical curve diagram of the proliferation of mouse intestinal cells after intragastric administration of the microcapsules containing Lactobacillus acidophilus prepared in Example 1.
  • Fig. 9 is a statistical histogram of the effect of the microcapsules containing Lactobacillus acidophilus prepared in Example 1 on the activity of small intestinal macrophages.
  • 10A is a representative flow chart of the effect of the microcapsules containing Lactobacillus acidophilus prepared in Example 1 on lymphocyte subsets.
  • 10B is a statistical diagram of the effect of the microcapsules containing Lactobacillus acidophilus prepared in Example 1 on helper T lymphocytes.
  • 10C is a statistical diagram of the effect of the microcapsules containing Lactobacillus acidophilus prepared in Example 1 on cytotoxic T lymphocytes.
  • Lactobacillus acidophilus and Bifidobacterium strains are used in the examples of this application, but those skilled in the art should understand that any other Lactobacillus acidophilus and Bifidobacterium strains can also implement this application. Lactobacillus acidophilus and bifidobacteria are not specifically limited.
  • a microcapsule containing Lactobacillus acidophilus was prepared according to the following method:
  • Lactobacillus acidophilus liquid obtained from the Fermented Food and Microbial Resources Development Research Laboratory (5) of Tianjin University of Science and Technology
  • the amount of Lactobacillus liquid is 10 9 CFU/mL
  • step (3) Set the suspension in step (2) at a gas velocity of 500L/h, and set the feed rate of the spray dryer to 2mL/min, and spray it into the 1mol/L calcium chloride solution through the nozzle of the spray dryer , Slowly stir the solidified droplets to form microcapsules;
  • Lactobacillus acidophilus preparation and detection of Lactobacillus acidophilus are as follows:
  • Optical microscope and scanning electron microscope were used to observe the morphology of Lactobacillus acidophilus.
  • the optical microscope characterization of Lactobacillus acidophilus is shown in Figure 1, and the scanning electron microscope characterization of Lactobacillus acidophilus is shown in Figure 2.
  • the Lactobacillus acidophilus was identified by the Gram staining method.
  • a microcapsule containing Lactobacillus acidophilus was prepared according to the following method:
  • Lactobacillus acidophilus liquid obtained from the Fermented Food and Microbial Resources Development Research Laboratory (5) of Tianjin University of Science and Technology
  • the amount of Lactobacillus liquid is 10 8 CFU/mL
  • step (3) Set the suspension in step (2) at a gas velocity of 450L/h, and set the feed rate of the spray dryer to 1mL/min, and spray 0.5mol/L calcium chloride solution through the nozzle of the spray dryer In the medium, slowly stir the solidified droplets to form microcapsules;
  • a microcapsule containing Lactobacillus acidophilus was prepared according to the following method:
  • Lactobacillus acidophilus liquid obtained from the Fermented Food and Microbial Resources Development Research Laboratory (5) of Tianjin University of Science and Technology
  • the amount of Lactobacillus liquid is 10 10 CFU/mL
  • step (3) Set the suspension in step (2) at a gas velocity of 550L/h, and set the feed rate of the spray dryer to 3mL/min, and spray 1.5mol/L calcium chloride solution through the nozzle of the spray dryer In the medium, slowly stir the solidified droplets to form microcapsules;
  • a microcapsule containing Lactobacillus acidophilus was prepared according to the following method:
  • Lactobacillus acidophilus liquid obtained from the Fermented Food and Microbial Resources Development Research Laboratory (5) of Tianjin University of Science and Technology
  • the amount of Lactobacillus liquid is 10 11 CFU/mL
  • step (3) Set the suspension in step (2) at a gas velocity of 500L/h, and set the feed rate of the spray dryer to 2mL/min, and spray it into the 1mol/L calcium chloride solution through the nozzle of the spray dryer , Slowly stir the solidified droplets to form microcapsules;
  • a microcapsule containing bifidobacteria was prepared according to the following method:
  • step (3) Set the suspension in step (2) at a gas velocity of 500L/h, and set the feed rate of the spray dryer to 2mL/min, and spray it into the 1mol/L calcium chloride solution through the nozzle of the spray dryer , Slowly stir the solidified droplets to form microcapsules;
  • the preparation method of this comparative example is the same as that of Example 1, except that the gas velocity of spraying the salt solution in step (3) is 300 L/h, and the feed rate of the spray dryer is 5 mL/min.
  • the preparation method of this comparative example is the same as that of Example 1, except that the gas velocity of spraying the ionic solution in step (3) is 600 L/h, and the feed rate of the spray dryer is 5 mL/min.
  • the preparation method of this comparative example is the same as that of Example 1, except that the gas velocity of spraying the salt solution in step (3) is 300 L/h, and the feed rate of the spray dryer is 0.5 mL/min.
  • the preparation method of this comparative example is the same as that of Example 1, except that the gas velocity of spraying the ionic solution in step (3) is 600 L/h, and the feed rate of the spray dryer is 0.5 mL/min.
  • step (1) dissolve the mixture of sodium alginate and modified starch in physiological saline, and the mass ratio of sodium alginate and modified starch is 1:1. Into a mixture of sodium alginate and starch.
  • the probiotic-loaded microgel particles prepared in Examples 1-5 are between 25-40 ⁇ m.
  • the process and process described in this application can control the size of the capsules, and obtain microcapsules with small particle diameters and easy absorption by the human body. , The overall performance of the microcapsules can be improved. It is explained that the preparation method described in this application uses sodium alginate as the wall material of the microcapsules, and the spray method combined with the ion solidification method to prepare the microcapsules. The two methods have a synergistic effect to achieve the microcapsules with good spherical shape, small particle size and good dispersion. It can meet the needs of subsequent animal in vivo evaluation.
  • the test method includes the steps: the probiotic-containing microcapsules prepared in Examples 1-5 and the The embedding rate of microcapsules prepared in proportions 1-5 is shown in Table 2:
  • the embedding rate of the probiotic-containing microcapsules prepared in Examples 1-5 is above 75%, which fully demonstrates that the preparation process of the present application controls the spraying of the core material during the formation of the microcapsules.
  • the gas velocity of the wall material suspension and the feed rate of the material liquid of the spray device can further increase the embedding rate of the microcapsules, and can meet the requirements of subsequent animal in vivo evaluation.
  • the morphology of the microcapsules carrying Lactobacillus acidophilus was observed using an optical microscope and a scanning electron microscope (the microscope model is JEOL (JEM-6700F)), and the results are shown in Figures 3 and 4.
  • An optical microscope with shooting function was used to observe, photograph and record the morphology of the microcapsules.
  • the optical microscope characterization of the microcapsules containing Lactobacillus acidophilus prepared in Example 1 is shown in FIG. 3.
  • the specific process is as follows: use a dropper to drop a small amount of sample on the glass slide, gently cover the cover glass, first find a suitable field of view under a low power lens, and then switch to a high power lens for microcapsule morphology observation, and use WV-
  • the CP230/G camera captures and records the state of the sample, and preliminarily evaluates the quality of the microcapsules through morphological observation. It can be seen from the microscopic photograph in Figure 3 that many rod-shaped bacteria are embedded in the microcapsules, indicating that the microcapsules have a relatively good embedding effect on the bacteria.
  • the morphology of the microcapsules carrying Lactobacillus acidophilus was observed with a scanning electron microscope.
  • FIG. 4 The scanning electron microscopic characterization of the microcapsules carrying Lactobacillus acidophilus prepared in Example 1 is shown in FIG. 4. It can be seen from the morphology of the microcapsules in Figure 4 that there are rod-shaped bacteria on the surface of the microcapsules, indicating that the microcapsules can encapsulate more bacteria particles.
  • mice (albino laboratory mice) were administered by intragastric administration for 14 days. The mice were dissected and their ileums were taken, slices were prepared, Ki-67 staining was performed by immunohistochemistry, photographed under a microscope, and fully automated quantification by Vectra III The pathology imaging analysis system performs data processing and statistics on the slices. Normal saline, Lactobacillus acidophilus liquid, and oral low, medium, and high-dose Lactobacillus acidophilus microcapsules (the bacterial content of each dose in the low, medium, and high-dose groups is 10 8 CFU, 10 9 CFU, and 10 10 CFU, respectively).
  • oral administration of low, medium and high doses of Lactobacillus acidophilus microcapsules can significantly promote the intestinal tract of mice Proliferation of epithelial cells; compared with the blank control group at 3 time points on the 7, 14 and 21 days of administration, the gavage bacteria liquid group, the low-dose bacteria-carrying microcapsule group, the middle-dose bacteria-carrying microcapsule group, and the bacteria-carrying group
  • the proliferation of intestinal epithelial cells of mice in the microcapsule high-dose group showed significant differences, indicating that the effect of the mice after taking the Lactobacillus acidophilus microcapsules was significant.
  • the BALB/C mice were given gastric Lactobacillus acidophilus microcapsules for 14 days, and the cervical vertebrae were dislocated to death.
  • the spleens were aseptically removed to prepare a spleen cell suspension.
  • the effect of intragastric administration of bacteria-loaded microcapsules on the phagocytic activity of mouse spleen macrophages was detected.
  • the results of the effect of the microcapsules containing Lactobacillus acidophilus on the activity of small intestinal macrophages prepared in Example 1 are shown in Figure 9.
  • the absorbance value of the ordinate in Figure 9 is the absorbance value at 570nm, which is related to the phagocytosis of macrophages. Activity is related.
  • the low, medium, and high on the abscissa represent the low, medium, and high doses of Lactobacillus acidophilus microcapsules (each dose in the low, medium, and high dose groups).
  • the bacterial content is 10 8 CFU, 10 9 CFU and 10 10 CFU respectively)
  • oral Lactobacillus acidophilus microcapsules can increase the phagocytic activity of mouse spleen macrophages.
  • mouse spleen macrophages The phagocytic activity of cells is dose-dependent, that is, the higher the bacterial content in the microcapsules, the better the phagocytic activity of mouse spleen macrophages.
  • the high dose of bacteriostatic microcapsules can extremely significantly increase the phagocytic ability of mouse spleen macrophages (p ⁇ 0.001), indicating that Lactobacillus acidophilus microcapsules have the ability to activate macrophages, thereby increasing The body's non-specific immune function.
  • lymphocytes can be recycled through lymphatic vessels, peripheral blood, etc., to perform functions such as cellular immunity and immune regulation.
  • the flow cytometry analyzer detects the lymphocyte subgroups according to the different surface markers of lymphocytes: lymphocytes mainly include T lymphocytes (CD3 + ), B lymphocytes (CD19 + ), and NK cells (CD16 + CD56 + ). T lymphocytes can be further tested for helper T lymphocytes (CD3 + CD4 + ) and inhibitory/cytotoxic T lymphocytes (CD3 + CD8 + ).
  • the low, medium, and high on the right represent and oral low, medium, and high-dose Lactobacillus acidophilus microcapsules (the bacterial content of each dose in the low, medium, and high-dose groups is 10 8 CFU, 10 9 CFU, and 10 10 CFU, respectively)
  • the effect of the microcapsules containing Lactobacillus acidophilus prepared in Example 1 on mouse helper T lymphocytes (CD3 + CD4 + ) is shown in Figure 10B; the microcapsules containing Lactobacillus acidophilus prepared in Example 1 affect The influence of inhibitory/cytotoxic T lymphocytes (CD3 + CD8 + ) is shown in Figure 10C.
  • the increase in the content of CD4 + T lymphocytes is beneficial to assist humoral immunity and mediate cellular immunity to regulate the immune effect of the body, while the increase in the content of CD8 + T lymphocytes is conducive to the improvement of the immune level of the body.

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Abstract

一种载益生菌的微胶囊的制备方法及由其制得的产品和应用，方法包括如下步骤：(1)将海藻酸钠溶于溶剂中，得到海藻酸钠溶液；(2)将益生菌与步骤(1)得到的海藻酸钠溶液混合均匀，得到益生菌悬浮液；和(3)向步骤(2)得到的益生菌悬浮液中喷入盐溶液，搅拌固化，得到所述载益生菌的微胶囊。该微胶囊用于制备保健食品、饮料食品、临床营养制剂、药物胶囊制剂或化妆品的用途。

Description

一种载益生菌的微胶囊的制备方法及由其制得产品和应用 技术领域
本申请属于微生物技术领域，具体涉及一种载益生菌的微胶囊的制备方法及由其制得产品和应用。
背景技术
益生菌是可改善宿主肠道微生态平衡，对宿主有益的活性微生物，定植于人体肠道、生殖系统内，能产生确切健康功效。当一定数量的益生菌被人体摄入后，将产生诸多促进健康的作用，包括控制肠道感染、增强免疫力、调节血浆及胆固醇水平、抗癌，对于患有乳糖不耐症的患者可以促进他们对乳糖的利用。近年来越来越多的研究证实益生菌能够抑制致病菌生长，消除致癌因子，维持肠道正常菌群，而且具有缓解乳糖不耐症，降低胆固醇，提高免疫力等重要生理功效。当益生菌缺乏时，就会导致肠道菌群失调、腐败细菌滋生、肠道蠕动减弱、免疫功能低下以及合成B族维生素功能下降等不利影响。
很多益生菌口服经过胃的强酸环境时容易失活，导致其在肠道发挥的活性作用较弱，为了最大限度保证益生菌在肠道中的活性，发挥肠道菌群调节作用的有益功效，将益生菌微胶囊化可以起到很好的隔离保护作用，使其到达肠道时发挥更好的功效。嗜酸乳杆菌，为乳杆菌属，革兰式阳性杆菌，广泛存在人体和一些动物肠道内。
CN104911171A公开了以海藻酸钠、明胶复合凹土制备益生菌微胶囊的方法，该方法首先是纯化凹土，其次将凹土与海藻酸钠、明胶混合成复合壁材，接着将已制备好的益生菌菌悬液在一定的条件下与上述复合壁材混合均匀，然后缓慢滴入CaCl 2中固化成型，最后真空冷冻法干燥，获得益生菌微胶囊，此方法制备得到的益生菌微胶囊包埋物质的活性较低和存活率较低。
CN102228235A公开了一种益生菌微胶囊的制备方法及其应用，采用将海藻酸钠溶液与变性淀粉溶液等体积混合，再与益生菌混合，通过喷雾器喷嘴喷入CaCl 2溶液中，静置固化后生理盐水洗涤过滤，这种方法虽然在一定程度增强了胶囊壁得机械强度，但是所得到的胶囊大小难于控制，很难应用于食品以及药品当中工业生产，而且加入变性淀粉后，微胶囊的pH敏感释放的特性减弱，导致其在浸入胃部后，部分微胶囊在胃部的酸性环境下表面无法变得致密，而在肠道的弱酸环境下又无法迅速崩解，释放出包埋的益生菌。
因此，开发一种能保持所包埋物质的活性且易于释放的载益生菌的微胶囊的制备方法，是本领域目前亟待解决的问题。
发明内容
本申请提供了一种载益生菌的微胶囊的制备方法及由其制得产品和应用。由所述方法制备得到的载益生菌的微胶囊具有胶囊球形佳、粒径可控、分散性 好，pH敏感性好，且在肠道易于释放的优点，可满足后续动物体内评价需求。
第一方面，本申请提供一种载益生菌的微胶囊的制备方法，所述方法包括如下步骤：
(1)将海藻酸钠溶于溶剂中，得到海藻酸钠溶液；
(2)将益生菌与步骤(1)得到的海藻酸钠溶液混合均匀，得到益生菌悬浮液；和
(3)将步骤(2)得到的益生菌悬浮液中喷入盐溶液，搅拌固化，得到所述载益生菌的微胶囊。
本申请采用海藻酸钠作为益生菌微胶囊的壁材，由于海藻酸钠具有良好的生物相容特性和生物可降解特性，海藻酸钠可在某些二价阳离子如钙、钡存在时吸收水分生成凝胶，可用于微胶囊的制备。另外，海藻酸盐微胶囊具有优良的pH敏感释放的特性，在酸性环境下表面更为致密，保护所包埋的益生菌，而在肠道的pH环境下，能够迅速崩解，释放出包埋的益生菌，发挥调节肠道菌群的作用。喷雾干燥法应用最广泛，具有操作简单，成本较低的优点。本申请所述的制备方法采用海藻酸钠作为微胶囊的壁材，喷射法结合离子固化法制备微胶囊，两种方法协同作用，以实现微胶囊球形佳，粒径可控，分散性好，可满足后续动物体内评价需求的目的。
在一个实施方案中，步骤(1)所述溶剂为水和/或氯化钠水溶液，优选为氯化钠水溶液。
在一个实施方案中，所述氯化钠水溶液的质量百分浓度为0.85-0.9％，例如可以是0.85％、0.86％、0.87％、0.88％、0.89％、0.9％。
在一个具体的实施方案中，步骤(1)所述溶剂为生理盐水。
在一个实施方案中，步骤(1)所述海藻酸钠溶液的质量百分浓度为0.5-3％，例如可以是0.5％、0.6％、0.7％、0.8％、0.9％、1％、1.2％、1.4％、1.6％、1.8％、1.9％、2％、2.2％、2.4％、2.6％、2.8％、3％。
在一个实施方案中，步骤(2)所述益生菌的加入量为10 8-10 11CFU/mL，例如可以是1×10 8CFU/mL、2×10 8CFU/mL、3×10 8CFU/mL、4×10 8CFU/mL、5×10 8CFU/mL、6×10 8CFU/mL、7×10 8CFU/mL、8×10 8CFU/mL、9×10 8CFU/mL、1×10 9CFU/mL、2×10 9CFU/mL、3×10 9CFU/mL、4×10 9CFU/mL、5×10 9CFU/mL、6×10 9CFU/mL、7×10 9CFU/mL、8×10 9CFU/mL、9×10 9CFU/mL、1×10 10CFU/mL、2×10 10CFU/mL、3×10 10CFU/mL、4×10 10CFU/mL、5×10 10CFU/mL、6×10 10CFU/mL、7×10 10CFU/mL、8×10 10CFU/mL、9×10 10CFU/mL、1×10 11CFU/mL。
在一个实施方案中，步骤(2)所述益生菌选自嗜酸乳杆菌、双歧杆菌或革兰氏阳性球菌中的任意一种或至少两种的组合。
在一个实施方案中，步骤(3)所述喷入盐溶液的方式为通过喷雾干燥器的喷嘴喷入。
在一个实施方案中，所述盐溶液为钙盐溶液。
在一个实施方案中，所述钙盐溶液的浓度为0.5-1.5mol/L，例如可以是0.5mol/L、0.6mol/L、0.7mol/L、0.8mol/L、0.9mol/L、1mol/L、1.1mol/L、1.2mol/L、1.3mol/L、1.4mol/L、1.5mol/L。
在一个实施方案中，所述钙盐溶液包括葡萄糖酸钙、磷酸氢钙、乳酸钙、硫酸钙或氯化钙中的任意一种或至少两种的组合，优选为氯化钙。
在一个实施方案中，所述喷入离子溶液的气速为450-550L/h，例如可以是450L/h、460L/h、470L/h、480L/h、490L/h、500L/h、510L/h、520L/h、530L/h、540L/h、550L/h。
在一个实施方案中，所述喷雾干燥器进料速度为1-3mL/min，例如可以是1mL/min、1.2mL/min、1.4mL/min、1.6mL/min、1.8mL/min、2mL/min、2.2mL/min、2.4mL/min、2.6mL/min、2.8mL/min、3mL/min。
本申请提供一种提高微胶囊包埋率及胃肠道敏感释放的海藻酸钙微胶囊制备工艺，该制备工艺通过控制微胶囊形成过程中的喷入含芯材的壁材悬浮液的气速和喷射装置的料液进料速度，进一步提高微胶囊包埋率，同时形成的海藻酸钙微胶囊具有在胃部酸性环境下形成致密保护层、不释放包埋物质，而到达呈中性至弱碱性的肠道后交联的钙离子价键能够迅速解离，释放微胶囊中包埋的芯材物质，使其具备良好的pH敏感释放行为。同时，通过喷嘴尺寸选择、喷射工艺控制，还能够控制胶囊大小，进而提高微胶囊的整体性能。
在一个实施方案中，步骤(3)后还包括步骤(4)：将步骤(3)得到的载益生菌的微胶囊洗涤、收集，悬浮保存在溶剂中。
在一个实施方案中，所述洗涤为离心洗涤。
在一个实施方案中，步骤(4)中所述溶剂为生理盐水。
在一个具体的实施方案中，所述方法包括如下步骤：
(1)将海藻酸钠溶于溶剂中，得到质量百分浓度为0.5-3％的海藻酸钠溶液；
(2)将益生菌与步骤(1)得到的海藻酸钠溶液缓慢搅拌混合均匀，所述益生菌的加入量为10 8-10 11CFU/mL，得到益生菌悬浮液；
(3)将步骤(2)得到的益生菌悬浮液，通过喷射装置的喷嘴喷入钙盐溶液中，所述钙盐溶液浓度为0.5-1.5mol/L，喷入气速为450-550L/h，喷雾干燥器进料速度为1-3mL/min，搅拌固化，得到载益生菌的微胶囊；和
(4)将步骤(3)得到的载益生菌的微胶囊进行离心洗涤、收集，悬浮保存在生理盐水中。
第二方面，本申请提供一种如第一方面所述载益生菌的微胶囊的制备方法制备得到的载益生菌的微胶囊。
在一个实施方案中，所述微胶囊的粒径为25-40μm，例如可以是25μm、26μm、27μm、28μm、29μm、30μm、31μm、32μm、34μm、35μm、36 μm、37μm、38μm、39μm、40μm。
在一个具体的实施方案中，所述微胶囊的粒径为30-35μm。
第三方面，本申请提供一种如第二方面所述载益生菌的微胶囊的用途，所述微胶囊用于制备保健食品、饮料食品、临床营养制剂、药物微囊制剂或化妆品。
相对于现有技术，本申请具有以下有益效果：
(1)本申请所述的载益生菌的微胶囊的制备方法，解决了传统方法制备益生菌微胶囊粒径大、球形不规整、分散性差、成本高、制备繁琐的问题。
(2)本申请的益生菌微胶囊粒径为25-40μm，pH敏感性佳，在酸性环境下表面更为致密，保护所包埋的益生菌，而在肠道的pH环境下，能够迅速崩解，释放出包埋的益生菌，可满足后续动物体内评价需求。
附图说明
图1为嗜酸乳杆菌光学显微镜表征图。
图2是嗜酸乳杆菌扫描电镜表征图。
图3是实施例1制备的载有嗜酸乳杆菌微胶囊光学显微镜表征图。
图4是实施例1制备的载有嗜酸乳杆菌微胶囊扫描电镜表征图。
图5是实施例1制备的载有嗜酸乳杆菌微胶囊的粒径及粒径分布图。
图6A是实施例1制备的载有嗜酸乳杆菌微胶囊在pH＝1.2的环境下表面的形态。
图6B是实施例1制备的载有嗜酸乳杆菌微胶囊在pH＝1.2的环境下表面的形态局部放大图。
图6C是实施例1制备的载有嗜酸乳杆菌微胶囊在pH＝6.8的环境下表面的形态。
图6D是实施例1制备的载有嗜酸乳杆菌微胶囊在pH＝6.8的环境下表面的形态局部放大图。
图6E是对比例5制备的载有嗜酸乳杆菌微胶囊在pH＝1.2的环境下表面的形态。
图6F是对比例5制备的载有嗜酸乳杆菌微胶囊在pH＝1.2的环境下表面的形态局部放大图。
图6G是对比例5制备的载有嗜酸乳杆菌微胶囊在pH＝6.8的环境下表面的形态。
图6H是对比例5制备的载有嗜酸乳杆菌微胶囊在pH＝6.8的环境下表面的形态局部放大图。
图7A是未给药的小鼠肠道细胞增殖的免疫组化图。
图7B是未给药的小鼠肠道细胞增殖的免疫组化图的局部放大图。
图7C是生理盐水灌胃后第7天小鼠肠道细胞增殖的免疫组化图。
图7D是嗜酸乳杆菌液灌胃后第7天小鼠肠道细胞增殖的免疫组化图。
图7E是实施例1制备的水灌载嗜酸乳杆菌微囊低剂量灌胃后第7天小鼠肠道细胞增殖的免疫组化图。
图7F是实施例1制备的水灌载嗜酸乳杆菌微囊中剂量灌胃后第7天小鼠肠道细胞增殖的免疫组化图。
图7G是实施例1制备的水灌载嗜酸乳杆菌微囊高剂量灌胃后第7天小鼠肠道细胞增殖的免疫组化图。
图7H是生理盐水灌胃后第14天小鼠肠道细胞增殖的免疫组化图。
图7I是嗜酸乳杆菌液灌胃后第14天小鼠肠道细胞增殖的免疫组化图。
图7J是实施例1制备的水灌载嗜酸乳杆菌微囊低剂量灌胃后第14天小鼠肠道细胞增殖的免疫组化图。
图7K是实施例1制备的水灌载嗜酸乳杆菌微囊中剂量灌胃后第14天小鼠肠道细胞增殖的免疫组化图。
图7L是实施例1制备的水灌载嗜酸乳杆菌微囊高剂量灌胃后第14天小鼠肠道细胞增殖的免疫组化图。
图7M是生理盐水灌胃后第21天小鼠肠道细胞增殖的免疫组化图。
图7N是嗜酸乳杆菌液灌胃后第21天小鼠肠道细胞增殖的免疫组化图。
图7O是实施例1制备的水灌载嗜酸乳杆菌微囊低剂量灌胃后第21天小鼠肠道细胞增殖的免疫组化图。
图7P是实施例1制备的水灌载嗜酸乳杆菌微囊中剂量灌胃后第21天小鼠肠道细胞增殖的免疫组化图。
图7Q是实施例1制备的水灌载嗜酸乳杆菌微囊高剂量灌胃后第21天小鼠肠道细胞增殖的免疫组化图。
图8A是实施例1制备的载有嗜酸乳杆菌微胶囊灌胃后小鼠肠道细胞增殖的统计柱状图。
图8B是实施例1制备的载有嗜酸乳杆菌微胶囊灌胃后小鼠肠道细胞增殖的统计曲线图。
图9是实施例1制备的载有嗜酸乳杆菌微胶囊对小肠巨噬细胞活性影响统计柱状图。
图10A是实施例1制备的载有嗜酸乳杆菌微胶囊对淋巴细胞亚群的影响的代表性流式图。
图10B是实施例1制备的载有嗜酸乳杆菌微胶囊对辅助性T淋巴细胞影响的统计图。
图10C是实施例1制备的载有嗜酸乳杆菌微胶囊对细胞毒性T淋巴细胞影响的统计图。
具体实施方式
下面通过具体实施方式来进一步说明本申请的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本申请，不应视为对本申请的具体限制。
本申请的实施例中使用了具体的嗜酸乳杆菌和双歧杆菌菌株，但是本领域技术人员应当理解，任何其他嗜酸乳杆菌和双歧杆菌菌株也都可以实施本申请。嗜酸乳杆菌和双歧杆菌不被具体限制。
实施例1
在本实施例中，根据如下方法制备了一种载嗜酸乳杆菌微胶囊：
(1)将海藻酸钠溶于生理盐水中，制备成海藻酸钠溶液，其中海藻酸钠溶液的浓度为2wt％；
(2)将嗜酸乳杆菌液(获自天津科技大学发酵食品与微生物资源开发研究室(5))与海藻酸钠溶液混合，缓慢搅拌，使其混合均匀形成悬浮液，其中加入的嗜酸乳杆菌液的量为10 9CFU/mL；
(3)将步骤(2)中的悬浮液，在500L/h气速下，喷雾干燥器的进料速度设置为2mL/min，通过喷雾干燥器的喷嘴喷入1mol/L氯化钙溶液中，缓慢搅拌固化液滴，形成微胶囊；
(4)离心洗涤去除多余的氯化钙和未固化的液滴，收集制备的微胶囊，悬浮保存在生理盐水中。
其中，所述嗜酸乳杆菌的准备及检测如下：
采用光学显微镜和扫描电子显微镜对嗜酸乳杆菌的形貌进行观察，嗜酸乳杆菌光学显微镜表征如图1所示，嗜酸乳杆菌扫描电镜表征如图2所示。通过革兰氏染色法对嗜酸乳杆菌进行鉴别，图1结果显示：包埋用的嗜酸乳杆菌为革兰式阳性菌；采用扫描电子显微镜观察了嗜酸乳杆菌的形貌，图2为放大5,000倍下的嗜酸乳杆菌的形貌，结果显示：嗜酸乳杆菌为杆状菌，杆的末端呈圆形，长度约为2-3μm，杆的截面直径约为600nm左右。
实施例2
在本实施例中，根据如下方法制备了一种载嗜酸乳杆菌微胶囊：
(1)将海藻酸钠溶于生理盐水中，制备成海藻酸钠溶液，其中海藻酸钠溶液的浓度为0.5wt％；
(2)将嗜酸乳杆菌液(获自天津科技大学发酵食品与微生物资源开发研究室(5))与海藻酸钠溶液混合，缓慢搅拌，使其混合均匀形成悬浮液，其中加入的嗜酸乳杆菌液的量为10 8CFU/mL；
(3)将步骤(2)中的悬浮液，在450L/h气速下，喷雾干燥器的进料速度设置为1mL/min，通过喷雾干燥器的喷嘴喷入0.5mol/L氯化钙溶液中，缓慢搅拌固化液滴，形成微胶囊；
(4)离心洗涤去除多余的氯化钙和未固化的液滴，收集制备的微胶囊，悬浮保存在生理盐水中。
其中，所述嗜酸乳杆菌的准备及检测方法同实施例1。
实施例3
在本实施例中，根据如下方法制备了一种载嗜酸乳杆菌微胶囊：
(1)将海藻酸钠溶于生理盐水中，制备成海藻酸钠溶液，其中海藻酸钠溶液的浓度为3wt％；
(2)将嗜酸乳杆菌液(获自天津科技大学发酵食品与微生物资源开发研究室(5))与海藻酸钠溶液混合，缓慢搅拌，使其混合均匀形成悬浮液，其中加入的嗜酸乳杆菌液的量为10 10CFU/mL；
(3)将步骤(2)中的悬浮液，在550L/h气速下，喷雾干燥器的进料速度设置为3mL/min，通过喷雾干燥器的喷嘴喷入1.5mol/L氯化钙溶液中，缓慢搅拌固化液滴，形成微胶囊；
(4)离心洗涤去除多余的氯化钙和未固化的液滴，收集制备的微胶囊，悬浮保存在生理盐水中。
其中，所述嗜酸乳杆菌的准备及检测方法同实施例1。
实施例4
在本实施例中，根据如下方法制备了一种载嗜酸乳杆菌微胶囊：
(1)将海藻酸钠溶于生理盐水中，制备成海藻酸钠溶液，其中海藻酸钠溶液的浓度为2wt％；
(2)将嗜酸乳杆菌液(获自天津科技大学发酵食品与微生物资源开发研究室(5))与海藻酸钠溶液混合，缓慢搅拌，使其混合均匀形成悬浮液，其中加入的嗜酸乳杆菌液的量为10 11CFU/mL；
(3)将步骤(2)中的悬浮液，在500L/h气速下，喷雾干燥器的进料速度设置为2mL/min，通过喷雾干燥器的喷嘴喷入1mol/L氯化钙溶液中，缓慢搅拌固化液滴，形成微胶囊；
(4)离心洗涤去除多余的氯化钙和未固化的液滴，收集制备的微胶囊，悬浮保存在生理盐水中。
其中，所述嗜酸乳杆菌的准备及检测方法同实施例1。
实施例5
在本实施例中，根据如下方法制备了一种载双歧杆菌微胶囊：
(1)将海藻酸钠溶于生理盐水中，制备成海藻酸钠溶液，其中海藻酸钠溶液的浓度为2wt％；
(2)将双歧杆菌液(获自科汉森(天津)食品添加剂有限公司)与海藻酸钠溶液混合，缓慢搅拌，使其混合均匀形成悬浮液，其中加入的双歧杆菌杆菌液的量为10 11CFU/mL；
(3)将步骤(2)中的悬浮液，在500L/h气速下，喷雾干燥器的进料速度设置为2mL/min，通过喷雾干燥器的喷嘴喷入1mol/L氯化钙溶液中，缓慢搅拌固化液滴，形成微胶囊；
(4)离心洗涤去除多余的氯化钙和未固化的液滴，收集制备的微胶囊，悬浮保存在生理盐水中。
对比例1
本对比例制备方法同实施例1，区别仅在于：步骤(3)所述喷入盐溶液的气速为300L/h，所述喷雾干燥器进料速度为5mL/min。
对比例2
本对比例制备方法同实施例1，区别仅在于：步骤(3)所述喷入离子溶液的气速为600L/h，所述喷雾干燥器进料速度为5mL/min。
对比例3
本对比例制备方法同实施例1，区别仅在于：步骤(3)所述喷入盐溶液的气速为300L/h，所述喷雾干燥器进料速度为0.5mL/min。
对比例4
本对比例制备方法同实施例1，区别仅在于：步骤(3)所述喷入离子溶液的气速为600L/h，所述喷雾干燥器进料速度为0.5mL/min。
对比例5
本对比例制备方法同实施例1，区别仅在于：步骤(1)将海藻酸钠和变性淀粉的混合物溶于生理盐水中，所述海藻酸钠和变性淀粉的质量比为1:1，制备成海藻酸钠和淀粉混合液。
试验例1
微胶囊的粒径测定
取实施例1-5制备的载益生菌微胶囊和对比例1-5制备的微胶囊重悬于生理盐水中，超声分散后，将其滴加到激光粒度仪的样品池中，进行体积平均粒径(d4,3)及粒径分布(span)的测定。微囊的粒径大小及粒径分布采用激光粒度仪进行测定，实施例1制备的载有嗜酸乳杆菌微胶囊的粒径及粒径分布图如图5所示，实施例1-5制备的载益生菌微胶囊和对比例1-5制备的微胶囊平均粒径大小如表1所示。
表1
Figure PCTCN2020109716-appb-000001
Figure PCTCN2020109716-appb-000002
由表1的测试数据可知，实施例1-5制备的载益生菌微胶颗粒在25-40μm之间，本申请所述流程工艺能够控制胶囊大小，获得颗粒粒径小便于人体吸收的微囊，微胶囊的整体性能得以提高。说明本申请所述的制备方法采用海藻酸钠作为微胶囊的壁材，喷雾法结合离子固化法制备微胶囊，两种方法协同作用，能够实现微胶囊球形佳、粒径小、分散性好，可满足后续动物体内评价需求的目的。
试验例2
包埋率测试
取实施例1-5制备的载益生菌微胶囊和对比例1-5制备的微胶囊进行包埋率测试，所述测试方法包括步骤：实施例1-5制备的载益生菌微胶囊和对比例1-5制备的微胶囊包埋率如表2所示：
表2
Figure PCTCN2020109716-appb-000003
由表2的测试数据可知，实施例1-5制备的载益生菌微胶囊包埋率在75％以上，这充分说明本申请的制备工艺通过控制微胶囊形成过程中的喷入含芯材的壁材悬浮液的气速和喷射装置的料液进料速度，能够进一步提高微胶囊包埋率，可满足后续动物体内评价需求。
试验例3
微胶囊的显微观察和pH敏感性测试
采用光学显微镜和扫描电子显微镜对载嗜酸乳杆菌微胶囊的形貌进行观察(显微镜型号为JEOL(JEM-6700F))，结果如图3和4所示。采用具有拍摄功能的光学显微镜对微胶囊的形态进行观察、拍照及记录，实施例1制备的载有嗜酸乳杆菌微胶囊光学显微镜表征如图3所示。具体过程如下：使用滴管滴加少量样品于载玻片上，轻轻盖上盖玻片，先在低倍镜下找到合适的视野，再转换到高倍镜进行微胶囊形态观察，同时用WV-CP230/G摄像头将样品状态拍摄记录，通过形态观察初步评价微胶囊的质量。由图3的显微镜照片可以看出，微囊中包埋了很多杆状菌，表明微囊对菌具有比较好的包埋效果。采用扫描电子显微镜观察了载嗜酸乳杆菌微胶囊的形貌，实施例1制备的载有嗜酸乳杆菌微胶囊扫描电镜表征如图4所示。由图4的微囊形貌可以看出，微囊表面有杆状菌的形态存在，表明微囊能够包埋较多的菌颗粒。
在电镜下观察不同pH值条件下，微胶囊表面的形态：实施例1制备的载有嗜酸乳杆菌微胶囊在pH＝1.2的环境下表面的形态及其局部放大图如图6A、图6B所示，实施例1制备的载有嗜酸乳杆菌微胶囊在pH＝6.8的环境下表面的形态及其局部放大图如图6C、图6D所示，对比例5制备的载有嗜酸乳杆菌微胶囊在pH＝1.2的环境下表面的形态及其局部放大图如图6E、图6F所示，对比例5制备的载有嗜酸乳杆菌微胶囊在pH＝6.8的环境下表面的形态及其局部放大图如图6G、图6H所示。
由图6A-图6H的对比可知，在电镜下观察不同pH值环境条件下，在酸性条件下，本申请实施例1制备的微胶囊的表面更加致密，表现出优良的pH值敏感性，能够有效的保护益生菌，在弱酸条件下胶囊由很快崩解迅速释放，便于肠道吸收；而对比例5加入淀粉做壁材制备的胶囊在酸性条件下pH值敏感性低，表面孔洞较大，无法有效的保护益生菌。
试验例4
实施例1制备的嗜酸乳杆菌微囊对肠道细胞增殖的影响
BALB/C小鼠(白变种实验室老鼠)连续灌胃给药14天后，解剖小鼠取其回肠，制备切片，利用免疫组化进行Ki-67染色，显微镜下拍照，通过Vectra III全自动定量病理成像分析系统对切片进行数据处理统计。生理盐水、嗜酸乳杆菌液以及口服低、中、高剂量的嗜酸乳杆菌微胶囊(低、中、高剂量组每剂菌含量分别为10 8CFU、10 9CFU和10 10CFU)第0、7、14和21天时小鼠回肠细胞增殖的免疫组化情况如图7A-Q所示，可以看出，随着给药天数的增加，与空白对照组(灌胃生理盐水组)相比，灌胃菌液组、载菌微胶囊低剂量组、载菌微胶囊中剂量组以及载菌微胶囊高剂量组的小鼠道内上皮细胞增殖均有增加，但是服用载菌微胶囊小鼠道内上皮细胞增殖相比灌胃菌液组更为明显。其中，以服用载菌微胶囊高剂量的小鼠肠道上皮细胞增殖最明显，说明载菌微胶囊高剂量服用效果最佳。
实施例1制备的载有嗜酸乳杆菌微胶囊灌胃后小鼠肠道细胞增殖的统计柱状图如图8A所示，实施例1制备的载有嗜酸乳杆菌微胶囊灌胃后小鼠肠道细胞增殖的统计曲线图如图8B所示，与空白对照组(灌胃生理盐水组)相比，口服低、中、高剂量的嗜酸乳杆菌微胶囊均能显著促进小鼠肠道上皮细胞的增殖；给药第7、14和21天3个时间点，与空白对照组相比，灌胃菌液组、载菌微胶囊低剂量组、载菌微胶囊中剂量组以及载菌微胶囊高剂量组小鼠肠道上皮细胞增殖的情况均呈现显著性差异，说明小鼠服用载嗜酸乳杆菌微胶囊后效果显著。
试验例5
实施例1制备的载嗜酸乳杆菌微囊对巨噬细胞吞噬活性测试
BALB/C小鼠灌胃嗜酸乳杆菌微胶囊14天后，颈椎脱臼致死，无菌摘取脾脏，制备成脾细胞悬液。检测灌胃载菌微胶囊后对小鼠脾脏巨噬细胞吞噬活性的影响。实施例1制备的载有嗜酸乳杆菌微胶囊对小肠巨噬细胞活性影响结果如图9所示，图9中纵坐标吸光度值为570nm处的吸光度值，这个值的大小与巨噬细胞吞噬活性相关，值越高，吞噬活性越强，其中，横坐标中的低、中、高分别代表以及口服低、中、高剂量的嗜酸乳杆菌微胶囊(低、中、高剂量组每剂菌含量分别为10 8CFU、10 9CFU和10 10CFU)与空白对照组相比，口服嗜酸乳杆菌微胶囊均能够提高小鼠脾脏巨噬细胞的吞噬活性，其中，小鼠脾脏巨噬细胞的吞噬活性呈现剂量依赖性，即载菌微囊中含菌量越高，小鼠脾脏巨噬细胞吞噬活性越好。与空白对照组相比，载菌微囊高剂量能够极其显著提高小鼠脾脏巨噬细胞的吞噬能力(p<0.001)，说明嗜酸乳杆菌微胶囊具有活化巨噬细胞的能力，从而能够提高机体的非特异性免疫功能。
试验例6
实施例1载嗜酸乳杆菌微囊对T淋巴细胞亚群的影响
成熟的T淋巴细胞可经淋巴管、外周血等进行再循环，发挥细胞免疫及免疫调节等功能。流式细胞分析仪根据淋巴细胞表面标志的不同来检测各淋巴细胞亚群：淋巴细胞主要包括T淋巴细胞(CD3 +)，B淋巴细胞(CD19 +)，NK细胞(CD16 +CD56 +)，其中T淋巴细胞可进一步测定辅助性T淋巴细胞(CD3 +CD4 +)和抑制性/细胞毒性T淋巴细胞(CD3 +CD8 +)。
实施例1制备的载有嗜酸乳杆菌微胶囊对淋巴细胞亚群的影响的代表性流式测试结果如图10A所示，其中，横坐标和纵坐标分别表示对应细胞亚群的细胞数，右侧低、中、高分别代表以及口服低、中、高剂量的嗜酸乳杆菌微胶囊(低、中、高剂量组每剂菌含量分别为10 8CFU、10 9CFU和10 10CFU)；实施例1制备的载有嗜酸乳杆菌微胶囊对小鼠辅助性T淋巴细胞(CD3 +CD4 +)的影响如图10B所示；实施例1制备的载有嗜酸乳杆菌微胶囊对抑制性/细胞毒性T淋巴细胞(CD3 +CD8 +)影响的如图10C所示。结果表明：与空白对照组相比，BALB/C小鼠在口服嗜酸乳杆菌微胶囊后，其外周血T淋巴细胞亚群中CD3 +CD4 +和CD3 +CD8 +淋巴细胞的含量呈现显著升高(p<0.05)。CD4 +T淋巴细胞含量的升高有利于辅助体液免疫，并且介导细胞免疫来调节机体的免疫作用， 而CD8 +T淋巴细胞的含量的升高，有利于机体免疫水平的提高。
申请人声明，本申请通过上述实施例来说明本申请的工艺方法，但本申请并不局限于上述工艺步骤，即不意味着本申请必须依赖上述工艺步骤才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请所选用原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (15)

  1. 一种载益生菌的微胶囊的制备方法，其包括如下步骤：
    (1)将海藻酸钠溶于溶剂中，得到海藻酸钠溶液；
    (2)将益生菌与步骤(1)得到的海藻酸钠溶液混合均匀，得到益生菌悬浮液；和
    (3)向步骤(2)得到的益生菌悬浮液中喷入盐溶液，搅拌固化，得到所述载益生菌的微胶囊。
  2. 根据权利要求1所述载益生菌的微胶囊的制备方法，其中，
    步骤(1)所述溶剂为水或氯化钠水溶液，优选为氯化钠水溶液，进一步优选为质量百分浓度为0.85-0.9％的氯化钠水溶液；或者
    步骤(1)所述溶剂为生理盐水。
  3. 根据权利要求1或2所述载益生菌的微胶囊的制备方法，其中，步骤(1)所述海藻酸钠溶液的质量百分浓度为0.5-3％。
  4. 根据权利要求1-3中任一项所述载益生菌的微胶囊的制备方法，其中，
    步骤(2)所述益生菌的加入量为10 8-10 11CFU/mL。
  5. 根据权利要求1-4中任一项所述载益生菌的微胶囊的制备方法，其中，步骤(2)所述益生菌选自嗜酸乳杆菌、双歧杆菌或革兰氏阳性球菌中的任意一种或至少两种的组合。
  6. 根据权利要求1-5中任一项所述载益生菌的微胶囊的制备方法，其中，步骤(3)所述喷入盐溶液的方式为通过喷雾干燥器的喷嘴喷入。
  7. 根据权利要求1-6中任一项所述载益生菌的微胶囊的制备方法，其中，所述盐溶液为钙盐溶液；
    优选地，所述钙盐溶液包括葡萄糖酸钙、磷酸氢钙、乳酸钙、硫酸钙或氯化钙中的任意一种或至少两种的组合，优选为氯化钙。
  8. 根据权利要求7中任一项所述载益生菌的微胶囊的制备方法，其中，所述钙盐溶液的浓度为0.5-1.5mol/L。
  9. 根据权利要求1-8中任一项所述载益生菌的微胶囊的制备方法，其中，
    所述喷入盐溶液的气速为450-550L/h；和/或
    所述喷雾干燥器进料速度为1-3mL/min。
  10. 根据权利要求1-9中任一项所述载益生菌的微胶囊的制备方法，其中，步骤(3)后还包括步骤(4)：将步骤(3)得到的载益生菌的微胶囊洗涤、收集，悬浮保存在溶剂中；
    优选地，所述洗涤为离心洗涤；
    优选地，步骤(4)中所述溶剂为生理盐水。
  11. 根据权利要求1-10中任一项所述载益生菌的微胶囊的制备方法，其中，所述方法包括如下步骤：
    (1)将海藻酸钠溶于溶剂中，得到质量百分浓度为0.5-3％的海藻酸钠溶液；
    (2)将益生菌与步骤(1)得到的海藻酸钠溶液缓慢搅拌混合均匀，所述益生菌的加入量为10 8-10 11CFU/mL，得到益生菌悬浮液；
    (3)将步骤(2)得到的益生菌悬浮液，通过喷雾干燥器的喷嘴喷入钙盐溶液，所述钙盐溶液浓度为0.5-1.5mol/L，喷入气速为450-550L/h，喷雾干燥器进料速度为1-3mL/min，搅拌固化，得到载益生菌的微胶囊；和
    (4)将步骤(3)得到的载益生菌的微胶囊进行离心洗涤、收集，悬浮保存在生理盐水中。
  12. 一种如权利要求1-11中任一项所述载益生菌的微胶囊的制备方法制备得到的载益生菌的微胶囊。
  13. 根据权利要求12所述的载益生菌的微胶囊，其中，所述微胶囊的粒径为25-40μm。
  14. 根据权利要求11或12所述的载益生菌的微胶囊，其中，所述微胶囊的粒径为30-35μm。
  15. 根据权利要求12-14中任一项所述载益生菌的微胶囊用于制备保健食品、饮料食品、临床营养制剂、药物微囊制剂或化妆品的用途。
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