Bovine colostrum immune globulin micro-capsule for improving antiviral capability, preparation method and application thereof

Abstract

The invention belongs to the technical field of bovine coloctrum immune globulin microcapsules, and particularly relates to a bovine coloctrum immune globulin microcapsule for improving antiviral ability, the bovine coloctrum immune globulin microcapsule comprises a capsule core material, an inner cladding layer coating the capsule core material, and an outer cladding layer coating the inner cladding layer; wherein the capsule core material contains bovine coloctrum; the inner cladding contains a stabilizer and lecithin; the outer cladding layer contains a cross-linking agent, sodium alginate and modified pectin. After the acetylation modified pectin is cured, the acetylation modified pectin can well maintain an encapsulation state in a gastric digestion stage, so that active factors such as immune globulin contained in bovine coloctrum are prevented from being damaged, and the antiviral capability of the bovine coloctrum is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of bovine colostrum immunoglobulin microcapsules, and specifically relates to a bovine colostrum immunoglobulin microcapsule for improving antiviral ability, a preparation method and an application thereof. Background Art

[0002] Bovine colostrum is a very special kind of "milk". It refers to the milk of a cow within 72 hours of giving birth. It is generally light yellow in color and has a certain milky smell and bitterness. It needs to be purified and processed before it can be used for consumption. Bovine colostrum is not only rich in nutrients, but also contains active factors such as immunoglobulins, lactoferrin, and cytokines that promote growth and development, and can provide balanced nutrition for the body. The nutrients in bovine colostrum have the following functions: (1) improve the body's antiviral ability; (2) regulate intestinal flora; (3) delay aging; (4) promote growth and development; (5) regulate blood sugar; (6) improve gastrointestinal function. However, the active factors such as immunoglobulins contained in bovine colostrum are easily destroyed. For example, they are often not resistant to gastric acid, oxidation, and high temperature. Summary of the invention

[0003] The purpose of the present invention is to provide a bovine colostrum immunoglobulin microcapsule for improving antiviral ability, a preparation method and application thereof, so as to solve the problems raised in the above background technology.

[0004] According to a first aspect of an embodiment of the present invention, a bovine colostrum immunoglobulin microcapsule for improving antiviral ability is provided, wherein the bovine colostrum immunoglobulin microcapsule comprises a core, an inner layer coating the core, and an outer layer coating the inner layer; wherein the core contains bovine colostrum; the inner layer contains a stabilizer and lecithin; and the outer layer contains a cross-linking agent, sodium alginate and modified pectin.

[0005] In one aspect of the embodiments of the present invention, the stabilizer is monoglyceride.

[0006] In one aspect of the embodiments of the present invention, the monoglyceride is glyceryl monolaurate, glyceryl monopalmitate, glyceryl monooleate, glyceryl monolinoleate, glyceryl monolinolenate or glyceryl monobehenate.

[0007] In one aspect of the embodiments of the present invention, preferably, the monoglyceride is glyceryl monolinoleate or glyceryl monobehenate. Further preferably, the monoglyceride is glyceryl monobehenate.

[0008] In one aspect of the embodiments of the present invention, the cross-linking agent is glutaraldehyde.

[0009] In one aspect of the embodiments of the present invention, the mass ratio of the monoglyceride to lecithin is (0.75-2):1.

[0010] In one aspect of the embodiments of the present invention, the modified pectin is acetylated modified pectin.

[0011] According to a second aspect of an embodiment of the present invention, there is provided a method for preparing the aforementioned bovine colostrum immunoglobulin microcapsules, comprising the following steps:

[0012] Step 1: dissolving monoglyceride and lecithin in edible oil, and then adding bovine colostrum to obtain the emulsion of step 1;

[0013] Step 2: preparing acetylated pectin;

[0014] Step 3: dissolving the acetylated pectin in water, and then adding citric acid, a cross-linking agent and sodium alginate to obtain the solution of step 3;

[0015] Step 4: Add the solution of step 3 dropwise to the emulsion of step 1 under stirring, continue stirring for 3-6 hours after the addition is complete, and let stand for 10-18 hours to obtain the emulsion of step 4;

[0016] Step 5: adding the emulsion of step 4 to the diluent containing the coagulant, and after standing and stratifying, taking the upper layer of liquid, and concentrating the upper layer of liquid to obtain the bovine colostrum immunoglobulin microcapsules.

[0017] In one aspect of the embodiments of the present invention, in step 1, the bovine colostrum may further contain one or more sterols; wherein the sterol is any one of cholesterol, brassicasterol, β-sitosterol, ergosterol, cynarinosterol or stigmasterol.

[0018] In one aspect of the embodiments of the present invention, preferably, the sterol is ergosterol or stigmasterol.

[0019] In one aspect of the embodiment of the present invention, based on the total mass of the bovine colostrum, the mass percentage of the sterol is 0.05%-0.5%, but is not limited thereto. Specifically, based on the total mass of the bovine colostrum, the sterol is ergosterol or stigmasterol, and the mass percentage of the sterol is 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5%.

[0020] In one aspect of the embodiments of the present invention, in step 1, the monoglyceride is glyceryl monolaurate, glyceryl monopalmitate, glyceryl monooleate, glyceryl monolinoleate, glyceryl monolinolenate or glyceryl monobehenate.

[0021] In one aspect of the embodiment of the present invention, preferably, in step 1, the monoglyceride is glyceryl monolinoleate or glyceryl monobehenate. Further preferably, in step 1, the monoglyceride is glyceryl monobehenate.

[0022] In one aspect of the embodiment of the present invention, in step 1, the mass ratio of the monoglyceride to lecithin is (0.75-2): 1. Preferably, in step 1, the mass ratio of the monoglyceride to lecithin is (1.25-1.5): 1.

[0023] In one aspect of the embodiment of the present invention, in step 1, the edible oil is one or more of soybean oil, corn oil, grape seed oil, sesame oil, olive oil, sweet almond oil, wheat germ oil, sunflower seed oil, sea buckthorn oil, and castor oil. Specifically, the edible oil is soybean oil.

[0024] In one aspect of the embodiments of the present invention, in step 1, the volume ratio of the edible oil to the bovine colostrum is 1:(3.5-6); preferably, in step 1, the volume ratio of the edible oil to the bovine colostrum is 1:(4.5-5).

[0025] In one aspect of the embodiments of the present invention, the pectin used in the present invention is orange peel pectin, grapefruit pectin or southern jujube pectin, but is not limited thereto. Preferably, the pectin is orange peel pectin.

[0026] In one aspect of the embodiments of the present invention, the acetylated pectin is prepared by the following steps:

[0027] Step 2-1: preparing extracted orange peel pectin;

[0028] Step 2-2: dissolving the extracted orange peel pectin in water, adding an alkali solution to adjust the pH value to 9.0, then adding acetic anhydride dropwise under stirring until the pH value is 7.5, and then reacting at 40° C.-55° C. for 1.5-3 hours to obtain the intermediate product of step 2-2;

[0029] Step 2-3: After centrifuging the intermediate product of step 2-2, the supernatant is collected, and then the supernatant is dialyzed, concentrated, alcohol-precipitated and freeze-dried to obtain the acetylated-modified orange peel pectin.

[0030] In one aspect of the embodiment of the present invention, in step 2-2, the alkaline solution is a dilute NaOH solution, but is not limited thereto. In one aspect of the embodiment of the present disclosure, specifically, in step 2-2, the alkaline solution is a 0.2 mol / L dilute NaOH solution.

[0031] In one aspect of an embodiment of the present invention, specifically, step 2-3: centrifuge the intermediate product of step 2-2, collect the supernatant, transfer the supernatant into a dialysis bag of 8000-14000u, dialyze for 24h, concentrate the supernatant after dialysis, and precipitate the concentrated product with alcohol at a volume ratio of 1:4 between the reaction solution and anhydrous ethanol; precipitate at 4°C for 8-16h, centrifuge and re-dissolve the precipitate in water; remove the ethanol, and freeze-dry to obtain the acetylated modified orange peel pectin.

[0032] In one aspect of the embodiments of the present invention, the extracted orange peel pectin is prepared by the following steps:

[0033] Step 3-1: Take a small piece of orange peel, add the small piece of orange peel to an ammonium oxalate solution, and heat under reflux at 65° C.-75° C. for 2-3 hours; after the heating is completed, centrifuge the obtained product, remove the supernatant, and obtain the precipitate of step 3-1;

[0034] Step 3-2: Slowly add 3-4 times the volume of anhydrous ethanol to the product of step 3-1, stir evenly and let stand for 8-16 hours, then centrifuge and freeze-dry the obtained product to obtain the extracted orange peel pectin.

[0035] In one aspect of the embodiment of the present invention, in step 3-1, the concentration of the ammonium oxalate solution is 30 mmol / L to 120 mmol / L; specifically, in step 3-1, the concentration of the ammonium oxalate solution is 0.1 mol / L.

[0036] In one aspect of an embodiment of the present invention, specifically, step 3-1: take small pieces of orange peel, add ammonium oxalate solution with a concentration of 0.1 mol / L, the solid-liquid ratio (g / mL) is 1:20, and heat to reflux at 70°C for 2.5 hours; after heating, filter out impurities with gauze, leaving the filtrate; centrifuge the filtrate at 3000r / min for 10 minutes, remove the supernatant, and obtain the precipitate of step 3-1.

[0037] In one aspect of the embodiments of the present invention, in step 3, the mass ratio of the acetylated pectin to sodium alginate is (4-8):1.

[0038] In one aspect of the embodiments of the present invention, in step 3, the mass ratio of the acetylated pectin to citric acid is (15-150):1; but not limited thereto.

[0039] In one aspect of an embodiment of the present invention, in step 3, the solution of step 3 may further include an osmotic pressure regulator, and the osmotic pressure regulator may be at least one of glucose, sucrose, mannitol, succinate, arginine, glycine, lysine and citrate, but is not limited thereto.

[0040] In one aspect of the embodiment of the present invention, in step 5, the coagulant is Na2SO4, and the diluent containing the coagulant is a 2% Na2SO4 solution.

[0041] In one aspect of an embodiment of the present invention, specifically, step 5: provide a diluent containing a coagulant, add the emulsion of step 4 to the diluent containing a coagulant, and after standing and stratifying, take the upper layer of liquid, and evaporate the upper layer of liquid under reduced pressure at room temperature to obtain a colloidal product, and spray-dry the colloidal product to obtain the bovine colostrum immunoglobulin microcapsules.

[0042] According to a second aspect of the embodiments of the present disclosure, there is provided a use of the aforementioned bovine colostrum immunoglobulin microcapsules or the bovine colostrum immunoglobulin microcapsules obtained by the aforementioned preparation method in the preparation of a pharmaceutical composition.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The preparation method of the bovine colostrum immunoglobulin microcapsules of the present invention comprises the following steps: first preparing an emulsion in which the bovine colostrum is coated with a stabilizer and lecithin, and then combining the stabilizer and acetylated pectin to obtain the bovine colostrum immunoglobulin microcapsules with pectin as an outer coating layer and the stabilizer and lecithin as an inner coating layer; after solidification, the acetylated pectin can better maintain an encapsulation state when being digested in the stomach, thereby preventing active factors such as immunoglobulins contained in the bovine colostrum from being destroyed, thereby improving the body's antiviral ability.

[0045] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. The relevant embodiments described herein are illustrative and are used to provide a basic understanding of the present invention. The embodiments of the present invention should not be interpreted as limiting the present invention.

[0047] For simplicity, only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, and any upper limit can be combined with any other upper limit to form an unspecified range. In addition, each separately disclosed point or single value can itself be combined as a lower limit or upper limit with any other point or single value or with other lower limits or upper limits to form an unspecified range.

[0048] In this article, the terms "comprises", "comprising" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device that includes a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of more restrictions, an element defined by the sentence "comprising a ..." does not exclude the presence of other identical elements in the process, method, article or device that includes the element.

[0049] In the description herein, unless otherwise specified, “above” and “below” include the number.

[0050] Unless otherwise specified, the terms used in the present invention have the commonly known meanings generally understood by those skilled in the art. Unless otherwise specified, the numerical values ​​of the various parameters mentioned in the present invention can be measured using various measurement methods commonly used in the art (for example, they can be tested according to the methods given in the embodiments of the present invention).

[0051] The term "about" is used to describe and illustrate small changes. When used in conjunction with an event or situation, the term may refer to an example in which the event or situation occurs precisely and an example in which the event or situation occurs very approximately. For example, when used in conjunction with a numerical value, the term may refer to a range of variation of less than or equal to ±10% of the numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%. In addition, amounts, ratios, and other numerical values ​​are sometimes presented in this article in a range format. It should be understood that such a range format is for convenience and simplicity, and should be flexibly understood to include not only numerical values ​​explicitly designated as range limits, but also all individual numerical values ​​or subranges encompassed within the range, as if each numerical value and subrange were explicitly designated.

[0052] A list of items connected by the terms "at least one of," "at least one of," "at least one of," or other similar terms may mean any combination of the listed items. For example, if items A and B are listed, the phrase "at least one of A and B" means only A; only B; or A and B. In another example, if items A, B, and C are listed, the phrase "at least one of A, B, and C" means only A; or only B; only C; A and B (excluding C); A and C (excluding B); B and C (excluding A); or all of A, B, and C. Item A may contain a single component or multiple components. Item B may contain a single component or multiple components. Item C may contain a single component or multiple components.

[0053] The present invention will be further described below in conjunction with the examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0054] Embodiment 1

[0055] 1. Preparation of pectin extracted with ammonium oxalate:

[0056] Take dry orange peel, cut it into small pieces, weigh 20g of small pieces of orange peel, add 0.1mol / L ammonium oxalate solution, the solid-liquid ratio (g / mL) is 1:20, and heat to reflux at 70°C for 2.5h; after heating, filter out impurities with gauze to leave the filtrate; centrifuge the filtrate at 3000r / min for 10min, remove the supernatant, and obtain a precipitate; slowly add 3 times the volume of anhydrous ethanol to the precipitate, stir evenly and let it stand for 12h, then centrifuge and freeze-dry the resulting product to obtain pectin extracted with ammonium oxalate.

[0057] 2. Preparation of acetylated pectin:

[0058] Weigh 5 g of pectin extracted with ammonium oxalate, dissolve it in 200 mL of deionized water, maintain magnetic stirring (400 r / min), add 0.5 mol / L NaOH solution to adjust the pH value to 9.0, stir for 30 min, add acetic anhydride to pH 7.5, and then react at 50°C for 2.5 h; after the reaction, centrifuge the reaction solution at 3000 r / min for 8 min, collect the supernatant after centrifugation, transfer the supernatant to a 8000-14000u dialysis bag, dialyze for 24 h, concentrate the supernatant after dialysis, and precipitate the concentrated product with alcohol at a volume ratio of 1:4 between the reaction solution and anhydrous ethanol; precipitate at 4°C for 12 h, re-dissolve the precipitate with water after centrifugation; after removing ethanol, freeze-dry to obtain acetylated pectin.

[0059] 3. Preparation of bovine colostrum immunoglobulin microcapsules:

[0060] 18 mL of bovine colostrum was measured, 0.45 g of lecithin and 0.64 g of monolaurin were dissolved in 3.6 mL of soybean oil, and ultrasonic treatment was performed at 200 kHz for 15 min, and then the bovine colostrum was quickly added, and magnetic stirring was performed at 1000 r / min for 5 min to obtain an emulsion containing bovine colostrum;

[0061] 2.5 g of the acetylated pectin prepared above was taken and dissolved in 25 mL of 40° C. deionized water, and then 0.1 g of a cross-linking agent glutaraldehyde, 0.125 g of citric acid and 0.4 g of sodium alginate were added and stirred for 15 min to obtain an aqueous solution containing acetylated pectin;

[0062] The emulsion containing bovine colostrum was stirred at a stirring speed of 500 r / min, and then an aqueous solution containing acetylated pectin was added dropwise thereto. After the addition, stirring was continued for 5 hours, and the mixture was allowed to stand for 12 hours to obtain an emulsion.

[0063] The above emulsion was added to 200 mL of 2% Na2SO4 solution, and after standing for stratification, the upper layer of liquid was taken, and the obtained upper layer of liquid was evaporated under reduced pressure in a rotary evaporator at room temperature to form a colloidal product, and then the colloidal product was freeze-dried to obtain the bovine colostrum immunoglobulin microcapsules of Example 1.

[0064] Embodiment 2

[0065] 1. Preparation of pectin extracted with ammonium oxalate:

[0066] Take dry orange peel, cut it into small pieces, weigh 20g of small pieces of orange peel, add 0.1mol / L ammonium oxalate solution, the solid-liquid ratio (g / mL) is 1:20, and heat to reflux at 70°C for 2.5h; after heating, filter out impurities with gauze to leave the filtrate; centrifuge the filtrate at 3000r / min for 10min, remove the supernatant, and obtain a precipitate; slowly add 3 times the volume of anhydrous ethanol to the precipitate, stir evenly and let it stand for 12h, then centrifuge and freeze-dry the resulting product to obtain pectin extracted with ammonium oxalate.

[0067] 2. Preparation of acetylated pectin:

[0068] Weigh 5 g of pectin extracted with ammonium oxalate, dissolve it in 200 mL of deionized water, maintain magnetic stirring (400 r / min), add 0.5 mol / L NaOH solution to adjust the pH value to 9.0, stir for 30 min, add acetic anhydride to pH 7.5, and then react at 50°C for 2.5 h; after the reaction, centrifuge the reaction solution at 3000 r / min for 8 min, collect the supernatant after centrifugation, transfer the supernatant to a 8000-14000u dialysis bag, dialyze for 24 h, concentrate the supernatant after dialysis, and precipitate the concentrated product with alcohol at a volume ratio of 1:4 between the reaction solution and anhydrous ethanol; precipitate at 4°C for 12 h, re-dissolve the precipitate with water after centrifugation; after removing ethanol, freeze-dry to obtain acetylated pectin.

[0069] 3. Preparation of bovine colostrum immunoglobulin microcapsules:

[0070] 18 mL of bovine colostrum was measured, 0.45 g of lecithin and 0.64 g of monopalmitin were dissolved in 3.6 mL of soybean oil, and ultrasonic treatment was performed at 200 kHz for 15 min, and then the bovine colostrum was quickly added, and magnetic stirring was performed at 1000 r / min for 5 min to obtain an emulsion containing bovine colostrum;

[0071] 2.5 g of the acetylated pectin prepared above was taken and dissolved in 25 mL of 40° C. deionized water, and then 0.1 g of a cross-linking agent glutaraldehyde, 0.125 g of citric acid and 0.4 g of sodium alginate were added and stirred for 15 min to obtain an aqueous solution containing acetylated pectin;

[0072] The emulsion containing bovine colostrum was stirred at a stirring speed of 500 r / min, and then an aqueous solution containing acetylated pectin was added dropwise thereto. After the addition, stirring was continued for 5 hours, and the mixture was allowed to stand for 12 hours to obtain an emulsion.

[0073] The aforementioned emulsion was added to 200 mL of 2% Na2SO4 solution, and after standing for stratification, the upper layer of liquid was taken, and the obtained upper layer of liquid was evaporated under reduced pressure in a rotary evaporator at room temperature to form a colloidal product, and then the colloidal product was freeze-dried to obtain the bovine colostrum immunoglobulin microcapsules of Example 2.

[0074] Embodiment 3

[0075] 1. Preparation of pectin extracted with ammonium oxalate:

[0076] Take dry orange peel, cut it into small pieces, weigh 20g of small pieces of orange peel, add 0.1mol / L ammonium oxalate solution, the solid-liquid ratio (g / mL) is 1:20, and heat to reflux at 70°C for 2.5h; after heating, filter out impurities with gauze to leave the filtrate; centrifuge the filtrate at 3000r / min for 10min, remove the supernatant, and obtain a precipitate; slowly add 3 times the volume of anhydrous ethanol to the precipitate, stir evenly and let it stand for 12h, then centrifuge and freeze-dry the resulting product to obtain pectin extracted with ammonium oxalate.

[0077] 2. Preparation of acetylated pectin:

[0078] Weigh 5 g of pectin extracted with ammonium oxalate, dissolve it in 200 mL of deionized water, maintain magnetic stirring (400 r / min), add 0.5 mol / L NaOH solution to adjust the pH value to 9.0, stir for 30 min, add acetic anhydride to pH 7.5, and then react at 50°C for 2.5 h; after the reaction, centrifuge the reaction solution at 3000 r / min for 8 min, collect the supernatant after centrifugation, transfer the supernatant to a 8000-14000u dialysis bag, dialyze for 24 h, concentrate the supernatant after dialysis, and precipitate the concentrated product with alcohol at a volume ratio of 1:4 between the reaction solution and anhydrous ethanol; precipitate at 4°C for 12 h, re-dissolve the precipitate with water after centrifugation; after removing ethanol, freeze-dry to obtain acetylated pectin.

[0079] 3. Preparation of bovine colostrum immunoglobulin microcapsules:

[0080] 18 mL of bovine colostrum was measured, 0.45 g of lecithin and 0.64 g of monoolein were dissolved in 3.6 mL of soybean oil, and ultrasonic treatment was performed at 200 kHz for 15 min, and then the bovine colostrum was quickly added, and magnetic stirring was performed at 1000 r / min for 5 min to obtain an emulsion containing bovine colostrum;

[0081] 2.5 g of the acetylated pectin prepared above was taken and dissolved in 25 mL of 40° C. deionized water, and then 0.1 g of a cross-linking agent glutaraldehyde, 0.125 g of citric acid and 0.4 g of sodium alginate were added and stirred for 15 min to obtain an aqueous solution containing acetylated pectin;

[0082] The emulsion containing bovine colostrum was stirred at a stirring speed of 500 r / min, and then an aqueous solution containing acetylated pectin was added dropwise thereto. After the addition, stirring was continued for 5 hours, and the mixture was allowed to stand for 12 hours to obtain an emulsion.

[0083] The aforementioned emulsion was added to 200 mL of 2% Na2SO4 solution, and after standing for stratification, the upper layer of liquid was taken, and the obtained upper layer of liquid was evaporated under reduced pressure in a rotary evaporator at room temperature to form a colloidal product, which was then freeze-dried to obtain the bovine colostrum immunoglobulin microcapsules of Example 3.

[0084] Embodiment 4

[0085] 1. Preparation of pectin extracted with ammonium oxalate:

[0086] Take dry orange peel, cut it into small pieces, weigh 20g of small pieces of orange peel, add 0.1mol / L ammonium oxalate solution, the solid-liquid ratio (g / mL) is 1:20, and heat to reflux at 70°C for 2.5h; after heating, filter out impurities with gauze to leave the filtrate; centrifuge the filtrate at 3000r / min for 10min, remove the supernatant, and obtain a precipitate; slowly add 3 times the volume of anhydrous ethanol to the precipitate, stir evenly and let it stand for 12h, then centrifuge and freeze-dry the resulting product to obtain pectin extracted with ammonium oxalate.

[0087] 2. Preparation of acetylated pectin:

[0088] Weigh 5 g of pectin extracted with ammonium oxalate, dissolve it in 200 mL of deionized water, maintain magnetic stirring (400 r / min), add 0.5 mol / L NaOH solution to adjust the pH value to 9.0, stir for 30 min, add acetic anhydride to pH 7.5, and then react at 50°C for 2.5 h; after the reaction, centrifuge the reaction solution at 3000 r / min for 8 min, collect the supernatant after centrifugation, transfer the supernatant to a 8000-14000u dialysis bag, dialyze for 24 h, concentrate the supernatant after dialysis, and precipitate the concentrated product with alcohol at a volume ratio of 1:4 between the reaction solution and anhydrous ethanol; precipitate at 4°C for 12 h, re-dissolve the precipitate with water after centrifugation; after removing ethanol, freeze-dry to obtain acetylated pectin.

[0089] 3. Preparation of bovine colostrum immunoglobulin microcapsules:

[0090] 18 mL of bovine colostrum was measured, 0.45 g of lecithin and 0.64 g of monolinoleylglycerol were dissolved in 3.6 mL of soybean oil, and ultrasonic treatment was performed at 200 kHz for 15 min, and then the bovine colostrum was quickly added, and magnetic stirring was performed at 1000 r / min for 5 min to obtain an emulsion containing bovine colostrum;

[0091] 2.5 g of the acetylated pectin prepared above was taken and dissolved in 25 mL of 40° C. deionized water, and then 0.1 g of a cross-linking agent glutaraldehyde, 0.125 g of citric acid and 0.4 g of sodium alginate were added and stirred for 15 min to obtain an aqueous solution containing acetylated pectin;

[0092] The emulsion containing bovine colostrum was stirred at a stirring speed of 500 r / min, and then an aqueous solution containing acetylated pectin was added dropwise thereto. After the addition, stirring was continued for 5 hours, and the mixture was allowed to stand for 12 hours to obtain an emulsion.

[0093] The aforementioned emulsion was added to 200 mL of 2% Na2SO4 solution, and after standing for stratification, the upper layer of liquid was taken, and the obtained upper layer of liquid was evaporated under reduced pressure in a rotary evaporator at room temperature to form a colloidal product, which was then freeze-dried to obtain the bovine colostrum immunoglobulin microcapsules of Example 4.

[0094] Embodiment 5

[0095] 1. Preparation of pectin extracted with ammonium oxalate:

[0096] Take dry orange peel, cut it into small pieces, weigh 20g of small pieces of orange peel, add 0.1mol / L ammonium oxalate solution, the solid-liquid ratio (g / mL) is 1:20, and heat to reflux at 70°C for 2.5h; after heating, filter out impurities with gauze to leave the filtrate; centrifuge the filtrate at 3000r / min for 10min, remove the supernatant, and obtain a precipitate; slowly add 3 times the volume of anhydrous ethanol to the precipitate, stir evenly and let it stand for 12h, then centrifuge and freeze-dry the resulting product to obtain pectin extracted with ammonium oxalate.

[0097] 2. Preparation of acetylated pectin:

[0098] Weigh 5 g of pectin extracted with ammonium oxalate, dissolve it in 200 mL of deionized water, maintain magnetic stirring (400 r / min), add 0.5 mol / L NaOH solution to adjust the pH value to 9.0, stir for 30 min, add acetic anhydride to pH 7.5, and then react at 50°C for 2.5 h; after the reaction, centrifuge the reaction solution at 3000 r / min for 8 min, collect the supernatant after centrifugation, transfer the supernatant to a 8000-14000u dialysis bag, dialyze for 24 h, concentrate the supernatant after dialysis, and precipitate the concentrated product with alcohol at a volume ratio of 1:4 between the reaction solution and anhydrous ethanol; precipitate at 4°C for 12 h, re-dissolve the precipitate with water after centrifugation; after removing ethanol, freeze-dry to obtain acetylated pectin.

[0099] 3. Preparation of bovine colostrum immunoglobulin microcapsules:

[0100] 18 mL of bovine colostrum was measured, 0.45 g of lecithin and 0.64 g of monolinolenic acid glyceride were dissolved in 3.6 mL of soybean oil, and ultrasonic treatment was performed at 200 kHz for 15 min, and then the bovine colostrum was quickly added, and magnetic stirring was performed at 1000 r / min for 5 min to obtain an emulsion containing bovine colostrum;

[0101] 2.5 g of the acetylated pectin prepared above was taken and dissolved in 25 mL of 40° C. deionized water, and then 0.1 g of a cross-linking agent glutaraldehyde, 0.125 g of citric acid and 0.4 g of sodium alginate were added and stirred for 15 min to obtain an aqueous solution containing acetylated pectin;

[0102] The emulsion containing bovine colostrum was stirred at a stirring speed of 500 r / min, and then an aqueous solution containing acetylated pectin was added dropwise thereto. After the addition, stirring was continued for 5 hours, and the mixture was allowed to stand for 12 hours to obtain an emulsion.

[0103] The aforementioned emulsion was added to 200 mL of 2% Na2SO4 solution, and after standing for stratification, the upper layer of liquid was taken, and the obtained upper layer of liquid was evaporated under reduced pressure in a rotary evaporator at room temperature to form a colloidal product, which was then freeze-dried to obtain the bovine colostrum immunoglobulin microcapsules of Example 5.

[0104] Embodiment 6

[0105] 1. Preparation of glyceryl monobehenate:

[0106] 10 mL of glycerol, 20 mL of acetone, 3 mL of cyclohexane as a water-carrying agent, and 0.25 g of toluenesulfonic acid as a catalyst are added to a 250 mL three-necked flask with a thermometer, a water separator, and a reflux condenser, stirring is started, and the temperature is raised to reflux, and the water-carrying agent takes out the water generated by the reaction from the reaction system. When the amount of water in the water separator remains substantially unchanged and the reaction temperature no longer rises, the condensation reaction is stopped, and 6.7 g of behenic acid is added after cooling, and the temperature is raised to reflux, and the water-carrying agent continues to take out the water generated in the reaction system. When the amount of water in the water separator is substantially constant, the esterification reaction is terminated, and the water-carrying agent and excess acetone are removed by vacuum distillation, and behenic acid isopropylidene glyceride is obtained after cooling;

[0107] Take 2 g of the prepared isopropylidene behenate, add 20 mL of 2 mol / L hydrochloric acid, stir and hydrolyze at room temperature for 1 h, remove the protecting group, filter and wash with water until neutral, and obtain monobehenate after drying.

[0108] 2. Preparation of pectin extracted with ammonium oxalate:

[0109] Take dry orange peel, cut it into small pieces, weigh 20g of small pieces of orange peel, add 0.1mol / L ammonium oxalate solution, the solid-liquid ratio (g / mL) is 1:20, and heat to reflux at 70°C for 2.5h; after heating, filter out impurities with gauze to leave the filtrate; centrifuge the filtrate at 3000r / min for 10min, remove the supernatant, and obtain a precipitate; slowly add 3 times the volume of anhydrous ethanol to the precipitate, stir evenly and let it stand for 12h, then centrifuge and freeze-dry the resulting product to obtain pectin extracted with ammonium oxalate.

[0110] 3. Preparation of acetylated pectin:

[0111] Weigh 5 g of pectin extracted with ammonium oxalate, dissolve it in 200 mL of deionized water, maintain magnetic stirring (400 r / min), add 0.5 mol / L NaOH solution to adjust the pH value to 9.0, stir for 30 min, add acetic anhydride to pH 7.5, and then react at 50°C for 2.5 h; after the reaction, centrifuge the reaction solution at 3000 r / min for 8 min, collect the supernatant after centrifugation, transfer the supernatant to a 8000-14000u dialysis bag, dialyze for 24 h, concentrate the supernatant after dialysis, and precipitate the concentrated product with alcohol at a volume ratio of 1:4 between the reaction solution and anhydrous ethanol; precipitate at 4°C for 12 h, re-dissolve the precipitate with water after centrifugation; after removing ethanol, freeze-dry to obtain acetylated pectin.

[0112] 4. Preparation of bovine colostrum immunoglobulin microcapsules:

[0113] 18 mL of bovine colostrum was measured, 0.45 g of lecithin and 0.64 g of monobehenyl glyceride were dissolved in 3.6 mL of soybean oil, and ultrasonic treatment was performed at 200 kHz for 15 min, and then the bovine colostrum was quickly added, and magnetic stirring was performed at 1000 r / min for 5 min to obtain an emulsion containing bovine colostrum;

[0114] 2.5 g of the acetylated pectin prepared above was taken and dissolved in 25 mL of 40° C. deionized water, and then 0.1 g of a cross-linking agent glutaraldehyde, 0.125 g of citric acid and 0.4 g of sodium alginate were added and stirred for 15 min to obtain an aqueous solution containing acetylated pectin;

[0115] The emulsion containing bovine colostrum was stirred at a stirring speed of 500 r / min, and then an aqueous solution containing acetylated pectin was added dropwise thereto. After the addition, stirring was continued for 5 hours, and the mixture was allowed to stand for 12 hours to obtain an emulsion.

[0116] The aforementioned emulsion was added to 200 mL of 2% Na2SO4 solution, and after standing for stratification, the upper layer of liquid was taken, and the obtained upper layer of liquid was evaporated under reduced pressure in a rotary evaporator at room temperature to form a colloidal product, which was then freeze-dried to obtain the bovine colostrum immunoglobulin microcapsules of Example 6.

[0117] Embodiment 7

[0118] The steps of Example 7 are basically the same as those of Example 1, except that the same molar amount of dilaurin is used to replace the monolaurin used in Example 1.

[0119] Embodiment 8

[0120] The steps of Example 8 are basically the same as those of Example 1, except that Example 8 uses the same molar amount of palmitic acid diglyceride to replace the monopalmitic acid glyceride used in Example 2.

[0121] Comparative Example 1

[0122] 1. Preparation of hot water extracted pectin:

[0123] Take dry orange peel, cut it into small pieces, weigh 20g of small pieces of orange peel, add deionized water, the solid-liquid ratio (g / mL) is 1:20, and heat under reflux at 70°C for 2.5h; after heating, filter out impurities with gauze to leave the filtrate; centrifuge the filtrate at 3000r / min for 10min, remove the supernatant, and obtain a precipitate; slowly add 3 times the volume of anhydrous ethanol to the precipitate, stir evenly and let it stand for 12h, then centrifuge and freeze-dry the resulting product to obtain pectin extracted by hot water.

[0124] 2. Preparation of acetylated pectin:

[0125] Weigh 5g of pectin extracted by hot water, dissolve it in 200mL of deionized water, maintain magnetic stirring (400r / min), add 0.5mol / L NaOH solution to adjust the pH value to 9.0, stir for 30min, add acetic anhydride to pH 7.5, and then react at 50°C for 2.5h; after the reaction, centrifuge the reaction solution at 3000r / min for 8min, collect the supernatant after centrifugation, transfer the supernatant to a 8000-14000u dialysis bag, dialyze for 24h, concentrate the supernatant after dialysis, and precipitate the concentrated product with alcohol at a volume ratio of 1:4 between the reaction solution and anhydrous ethanol; precipitate at 4°C for 12h, re-dissolve the precipitate with water after centrifugation; after removing ethanol, freeze-dry to obtain acetylated pectin.

[0126] 3. Preparation of bovine colostrum immunoglobulin microcapsules:

[0127] 18 mL of bovine colostrum was measured, 0.45 g of lecithin and 0.64 g of monolaurin were dissolved in 3.6 mL of soybean oil, and ultrasonic treatment was performed at 200 kHz for 15 min, and then the bovine colostrum was quickly added, and magnetic stirring was performed at 1000 r / min for 5 min to obtain an emulsion containing bovine colostrum;

[0128] 2.5 g of the acetylated pectin prepared above was taken and dissolved in 25 mL of 40° C. deionized water, and then 0.1 g of a cross-linking agent glutaraldehyde, 0.125 g of citric acid and 0.4 g of sodium alginate were added and stirred for 15 min to obtain an aqueous solution containing acetylated pectin;

[0129] The emulsion containing bovine colostrum was stirred at a stirring speed of 500 r / min, and then an aqueous solution containing acetylated pectin was added dropwise thereto. After the addition, stirring was continued for 5 hours, and the mixture was allowed to stand for 12 hours to obtain an emulsion.

[0130] The above emulsion was added to 200 mL of 2% Na2SO4 solution, and after standing for stratification, the upper layer of liquid was taken, and the obtained upper layer of liquid was evaporated under reduced pressure in a rotary evaporator at room temperature to form a colloidal product, and then the colloidal product was freeze-dried to obtain the bovine colostrum immunoglobulin microcapsules of Comparative Example 1.

[0131] Comparative Example 2

[0132] 1. Preparation of lye-extracted pectin:

[0133] Take dry orange peel, cut it into small pieces, weigh 20g of small pieces of orange peel, add a NaOH solution with a concentration of 0.3mol / L, the solid-liquid ratio (g / mL) is 1:20, and heat to reflux at 70°C for 2.5h; after the heating is completed, filter out impurities with gauze to leave the filtrate; centrifuge the filtrate at 3000r / min for 10min, remove the supernatant, and obtain a precipitate; slowly add 3 times the volume of anhydrous ethanol to the precipitate, stir evenly and let it stand for 12h, and then centrifuge and freeze-dry the resulting product to obtain pectin extracted with alkali solution.

[0134] The steps of preparing acetylated pectin and preparing bovine colostrum immunoglobulin microcapsules in Comparative Example 2 are the same as those in Example 1.

[0135] Comparative Example 3

[0136] 1. Preparation of acid-extracted pectin:

[0137] Take dry orange peel, cut it into small pieces, weigh 20g of small pieces of orange peel, add 0.25mol / L HCl solution, the solid-liquid ratio (g / mL) is 1:20, and heat to reflux at 50°C for 2.5h; after heating, filter out impurities with gauze to leave the filtrate; centrifuge the filtrate at 3000r / min for 10min, remove the supernatant, and obtain a precipitate; slowly add 3 times the volume of anhydrous ethanol to the precipitate, stir evenly and let it stand for 12h, then centrifuge and freeze-dry the resulting product to obtain acid-extracted pectin.

[0138] The steps of preparing acetylated pectin and preparing bovine colostrum immunoglobulin microcapsules in Comparative Example 3 are the same as those in Example 1.

[0139] Comparative Example 4

[0140] Comparative Example 4 The steps for preparing pectin extracted with ammonium oxalate were the same as those in Example 1, but it was directly used for preparing bovine colostrum immunoglobulin microcapsules without acetylation modification.

[0141] In vitro digestion simulation experiment:

[0142] 1. Preparation of digestive fluid: The composition of simulated gastric fluid and simulated intestinal fluid is shown in Table 1.

[0143] Table 1

[0144] Components Gastric juice Intestinal fluid 0.5M KCl / mL 6.9 6.8 <![CDATA[0.5M KH2PO4 / mL]]> 0.9 0.8 <![CDATA[1M NaHCO3 / mL]]> 12.5 42.5 2M NaCl / mL 11.8 9.6 <![CDATA[0.15MgCl2 / mL]]> 0.4 1.1 <![CDATA[0.5M(NH4)2CO3 / mL]]> 0.5 -

[0145] Weigh 0.5 g of each sample of Examples 1 to 8 and Comparative Examples 1 to 4, and add them to 10 mL of simulated gastric fluid or simulated intestinal fluid, respectively; wherein, 1000 U / mg of pepsin is added to the simulated gastric fluid and the pH is adjusted to 2.1 using hydrochloric acid, 1000 U / mg of compound enzyme (trypsin: amylase = 1:1) is added to the simulated intestinal fluid and the pH value is adjusted to 7.0, and then the retention rate of immunoglobulin G is measured at 15 min, 60 min and 120 min, respectively. The results of the retention rate of immunoglobulin G in simulated gastric fluid are shown in Table 2, and the results of the retention rate of immunoglobulin G in simulated intestinal fluid are shown in Table 3.

[0146] Table 2

[0147] Example 15min retention rate 60min retention rate 120min retention rate Embodiment 1 67.5％ 58.4％ 49.5％ Embodiment 2 66.7％ 56.4％ 47.1％ Embodiment 3 65.8％ 56.0％ 47.6％ Embodiment 4 70.4％ 61.5％ 55.8％ Embodiment 5 69.3％ 60.7％ 56.2％ Embodiment 6 75.8％ 70.4％ 64.9％ Embodiment 7 60.4％ 51.7％ 41.8％ Embodiment 8 59.5％ 49.0％ 38.1％ Comparative Example 1 65.1％ 59.4％ 47.6％ Comparative Example 2 65.0％ 59.7％ 47.1％ Comparative Example 3 64.8％ 58.9％ 46.5％ Comparative Example 4 52.4％ 39.2％ 30.7％

[0148] Table 3

[0149] Example 15min retention rate 60min retention rate 120min retention rate Embodiment 1 20.8％ 14.1％ 8.9％ Embodiment 2 24.1％ 18.2％ 11.7％ Embodiment 3 26.0％ 19.5％ 12.4％ Embodiment 4 19.7％ 13.5％ 8.1％ Embodiment 5 20.4％ 14.0％ 10.2％ Embodiment 6 16.7％ 10.8％ 4.5％ Embodiment 7 20.9％ 14.3％ 9.0％ Embodiment 8 24.3％ 18.1％ 11.7％ Comparative Example 1 36.8％ 27.8％ 22.4％ Comparative Example 2 34.7％ 25.1％ 20.8％ Comparative Example 3 26.8％ 20.2％ 17.7％ Comparative Example 4 21.0％ 14.5％ 9.1％

[0150] The retention rate of immunoglobulin G in simulated gastric fluid needs to be as high as possible, so that the activity of bovine colostrum can be retained in the human stomach for as long as possible, while the retention rate of immunoglobulin G in simulated intestinal fluid is as low as possible, so as to promote absorption in the intestine. The preparation method of bovine colostrum immunoglobulin microcapsules of the present invention is to first prepare an emulsion in which bovine colostrum is coated with stabilizers and lecithin, and then combine stabilizers and acetylated pectin to obtain bovine colostrum immunoglobulin microcapsules with pectin as the outer layer and stabilizers and lecithin as the inner layer. Acetylated pectin will solidify under strong acidic conditions so that the structure can be better retained without being destroyed; and the pectin obtained by ammonium oxalate extraction has a higher protein content than pectin extracted by other methods, so it is easy to be affected by enzymes in the intestinal environment and collapse in structure; therefore, the bovine colostrum immunoglobulin microcapsules prepared by the present invention can keep the structure intact for a long time in the gastric environment, thereby retaining the activity of bovine colostrum, and are easy to absorb in the intestinal environment.

[0151] Those skilled in the art will readily appreciate other embodiments of the present invention after considering the specification and practicing the disclosure disclosed herein. The present invention is intended to cover any variations, uses or adaptations of the present invention that follow the general principles of the present invention and include common knowledge or customary technical means in the art that are not disclosed by the present invention.

Claims

1. A bovine colostrum immunoglobulin microcapsule for improving antiviral ability, characterized in that: The bovine colostrum immunoglobulin microcapsule comprises a core, an inner layer covering the core, and an outer layer covering the inner layer; The core material contains bovine colostrum; the inner layer contains a stabilizer and lecithin; and the outer layer contains a cross-linking agent, sodium alginate and modified pectin.

2. The bovine colostrum immunoglobulin microcapsule according to claim 1, characterized in that: The stabilizer is monoglyceride of fatty acid.

3. The bovine colostrum immunoglobulin microcapsule according to claim 2, characterized in that: The mono fatty acid glyceride is glyceryl monolaurate, glyceryl monopalmitate, glyceryl monooleate, glyceryl monolinoleate, glyceryl monolinolenate or glyceryl monobehenate.

4. The bovine colostrum immunoglobulin microcapsule according to claim 2, characterized in that: The mass ratio of the monoglyceride to lecithin is (0.75-2):

1.

5. The bovine colostrum immunoglobulin microcapsule according to claim 1, characterized in that: The cross-linking agent is glutaraldehyde.

6. The bovine colostrum immunoglobulin microcapsule according to claim 1, characterized in that: The modified pectin is acetylated pectin.

7. A method for preparing the bovine colostrum immunoglobulin microcapsules according to any one of claims 1 to 6, characterized in that: The following steps are involved: Step 1: dissolving monoglyceride and lecithin in edible oil, and then adding bovine colostrum to obtain the emulsion of step 1; wherein the edible oil is one or more of soybean oil, corn oil, grape seed oil, sesame oil, olive oil, sweet almond oil, wheat germ oil, sunflower seed oil, sea buckthorn oil, and castor oil; Step 2: preparing acetylated pectin; Step 3: dissolving the acetylated pectin in water, and then adding citric acid, glutaraldehyde and sodium alginate to obtain the solution of step 3; Step 4: Add the solution of step 3 dropwise to the emulsion of step 1 under stirring, continue stirring for 3-6 hours after the addition is complete, and let stand for 10-18 hours to obtain the emulsion of step 4; Step 5: adding the emulsion of step 4 to a diluent containing a coagulant, and after standing and stratifying, taking the upper layer of liquid, and concentrating the upper layer of liquid to obtain the bovine colostrum immunoglobulin microcapsules; wherein the coagulant is Na2SO4, and the diluent containing the coagulant is a 2% Na2SO4 solution.

8. The method for preparing bovine colostrum immunoglobulin microcapsules according to claim 6, characterized in that: The acetylated pectin is prepared by the following steps: Step 2-1: preparing extracted orange peel pectin; Step 2-2: dissolving the extracted orange peel pectin in water, adding an alkali solution to adjust the pH value to 9.0, then adding acetic anhydride dropwise under stirring until the pH value is 7.5, and then reacting at 50° C. for 1.5-3 hours to obtain the intermediate product of step 2-2; wherein the alkali solution is a 0.2 mol / L NaOH dilute solution; Step 2-3: After centrifuging the intermediate product of step 2-2, the supernatant is collected, and then the supernatant is dialyzed, concentrated, alcohol-precipitated and freeze-dried to obtain the acetylated-modified orange peel pectin.

9. The method for preparing bovine colostrum immunoglobulin microcapsules according to claim 6, characterized in that: At least one of the following conditions is met: (1) In step 1, the mass ratio of monoglyceride to lecithin is (1.4-1.5):1; (2) In step 1, the volume ratio of the edible oil to the bovine colostrum is 1:5; (3) In step 3, the mass ratio of the acetylated tangerine peel pectin to sodium alginate is (6-6.5):1; (4) In step 5, the upper layer liquid obtained by static stratification is concentrated and then spray-dried to obtain the bovine colostrum immunoglobulin microcapsule solid.

10. Use of the bovine colostrum immunoglobulin microcapsules according to any one of claims 1 to 6 or the bovine colostrum immunoglobulin microcapsules prepared by the preparation method according to any one of claims 7 to 9 in preparing a pharmaceutical composition.