CN111821421B - Intestinal sustained-release bovine colostrum sea cucumber peptide chewable tablet and preparation method thereof - Google Patents

Abstract

Translated fromChinese

本发明提供一种肠道缓释牛初乳海参肽咀嚼片及其制备方法，咀嚼片中包括如下成分：酪蛋白磷酸肽、海参肽、牛初乳粉、大豆肽、浓缩乳清蛋白、叶黄素酯、维生素C、果蔬粉、抗性糊精、乳糖、赤藓糖醇、山梨糖醇、DL‑苹果酸、低聚木糖、硬脂酸镁；还包括靶向缓释生物相容微囊，所述靶向缓释生物相容微囊，包括：聚碳酸酯薄膜、牛血清白蛋白冻干粉、氨基酸、壳寡糖、卵磷脂、聚(2‑乙基‑2‑恶唑啉)接枝葡聚糖纳米颗粒。本发明提供的制备方法和成分制备的肠道缓释牛初乳海参肽咀嚼片能够在肠道内释放具有质量和营养作用的活性肽，能够穿过血脑屏障作用于脑细胞修复心脑血管和氧化应激后损伤，辅助在胃内消化牛初乳粉和浓缩乳清蛋白进而增强人体免疫力。

The invention provides an intestinal sustained-release bovine colostrum sea cucumber peptide chewable tablet and a preparation method thereof. The chewable tablet includes the following components: casein phosphopeptide, sea cucumber peptide, bovine colostrum powder, soybean peptide, concentrated whey protein, leaf Flavin esters, vitamin C, fruit and vegetable powder, resistant dextrin, lactose, erythritol, sorbitol, DL-malic acid, xylo-oligosaccharides, magnesium stearate; also includes targeted sustained-release biocompatibility Microcapsules, the targeted slow-release biocompatible microcapsules include: polycarbonate film, bovine serum albumin lyophilized powder, amino acid, chitosan oligosaccharide, lecithin, poly(2-ethyl-2-oxazole) morpholino) grafted dextran nanoparticles. The intestinal slow-release bovine colostrum sea cucumber peptide chewable tablet prepared by the preparation method and composition provided by the invention can release active peptides with quality and nutritional effects in the intestinal tract, and can pass through the blood-brain barrier and act on brain cells to repair cardiovascular and cerebrovascular diseases. After oxidative stress, it assists the digestion of bovine colostrum powder and whey protein concentrate in the stomach to enhance human immunity.

Description

Intestinal slow-release bovine colostrum and sea cucumber peptide chewable tablet and preparation method thereof

Technical Field

The invention belongs to the technical field of compound biology, and particularly relates to an intestinal tract sustained-release bovine colostrum and sea cucumber peptide chewable tablet and a preparation method thereof.

Background

Type 2 diabetes is an intractable chronic disease, no specific treatment method is available at present, and the general onset needs lifetime medication.Type 2 diabetes can lead to a variety of complications, such as neurological, cardiovascular and cerebrovascular complications, and can also lead to death of the patient. The "peptide" is a substance between protein and amino acid, is a compound formed by connecting two or more amino acids by peptide bonds, is a core substance of cells, plays an important physiological role in human body and plays a physiological function. Various cell functions and all life behaviors in a human body, such as growth, development, multiplication, metabolism, operation and the like, can be realized only by peptides, and the peptides control the synthesis quantity, quality and speed of proteins; controlling human diseases and aging is a key material determining the quality of human life.

Cerebrovascular disease refers to transient or persistent local and diffuse brain injury caused by pathological changes such as cerebral atherosclerosis, thrombosis, stenosis, occlusion, aneurysm and the like, has the characteristics of high disability rate and high fatality rate, and can seriously affect the life quality and life safety of patients. However, the prior art lacks chewable tablets which can effectively treat the cell oxidative stress caused bytype 2 diabetes or cardiovascular and cerebrovascular diseases and chewable tablets which can slowly release peptide compositions with therapeutic and nutritional effects in intestinal tracts.

Disclosure of Invention

Aiming at the defects, the invention provides the intestinal tract slow-release bovine colostrum sea cucumber peptide chewable tablet which can release casein phosphopeptide, sea cucumber peptide and soybean peptide with quality and nutrition effects in the intestinal tract, can pass through a blood brain barrier to act on brain cells to repair cardiovascular and cerebrovascular sequelae and damage after cell oxidative stress, and can assist bovine colostrum powder and concentrated whey protein digested in the stomach to further enhance the immunity of the human body, and the preparation method thereof.

The invention provides the following technical scheme: an intestinal slow-release bovine colostrum and sea cucumber peptide chewable tablet, each 1g of the chewable tablet comprises the following components:

1-15 mg of casein phosphopeptide;

1mg to 50mg of sea cucumber peptide;

bovine colostrum powder 5-50 mg;

5-70 mg of soybean peptide;

4mg to 114mg of concentrated whey protein;

1 mg-40 mg of lutein ester;

1 mg-30 mg of vitamin C;

10 mg-100 mg of fruit and vegetable powder;

1mg to 5mg of resistant dextrin;

1 mg-4 mg of lactose;

erythritol 0.5-10 mg;

sorbitol 0.5-15 mg;

0.05 mg-1 mg of DL-malic acid;

0.6 mg-1 mg of xylo-oligosaccharide;

magnesium stearate 0.5-1 mg.

Further, each 1g of chewable tablet also comprises 20-30 mg of targeted sustained-release biocompatible microcapsule, and the targeted sustained-release biocompatible microcapsule comprises the following components in parts by weight: 20 to 30 parts of polycarbonate film with the aperture of 200 to 300nm and the thickness of 10 to 12 mm; 10-15 parts of bovine serum albumin freeze-dried powder; 10-15 parts of amino acid; 5-10 parts of chitosan oligosaccharide; 5-10 parts of lecithin; 20-30 parts of poly (2-ethyl-2-oxazoline) grafted glucan nano particles and 0.05-0.1 part of N, N-dimethylformamide; the poly (2-ethyl-2-oxazoline) grafted glucan nanoparticle comprises the following components in parts by weight:

2.5-5 parts of poly (2-ethyl-2-oxazoline); 0.2 to 0.5 portion of succinic anhydride; 0.5 to 1 portion of 3,3' -dithiopropionic acid; 1-1.5 parts of N- (3- (dimethylamino) propyl) -N-ethyl carbodiimide hydrochloride; 2-2.5 parts of 4- (dimethylamino) pyridine; 3-6 parts of glucan;

the preparation method of the poly (2-ethyl-2-oxazoline) grafted glucan nanoparticle comprises the following steps:

m1: dissolving the weight component of poly (2-ethyl-2-oxazoline) and the weight component of succinic anhydride in dichloromethane to form a mixed solution of poly (2-ethyl-2-oxazoline) organic solution with the concentration of 5mmol/L to 10mmol/L and succinic anhydride organic solution with the concentration of 30mmol/L to 50mmol/L, adding the weight component of 4- (dimethylamino) pyridine into the mixed solution, and stirring and reacting for 12h to 24h at the rotating speed of 75rpm to 150rpm and the temperature of 25 ℃ to 28 ℃;

m2: mixing the mixture obtained in the M1 step with diethyl ether at a weight-to-volume ratio of 1:8 for reaction for 10-15 min, precipitating in the diethyl ether to obtain a crude poly (2-ethyl-2-oxazoline) product with terminal amination and carboxylation, and drying the crude poly (2-ethyl-2-oxazoline) product with terminal amination and carboxylation under vacuum condition for 18-36 h to obtain crude poly (2-ethyl-2-oxazoline) product powder with terminal amination and carboxylation;

m3: dissolving the dextran of the weight component in dimethyl sulfoxide, dissolving the dextran in the dimethyl sulfoxide by microwave for 3 to 4 hours under the condition of 10 to 20mHz, then adding the crude product powder of the poly (2-ethyl-2-oxazoline) obtained in the M2 step and half of the N- (3- (dimethylamino) propyl) -N-ethylcarbodiimide hydrochloride of the weight component, stirring the mixture for 30 to 60 minutes at the temperature of between 28 and 32 ℃, adding the 3,3' -dithiopropionic acid of the weight component and the remaining half of the N- (3- (dimethylamino) propyl) -N-ethylcarbodiimide hydrochloride of the weight component, stirring the mixture for 30 to 60 minutes at the temperature of between 28 and 32 ℃, dialyzing the obtained mixture for 1 to 2 hours under a dialysis membrane with the molecular pore size of between 3000 and 3500Da, and (3) freeze-drying at-100 ℃ to obtain the poly (2-ethyl-2-oxazoline) grafted glucan nano-particles.

Further, the preparation method of the targeted sustained-release biocompatible microcapsule comprises the following steps:

a1: dissolving the amino acid and the bovine serum albumin freeze-dried powder in the weight components in distilled water, fully stirring and hydrating for 20-30 min at 80-100 rpm, filtering the obtained mixed solution under a polyvinylidene fluoride filter with the particle size of 0.35-0.45 μm, and taking the filtered supernatant to obtain the amino acid-bovine serum albumin mixed supernatant;

a2: dissolving the weight component of the chitosan oligosaccharide and the weight component of the lecithin in distilled water to form a chitosan oligosaccharide solution with the concentration of 1 mmol/L-5 mmol/L and a lecithin solution with the concentration of 3 mmol/L-8 mmol/L;

a3: dissolving the polycarbonate film of the weight component in a phosphate buffer solution containing sodium chloride with the concentration of 0.13-0.15M, and impregnating and permeating the poly (2-ethyl-2-oxazoline) grafted glucan nano-particles of the weight component at the rate of 9-11 mL/hThen the mixed supernatant of the amino acid and the bovine serum albumin obtained in the step A1 is impregnated and permeated into the polycarbonate film at the speed of 9-11 mL/h and the thickness of the film is 5cm²Standing for 3-5 min under the nitrogen flow with the blowing speed of/L; impregnating the chitosan oligosaccharide solution obtained in the step A2 at the rate of 9-11 mL/h through the polycarbonate film at the depth of 5cm²Standing for 3-5 min under nitrogen gas flow at blow rate of/L, impregnating the lecithin solution obtained in the step A2 at the rate of 9-11 mL/h through the polycarbonate film, continuously dripping N, N-dimethylformamide with the weight component in the impregnation process of the lecithin solution, and after the impregnation is finished, keeping the concentration of N, N-dimethylformamide at 5cm²Standing for 3-5 min under the nitrogen flow with the blowing speed of L, and drying the product in vacuum for 10-15 min to obtain the targeted sustained-release biocompatible microcapsule.

Further, various fruit and vegetable powders in the fruit and vegetable powder comprise the following components in parts by mass: 5-15% of kiwi fruit powder, 5-15% of tomato powder, 5-15% of blackberry powder, 5-15% of pumpkin powder, 5-15% of carrot powder, 5-15% of mushroom powder, 5-15% of mint powder and the balance of green apple powder.

Further, the preparation method of the sea cucumber peptide comprises the following steps:

1) adding 300-500 parts of sea cucumber, 30-40 parts of alkaline protease and 50-60 parts of subtilisin into a phosphate buffer solution, preserving at 40-55 ℃ for 3-4 h, and keeping the pH value of the solution at 7.5-8.5;

2) after the hydrolysis in the step 1) is finished, the temperature is raised to 95 ℃ and kept for 10-15 min to inactivate the enzyme;

3) centrifuging the substance obtained in the step 2) at 0-4 ℃ and 7000-8000 rpm for 25-30 min to obtain a soluble protein hydrolysate;

4) carrying out further enzymolysis on the soluble protein obtained in the step 3) and 20-25 parts of alkaline protease at 35-45 ℃ for 30-60 min, centrifuging for 15-20 min at the temperature of 0-4 ℃ and the rotating speed of 10000 Xg-12000 Xg by adopting an Amicon ultracentrifugal filter, and taking the upper-layer minced mixed solution as micromolecular sea cucumber peptide;

5) freezing and vacuum-drying the small-molecular sea cucumber peptide obtained in the step 4) to obtain sea cucumber peptide powder.

Further, the weight-to-volume ratio of the sea to the phosphate buffer solution in the step 1) is (2:1) - (2.5: 1).

Further, the molecular weight of the small molecular sea cucumber peptide obtained in the step 4) is 15 KDa-20 KDa.

The invention also provides a preparation method of the intestinal tract sustained-release bovine colostrum and sea cucumber peptide chewable tablet, which comprises the following steps:

s1: dissolving the casein phosphopeptide, the sea cucumber peptide and the soybean peptide in the volume ratio of (1:2) - (3:5) to the NaCl solution with the concentration of 0.15M, and stirring for 30-40 min at the rotating speed of 100-150 rpm and the temperature of 15-20 ℃;

s2: adding the targeted sustained-release biocompatible microcapsules into the mixture obtained in the step S1, stirring at the rotating speed of 150-200 rpm and the temperature of 26-28 ℃ for 15-20 min, and continuously dropwise adding an ethanol solution during stirring to obtain a targeted sustained-release biocompatible microencapsulated peptide composition;

s3: vacuum freeze-drying the targeted sustained-release biocompatible microencapsulated peptide composition obtained in the step S2 at a vacuum degree of 0.02 MPa-0.05 MPa and a temperature of-4 ℃ to-2 ℃ to obtain sustained-release peptide composition freeze-dried powder;

s4: dissolving the bovine colostrum powder, the concentrated whey protein and the freeze-dried powder of the sustained-release peptide composition obtained in the step S3 in glycerol and distilled water in a volume ratio of 2: 3-4: 5, stirring at a rotating speed of 200-250 rpm for 10-15 min, adding the fruit and vegetable powder, continuously stirring for 10-15 min, and sieving with a 5-10 mesh sieve to obtain wet particles;

s5: mixing said weight component of lutein ester, said weight component of vitamin C, said weight component of DL-malic acid, said weight component of lactose, said weight component of erythritol, said weight component of sorbitol and said weight componentDissolving the xylo-oligosaccharide in 100-150 ml of distilled water, uniformly and rotatably spraying wet particles obtained in the step S4 at the spraying rate of 1.5-2.0 ml/min and the atomizing pressure of 1.5-2.0 MPa, uniformly mixing the wet particles and the wet particles, and then uniformly mixing the wet particles and the wet particles by 5cm²/L～10cm²Blowing nitrogen for 10min at a/L speed;

s6: and uniformly stirring the resistant dextrin and the magnesium stearate of the weight components with the granules obtained in the step S5, and tabletting to obtain 1g of the intestinal slow-release bovine colostrum and sea cucumber peptide chewable tablet.

The invention has the beneficial effects that:

1. the fruit and vegetable powder added in equal proportion can be used for enhancing the peristalsis of gastrointestinal tracts and the fruit taste of the chewable tablets, improving the mouthfeel and avoiding constipation caused by eating the chewable tablets, and the various fruit and vegetable powder contains various celluloses, vitamins and minerals, so that the deficiency of various trace elements or certain nutrient substances which are lacked by diseases of human bodies can be effectively supplemented.

2. In the process of manufacturing the chewable tablet, small molecular peptide with peptide activity, namely casein phosphopeptide, sea cucumber peptide and soybean peptide, is directly combined with a framework of glucan of poly (2-ethyl-2-oxazoline) grafted glucan through a targeting sustained-release biocompatible microcapsule, or can be wrapped in micelle of a glucan hydrophobic core, then the micelle is attached to the surface of a polycarbonate film, a bovine serum albumin/amino acid wrapping layer is attached to the outer layer, a chitosan oligosaccharide-lecithin double-layer film is attached to the outermost layer, the active small peptide can be effectively wrapped by grease through the chitosan oligosaccharide-lecithin, the chitosan oligosaccharide-lecithin double-layer film has hydrophilic lipophilicity, after the chewable tablet enters the stomach, the chitosan oligosaccharide-lecithin double-layer film, bovine colostrum powder, concentrated whey protein and the like are digested by pepsin and other enzymes, the covalent bond between the chitosan oligosaccharide and the lecithin is untied, leaving the polycarbonate film of poly (2-ethyl-2-oxazoline) grafted glucan nano particles with chitosan oligosaccharide and bovine serum albumin/amino acid wrapping layers with amino, feeding the polycarbonate film, bovine colostrum powder and concentrated whey protein which are primarily decomposed into an intestinal tract, feeding the amino in chitosan oligosaccharide molecules into a neutral pH environment of the intestinal tract from a strong acid environment in the stomach, and feeding the chitosan oligosaccharide molecules into the neutral pH environment of the intestinal tractThe amino group on the surface of the sugar is changed to NH3⁺The pH around the polycarbonate film of poly (2-ethyl-2-oxazoline) grafted glucan nano particles with chitosan oligosaccharide with amino and a bovine serum albumin/amino acid wrapping layer is smaller than the pKa of the polycarbonate film, so that the ionic electrostatic repulsion between network structures is reduced, the whole is decomposed, and the small peptide of the bovine serum albumin/amino acid wrapping layer is released.

3. Bovine serum albumin contains two tryptophan residues: trp134 is positioned on the surface of a molecule, Trp 213 is positioned in a hydrophobic sac of natural bovine serum albumin, and immunoglobulin IgG in bovine colostrum is hydrophobic, so that the activity and stability of active small peptide attached to poly (2-ethyl-2-oxazoline) grafted glucan nanoparticles wrapped inside the bovine colostrum can be ensured; part of bovine serum albumin/amino acid can be decomposed in the intestinal tract, and absorbed by the intestinal tract together with bovine colostrum powder and concentrated serum protein;

due to the fact that the bovine serum albumin, the Trp134 tryptophan residue with hydrophobic property located on the surface, and the other part of bovine serum albumin/amino acid can carry casein phosphopeptide, sea cucumber peptide and soybean peptide which are wrapped by the poly (2-ethyl-2-oxazoline) grafted glucan nanoparticles to pass through a blood brain barrier, enter the brain and repair cell damage caused by heart and brain blood vessels due totype 2 diabetes and cell damage caused by oxidative stress to repair and nourish nerve cells.

4. The targeting sustained-release biocompatible microcapsule provided by the application adopts 4- (dimethylamino) pyridine and N- (3- (dimethylamino) propyl) -N-ethylcarbodiimide hydrochloride as coupling agents, electrostatic attraction is formed by carboxyl on poly (2-ethyl-2-oxazoline) and hydroxyl on glucan, aminated and carboxylated poly (2-ethyl-2-oxazoline) are grafted to glucan with hydroxyl to form nano-particles with a poly (2-ethyl-2-oxazoline) grafted glucan shell structure, different core filling amounts of the nano-particles can be adjusted by adding 3,3' -dithiopropionic acid, and different drug loading amounts are further ensured, the glucan is a hydrophilic substance, and a mixture of various active peptides can be directly combined with the skeleton of the glucan, it can also be encapsulated in micelles with a hydrophobic core of dextran.

5. The health-care food has a good repairing effect on cell oxidative stress, has an obvious effect of reducing blood sugar and blood pressure for high-sugar supplementary food, can enhance the immunity of a patient, supplements various peptides and various macronutrients, macroelements and trace elements, adjusts and restores the functions of an organism, improves sleep, achieves the aims of reducing toxicity and improving efficiency, has no toxic or side effect, and can be eaten for a long time.

Drawings

FIG. 1 is a bar graph of the mean blood glucose on the 0 th balance of each group in effect example 1 of the present invention;

FIG. 2 is a bar graph of the mean glycaemic intensity of 14 th balance of each group in effect example 1 of the present invention;

FIG. 3 is a 21 st balance glycemic histogram for each group of example 1 of the present invention;

FIG. 4 is a column of the mean blood glucose of the 28 th balance of each group in effect example 1 of the present invention;

FIG. 5 is a column of the 31 st balance's mean blood glucose for each group in effect example 1 of the present invention;

FIG. 6 is a bar graph showing the glucose lowering rate at day 14 for each group in effect example 1 of the present invention;

FIG. 7 is a bar graph showing the glucose lowering rate at day 21 for each group in effect example 1 of the present invention;

FIG. 8 is a bar graph of the glucose lowering rate at day 28 for each group in effect example 1 of the present invention;

FIG. 9 is a bar graph showing the glucose lowering rate at day 31 in each group in effect example 1 of the present invention;

FIG. 10 is a bar graph comparing the effects on DON-induced IPEC-J2 cell inflammation and apoptosis-related genes in effect example 2 of the present invention;

FIG. 11 is a histogram of the MDA content in serum of each group in effect example 3 of the present invention;

FIG. 12 shows serum H in each group in example 3 in which the effects of the present invention are exhibited₂O₂A content bar graph;

FIG. 13 is a bar graph showing the 8-OHDG content in liver of each group in example 3, which is an effect of the present invention;

FIG. 14 is a bar graph showing the carbonyl PC content in liver proteins of each group in example 3 of the present invention;

FIG. 15 is a histogram of GSH-Px activity of each group in Effect example 3 of the invention;

fig. 16 is a graph showing the effect of the chewable tablet provided by the invention after 1 month of eating in example 1 of clinical application of the invention;

FIG. 17 is a graph showing the effect of the chewable tablet provided by the invention onpatients 2 years after eating the chewable tablet in clinical application example 1 of the invention;

fig. 18 is a blood chemistry test report sheet of a patient who has eaten the chewable tablet provided by the present invention together with glibenclamide, rehmannia glutinosa pills and insulin for 1.5 months in clinical application example 1;

FIG. 19 is a blood glucose test report of a patient at the time of diagnosis in accordance with example 2 of the clinical application of the present invention;

fig. 20 is a blood glucose test report form of a patient who has consumed the chewable tablet provided by the present invention for 2 weeks according to example 2 of the clinical application of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The lutein ester is derived from marigold extract, and other raw materials are all purchased from the market. The amino acid used in the targeted sustained-release biocompatible microcapsule of the invention can be lysine, aspartic acid, tryptophan, proline, or a single amino acid polymer or any of several amino acid polymers of the above amino acids.

Example 1

The intestinal slow-release chewable tablet containing bovine colostrum and sea cucumber peptide provided by the embodiment comprises the following components in 1g of chewable tablet:

1mg of casein phosphopeptide;

50mg of sea cucumber peptide;

bovine colostrum powder 5 mg;

70mg of soybean peptide;

concentrated whey protein 114 mg;

lutein ester 1 mg;

1mg of vitamin C;

10mg of fruit and vegetable powder;

1mg of resistant dextrin;

4mg of lactose;

erythritol 0.5 mg;

sorbitol 15 mg;

1mg of DL-malic acid;

1mg of xylo-oligosaccharide;

magnesium stearate 0.5 mg;

the targeted slow release biocompatible microcapsule is 20 mg;

the targeted sustained-release biocompatible microcapsule comprises the following components in parts by weight: 20 parts of a polycarbonate film with the aperture of 200nm and the thickness of 10 mm; 15 parts of bovine serum albumin freeze-dried powder; 10 parts of amino acid; 5 parts of chitosan oligosaccharide; 10 parts of lecithin; 30 parts of poly (2-ethyl-2-oxazoline) grafted glucan nanoparticles; 0.05 part of N, N-dimethylformamide.

The poly (2-ethyl-2-oxazoline) grafted glucan nanoparticle comprises the following components in parts by weight:

2.5 parts of poly (2-ethyl-2-oxazoline); 0.2 part of succinic anhydride; 0.5 part of 3,3' -dithiopropionic acid; 1 part of N- (3- (dimethylamino) propyl) -N-ethyl carbodiimide hydrochloride; 2 parts of 4- (dimethylamino) pyridine; and 3 parts of glucan.

The preparation method of the poly (2-ethyl-2-oxazoline) grafted glucan nanoparticle comprises the following steps:

m1: dissolving 2.5 parts of poly (2-ethyl-2-oxazoline) and 0.2 part of succinic anhydride in methylene chloride to form a mixed solution of a poly (2-ethyl-2-oxazoline) organic solution with a concentration of 5mmol/L and a succinic anhydride organic solution with a concentration of 30mmol/L, adding 2 parts of 4- (dimethylamino) pyridine to the mixed solution, and stirring and reacting at a rotation speed of 75rpm and a temperature of 25 ℃ for 12 hours;

m2: mixing the mixture obtained in the M1 step with diethyl ether at a weight-to-volume ratio of 1:8 for reaction for 10min, precipitating in the diethyl ether to obtain a crude poly (2-ethyl-2-oxazoline) product with terminal amination and carboxylation, and drying the crude poly (2-ethyl-2-oxazoline) product with terminal amination and carboxylation under vacuum for 18h to obtain crude poly (2-ethyl-2-oxazoline) product powder with terminal amination and carboxylation;

m3: dissolving 3 parts of glucan in dimethyl sulfoxide, dissolving the glucan in microwave for 3 hours under the condition of 10mHz, adding crude poly (2-ethyl-2-oxazoline) powder obtained in the M2 step and 0.5 part of N- (3- (dimethylamino) propyl) -N-ethylcarbodiimide hydrochloride, stirring the mixture at 28 ℃ for 60 minutes, adding 0.5 part of 3,3' -dithiopropionic acid and 0.5 part of N- (3- (dimethylamino) propyl) -N-ethylcarbodiimide hydrochloride, stirring the mixture at 32 ℃ for 30 minutes, dialyzing the obtained mixture under a dialysis membrane with the molecular size of 3000Da for 1 hour, and freeze-drying the dialyzed mixture at-100 ℃ to obtain the poly (2-ethyl-2-oxazoline) grafted glucan nanoparticles.

The preparation method of the targeted sustained-release biocompatible microcapsule comprises the following steps:

a1: dissolving 10 parts of amino acid and 15 parts of bovine serum albumin freeze-dried powder in distilled water, fully stirring and hydrating for 30min at 80rpm, filtering the obtained mixed solution under a 0.35-micron polyvinylidene fluoride filter, and taking the filtered supernatant to obtain amino acid-bovine serum albumin mixed supernatant;

a2: dissolving 5 parts of chitosan oligosaccharide and 10 parts of lecithin in distilled water to form a chitosan oligosaccharide solution with the concentration of 1mmol/L and a lecithin solution with the concentration of 8 mmol/L;

a3: dissolving 20 parts of polycarbonate film in a phosphate buffer solution containing sodium chloride at a concentration of 0.13M, and impregnating 30 parts of poly (2-ethyl-2-oxazoline) grafted glucan nanoparticles through the polycarbonate film at a rate of 9mL/h to make the polycarbonate film positively charged;

then soaking the mixed supernatant of the amino acid and the bovine serum albumin obtained in the step A1 at a rate of 9mL/h to penetrate through the polycarbonate film so that the polycarbonate film is negatively charged at 5cm²Standing for 3min under the nitrogen flow with the blowing speed of/L;

impregnating the chitosan oligosaccharide solution obtained in the step A2 at a rate of 9mL/h through a polycarbonate film at a rate of 5cm²Standing under nitrogen flow at blowing rate/L for 3min, soaking the lecithin solution obtained in A2 at a rate of 9mL/hA carbonic ester film, 0.05 part of N, N-dimethylformamide is continuously dripped in the process of dipping lecithin solution, and the thickness of the film is 5cm after dipping is finished²And standing for 3min under the blowing speed of nitrogen gas flow, and drying the product in vacuum for 10min to obtain the targeted slow-release biocompatible microcapsule.

After the chitosan oligosaccharide solution is soaked, N-dimethylformamide is dropwise added along with the soaking of lecithin solution, the chitosan oligosaccharide and lecithin form a polyelectrolyte double-layer membrane on the surface of the polycarbonate membrane by utilizing the electrostatic interaction between strong charges, hydroxyl in the chitosan oligosaccharide molecule and carbonyl on the lecithin form carboxyl under the action of the N, N-dimethylformamide, and then the electrostatic interaction is formed through the attraction of positive and negative charges, so that the polyelectrolyte chitosan oligosaccharide-lecithin double-layer membrane is formed and attached to the surface of the polycarbonate membrane.

The fruit and vegetable powder comprises the following components in parts by mass: the tomato and blackberry mixed green apple powder comprises, by weight, 5% of kiwi fruit powder, 15% of tomato powder, 5% of blackberry powder, 5% of pumpkin powder, 15% of carrot powder, 15% of mushroom powder, 15% of mint powder and the balance green apple powder.

The preparation method of the sea cucumber peptide comprises the following steps:

1) adding 300 parts of sea cucumber, 30 parts of alkaline protease and 50 parts of subtilisin into a phosphate buffer solution, storing at 40 ℃ for 4 hours, keeping the pH of the solution at 7.5, and adding the phosphate buffer solution according to the weight-volume ratio of the sea cucumber to the phosphate buffer solution of 2: 1;

2) after the hydrolysis in the step 1) is finished, the enzyme is inactivated by raising the temperature to 95 ℃ and keeping the temperature for 10 min;

3) centrifuging the substance obtained in step 2) at 0 deg.C and 7000rpm for 30min to obtain soluble protein hydrolysate;

4) carrying out further enzymolysis on the soluble protein obtained in the step 3) and 20 parts of alkaline protease at 35 ℃ for 60min, centrifuging for 20min at 4 ℃ and 10000 Xg of rotation speed by adopting an Amicon ultracentrifugal filter, and taking the upper-layer minced mixed solution as micromolecule sea cucumber peptide with the molecular weight of 15 KDa;

5) freezing and vacuum drying the small molecular sea cucumber peptide obtained in the step 4) to obtain sea cucumber peptide powder.

The embodiment also provides a preparation method of the intestinal slow-release bovine colostrum and sea cucumber peptide chewable tablet, which comprises the following steps:

s1: dissolving 1mg casein phosphopeptide, 50mg sea cucumber peptide and 70mg soybean peptide in a volume ratio of 1:2 ethanol to 0.15M NaCl solution, and stirring at 100rpm and 20 deg.C for 30 min;

s2: adding 20mg of targeted sustained-release biocompatible microcapsule into the mixture obtained in the step S1, stirring at the rotating speed of 150rpm and the temperature of 28 ℃ for 15min, and continuously dropwise adding an ethanol solution in the stirring process to obtain a targeted sustained-release biocompatible microcapsule-encapsulated peptide composition;

s3: vacuum freeze-drying the targeting sustained-release biocompatible microencapsulated peptide composition obtained in the step S2 at a vacuum degree of 0.02MPa and a temperature of-4 ℃ to obtain sustained-release peptide composition freeze-dried powder;

s4: dissolving 5mg of bovine colostrum powder, 114mg of concentrated whey protein and the slow-release peptide composition freeze-dried powder obtained in the step S3 in glycerol and distilled water with the volume ratio of 2:3, stirring for 15min at the rotating speed of 200rpm, adding 10mg of fruit and vegetable powder, continuously stirring for 10min, and sieving with a 5-mesh sieve to obtain wet granules;

s5: dissolving 1mg of lutein ester, 1mg of vitamin C, 1mg of DL-malic acid, 4mg of lactose, 0.5mg of erythritol, 15mg of sorbitol and 1mg of xylo-oligosaccharide in 100ml of distilled water, uniformly and rotatably spraying the wet granules obtained in the step S4 at a spraying rate of 1.5ml/min and an atomizing pressure of 1.5MPa, uniformly mixing the wet granules and the wet granules, and then uniformly mixing the wet granules with 5cm of granules²Blowing nitrogen for 10min at a/L speed;

s6: and uniformly stirring 1mg of resistant dextrin and 0.5mg of magnesium stearate with the granules obtained in the step S5, and tabletting to obtain 1g of intestinal slow-release bovine colostrum sea cucumber peptide chewable tablets.

Example 2

The intestinal slow-release chewable tablet containing bovine colostrum and sea cucumber peptide provided by the embodiment comprises the following components in 1g of chewable tablet:

casein phosphopeptide 15 mg;

1mg of sea cucumber peptide;

bovine colostrum powder 50 mg;

70mg of soybean peptide;

4mg of concentrated whey protein;

lutein ester 40 mg;

30mg of vitamin C;

100mg of fruit and vegetable powder;

resistant dextrin 5 mg;

1mg of lactose;

erythritol 10 mg;

sorbitol 0.5 mg;

0.05mg of DL-malic acid;

0.6mg of xylo-oligosaccharide;

magnesium stearate 1 mg;

30mg of targeted sustained-release biocompatible microcapsule;

the targeted sustained-release biocompatible microcapsule comprises the following components in parts by weight: 30 parts of a polycarbonate film with the aperture of 300nm and the thickness of 12 mm; 10 parts of bovine serum albumin freeze-dried powder; 15 parts of amino acid; 10 parts of chitosan oligosaccharide; 5 parts of lecithin; 20 parts of poly (2-ethyl-2-oxazoline) grafted glucan nanoparticles; 0.1 part of N, N-dimethylformamide.

The poly (2-ethyl-2-oxazoline) grafted glucan nanoparticle comprises the following components in parts by weight:

5 parts of poly (2-ethyl-2-oxazoline); 0.5 part of succinic anhydride; 1 part of 3,3' -dithiopropionic acid; 1.5 parts of N- (3- (dimethylamino) propyl) -N-ethylcarbodiimide hydrochloride; 2.5 parts of 4- (dimethylamino) pyridine; and 6 parts of glucan.

The preparation method of the poly (2-ethyl-2-oxazoline) grafted glucan nanoparticle comprises the following steps:

m1: dissolving 5 parts of poly (2-ethyl-2-oxazoline) and 0.5 part by weight of succinic anhydride in methylene chloride to form a mixed solution of a poly (2-ethyl-2-oxazoline) organic solution with a concentration of 10mmol/L and a succinic anhydride organic solution with a concentration of 50mmol/L, adding 2.5 parts of 4- (dimethylamino) pyridine to the mixed solution, and stirring and reacting at a rotation speed of 150rpm and a temperature of 28 ℃ for 24 hours;

m2: mixing the mixture obtained in the M1 step with diethyl ether at a weight-to-volume ratio of 1:8 for reaction for 15min, precipitating in diethyl ether to obtain a crude poly (2-ethyl-2-oxazoline) product with terminal amination and carboxylation, and drying the crude poly (2-ethyl-2-oxazoline) product with terminal amination and carboxylation under vacuum for 36h to obtain crude poly (2-ethyl-2-oxazoline) product powder with terminal amination and carboxylation;

m3: 6 parts of glucan is dissolved in dimethyl sulfoxide, microwave dissolution is carried out for 4 hours under the condition of 20mHz, then poly (2-ethyl-2-oxazoline) crude product powder obtained in the M2 step and 0.75 part of N- (3- (dimethylamino) propyl) -N-ethylcarbodiimide hydrochloride are added, after stirring for 30 minutes at 32 ℃, 1 part of 3,3' -dithiopropionic acid and 0.75 part of N- (3- (dimethylamino) propyl) -N-ethylcarbodiimide hydrochloride are added, after stirring for 60 minutes at 28 ℃, the obtained mixture is dialyzed for 2 hours under a dialysis membrane with the molecular pore size of 3500Da, and freeze-drying is carried out at-100 ℃ to obtain the poly (2-ethyl-2-oxazoline) grafted glucan nano-particles.

The preparation method of the targeted sustained-release biocompatible microcapsule comprises the following steps:

a1: dissolving 15 parts of amino acid and 10 parts of bovine serum albumin freeze-dried powder in distilled water, fully stirring and hydrating for 20min at 100rpm, filtering the obtained mixed solution under a 0.45-micron polyvinylidene fluoride filter, and taking the filtered supernatant to obtain amino acid-bovine serum albumin mixed supernatant;

a2: dissolving 10 parts of chitosan oligosaccharide and 5 parts of lecithin in distilled water to form a chitosan oligosaccharide solution with the concentration of 5mmol/L and a lecithin solution with the concentration of 3 mmol/L;

a3: dissolving 30 parts of polycarbonate film in a phosphate buffer solution containing sodium chloride at a concentration of 0.15M, and impregnating 20 parts of poly (2-ethyl-2-oxazoline) grafted glucan nanoparticles through the polycarbonate film at a rate of 11mL/h to make the polycarbonate film positively charged;

then soaking the mixed supernatant of the amino acid and the bovine serum albumin obtained in the step A1 at a rate of 11mL/h to penetrate through the polycarbonate film so that the polycarbonate film is negatively charged at 5cm²Standing for 5min under the nitrogen flow with the blowing speed of/L;

obtained by the step A2The chitosan oligosaccharide solution impregnated through the polycarbonate film at a rate of 11mL/h at 5cm²Standing for 5min under nitrogen flow at blowing rate of L, soaking the lecithin solution obtained in the step A2 at a rate of 11mL/h to penetrate through the polycarbonate film, continuously dropwise adding 0.1 part of N, N-dimethylformamide during the soaking of the lecithin solution, and soaking for 5cm after the soaking is finished²And standing for 5min under the nitrogen flow with the blowing speed of L, and drying the product in vacuum for 15min to obtain the targeted slow-release biocompatible microcapsule.

The fruit and vegetable powder comprises the following components in parts by mass: the tomato and blackberry mixed green apple powder comprises, by weight, 15% of kiwi fruit powder, 5% of tomato powder, 15% of blackberry powder, 15% of pumpkin powder, 5% of carrot powder, 5% of mushroom powder, 5% of mint powder and the balance green apple powder.

The preparation method of the sea cucumber peptide comprises the following steps:

1) adding 500 parts by weight ofsea cucumber participation 40 parts by weight of alkaline protease and 60 parts by weight of subtilisin into a phosphate buffer solution, preserving for 3 hours at 55 ℃, keeping the pH of the solution at 8.5, and adding the phosphate buffer solution according to the weight-volume ratio of the sea cucumber to the phosphate buffer solution of 2.5: 1;

2) after the hydrolysis in step 1) is completed, inactivating the enzyme by raising the temperature to 95 ℃ and keeping the temperature for 15 min;

3) centrifuging the substance obtained in step 2) at 4 deg.C and 8000rpm for 25min to obtain soluble protein hydrolysate;

4) carrying out further enzymolysis on the soluble protein obtained in the step 3) and 25 parts of alkaline protease at 45 ℃ for 30min, centrifuging for 15min at the temperature of 0 ℃ and the rotating speed of 12000 Xg by using an Amicon ultracentrifugal filter, and taking the upper-layer minced mixed solution as micromolecule sea cucumber peptide with the molecular weight of 20 KDa;

5) freezing and vacuum drying the small molecular sea cucumber peptide obtained in the step 4) to obtain sea cucumber peptide powder.

The embodiment also provides a preparation method of the intestinal slow-release bovine colostrum and sea cucumber peptide chewable tablet, which comprises the following steps:

s1: dissolving casein phosphopeptide 15mg, Stichopus japonicus peptide 1mg, and soybean peptide 70mg in 3:5 ethanol and 0.15M NaCl solution, and stirring at 150rpm and 15 deg.C for 40 min;

s2: adding 30mg of targeted sustained-release biocompatible microcapsule into the mixture obtained in the step S1, stirring at the rotating speed of 200rpm and the temperature of 26 ℃ for 20min, and continuously dropwise adding an ethanol solution in the stirring process to obtain a targeted sustained-release biocompatible microcapsule-encapsulated peptide composition;

s3: vacuum freeze-drying the targeting sustained-release biocompatible microencapsulated peptide composition obtained in the step S2 at a vacuum degree of 0.05MPa and a temperature of-2 ℃ to obtain sustained-release peptide composition freeze-dried powder;

s4: dissolving 50mg of bovine colostrum powder, 4mg of concentrated whey protein and the slow-release peptide composition freeze-dried powder obtained in the step S3 in glycerol and distilled water with the volume ratio of 4:5, stirring at the rotating speed of 250rpm for 10min, adding 100mg of fruit and vegetable powder, continuously stirring for 15min, and sieving with a 10-mesh sieve to obtain wet granules;

s5: dissolving lutein ester 40mg, vitamin C30 mg, DL-malic acid 0.05mg, lactose 1mg, erythritol 10mg, sorbitol 0.5mg and xylo-oligosaccharide 0.6mg in distilled water 150ml, uniformly rotary spraying the wet granules obtained in step S4 at a spraying rate of 2.0ml/min and an atomizing pressure of 2.0MPa, uniformly mixing with the wet granules, and then uniformly mixing with the wet granules at a speed of 10cm²Blowing nitrogen for 10min at a/L speed;

s6: and uniformly stirring 5mg of resistant dextrin and 1mg of magnesium stearate with the granules obtained in the step S5, and tabletting to obtain 1g of intestinal slow-release bovine colostrum sea cucumber peptide chewable tablets.

Example 3

The intestinal slow-release chewable tablet containing bovine colostrum and sea cucumber peptide provided by the embodiment comprises the following components in 1g of chewable tablet:

casein phosphopeptide 8 mg;

25mg of sea cucumber peptide;

bovine colostrum powder 27.5 mg;

37.5mg of soybean peptide;

concentrated whey protein 59 mg;

lutein ester 20.5 mg;

15mg of vitamin C;

55mg of fruit and vegetable powder;

3mg of resistant dextrin;

lactose 2.5 mg;

erythritol 5.25 mg;

sorbitol 7.75 mg;

0.5mg of DL-malic acid;

0.8mg of xylo-oligosaccharide;

magnesium stearate 0.75 mg;

15mg of targeted sustained-release biocompatible microcapsule;

the targeted sustained-release biocompatible microcapsule comprises the following components in parts by weight: 25 parts of a polycarbonate film with the aperture of 250nm and the thickness of 11 mm; 12.5 parts of bovine serum albumin freeze-dried powder; 12.5 parts of amino acid; 7.5 parts of chitosan oligosaccharide; 7.5 parts of lecithin; 25 parts of poly (2-ethyl-2-oxazoline) grafted glucan nanoparticles; 0.075 part of N, N-dimethylformamide.

The poly (2-ethyl-2-oxazoline) grafted glucan nanoparticle comprises the following components in parts by weight:

3.75 parts of poly (2-ethyl-2-oxazoline); 0.375 part of succinic anhydride; 0.75 part of 3,3' -dithiopropionic acid; 1.25 parts of N- (3- (dimethylamino) propyl) -N-ethylcarbodiimide hydrochloride; 2.25 parts of 4- (dimethylamino) pyridine; and 4.5 parts of glucan.

The preparation method of the poly (2-ethyl-2-oxazoline) grafted glucan nanoparticle comprises the following steps:

m1: dissolving 3.75 parts of poly (2-ethyl-2-oxazoline) and 0.375 part of succinic anhydride in methylene chloride to form a mixed solution of a poly (2-ethyl-2-oxazoline) organic solution with a concentration of 7.5 mmol/L and a succinic anhydride organic solution with a concentration of 40mmol/L, adding 2.25 parts of 4- (dimethylamino) pyridine to the mixed solution, and stirring at a rotation speed of 112.5rpm and a temperature of 27 ℃ for reaction for 18 hours;

m2: mixing the mixture obtained in the M1 step with diethyl ether at a weight-to-volume ratio of 1:8 for reaction for 12min, precipitating in diethyl ether to obtain a crude poly (2-ethyl-2-oxazoline) product with the amino and carboxyl at the tail end, and drying the crude poly (2-ethyl-2-oxazoline) product with the amino and carboxyl at the tail end for 27h under vacuum to obtain crude poly (2-ethyl-2-oxazoline) product powder with the amino and carboxyl at the tail end;

m3: 4.5 parts of glucan is dissolved in dimethyl sulfoxide, microwave dissolution is carried out for 3.5h under the condition of 15mHz, then, crude product powder of poly (2-ethyl-2-oxazoline) obtained in the M2 step and 0.625 part of N- (3- (dimethylamino) propyl) -N-ethylcarbodiimide hydrochloride are added, after stirring for 45min at 30 ℃, 0.75 part of 3,3' -dithiopropionic acid and 0.625 part of N- (3- (dimethylamino) propyl) -N-ethylcarbodiimide hydrochloride are added, after stirring for 45min at 30 ℃, the obtained mixture is dialyzed for 1.5h under a dialysis membrane with the molecular pore size of 3200Da, and freeze-drying is carried out at-100 ℃ to obtain the poly (2-ethyl-2-oxazoline) grafted glucan nano particles.

The preparation method of the targeting sustained-release biocompatible microcapsule comprises the following steps:

a1: dissolving 12.5 parts of amino acid and 12.5 parts of bovine serum albumin freeze-dried powder in distilled water, fully stirring and hydrating for 25min at 90rpm, filtering the obtained mixed solution under a 0.40-micron polyvinylidene fluoride filter, and taking the filtered supernatant to obtain amino acid-bovine serum albumin mixed supernatant;

a2: dissolving 7.5 parts of chitosan oligosaccharide and 7.5 parts of lecithin in distilled water to form a chitosan oligosaccharide solution with the concentration of 3mmol/L and a lecithin solution with the concentration of 5.5 mmol/L;

a3: dissolving 25 parts of polycarbonate film in a phosphate buffer solution containing sodium chloride at a concentration of 0.14M, impregnating 25 parts of poly (2-ethyl-2-oxazoline) grafted glucan nanoparticles through the polycarbonate film at a rate of 10mL/h, so that the polycarbonate film is positively charged;

then soaking the mixed supernatant of the amino acid and the bovine serum albumin obtained in the step A1 at a rate of 10mL/h to permeate the polycarbonate film so that the polycarbonate film is negatively charged at 5cm²Standing for 4min under the nitrogen flow with the blowing speed of/L;

impregnating the chitosan oligosaccharide solution obtained in the step A2 at a rate of 10mL/h through a polycarbonate film at a rate of 5cm²Standing for 4min under nitrogen flow at blowing rate, soaking the lecithin solution obtained in the step A2 at a rate of 10mL/h to penetrate through the polycarbonate film, and continuously dropwise adding 0.075 part in the soaking process of the lecithin solutionN, N-dimethylformamide (5 cm) after completion of the impregnation²Standing for 4min under the blowing speed of nitrogen gas flow, and drying the product in vacuum for 13min to obtain the targeted sustained-release biocompatible microcapsule.

The fruit and vegetable powder comprises the following components in parts by mass: 10% of kiwi fruit powder, 10% of tomato powder, 10% of blackberry powder, 10% of pumpkin powder, 10% of carrot powder, 10% of mushroom powder, 10% of mint powder and the balance of green apple powder.

The preparation method of the sea cucumber peptide comprises the following steps:

1) adding 400 parts by weight of sea cucumber 35 parts by weight of alkaline protease and 55 parts by weight of subtilisin into a phosphate buffer solution, preserving at 47.5 ℃ for 3.5h, keeping the pH of the solution at 8, and adding the phosphate buffer solution according to the weight-volume ratio of the sea cucumber to the sea cucumber being 2.25: 1;

2) after the hydrolysis in step 1) is completed, the enzyme is inactivated by raising the temperature to 95 ℃ and keeping the temperature for 13 min;

3) centrifuging the substance obtained in step 2) at 2 deg.C and 7500rpm for 28min to obtain soluble protein hydrolysate;

4) carrying out further enzymolysis on the soluble protein obtained in the step 3) and 23 parts of alkaline protease at 40 ℃ for 45min, centrifuging for 18min at the temperature of 2 ℃ and the rotating speed of 11000 Xg by adopting an Amicon ultracentrifugal filter, and taking the upper-layer minced mixed solution as micromolecule sea cucumber peptide with the molecular weight of 18 KDa;

5) freezing and vacuum drying the small molecular sea cucumber peptide obtained in the step 4) to obtain sea cucumber peptide powder.

The embodiment also provides a preparation method of the intestinal slow-release bovine colostrum and sea cucumber peptide chewable tablet, which comprises the following steps:

s1: dissolving casein phosphopeptide 8mg, Stichopus japonicus peptide 25mg, and soybean peptide 37.5mg in ethanol and NaCl solution at volume ratio of 11:50 and concentration of 0.15M, and stirring at 125rpm and 18 deg.C for 35 min;

s2: adding 15mg of targeted sustained-release biocompatible microcapsule into the mixture obtained in the step S1, stirring at the rotating speed of 180rpm and the temperature of 27 ℃ for 18min, and continuously dropwise adding an ethanol solution in the stirring process to obtain a targeted sustained-release biocompatible microcapsule-encapsulated peptide composition;

s3: vacuum freeze-drying the targeted sustained-release biocompatible microencapsulated peptide composition obtained in the step S2 at a vacuum degree of 0.03MPa and a temperature of-3 ℃ to obtain sustained-release peptide composition freeze-dried powder;

s4: dissolving 27.5mg of bovine colostrum powder, 59mg of concentrated whey protein and the slow-release peptide composition freeze-dried powder obtained in the step S3 in glycerol and distilled water with the volume ratio of 7:10, stirring for 13min at the rotating speed of 225rpm, adding the fruit and vegetable powder with the weight components, continuously stirring for 13min, and sieving with an 8-mesh sieve to obtain wet granules;

s5: dissolving 20.5mg of lutein ester, 15mg of vitamin C, 0.5mg of DL-malic acid, 2.5mg of lactose, 5.25mg of erythritol, 7.75mg of sorbitol and 0.8mg of xylo-oligosaccharide in 125ml of distilled water, uniformly and rotatably spraying the wet granules obtained in the step S4 at a spraying rate of 1.8ml/min and an atomizing pressure of 1.8MPa, uniformly mixing the wet granules and the wet granules, and then uniformly mixing the wet granules and 8cm of wet granules²Blowing nitrogen for 10min at a/L speed;

s6: and uniformly stirring 3mg of resistant dextrin and 0.75mg of magnesium stearate with the granules obtained in the step S5, and tabletting to obtain 1g of intestinal slow-release bovine colostrum sea cucumber peptide chewable tablets.

Effect example 1

A KM mouse is taken as an experimental model object, 30 KM mice are taken, 27 KM mice are taken to be injected with alloxan in the abdominal cavity to prepare a diabetes model mouse, and after stimulation is completed, the mice with the fasting blood glucose of 11 mM-30 mM are tested to be successfully modeled.

The successfully molded 27 mice were randomly divided into 9 groups, 3 unmodeled mice were used as the group (1) control group, and all 30 mice were divided into 10 groups as follows:

(1) control group (physiological saline)

(2) Model group (physiological saline)

(3) Glibenclamide treatment group

(4) Glibenclamide and phosphopeptide treatment group

(5) Glibenclamide and six-ingredient rehmannia pill treatment group

(6) Glibenclamide, phosphopeptide and six-ingredient rehmannia pill treatment group

(7) Glibenclamide decreasing treatment group

(8) Glibenclamide degressive + phosphopeptide treatment group

(9) Glibenclamide degressive and six-ingredient rehmannia pill treatment group

(10) Glibenclamide degressive + phosphorus ginseng peptide + six-ingredient rehmannia pill treatment group

The administration mode comprises the following steps: the treatment groups (3) to (10) are all administered according to 10mg/kg of glibenclamide, 1.6g/kg of the water dispersion of the phosphopeptide chewable tablet and 0.4g/kg of liuwei Dihuang pills, the glibenclamide with the concentration is firstly administered for 14 days, then the corresponding liuwei Dihuang pills or/and the phosphopeptide are respectively added according to the components for treatment for 7 days, and the glibenclamide with the concentration is continuously administered during the treatment period.

After 7 days of treatment, the administration was continued in groups (3) to (6), and the administration concentration of glyburide was decreased by 10% every day from day 22 to day 28 in groups (7) to (10) until the administration concentration of glyburide became 30% at day 28, and the administration was continued at the administration concentration of glyburide at day 28 from day 29 to day 31, and the administration was terminated after the completion of the administration on day 31.

Blood was collected by decapitation ondays 0, 14, 21, 28, and 31 of the administration, and the blood glucose levels of the groups were measured, and the average values and the blood glucose reduction rates were calculated, and the results are shown in tables 1 to 2, and fig. 1 to 9.

TABLE 1 mean blood glucose (mM) values from tail-off blood collection

	(1)	(2)	(3)	(4)	(5)	(6)	(7)	(8)	(9)	(10)
											Day 0	6.27	15.87	16.77	16.13	16.97	16.23	16.63	15.87	16.73	15.93
Day 14	6.30	16.27	8.03	7.90	7.67	8.17	7.73	8.10	7.93	8.10
											Day 21	6.57	16.87	8.27	8.37	8.63	8.53	8.03	8.77	8.07	8.27
Day 28	6.40	18.50	8.03	8.20	8.50	7.83	14.43	10.77	15.17	10.63
											Day 31	6.27	18.60	8.70	8.13	8.87	7.77	15.33	8.83	15.70	7.67

TABLE 2 sugar reduction rates of the groups

The experimental result shows that after the dosage of the glibenclamide is reduced to 30% of the effective dosage, the phosphorus-containing ginseng peptide chewable tablet dispersing agent provided by the application is continuously given with a proper amount, so that the increase of blood sugar caused by the reduction of the dosage of the glibenclamide can be relieved, and the stability of the blood sugar can be maintained.

After the dosage of the glibenclamide is reduced to 30 percent of the effective dosage, only a proper amount of pills of six ingredients with rehmannia are continuously taken alone, so that the blood sugar rise caused by the reduction of the dosage of the glibenclamide cannot be effectively relieved. Thus, the efficacious effect of the phosphopeptide chewable tablets claimed in the present application on lowering blood glucose was demonstrated.

Effect example 2

The IPEC-J2 is stimulated to be inflammatory and apoptotic by DON, and the phosphorus ginseng peptide chewable tablet water dispersant CAP is adopted to carry out the determination of the effect of the relieving experiment.

The IPEC-J2 cell line was supplied by the department of Oncoinhibian university of transportation. IPEC-J2 cell culture medium is HGDMEM medium containing 10% FBS and 1% penicillin-streptomycin, and is inoculated to 25cm²The cells were cultured in a flask and placed in an incubator containing 5% CO2 at 37 ℃. When the cell fusion rate reaches 80% -90%, the cells are respectively inoculated in a 96-well plate and a 6-well plate for 24h, and then different treatments are carried out.

DON purity > 99% was purchased from Sigma-Aldrich (USA); phosphate Buffered Saline (PBS), 0.25% pancreatin-ethylenediaminetetraacetic acid (EDTA), penicillin-streptomycin (10,000 units/10,000. mu.g/ml), dimethyl sulfoxide (DMSO) was purchased from Beijing Solibao Bio Inc.; high glucose medium (HGDMEM) and Fetal Bovine Serum (FBS) were purchased from Biological Industries, Inc. (Israel); trizol reagent was purchased from Invitrogen corporation (USA); the reverse transcription kit and the TB GREEN kit were purchased from TaKaRa (Chinese Dalian).

IPEC-J2 cells were seeded in a 6-well plate at a density of 5X 105 cells/well until the cell fusion rate reached 80%, and after passing through the above four treatment groups (blank vs. illumination, 0.5. mu.g/mL DON, 20. mu.g/mL phosphopeptide chewable tablet aqueous dispersion + 0.5. mu.g/mL DON), total RNA was extracted using Trizol, and the RNA pellet was dissolved in 50. mu.L RNase-free water and stored at-80 ℃. The quality and purity of the RNA were checked using an Agilent 2100 bioanalyzer and the integrity of the RNA was checked using agarose gel electrophoresis. Using the TB GREEN kit, about 1. mu.g of total RNA in each sample was subjected to reverse transcription at 42 ℃ to remove gDNA by 2g, thereby producing cDNA. Using CFX Connect^TMThe real-time PCR detection system performs real-time PCR. PCR conditions were 95 ℃ pre-denaturation for 300s, followed by 38 cycles of 95℃ 20s, 60 ℃ 30s and 72 ℃ 30 s. Relative expression levels of each inflammation-related or oxidative stress gene were analyzed by 2- Δ Δ CT method using GAPDH as an internal reference gene, and primers for all genes are listed in table 3.

Primer sequences for the genes of Table 3

The experimental results are shown in fig. 10, in which: (A) mRNA expression of inflammation-associated genes; (B) mRNA expression of apoptosis-related genes; (C) ratio of Bax/Bcl-2. Data for each group are expressed as mean ± standard deviation (n ═ 3), with different letters on the bars indicating significant differences (P <0.05) and the same letters indicating insignificant differences (P > 0.05).

Addition of DON can significantly up-regulate relative mRNA expression of IL-6, IL-8, TNF-alpha, Bax and caspase-3 genes in cells (P <0.05), and significantly down-regulate mRNA expression of Bcl-2 (P < 0.05). The addition of the phosphopeptide chewable tablet dispersant CAP can obviously lower the relative mRNA expression of IL-6, IL-8, TNF-alpha, Bax and caspase-3 genes, the Bax/Bcl-2 ratio (P <0.05) and up-regulate the expression abundance of the mRNA of the Bcl-2 gene (P < 0.05).

Experimental results prove that the phosphopeptide chewable tablet dispersing agent provided by the application can relieve and alleviate the toxicity of IPEC-J2 cells induced by DON, and improve the repair capacity of oxidative stress damaged cells.

Effect example 3

In this study, 45 SPF-rated male mice were randomly divided into five groups, namely, a control group (a), a diquat treatment group (B), a phsophagin chewable tablet dispersant low dose group (50mg/kg D) (C) provided herein, a phsophagin chewable tablet dispersant medium dose group (100mg/kg D) (D) provided herein, and a phsophagin chewable tablet dispersant high dose group (300mg/kg D) (E) provided herein, each group having three cages, and each cage having three mice. The rats are weighed and then are irrigated with stomach in the squirrel cage sequence, the control group and the diquat treatment group are irrigated with 20ml/kg x d normal saline, and the composite polypeptide group is irrigated with composite polypeptide with different dosages. After three weeks of continuous feeding to mice, on day 22, four groups were challenged with diquat (i.p.) for six hours except for the control group, and then serum and liver samples of the mice were collected.

Detecting the MDA content and H content of oxidative stress metabolite malondialdehyde of mice in a control group (A), a diquat treatment group (B), a phosphorus ginseng peptide chewable tablet dispersant low-dose group (C), a medium-dose group (D) and a high-dose group (E)₂O₂The results are shown in FIGS. 11-12.

The effects of the control group (a), the diquat treatment group (B), the phsophatide chewable tablet dispersant low dose group (C), the medium dose group (D), and the high dose group (E) provided herein on the activity levels of the proteins carbonyl PC, 8-hydroxydeoxyguanosine (8-OHDG), and antioxidant enzyme GSH-Px in the liver of mice were examined, and the results are shown in fig. 13 to 15.

Compared with a diquat treatment group, the phosphopeptide chewable tablet dispersant provided by the application can obviously reduce the content of Malondialdehyde (MDA) in serum and enhance the activity of superoxide anion (P is less than 0.05), and the higher the dosage, the better the effect is; the phosphopeptide chewable tablet dispersing agent provided by the application can obviously reduce the content of 8-hydroxydeoxyguanosine (8-OHDG) and the content of Protein Carbonyl (PC) in the liver, enhance the activity of glutathione peroxidase (GSH-Px) in the liver (P is less than 0.05), has better effect of medium dosage, and has no obvious influence on the content activity of Malondialdehyde (MDA) (P is more than 0.05).

In summary, the application provides an effect of the phosphopeptide chewable tablet dispersant on the oxidative stress of mice induced by diquat. The phospho-ginseng peptide chewable tablet dispersing agent provided by the application has a certain relieving effect on oxidative stress of a mouse organism, can reduce the content of oxidative stress metabolites and improve the activity of antioxidant enzymes, thereby effectively relieving oxidative damage of tissues and achieving the effect of relieving oxidative stress. The high dosage is safe and harmless to the organism.

Clinical application example 1

The disease is diagnosed astype 2 diabetes mellitus in 2016 (7 months) after birth of the year Zhongxian, male and 1960, height of 178cm and weight of 180 kg; under the condition of not taking hypoglycemic drugs and insulin, the blood sugar level before meal is 15-18mmol/L, and theblood sugar level 2 hours after meal is 22-27 mmol/L; when the hypoglycemic agent and insulin are taken, the blood sugar level before meal is 6-10mmol/L, and the blood sugar level after 2 hours is 12-15 mmol/L.

After the diagnosis is confirmed and the medicines of insulin and glibenclamide are used, the body ulcer spreads to the whole body (except the face), the blood sugar index rises again, the injection amount of the insulin is increased to 60 units, the wound can not heal and expands along with time, the severe bleeding of the wound is accompanied, and 3 grades of hypertension, gout and double renal failure are also developed as complications.

After eating the intestinal canal sustained-release bovine colostrum sea cucumber peptide chewable tablet provided by the invention, the blood sugar level is reduced to 5-7mmol/L before meal and 7-9mmol/L after meal, the insulin injection amount is reduced to 20 units per day, 1 glibenclamide tablet has the advantages that the wound is gradually healed, the blood pressure is close to normal, the body mental state is improved, and the waist is powerful; as shown in fig. 16 and 17, compared with 1 month after eating the chewable tablet provided by the invention for 2 years, the skin ulceration is obviously improved; as shown in fig. 18, a blood chemistry test report that patients who take the chewable tablets provided by the present invention and have been supplemented with glibenclamide, rehmannia glutinosa pills, and insulin injected for 1.5 months shows that the blood sugar level is maintained at 3.9-6.1 mmol/L, which can effectively alleviate complications caused by excessive or long-term use of hypoglycemic drugs and insulin, effectively recover organ functions, and is beneficial to maintaining body function stability.

Clinical application example 2

Mr. Li, man, and the person who lives in 1962, diagnosed thetype 2 diabetes in the national hospital of Ningguo, Anhui, in 7.2018, as shown in FIG. 19, which is a blood glucose test report at the time of diagnosis, showing that fasting blood glucose is 8.77mmol/L, without western medicine means, by controlling the blood glucose level of diet, fasting blood glucose is more than 10mmol/L in 6.2019, as shown in FIG. 20, after eating the chewable tablet provided by the present invention for two weeks in 6.2019, the hospital checks that the blood glucose level is 6.38mmol/L again, and western medicine and other means are not taken during the period of the patient.

While the invention has been described with reference to a preferred embodiment, various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention. It is intended that the invention not be limited to the particular embodiments disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.

Claims (8)

1. The intestinal slow-release bovine colostrum and sea cucumber peptide chewable tablet is characterized in that each 1g of the chewable tablet comprises the following components:

casein phosphopeptide 8 mg;

25mg of sea cucumber peptide;

bovine colostrum powder 27.5 mg;

37.5mg of soybean peptide;

concentrated whey protein 59 mg;

lutein ester 20.5 mg;

vitamin C15 mg;

55mg of fruit and vegetable powder;

3mg of resistant dextrin;

lactose 2.5 mg;

erythritol 5.25 mg;

sorbitol 7.75 mg;

0.5mg of DL-malic acid;

0.8mg of xylo-oligosaccharide;

magnesium stearate 0.75 mg;

each 1g of chewable tablet also comprises 15mg of targeted sustained-release biocompatible microcapsule, and the targeted sustained-release biocompatible microcapsule comprises the following components in parts by weight: 25 parts of polycarbonate film with the aperture of 250nm and the thickness of 11 mm; 12.5 parts of bovine serum albumin freeze-dried powder; 12.5 parts of amino acid; 7.5 parts of chitosan oligosaccharide; 7.5 parts of lecithin; 25 parts of poly (2-ethyl-2-oxazoline) grafted glucan nanoparticles; 0.075 part of N, N-dimethylformamide;

the small molecular peptide with peptide activity, namely casein phosphopeptide, sea cucumber peptide and soybean peptide, is directly combined with a glucan skeleton of poly (2-ethyl-2-oxazoline) grafted glucan through a targeted slow release biocompatible microcapsule, can also be wrapped in a micelle of a glucan hydrophobic core, is attached to the surface of a polycarbonate film, is attached to a bovine serum albumin/amino acid wrapping layer at the outer layer, is attached to a chitosan oligosaccharide-lecithin double-layer film at the outermost layer, can effectively wrap active small peptide through chitosan oligosaccharide-lecithin, and has hydrophile lipophilicity.

2. The chewable tablet of bovine colostrum and sea cucumber peptide with sustained release in intestinal tract of claim 1, wherein the poly (2-ethyl-2-oxazoline) grafted glucan nanoparticle comprises the following components by weight:

3.75 parts of poly (2-ethyl-2-oxazoline); 0.375 part of succinic anhydride; 0.75 parts of 3,3' -dithiopropionic acid; 1.25 parts of N- (3- (dimethylamino) propyl) -N-ethylcarbodiimide hydrochloride; 2.25 parts of 4- (dimethylamino) pyridine; 4.5 parts of glucan;

the preparation method of the poly (2-ethyl-2-oxazoline) grafted glucan nanoparticle comprises the following steps:

m1: dissolving the weight component of poly (2-ethyl-2-oxazoline) and the weight component of succinic anhydride in dichloromethane to form a mixed solution of a poly (2-ethyl-2-oxazoline) organic solution with the concentration of 7.5 mmol/L and a succinic anhydride organic solution with the concentration of 40mmol/L, adding the weight component of 4- (dimethylamino) pyridine to the mixed solution, and stirring and reacting at the rotating speed of 112.5rpm and the temperature of 27 ℃ for 18 hours;

m2: mixing the mixture obtained in the M1 step with diethyl ether at a weight-to-volume ratio of 1:8 for reaction for 12min, precipitating in the diethyl ether to obtain a crude poly (2-ethyl-2-oxazoline) product subjected to terminal amination and carboxylation, and drying the crude poly (2-ethyl-2-oxazoline) product subjected to terminal amination and carboxylation for 27h under vacuum conditions to obtain crude poly (2-ethyl-2-oxazoline) product powder subjected to terminal amination and carboxylation;

m3: dissolving the dextran with the weight components in dimethyl sulfoxide, dissolving for 3.5h under the condition of 15mHz by microwave, then, the crude powder of poly (2-ethyl-2-oxazoline) obtained in the M2 step, half the weight of N- (3- (dimethylamino) propyl) -N-ethylcarbodiimide hydrochloride as the above-mentioned component were added thereto, and after stirring at 30 ℃ for 45 minutes, adding the weight component of 3,3' -dithiopropionic acid and the rest half of the weight component of N- (3- (dimethylamino) propyl) -N-ethyl carbodiimide hydrochloride, stirring at 30 ℃ for 45min, the resulting mixture was dialyzed under a dialysis membrane of 3200Da molecular pore size for 1.5h, and lyophilized at-100 ℃ to obtain poly (2-ethyl-2-oxazoline) grafted dextran nanoparticles.

3. The intestinal tract sustained-release chewable tablet containing bovine colostrum and sea cucumber peptide as claimed in claim 1, wherein the preparation method of the targeted sustained-release biocompatible microcapsule comprises the following steps:

a1: dissolving the amino acid and the bovine serum albumin freeze-dried powder in the weight components in distilled water, fully stirring and hydrating for 25min at 90rpm, filtering the obtained mixed solution under a 0.40-micron polyvinylidene fluoride filter, and taking the filtered supernatant to obtain the amino acid-bovine serum albumin mixed supernatant;

a2: dissolving the weight component of the chitosan oligosaccharide and the weight component of the lecithin in distilled water to form a chitosan oligosaccharide solution with the concentration of 3mmol/L and a lecithin solution with the concentration of 5.5 mmol/L;

a3: dissolving the weight component of polycarbonate film in phosphate buffer solution containing sodium chloride with concentration of 0.14M, impregnating the weight component of poly (2-ethyl-2-oxazoline) grafted glucan nanoparticles through the polycarbonate film at a rate of 10mL/h, and then impregnating the amino acid-bovine serum albumin mixed supernatant obtained in the step A1 through the polycarbonate film at a rate of 10mL/h at a rate of 5cm²Standing for 4min under the nitrogen flow with the blowing speed of/L; impregnating the chitosan oligosaccharide solution obtained in the step A2 at a rate of 10mL/h through the polycarbonate film at a rate of 5cm²Standing for 4min under nitrogen gas flow at a blowing rate of/L, impregnating the lecithin solution obtained in the step A2 at a rate of 10mL/h through the polycarbonate film, continuously dropwise adding N, N-dimethylformamide as a weight component during the impregnation of the lecithin solution, and after the impregnation is finished, keeping the concentration of N, N-dimethylformamide at 5cm²And standing for 4min under the blowing speed of nitrogen gas flow, and drying the product in vacuum for 13min to obtain the targeted slow-release biocompatible microcapsule.

4. The intestinal tract sustained-release bovine colostrum and sea cucumber peptide chewable tablet according to claim 1, wherein the fruit and vegetable powder comprises the following components in parts by mass: 10% of kiwi fruit powder, 10% of tomato powder, 10% of blackberry powder, 10% of pumpkin powder, 10% of carrot powder, 10% of mushroom powder, 10% of mint powder and the balance of green apple powder.

5. The intestinal tract sustained-release chewable tablet containing bovine colostrum and sea cucumber peptide as claimed in claim 1, wherein the preparation method of the sea cucumber peptide comprises the following steps:

1) adding 400 parts by weight of sea cucumber 35 parts by weight of alkaline protease and 55 parts by weight of subtilisin into a phosphate buffer solution, preserving at 47.5 ℃ for 3.5h, and keeping the pH value of the solution at 8;

2) after the hydrolysis in the step 1) is finished, the enzyme is inactivated by raising the temperature to 95 ℃ and keeping the temperature for 13 min;

3) centrifuging the substance obtained in step 2) at 2 ℃ and 7500rpm for 28min to obtain a soluble protein hydrolysate;

4) carrying out further enzymolysis on the soluble protein obtained in the step 3) and 23 parts of alkaline protease at 40 ℃ for 45min, centrifuging for 18min at the temperature of 2 ℃ and the rotating speed of 11000 Xg by adopting an Amicon ultracentrifugal filter, and taking the upper-layer minced mixed solution as micromolecular sea cucumber peptide;

5) freezing and vacuum-drying the small-molecular sea cucumber peptide obtained in the step 4) to obtain sea cucumber peptide powder.

6. The chewable tablet of colostrum and sea cucumber peptide for intestinal slow release of claim 5, wherein the weight-to-volume ratio of the sea component to the phosphate buffer solution in the step 1) is 2.25: 1.

7. The chewable tablet of colostrum and sea cucumber peptide for intestinal slow release of claim 5, wherein the molecular weight of the small molecular sea cucumber peptide obtained in step 4) is 18 KDa.

8. The preparation method of the intestinal tract sustained-release chewable tablet containing bovine colostrum and sea cucumber peptide according to any one of claims 1 to 7, which is characterized by comprising the following steps:

s1: dissolving the casein phosphopeptide, the sea cucumber peptide and the soybean peptide in a volume ratio of 11:50 and 0.15M NaCl solution, and stirring at 125rpm and 18 ℃ for 35 min;

s2: adding the targeted sustained-release biocompatible microcapsules of the weight components into the mixture obtained in the step S1, stirring at the rotating speed of 180rpm and the temperature of 27 ℃ for 18min, and continuously dropwise adding an ethanol solution in the stirring process to obtain a targeted sustained-release biocompatible microencapsulated peptide composition;

s3: vacuum freeze-drying the targeted sustained-release biocompatible microencapsulated peptide composition obtained in the step S2 at a vacuum degree of 0.03MPa and a temperature of-3 ℃ to obtain sustained-release peptide composition freeze-dried powder;

s4: dissolving the bovine colostrum powder, the concentrated whey protein and the freeze-dried powder of the sustained-release peptide composition obtained in the step S3 in glycerol and distilled water in a volume ratio of 7:10, stirring for 13min at a rotating speed of 225rpm, adding the fruit and vegetable powder, continuously stirring for 13min, and sieving with an 8-mesh sieve to obtain wet granules;

s5: dissolving the lutein ester, the vitamin C, the DL-malic acid, the lactose, the erythritol, the sorbitol and the xylo-oligosaccharide in 125ml of distilled water, uniformly and rotatably spraying the wet granules obtained in the step S4 at a spraying rate of 1.8ml/min and an atomizing pressure of 1.8MPa, uniformly mixing the wet granules and the wet granules, and uniformly mixing the wet granules by 8cm²Blowing nitrogen for 10min at a/L speed;

s6: and uniformly stirring the resistant dextrin and the magnesium stearate of the weight components with the granules obtained in the step S5, and tabletting to obtain 1g of the intestinal slow-release bovine colostrum and sea cucumber peptide chewable tablet.

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