CN115969961B

CN115969961B - Calcium, VD and protein peptide composition for improving bone mineral density and preparation method and application thereof

Info

Publication number: CN115969961B
Application number: CN202310259063.8A
Authority: CN
Inventors: 董帅; 陈雯菲
Original assignee: Beijing Langdi Pharmaceutical Co ltd
Current assignee: Beijing Langdi Pharmaceutical Co ltd
Priority date: 2023-03-17
Filing date: 2023-03-17
Publication date: 2023-05-12
Anticipated expiration: 2043-03-17
Also published as: CN115969961A

Abstract

The invention provides a calcium, VD and protein peptide composition for improving bone density, which is prepared by extracting kudzuvine root, malaytea scurfpea fruit, epimedium herb and glossy privet fruit with boiling water, filtering, drying filtrate to obtain a traditional Chinese medicine water extract, mixing filter residues with bone collagen, a carbon source and a nitrogen source to obtain a traditional Chinese medicine-protein peptide culture medium, inoculating activated lactobacillus plantarum and lactobacillus salivarius, fermenting, adding a calcium ion solution into the product after phosphorylation reaction to obtain a phosphorylated calcium peptide fermentation compound, and uniformly mixing the phosphorylated calcium peptide fermentation compound with the traditional Chinese medicine water extract, vitamin D1, vitamin D3, bisphosphonate, calcitonin, sea cucumber saponin and ginsenoside Rg3 to prepare microcapsules, thus obtaining the calcium, VD and protein peptide composition for improving bone density. The composition prepared by the invention contains abundant micromolecular substances, has higher absorption rate, has the effects of improving intestinal permeability, improving bone density, promoting osteoblast proliferation and differentiation and intestinal wall cells to absorb calcium, and improving osteoporosis, and has wide application prospect.

Description

Calcium, VD and protein peptide composition for improving bone mineral density and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a calcium, VD and protein peptide composition for improving bone mineral density, and a preparation method and application thereof.

Background

70% -80% of the organic matters in the bones are collagen, and when the bones are generated, enough collagen fibers must be synthesized to form the framework of the bones. Thus, collagen is known as "bone in bone". Loss or lack of collagen can cause diseases such as osteoporosis. Tendons—the components that make up tendons where bones are associated with muscles 80% are collagen, and the lack of collagen results in weakness of tendons and muscles.

With the increase of age, collagen loss and insufficient nutrition supply in various aspects of the body, the insufficient nutrition supply of cartilage can lead to the increase of inorganic matters in bones, further the decrease of the elasticity and toughness of the bones, the unavoidable degradation and abrasion of articular cartilage, the joint always in a motion state, the more serious abrasion of the cartilage, the direct contact of two bones, the inflammation initiation and the final bone joint diseases. Osteoarthritis is frequently found in the middle-aged and elderly, and has a very high disability rate, called "cancer that is not dead". And according to the data, along with the gradual increase of the aging degree of people, the osteoporosis gradually becomes a key factor of the healthy life of the people. In addition, fracture is one of the important factors of bone injury, and fracture refers to a complete or partial fracture of bone or bone structure, which is often found in children and the elderly, and sometimes occurs in young and middle-aged people.

The traditional bone joint clinical medicine is mainly used by combining pain relieving and non-steroidal anti-inflammatory drugs, but excessive medicine treatment has a series of side effects, so that not only can the liver and kidney metabolism burden be increased, but also medicine dependence effect can be generated, and a great health potential safety hazard exists; but also has the treatment methods of joint replacement, reparative treatment, basic treatment, exercise support and the like, but has the defects of high cost, temporary treatment, undefined treatment effect and the like.

While fracture is usually accompanied by important vascular injury, peripheral tissue injury and peripheral nerve injury, healing of fracture is generally divided into a granulation tissue repair period, an original callus formation period, an mature bone plate period and a shaping period. The granulomatous tissue repair period is the destruction of the normal structure of the bone and surrounding soft tissue after fracture, hematoma formation, removal and organization of hematoma, granulomatous tissue formation, primary connection of broken ends of the bone, and completion of the process within two to three weeks after fracture. The original callus formation phase, i.e. new bone formation after fracture. Starting seven to ten days after the fracture and continuing until the fracture heals completely. Mature bone plate period is the transformation of the original callus into strong plate-like bone, which takes 8 to 12 weeks. The shaping period is shaping in the fracture healing process, so that the bone healed by the fracture end is firm and powerful, and the fracture line disappears and recovers the normal structure before the fracture end is healed, usually for months or even years. Most of fracture repair needs to be carried out in bed, so that not only is the activity of nerves and muscles greatly reduced, but also the blood circulation is not smooth, the fracture healing is not facilitated, the repair time is too long, and complications such as hemorrhoids, deep vein thrombosis of lower limbs, joint stiffness and the like can also occur.

With the improvement of the living standard of people and the increasing of nutrition and health care consciousness, people pursue foods beneficial to self health and accept the health preserving mode of 'diet and diet therapy', and the diet therapy health preserving becomes a focus of attention. The health care purpose is achieved through the dietetic invigoration health care, and the rehabilitation effect is promoted through the dietetic invigoration, so that the rehabilitation time is shortened.

Bone peptide is collagen peptide, which is prepared by taking bone gelatin as a raw material and applying an enzymolysis process and controlling reaction conditions. The collagen can effectively inhibit the decomposition of collagen in bones and cartilage meat, accelerate the increase of bone density, prevent and treat degenerative arthritis, quickly diminish inflammation, relieve pain, detumescence, gradually repair articular cartilage and promote the formation of new bones, effectively prevent the degenerative arthritis, protect arthritic patients, be used for helping the treatment of arthritic patients, effectively improve joint pain, accelerate the repair of broken bones and the recovery of fracture. In the existing practical production, products such as bone peptide tablets, bone peptide injection, bone peptide beverage and the like are also available, but the existing bone peptide beverage products have great optimization space in the aspects of absorption rate, human body utilization rate, effect and the like.

Chinese patent application CN107630061a discloses a method for preparing yak bone type i collagen, which comprises the following steps: step one: the frozen yak bone raw material is subjected to optimization and cleaning and then crushed by a hard bone crusher to prepare a yak bone crushed material; step two: washing and impurity-removing the broken yak bones; step three: degreasing the cleaned and decontaminated broken yak bones with diethyl ether, and decalcifying with EDTA or HCl to obtain defatted and decalcified yak bones; step four: dissolving defatted and decalcified yak bones by using acetic acid, and adding pepsin for enzymolysis treatment; step five: adding NaCl solution into the supernatant obtained after enzymolysis for salting out; step six: centrifuging the salted-out product to obtain a precipitate; step seven: and freeze-drying the precipitate to obtain the yak bone type I collagen finished product.

Chinese patent CN105753972B discloses a method for coproducing calcium chelate of yak bone collagen polypeptide and bone polypeptide, which comprises the following steps: step one, extracting collagen polypeptide with molecular weight of 2kDa-4kDa and other collagen polypeptides from yak bones; step two, chelating the collagen polypeptide with the molecular weight of 2kDa-4kDa obtained in the step one with Ca < 2+ > to obtain the yak bone collagen polypeptide chelated calcium, wherein the temperature is 50-55 ℃ and the pH value is 6.8-7.2 in the chelating process, and the chelating time is 1.5-2 hours; and thirdly, concentrating the other collagen polypeptides obtained in the first step to obtain bone polypeptides.

Chinese patent CN103783254B discloses a preparation method of yak bone collagen peptide, which comprises the steps of pretreatment of raw materials, gelatin extraction, separation of collagen extract, compound enzymolysis, enzyme deactivation and sterilization, separation of peptide liquid, decolorization and deodorization, concentration and drying. The complex enzyme adopted in the complex enzymolysis is composed of protease, amylase, pectase, cellulase and xylanase. The method has the advantages that the gelatin extraction process is simple, and the grease and the gelatin are completely separated by utilizing the flow of the mixed solution; the enzymolysis temperature is 48-52 ℃ and the enzymolysis time is 4-6 hours.

Chinese patent application CN102229971a discloses a method for preparing collagen peptide from fresh yak bone, said method comprising the steps of: 1) Enzymolysis: coarsely crushing fresh bone of the pulped yak, adding water to disperse, adding a corresponding amount of enzyme and buffer solution, stirring at constant temperature for enzymolysis, and filtering with gauze to obtain primary enzymolysis solution; 2) And (3) secondary enzymolysis: adding another enzyme into the primary enzymolysis liquid, stirring at constant temperature for enzymolysis, and heating to inactivate the enzyme to obtain a secondary enzymolysis liquid; 3) And (3) filtering: adding a flocculating agent into the secondary enzymolysis liquid, stirring to precipitate impurities, and filtering with a filter cloth to obtain filtrate; 4) Ultrafiltration: removing impurities from the filtrate by an ultrafiltration membrane, and slightly concentrating the filtrate to obtain concentrated solution; 5) Spray drying: spray drying the concentrated solution to obtain the final product.

Chinese patent CN105018554B discloses a small molecule bovine bone collagen peptide and a preparation method thereof, the preparation method sequentially comprises the following steps: crushing, extracting and separating, carrying out enzymolysis, carrying out composite filtration, concentrating and drying; the enzymolysis process is as follows: adding collagenase into bovine collagen extract for primary enzymolysis; after primary enzymolysis, regulating the pH value of the primary enzymolysis liquid to 6-7 by using mixed acid, and then adding proline protease for secondary enzymolysis, wherein the secondary enzymolysis time is 2-4 h, and the addition amount of the proline protease is 0.1-0.3% of the mass of bovine bone collagen.

Chinese patent application CN108208851a discloses a five-membered composite small molecule collagen peptide powder, which is prepared from the following components in percentage by weight: 10% -50% of bovine collagen peptide, 10% -50% of fish collagen peptide, 10% -30% of walnut peptide, 5% -20% of wheat peptide and 5% -20% of corn peptide, and five collagen peptide powders of bovine collagen peptide, deep sea cod skin collagen peptide, walnut peptide, wheat peptide and corn peptide are adopted to prepare five-membered compound micromolecular collagen peptide powder. CN107648205a discloses a collagen peptide dressing for promoting wound healing, the mass percentages of the components are as follows: 50-70% of bovine bone collagen oligopeptide, 0.5-5% of modified chitosan solution, 5-20% or 1-5% of sea cucumber oligopeptide or cecropin B, 1-5% of ginseng oligopeptide, 0-20% of humectant and the balance of solvent.

The method comprises the steps of 'research on nutrition quality evaluation of bovine bones and efficacy of yak bone collagen peptide', gu Wei, gansu agricultural university, 2017, collecting four parts of humerus, femur, backbone and rib of 4 regional yaks and 7 main regional bovine species in China as samples for nutrition component detection, using main components, clustering and discriminant analysis methods for nutrition quality evaluation of bovine bone samples, using Gannan yak bones as raw materials, researching the structures and compositions of yak bone collagen prepared by an acid method and yak bone collagen peptide prepared by enzymolysis, and using human osteoblasts as in vitro models for nutrition efficacy evaluation of yak bone collagen peptide.

"collagen peptide-containing serum promotes osteoblast proliferation and differentiation", liu Junli et al, journal of osteoporosis in China, 2018 (6), illustrates the effect of bovine bone CP compound serum on osteoblast proliferation and differentiation by preparing bovine bone CP compound rat serum at 3%, 6%, 10% serum concentrations in experimental and untreated control groups treated with bovine bone CP compound, adding the serum to serum-free DMEM solution, and measuring the effect of bovine bone CP serum on MC3T3-E1 cell proliferation and cell cycle by MTT method and flow cytometry, respectively.

However, in the existing technology for preparing collagen peptide from yak bone, the method for extracting and enzymolysis of common cow bone is mostly used or carried, and differences between the yak bone and the common cow bone are ignored, for example, because the special growth condition of the yak is that the calcium content of the yak bone is obviously higher than that of the common raising cow and the bone bonding strength is obviously higher, the mineral removing agent for the common raising cow is difficult to effectively remove the calcium in the yak bone.

Disclosure of Invention

The invention aims to provide a calcium, VD and protein peptide composition for improving bone mineral density, a preparation method and application thereof, wherein the preparation method is simple, the raw material sources are wide, the composition has the effects of improving intestinal permeability, reducing intestinal inflammation, improving bone mineral density, promoting osteoblast proliferation and differentiation, reducing bone resorption, continuously forming new bone, promoting the calcium absorption capacity of intestinal wall cells and improving osteoporosis, and has wide application prospect.

The technical scheme of the invention is realized as follows:

the invention provides a preparation method of a calcium, VD and protein peptide composition for improving bone density, which comprises the steps of extracting kudzuvine root, malaytea scurfpea fruit, epimedium herb and glossy privet fruit with boiling water, filtering, drying filtrate to obtain a traditional Chinese medicine water extract, mixing filter residues with collagen, a carbon source and a nitrogen source to obtain a traditional Chinese medicine-protein peptide culture medium, inoculating activated lactobacillus plantarum and lactobacillus salivarius, fermenting, adding a calcium ion solution into the product to obtain a phosphorylated calcium peptide fermentation compound, uniformly mixing the phosphorylated calcium peptide fermentation compound with the traditional Chinese medicine water extract, vitamin D1, vitamin D3, bisphosphonate, calcitonin, sea cucumber saponin and ginsenoside Rg3, and preparing microcapsules to obtain the calcium, VD and protein peptide composition for improving bone density.

As a further improvement of the invention, the method comprises the following steps:

s1, activation of probiotics: marking and activating lactobacillus plantarum and lactobacillus salivarius in a Gao's medium respectively, and culturing to obtain strain seed liquid;

s2, preparing a traditional Chinese medicine water extract: cleaning radix Puerariae, fructus Psoraleae, herba Epimedii and fructus Ligustri Lucidi, drying, pulverizing to obtain Chinese medicinal powder, boiling with water, extracting, filtering, mixing filtrates, concentrating, drying to obtain Chinese medicinal water extract, and filtering residue;

s3, preparing a traditional Chinese medicine-protein peptide culture medium: mixing the filter residue, bone collagen, carbon source and nitrogen source in the step S2, adding into sterile water, stirring and mixing uniformly, and sterilizing to obtain a traditional Chinese medicine-protein peptide culture medium;

s4, fermenting: inoculating lactobacillus plantarum and lactobacillus salivarius strain seed liquid prepared in the step S1 into the traditional Chinese medicine-protein peptide culture medium prepared in the step S3, and fermenting and culturing to obtain a fermentation product;

s5, preparing a phosphorylation fermentation product: adding sodium trimetaphosphate into the fermentation product obtained in the step S4, adjusting the pH value, and heating for reaction to obtain a phosphorylated fermentation product;

s6, preparation of a phosphorylated calcium peptide fermentation complex: adding a calcium ion solution into the phosphorylated fermentation product prepared in the step S5, stirring for reaction, and freeze-drying to obtain a phosphorylated calcium peptide fermentation compound;

S7, preparation of a vitamin D composition: uniformly mixing vitamin D1 and vitamin D3 to obtain vitamin D composition;

s8, preparation of an osteoclast inhibitor: uniformly mixing bisphosphonate and calcitonin to prepare the osteoclast inhibitor;

s9, preparation of an active composition: uniformly mixing sea cucumber saponin and ginsenoside Rg3 to obtain an active composition;

s10, preparation of a calcium, VD and protein peptide composition for improving bone mineral density: adding the traditional Chinese medicine water extract prepared in the step S2, the phosphorylated calcium peptide fermentation complex prepared in the step S6, calcium citrate, vitamin D composition, osteoclast inhibitor and active composition into water, stirring and mixing uniformly, adding sodium alginate, sodium carboxymethylcellulose and carboxymethyl pachyman, stirring and dissolving to obtain a water phase; adding the water phase into edible oil, emulsifying by a rapid membrane, adding metal ion solution, solidifying at normal temperature, filtering, and freeze drying to obtain calcium, VD, and protein peptide composition for improving bone density.

As a further improvement of the invention, the condition of the activation culture in the step S1 is under the micro-anoxic condition, the temperature is 36-38 ℃, the time is 18-24 hours, the rotating speed is 50-80r/min, and the micro-anoxic condition is 5-7% CO ₂ 、7-10%O ₂ The balance is N ₂ Wherein% is volume percent; in the step S2, the mass ratio of the kudzuvine root, the malaytea scurfpea fruit, the epimedium herb and the glossy privet fruit is 5-10:3-5:5-7:1-3, the solid-liquid ratio of the Chinese medicinal powder and the water is 1:5-10g/mL, the times of boiling extraction are 2-3 times, and the time is 3-5h.

As a further improvement of the invention, in the step S3, the mass ratio of the filter residue, the bone collagen, the carbon source, the nitrogen source and the sterile water is 10-15:7-10:5-12:4-7:150-200; the inoculum sizes of lactobacillus plantarum and lactobacillus salivarius strain seed solutions in the step S4 are 3-5% and 2-4% respectively, the conditions of fermentation culture are under micro-anoxic conditions, the temperature is 36-38 ℃, the time is 48-72h, the rotating speed is 50-70r/min, and the micro-anoxic conditions are 5-7% CO ₂ 、7-10%O ₂ The balance is N ₂ Wherein% is by volume.

As a further improvement of the invention, the mass ratio of the fermentation product to the sodium trimetaphosphate in the step S5 is 100:7-12, the pH value is adjusted to 8-9, the temperature of the heating reaction is 35-45 ℃ and the time is 50-70min; the mass ratio of the phosphorylated fermentation product to the calcium ion solution in the step S6 is 10:5-7, wherein the calcium ion solution is 40-50wt% of calcium chloride solution, and the stirring reaction time is 30-50min.

As a further improvement of the invention, the mass ratio of the vitamin D1 to the vitamin D3 in the step S7 is 1-3:5; the mass ratio of the bisphosphonate to the calcitonin in the step S8 is 3-5:2; in the step S9, the mass ratio of the sea cucumber saponin to the ginsenoside Rg3 is 4-7:5.

As a further improvement of the invention, in the step S10, the mass ratio of the traditional Chinese medicine water extract, the calcium phosphate peptide fermentation complex, the calcium citrate, the vitamin D composition, the osteoclast inhibitor, the active composition, the sodium alginate, the sodium carboxymethyl cellulose and the carboxymethyl pachyman is 4-6:10:50-70:0.0001-0.001:0.01-0.02:0.2-0.4:10-12:5-7:4-6, the mass ratio of the water phase to the edible oil is 3-5:5-7, the pore diameter of the rapid membrane is 1-3mm, the metal ion solution is a solution containing 3-5wt% of calcium chloride, ferric chloride or aluminum chloride, and the normal temperature curing time is 20-30min.

As a further improvement of the invention, the method specifically comprises the following steps:

s1, activation of probiotics: marking lactobacillus plantarum and lactobacillus salivarius in a Gao's medium respectively, and carrying out activation culture for 18-24 hours at 36-38 ℃ and 50-80r/min under a micro-anoxic condition to obtain strain seed liquid;

The micro-anoxic condition is 5-7% CO ₂ 、7-10%O ₂ The balance is N ₂ Wherein% is volume percent;

s2, preparing a traditional Chinese medicine water extract: cleaning 5-10 parts by weight of kudzuvine root, 3-5 parts by weight of fructus psoraleae, 5-7 parts by weight of epimedium and 1-3 parts by weight of glossy privet fruit, drying, crushing to obtain traditional Chinese medicine powder, adding water, boiling and extracting for 2-3 times, each time for 3-5 hours, wherein the solid-to-liquid ratio of the traditional Chinese medicine powder to the water is 1:5-10g/mL, filtering, combining the filtrates, concentrating, drying to obtain a traditional Chinese medicine water extract, and reserving filter residues;

s3, preparing a traditional Chinese medicine-protein peptide culture medium: mixing 10-15 parts by weight of filter residues in the step S2, 7-10 parts by weight of bone collagen, 5-12 parts by weight of carbon source and 4-7 parts by weight of nitrogen source, adding into 150-200 sterile water, stirring and mixing uniformly, and sterilizing to obtain a traditional Chinese medicine-protein peptide culture medium;

preferably, the carbon source is selected from at least one of glucose, maltose, isomaltose, fructose, sucrose, arabinose, fructo-oligosaccharide, xylo-oligosaccharide and agar, the nitrogen source is selected from at least one of peptone, fish meal, urea, amino acid, nitrate and ammonium salt, the nitrate is selected from at least one of sodium nitrate, potassium nitrate, magnesium nitrate, aluminum nitrate, zinc nitrate, ferric nitrate, copper nitrate and manganese nitrate, and the ammonium salt is selected from at least one of ammonium chloride, ammonium sulfate and ammonium nitrate; the amino acid is at least one selected from glycine, serine, threonine, valine, tryptophan, leucine, alanine, cysteine, methionine, lysine, isoleucine and phenylalanine.

S4, fermenting: inoculating lactobacillus plantarum and lactobacillus salivarius strain seed liquid prepared in the step S1 into the traditional Chinese medicine-protein peptide culture medium prepared in the step S3, wherein the inoculum size is 3-5% and 2-4% respectively, and fermenting and culturing for 48-72h at 36-38 ℃ and 50-70r/min under a micro-anoxic condition to obtain a fermentation product;

s5, preparing a phosphorylation fermentation product: adding 7-12 parts by weight of sodium trimetaphosphate into 100 parts by weight of the fermentation product prepared in the step S4, adjusting the pH value to 8-9, heating to 35-45 ℃, and reacting for 50-70min to prepare a phosphorylated fermentation product;

s6, preparation of a phosphorylated calcium peptide fermentation complex: adding 5-7 parts by weight of 40-50wt% calcium chloride solution into 10 parts by weight of the phosphorylated fermentation product prepared in the step S5, stirring and reacting for 30-50min, and freeze-drying to obtain a phosphorylated calcium peptide fermentation compound;

s7, preparation of a vitamin D composition: uniformly mixing 1-3 parts by weight of vitamin D1 and 5 parts by weight of vitamin D3 to prepare a vitamin D composition;

s8, preparation of an osteoclast inhibitor: uniformly mixing 3-5 parts by weight of bisphosphonate and 2 parts by weight of calcitonin to prepare an osteoclast inhibitor;

Preferably, the bisphosphonate is selected from at least one of etidronate sodium, clodronate sodium, pamidronate sodium, tiludronate sodium, alendronate sodium, olpadronate sodium, risedronate sodium, ibandronate sodium, zoledronic acid.

S9, preparation of an active composition: uniformly mixing 4-7 parts by weight of sea cucumber saponin and 5 parts by weight of ginsenoside Rg3 to prepare an active composition;

s10, preparation of a calcium, VD and protein peptide composition for improving bone mineral density: adding 4-6 parts by weight of the traditional Chinese medicine water extract prepared in the step S2, 10 parts by weight of the phosphorylated calcium peptide fermentation compound prepared in the step S6, 50-70 parts by weight of calcium citrate, 0.0001-0.001 part by weight of the vitamin D composition prepared in the step S7, 0.01-0.02 part by weight of the osteoclast inhibitor prepared in the step S8 and 0.2-0.4 part by weight of the active composition prepared in the step S9 into water, stirring and mixing uniformly, adding 10-12 parts by weight of sodium alginate, 5-7 parts by weight of sodium carboxymethylcellulose and 4-6 parts by weight of carboxymethyl pachyman, stirring and dissolving to obtain a water phase; adding 30-50 parts by weight of water phase into 50-70 parts by weight of edible oil, emulsifying by a rapid membrane with a pore diameter of 1-3mm, adding 10-20 parts by weight of calcium chloride, ferric chloride or aluminum chloride solution containing 3-5wt%, solidifying at normal temperature for 20-30min, filtering, and freeze-drying to obtain the calcium, VD and protein peptide composition for improving bone density.

Preferably, the edible oil is at least one selected from peanut oil, corn oil, soybean oil, rapeseed oil, sesame oil, linseed oil, olive oil and sunflower seed oil.

The invention further provides a calcium, VD and protein peptide composition for improving bone mineral density, which is prepared by the preparation method.

The invention further provides application of the calcium, VD and protein peptide composition for improving bone mineral density in preparing products for treating and preventing osteoporosis.

The invention has the following beneficial effects:

the senile osteoporosis is mainly a senile systemic hypofunction patient, the metabolism of each organ in the body is slow or abnormal along with the aging, the synthesis and secretion of kidney hydroxylase are reduced, the low metabolism state can reduce the enzyme activity, the generation of active vitamin D1 and vitamin D3 is not favored, the calcium absorption capacity of intestinal wall cells is reduced, and the calcium absorption capacity of human bodies is reduced, so that the invention has the effects of well promoting the calcium absorption capacity of the intestinal wall cells by adding the mixture of the supplementary vitamin D1 and the vitamin D3, and the addition of the two has the synergistic effect.

The osteoclast inhibitor comprises bisphosphonate and calcitonin, which can effectively block the activation of osteoclasts and induce the osteoclasts to undergo apoptosis, and the calcitonin can directly inhibit the physiological actions of the osteoclasts, both of which can slow down the decomposition and loss of bones and fully stimulate the generation of new bones, thereby effectively relieving the occurrence of senile osteoporosis and having a synergistic action.

The calcium agent and vitamin D are typical bone mineralization promoting drugs, the former is an important component for forming bone, the latter can directly promote calcium and phosphorus absorption, and free calcium and phosphorus components from bone into peripheral blood, so that blood calcium and blood phosphorus return to normal values, mineralization is returned, new bone is formed continuously, and osteoporosis can be relieved greatly. Therefore, the vitamin D and a large amount of organic calcium (calcium citrate and calcium phosphate peptide fermentation complex) are supplemented, and the vitamin D and the calcium phosphate peptide fermentation complex have good interaction effect.

Protein phosphorylation is an effective means for improving protein function, and the introduction of phosphate groups can promote calcium absorption of organisms, because the side chains of protein amino acids are added with a phosphate group with strong negative electricity, and esterification reaction is easy to occur, so that the configuration, activity and interaction force with other molecules of the protein are changed. Phosphorylation can also enhance the solubility of calcium phosphate, promoting calcium absorption by organisms. The phosphoserine residue of the phosphoprotein peptide can be combined with calcium ions to form a soluble compound to prevent calcium precipitation in the small intestine, and the prepared phosphoprotein peptide can increase the chelation site of polypeptide and metal ions, improve the binding activity of metal ions and improve the functional characteristics of the phosphoprotein peptide.

Furthermore, based on the prepared phosphorylated fermentation product, the calcium ion solution is added to prepare the phosphorylated protein peptide chelated calcium, so that the product formed by combining the phosphorylated protein peptide and calcium ions in the phosphorylated protein peptide chelated calcium through coordinate covalent bonds has the advantages of quick absorption, strong nutrition, high biological potency, antioxidation, antibiosis, blood fat reduction, immunoregulation and other activities. The binding sites for calcium ions are mainly carboxyl groups of aspartic acid and glutamic acid. The phosphorylated protein peptide also forms a specific spatial structure after being combined with calcium ions, thereby promoting calcium absorption. When the calcium ions and the small peptide are chelated, the calcium supplementing effect is better, and the calcium ions exist in a chelated state in the small intestine to accelerate the absorption of the small intestine to the peptide, so that the calcium supplementing effect of the phosphorylated protein peptide chelated calcium is better.

The active composition prepared by the invention comprises sea cucumber saponin and ginsenoside Rg3 which have similar structures and have the effects of promoting osteoblast proliferation and differentiation, reducing bone resorption and improving osteoporosis. Sea cucumber saponins can also reduce urinary calcium and urinary phosphorus concentration, reduce bone mineral loss, enhance bone density and bone mineralization deposition, improve osteoporosis, and have synergistic effect by adding the sea cucumber saponins.

The estrogen can inhibit bone resorption and promote bone formation, has obvious bone protection effect, and the pueraria root added in the traditional Chinese medicine composition contains abundant pueraria root isoflavone due to the estrogen effect of isoflavone compounds after being extracted by water. Puerarin can also exert greater physiological effects with smaller amounts of estrogen by increasing estrogen receptor levels on osteoblasts. The fructus psoraleae contains abundant isopsoralen, can promote the proliferation of osteoblasts, inhibit the apoptosis of osteoblasts, promote the proliferation and differentiation of bone marrow mesenchymal stem cells cultured in vitro, reduce calcium salt deposition, and has good anti-osteoporosis effect. Radix Puerariae is sweet, pungent and flat, and has the effects of relieving exterior syndrome, fever, pain in the neck and back, and diabetes due to yin deficiency; epimedium herb Wen Weixin is sweet and can tonify kidney yang and strengthen tendons and bones; fructus Psoraleae has effects of replenishing essence, nourishing marrow, replenishing kidney and invigorating spleen; fructus Ligustri Lucidi has effects of invigorating qi, promoting blood circulation, nourishing liver and kidney, and strengthening waist and knee; the medicines are used together to tonify spleen and kidney, strengthen tendons and bones, activate blood and remove meridian obstruction, and can treat osteoporosis.

The probiotics include lactobacillus plantarum and lactobacillus salivarius fermentation, wherein the lactobacillus plantarum can reduce the expression of inflammatory factors such as TNF-alpha and IL-1b, increase the expression of substances with the function of inhibiting osteolysis, and reduce bone mass loss; lactobacillus salivarius can achieve increased calcium ion absorption by promoting calcium ion absorption by intestinal epithelial cells. Under the synergistic effect of the two, the composition can also improve intestinal permeability, reduce intestinal inflammation and improve bone density.

Through the mixed embedding of sodium alginate, sodium carboxymethyl cellulose and carboxymethyl pachyman, after metal ion crosslinking, the formed microcapsule shell layer has good pH responsiveness, and can keep a relatively complete form under the acidic condition of low pH, so that active ingredients such as probiotics, phosphorylated calcium peptide fermentation complex and the like are prevented from being digested and deactivated by gastric acid, the active ingredients can be conveyed to intestinal tracts in a targeted manner, after the microcapsule form is broken, the shell material can be used as a prebiotic substance, the growth and the colonization of probiotics are promoted, the action of the probiotics is promoted, and the absorption of other active ingredients by small intestines is promoted.

The calcium, VD and protein peptide composition for improving bone mineral density, which is prepared by the invention, has the advantages of simple preparation method, wide raw material sources, capability of improving intestinal permeability, reducing intestinal inflammation, improving bone mineral density, promoting osteoblast proliferation and differentiation, reducing bone resorption, continuously forming new bone, promoting the calcium absorption capacity of intestinal wall cells and improving osteoporosis, and has wide application prospect.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.

FIG. 1 is a graph showing the comparative survival rate in test example 1 of the present invention;

FIG. 2 is a graph showing the comparison of the release rate in test example 1 of the present invention.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Lactobacillus plantarum, 150 hundred million cfu/g, purchased from Weifang Rui Chen Biotechnology Co., ltd; lactobacillus salivarius, 350 hundred million cfu/g, purchased from Guangzhou Huijia biotechnology limited; bone collagen with purity >99% is purchased from Jiangsu Miao biosciences, inc.; carboxymethyl pachyman with purity >98% is purchased from Hunan Tokay pharmaceutical Co.

Example 1

The embodiment provides a preparation method of a calcium, VD and protein peptide composition for improving bone mineral density, which specifically comprises the following steps:

s1, activation of probiotics: marking lactobacillus plantarum and lactobacillus salivarius in a Gao's medium respectively, and carrying out activation culture for 18h at 36 ℃ and 50r/min under a micro-anoxic condition to obtain strain seed liquid;

The micro-anoxic condition is 5 percent CO ₂ 、7%O ₂ The balance is N ₂ Wherein% is volume percent;

s2, preparing a traditional Chinese medicine water extract: cleaning 5 parts by weight of radix puerariae, 3 parts by weight of fructus psoraleae, 5 parts by weight of epimedium and 1 part by weight of glossy privet fruit, drying, crushing to obtain traditional Chinese medicine powder, adding water, boiling and extracting for 2 times, each time for 3 hours, wherein the solid-to-liquid ratio of the traditional Chinese medicine powder to the water is 1:5g/mL, filtering, merging filtrate, concentrating, drying at 105 ℃ for 2 hours to obtain a traditional Chinese medicine water extract, and reserving filter residues;

s3, preparing a traditional Chinese medicine-protein peptide culture medium: mixing 10 parts by weight of filter residues in the step S2, 7 parts by weight of bone collagen, 2 parts by weight of fructose, 2 parts by weight of sucrose, 1 part by weight of arabinose, 3 parts by weight of fish meal and 1 part by weight of ammonium nitrate, adding the mixture into 150 sterile water, stirring and mixing for 15min, and sterilizing by ultraviolet rays to obtain a traditional Chinese medicine-protein peptide culture medium;

s4, fermenting: inoculating lactobacillus plantarum and lactobacillus salivarius strain seed liquid prepared in the step S1 into the traditional Chinese medicine-protein peptide culture medium prepared in the step S3, wherein the inoculum size is 3% and 2% respectively, and fermenting and culturing for 48 hours at 36 ℃ and 50r/min under a micro-anoxic condition to obtain a fermentation product;

S5, preparing a phosphorylation fermentation product: adding 7 parts by weight of sodium trimetaphosphate into 100 parts by weight of the fermentation product prepared in the step S4, adjusting the pH value to 8, heating to 35 ℃, and reacting for 50min to prepare a phosphorylated fermentation product;

s6, preparation of a phosphorylated calcium peptide fermentation complex: adding 5 parts by weight of 40wt% calcium chloride solution into 10 parts by weight of the phosphorylated fermentation product prepared in the step S5, stirring and reacting for 30min, and freeze-drying to obtain a phosphorylated calcium peptide fermentation compound;

s7, preparation of a vitamin D composition: mixing 1 part by weight of vitamin D1 and 5 parts by weight of vitamin D3 for 15min under stirring to obtain a vitamin D composition;

s8, preparation of an osteoclast inhibitor: stirring and mixing 3 parts by weight of sodium tiludronate and 2 parts by weight of calcitonin for 15min to prepare an osteoclast inhibitor;

s9, preparation of an active composition: stirring and mixing 4 parts by weight of sea cucumber saponin and 5 parts by weight of ginsenoside Rg3 for 15min to obtain an active composition;

s10, preparation of a calcium, VD and protein peptide composition for improving bone mineral density: adding 4 parts by weight of the traditional Chinese medicine water extract prepared in the step S2, 10 parts by weight of the phosphorylated calcium peptide fermentation complex prepared in the step S6, 50 parts by weight of calcium citrate, 0.0001 part by weight of the vitamin D composition prepared in the step S7, 0.01 part by weight of the osteoclast inhibitor prepared in the step S8 and 0.2 part by weight of the active composition prepared in the step S9 into water, stirring and mixing for 20min, adding 10 parts by weight of sodium alginate, 5 parts by weight of sodium carboxymethylcellulose and 4 parts by weight of carboxymethyl pachyman, and stirring and dissolving to obtain a water phase; adding 30 parts by weight of water phase into 50 parts by weight of peanut oil, emulsifying by a rapid membrane with a pore diameter of 1mm, adding 10 parts by weight of calcium chloride, ferric chloride or aluminum chloride solution containing 3wt% and solidifying for 20min at normal temperature, filtering, and freeze-drying to obtain the calcium, VD and protein peptide composition for improving bone density.

Example 2

s1, activation of probiotics: marking lactobacillus plantarum and lactobacillus salivarius in a Gao's medium respectively, and carrying out activation culture for 24 hours at 38 ℃ and 80r/min under a micro-anoxic condition to obtain strain seed liquid;

the micro-anoxic condition is 7 percent CO ₂ 、10%O ₂ The balance is N ₂ Wherein% is volume percent;

s2, preparing a traditional Chinese medicine water extract: cleaning 10 parts by weight of radix puerariae, 5 parts by weight of fructus psoraleae, 7 parts by weight of epimedium and 3 parts by weight of glossy privet fruit, drying, crushing to obtain traditional Chinese medicine powder, adding water, boiling and extracting for 3 times, each time for 5 hours, wherein the solid-to-liquid ratio of the traditional Chinese medicine powder to the water is 1:10g/mL, filtering, merging filtrate, concentrating, drying at 105 ℃ for 2 hours to obtain a traditional Chinese medicine water extract, and reserving filter residues;

s3, preparing a traditional Chinese medicine-protein peptide culture medium: mixing 15 parts by weight of filter residues in the step S2, 10 parts by weight of bone collagen, 5 parts by weight of maltose, 5 parts by weight of isomaltose, 2 parts by weight of fructose, 2 parts by weight of fish meal, 2 parts by weight of urea, 2 parts by weight of zinc nitrate and 1 part by weight of ferric nitrate, adding into 200 sterile water, stirring and mixing for 15min, and sterilizing by ultraviolet rays to obtain a traditional Chinese medicine-protein peptide culture medium;

S4, fermenting: inoculating lactobacillus plantarum and lactobacillus salivarius strain seed liquid prepared in the step S1 into the traditional Chinese medicine-protein peptide culture medium prepared in the step S3, wherein the inoculum size is 5% and 4% respectively, and fermenting and culturing for 72h at 38 ℃ and 70r/min under a micro-anoxic condition to obtain a fermentation product;

s5, preparing a phosphorylation fermentation product: adding 12 parts by weight of sodium trimetaphosphate into 100 parts by weight of the fermentation product prepared in the step S4, adjusting the pH value to 9, heating to 45 ℃, and reacting for 70 minutes to prepare a phosphorylated fermentation product;

s6, preparation of a phosphorylated calcium peptide fermentation complex: adding 7 parts by weight of 50wt% calcium chloride solution into 10 parts by weight of the phosphorylated fermentation product prepared in the step S5, stirring and reacting for 50min, and freeze-drying to obtain a phosphorylated calcium peptide fermentation compound;

s7, preparation of a vitamin D composition: mixing 3 parts by weight of vitamin D1 and 5 parts by weight of vitamin D3 for 15min under stirring to obtain a vitamin D composition;

s8, preparation of an osteoclast inhibitor: stirring and mixing 5 parts by weight of risedronate sodium and 2 parts by weight of calcitonin for 15min to prepare an osteoclast inhibitor;

s9, preparation of an active composition: stirring and mixing 7 parts by weight of sea cucumber saponin and 5 parts by weight of ginsenoside Rg3 for 15min to obtain an active composition;

S10, preparation of a calcium, VD and protein peptide composition for improving bone mineral density: adding 6 parts by weight of the traditional Chinese medicine water extract prepared in the step S2, 10 parts by weight of the phosphorylated calcium peptide fermentation complex prepared in the step S6, 70 parts by weight of calcium citrate, 0.001 part by weight of the vitamin D composition prepared in the step S7, 0.02 part by weight of the osteoclast inhibitor prepared in the step S8 and 0.4 part by weight of the active composition prepared in the step S9 into water, stirring and mixing for 20min, adding 12 parts by weight of sodium alginate, 7 parts by weight of sodium carboxymethyl cellulose and 6 parts by weight of carboxymethyl pachyman, and stirring and dissolving to obtain a water phase; adding 50 parts by weight of water phase into 70 parts by weight of soybean oil, emulsifying by a rapid membrane with a pore diameter of 3mm, adding 20 parts by weight of calcium chloride, ferric chloride or aluminum chloride solution containing 5wt% and solidifying for 30min at normal temperature, filtering, and freeze-drying to obtain the calcium, VD and protein peptide composition for improving bone density.

Example 3

s1, activation of probiotics: marking lactobacillus plantarum and lactobacillus salivarius in a Gao's medium respectively, and carrying out activation culture for 21h at 37 ℃ and 65r/min under a micro-anoxic condition to obtain strain seed liquid;

The micro-anoxic condition is 6 percent CO ₂ 、8.5%O ₂ The balance is N ₂ Wherein% is volume percent;

s2, preparing a traditional Chinese medicine water extract: cleaning 7 parts by weight of radix puerariae, 4 parts by weight of fructus psoraleae, 6 parts by weight of epimedium and 2 parts by weight of glossy privet fruit, drying, crushing to obtain traditional Chinese medicine powder, adding water, boiling and extracting for 3 times, 4 hours each time, wherein the solid-to-liquid ratio of the traditional Chinese medicine powder to the water is 1:7g/mL, filtering, merging filtrate, concentrating, drying at 105 ℃ for 2 hours to obtain a traditional Chinese medicine water extract, and reserving filter residues;

s3, preparing a traditional Chinese medicine-protein peptide culture medium: mixing 12 parts by weight of filter residues in the step S2, 8.5 parts by weight of bone collagen, 5 parts by weight of glucose, 2 parts by weight of maltose, 3 parts by weight of peptone and 2 parts by weight of urea, adding the mixture into 170 sterile water, stirring and mixing for 15min, and sterilizing by ultraviolet rays to obtain a traditional Chinese medicine-protein peptide culture medium;

s4, fermenting: inoculating lactobacillus plantarum and lactobacillus salivarius strain seed liquid prepared in the step S1 into the traditional Chinese medicine-protein peptide culture medium prepared in the step S3, wherein the inoculum size is 4% and 3% respectively, and fermenting and culturing for 56h at 37 ℃ and 60r/min under a micro-anoxic condition to obtain a fermentation product;

S5, preparing a phosphorylation fermentation product: adding 10 parts by weight of sodium trimetaphosphate into 100 parts by weight of the fermentation product prepared in the step S4, adjusting the pH value to 8.5, heating to 40 ℃, and reacting for 60 minutes to prepare a phosphorylated fermentation product;

s6, preparation of a phosphorylated calcium peptide fermentation complex: adding 6 parts by weight of 45wt% calcium chloride solution into 10 parts by weight of the phosphorylated fermentation product prepared in the step S5, stirring and reacting for 40min, and freeze-drying to obtain a phosphorylated calcium peptide fermentation compound;

s7, preparation of a vitamin D composition: mixing 2 parts by weight of vitamin D1 and 5 parts by weight of vitamin D3 for 15min under stirring to obtain a vitamin D composition;

s8, preparation of an osteoclast inhibitor: stirring and mixing 4 parts by weight of olpadronate sodium and 2 parts by weight of calcitonin for 15min to prepare an osteoclast inhibitor;

s9, preparation of an active composition: stirring and mixing 5 parts by weight of sea cucumber saponin and 5 parts by weight of ginsenoside Rg3 for 15min to obtain an active composition;

s10, preparation of a calcium, VD and protein peptide composition for improving bone mineral density: adding 5 parts by weight of the traditional Chinese medicine water extract prepared in the step S2, 10 parts by weight of the phosphorylated calcium peptide fermentation complex prepared in the step S6, 60 parts by weight of calcium citrate, 0.0005 part by weight of the vitamin D composition prepared in the step S7, 0.015 part by weight of the osteoclast inhibitor prepared in the step S8 and 0.3 part by weight of the active composition prepared in the step S9 into water, stirring and mixing for 20min, adding 11 parts by weight of sodium alginate, 6 parts by weight of sodium carboxymethyl cellulose and 5 parts by weight of carboxymethyl pachyman, and stirring and dissolving to obtain a water phase; adding 40 parts by weight of water phase into 60 parts by weight of rapeseed oil, emulsifying by a rapid membrane with the pore diameter of 2mm, adding 15 parts by weight of calcium chloride, ferric chloride or aluminum chloride solution containing 4wt% and solidifying for 25min at normal temperature, filtering, and freeze-drying to obtain the calcium, VD and protein peptide composition for improving bone density.

Comparative example 1

In contrast to example 3, in step S2, radix Puerariae was not added.

The method comprises the following steps:

s2, preparing a traditional Chinese medicine water extract: washing, drying and crushing 11 parts by weight of fructus psoraleae, 6 parts by weight of epimedium and 2 parts by weight of glossy privet fruit to obtain traditional Chinese medicine powder, adding water, boiling and extracting for 3 times, 4 hours each time, wherein the solid-liquid ratio of the traditional Chinese medicine powder to the water is 1:7g/mL, filtering, merging filtrate, concentrating, drying at 105 ℃ for 2 hours to obtain a traditional Chinese medicine water extract, and reserving filter residues.

Comparative example 2

The difference from example 3 is that no fructus Psoraleae was added in step S2.

The method comprises the following steps:

s2, preparing a traditional Chinese medicine water extract: cleaning, drying and crushing 11 parts by weight of kudzuvine root, 6 parts by weight of epimedium herb and 2 parts by weight of glossy privet fruit to obtain traditional Chinese medicine powder, adding water, boiling and extracting for 3 times, 4 hours each time, wherein the solid-to-liquid ratio of the traditional Chinese medicine powder to the water is 1:7g/mL, filtering, merging filtrate, concentrating, drying at 105 ℃ for 2 hours to obtain a traditional Chinese medicine water extract, and reserving filter residues.

Comparative example 3

The difference from example 3 is that no filter residue was added in step S3.

The method comprises the following steps:

s3, preparing a traditional Chinese medicine-protein peptide culture medium: mixing 20.5 parts by weight of bone collagen, 5 parts by weight of glucose, 2 parts by weight of maltose, 3 parts by weight of peptone and 2 parts by weight of urea, adding into 170 sterile water, stirring and mixing for 15min, and sterilizing by ultraviolet rays to obtain the traditional Chinese medicine-protein peptide culture medium.

Comparative example 4

The difference compared to example 3 is that no collagen is added in step S3.

The method comprises the following steps:

s3, preparing a traditional Chinese medicine-protein peptide culture medium: mixing 20.5 parts by weight of filter residues in the step S2, 5 parts by weight of glucose, 2 parts by weight of maltose, 3 parts by weight of peptone and 2 parts by weight of urea, adding into 170 sterile water, stirring and mixing for 15min, and sterilizing by ultraviolet rays to obtain the traditional Chinese medicine-protein peptide culture medium.

Comparative example 5

The difference compared to example 3 is that lactobacillus plantarum was not inoculated in step S4.

The method comprises the following steps:

s4, fermenting: inoculating lactobacillus salivarius strain seed liquid prepared in the step S1 into the traditional Chinese medicine-protein peptide culture medium prepared in the step S3, and fermenting and culturing for 56h at 37 ℃ under a micro-anoxic condition to obtain a fermentation product;

the micro-anoxic condition is 6 percent CO ₂ 、8.5%O ₂ The balance is N ₂ Wherein% is by volume.

Comparative example 6

The difference compared to example 3 is that lactobacillus salivarius was not inoculated in step S4.

The method comprises the following steps:

s4, fermenting: inoculating lactobacillus plantarum strain seed liquid prepared in the step S1 into the traditional Chinese medicine-protein peptide culture medium prepared in the step S3, and fermenting and culturing for 56 hours at 37 ℃ and 60r/min under a micro-anoxic condition to obtain a fermentation product;

Comparative example 7

In comparison with example 3, the difference is that step S4 is not performed and the bone collagen is directly added to step S5.

The method comprises the following steps:

s1, preparing a traditional Chinese medicine water extract: cleaning 7 parts by weight of radix puerariae, 4 parts by weight of fructus psoraleae, 6 parts by weight of epimedium and 2 parts by weight of glossy privet fruit, drying, crushing to obtain traditional Chinese medicine powder, adding water, boiling and extracting for 3 times, each time for 4 hours, wherein the solid-to-liquid ratio of the traditional Chinese medicine powder to the water is 1:7g/mL, filtering, merging filtrate, concentrating, and drying at 105 ℃ for 2 hours to obtain a traditional Chinese medicine water extract;

s2, preparing phosphorylated bone collagen: adding 10 parts by weight of sodium trimetaphosphate into 10 parts by weight of bone collagen, adjusting the pH value to 8.5, heating to 40 ℃, and reacting for 60min to obtain phosphorylated bone collagen;

s3, preparation of a phosphorylated calcium peptide complex: adding 6 parts by weight of 45wt% calcium chloride solution into 10 parts by weight of the phosphorylated bone collagen prepared in the step S5, stirring and reacting for 40min, and freeze-drying to obtain a phosphorylated calcium peptide compound;

s4, preparation of a vitamin D composition: mixing 2 parts by weight of vitamin D1 and 5 parts by weight of vitamin D3 for 15min under stirring to obtain a vitamin D composition;

S5, preparation of an osteoclast inhibitor: stirring and mixing 4 parts by weight of olpadronate sodium and 2 parts by weight of calcitonin for 15min to prepare an osteoclast inhibitor;

s6, preparation of an active composition: stirring and mixing 5 parts by weight of sea cucumber saponin and 5 parts by weight of ginsenoside Rg3 for 15min to obtain an active composition;

s7, preparation of calcium, VD and protein peptide composition for improving bone mineral density: adding 5 parts by weight of the traditional Chinese medicine water extract prepared in the step S1, 10 parts by weight of the phosphorylated calcium peptide compound prepared in the step S3, 60 parts by weight of calcium citrate, 0.0005 part by weight of the vitamin D composition prepared in the step S4, 0.015 part by weight of the osteoclast inhibitor prepared in the step S5 and 0.3 part by weight of the active composition prepared in the step S6 into water, stirring and mixing for 20min, adding 11 parts by weight of sodium alginate, 6 parts by weight of sodium carboxymethyl cellulose and 5 parts by weight of carboxymethyl pachyman, and stirring and dissolving to obtain a water phase; adding 40 parts by weight of water phase into 60 parts by weight of rapeseed oil, emulsifying by a rapid membrane with the pore diameter of 2mm, adding 15 parts by weight of calcium chloride, ferric chloride or aluminum chloride solution containing 4wt% and solidifying for 25min at normal temperature, filtering, and freeze-drying to obtain the calcium, VD and protein peptide composition for improving bone density.

Comparative example 8

In comparison with example 3, the difference is that step S5 is not performed.

The method comprises the following steps:

the micro-anoxic condition is 6 percent CO ₂ 、8.5%O ₂ The balance is N ₂ Wherein% is volume percentRatio of;

s5, preparing a calcium peptide fermentation complex: adding 6 parts by weight of 45wt% calcium chloride solution into 10 parts by weight of the fermentation product prepared in the step S4, stirring and reacting for 40min, and freeze-drying to obtain a calcium peptide fermentation compound;

s6, preparation of a vitamin D composition: mixing 2 parts by weight of vitamin D1 and 5 parts by weight of vitamin D3 for 15min under stirring to obtain a vitamin D composition;

s7, preparation of an osteoclast inhibitor: stirring and mixing 4 parts by weight of olpadronate sodium and 2 parts by weight of calcitonin for 15min to prepare an osteoclast inhibitor;

s8, preparation of an active composition: stirring and mixing 5 parts by weight of sea cucumber saponin and 5 parts by weight of ginsenoside Rg3 for 15min to obtain an active composition;

s9, preparation of calcium, VD and protein peptide composition for improving bone mineral density: adding 5 parts by weight of the traditional Chinese medicine water extract prepared in the step S2, 10 parts by weight of the calcium peptide fermentation compound prepared in the step S5, 60 parts by weight of calcium citrate, 0.0005 part by weight of the vitamin D composition prepared in the step S6, 0.015 part by weight of the osteoclast inhibitor prepared in the step S7 and 0.3 part by weight of the active composition prepared in the step S8 into water, stirring and mixing for 20min, adding 11 parts by weight of sodium alginate, 6 parts by weight of sodium carboxymethyl cellulose and 5 parts by weight of carboxymethyl pachyman, and stirring and dissolving to obtain a water phase; adding 40 parts by weight of water phase into 60 parts by weight of rapeseed oil, emulsifying by a rapid membrane with the pore diameter of 2mm, adding 15 parts by weight of calcium chloride, ferric chloride or aluminum chloride solution containing 4wt% and solidifying for 25min at normal temperature, filtering, and freeze-drying to obtain the calcium, VD and protein peptide composition for improving bone density.

Comparative example 9

In comparison with example 3, the difference is that step S6 is not performed.

The method comprises the following steps:

s9, preparation of calcium, VD and protein peptide composition for improving bone mineral density: adding 5 parts by weight of the traditional Chinese medicine water extract prepared in the step S2, 10 parts by weight of the phosphorylated fermentation product prepared in the step S5, 60 parts by weight of calcium citrate, 0.0005 part by weight of the vitamin D composition prepared in the step S6, 0.015 part by weight of the osteoclast inhibitor prepared in the step S7 and 0.3 part by weight of the active composition prepared in the step S8 into water, stirring and mixing for 20min, adding 11 parts by weight of sodium alginate, 6 parts by weight of sodium carboxymethyl cellulose and 5 parts by weight of carboxymethyl pachyman, and stirring and dissolving to obtain a water phase; adding 40 parts by weight of water phase into 60 parts by weight of rapeseed oil, emulsifying by a rapid membrane with the pore diameter of 2mm, adding 15 parts by weight of calcium chloride, ferric chloride or aluminum chloride solution containing 4wt% and solidifying for 25min at normal temperature, filtering, and freeze-drying to obtain the calcium, VD and protein peptide composition for improving bone density.

Comparative example 10

In comparison with example 3, the difference is that vitamin D1 is not added in step S7.

The method comprises the following steps:

s7, preparation of a vitamin D composition: 7 parts by weight of vitamin D3 was stirred and mixed for 15 minutes to prepare a vitamin D composition.

Comparative example 11

In comparison with example 3, the difference is that vitamin D3 is not added in step S7.

The method comprises the following steps:

s7, preparation of a vitamin D composition: 7 parts by weight of vitamin D1 was stirred and mixed for 15 minutes to prepare a vitamin D composition.

Comparative example 12

In comparison with example 3, the difference is that no sodium olpadronate was added in step S8.

The method comprises the following steps:

s8, preparation of an osteoclast inhibitor: mixing 6 parts by weight of calcitonin for 15min to obtain the osteoclast inhibitor.

Comparative example 13

In comparison with example 3, the difference is that no calcitonin is added in step S8.

The method comprises the following steps:

s8, preparation of an osteoclast inhibitor: 6 parts by weight of olpadronate sodium is stirred and mixed for 15min to prepare the osteoclast inhibitor.

Comparative example 14

Compared with example 3, the difference is that no sea cucumber saponin was added in step S9.

The method comprises the following steps:

s9, preparation of an active composition: 10 parts by weight of ginsenoside Rg3 is stirred and mixed for 15min to prepare the active composition.

Comparative example 15

The difference from example 3 is that ginsenoside Rg3 is not added in step S9.

The method comprises the following steps:

s9, preparation of an active composition: 10 parts by weight of sea cucumber saponin is stirred and mixed for 15min to prepare the active composition.

Comparative example 16

In comparison with example 3, the difference is that the phosphorylated calcium peptide fermentation complex is not added in step S10.

The method comprises the following steps:

s10, preparation of a calcium, VD and protein peptide composition for improving bone mineral density: adding 5 parts by weight of the traditional Chinese medicine water extract prepared in the step S2, 60 parts by weight of calcium citrate, 0.0005 part by weight of the vitamin D composition prepared in the step S7, 0.015 part by weight of the osteoclast inhibitor prepared in the step S8 and 0.3 part by weight of the active composition prepared in the step S9 into water, stirring and mixing for 20min, adding 11 parts by weight of sodium alginate, 6 parts by weight of sodium carboxymethylcellulose and 5 parts by weight of carboxymethyl pachyman, and stirring and dissolving to obtain a water phase; adding 40 parts by weight of water phase into 60 parts by weight of rapeseed oil, emulsifying by a rapid membrane with the pore diameter of 2mm, adding 15 parts by weight of calcium chloride, ferric chloride or aluminum chloride solution containing 4wt% and solidifying for 25min at normal temperature, filtering, and freeze-drying to obtain the calcium, VD and protein peptide composition for improving bone density.

Comparative example 17

The difference compared to example 3 is that the vitamin D composition is not added in step S10.

The method comprises the following steps:

s10, preparation of a calcium, VD and protein peptide composition for improving bone mineral density: adding 5 parts by weight of the traditional Chinese medicine water extract prepared in the step S2, 10 parts by weight of the phosphorylated calcium peptide fermentation complex prepared in the step S6, 60 parts by weight of calcium citrate, 0.015 part by weight of the osteoclast inhibitor prepared in the step S8 and 0.3 part by weight of the active composition prepared in the step S9 into water, stirring and mixing for 20min, adding 11 parts by weight of sodium alginate, 6 parts by weight of sodium carboxymethylcellulose and 5 parts by weight of carboxymethyl pachyman, and stirring and dissolving to obtain a water phase; adding 40 parts by weight of water phase into 60 parts by weight of rapeseed oil, emulsifying by a rapid membrane with the pore diameter of 2mm, adding 15 parts by weight of calcium chloride, ferric chloride or aluminum chloride solution containing 4wt% and solidifying for 25min at normal temperature, filtering, and freeze-drying to obtain the calcium, VD and protein peptide composition for improving bone density.

Comparative example 18

The difference compared to example 3 is that no calcium citrate is added in step S10.

The method comprises the following steps:

s10, preparation of a calcium, VD and protein peptide composition for improving bone mineral density: adding 5 parts by weight of the traditional Chinese medicine water extract prepared in the step S2, 10 parts by weight of the phosphorylated calcium peptide fermentation complex prepared in the step S6, 0.0005 part by weight of the vitamin D composition prepared in the step S7, 0.015 part by weight of the osteoclast inhibitor prepared in the step S8 and 0.3 part by weight of the active composition prepared in the step S9 into water, stirring and mixing for 20min, adding 11 parts by weight of sodium alginate, 6 parts by weight of sodium carboxymethylcellulose and 5 parts by weight of carboxymethyl pachyman, stirring and dissolving to obtain a water phase; adding 40 parts by weight of water phase into 60 parts by weight of rapeseed oil, emulsifying by a rapid membrane with the pore diameter of 2mm, adding 15 parts by weight of calcium chloride, ferric chloride or aluminum chloride solution containing 4wt% and solidifying for 25min at normal temperature, filtering, and freeze-drying to obtain the calcium, VD and protein peptide composition for improving bone density.

Comparative example 19

The difference from example 3 is that no osteoclast inhibitor was added in step S10.

The method comprises the following steps:

s10, preparation of a calcium, VD and protein peptide composition for improving bone mineral density: adding 5 parts by weight of the traditional Chinese medicine water extract prepared in the step S2, 10 parts by weight of the phosphorylated calcium peptide fermentation complex prepared in the step S6, 60 parts by weight of calcium citrate, 0.0005 part by weight of the vitamin D composition prepared in the step S7 and 0.3 part by weight of the active composition prepared in the step S9 into water, stirring and mixing for 20min, adding 11 parts by weight of sodium alginate, 6 parts by weight of sodium carboxymethylcellulose and 5 parts by weight of carboxymethyl pachyman, and stirring and dissolving to obtain a water phase; adding 40 parts by weight of water phase into 60 parts by weight of rapeseed oil, emulsifying by a rapid membrane with the pore diameter of 2mm, adding 15 parts by weight of calcium chloride, ferric chloride or aluminum chloride solution containing 4wt% and solidifying for 25min at normal temperature, filtering, and freeze-drying to obtain the calcium, VD and protein peptide composition for improving bone density.

Comparative example 20

The difference compared to example 3 is that no active composition is added in step S10.

The method comprises the following steps:

s10, preparation of a calcium, VD and protein peptide composition for improving bone mineral density: adding 5 parts by weight of the traditional Chinese medicine water extract prepared in the step S2, 10 parts by weight of the phosphorylated calcium peptide fermentation complex prepared in the step S6, 60 parts by weight of calcium citrate, 0.0005 part by weight of the vitamin D composition prepared in the step S7 and 0.015 part by weight of the osteoclast inhibitor prepared in the step S8 into water, stirring and mixing for 20min, adding 11 parts by weight of sodium alginate, 6 parts by weight of sodium carboxymethylcellulose and 5 parts by weight of carboxymethyl pachyman, stirring and dissolving to obtain a water phase; adding 40 parts by weight of water phase into 60 parts by weight of rapeseed oil, emulsifying by a rapid membrane with the pore diameter of 2mm, adding 15 parts by weight of calcium chloride, ferric chloride or aluminum chloride solution containing 4wt% and solidifying for 25min at normal temperature, filtering, and freeze-drying to obtain the calcium, VD and protein peptide composition for improving bone density.

Comparative example 21

The difference from example 3 is that the water extract of the traditional Chinese medicine is not added in step S10.

The method comprises the following steps:

s10, preparation of a calcium, VD and protein peptide composition for improving bone mineral density: adding 10 parts by weight of the phosphorylated calcium peptide fermentation complex prepared in the step S6, 60 parts by weight of calcium citrate, 0.0005 part by weight of the vitamin D composition prepared in the step S7, 0.015 part by weight of the osteoclast inhibitor prepared in the step S8 and 0.3 part by weight of the active composition prepared in the step S9 into water, stirring and mixing for 20min, adding 11 parts by weight of sodium alginate, 6 parts by weight of sodium carboxymethylcellulose and 5 parts by weight of carboxymethyl pachyman, stirring and dissolving to obtain a water phase; adding 40 parts by weight of water phase into 60 parts by weight of rapeseed oil, emulsifying by a rapid membrane with the pore diameter of 2mm, adding 15 parts by weight of calcium chloride, ferric chloride or aluminum chloride solution containing 4wt% and solidifying for 25min at normal temperature, filtering, and freeze-drying to obtain the calcium, VD and protein peptide composition for improving bone density.

Comparative example 22

The difference compared to example 3 is that step S10 is not microencapsulated.

The method comprises the following steps:

s10, preparation of a calcium, VD and protein peptide composition for improving bone mineral density: 5 parts by weight of the aqueous extract of the traditional Chinese medicine prepared in the step S2, 10 parts by weight of the phosphorylated calcium peptide fermentation complex prepared in the step S6, 60 parts by weight of calcium citrate, 0.0005 part by weight of the vitamin D composition prepared in the step S7, 0.015 part by weight of the osteoclast inhibitor prepared in the step S8 and 0.3 part by weight of the active composition prepared in the step S9 are stirred and mixed for 20 minutes to obtain the calcium, VD and protein peptide composition for improving bone density.

Test example 1

0.5g of the calcium, VD and protein peptide compositions for improving bone density prepared in the examples 1-3 and the comparative example 22 are respectively added into 10mL of artificial simulated gastric fluid and 10mL of artificial simulated intestinal fluid to react for 2h and 3h at 37 ℃ and 70r/min, and in addition, the same amount of probiotics for improving intestinal flora balance is added into 10mL of artificial simulated gastric fluid, and after reacting for 2h at 37 ℃ and 70r/min, the mixture is centrifuged, 10mL of artificial simulated intestinal fluid is added, and the reaction is continued for 3h at 37 ℃. Probiotic colony cell counts were performed after the end of the reaction.

Gastric juice is simulated manually: 16.4mL of diluted hydrochloric acid is taken, about 800mL of water is added, the pH is adjusted to 2.0, 10g of pepsin is added, and after stirring evenly, water is added to fix the volume to 1000mL.

Manually simulating intestinal juice: 6.8g of monopotassium phosphate, 500mL of distilled water is added for dissolution, and 0.4% sodium hydroxide is used for regulating the pH to 6.8; sterilizing at 120deg.C for 20min; 10g of trypsin, adding a proper amount of sterile water to dissolve, mixing the two solutions, and adding water to fix the volume to 1000mL.

Survival was calculated according to the following formula:

survival (%) =n _t /N ₀ ×100%

Wherein N is _t For the concentration of probiotics (cfu/g) that survived a certain time of incubation in vitro, N ₀ Is the original concentration (cfu/g) of probiotics.

The release rate was calculated according to the following formula:

Release rate (%) = (W) _t -W ₀ ）/W ₀ ×100%

In which W is _t Initial weight for sample; w (W) ₀ The samples were weighed after incubation in vitro for a certain period of time.

The results are shown in FIGS. 1 and 2.

1-2, it can be seen that the calcium, VD and protein peptide compositions for improving bone density prepared in examples 1-3 of the present invention can maintain a better form in artificial simulated gastric fluid, and collapse to release the contents in artificial simulated intestinal fluid. According to the invention, through the mixed embedding of sodium alginate, sodium carboxymethyl cellulose and carboxymethyl pachyman, after metal ion crosslinking, the formed microcapsule shell has good pH responsiveness, and can keep a relatively complete form under an acidic condition with low pH, so that active ingredients such as probiotics, phosphorylated calcium peptide fermentation complex and the like are prevented from being digested and deactivated by gastric acid, the active ingredients can be conveyed to intestinal tracts in a targeted manner, after the microcapsule form is broken, the shell material can be used as a prebiotic substance, the growth and the colonization of probiotics are promoted, the action of the probiotics is promoted, and the absorption of other active ingredients by small intestines is promoted.

Test example 2

280 weaned SPF-class female rats of 3-4 weeks of birth were randomly divided into 28 groups according to mass, each group comprising 10 normal control group, low-calcium control group, calcium carbonate group, examples 1-3 group, and comparative examples 1-22 group. In addition to the normal control group, the other groups were fed with low-calcium feed continuously to prepare low-bone-mass model rats, while each group was fed with the corresponding test article, the calcium carbonate group was fed with 150mg/kg of calcium carbonate, the groups of examples 1-3 and comparative examples 1-22 were fed with the corresponding calcium, VD, protein peptide compositions for improving bone density, 150mg/kg of distilled water, and the low-calcium control group was fed with the normal feed and the corresponding distilled water for 13 weeks.

The formula of the low-calcium feed comprises the following components: 15% of soybean powder, 10% of casein, 4% of soybean oil, 54% of flour, 2% of cellulose, 1% of vitamin, 2.6% of calcium-free mineral salt, 0.2% of methionine, 0.2% of choline chloride and 11% of starch; and (5) radiation sterilization.

Measurement of body length and constitution: after the rats fasted for 12 hours, the body length and mass were measured 1 time at week 13. The results are shown in Table 1.

TABLE 1

Group of	Body mass (g)	Body length (cm)
			Normal control group	317.2±22.1	24.2±1.4
Low-calcium control group	282.5±19.5 ^*	20.1±1.2 ^*
			Calcium carbonate group	291.4±21.4 ^#	21.9±1.5 ^#
Example 1 group	303.2±22.4 ^#	23.1±1.9 ^#
			Example 2 group	304.1±19.5 ^#	23.2±2.1 ^#
Example 3 group	305.0±15.7 ^#	23.4±2.0 ^#
			Comparative example 1 group	298.7±17.2	22.8±1.6
Comparative example 2 group	297.5±23.1	22.6±1.4
			Comparative example 3 group	295.6±22.4	22.4±1.2
Comparative example 4 group	295.0±19.8	22.3±1.6
			Comparative example 5 group	296.8±20.4	22.7±1.9
Comparative example 6 group	297.0±18.9	22.6±1.8
			Comparative example 7 group	295.4±16.7	22.4±1.2
Comparative example 8 group	298.9±17.2	22.9±1.1
			Comparative example 9 group	296.7±15.9	22.6±1.4
Comparative example 10 group	296.5±18.4	22.5±1.7
			Comparative example 11 group	295.8±19.0	22.4±1.5
Comparative example 12 group	297.4±14.7	22.7±2.0
			Comparative example 13 group	297.2±21.0	22.6±2.1
Comparative example 14 group	295.3±20.3	22.4±1.9
			Comparative example 15 group	294.9±19.8	22.3±2.2
Comparative example 16 group	292.3±18.9	22.1±1.0
			Comparative example 17 group	295.0±19.2	22.3±0.9
Comparative example 18 groups	292.0±17.8	22.0±1.2
			Comparative example 19 group	296.5±19.5	22.5±1.4
Comparative example 20 group	293.6±20.1	22.1±1.2
			Comparative example 21 group	294.2±20.3	22.2±1.3
Comparative example 22 group	295.1±19.8	22.4±1.5

Annotation: * P <0.05 for comparison to the normal control group; # is P <0.05 compared to the low calcium control group.

As can be seen from the above table, the calcium, VD and protein peptide compositions for improving bone mineral density prepared in examples 1-3 of the present invention can significantly promote growth of rat body.

Measurement of lumbar vertebra density: the lumbar bone density of the rat was measured by a dual-energy X-ray bone densitometer 1d before the end of the test.

Determination of femoral fracture force: after the test is finished, taking out the left femur and placing the left femur in a refrigerator, placing the position of the femoral head with the concave surface facing downwards and the femoral head end facing forwards on two supporting points with the span of 20mm by using a medicine packaging performance tester, and applying a pressing head (with the thickness of 1.2 mm) to a specimen by 20mm & min downwards right above the middle position of the femur length ^-1 Until the femur breaks. The maximum force value is recorded and calculated, and the maximum force which can be born before fracture of the femur is the maximum fracture force.

Determination of femur calcium content: the bone calcium content was determined after digestion of the constant weight left femur by atomic absorption.

The results are shown in Table 2.

Group of	Lumbar bone Density (g/cm) ² ）	Maximum breaking force (N)	Bone calcium content (mg/g)
				Normal control group	0.242±0.021	141.2±13.02	307.2±25.72
Low-calcium control group	0.115±0.011 ^*	65.7±5.28 ^*	238.1±18.21 ^*
				Calcium carbonate group	0.154±0.021 ^#	120.2±10.21 ^#	274.5±22.12 ^#
Example 1 group	0.224±0.024 ^#	128.9±11.24 ^#	291.4±20.14 ^#
				Example 2 group	0.226±0.029 ^#	129.5±10.58 ^#	292.1±21.42 ^#
Example 3 group	0.234±0.027 ^#	130.2±13.21 ^#	294.5±22.82 ^#
				Comparative example 1 group	0.188±0.022	126.7±12.44	288.4±20.11
Comparative example 2 group	0.184±0.026	127.0±12.08	289.0±21.78
				Comparative example 3 group	0.176±0.029	125.5±11.27	287.2±21.48
Comparative example 4 group	0.168±0.033	124.9±11.50	286.1±22.31
				Comparative example 5 group	0.173±0.031	125.2±12.42	287.4±19.22
Comparative example 6 group	0.171±0.029	125.0±12.11	287.0±20.33
				Comparative example 7 group	0.166±0.030	124.6±13.04	286.7±21.41
Comparative example 8 group	0.162±0.028	122.5±13.10	284.7±20.38
				Comparative example 9 group	0.160±0.027	121.7±12.57	278.5±19.27
Comparative example 10 group	0.169±0.029	124.8±12.11	281.0±20.47
				Comparative example 11 group	0.171±0.025	125.4±12.88	280.2±21.31
Comparative example 12 group	0.175±0.024	125.7±12.47	288.0±20.38
				Comparative example 13 group	0.178±0.019	126.0±11.38	288.5±21.48
Comparative example 14 group	0.172±0.027	125.1±11.30	284.2±19.58
				Comparative example 15 group	0.174±0.030	125.5±11.58	283.7±18.39
Comparative example 16 group	0.159±0.028	121.2±12.17	276.4±17.09
				Comparative example 17 group	0.167±0.025	124.2±12.35	279.4±20.12
Comparative example 18 groups	0.164±0.021	122.5±10.21	278.2±18.92
				Comparative example 19 group	0.172±0.022	125.2±11.25	287.3±19.24
Comparative example 20 group	0.170±0.025	124.7±11.49	282.5±20.15
				Comparative example 21 group	0.162±0.029	125.0±10.28	285.8±19.28
Comparative example 22 group	0.165±0.031	124.9±11.32	279.7±19.42

From the above table, the compositions of calcium, VD and protein peptide prepared in examples 1-3 of the present invention can significantly improve bone density, increase bone calcium content and significantly improve maximum breaking force.

In comparative examples 1 and 2, as compared with example 3, no radix Puerariae or fructus Psoraleae was added in step S2. In comparative example 21, in contrast to example 3, no aqueous extract of the traditional Chinese medicine was added in step S10. In comparative examples 3 and 4, no filter residue or collagen was added in step S3, as compared with example 3. The Chinese medicinal composition has the advantages of reduced body quality, shortened body length, reduced bone density and reduced maximum breaking force, and the kudzuvine root added in the Chinese medicinal composition contains abundant kudzuvine root isoflavone due to the estrogen action of isoflavone compounds after being extracted by water. Puerarin can also exert greater physiological effects with smaller amounts of estrogen by increasing estrogen receptor levels on osteoblasts. The fructus psoraleae contains abundant isopsoralen, can promote the proliferation of osteoblasts, inhibit the apoptosis of osteoblasts, promote the proliferation and differentiation of bone marrow mesenchymal stem cells cultured in vitro, reduce calcium salt deposition, and has good anti-osteoporosis effect. Radix Puerariae is sweet, pungent and flat, and has the effects of relieving exterior syndrome, fever, pain in the neck and back, and diabetes due to yin deficiency; epimedium herb Wen Weixin is sweet and can tonify kidney yang and strengthen tendons and bones; fructus Psoraleae has effects of replenishing essence, nourishing marrow, replenishing kidney and invigorating spleen; fructus Ligustri Lucidi has effects of invigorating qi, promoting blood circulation, nourishing liver and kidney, and strengthening waist and knee; the medicines are used together to tonify spleen and kidney, strengthen tendons and bones, activate blood and remove meridian obstruction, and can treat osteoporosis.

Comparative examples 5 and 6 in comparison with example 3, lactobacillus plantarum or lactobacillus salivarius was not inoculated in step S4. Comparative example 7 in comparison with example 3, step S4 was not performed and bone collagen was directly added to step S5. The invention is fermented by probiotics including lactobacillus plantarum and lactobacillus salivarius, wherein the lactobacillus plantarum can reduce the expression of inflammatory factors such as TNF-alpha and IL-1b, increase the expression of substances with the function of inhibiting osteolysis and reduce the loss of bone mass; lactobacillus salivarius can achieve increased calcium ion absorption by promoting calcium ion absorption by intestinal epithelial cells. Under the synergistic effect of the two, the composition can also improve intestinal permeability, reduce intestinal inflammation and improve bone density.

Comparative example 8 compared to example 3, step S5 was not performed. The bone density is reduced, the maximum breaking force is reduced, protein phosphorylation is an effective means for improving protein functions, and the introduction of phosphate groups can promote the absorption of calcium by organisms, because the side chains of protein amino acids are added with a phosphate group with strong negative electricity, and esterification reaction is easy to occur, so that the configuration, activity and interaction force with other molecules of the protein are changed. Phosphorylation can also enhance the solubility of calcium phosphate, promoting calcium absorption by organisms. The phosphoserine residue of the phosphoprotein peptide can be combined with calcium ions to form a soluble compound to prevent calcium precipitation in the small intestine, and the prepared phosphoprotein peptide can increase the chelation site of polypeptide and metal ions, improve the binding activity of metal ions and improve the functional characteristics of the phosphoprotein peptide.

Comparative example 9 compared to example 3, step S6 was not performed. The invention prepares the phosphorylated protein peptide chelated calcium by adding calcium ion solution based on the prepared phosphorylated fermentation product, so that the product formed by combining the phosphorylated protein peptide and calcium ions in the phosphorylated protein peptide chelate with coordinate covalent bonds has the advantages of quick absorption, strong nutrition, high biological potency, antioxidation, antibiosis, blood fat reduction, immunoregulation and the like. The binding sites for calcium ions are mainly carboxyl groups of aspartic acid and glutamic acid. The phosphorylated protein peptide also forms a specific spatial structure after being combined with calcium ions, thereby promoting calcium absorption. When the calcium ions and the small peptide are chelated, the calcium supplementing effect is better, and the calcium ions exist in a chelated state in the small intestine to accelerate the absorption of the small intestine to the peptide, so that the calcium supplementing effect of the phosphorylated protein peptide chelated calcium is better.

Comparative example 16 in contrast to example 3, no phosphorylated calcium peptide fermentation complex was added in step S10. The quality of the body is obviously reduced, the length of the body is obviously shortened, the bone density is obviously reduced, the maximum breaking force is reduced, and the bone calcium content is reduced.

In comparative examples 10 and 11, vitamin D1 or vitamin D3 was not added in step S7, as compared with example 3. Comparative example 17 in contrast to example 3, no vitamin D composition was added in step S10. The invention aims to solve the problems that the body quality is reduced, the length is shortened, the bone density is reduced, the maximum breaking force is reduced, the bone calcium content is reduced, the body of an elderly patient is in a calcium deficiency sign, and the calcium content in ingested foods is low, and the calcium absorption capacity of the body is reduced, so that the satisfactory effect is difficult to obtain by simple calcium supplementing treatment, the elderly osteoporosis is mainly a aged systemic hypofunction patient, the metabolism of each organ in the body is slow or abnormal along with the age, the synthesis and secretion of kidney hydroxylase are reduced, the enzyme activity is reduced in a low metabolism state, the generation of active vitamin D1 and vitamin D3 is not facilitated, the calcium absorption capacity of intestinal wall cells is reduced, and the calcium absorption capacity of the human body is insufficient.

Comparative example 18 in contrast to example 3, no calcium citrate was added in step S10. The bone mineral composition has the advantages of reduced body mass, shortened body length, reduced bone density, reduced maximum breaking force, obviously reduced bone calcium content, and calcium agent and vitamin D are typical bone mineralization promoting medicines, the calcium agent and vitamin D are important components for forming bone, and the calcium agent and vitamin D can directly promote calcium and phosphorus absorption, and free calcium and phosphorus components from the bone into peripheral blood, so that calcium and phosphorus in blood return to normal values, mineralization is converted, new bone is formed continuously, and osteoporosis can be greatly relieved. Therefore, the vitamin D and a large amount of organic calcium (calcium citrate and calcium phosphate peptide fermentation complex) are supplemented, and the vitamin D and the calcium phosphate peptide fermentation complex have good interaction effect.

In comparative examples 12 and 13, sodium olpadronate or calcitonin was not added in step S8, as compared with example 3. In comparative example 19, in contrast to example 3, no osteoclast inhibitor was added in step S10. The osteoclast inhibitor comprises bisphosphonate and calcitonin, which can effectively block the activation of osteoclasts and induce the osteoclasts to undergo apoptosis, and the calcitonin can directly inhibit the physiological action of the osteoclasts, both of which can slow down the decomposition and loss of bone and fully stimulate the generation of new bone, thereby effectively relieving the occurrence of senile osteoporosis and having a synergistic action.

In comparative examples 14 and 15, no sea cucumber saponin or ginsenoside Rg3 was added in step S9, as compared with example 3. Comparative example 20 in contrast to example 3, no active composition was added in step S10. The active composition prepared by the invention comprises sea cucumber saponin and ginsenoside Rg3 which have similar structures and have the effects of promoting osteoblast proliferation and differentiation, reducing bone resorption and improving osteoporosis. Sea cucumber saponins can also reduce urinary calcium and urinary phosphorus concentration, reduce bone mineral loss, enhance bone density and bone mineralization deposition, improve osteoporosis, and have synergistic effect by adding the sea cucumber saponins.

Comparative example 22 in comparison with example 3, step S10 was not microencapsulated. The body quality is reduced, the body length is shortened, the bone density is reduced, the maximum breaking force is reduced, and the bone calcium content is reduced. According to the invention, through the mixed embedding of sodium alginate, sodium carboxymethyl cellulose and carboxymethyl pachyman, after metal ion crosslinking, the formed microcapsule shell has good pH responsiveness, and can keep a relatively complete form under an acidic condition with low pH, so that active ingredients such as probiotics, phosphorylated calcium peptide fermentation complex and the like are prevented from being digested and deactivated by gastric acid, the active ingredients can be conveyed to intestinal tracts in a targeted manner, after the microcapsule form is broken, the shell material can be used as a prebiotic substance, the growth and the colonization of probiotics are promoted, the action of the probiotics is promoted, and the absorption of other active ingredients by small intestines is promoted.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims

1. A preparation method of a calcium, VD and protein peptide composition for improving bone density is characterized by extracting radix puerariae, fructus psoraleae, epimedium and glossy privet fruit with boiling water, filtering, drying filtrate to obtain a traditional Chinese medicine water extract, mixing filter residues with collagen, a carbon source and a nitrogen source to obtain a traditional Chinese medicine-protein peptide culture medium, inoculating activated lactobacillus plantarum and lactobacillus salivarius, fermenting, carrying out phosphorylation reaction on the product, adding a calcium ion solution to obtain a phosphorylated calcium peptide fermentation compound, uniformly mixing the phosphorylated calcium peptide fermentation compound with the traditional Chinese medicine water extract, calcium citrate, vitamin D1, vitamin D3, bisphosphonate, calcitonin, sea cucumber saponin and ginsenoside Rg3, and preparing a microcapsule to obtain the calcium, VD and protein peptide composition for improving bone density.

2. The method of manufacturing according to claim 1, comprising the steps of:

3. The method according to claim 2, wherein the conditions of the activation culture in step S1 are micro-anoxic conditions of 5-7% CO, at 36-38deg.C for 18-24h and at a rotational speed of 50-80r/min ₂ 、7-10%O ₂ The balance is N ₂ Wherein% is volume percent; in the step S2, the mass ratio of the kudzuvine root, the malaytea scurfpea fruit, the epimedium herb and the glossy privet fruit is 5-10:3-5:5-7:1-3, the solid-liquid ratio of the Chinese medicinal powder and the water is 1:5-10g/mL, the times of boiling extraction are 2-3 times, and the time is 3-5h.

4. The method according to claim 2, wherein the mass ratio of the filter residue, the bone collagen, the carbon source, the nitrogen source and the sterile water in the step S3 is 10-15:7-10:5-12:4-7:150-200; the inoculum sizes of lactobacillus plantarum and lactobacillus salivarius strain seed solutions in the step S4 are 3-5% and 2-4% respectively, the conditions of fermentation culture are under micro-anoxic conditions, the temperature is 36-38 ℃, the time is 48-72h, the rotating speed is 50-70r/min, and the micro-anoxic conditions are 5-7% CO ₂ 、7-10%O ₂ The balance is N ₂ Wherein% is by volume.

5. The preparation method according to claim 2, wherein the mass ratio of the fermentation product to sodium trimetaphosphate in the step S5 is 100:7-12, the pH value is adjusted to 8-9, the temperature of the heating reaction is 35-45 ℃, and the time is 50-70min; the mass ratio of the phosphorylated fermentation product to the calcium ion solution in the step S6 is 10:5-7, wherein the calcium ion solution is 40-50wt% of calcium chloride solution, and the stirring reaction time is 30-50min.

6. The preparation method according to claim 2, wherein the mass ratio of vitamin D1 to vitamin D3 in step S7 is 1-3:5; the mass ratio of the bisphosphonate to the calcitonin in the step S8 is 3-5:2; in the step S9, the mass ratio of the sea cucumber saponin to the ginsenoside Rg3 is 4-7:5.

7. The preparation method according to claim 2, wherein in the step S10, the mass ratio of the water extract of the traditional Chinese medicine, the calcium phosphate peptide fermentation complex, the calcium citrate, the vitamin D composition, the osteoclast inhibitor, the active composition, the sodium alginate, the sodium carboxymethyl cellulose and the carboxymethyl pachyman is 4-6:10:50-70:0.0001-0.001:0.01-0.02:0.2-0.4:10-12:5-7:4-6, the mass ratio of the water phase to the edible oil is 3-5:5-7, the pore diameter of the rapid membrane is 1-3mm, the metal ion solution is a solution containing 3-5wt% of calcium chloride, ferric chloride or aluminum chloride, and the room temperature curing time is 20-30min.

8. The preparation method according to claim 2, characterized by comprising the following steps:

9. A calcium, VD, protein peptide composition for improving bone density prepared by the preparation method of any one of claims 1 to 8.

10. Use of a calcium, VD, protein peptide composition for improving bone mineral density according to claim 9 for the preparation of a product for the treatment and prevention of osteoporosis.