Nutrition enhancer beneficial to bone health of middle-aged and elderly people, preparation method of nutrition enhancer and application of nutrition enhancer in preparation of medial care food

Abstract

The invention provides a nutrition enhancer beneficial to bone health of middle-aged and elderly people, a preparation method of the nutrition enhancer and application of the nutrition enhancer in preparation of medial care food, and belongs to the field of nutritious food. The nutrient supplement comprises a plant fermentation product, osteopontin peptide, collagen peptide, N-acetylglucosamine and calcium, vitamins and mineral substances can be added, and the plant fermentation product is obtained by performing anaerobic fermentation on rhizoma drynariae and chestnuts through a complex microbial inoculant composed of lactobacillus reuteri and bifidobacterium longum subsp. Longum; the collagen peptide is prepared by performing enzymolysis on bovine bones through alkaline protease and flavourzyme; the hydrogel is prepared by adopting a sodium alginate-calcium chloride-carboxymethyl chitosan micro-capsule embedding process. An in-vitro experiment shows that the nutrition enhancer can remarkably promote osteoblast proliferation, differentiation and mineralization; animal experiments show that the bone mineral density of osteoporosis model mice can be effectively improved. The nutrition enhancer disclosed by the invention is suitable for preparing care food and bone health products, and has a remarkable effect of improving bone health of middle-aged and elderly people.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nutritional food, and belongs to the technical field of A23L33 / 00 in the IPC classification. Specifically, the present application relates to a nutritional fortifier beneficial to the bone health of the middle-aged and the elderly, a preparation method thereof, and an application of the nutritional fortifier in the preparation of care food. BACKGROUND

[0002] With the acceleration of the global population aging process, the health problems of the middle-aged and the elderly have attracted increasing attention. Bone health is one of the key factors affecting the quality of life of the middle-aged and the elderly. With the increase of age, the bone mass in the skeleton gradually decreases, the bone density decreases, the bone cortex thins, and the microstructure of the skeleton is damaged, thereby reducing the strength and toughness of the skeleton, increasing the bone fragility, and significantly increasing the risk of bone fracture. At the same time, osteoporosis can also cause bone pain and muscle weakness, and seriously affect the daily activity ability and self-care ability of the middle-aged and the elderly.

[0003] At present, the methods for improving the bone health of the middle-aged and the elderly mainly include dietary adjustment, exercise, and the use of nutritional supplements. However, these methods have many limitations in practical application. In terms of diet, the gastrointestinal absorption capacity of the middle-aged and the elderly weakens, and the absorption efficiency of nutrients such as calcium and iron decreases. It is difficult to meet the large amount of nutrients required for bone health by simply relying on daily dietary intake. For example, it is commonly believed in the traditional concept that calcium can be supplemented by drinking bone soup, but in fact the calcium content in bone soup is very low and difficult to be effectively absorbed by the human body. Although exercise is beneficial to bone health, it is difficult to implement for the middle-aged and the elderly with decreased physical function and poor mobility, and the effect of exercise varies greatly among individuals.

[0004] The invention patent with the publication number CN104582506B and the classification number A23L33 / 00 discloses a nutritional composition for promoting musculoskeletal health in patients with inflammatory bowel disease. The nutritional composition comprises casein, vitamin K with a ratio of vitamin K1:K2 of 3:1 to 1:3, and provides 3.5-20 μg / 100kcal of the nutritional composition of vitamin K, vitamin D and α-linolenic acid. A pharmaceutical preparation, a nutritional preparation, a tube feeding preparation, a dietary supplement, a functional food, a beverage product or a combination thereof comprising the nutritional composition is also disclosed. A method for improving musculoskeletal health is also disclosed.

[0005] The application patent with the publication number CN109700009A and the classification number A23L33 / 00 discloses a natural nutrient for repairing the bone function of the osteoporosis population and a preparation method thereof, the preparation method comprising the following steps: step 1, the washed moringa seeds and the cleaned pueraria are respectively sent into a crushing device to be crushed, and the fermentation raw materials are obtained by mixing the crushed moringa seeds and the crushed pueraria according to a proportion; step 2, the strain stock solution obtained by mixing the bacillus subtilis, the saccharomyces cerevisiae, the lactobacillus plantarum and the lactobacillus fermentum according to a proportion is inoculated into the fermentation raw materials, and the fermentation is carried out after adding sugar and inorganic salt; step 3, the product after fermentation is directly dried, and the fermentation finished product A is obtained by crushing and sieving; or the fermentation finished product B is obtained by sterilizing the liquid obtained by centrifugal separation with an appropriate amount of water; or the fermentation finished product C is obtained by further concentrating and drying.

[0006] The application patent with the publication number CN106235311A and the classification number A23L33 / 00 discloses a composition with the function of promoting the health of the skeletal system and the application thereof. The composition comprises skimmed milk powder, fructo-oligosaccharide, yak bone, orange fruit powder, D-glucosamine hydrochloride, bone collagen protein, chondroitin sulfate, epimedium extract, rhizoma drynaria extract, mushroom powder, magnesium carbonate, casein phosphopeptide, vitamin K2, and has a significant preventive and therapeutic effect on the osteoporosis patients and the people with skeletal system disorders such as joint stiffness, arthritis, hyperostosis, joint pain and muscle spasm, can increase the bone density and promote the health of the skeletal system.

[0007] In the aspect of nutritional supplements, common calcium and vitamin D supplements have no obvious effect on the bone health if the body has enough calcium and vitamin D. Moreover, the utilization rate of ordinary calcium agents is usually low. Meanwhile, the diet of some old people who need care usually has special requirements, that is, the diet should be easy to digest and absorb and rich in comprehensive nutrition to maintain the body function and promote the bone health. However, there is a lack of nutritional supplements and the corresponding care food specially for the bone health of the care population in the market. Therefore, it is of great practical significance and urgent market demand to develop a nutritional supplement which can effectively improve the bone health of the old people, is easy to absorb and is suitable for the care food. SUMMARY

[0008] The present application aims to provide a nutritional supplement which is beneficial to the bone health of the old people, a preparation method thereof and the application in the preparation of care food. The nutritional supplement is easy to digest and absorb, can promote the proliferation, differentiation and mineralization of the osteoblasts and improve the bone density.

[0009] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions.

[0010] The present application provides a kind of nutrition fortifier for the health of old bone, including the following weight parts of components: plant fermentation 20-30 parts, bone bridge protein peptide 2-5 parts, collagen peptide 15-25 parts, N-acetyl glucosamine 5-15 parts and calcium 20-40 parts;The plant fermentation is obtained by composite bacterial agent anaerobic fermentation of rhizoma drynariae and chestnut.

[0011] Preferably, the composite bacterial agent is composed of Lactobacillus reuteri and Bifidobacterium longum subsp.

[0012] More preferably, the collagen peptide is obtained by enzymatic hydrolysis of bovine bone.

[0013] Preferably, the calcium is one or more of calcium carbonate, calcium phosphate, calcium citrate, calcium lactate.

[0014] Preferably, it further includes vitamins and minerals.

[0015] More preferably, the vitamin is one or more of vitamin D3, vitamin K2, vitamin C, vitamin B12.

[0016] More preferably, the mineral is magnesium glycinate, zinc citrate.

[0017] The present application also provides a preparation method of the above-mentioned nutrition fortifier, comprising: mixing the plant fermentation, bone bridge protein peptide, collagen peptide and N-acetyl glucosamine with sodium alginate solution, dropping into calcium chloride solution to solidify into microspheres, then reacting with chitosan solution to obtain microcapsules, mixing with calcium to obtain the nutrition fortifier.

[0018] The present application also provides the application of the above-mentioned nutrition fortifier in the preparation of intermediate care food.

[0019] The present application also provides the application of the above-mentioned nutrition fortifier in the preparation of bone health products.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] The present application provides a kind of nutrient fortifier for the health of old bone, including plant fermentation, bone bridge protein peptide, collagen peptide, N-acetyl glucosamine and calcium, also can add vitamin and mineral, plant fermentation is obtained by the anaerobic fermentation of complex microbial inoculum of Rhizoma Drynariae, chestnut by Lactobacillus reuteri and Bifidobacterium longum longum subsp.; Collagen peptide is made by bovine bone by alkaline protease and flavour protease enzymolysis; It is made by sodium alginate-calcium chloride-carboxymethyl chitosan microencapsulation process.In vitro experiment shows that the present application nutrient fortifier can significantly promote osteoblast proliferation, differentiation and mineralization.Animal experiments show that it can effectively improve the bone density of osteoporosis model mice.The present application nutrient fortifier is suitable for the preparation of care food and bone health product, and has remarkable effect on improving the health of old bone, and is suitable as dietary supplement for the health of old bone. DETAILED DESCRIPTION

[0022] The present application provides a kind of nutrient fortifier for the health of old bone, including the following weight parts of components: plant fermentation 20-30 parts, bone bridge protein peptide 2-5 parts, collagen peptide 15-25 parts, N-acetyl glucosamine 5-15 parts and calcium 20-40 parts;The plant fermentation is obtained by the anaerobic fermentation of complex microbial inoculum of Rhizoma Drynariae and chestnut.

[0023] The complex microbial inoculum is composed of Lactobacillus reuteri and Bifidobacterium longum longum subsp.;Lactobacillus reuteri is Lactobacillus reuteri CICC ® 6121, Bifidobacterium longum longum subsp. is Bifidobacterium longum longum subsp. CICC ® 24934, from China Industrial Microbial Strain Preservation and Management Center.The preparation method of the plant fermentation preferably includes: respectively crushing Rhizoma Drynariae leaves and chestnuts, passing through 150-250 mesh screen, obtaining material powder, adding deionized water according to solid-liquid ratio 1:20-30 g / mL, inoculating complex microbial inoculum, fermenting at 35-40 DEG C, pH value 6.5-7 for 30-40 h, filtering, sterilizing, concentrating the filtrate under reduced pressure, drying to obtain Rhizoma Drynariae extract;Among them, the complex microbial inoculum is preferably composed of Lactobacillus reuteri and Bifidobacterium longum longum subsp. according to mass ratio 1-3:2-5, more preferably 2:3, the number of Lactobacillus reuteri is preferably 3-8 hundred million cfu / g, more preferably 5 hundred million cfu / g, the number of Bifidobacterium longum longum subsp. is preferably 2-5 hundred million cfu / g, more preferably 3 hundred million cfu / g, and the inoculation amount of complex microbial inoculum is preferably 3%-8% of the weight of material powder, more preferably 5%.

[0024] The bone glue is warm in flavor, bitter in taste, and belongs to liver and kidney channels, and can significantly improve the absorption capacity of bone to minerals such as calcium and phosphorus, effectively improve the blood calcium and blood phosphorus levels, and provide sufficient raw materials for bone calcification and bone formation through the overall regulation of kidney and essence, strong muscles and bones. The chestnut is sweet in taste, warm in flavor, and belongs to spleen, stomach and kidney channels, and can provide essential minerals for bones, reduce the damage of oxidative stress to osteoblasts, and promote the synthesis of collagen. The present application can decompose macromolecular substances in bone glue and chestnut through anaerobic fermentation process, release more small molecular active ingredients, and transform the components of bone glue and chestnut to produce metabolites with biological activity, thereby further enhancing the effect of promoting bone health.

[0025] The OPN peptide regulates mineralization filling, and the combination of the two can promote the ALP activity of osteoblasts.

[0026] The collagen peptide is obtained by enzymatic hydrolysis of bovine bone. The preparation method of the collagen peptide preferably comprises the following steps: crushing bovine bone, mixing with water, treating at 110-120 DEG C, 0.1-0.3 MPa for 20-30 min, cooling to 30-40 DEG C, removing the oil phase part, adding alkaline protease, enzymatic hydrolysis at 60-65 DEG C, pH 8.0-8.5 for 1.5-2.5 h, after enzyme inactivation, adding flavor protease, enzymatic hydrolysis at 50-55 DEG C, pH 6-7 for 1-2 h, after enzyme inactivation, filtering with a nanofiltration ceramic membrane with a molecular weight cut-off of 800-1000 Da, concentrating and drying the permeate to obtain the collagen peptide. The alkaline protease and flavor protease are purchased from NOVONESIS / NO and Xin Yuan brand flagship store, and the brand is Vinoxin, and the serial number is Alcalase 2.4L FG, Flavourzyme 500MG respectively. The collagen peptide prepared by the present application can directly provide bone matrix raw materials, stimulate osteoblast activity, and promote calcium deposition and mineralization.

[0027] The calcium is preferably one or more of calcium carbonate, calcium phosphate, calcium citrate, and calcium lactate.

[0028] The present application also includes vitamins and minerals, the vitamins are preferably one or more of vitamin D3, vitamin K2, vitamin C, and vitamin B12, and the minerals are preferably magnesium glycinate and zinc citrate.

[0029] The present application also provides a preparation method of the above-mentioned nutritional supplement, comprising: mixing plant fermentation, osteonectin peptide, collagen peptide and N-acetyl glucosamine with sodium alginate solution, dropping into calcium chloride solution to solidify into microspheres, reacting with chitosan solution to obtain microcapsules, and mixing with calcium to obtain the nutritional supplement.

[0030] The present invention also provides the application of the above-mentioned nutritional fortifier in the preparation of care foods.

[0031] This invention also provides the application of the above-mentioned nutritional fortifiers in the preparation of bone health products; the assisted living food is preferably a liquid, semi-liquid, or easily swallowed solid food. The components of this invention have small molecular weights, which is beneficial for digestion and absorption by middle-aged and elderly people.

[0032] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0033] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0034] Unless otherwise specified, the following embodiments are all conventional methods.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0036] Limosilactobacillus reuteri CICC ® 6121, *Bifidobacterium longum* subsp. *Longum* CICC ® 24934, purchased from the China Industrial Microbial Culture Collection Center. *Lactobacillus delbrueckii* CICC ® 6289, Bifidobacterium animalis CICC ® Item 24931 was purchased from the China Industrial Microbial Culture Collection Center. Alkaline protease and flavor protease were both purchased from the NOVONESIS flagship store, both branded as Vinoxin, with catalog numbers Alcalase 2.4L FG and Flavorzyme 500MG respectively. Papain was purchased from Shanxi Shangda Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g, catalog number SD-MGDBM. Aminopeptidase was purchased from Ningxia Xiasheng Industrial Group Co., Ltd., with an enzyme activity of 5,000 U / g, catalog number FDG-2251.

[0037] Example 1

[0038] Nutritional fortification agents that are beneficial to bone health in middle-aged and elderly people

[0039] S1. Plant fermentation products

[0040] The leaves of Drynaria fortunei and chestnuts were crushed and passed through a 200-mesh sieve to obtain material powder. Deionized water was added at a material-to-liquid ratio of 1:25 g / mL, and compound microbial agent was inoculated. Fermentation was carried out at 37℃ and pH 6.8 for 36 hours. The mixture was filtered, sterilized, and the filtrate was concentrated under reduced pressure and dried to a water content of 5 wt% to obtain plant fermentation product.

[0041] The weight ratio of Drynaria fortunei leaves to chestnuts is 1:2; the compound microbial agent is composed of Lactobacillus reuteri and Bifidobacterium longum subsp. longum. The Lactobacillus reuteri in the compound microbial agent has a bacterial count of 500 million CFU / g and the Bifidobacterium longum subsp. longum has a bacterial count of 300 million CFU / g. The inoculation amount of the compound microbial agent is 5% of the weight of the material powder.

[0042] S2. Collagen Peptides

[0043] Bovine bones were crushed and passed through a 100-mesh sieve to obtain bovine bone powder. The powder was mixed with water at a ratio of 1:15 g / mL and treated at 115℃ and 0.2 MPa for 25 min. After cooling to 35℃, the oil phase was removed. Alcalase 2.4 L FG was added and enzymatically hydrolyzed at 62℃ and pH 8.2 for 2 h. After enzyme inactivation, flavor enzyme 500 mg was added and enzymatically hydrolyzed at 52℃ and pH 6.5 for 1.5 h. After enzyme inactivation, the mixture was filtered using a nanofiltration ceramic membrane with a molecular weight cutoff of 800 Da. The permeate was concentrated and dried to obtain collagen peptides.

[0044] The amount of alkaline protease added was 1.2% of the weight of the beef bone meal, and the amount of flavor protease added was 0.8% of the weight of the beef bone meal.

[0045] S3. Preparation of nutritional fortifiers beneficial to bone health in middle-aged and elderly people

[0046] Accurately weigh 25g of plant fermentation product prepared by S1, 3g of osteopontin peptide, 20g of collagen peptide prepared by S2, 10g of N-acetylglucosamine, 15g of calcium phosphate, 15g of calcium citrate, 25μg of vitamin D3, 100μg of vitamin K2 (MK-7), 100mg of vitamin C, 2.4μg of vitamin B12, 150mg of magnesium glycine, and 10mg of zinc citrate.

[0047] Sodium alginate was dissolved in deionized water to obtain a 20 mg / mL sodium alginate solution; calcium chloride was dissolved in deionized water to obtain a 50 mg / mL calcium chloride solution; carboxymethyl chitosan was dissolved in deionized water to obtain a 10 mg / mL carboxymethyl chitosan solution.

[0048] Plant fermentation products, osteopontin peptides, collagen peptides prepared by S2, and N-acetylglucosamine were mixed and 20 mL of sodium alginate solution was added. The mixture was homogenized at 10,000 rpm for 2 min to obtain a mixed slurry. This slurry was then added dropwise to a calcium chloride solution and magnetically stirred at 200 rpm for 30 min to solidify. The mixture was filtered, washed three times with deionized water, and the microspheres were collected. Carboxymethyl chitosan solution was added at a ratio of 1:10 g / mL, and the mixture was shaken at 120 rpm and 25℃ for 40 min. The mixture was then centrifuged at 3000 rpm for 5 min to collect the microcapsules. The microcapsules were freeze-dried to a water content of 5 wt% to obtain dried microcapsules. The dried microcapsules were then mixed with calcium phosphate, calcium citrate, vitamin D3, vitamin K2 (MK-7), vitamin C, vitamin B12, magnesium glycine, and zinc citrate at 20 rpm for 30 min and sterilized to obtain a nutrient fortifier.

[0049] Example 2

[0050] S1. Plant fermentation products

[0051] The leaves of Drynaria fortunei and chestnuts were crushed and passed through a 150-mesh sieve to obtain material powder. Deionized water was added at a material-to-liquid ratio of 1:20 g / mL, and compound microbial agent was inoculated. Fermentation was carried out at 34℃ and pH 6.5 for 40 hours. The mixture was filtered, sterilized, and the filtrate was concentrated under reduced pressure and dried to a water content of 3wt% to obtain plant fermentation product.

[0052] The weight ratio of Drynaria fortunei leaves to chestnuts is 1:1; the compound microbial agent is composed of Lactobacillus reuteri and Bifidobacterium longum subsp. longum. The Lactobacillus reuteri in the compound microbial agent has a bacterial count of 300 million CFU / g and the Bifidobacterium longum subsp. longum has a bacterial count of 200 million CFU / g. The inoculation amount of the compound microbial agent is 8% of the weight of the material powder.

[0053] S2. Collagen Peptides

[0054] Bovine bones were crushed and passed through an 80-mesh sieve to obtain bovine bone powder. The powder was mixed with water at a ratio of 1:12 g / mL and treated at 110℃ and 0.1 MPa for 30 min. After cooling to 30℃, the oil phase was removed. Alcalase 2.4 L FG was added and enzymatically hydrolyzed at 60℃ and pH 8.0 for 2.5 h. After enzyme inactivation, flavor enzyme 500 mg was added and enzymatically hydrolyzed at 50℃ and pH 6 for 2 h. After enzyme inactivation, the mixture was filtered using a nanofiltration ceramic membrane with a molecular weight cutoff of 800 Da. The permeate was concentrated and dried to obtain collagen peptides.

[0055] The amount of alkaline protease added is 1% of the weight of the beef bone meal, and the amount of flavor protease added is 0.5% of the weight of the beef bone meal.

[0056] S3. Preparation of nutritional fortifiers beneficial to bone health in middle-aged and elderly people

[0057] Accurately weigh 20g of plant fermentation product prepared by S1, 2g of osteopontin peptide, 15g of collagen peptide prepared by S2, 5g of N-acetylglucosamine, 10g of calcium phosphate, 10g of calcium citrate, 22μg of vitamin D3, 80μg of vitamin K2 (MK-7), 80mg of vitamin C, 2μg of vitamin B12, 100mg of magnesium glycine, and 8mg of zinc citrate.

[0058] Sodium alginate was dissolved in deionized water to obtain a sodium alginate solution of 18 mg / mL; calcium chloride was dissolved in deionized water to obtain a calcium chloride solution of 45 mg / mL; carboxymethyl chitosan was dissolved in deionized water to obtain a carboxymethyl chitosan solution of 8 mg / mL.

[0059] Plant fermentation products, osteopontin peptides, collagen peptides prepared by S2, and N-acetylglucosamine were mixed and 18 mL of sodium alginate solution was added. The mixture was homogenized at 8000 rpm for 5 min to obtain a mixed slurry. This slurry was then added dropwise to a calcium chloride solution and magnetically stirred at 180 rpm for 40 min to solidify. The mixture was filtered, washed twice with deionized water, and the microspheres were collected. Carboxymethyl chitosan solution was added at a ratio of 1:8 g / mL, and the mixture was shaken at 100 rpm and 22℃ for 50 min. The mixture was then centrifuged at 2500 rpm for 3 min to collect the microcapsules. The microcapsules were freeze-dried to a water content of 4 wt% to obtain dried microcapsules. The dried microcapsules were then mixed with calcium phosphate, calcium citrate, vitamin D3, vitamin K2 (MK-7), vitamin C, vitamin B12, magnesium glycine, and zinc citrate at 18 rpm for 40 min and sterilized to obtain a nutrient fortifier.

[0060] Example 3

[0061] S1. Plant fermentation products

[0062] The leaves of Drynaria fortunei and chestnuts were crushed and passed through a 250-mesh sieve to obtain material powder. Deionized water was added at a material-to-liquid ratio of 1:30 g / mL, and compound bacterial agent was inoculated. Fermentation was carried out at 40℃ and pH 7 for 30 hours. The mixture was filtered, sterilized, and the filtrate was concentrated under reduced pressure and dried to a water content of 3wt% to obtain plant fermentation product.

[0063] The weight ratio of Drynaria fortunei leaves to chestnuts is 1:3; the compound microbial agent is composed of Lactobacillus reuteri and Bifidobacterium longum subsp. longum. The Lactobacillus reuteri in the compound microbial agent has a bacterial count of 800 million CFU / g and the Bifidobacterium longum subsp. longum has a bacterial count of 500 million CFU / g. The inoculation amount of the compound microbial agent is 3% of the weight of the material powder.

[0064] S2. Collagen Peptides

[0065] Bovine bones were pulverized and passed through a 120-mesh sieve to obtain bovine bone powder. This powder was mixed with water at a ratio of 1:10 g / mL and treated at 120℃ and 0.3 MPa for 20 min. After cooling to 40℃, the oil phase was removed. Alcalase 2.4 L FG was added, and the mixture was enzymatically hydrolyzed at 65℃ and pH 8.5 for 1.5 h. After enzyme inactivation, flavorzyme 500 mg was added, and the mixture was enzymatically hydrolyzed at 55℃ and pH 7 for 1 h. After enzyme inactivation, the mixture was filtered through a nanofiltration ceramic membrane with a molecular weight cutoff of 1000 Da. The permeate was concentrated and dried to obtain collagen peptides.

[0066] The amount of alkaline protease added is 1.5% of the weight of the beef bone meal, and the amount of flavor protease added is 1% of the weight of the beef bone meal.

[0067] S3. Preparation of nutritional fortifiers beneficial to bone health in middle-aged and elderly people

[0068] Accurately weigh 30g of plant fermentation product prepared by S1, 5g of osteopontin peptide, 25g of collagen peptide prepared by S2, 15g of N-acetylglucosamine, 20g of calcium phosphate, 20g of calcium citrate, 28μg of vitamin D3, 120μg of vitamin K2 (MK-7), 120mg of vitamin C, 2.5μg of vitamin B12, 160mg of magnesium glycine, and 15mg of zinc citrate.

[0069] Sodium alginate was dissolved in deionized water to obtain a 22 mg / mL sodium alginate solution; calcium chloride was dissolved in deionized water to obtain a 52 mg / mL calcium chloride solution; carboxymethyl chitosan was dissolved in deionized water to obtain a 12 mg / mL carboxymethyl chitosan solution.

[0070] Plant fermentation products, osteopontin peptides, collagen peptides prepared by S2, and N-acetylglucosamine were mixed and added to 30 mL of sodium alginate solution. The mixture was homogenized at 11,000 rpm for 1.5 min to obtain a mixed slurry. This slurry was then added dropwise to calcium chloride solution and magnetically stirred at 250 rpm for 28 min to solidify. The mixture was filtered, washed four times with deionized water, and the microspheres were collected. Carboxymethyl chitosan solution was added at a ratio of 1:12 g / mL, and the mixture was shaken at 150 rpm and 28℃ for 36 min. The mixture was then centrifuged at 3500 rpm for 4 min to collect the microcapsules. The microcapsules were freeze-dried to a water content of 4 wt% to obtain dried microcapsules. The dried microcapsules were then mixed with calcium phosphate, calcium citrate, vitamin D3, vitamin K2 (MK-7), vitamin C, vitamin B12, magnesium glycine, and zinc citrate at 50 rpm for 28 min and sterilized to obtain a nutrient fortifier.

[0071] Comparative Example 1

[0072] The specific implementation method is the same as in Example 1, except that 25 parts of plant fermentation material are discarded, the weight of collagen peptides is adjusted to 45 parts, and step S1 is deleted.

[0073] Comparative Example 2

[0074] The specific implementation method is the same as in Example 1, except that 20 parts of collagen peptides are discarded, the weight of plant fermentation product is adjusted to 45 parts, and step S2 is deleted.

[0075] Comparative Example 3

[0076] The specific implementation method is the same as in Example 1, except that step S1 is as follows:

[0077] The leaves of Drynaria fortunei and chestnuts were crushed and passed through a 200-mesh sieve to obtain material powder. Deionized water was added at a material-to-liquid ratio of 1:25 g / mL, and compound microbial agent was inoculated. Fermentation was carried out at 37℃ and pH 6.8 for 36 hours. The mixture was filtered, sterilized, and the filtrate was concentrated under reduced pressure and dried to a water content of 5 wt% to obtain plant fermentation product.

[0078] The weight ratio of Drynaria fortunei leaves to chestnuts is 1:2; the compound microbial agent is composed of Lactobacillus delbrueckii and Bifidobacterium animalis in a mass ratio of 2:3, with Lactobacillus delbrueckii having a bacterial count of 500 million CFU / g and Bifidobacterium animalis having a bacterial count of 300 million CFU / g, and the inoculation amount of the compound microbial agent is 5% of the weight of Drynaria fortunei powder.

[0079] Comparative Example 4

[0080] The specific implementation method is the same as in Example 1, except that step S2 is as follows:

[0081] Bovine bones were pulverized and passed through a 100-mesh sieve to obtain bovine bone powder. This powder was mixed with water at a ratio of 1:15 g / mL and treated at 115℃ and 0.2 MPa for 25 min. After cooling to 35℃, the oil phase was removed. Papain was added, and the mixture was enzymatically hydrolyzed at 62℃ and pH 7 for 2 h. After enzyme inactivation, aminopeptidase was added, and the mixture was enzymatically hydrolyzed at 52℃ and pH 7.5 for 1.5 h. After enzyme inactivation, the mixture was filtered through a nanofiltration ceramic membrane with a molecular weight cutoff of 800 Da. The permeate was concentrated and dried to obtain collagen peptides. The amount of papain added was 1.2% of the weight of the bovine bone powder, and the amount of aminopeptidase added was 0.8% of the weight of the bovine bone powder.

[0082] Comparative Example 5

[0083] The specific implementation method is the same as in Example 1. The difference is that the sodium alginate-calcium chloride-carboxymethyl chitosan microencapsulation step is omitted in step S3. Instead, the plant fermentation product, osteopontin peptide, collagen peptide prepared in S2, N-acetylglucosamine, calcium phosphate, calcium citrate, vitamin D3, vitamin K2 (MK-7), vitamin C, vitamin B12, magnesium glycine and zinc citrate are directly mixed at 20 rpm for 30 min and sterilized to obtain the nutrient fortifier.

[0084] Experimental Example 1

[0085] In vitro experiments

[0086] Effects of osteopontin peptides and / or Drynaria fortunei extract on osteogenic activity

[0087] (1) Cell proliferation

[0088] The experiment was divided into a blank group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group, and Comparative Example 5 group.

[0089] Each group will use MC3T3-E1 cells at a rate of 5 × 10⁻⁶. 3 Cells were seeded at a density of [insert density here] in 96-well cell culture plates. After cell attachment, the culture medium was aspirated, and each well was incubated with complete culture medium containing 50 μg / mL of the test sample for 48 h. After removing the complete culture medium, 100 µL of 0.5 mg / mL MTT solution was added to each well for 4 h. After removing the MTT solution, 150 µL of DMSO solution was added. The control group was treated with complete culture medium without any added components. The absorbance was measured at 570 nm using a microplate reader, and the cell proliferation rate was calculated. Each test was performed in triplicate. The specific results are shown in Table 1.

[0090] The samples to be tested were the nutrient fortifiers of Examples 1-3 and Comparative Examples 1-5.

[0091] Cell proliferation rate (%) = (absorbance of sample to be tested - absorbance of blank group) / absorbance of blank group × 100%.

[0092] Table 1. Effects of different nutrient fortifiers on the proliferation of MC3T3-E1 cells.

[0093]

[0094] As shown in Table 1, the cell proliferation rate of Examples 1-3 was significantly higher than that of Comparative Examples 1-5, indicating that the nutrient fortifier of the present invention can significantly improve osteoblast proliferation. Examples 1 and Comparative Examples 1-2 show that the combination of plant fermentation products and collagen peptides has a significant promoting effect on osteoblast proliferation. Examples 1 and Comparative Examples 2-5 show that the selection of compound microbial agents, proteases, and microencapsulation technology are beneficial to promoting cell proliferation.

[0095] (2) Cell differentiation

[0096] The experiment was divided into a blank group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group, and Comparative Example 5 group.

[0097] Each group of MC3T3-E1 cells was fed with 1×10 5 Cells / wells were cultured in 24-well plates until adherent. Complete culture medium was removed, and the cells were cultured for 10 days in differentiation medium containing 50 μg / mL of the test sample (10% fetal bovine serum + 50 µg / mL L-ascorbic acid + 10 mM β-glycerophosphate), with fresh medium replaced every 2 days. After 10 days, ALP activity was measured according to the ALP assay kit instructions, and the ALP activity ratio of the test samples was calculated. Each test was performed in triplicate. Specific results are shown in Table 2.

[0098] The samples to be tested were the nutrient fortifiers of Examples 1-3 and Comparative Examples 1-5.

[0099] ALP activity ratio (%) = ALP activity of the test sample group ÷ ALP activity of the blank group × 100%.

[0100] Table 2. Effects of nutritional fortifiers on MC3T3-E1 cell differentiation

[0101]

[0102] Table 2 shows that the ALP activity ratios in Examples 1-3 were significantly higher than those in Comparative Examples 1-5. Data from Examples 1 and Comparative Examples 1-2 indicate that the synergistic effect of plant fermentation products and collagen peptides is crucial for promoting cell differentiation. Data from Examples 1 and Comparative Examples 3-5 show that selecting appropriate compound bacterial agents, proteases, and microencapsulation processes has a positive effect on enhancing the cell differentiation promotion effect. The Example groups showed significantly better effects in promoting MC3T3-E1 cell differentiation than the Comparative Examples, with Example 1 showing the best results.

[0103] (3) Cellular mineralization

[0104] The experiment was divided into a blank group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group, and Comparative Example 5 group.

[0105] Each group of MC3T3-E1 cells was fed with 1×10 5 Cells / wells were cultured in 24-well plates until adherent. Complete culture medium was removed, and the cells were cultured for 21 days in differentiation medium containing 50 μg / mL of the test sample (10% fetal bovine serum + 50 µg / mL L-ascorbic acid + 10 mM β-glycerophosphate), with fresh medium replaced every 2 days. After 21 days, the cells were washed, fixed with 4% paraformaldehyde, stained with alizarin red, washed twice with deionized water, dried at 37°C, and dissolved in 10% hexadecylpyridine chloride solution with shaking for 30 min. The absorbance was measured at 562 nm using a microplate reader, and the mineralization rate was calculated. Each test was performed in triplicate. Specific results are shown in Table 3.

[0106] The samples to be tested were the nutrient fortifiers of Examples 1-3 and Comparative Examples 1-5.

[0107] Cell mineralization rate (%) = absorbance of the sample to be tested ÷ absorbance of the blank group × 100%.

[0108] Table 3. Effects of nutritional fortifiers on mineralization of MC3T3-E1 cells.

[0109]

[0110] Table 3 shows that the cell mineralization rate of Examples 1-3 was significantly higher than that of Comparative Examples 1-5. Data from Examples 1 and Comparative Examples 1-2 indicate a synergistic effect between plant fermentation products and collagen peptides, effectively promoting bone matrix mineralization. Data from Examples 1 and Comparative Examples 3-5 show that the original compound bacterial agent, protease selection, and microencapsulation process help enhance the ability of nutrient fortifiers to promote cell mineralization. The Example groups showed better performance in promoting MC3T3-E1 cell mineralization, with Example 1 showing the best effect.

[0111] Experimental Example 2

[0112] animal experiments

[0113] One hundred and ten healthy, non-pregnant, 12-week-old female SPF-grade C57BL / 6 mice were selected and, after one week of acclimatization, were randomly divided into 11 groups: sham-operated group, model group, positive control group, and experimental group (Examples 1-3, Comparative Examples 1-5), with ten mice in each group. There were no significant differences in weight, health status, etc. among the groups, so intergroup experiments could be conducted.

[0114] Mice in each group were anesthetized by intraperitoneal injection of 3% sodium pentobarbital at a dose of 40 mg / kg. After fixation, the back hair was removed, and a 0.5-1 cm skin incision was made to expose the ovary. After ligation of the fallopian tubes, the ovary was removed. In the sham surgery group, only an equal weight of adipose tissue was removed. Postoperatively, antibiotics were administered at a dose of 50,000 U / kg for 3 consecutive days to prevent infection.

[0115] Three days later, the positive control group was administered alendronate sodium by gavage at a dose of 15 mg / kg, the experimental group was administered the nutritional fortifiers of Examples 1-3 and Comparative Examples 1-5 by gavage at a dose of 250 mg / kg, and the sham-operated group and the model group were administered 0.5% sodium carboxymethyl cellulose by gavage at a dose of 250 mg / kg. The gavage was continued for 12 weeks.

[0116] Bone mineral density (BMD) of the femur in mice administered via gavage at weeks 6 and 12 was measured using a dual-energy X-ray absorptiometry (DXA) system. Mice were anesthetized and their femurs were scanned in a prone position to measure BMD. Each measurement was repeated three times. The specific results are shown in Table 4.

[0117] Table 4. Effects of nutritional fortifiers on bone mineral density in each group

[0118]

[0119] As shown in Table 4, at weeks 6 and 12, the bone mineral density (BMD) of the model group was significantly lower than that of the sham-operated group, indicating the successful establishment of the osteoporosis model in mice. The BMD of the example group at week 12 was close to that of the positive control group and significantly higher than that of the model group, indicating that the nutritional fortifier of this invention can effectively improve osteoporosis. The BMD of Example 1-3 groups at week 12 was higher than that of Comparative Example 1-5 groups, and showed an increasing trend over time, indicating that the nutritional fortifier of this invention can effectively improve the BMD of osteoporosis model mice. The lower BMD of Comparative Example 1-2 groups further demonstrates the necessity of the synergistic effect of plant fermentation products and collagen peptides. The BMD of Comparative Example 3-5 groups was lower than that of the example group, indicating that appropriate compound bacterial agents, proteases, and microencapsulation processes have a positive effect on improving BMD. The example groups showed better results than the comparative example groups in improving BMD, with Example 1 showing a particularly outstanding effect.

[0120] In summary, the combined use of plant fermentation products and collagen peptides in the nutritional fortifier of this invention has a significant synergistic effect on promoting osteoblast proliferation, differentiation, and mineralization; neither can be omitted. The combination of *Lactobacillus reuteri* and *Bifidobacterium longum* subsp. *longum* is more effective than other bacterial species. The specific combination of alkaline protease and flavor protease is more efficient. The microencapsulation process using sodium alginate-calcium chloride-carboxymethyl chitosan significantly improves the stability and bioavailability of the active ingredients. In vitro experiments (cell proliferation, differentiation, mineralization) and animal experiments (bone density improvement) show consistent results, verifying the significant anti-osteoporosis effect of the nutritional fortifier. The nutritional fortifier of this invention has an effect close to that of the positive control drug alendronate sodium and may have better safety, making it suitable as a dietary supplement for the bone health of middle-aged and elderly people.

[0121] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nutritional fortifier beneficial to bone health in middle-aged and elderly people, characterized in that, It includes the following components by weight: 20-30 parts plant ferment, 2-5 parts osteopontin peptide, 15-25 parts collagen peptide, 5-15 parts N-acetylglucosamine and 20-40 parts calcium. The plant ferment is obtained by anaerobic fermentation of Drynaria fortunei and chestnuts using a compound microbial agent.

2. The nutrient fortifier according to claim 1, characterized in that, The compound bacterial agent consists of *Lactobacillus reuteri* and *Bifidobacterium longum* subsp. *longum*.

3. The nutrient fortifier according to claim 1 or 2, characterized in that, The collagen peptides were obtained by enzymatic hydrolysis of bovine bones.

4. The nutrient fortifier according to claim 1, characterized in that, The calcium is one or more of calcium carbonate, calcium phosphate, calcium citrate, and calcium lactate.

5. The nutrient fortifier according to claim 1, characterized in that, It also includes vitamins and minerals.

6. The nutrient fortifier according to claim 4, characterized in that, The vitamin is one or more of vitamin D3, vitamin K2, vitamin C, and vitamin B12.

7. The nutrient fortifier according to claim 6, characterized in that, The minerals are magnesium glycine and zinc citrate.

8. A method for preparing a nutrient fortifier according to any one of claims 1-7, characterized in that, include: Plant fermentation products, osteopontin peptides, collagen peptides, and N-acetylglucosamine were mixed with sodium alginate solution, and then calcium chloride solution was added dropwise to solidify and form microspheres. These microspheres were then reacted with chitosan solution to obtain microcapsules, which were then mixed with calcium to obtain a nutrient fortifier.

9. The use of the nutritional fortifier according to any one of claims 1-7 in the preparation of care foods.

10. The use of the nutritional fortifier according to any one of claims 1-7 in the preparation of bone health products.

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