Composition for nourishing tendons and strengthening bones and preparation method thereof

Abstract

The invention relates to the technical field of functional food, in particular to a tendon-nourishing and bone-strengthening composition and a preparation method thereof, and the tendon-nourishing and bone-strengthening composition comprises a fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer, clover leaf saponin, a selenium yeast-pachymaran compound, glucosamine, compound nuts, vitamin D, a beef-pork compound, a traditional Chinese medicine compound and cheese; the three functional components of the fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer, the clover leaf saponin and the selenium yeast-pachymaran compound cooperate to effectively block the accumulation and toxic effect of harmful heavy metals in the Chinese herbal medicines in the human body, and normal absorption of trace elements necessary for the human body is not interfered at the same time.

Description

Technical Field

[0001] This invention relates to the field of functional food technology, specifically to a composition for nourishing tendons and bones and its preparation method. Background Technology

[0002] Traditional Chinese medicinal herbs are highly susceptible to heavy metal contamination during growth, harvesting, processing, and storage. Sources of contamination primarily include industrial wastewater discharge, polluted soil, atmospheric deposition, and the improper use of pesticides and fertilizers. Common pollutants include lead, arsenic, mercury, cadmium, and chromium. The presence of these heavy metals not only reduces product quality but also poses a threat to human health. Heavy metals can accumulate in the body over a long period, causing irreversible damage to the liver, kidneys, nervous system, and hematopoietic system. Lead and cadmium, in particular, directly interfere with bone calcification, competing with calcium ions for bone matrix binding sites, thus inducing osteoporosis—contrary to the intended therapeutic effects of bone-strengthening and muscle-nourishing compositions. Furthermore, heavy metals may antagonize the active ingredients in the composition, reducing efficacy or triggering toxic side effects, and causing the product to fail to meet relevant health food standards, thus affecting its legal market entry and promotion.

[0003] Currently, existing bone-strengthening and muscle-nourishing compositions generally lack a systematic solution for heavy metal pollution. Some studies have attempted to use single chelating agents to adsorb heavy metals, but the chelation selectivity of single components is limited, making it difficult to effectively remove harmful heavy metals while retaining essential trace elements such as iron, zinc, and copper. There is a risk of over-chelation, and the removal efficiency still has significant room for improvement. Therefore, how to achieve efficient and selective removal of harmful heavy metals while ensuring the integrity of bone-strengthening and muscle-nourishing effects is a pressing technical challenge in this field. Summary of the Invention

[0004] (1) Technical problems to be solved The purpose of this invention is to provide a composition for nourishing tendons and bones and its preparation method, which can effectively block the accumulation and toxic effects of harmful heavy metals in traditional Chinese medicine in the human body, while not interfering with the normal absorption of essential trace elements in the human body.

[0005] (2) Technical solution To achieve the above objectives, on the one hand, the present invention provides a composition for nourishing tendons and bones, comprising the following parts by weight: 10-30 parts of fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer, 20-40 parts of alfalfa leaf saponins, 20-40 parts of selenium yeast-Poria cocos polysaccharide complex, 5-10 parts of glucosamine, 1-3 parts of compound nuts, 20-40 parts of vitamin D, 5-15 parts of beef-pork complex, 10-20 parts of traditional Chinese medicine complex, and 20-60 parts of cheese; The organic selenium content in the selenium yeast-Poria cocos polysaccharide complex is 100~150μg / g. The recommended daily intake of the composition of the present invention is 10g, so that the daily intake of selenium in the composition does not exceed 400μg, which meets the tolerable upper intake level of dietary selenium stipulated by the Chinese Nutrition Society.

[0006] Furthermore, the vitamin D is food-grade vitamin D3 microencapsulated powder with a vitamin D3 content of 1000 IU / g (i.e., 25 μg / g).

[0007] Furthermore, the preparation method of the selenium yeast-Poria cocos polysaccharide complex includes the following steps: S11. Inoculate the brewer's yeast into yeast extract peptone glucose medium, add food-grade sodium selenite, and carry out aerobic fermentation. After fermentation, centrifuge to collect the cells, wash with purified water, freeze dry, and obtain selenium yeast powder. S12. Weigh out Poria cocos powder, add purified water, stir magnetically and filter, and collect the filtrate; add purified water to the residue again, repeat the above extraction operation once, combine the two filtrates, centrifuge and collect the supernatant to obtain Poria cocos polysaccharide extract. S13. Concentrate the extract of Poria cocos polysaccharide under reduced pressure, slowly add food-grade succinic anhydride to the concentrate, and simultaneously add 5% sodium hydroxide solution to maintain the pH of the system at 8.0~9.0. React at 30℃. After the reaction is completed, adjust the pH to 6.8~7.2 with 0.1mol / L food-grade citric acid, dialyze, and obtain carboxylated Poria cocos polysaccharide solution. S14. The carboxylated Poria cocos polysaccharide solution and the selenium yeast powder obtained in step S11 are mixed at a mass ratio of 1:1, stirred, then dialyzed, filtered, and freeze-dried to obtain the selenium yeast-Poria cocos polysaccharide complex.

[0008] Furthermore, the preparation method of the alfalfa leaf saponins includes the following steps: S21. Dry alfalfa leaves and pulverize them. Reflux them in a Soxhlet extractor with food-grade n-hexane. Place the extracted residue in a ventilated place to air dry to obtain defatted leaf powder. S22. Add food-grade ethanol to defatted leaf powder, extract with ultrasound, filter the extract, concentrate the filtrate to one-fifth of the original volume by rotary evaporation, add an equal volume of purified water, let it cool down and stand at 4℃ to precipitate a white flocculent precipitate, filter and collect the precipitate to obtain the first solid. S23. Add food-grade ethanol to the first solid to redissolve it, then extract it three times with food-grade ethyl acetate, separate and discard the ethyl acetate layer, retain the aqueous layer, then extract it three times with food-grade n-butanol, combine the n-butanol layers, wash with a small amount of purified water to obtain the n-butanol phase rich in saponins; S24. Pack the D101 macroporous adsorption resin column, activate it sequentially with distilled water and food-grade ethanol, evaporate the n-butanol phase obtained in step S23 to dryness and then reconstitute it onto the sample, elute sequentially with purified water, 30% ethanol, 50% ethanol, 70% ethanol and 95% ethanol gradient, collect each eluent, concentrate and combine them by rotary evaporation to obtain saponin concentrate. S25. Add food-grade acetone to the saponin concentrate, let stand at 4°C, filter and collect the precipitate, freeze dry to obtain alfalfa leaf saponins.

[0009] Furthermore, the preparation method of the fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer includes the following steps: S31. After drying fresh mulberry leaves, crush them and add purified water to prepare a substrate solution. Adjust the pH to 6.8~7.0, inoculate with Bacillus subtilis, and ferment aerobicly at 37℃ and 150rpm. After fermentation, inactivate the bacteria at 85℃ to obtain an inactivated fermentation broth. S32. Add ethanol to the inactivated fermentation broth to a final concentration of 65%, extract with ultrasonic assistance, repeat 3 times, filter, and concentrate by rotary evaporation to obtain fermented mulberry leaf polyphenol extract for later use. S33. After cleaning the body wall of the sea cucumber, add purified water, then add food-grade alkaline protease, enzymatically hydrolyze, centrifuge to collect the supernatant, add ethanol, filter to collect the precipitate, redissolve the precipitate in purified water, dialyze, freeze dry to obtain sea cucumber glycosaminoglycan powder. S34. Dissolve the fermented mulberry leaf polyphenol extract obtained in step S32 and the sea cucumber glycosaminoglycan powder obtained in step S33 in phosphate buffer at a mass ratio of 1:1, add food-grade laccase, stir, and after the reaction is completed, incubate at 85℃ to inactivate the enzyme, dialyze, filter, and freeze dry to obtain fermented mulberry leaf polyphenol-sea cucumber glycosaminoglycan polymer.

[0010] Furthermore, the compound nuts are made by mixing almonds, sunflower seeds, chia seeds and hazelnuts in a mass ratio of 2:1:1:1.

[0011] Furthermore, the herbal compound is composed of Eucommia ulmoides, Achyranthes bidentata, Dipsacus asper, and Lycium barbarum in a mass ratio of 3:1:1:1.

[0012] Furthermore, the mass ratio of beef to pork in the beef-pork complex is 3:1.

[0013] On the other hand, the present invention also provides a method for preparing a composition for nourishing tendons and strengthening bones, comprising the following steps: S41. Place the selenium yeast-Poria cocos polysaccharide complex, fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer, alfalfa leaf saponins, glucosamine, compound nuts, and traditional Chinese medicine complex in a V-type mixer, mix evenly, and sieve to obtain the first mixed dry powder. S42. Add purified water to the first mixed dry powder, disperse evenly, heat in a water bath at 80~85℃ for 30min, filter while hot through a 0.45μm food-grade filter membrane, collect the filtrate, concentrate by rotary evaporation, and freeze dry to obtain a mixed dry powder after heavy metal removal; dissolve vitamin D in food-grade medium-chain triglyceride oil, spray evenly on the surface of the above mixed dry powder, mix evenly to obtain a second mixed dry powder; S43. The beef-pork complex is mixed with cheese, and food-grade medium-chain triglyceride oil is added. The mixture is dissolved in a water bath at 60°C to form a paste. The paste is then added to a second mixed dry powder to obtain the final mixture. The mixture is then vacuum dried, ground, and sieved to obtain a composition for nourishing tendons and bones.

[0014] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1. This invention introduces three functional components—selenium yeast-Poria cocos polysaccharide complex, alfalfa leaf saponins, and fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer—to construct a heavy metal removal system. The three components work synergistically to achieve a removal rate of over 88% for harmful heavy metals such as lead, mercury, and cadmium, which is significantly better than control schemes with single components or lacking any component. This effectively solves the problem of heavy metal residues in Chinese medicinal materials, which restricts the safety of bone and tendon strengthening products.

[0015] 2. The three functional components introduced in this invention effectively remove harmful heavy metals while having almost no interference with essential trace elements such as iron, zinc, and copper. Simultaneously, the chondroitin sulfate unit in the fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer helps promote cartilage matrix synthesis and repair; alfalfa leaf saponins have anti-inflammatory activity, helping to reduce inflammatory responses in joint tissues; and the organic selenium and poria polysaccharides in the selenium yeast-poria polysaccharide complex have antioxidant and immunomodulatory activities. These effects, combined with the synergistic effects of glucosamine, traditional Chinese medicine complexes, vitamin D, and cheese components in the composition, fully achieve the overall goal of nourishing tendons and bones while ensuring food safety. Detailed Implementation

[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Example 1: This example discloses a composition for nourishing tendons and bones, comprising the following parts by weight: 20 parts fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer, 30 parts alfalfa leaf saponins, 30 parts selenium yeast-Poria cocos polysaccharide complex, 8 parts glucosamine, 2 parts compound nuts, 30 parts vitamin D, 10 parts beef-pork complex, 15 parts traditional Chinese medicine complex, and 40 parts cheese; The organic selenium content in the selenium yeast-Poria cocos polysaccharide complex is 100~150μg / g. The recommended daily intake of the composition of the present invention is 10g, so that the daily intake of selenium in the composition does not exceed 400μg, which meets the tolerable upper intake level of dietary selenium stipulated by the Chinese Nutrition Society.

[0018] The vitamin D is food-grade vitamin D3 microencapsulated powder with a vitamin D3 content of 1000 IU / g (i.e., 25 μg / g).

[0019] The preparation method of the selenium yeast-Poria cocos polysaccharide complex includes the following steps: S11. Add Saccharomyces cerevisiae (with a viable count of not less than 10). 8 (CFU / g) was inoculated into yeast extract peptone glucose medium, and food-grade sodium selenite was added at a concentration of 2.0 mg / L. The mixture was then fermented aerobicly at 28℃ and 180 rpm for 60 h to allow the yeast to convert inorganic selenium into an organic selenium form, mainly selenomethionine. After fermentation, the mixture was centrifuged at 4500 rpm for 10 min, and the cells were collected. The cells were washed three times with purified water and then freeze-dried at -45℃ to obtain selenium yeast powder with a selenium content of 200~300 μg / g dry weight. S12. Weigh 500g of Poria cocos powder, add 1000mL of purified water, place on a magnetic stirrer and stir at 400rpm and 95℃ for 3h, then filter and collect the filtrate; add another 1000mL of purified water to the filter residue, repeat the above extraction operation once, combine the two filtrates, centrifuge at 6500rpm for 15min, collect the supernatant, and obtain Poria cocos polysaccharide extract; S13. Place the Poria cocos polysaccharide extract in a rotary evaporator and concentrate it to 450 mL under reduced pressure at 55 °C. Slowly add food-grade succinic anhydride (mass ratio of 0.3:1 to Poria cocos polysaccharide) to the concentrate, while simultaneously adding 5% sodium hydroxide solution to maintain the pH of the system at 8.0~9.0. React at 30 °C for 2.5 h to allow the succinic anhydride to undergo an esterification reaction with the hydroxyl groups of the polysaccharide to introduce carboxyl functional groups. After the reaction is complete, adjust the pH to 6.8~7.2 with 0.1 mol / L food-grade citric acid. Transfer the solution to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze it in purified water for 24 h (changing the dialysate every 8 h) to obtain a carboxylated Poria cocos polysaccharide solution. S14. The carboxylated Poria cocos polysaccharide solution and the selenium yeast powder obtained in step S11 were mixed at a mass ratio of 1:1 and gently stirred at 40°C for 3 hours to allow the Poria cocos polysaccharide chains to associate with the selenium yeast protein components through hydrogen bonding and electrostatic interaction. The mixture was then transferred to a dialysis bag and dialyzed in ultrapure water for 48 hours (with water changed every 8 hours). After dialysis, the dispersion was filtered through a 0.45 μm food-grade filter membrane and freeze-dried for 42 hours to obtain the selenium yeast-Poria cocos polysaccharide complex.

[0020] The preparation method of the alfalfa leaf saponins includes the following steps: S21. Weigh 30g of alfalfa leaves and dry them at 55℃. Then, pulverize them to 40-60 mesh using a pulverizer. Reflux them in a Soxhlet extractor with 100mL of food-grade n-hexane for 8 hours. Place the extracted residue in a ventilated place to air dry thoroughly to remove residual solvent and obtain defatted leaf powder. S22. Add 500 mL of food-grade ethanol to defatted leaf powder, place it in an ultrasonic extractor for ultrasonic-assisted extraction 3 times (30 min each time, temperature 55℃), take out the extract and filter it, concentrate the filtrate to one-fifth of the original volume on a rotary evaporator at 40℃, add an equal volume of purified water, let it cool down and let it stand at 4℃ for 12 h to precipitate a white flocculent precipitate, filter and collect the precipitate to obtain the first solid; S23. Add 50 mL of food-grade ethanol to the first solid to redissolve it, then extract it three times with food-grade ethyl acetate (50 mL each time), separate and discard the ethyl acetate layer (to remove non-saponin impurities such as chlorophyll and fat-soluble pigments), retain the aqueous layer, and then extract it three times with food-grade n-butanol (50 mL each time). Combine the n-butanol layers and wash them with a small amount of purified water to obtain the n-butanol phase rich in saponins. S24. Pack the D101 macroporous adsorption resin column, activate it sequentially with distilled water and food-grade ethanol, evaporate the n-butanol phase obtained in step S23 to dryness and then reconstitute it onto the sample, elute sequentially with purified water, 30% ethanol, 50% ethanol, 70% ethanol and 95% ethanol gradient, collect each eluent, concentrate and combine them by rotary evaporation to obtain saponin concentrate. S25. Add 150 mL of food-grade acetone to the saponin concentrate, let it stand at 4 °C for 12 h, filter and collect the precipitate, place the precipitate in a freeze dryer and dry at -75 °C for 22 h to obtain alfalfa leaf saponins.

[0021] The residual ethanol content in the obtained alfalfa leaf saponins does not exceed 50 mg / kg, and the residual n-hexane content does not exceed 1 mg / kg. The above solvents are used as food processing aids, which complies with the relevant provisions on food processing aid residues in Appendix C of the National Food Safety Standard for the Use of Food Additives (GB 2760-2014).

[0022] The preparation method of the fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer includes the following steps: S31. Dry fresh mulberry leaves at 55℃ and then pulverize them to 40-60 mesh. Prepare a substrate solution with purified water at a material-to-liquid ratio of 1:10 (mass-volume ratio, g / mL), adjust the pH to 6.8-7.0, and inoculate with Bacillus subtilis (CICC10025) until the bacterial concentration is approximately 10. 7 CFU / mL, aerobic fermentation at 37℃ and 150rpm for 60h, and after fermentation, inactivation at 85℃ for 15min to obtain inactivated fermentation broth; S32. Add ethanol to the inactivated fermentation broth to a final concentration of 65%, extract with ultrasonic assistance for 30 min, repeat 3 times, filter, and concentrate by rotary evaporation at 40℃ to obtain fermented mulberry leaf polyphenol extract for later use. S33. After cleaning the sea cucumber body wall, add 10 times the amount of purified water, then add food-grade alkaline protease (0.15 AU / g substrate) and enzymatically hydrolyze at 50℃ for 5 hours to remove protein. Centrifuge and collect the supernatant, add 3 times the volume of ethanol, filter and collect the precipitate, redissolve the precipitate in purified water, transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze for 24 hours. Freeze dry to obtain sea cucumber glycosaminoglycan powder; the proportion of chondroitin sulfate units in the obtained sea cucumber glycosaminoglycan powder is not less than 82%, which is verified by the uronic acid content detection method. S34. The fermented mulberry leaf polyphenol extract obtained in step S32 and the sea cucumber glycosaminoglycan powder obtained in step S33 were dissolved in phosphate buffer (pH 6.8~7.0) at a mass ratio of 1:1. Food-grade laccase (derived from Trametesversicolor, at a dosage of 0.8 U / mL) was added, and the mixture was gently stirred at 28℃ for 7 h. After the reaction was completed, the enzyme was inactivated by incubation at 85℃ for 10 min. The solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in ultrapure water for 48 h (with water changed every 8 h). The dialysate was filtered through a 0.45 μm food-grade filter membrane and freeze-dried to obtain the fermented mulberry leaf polyphenol-sea cucumber glycosaminoglycan polymer.

[0023] The compound nuts are made by mixing almonds, sunflower seeds, chia seeds and hazelnuts in a mass ratio of 2:1:1:1.

[0024] The herbal compound is composed of Eucommia ulmoides, Achyranthes bidentata, Dipsacus asper, and Lycium barbarum in a mass ratio of 3:1:1:1.

[0025] The preparation method of the traditional Chinese medicine complex includes the following steps: weigh out Eucommia ulmoides, Achyranthes bidentata, Dipsacus asper, and Lycium barbarum respectively, pulverize them to 40-60 mesh, mix them evenly in a mass ratio of 3:1:1:1, add 10 times the amount of 0.1mol / L food-grade citric acid solution, stir and soak at room temperature for 2 hours, filter and collect the medicinal materials, wash them repeatedly with purified water until the pH value of the filtrate is 6.5-7.0, dry the washed medicinal materials at 55℃ to constant weight, and obtain the traditional Chinese medicine complex.

[0026] The beef-pork complex has a beef to pork mass ratio of 3:1.

[0027] The method for preparing the beef-pork complex is as follows: Fresh beef and pork are selected, visible fat and fascia are removed, and they are cut into pieces. They are then sterilized in a high-pressure steam sterilizer at 121℃ for 20 minutes. After cooling, they are vacuum dried at 55℃ until the moisture content is less than 5%. They are then pulverized to 80~100 mesh and mixed evenly at a mass ratio of 3:1 to obtain the beef-pork complex.

[0028] The preparation method of the composition for nourishing tendons and strengthening bones includes the following steps: S41. Place the selenium yeast-Poria cocos polysaccharide complex, fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer, alfalfa leaf saponins, glucosamine, compound nuts, and traditional Chinese medicine complex in a V-type mixer and mix at 200 rpm for 25 min until uniform. Then, sieve through a 90-mesh sieve to obtain the first mixed dry powder. S42. Add 300 mL of purified water to the first mixed dry powder, disperse it evenly, and heat it in a water bath at 80~85℃ for 30 min to allow the fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer and heavy metal chelate to form an insoluble precipitate in the aqueous phase; filter it while hot through a 0.45 μm food-grade filter membrane, collect the filtrate, concentrate it to about 50 mL by rotary evaporation under reduced pressure at 55℃, and freeze-dry it at -45℃ for 48 hours to obtain the mixed dry powder after removing heavy metals; dissolve vitamin D in 15 mL of food-grade medium-chain triglyceride oil, spray it evenly on the surface of the above mixed dry powder, and mix it in a V-type mixer at 200 rpm for 10 min to obtain the second mixed dry powder; S43. Mix beef-pork complex with cheese, add 15 mL of food-grade medium-chain triglyceride oil, dissolve in a water bath at 60°C to form a paste, then add to the second mixed dry powder to obtain the final mixture, place in a vacuum drying oven and dry at 60°C for 12 h, grind, and sieve through a 90-mesh sieve to obtain a composition for nourishing tendons and strengthening bones.

[0029] Example 2: This example is based on Example 1, but differs from Example 1 in that it includes the following components by weight: 10 parts fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer, 20 parts alfalfa leaf saponins, 20 parts selenium yeast-Poria cocos polysaccharide complex, 5 parts glucosamine, 1 part compound nuts, 20 parts vitamin D, 5 parts beef-pork complex, 10 parts traditional Chinese medicine complex, and 20 parts cheese. Other components and preparation methods are the same as in Example 1.

[0030] Example 3: This example is based on Example 1, but differs from Example 1 in that it includes the following components by weight: 30 parts fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer, 40 parts alfalfa leaf saponins, 40 parts selenium yeast-Poria cocos polysaccharide complex, 10 parts glucosamine, 3 parts compound nuts, 40 parts vitamin D, 15 parts beef-pork complex, 20 parts traditional Chinese medicine complex, and 60 parts cheese. Other components and preparation methods are the same as in Example 1.

[0031] Comparative Example 1: This comparative example differs from Example 1 in that, in preparing the selenium yeast-Poria cocos polysaccharide complex, the addition of food-grade succinic anhydride and the carboxylation reaction in step S13 are omitted. After the Poria cocos polysaccharide extract is concentrated under reduced pressure, it is not subjected to carboxylation modification treatment, and the pH is directly adjusted to 6.8-7.2. After dialysis, an uncarboxylated Poria cocos polysaccharide solution is obtained, which is then compounded with selenium yeast powder according to step S14. Other components and preparation methods are the same as in Example 1.

[0032] Comparative Example 2: This comparative example is based on Example 1, but differs from Example 1 in that, in the preparation of fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer, the fermentation treatment of mulberry leaves by Bacillus subtilis in step S31 is omitted. Instead, unfermented dried mulberry leaf powder is directly added to 65% ethanol at the same material-liquid ratio for ultrasonic extraction. The resulting unfermented mulberry leaf polyphenol extract is used in subsequent steps. The remaining steps are the same as in Example 1, and the other components and preparation methods are the same as in Example 1.

[0033] Comparative Example 3: This comparative example differs from Example 1 in that, in preparing the fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer, the oxidative coupling reaction catalyzed by food-grade laccase in step S34 is omitted. The fermented mulberry leaf polyphenol extract and sea cucumber glycosamine powder are dissolved in phosphate buffer at a mass ratio of 1:1, and laccase is not added. The mixture is then gently stirred at 28°C for 7 hours. The remaining steps are the same as in Example 1, thus replacing the covalently coupled polymer with a simple physical mixture of the two. Other components and preparation methods are the same as in Example 1.

[0034] Comparative Example 4: This comparative example is based on Example 1, but differs from Example 1 in that the treatment of heating in a water bath at 80~85℃ for 30 minutes is omitted in step S42 of preparing the composition. After the first mixed dry powder is fully dispersed in purified water, it is directly filtered through a 0.45μm filter membrane. Other components and preparation methods are the same as in Example 1.

[0035] Comparative Example 5: This comparative example is based on Example 1, but differs from Example 1 in that it does not include alfalfa leaf saponins. Other components and preparation methods are the same as in Example 1.

[0036] Comparative Example 6: This comparative example is based on Example 1, but differs from Example 1 in that it does not include the selenium yeast-Poria cocos polysaccharide complex. Other components and preparation methods are the same as in Example 1.

[0037] Comparative Example 7: This comparative example is based on Example 1, but differs from Example 1 in that it does not include fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer. Other components and preparation methods are the same as in Example 1.

[0038] Comparative Example 8: This comparative example is based on Example 1, but differs from Example 1 in that it does not include selenium yeast-Poria cocos polysaccharide complex, alfalfa leaf saponins, and fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer. Other components and preparation methods are the same as in Example 1.

[0039] Experimental verification: Experiment 1: Verify the removal efficiency and selectivity of the composition for heavy metal removal. The results are shown in Table 1.

[0040] Test samples: Examples 1-3 and Comparative Examples 1-8, with 3 parallel samples prepared for each sample.

[0041] Pollution model preparation: Add Pb-containing compounds to the first mixed dry powder obtained in step S41 at a ratio of 2-3 times the heavy metal limit standard for health foods (GB 16740-2014). 2+ (1000μg / g), Hg 2+ (600μg / g), Cd 2+ (800μg / g), Fe 3+ (100μg / g), Zn 2+ (60μg / g), Cu 2+ After the mixed standard solution of (40 μg / g) was fully adsorbed and equilibrated for 24 h, each sample was processed according to the corresponding S42 step (if the process was changed, follow the instructions of each comparative example), the filtrate and the retentate were collected by filtration, and the concentration of each metal element was determined.

[0042] Detection indicators and methods: Removal rate of harmful heavy metals: Pb before and after filtration was determined by inductively coupled plasma mass spectrometry (ICP-MS, according to GB 5009.268). 2+ Hg 2+ Cd 2+ Concentration, removal rate (%) = (initial concentration − filtrate concentration after filtration) / initial concentration × 100%.

[0043] Essential trace element retention rate: Simultaneous determination of Fe before and after filtration 3+ Zn 2+ Cu 2+Concentration, retention rate (%) = concentration of filtrate after filtration / initial concentration × 100%, to evaluate the selective retention capacity of the system for essential trace elements. The higher the retention rate, the less interference from essential elements.

[0044] Table 1. Removal rate of harmful heavy metals and retention rate of essential trace elements for each sample: It should be noted that in Comparative Example 4, the heating step was omitted. Although the heavy metals chelated with the functional components, the complex did not transform into an insoluble precipitate and could not be retained by the filter membrane, resulting in the lowest removal rate. In Comparative Example 6, the selenium-yeast-Poria cocos polysaccharide complex was missing, and the system lost its selective chelating ability for soft acidic metals, Hg... 2+ The removal rate decreased particularly significantly, and alfalfa leaf saponins and polyphenols also non-selectively chelated essential trace elements, resulting in a simultaneous and substantial decrease in the retention rates of Fe, Zn, and Cu.

[0045] Based on the data in Table 1, it can be concluded that the three functional components of this invention have a highly efficient and selective removal capability for harmful heavy metals. The absence of each core component and key preparation step leads to a significant decrease in removal efficiency. Among them, the performance degradation is most significant when the heating precipitation step is omitted (Comparative Example 4) and the selenium yeast-Poria cocos polysaccharide complex is missing (Comparative Example 6). Although the blank system (Comparative Example 8) has the highest retention rate of essential elements, the removal rate of harmful heavy metals is extremely low, which fully demonstrates the necessity of the synergistic effect of the three functional components.

[0046] Experiment 2: Verify the promoting effect of the composition on bone and cartilage health. The results are shown in Table 2.

[0047] Test samples: Examples 1-3, with blank control group, model control group and positive control group, 10 animals in each group.

[0048] Animal model: Sixty SPF-grade male SD rats, weighing 180–220 g, were randomly divided into six groups of ten each: blank control group, model control group, Example 1 group, Example 2 group, Example 3 group, and positive control group (calcium carbonate 300 mg / kg·d + vitamin D3 300 IU / kg·d administered by gavage). Except for the blank control group, all groups of rats were administered retinoic acid 80 mg / kg·d by gavage for 14 consecutive days to establish an osteoporosis model. During the modeling process, each treatment group was administered the corresponding combination by gavage at 3.0 g / kg·d (converted to the recommended daily intake), while the blank control group and model control group were given an equal volume of distilled water. After 28 days of administration, samples were collected 24 hours after the last administration for testing.

[0049] Detection indicators and methods: Bone mineral density (BMD, g / cm²): The femur was scanned using dual-energy X-ray absorptiometry (DXA) to determine bone mineral content and bone density. The degree of BMD reduction reflects the degree of osteoporosis.

[0050] Serum alkaline phosphatase (ALP, U / L): Measured using the rate method (fully automated biochemical analyzer). Elevated ALP reflects accelerated bone turnover and should be significantly elevated after modeling.

[0051] Serum osteocalcin (OC, ng / mL): Measured by enzyme-linked immunosorbent assay (ELISA). OC is an indicator of bone formation, and should be significantly reduced in the model group due to inhibition of bone synthesis metabolism.

[0052] Serum type I collagen C-terminal peptide (CTX-I, ng / mL): Measured by ELISA. CTX-I is an indicator of bone resorption, and should be significantly elevated in the model group.

[0053] Maximum load on the femur (N): The left femur was subjected to a three-point bending mechanical test (universal testing machine, span 20 mm, loading rate 2 mm / min). The maximum load reflects the mechanical strength of the bone and is the most direct physical indicator for evaluating bone health.

[0054] Table 2. Results of bone health-related indicators in rats of each group: Based on the data in Table 2, it can be concluded that the composition of the present invention can effectively improve bone mineral density, bone formation / resorption marker levels, and femoral mechanical strength in retinoic acid-induced osteoporotic rats.

[0055] Experiment 3: Verify the safety of the final composition.

[0056] The final products of the bone-strengthening and muscle-nourishing compositions prepared in Examples 1-3 were subjected to full-item testing for safety indicators such as heavy metal residues, microbial indicators, solvent residues, and acute toxicity in accordance with GB 16740-2014 "National Food Safety Standard for Health Food" and related national food safety standards. The results showed that all indicators of the final products of Examples 1-3 met the requirements of national food safety standards, and the products were safe.

[0057] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A composition for nourishing tendons and strengthening bones, characterized in that, The product comprises the following components by weight: 10-30 parts fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer, 20-40 parts alfalfa leaf saponins, 20-40 parts selenium yeast-Poria cocos polysaccharide complex, 5-10 parts glucosamine, 1-3 parts complex nuts, 20-40 parts vitamin D, 5-15 parts beef-pork complex, 10-20 parts traditional Chinese medicine complex, and 20-60 parts cheese. The organic selenium content in the selenium yeast-Poria cocos polysaccharide complex is 100~150μg / g.

2. The composition for nourishing tendons and strengthening bones according to claim 1, characterized in that, The preparation method of the selenium yeast-Poria cocos polysaccharide complex includes the following steps: S11. Inoculate the brewer's yeast into yeast extract peptone glucose medium, add food-grade sodium selenite, and carry out aerobic fermentation. After fermentation, centrifuge to collect the cells, wash with purified water, freeze dry, and obtain selenium yeast powder. S12. Weigh out Poria cocos powder, add purified water, stir magnetically and filter, and collect the filtrate; add purified water to the residue again, repeat the above extraction operation once, combine the two filtrates, centrifuge and collect the supernatant to obtain Poria cocos polysaccharide extract. S13. Concentrate the extract of Poria cocos polysaccharide under reduced pressure, slowly add food-grade succinic anhydride to the concentrate, and simultaneously add 5% sodium hydroxide solution to maintain the pH of the system at 8.0~9.

0. React at 30℃. After the reaction is completed, adjust the pH to 6.8~7.2 with 0.1mol / L food-grade citric acid, dialyze, and obtain carboxylated Poria cocos polysaccharide solution. S14. The carboxylated Poria cocos polysaccharide solution and the selenium yeast powder obtained in step S11 are mixed at a mass ratio of 1:1, stirred, then dialyzed, filtered, and freeze-dried to obtain the selenium yeast-Poria cocos polysaccharide complex.

3. The composition for nourishing tendons and strengthening bones according to claim 1, characterized in that, The preparation method of the alfalfa leaf saponins includes the following steps: S21. Dry alfalfa leaves and pulverize them. Reflux them in a Soxhlet extractor with food-grade n-hexane. Place the extracted residue in a ventilated place to air dry to obtain defatted leaf powder. S22. Add food-grade ethanol to defatted leaf powder, extract with ultrasound, filter the extract, concentrate the filtrate to one-fifth of the original volume by rotary evaporation, add an equal volume of purified water, let it cool down and stand at 4℃ to precipitate a white flocculent precipitate, filter and collect the precipitate to obtain the first solid. S23. Add food-grade ethanol to the first solid to redissolve it, then extract it three times with food-grade ethyl acetate, separate and discard the ethyl acetate layer, retain the aqueous layer, then extract it three times with food-grade n-butanol, combine the n-butanol layers, wash with a small amount of purified water to obtain the n-butanol phase rich in saponins; S24. Pack the D101 macroporous adsorption resin column, activate it sequentially with distilled water and food-grade ethanol, evaporate the n-butanol phase obtained in step S23 to dryness and then reconstitute it onto the sample, elute sequentially with purified water, 30% ethanol, 50% ethanol, 70% ethanol and 95% ethanol gradient, collect each eluent, concentrate and combine them by rotary evaporation to obtain saponin concentrate. S25. Add food-grade acetone to the saponin concentrate, let stand at 4°C, filter and collect the precipitate, freeze dry to obtain alfalfa leaf saponins.

4. The composition for nourishing tendons and strengthening bones according to claim 1, characterized in that, The preparation method of the fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer includes the following steps: S31. After drying fresh mulberry leaves, crush them and add purified water to prepare a substrate solution. Adjust the pH to 6.8~7.0, inoculate with Bacillus subtilis, and ferment aerobicly at 37℃ and 150rpm. After fermentation, inactivate the bacteria at 85℃ to obtain an inactivated fermentation broth. S32. Add ethanol to the inactivated fermentation broth to a final concentration of 65%, extract with ultrasonic assistance, repeat 3 times, filter, and concentrate by rotary evaporation to obtain fermented mulberry leaf polyphenol extract for later use. S33. After cleaning the body wall of the sea cucumber, add purified water, then add food-grade alkaline protease, enzymatically hydrolyze, centrifuge to collect the supernatant, add ethanol, filter to collect the precipitate, redissolve the precipitate in purified water, dialyze, freeze dry to obtain sea cucumber glycosaminoglycan powder. S34. Dissolve the fermented mulberry leaf polyphenol extract obtained in step S32 and the sea cucumber glycosaminoglycan powder obtained in step S33 in phosphate buffer at a mass ratio of 1:1, add food-grade laccase, stir, and after the reaction is completed, incubate at 85℃ to inactivate the enzyme, dialyze, filter, and freeze dry to obtain fermented mulberry leaf polyphenol-sea cucumber glycosaminoglycan polymer.

5. The composition for nourishing tendons and strengthening bones according to claim 1, characterized in that, The compound nuts are made by mixing almonds, sunflower seeds, chia seeds and hazelnuts in a mass ratio of 2:1:1:

1.

6. The composition for nourishing tendons and strengthening bones according to claim 1, characterized in that, The herbal compound is composed of Eucommia ulmoides, Achyranthes bidentata, Dipsacus asper, and Lycium barbarum in a mass ratio of 3:1:1:

1.

7. The composition for nourishing tendons and bones according to claim 1, characterized in that, The beef-pork complex has a beef to pork mass ratio of 3:

1.

8. A method for preparing a bone-strengthening and tendon-nourishing composition according to any one of claims 1 to 7, characterized in that, Includes the following steps: S41. Place the selenium yeast-Poria cocos polysaccharide complex, fermented mulberry leaf polyphenol-sea cucumber glycosamine polymer, alfalfa leaf saponins, glucosamine, compound nuts, and traditional Chinese medicine complex in a V-type mixer, mix evenly, and sieve to obtain the first mixed dry powder. S42. Add purified water to the first mixed dry powder, disperse evenly, heat in a water bath at 80~85℃ for 30min, filter while hot through a 0.45μm food-grade filter membrane, collect the filtrate, concentrate by rotary evaporation, and freeze dry to obtain a mixed dry powder after heavy metal removal; dissolve vitamin D in food-grade medium-chain triglyceride oil, spray evenly on the surface of the above mixed dry powder, mix evenly to obtain a second mixed dry powder; S43. The beef-pork complex is mixed with cheese, and food-grade medium-chain triglyceride oil is added. The mixture is dissolved in a water bath at 60°C to form a paste. The paste is then added to a second mixed dry powder to obtain the final mixture. The mixture is then vacuum dried, ground, and sieved to obtain a composition for nourishing tendons and bones.

Images