Rosa roxburghii tratt fruit composite beverage for enhancing bone mineral density and preparation method thereof

Abstract

The invention belongs to the technical field of food processing, and particularly relates to a rosa roxburghii tratt fruit composite beverage for enhancing bone mineral density and a preparation method thereof. The beverage is prepared by taking roxburgh rose juice as a core component, combining a wolfberry extract, a Chinese date extract, mulberry pulp, black sesame hydrolysate, a raspberry extract, calcium gluconate and lophatherum gracile powder and performing a multi-stage compound fermentation process. Rosa roxburghii tratt in the formula is rich in vitamin C and SOD, and cooperates with antioxidant activity of wolfberry and jujube and polyphenol substances of mulberry and raspberry to jointly inhibit bone oxidative damage; peptide fragments released by the black sesame hydrolysate are combined with calcium gluconate, so that the bioavailability of calcium is improved; and the lophatherum gracile powder regulates intestinal flora and further promotes calcium absorption. The composite beverage can effectively enhance the bone mineral density, and a safe and effective solution is provided for preventing and improving bone problems such as osteoporosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of food processing, and particularly relates to a Rosa roxburghii composite drink for enhancing bone density and a preparation method thereof. Background Art

[0002] With the aggravation of population aging, osteoporosis has become a global health problem. The core of bone health lies in maintaining bone density, and calcium absorption efficiency, collagen synthesis, and antioxidant capacity are key factors affecting bone metabolism. Currently, the mainstream solutions mainly focus on calcium supplementation, such as calcium carbonate, calcium lactate, etc., but they have problems such as low bioavailability (about 30%-40%), dependence on the gastric acid environment, and easy induction of constipation. At the same time, vitamin D3 is widely used as a calcium absorption promoter, but excessive intake may lead to the risk of hypercalcemia. In recent years, plant-derived ingredients have attracted much attention due to their natural safety, but existing products are mostly limited to single-component supplementation (such as soy isoflavones) and lack a multi-target synergistic mechanism.

[0003] Rosa roxburghii Tratt., as a plant with both medicinal and edible uses, is rich in active substances such as vitamin C (with an average content of about 2300 mg / 100 g, which is 50 times that of citrus), superoxide dismutase (SOD), and Rosa roxburghii polysaccharide. Research shows that vitamin C can promote the differentiation of osteoblasts and inhibit the activity of osteoclasts, while SOD can scavenge free radicals and reduce the damage of oxidative stress to bone tissue. However, Rosa roxburghii fruits have a strong sour and astringent taste and poor stability, and direct processing easily leads to the degradation of active ingredients. Existing technologies mostly use high-temperature sterilization or add flavoring agents, which instead destroy their nutritional characteristics. For example, patent CN201810908085.1 uses a high-temperature concentration process, resulting in a vitamin C loss rate of more than 40%; another type of solution (such as CN202010976547.0) improves the taste by adding sucrose, but increases the metabolic burden and is not suitable for diabetic patients.

[0004] In the field of composite formulations, existing bone health drinks mostly adopt the basic framework of "calcium + vitamin D", supplemented with collagen or probiotics. For example, CN202410586438.6 discloses a collagen calcium drink, but it relies on animal-derived calcium (bone meal) and collagen, which has an allergy risk and does not meet the vegetarian requirements. Patent CN202110789854.2 combines probiotics with calcium, but does not solve the problem of the survival rate of the bacterial population in the gastric acid environment (usually less than 20%).

[0005] It is worth noting that the synergistic effects of Rosa roxburghii tratt with specific components have not been fully explored: Rosa roxburghii tratt polysaccharides can regulate the intestinal flora and improve the absorption rate of minerals; its flavonoids combined with collagen peptides may enhance osteogenic activity through the Wnt / β-catenin pathway. However, the existing compound formula design lacks research on the interaction of components, resulting in insufficient efficacy superposition.

[0006] Therefore, there is an urgent need to develop a compound drink with Rosa roxburghii tratt as the core, which can achieve efficient calcium absorption, promote collagen synthesis and inhibit oxidative damage through scientific proportioning and innovative processes. Summary of the Invention

[0007] The purpose of the present invention is to provide a Rosa roxburghii tratt compound drink for enhancing bone density and its preparation method. By improving the formula design and preparation process, the prepared Rosa roxburghii tratt compound drink effectively enhances bone density, providing a safe and effective solution for preventing and improving bone problems such as osteoporosis.

[0008] In order to achieve the above purpose, the present invention provides the following technical solutions:

[0009] In the first aspect of the present invention, a Rosa roxburghii tratt compound drink for enhancing bone density is provided. Calculated by weight, the raw materials for preparing the compound drink include: 45-65 parts of Rosa roxburghii tratt fruit juice, 5-10 parts of wolfberry extract, 5-10 parts of jujube extract, 3-8 parts of mulberry fruit pulp, 2-5 parts of black sesame hydrolyzate, 2-6 parts of raspberry extract, 2-6 parts of calcium gluconate, and 1-4 parts of lophatherum gracile powder.

[0010] Further, the Rosa roxburghii tratt fruit juice is obtained by washing, crushing, juicing, and filtering fresh Rosa roxburghii tratt fruits.

[0011] Further, the wolfberry extract is prepared according to the following method:

[0012] (1) Soak wolfberries in purified water, and crush the wolfberries after soaking;

[0013] (2) Add purified water to the crushed wolfberries, heat to 55-75 °C and extract for 2-4 hours to obtain a wolfberry extract;

[0014] (3) Centrifuge the wolfberry extract to remove residues, and concentrate to a dry matter content of 15-20% to obtain the wolfberry extract.

[0015] Further, the jujube extract is prepared according to the following method:

[0016] (1) Soak jujubes in purified water, and crush the jujubes after soaking;

[0017] (2) Add purified water to the crushed jujubes, heat to 55-75 °C and extract for 2-4 hours to obtain a jujube extract;

[0018] (3) Centrifuge the jujube extract to remove residues, and concentrate it to a dry matter content of 15 - 20% to obtain the jujube extract.

[0019] Further, the mulberry fruit pulp is obtained by washing and crushing mature and plump mulberry fruits.

[0020] Further, the black sesame hydrolyzate is prepared according to the following method:

[0021] (1) Wash, dry, and crush the black sesame.

[0022] (2) Add pure water to the crushed black sesame at a mass ratio of 1:(8 - 10), heat at 80 - 90°C for 10 - 20 minutes, and then cool to obtain the black sesame extract.

[0023] (3) Add protease to the black sesame extract, with the addition amount of protease in the black sesame extract being 70 - 110 U / mL, and heat to 40 - 50°C for hydrolysis for 1 - 3 hours.

[0024] (4) After the hydrolysis is completed, filter the hydrolyzate and concentrate it to a dry matter content of 20 - 25% to obtain the black sesame hydrolyzate.

[0025] Further, the raspberry extract is prepared according to the following method:

[0026] (1) Wash and crush the raspberries, add pure water, and perform ultrasonic extraction, with the extraction temperature controlled at 40 - 50°C and the time being 20 - 40 minutes to obtain the raspberry extract.

[0027] (2) Centrifuge the raspberry extract to remove residues, and concentrate it to a dry matter content of 20 - 25% to obtain the raspberry extract.

[0028] Further, the lophatherum gracile powder can be directly purchased, or obtained by washing, drying, and crushing fresh lophatherum gracile.

[0029] In the formula of the present invention, Rosa roxburghii Tratt juice is used as the core ingredient, which is rich in vitamin C and SOD. Vitamin C can directly promote the differentiation of osteoblasts, and SOD has the function of scavenging free radicals to reduce bone oxidative damage. The extracts of Lycium barbarum and Chinese date are rich in polysaccharides and cyclic adenosine monophosphate, which can indirectly enhance bone metabolism by regulating the immune and endocrine systems. The anthocyanins and polyphenols in mulberry fruit pulp and Rubus idaeus L. extract can inhibit the activity of osteoclasts and reduce bone loss. The hydrolyzate of black sesame seeds releases calcium chelating peptides after enzymatic hydrolysis, which form soluble complexes with calcium gluconate to improve the intestinal absorption efficiency of calcium. The powder of Lophatherum gracile contains flavonoids, which enhance the activity of mineral absorption channels by regulating the intestinal flora structure. Each component in the formula acts synergistically through multiple targets: vitamin C combines with calcium chelating peptides to improve calcium solubility, polysaccharides and polyphenols synergistically inhibit the interference of inflammatory factors on bone metabolism, and the metabolites of probiotics (such as short-chain fatty acids) further optimize the intestinal absorption environment. The synergistic effect of each component jointly improves the effect of the compound drink in enhancing bone density.

[0030] The second aspect of the present invention provides a preparation method of the above-mentioned Rosa roxburghii Tratt compound drink for enhancing bone density, which includes the following steps:

[0031] (1) Mix Rosa roxburghii Tratt juice, Lycium barbarum extract, and Chinese date extract, inoculate compound fermenting bacteria 1 into the mixture for fermentation, and filter to obtain fermentation broth 1;

[0032] (2) Mix mulberry fruit pulp, black sesame seed hydrolyzate, and Rubus idaeus L. extract, inoculate Saccharomyces cerevisiae into the mixture for fermentation, and filter to obtain fermentation broth 2;

[0033] (3) Mix fermentation broth 1 and fermentation broth 2, add Lophatherum gracile powder, then inoculate compound fermenting bacteria 2 for secondary fermentation, and filter to obtain fermentation broth 3;

[0034] (4) Add calcium gluconate to fermentation broth 3, stir to dissolve, and perform pasteurization at 80 - 85 °C for 15 - 20 seconds to obtain the above-mentioned Rosa roxburghii Tratt compound drink for enhancing bone density.

[0035] Further, the compound fermenting bacteria 1 in step (1) includes Lactobacillus rhamnosus, Bifidobacterium lactis, and Lactobacillus acidophilus. Among them, the concentration of Lactobacillus rhamnosus in the mixture is (10 7 -10 8 ) CFU / mL, the concentration of Bifidobacterium lactis in the mixture is (10 6 -10 7 ) CFU / mL, and the concentration of Lactobacillus acidophilus in the mixture is (10 6 -10 7 ) CFU / mL.

[0036] Further, the concentration of Saccharomyces cerevisiae in the mixture in step (2) is (106 -10 8 ) CFU / mL.

[0037] Furthermore, the composite fermenting bacterium 2 described in step (3) includes Lactobacillus plantarum and Lactobacillus casei. Among them, the concentration of Lactobacillus plantarum in the mixture is (10 6 -10 7 ) CFU / mL, and the concentration of Lactobacillus casei in the mixture is (10 7 -10 8 ) CFU / mL.

[0038] Furthermore, the fermentation temperature in step (1) is 36 - 38 °C, and the fermentation time is 18 - 24 hours; the fermentation temperature in step (2) is 26 - 30 °C, and the fermentation time is 12 - 18 hours; the fermentation temperature in step (3) is 28 - 32 °C, and the fermentation time is 6 - 8 hours.

[0039] The present invention further enhances the functional activity of each component through a staged fermentation process. In the first stage, Lactobacillus rhamnosus, Bifidobacterium lactis, and Lactobacillus acidophilus are used to degrade Rosa roxburghii Tratt pectin, release bound vitamin C, and simultaneously produce lactic acid to lower the pH and inhibit harmful bacteria. In the second stage, Saccharomyces cerevisiae is used to ferment the mixture of mulberry, black sesame, and raspberry, producing ethanol and ester substances, which can stabilize polyphenol components and enhance the flavor. In the third stage, Lactobacillus plantarum and Lactobacillus casei are used to further decompose macromolecular polysaccharides into oligosaccharides to promote the prebiotic effect. During the fermentation process, the organic acids produced by the microbial metabolism can form soluble salts with calcium, avoiding precipitation and improving the stability of the drink. At the same time, the biological activity of polyphenol substances is further enhanced after microbial transformation.

[0040] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0041] The Rosa roxburghii Tratt composite drink for enhancing bone density provided by the present invention takes Rosa roxburghii Tratt juice as the core component, and is prepared by a multi-stage composite fermentation process with the addition of wolfberry extract, jujube extract, mulberry fruit pulp, black sesame hydrolyzate, raspberry extract, calcium gluconate, and lophatherum gracile powder. The Rosa roxburghii Tratt in the drink is rich in vitamin C and SOD, which synergistically inhibit bone oxidative damage with the antioxidant activity of wolfberry and jujube, and the polyphenol substances of mulberry and raspberry. The peptide segments released by the black sesame hydrolyzate combine with calcium gluconate to improve the bioavailability of calcium. The lophatherum gracile powder regulates the intestinal flora and further promotes calcium absorption. Verified by animal experiments, the drink of the present invention can significantly increase the trabecular bone thickness, number, and surface area, reduce the trabecular bone spacing, improve the bone tissue structure, and significantly increase the calcium absorption rate, thereby effectively enhancing bone density and providing a safe and effective solution for preventing and improving bone problems such as osteoporosis. Description of the Drawings

[0042] Figure 1 Comparison charts of trabecular bone thickness, trabecular bone number, trabecular bone separation, and bone surface area of rats in the normal group, model group, calcium gluconate group, low-dose group, medium-dose group, and high-dose group.

[0043] Figure 2 Micro-CT analysis charts of the femurs of rats in the normal group, model group, calcium gluconate group, low-dose group, medium-dose group, and high-dose group.

[0044] Figure 3 Hematoxylin-eosin staining section charts of the femurs of rats in the normal group, model group, calcium gluconate group, low-dose group, medium-dose group, and high-dose group.

[0045] Figure 4 Micro-CT analysis charts of the femurs of rats in Comparative Example 1 group.

[0046] Figure 5 Hematoxylin-eosin staining section charts of the femurs of rats in Comparative Example 1 group. Detailed implementation manners

[0047] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0048] It is worth noting that the raw materials used in the embodiments are all ordinary commercially available products without special instructions. The following are exemplary descriptions:

[0049] Neutral protease, purchased from Shanghai Yuanye Bio-Technology Co., Ltd., with an enzyme activity of 90 U / mL.

[0050] Lactobacillus rhamnosus, purchased from Beijing Zhongke Quality Inspection Biotechnology Co., Ltd., No. ZKCC 15463.

[0051] Bifidobacterium lactis, purchased from Inner Mongolia Puze Bioproducts Co., Ltd., No. CGMCC 4473.

[0052] Lactobacillus acidophilus, purchased from Inner Mongolia Puze Bioproducts Co., Ltd., No. CGMCC 4472

[0053] Saccharomyces cerevisiae, purchased from the China General Microbiological Culture Collection Center, No. CGMCC No.3217.

[0054] Lactobacillus plantarum, purchased from the China Center for Type Culture Collection, No. CCTCC AB2010210.

[0055] Lactobacillus casei, purchased from Shanghai Center for Preservation of Microorganisms, No.: SHBCC D24737.

[0056] Example 1

[0057] This example provides a compound Rosa roxburghii drink for enhancing bone density. By weight, the raw materials for preparing the compound drink include: 55 parts of Rosa roxburghii fruit juice, 7 parts of wolfberry extract, 8 parts of jujube extract, 5 parts of mulberry fruit pulp, 3 parts of black sesame hydrolyzate, 4 parts of raspberry extract, 4 parts of calcium gluconate, and 2 parts of lophatherum gracile powder.

[0058] The Rosa roxburghii fruit juice is prepared as follows: The fresh Rosa roxburghii fruits are rinsed 3 times with flowing water to remove surface impurities, and then crushed to a particle size of ≤2 mm using a fruit and vegetable crusher. The crushed fruits are pressed using a juicer, and the juice is collected and filtered through a 200-mesh filter cloth to remove the fruit residue, obtaining the Rosa roxburghii fruit juice.

[0059] The wolfberry extract is prepared as follows:

[0060] (1) Wolfberries and purified water are mixed at a mass ratio of 1:5, soaked for 2 hours until softened, and then crushed to a particle size of ≤0.5 mm using a colloid mill.

[0061] (2) Add 10 times the mass of purified water to the crushed wolfberries, heat to 65°C, and stir and extract at a constant temperature for 3 hours to obtain a wolfberry extract solution.

[0062] (3) Centrifuge the wolfberry extract solution at 4000 r / min for 15 minutes, discard the precipitate, and concentrate it to a dry matter content of 18% using a vacuum concentrator (temperature 60°C, vacuum degree -0.08 MPa) to obtain the wolfberry extract.

[0063] The jujube extract is prepared as follows:

[0064] (1) Jujubes and purified water are mixed at a mass ratio of 1:4, soaked for 3 hours until swollen, and then crushed to a particle size of ≤1 mm using a hammer mill.

[0065] (2) Add 8 times the mass of purified water to the crushed jujubes, extract at 70°C for 2.5 hours to obtain a jujube extract solution.

[0066] (3) Centrifuge the jujube extract solution at 3500 r / min for 20 minutes to remove the fiber residue, and vacuum concentrate it to a dry matter content of 17% under the conditions of a temperature of 55°C and a vacuum degree of -0.07 MPa to obtain the jujube extract.

[0067] The mulberry fruit pulp is prepared as follows: The fresh mulberries are soaked in 0.1% brine for 10 minutes, then rinsed 3 times with flowing water, and crushed into a pulp using a crusher to obtain the mulberry fruit pulp.

[0068] The black sesame hydrolyzate is prepared according to the following method:

[0069] (1) Wash the black sesame, dry it at 60°C for 3 hours, and then grind it to a particle size ≤ 0.3 mm;

[0070] (2) Add pure water to the ground black sesame according to a mass ratio of 1:9, heat it at 85°C for 15 minutes, and cool it to 40°C to obtain the black sesame extract;

[0071] (3) Add neutral protease (enzyme activity 90 U / mL) to the black sesame extract, hydrolyze it at 45°C for 2 hours, and heat it at 80°C for 10 minutes to terminate the reaction;

[0072] (4) After hydrolysis, filter the hydrolyzate, and under the conditions of a vacuum degree of -0.09 MPa and a temperature of 50°C, vacuum concentrate it to a dry matter content of 22% to obtain the black sesame hydrolyzate.

[0073] The raspberry extract is prepared according to the following method:

[0074] (1) Wash the raspberries, crush them with a colloid mill to a particle size ≤ 0.5 mm, add pure water according to a mass ratio of 1:8, and extract them by ultrasonic wave (power 300 W, frequency 40 kHz) at 45°C for 30 minutes to obtain the raspberry extract;

[0075] (2) Centrifuge the raspberry extract at 5000 r / min for 10 minutes to remove the fruit residue, and concentrate it to a dry matter content of 23% under the conditions of a temperature of 50°C and a vacuum degree of -0.08 MPa to obtain the raspberry extract.

[0076] The lophatherum gracile powder is prepared according to the following method: Rinse the fresh lophatherum gracile with clear water twice, drain it, dry it in hot air at 60°C until the water content ≤ 8%, and then grind it into a fine powder of 200 mesh with an ultrafine grinder to obtain the lophatherum gracile powder.

[0077] The preparation method of the rosa roxburghii tratt compound drink for enhancing bone density includes the following steps:

[0078] (1) Mix the rosa roxburghii tratt juice, wolfberry extract, and jujube extract, place them in a fermentation tank, stir and mix evenly, inoculate the mixture with compound fermentation bacteria 1 (Lactobacillus rhamnosus 1×10 7 CFU / mL, Bifidobacterium lactis 5×10 6 CFU / mL, Lactobacillus acidophilus 5×10 6 CFU / mL) for fermentation, ferment at a constant temperature of 37°C for 20 hours, and after fermentation, filter it with a 200-mesh filter cloth to obtain fermentation broth 1;

[0079] (2) Mix the mulberry pulp, black sesame hydrolyzate, and raspberry extract, place them in a fermentation tank, stir and mix evenly, inoculate the mixture with Saccharomyces cerevisiae (1×107 Perform fermentation at 28 °C for 15 hours at a concentration of

[0080] (3) Mix fermentation broth 1 and fermentation broth 2, add powder of lophatherum gracile, and then inoculate with compound fermentation bacteria 2 (Lactobacillus plantarum 5×10 6 CFU / mL, Lactobacillus casei 1×10 7 CFU / mL) for secondary fermentation. Ferment at a constant temperature of 30 °C for 7 hours, and after fermentation, filter with a 0.45 μm microporous membrane to obtain fermentation broth 3;

[0081] (4) Add calcium gluconate to fermentation broth 3, stir and dissolve at 100 r / min for 20 minutes, and perform pasteurization at 83 °C for 18 seconds to obtain the prepared rose roxburghii compound drink for enhancing bone density. Sterilely fill the drink into an aluminum-plastic composite bag and store it refrigerated at 4 °C.

[0082] Comparative Example 1

[0083] This comparative example provides a rose roxburghii compound drink for enhancing bone density, which is different from Example 1 in that the preparation method of the rose roxburghii compound drink for enhancing bone density includes the following steps:

[0084] (1) Mix rose roxburghii fruit juice, wolfberry extract, jujube extract, mulberry fruit pulp, black sesame hydrolyzate, and raspberry extract, place them in a fermentation tank, stir and mix evenly, and inoculate the mixture with compound fermentation bacteria 1 (Lactobacillus rhamnosus 1×10 7 CFU / mL, Bifidobacterium lactis 5×10 6 CFU / mL, Lactobacillus acidophilus 5×10 6 CFU / mL) for fermentation. Ferment at a constant temperature of 37 °C for 20 hours, and after fermentation, filter with a 200-mesh filter cloth to obtain fermentation broth 1;

[0085] (2) Add powder of lophatherum gracile to fermentation broth 1, and then inoculate with compound fermentation bacteria 2 (Lactobacillus plantarum 5×10 6 CFU / mL, Lactobacillus casei 1×10 7 CFU / mL) for secondary fermentation. Ferment at a constant temperature of 30 °C for 7 hours, and after fermentation, filter with a 0.45 μm microporous membrane to obtain fermentation broth 2;

[0086] (3) Add calcium gluconate to fermentation broth 2, stir and dissolve at 100 r / min for 20 minutes, and perform pasteurization at 83 °C for 18 seconds to obtain the prepared rose roxburghii compound drink for enhancing bone density. Sterilely fill the drink into an aluminum-plastic composite bag and store it refrigerated at 4 °C.

[0087] Performance Test 1 Stability Experiment

[0088] The Rosa roxburghii composite drinks prepared in Example 1 and Comparative Example 1 were respectively subjected to high-temperature stability and motion stability tests to observe whether there was stratification, precipitation, and color change in the appearance. The results are shown in Table 1.

[0089] High-temperature stability test conditions: Store at 55 °C for 30 days.

[0090] Motion stability test conditions: Store in a shaker at 25 °C with a shaking speed of 200 r / min for 30 days.

[0091] Table 1 Results of the stability experiment

[0092] High-temperature stability Motion stability Example 1 No change No change Comparative Example 1 Slight discoloration and a small amount of precipitation Slight discoloration

[0093] The above experimental results show that the Rosa roxburghii composite drink prepared by the present invention has excellent stability and can remain stable without color change and precipitation for a long time in high-temperature and motion environments. In the preparation process of Comparative Example 1, the fermentation steps were combined, and the independent fermentation stage of mulberry-black sesame-fructus rubi was cancelled. The beer yeast could not fully metabolize the tannins in the mulberry, and the residual tannins combined with calcium to form insoluble complexes, resulting in slight precipitation. At the same time, this fermentation method affected the metabolic pathway of the flora, and some intermediate products, such as ethanol, were not fully consumed, accelerating the oxidation and color change.

[0094] Performance test 2

[0095] 2.1 Animal operations

[0096] All animal operations were carried out in accordance with the Guidelines for Laboratory Animal Management of the China Animal Health and Epidemiology Center (www.cahec.cn) and approved by the Animal Ethics Committee of Guizhou Medical University (Approval No.: 060960956).

[0097] SD rats, weighing 180 - 220 g, were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Certification No.: SCXK(Beijing)2023 - 0013).

[0098] Before the experiment, SD rats were acclimatized in an environment of 24 ± 2 °C for 1 week. Seventy SD rats were randomly divided into 7 groups: normal control group (NC), model control group (MC), low, medium, and high-dose RRFP (the Rosa roxburghii Tratt composite drink of Example 1) (RRFL-Ca, RRFM-Ca, RRFH-Ca), CaCO3 group, and Comparative Example 1 group, with 10 rats in each group. RRFP and CaCO3 suspensions were prepared with sodium carboxymethylcellulose. Except for the NC group, the rats in the other groups were intraperitoneally injected with d-galactose at 300 mg / kg to induce, and fed with a low-calcium diet (the calcium content of the rat diet was 150 mg / kg) for 6 weeks. Meanwhile, the NC group and the MC group were intragastrically administered 0.5% sodium carboxymethylcellulose; the RRFL-Ca group, the RRFM-Ca group, and the RRFH-Ca group were respectively administered 10 mg / kg, 20 mg / kg, and 40 mg / kg RRFP; the CaCO3 group was administered 150 mg / kg CaCO3; the Comparative Example 1 group was administered the Rosa roxburghii Tratt composite drink prepared in Comparative Example 1 at 40 mg / kg. The NC group was fed with a normal diet (calcium content was 12 g / kg), and the MC, RRFL-Ca, RRFM-Ca, RRFH-Ca, CaCO3 groups, and the Comparative Example 1 group were fed with a low-calcium diet.

[0099] 2.2 Sample collection

[0100] After completing the 6-week treatment protocol, urine and fecal samples were collected. All SD rats were fasted for 12 hours, then anesthetized, euthanized, and dissected to collect blood and femur samples. The muscle components of the left and right femurs were carefully cleaned. The left femur was used for bone density measurement, while the right femur was stored in 4% paraformaldehyde solution for subsequent histopathological analysis.

[0101] 2.3 Study on calcium absorption and balance

[0102] The calcium contents in the feces and urine of SD rats were accurately measured. The calcium absorption rate and calcium balance were calculated using the following formulas:

[0103] Calcium absorption rate (%) = (calcium intake - (fecal calcium + urinary calcium)) / calcium intake × 100%

[0104] Calcium balance = calcium intake - (urinary calcium + fecal calcium)

[0105] The results are shown in Table 2:

[0106] Table 2 Body weight, calcium intake, calcium balance, and apparent calcium absorption rate of rats

[0107]

[0108]

[0109] Note: *p < 0.05 compared with the NC group, #p < 0.05 compared with the CaCO3 group.

[0110] The results showed that the apparent calcium absorption rates of the RRFL-Ca, RRFM-Ca, and RRFH-Ca rat groups were 69.18 ± 5.33%, 72.98 ± 4.76%, and 75.47 ± 3.23% respectively, and these values were significantly higher than those of the CaCO3 group (p < 0.05). Compared with traditional calcium carbonate supplements, the Rosa roxburghii compound drink prepared by the present invention can significantly improve the calcium absorption efficiency of rats, providing a more effective solution for preventing and improving bone problems such as osteoporosis. The apparent calcium absorption rate value of Comparative Example 1 was 71.75 ± 4.82%, which was significantly lower than that of the RRFH-Ca group, indicating that under the same gavage volume, the Rosa roxburghii compound drink in Comparative Example 1 had a less effective improvement on the calcium absorption efficiency of rats than that in Example 1.

[0111] 2.4 Histological evaluation by micro-computed tomography (micro-CT)

[0112] The samples were scanned using μCT (Inveon, Siemens, Erlangen, Germany) with an effective pixel size of 8.82 μm, 80 kV / 500 μA, an exposure time of 1500 ms, and 360 rotations. A standard cylindrical (diameter 3 mm × height 5 mm) volume of interest (VOI) was established and CPCs were placed in it. In this standardized VOI, bone regeneration indices were detected, including bone volume (BV), bone surface (BS), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). The thigh bones were weighed and measured, and then fixed in 10% formalin at 4 °C overnight. The femoral samples were decalcified in 8% formic acid / hydrochloric acid solution for 24 hours. Subsequently, the samples were briefly washed and neutralized in ammonia for 30 minutes. The tissues were dehydrated, automatically processed, paraffin-embedded, and sectioned at 4 μm. The sections were stained with hematoxylin-eosin for morphological evaluation.

[0113] Compared with the MC group, the Tb.Th values (trabecular thickness) of the RRFL-Ca, RRFM-Ca, and RRFH-Ca groups of SD rats increased by 25.93%, 37.04%, and 48.15% respectively ( Figure 1 A). Similarly, the Tb.N (trabecular number) increased by 8.12%, 15.56%, and 21.26% respectively ( Figure 1 B), while the BS (bone surface area) increased by 15.47%, 25.92%, and 37.23% respectively ( Figure 1 C). On the contrary, the Tb.Sp (trabecular separation) decreased by 15.79%, 26.31%, and 36.84% respectively ( Figure 1 D).

[0114] The three-dimensional simulation of the femur is as Figure 2 shown. The HE staining results showed that the trabecular bone in the NC group was intact, regularly arranged, and dense; the trabecular bone in the MC group was sparse, thinned, and even fragmented, with a disordered structure and fragments. It is worth noting that a significant improvement was observed in the RRFP group ( Figure 3 ).

[0115] Figure 4 It is the three-dimensional structure diagram of the femur of the rats in Comparative Example 1 group. Figure 5 It is the histological section staining diagram of the femur of the rats in Comparative Example 1 group. As can be seen from the figure, the trabecular bone structure in the Comparative Example 1 group was improved, but the arrangement and density of the trabecular bone were still not as good as those in the RRFP group, there was a disordered bone tissue structure, and the integrity and density of the trabecular bone were not as good as those in the RRFP group.

[0116] The above results indicate that the Rosa roxburghii complex drink of the present invention can not only improve the calcium absorption rate, but also effectively promote the regeneration and repair of bone tissue, significantly improve the bone tissue structure of osteoporotic rats, and provide strong evidence for the prevention and treatment of osteoporosis.

[0117] The above is the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle described in the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A Rosa roxburghii composite drink for enhancing bone density, characterized in that, By weight parts, the raw materials for preparing the compound beverage include: 45 - 65 parts of Rosa roxburghii fruit juice, 5 - 10 parts of wolfberry extract, 5 - 10 parts of jujube extract, 3 - 8 parts of mulberry fruit pulp, 2 - 5 parts of black sesame hydrolyzate, 2 - 6 parts of raspberry extract, 2 - 6 parts of calcium gluconate, and 1 - 4 parts of lophatherum gracile powder.

2. The compound beverage according to claim 1, wherein The wolfberry extract is prepared according to the following method: (1) Soak wolfberries in purified water, and crush the wolfberries after soaking. (2) Add purified water to the crushed wolfberries, heat to 55 - 75 °C and extract for 2 - 4 hours to obtain a wolfberry extract solution. (3) Centrifuge the wolfberry extract solution to remove residues, and concentrate to a dry matter content of 15 - 20% to obtain the wolfberry extract.

3. The compound drink according to claim 1, characterized in that, The jujube extract is prepared according to the following method: (1) Soak jujubes in purified water, and crush the jujubes after soaking. (2) Add purified water to the crushed jujubes, heat to 55 - 75 °C and extract for 2 - 4 hours to obtain a jujube extract solution. (3) Centrifuge the jujube extract solution to remove residues, and concentrate to a dry matter content of 15 - 20% to obtain the jujube extract.

4. The compound beverage according to claim 1, wherein The black sesame hydrolyzate is prepared according to the following method: (1) Wash, dry, and crush black sesame. (2) Add purified water to the crushed black sesame according to a mass ratio of 1:(8 - 10), heat at 80 - 90 °C for 10 - 20 minutes, and cool to obtain a black sesame extract solution. (3) Add protease to the black sesame extract solution, and the addition amount of protease in the black sesame extract solution is 70 - 110 U / mL, heat to 40 - 50 °C and hydrolyze for 1 - 3 hours. (4) After hydrolysis, filter the hydrolyzate and concentrate to a dry matter content of 20 - 25% to obtain the black sesame hydrolyzate.

5. The compound beverage according to claim 1, wherein The raspberry extract is prepared according to the following method: (1) Wash and crush raspberries, add purified water, and perform ultrasonic extraction. Control the extraction temperature at 40 - 50 °C for 20 - 40 minutes to obtain a raspberry extract solution. (2) Centrifuge the raspberry extract solution to remove residues, and concentrate to a dry matter content of 20 - 25% to obtain the raspberry extract.

6. The preparation method of the Rosa roxburghii compound beverage for enhancing bone density according to any one of claims 1 - 5, comprising the following steps: (1) Mix Rosa roxburghii fruit juice, wolfberry extract, and jujube extract, inoculate compound fermentation bacteria 1 into the mixture for fermentation, and filter to obtain fermentation liquid 1. (2) Mix mulberry fruit pulp, black sesame hydrolyzate, and raspberry extract, inoculate Saccharomyces cerevisiae into the mixture for fermentation, and filter to obtain fermentation liquid 2. (3) Mix fermentation liquid 1 and fermentation liquid 2, add lophatherum gracile powder, and then inoculate compound fermentation bacteria 2 for secondary fermentation, and filter to obtain fermentation liquid 3. (4) Add calcium gluconate to fermentation liquid 3, stir to dissolve, and perform pasteurization at 80 - 85 °C for 15 - 20 seconds to obtain the Rosa roxburghii compound beverage for enhancing bone density.

7. The preparation method according to claim 6, characterized in that, The compound fermenting bacteria 1 described in step (1) includes Lactobacillus rhamnosus, Bifidobacterium lactis and Lactobacillus acidophilus. Among them, the concentration of Lactobacillus rhamnosus in the mixture is (10 7 -10 8 ) CFU / mL, the concentration of Bifidobacterium lactis in the mixture is (10 6 -10 7 ) CFU / mL, and the concentration of Lactobacillus acidophilus in the mixture is (10 6 -10 7 ) CFU / mL.

8. The preparation method according to claim 6, characterized in that, The concentration of the brewer's yeast in the mixture in step (2) is (10 6 -10 8 ) CFU / mL.

9. The preparation method according to claim 6, characterized in that, The composite fermenting bacterium 2 described in step (3) includes Lactobacillus plantarum and Lactobacillus casei. Among them, the concentration of Lactobacillus plantarum in the mixture is (10 6 -10 7 ) CFU / mL, and the concentration of Lactobacillus casei in the mixture is (10 7 -10 8 ) CFU / mL.

10. The preparation method according to claim 6, characterized in that, The fermentation temperature in step (1) is 36 - 38 °C, and the fermentation time is 18 - 24 hours; the fermentation temperature in step (2) is 26 - 30 °C, and the fermentation time is 12 - 18 hours; the fermentation temperature in step (3) is 28 - 32 °C, and the fermentation time is 6 - 8 hours.

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