Liquid dairy product for improving bone and joint health and preparation method thereof

Abstract

The present invention relates to the technical field of dairy products, specifically disclosing a liquid dairy product for improving bone and joint health and a preparation method thereof. The liquid dairy product of the present invention comprises: animal milk, hyaluronate, colostrum basic protein, and N-acetylglucosamine; the mass ratio of hyaluronate, colostrum basic protein, and N-acetylglucosamine is (1-2):(1-2):(1-8). The present invention enables the successful application of N-acetylglucosamine in liquid milk systems. Without the addition of stabilizers or emulsifiers, the system is stable at room temperature for six months, has no unpleasant flavor, and promotes bone and joint health.

Description

Technical Field

[0001] The present invention relates to the technical field of dairy products, in particular to a liquid dairy product for improving bone and joint health and a preparation method thereof. Background Art

[0002] With the increasing aging of the population, there is an urgent need to address the nutritional bottleneck of foods specifically for middle-aged and elderly people. Based on the objective nutritional gaps among middle-aged and elderly people, such as insufficient dietary calcium intake and joint and muscle degeneration, as well as the subjective needs of middle-aged and elderly consumers, it is necessary to design and develop native high-calcium activity-fortified milk for middle-aged and elderly people to promote healthy aging.

[0003] Milk and dairy products are rich in calcium and are highly absorbable, making them the best source of dietary calcium. They can effectively address low dietary calcium intake and promote bone health. Adding ingredients that promote joint and muscle health to milk could potentially create products more suitable for middle-aged and elderly individuals.

[0004] In addition, consumers prefer green foods that are nutritious and contain fewer additives. Although the types and amounts of emulsifiers and stabilizers added to functional milk currently meet the requirements, it is still necessary to keep the labels as clean as possible based on the concept of green, low-carbon and healthy living.

[0005] Components known to benefit joint and muscle health are prone to stability issues when used in functional milk formulations, thus impacting the product's shelf life. To prevent quality issues such as fat bubbling, protein precipitation, and protein denaturation in formula milk products with a six-month shelf life at room temperature, various thickeners and stabilizers are often added. However, this approach aligns with the trend toward "minimum additives" in product design. Therefore, further research is needed to achieve products that balance efficacy and stability. Summary of the Invention

[0006] One of the objectives of the present invention is to provide a liquid dairy product that is stable and has the effect of improving joint health without adding additional emulsifiers and stabilizers.

[0007] The present invention provides a liquid dairy product comprising: animal milk, hyaluronate, colostrum basic protein and N-acetylglucosamine; the mass ratio of the hyaluronate, colostrum basic protein and N-acetylglucosamine is (1-2): (1-2): (1-8).

[0008] N-acetylglucosamine (NAG, relative molecular mass 221.21) is a derivative of glucosamine. It exists in organisms as a functional monosaccharide and is widely found in nature. It has good bioavailability and can help maintain joint health. However, the present study found that N-acetylglucosamine is prone to agglomeration and protein denaturation when directly dissolved in milk (possibly due to the protein and Ca in milk). 2+ , fat, etc.), is difficult to disperse and dissolve, and has poor thermal stability. After sterilization processes such as pasteurization and ultra-high temperature sterilization, problems such as tube sticking and precipitation may occur, even preventing continuous production and filling. Furthermore, during storage, fat may float or protein may precipitate, affecting the stability of the entire liquid dairy product, making it impossible to guarantee a six-month shelf life at room temperature. Adding conventional stabilizers / emulsifiers can help alleviate this problem, but it does not meet consumer expectations for products with "few additives" or "no additives." Furthermore, N-acetylglucosamine has a fermented flavor, and adding it directly to milk can cause an unpleasant flavor, making it difficult for consumers to accept.

[0009] Hyaluronates (such as sodium hyaluronate) have the effect of protecting joints, but when added directly to neutral milk, the polyhydroxyl interactions within its molecules will form a dense triple helix structure, which is easy to agglomerate with milk protein. After sterilization processes such as pasteurization and ultra-high temperature sterilization, it is more likely to affect the stability of the product system.

[0010] To this end, the present invention has discovered that when hyaluronate, colostrum alkaline protein and N-acetylglucosamine are compounded in a specific ratio, the benefits of promoting bone and joint health can be achieved without the presence of food additives such as stabilizers, acid-base regulators and emulsifiers. The system can also be stable at room temperature for 6 months, avoiding acid denaturation of milk protein and enhancing the flavor and taste, thereby enabling N-acetylglucosamine and hyaluronate to be successfully applied in liquid milk systems.

[0011] The liquid dairy product of the present invention does not include other emulsifiers and stabilizers.

[0012] In the present invention, animal milk refers to the milk of mammals such as cow's milk, goat's milk, camel's milk, deer's milk, donkey's milk, and horse's milk.

[0013] The liquid dairy product of the present invention is suitable for people other than infants, children, pregnant women and lactating women.

[0014] Preferably, in the liquid dairy product of the present invention, the mass ratio of hyaluronate, colostrum basic protein and N-acetylglucosamine is 1:2:8.

[0015] The liquid dairy product of the present invention comprises, per 1000 parts by weight, 0.06-0.2 parts by weight of hyaluronate, 0.06-0.25 parts by weight of colostrum basic protein and 0.1-0.5 parts by weight of N-acetylglucosamine.

[0016] Preferably, the liquid milk of the present invention has a protein content of ≥3.0 g / 100 mL, a calcium content of ≥100 mg / 100 mL, and a shelf life of 6 months when stored at room temperature.

[0017] In the liquid dairy product of the present invention, the hyaluronate is sodium hyaluronate. Preferably, the molecular weight of the sodium hyaluronate is 80,000-400,000 Da, more preferably 90,000-230,000 Da.

[0018] The hyaluronate of the present invention may also be potassium hyaluronate.

[0019] The liquid dairy product of the present invention also includes other functional components, which include one or more of calcium β-hydroxy-β-methylbutyrate, lactase, vitamin D, vitamin A, vitamin E, zinc salt, milk mineral salt, milk basic protein (MBP), magnesium salt, and phosphate salt.

[0020] The liquid dairy product of the present invention may include, in addition to the main active ingredients of hyaluronate, colostrum basic protein and N-acetylglucosamine, other ingredients beneficial to the animal body, preferably ingredients beneficial to bones and muscles.

[0021] In the liquid dairy product of the present invention, when the liquid dairy product further includes calcium β-hydroxy-β-methylbutyrate, the mass ratio of hyaluronate to calcium β-hydroxy-β-methylbutyrate is (0.6-2):(1-2).

[0022] The present invention preferably adds calcium β-hydroxy-β-methylbutyrate (CaHMB) to milk protein to promote muscle synthesis. However, CaHMB has a bitter taste, and adding it directly to milk can easily cause an unpleasant flavor and be difficult for consumers to accept. Research by the present invention has found that combining it with a hyaluronate (e.g., sodium hyaluronate) in a specific ratio can alleviate the astringency associated with CaHMB.

[0023] The liquid dairy product of the present invention comprises 0.5-2.5 parts by weight (preferably 0.5-0.55 parts by weight) of the other functional components per 1000 parts by weight.

[0024] The present invention also provides a method for preparing the liquid dairy product without other functional components, comprising:

[0025] (1) Premixing hyaluronate, colostrum basic protein, and N-acetylglucosamine, and then fully mixing with a portion of the formula amount of animal milk to obtain a liquid feed;

[0026] (2) mixing the liquid feed with the remaining amount of animal milk, homogenizing, and sterilizing;

[0027] Preferably, in step (1), the mass of the partial formula amount of animal milk is 10-30 times the total mass of hyaluronate, colostrum basic protein and N-acetylglucosamine;

[0028] And / or, in step (1), the method for fully mixing is: stirring at 40-50°C for 3-5 minutes, then heating to 70-75°C and stirring for 10-15 minutes;

[0029] And / or, in step (2), the homogenization and sterilization method is: perform a first homogenization at 50-80°C, a total homogenization pressure of 150-170 bar, and a secondary pressure of 30-40 bar, then perform pasteurization, cool to 1-7°C after pasteurization, and then perform a second homogenization at 60-90°C, a total homogenization pressure of 220-240 bar, and a secondary pressure of 40-50 bar, and then perform ultra-high temperature sterilization;

[0030] The pasteurization temperature is 85±2°C and the time is 15s~20s, and the ultra-high temperature sterilization temperature is 137±2°C and the time is 4~6s.

[0031] The present invention also provides a method for preparing the above-mentioned liquid dairy product containing other functional components. When the other functional components do not include lactase, the method comprises:

[0032] (1) After premixing hyaluronate, colostrum basic protein and N-acetylglucosamine, thoroughly mix with other functional components that do not contain lactase and a portion of the formula amount of animal milk;

[0033] (2) Add the remaining amount of animal milk and then homogenize and sterilize;

[0034] Preferably, in step (1), the mass of the partial formula amount of animal milk is 10-30 times the total mass of hyaluronate, colostrum basic protein, N-acetylglucosamine and other effective components without lactase;

[0035] When the other functional component is only lactase, the method comprises:

[0036] (A) Premixing hyaluronate, colostrum basic protein, and N-acetylglucosamine, and then thoroughly mixing with a portion of the formula amount of animal milk;

[0037] (B) adding the remaining amount of animal milk and then homogenizing and sterilizing;

[0038] (C) Adding lactase;

[0039] Preferably, in step (A), the mass of the partial formula amount of animal milk is 10-30 times the total mass of hyaluronate, colostrum basic protein and N-acetylglucosamine;

[0040] Alternatively, the method comprises:

[0041] (a) premixing hyaluronate, colostrum basic protein, and N-acetylglucosamine, and then preliminarily mixing with a portion of the formulated amount of animal milk to obtain a premixed liquid;

[0042] (b) mixing the primary mixed liquid with lactase, performing enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, performing enzyme inactivation and secondary mixing;

[0043] (c) adding the remaining amount of animal milk to the mixture and then homogenizing and sterilizing the mixture;

[0044] Preferably, in step (a), the mass of the partial formula amount of animal milk is 10-30 times the total mass of hyaluronate, colostrum basic protein, N-acetylglucosamine and lactase;

[0045] When the other efficacy components include lactase and other components, the method comprises:

[0046] (1) premixing hyaluronate, colostrum basic protein, and N-acetylglucosamine, and then thoroughly mixing with other functional components that do not contain lactase and a portion of the formula amount of animal milk;

[0047] (II) adding the remaining amount of animal milk and then homogenizing and sterilizing;

[0048] (III) adding lactase;

[0049] Preferably, in step (I), the mass of the partial formula amount of animal milk is 10-30 times the total mass of hyaluronate, colostrum basic protein, N-acetylglucosamine and other effective components without lactase;

[0050] Alternatively, the method comprises:

[0051] (i) premixing hyaluronate, colostrum basic protein, and N-acetylglucosamine, and then preliminarily mixing with other functional components that do not contain lactase and a portion of the formula amount of animal milk;

[0052] (ii) adding lactase for enzymatic hydrolysis, and then inactivating the enzyme and performing secondary mixing after the enzymatic hydrolysis is completed;

[0053] (iii) adding the remaining amount of animal milk to the mixture and then homogenizing and sterilizing the mixture;

[0054] Preferably, in step (i), the mass of the partial formula amount of animal milk is 10-30 times the total mass of hyaluronate, colostrum basic protein, N-acetylglucosamine, other effective components excluding lactase, and lactase.

[0055] In the above method of the present invention, in steps (1), (A), and (1), the method of fully mixing is as described above;

[0056] and / or, in steps (2), (B), (c), (III) and (iii), the homogenization and sterilization methods are as described above;

[0057] And / or, in steps (a) and (i), the preliminary mixing method is: stirring at 40-50°C for 3-5 minutes, in steps (b) and (ii), the enzymatic hydrolysis temperature is 55-60°C, and the secondary mixing method is: stirring at 70-75°C for 10-15 minutes.

[0058] Preferably, the premix obtained by premixing hyaluronate, colostrum basic protein and N-acetylglucosamine is added at a rate of ≤1 kg / min when mixed with other components to facilitate better dispersion and dissolution.

[0059] Preferably, nitrogen is added during filling of the liquid milk of the present invention.

[0060] As a specific embodiment, the preparation method of the liquid milk of the present invention is as follows:

[0061] (1) Weighing / mixing: premix hyaluronate, colostrum basic protein, and N-acetylglucosamine in proportion;

[0062] (2) Use a high-efficiency online mixer (mixing shear) to mix the materials: add raw milk to the mixing tank (the amount of milk should be 10-30 times the total mass of the raw materials to be mixed and not less than the stirring liquid level), start stirring, heat it to 40-50℃ and then start the high-efficiency online mixer. In the circulation state, slowly add the premixed materials and other raw materials (if any) to the high-efficiency online mixer and stir at a constant temperature for 3-5 minutes, then continue to heat it to 70-75℃ and continue stirring for 10-15 minutes to ensure that all the raw materials are evenly dissolved; take an appropriate amount of liquid and place it in a steel basin, observe it under natural light or light, and the color of the liquid should be uniform and the texture should be free of particles.

[0063] (3) Online mixing: The remaining raw milk is mixed with the mixed liquid online and then transferred to the pasteurization system. Before the end of pasteurization, the mixed liquid in the milk silo or batching tank must be added.

[0064] (4) Pasteurization: preheating temperature 50~80℃. Flash evaporation (falling film) equipment parameters are adjusted according to the physical and chemical indicators of the semi-finished products of the corresponding products.

[0065] Homogenization temperature: 50-80°C (preferably 65-75°C). Total homogenization pressure: 150-170 bar (secondary pressure: 30-40 bar). Pasteurization temperature: 85±2°C, time: 15-20 seconds, then cool to 1-7°C.

[0066] (5) Sterilization process: preheating by plate heat exchanger (60~90℃), homogenization (60~90℃, total pressure 220~240bar, secondary pressure 50~60bar), ultra-high temperature sterilization (137±2℃, 4~6s), cooling to 15~30℃;

[0067] (6) Aseptic filling process: The aseptic tank is cooled to 15℃~30℃ for filling and filled with nitrogen (the air pressure setting parameter is 2.8 bar);

[0068] (7) Finished product: The product is packaged in light-blocking and oxygen-blocking paper.

[0069] The present invention also provides the use of the liquid dairy product or the liquid dairy product prepared by the method in the preparation of products for improving bone and joint health.

[0070] The beneficial effects of the present invention are at least:

[0071] The present invention can successfully apply N-acetylglucosamine to a liquid milk system. Without adding stabilizers or emulsifiers, the system is stable at room temperature for 6 months and has no unpleasant flavor. The obtained liquid milk can promote calcium absorption while inducing the osteoblast mineralization process to form bone cells, which is beneficial to the health of bones and joints. DETAILED DESCRIPTION

[0072] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0073] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources or prepared according to conventional methods in the art. The raw cow's milk in the present invention complies with the provisions of GB 19301. The purity of sodium hyaluronate is ≥95%. The content of β-hydroxy-β-methylbutyric acid is 80.5 g / 100 g. The lactase used in Example 4 of the present invention was purchased from Novozymes (China) Biotechnology Co., Ltd., and its enzyme activity was 15763 SDLU / g.

[0074] Example 1

[0075] This embodiment provides a liquid milk comprising raw cow's milk, hyaluronate, glucosamine, and colostrum basic protein. The hyaluronate is sodium hyaluronate with a molecular weight of 90,000 Da, and the glucosamine is N-acetylglucosamine. Per 1,000 parts by mass of the product, the sodium hyaluronate content is 0.0625 parts by mass, the N-acetylglucosamine content is 0.50 parts by mass, the colostrum basic protein content is 0.125 parts by mass, and the balance is raw cow's milk.

[0076] The preparation method of the liquid milk comprises the following specific steps:

[0077] In a mixing tank (pot), mix 25 times the total weight of the premix (sodium hyaluronate, N-acetylglucosamine, colostrum basic protein) in raw milk, i.e., approximately 17.19 parts by mass of raw milk. Start stirring: heat the raw milk to 48.1°C and add the premix (sodium hyaluronate: colostrum basic protein: N-acetylglucosamine = 1:2:8) in a circulating state at a rate of ≤1kg / min. After stirring at a constant temperature for 5 minutes, continue heating to 70.1°C and continue stirring for 10 minutes to ensure that all the raw materials are dissolved.

[0078] The remaining raw milk in the formula is mixed with the mixed liquid online and then transferred to the pasteurization system for the first homogenization, pasteurization, and first cooling. Before the end of pasteurization, the mixed liquid in the milk silo or batching tank must be added. The specific process is: the liquid is preheated to 60.8℃ through a plate heat exchanger, and the first homogenization is carried out at a temperature of 60.8℃. The total pressure of the first homogenization is 161 bar (secondary pressure 34.8bar); after the first homogenization is completed, pasteurization is carried out at a temperature of 84.7℃ for 15s, and the first cooling to 5.0℃ is completed after pasteurization;

[0079] The liquid after the first cooling was preheated to 63.4°C via a plate heat exchanger and homogenized for the second time at 63.4°C. The total pressure of the second homogenization was 223 bar (secondary pressure 48.3 bar). After the second homogenization, ultra-high temperature sterilization was performed at 137°C for 4 seconds.

[0080] The liquid was cooled to 20°C for filling and filled with nitrogen (the air pressure setting parameter was 2.8 bar).

[0081] The product indicators were tested and confirmed to have a protein content of 3.60 g / 100 mL, a calcium content of 120 mg / 100 mL, a pH of 6.66, and commercial sterility.

[0082] Example 2

[0083] This embodiment provides a liquid milk, the raw materials for its preparation are the same as those in Example 1, except that per 1000 parts by mass of the product, the content of sodium hyaluronate is 0.20 parts by mass, the content of N-acetylglucosamine is 0.10 parts by mass, the content of colostrum basic protein is 0.10 parts by mass, and the balance is raw cow's milk.

[0084] The specific preparation method is the same as that of Example 1. The product indicators were tested, confirming that the protein content of the finished product was 3.65 g / 100 mL, the calcium content was 120 mg / 100 mL, the pH of the finished product was 6.67, and the finished product was commercially sterile.

[0085] Example 3

[0086] This embodiment provides a liquid milk, the raw materials for its preparation are the same as those in Example 1, except that per 1000 parts by mass of the product, the content of sodium hyaluronate is 0.086 parts by mass, the content of N-acetylglucosamine is 0.4295 parts by mass, the content of colostrum basic protein is 0.172 parts by mass, and the remainder is raw cow's milk.

[0087] The specific preparation method is the same as that of Example 1. The product indicators were tested, confirming that the protein content of the finished product was 3.67 g / 100 mL, the calcium content was 121 mg / 100 mL, the pH of the finished product was 6.78, and the finished product was commercially sterile.

[0088] Example 4

[0089] The present embodiment provides a liquid milk, the raw materials including: hyaluronate, glucosamine, colostrum alkaline protein, milk alkaline protein, lactase, β-hydroxy-β-methylbutyrate calcium, and raw cow's milk. Among them, the hyaluronate is sodium hyaluronate with a molecular weight of 230,000 Da, and the glucosamine is N-acetylglucosamine. Per 1000 parts by mass of the product, the content of sodium hyaluronate is 0.0625 parts by mass, the content of colostrum alkaline protein is 0.0625 parts by mass, the content of N-acetylglucosamine is 0.50 parts by mass, the content of β-hydroxy-β-methylbutyrate calcium is 0.20 parts by mass, the content of milk alkaline protein is 0.2 parts by mass, the content of lactase is 1.0 parts by mass, and the balance is raw cow's milk.

[0090] The preparation method of the liquid milk comprises the following specific steps:

[0091] In a mixing tank (pot), mix the premix (sodium hyaluronate, N-acetylglucosamine, colostrum alkaline protein), lactase, milk alkaline protein, and 15 times the total weight of raw milk of β-hydroxy-β-methylbutyrate calcium. Start stirring: heat the raw milk to 45.0°C, add the premix (sodium hyaluronate: colostrum alkaline protein: N-acetylglucosamine = 1:1:8), milk alkaline protein, and β-hydroxy-β-methylbutyrate calcium in a circulation state, with the premix addition rate ≤ 1kg / min. After constant temperature stirring for 5 minutes, continue to heat to 60.0°C, add lactase, and after enzymatic hydrolysis for 30 minutes, continue to heat to 75.0°C, and continue stirring for 10 minutes to ensure that all the raw materials are dissolved and the lactase is inactivated.

[0092] The remaining raw milk from the formula is transferred to the pasteurization system, and then the prepared liquid is added online to the raw milk for the first homogenization, pasteurization, and first cooling. The specific process is as follows: the liquid is preheated to 74.0°C via a plate heat exchanger, and the first homogenization is carried out at 74.0°C under the condition of a total pressure of 168 bar (secondary pressure 35.1 bar). After the first homogenization, it is pasteurized at 84.6°C for 15 seconds, and then cooled to 4.0°C after pasteurization.

[0093] The liquid after the first cooling was preheated to 80°C through a plate heat exchanger, and then homogenized for the second time at 80°C. The total pressure of the second homogenization was 240 bar (secondary pressure 49.3 bar). After the second homogenization, ultra-high temperature sterilization was carried out at 137°C for 6 seconds.

[0094] The liquid was cooled to 25°C for filling using a sterile tank filling machine and then filled with nitrogen (the air pressure setting parameter was 2.8 bar).

[0095] The product indicators were tested and confirmed to have a protein content of 3.65 g / 100 mL, a calcium content of 125 mg / 100 mL, a pH of 6.70, and commercial sterility.

[0096] Comparative Example 1

[0097] This comparative example provides a liquid milk, the preparation method and raw materials of which are basically the same as those in Example 1, except that, per 1000 parts by mass of the product, the content of sodium hyaluronate is 0.0625 parts by mass, the content of N-acetylglucosamine is 0.1875 parts by mass, the content of colostrum basic protein is 0.4375 parts by mass, and the balance is raw cow's milk.

[0098] Comparative Example 2

[0099] This comparative example provides a liquid milk, the preparation method and raw materials of which are basically the same as those of Example 1, except that casein phosphopeptide is used instead of colostrum basic protein.

[0100] Comparative Example 3

[0101] This comparative example provides a liquid milk, the preparation method and raw materials of which are basically the same as those of Example 1, except that glucosamine hydrochloride is used instead of N-acetylglucosamine.

[0102] Experimental example

[0103] This experimental example tested the liquid milk prepared in the above examples and comparative examples, as follows:

[0104] 1. Clarification index test method: using LUMiSizer X65 stability analyzer, light factor 1.00, rotation speed 4000 rpm, temperature 25 ° C, profile 300, time interval 10 s.

[0105] 2. Particle size analysis: Median diameter (μm) test method: Use LA 960 laser particle size analyzer with a transmittance of 70%~90% to obtain the D50 median diameter index.

[0106] 3. Centrifugal Sedimentation Rate (%) Test Method: Weigh 50g of sample and place it in a centrifuge tube. Centrifuge at 3500rpm for 15min. Remove the supernatant from the centrifuge tube and place it in a 37℃ incubator to dry for 2min. Calculate the amount of sedimentation (xg). Centrifugal Sedimentation Rate = x / 50×100%.

[0107] 4. Microbiological Determination Method (Commercial Sterility): Take one sample from each batch and store it in a refrigerator at 2°C to 5°C as a control. Keep the remaining samples at 36°C ± 1°C for 10 days. Then, perform sensory, pH, and smear staining microscopy examinations. If there is no sign of microbial proliferation, the product is deemed commercially sterile. For detailed testing methods, see GB 4789.26.

[0108] Sensory evaluation method: Professional sensory training personnel (n=50 people), who are in good health, have no lactose intolerance, and usually drink milk, were organized to conduct a sensory evaluation of commercially sterilized products. The maximum score is 100 points. The evaluation criteria are shown in Table 1. The final score is the average.

[0109] Table 1 Product sensory evaluation standards

[0110]

[0111] 5. Bone density function evaluation method:

[0112] (1) The rat experiment refers to the "Health Food Function Test and Evaluation Method (2023)" to test bone density and urine calcium content.

[0113] (2) Zebrafish test: A wild-type AB strain was used to induce a zebrafish osteoporosis model with dexamethasone. During model pre-construction, a 30-fish / 25 mL system was used. After 96 hours of treatment, there was a significant difference in skull fluorescence intensity between the positive control group (alendronate 5.00 μg / mL + dexamethasone 2 μM) and the model control group (dexamethasone 2 μM) (p ≤ 0.05). There was also a significant difference in skull fluorescence intensity between the normal control group (distilled water) and the model control group (p ≤ 0.05). Statistical differences between the two groups indicate successful modeling, indicating that the concentrations of the substances used in each group are appropriate and can be used for formal experiments.

[0114] ① Experimental groups: Example 1, Example 2, Example 3, Example 4, Comparative Example 1, Comparative Example 2, and Comparative Example 3. A normal control group (distilled water), a model control group (dexamethasone 2 μM), and a positive control group (alendronate sodium 5.00 μg / mL) were also included. The experimental system consisted of 30 animals per 25 mL volume.

[0115] ② Experimental method: Add the test sample according to the group setting, and change the liquid every day. Except for the normal control group, the other experimental groups were given an additional water-soluble dexamethasone 2μM to establish a zebrafish osteoporosis model. Each embodiment and comparative example was based on the conversion method of zebrafish to human dose for efficacy evaluation in patent CN113496071 A. The feeding experiment was carried out using milk instead of water environment, with a feeding concentration of 266μg / mL and feeding for 96h. After the experiment, the zebrafish in each experimental group were stained with alizarin red. Subsequently, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. The data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. The fluorescence intensity of the zebrafish skull was analyzed, and the statistical analysis results of this indicator were used to evaluate the efficacy of the sample in increasing skull bone density.

[0116] ③Evaluation index: Analyze the fluorescence intensity of the zebrafish skull and use the statistical analysis results of this index to evaluate the efficacy of the sample in increasing bone density.

[0117] Bone density function evaluation: (1) Rat experiment: The bone calcium content or bone density is significantly higher than that of the low calcium control group (p ≤ 0.05, the difference is statistically significant) and not lower than that of the corresponding dose of calcium carbonate control group. The calcium absorption rate is not lower than that of the calcium carbonate control group. It can be determined that the test sample has the effect of improving bone density. (2) Zebrafish experiment: Compared with the model control group, the fluorescence intensity of the skull and the fluorescence intensity of osteogenesis are significantly increased (p ≤ 0.05, the difference is statistically significant).

[0118] 6. Joint function evaluation method:

[0119] Zebrafish were tested using a transgenic osteogenic green fluorescent strain (cy25) and a dexamethasone-induced zebrafish cartilage injury model. The model was pre-established using 30 zebrafish per 25 mL system. After 96 hours of treatment, significant differences in cartilage fluorescence intensity and type II collagen content were observed between the positive control group (chondroitin sulfate A sodium salt 1000 μg / mL + dexamethasone 25 μM) and the model control group (dexamethasone 25 μM) (p ≤ 0.05). Significant differences in skull fluorescence intensity and osteoblast fluorescence intensity were also observed between the normal control group (distilled water) and the model control group (p ≤ 0.05). Statistically significant differences between the two groups indicated successful model establishment.

[0120] ① Experimental groups: Example 1, Example 2, Example 3, Example 4, Comparative Example 1, Comparative Example 2, and Comparative Example 3. A normal control group (distilled water), a model control group (using 25 μM dexamethasone), and a positive control group (chondroitin sulfate A sodium salt 1000 μg / mL) were also included. The experimental system consisted of 30 animals per 25 mL volume.

[0121] ② Experimental Methods: Test samples were added according to group settings, and the solution was changed daily. Except for the normal control group, all other experimental groups were additionally administered 25 μM dexamethasone in water to establish a zebrafish cartilage injury model. In each example and comparative example, the feeding experiment was conducted using milk instead of distilled water, using a concentration of 266 μg / mL, for 96 hours, following the zebrafish-to-human dosage conversion method described in Patent CN113496071 A for efficacy evaluation. After the experiment, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. Zebrafish cartilage fluorescence intensity (1,000,000 pixels) was analyzed, and statistical analysis of this indicator was used to evaluate the cartilage injury protective efficacy of the samples. Zebrafish samples were collected according to the Zebrafish Col II ELISA kit instructions, and data were collected using a multi-function microplate reader to analyze type II collagen content (ng / mg protein).

[0122] ③Evaluation indicators: cartilage fluorescence intensity and type II collagen content.

[0123] Joint function evaluation: In zebrafish experiments, compared with the model control group, the fluorescence intensity of cartilage and the content of type II collagen were significantly improved (p≤0.05 was statistically significant).

[0124] Here are the results:

[0125] 1. The sensory evaluation results are shown in Table 2.

[0126] Table 2 Sensory evaluation results of products

[0127]

[0128] 2. The results of bone density indicators are shown in Table 3.

[0129] Table 3 Bone density test results

[0130]

[0131] 3. The results of calcium absorption rate for promoting calcium absorption are shown in Table 4.

[0132] Table 4

[0133]

[0134] 4. Contributes to joint health. The results of the zebrafish test are shown in Table 5.

[0135] Table 5

[0136]

[0137] 5. The above test results and stability data are summarized in Table 6.

[0138] Table 6

[0139]

[0140] In the table, clarification index refers to the test value of the offline sample, Δclarification index is the test value of the sample placed at room temperature for 6 months - the test value of the offline sample (absolute value), particle size median diameter (D50) refers to the test value of the offline sample, Δparticle size median diameter is the test value of the sample placed at room temperature for 6 months - the test value of the offline sample (absolute value), centrifugal sedimentation rate refers to the test value of the offline sample, Δcentrifugal sedimentation rate is the test value of the sample placed at room temperature for 6 months - the test value of the offline sample (absolute value).

[0141] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A liquid dairy product, characterized in that: include: Animal milk, hyaluronate, colostrum basic protein and N-acetylglucosamine; the mass ratio of hyaluronate, colostrum basic protein and N-acetylglucosamine is (1-2): (1-2): (1-8); The preparation method of the liquid dairy product comprises: (1) Premixing hyaluronate, colostrum basic protein, and N-acetylglucosamine, and then fully mixing with a portion of the formula amount of animal milk to obtain a liquid feed; (2) The feed liquid is mixed with the remaining amount of animal milk, and then homogenized and sterilized.

2. The liquid dairy product according to claim 1, characterized in that The mass ratio of the hyaluronate, colostrum basic protein and N-acetylglucosamine is 1:2:

8.

3. The liquid dairy product according to claim 1, characterized in that The hyaluronate is sodium hyaluronate.

4. The liquid dairy product according to any one of claims 1 to 3, characterized in that The invention also includes other functional components, which include one or more of calcium β-hydroxy-β-methylbutyrate, lactase, vitamin D, vitamin A, vitamin E, zinc salt, milk mineral salt, milk basic protein, magnesium salt, and phosphorus salt.

5. The liquid dairy product according to claim 4, characterized in that The other functional components are included in an amount of 0.5-2.5 parts by weight per 1000 parts by weight.

6. The liquid dairy product according to claim 4, characterized in that When the liquid dairy product further comprises calcium β-hydroxy-β-methylbutyrate, the mass ratio of hyaluronate to calcium β-hydroxy-β-methylbutyrate is (0.6-2):(1-2).

7. A method for preparing the liquid dairy product according to any one of claims 1 to 3, characterized in that: include: (1) Premixing hyaluronate, colostrum basic protein, and N-acetylglucosamine, and then fully mixing with a portion of the formula amount of animal milk to obtain a liquid feed; (2) The feed liquid is mixed with the remaining amount of animal milk, and then homogenized and sterilized.

8. The method according to claim 7, characterized in that In step (1), the mass of the partial formula amount of animal milk is 10-30 times the total mass of hyaluronate, colostrum basic protein and N-acetylglucosamine; And / or, in step (1), the method for fully mixing is: stirring at 40-50°C for 3-5 minutes, then heating to 70-75°C and stirring for 10-15 minutes; And / or, in step (2), the homogenization and sterilization method is: perform a first homogenization at 50-80°C, a total homogenization pressure of 150-170 bar, and a secondary pressure of 30-40 bar, then perform pasteurization, cool to 1-7°C after pasteurization, and then perform a second homogenization at 60-90°C, a total homogenization pressure of 220-240 bar, and a secondary pressure of 40-50 bar, and then perform ultra-high temperature sterilization; The pasteurization temperature is 83-87°C and the time is 15s-20s, and the ultra-high temperature sterilization temperature is 137±2°C and the time is 4-6s.

9. A method for preparing the liquid dairy product according to any one of claims 4 to 6, characterized in that: When the other functional components do not include lactase, the method comprises: (1) After premixing hyaluronate, colostrum basic protein and N-acetylglucosamine, thoroughly mix with other functional components that do not contain lactase and a portion of the formula amount of animal milk; (2) Add the remaining amount of animal milk and then homogenize and sterilize; When the other functional component is only lactase, the method comprises: (A) Premixing hyaluronate, colostrum basic protein, and N-acetylglucosamine, and then thoroughly mixing with a portion of the formula amount of animal milk; (B) adding the remaining amount of animal milk and then homogenizing and sterilizing; (C) Adding lactase; Alternatively, the method comprises: (a) premixing hyaluronate, colostrum basic protein, and N-acetylglucosamine, and then preliminarily mixing with a portion of the formulated amount of animal milk to obtain a premixed liquid; (b) mixing the primary mixed liquid with lactase, performing enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, performing enzyme inactivation and secondary mixing; (c) adding the remaining amount of animal milk to the mixture and then homogenizing and sterilizing the mixture; When the other efficacy components include lactase and other components, the method comprises: (1) premixing hyaluronate, colostrum basic protein, and N-acetylglucosamine, and then thoroughly mixing with other functional components that do not contain lactase and a portion of the formula amount of animal milk; (II) adding the remaining amount of animal milk and then homogenizing and sterilizing; (III) adding lactase; Alternatively, the method comprises: (i) premixing hyaluronate, colostrum basic protein, and N-acetylglucosamine, and then preliminarily mixing with other functional components that do not contain lactase and a portion of the formula amount of animal milk; (ii) adding lactase for enzymatic hydrolysis, and then inactivating the enzyme and performing secondary mixing after the enzymatic hydrolysis is completed; (iii) Add the remaining amount of animal milk and then homogenize and sterilize.

10. The method according to claim 9, characterized in that When the other functional components do not include lactase, in step (1) of the method, the mass of the partial formula amount of animal milk is 10-30 times the total mass of hyaluronate, colostrum alkaline protein, N-acetylglucosamine and the other functional components that do not contain lactase; When the other active ingredient is only lactase, in step (A) of the method, the mass of the animal milk in the partial formula is 10-30 times the total mass of hyaluronate, colostrum basic protein and N-acetylglucosamine; in step (a) of the method, the mass of the animal milk in the partial formula is 10-30 times the total mass of hyaluronate, colostrum basic protein, N-acetylglucosamine and lactase; When the other functional components include lactase and other components, in step (I) of the method, the mass of the animal milk in the partial formula is 10-30 times the total mass of hyaluronate, colostrum alkaline protein, N-acetylglucosamine and other functional components without lactase; in step (i) of the method, the mass of the animal milk in the partial formula is 10-30 times the total mass of hyaluronate, colostrum alkaline protein, N-acetylglucosamine, other functional components without lactase and lactase.

11. The method according to claim 9 or 10, characterized in that In steps (1), (A), and (1), the method of thorough mixing is as described in claim 8; and / or, in steps (2), (B), (c), (III) and (iii), the homogenization and sterilization methods are as described in claim 8; And / or, in steps (a) and (i), the preliminary mixing method is: stirring at 40-50°C for 3-5 minutes, in steps (b) and (ii), the enzymatic hydrolysis temperature is 55-60°C, and the secondary mixing method is: stirring at 70-75°C for 10-15 minutes.

12. Use of the liquid dairy product according to any one of claims 1 to 6 or the liquid dairy product prepared by the method according to any one of claims 7 to 11 in the preparation of a product for improving bone and joint health.

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