Collagen product as well as preparation method and application thereof

Abstract

The invention relates to the technical field of collagen preparation, in particular to a collagen product as well as a preparation method and application thereof. The collagen product prepared by the invention overcomes the problems of poor dissolution effect in cold water, poor fat solubility and poor emulsifying property. The preparation method comprises the following steps: crushing bovine bones, and performing collagen extraction, enzymolysis treatment, glycosylation treatment, fatty acid amidation treatment, polyphenol grafting modification and colloid compounding to obtain the collagen product. During glycosylation treatment, the cold water dissolving effect is improved by adjusting the variety of reducing sugar, the reaction temperature and time and grafting bone peptide molecules to sugar chains, and a flavor substance is generated by the Maillard reaction in the glycosylation treatment, so that the effect of covering the fishy smell of cattle bones is achieved; relevant parameters are adjusted through fatty acid amidation treatment, the bone peptide is endowed with liposolubility and emulsibility, and the solubility and emulsibility of the collagen product are synergistically improved through colloid compounding, optimization of the use amount of various colloids and control of reaction conditions.

Description

Technical Field

[0001] The present invention relates to the technical field of collagen preparation, and specifically to a collagen product, a preparation method thereof and an application thereof. Background Art

[0002] Collagen, as a key protein widely present in animal connective tissues, occupies an extremely important position in multiple fields of food and industry. The addition of collagen can improve the texture of food, endow food with better taste, enhance elasticity in meat products, improve stability in dairy products, and greatly increase the nutritional value of food, thus being highly favored by the food industry.

[0003] However, it cannot be ignored that there are still many intractable problems with existing collagen products. In terms of solubility, traditional collagen products have good solubility in hot water, but show obvious deficiencies in cold water; in the actual application of instant bone broth, cold drinks and other products, it is difficult to achieve uniform dissolution in a short time, seriously affecting the instant solubility and taste of the products. This defect limits its wide application in scenarios that require rapid dissolution or low-temperature processing; in the prior art, high-molecular proteins are enzymatically hydrolyzed, but the enzymatic hydrolysis is incomplete, and the enzymes used in enzymatic hydrolysis have an adverse effect on the flavor of collagen products; at the same time, the fluidity becomes poor after dissolution in cold water, which greatly increases the operation difficulty, prolongs the production cycle and reduces the production efficiency in the actual production process, such as the automated filling process.

[0004] Poor fat solubility results in its inability to effectively blend with fat-soluble components. When making bone broth products containing oil, it is impossible to evenly disperse the oil, and layering is very likely to occur. Poor emulsifying property makes it difficult to effectively stabilize oil droplets when constructing an emulsion system, greatly increasing the risk of emulsion demulsification, seriously affecting the stability and shelf life of the product; these problems are like barriers, hindering the in-depth application and development of collagen products in more fields. There is an urgent need to use innovative technologies to significantly improve its solubility and flow rate in cold water, while enhancing its fat solubility and emulsifying property, so as to fully exert the superior performance of the protein after biological modification.

[0005] Therefore, a collagen product, a preparation method thereof and an application thereof are proposed. Summary of the Invention

[0006] The object of the present invention is to provide a collagen product, its preparation method and application. The collagen product is prepared through the processes of crushing bovine bones, followed by collagen extraction, enzymatic hydrolysis, glycosylation, fatty acid amidation, polyphenol graft modification, and colloid compounding. During glycosylation, by adjusting the type of reducing sugar, reaction temperature and time, sugar chains are grafted onto the osteopeptide molecules, improving the cold water solubility. During glycosylation, Maillard reaction generates flavor substances, masking the fishy smell of bovine bones. By adjusting relevant parameters during fatty acid amidation, the osteopeptide is endowed with liposolubility and emulsifying property. Then, through colloid compounding, the dosages of various colloids are optimized and the reaction conditions are controlled to synergistically enhance the solubility and emulsifying property of the collagen product. In addition, degreasing treatment and polyphenol graft modification are optimized to reduce the interference of fat oxidation and synergistically enhance the antioxidant capacity, so that the product has good antioxidant property and no precipitation after 6 months of storage.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a preparation method of a collagen product, and the preparation method is as follows:

[0009] S1 Collagen extraction

[0010] S2 Enzymatic hydrolysis

[0011] Papain and neutral protease are added to bovine bone blocks at a ratio of 2 - 5:1, and enzymatic hydrolysis reaction is carried out at 37 - 42 °C for 4 - 6 h, with an enzymatic hydrolysis rotation speed of 150 - 200 r / min to obtain enzymatically hydrolyzed collagen powder;

[0012] S3 Glycosylation

[0013] Maltose, glucose and lactose are added at a ratio of 1 - 2:1:2 - 3, and the reaction is carried out at 40 - 60 °C for 4 - 7.5 h to obtain a glycosylated osteopeptide solution;

[0014] S4 Fatty acid amidation

[0015] 9.5% - 14.0% fatty acid is added to the osteopeptide solution; 0.3% - 0.6% lipase is added at 25 - 40 °C, and the reaction is carried out at a rotation speed of 100 - 150 r / min to obtain a fatty acid amidation - modified osteopeptide solution;

[0016] S5 Polyphenol graft modification

[0017] 2% - 3% genipin is added, and after the reaction, 4% - 8% tea polyphenols are added, and the stirring reaction is carried out at 30 - 35 °C for 18 - 24 h; polyphenol - grafted modified osteopeptide powder is obtained;

[0018] S6 Colloid compounding

[0019] Add 0.1%-0.4% of hydrocolloid and deionized water, and freeze-dry to obtain a collagen product; the hydrocolloid is obtained by mixing xanthan gum, sodium caseinate, and gum arabic in a ratio of 1:1-2:1-3.

[0020] Freeze-drying first freezes the solution and then sublimes the ice under vacuum conditions, which can effectively retain the activity of collagen.

[0021] Preferably, the collagen extraction in S1 is divided into raw material pretreatment, demineralization treatment, and degreasing treatment; the soaking time for degreasing treatment is 8-12h.

[0022] During the demineralization treatment, hydrochloric acid reacts with the minerals in bovine bone to dissolve them, thereby achieving the purpose of demineralization; the reaction principle is acid-base neutralization, and the hydrogen ions in hydrochloric acid react with the metal ions in the bone minerals to undergo a displacement reaction. For example, calcium carbonate reacts with hydrochloric acid to form calcium chloride, carbon dioxide, and water; through the demineralization treatment, the interference of minerals in the subsequent extraction process on collagen can be reduced, and the purity of collagen can be improved.

[0023] The ethanol used in the degreasing treatment process can dissolve the fat in bovine bone to achieve degreasing; the degreased bovine bone can reduce the influence of fat on the extraction and quality of collagen and prevent the generation of peculiar smells due to fat oxidation.

[0024] Preferably, for the enzymatic hydrolysis treatment in S2, centrifugation and ultrafiltration treatments are also carried out after enzymatic hydrolysis; the centrifugation speed is 5000r / min, and the centrifugation time is 15-20min to remove the unreacted solid residues; the supernatant is ultrafiltered using an ultrafiltration membrane with a molecular weight cut-off of 20-30kDa to obtain a collagen solution; the collagen solution is concentrated and dried to obtain enzymatically hydrolyzed collagen powder.

[0025] The ultrafiltration process is based on the difference in molecular size. Under pressure drive, small molecule substances pass through the ultrafiltration membrane, while collagen is retained, thereby achieving separation and purification. Papain and neutral protease in the complex enzyme can specifically cut the peptide bonds in the collagen molecule and decompose it into smaller peptide segments, thereby achieving the efficient extraction of collagen from bovine bone.

[0026] Preferably, the fatty acid is obtained by mixing linoleic acid and fish oil in a mass ratio of 1-2:1-3.

[0027] Preferably, the mass ratio of deionized water to the bone peptide powder grafted with polyphenols in S6 is 6-9:10; the dissolution temperature of the hydrocolloid in deionized water is 50-60°C.

[0028] The present invention provides a collagen product obtained by the above preparation method. The raw materials for preparing the collagen product include bovine bone blocks, papain, neutral protease, maltose, glucose, lactose, linoleic acid, fish oil, genipin, tea polyphenols, xanthan gum, sodium caseinate and arabic gum.

[0029] The present invention also provides an application of a collagen product obtained by the above preparation method. The collagen product is applied to the preparation of bone broth; calculated in grams per milliliter, the collagen product is mixed and dissolved with cold water in a ratio of 1:5 to obtain bone broth.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] 1. In the enzymatic hydrolysis treatment, the enzyme quality, temperature, time and rotation speed are accurately regulated. Under the appropriate pH value, the compound protease hydrolyzes macromolecular collagen into small peptide segments. The peptide chain is shortened, hydrophilic groups are exposed, and the solubility is greatly improved. At the same time, the short peptide structure also promotes intestinal absorption; during the glycosylation treatment, the type of reducing sugar, reaction temperature and time are adjusted. The bone peptide molecules graft sugar chains, increasing steric hindrance, reducing the interaction between peptide chains, improving the cold water dissolution effect, and enhancing the solubility and fluidity in cold water; at the same time, the Maillard reaction generates flavor substances, covering the fishy smell of bone broth, improving the sensory quality, and lactose in the reducing sugar also helps to remove the fishy smell.

[0032] 2. The performance of the collagen product is improved by S4 fatty acid amidation treatment and S6 colloid compounding; during the fatty acid amidation treatment, the dosage ratio of fatty acids, the dosage of fatty acids in bone peptides and the lipase hydrolysis temperature are adjusted. Lipase catalyzes the reaction of oleic acid and bone peptides, introducing hydrophobic groups, endowing bone peptides with liposolubility, enabling them to encapsulate fat-soluble nutrients, and acting as an emulsifier in the oil-water system; during colloid compounding, xanthan gum, sodium caseinate and arabic gum are selected, and the proportions of the three, the overall dosage, the dosage of deionized water and the dissolution temperature are optimized; the three colloids cooperate with fatty acid amidation. The xanthan gum network structure encapsulates fat-soluble droplets, sodium caseinate enhances liposolubility and emulsifying properties, and arabic gum reduces the oil-water interfacial tension, jointly enhancing liposolubility and emulsifying properties.

[0033] 3. By optimizing the degreasing treatment of S1 collagen extraction and S5 polyphenol graft modification, the collagen product is endowed with good antioxidant and stability; the degreasing treatment removes bovine bone fat and reduces the interference of fat oxidation; during polyphenol graft modification, the dosages of genipin and tea polyphenols, reaction temperature and time are adjusted. Genipin promotes the covalent connection between tea polyphenols and bone peptides, and the rich phenolic hydroxyl structure of tea polyphenols endows strong antioxidant properties. The two cooperate to enhance the antioxidant capacity of bone broth, and genipin itself also has antioxidant effects; after this treatment, the initial DPPH free radical scavenging rate and lipid peroxidation inhibition rate of the product are high, and good antioxidant properties are still maintained after 6 months of storage, and no precipitation occurs. Description of the Drawings

[0034] Figure 1 This is the test result graph of the dissolution performance of the collagen product of the present invention;

[0035] Figure 2 This is the flowchart of the preparation method of the collagen product of the present invention;

[0036] Figure 3 This is the finished product graph of the collagen product of the present invention. Detailed implementation manners

[0037] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all 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.

[0038] Please refer to Figures 1 to 3 , the present invention provides a collagen product, its preparation method and application, and the technical solutions are as follows:

[0039] Example 1

[0040] The preparation method of the collagen product is as follows:

[0041] S1 Collagen extraction

[0042] Raw material pretreatment: Select fresh bovine bones, remove the attached tissues such as muscles and fats, rinse them with clean water, and remove the surface impurities and blood; break the bovine bones into small pieces for subsequent processing, and the average diameter of the broken bone pieces is 2.5 cm.

[0043] Demineralization treatment: Immerse 1 Kg of the broken bovine bone pieces in a 1 mol / L hydrochloric acid solution, and the solid-liquid ratio of the bone pieces to the hydrochloric acid solution is 1:10 (g / mL), and the soaking time is 20 h;

[0044] Degreasing treatment: Rinse the demineralized bovine bone pieces with deionized water until neutral, and then soak them in an ethanol solution with a volume fraction of 95%, and the solid-liquid ratio is 1:5 (g / mL), and the soaking time is 10 h to obtain degreased bovine bone pieces.

[0045] S2 Enzymatic hydrolysis treatment

[0046] Enzymatic extraction: 800 g of defatted bovine bone blocks were placed into a reaction kettle, and deionized water was added to completely submerge the bone blocks, with a liquid-solid ratio of 8:1 (mL / g). A composite enzyme composed of food-grade papain and neutral protease (subtilisin) was added, and the addition amount of the composite enzyme was 1.5% of the mass of the bone blocks. The mass ratio of papain to neutral protease was 3:1. The enzymatic reaction was carried out at 40 °C for 5 h, with continuous stirring during the reaction at a rotation speed of 180 r / min, and the pH was maintained at 7.3 during the enzymatic hydrolysis.

[0047] Separation and purification: After the enzymatic reaction ended, the reaction solution was centrifuged at a rotation speed of 5000 r / min for 18 min to remove the unreacted solid residues. The supernatant was processed by ultrafiltration technology using an ultrafiltration membrane with a molecular weight cut-off of 30 kDa to remove small molecule impurities and enzymes, obtaining a high-purity collagen solution.

[0048] Concentration and drying: The purified collagen solution was vacuum concentrated. Under the conditions of a vacuum degree of 0.08 MPa and a temperature of 50 °C, the collagen solution was concentrated to 820 mL. Then, the concentrated solution was dried into a powder by freeze-drying technology, obtaining enzymatically hydrolyzed collagen powder extracted from fresh bovine bone.

[0049] S3 Glycosylation treatment

[0050] Take 50 g of enzymatically hydrolyzed collagen powder; select food-grade reducing sugars (maltose, glucose, and lactose, with a dosage ratio of 1:1:2) as the reducing sugars, and their dosage is 10% of the mass of the enzymatically hydrolyzed collagen powder; prepare 200 mL of deionized water for dissolving the raw materials; add the bone peptide solution and maltose to the deionized water in sequence and stir evenly to completely dissolve the solid substances; place the reaction system in a constant temperature water bath at 60 °C for 4 h; stir once every 30 min during the reaction to ensure uniform reaction; after the reaction ends, quickly cool the reaction solution to room temperature, and use dialysis to remove the unreacted raw materials and small molecule impurities; the molecular weight cut-off of the dialysis bag is 1 kDa, and the dialysis time is 24 h, with the dialysis solution being changed multiple times during this period; the solution after dialysis is vacuum concentrated to obtain 55 g of glycosylated bone peptide solution.

[0051] S4 Fatty acid amidation treatment

[0052] Take 30 g of glycosylated osteopeptide solution; take food-grade linoleic acid and fish oil as fatty acids. The mixing mass ratio of linoleic acid and fish oil is 1:1, and the total amount of the two is 12.5% of the mass of the osteopeptide solution, that is, 3.6 g; prepare food-grade lipase and add it according to 0.5% of the mass of the osteopeptide; select 300 mL of food-grade absolute ethanol as the reaction solvent; add the osteopeptide solution and linoleic acid into absolute ethanol in sequence and stir to make them evenly dispersed; under the constant temperature condition of 40 °C, add lipase and the reaction lasts for 5 h; during the reaction, magnetic stirring is adopted with a rotation speed of 120 r / min to ensure the homogeneity of the reaction system; after the reaction, remove the lipase by filtration; the filtrate is evaporated to remove absolute ethanol by a rotary evaporator under the conditions of 45 °C and a vacuum degree of 0.08 MPa; the remaining product is washed 5 times with food-grade ethanol to remove unreacted linoleic acid; finally, remove ethanol to obtain a fatty acid amidated modified osteopeptide solution.

[0053] S5 Polyphenol grafting modification

[0054] Take 20 g of the fatty acid amidated modified osteopeptide solution after enzymatic hydrolysis. Select tea polyphenol as the grafting raw material, and its dosage is 5% of the mass of the fatty acid amidated modified osteopeptide solution, that is, 1 g. The dosage of genipin is 2.5% of the mass of the fatty acid amidated modified osteopeptide solution; the reaction solvent is 200 mL of phosphate buffer solution with a pH value of 6.5. Dissolve the osteopeptide in the buffer solution and stir evenly; under the constant temperature condition of 30 °C, add accurately weighed genipin and stir for 20 min to make it dissolve evenly, then add tea polyphenol and continuously stir and react for 20 h; after the reaction, remove unreacted small molecule substances and impurities from the reaction solution by ultrafiltration (cut-off molecular weight is 3 kDa). The ultrafiltered solution is dialyzed for 48 h with a dialysis bag (cut-off molecular weight is 1 kDa) to further purify the product. Finally, freeze-dry the dialyzed solution to obtain polyphenol grafted modified osteopeptide powder.

[0055] S6 Colloid compounding

[0056] Select food-grade xanthan gum, sodium caseinate, and gum arabic as hydrocolloids. The dosage ratio of the above three substances is 1:1:1, and the addition amount of the hydrocolloid is 0.2% of the mass of the polyphenol-grafted bone peptide powder; Prepare an appropriate amount of deionized water for dissolving the bone broth powder and the hydrocolloid; The pH of the system is 7.5. Slowly add the hydrocolloid to 16 mL of deionized water while stirring, and control the rotation speed at 200 - 300 r / min to fully dissolve the xanthan gum; Since the dissolution rate of the hydrocolloid is slow, heat it to 55 °C to accelerate the dissolution process, but it needs to be cooled to room temperature after dissolution; Add 20 g of polyphenol-grafted bone peptide powder to the dissolved hydrocolloid solution and continue stirring for 20 min to fully mix the bone broth powder and the colloid solution evenly; Freeze-dry the evenly mixed bone broth mixture, then grind it and pass through a 200-mesh sieve to obtain a bone broth powder product with a compounded hydrocolloid, that is, a collagen product. The process of the above preparation method is as Figure 2 shown; The finished product diagram of the collagen product is as Figure 3 shown.

[0057] Examples 2 - 4

[0058] Different from Example 1, the processing conditions in the S2 and S3 stages are changed, as shown in Table 1 specifically.

[0059] Table 1 S2 Enzymatic Hydrolysis Treatment and S3 Glycosylation Treatment Conditions

[0060]

[0061] Comparative Example 1

[0062] Different from Example 1, only papain is added.

[0063] Comparative Example 2

[0064] Different from Example 1, only subtilisin is added.

[0065] Comparative Example 3

[0066] Different from Example 1, an ultrafiltration membrane with a cut-off molecular weight of 100 kDa is used.

[0067] Comparative Example 4

[0068] Different from Example 1, no enzymatic hydrolysis treatment is carried out.

[0069] Comparative Example 5

[0070] Different from Example 1, only glucose is used as the reducing sugar.

[0071] Comparative Example 6

[0072] Different from Example 1, no S3 glycosylation treatment is carried out.

[0073] Experimental Example 1

[0074] The collagen products obtained in Examples 1-4 and Comparative Examples 1-6 were tested for solubility and flow properties. The specific test methods are as follows: The complete dissolution time was observed using a stopwatch. The dissolution rate was determined based on the principle of the drying and weighing method in GB 5009.3-2016 "National Food Safety Standard - Determination of Moisture in Foods", measuring the mass of the solution before and after dissolution. The complete dissolution time refers to the time required to add cold water (15°C) to the bone broth powder, with 100 g of bone broth powder corresponding to 500 mL of cold water, and stirring until no visible particles remained, and the unit is seconds. The dissolution rate was determined by calculating the ratio of the difference in solution mass before and after dissolution to the initial mass of the bone broth powder, reflecting the degree of dissolution of the bone broth powder in warm water.

[0075] The flow property test method is as follows: The viscosity of the bone broth solution was measured using a viscometer, and the unit is mPa·s. Viscosity reflects the ease of flow of the solution. The lower the viscosity, the better the fluidity. At the same time, the flow rate and shape of the solution on an inclined plane can be observed. The flow rate and shape were observed and recorded on a plane with a fixed inclination angle of 30°, which is an auxiliary index for the flow property test. The final test results are shown in Table 2 and Figure 1 as follows.

[0076] Table 2 Test Results of Solubility and Fluidity

[0077]

[0078] The collagen product prepared by the present invention was mixed and dissolved with warm water. Table 2 and Figure 1It is shown that under the conditions of Examples 1-4, the dissolution time is 32-36 s, the dissolution rate is 97.6%-98.3%, the viscosity is 22-26 mPa·s, and all of them flow smoothly and evenly. By enzymatically hydrolyzing the collagen extracted from bovine bone, adjusting the quality of the enzyme used for enzymatic hydrolysis, the enzymatic hydrolysis temperature, the enzymatic hydrolysis time and the enzymatic hydrolysis rotation speed, and treating the centrifugation time for separation and purification and the molecular weight intercepted by protein peptides, the protease can specifically recognize and cleave the peptide bonds in the bone collagen molecule under the pH value and temperature conditions of the present invention, gradually hydrolyzing the macromolecular collagen into small peptide segments. As the hydrolysis progresses, the peptide chain length gradually shortens, and the exposure of hydrophilic groups increases, thus significantly improving the solubility of the peptide; and the short peptide structure can more effectively bind to the transport proteins on the surface of intestinal epithelial cells, promoting the absorption process; it can be quickly dispersed in water, improving the solubility. Then, the S3 glycosylation treatment step is carried out. By adjusting the types of reducing sugars used and the reaction temperature and time, finally, the collagen product has good solubility and fluidity in cold water; the amino group in the bone peptide molecule undergoes a condensation reaction with the carbonyl group of glucose to form a Schiff base, and then through a series of complex rearrangement, cyclization and other reactions, a sugar chain is grafted onto the bone peptide molecule; the presence of the sugar chain increases the steric hindrance between bone peptide molecules, reducing the interaction between peptide chains, thereby improving the dissolution effect in cold water; at the same time, a series of volatile flavor substances such as furans and pyrazines are generated during the Maillard reaction, and these substances endow the bone broth with a unique caramel flavor, effectively masking the original fishy smell of the bone broth and significantly improving the sensory quality of the bone broth; the use of lactose in the reducing sugar also partially covers the original fishy smell of the bone broth.

[0079] Comparative Examples 1 and 2, compared with Example 1, use of composite enzymes, the dissolution time is prolonged, the multiple enzymes in the composite enzymes work synergistically, and can more comprehensively cut collagen peptide bonds, while the single papain has a limited range of action on the substrate, resulting in a relatively small number of small molecule peptides generated by hydrolysis, strong intermolecular interactions, and a longer time required to disperse in water; the composite enzyme can act on collagen from different sites, making the hydrolysis more complete and generating more soluble small molecule peptides. When papain is used alone, the degree of hydrolysis is limited, and some collagen cannot be fully converted into a soluble form. There are more peptides with larger molecular weights in the liquid, and the molecules are entangled and hindered with each other, which increases the internal friction when the solution flows, increases the viscosity, and reduces the flow rate. The molecular weight cutoff of Comparative Example 3 was too large, and the solubility and fluidity of the collagen product were reduced; Comparative Example 4 was not subjected to enzymatic hydrolysis and Comparative Example 6 was not subjected to glycosylation, both of which reduced the solubility and fluidity of the collagen product in cold water; Comparative Example 5 only used glucose as a reducing sugar, which had no effect on the solubility and fluidity, but the final product had a fishy smell and poor flavor. Enzymatic hydrolysis and glycosylation treatments jointly improved the solubility and fluidity of the collagen product.

[0080] Embodiment 5-8

[0081] Different from Example 1, the method of S4 fatty acid amidation treatment is changed, as shown in Table 3. The two substances in Table 3 are linoleic acid and fish oil.

[0082] Table 3S4 Fatty acid amidation treatment method

[0083]

[0084] Comparative Example 7

[0085] In contrast to Example 1, only linoleic acid was used as fatty acid.

[0086] Comparative Example 8

[0087] In contrast to Example 1, only fish oil was used as fatty acid.

[0088] Comparative Example 9

[0089] The difference from Example 1 is that the total amount of linoleic acid and fish oil is 20.0% of the mass of osteopeptide.

[0090] Comparative Example 10

[0091] The difference from Example 1 is that the reaction temperature for adding lipase is 60°C.

[0092] Experimental Example 2

[0093] The fat solubility and emulsifying properties of the collagen products prepared in Example 1, Examples 5 - 8 and Comparative Examples 7 - 10 were tested. The test method for fat solubility is as follows: Accurately weigh 0.5 g of the collagen product dissolved in cold water, place it in a stoppered test tube, add 10 mL of n - hexane (a commonly used non - polar fat - soluble solvent), tightly stopper the test tube and shake it vigorously for 1 min to fully mix the sample with n - hexane; place the test tube in a constant - temperature shaker and shake it at a speed of 150 r / min at 25 °C for 30 min; take out the test tube and let it stand for 15 min to precipitate the undissolved substances; use a pipette to draw the supernatant and test the content of the collagen product in it using a Kjeldahl nitrogen analyzer. Calculate the fat solubility by calculating the percentage of the mass of the collagen product dissolved in n - hexane in the initially weighed mass.

[0094] The test method for emulsifying properties is as follows: Prepare the oil phase (such as using soybean oil as the simulated oil phase) and the water phase (an aqueous solution containing 0.5% of the collagen product dissolved in cold water); mix the oil phase and the water phase in a 1:1 volume ratio in the container of a high - speed homogenizer and homogenize it at a speed of 10,000 r / min for 5 min to fully emulsify the oil and water to form an emulsion; take 10 mL of the emulsion and place it in a centrifuge tube, centrifuge it at a speed of 3,000 r / min for 10 min, and observe the layering of the emulsion; calculate the emulsion stability index (ESI) by measuring the percentage of the volume of the lower aqueous phase in the total volume after centrifugation. ESI=(1 - volume of the lower aqueous phase / total volume)×100%. At the same time, use a laser particle size analyzer to measure the average particle size of the oil droplets in the emulsion to evaluate the emulsifying effect. The smaller the average particle size of the oil droplets, the better the emulsifying effect. The final test results are shown in Table 4.

[0095] Table 4 Test results of fat solubility and emulsifying properties of Example 1, Examples 5 - 8 and Comparative Examples 7 - 10

[0096] Example Dissolution rate (%) ESI (%) Average particle size (μm) Example 1 35.1 72.6 4.0 Example 5 35.7 73.4 4.4 Example 6 34.0 71.5 3.6 Example 7 37.5 72.3 3.8 Example 8 36.1 73.0 4.2 Comparative Example 7 31.1 68.0 4.0 Comparative Example 8 32.7 69.1 4.2 Comparative Example 9 39.2 75.8 3.3 Comparative Example 10 27.9 65.4 5.4

[0097] The collagen products prepared by the present invention, under the conditions of Examples 1, 5 - 8, have a dissolution rate of 34.0% - 37.5%, an ESI of 71.5% - 73.4%, and an average particle size of 3.6 - 4.4 μm. In the production process of the present invention, through S4 fatty acid amidation treatment, by adjusting the dosage ratio of fatty acids, adjusting the dosage of fatty acids in osteopeptides, and controlling the enzymatic hydrolysis temperature after adding lipase, the enzymatic hydrolysis conditions are controlled, so that the collagen products have good fat solubility and emulsifying properties; lipase catalyzes the esterification reaction between the carboxyl group of oleic acid and the amino group in the osteopeptide molecule to form an amide bond, realizing the grafting of fatty acids onto the osteopeptide molecule, and changing the lipophilicity of osteopeptides by introducing hydrophobic groups; after fatty acid amidation treatment, the hydrophobic fatty acid groups endow osteopeptides with fat solubility, enabling them to stably encapsulate fat-soluble nutrients such as vitamin A and vitamin D, avoiding the loss of vitamins; one end of the modified osteopeptide molecule is a hydrophilic peptide chain part, and the other end is a hydrophobic fatty acid part, which can reduce the surface tension of the oil-water interface in the oil-water system and play the role of a natural emulsifier.

[0098] In Comparative Example 7 and Comparative Example 8, only one kind of fatty acid was used, resulting in a decrease in the dissolution rate in n-hexane and a decrease in stability compared to Example 1, and no obvious change in the average particle size; in Comparative Example 7, only linoleic acid was used as the fatty acid, resulting in a decrease in fat solubility. Linoleic acid has a specific carbon chain length and unsaturated bond structure, which can react with the amino group in the osteopeptide molecule to form an amide bond and introduce hydrophobic groups. However, due to the lack of the synergistic effect of other fatty acids, an optimal hydrophobic structure may not be formed, resulting in a less effective improvement in fat solubility than the case of the combined action of multiple fatty acids in Example 1; the emulsifier structure formed by a single fatty acid is relatively simple, with limited arrangement and stabilizing effects at the oil-water interface, and it is difficult to effectively reduce the surface tension of the oil-water interface like the synergistic modification of multiple fatty acids, resulting in a decrease in emulsion stability; in Comparative Example 8, only oleic acid was used. Although the fatty acid types in fish oil are rich, due to the inconsistent content ratio of some fatty acids with the ratio of the combined multiple fatty acids in Example 1, the effect of the introduced hydrophobic groups changes, and the fat solubility and emulsifying properties are lower than those in Example 1, but better than the fat solubility and emulsifying properties in Comparative Example 7. In Comparative Example 9, the content of fatty acids increased, introducing more hydrophobic groups and improving fat solubility. However, excessive fatty acids led to over-modification of the osteopeptide molecular structure, enhancing the interaction between osteopeptide molecules, and other properties such as the solubility of osteopeptides in water may be negatively affected to a certain extent; the collagen products obtained in Comparative Example 9 were tested according to the method of Experimental Example 1. The results showed that the enzymatic hydrolysis time was 86 s, the dissolution rate was 70.6%, the viscosity was 68 mPa·s, and it was difficult to flow. In Comparative Example 10, the reaction temperature of lipase was too high, which was not conducive to the action of lipase, so the fat solubility and emulsifying properties decreased.

[0099] Examples 9 - 11

[0100] Different from Example 7, the conditions for the colloidal compounding of S6 were changed, as specifically shown in Table 5. The three substances in Table 5 are xanthan gum, sodium caseinate, and gum arabic respectively.

[0101] Table 5 Conditions for the Colloidal Compounding of S6

[0102]

[0103]

[0104] Comparative Example 11

[0105] Different from Example 7, only xanthan gum was used as the hydrophilic colloid.

[0106] Comparative Example 12

[0107] Different from Example 7, only sodium caseinate was used as the hydrophilic colloid.

[0108] Comparative Example 13

[0109] Different from Example 7, only gum arabic was used as the hydrophilic colloid.

[0110] Comparative Example 14

[0111] Different from Example 7, the colloidal compounding process of S6 was not carried out, and the osteopeptide solution obtained in step S5 was directly freeze-dried.

[0112] Comparative Example 15

[0113] Different from Example 7, the fatty acid amidation process of S4 was not carried out.

[0114] Comparative Example 16

[0115] Different from Example 7, the amount of deionized water used was 40 parts.

[0116] Experimental Example 3

[0117] The collagen products prepared in Example 7, Examples 9-11, and Comparative Examples 11-16 were subjected to liposolubility and emulsifying property tests. The specific test method was carried out according to the method of Experimental Example 2, and the final test results are shown in Table 5.

[0118] The collagen products prepared by the present invention, under the conditions of Example 7 and Examples 9-11, have a dissolution rate of 36.8%-37.9%, an ESI of 71.9%-72.7%, and an average particle size of 3.8-4.2 μm. In the S6 colloid compounding step, by using xanthan gum, sodium caseinate, and gum arabic as hydrophilic colloids, adjusting the proportions of the above three substances, and adjusting the overall dosage of the hydrophilic colloids, while adjusting the dosage of deionized water and the dissolution temperature, the collagen products are made to have good fat solubility and emulsifying properties; xanthan gum itself does not directly improve fat solubility, but it can interact with osteopeptides with fat solubility after fatty acid amidation modification by forming a three-dimensional network structure. The hydrophilic groups on its molecular chain combine with water molecules to wrap the droplets containing fat-soluble osteopeptides, indirectly promoting the dispersion of these droplets in water, thereby macroscopically enhancing the stability of the collagen products in a fat-containing system and better exhibiting fat solubility; the three-dimensional network structure can wrap the oil droplets, further restricting the movement of the oil droplets and enhancing the stability of the emulsion. In bone broth emulsion, xanthan gum can prevent the oil droplets from floating or coalescing, making the emulsion texture more uniform. Sodium caseinate has amphiphilicity, and its hydrophobic region can interact with osteopeptides with hydrophobic groups introduced by fatty acid amidation modification, enhancing the affinity between osteopeptides and fat-soluble substances; it can form micelle structures in solution, which can wrap fat-soluble components, promote their dispersion in water, and improve the fat solubility of the collagen products; and it can form a protein film with a certain strength at the oil-water interface; one end of its molecule with a hydrophilic region faces the aqueous phase, and the other end with a hydrophobic region faces the oil phase, arranging directionally at the oil-water interface, reducing the surface tension of the oil-water interface, forming a stable emulsifying film, preventing the aggregation of oil droplets, and significantly improving the emulsifying stability and emulsifying efficiency. The lipophilic groups in the molecular structure of gum arabic improve the fat solubility of the collagen products; it can quickly form an elastic film at the oil-water interface, effectively reducing the surface tension of the oil-water interface, evenly dispersing and stably wrapping the oil droplets, and preventing the aggregation of oil droplets through steric hindrance and electrostatic repulsion, etc., improving the emulsifying stability. The above three substances act synergistically, and synergistically with fatty acid amidation treatment, jointly improving the fat solubility and emulsifying properties of the collagen products. Comparative Examples 11-13 only use one substance as the hydrophilic colloid respectively, and the dissolution rate and stability are lower than those of Example 7. Comparative Example 14 does not carry out the colloid compounding process, and the fat solubility and emulsifying properties further decline. Without the action of the hydrophilic colloid, the osteopeptides with fat solubility after fatty acid amidation modification cannot be stably dispersed in the fat-containing system with the help of the colloid in subsequent applications. During the dissolution process, the osteopeptides are prone to aggregation and it is difficult to fully exert their fat solubility, resulting in a significant decline in the fat solubility performance of the collagen products; and lacking the functions of the hydrophilic colloid such as increasing the solution viscosity and forming a stable interfacial film, when forming an emulsion, it is impossible to effectively prevent the aggregation and stratification of oil droplets, so the performance of the collagen products declines.In Comparative Example 15, the fatty acid amidation process of S4 was not carried out, and no hydrophobic group was introduced into the osteopeptide molecule, so it did not have liposolubility itself. Even if a hydrocolloid was added, the osteopeptide could not be dispersed in the oil, and the liposolubility of the collagen product was almost lost; without the osteopeptide after fatty acid amidation modification as a natural emulsifier component, it was difficult to form a stable emulsion only relying on the emulsifying ability of the hydrocolloid itself. The hydrocolloid could not effectively reduce the surface tension at the oil-water interface, and a uniform and stable emulsification system could not be formed during oil-water mixing, resulting in serious lack of emulsifying property. Therefore, the liposolubility and emulsifying property were the worst. In Comparative Example 16, the dosage of deionized water was too high, resulting in a decrease in liposolubility and stability; moreover, for the actually obtained collagen product, after being dissolved in cold water, its effective concentration decreased and the richness of the bone broth decreased.

[0119] Examples 12 - 14

[0120] Different from Example 11, the process of polyphenol graft modification in S5 and the conditions of degreasing treatment in S1 collagen extraction were changed, as shown in Table 6 specifically.

[0121] Comparative Example 17

[0122] Different from Example 11, degreasing treatment was not carried out during the S1 collagen extraction process.

[0123] Comparative Example 18

[0124] Different from Example 11, genipin was not used in the S5 polyphenol graft modification.

[0125] Comparative Example 19

[0126] Different from Example 11, tea polyphenol was not used in the S5 polyphenol graft modification.

[0127] Comparative Example 20

[0128] Different from Example 11, the reaction time was 5 h.

[0129] Table 6 The process of S5 polyphenol graft modification and the conditions of S1 collagen extraction

[0130]

[0131] Experimental Example 4

[0132] The collagen products prepared in Examples 11 - 14 and Comparative Examples 17 - 20 were subjected to stability tests, mainly including the determination of antioxidant stability and lipid peroxidation inhibition rate; the antioxidant test was mainly characterized by the DPPH free radical scavenging rate, and the higher the scavenging rate, the better the antioxidant effect. Accurately weigh 0.1 g of the collagen products obtained by different treatments, add them to 10 mL of absolute ethanol, and ultrasonically oscillate for 30 min to fully dissolve or disperse them; take 2 mL of this solution, add 2 mL of a DPPH ethanol solution with a concentration of 0.2 mmol / L, mix well, and let it stand in the dark for 30 min; use a spectrophotometer to measure the absorbance A_sample at a wavelength of 517 nm, use 2 mL of absolute ethanol instead of the sample solution to measure the blank absorbance A_blank; use 2 mL of absolute ethanol instead of the DPPH solution to measure the sample background absorbance A_background; DPPH free radical scavenging rate (%) = [1 - (A_sample - A_background) / A_blank] × 100%.

[0133] The method for determining the lipid peroxidation inhibition rate is as follows: Linoleic acid was used as a substrate to simulate the lipid system; 0.1 g of the collagen product was added to a mixed solution containing 0.1 mol / L phosphate buffer (pH 7.0), 0.05 mol / L linoleic acid, and 0.2 mol / L ethanol, with a total volume of 10 mL, and the reaction was carried out in the dark at 37 °C in a constant temperature incubator for 3 days. Take an appropriate amount of the reaction solution every 24 h, and use the thiobarbituric acid method to determine the content of malondialdehyde (MDA); lipid peroxidation inhibition rate (%) = [1 - (MDA_sample - MDA_blank) / MDA_control] × 100%, where MDA_sample is the MDA content after adding the collagen product, MDA_blank is the MDA content of the blank group without adding any antioxidant, and MDA_control is the MDA content of the control group only adding linoleic acid and buffer solution.

[0134] And the stability of the collagen products after 6 months of storage was tested. Visually observe whether there is precipitation. The final test results are shown in Table 7.

[0135] Table 7 Stability test results of the collagen products in Examples 11 - 14 and Comparative Examples 17 - 20

[0136]

[0137] The collagen products prepared by the present invention, under the conditions of Examples 11-14, the DPPH radical scavenging rate is 87.5%-89.4%, and the DPPH radical scavenging rate after 6 months of storage is 82.1%-85.9%; the lipid peroxidation inhibition rate is 73.1%-74.5%, and the lipid peroxidation inhibition rate after 6 months of storage is 66.8%-69.2%. And after 6 months of storage, after being dissolved in cold water and allowed to stand, no precipitation occurs. In Examples 11-14, by adjusting the degreasing treatment conditions for S1 collagen extraction, the amounts of genipin and tea polyphenols used in S5 polyphenol graft modification, and adjusting the reaction temperature and reaction time, the collagen has good antioxidant effects, and has good antioxidant properties and good stability after 6 months of storage; the active groups in the genipin molecule react with the phenolic hydroxyl groups of tea polyphenols and the amino groups in the osteopeptide molecule, promoting the formation of stable covalent bonds between the two, realizing the grafting of tea polyphenols on the osteopeptide molecule. Tea polyphenols have a rich phenolic hydroxyl structure and are highly efficient antioxidants. After being grafted onto the osteopeptide molecule, the two act synergistically, significantly enhancing the antioxidant capacity of bone broth. Genipin is the hydrolysis product of geniposide and also has good antioxidant effects itself; the degreasing treatment can remove the fat in bovine bone and reduce the interference of fat oxidation on the antioxidant performance of collagen products; the above conditions work together to improve the antioxidant and stability of collagen products. In Comparative Example 17, degreasing treatment was not carried out, and the fat in bovine bone was oxidized during subsequent processing and storage, generating oxidation products such as free radicals, interfering with the test results of the antioxidant performance of collagen products themselves. After 6 months of storage, a large amount of fat oxidation led to precipitation, a significant increase in acid value, and a decrease in DPPH radical scavenging rate and lipid peroxidation inhibition rate; in Comparative Examples 18 and 19, genipin and tea polyphenols were not used for treatment respectively. First, the antioxidant effects they possess cannot be achieved. Second, without adding genipin, tea polyphenols cannot be connected to polypeptides by chemical bonds, and without adding tea polyphenols, the antioxidant stability also decreases. The reaction time in Comparative Example 20 was too short, and the grafting reaction was incomplete. After 6 months of storage, the antioxidant stability decreased greatly, and a large amount of precipitation occurred.

[0138] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a collagen product, characterized in that: The preparation method is as follows: S1 Collagen Extraction S2 enzymatic treatment Add papain and neutral protease to the bovine bone block, perform enzymatic hydrolysis at 37-42°C for 4-6 hours, and the speed of enzymatic hydrolysis is 150-200 r / min to obtain enzymatic hydrolyzed collagen powder; S3 glycosylation processing Add maltose, glucose and lactose, react at 40-60°C for 4-7.5h to obtain a glycosylated osteopeptide solution; S4 fatty acid amidation treatment 9.5%-14.0% fatty acid is added to the osteopeptide solution; 0.3%-0.6% lipase is added at 25-40° C. and the reaction is carried out at a speed of 100-150 r / min to obtain a fatty acid amidation-modified osteopeptide solution; S5 polyphenol grafting modification Add 2%-3% genipin, add 4%-8% tea polyphenols after the reaction, and stir and react at 30-35° C. for 18-24 hours to obtain polyphenol grafted modified bone peptide powder; S6 colloid compound 0.1%-0.4% of hydrophilic colloid and deionized water are added, and freeze-dried to obtain the collagen product; the hydrophilic colloid is obtained by mixing xanthan gum, sodium caseinate and gum arabic in a ratio of 1:1-2:1-3.

2. The method for preparing a collagen product according to claim 1, characterized in that: S1 The collagen extraction is divided into raw material pretreatment, demineralization treatment and degreasing treatment; the soaking time of the degreasing treatment is 8-12h.

3. The method for preparing a collagen product according to claim 1, characterized in that: In S2, the enzymatic hydrolysis treatment is performed, by mass ratio, with the ratio of papain to neutral protease being 2-5:1; the ratio of maltose, glucose and lactose being 1-2:1:2-3; after enzymatic hydrolysis, centrifugation and ultrafiltration treatment are also performed; the centrifugal speed is 5000r / min, and the centrifugation time is 15-20min to remove unreacted solid residue; the supernatant is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 20-30kDa to obtain a collagen solution; the collagen solution is concentrated and dried to obtain the enzymatically hydrolyzed collagen powder.

4. The method for preparing a collagen product according to claim 1, characterized in that: The fatty acid is obtained by mixing linoleic acid and fish oil in a mass ratio of 1-2:1-3; the addition percentage of the fatty acid is the mass percentage of the osteopeptide solution; and the addition percentage of the lipase is the mass percentage of the osteopeptide solution.

5. The method for preparing a collagen product according to claim 1, characterized in that: The mass ratio of the deionized water to the polyphenol grafted modified bone peptide powder in S6 is 6-9:10; the dissolution temperature of the hydrophilic colloid in the deionized water is 50-60°C.

6. A collagen product obtained by the preparation method according to claim 1, characterized in that: The raw materials for preparing the collagen product include bovine bone block, papain, neutral protease, maltose, glucose, lactose, linoleic acid, fish oil, genipin, tea polyphenols, xanthan gum, sodium caseinate and gum arabic.

7. An application of the collagen product obtained by the preparation method according to claim 1, characterized in that: The application of the collagen product in the preparation of bone broth: the collagen product and cold water are mixed and dissolved in a ratio of 1:5 in grams per milliliter to obtain bone broth.

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