Glycosylated bovine collagen antioxidant peptide, and preparation method and application thereof

Abstract

The present application relates to the field of food processing and biotechnology, and in particular to a glycosylated bovine bone collagen antioxidant peptide, a preparation method and application thereof. The glycosylated bovine bone collagen antioxidant peptide is prepared by enzymolysis and Maillard reaction using Yanbian yellow cattle bone as raw material, and the core antioxidant peptide sequence is shown as SEQ ID NO: 1-7. The present application uses Maillard reaction to endow the Yanbian yellow cattle bone collagen peptide with excellent caramel aroma, meat aroma and salty and fresh characteristics, and the antioxidant mechanism of the antioxidant peptide is jointly confirmed through multiscale simulation and in vitro experiment. Therefore, the present application integrates a set of high-value utilization route of bovine bone by using enzymolysis and Maillard modification, realizes the transformation of waste into treasure of slaughter by-products, and meets the green and sustainable development concept.

Description

Technical Field

[0001] This invention relates to the fields of food processing and biotechnology, specifically to a glycosylated bovine bone collagen antioxidant peptide, its preparation method, and its application. More specifically, it relates to a glycosylated bovine bone collagen antioxidant peptide prepared from Yanbian yellow cattle bone through a combination of enzymatic hydrolysis and Maillard reaction, and its application in functional food ingredients and natural antioxidants. Background Technology

[0002] my country is a major producer of livestock and poultry bone resources globally, with an annual output exceeding 17 million tons, of which approximately 5 million tons are bovine bones. However, the current comprehensive utilization rate of bovine bones is less than 10%, with large quantities of collagen-rich bone resources being subjected to low-value processing or directly discarded. This not only causes serious resource waste but also creates environmental pressure. Therefore, developing high-value utilization pathways for bovine bone resources has significant research importance and practical value.

[0003] Bovine bones contain over 20% collagen and are rich in glycine, proline, and hydroxyproline, making them an excellent raw material for preparing bioactive peptides. Studies have shown that bovine bone collagen peptides possess various biological activities, including antioxidant activity, inhibition of angiotensin-converting enzyme (ACE), and promotion of bone proliferation. However, collagen peptides prepared by traditional enzymatic hydrolysis methods generally suffer from flavor defects such as a strong bitter taste and residual fishy odor, and their antioxidant activity is limited, severely restricting their further application in food and functional products.

[0004] The Maillard reaction (MR) is a food-grade, green modification technology that effectively improves the flavor characteristics and enhances the antioxidant activity of protein hydrolysates through the condensation reaction between reducing sugars and amino compounds. Studies have shown that the Maillard reaction can significantly enhance the free radical scavenging and metal ion chelating abilities of peptides, while generating characteristic flavor compounds such as pyrazines and furans, imparting pleasant flavors such as roasted, caramel, and meaty aromas to the products.

[0005] The progress and product characteristics of the Maillard reaction are highly dependent on key parameters such as reaction temperature, time, and peptide-to-glycan ratio. Too low a temperature, insufficient reaction time, or an excessively high peptide-to-glycan ratio will lead to incomplete reaction, a lighter color, and an inability to form the typical "meaty" base. Too high a temperature or too long a reaction time will result in over-reaction, flavor degradation, and increased safety risks. Therefore, the reaction process must be strictly monitored.

[0006] However, there are few studies on Maillard reaction modification of bovine bone collagen peptides, especially the structure-activity relationship between changes in peptide molecular structure and enhanced antioxidant activity during the reaction is still unclear. Key active peptides lack precise identification, and their antioxidant mechanism and the contribution of key active sites still need to be systematically elucidated.

[0007] To address the aforementioned issues, computer-aided simulation techniques such as molecular docking, molecular dynamics simulation, and quantum chemistry have been increasingly applied to the screening and mechanism analysis of bioactive peptides in recent years. These techniques can reveal the binding modes, dynamic stability, and electron donor characteristics of peptides to target proteins at the molecular and electronic levels, providing a theoretical basis for the rational design of highly active antioxidant peptides.

[0008] Therefore, this study aims to develop a method for preparing collagen peptides using bovine bone by-products as raw materials, which can optimize flavor and enhance antioxidant activity, and to systematically elucidate its molecular mechanism of action, in order to provide technical support for the high-value utilization of bovine bone resources and the development of natural antioxidants. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a glycosylated bovine collagen antioxidant peptide, its preparation method, and its application. This glycosylated bovine collagen antioxidant peptide possesses excellent free radical scavenging ability. Furthermore, the preparation method provided by the present invention utilizes enzymatic hydrolysis and Maillard reaction for synergistic modification, which is highly efficient and controllable. The prepared product combines flavor enhancement and antioxidant capacity.

[0010] To achieve this objective, the present invention adopts the following technical solution:

[0011] In a first aspect, the present invention provides a glycosylated bovine collagen antioxidant peptide, said antioxidant peptide comprising the peptide sequence as described in any one of SEQ ID NO:1-7.

[0012] Among them, SEQ ID NO:1 is GPSPGPGPSPG;

[0013] SEQ ID NO:2 is GPSGPAGPTG;

[0014] SEQ ID NO:3 is GPPGSAGVPGVPG;

[0015] SEQ ID NO:4 is LPAGGDLTDFLK;

[0016] SEQ ID NO:5 is GPVGPTGPVG;

[0017] SEQ ID NO:6 is AGGDLTDFLK;

[0018] SEQ ID NO:7 is IDGRPGPIGP.

[0019] In this invention, the glycosylated bovine bone collagen antioxidant peptide is prepared by enzymatic hydrolysis and Maillard reaction using Yanbian yellow cattle bone as raw material. The Maillard reaction product is identified to contain antioxidant peptides such as SEQ ID NO:1-7.

[0020] Preferably, the glycosylated bovine collagen antioxidant peptide comprises the peptide sequence as described in any one of SEQ ID NO:1-3.

[0021] Among them, SEQ ID NO:1 is GPSPGPGPSPG;

[0022] SEQ ID NO:2 is GPSGPAGPTG;

[0023] SEQ ID NO:3 is GPGGSAGVPGVPG.

[0024] In this invention, the DPPH radical scavenging rates of GPSPGPGPSPG, GPSGPAGPTG, and GPPGSAGVPGVPG were 44.17±0.96%, 34.66±2.80%, and 39.54±3.32%, respectively; the hydroxyl radical scavenging rates were 50.26±1.60%, 38.89±1.50%, and 37.63±3.00%, respectively; and the superoxide anion radical scavenging rates were 49.58±0.72%, 54.17±2.60%, and 51.67±3.61%, respectively.

[0025] In a second aspect, the present invention provides a method for preparing glycosylated bovine collagen antioxidant peptides as described in the first aspect, comprising the following steps:

[0026] (1) Using Yanbian cattle bones as raw material, extract the collagen from the Yanbian cattle bones to obtain bovine bone collagen;

[0027] (2) The bovine bone collagen powder is then enzymatically hydrolyzed using flavor protease to obtain bovine bone collagen peptides;

[0028] (3) The bovine bone collagen peptides are mixed with reducing sugars and subjected to Maillard reaction to obtain the glycosylated bovine bone collagen antioxidant peptides.

[0029] As a preferred technical solution of the present invention, the extraction operation in step (1) includes: crushing, defatting, decalcification and deproteinization.

[0030] Preferably, the extraction process includes: pulverization, defatting with n-hexane solution, decalcification with EDTA solution, and deproteinization with NaOH solution.

[0031] Preferably, in step (1), the degreasing conditions are: treatment with 10% (v / v) n-hexane solution at 4°C for 48 hours, with a material-to-liquid ratio of 1:10 (g / mL), and the extract is replaced every 12 hours.

[0032] Preferably, in step (1), the decalcification conditions are as follows: 0.5 M EDTA solution is used to treat at 4°C for 48 h, the material-to-liquid ratio is 1:10 (g / mL), and the extract is replaced every 12 h.

[0033] Preferably, in step (1), the deproteinization conditions are: treatment with 0.1 M NaOH solution at 4 °C for 8 h, and the material-to-liquid ratio is 1:10 (g / mL).

[0034] Preferably, in step (1), the pepsin extraction conditions are as follows: bone powder is immersed in a 0.5 M acetic acid solution containing 4% (w / w) pepsin at a material-to-liquid ratio of 1:10 (g / mL), and extracted with magnetic stirring at room temperature for 24 h, and the extraction is repeated 3 times.

[0035] Preferably, in step (1), the salting-out and dialysis conditions are as follows: NaCl is added to the combined supernatant until the final concentration is 0.9 M, the pH is adjusted to 7.0 using Tris buffer, and the mixture is allowed to stand overnight for salting-out; the precipitate is collected by centrifugation and reconstituted with 0.5 M acetic acid solution, and then dialyzed with 0.1 M acetic acid solution and pure water for 1 day each.

[0036] As a preferred technical solution of the present invention, the amount of flavor protease added in step (2) is 1%-5% of the bovine bone collagen protein, for example, it can be 1%, 2%, 3%, 4% or 5%; the enzyme activity of the flavor protease in step (2) is 10-50 U / mg, for example, it can be 10 U / mg, 15 U / mg, 20 U / mg, 25 U / mg, 30 U / mg, 35 U / mg, 40 U / mg, 45 U / mg or 50 U / mg, etc.

[0037] Preferably, in step (2), the amount of flavor protease added is 2% of the bovine bone collagen protein, and the enzyme activity is 20 U / mg.

[0038] Preferably, the enzymatic hydrolysis temperature in step (2) is 40℃-55℃, for example, it can be 40℃, 42℃, 43℃, 44℃, 45℃, 48℃, 50℃, 52℃, 53℃ or 55℃; the constant temperature water bath enzymatic hydrolysis time in step (2) is 4-8 h, for example, it can be 4 h, 4.5 h, 5 h, 5.5 h, 6 h, 6.5 h, 7 h, 7.5 h or 8 h, etc.

[0039] Preferably, the conditions for the directional enzymatic hydrolysis are: a material-to-liquid ratio of 1:10 (g / mL), an enzymatic hydrolysis temperature of 50℃, and a constant temperature water bath enzymatic hydrolysis time of 6 h.

[0040] As a preferred technical solution of the present invention, the reducing sugar in step (3) is xylose.

[0041] Preferably, the mass ratio of bovine bone collagen peptide to xylose is 5:1-5:3, more preferably 5:2, and the solid-liquid ratio is 1:10 (w / v).

[0042] Preferably, the bovine bone collagen peptides are covalently bound to xylose, with a grafting degree of 20%-50%.

[0043] As a preferred embodiment of the present invention, the Maillard reaction is carried out under oil bath conditions.

[0044] Preferably, the Maillard reaction temperature is 80℃-140℃, for example, it can be 80℃, 90℃, 100℃, 110℃, 120℃, 130℃ or 140℃, etc., preferably 110℃.

[0045] Preferably, the Maillard reaction time is 50-90 min, for example, 50 min, 60 min, 70 min, 80 min or 90 min, preferably 70 min.

[0046] Preferably, in step (3), the conditions for the heat-induced Maillard reaction are as follows: the reducing sugar is xylose, the mass ratio of BBCP to xylose is 5:2, the solid-liquid ratio is 1:10 (w / v), the oil bath constant temperature heating reaction temperature is 110℃, and the reaction time is 70min.

[0047] As a preferred technical solution of the present invention, the Maillard reaction product of bovine bone collagen peptides with both flavor enhancement and antioxidant functions can be prepared by the following method:

[0048] (1) Extraction and purification of bovine bone collagen: Fresh bovine bones were taken, and after removing tendons and meat, they were coarsely crushed. The bone powder was defatted with hexane solution, decalcified with EDTA solution, and deproteinized with NaOH solution in sequence. After each step, it was washed with distilled water until neutral, dried, and finely crushed. The bone powder was immersed in an acetic acid solution containing pepsin according to the material-to-liquid ratio and magnetically stirred for extraction. The supernatant was collected by centrifugation and the extraction was repeated. The supernatants were combined and salted out with NaCl overnight. The precipitate was collected by centrifugation, redissolved with acetic acid, dialyzed with acetic acid and pure water, and freeze-dried to obtain purified bovine bone collagen.

[0049] (2) Targeted enzymatic hydrolysis: The above bovine bone collagen was mixed with water, flavored protease was added, and targeted compound enzymatic hydrolysis was carried out in a constant temperature water bath. After enzymatic hydrolysis, the enzyme was inactivated by heating and freeze-dried to obtain bovine bone collagen peptide (BBCP) powder.

[0050] (3) Maillard reaction synergistic modification: The above-mentioned BBCP powder and xylose were mixed in proportion, dissolved in pure water at a certain solid-liquid ratio, sealed and placed in an oil bath for constant temperature heating to induce the Maillard reaction. After the reaction was completed, the reaction was terminated by rapid cooling in an ice bath, thus obtaining the bovine bone collagen peptide Maillard reaction product (BBCP MRPs) with both flavor enhancement and antioxidant functions. The structure before and after the Maillard reaction was characterized by grafting degree determination, molecular weight distribution, infrared, ultraviolet, fluorescence spectroscopy and circular dichroism spectroscopy.

[0051] This invention also includes component fractionation, core peptide identification, and multi-scale analysis of the product obtained in step (3). Specifically, this includes:

[0052] The product obtained in step (3) was subjected to ultrafiltration fractionation to separate the core components with a strength of <3 kDa. The volatile flavor profile was reconstructed using GC-IMS, and the core peptide sequence was accurately identified using LC-MS / MS. The antioxidant mechanism of the core peptide was analyzed by combining network pharmacology, molecular docking, molecular dynamics simulations, and quantum chemical calculations.

[0053] Thirdly, the Maillard reaction products protected by this invention, in addition to antioxidant peptides, also contain a variety of volatile organic compounds. The volatile organic compounds mentioned in this invention include at least 9 aldehydes, 5 ketones, 6 furans, 2 pyrazines and 1 alcohol.

[0054] Before the Maillard reaction, the sample was dominated by alcohols and aldehydes; after the Maillard reaction, the content of alcohols and aldehydes decreased, while the content of ketones, furans, and pyrazines increased. Among these, the small molecule components <3 kDa were further enriched with ketone and furan flavor compounds. Ketones are important flavor precursors in the Maillard reaction system, while furans and pyrazines are typical flavor compounds generated by the Maillard reaction. 2,3-Pentanedione, 2,3-Butanedione, 2-Furfural, 2,5-Dimethylfuran, 2-Ethylfuran, and 2-Methylpyrazine are characteristic compounds generated after the Maillard reaction, which can impart caramel, meat, nutty, and roasted aromas.

[0055] Fourthly, the present invention also claims the use of the bovine bone collagen peptide Maillard reaction product or the core antioxidant polypeptide in the preparation of bifunctional food ingredients.

[0056] Preferably, the dual-function food ingredient is a natural seasoning ingredient that has both antioxidant activity and saltiness-enhancing and umami-enhancing characteristics.

[0057] More preferably, in the flavoring ingredients, the relative content of newly generated characteristic volatile flavor compounds such as 2,3-Pentadione and 2-Ethyl-5-methylpyrazine is significantly positively correlated with the antioxidant activity of the peptide.

[0058] Preferably, the health food is a health food used to prevent tissue damage caused by oxidative stress in the body.

[0059] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0060] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0061] (1) Clear mechanism: The antioxidant mechanism of the product of this invention has been confirmed by multi-scale simulation and in vitro experiments. GPSPGPGPSPG exhibits top-notch microscopic electron-donating activity due to its excellent electronic structure and lowest band gap. GPSGPAGPTG and GPPGSAGVPGVPG rely on the excellent spatial skeleton flexibility to form a very stable dynamic binding with antioxidant receptors such as MMP9, MMP2 and CASP3 in vivo, and achieve efficient free radical scavenging through the hydrogen atom transfer mechanism mediated by proline and aspartic acid residues.

[0062] (2) Product advantages: Traditional thermally modified or enzymatically hydrolyzed products are often accompanied by bitterness or unpleasant taste. The product of this invention successfully breaks through this bottleneck, using the Maillard reaction to endow the product with excellent caramel aroma, meat aroma, and saltiness-enhancing and umami-enhancing characteristics. These characteristic flavor substances are highly enriched in the <3 kDa small molecule components and show a strong positive correlation with antioxidant activity, achieving a synergistic enhancement of flavor quality and antioxidant potential.

[0063] (3) High-value utilization of resources: Traditional antioxidant peptides are mostly derived from high-value plants and animals, resulting in high production costs. Beef bones, as a major by-product of meat processing, are often discarded at low value, causing serious resource waste and environmental pressure. This invention integrates a set of high-value utilization pathways of "directed enzymatic hydrolysis-Maillard modification", realizing the transformation of slaughter by-products into valuable resources, which is in line with the concept of green and sustainable development. Attached Figure Description

[0064] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0065] Figure 1 The figures shown in Example 1 are the browning index changes of bovine bone collagen peptide Maillard reaction products and the influence of electronic tongue response values. Figures A, B, and C show the browning index under different reaction temperatures, times, and peptide-to-glycan ratios, while figures D, E, and F show the influence of different reaction temperatures, times, and peptide-to-glycan ratios on the electronic tongue response values.

[0066] Figure 2 The following are structural characterization diagrams of BBCP and BBCP MRPs in Example 2; where Figure A is a comparison of grafting degree, Figure B is a comparison of hydrolysis degree, Figure C is an infrared spectrum, Figure D is an ultraviolet spectrum, Figure E is a fluorescence spectrum, Figure F is a circular dichroism chromatogram, and Figure G is a circular dichroism chromatogram. Figure 2 Estimated results of the content of hierarchical structures.

[0067] Figure 3 The graphs show a comparison of the free radical scavenging capacity and total reducing capacity of BBCP, BBCP MRPs and their ultrafiltration fractionated peptide components in Example 3; where Figure A is a comparison of DPPH free radical scavenging capacity, Figure B is a comparison of hydroxyl free radical scavenging capacity, Figure C is a comparison of superoxide anion free radical scavenging capacity, and Figure D is a comparison of total reducing capacity; different letters indicate significant differences between groups (P < 0.05).

[0068] Figure 4 The figures for Example 4 show the total amino acid composition and GC-IMS spectra of BBCP, BBCP MRPs, and their <3 kDa ultrafiltration peptide components; Figure A is the amino acid composition analysis diagram, Figure B is the volatile organic compound fingerprint spectrum, Figure C shows the changes in volatile organic compounds among the samples, Figure D shows the relative content of volatile organic compounds, and Figure E shows the trend of volatile organic compound peak intensity.

[0069] Figure 5 The figures shown are molecular docking and molecular dynamics simulation diagrams of the seven antioxidant peptides in Example 5. Figure A shows the binding affinity analysis results, Figure B shows the visualization analysis results of the nine complexes with the lowest binding energies, Figure C shows the root mean square deviation of the nine complexes with the lowest binding energies, Figure D shows the radius of gyration of the nine complexes with the lowest binding energies, Figure E shows the solvent-accessible surface area of ​​the nine complexes with the lowest binding energies, and Figure F shows the number of hydrogen bonds in the nine complexes with the lowest binding energies.

[0070] Figure 6 The above are the HOMO and LUMO orbitals and electron cloud density distribution diagrams of the antioxidant peptides in Example 5; where diagrams A-G correspond to SEQ ID No: 1-7 respectively.

[0071] Figure 7 The above figures show a comparison of the antioxidant activities of the seven antioxidant peptides in Example 6; Figure A shows a comparison of DPPH free radical scavenging rates, Figure B shows a comparison of hydroxyl free radical scavenging rates, and Figure C shows a comparison of superoxide anion free radical scavenging rates. Different letters indicate significant differences between the groups (P < 0.05). Detailed Implementation

[0072] The technical solution of the present invention will be further illustrated below through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be determined by the claims.

[0073] In the following embodiments, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the art; unless otherwise specified, all experimental methods and techniques used were conventional methods and techniques in the art.

[0074] In the following examples, the flavor protease was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Unless otherwise specified, all other reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and techniques used were conventional methods and techniques in the field.

[0075] Example 1

[0076] Preparation of Maillard reaction products of bovine bone collagen peptides

[0077] (1) Preparation of bovine bone collagen peptides: Fresh Yanbian yellow cattle bones (purchased from Niuli Yellow Cattle Specialty Store, Yanji City, Yanbian Korean Autonomous Prefecture, Jilin Province) were deboned and the tendons and meat were removed. They were then coarsely crushed using a bone crusher and successively degreased, decalcified, and deproteinized using 10% n-hexane solution (4℃, 48h, material-to-liquid ratio 1:10, liquid changed every 12h), 0.5 M EDTA (4℃, 48h, material-to-liquid ratio 1:10, liquid changed every 12h), and 0.1 M NaOH (4℃, 8h, material-to-liquid ratio 1:10). After each step, the bones were washed with distilled water until neutral. After drying at 60℃ for 6h, they were finely crushed using a grinder.

[0078] Bone meal was added to 0.5 M acetic acid containing 4% pepsin at a material-to-liquid ratio of 1:10, and the mixture was magnetically stirred for 24 h. The supernatant was collected by centrifugation, and the extraction was repeated 3 times. The supernatants were then combined.

[0079] Precipitation was performed overnight with 0.9 M NaCl (pH adjusted to 7.0 with Tris), the precipitate was collected by centrifugation, redissolved with 0.5 M acetic acid, dialyzed with 0.1 M acetic acid and pure water for 1 day each, and then lyophilized to obtain bovine bone collagen.

[0080] (2) Enzymatic hydrolysis: Take collagen, add water at a ratio of 1:10 (g / mL) and mix well. Add flavor protease at 2% of the collagen protein mass and hydrolyze in a 50℃ water bath for 6 h. After hydrolysis, heat to inactivate the enzyme and freeze-dry the hydrolysate to obtain bovine bone collagen peptide (BBCP).

[0081] (3) Maillard reaction: Bovine bone collagen peptides and xylose were mixed in proportion and dissolved in pure water at a solid-liquid ratio of 1:10 (w / v). The mixture was then sealed with a high-temperature resistant tissue culture sealing film and placed in an oil bath for constant temperature heating.

[0082] The Maillard reaction process parameters, including heating temperature (80℃, 95℃, 110℃, 125℃, and 140℃), time (50 min, 60 min, 70 min, 80 min, and 90 min), and peptide-to-glycine ratio (5:1, 10:3, 5:2, 2:1, and 5:3), were optimized using the browning index and electronic tongue response value. Bovine bone collagen peptide Maillard reaction products (BBCPMRPs) were prepared according to the above process conditions, lyophilized, and stored at -20℃.

[0083] (4) Browning index and electronic tongue determination: The BBCP MRPs solution was diluted 20 times and the absorbance at 294 nm and 420 nm was measured using a full-wavelength microplate reader.

[0084] Dissolve 0.1 g of sodium chloride and 0.4 g of monosodium glutamate in 80 mL of pure water to prepare a umami base solution. After centrifugation, collect the supernatant, dilute it to 80 mL with the umami base solution, transfer it to a test vial, and measure it using a taste analysis system.

[0085] (5) Measurement results

[0086] Figure 1 The effects of reaction temperature, time, and peptide-to-glycan ratio on the browning index and electronic tongue response value of bovine bone collagen peptide Maillard reaction products were demonstrated.

[0087] like Figure 1 As shown in Figures A and D, as the temperature increases, A 294 and A 420 Overall increase, A at 125℃ 294 A slight decrease indicates that the Maillard reaction intermediates are rapidly transforming into the final product at this point, where the saltiness is optimally enhanced, but the umami flavor is below 110°C. At 140°C, both increase sharply, while umami, saltiness, and richness decrease significantly, indicating that excessively high temperatures can lead to over-reaction and flavor deterioration.

[0088] like Figure 1 As shown in Figures B and E, with the extension of reaction time, A 294 With A 420 A gradually increased, and at 70 min A 294 The reaction stabilizes at this point, where the saltiness, umami, and richness of the product reach their optimal values, indicating that this is a suitable endpoint for the Maillard reaction. Extending the reaction time leads to the formation of excessive end products, resulting in a decrease in saltiness, umami, and richness.

[0089] like Figure 1 As shown in Figures C and F, with the increase of the peptide-to-glycan ratio, A 294 and A 420The trend shows an initial rapid increase followed by a gradual leveling off and a slight decrease. When the peptide-to-glycan ratio is 5:2, the product exhibits the highest saltiness and richness. Further increasing the ratio leads to a slight decrease in saltiness and a significant enhancement in umami, attributed to the excessive addition of xylose, which did not fully participate in the reaction.

[0090] In summary, the optimal process conditions are: temperature 110℃, time 70 min, and peptide-glycan ratio 5:2.

[0091] Under this condition, A 294 Higher levels of these substances are beneficial for the formation of Amadori products and aldehyde / ketone intermediates with antioxidant potential, and A... 420 The color of the product did not rise sharply, and it remained stable without excessive caramelization. Electronic tongue results showed that the saltiness was significantly enhanced after the reaction, and the umami and richness were also improved, indicating that the Maillard reaction not only imparts antioxidant activity but also improves flavor.

[0092] Example 2

[0093] To demonstrate that the Maillard reaction occurred, the product obtained in Example 1 was subjected to structural characterization analysis.

[0094] (1) Grafting degree detection

[0095] Grafting degree (DG) is an important core indicator for evaluating the progress of Maillard reaction and the degree of covalent binding between proteins / peptides and sugar molecules.

[0096] Detection method: Mix 1 mL of OPA reagent with 50 μL (1 mg / mL) of sample solution, react in a 37℃ water bath in the dark for 3 min, and measure the absorbance at 340 nm. Use distilled water as a blank group to obtain the grafting degree DG.

[0097] like Figure 2 As shown in Figure A, the grafting degree of the control group was extremely low, only 1.71% ± 0.78%, indicating that there was essentially no covalent binding between the peptide and the reducing sugar in the unreacted initial system. The grafting degree of the BBCP MRPs increased significantly to 35.67% ± 1.37%, confirming that the early condensation of the Maillard reaction occurred efficiently between the bovine bone collagen peptide and the reducing sugar.

[0098] (2) Molecular weight distribution

[0099] To assess the degree of peptide crosslinking and degradation induced by the Maillard reaction, the molecular weight distribution of the samples before and after the reaction was determined.

[0100] A liquid chromatography system (1260 Infinity II, Agilent, USA) with a VWD detector and Openlab CDS Data Analysis 2.7 data processing software was used.

[0101] Molecular weight distribution was determined using a TSKgel G2000 SWXL gel chromatography column (300 mm × 7.8 mm, 5 μm), with acetonitrile-water-trifluoroacetic acid (45:55:0.1, v / v / v) as the mobile phase, a flow rate of 0.5 mL / min, a column temperature of 30℃, a detection wavelength of 220 nm, and an injection volume of 10 μL.

[0102] like Figure 2 As shown in Figure B, both samples before and after the Maillard reaction exhibited a high degree of hydrolysis, with molecular weights mainly concentrated in the <3 kDa region. After the Maillard reaction, the proportions of both low molecular weight (<1 kDa) and high molecular weight (>5 kDa) regions increased. This phenomenon can be attributed to the synergistic effect of degradation and polymerization during the reaction. On the one hand, continuous high-temperature treatment induced thermal cleavage of the peptide chain and typical Strecker degradation, leading to the conversion of medium- and long-chain peptides into smaller molecular weight fragments; on the other hand, the covalent bonding between the amino groups on the peptide side chains and the reducing sugars promoted the formation of high molecular weight compounds.

[0103] (3) Infrared spectrum

[0104] To directly confirm the primary structure modification and functional group changes induced by the Maillard reaction, Fourier transform infrared spectroscopy analysis was performed.

[0105] The sample was mixed with KBr at a ratio of 1:100 and analyzed using an FTIR spectrometer at 4000–400 cm⁻¹. -1 Scan within the range, with a resolution of 4 cm. -1 .

[0106] like Figure 2 As shown in Figure C, BBCP is at 3335.04 cm. -1 An absorption peak appears at [value missing], attributed to the NH stretching vibration, representing the free amino group (-NH2) in the peptide or amino acid. After the Maillard reaction, the intensity of this absorption peak significantly decreases, indicating that a large amount of the free amino group has been consumed; simultaneously, an absorption peak appears at a higher wavenumber (3405.51 cm⁻¹). -1 A new broad peak appears at 2937.71 cm⁻¹, which is attributed to the newly formed -NH₄⁻ or -OH⁻ after the reaction. The second peak is located at 2937.71 cm⁻¹. -1 The value at 1600-1700 cm⁻¹ represents the stretching vibration of the methylene group (-CH₂-). The characteristic absorption peak appears at 1600-1700 cm⁻¹. -1 and 1500-1550 cm -1 Within this range, these correspond to the C=O stretching vibration of the amide I band and the NH bending vibration of the amide II band, respectively. After the Maillard reaction, the intensities of both peaks decreased significantly, with the amide II band (1542.75 cm⁻¹) showing the most significant decrease. -1The peak of ) shows a significant shift, which is closely related to the formation of Schiff base (C=N). This is similar to the peak of BBCP at 1084.77 cm⁻¹. -1 Compared to the absorption peak at the same location, the peak of MRPs showed a red shift and a higher peak absorption intensity, confirming the occurrence of the glycosylation reaction.

[0107] Furthermore, following the Maillard reaction, CN stretching and NH bending (1220-1400 cm) attributed to amide III vibrations were observed. -1 The bands of the ) show dispersion and broadening, which may be due to the deamidation reaction or Strecker degradation that may occur in the late Maillard reaction, which changes the strength of the CN bond and the state of NH.

[0108] (4) Ultraviolet spectrum

[0109] To monitor structural changes and the formation of characteristic chromophores during the Maillard reaction, the UV-Vis absorption spectra of the samples were recorded.

[0110] The UV-Vis spectra of BBCP and BBCP MRPs (1 mg / mL) in the wavelength range of 200–500 nm were recorded using a spectrophotometer.

[0111] like Figure 2 As shown in Figure D, BBCP MRPs exhibit a significant absorption peak near 294 nm, which is associated with the formation of heterocyclic compounds and dicarbonyl compounds such as ketones and aldehydes. Furthermore, in the 320-420 nm range, BBCP MRPs show higher absorbance than BBCP, confirming the formation of brown polymers such as melanoidins.

[0112] (5) Fluorescence spectrum

[0113] Fluorescence spectroscopy provides insights into the spectral properties, concentration, and intensity of fluorescent compounds produced during the reaction.

[0114] Fluorescence spectroscopy analysis was performed on BBCP and BBCP MRPs (1 mg / mL). The excitation wavelength was fixed at 347 nm, and emission spectroscopy scans were performed in the range of 400–600 nm.

[0115] like Figure 2 Figure E shows that BBCP MRPs exhibit significantly stronger fluorescence intensity in the wavelength range of 400-550 nm. This fluorescence enhancement can be attributed to the generation of intermediate Maillard reaction products with inherent fluorescence properties.

[0116] (6) Circular dichroism

[0117] To elucidate the effect of the Maillard reaction on the secondary structure of peptides, circular dichroism chromatography was performed.

[0118] Using a quartz cuvette with an optical path of 1 mm, far-ultraviolet CD spectra of 180-280 nm were collected at 25℃ with a scanning speed of 200 nm / min and a bandwidth of 0.5 nm. Secondary structure content was calculated by fitting using CDNN software.

[0119] like Figure 2 The secondary structure changes shown in Figure F further confirm that the Maillard reaction reduces ordered conformations and increases disordered structures. The secondary structure content of BBCPs and BBCP MRPs was estimated using the CD secondary structure analysis tool. Figure 2 (See Figure G). Compared with BBCP, the α-helix content of BBCP MRPs decreased significantly from 30.20% to 12.99%, the β-sheet content increased from 0.63% to 4.76%, the antiparallel β-sheet and β-turn contents decreased slightly, and the random coil content increased significantly.

[0120] The above results indicate that the Maillard reaction promotes the conversion of the α-helix to random coils and β-sheets. This is because, during the Maillard reaction, the reducing sugar covalently binds to the free amino groups of the peptide, and the resulting glycosylated products disrupt the original hydrogen bond network of the α-helix, thereby promoting the peptide conformational shift towards random coils and β-sheets.

[0121] Example 3

[0122] To evaluate the antioxidant activity of BBCP before and after Maillard reaction and to screen for the component with the best activity, the free radical scavenging ability and reducing power of BBCP, BBCP MRPs and ultrafiltration components were measured.

[0123] BBCP MRPs were reconstituted with pure water, and the supernatant was collected after centrifugation. The supernatant was then subjected to ultrafiltration fractionation using 10 kDa and 3 kDa ultrafiltration centrifuge tubes to obtain three fractions: <3 kDa, 3-10 kDa, and >10 kDa.

[0124] The antioxidant capacity of the three ultrafiltration components, BBCP, and BBCP MRPs was determined using chemical methods, specifically including:

[0125] (1) Determination of DPPH free radical scavenging activity: The activity was determined using a commercially available kit.

[0126] like Figure 3As shown in Figure A, the DPPH radical scavenging rate of BBCP was only 10.58±1.92%. After Maillard reaction, the scavenging rate of BBCP MRPs increased to 42.68±3.87%, showing a significant increase. Further ultrafiltration separation revealed the highest scavenging rate for the <3 kDa fraction, reaching 49.60±2.96%, indicating that low molecular weight peptides are the main contributing components to this activity.

[0127] (2) Determination of hydroxyl radical scavenging activity: The activity was determined using a commercially available kit.

[0128] like Figure 3 As shown in Figure B, in terms of hydroxyl radical scavenging, BBCP scavenging rate was 57.05±0.21%, which increased to 71.52±0.29% after the reaction. The <3 kDa component further increased to 88.87±0.14%, making the most significant contribution.

[0129] (3) Determination of superoxide anion free radical scavenging ability: The pyrogallol auto-oxidation method was used for determination.

[0130] like Figure 3 As shown in Figure C, the superoxide anion scavenging capacity did not change much before and after the reaction. Among them, the scavenging rate of the <3 kDa component was 69.84±2.75%, which was higher than that of other components.

[0131] (4) Determination of reducing power: The iron reduction method was used for determination.

[0132] like Figure 3 As shown in Figure D, BBCP exhibits extremely low ferric reducing power (0.01±0.00), which significantly increases after Maillard reaction (0.30±0.01), with the <3 kDa fraction showing the strongest activity (0.46±0.00). These results indicate that the antioxidant activity of BBCP MRPs is significantly higher than that of BBCP, suggesting that the Maillard reaction significantly enhances the free radical scavenging ability of the peptides.

[0133] The antioxidant activities of different molecular weight components vary significantly, with the low molecular weight components (<3 kDa) exhibiting the most prominent antioxidant activity and being the key research target for subsequent screening of active peptides.

[0134] Example 4

[0135] To elucidate the compositional characteristics of amino acids and volatile flavor compounds related to flavor formation in the samples, the total amino acid composition was determined and volatile compounds were analyzed.

[0136] (1) Total amino acid composition analysis

[0137] Given that the degradation and transformation of amino acids during the Maillard reaction are the fundamental driving force for flavor formation, total amino acid analysis was conducted to monitor the changes in amino acid composition.

[0138] The sample was hydrolyzed with 6 mol / L hydrochloric acid at 105℃ for 24 hours, neutralized, brought to a final volume, and centrifuged. The supernatant was used for derivatization, and then C0.05 was used. 18 The chromatographic column (250 mm × 4.6 mm, 0.5 μm) was used for detection by high performance liquid chromatography.

[0139] The chromatographic conditions were: column temperature 38℃, detection wavelength 360 nm, flow rate 1.0 mL / min, and injection volume 20 μL.

[0140] Amino acid composition analysis showed that ( Figure 4 (Figure A) The total amino acid content of BBCP MRPs decreased significantly compared to BBCP, with lysine (Lys) showing the largest decrease (29.3%), confirming that its ε-amino group is the most active site in the Maillard reaction. The simultaneous decrease in arginine (Arg) promoted the formation of antioxidant heterocyclic compounds (such as pyrazines). Studies have shown that arginine and lysine are the main amino acids involved in the Maillard reaction.

[0141] In addition, the proportions of aspartic acid (Asp) and glutamic acid (Glu) were increased in BBCP MRPs and <3 kDa components, which is consistent with the enhanced umami flavor and the synergistic effect of saltiness and umami.

[0142] Although the triple helix structure of collagen is partially damaged due to a slight decrease in Pro and Hyp, the <3 kDa component still retains amino acids such as Gly, Pro, Ala, Glu, and Arg. The Hyp content is still relatively high in the low molecular weight component, indicating that this component is a functional product with collagen structural characteristics.

[0143] (2) GC-IMS detection

[0144] To comprehensively construct a volatile flavor fingerprint spectrum and capture the evolution of aromatic compounds induced by Maillard reactions, GC-IMS technology was used for analysis.

[0145] Volatile compounds were analyzed using gas chromatography-ion mobility spectrometry (GC-IMS). Samples were prepared as 30 mg / mL solutions with pure water. 2 mL of each solution was placed in a 20 mL headspace vial, and three replicates were prepared for each sample. The injection volume was 500 μL, and the mixture was incubated at 60 °C for 15 min with a stirring speed of 500 r / min.

[0146] Figure 4 Figure B, using BBCP1 as a reference, shows the differential volatile organic compound fingerprints obtained by GC-IMS. This fingerprint spectrum ( Figure 4 Figure C shows the changes in volatile organic compounds among the samples, with colors ranging from blue to red representing increasing concentrations. A total of 23 compounds were identified, including 9 aldehydes, 5 ketones, 6 furans, 2 pyrazines, and 1 alcohol.

[0147] Figure 4 Figures D and E show the relative content and peak intensity trends of five major volatile flavor compounds in BBCP, BBCP MRPs, and BBCP MRPs <3 kDa, respectively.

[0148] Before the Maillard reaction, the sample was dominated by alcohols and aldehydes; after the Maillard reaction, the contents of alcohols and aldehydes decreased, while the contents of ketones, furans, and pyrazines increased. Among them, the small molecule components <3 kDa were further enriched with ketone and furan flavor compounds.

[0149] Ketones are important flavor precursors in the Maillard reaction system, with furans and pyrazines being typical flavor compounds generated by the Maillard reaction. 2,3-Pentadione, 2,3-Butanedione, 2-furaldehyde, 2,5-Dimethylfuran, 2-Ethylfuran, and 2-Methylpyrazine are characteristic compounds generated after the Maillard reaction, contributing caramel, meat, nutty, and roasted aromas.

[0150] Example 5

[0151] To elucidate the molecular composition, binding mechanism, and electronic structure characteristics of Maillard reaction products, the compositional spectra of the samples were determined by LC-MS / MS. The binding modes of the active molecules to the target receptor and the conformational stability of the complex were determined by molecular docking and molecular dynamics simulations, respectively. The frontier molecular orbital energy levels and electrostatic potential distributions of the key products were determined by quantum chemical calculation methods.

[0152] (1) Liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis

[0153] The peptide sequences of <3 kDa BBCP MRPs components were identified by LC-MS / MS. After desalting with ZipTip C18, the peptides were eluted with 60% ACN / 0.1% TFA and then vacuum dried.

[0154] Separation was performed using liquid chromatography and a PepMap RSLC C18 column (75 μm × 150 mm, 2 μm). The mobile phases were 0.1% formic acid-2% ACN and 0.1% formic acid-ACN, with a flow rate of 300 nL / min and gradient elution for 60 min.

[0155] High-resolution mass spectrometry was used for analysis. The raw mass spectrometry data were analyzed by database retrieval using PEAKS Studio software. The database searched was the UniProtKB bovine species protein database (Proteome ID: UP000009136).

[0156] (2) Computer screening of antioxidant peptides

[0157] The ToxinPred and AllerTOP v2.1 tools were used to screen bioactive peptides (Peptide Ranker score > 0.5) for safety. The non-toxic and non-allergenic sequences obtained from the screening were further used to predict their antioxidant activity using the deep learning tools AnOxPP-1.0 (http: / / www.cqudfbp.net / AI-Tools / AnOxPP / mode / modeInput.jsp) and AnOxPePred-1.0 (https: / / services.healthtech.dtu.dk / services / AnOxPePred-1.0 / ).

[0158] The prediction results are shown in Table 1.

[0159] Table 1. Calculation and screening of antioxidant peptide sequences and their bioactivity.

[0160] (3) Molecular docking and molecular dynamics simulation

[0161] To assess the binding affinity between the screened peptides and candidate target proteins and to explore their specific intermolecular interactions, molecular docking analysis was performed. Simultaneously, to further evaluate the structural stability and dynamic behavior of the peptide-receptor complex under simulated physiological conditions, molecular dynamics simulations were conducted.

[0162] Seven peptides were used to perform molecular docking with six core targets (Table 2).

[0163] Table 2. Core targets of antioxidant peptides in regulating oxidative stress

[0164] The 3D structures of the ligands were prepared using ChemOffice software, and the 2D structure files were sourced from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). High-resolution crystal structures were obtained from the RCSB PDB database (https: / / www.rcsb.org / ) and processed using PyMOL software as acceptors. Docking operations were performed using AutoDock Vina 1.5.6 software, and hydrogenation, dehydration, and grid cell configuration were performed using AutoDock Tools 1.5.7 software.

[0165] The optimal conformation was determined based on the binding score, and the top five complexes were visualized using PyMOL and Discovery Studio 2019 software. Molecular dynamics simulations were performed for 100 ns using Gromacs 2022 software. The complexes were solvated in a water tank with periodic boundary conditions set at 1.2 nm. Electrostatic interactions were managed using the PME and Verlet algorithms. NVT and NPT equilibrations were performed sequentially (100 ps, ​​τ = 0.1 ps), followed by a 100 ns production run at 310 K and 1 bar.

[0166] Figure 5 As shown in Figure A, all seven antioxidant peptides exhibited good binding affinity to the six core targets. Visual analysis was performed on the nine complexes with the lowest binding energies. Figure 5 (Figure B) The results show that the peptides exhibit extremely strong binding affinity to the MMP2, MMP9, and CASP3 receptors. Among all complexes, hydrogen bonds are the most numerous and widely distributed, serving as the primary driving force for stable binding, followed by hydrophobic interactions, which are particularly prominent in the MMP9 and MMP2 complexes. Key binding sites are mainly concentrated on histidine (His) and tyrosine (Tyr) residues.

[0167] Root mean square deviation (RMSD) is a good indicator of the conformational stability of proteins and ligands. For example... Figure 5 As shown in Figure C, except for the MMP2-GPVGPTGPVG complex which showed a slight upward trend and expansion during the simulation, the other complex systems reached equilibrium within 5-90 ns, and the final RMSD fluctuation range was within 2 Å, indicating high binding stability.

[0168] Regarding the radius of gyration (Rg), except for MMP2-GPVGPTGPVG, the other complexes exhibited relatively stable fluctuations during motion, indicating that the small molecule-target protein complexes did not undergo significant expansion or contraction during movement. MMP2-GPVGPTGPVG may have experienced a conformational change or significant oscillations in the flexible loop region near the binding pocket, but subsequently returned to stability (e.g., ...). Figure 5 (As shown in Figure D).

[0169] The solvent-accessible surface area (SASA) of all complexes remained stable within 100 ns with very small fluctuations, indicating that all complexes formed after binding to the target protein exhibited kinetic stability in aqueous solution (e.g., ...). Figure 5 (As shown in Figure E).

[0170] like Figure 5 As shown in Figure F, the interaction patterns between different peptide sequences and target proteins differ significantly. GPGGSAGVPGVPG exhibits the strongest polar interaction when binding to MMP9, MMP2, and CASP3; in the later stages of the simulation, the number of hydrogen bonds between this peptide and the target protein remains stable at 4-6 with low fluctuation frequency. In the MMP9-IDGRPGPIGP and MMP2-IDGRPGPIGP systems, the number of hydrogen bonds shows a clear stepwise increase over time: the number of hydrogen bonds is low in the early stages of the simulation, and as the simulation progresses, the peptide gradually penetrates deeper into the protein binding pocket through conformational adjustments, resulting in a significant increase in the number of hydrogen bonds that tends to reach equilibrium, suggesting a high degree of structural complementarity between the peptide and the protein.

[0171] In summary, the peptides exhibited strong binding affinity to the MMP2, MMP9, and CASP3 receptors. Hydrogen bonding and hydrophobic interactions were the dominant forces driving stable binding, with His and Tyr residues serving as key binding sites. Molecular dynamics simulations verified the dynamic stability of the complexes; except for MMP2-GPVGPTGPVG, all other complexes showed stable binding.

[0172] (4) Quantum Chemistry

[0173] To predict active sites and reveal the submolecular mechanism by which active peptides scavenge free radicals, frontier molecular orbital theory was applied.

[0174] All density functional theory (DFT) calculations were performed using Gaussian 16 Rev. C.01 software. The B3LYP functional with Grimme's D3BJ dispersion correction was used, and structural optimization was performed using the def2-SVP basis set. Resonant vibrational frequencies were calculated at the same level to confirm the existence of imaginary modes. Single-point energies were refined using the def2-TZVP basis set. Intermolecular interaction analysis and visualization based on the highest occupied molecular orbital (HOMO) / lowest unoccupied molecular orbital (LUMO) were performed using Multiwfn and VMD software.

[0175] The highest occupied molecular orbital energy level (E HOMO The energy gap (EHOMO) is a key electronic parameter for measuring the antioxidant activity of peptides; a high EHOMO value lowers the energy barrier for electron transitions from neutral molecules to ionized states, thereby enhancing the reactivity of peptides as electron donors. gap E represents the chemical stability and reactivity of a molecule. gap The smaller the size, the lower the energy required for the molecule to be in an excited state, and the easier it is for electronic transitions to occur.

[0176] like Figure 6 As shown, GPSPGPGPSPG (Figure A) E gap Lowest and E HOMO A high value indicates that it is in a state of extremely high reactivity, with electrons readily transitioning from the HOMO to the LUMO, making it a peptide with the strongest potential antioxidant activity. In free radical scavenging experiments, this peptide typically exhibits the strongest electron-donating ability or the fastest transient reaction rate.

[0177] The E values ​​of LPAGGDLTDFLK (D diagram), GPVGPTGPVG (E diagram), and AGGDLTDFLK (F diagram) HOMO The value is high and E gap The lower concentration also indicates superior potential antioxidant activity. The remaining sequence E... gap and E HOMO The values ​​are shown in GPSGPAGPTG (Figure B), GPPGSAGVPGVPG (Figure C), and IDGRPGPIGP (Figure G).

[0178] The electron cloud in the peptide is mainly concentrated near proline (Pro), aspartic acid (Asp), and some aromatic residues, indicating that these regions are the main active sites for free radical scavenging reactions. Among them, the side chain of Pro forms a unique five-membered heterocyclic structure with the nitrogen atom in the backbone, and its steric strain and relatively low ionization energy make it the most critical electron donor site. The carboxyl oxygen in the Asp side chain contains abundant lone pair electrons, which can donate electrons to free radicals through the single electron transfer (SET) mechanism; aromatic amino acids have a highly delocalized π-conjugated electron system, which makes it very easy for electrons to delocalize and transfer to free radicals, and the apparent free radicals formed after losing electrons can remain relatively stable through the conjugation effect, thereby terminating the free radical chain reaction.

[0179] In summary, GPSPGPGPSPG has the lowest E gap And the highest E HOMO Propionyl groups (Pro), Asp, and aromatic amino acids are the most active electron donor peptides. These peptides primarily quench free radicals through hydrogen atom transfer (HAT) and single electron transfer (SET) mechanisms, with Pro-rich peptides exhibiting the best antioxidant potential due to their unique electron donor structure.

[0180] Example 6

[0181] To evaluate the potential antioxidant efficacy of the seven screened peptides and verify their free radical scavenging ability, their free radical scavenging ability was determined by chemical methods.

[0182] like Figure 7 As shown, Figure A is a comparison of the DPPH free radical scavenging rate of seven antioxidant peptides, Figure B is a comparison of the hydroxyl free radical scavenging rate of seven antioxidant peptides, and Figure C is a comparison of the superoxide anion free radical scavenging rate of seven antioxidant peptides.

[0183] The DPPH radical scavenging rate, hydroxyl radical scavenging rate, and superoxide anion radical scavenging rate of GPSPGPGPSPG, GPSGPAGPTG, and GPPGSAGVPGVPG (as shown in Table 3 below) were significantly higher than those of other peptides.

[0184] Table 3

[0185] GPSPGPGPSPG peptides have the lowest E gap (0.1001 eV) gives it extremely high chemosensitivity and reaction rate upon contact with free radicals, thus exhibiting excellent free radical scavenging ability. GPSGPAGPTG and GPPGSAGVPGVPG have E values ​​of [missing value]. gapAlthough relatively high, molecular dynamics analysis revealed that these two peptides exhibit stable radius of gyration (Rg) values, minimal fluctuations in solvent accessible surface area (SASA), and the most abundant hydrogen bonds, demonstrating excellent binding stability and a strong inhibitory effect on oxidative stress-related enzymes. Furthermore, they possess typical collagen repeating motifs.

[0186] Studies have shown that proline residues, due to their unique pyrrolidine ring structure, can neutralize free radicals through a stable hydrogen atom transfer (HAT) mechanism. AGGDLTDFLK ranks in the upper-middle range among various antioxidant indicators. Compared to LPAGGDLTDFLK, it removes the N-terminal leucine and proline, which may reduce steric hindrance, resulting in its higher E6 content. HOMO The electron cloud of the orbital is more readily accessible to free radical acceptors, thus outperforming LPAGGDLTDFLK in all metrics compared to its similar sequence. GPVGPTGPVG and IDGRPGPIGP perform worse in some metrics: IDGRPGPIGP has a higher E gap And E HOMO Lower; GPVGPTGPVG, although E gap It is moderate, but it exhibits poor receptor binding ability in molecular dynamics simulations.

[0187] In summary, GPSPGPGPSPG, GPSGPAGPTG, and GPPGSAGVPGVPG are the three most promising antioxidant peptides. Among them, GPSPGPGPSPG exhibits the best electron donor activity due to its lowest Egap; GPSGPAGPTG and GPPGSAGVPGVPG achieve efficient free radical scavenging through stable target binding and a proline-mediated HAT mechanism. The differences in peptide activity are mainly regulated by electronic structure, spatial conformation, and binding stability.

[0188] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A glycosylated bovine collagen antioxidant peptide, characterized in that, The glycosylated bovine collagen antioxidant peptide comprises a peptide sequence as shown in any one of SEQ ID NO:1-7; Among them, SEQ ID NO:1 is GPSPGPGPSPG; SEQ ID NO:2 is GPSGPAGPTG; SEQ ID NO:3 is GPPGSAGVPGVPG; SEQ ID NO:4 is LPAGGDLTDFLK; SEQ ID NO:5 is GPVGPTGPVG; SEQ ID NO:6 is AGGDLTDFLK; SEQ ID NO:7 is IDGRPGPIGP.

2. The glycosylated bovine collagen antioxidant peptide according to claim 1, characterized in that, The glycosylated bovine collagen antioxidant peptide comprises the peptide sequence as described in any one of SEQ ID NO:1-3; Among them, SEQ ID NO:1 is GPSPGPGPSPG; SEQ ID NO:2 is GPSGPAGPTG; SEQ ID NO:3 is GPGGSAGVPGVPG.

3. A method for preparing the glycosylated bovine collagen antioxidant peptide as described in claim 1 or 2, characterized in that, The preparation method includes the following steps: (1) Using Yanbian cattle bones as raw material, extract the collagen from the Yanbian cattle bones to obtain bovine bone collagen; (2) The bovine bone collagen powder is then enzymatically hydrolyzed using flavor protease to obtain bovine bone collagen peptides; (3) The bovine bone collagen peptides are mixed with reducing sugars and subjected to Maillard reaction to obtain the glycosylated bovine bone collagen antioxidant peptides.

4. The preparation method according to claim 3, characterized in that, The extraction operations described in step (1) include: crushing, defatting, decalcification, and deproteinization; Preferably, the extraction process includes: pulverization, defatting with n-hexane solution, decalcification with EDTA solution, and deproteinization with NaOH solution.

5. The preparation method according to claim 3, characterized in that, The amount of flavor protease added in step (2) is 1%-5% of the bovine bone collagen protein. Preferably, the enzyme activity of the flavor protease in step (2) is 10-50 U / mg; Preferably, the enzymatic hydrolysis temperature in step (2) is 40-55℃, and the enzymatic hydrolysis time in a constant temperature water bath is 4-8 h.

6. The preparation method according to claim 3, characterized in that, The reducing sugar in step (3) is xylose and / or glucose, preferably xylose; Preferably, the mass ratio of bovine bone collagen peptide to xylose is 5:1-5:3, more preferably 5:2, and the solid-liquid ratio is 1:10 (w / v). Preferably, the bovine bone collagen peptides are covalently bound to xylose, with a grafting degree of 20%-50%.

7. The preparation method according to claim 3, characterized in that, The Maillard reaction was carried out under oil bath conditions; Preferably, the Maillard reaction is carried out at a temperature of 80°C-140°C, more preferably at 110°C, and for a reaction time of 50-90 min, more preferably 70 min.

8. A Maillard reaction product of bovine bone collagen peptides, characterized in that, The Maillard reaction product is prepared by the preparation method according to any one of claims 3-7; The Maillard reaction products include glycosylated bovine collagen antioxidant peptides and volatile flavor compounds; The glycosylated bovine collagen antioxidant peptide comprises a peptide sequence as shown in any one of SEQ ID NO:1-7; The volatile flavor compounds include 2,3-pentanedione, 2,3-butanedione, 2-furfural, 2,5-dimethylfuran, 2-ethylfuran, and 2-methylpyrazine.

9. The application of the glycosylated bovine collagen antioxidant peptide as described in claim 1 or 2, or the Maillard reaction product of the bovine collagen peptide as described in claim 8, in the preparation of food or food ingredients; The food mentioned is an antioxidant food; The food ingredients are those with antioxidant, salt-enhancing, or flavor-enhancing properties.

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