Preparation method of non-browning high grafting degree glycosylated collagen peptide

Abstract

This invention discloses a method for preparing glycosylated collagen peptides with high grafting degree and no browning, belonging to the field of peptide preparation technology. The method includes the following steps: adding reducing sugar (peptide-to-sugar molar ratio 1:0.5~1.5) to a high-concentration collagen peptide solution and stirring until completely dissolved; adjusting the pH of the solution to 6.0~8.0 and reacting at 70℃~90℃ for 15~60 min; after drying, placing the dried product in a ball mill and ball milling at 200~600 r / min for 30~240 min, finally obtaining a glycosylated collagen peptide product with high grafting degree and no browning. This invention achieves green, efficient, and high-grafting degree modification of collagen peptides by precisely controlling the hydrothermal reaction conditions and combining the mechanochemical effect of ball milling. It also completely solves the technical problems of low grafting degree and severe browning in traditional Maillard reactions. The oral bioavailability of the obtained glycosylated collagen peptides is significantly improved, which helps to promote the application and industrialization of collagen peptides in high-value-added fields.

Description

Technical Field

[0001] This invention belongs to the field of peptide technology, specifically relating to a method for preparing a browning-free, highly grafted glycosylated collagen peptide. Background Technology

[0002] Collagen peptides, as important bioactive substances, have attracted widespread attention due to their small molecular weight and easy absorption. They possess various important physiological activities, including antioxidant, antihypertensive, immunomodulatory, antibacterial, UV-inhibiting, and skin-protective activities, and have broad application prospects in pharmaceuticals, cosmetics, health foods, and food additives. However, the bioavailability of collagen peptides after oral ingestion is generally low, which is related to the hydrolytic action by a variety of peptidases abundant in the digestive tract, intestinal epithelium, and blood. This bottleneck severely restricts the efficacy and commercial application of collagen peptide products.

[0003] To address the aforementioned issues, peptide chemical modification is considered an effective technique. These methods utilize specific functional groups (such as amino, carboxyl, and thiol groups) on amino acid residues to chemically react with modifying agents, purposefully altering the structure of peptide molecules and thus endowing them with new and superior physicochemical properties or biological functions. Peptide chemical modification includes PEGylation, fatty acid modification, glycosylation, and phosphorylation. Among these, glycosylation has been shown to enhance peptide tolerance to digestive enzymes and improve transmembrane transport efficiency through intestinal epithelial cell glucose transporters.

[0004] Non-enzymatic glycosylation (Maillard reaction) utilizes the spontaneous reaction between the free amino groups at the ends of peptides and the carbonyl groups of reducing sugars. It can be carried out under controlled and safe conditions, aligning with the current trend and requirements of the food industry for natural "clean-label" ingredients. However, a technical drawback of this method is that increased grafting degree is often accompanied by a deep Maillard reaction, leading to darker product color and severe browning, affecting product appearance and quality. Therefore, how to achieve high grafting degree while effectively inhibiting browning has become a critical technical challenge that urgently needs to be overcome in this field. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing glycosylated collagen peptides with high grafting degree and no browning.

[0006] To address the aforementioned technical problems, this invention provides an application, specifically the application of reducing sugars in the preparation of glycosylated collagen peptides with high grafting degree and no browning.

[0007] Furthermore, the method for preparing browning-free, high-grafting-degree glycosylated collagen peptides provided by the present invention includes the following steps:

[0008] (1) Add reducing sugar (peptide-sugar molar ratio 1:0.5~1.5) to a high concentration solution of collagen peptides (10%~40%, w / v) and stir until completely dissolved;

[0009] (2) Adjust the pH of the solution to 6.0~8.0 and react at 70℃~90℃ for 15~60 min;

[0010] (3) After drying, the dried product is placed in a ball mill and milled at a speed of 200~600 r / min for 30~240 min to finally obtain a glycosylated collagen peptide product with high grafting degree and no browning.

[0011] The reducing sugars described in this invention include one or any combination of glucose, fructose, galactose, mannose, ribose, xylose, allulose, tagatose, arabinose, lactose, maltose, and isomaltulose.

[0012] Preferably, the wet heat reaction conditions are: peptide-to-glucose molar ratio of 1:1, initial pH of 8, peptide concentration of 40%, temperature of 90℃, and reaction time of 30 min. At this time, the system shows no browning and has the highest grafting degree.

[0013] Preferably, the increase in grafting degree is greatest when the ball milling reaction conditions are 400 r / min and 90 min.

[0014] Beneficial effects

[0015] This invention first controls the reaction at the initial stage using a wet heat method, and then promotes glycopeptide bond formation through ball milling mechanical energy, thus solving the problem of traditional Maillard reactions struggling to balance grafting degree and color. Furthermore, the process can be integrated with existing collagen peptide production lines, enabling large-scale industrial production without the need for additional complex equipment.

[0016] In this invention, the collagen peptides modified by glycosylation have higher bioavailability compared to unmodified collagen peptides. Attached Figure Description

[0017] The specific embodiments and beneficial effects of the present invention will be further described in detail below with reference to the accompanying drawings.

[0018] Figure 1 Grafting degree (a) and browning degree (b) of fish collagen peptides (FCP) and glycosylated products under different pH conditions with reaction time. Using the product obtained at an initial pH of 6 and a reaction time of 4 h as a base, the grafting degree fold (c) and browning degree fold (d) of FCP glycosylated products under different pH conditions.

[0019] Figure 2The grafting degree (a) and browning degree (b) of fish collagen peptides (FCP) and their glycosylated products under different peptide concentrations changed with reaction time. Using the product obtained at a peptide concentration of 10% and a reaction time of 4 h as a base, the grafting degree fold (c) and browning degree fold (d) of the FCP glycosylated products under different peptide concentrations were also analyzed.

[0020] Figure 3 The changes in grafting degree (a) and browning degree (b) of fish collagen peptides (FCP) and glycosylated products with reaction time under different reaction temperatures. Based on a reaction temperature of 70℃ - 4 h, the grafting degree fold (c) and browning degree fold (d) of FCP glycosylated products under each reaction temperature.

[0021] Figure 4 Effects of reaction time on grafting degree (a) and browning degree (b) of fish collagen peptides (FCP), and effect of cysteine ​​addition time on browning intensity of FCP (c).

[0022] Figure 5 The effect of ball milling time on the grafting degree of fish collagen (FCP).

[0023] Figure 6 Oral ingestion of fish collagen peptides (FCP) and plasma peptide-bound hydroxyproline concentration-time curves after glycosylation of FCP.

[0024] Figure 7 Oral intake of fish collagen peptides (FCP), iAUC (a) and bioavailability (b) of rat plasma after glycosylation of FCP. Detailed Implementation

[0025] Example 1

[0026] This embodiment provides a wet-heat method for preparing collagen peptide glycosylation products. The specific technical steps are as follows:

[0027] Accurately weigh fish collagen peptides (FCP) and glucose according to a peptide-to-glucose molar ratio of 1:1. Dissolve FCP in water to prepare a solution with a mass concentration of 10%-40%. Add the weighed glucose and stir until fully dissolved. Then adjust the pH of the solution to 6.0-10.0 and react at 70℃-100℃ for 0-360 min.

[0028] The grafting degree of FCP was determined using the OPA method. The OPA reagent consisted of 7.62 g sodium tetraborate decahydrate and 100 mg sodium dodecyl sulfate dissolved in 150 mL of ultrapure water, and 160 mg OPA dissolved in 4 mL of ethanol. After mixing all the solutions, 176 mg dithiothreitol (DTT) was added to the above solution, and the volume was adjusted to 200 mL with deionized water. The OPA reagent was prepared fresh and stored protected from light. 400 μl of sample was mixed with 3 mL of OPA reagent for 5 seconds, reacted at room temperature for 2 min, and the absorbance at 340 nm was measured using a microplate reader. This process was repeated three times. Serine solutions (0, 0.0625, 0.125, 0.25, 0.5 mg / mL) were prepared using serine as a standard. The solutions were then reacted with OPA reagent according to the above method, and the absorbance was measured to obtain the standard curve y = 3.1431x + 0.116 (R² = 0.9996).

[0029]

[0030] In the formula, DG is the grafting degree (%); A1 is the absorbance at 340 nm after the sample reaction; and A0 is the absorbance at 340 nm before the sample reaction.

[0031] The browning degree of the samples was determined using a UV spectrophotometer.

[0032] Figure 1 The effect of different initial pH values ​​on the grafting degree and browning degree of FCP was shown. Grafting degree and browning degree increased with increasing pH; however, with further increases in pH, the reaction rate became too fast, and the difficulty in controlling the reaction process increased significantly. Therefore, pH = 8 was selected as the optimal condition. Figure 2 The study demonstrated the effect of peptide concentration variations on FCP grafting degree and browning degree. Increasing substrate concentration not only provides more reaction sites, but also, in the industrial spray-drying production of collagen peptide powder, the concentration is typically controlled at 40%. This concentration parameter effectively reduces the amount of water evaporated during the spray-drying stage, improving production efficiency and lowering production costs. Therefore, a peptide concentration of 40% is selected as the optimal condition. Figure 3 The effect of temperature variation on FCP grafting degree and browning degree is shown. 90℃ can be regarded as the critical temperature for glycosylation modification, which can both enhance grafting efficiency through molecular thermal motion and effectively inhibit excessive browning. Therefore, 90℃ is selected as the optimal condition. Figure 4The effect of reaction time on the grafting degree and browning degree of collagen peptides was shown. A reaction time of 30 min resulted in the highest accumulation of the Maillard first-stage product, i.e., the glycosylated modification product, yielding glycosylated FCP with a grafting degree of 22.78 ± 1.36%. In summary, the optimal wet-heat method conditions in this embodiment are: pH = 8, glycopeptide ratio 1:1 (n / n), peptide concentration 40%, and reaction time 30 min, yielding glycopeptides with a grafting degree of 22.78 ± 1.36%.

[0033] Example 2

[0034] This embodiment provides a ball milling method for preparing collagen peptide glycosylation products. The specific technical steps are as follows:

[0035] The Maillard reaction of FCP with glucose was carried out under the humid heat conditions of Example (1), and after cooling, it was spray-dried at an inlet temperature of 180°C and an outlet temperature of 90°C. The dried product was then transferred to a ball mill and ball-milled for 0, 15, 30, 60, 120, 240 and 360 min at a speed of 400 rpm. Figure 5 When the ball milling time is 90 min, the grafting degree reaches 34.41 ± 1.05%.

[0036] Example 3

[0037] This embodiment provides a method for determining the oral bioavailability of collagen peptides. Using glycosylated FCP and FCP prepared under the optimal process conditions in Example 2 as the research subjects, this embodiment employs a rat animal model to determine the oral bioavailability of collagen peptides. The specific technical steps are as follows:

[0038] Male SD rats (8-9 weeks old) were acclimatized for one week with a standardized diet (AIN-93M) and free access to drinking water. The SD rats were randomly divided into a control group (physiological saline), a test group (FCP and glycosylated FCP), an oral reference preparation group (GPH(ig)), and an injection reference preparation group (GPH(iv)), with 6 rats in each group. The gavage dose was calculated based on a hydroxyproline content of 82 mg / kg. Rats were fasted overnight and had free access to drinking water before the experiment. The following morning, the corresponding drugs were administered by gavage. Blood samples were collected from the tail at 0 h, 0.5 h, 1 h, 2 h, 4 h, and 8 h after gavage, using anticoagulant tubes. After the blood samples were allowed to stand at 4°C for 30 min, they were centrifuged at 3000 rpm for 15 min. The supernatant was used to determine the peptide-bound hydroxyproline content, and pharmacokinetic curves were plotted to calculate bioavailability.

[0039] Relative bioavailability of oral peptides: The relative amount of hydroxyproline-containing dipeptides / tripeptides that enter the circulation in vivo, obtained by oral gavage with GPH as a reference.

[0040] Absolute bioavailability of oral peptides: The relative amount of hydroxyproline-containing dipeptides / tripeptides that enter the circulation in vivo, obtained with intravenous GPH as a reference.

[0041] Calculation formula:

[0042]

[0043]

[0044]

[0045]

[0046]

[0047] In the formula, F r F represents the relative oral bioavailability. s The absolute oral bioavailability is represented by AUC. The areas under the plasma concentration-time curves for the test formulation, oral reference formulation, injectable reference formulation, and blank control are the test formulation group, oral reference formulation group, injectable reference formulation group, and blank control, respectively. The areas under the curve for iAUC of the test formulation, oral reference formulation, injectable reference formulation group, and blank control are the test formulation group, oral reference formulation group, injectable reference formulation group, and blank control, respectively.

[0048] Figure 6 The results showed that, 1 h after administration, the peak concentration of peptide-bound hydroxyproline in the plasma of rats administered glycosylated FCP by gavage was 4.85 μg / mL higher than that in the FCP group by gavage; the AUC of the glycosylated FCP group was 17.43 h·μg / mL higher than that of FCP. Under the same administration conditions, oral glycosylated FCP was absorbed by the body to a greater extent than oral FCP. Figure 7 The relative bioavailability of FCP and glycosylated FCP were 43.86 ± 9.80% and 78.26 ± 13.49%, respectively, and the absolute bioavailability were 4.37 ± 0.97% and 7.81 ± 1.34%, respectively. The bioavailability of glycosylated FCP was 1.78 times that of FCP. The results indicate that glycosylation modification improves the bioavailability of collagen peptides.

[0049] This invention addresses the current state of industry technology by precisely controlling the wet-heat reaction conditions and combining them with the mechanochemical effect of ball milling. This achieves green, efficient, and high-grafting-degree modification of collagen peptides, while completely solving the technical problems of low grafting degree and severe browning of products in traditional Maillard reactions. The oral bioavailability of the obtained glycosylated collagen peptides is significantly improved, which helps to promote the application and industrialization of collagen peptides in high-value-added fields.

Claims

1. A method for preparing a browning-free, high-grafting-degree glycosylated collagen peptide, characterized in that, Includes the following steps: (1) Add glucose to a high-concentration collagen peptide solution and stir until completely dissolved; (2) Adjust the pH of the solution to 6.0~8.0 and react at 70℃~90℃ for 15~60 min; (3) After drying, the dried product is placed in a ball mill and ball milled at a speed of 400 r / min for 30 to 240 min to finally obtain glycosylated collagen peptides with high grafting degree and no browning. The mass-volume ratio of the high-concentration collagen peptide solution in step (1) is 10%~40%; the molar ratio of collagen peptide to glucose in step (1) is 1:0.5~1.5; The oral bioavailability of the glycosylated collagen peptides prepared by the method described above is significantly improved.

2. The method for preparing a browning-free, high-grafting-degree glycosylated collagen peptide according to claim 1, characterized in that, In step (1), the molar ratio of collagen peptides to glucose is 1:

1.

3. The method for preparing a browning-free, high-grafting-degree glycosylated collagen peptide according to claim 1, characterized in that, In step (2), the pH of the solution is adjusted to 8.

0.

4. The method for preparing a browning-free, high-grafting-degree glycosylated collagen peptide according to claim 1, characterized in that, The ball milling time in step (3) is 90 min.

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