Lactoferrin-polyphenol-polysaccharide ternary covalent complex, and preparation method and application thereof

Abstract

The application discloses a kind of lactoferrin-polyphenol-polysaccharide ternary covalent complex and its preparation method and application.The complex is formed by lactoferrin, epigallocatechin gallate and maltodextrin through oxidation-glycosylation double covalent modification.The preparation method comprises the following steps: first, lactoferrin is covalently combined with epigallocatechin gallate through oxidation reaction, then covalently combined with maltodextrin through Maillard reaction after dialysis purification, and finally freeze-dried to obtain the complex.The complex has significantly enhanced antioxidant activity and surface hydrophilicity, can effectively protect lactoferrin and release epigallocatechin gallate in simulated gastrointestinal digestion, can regulate intestinal flora short-chain fatty acid metabolism, and can significantly reduce water loss when used for fresh-cut apple preservation.The application solves the problems of poor stability and single function of lactoferrin, and can be widely applied in the fields of functional food, dietary supplements and fruit and vegetable preservation.

Description

Technical Field

[0001] This invention relates to the field of food processing and functional food technology, specifically to a lactoferrin-polyphenol-polysaccharide ternary covalent complex, its preparation method, and its application. Background Technology

[0002] Lactoferrin is a milk-derived bioactive protein with multiple biological functions, including immunomodulation, antibacterial activity, and antioxidant activity. However, its high sensitivity to environmental conditions (such as pH, temperature, ionic strength, and enzymes) limits its application stability. Existing technologies can enhance its functional activity through modification, but the effects are limited. Currently, few studies have constructed ternary complexes with polyphenols and polysaccharides using an oxidation-glycosylation dual covalent modification strategy, and systematically investigated their synergistic effects on digestive stability, intestinal flora metabolism regulation, and fruit and vegetable preservation.

[0003] Specifically, the existing technology has the following problems: (1) Lactoferrin has poor stability and is easily affected by factors such as pH, temperature, ionic strength and enzymes during processing and storage, which can cause structural changes or loss of function, thus limiting its direct application in food systems. (2) There is a lack of systematic research on the digestive properties, intestinal metabolic regulation and food preservation function of ternary covalent complexes. Existing research on lactoferrin modification is mostly focused on the characterization of physicochemical properties, making it difficult to fully evaluate its application potential as a functional food ingredient. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a lactoferrin-polyphenol-polysaccharide ternary covalent complex, its preparation method, and its application.

[0005] The specific technical solution of the present invention is as follows: This invention provides a lactoferrin-polyphenol-polysaccharide ternary covalent complex, which is formed by the dual covalent modification of lactoferrin, epigallocatechin gallate, and maltodextrin through oxidation and glycosylation. The lactoferrin covalently binds to epigallocatechin gallate through an oxidation reaction and covalently binds to maltodextrin through a Maillard reaction, forming a ternary covalent network structure in which epigallocatechin gallate and maltodextrin jointly modify lactoferrin.

[0006] The preparation method of the ternary covalent complex includes the following steps: (1) Prepare a 0.4% (w / v) solution of lactoferrin with distilled water and hydrate it overnight at 4°C; prepare a 0.05% (w / v) stock solution of epigallocatechin gallate with distilled water; mix the lactoferrin solution and the epigallocatechin gallate stock solution at a volume ratio of 1:1, adjust the pH to 9.0, and stir the mixture at 25°C for 24 hours in the presence of air to obtain the lactoferrin-epigallocatechin gallate reaction solution; (2) Transfer the reaction solution obtained in step (1) to a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and dialyze with deionized water for 48 hours, changing the water every 6-8 hours during the process, to obtain a purified lactoferrin-epigallocatechin gallate divalent complex. (3) Add maltodextrin to the lactoferrin-epigallocatechin gallate binary covalent complex obtained in step (2), with a mass ratio of lactoferrin to maltodextrin of 3:4. Stir at room temperature for 2 hours and hydrate overnight at 4°C. Adjust the pH to 11.0 and incubate at 75°C for 6 hours to obtain the lactoferrin-epigallocatechin gallate-maltodextrin ternary covalent complex reaction solution. (4) The reaction solution obtained in step (3) is freeze-dried to obtain lactoferrin-epigallocatechin gallate-maltodextrin ternary covalent complex.

[0007] The present invention also provides an application of the above-mentioned lactoferrin-polyphenol-polysaccharide ternary covalent complex in the preparation of dietary supplements or fruit and vegetable preservatives with antioxidant and intestinal flora regulation functions.

[0008] The application method of the ternary covalent complex is as follows: the complex is used for the preservation treatment of fresh-cut fruits, specifically by soaking the fresh-cut fruits in a solution containing the complex for 1 hour, taking them out and draining them, which can form a dense protective film on the surface of the fruits and vegetables and reduce water evaporation; or the complex is used as a functional food ingredient to prepare dietary supplements with antioxidant and intestinal flora regulation functions.

[0009] The present invention also provides the application of the above-mentioned lactoferrin-polyphenol-polysaccharide ternary covalent complex in the preparation of oral sustained-release formulations of active ingredients with digestive stability protection function.

[0010] The beneficial effects of this invention are as follows: (1) This invention successfully constructed a lactoferrin-epigallocatechin gallate-maltodextrin ternary covalent complex through an oxidation-glycosylation dual covalent modification strategy. Compared with unmodified lactoferrin, the surface hydrophilicity of this complex is significantly improved, while the DPPH radical scavenging activity, ABTS radical scavenging activity and FRAP iron reduction capacity are all significantly enhanced.

[0011] (2) In simulated gastrointestinal digestion experiments, the digestibility of lactoferrin in the ternary covalent complex of the present invention was significantly reduced (final digestibility was about 46-48%), and the release of epigallocatechin gallate was slow, indicating that the complex can effectively protect lactoferrin from hydrolysis by digestive enzymes and can also serve as a slow-release delivery carrier for epigallocatechin gallate.

[0012] (3) In in vitro fecal fermentation experiments, the ternary covalent complex of the present invention effectively regulates the metabolic production of short-chain fatty acids (such as acetic acid, propionic acid, butyric acid and total acid) under different intestinal flora backgrounds, showing flora-dependent metabolic regulation characteristics.

[0013] (4) In the application of fresh-cut apple preservation, the amount of moisture loss of apple slices treated with the ternary covalent complex of the present invention is significantly lower than that of the distilled water treatment group and the lactoferrin treatment group alone, and the preservation effect remains stable for 6 hours. Attached Figure Description

[0014] Figure 1 The graph shows the changes in lactoferrin digestibility and epigallocatechin gallate release rate of the ternary covalent complex of the present invention during simulated gastrointestinal digestion.

[0015] In this figure, A represents the changes in gastrointestinal digestibility of the bovine lactoferrin group. B is a graph showing the changes in gastrointestinal digestibility in the recombinant human lactoferrin group. C is a graph showing the change in the release rate of epigallocatechin gallate ester in the ternary covalent complex during gastrointestinal digestion.

[0016] Figure 2 This is a graph showing the weight change trend of fresh-cut apples after treatment with the ternary covalent compound of the present invention.

[0017] Among them, A to F are heatmaps showing the weight changes of apple slices in each group over 1 to 6 hours. G is a graph showing the overall weight change trend of apple slices in each group. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can understand it. All reagents used in the following embodiments are commercially available analytical grade, and the lactoferrin, epigallocatechin gallate, and maltodextrin used were obtained through commercial channels.

[0019] Example 1: Preparation of a lactoferrin-epigallocatechin gallate-maltodextrin ternary covalent complex This embodiment provides a method for preparing a lactoferrin-epigallocatechin gallate-maltodextrin ternary covalent complex, the specific steps of which are as follows: (1) Prepare a 0.4% (w / v) solution of lactoferrin with distilled water and stir overnight at 4°C to ensure full hydration. At the same time, prepare a 0.05% (w / v) stock solution of epigallocatechin gallate with distilled water. Mix the above lactoferrin solution and epigallocatechin gallate stock solution at a volume ratio of 1:1, adjust the pH of the mixture to 9.0, and stir at 25°C for 24 hours in the presence of air to obtain the lactoferrin-epigallocatechin gallate reaction solution.

[0020] (2) Transfer the reaction solution obtained in step (1) to a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and dialyze with deionized water for 48 hours, changing the deionized water every 6-8 hours to remove unreacted free epigallocatechin gallate. After dialysis, collect the liquid in the bag to obtain the purified lactoferrin-epigallocatechin gallate binary covalent complex solution.

[0021] (3) Add maltodextrin to the lactoferrin-epigallocatechin gallate binary covalent complex solution obtained in step (2), with a mass ratio of lactoferrin to maltodextrin of 3:4. Stir magnetically at room temperature for 2 hours to ensure thorough mixing, and then allow to stand overnight at 4°C for hydration. Adjust the pH of the mixture to 11.0 and incubate in a 75°C constant temperature water bath for 6 hours to obtain the lactoferrin-epigallocatechin gallate-maltodextrin ternary covalent complex reaction solution.

[0022] (4) Freeze-dry the reaction solution obtained in step (3) to obtain lactoferrin-epigallocatechin gallate-maltodextrin ternary covalent complex powder, labeled as LF-EGCG-Mal.

[0023] Example 2: Determination of the surface hydrophobicity of ternary covalent complexes This embodiment uses the ANS fluorescent probe method to determine the surface hydrophobicity of a ternary covalent complex. The specific procedure is as follows: 8 mmol / L ANS was dissolved in 10 mmol / L PBS buffer (pH 7.4) to prepare the fluorescent probe solution. The sample to be tested was serially diluted with 1×PBS buffer. 2 mL of the diluted sample solution was taken, and 10 μL of the ANS probe solution was added. After thorough mixing, the mixture was reacted in the dark for 10 minutes. The fluorescence intensity was measured using a fluorescence spectrophotometer at an excitation wavelength of 390 nm and an emission wavelength of 470 nm (both excitation and emission slit widths were 5 nm). A linear regression line was plotted between fluorescence intensity and protein concentration; the slope of the resulting line represents the surface hydrophobicity index.

[0024] The results showed that the surface hydrophobicity index of LF-EGCG-Mal was significantly lower than that of lactoferrin, indicating that the surface hydrophilicity of lactoferrin was enhanced after ternary covalent modification.

[0025] Example 3: Determination of the antioxidant activity of the ternary covalent complex This embodiment employs the DPPH free radical scavenging method and ABTS. + The antioxidant activity of the ternary covalent complex was comprehensively evaluated using a combination of free radical scavenging and FRAP iron reduction assays. DPPH method: The absorbance at 517 nm was measured after reacting the sample with 0.04 mg / mL DPPH anhydrous ethanol solution for 1 h. ABTS + Method 1: First, a 1:1 mixture of 7 mmol / L ABTS and 2.45 mmol / L K₂S₂O₈ was prepared and reacted in the dark for 16 h. Then, the mixture was diluted to a concentration of 0.70 ± 0.01 at 734 nm and reacted with the sample for 1 h before measurement. FRAP Method: A working solution of 300 mmol / L acetate buffer (pH 3.6), 10 mmol / L TPTZ, and 20 mmol / L FeCl₃ in a 10:1:1 ratio was reacted with the sample for 1 h before measuring the absorbance at 593 nm. The results showed that, compared with unmodified lactoferrin, LF-EGCG-Mal exhibited higher DPPH free radical scavenging activity and increased ABTS activity. + Both free radical scavenging activity and FRAP iron reduction ability were significantly improved.

[0026] Example 4: Determination of in vitro digestion properties of ternary covalent complexes This embodiment evaluates the digestive stability of a ternary covalent complex in a simulated gastrointestinal digestion process using an in vitro digestion model. Gastric and intestinal digestive solutions were prepared using 1.25× electrolyte stock solution. For the simulated gastric phase, 5 mL of the sample solution was mixed with 5 mL of simulated gastric juice containing pepsin and shaken at 37°C for 2 h. For the simulated intestinal phase, the pH of the gastric digestive solution was adjusted to 7.0, and 10 mL of simulated intestinal juice containing trypsin was added, followed by shaking at 37°C for 2 h. Samples were taken every 30 min during digestion. The content of lactoferrin in the digestive solution supernatant was determined using the BCA method, and the content of epigallocatechin gallate was determined using the Folin-Ciocalteu method.

[0027] like Figure 1 As shown, the digestibility of each sample gradually increased with increasing digestion time. The gastric digestibility of bovine lactoferrin and recombinant human lactoferrin was 66.67% and 62.27%, respectively, while the intestinal digestibility reached 79.83% and 88.72%, respectively. However, the lactoferrin digestibility of the lactoferrin-epigallocatechin gallate-maltodextrin ternary covalent complex was significantly reduced, with the final digestibility of the bovine lactoferrin complex and the recombinant human lactoferrin complex being only 46.50% and 47.88%, respectively.

[0028] also, Figure 1 Epigallocatechin gallate in C is released slowly throughout the gastrointestinal digestion process. The final release rates of bovine lactoferrin complex and recombinant human lactoferrin complex were 64.04% and 65.71%, respectively, with no significant difference between the two.

[0029] Example 5: Effects of ternary covalent complex on short-chain fatty acid metabolism in gut microbiota This study evaluated the effects of a ternary covalent complex on short-chain fatty acid metabolism in the gut microbiota of different hosts through an in vitro fecal fermentation experiment. Fresh feces were collected from SPF-grade BALB / c mice, C57BL / 6 mice, and KM mice, and fecal suspensions were prepared. The fecal slurry was inoculated into basal culture medium at a concentration of 10% (v / v), and 10% of the sample solution was also inoculated. The control group consisted of lactoferrin sample solution without the addition of epigallocatechin gallate and maltodextrin. All samples were anaerobic and statically cultured for 72 h. The fermentation broth was collected at 0 h, 24 h, 48 h, and 72 h, and the supernatant was obtained by centrifugation.

[0030] In different host fecal fermentation systems, the ternary covalent complex exhibited gut microbiota-dependent regulation of short-chain fatty acid metabolism. In BALB / c and C57BL / 6 mouse fermentation systems, the production of acetic acid, propionic acid, butyric acid, and total acid showed similar increasing trends; in the KM mouse fermentation system, propionic acid and butyric acid levels significantly increased after 48 h. These results indicate that the ternary covalent complex of this invention can be metabolized and utilized by gut microbiota, regulating short-chain fatty acid production.

[0031] Example 6: Application of ternary covalent complex in the preservation of fresh-cut apples This embodiment evaluates the effect of a ternary covalent complex on the water retention of fresh-cut apples. Fresh apples were washed, disinfected, dried, peeled, cored, and sliced ​​into pieces approximately 1 cm thick. The slices were then immersed for 1 hour each in distilled water, lactoferrin, and a lactoferrin-epigallocatechin gallate-maltodextrin ternary covalent complex solution. After immersion, the apples were drained and immediately weighed to obtain the initial weight W0. The weights were recorded as W after each 1-hour interval. t Calculate the weight change ΔW = W0 - W t .

[0032] like Figure 2As shown, the weight of apple slices in each group gradually decreased over time. The distilled water treatment group experienced the greatest water loss, with the largest loss occurring after 6 hours. Compared to the lactoferrin treatment group, the lactoferrin-epigallocatechin gallate-maltodextrin ternary covalent complex treatment group showed less water loss. The results indicate that the ternary covalent complex of this invention can form a dense protective film on the surface of fresh-cut apples, effectively reducing water evaporation.

[0033] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A lactoferrin-polyphenol-polysaccharide ternary covalent complex, characterized in that, The complex is formed by the dual covalent modification of lactoferrin, epigallocatechin gallate, and maltodextrin through oxidation-glycosylation. Lactoferrin covalently binds to epigallocatechin gallate through oxidation and to maltodextrin through Maillard reaction, forming a ternary covalent network structure in which epigallocatechin gallate and maltodextrin jointly modify lactoferrin.

2. The method for preparing the lactoferrin-polyphenol-polysaccharide ternary covalent complex according to claim 1, characterized in that, Includes the following steps: (1) Prepare a 0.4% (w / v) solution of lactoferrin with distilled water and hydrate it overnight at 4°C; prepare a 0.05% (w / v) stock solution of epigallocatechin gallate with distilled water; mix the lactoferrin solution and the epigallocatechin gallate stock solution at a volume ratio of 1:1, adjust the pH to 9.0, and stir the mixture at 25°C for 24 hours in the presence of air to obtain the lactoferrin-epigallocatechin gallate reaction solution; (2) The reaction solution obtained in step (1) was transferred to a dialysis bag for dialysis to obtain a purified lactoferrin-epigallocatechin gallate divalent complex. (3) Add maltodextrin to the lactoferrin-epigallocatechin gallate binary covalent complex obtained in step (2), with a mass ratio of lactoferrin to maltodextrin of 3:

4. Stir at room temperature for 2 hours and hydrate overnight at 4°C. Adjust the pH to 11.0 and incubate at 75°C for 6 hours to obtain the lactoferrin-epigallocatechin gallate-maltodextrin ternary covalent complex reaction solution. (4) The reaction solution obtained in step (3) is freeze-dried to obtain lactoferrin-epigallocatechin gallate-maltodextrin ternary covalent complex.

3. The preparation method according to claim 2, characterized in that, The molecular weight cutoff of the dialysis bag in step (2) is 8000-14000 Da, the dialysis time is 48 hours, and the water change interval is 6-8 hours.

4. The use of the lactoferrin-polyphenol-polysaccharide ternary covalent complex according to claim 1 in the preparation of dietary supplements or fruit and vegetable preservatives with antioxidant and intestinal flora regulation functions.

5. The application according to claim 4, characterized in that, The method of applying the fruit and vegetable preservative is as follows: soak fresh-cut fruit in a solution containing the compound for 1 hour, take it out and drain it, and a protective film will be formed on the surface of the fruit and vegetables.

6. The use of the lactoferrin-polyphenol-polysaccharide ternary covalent complex according to claim 1 in the preparation of an oral sustained-release formulation of an active ingredient with digestive stability protection function.

Images