Protein compound for relieving intestinal inflammation as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of food and medicine, and particularly relates to a protein compound for relieving intestinal inflammation and a preparation method and application thereof. The protein compound for relieving intestinal inflammation provided by the invention is mainly prepared by compounding milk-derived osteopontin (OPN) and alpha-lactalbumin (alpha-LA). Compared with single OPN or alpha-LA, the protein complex provided by the invention shows a remarkable effect of promoting proliferation of intestinal epithelial cells, and effectively improves the physical barrier function of intestinal tracts and relieves intestinal inflammation damage caused by lipopolysaccharide by enhancing expression of tight junction protein. Therefore, the invention proves that the OPN-alpha-LA compound has a remarkable effect of relieving intestinal inflammation, which not only is beneficial to promoting the in-depth study of OPN and the compound thereof in regulating intestinal mucosal immunity in the functional protein field, but also can lay a scientific foundation for developing novel functional foods or novel enteritis medicines by using the active protein as a raw material.

Description

Technical Field

[0001] The invention belongs to the technical field of food and medicine, and specifically relates to a protein complex for alleviating intestinal inflammation, and a preparation method and application thereof. Background Art

[0002] In recent years, with the continuous deepening of research on the active ingredients of bovine milk, the interactions and health benefits of milk-derived active proteins have gradually attracted widespread attention. As an active protein, milk-derived osteopontin (OPN) can not only promote nutrient absorption, intestinal and cognitive development, but also has multiple physiological activities such as immunomodulation and antibacterial. The open domain structure of OPN provides certain possibilities for its interaction with other active molecules. For example, studies have shown that the interaction between OPN and integrins is of great significance in cell biology. They regulate the biological behavior of cells through complex signal transduction networks and participate in the occurrence and development of various diseases. Studies have shown that OPN and lactoferrin (LF) can form a complex and show more significant effects in promoting in vitro intestinal development than using them alone. The above interactions not only reveal the complexity of the interactions between milk-derived active protein components, but also provide new ideas for the research and development of infant formula.

[0003] α-Lactalbumin (α-LA) is a key component of bovine whey protein and has multiple biological functions, including immunomodulation, antiviral, and antioxidant activity. These functions make α-LA also important in the field of infant nutrition and health.

[0004] At present, although the functional activities of OPN and α-LA have been reported to some extent, the research on their interaction is relatively scarce. At present, there is no research on whether OPN and α-LA can form a complex and whether there is a synergistic effect between them. Summary of the invention

[0005] In order to overcome the deficiencies in the prior art, the first purpose of the present invention is to provide a protein complex for alleviating intestinal inflammation, which is a compound of OPN and α-LA. Compared with single OPN, α-LA, and a simple combination of OPN and α-LA, it has a better synergistic immune effect and can effectively alleviate intestinal inflammation by promoting intestinal epithelial cell proliferation.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned protein complex for alleviating intestinal inflammation, which can achieve effective compounding of OPN and α-LA, and the prepared complex has an effective effect of alleviating intestinal inflammation.

[0007] The third object of the present invention is to provide the use of the above protein complex for alleviating intestinal inflammation.

[0008] To achieve the above object, the technical solution adopted by the present invention is: A protein complex for alleviating intestinal inflammation, wherein the protein complex is mainly obtained by compounding milk-derived osteopontin and alpha-lactalbumin under the condition of pH 4.0-4.5.

[0009] The protein complex provided by the present invention has a synergistic effect on relieving intestinal inflammation, which is significantly higher than the single use of OPN or α-LA, and is also better than the combination of OPN and α-LA. In addition, experiments have confirmed that after OPN and α-LA are combined, they can effectively regulate the expression level of Wnt / β-catenin signaling pathway and tight junction proteins in cells induced by LPS, achieving a significant effect of reducing intestinal inflammation.

[0010] The present invention does not specifically limit the original source of the milk-derived osteopontin used in the protein compounding, and the source of the osteopontin only needs to be a common milk source. As a preferred solution, the milk-derived osteopontin is bovine milk osteopontin or sheep milk osteopontin.

[0011] As a preferred embodiment, the mass ratio of milk-derived osteopontin to α-lactalbumin is (0.8-1.2):(0.8-1.2).

[0012] As a further preferred embodiment, the mass ratio of milk-derived osteopontin to α-lactalbumin is 1:1.

[0013] The method for preparing the above-mentioned protein complex for alleviating intestinal inflammation comprises the following steps: The milk-derived osteopontin is mixed in a liquid matrix to obtain a milk-derived osteopontin solution; α-lactalbumin is added to the milk-derived osteopontin solution and mixed, and the pH of the mixed system is adjusted to 4.0-4.5 to obtain a protein complex for relieving intestinal inflammation.

[0014] The preparation method provided by the present invention combines OPN and α-LA with each other through hydrophobic forces under a specific pH range to form a complex. Compared with single OPN and α-LA, the complex has a better synergistic immune effect, can effectively enhance intestinal cell immunity, strengthen the intestinal physical barrier, and alleviate LPS damage to the intestine through the promotion of intestinal epithelial cell proliferation, thereby protecting the health of the intestine.

[0015] As a preferred solution, the liquid matrix is ​​ultrapure water or deionized water.

[0016] As a preferred solution, the concentration of milk-derived osteopontin in the milk-derived osteopontin solution is 0.4 w / v% to 1.0 w / v%, more preferably 0.5 w / v%.

[0017] The application of the above protein complex for relieving intestinal inflammation is used in the preparation of food, health products or medicines for relieving intestinal inflammation.

[0018] As a preferred solution, the food is a dairy product; the health product is a nutritional supplement; and the medicine is an oral preparation. Furthermore, the dairy product can be formula milk powder, milk powder, liquid milk, cheese, condensed milk, etc. commonly used in the art.

[0019] As a preferred solution, the drug includes a protein complex for relieving intestinal inflammation and a pharmaceutically acceptable excipient. Further, the present invention does not specifically limit the types of excipients, and technicians can routinely select excipients according to the dosage form of the drug.

[0020] The technical solution of the present invention has the following advantages and beneficial effects: OPN and α-LA are both proteins with significant biological activity. There are no research reports on the combination of these two proteins and their functional applications. The present invention combines OPN and α-LA for the first time, and confirms the synergistic effect and mechanism of action between the two through experiments. The experiment shows that OPN and α-LA are combined with each other through hydrophobic forces to form a novel structure of OPN-α-LA complex. Compared with a single OPN, α-LA or a combination of the two, the OPN-α-LA complex of the present invention can alleviate intestinal inflammatory damage induced by lipopolysaccharide (LPS), improve the survival rate of damaged cells, and effectively reduce the release of lactate dehydrogenase (LDH) in damaged cells, alleviate the excessive secretion of pro-inflammatory factors caused by LPS, and promote the increase of anti-inflammatory factors. In addition, the complex significantly improves the intestinal barrier function by significantly upregulating the expression levels of three tight junction proteins. And the complex can effectively promote the proliferation of intestinal epithelial cells by regulating the expression of key genes in the Wnt / β-catenin signaling pathway, thereby significantly alleviating the damage of LPS to the intestine. The combined results showed that OPN-α-LA complex has a significant effect in alleviating intestinal inflammation.

[0021] In the above study of the present invention, by verifying that the OPN-α-LA complex promotes the proliferation of intestinal epithelial cells and enhances the expression of tight junction proteins, it effectively improves the physical barrier function of the intestine and alleviates the related mechanism of intestinal inflammatory damage caused by LPS. This is conducive to promoting in-depth research in the field of functional proteins on the role of OPN and its complexes in regulating intestinal mucosal immunity. It can also lay a scientific foundation for the development of new functional foods (infant formula, etc.) or drugs for preventing and treating intestinal inflammation using these active proteins as raw materials. At the same time, it also opens up a broader field for the application of OPN and provides a diversified research perspective. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the energy change result of the binding process of α-LA and OPN at pH 4 in the isothermal titration test of Experimental Example 1 of the present invention; Figure 2 This is the energy change result of the binding process of α-LA and OPN at pH 4.5 in the isothermal titration test of Experimental Example 1 of the present invention; Figure 3 This is a fluorescence spectrum of OPN-α-LA during the titration process of Test Example 1 of the present invention; Figure 4 The circular dichroism spectra and secondary structure diagrams of α-LA, OPN and OPN-α-LA in Experimental Example 1 of the present invention are shown; Figure 5 The Fourier transform infrared spectra test results of α-LA, OPN and OPN-α-LA in Experimental Example 1 of the present invention; Figure 6 This is the test result of the toxic effect of LPS on CCD841 CoN cells in Experimental Example 2 of the present invention; Figure 7 The results of the test of the proliferation effects of α-LA, OPN and OPN-α-LA on CCD841 CoN cells in Experimental Example 2 of the present invention; Figure 8 The effects of α-LA, OPN and OPN-α-LA on the viability of CCD841 CoN cells induced by LPS in Experimental Example 2 of the present invention; Fig. 9 This is the effect of α-LA, OPN and OPN-α-LA on the LDH content in LPS-induced CCD841 CoN cells in Experimental Example 2 of the present invention; Fig.10 The effects of the OPN-α-LA complex of Example 1 and the simple composition of OPN and α-LA of Comparative Example 1 on the LDH content in LPS-induced CCD841 CoN cells in Experimental Example 2 of the present invention; Fig.11This is the effect of α-LA, OPN and OPN-α-LA on the content of inflammatory factors in LPS-induced CCD841 CoN cells in Experimental Example 2 of the present invention; Fig.12 The results of the test of the expression of tight junction proteins at the protein level in LPS-induced CCD841 CoN cells by α-LA, OPN and OPN-α-LA in Experimental Example 2 of the present invention; Fig.13 The results of the expression of the Wnt / β-catenin signaling pathway at the gene level in CCD841 CoN cells induced by LPS by α-LA, OPN and OPN-α-LA in Experimental Example 2 of the present invention; Fig.14 These are the results of the expression of the Wnt / β-catenin signaling pathway at the protein level in LPS-induced CCD841 CoN cells by α-LA, OPN and OPN-α-LA in Experimental Example 2 of the present invention. DETAILED DESCRIPTION

[0023] The technical scheme and technical effects of the present invention will be clearly and completely described below in conjunction with specific embodiments and test examples. However, those skilled in the art should understand that the embodiments are only used to illustrate the technical scheme of the present invention and should not be regarded as limiting the scope of protection of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the raw materials used are commonly used in the art, available to the public or commercially available, unless otherwise specified. In the present invention, "room temperature" has the conventional meaning in the art, that is, 25±5°C.

[0024] In the following embodiments of the present invention, the α-lactalbumin (α-LA) and milk-derived osteopontin (OPN) used can be obtained from common commercial channels, or can be prepared by themselves according to the methods of the prior art. The present invention does not specifically limit the source of the raw materials, and the selection of raw materials by technicians only needs to be within the preparation concept and ratio of the composite of the present invention to achieve the technical effect of the present invention.

[0025] Furthermore, in the embodiments and comparative examples of the present invention, the α-lactalbumin (α-LA) used is derived from bovine milk, which is separated, purified and structurally identified by referring to the prior art 1 (Study on the Interaction and Antibacterial Activity of α-lactalbumin-carvacrol / thymol Non-covalent Complex [D]. Jilin University, 2023). After testing, the purity of the prepared α-lactalbumin is 89%-91%. Milk-derived osteopontin (OPN) is derived from bovine milk and is bovine milk osteopontin, which is separated, purified and structurally identified by referring to the prior art 2 (Study on the Interaction between Lactoferrin and Osteopontin and the Biological Activity Function of Its Complex [D]. Zhejiang University, 2019) by referring to the commercially available bovine milk. After testing, the purity of the prepared milk-derived osteopontin is 94%-96%.

[0026] In other embodiments of the present invention, milk-derived osteopontin may also be obtained from other milk sources, such as sheep milk osteopontin. In other embodiments, α-lactalbumin and milk-derived osteopontin are directly purchased from commercial channels, and their biological activity and preparation effect are equivalent to those of the present invention. Example

[0027] This embodiment provides a protein complex for relieving intestinal inflammation, which is mainly obtained by compounding milk-derived osteopontin (OPN) and α-lactalbumin (α-LA). During compounding, the mass ratio of OPN to α-LA is 1:1.

[0028] The method for preparing the above-mentioned protein complex for alleviating intestinal inflammation comprises the following steps: At room temperature, OPN was weighed and dissolved in ultrapure water to prepare a solution with an OPN concentration of 0.5 w / v%, and magnetic stirring was performed for 2 hours to fully hydrate the OPN to obtain an OPN solution. α-LA was then dissolved in the OPN solution at a specific mass ratio (OPN to α-LA mass ratio of 1:1), and magnetic stirring was performed for 20 minutes to fully dissolve the α-LA. After dissolution, the pH of the system was adjusted to 4.5 to obtain a protein complex for relieving intestinal inflammation (referred to as OPN-α-LA, the same below). Example

[0029] This embodiment provides a protein complex for relieving intestinal inflammation, which is mainly obtained by compounding milk-derived osteopontin (OPN) and α-lactalbumin (α-LA). During compounding, the mass ratio of OPN to α-LA is 1:1.

[0030] The method for preparing the above-mentioned protein complex for alleviating intestinal inflammation comprises the following steps: At room temperature, OPN was weighed and dissolved in ultrapure water to prepare a solution with an OPN concentration of 0.5 w / v%, and magnetic stirring was performed for 2 hours to fully hydrate the OPN to obtain an OPN solution. α-LA was then dissolved in the OPN solution at a specific mass ratio (the mass ratio of OPN to α-LA was controlled to be 1:1), and magnetic stirring was performed for 20 minutes to fully dissolve the α-LA. After dissolution, the pH of the system was adjusted to 4.0 to obtain a protein complex for alleviating intestinal inflammation.

[0031] Comparative Example 1 This comparative example provides a simple composition of OPN and α-LA, which is prepared by directly mixing OPN and α-LA without adjusting pH during preparation. The specific preparation method is: at room temperature, weigh OPN and dissolve it in ultrapure water to prepare a solution with an OPN concentration of 0.5w / v%, and stir it magnetically for 2 hours to fully hydrate OPN to obtain an OPN solution; then dissolve α-LA in the OPN solution at a specific mass ratio (control the mass ratio of OPN to α-LA to be 1:1), and stir it magnetically for 20 minutes to fully dissolve α-LA, so as to obtain a simple composition of OPN and α-LA.

[0032] The application effect of the technical solution of the present invention is described below in conjunction with test examples.

[0033] Experimental Example 1: Physicochemical structural characteristics of proteins and complexes The present invention first conducts an isothermal titration calorimetric test on the two protein raw materials (OPN and α-LA) involved in the present invention to examine the binding affinity of the two. Specifically, when conducting an isothermal titration test on the two proteins, the titrant is selected as OPN (concentration 0.1mM, dissolved in ultrapure water); the titrant is α-LA (concentration 1mM, dissolved in ultrapure water) and the titrant is titrated at pH 4 and pH 4.5 of the titration system, and the energy generated during the binding process is collected and detected to evaluate the binding affinity. The energy change results at different pH are shown in Figure 2. Figure 1 (pH 4) and Figure 2 (pH 4.5) as shown.

[0034] Depend on Figure 1 as well as Figure 2 It can be seen that OPN and α-LA have strong binding ability under pH 4 and 4.5 conditions, the heat generated by the combination of the two is large, and the protein binding process is an endothermic reaction. This shows that OPN and α-LA can bind under both pH conditions, especially at pH 4.5, with a dissociation constant of 4.80×10 -7 , at this time the binding affinity is stronger.

[0035] Furthermore, the present invention tests the fluorescence intensity of the α-LA and OPN complex system (OPN-α-LA) formed when the α-LA concentration is increased from 0 μg / mL to 980 μg / mL during the titration process at pH 4.5. The results are as follows: Figure 3 shown.

[0036] Depend on Figure 3 It can be seen that with the addition of α-LA, the fluorescence intensity of OPN decreases regularly, indicating that the increase in α-LA concentration leads to concentration-dependent quenching of the intrinsic fluorescence of OPN, accompanied by a blue shift of the maximum emission peak from 345 to 325 nm. This shows that there is an interaction between α-LA and OPN, and the conformation of the two changes after binding, which is the cause of the fluorescence quenching of OPN. Further calculations show that K SV is 1.1672×10 4 L / mol, and then calculated according to the formula, kq is 1.1672×10 12 L / s / mol, due to kq>2×10 10 L / s / mol, indicating that the interaction is static, and Kq increases with the increase of α-LA concentration, indicating that the interaction between the two is a static quenching process. Furthermore, according to the Van't Hoff equation ΔG = ΔH-TΔS and ΔG = -RTlnKa, lnKa is plotted against 1 / T and linear regression is performed to calculate ΔH>0, ΔS>0, and ΔG<0. This result is consistent with the results of the isothermal titration calorimetry test, which proves that the interaction between the complex formed by α-LA and OPN is a hydrophobic interaction.

[0037] Furthermore, the present invention characterized single α-LA, single OPN and the OPN-α-LA complex prepared in Example 1 by circular dichroism spectroscopy. The results are as follows: Figure 4 A and 4B. Among them, Figure 4 A is the circular dichroic spectra of α-LA, OPN, and OPN-α-LA; Figure 4 B is the secondary structure percentage of α-LA, OPN, and OPN-α-LA.

[0038] Depend on Figure 4 A and Figure 4B shows that the circular dichroism spectrum of the complex obtained by the combination of α-LA and OPN has changed compared with that of single α-LA and OPN, which also indicates that OPN-α-LA has interacted. Compared with OPN, the content of α-helix in the OPN-α-LA complex decreased by 1.6%, β-fold increased by 1.7%, β-turn content increased by 0.4%, and random coil decreased by 2.2%; compared with α-LA, the content of α-helix in the OPN-α-LA complex decreased by 8.3%, β-turn content increased by 2.7%, random coil decreased by 1.8%, and β-fold increased by 7.6%. The decrease in the content of α-helix and the increase in the content of β-fold of OPN and α-LA indicate that the molecular structure of the protein is fully stretched, turning from order to disorder, which is conducive to the improvement of the functional properties of the protein.

[0039] Furthermore, the present invention characterized single α-LA, single OPN and the OPN-α-LA complex of Example 1 by Fourier transform infrared spectroscopy. The results are as follows: Figure 5 As shown in A~5E. Among them, Figure 5 A is the Fourier transform infrared spectra of α-LA, OPN, and OPN-α-LA; Figure 5 B is the Gaussian fitting infrared spectrum of α-LA amide I band; Figure 5 C is the Gaussian fitting infrared spectrum of the amide I band of OPN; Figure 5 D is the Gaussian fitting infrared image of OPN-α-LA amide I band; Figure 5 E is the secondary structure percentage diagram of α-LA, OPN, and OPN-α-LA.

[0040] Depend on Figure 5 A~5E show that compared with single OPN and α-LA, the characteristic peaks of amide I, II and III have red-shifted to a certain extent after OPN and α-LA form a complex, indicating that there is a hydrophobic interaction between OPN and α-LA molecules. In addition, the characteristic absorption band of hydroxyl group shifts from 3304cm −1 Move to 3298cm −1 , indicating that hydrogen bonds between OPN and α-LA molecules participate in the interaction. OPN at 2962cm -1 A peak appears at 2965 cm-1 in the composite. -1, corresponding to -CH3 vibration, indicating the presence of hydrophobic forces between the complexes. These findings together verify the interaction between the OPN and α-LA complexes and demonstrate that hydrophobic interactions and partial hydrogen bonds are involved in the formation of the OPN and α-LA complexes. Since the band corresponding to amide I depends on the secondary structure of the protein backbone, it can also be used for quantitative analysis of different secondary structures. Compared with α-LA, the α-helix content of the OPN-α-LA complex decreased by 7.4%, the β-turn content increased by 2.23%, and the β-sheet content increased by 4.59%. Consistent with the circular dichroism data, the α-helix content decreased and the β-sheet content increased, further demonstrating the changes in the α-LA structure and the formation of the complex.

[0041] Experimental Example 2: Cell Model Construction and Application Effect Testing (1) Culture medium preparation: Add 10 m / v% FBS and 1 m / v% penicillin-streptomycin solution (double antibody) to DMEM culture medium, filter and sterilize with a 0.22 μm filter, and store at 4°C until use. Before use, place in a 37°C water bath and gently vortex to dissolve the solute evenly.

[0042] (2) Preparation of OPN-α-LA complex: Weigh 100 mg of OPN and dissolve it in 10 mL of ultrapure water. After OPN is fully hydrated, dissolve 100 mg of α-LA in the above solution at a mass ratio of OPN to α-LA of 1:1. Stir magnetically for 20 min to fully dissolve it. After it is evenly dissolved, adjust the pH to 4.5 to prepare a 10 mg / mL OPN-α-LA complex stock solution. Filter and sterilize with a 0.22 μm filter. Use DMEM medium as the diluent to dilute the concentration (1, 10, 50, 100, 200 μg / mL). Dilute it before use.

[0043] (3) Cell culture: CCD841 CoN normal human colon epithelial cell line was cultured in DMEM medium. In specific operations, CCD841 CoN cells were plated in 96-well plates and 25 cm 2 The cells were cultured in culture dishes at 5×10 3 / mL density was inoculated in 96-well plates (100 μL per well) and 1×10 4 / mL density was inoculated on 25cm 2 Cell flask (5 mL per well), set aside after cells adhere to the wall.

[0044] (4) Establishment of intestinal epithelial cell inflammation model: Lipopolysaccharide (LPS) was prepared into a 1 mg / mL storage solution, and after sterilization by 0.22 μm filtration, it was divided into appropriate amounts and stored at 4°C in the dark. If used for a long time, it should be stored at -20°C in the dark to avoid repeated freezing and thawing. Before use, gently vortex in a 37°C water bath to dissolve the solute evenly. LPS was added to the cells that had been attached in step (3) for inflammation treatment to construct a cell inflammation model.

[0045] In this experiment, cells were cultured at 5×10 3 / mL was inoculated in a 96-well plate, and 1×10 4 / mL density was inoculated on 25cm 2 In the cell flask, LPS was induced to establish the intestinal barrier dysfunction model. First, the toxic effect of LPS at different concentrations (1, 10, 20, 50, 100 μg / mL) on CCD841 CoN cells was investigated. Figure 6 shown.

[0046] Depend on Figure 6 It can be seen that LPS has a toxic effect on CCD841 CoN cells, and the cell survival rate gradually decreases with the increase of LPS treatment time and concentration. After treating the cells for 24 hours, the cell survival rate was 66.57% when the LPS concentration was 100 μg / mL, which was significantly reduced by 33.43% compared with the control group. Compared with treating the cells for 12 hours, the survival rate of the cells treated with 100 μg / mL LPS was lower after 24 hours, which indicates that the toxic effect of LPS on CCD841 CoN cells is time- and dose-dependent. Therefore, in the subsequent experiments of the present invention, the LPS concentration for LPS-induced CCD841 CoN cell damage was selected as 100 μg / mL, and the treatment time was 24 hours.

[0047] Furthermore, in the present invention, different doses (1, 10, 50, 100, 200 μg / mL) of single protein or complex (α-LA, OPN, OPN-α-LA) were added to 96-well plates seeded with CCD841 CoN cells (without LPS induction), and cultured anaerobically at 37°C for 12 h and 24 h to evaluate the effects of α-LA, OPN, and OPN-α-LA on the proliferation of CCD841 CoN cells. The results are as follows: Figure 7 A and 7B.

[0048] Depend on Figure 7 A and Figure 7B shows that, in general, the cell survival rate at each dose and culture time was greater than 100%, indicating that under this condition, α-LA, OPN, and OPN-α-LA proteins did not have toxic effects on cells. Among them, compared with α-LA and OPN, the cell survival rate of OPN-α-LA was significantly higher than that of the control group. Considering that the cell survival rate was significantly higher than that of the control group at an action time of 24h, the action time of 24h was selected for subsequent experimental analysis.

[0049] Furthermore, the present invention used different doses (1, 10, 50, 100, 200 μg / mL) of single protein or complex (α-LA, OPN, OPN-α-LA) to act on LPS-induced (100 μg / mL, 24 h) CCD841 CoN cells for 24 h to evaluate the effects of α-LA, OPN, and OPN-α-LA on the viability of LPS-induced CCD841 CoN cells. The results are as follows: Figure 8 shown.

[0050] Depend on Figure 8 It can be seen that α-LA, OPN, and OPN-α-LA can all improve the survival rate of cells to a certain extent. Compared with α-LA and OPN, OPN-α-LA showed a stronger effect in promoting cell proliferation. Considering that when the protein concentration was lower than 10μg / mL, the survival rate of damaged cells did not increase significantly, and the binding protein did not have a significant effect on cell proliferation, the α-LA, OPN, and OPN-α-LA protein concentrations of 10-200μg / mL were selected for subsequent experimental analysis.

[0051] LPS stimulation of normal cells may cause cell damage, produce inflammation and increase the release of lactate dehydrogenase (LDH) in cells. Therefore, LDH is one of the indicators for evaluating inflammation. The present invention used different doses (10, 50, 100, 200 μg / mL) of single protein or complex (α-LA, OPN, OPN-α-LA) to act on LPS-induced (100 μg / mL, 24h) CCD841 CoN cells for 24h to evaluate the effects of α-LA, OPN, and OPN-α-LA on the LDH content in LPS-induced CCD841 CoN cells. The results are as follows: Fig. 9 shown.

[0052] Depend on Fig. 9It can be seen that α-LA, OPN, and OPN-α-LA can effectively reduce the release of LDH in damaged cells under the condition of high-dose 200μg / mL pretreatment, and the amount of LDH released in cells gradually decreases with the extension of the action time. Compared with α-LA and OPN, OPN-α-LA can more effectively reduce the release of LDH in cells, which is consistent with the changing trend of its promoting effect on the proliferation of damaged cells, indicating that OPN-α-LA can promote the relief of cellular inflammation.

[0053] Further, under the same conditions as above, the present invention compares the effects of the OPN-α-LA complex formed in Example 1 of the present invention and the simple composition of OPN and α-LA provided in Comparative Example 1. The results are as follows: Fig.10 As shown. Fig.10 It can be seen that compared with the simple combination of OPN and α-LA, the OPN-α-LA complex significantly reduced the LDH content in inflammatory cells. This result shows that when OPN and α-LA are combined in the form of a complex, the specific changes in their molecular structure can enhance the synergistic effect between the two, thereby having a more positive impact on cells and effectively reducing cell damage.

[0054] Furthermore, the present invention evaluated the effects of α-LA, OPN, and OPN-α-LA on the content of inflammatory factors in LPS-induced CCD841 CoN cells by treating LPS-induced (100 μg / mL, 24 h) CCD841 CoN cells with a single protein or complex (α-LA, OPN, OPN-α-LA) at a dose of 200 μg / mL for 24 h. The results are as follows: Fig.11 A~11D. Among them, Fig.11 A~11D are the content determination results of TNF-α, IL-6, IL-8 and IL-10 respectively.

[0055] Depend on Fig.11 From A to 11D, it can be seen that α-LA, OPN, and OPN-α-LA can alleviate the excessive secretion of pro-inflammatory factors TNF-α, IL-6, and IL-8 caused by LPS after acting on cells, and can promote the increase of anti-inflammatory factor IL-10. Among them, the effect of OPN-α-LA is more obvious than that of α-LA and OPN.

[0056] Furthermore, the present invention evaluated the effects of α-LA, OPN, and OPN-α-LA on the expression of tight junction proteins at the protein level in LPS-induced CCD841 CoN cells treated with a single protein or complex (α-LA, OPN, and OPN-α-LA) at a dose of 200 μg / mL for 24 hours. The results are as follows: Fig.12 shown.

[0057] Depend on Fig.12 It can be seen that α-LA, OPN, and OPN-α-LA enhanced the expression of tight junction proteins (ZO-1, Claudin-1, and Occludin) induced by LPS at the protein level, and these results indicate that α-LA, OPN, and OPN-α-LA all increased the expression of tight junction proteins; among them, OPN-α-LA, compared with α-LA and OPN, can better enhance the intestinal physical barrier and alleviate LPS-induced intestinal inflammatory damage by increasing the expression of tight junction proteins.

[0058] Furthermore, the present invention used RT-qPCR to analyze the expression of key genes in the Wnt / β-catenin signaling pathway. Fig.13 shown.

[0059] Depend on Fig.13 It can be seen that compared with the control group, the expression of intracellular β-catenin, c-Myc and Cyclin D1 genes were significantly upregulated, and the expression of GSK-3β gene was significantly downregulated. Compared with the OPN and α-LA groups, the OPN-α-LA group was the most significant. This shows that OPN-α-LA can better regulate the expression changes of key genes in the Wnt / β-catenin pathway at the mRNA level and alleviate LPS damage to the intestine by promoting intestinal epithelial cell proliferation.

[0060] Furthermore, the present invention analyzed the expression of key genes in the Wnt / β-catenin signaling pathway at the protein level. Fig.14 shown.

[0061] Depend on Fig.14 It can be seen that the expression at the protein level is consistent with the change trend of gene expression. α-LA, OPN, and OPN-α-LA can all increase the expression of β-catenin, c-Myc, and Cyclin D1 genes in the Wnt / β-catenin pathway, and downregulate the expression of GSK-3β genes, among which the OPN-α-LA group is the most significant. This shows that α-LA, OPN, and OPN-α-LA can regulate the expression changes of key genes in the Wnt / β-catenin pathway at the protein level, and the promotion of intestinal epithelial cell proliferation may be achieved through the Wnt / β-catenin signaling pathway, promoting cell proliferation and alleviating LPS-induced intestinal inflammatory damage.

[0062] In summary, the protein complex for relieving intestinal inflammation provided by the present invention combines OPN and α-LA for the first time, and the synergistic effect and its mechanism of action between the two are confirmed by experiments. The experiment shows that OPN and α-LA are combined with each other through hydrophobic forces to form a novel structure of OPN-α-LA complex. Compared with a single OPN, α-LA or a simple combination of the two, the OPN-α-LA complex can relieve LPS-induced intestinal inflammatory damage, improve the survival rate of damaged cells, and effectively reduce the release of lactate dehydrogenase (LDH) in damaged cells, relieve the excessive secretion of pro-inflammatory factors caused by LPS, and promote the increase of anti-inflammatory factors. In addition, the complex significantly improves the intestinal barrier function by upregulating the expression of three tight junction proteins. By regulating the expression changes of key genes in the Wnt / β-catenin signaling pathway at the protein level, the complex effectively alleviates LPS damage to the intestine by promoting the proliferation of intestinal epithelial cells. The comprehensive results show that the OPN-α-LA complex has a significant effect in relieving intestinal inflammation.

[0063] Therefore, the above research of the present invention is conducive to promoting the further development of OPN and its complexes in regulating intestinal mucosal immunity in the field of functional proteins, and can also lay a scientific foundation for the development of new functional foods (infant formula milk powder) or drugs for preventing and treating intestinal inflammation using these active proteins as raw materials. At the same time, it also opens up a broader field for the application of OPN and provides a diversified research perspective. For example, the application of OPN-α-LA complex in infant formula milk powder can not only better meet the complex nutritional and functional needs of infants and young children, but also provide a new idea for raw material innovation and product upgrading of infant formula milk powder. This innovative protein complex is expected to significantly improve the nutritional value and health benefits of formula milk powder, and provide more comprehensive protection for the healthy growth of infants and young children.

[0064] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A protein complex for alleviating intestinal inflammation, characterized in that: The protein complex is mainly obtained by compounding milk-derived osteopontin and alpha-lactalbumin under the condition of pH 4.0-4.

5.

2. The protein complex for alleviating intestinal inflammation according to claim 1, characterized in that: The milk-derived osteopontin is bovine milk osteopontin or sheep milk osteopontin.

3. The protein complex for alleviating intestinal inflammation according to claim 1, characterized in that: The mass ratio of milk-derived osteopontin to α-lactalbumin is (0.8~1.2):(0.8~1.2).

4. The protein complex for alleviating intestinal inflammation according to claim 3, characterized in that: The mass ratio of milk-derived osteopontin to α-lactalbumin is 1:

1.

5. A method for preparing a protein complex for relieving intestinal inflammation according to any one of claims 1 to 4, characterized in that: The following steps are involved: Mixing milk-derived osteopontin in the liquid matrix to obtain a milk-derived osteopontin solution; α-lactalbumin is added to the milk-derived osteopontin solution and mixed evenly, and the pH of the mixed system is adjusted to 4.0-4.5 to obtain a protein complex for alleviating intestinal inflammation.

6. The method for preparing a protein complex for alleviating intestinal inflammation according to claim 5, characterized in that: The liquid matrix is ​​ultrapure water or deionized water.

7. The method for preparing a protein complex for alleviating intestinal inflammation according to claim 5, characterized in that: In the milk-derived osteopontin solution, the concentration of milk-derived osteopontin is 0.4 w / v% to 1.0 w / v%.

8. A use of the protein complex for relieving intestinal inflammation as claimed in any one of claims 1 to 4, characterized in that: Application in the preparation of food, health products or medicines for relieving intestinal inflammation.

9. The use of the protein complex for alleviating intestinal inflammation according to claim 8, characterized in that: The food is a dairy product; the health product is a nutritional supplement; and the medicine is an oral preparation.

10. The use of the protein complex for alleviating intestinal inflammation according to claim 9, characterized in that: The medicine comprises a protein complex for alleviating intestinal inflammation and pharmaceutically acceptable excipients.

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