Lactoferrin and osteopontin compound with function of improving bone health and preparation method of lactoferrin and osteopontin compound

Abstract

The invention discloses a preparation technology of a lactoferrin and osteopontin compound capable of improving bone health, and belongs to the technical field of food processing. In the compound, the mass ratio of lactoferrin to osteopontin is (3-5): 1, and the pH is 4-5.5. In a zebra fish model, after intervention of the lactoferrin, the osteopontin, the compound or the mixture, compared with a mixture group, the compound group of the lactoferrin and the osteopontin can obviously improve the fluorescence intensity of zebra fish spines, the relative bone mineral density is improved by 31.12%, and compared with a blank group, the relative bone mineral density is improved by 2.39 times. Therefore, the lactoferrin and osteopontin compound with the function of improving bone health can be prepared, and the lactoferrin and osteopontin compound has good application potential.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food processing, and particularly relates to a lactoferrin and osteopontin complex with the function of improving bone health and a preparation method thereof. BACKGROUND

[0002] Bone is a highly ordered and dynamic organ with multiple mechanical, biological and chemical functions. In order to maintain the integrity of the bone structure, the organism needs to continuously regulate the bone homeostasis throughout its life, and bone remodeling is the main process of regulating bone homeostasis, which determines the balance between bone formation and resorption. Bone remodeling is mainly participated by osteoblasts and osteoclasts, osteoclasts are responsible for the absorption of old bone, and osteoblasts are responsible for the generation of new bone.

[0003] Under normal physiological conditions in adults, the interaction between osteoblasts and osteoclasts is in a steady state, thereby maintaining the relative stability of the bone. When the balance is broken, osteoporosis or osteosclerosis occurs. When the bone resorption rate is greater than the bone formation rate, osteoporosis occurs. Bone density reduction and bone microstructure destruction are typical characteristics of osteoporosis, and the most serious harm of osteoporosis is the significant increase in the incidence of bone fracture. With the aggravation of population aging, the problem of bone loss represented by osteoporosis (OP) is becoming increasingly prominent. OP and its complications such as fragility fracture and activity limitation seriously reduce the quality of life of the elderly and increase the medical burden of families and society.

[0004] Milk and related products have always been important traditional diets. Dairy products contain rich minerals and high-quality proteins, which are very important for bone health, and the consumption of dairy products is beneficial to bone health. As a whole or single active ingredient, milk protein not only can serve as a mineral carrier to enhance the bioavailability of minerals, but also can affect bone metabolism and mediate bone remodeling at different stages.

[0005] Lactoferrin (LF) plays an important role in inducing osteoblast growth and activity and promoting bone growth in vivo. LF promotes osteoblast phenotype maturation by playing a role in promoting mitosis, anti-apoptosis and driving differentiation of precursor cells, expands the number of osteoblasts in the original osteoblast pool, and inhibits the occurrence of osteoclasts in a dose-dependent manner, and promotes the proliferation of osteoblasts. Osteopontin (OPN) contains a large number of negatively charged amino acids and phosphorylation modification sites, can combine with calcium ions in milk to form a water-soluble complex, and together with casein and other substances inhibits the precipitation of calcium phosphate in milk. OPN can also act as a decoy receptor to inhibit the proliferation and differentiation of osteoclasts. At the same time, the LF and OPN complex can resist the digestion of the gastrointestinal tract better than the monomer, and can better play a role in the intestinal tract. Therefore, the preparation of LF and OPN complex which can improve bone density has important significance for improving bone health. SUMMARY

[0006] This invention provides a method for preparing a lactoferrin (LF) and osteopontin (OPN) complex. Based on optimized preparation conditions, the structure of the prepared substance is characterized. The aim is to obtain a substance that can improve bone health, especially the LF-OPN complex, which can significantly promote bone development. The specific technical solution is as follows:

[0007] S1. To achieve the above objective, the present invention provides an application of a complex in improving bone health function, wherein the active ingredients of the complex are lactoferrin (LF) and osteopontin (OPN).

[0008] S2. Combine lactoferrin (LF) with osteopontin (OPN).

[0009] Preferably, the pH corresponding to the complex formed by lactoferrin (LF) and osteopontin (OPN) in S2 is 3-9.

[0010] Furthermore, in the application described in S2, the mass ratio of lactoferrin (LF):osteopontin (OPN) in the complex is (1~9):1, and the pH is 3~9.

[0011] Preferably, in the application described in S2, the mass ratio of lactoferrin (LF):osteopontin (OPN) in the complex is (3-5):1, and the pH is 4-5.5.

[0012] In another specific embodiment, the application wherein the lactoferrin (LF) and osteopontin (OPN) in the complex are provided as powders prepared from raw materials.

[0013] In addition, according to another aspect of the invention, there is also an application of a mixture in improving bone health, wherein the active ingredient of the composition is a mixture of lactoferrin (LF) and osteopontin (OPN).

[0014] Preferably, the mass ratio of lactoferrin (LF) to osteopontin (OPN) in S2 is (3-5):1.

[0015] The present invention also provides the application of lactoferrin (LF) and osteopontin (OPN) in improving bone health, mainly reflected in a significant increase in bone fluorescence intensity and bone density.

[0016] Preferably, the animal is a zebrafish, and the product contains a lactoferrin (LF) and osteopontin (OPN) complex at a mass of 0.125 mg / mL.

[0017] Mechanism studies of this invention show that the lactoferrin (LF) and osteopontin (OPN) complex in the product reflects increased bone density through increased fluorescence intensity in vertebral bones.

[0018] Therefore, the present invention provides the application of the lactoferrin (LF) and osteopontin (OPN) complex in improving bone health.

[0019] The beneficial effects of this invention are as follows: This invention provides a lactoferrin (LF) and osteopontin (OPN) complex that improves bone health and increases bone density. The preparation method of this complex is simple, the reaction conditions are mild, and it has good application potential and value. Attached Figure Description

[0020] Figure 1 This is a diagram of the potential and theoretical interaction intensity in Embodiment 1 of the present invention.

[0021] Figure 2 This is a turbidity change graph for Example 2 of the present invention.

[0022] Figure 3 These are particle size potential diagrams for Examples 3-6 of the present invention.

[0023] Figure 4 These are the Fourier transform infrared spectra of embodiments 3-6 of the present invention.

[0024] Figure 5 These are circular dichroism chromatograms of Examples 3-6 of the present invention.

[0025] Figure 6 These are the fluorescence spectra of Examples 3-6 of the present invention.

[0026] Figure 7 These are the Raman spectra of Examples 3-6 of the present invention.

[0027] Figure 8 These are scanning electron microscope images of embodiments 3-6 of the present invention.

[0028] Figure 9 This is a safety assessment of zebrafish in Examples 3-6 of the present invention.

[0029] Figure 10 The relative bone density of the zebrafish vertebrae in Examples 3-6 of this invention. Detailed Implementation

[0030] The specific embodiments of the present invention will be described in detail below with reference to specific examples and accompanying drawings, but the present invention is not limited to the embodiments.

[0031] Example 1

[0032] A lactoferrin (LF) and osteopontin (OPN) complex with bone health-improving functions and its preparation method, wherein the effective components of the complex for improving bone density are LF and OPN, and the method comprises the following steps:

[0033] (1) Preparation of lactoferrin (LF): Dissolve LF in deionized water to prepare a solution with a concentration of 1 mg / mL. Stir on a magnetic stirrer for 2 hours, then refrigerate at 4°C for 18 hours to completely dissolve the LF solution.

[0034] (2) Preparation of osteopontin (OPN): Dissolve OPN in deionized water to prepare a solution with a concentration of 1 mg / mL. Stir on a magnetic stirrer for 2 hours, then refrigerate at 4°C for 18 hours to completely dissolve the OPN solution.

[0035] (3) The above solutions were treated by adjusting the pH of LF and OPN to 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, and 11.0, respectively.

[0036] (4) Potential determination of the above solutions: Three parallel samples were set up for each sample. The potential of the sample was detected by a laser particle size analyzer and the theoretical interaction strength (SEI) was calculated.

[0037] Example 2

[0038] A lactoferrin (LF) and osteopontin (OPN) complex with bone health-improving functions and its preparation method, wherein the effective components of the complex for improving bone density are LF and OPN, and the method comprises the following steps:

[0039] (1) Preparation of lactoferrin (LF): Dissolve LF in deionized water to prepare a solution with a concentration of 1 mg / mL. Stir on a magnetic stirrer for 2 hours, then refrigerate at 4°C for 18 hours to completely dissolve the LF solution.

[0040] (2) Preparation of osteopontin (OPN): Dissolve OPN in deionized water to prepare a solution with a concentration of 1 mg / mL. Stir on a magnetic stirrer for 2 hours, then refrigerate at 4°C for 18 hours to completely dissolve the OPN solution.

[0041] (3) The above solution was treated by mixing LF and OPN in a mass ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, and 9:1, and then adjusting the pH to 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, and 11.0, respectively.

[0042] (4) Turbidity determination of the above solution: Three replicates were set for each sample, and OD was detected using an enzyme-linked immunosorbent assay (ELISA) reader. 600nm The turbidity of the sample.

[0043] Example 3

[0044] A lactoferrin (LF) and osteopontin (OPN) complex with bone health-improving functions and its preparation method, wherein the effective component for improving bone density is LF, comprising the following steps:

[0045] (1) Preparation of lactoferrin (LF): Dissolve LF in deionized water to prepare a solution with a concentration of 1 mg / mL. Stir on a magnetic stirrer for 2 hours, then refrigerate at 4°C for 18 hours to completely dissolve the LF solution.

[0046] (2) Vacuum freeze drying: After the target protein sample is pre-frozen at -80℃ for 24 hours, it is freeze-dried using a freeze drying device to remove moisture.

[0047] The lactoferrin (LF) manufactured in this embodiment is rehydrated and reconstituted using deionized water to restore it to its usable state.

[0048] Example 4

[0049] A lactoferrin (LF) and osteopontin (OPN) complex with bone health-improving functions and its preparation method, wherein the effective component for improving bone density is OPN, comprising the following steps:

[0050] (1) Preparation of osteopontin (OPN): Dissolve OPN in deionized water to prepare a solution with a concentration of 1 mg / mL. Stir on a magnetic stirrer for 2 hours, then refrigerate at 4°C for 18 hours to completely dissolve the OPN solution.

[0051] (2) Vacuum freeze drying: After the target protein sample is pre-frozen at -80℃ for 24 hours, it is freeze-dried using a freeze drying device to remove moisture.

[0052] In this embodiment, the osteopontin (OPN) manufactured is rehydrated and reconstituted using deionized water to restore it to its usable state.

[0053] Example 5

[0054] A lactoferrin (LF) and osteopontin (OPN) complex with bone health-improving functions and its preparation method, wherein the effective components of the complex for improving bone density are LF and OPN, and the method comprises the following steps:

[0055] (1) Preparation of lactoferrin (LF): Dissolve LF in deionized water to prepare a solution with a concentration of 1 mg / mL. Stir on a magnetic stirrer for 2 hours, then refrigerate at 4°C for 18 hours to completely dissolve the LF solution.

[0056] (2) Osteopontin (OPN) preparation: Dissolve OPN in deionized water to prepare a solution with a concentration of 1 mg / mL. Completely dissolve OPN to obtain OPN solution.

[0057] (3) Preparation of lactoferrin (LF) and osteopontin (OPN) complex: The complex was stirred on a magnetic stirrer for 2 hours under the following conditions: LF:OPN mass ratio of (3-5):1 and pH of 4-5.5. Then it was refrigerated at 4℃ for 18 hours to obtain the LF-OPN complex.

[0058] (4) Vacuum freeze drying: After the target protein sample is pre-frozen at -80℃ for 24 hours, it is freeze-dried using a freeze-drying device to remove moisture.

[0059] The lactoferrin (LF) and osteopontin (OPN) complex manufactured in this embodiment is rehydrated and reconstituted using deionized water to restore it to its usable state.

[0060] Example 6

[0061] A lactoferrin (LF) and osteopontin (OPN) complex with bone health-improving functions and its preparation method, wherein the effective components for improving bone density are LF and OPN, and the method comprises the following steps:

[0062] (1) Preparation of lactoferrin (LF): Dissolve LF in deionized water to prepare a solution with a concentration of 1 mg / mL. Stir on a magnetic stirrer for 2 hours, then refrigerate at 4°C for 18 hours to completely dissolve the LF solution.

[0063] (2) Osteopontin (OPN) preparation: Dissolve OPN in deionized water to prepare a solution with a concentration of 1 mg / mL. Completely dissolve OPN to obtain OPN solution.

[0064] (3) Preparation of lactoferrin (LF) and osteopontin (OPN) composition: The LF+OPN composition was prepared by stirring on a magnetic stirrer for 2 hours under the condition that the mass ratio of LF:OPN was (3~5):1, and then refrigerated at 4℃ for 18 hours to obtain the LF+OPN composition.

[0065] (4) Vacuum freeze drying: After the target protein sample is pre-frozen at -80℃ for 24 hours, it is freeze-dried using a freeze-drying device to remove moisture.

[0066] The lactoferrin (LF) and osteopontin (OPN) mixture manufactured in this embodiment is rehydrated and reconstituted with deionized water to restore it to its usable state.

[0067] The methods for material characterization and functional verification of Examples 3-6 are as follows:

[0068] (1) Measurement of particle size potential

[0069] Take an appropriate amount of the samples prepared in Examples 3-6 and dissolve them in deionized water. Use a laser particle size analyzer to detect the particle size potential of the samples.

[0070] (2) Fourier transform infrared spectrum

[0071] Accurately weigh 1 mg of the samples prepared in Examples 3-6 and mix with 100 mg of potassium bromide. After tableting, analyze the mixture using a Fourier transform infrared spectroscopy (FTIR) instrument at 4000 cm⁻¹. -1 -400 cm -1 The instrument performs scanning within a certain range, with a resolution of 4 cm. -1 64 scans.

[0072] (3) Circular dichroism

[0073] Accurately weigh a certain mass of the samples prepared in Examples 3-6 and prepare a solution with a concentration of 0.2 mg / mL. Use ultrapure water as a blank control group and detect changes in secondary structure using circular dichroism chromatograph.

[0074] (4) Fluorescence spectrum

[0075] Accurately weigh a certain mass of the samples prepared in Examples 3 to 6, prepare a solution of 0.2 mg / mL, measure its emission spectrum at an excitation wavelength of 280 nm, and record the emission wavelength between 300 and 450 nm.

[0076] (5) Raman spectroscopy

[0077] Weigh a certain mass of the samples prepared in Examples 3-6 and record the values ​​at 400–3200 cm⁻¹ using a Raman spectrometer. −1 Raman spectra within the range.

[0078] (6) Scanning electron microscope

[0079] A certain mass of the samples prepared in Examples 3 to 6 were accurately weighed and uniformly attached to a sample column with carbon conductive adhesive. The sample was then placed in a vacuum sputtering instrument to deposit a thin metal film. The scanning conditions were: accelerating voltage 15.0 kV, beam current 6.9×10−2mA, working distance 6.7mm. The morphology was observed and images were acquired.

[0080] (7) Safety assessment of zebrafish

[0081] Accurately weigh a certain mass of the samples prepared in Examples 3-6 and prepare concentrations of 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, and 2 mg / mL, respectively. Zebrafish juvenile embryos cultured for 3 days post-fertilization (dpf) were selected and intervened until 7 days post-fertilization (dpf), then calcein staining was performed, and the embryos were observed and measured under a microscope.

[0082] The results show that the optimal compounding conditions for the present invention are a lactoferrin (LF) to osteopontin (OPN) mass ratio of (3-5):1 and a pH of 4-5.5. After administration of the compound, the fluorescence intensity of zebrafish vertebrae was significantly increased, indicating a significant increase in bone density. Compared to the mixture group, bone density increased by 31.12%, demonstrating a significant function in improving bone health.

[0083] This invention illustrates, through the above embodiments, a lactoferrin (LF) and osteopontin (OPN) complex with bone health-improving functions and its preparation method. However, the above description is merely the preferred embodiment of the invention. It should be noted that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the invention. For those skilled in the art, several modifications can be made without departing from the principle of the invention, and these modifications should also be considered within the scope of protection of the invention.

Claims

1. A lactoferrin and osteopontin complex having a function of improving bone health and a method of preparing the same, characterized by comprising the steps of: Comprising the following steps: ​ (1) Solution preparation: lactoferrin (LF) and osteopontin (OPN) are dissolved in deionized water respectively, and configured into a solution with a concentration of 1 mg / mL, and the proteins are completely dissolved to obtain an LF solution and an OPN solution; (2) Complex preparation: lactoferrin (LF) and osteopontin (OPN) are combined at a mass ratio of (3-5):1 under the condition of pH 4-5.5, and then freeze-dried using a freeze-drying device to obtain a complex powder.

2. The solution formulation method according to claim 1, characterized by: In the step (1), the lactoferrin (LF) and osteopontin (OPN) are stirred on a magnetic stirrer for 2 h, and then stored in a refrigerator at 4°C for 18 h.

3. The method of claim 1, wherein: In the step (2), after the lactoferrin (LF) and osteopontin (OPN) are combined, they are stirred on a magnetic stirrer for 2 h, and then stored in a refrigerator at 4°C for 18 h.

4. The method of claim 1, wherein: The preparation method is suitable for lactoferrin and osteopontin complex with the function of improving bone health.

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