Costrum basic protein and preparation method thereof

Abstract

The invention relates to the field of food, in particular to colostrum basic protein as well as a preparation method and application thereof. The colostrum basic protein of the present invention can increase bone mass, increase bone density, promote skeletal development, promote skeletal regeneration or repair, promote calcium absorption, promote bone collagen production, and / or prevent and / or treat bone related diseases (such as osteoporosis, osteoporosis, rickets, osteoarthrosis).

Description

Technical Field

[0001] The invention belongs to the field of food, and in particular relates to colostrum alkaline protein and a preparation method and application thereof. Background Art

[0002] Colostrum Basic Protein (CBP) is a new resource food raw material approved by Announcement No. 12 in 2009. The production process of colostrum basic protein new food raw material announcement is as follows: "It is made from bovine colostrum, sterilized, defatted, centrifuged, casein, α-lactalbumin, β-lactoglobulin removed, microfiltered, ultrafiltered, freeze-dried, etc."

[0003] Colostrum and its extracts have been reported to promote bone density. Most studies believe that the main mechanism comes from milk calcium. Some studies also suggest that it activates osteoblast activity and increases alkaline phosphatase activity, which is beneficial to the increase of bone density and height growth. Lee et al. published a study in 2007 titled "Effects of Colostrum Basic Protein from Colostrum WheyProtein: Increased in Osteoblast Proliferation and Bone Metabolism" and reported that CBP (1, 10, 100 μg / mL) treatment did increase the cell proliferation of osteoblast MC3T3 cells in a dose-dependent manner. After CBP treatment, alkaline phosphatase activity (a marker of osteoblast phenotype) in cells also increased in a dose-dependent manner; however, there was no significant difference in the concentration of osteocalcin in serum. Compared with ovariectomized rats fed a normal diet, femoral bone density of ovariectomized rats fed 1% and 10% CBP increased significantly; however, there was no significant difference in the total crude protein and calcium content of bones.

[0004] However, while colostrum stimulates bone density and bone length, it may be detrimental to bone toughness, increase the risk of fractures, and even contain certain potential health risk factors.

[0005] For example, colostrum is rich in growth factors such as IGF-1. Studies have shown that IGF-1 in colostrum may induce an increase in IGF-1 levels in the human body, thereby stimulating longitudinal bone growth and increasing bone density. However, IGF-1 in cow's milk is also related to the occurrence and development of various cancers, which may bring health risks or even harm.

[0006] In addition, studies have also shown that colostrum can stimulate an increase in NTx of type I collagen in serum. Most of the serum NTx is excreted from the body through urine, resulting in the loss of bone collagen. Bone collagen is an important component of the human skeleton. Calcium in the bone is deposited in the form of hydroxyapatite and fixed with bone collagen as an adhesive. Bone collagen is like a net full of small holes in the bone, firmly locking calcium. Summary of the Invention

[0007] To overcome the above problems, the present invention provides a colostrum basic protein, which can promote bone collagen synthesis and bone calcium absorption, and ultimately show an increase in bone density and bone length.

[0008] In a first aspect of the present invention, there is provided a colostrum basic protein, wherein the protein content ≥ 80%, the content of components with a molecular weight of 1 - 30 KDa ≥ 50%, and the IgG content ≥ 10%.

[0009] In some embodiments, in the colostrum basic protein, the content ratio of the molecular weight of 1 KDa - 6 KDa ≤ 10%, and the content ratio of the molecular weight of 6 KDa - 30 KDa ≥ 40%.

[0010] In some embodiments, in the colostrum basic protein, the IGF-1 content ≤ 0.008‰.

[0011] In some embodiments, in the colostrum basic protein, the IgG content is 10% - 20%.

[0012] In some embodiments, in the colostrum basic protein, the IgG content is 12% - 18%.

[0013] In some embodiments, in the colostrum basic protein, the IgG content is 12% - 17%.

[0014] In some embodiments, in the colostrum basic protein, the IgG content is 15%.

[0015] In some embodiments, in the colostrum basic protein, the content ratio of the molecular weight of 1 KDa - 6 KDa is 3% - 10%.

[0016] In some embodiments, in the colostrum basic protein, the content ratio of the molecular weight of 6 KDa - 30 KDa is 40% - 70%.

[0017] In some embodiments, in the colostrum basic protein, the content ratio of the molecular weight of 1 KDa - 6 KDa is 3% - 10%, and the content ratio of the molecular weight of 6 KDa - 30 KDa is 40% - 70%.

[0018] In some embodiments, in the colostrum basic protein, the content ratio of the molecular weight of 1 KDa to 6 KDa is 3% to 9%.

[0019] In some embodiments, in the colostrum basic protein, the content ratio of the molecular weight of 1 KDa to 6 KDa is 3% to 8%.

[0020] In some embodiments, in the colostrum basic protein, the content ratio of the molecular weight of 1 KDa to 6 KDa is 6% to 8%.

[0021] In some embodiments, in the colostrum basic protein, the content ratio of the molecular weight of 6 KDa to 30 KDa is 45% to 70%.

[0022] In some embodiments, in the colostrum basic protein, the content ratio of the molecular weight of 6 KDa to 30 KDa is 50% to 70%.

[0023] In some embodiments, in the colostrum basic protein, the content ratio of the molecular weight of 6 KDa to 30 KDa is 55% to 70%.

[0024] In some embodiments, in the colostrum basic protein, the IGF-1 content ≤ 0.006‰.

[0025] In some embodiments, in the colostrum basic protein, the IGF-1 content ≤ 3×10^(-4)‰.

[0026] In some embodiments, in the colostrum basic protein, the IGF-1 content ≤ 3×10^(-5)‰.

[0027] In some embodiments, in the colostrum basic protein, the IGF-1 content ≤ 3×10^(-6)‰.

[0028] In some embodiments, in the colostrum basic protein, the IgG content is 15%.

[0029] In some embodiments, in the colostrum basic protein, the content of the molecular weight of 1 KDa to 6 KDa is 6.9%, and the content of the molecular weight of 6 KDa to 30 KDa is 60.8%.

[0030] In some embodiments, in the colostrum basic protein, the IGF-1 content ≤ 1.5×10^(-6)‰.

[0031] In some embodiments, the colostrum basic protein is prepared by a method comprising the following steps:

[0032] (1) Provide a raw material of colostrum basic protein,

[0033] (2) Hydrolyze the colostrum basic protein raw material with alkaline protease under ultra-high pressure.

[0034] In some embodiments, the colostrum basic protein raw material described in step (1) is commercially available colostrum basic protein, or is obtained by using bovine colostrum as the raw material, through sterilization, defatting, centrifugal separation, removal of casein, α-lactalbumin, β-lactoglobulin, microfiltration, and ultrafiltration.

[0035] In some embodiments, step (2) has one or more of the following technical features:

[0036] a) The mass ratio of the alkaline protease to the colostrum basic protein raw material is 1:80 - 150.

[0037] b) The ultra-high pressure is ≥100 MPa.

[0038] c) The hydrolysis is carried out at 36°C - 38°C for 1 - 4 hours.

[0039] d) In the hydrolysis system, the concentration of the colostrum basic protein raw material is 8 - 20 mg / mL.

[0040] In some embodiments, the mass ratio of the alkaline protease to the colostrum basic protein raw material is 1:90 - 120.

[0041] In some embodiments, the mass ratio of the alkaline protease to the colostrum basic protein raw material is 1:95 - 110.

[0042] In some embodiments, the mass ratio of the alkaline protease to the colostrum basic protein raw material is 1:100.

[0043] In some embodiments, the ultra-high pressure is 100 - 120 MPa.

[0044] In some embodiments, the hydrolysis is carried out at 37°C for 1.5 - 2.5 hours.

[0045] In some embodiments, the hydrolysis is carried out at 37°C for 2 hours.

[0046] In some embodiments, in the hydrolysis system, the concentration of the colostrum basic protein raw material is 8 - 15 mg / mL.

[0047] In some embodiments, in the hydrolysis system, the concentration of the colostrum basic protein raw material is 9 - 13 mg / mL.

[0048] In some embodiments, in the hydrolysis system, the concentration of the colostrum basic protein raw material is 10 mg / mL.

[0049] In some embodiments, after step (2), it further includes inactivating the enzyme by heat treatment.

[0050] In some embodiments, after step (2), it further includes drying.

[0051] In some embodiments, the drying is freeze-drying.

[0052] In some embodiments, the drying is vacuum freeze-drying;

[0053] In some embodiments, the heat treatment is to keep warm at 100 - 120 °C for 8 - 20 min.

[0054] In some embodiments, the heat treatment is to keep warm at 100 - 110 °C for 8 - 15 min.

[0055] In some embodiments, the heat treatment is to keep warm at 100 °C for 10 min.

[0056] The second aspect of the present invention provides a method for preparing the colostrum basic protein described in the first aspect of the present invention, which includes ultra-high pressure enzymatic hydrolysis of the colostrum basic protein raw material.

[0057] In some embodiments, the method includes the following steps:

[0058] (1) Provide a colostrum basic protein raw material,

[0059] (2) Enzymatically hydrolyze the colostrum basic protein raw material with alkaline protease under ultra-high pressure.

[0060] In some embodiments, the colostrum basic protein raw material in step (1) is commercially available colostrum basic protein, or is obtained by using bovine colostrum as a raw material, through sterilization, degreasing, centrifugal separation, removing casein, α-lactalbumin, β-lactoglobulin, microfiltration, and ultrafiltration.

[0061] In some embodiments, step (2) has one or more of the following technical features:

[0062] a) The mass ratio of the alkaline protease to the colostrum basic protein raw material is 1:80 - 150,

[0063] b) The ultra-high pressure is ≥100 MPa,

[0064] c) The enzymatic hydrolysis is to hydrolyze for 1 - 4 hours at 36 °C - 38 °C,

[0065] d) In the enzymatic hydrolysis system, the concentration of the colostrum basic protein raw material is 8 - 20 mg / mL.

[0066] In some embodiments, the mass ratio of the alkaline protease to the colostrum basic protein raw material is 1:90 - 120.

[0067] In some embodiments, the mass ratio of the alkaline protease to the colostrum basic protein raw material is 1:95 - 110.

[0068] In some embodiments, the mass ratio of the alkaline protease to the colostrum basic protein raw material is 1:100.

[0069] In some embodiments, the ultra-high pressure is 100 - 120 MPa.

[0070] In some embodiments, the enzymatic hydrolysis is carried out at 37°C for 1.5 - 2.5 hours.

[0071] In some embodiments, the enzymatic hydrolysis is carried out at 37°C for 2 hours.

[0072] In some embodiments, in the enzymatic hydrolysis system, the concentration of the colostrum basic protein raw material is 8 - 15 mg / mL.

[0073] In some embodiments, in the enzymatic hydrolysis system, the concentration of the colostrum basic protein raw material is 9 - 13 mg / mL.

[0074] In some embodiments, in the enzymatic hydrolysis system, the concentration of the colostrum basic protein raw material is 10 mg / mL.

[0075] In some embodiments, after step (2), it further includes inactivating the enzyme by heat treatment.

[0076] In some embodiments, after step (2), it further includes drying.

[0077] In some embodiments, the drying is freeze-drying.

[0078] In some embodiments, the drying is vacuum freeze-drying.

[0079] In some embodiments, the heat treatment is carried out at 100 - 120°C for 8 - 20 min.

[0080] In some embodiments, the heat treatment is carried out at 100 - 110°C for 8 - 15 min.

[0081] In some embodiments, the heat treatment is carried out at 100°C for 10 min.

[0082] The third aspect of the present invention provides a dairy product, which includes the colostrum basic protein described in the first aspect of the present invention.

[0083] In some embodiments, the dairy product further includes calcium and / or vitamin D.

[0084] In some embodiments, the vitamin D is vitamin D2 and / or vitamin D3.

[0085] In some embodiments, the dairy product is a solid dairy product or a liquid dairy product.

[0086] In some embodiments, the solid dairy product is selected from formula milk powder and soy-based milk powder.

[0087] In some embodiments, the solid dairy product is selected from infant formula milk powder, children's formula milk powder, adult formula milk powder, and middle-aged and elderly formula milk powder.

[0088] In some embodiments, the liquid dairy product is selected from modified milk, milk beverage, and soy milk.

[0089] The fourth aspect of the present invention provides the use of the colostrum basic protein described in the first aspect of the present invention in the preparation of a product.

[0090] In some embodiments, the product is a dairy product.

[0091] In some embodiments, the dairy product is a solid dairy product or a liquid dairy product.

[0092] In some embodiments, the solid dairy product is selected from formula milk powder and soy-based milk powder.

[0093] In some embodiments, the solid dairy product is selected from infant formula milk powder, children's formula milk powder, adult formula milk powder, and middle-aged and elderly formula milk powder.

[0094] In some embodiments, the liquid dairy product is selected from modified milk, milk beverage, and soy milk.

[0095] In some embodiments, the product is a product for increasing bone mass, increasing bone density, promoting bone development, promoting bone regeneration or repair, promoting calcium absorption, promoting bone collagen production, and / or preventing and / or treating bone-related diseases.

[0096] In some embodiments, the bone-related diseases are osteoporosis, osteopenia, rickets, or osteoarthropathy.

[0097] The beneficial effects achieved by the present invention:

[0098] (1) A colostrum basic protein with specific characteristics is obtained. This colostrum basic protein can improve the survival rate of osteocytes, promote bone collagen synthesis and bone calcium absorption, and increase bone density and bone length;

[0099] (2) A method for preparing the colostrum basic protein of the present invention is provided. This method can increase the IgG content in the colostrum basic protein and optimize the content ratio of each molecular weight in IgG;

[0100] (3) provides products containing the colostrum basic protein of the present invention, such as dairy products, which can promote collagen synthesis, calcium absorption in bones, increase bone density and bone length, and / or prevent and / or treat bone-related diseases. Description of the Drawings

[0101] Figure 1 . Cell viability of hFOB1.19 human osteoblasts after being treated with CBP of the present invention at different concentrations for 72 hours.

[0102] Figure 2 . Cell viability of hFOB1.19 human osteoblasts after being treated with CBP of the present invention and commercially available CBP for 72 hours.

[0103] Figure 3 . Morphological changes of hFOB1.19 human osteoblasts after being treated with CBP of the present invention at different concentrations.

[0104] Figure 4 . Effect of CBP of the present invention on the expression level of bone collagen in hFOB1.19 human osteoblasts.

[0105] Figure 5 . Effect of CBP of the present invention and commercially available CBP on the expression level of bone collagen in hFOB1.19 human osteoblasts.

[0106] Figure 6 . Effect of CBP of the present invention on the expression level of human alkaline phosphatase in hFOB1.19 human osteoblasts.

[0107] Figure 7 . Effect of CBP of the present invention and commercially available CBP on the expression level of human alkaline phosphatase in hFOB1.19 human osteoblasts.

[0108] Figure 8 . Effect of CBP of the present invention on the content of bone collagen in zebrafish.

[0109] Figure 9 . Effect of CBP of the present invention and commercially available CBP on the content of bone collagen in zebrafish.

[0110] Figure 10 . Effect of CBP of the present invention on human alkaline phosphatase in zebrafish.

[0111] Figure 11 . Effect of CBP of the present invention and commercially available CBP on the content of human alkaline phosphatase in zebrafish.

[0112] Figure 12 . Effect of CBP of the present invention on calcium elements in zebrafish.

[0113] Figure 13. Influence of the CBP of the present invention and commercially available CBP on the calcium element content in the whole body of zebrafish.

[0114] Figure 14 . Development status of alizarin red staining of zebrafish body skeleton.

[0115] Figure 15 . Development status of alizarin red staining of zebrafish tail skeleton.

[0116] Figure 16 . Influence of the CBP of the present invention on the development of zebrafish thoracic vertebrae.

[0117] Figure 17 . Influence of the CBP of the present invention on the development of zebrafish caudal fin.

[0118] Figure 18 . Histogram of zebrafish body length statistics.

[0119] Figure 19 . Influence of the CBP of the present invention and commercially available CBP on the body length of zebrafish.

[0120] Note: The blank group, Control, and 0 μg / mL all represent the blank group; unless otherwise specified, the p-value is the comparison result with the blank group, where ns indicates no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001. Detailed implementation manners

[0121] The following will describe the implementation schemes of the present invention in detail in conjunction with the embodiments. However, those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0122] Example 1 Preparation of CBP

[0123] Using bovine colostrum as raw material, after sterilization, defatting, centrifugal separation, removing casein, α-lactalbumin, and β-lactoglobulin, a protein liquid is obtained through microfiltration and ultrafiltration, with a protein concentration of 10 mg / mL. Alkaline protease (purchased from Yuanye Bio, product number S10154) is used to hydrolyze at a ratio of enzyme:substrate (E:S) of 1:100 under 100 MPa of ultra-high pressure at 37 °C for 2 hours. After digestion, the enzyme is inactivated by heat treatment (100 °C, 10 min), and then vacuum freeze-drying is used to complete the preparation to obtain CBP. After detection, the protein content in the obtained CBP is ≥80%, the content of components with a molecular weight of 1 - 30 KDa is ≥50%, the IgG content is 15 g / 100 g, IGF-1 is not detected (detection limit is 1.5 ng / g), the content of components with a molecular weight of 1 KDa - 6 KDa is 6.9%, and the content of components with a molecular weight of 6 KDa - 30 KDa is 60.8%.

[0124] Example 2 Biological Test

[0125] 1. Test Materials

[0126] 1.1 Test Cells

[0127] The cells used in this test are human osteoblast hFOB1.19, source: American Type Culture Collection #: C15075953.

[0128] 1.1.1 Main Instruments and Reagents

[0129] 1.1.2 Main Test Instruments: Carbon dioxide incubator (Sanyo MCO-15AC, Japan), SW-CJ cell culture super clean bench (Suzhou Purification Co., Ltd.), TGL-16 high-speed refrigerated centrifuge (Xiangyi Centrifuge Instrument Co., Ltd.), Multiskan TM GO microplate reader (Thermo Fisher Scientific (China) Co., Ltd.), SQP ten-thousandth electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.).

[0130] 1.1.3 Main Test Reagents: High-glucose DMEM medium (Cytiva AJ30728130, USA), phosphate buffer solution (PBS) (Wuhan Boster 700857135), 10% fetal bovine serum (Zhejiang Sijiqing 23020701), trypsin (Sigma 20201031-0404, USA), thiazolyl blue (Aladdin J2106161), bone collagen ELISA detection kit (Jingmei JM-08102F1), alkaline phosphatase ELISA detection kit (Xinquan Technology 04 / 2023).

[0131] 1.2 Test Zebrafish

[0132] The zebrafish used in this experiment were AB strain zebrafish, sourced from the National Zebrafish Resource Center.

[0133] 1.2.1 Main Instruments and Reagents

[0134] 1.2.2 Main experimental instruments: Z-A-D5 five-layer single-row independent culture unit (Shanghai Haisheng Biological Experimental Equipment Co., Ltd.), SZ680 continuous variable magnification stereomicroscope (Chongqing Optoelectronic Instrument Co., Ltd.), ZXSD-A1090 biochemical incubator (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.), SQP one-ten-thousandth electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.).

[0135] 1.2.3 Main experimental reagents: H 2 O 2 , NaOH, ethanol, alizarin (Maclean, Lot#: C15075953), paraformaldehyde (Maclean, Lot#: C10060370).

[0136] 1.3 Test reference substances

[0137] Commercially available CBP: Protein content ≥ 80%, component content with molecular weight of 1 - 30KDa ≥ 50%, IgG = 8.6g / 100g, IGF-1 8.2μg / g, 1KDa - 6KDa in IgG = 38.6%, 6KDa - 30KDa = 21.8%.

[0138] 2. Test methods

[0139] 2.1 Cell experiment grouping and control

[0140] Experimental group (CBP prepared in Example 1 of the present invention): IgG = 15g / 100g, IGF-1 not detected, 1KDa - 6KDa in IgG = 6.9%, 6KDa - 30KDa = 60.8%;

[0141] Control group (commercially available CBP): IgG = 8.6g / 100g, IGF-1 8.2μg / g, 1KDa - 6KDa in IgG = 38.6%, 6KDa - 30KDa = 21.8%;

[0142] Blank group: High-glucose DMEM medium.

[0143] 2.2 Main operation steps of cell experiment

[0144] 2.2.1 Effect of CBP on the proliferation of hFOB1.19 human osteoblasts

[0145] Inoculate hFOB1.19 human osteoblasts in a 96-well plate (about 5×10 3(cells / well), and cultured in DMEM medium containing 10% (v / v) fetal bovine serum at a temperature of 37°C and a concentration of 5% CO 2 hFOB1.19 cells were co-cultured with CBP prepared in Example 1 of the present invention at different concentrations for 72 h. In addition, commercially available CBP was co-cultured with hFOB1.19 cells at a concentration of 200 μg / mL for 72 h. MTT (5 mg / mL) was added to each well and incubated for 4 h. Finally, the remaining formazan was dissolved with DMSO, and the absorbance was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The cell viability of each treatment group was calculated. At the same time, observations were made under a microscope every 24 h, and photographs were taken at 72 h for recording.

[0146] 2.2.2 Effect of CBP on the expression levels of bone collagen and human alkaline phosphatase in hFOB1.19 human osteoblasts

[0147] hFOB1.19 human osteoblasts were seeded in 6-well plates (about 1×10 4 (cells / well), and cultured in DMEM medium containing 10% (v / v) fetal bovine serum at a temperature of 37°C and a concentration of 5% CO 2 hFOB1.19 cells were co-cultured with CBP prepared in Example 1 of the present invention at different concentrations for 72 h. In addition, commercially available CBP was co-cultured with hFOB1.19 cells at a concentration of 200 μg / mL for 72 h. Then the cells were collected, resuspended with 1 mL of PBS, sonicated at 20 w for 2 min (5 s on, 5 s off) in an ice bath, and then centrifuged at 3000 rpm for 10 min. The supernatant was taken, and the total protein of the sample was quantified with a BCA reagent. Then the target protein in each sample was detected with an ELISA kit.

[0148] 2.3 Data statistics

[0149] GraphPad Prism 8.0 software was used to statistically process the data. The experimental data were all expressed as mean ± SD, and one-way analysis of variance or t-test statistical methods were used to analyze the data.

[0150] 2.4 Judgment basis

[0151]

[0152] 2.5 Zebrafish grouping and control

[0153] Experimental group: CBP prepared in Example 1 of the present invention was added to the culture water;

[0154] Control group: Commercially available CBP was added to the culture water;

[0155] Blank group: Cultured water, which is composed of deionized water (purified from tap water by a water purifier), sodium bicarbonate and sea salt, is isotonic with zebrafish, at a temperature of about 27.5 °C, with a conductivity of 500 - 800 and a pH of about 7 - 8.

[0156] 2.5.1 Determination of the content of bone collagen in zebrafish

[0157] Select zebrafish larvae (15 days old) at the critical stage of skeletal growth and development as the experimental subjects, with 10 larvae in each group, a total of 4 groups: blank group, control group (commercially available CBP, 100 μg / mL), experimental group 1 (the CBP of the present invention, 50 μg / mL), and experimental group 2 (the CBP of the present invention, 100 μg / mL). After continuous treatment for 15 days, dissect the zebrafish, weigh the mass of the zebrafish skeleton, ultrasonically disrupt the bone tissue, take the supernatant solution, and then use a bone collagen kit to detect the change in the content of bone collagen in the zebrafish skeleton by ELISA method.

[0158] 2.5.2 Human alkaline phosphatase (ALP) in zebrafish

[0159] Select zebrafish larvae (15 days old) at the critical stage of skeletal growth and development as the experimental subjects, with 10 larvae in each group, a total of 4 groups: blank group, control group (commercially available CBP, 100 μg / mL), experimental group 1 (the CBP of the present invention, 50 μg / mL), and experimental group 2 (the CBP of the present invention, 100 μg / mL). After continuous treatment for 15 days, weigh the extracted zebrafish skeleton tissue, ultrasonically disrupt it with 1 mL ddH 2 O, detect the total protein content with a BCA kit, and then detect the ALP enzyme content in the skeleton tissue per unit mass with an ELISA kit. Analyze the change of this enzyme in the zebrafish skeleton.

[0160] 2.5.3 Determination of the content of calcium element in zebrafish

[0161] Select zebrafish larvae (15 days old) at the critical stage of skeletal growth and development as the experimental subjects, with 10 larvae in each group, a total of 4 groups: blank group, control group (commercially available CBP, 100 μg / mL), experimental group 1 (the CBP of the present invention, 50 μg / mL), and experimental group 2 (the CBP of the present invention, 100 μg / mL). After continuous treatment for 15 days, dissect the zebrafish, weigh the mass of the zebrafish skeleton, carry out nitrification, and then make up the volume to 5 mL with distilled water, and use ICP to detect the calcium ion concentration in the skeleton per unit mass.

[0162] 2.5.4 Development of the body and tail bones of zebrafish

[0163] Take 15-day-old zebrafish larvae, divide them into 4 groups, with 5 zebrafish in each group. For the first group, directly place the zebrafish at -20°C for 20 min. After freezing and sacrificing, place them in 4% paraformaldehyde for fixation. The second group is the blank group, cultured in normal culture water. The third group is the low-dose experimental group, cultured in culture water containing 50 μg / mL CBP. The fourth group is the high-dose experimental group, cultured in culture water containing 100 μg / mL CBP. Continuously culture for 15 days, place at -20°C for 20 min, after freezing and sacrificing, place them in 4% paraformaldehyde for fixation. Wash 3 times with PBS, and use 3% H 2 O 2 / 0.5% NaOH for decolorization for 2 h. Dehydrate step by step with 60% ethanol, 80% ethanol, and 100% ethanol. Stain each group of zebrafish with 0.5% alizarin staining solution for 6 h, wash 3 times with PBS, and take pictures under a microscope.

[0164] 2.5.5 Growth and development of zebrafish thorax and fins

[0165] Take 15-day-old zebrafish larvae, divide them into 2 groups, with 5 zebrafish in each group. The first group is the blank group, cultured in normal culture water. The second group is the experimental group, cultured in culture water containing 100 μg / mL CBP. Continuously culture for 15 days, place at -20°C for 20 min, after freezing and sacrificing, place them in 4% paraformaldehyde for fixation, embed in paraffin, and section. Use hematoxylin-eosin staining (HE staining method), and use the Thermo Shandon Varistain Gemini fully automatic staining machine to dewax, hydrate, dehydrate, and clear according to the following conditions. Dehydration steps: successively use xylene (I) for 15 min; xylene (II) for 15 min; 100% ethanol (I) for 15 min; 100% ethanol (II) for 5 min; 95% ethanol for 5 min; 90% ethanol for 5 min; 80% ethanol for 5 min; 70% ethanol for 5 min; distilled water for 3 min; 0.5% hematoxylin for 5 min; rinse with running water for 2 min; soak in hydrochloric acid alcohol for 10 s; rinse with running water for 40 min; rinse with distilled water for 3 min; soak in 0.05% eosin for 4 min; 80% ethanol for 2 min; 90% ethanol for 2 min; 95% ethanol for 2 min; 100% ethanol (I) for 3 min; 100% ethanol (II) for 3 min; xylene (I) for 5 min; xylene (II) for 5 min; xylene (III) for 5 min. Then use the Thermo Shandon Consul fully automatic cover slip machine to cover slip routinely, and then use a microscope to observe the growth and development of the zebrafish thorax and fins.

[0166] 2.5.6 Zebrafish body length measurement

[0167] Take 15-day-old zebrafish larvae, divide them into 5 groups with 5 zebrafish in each group. For the first group, directly measure the body length of the zebrafish; the second group is the blank group, cultured in normal breeding water; the third group is the low-dose experimental group, cultured in breeding water with 50 μg / mL CBP; the fourth group is the high-dose experimental group, cultured in breeding water with 100 μg / mL CBP; the fifth group is the control group, cultured in breeding water with 100 μg / mL commercially available CBP. Continuously culture for 15 days, place at -20 °C for 20 min, after freezing and sacrificing, place under a microscope for photographing, and use Image J to measure the body length of zebrafish in each group.

[0168] 2.6 Zebrafish data statistics

[0169] Use GraphPad Prism 8.0 software to statistically process the data. The experimental data are all expressed as mean ± SEM, and statistical methods such as one-way ANOVA are used to statistically analyze the data.

[0170] 2.7 Quantitative determination basis for zebrafish experiments

[0171]

[0172]

[0173] 3. Experimental results

[0174] 3.1 Effect of CBP on the proliferation of hFOB1.19 human osteoblasts

[0175] The effect of CBP of the present invention on the proliferation of hFOB1.19 human osteoblasts is shown in Table 1, Figure 1 and Figure 3 As shown. With the increase in the concentration of CBP, the growth state of hFOB1.19 human osteoblasts is good, and the cell gaps are getting closer and closer. According to the MTT results, it is also found that with the increase in the concentration of CBP, the survival rate of hFOB1.19 human osteoblasts also increases. When the CBP concentration reaches 50 μg / mL, its cell survival rate is 102.3%, indicating that when the CBP concentration is greater than 50 μg / mL, it can promote the proliferation of hFOB1.19 human osteoblasts. In addition, when the CBP concentration reaches 200 μg / mL, its cell survival rate is 108.4%, and when the CBP concentration reaches 400 μg / mL, its cell survival rate is 107.1%, indicating that when the CBP concentration of the present invention is 200 μg / mL, it has a better effect on promoting the proliferation of hFOB1.19 human osteoblasts, and when the CBP concentration is greater than 200 μg / mL, there is no significant increase in the efficiency of cell proliferation.

[0176] Table 1. Cell survival rate after treating hFOB1.19 human osteoblasts with different concentrations of CBP of the present invention for 72 hours

[0177]

[0178] In this invention, CBP (experimental group) and traditional CBP (control group) were co-cultured with hFOB1.19 cells at a concentration of 200 μg / mL for 72 hours. The comparison of their effects on the proliferation of hFOB1.19 human osteoblasts is shown in Table 2 and Figure 2 as follows. The results showed that there were significant differences between the experimental group and the blank group, while there were no significant differences between the control group and the blank group. It can be seen that the CBP of this invention has a better effect on the proliferation of hFOB1.19 human osteoblasts than the commercially available CBP obtained by traditional processes.

[0179] Table 2. Cell viability of hFOB1.19 human osteoblasts after being treated with CBP of this invention and commercially available CBP for 72 hours

[0180]

[0181] 3.2 Effect of CBP on the expression level of ossein in hFOB1.19 human osteoblasts

[0182] The effect of CBP of this invention on the expression level of ossein in hFOB1.19 human osteoblasts is shown in Table 3 and Figure 4 as follows. The CBP of this invention effectively promotes the expression of human ossein with the increase of concentration; the CBP of this invention shows an obvious promoting effect on the expression of ossein with the increase of concentration.

[0183] Table 3. Effect of CBP of this invention on the expression level of ossein in hFOB1.19 human osteoblasts

[0184]

[0185] Note: One-way ANOVA was used, and Control was compared with 100 μg / mL, 200 μg / mL, and 400 μg / mL respectively for testing. The P values were 0.0186, 0.0020, and 0.0002; t-test analysis was used. When comparing 100 μg / mL with 200 μg / mL for testing, the P value was 0.1543. When comparing 200 μg / mL with 400 μg / mL for testing, the P value was 0.0334.

[0186] In this invention, CBP (experimental group) and commercially available CBP (control group) were co-cultured with hFOB1.19 cells at a concentration of 200 μg / mL for 72 hours. The comparison of their effects on the proliferation of hFOB1.19 human osteoblasts is shown in Table 4 and Figure 5As shown in the figure. The results show that there is a significant difference between the experimental group and the blank group, while there is no significant difference between the control group and the blank group. It can be seen that the promoting effect of the CBP of the present invention on the expression of human bone collagen (Ossein) in hFOB1.19 is better than that of the commercially available CBP obtained by traditional processes.

[0187] Table 4. Effects of CBP of the present invention and commercially available CBP on the expression level of bone collagen in hFOB1.19 human osteoblasts

[0188]

[0189] Note: One-way ANOVA multiple comparisons were performed on the control group and the experimental group with the blank group respectively. Compared with the blank group, the P values of the control group and the experimental group were 0.1351 and 0.0020 respectively.

[0190] 3.3 Effects of CBP on the expression level of human alkaline phosphatase (ALP) in hFOB1.19 human osteoblasts

[0191] The effects of CBP of the present invention on the expression level of human alkaline phosphatase (ALP) in hFOB1.19 human osteoblasts are shown in Table 5 and Figure 6 as shown. The results show that CBP of the present invention effectively promotes the expression of human alkaline phosphatase (ALP) with the increase of concentration. However, compared with the blank group (0 μg / mL), there is no significant difference in the experimental groups with concentrations of 25 - 200 μg / mL. Only when the concentration reaches 400 μg / mL is there a significant difference.

[0192] Table 5. Effects of CBP of the present invention on the expression level of human alkaline phosphatase in hFOB1.19 human osteoblasts

[0193]

[0194] CBP of the present invention (experimental group) and traditional CBP (control group) were co-cultured with hFOB1.19 cells at a concentration of 200 μg / mL for 72 h. The comparison of the effects on the expression level of human alkaline phosphatase (ALP) in hFOB1.19 human osteoblasts is shown in Table 6 and Figure 7 as shown. The results show that the promoting effect of CBP of the present invention on the expression of human alkaline phosphatase (ALP) in hFOB1.19 human osteoblasts is better than that of the commercially available CBP obtained by traditional processes. However, compared with the blank group, there is no significant difference between CBP of the present invention and the commercially available CBP obtained by traditional processes, further indicating that CBP of the present invention cannot significantly promote the expression of human alkaline phosphatase in hFOB1.19 human osteoblasts.

[0195] Table 6. Effects of CBP of the present invention and commercially available CBP on the expression level of human alkaline phosphatase in hFOB1.19 human osteoblasts

[0196]

[0197] The above cell experiment results show that the CBP of the present invention can promote collagen synthesis in a dose-dependent manner, while the commercially available CBP obtained by traditional processes does not have this effect.

[0198] 3.4 Determination of the content of bone collagen in zebrafish

[0199] The effect of the CBP of the present invention on the content of bone collagen in zebrafish is shown in Table 7 and Figure 8 as follows. The contents of bone collagen in zebrafish in the 50 μg / mL and 100 μg / mL concentration groups were 13.12 ± 0.42 μg / mL and 16.88 ± 1.78 μg / mL, respectively, both higher than that of the blank group (11.43 ± 0.83 μg / mL), and there was a significant difference between the 100 μg / mL concentration group and the blank group (p < 0.05), indicating that the CBP of the present invention at 100 μg / mL can effectively promote the production of bone collagen in zebrafish.

[0200] Table 7 Effect of the CBP of the present invention on the content of bone collagen in zebrafish

[0201]

[0202] The CBP of the present invention (experimental group) and commercially available CBP (control group) were continuously treated with zebrafish larvae (15 days old) at a concentration of 100 μg / mL for 15 days, and the comparison of the effects on the content of bone collagen in zebrafish is shown in Table 8 and Figure 9 as follows. There was a significant difference between the experimental group and the blank group (p < 0.001), while there was no significant difference between the control group and the blank group. It can be seen that the CBP of the present invention has a better promoting effect on the production of bone collagen in zebrafish than the commercially available CBP obtained by traditional processes.

[0203] Table 8 Effects of the CBP of the present invention and commercially available CBP on the content of bone collagen in zebrafish

[0204]

[0205] 3.5 Determination of the content of human alkaline phosphatase (ALP) in zebrafish

[0206] The effect of the CBP of the present invention on the content of human alkaline phosphatase (ALP) in zebrafish is shown in Table 9 and Figure 10 as follows. The contents of human alkaline phosphatase in zebrafish in the 50 μg / mL and 100 μg / mL concentration groups were 13.85 ± 0.52 pg / mL and 15.00 ± 0.74 pg / mL, respectively, both higher than that of the blank group (12.91 ± 0.85 pg / mL), but the difference was not significant (p > 0.05), indicating that the CBP of the present invention did not significantly promote the increase in the content of human alkaline phosphatase in zebrafish.

[0207] Table 9. Effects of CBP of the present invention on the content of human alkaline phosphatase in zebrafish

[0208]

[0209] The CBP of the present invention (experimental group) and commercially available CBP (control group) were continuously treated with zebrafish larvae (15-day-old) at a concentration of 100 μg / mL for 15 days, and the effects on the content of human alkaline phosphatase in zebrafish are compared in Table 10 and Figure 11 as shown. The results show that although the promoting effect of the experimental group on the expression of human alkaline phosphatase (ALP) in zebrafish is better than that of the commercially available CBP obtained by the traditional process, there is no significant difference between the two compared with the blank group, further indicating that the CBP of the present invention cannot significantly promote the increase in the content of human alkaline phosphatase in zebrafish.

[0210] Table 10. Effects of CBP of the present invention and commercially available CBP on the content of human alkaline phosphatase in zebrafish

[0211]

[0212]

[0213] 3.6 Determination of calcium element content in zebrafish

[0214] The effects of CBP of the present invention on the calcium element content in zebrafish are shown in Table 11 and Figure 12 as shown. The calcium element contents of zebrafish in the 50 μg / mL and 100 μg / mL concentration groups are 7.69 ± 0.26 pg / g and 9.53 ± 0.27 pg / g respectively, both higher than those of the blank group (5.85 ± 0.34 pg / g), and the difference is significant (p < 0.05), indicating that the CBP of the present invention can effectively promote the increase in the calcium element content in zebrafish.

[0215] Table 11. Effects of CBP of the present invention on the calcium element content in the whole body of zebrafish

[0216]

[0217] The CBP of the present invention (experimental group) and commercially available CBP (control group) were continuously treated with zebrafish larvae (15-day-old) at a concentration of 100 μg / mL for 15 days, and the effects on the calcium element content in the whole body of zebrafish are compared in Table 12 and Figure 13 as shown. There are significant differences between the experimental group, the control group and the blank group (p < 0.001), and there are also significant differences between the experimental group and the control group. It can be seen that the promoting effect of the CBP of the present invention on the calcium element content in the whole body of zebrafish is better than that of the commercially available CBP obtained by the traditional process.

[0218] Table 12. Effects of the CBP of the present invention and commercially available CBP on the total calcium content in zebrafish

[0219]

[0220] 3.7 Somatic skeletal development of zebrafish

[0221] The effects of the CBP of the present invention at 50 μg / mL and 100 μg / mL on the somatic skeletal development of zebrafish are as Figure 14 shown. According to the experimental results, carefully observing the somatic skeletal development of zebrafish, it can be seen that the somatic skeletons of zebrafish at 15 days old in the first group are not yet mature and do not form clear nodules, while the somatic skeletons of zebrafish at 30 days old in the second group show obvious nodules in their development. The somatic skeletons of zebrafish in the third and fourth groups after treatment with 50 μg / mL and 100 μg / mL of CBP are fully developed, with the spinal column developed completely and the nodules clear. This shows that the CBP of the present invention can promote the somatic skeletal development of zebrafish.

[0222] 3.8 Caudal skeletal development of zebrafish

[0223] The effects of the CBP of the present invention at 50 μg / mL and 100 μg / mL on the caudal skeletal development of zebrafish are as Figure 15 shown. According to the experimental results, carefully observing the caudal skeletal development of zebrafish, it can be observed that the caudal skeletons of zebrafish at 15 days old in the first group are not yet mature and do not form clear nodules, and the caudal nodules of the small fish at 30 days old in the second group are generated, but there is still a section at the very end that is not fully developed. While the caudal skeletons of small fish in the third and fourth groups treated with 50 μg / mL and 100 μg / mL of CBP are fully developed, with the caudal spinal column and caudal fin developed completely and the nodules clear. This shows that the CBP of the present invention can promote the caudal skeletal development of zebrafish.

[0224] 3.9 Thoracic vertebral morphology development of zebrafish

[0225] The effect of 100 μg / mL of the CBP of the present invention on the thoracic vertebral development of zebrafish is as Figure 16 shown. According to the experimental results, it is shown that the maturity of the thoracic vertebrae of zebrafish in the 100 μg / mL CBP treatment group is significantly better than that of zebrafish in the blank group, indicating that the CBP of the present invention has a better effect on promoting the growth and development of zebrafish thoracic vertebrae.

[0226] 3.10 Fin growth and development of zebrafish

[0227] The effect of 100 μg / mL of the CBP of the present invention on the caudal fin development of zebrafish is as Figure 17 shown. According to the experimental results, it is shown that the growth length of the caudal fins of zebrafish in the 100 μg / mL CBP treatment group is significantly better than that of zebrafish in the blank group, indicating that the CBP of the present invention has a better effect on promoting the growth and development of zebrafish caudal fins.

[0228] 3.11 Zebrafish Body Length Growth and Development

[0229] The effect of the CBP of the present invention on the body length of zebrafish is shown in Table 13 and Figure 18 as follows. The body length of zebrafish in the blank group was measured to be 114.31 ± 10.51. After 15 days of treatment with 50 μg / mL and 100 μg / mL CBP, the body lengths of zebrafish were 155.56 ± 3.87 and 185.91 ± 10.38 respectively, which increased compared with the blank group, and the differences were significant; indicating that the CBP of the present invention has a significant promoting effect on the body length of zebrafish.

[0230] Table 13. Effect of CBP of the Present Invention on Zebrafish Body Length

[0231]

[0232] The CBP of the present invention (experimental group) and commercially available CBP (control group) were continuously treated with zebrafish larvae (15-day-old) at a concentration of 100 μg / mL for 15 days, and the comparison of the effects on zebrafish body length is shown in Table 14 and Figure 19 as follows. The results show that the CBP of the present invention can significantly promote the growth and development of zebrafish body length, and is superior to commercially available CBP.

[0233] Table 14. Effects of CBP of the Present Invention and Commercially Available CBP on Zebrafish Body Length

[0234]

[0235] The above zebrafish experiments show that the CBP of the present invention promotes the synthesis of collagen and the absorption of bone calcium, and finally shows that the development status of the spine, thoracic vertebrae, and caudal vertebrae of zebrafish intervened by the CBP of the present invention is better, the caudal fin is longer, and the body length is longer, which is superior to commercially available CBP.

[0236] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: it is still possible to modify the specific implementation manners of the present invention or perform equivalent replacements for some technical features; without departing from the spirit of the technical solutions of the present invention, they should all be covered within the scope of the technical solutions claimed by the present invention.

Claims

1. A colostrum basic protein, wherein, the protein content is ≥ 80%, the content of components with a molecular weight of 1 - 30 KDa is ≥ 50%, and the IgG content is ≥ 10%; Preferably, the content ratio of 1 KDa - 6 KDa molecular weight is ≤ 10%, and the content ratio of 6 KDa - 30 KDa molecular weight is ≥ 40%; More preferably, the IGF - 1 content is ≤ 0.008‰.

2. The colostrum basic protein according to claim 1, wherein, the IgG content is 10% - 20% (such as 12% - 18%, 12% - 17%, 15%); Preferably, the content ratio of 1 KDa - 6 KDa molecular weight is 3% - 10% (such as 3% - 9%, 3% - 8%, 6% - 8%), and the content ratio of 6 KDa - 30 KDa molecular weight is 40% - 70% (such as 45% - 70%, 50% - 70%, 55% - 70%); More preferably, the IGF - 1 content is ≤ 0.006‰ (such as ≤ 3×10^(-4)‰, ≤ 3×10^(-5)‰, ≤ 3×10^(-6)‰).

3. The colostrum basic protein according to claim 1 or 2, wherein, the IgG content is 15%; Preferably, the content of 1 KDa - 6 KDa molecular weight is 6.9%, and the content of 6 KDa - 30 KDa molecular weight is 60.8%; More preferably, the IGF - 1 content is ≤ 1.5×10^(-6)‰.

4. The colostrum basic protein according to any one of claims 1 - 3, which is prepared by a method comprising the following steps: (1) Provide a colostrum basic protein raw material, (2) Enzymatically hydrolyze the colostrum basic protein raw material with alkaline protease under ultra - high pressure; Preferably, the colostrum basic protein raw material in step (1) is a commercially available colostrum basic protein, or is obtained by using bovine colostrum as a raw material, through sterilization, degreasing, centrifugal separation, removing casein, α - lactalbumin, β - lactoglobulin, microfiltration, and ultrafiltration; Preferably, step (2) has one or more of the following technical features: a) The mass ratio of the alkaline protease to the colostrum basic protein raw material is 1:80 - 150, preferably 1:90 - 120, more preferably 1:95 - 110, and even more preferably 1:100, b) The ultra - high pressure is ≥ 100 MPa, such as 100 - 120 MPa, c) The enzymatic hydrolysis is carried out at 36℃ - 38℃ for 1 - 4 hours, more preferably at 37℃ for 1.5 - 2.5 hours, such as 2 hours, d) In the enzymatic hydrolysis system, the concentration of the colostrum basic protein raw material is 8 - 20 mg / mL, preferably 8 - 15 mg / mL, more preferably 9 - 13 mg / mL, and even more preferably 10 mg / mL; Preferably, after step (2), it also includes inactivating the enzyme by heat treatment; Optionally, it also includes drying, such as freeze - drying, preferably vacuum freeze - drying; Preferably, the heat treatment is carried out at 100 - 120℃ for 8 - 20 min, preferably at 100 - 110℃ for 8 - 15 min, and more preferably at 100℃ for 10 min.

5. A method for preparing the colostrum basic protein according to any one of claims 1-4, which comprises subjecting the colostrum basic protein raw material to ultra-high pressure enzymatic hydrolysis.

6. The method according to claim 5, which comprises the following steps: (1) Provide a colostrum basic protein raw material, (2) Enzymatically hydrolyze the colostrum basic protein raw material with alkaline protease under ultra-high pressure; Preferably, the colostrum basic protein raw material described in step (1) is commercially available colostrum basic protein, or is obtained by using bovine colostrum as a raw material, followed by sterilization, defatting, centrifugal separation, removal of casein, α-lactalbumin, β-lactoglobulin, microfiltration, and ultrafiltration; Preferably, step (2) has one or more of the following technical features: a) The mass ratio of the alkaline protease to the colostrum basic protein raw material is 1:80-150, preferably 1:90-120, more preferably 1:95-110, and even more preferably 1:100, b) The ultra-high pressure is ≥100 MPa, such as 100-120 MPa, c) The enzymatic hydrolysis is carried out at 36°C - 38°C for 1-4 hours, more preferably at 37°C for 1.5-2.5 hours, such as 2 hours, d) In the enzymatic hydrolysis system, the concentration of the colostrum basic protein raw material is 8-20 mg / mL, preferably 8-15 mg / mL, more preferably 9-13 mg / mL, and even more preferably 10 mg / mL; Preferably, after step (2), it further comprises inactivating the enzyme by heat treatment; Optionally, it further comprises drying, such as freeze-drying, preferably vacuum freeze-drying; Preferably, the heat treatment is carried out at 100-120°C for 8-20 min, preferably at 100-110°C for 8-15 min, and more preferably at 100°C for 10 min.

7. A dairy product, which comprises the colostrum basic protein according to any one of claims 1-4; Preferably, it further comprises calcium and / or vitamin D (such as vitamin D2 and / or vitamin D3); Preferably, the dairy product is a solid dairy product or a liquid dairy product. Preferably, the solid dairy product is selected from formula milk powder and soy-based milk powder, preferably from infant formula milk powder, children's formula milk powder, adult formula milk powder, and middle-aged and elderly formula milk powder; preferably, the liquid dairy product is selected from formulated milk, milk beverage, and soy milk.

8. Use of the colostrum basic protein according to any one of claims 1-4 in the preparation of a product; Preferably, the product is a dairy product; Preferably, the dairy product is a solid dairy product or a liquid dairy product. Preferably, the solid dairy product is selected from formula milk powder and soy-based milk powder, preferably from infant formula milk powder, children's formula milk powder, adult formula milk powder, and middle-aged and elderly formula milk powder; preferably, the liquid dairy product is selected from formulated milk, milk beverage, and soy milk; Preferably, the product is a product for increasing bone mass, increasing bone density, promoting bone development, promoting bone regeneration or repair, promoting calcium absorption, promoting bone collagen production, and / or preventing and / or treating bone-related diseases (such as osteoporosis, osteopenia, rickets, osteoarthropathy).

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