Preparation method of colostrum immune protein extract with tumor inhibition effect

Abstract

The invention discloses a preparation method of a colostrum immune protein extract with a tumor inhibition effect. The preparation method comprises the following steps: (1) selecting raw materials; (2) flocculating casein; (3) removing sugar; (4) filtering with an ultrafiltration membrane; (5) directional hydrolysis; (6) cation exchange resin adsorption; (7) desalting; (8) freeze drying; the extract provided by the invention enriches hydrolytic fragments of colostrum immune protein rich in hydrophobic amino acid and alkaline amino acid, and shows stronger anti-oxidation, immunological enhancement and tumor inhibition activities. Different from common colostrum immune protein, the extract exerts anti-tumor activity which is a combined effect of dual effects of immune enhancement and tumor cell growth inhibition.

Description

Technical Field

[0001] The invention relates to the field of food nutrition and functional factors, in particular to a method for preparing a colostrum immune protein extract with tumor inhibition efficacy. Background Art

[0002] Colostrum refers to the milk secreted by cows within three days after calving, and its composition is very different from that of normal milk. From a nutritional point of view, colostrum is rich in proteins with immune-enhancing activity, including immunoglobulins (such as IgG, IgA, IgM) and lactoferrin, and its content is much higher than that of normal milk. These immune proteins can help newborn calves resist harmful substances such as external pathogens, and have multiple functions such as antibacterial, antiviral, and antioxidant.

[0003] From a biological perspective, colostrum immune proteins are an important natural defense for newborns against external pathogens. For example, immunoglobulins (Ig), including IgG, IgA and IgM, can specifically recognize and bind to pathogens such as bacteria and viruses, prevent them from invading body cells, and activate the body's immune defense mechanisms, such as the complement system and phagocytes, to eliminate pathogens. Lactoferrin has multiple functions such as antibacterial, antiviral, and antioxidant. It can chelate iron ions, making it impossible for microorganisms that rely on iron ions to obtain sufficient iron nutrition and inhibit their growth and reproduction. It plays an important role in preventing and treating gastrointestinal infections.

[0004] As people pay more and more attention to health, the demand for natural products with immune regulatory functions is growing. Traditional drug treatments often have problems such as large side effects or short-lasting effects when dealing with some chronic diseases and immune dysfunction. As a natural bioactive substance, the safety and effectiveness of colostrum immune protein are gradually recognized. In the food industry, consumers' pursuit of functional foods has prompted companies to explore the use of colostrum immune protein to develop new products. In the pharmaceutical industry, colostrum immune protein is also regarded as a potential drug raw material or auxiliary therapeutic ingredient for the development of innovative drugs for the treatment of immune-related diseases. According to market research institutions, the global colostrum immune protein health care product market will continue to grow in the next few years, with an annual compound growth rate expected to reach 10%-15%. Summary of the invention

[0005] In view of the above problems existing in the prior art, the present invention provides a method for preparing a colostrum immune protein extract with tumor inhibition effect. The present invention utilizes membrane separation, protease directional hydrolysis, cation exchange resin adsorption and other technical means to realize the preparation of a colostrum immune protein extract with strong tumor inhibition activity. The extract is characterized in that it is enriched with hydrolysis fragments rich in hydrophobic amino acids and rich in basic amino acids of colostrum immune protein, and exhibits stronger antioxidant, immune enhancement and tumor inhibition activity. Different from ordinary colostrum immune protein, the anti-tumor activity of the extract is the result of the combined effect of its dual effects of immune enhancement and tumor cell growth inhibition.

[0006] The technical solution of the present invention is as follows:

[0007] The first object of the present invention is to provide a method for preparing a colostrum immune protein extract having tumor inhibition efficacy, comprising the following steps:

[0008] (1) Raw material selection: select bovine colostrum collected within 3 days of calving;

[0009] (2) flocculating casein, adding rennet to bovine colostrum, hydrolyzing, filtering, and collecting whey;

[0010] (3) removing sugar by filtering the whey liquid with a nanofiltration membrane, repeatedly washing and filtering until the lactose content in the retentate is less than 0.1%, and collecting the retentate;

[0011] (4) filtering the retentate collected in step (3) through an ultrafiltration membrane, filtering the retentate through an ultrafiltration membrane, and repeatedly washing the retentate until the total content of lactalbumin and lactoglobulin in the retentate is less than 10%, and collecting the retentate;

[0012] (5) directional hydrolysis, mixing the composite enzyme composed of papain and trypsin with the retentate obtained in step (4), and hydrolyzing to obtain a directional hydrolyzate;

[0013] (6) Adsorption by cation exchange resin, collecting target and non-target components by step elution, first using pH 7.0 phosphate buffer containing 0.5-1.0% NaCl, and mixing with the penetration solution to obtain component 1; then using pH 8.0 phosphate buffer containing 2-4% NaCl for a second elution to elute colostrum immune protein component 2 with a high positive charge density;

[0014] (7) Desalting: using a rolled nanofiltration membrane to desalt and concentrate the collected colostrum immune protein component 1 and component 2, respectively, and repeatedly adding water for filtration until the salt content accounts for less than 0.5% of the total solids;

[0015] (8) Freeze drying: freeze drying the desalted and concentrated colostrum immune protein component. Colostrum immune protein component 2 is a colostrum immunoglobulin extract with tumor inhibitory activity; colostrum immune protein component 1 is a common immune-enhancing high-end ingredient.

[0016] In one embodiment of the present invention, in step (1), the bovine colostrum has a high level of immunoglobulin and lactoferrin immune proteins.

[0017] In one embodiment of the present invention, in step (2), the amount of rennet used is 0.05% of the mass of bovine colostrum; the hydrolysis conditions are: pH 6.0-6.5, temperature 50-65°C, and time 0.5-1h.

[0018] In one embodiment of the present invention, in step (2), filtration is performed after hydrolysis to separate the flocculated casein, and the whey liquid contains immunoglobulin and lactoferrin, two proteins with immune-enhancing effects.

[0019] In one embodiment of the present invention, in step (3), the molecular weight cutoff of the nanofiltration membrane is 1000 Da.

[0020] In one embodiment of the present invention, in step (3), the lactose is removed in order to effectively protect the structure of the active protein in the whey liquid and to increase the concentration of the active immunoglobulin and lactoferrin. After filtering 80% of the water by nanofiltration, an equal volume of water is added for filtration again until the lactose content in the retentate is less than 0.1%. The colostrum immune protein solution after the lactose removal and concentration is collected. At this time, the main components in the collected retentate are immunoglobulin, lactoferrin, lactalbumin and lactoglobulin. The molecular weights of these proteins are immunoglobulin>lactoferrin>lactoglobulin>lactalbumin.

[0021] In one embodiment of the present invention, in step (4), the molecular weight cutoff of the ultrafiltration membrane is 80-100 kDa; in order to further increase the content of immunoglobulin and lactoferrin, a higher immune protein content is obtained.

[0022] In one embodiment of the present invention, in step (5), the mass ratio of papain to trypsin is 1:0.5-2; the dosage of the complex enzyme is 2000-4000 U / g, based on the relative dosage of protein; the hydrolysis temperature is 45-55° C., and the time is 1-4 h.

[0023] In one embodiment of the present invention, in step (5), the two enzymes are characterized in that they can directionally hydrolyze the hydrophobic amino acid termini and basic amino acid termini in the amino acid peptide chains of proteins. The hydrophobic amino acids are the key to determining the antioxidant and immune-enhancing activities of protein hydrolysates. The hydrolysis of the basic amino acid termini can give the polypeptide a higher positive charge density. The positive charge density of the polypeptide can effectively improve its affinity with bacteria and viral cells, and exhibit stronger antiviral, antibacterial and antitumor activities.

[0024] In one embodiment of the present invention, in step (6), the cation exchange resin is one or more of polystyrene sulfonic acid resin, perfluorosulfonic acid resin, acrylic resin, and macroporous cation exchange resin; the adsorption conditions are pH 6.5-8.0, the adsorption temperature is 30-45°C, and the adsorption solid-liquid mass ratio is 1:5-10.

[0025] In one embodiment of the present invention, in step (7), the molecular weight cut-off of the spiral nanofiltration membrane is 200-400 Da.

[0026] The second object of the present invention is to provide a colostrum immune protein extract prepared by the above method.

[0027] In one embodiment of the present invention, the yield of colostrum immune protein is high, the obtained extract has a strong positive charge, strong antioxidant capacity, and exhibits a good tumor inhibition effect.

[0028] The colostrum immune protein extract in the present invention is enriched with hydrolyzed fragments of colostrum immune protein rich in hydrophobic amino acids and basic amino acids, showing stronger antioxidant, immune enhancement and tumor inhibition activities. Different from ordinary colostrum immune protein, the extract exerts anti-tumor activity as a result of the combined effects of its dual effects of immune enhancement and tumor cell growth inhibition.

[0029] The third object of the present invention is to provide an application of the colostrum immune protein extract for preparing a tumor inhibitor.

[0030] The beneficial technical effects of the present invention are:

[0031] The present invention utilizes membrane separation, protease directional hydrolysis, cation exchange resin adsorption and other technical means to realize the preparation of colostrum immune protein extract with strong tumor inhibition activity. The extract is characterized in that it is enriched with hydrolysis fragments rich in hydrophobic amino acids and rich in basic amino acids of colostrum immune protein, and exhibits stronger antioxidant, immune enhancement and tumor inhibition activities. Different from ordinary colostrum immune protein, the extract exerts anti-tumor activity as a result of the combined effects of its dual effects of immune enhancement and tumor cell growth inhibition. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1Serum cytokines of tumor mice in different drug administration groups;

[0033] Figure 2 p38MAPK gene and PI3KCA gene of tumor mice in different drug administration groups;

[0034] Figure 3 VEGF protein, NF-κB protein and P-NF-κB protein in tumor mice in different drug administration groups. DETAILED DESCRIPTION

[0035] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0036] Embodiment 1:

[0037] (1) Raw material selection: The raw material referred to in this method is bovine colostrum collected within 3 days of calving, which contains high levels of immunoglobulin and lactoferrin immune proteins.

[0038] (2) Rennet-induced flocculation: Rennet was used to induce casein flocculation in colostrum. The pH value of rennet hydrolysis was 6.0, the hydrolysis temperature was 50°C, and the hydrolysis time was 0.5 h. After hydrolysis, the flocculated casein was filtered and separated, and the whey was collected to further extract immunoglobulin and lactoferrin, two proteins with immune-enhancing effects.

[0039] (3) Lactose removal: In order to effectively protect the structure of active proteins in whey, a 1000Da nanofiltration membrane is used to separate lactose from active proteins to increase the concentration of active immunoglobulins and lactoferrin. After 80% of the water is filtered out by nanofiltration, an equal volume of water is added and washed again until the lactose content in the retentate is <0.1%.

[0040] (4) Ultrafiltration membrane filtration: In order to further increase the content of immunoglobulin and lactoferrin and obtain a higher immune protein content, an ultrafiltration membrane with a molecular weight cutoff of 100 kDa is used to filter the lactalbumin and lactoglobulin in the whey solution, and the filtration is repeated until the lactalbumin and lactoglobulin in the retentate account for less than 10% of the total solid content.

[0041] (5) Protease-directed hydrolysis: The bovine colostrum immunologically active protein obtained in step (4) was hydrolyzed using papain and trypsin, with a total amount of papain and trypsin added being 3000 U / g, a ratio of 1:2, a hydrolysis temperature of 50° C., and a hydrolysis time of 2 h.

[0042] (6) Cation exchange resin adsorption: In order to further enrich colostrum immunoglobulins with positive charge characteristics, cation exchange resin was used for adsorption extraction. The cation exchange resin material used was a macroporous cation exchange resin. The adsorption conditions were pH 6.5, adsorption temperature 30°C, and adsorption solid-liquid mass ratio was 1:8. The target and non-target components were collected by step-by-step elution. First, a pH 7.0 phosphate buffer containing 0.75% NaCl was used. After elution, it was mixed with the penetration solution to obtain component 1; then a pH 8.0 phosphate buffer containing 4% NaCl was used for a second elution to elute the colostrum immune protein component 2 with a high positive charge density.

[0043] (7) Desalting: Using a rolled nanofiltration membrane with a molecular weight cutoff of 300 Da, the collected colostrum immune protein component 1 and component 2 were desalted and concentrated respectively, and water was repeatedly added for filtration until the salt content accounted for less than 0.5% of the total solids.

[0044] (8) Freeze drying: The desalted and concentrated colostrum immune protein fraction is freeze dried. Colostrum immune protein fraction 2 is a colostrum immunoglobulin extract with tumor inhibitory activity; colostrum immune protein fraction 1 is a common immune-enhancing high-end ingredient.

[0045] Embodiment 2:

[0046] Step (5) protease-directed hydrolysis: The bovine colostrum immunologically active protein obtained in step (4) is hydrolyzed using papain and trypsin, with the total amount of papain and trypsin added being 4000 U / g, the ratio being 2:1, the hydrolysis temperature being 50° C., and the hydrolysis time being 2 h.

[0047] The other steps are the same as in Example 1.

[0048] Embodiment 3:

[0049] Step (6) cation exchange resin adsorption: The cation exchange resin material used is a macroporous cation exchange resin, the adsorption conditions are pH 6.5, the adsorption temperature is 30°C, the adsorption solid-liquid mass ratio is 1:5, and the target and non-target components are collected by step-by-step elution. First, a pH 7.0 phosphate buffer containing 0.5% NaCl is used, and after elution, it is mixed with the penetration liquid to obtain component 1; then a pH 8.0 phosphate buffer containing 3% NaCl is used for a second elution to elute the colostrum immune protein component 2 with a high positive charge density.

[0050] The other steps are the same as in Example 1.

[0051] Comparative Example 1:

[0052] Step (5) protease-directed hydrolysis: The bovine colostrum immunologically active protein obtained in step (4) is hydrolyzed using papain and trypsin, with the total amount of papain and trypsin added being 5000 U / g, the ratio being 1:2, the hydrolysis temperature being 50° C., and the hydrolysis time being 2 h.

[0053] The other steps are the same as in Example 1.

[0054] Comparative Example 2:

[0055] Steps (1) to (5) are the same as those in Example 1. Steps (6) and (7) are deleted, and the sample is freeze-dried directly after directed enzymatic hydrolysis to obtain a comparative sample.

[0056] Comparative Example 3:

[0057] Step (5) in the embodiment is deleted, and the other steps are the same as those in embodiment 1.

[0058] Experimental methods

[0059] (1) Protein yield of component 2

[0060] The total protein content m in the crude bovine colostrum immune protein obtained in step (4) was determined by Kjeldahl method. 0 , and the total protein content m in the fraction 2 eluted in step (6) 1 , using the formula m 0 / m 1 *100 to calculate protein yield.

[0061] Weigh 1.0g of solid sample accurately, transfer it into a dry 100ml or 500ml nitrogen determination bottle, add 0.2g of copper sulfate, 6g of potassium sulfate and 20ml of sulfuric acid, shake it slightly and place a small funnel at the mouth of the bottle, place the bottle at a 45-degree angle on an asbestos net with small holes, heat it with a low fire, wait until the contents are completely carbonized and the foam stops completely, increase the firepower, and keep the liquid in the bottle slightly boiling until the liquid is blue-green, clear and transparent, and continue heating for 0.5 hours. Remove and cool, carefully add 20ml of water, cool it, transfer it into a 100ml volumetric flask, and wash the nitrogen determination bottle with a small amount of water, add the washing liquid into the volumetric flask, add water to the scale, mix well and set aside. Perform a reagent blank test at the same time.

[0062] Add a few drops of methyl red indicator and a few milliliters of sulfuric acid to about 2 / 3 of the water in the steam generator to keep the water acidic, and add a few glass beads to prevent violent boiling.

[0063] Add 10ml of 2% boric acid solution and 1 drop of mixed indicator to the receiving bottle, and insert the lower end of the condenser under the liquid surface. Pipette 10.0ml of sample digestion solution from the small glass cup into the reaction chamber, and wash the small beaker with 10ml of water to make it flow into the reaction chamber, and plug the rod-shaped glass stopper of the small glass cup. Pour 10ml of 40% sodium hydroxide solution into the small glass cup, lift the glass stopper to make it slowly flow into the reaction chamber, and do not plug the glass cover immediately. It should be rinsed with distilled water first and then covered, and water should be added to the small glass cup to prevent air leakage. Clamp the spiral clamp and start distillation. Steam is passed into the reaction chamber to make ammonia pass through the condenser and enter the receiving bottle. Distill for 5 minutes. Move the receiving bottle so that the lower end of the condenser leaves the liquid dish, distill for another 1 minute, and then rinse the outside of the lower end of the condenser with a small amount of water.

[0064] Titration: Remove the receiving bottle and titrate with 0.05mol / L sulfuric acid or 0.05mol / L hydrochloric acid standard solution until gray or blue-purple is the end point. At the same time, take 10.0ml of reagent blank digestion solution and titrate according to the above operation.

[0065] The absolute mass of protein in the sample was calculated according to the following formula:

[0066] m=(VV 0 )×N×0.014×F

[0067] V is the volume of sulfuric acid or hydrochloric acid standard solution consumed by the sample;

[0068] V 0 The volume of sulfuric acid or hydrochloric acid standard solution consumed for the reagent blank;

[0069] N is the equivalent concentration of sulfuric acid or hydrochloric acid standard solution;

[0070] F is the factor for converting nitrogen to protein (calculated as 6.25).

[0071] (2) Surface charge

[0072] The collected colostrum immune protein components were prepared into a solution with a concentration of 1 mg / mL, 1 mL of the sample was added to the measuring cell, and the measurement was performed after stabilization at 25°C for 120 seconds. The refractive index of the sample was 1450 and the absorbance was 0.001. In order to exclude large insoluble protein aggregates, all samples were filtered through a 0.45 μm water membrane and the surface charge was re-measured.

[0073] (3) Surface hydrophobicity

[0074] ANS was used as a fluorescent probe to add to the collected colostrum immune protein component solution. The solution concentration was 1 mg / mL. 20 μL of ANS solution was added to 4 mL of sample solution. The reaction was carried out in the dark for 3 min, and then the fluorescence spectrum of the sample from 400 to 600 nm was scanned using an F-7000 fluorescence spectrometer (Hitachi, Tokyo, Japan) at an excitation wavelength of 390 nm. The excitation and emission slits were both set to 5.0 nm. The fluorescence intensity at the excitation wavelength of 480 nm was read as a comparable quantitative indicator of surface hydrophobicity.

[0075] (4) Antioxidant

[0076] ①ABTS free radical scavenging rate

[0077] Prepare ABTS into a 6.94mmol / L aqueous solution and potassium persulfate into a 2.6mmol / L aqueous solution. Place the mixed solution in a cool place for 12 to 16 hours before use to allow the two to react completely. Then dilute the original solution with 95% ethanol to obtain a dilution whose absorbance should be between 0.70±0.02. During the measurement, take 3.6ml of the dilution and 0.4ml of the sample solution, replace the sample solution with deionized water for the blank sample, mix well, react at room temperature in the dark for 5 minutes, and measure the absorbance at a wavelength of 734nm.

[0078] The calculation formula of ABTS clearance rate (%) is:

[0079]

[0080] A 0 Indicates the absorbance of the blank sample, A 1 Indicates the absorbance of the sample solution.

[0081] ②Superoxide anion Clearance

[0082] The determination of clearance rate was based on the method of Xu Shenhong et al. and slightly modified. 1.5 ml of Tris-HCl buffer (0.1 mol / L, containing 2 mmol / L EDTA) with pH = 8.2 and sample solution were added to the test tube, and distilled water was used to replace the blank tube. After 20 minutes of constant temperature at 25°C, pyrogallol (6 mmol / L) preheated at 25°C was added, and 10 mmol / L hydrochloric acid was used to replace the blank tube. The mixture was quickly shaken and immediately poured into a cuvette with a light path of 1 cm. The absorbance was measured every 0.5 min at a wavelength of 420 nm for a total of 2 minutes, and the rate of change of the absorbance value of the solution over time was calculated.

[0083] (5) Construction of tumor-bearing mouse model, expression of immune factors and tumor factors

[0084] Preparation of tumor-bearing model: After 1 week of adaptation, 2×10 6 The tumor cells were subcutaneously inoculated with cells. After the tumor mass was touched one week later, the downstream experiment was started. The samples tested were colostrum immune protein component 1 and component 2 obtained according to Example 1, counted as sample No. 1 and sample No. 2, respectively. The mice with successful modeling were randomly divided into low, medium and high dose groups of sample No. 1 and sample No. 2; tumor model group; healthy mouse group, 4 mice in each group. The tumor model group and the healthy mouse group were gavaged with normal saline during the experiment, and the low, medium and high dose groups of sample No. 1 and sample No. 2 were gavaged with samples of different concentrations (50, 100, 200 mg / kg), and the administration was continued for 4 weeks. The weight of the mice was recorded daily, and the general performance of the mice, such as diet, drinking water, and activity status, was observed.

[0085] Serum cellular immune factor content detection:

[0086] 24 hours after the last administration, blood was collected from mice, left to stand for 2 hours, and centrifuged at 3000 r / min for 10 minutes at 4°C to separate serum.

[0087] 1. Detection of IFN-γ

[0088] (1) Dilute the standard to 1200 ng / L, 800 ng / L, 400 ng / L, 200 ng / L, and 100 ng / L, add 50 μl to each well, and set up a blank well to prepare a standard curve.

[0089] (2) Add 10 μl of the serum to be tested to each well, then add 40 μl of sample diluent and mix well. (Equivalent to diluting the serum to be tested 5 times)

[0090] (3) Seal the plate with a sealing film and incubate at 37°C for 30 min. Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let stand for 30 seconds, then discard. Repeat this process 5 times and pat dry.

[0091] (4) Add 50 μl of enzyme-labeled reagent to each well, except for the blank well. Seal the plate with a sealing film and incubate at 37°C for 30 min. Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds, then discard it. Repeat this process 5 times and pat dry.

[0092] (5) First add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake to mix, and incubate at 37°C in the dark for 15 min. Add 50 μl of stop solution to each well to terminate the reaction.

[0093] (6) Using blank as the zero value, measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm.

[0094] 2. Detection of TNF-α

[0095] The specific operation steps are the same as above, except that the kit used is changed to a TNF-α detection kit.

[0096] 3. Detection of IL-6

[0097] The specific operation steps are the same as above, except that the kit used is changed to an IL-6 detection kit.

[0098] Detection of p38MAPK and PI3KCA expression at the mRNA level in tumor tissue

[0099] 1. Extraction of total RNA from tissues

[0100] (1) Tumor tissue was placed in a 700 μl Trizol homogenizer tube and homogenized on ice for 30 seconds;

[0101] (2) Lyse in a clean bench for 15 min, then centrifuge at 12,000 rpm for 15 min;

[0102] (3) Aspirate the supernatant into a new 1.5 mL centrifuge tube, add 200 μL of chloroform, shake well, let stand at room temperature for 5 min, and centrifuge at 4°C, 12,000 rpm for 15 min;

[0103] (4) Aspirate the supernatant into a new 1.5 mL centrifuge tube, add an equal volume of isopropanol, mix well, let stand at room temperature for 15 min, centrifuge at 4 °C, 12000 r / min for 15 min, and discard the supernatant;

[0104] (5) Add 1 mL of 75% anhydrous ethanol to rinse the precipitate, centrifuge at 4°C, 12,000 rpm for 5 min, and discard the supernatant;

[0105] (6) Add 1 mL of anhydrous ethanol to rinse the precipitate, centrifuge at 4°C, 12,000 rpm for 5 min, discard the supernatant, and dry at room temperature for 5 min;

[0106] (7) Add 20 μL of DEPC water to dissolve the RNA and store it in a -80°C refrigerator until use.

[0107] 2. Reverse transcription

[0108] Table 1

[0109]

[0110]

[0111] Reaction procedure: 42°C, 60 minutes; 70°C, 5 minutes; store at -20°C.

[0112] 3. Real-time PCR amplification

[0113] Primer sequences:

[0114] Table 2

[0115]

[0116] Reaction system:

[0117] Table 3

[0118] SYBRGreen Mix: 10μL Upstream primer F: 1μL Downstream primer R: 1μL <![CDATA[ddH 2 O:]]> 11μL cDNA Template: 2μL Total volume: 25μL

[0119] Reaction procedure: 95°C, 10 min; (95°C, 15 sec; 55°C, 45 sec)×40; 95°C, 15 sec; 60°C, 1 min; 95°C, 15 sec; 60°C, 15 sec.

[0120] Detection of VEGF and NF-κB protein expression in tumor tissue

[0121] Table 4

[0122]

[0123]

[0124] 1. Protein quantification

[0125] (1) Drawing of standard curve: Take an ELISA plate and add reagents according to the table below.

[0126] Table 5

[0127] Hole No. 1 2 3 4 5 6 7 8 Protein standard solution (μl) 0 2 4 6 8 12 16 20 Deionized water (μl) 20 18 16 14 12 8 4 0 Protein concentration (μg / μl) 0 0.05 0.1 0.15 0.2 0.3 0.4 0.5

[0128] (2) According to the number of samples, prepare an appropriate amount of BCA working solution by mixing BCA reagent A and reagent B in a volume ratio of 50:1 and mix thoroughly; add 180 μl of BCA working solution to each well.

[0129] (3) Place the ELISA plate on an oscillator for 30 seconds, place at 37°C for 30 minutes, and then measure the absorbance at 562 nm. Draw a standard curve with the absorbance value as the horizontal axis and the protein concentration (μg / μl) as the vertical axis;

[0130] (4) Add 2 μl of the protein to be tested and 18 μl of PBS (diluted 10 times) to the ELISA plate, add 180 μl of BCA working solution, place the ELISA plate on an oscillator for 30 seconds, incubate at 37°C for 30 minutes, and then measure the absorbance at 562 nm. Duplicate wells are made for each sample.

[0131] (5) Based on the absorbance value of the sample, the corresponding protein concentration (μg / μl) can be found on the standard curve and multiplied by the sample dilution factor (10) to obtain the actual sample concentration (μg / μl).

[0132] 2. Preparation of SDS-PAGE gel

[0133] Bottom layer glue:

[0134] Table 6

[0135]

[0136] Upper glue:

[0137] Table 7

[0138]

[0139] 3. Sample loading and electrophoresis

[0140] (1) The amount of protein loaded in each well is 20 μg. According to the protein quantification results, take the required protein and add an appropriate amount of loading buffer. After boiling in a water bath for 10 minutes, centrifuge and take the supernatant for loading. Place the prepared PAGE gel in the electrophoresis tank and add an appropriate amount of electrophoresis buffer. Slowly add the prepared sample to the corresponding well with a pipette gun.

[0141] (2) 80V for 20 min for the upper gel layer and 120V for 60 min for the lower gel layer. When the dye reaches the bottom of the gel, cut off the power supply, stop electrophoresis, and proceed to the next step of membrane transfer.

[0142] 4. Transfer

[0143] For semi-dry transfer, the sandwich is arranged as: / filter paper / gel / membrane / filter paper, soaked with electrotransfer buffer, and placed directly between the positive and negative electrodes of the electrotransfer instrument. The gel is placed on the negative electrode and the membrane is placed on the positive electrode. The semi-dry electrotransfer buffer is: 48mM Tris, 39mMglycine, 0.04% SDS, 20% methanol. Transfer at 25V for 30min. Soak the PVDF membrane in methanol for 2min before transfer, and then incubate in ice-cold electrotransfer buffer for 5min. The gel also needs to be balanced in ice-cold electrotransfer buffer for 3-5min, otherwise the bands will be deformed during transfer.

[0144] 5. Membrane blocking and antibody incubation

[0145] (1) Blocking: Block with 5% skimmed milk powder at room temperature for 1 h.

[0146] (2) Dilute the primary antibody in blocking solution and incubate the membrane at room temperature for 2 h.

[0147] (3) The membrane incubated with primary antibody was washed three times with TBST, each time for 5 min. Then, HRP-labeled secondary antibody was diluted 1:1000 according to the dosage and incubated with the membrane at 37°C for 1 h. It was then washed three times with TBST, each time for 5 min.

[0148] (4) Take equal amounts of ECL luminescent solution A and B, mix them evenly, and apply them to the front of the membrane in a dark room away from light for 5 minutes. Pour out the color developing solution, carefully absorb the color developing solution with paper, and cover it with a flat layer of transparent paper. Place it in the imaging system for scanning.

[0149] Experimental Results

[0150] Table 8

[0151]

[0152] From the above results, it can be seen that compared with Example 1, Example 2 increases the amount of enzyme added, which has a positive effect on increasing the protein yield of component 1, improving the surface charge and surface hydrophobicity, and the ABTS free radical scavenging rate and the superoxide anion free radical scavenging rate are basically the same. Example 3 reduces the concentration of the washing salt in step (6), and the protein yield of component 1 is significantly improved, indicating that more proteins with relatively weak adsorption enter component 2, which increases the protein content in component 2. The surface positive charge of this part of protein is relatively low, so the surface charge and surface hydrophobicity of the component 2 finally obtained are lower than those in Example 1, and the ABTS free radical scavenging rate and the superoxide anion free radical scavenging rate are also slightly lower than the component 2 collected in Example 1. It shows that the directional hydrolysis conditions in step (5) and the elution conditions in step (6) have a certain influence on the protein composition, charged properties, surface hydrophobicity, and free radical scavenging ability of component 2. The hydrolysis conditions and elution conditions used in the present invention can effectively achieve the ABTS free radical scavenging rate and superoxide anion free radical scavenging rate of the bovine colostrum immune protein extract greater than 90%.

[0153] In comparison, the ABTS free radical scavenging rate and superoxide anion free radical scavenging rate of the comparative sample were significantly decreased.

[0154] Compared with Example 1, Comparative Example 1 increases the amount of protease added, which has little effect on the protein yield and surface charge of Component 2, but significantly reduces the surface hydrophobicity, indicating that excessive enzyme addition will cause damage to the hydrophobic microregions on the protein peptide segment, resulting in a decrease in the free radical scavenging ability.

[0155] Compared with Example 1, Comparative Example 2 deleted step (6) of cation exchange resin adsorption, that is, the components with different charges of the bovine colostrum immune protein after hydrolysis were not separated. The mixture was characterized by low surface charge and hydrophobicity, and the ABTS free radical scavenging rate and superoxide anion free radical scavenging rate were only about half of the component 2 separated in Example 1. It can be seen that the use of cationic adsorption resin to separate and enrich the components with high positive charge density in the colostrum immune protein hydrolysate proposed by the present invention is effective in improving antioxidant properties.

[0156] Compared with Example 1, Comparative Example 3 deleted step (5) of directional enzymatic hydrolysis, and the yield of component 2 was greatly reduced. This is because the peptides with positive points in colostrum immunoglobulin were not released, the unhydrolyzed immunoglobulin molecules were all negatively charged, and lactoferrin was positively charged. Therefore, component 2 obtained according to the method of Comparative Example 3 was mainly composed of lactoferrin, and its surface hydrophobicity, ABTS free radical scavenging rate and superoxide anion free radical scavenging rate were all lower than those of the extract after hydrolysis, which must be related to the degree of exposure of the active sites before and after hydrolysis. It can be seen that the qualitative enzymatic hydrolysis method proposed in the present invention is effective for enriching high positive charge density and high antioxidant activity peptides in bovine colostrum immune proteins.

[0157] In order to further illustrate that the colostrum immune protein extract obtained by the present invention has a high anti-tumor function, the immune indicators and anti-tumor indicators of component 1 and component 2 obtained in Example 1 were compared to further demonstrate the effectiveness and creativity of the method for preparing the colostrum immune protein extract with tumor inhibitory activity designed by the present invention.

[0158] Immune indicators:

[0159] (1) IFN-γ: Interferon-γ is a soluble dimeric cytokine that is mainly secreted by natural killer (NK) cells and natural killer T cells (NKT) cells and plays a role in innate immunity. After mice develop tumors, the IFN-γ content in the serum will increase under the action of the immune system. Therefore, if the drug has an anti-tumor effect after use, the IFN-γ content in the serum of tumor mice will decrease. Figure 1 As can be seen from a, there is no significant difference in IFN-γ content between component 1 and component 2 in the medium and low dose administration groups, and in the high dose administration group, the IFN-γ content of component 2 drug group is significantly lower, indicating that it has stronger anti-tumor function.

[0160] (2) TNF-α: TNF-α is a type of tumor necrosis factor that can kill certain tumor cells or inhibit their proliferation both in vivo and in vitro. After mice develop tumors, the TNF-α content in their serum will increase under the action of the immune system. If the drug has an anti-tumor effect, the TNF-α content in the serum of tumor mice will decrease. Figure 1As can be seen from b, there is no significant difference in TNF-α content between component 1 and component 2 in the medium and low dose administration groups, and the TNF-α content of component 2 in the high dose administration group is significantly lower, indicating that it has stronger anti-tumor function.

[0161] (3) IL-6: Interleukin-6 is a multifunctional cytokine with a wide range of functions. Its dysregulated expression can cause many diseases. Its clinical manifestation is mainly an increase in IL-6 levels during the onset of the disease. After mice develop tumors, the IL-6 content in the serum will increase under the action of the immune system. Therefore, if the drug has an anti-tumor effect, the IL-6 content in the serum of tumor mice will decrease. Figure 1 As can be seen from c, there is no significant difference in IL-6 content between component 1 and component 2 in the medium and low dose administration groups. In the high dose administration group, the IL-6 content of component 2 drug group is significantly lower, indicating that it has stronger anti-tumor function.

[0162] Anti-tumor index:

[0163] (1) p38MAPK gene: MAPK is a very conservative serine / threonine protein kinase and a major group of signaling molecules in the signal transduction process. It plays an important role in the development and occurrence of diseases. Tumor and inflammatory stimulation can induce the activation of P38 in endogenous immune cells such as monocytes, endothelial cells and neutrophils. After activation, P38 undergoes nuclear translocation and phosphorylates and activates many protein kinases and transcription factors, leading to cell apoptosis. Therefore, if the drug has an anti-tumor effect after use, the expression of the p38MAPK gene in tumor mice will increase, promoting tumor apoptosis. Figure 2 a It can be seen that in the medium and high dose administration groups, the p38MAPK gene expression level in the component 2 administration group was significantly higher than that in the component 1 administration group, indicating that it has a stronger anti-tumor function.

[0164] (2) PI3KCA gene: This gene is a proto-oncogene, and its mutation can not only reduce cell apoptosis but also promote tumor infiltration and increase the activity of its downstream kinase PI3Ks. Therefore, if the drug has an anti-tumor effect, the expression of PI3KCA gene in tumor mice will be reduced, inhibiting tumor infiltration and metastasis. Figure 2 b It can be seen that in the low-, medium- and high-dose administration groups, the PI3KCA gene expression level in the component 2 administration group was significantly lower than that in the component 1 administration group, indicating that it has a stronger anti-tumor function.

[0165] (3) VEGF protein: Vascular endothelial growth factor is a highly specific vascular endothelial cell growth factor that promotes vascular permeability, extracellular matrix degeneration, endothelial cell migration, proliferation and angiogenesis. It is involved in the onset and progression of many angiogenesis-dependent diseases, such as tumors. Therefore, if the drug has an anti-tumor effect, the expression of VEGF protein in tumor mice will decrease. Figure 3 a It can be seen that in the high-dose administration group, the VEGF protein expression level in the component 2 administration group was significantly lower than that in the component 1 administration group, indicating that it has a stronger anti-tumor function.

[0166] (4) NF-κB protein: Nuclear factor κB, which is involved in the cell's response to external stimuli. Misregulation of NF-κB can cause autoimmune diseases, chronic inflammation, and many cancers. Continuously activated NF-κB can increase the levels of some cytokines that promote tumor growth and cause abnormal activity of proteins related to the cell cycle. Therefore, if the drug has an anti-tumor effect, the expression of NF-κB protein in tumor mice will decrease. Figure 3 a It can be seen that in the high-dose administration group, the expression level of NF-κB protein in the component 2 administration group was significantly lower than that in the component 1 administration group, indicating that it has a stronger anti-tumor function.

[0167] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a colostrum immune protein extract having tumor suppressive effect, characterized in that: The preparation method comprises the following steps: (1) Raw material selection: select bovine colostrum collected within 3 days of calving; (2) flocculating casein, adding rennet to bovine colostrum, hydrolyzing, filtering, and collecting whey; (3) removing sugar by filtering the whey liquid with a nanofiltration membrane, repeatedly washing and filtering until the lactose content in the retentate is less than 0.1%, and collecting the retentate; (4) filtering the retentate collected in step (3) through an ultrafiltration membrane, filtering the retentate through an ultrafiltration membrane, and repeatedly washing the retentate until the total content of lactalbumin and lactoglobulin in the retentate is less than 10%, and collecting the retentate; (5) directional hydrolysis, mixing the composite enzyme composed of papain and trypsin with the retentate obtained in step (4), and hydrolyzing to obtain a directional hydrolyzate; (6) Adsorption by cation exchange resin, collecting target and non-target components by step elution, first using pH 7.0 phosphate buffer containing 0.5-1.0% NaCl, and mixing with the penetration solution to obtain component 1; then using pH 8.0 phosphate buffer containing 2-4% NaCl for a second elution to elute colostrum immune protein component 2 with a high positive charge density; (7) Desalting: using a rolled nanofiltration membrane to desalt and concentrate the collected colostrum immune protein component 1 and component 2, respectively, and repeatedly adding water for filtration until the salt content accounts for less than 0.5% of the total solids; (8) Freeze drying: freeze drying the desalted and concentrated colostrum immune protein component. Colostrum immune protein component 2 is a colostrum immunoglobulin extract with tumor inhibitory activity; colostrum immune protein component 1 is a common immune-enhancing high-end ingredient.

2. The preparation method according to claim 1, characterized in that: In step (2), the amount of rennet used is 0.05% of the mass of bovine colostrum; the hydrolysis conditions are: pH 6.0-6.5, temperature 50-65° C., and time 0.5-1 h.

3. The preparation method according to claim 1, characterized in that: In step (3), the molecular weight cut-off of the nanofiltration membrane is 1000Da.

4. The preparation method according to claim 1, characterized in that: In step (4), the molecular weight cut-off of the ultrafiltration membrane is 80-100 kDa.

5. The preparation method according to claim 1, characterized in that: In step (5), the mass ratio of papain to trypsin is 1:0.5-2; the dosage of the complex enzyme is 2000-4000 U / g protein; the hydrolysis temperature is 45-55° C., and the time is 1-4 h.

6. The preparation method according to claim 1, characterized in that: In step (6), the cation exchange resin is one or more of polystyrene sulfonic acid resin, perfluorosulfonic acid resin, acrylic resin, and macroporous cation exchange resin; the adsorption conditions are pH 6.5-8.0, the adsorption temperature is 30-45°C, and the adsorption solid-liquid mass ratio is 1:5-10.

7. The preparation method according to claim 1, characterized in that: In step (7), the molecular weight cut-off of the rolled nanofiltration membrane is 200-400 Da.

8. A colostrum immune protein extract obtained by the method according to any one of claims 1 to 7.

9. The colostrum immune protein extract according to claim 8, characterized in that: The yield of colostrum immune protein is high, the obtained extract has a strong positive charge, strong antioxidant capacity, and shows a good tumor inhibition effect.

10. A use of the colostrum immune protein extract according to claim 8, characterized in that: Used in the preparation of tumor inhibitors.

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