A method for preparing lactoferrin

Abstract

The present invention belongs to the technical field of lactoferrin preparation, and specifically relates to a preparation method of lactoferrin. After degreasing, casein precipitation, and ultrafiltration treatment, a permeate is obtained. An amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex is added to the permeate, stirred for 2.5 h, and then centrifuged at a speed of 1000 rpm for 5 min. An absolute ethanol solution is added to the supernatant, ultrasonically treated at room temperature for 10 min, and then dialyzed with deionized water. After dialysis, the obtained mixture is centrifuged and freeze-dried to obtain lactoferrin. In the present invention, the amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex is used to extract lactoferrin therein, separating lactoferrin from other substances, and the separation and purification effect is better. By adding an absolute ethanol solution, an amphoteric alkali lignin-sodium dodecylbenzenesulfonate particle coated with lactoferrin is formed, which can be directly added to yogurt without further elution.

Description

Technical Field

[0001] The present invention belongs to the technical field of lactoferrin preparation, and particularly relates to a method for preparing lactoferrin. Background Art

[0002] Lactoferrin is an important protein found in milk, a bioactive protein widely present in biological secretions, and has a variety of physiological activities, including immunomodulatory effects, anti-inflammatory effects, etc. It plays an important role in disease prevention and treatment, nutritional supplementation, food and drug development, etc.

[0003] Milk-containing beverages are a common type of nutritious beverage with rich flavors and certain health functions. Especially fermented lactic acid bacteria yogurt, the active lactic acid bacteria contained therein can inhibit the growth of harmful bacteria in the intestine, regulate the balance of the intestinal microecology, and enhance the human body's immunity.

[0004] Yogurt containing lactoferrin has gradually been sought after by people. However, most of the existing lactoferrin is separated and purified by affinity chromatography or ion exchange chromatography. This method has a high purity for purifying lactoferrin, but it is necessary to first extract and enrich lactoferrin with a chromatographic column, and then elute the lactoferrin on the chromatographic column with an eluent before it can be obtained, and the operation process is complex.

[0005] Moreover, after lactoferrin is added to yogurt, it is easily digested by pepsin in the stomach, and the content of lactoferrin entering the intestine is less. And the concentration of lactoferrin and its efficacy show a positive dose-effect relationship. Therefore, the effect of lactoferrin is not obvious. On the other hand, after being digested by pepsin, the integrity of lactoferrin will be damaged, and the structure, surface charge and structural integrity of lactoferrin will all affect the interaction between lactoferrin and its receptor, thereby affecting the biological activity of lactoferrin. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for preparing lactoferrin to solve the problems raised in the above background art.

[0007] To achieve the above technical purpose, the technical solution of the present invention is as follows:

[0008] A method for preparing lactoferrin includes the following steps:

[0009] S1, degreasing;

[0010] Centrifuge and separate colostrum to remove the upper layer of fat;

[0011] S2, casein precipitation;

[0012] Add dilute hydrochloric acid to defatted bovine colostrum, adjust the pH of the solution to 4.6, heat it in a water bath to 40 °C, keep it warm for 30 min, then centrifuge it at a high speed of 12,000 r / min for 30 min at 40 °C, retain the upper whey protein, and filter and remove impurities from the upper whey protein to obtain a crude sample;

[0013] S3. Ultrafiltration treatment;

[0014] Dilute the crude sample with distilled water and perform ultrafiltration after dilution to obtain a permeate;

[0015] S4. Extraction and purification;

[0016] Add an amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex to the permeate, stir for 2.5 h, then centrifuge at a speed of 1000 rpm for 5 min, add an absolute ethanol solution to the supernatant, ultrasonicate at room temperature for 10 min, then transfer it to a dialysis bag and dialyze it in deionized water for 60 h with a cut-off molecular weight of 100 Da. After dialysis, separate the solid matter and freeze-dry it to obtain lactoferrin.

[0017] As a further improvement, the preparation method of the amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex is as follows: dissolve amphoteric alkali lignin in dilute hydrochloric acid, then add sodium dodecylbenzenesulfonate to it, adjust the pH to 3 with dilute hydrochloric acid, then react at 45 °C for 2 h, then continue to add dilute hydrochloric acid to it while stirring, separate the precipitate and freeze-dry it to obtain the amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex, and the mass ratio of the amphoteric alkali lignin to the sodium dodecylbenzenesulfonate is 1:0.8.

[0018] As a further improvement, the preparation method of the amphoteric alkali lignin is as follows: disperse alkali lignin in deionized water, adjust the pH of the solution to 12, then heat the solution to 85 °C, add 3-chloro-2-hydroxypropyltrimethylammonium chloride to it, maintain the pH of the solution at 12, after reacting for 4 h, cool the product to room temperature, then dialyze it with a dialysis bag for 7 days, and the obtained solid matter is freeze-dried to obtain amphoteric alkali lignin.

[0019] As a further improvement, the mass ratio of the alkali lignin to the 3-chloro-2-hydroxypropyltrimethylammonium chloride is 1:2.

[0020] As a further improvement, the cut-off molecular weight during dialysis is 1000 Da.

[0021] As a further improvement, the alkali lignin is obtained by methanol fractional extraction.

[0022] As a further improvement, in step S4, 50 g of the amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex is added to every 10 mL of the permeate.

[0023] As a further improvement, in step S4, the volume ratio of the added amount of the absolute ethanol solution to the volume of the supernatant is 1:2.

[0024] As a further improvement, in step S3, the volume ratio of the crude sample to the distilled water is 1:1. Ultrafiltration is carried out using a 100KDa Pellicon XL ultrafiltration membrane. The ultrafiltration temperature is 34.7°C, and the ultrafiltration pressure is 0.28 MPa.

[0025] As a further improvement, the specific method for methanol fractional extraction is as follows: Mix lignin with a methanol solution, and then magnetically stir at a rotation speed of 300 rpm for 30 min to fully dissolve the lignin. After filtration, a lignin methanol solution is obtained. Then, the methanol solution in the lignin methanol solution is removed by rotary evaporation, volatilized at room temperature for 2 h, and then vacuum dried to obtain alkali lignin.

[0026] Due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows:

[0027] A preparation method of lactoferrin provided by the present invention. After defatting, casein precipitation and ultrafiltration treatment of colostrum, lactoferrin therein is extracted by an amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex, and lactoferrin is separated from other substances, and the separation and purification effect is better.

[0028] In the preparation process of the amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex in the present invention, by adjusting the mass ratio of amphoteric alkali lignin to sodium dodecylbenzenesulfonate to 1:0.8, amphoteric alkali lignin and sodium dodecylbenzenesulfonate are complexed, and the surface of the obtained amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex is negatively charged.

[0029] In the permeate obtained after ultrafiltration treatment, lactoferrin is positively charged, and except for lactoferrin, other proteins are negatively charged. The amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex with a negatively charged surface binds to the positively charged lactoferrin to separate lactoferrin from other substances.

[0030] By adding an absolute ethanol solution, the dodecyl long chain in the amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex wraps outward, and lactoferrin is wrapped inside to form an amphoteric alkali lignin-sodium dodecylbenzenesulfonate particle coated with lactoferrin. This particle can be directly added to yogurt, and there is no need to elute lactoferrin again, and the purification process of lactoferrin is more convenient.

[0031] Moreover, the obtained particles do not decompose under acidic conditions but decompose under alkaline conditions. When added to yogurt, they will not be decomposed by pepsin under the acidic conditions in the stomach, and lactoferrin will not be damaged. After entering the intestine, the decomposition rate reaches 95.3% within 24 hours. In the intestine, lactoferrin is decomposed and released, binds to the lactoferrin receptor on intestinal epithelial cells, and improves the absorption rate of lactoferrin by the human body.

[0032] Since there is a positive dose-effect relationship between the concentration of lactoferrin and its efficacy, when the added amount of lactoferrin is the same, the higher the absorption rate, the better the efficacy it exerts. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a graph showing the retention rate of lactoferrin during the simulated gastric juice digestion process in Example 4;

[0034] Figure 2 It is a graph showing the cumulative release rate of lactoferrin during the simulated intestinal juice digestion process in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0035] The technical solutions of the present invention will be clearly and completely described below in conjunction with the specific embodiments. However, those skilled in the art will understand that the following described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments, and are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the scope of protection of the present invention. For those conditions not specified in the embodiments, they shall be carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0036] Example 1 A preparation method of lactoferrin specifically includes the following steps:

[0037] S1. Defatting;

[0038] Put the bovine colostrum into a centrifuge tube and centrifuge it at 4°C and a speed of 12,000 r / min for 30 min by a high-speed refrigerated centrifuge to remove the upper layer of fat;

[0039] S2. Casein precipitation;

[0040] Drop 1 mol / L dilute hydrochloric acid into the defatted bovine colostrum, stir evenly, adjust the pH of the bovine colostrum solution to 4.6, heat it in a water bath to 40°C, keep it warm for 30 min, and then centrifuge it at 40°C and a speed of 12,000 r / min for 30 min to retain the upper layer of whey protein;

[0041] S3. Filtration and impurity removal;

[0042] Pass the upper-layer whey protein solution through a medium-speed qualitative filter paper to remove the residual casein in the solution, and then filter it through a 0.45-μm microporous membrane to remove dust and bacteria in the solution, obtaining a crude sample;

[0043] S4. Ultrafiltration treatment;

[0044] Add distilled water filtered through a 0.22-μm microporous membrane to the crude sample, with the volume ratio of the crude sample to distilled water being 1:1, and then perform ultrafiltration to obtain a permeate. The ultrafiltration uses a 100KDa Pellicon XL ultrafiltration membrane, the ultrafiltration temperature is 34.7 °C, and the ultrafiltration pressure is 0.28 MPa;

[0045] S5. Extraction and purification;

[0046] Add an amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex to the permeate. Specifically, add 50 g of the amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex to every 10 mL of the permeate, stir for 2.5 h, then centrifuge at a speed of 1000 rpm for 5 min. Then add an anhydrous ethanol solution to the supernatant, ultrasonicate at room temperature for 10 min, then transfer it to a dialysis bag and dialyze in deionized water for 60 h with a molecular weight cut-off of 100 Da. After dialysis, separate the solid matter, and freeze-dry the obtained solid matter at -10 °C for 10 min to obtain lactoferrin. The addition amount of the anhydrous ethanol solution is in a volume ratio of 1:2 to the volume of the supernatant.

[0047] Example 2 This example provides a preparation method for the amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex in Example 1, which specifically includes the following steps;

[0048] S11. Fractional extraction of alkali lignin;

[0049] Mix lignin with a methanol solution with a volume fraction of 80% at room temperature. Add 2 g of lignin to every 10 mL of the methanol solution, magnetically stir at a speed of 300 rpm for 30 min, filter to obtain a lignin methanol solution. After removing the methanol solution from the lignin methanol solution by rotary evaporation, let it volatilize at room temperature for 3 h, and then dry it to constant weight in a vacuum drying oven to obtain alkali lignin;

[0050] S12. Preparation of amphoteric alkali lignin;

[0051] Under normal pressure, 10 g of alkali lignin was dispersed in 100 mL of deionized water, and then the pH of the solution was adjusted to 12 using 20 wt% sodium hydroxide solution. The solution was heated to 85 °C, and 20 g of 3-chloro-2-hydroxypropyltrimethylammonium chloride was added dropwise to the alkali lignin solution while adding 20 wt% sodium hydroxide solution to maintain the pH of the solution at 12. After reacting for 4 h, the product was cooled to room temperature and then dialyzed in a dialysis bag for 7 days with a molecular weight cut-off of 1000 Da. The obtained solid was freeze-dried to obtain amphoteric alkali lignin, where the mass ratio of alkali lignin to 3-chloro-2-hydroxypropyltrimethylammonium chloride was 1:2;

[0052] S13. Preparation of amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex;

[0053] 10 g of amphoteric alkali lignin was dissolved in dilute hydrochloric acid, and then 8 g of sodium dodecylbenzenesulfonate was added thereto. The pH was adjusted to 3 with dilute hydrochloric acid, and complexation was carried out at 45 °C for 2 h. Then, 50 mL of dilute hydrochloric acid was continuously added thereto while stirring. The precipitate was separated and freeze-dried at -10 °C for 30 min to obtain an amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex, where the mass ratio of amphoteric alkali lignin to sodium dodecylbenzenesulfonate was 1:0.8.

[0054] Comparative Example 1 This comparative example provides a method for preparing an amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex. The specific steps are the same as those in Example 2, except that in this comparative example, the mass ratio of amphoteric alkali lignin to sodium dodecylbenzenesulfonate is 1:0.4.

[0055] Comparative Example 2 This comparative example provides a method for preparing lactoferrin. The specific steps are the same as those in Example 1, except that the amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex used in this comparative example is prepared by the method in Comparative Example 1.

[0056] Comparative Example 3 This comparative example provides another method for preparing lactoferrin. The specific method is the same as that in Example 1, except that the obtained permeate in this comparative example is extracted and purified using a cation exchange resin, and the specific method is the prior art.

[0057] Example 3 Three portions of bovine colostrum, 200 mL each, were taken and denoted as the experimental group, control group 1, and control group 2, respectively. Lactoferrin was prepared by the preparation methods in Example 1, Comparative Example 2, and Comparative Example 3, respectively. Then, the concentration of lactoferrin in the permeate during the preparation of lactoferrin in the three groups was measured by ELISA and denoted as C1, and the volume of the permeate was V1.

[0058] Then, the ELISA method was used to determine the lactoferrin concentration in the dialysis fluid obtained during the extraction and purification processes of the experimental group and control group 1; the ELISA method was used to determine the lactoferrin concentration in the eluate obtained during the purification process of control group 2 using cation exchange resin, denoted as C2, and the volumes of both the dialysis fluid and the eluate were denoted as V2.

[0059] Based on the data obtained above, the recovery rates of lactoferrin in the three groups of samples were calculated respectively. The results are shown in Table 1. Among them, the calculation method for the experimental group and control group 1 was recovery rate (%) = (C1×V1 - C2×V2) / (C1×V1) × 100%; the calculation method for control group 2 was recovery rate (%) = (C2×V2) / (C1×V1) × 100%.

[0060]

[0061] In this example, by measuring the recovery rate of lactoferrin using the lactoferrin preparation methods in Example 1, Comparative Example 2, and Comparative Example 3, and comparing the experimental group and control group 2, it was verified that the extraction and purification effect of lactoferrin using the amphoteric alkali lignin - sodium dodecylbenzenesulfonate complex in the present invention was better.

[0062] And by comparing the results of the experimental group and control group 1, it was shown that during the preparation of the amphoteric alkali lignin - sodium dodecylbenzenesulfonate in the present invention, when the mass ratio of amphoteric alkali lignin to sodium dodecylbenzenesulfonate was 1:0.8, the obtained amphoteric alkali lignin - sodium dodecylbenzenesulfonate complex had a good extraction effect on lactoferrin, while when the mass ratio of amphoteric alkali lignin to sodium dodecylbenzenesulfonate was 1:0.4, the amphoteric alkali lignin - sodium dodecylbenzenesulfonate complex had almost no extraction effect on lactoferrin.

[0063] This was because when the mass ratio of amphoteric alkali lignin to sodium dodecylbenzenesulfonate was 1:0.4, the surface of the obtained amphoteric alkali lignin - sodium dodecylbenzenesulfonate complex showed a positive charge and could not bind to the positively charged lactoferrin. While when the mass ratio of amphoteric alkali lignin to sodium dodecylbenzenesulfonate was 1:0.8, the surface of the obtained amphoteric alkali lignin - sodium dodecylbenzenesulfonate complex showed a negative charge and could bind to the positively charged lactoferrin to separate and purify lactoferrin.

[0064] Example 4 In this example, the digestion of the lactoferrin prepared in the present invention in simulated gastric juice and simulated intestinal juice was verified through digestion simulation.

[0065] Prepare simulated gastric juice and simulated intestinal juice according to Table 2, and adjust the pH of the simulated gastric juice and simulated intestinal juice to 3.0 and 7.4 respectively using hydrochloric acid and sodium hydroxide.

[0066]

[0067] 20 mg of lactoferrin prepared in Example 1 was added to 20 mL of simulated gastric juice as the experimental group, and ordinary lactoferrin was used as the control for simulated digestion. Each sample was placed in a shaker and digested at 37 °C and 100 r / min for 30 min, 60 min, 90 min, and 120 min respectively. After the simulated digestion at each stage was completed, sodium hydroxide solution was immediately added to destroy the outer shell structure of the microparticles, completely release lactoferrin, measure the amount of lactoferrin, and calculate the retention rate of lactoferrin.

[0068] In addition, 20 mg of lactoferrin prepared in Example 1 was placed in 20 mL of simulated intestinal juice and shaken and released in a shaker. 0.5 mL of the supernatant was taken every 5 min to measure the content of lactoferrin therein. After each sampling, 0.5 mL of simulated intestinal juice was added to maintain a constant solution volume. The cumulative release rate of lactoferrin during the release process was calculated based on the amount of lactoferrin completely dissolved at the end.

[0069] As Figure 1 shown, it is a graph of the retention rate results of lactoferrin during simulated gastric juice digestion. It can be seen from Figure 1 that after simulated gastric juice digestion for 30 min, 60 min, 90 min, and 120 min, the retention rates of lactoferrin were 96.43%, 87.52%, 83.07%, and 80.26% respectively. This is because during the preparation process of lactoferrin, most of the lactoferrin was encapsulated inside the amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex, and only a small amount of lactoferrin adhered to the surface of the microparticles. The lactoferrin encapsulated by the amphoteric alkali lignin-sodium dodecylbenzenesulfonate complex was basically not released under the addition of acidic simulated gastric juice.

[0070] As Figure 2 shown, it is a graph of the cumulative release rate results of lactoferrin during simulated intestinal juice digestion. It can be seen from Figure 2 that in simulated intestinal juice, the release rate of lactoferrin is very fast. Within 15 min, 90% of the lactoferrin was released, and complete release of lactoferrin could be achieved within 20 min.

[0071] The specific embodiments of the present invention described above do not constitute a limitation on the protection scope of the present invention. Any other corresponding changes and deformations made according to the technical concept of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. A method for preparing lactoferrin, characterized in that: The following steps are involved: S1, degreasing; Centrifuge the bovine colostrum to remove the upper fat layer; S2, casein precipitation; Add dilute hydrochloric acid to the defatted bovine colostrum to adjust the pH of the solution to 4.6, heat the solution to 40°C in a water bath, keep the solution warm for 30 minutes, and then centrifuge the solution at 40°C for 30 minutes at a centrifugal speed of 12,000 r / min, retain the upper whey protein, and filter and remove impurities from the upper whey protein to obtain a crude sample; S3, ultrafiltration treatment; The crude sample is diluted with distilled water, and after dilution, ultrafiltration is performed to obtain a permeate; S4, extraction and purification; Amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex was added to the permeate, stirred for 2.5 hours, and then centrifuged at 1000 rpm for 5 minutes. Anhydrous ethanol solution was added to the supernatant, and ultrasonicated for 10 minutes at room temperature. Then, the supernatant was transferred to a dialysis bag and dialyzed in deionized water for 60 hours with a molecular weight cutoff of 100 Da. After the dialysis was completed, the solid matter was separated and freeze-dried to obtain lactoferrin; The preparation method of the amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex is as follows: dissolving the amphoteric alkali lignin in dilute hydrochloric acid, then adding sodium dodecylbenzene sulfonate thereto, adjusting the pH to 3 with dilute hydrochloric acid, then reacting at 45° C. for 2 hours, continuing to add dilute hydrochloric acid thereto while stirring, separating the precipitate and freeze-drying to obtain the amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex, wherein the mass ratio of the amphoteric alkali lignin to the sodium dodecylbenzene sulfonate is 1:0.8; The preparation method of the amphoteric alkali lignin is as follows: alkali lignin is dispersed in deionized water, the pH of the solution is adjusted to 12, then the solution is heated to 85°C, 3-chloro-2-hydroxypropyltrimethylammonium chloride is added thereto, the pH of the solution is maintained at 12, the product is cooled to room temperature after reacting for 4 hours, and then dialyzed with a dialysis bag for 7 days, and the obtained solid material is freeze-dried to obtain the amphoteric alkali lignin, and the molecular weight cutoff during the dialysis process is 1000Da.

2. The method for preparing lactoferrin according to claim 1, characterized in that The mass ratio of the alkali lignin to the 3-chloro-2-hydroxypropyltrimethylammonium chloride is 1:

2.

3. The method for preparing lactoferrin according to claim 2, characterized in that In step S4, 50 g of amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex was added to every 10 mL of permeate.

4. The method for preparing lactoferrin according to claim 1, characterized in that In step S4, the volume ratio of the added amount of the anhydrous ethanol solution to the supernatant is 1:

2.

5. The method for preparing lactoferrin according to claim 1, characterized in that In step S3, the volume ratio of the crude sample to the distilled water is 1:1, the ultrafiltration uses a 100 KDa Pellicon XL ultrafiltration membrane, the ultrafiltration temperature is 34.7° C., and the ultrafiltration pressure is 0.28 MPa.

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