Preparation method of lactoferrin

Abstract

The invention belongs to the technical field of preparation of lactoferrin, and particularly relates to a preparation method of lactoferrin, which comprises the following steps: degreasing, precipitating casein, and carrying out ultrafiltration treatment to obtain permeate, adding an amphoteric alkali lignin-sodium dodecyl benzene sulfonate complex into the permeate, stirring for 2.5 hours, centrifuging at the rotating speed of 1000rpm for 5 minutes, adding an absolute ethyl alcohol solution into the supernate, and centrifuging at the rotating speed of 1000rpm for 5 minutes to obtain the lactoferrin. And performing ultrasonic treatment at room temperature for 10 minutes, dialyzing with deionized water, centrifuging the obtained mixture after dialysis, and performing freeze drying to obtain the lactoferrin. Lactoferrin in the amphoteric alkali lignin-sodium dodecyl benzene sulfonate complex is extracted, the lactoferrin and other substances are separated, the separation and purification effect is better, and the amphoteric alkali lignin-sodium dodecyl benzene sulfonate particles coated with the lactoferrin are formed by adding the absolute ethyl alcohol solution. The product can be directly added into yoghourt without elution.

Description

Technical Field

[0001] The 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. It is a biologically active protein widely found in biological secretions and has multiple physiologically active functions, including immunomodulatory and anti-inflammatory effects. It plays an important role in disease prevention and treatment, nutritional supplementation, food and drug development, etc.

[0003] Milk-containing beverages are a common nutritional beverage with rich flavors and certain health functions, especially fermented lactic acid bacteria yogurt. The active lactic acid bacteria it contains can inhibit the growth of harmful bacteria in the intestines, regulate the balance of intestinal microecology, and enhance the body's immunity.

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

[0005] Moreover, after lactoferrin is added to yogurt, it is easily digested by pepsin in the stomach, and the amount of lactoferrin entering the intestine is relatively small. The concentration and efficacy of lactoferrin 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 destroyed, and the structure, surface charge and structural integrity of lactoferrin will affect the interaction between lactoferrin and its receptor, thereby affecting the biological activity of lactoferrin. Summary of the invention

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

[0007] In order to achieve the above technical objectives, the technical solution of the present invention is: A method for preparing lactoferrin comprises the following steps: 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, ultrasonicated at room temperature for 10 minutes, and then transferred to a dialysis bag and dialyzed in deionized water for 60 hours with a molecular weight cutoff of 100 Da. After the dialysis, the solid matter was separated and freeze-dried to obtain lactoferrin.

[0008] As a further improvement, the preparation method of the amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex is: dissolving the amphoteric alkali lignin in dilute hydrochloric acid, then adding sodium dodecylbenzene sulfonate thereto, adjusting the pH to 3 with dilute hydrochloric acid, and then reacting at 45°C for 2h, and then continuing to add dilute hydrochloric acid thereto while stirring, separating the precipitate and freeze-drying it to obtain the amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex, and the mass ratio of the amphoteric alkali lignin to the sodium dodecylbenzene sulfonate is 1:0.8.

[0009] As a further improvement, 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, and then the solution is heated to 85°C, 3-chloro-2-hydroxypropyltrimethylammonium chloride is added thereto, and the pH of the solution is maintained at 12. After reacting for 4 hours, the product is cooled to room temperature, and then dialyzed with a dialysis bag for 7 days. The obtained solid substance is freeze-dried to obtain amphoteric alkali lignin.

[0010] As a further improvement, the mass ratio of the alkali lignin to the 3-chloro-2-hydroxypropyltrimethylammonium chloride is 1:2.

[0011] As a further improvement, the molecular weight cut-off during dialysis was 1000 Da.

[0012] As a further improvement, the alkali lignin is obtained by fractional extraction with methanol.

[0013] As a further improvement, in step S4, 50 g of amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex is added to every 10 mL of permeate.

[0014] As a further improvement, in step S4, the volume ratio of the added amount of the anhydrous ethanol solution to the supernatant is 1:2.

[0015] As a further improvement, in step S3, the volume ratio of the crude sample to the distilled water is 1:1, the ultrafiltration uses a 100KDa Pellicon XL ultrafiltration membrane, the ultrafiltration temperature is 34.7°C, and the ultrafiltration pressure is 0.28MPa.

[0016] As a further improvement, the specific method of the methanol graded extraction is: mixing lignin and methanol solution, and then magnetically stirring at a rotation speed of 300 rpm for 30 minutes to fully dissolve the lignin, filtering to obtain a lignin methanol solution, and then removing the methanol solution from the lignin methanol solution by rotary evaporation, volatilizing at room temperature for 2 hours, and then vacuum drying to obtain alkaline lignin.

[0017] Due to the adoption of the above technical solution, the beneficial effects of the present invention are: The invention provides a method for preparing lactoferrin. After bovine colostrum is defatted, casein is precipitated and ultrafiltered, lactoferrin therein is extracted through an amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex, and lactoferrin and other substances are separated, so that the separation and purification effect is better.

[0018] In the preparation process of the amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex of the present invention, the mass ratio of the amphoteric alkali lignin and the sodium dodecylbenzene sulfonate is adjusted to 1:0.8, and the amphoteric alkali lignin and the sodium dodecylbenzene sulfonate are complexed, so that the surface of the obtained amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex is negatively charged.

[0019] In the permeate obtained after ultrafiltration treatment, lactoferrin is positively charged, and other proteins except lactoferrin are negatively charged. The negatively charged amphoteric alkaline lignin-sodium dodecylbenzene sulfonate complex combines with the positively charged lactoferrin to separate lactoferrin from other substances.

[0020] By adding anhydrous ethanol solution, the dodecyl long chains in the amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex are coated outwardly, and the lactoferrin is coated inside, forming an amphoteric alkali lignin-sodium dodecylbenzene sulfonate microparticle coated with lactoferrin. The microparticle can be directly added to yogurt without eluting the lactoferrin, and the purification process of lactoferrin is simpler.

[0021] The obtained microparticles do not decompose under acidic conditions but decompose under alkaline conditions. When added to yogurt and consumed, they will not be decomposed by pepsin under acidic conditions in the stomach and will not destroy lactoferrin. After entering the intestine, the decomposition rate reaches 95.3% within 24 hours. Lactoferrin is decomposed and released in the intestine, and is bound by the lactoferrin receptors of intestinal epithelial cells, thereby improving the body's absorption rate of lactoferrin.

[0022] Since the concentration and efficacy of lactoferrin show a positive dose-response relationship, when the amount of lactoferrin added is the same, the higher the absorption rate, the better the efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a graph showing the retention rate of lactoferrin during simulated gastric juice digestion in Example 4; Figure 2 This is a graph showing the cumulative release rate of lactoferrin during the simulated intestinal fluid digestion process in Example 4. DETAILED DESCRIPTION

[0024] The technical scheme of the present invention will be clearly and completely described below in conjunction with specific embodiments, but it will be appreciated by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work, all belong to the scope of protection of the present invention. If specific conditions are not indicated in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0025] Example 1 A method for preparing lactoferrin comprises the following steps: S1, degreasing; Place the colostrum in a centrifuge tube and centrifuge it in a high-speed refrigerated centrifuge at 4°C and 12,000 r / min for 30 min to remove the upper fat layer; S2, casein precipitation; Add 1 mol / L dilute hydrochloric acid to the defatted colostrum, stir evenly, adjust the pH of the colostrum solution to 4.6, heat to 40°C in a water bath, keep warm for 30 minutes, and then centrifuge at 40°C and 12000 r / min for 30 minutes to retain the upper whey protein; S3, filtering and removing impurities; The upper whey protein solution was passed through a medium-speed qualitative filter paper to remove the residual casein in the solution, and then filtered through a 0.45 μm microporous filter membrane to remove dust and bacteria in the solution to obtain a crude sample; S4, ultrafiltration treatment; Distilled water filtered through a 0.22 μm microporous membrane was added to the crude sample, the volume ratio of the crude sample to the distilled water was 1:1, and then the sample was ultrafiltered to obtain a permeate, the ultrafiltration used a 100 KDa Pellicon XL ultrafiltration membrane, the ultrafiltration temperature was 34.7 °C, and the ultrafiltration pressure was 0.28 MPa; S5, extraction and purification; Add amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex to the permeate, specifically, add 50g of amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex to every 10mL of permeate, stir for 2.5h, centrifuge at 1000rpm for 5min, then add anhydrous ethanol solution to the supernatant, ultrasonicate for 10min at room temperature, then transfer to a dialysis bag, dialyze in deionized water for 60h, with a molecular weight cutoff of 100Da, separate the solid matter after the dialysis, freeze-dry the obtained solid matter at -10°C for 10min to obtain lactoferrin, and the volume ratio of the added anhydrous ethanol solution to the supernatant is 1:2.

[0026] Example 2 This example provides a method for preparing the amphoteric alkaline lignin-sodium dodecylbenzene sulfonate complex in Example 1, which specifically includes the following steps: S11, fractional extraction of alkali lignin; Lignin and a methanol solution with a volume fraction of 80% were mixed at room temperature, 2 g of lignin was added to every 10 mL of the methanol solution, and the mixture was magnetically stirred at a speed of 300 rpm for 30 min. The lignin methanol solution was filtered to obtain the lignin methanol solution, and the methanol solution was removed by rotary evaporation of the lignin methanol solution, and the solution was allowed to evaporate at room temperature for 3 h, and then dried in a vacuum drying oven to constant weight to obtain alkaline lignin; S12, preparation of amphoteric alkaline lignin; Under normal pressure, 10 g of alkali lignin was dispersed in 100 mL of deionized water, and then a 20 wt % sodium hydroxide solution was used to adjust the pH of the solution to 12. The solution was heated to 85 ° C. 20 g of 3-chloro-2-hydroxypropyltrimethylammonium chloride was added dropwise to the alkali lignin solution, and a 20 wt % sodium hydroxide solution was added at the same time to maintain the pH of the solution at 12. After reacting for 4 hours, the product was cooled to room temperature and then placed in a dialysis bag for 7 days with a molecular weight cutoff of 1000 Da. The obtained solid was freeze-dried to obtain amphoteric alkali lignin, wherein the mass ratio of alkali lignin to 3-chloro-2-hydroxypropyltrimethylammonium chloride was 1:2; S13, preparation of amphoteric alkaline lignin-sodium dodecylbenzene sulfonate complex; 10 g of amphoteric alkali lignin was dissolved in dilute hydrochloric acid, and then 8 g of sodium dodecylbenzene sulfonate was added thereto, the pH was adjusted to 3 with dilute hydrochloric acid, complexed at 45°C for 2 hours, and then 50 mL of dilute hydrochloric acid was continued to be added thereto while stirring, the precipitate was separated, and freeze-dried at -10°C for 30 minutes to obtain an amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex, wherein the mass ratio of the amphoteric alkali lignin to the sodium dodecylbenzene sulfonate was 1:0.8.

[0027] Comparative Example 1 This comparative example provides a method for preparing an amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex. The specific steps are the same as those in Example 2, except that the mass ratio of the amphoteric alkali lignin to the sodium dodecylbenzene sulfonate in this comparative example is 1:0.4.

[0028] 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 dodecylbenzene sulfonate complex used in this comparative example is obtained by the preparation method in Comparative Example 1.

[0029] Comparative Example 3 This comparative example provides another method for preparing lactoferrin. The specific method is the same as that of Example 1, except that in this comparative example, a cation exchange resin is used to extract and purify the obtained permeate. The specific method is the prior art.

[0030] Example 3 Take 3 portions of bovine colostrum, each with 200 mL, and record them as experimental group, control group 1 and control group 2 respectively. Prepare lactoferrin by the preparation methods in Example 1, comparative example 2 and comparative example 3 respectively. Then, ELISA method is used to determine the concentration of lactoferrin in the permeate during the preparation process of lactoferrin in the three groups, which is recorded as C1 and the volume of the permeate is V1.

[0031] Then, the ELISA method was used to determine the lactoferrin concentration in the dialysate obtained during the extraction and purification process of the experimental group and the control group 1; the ELISA method was used to determine the lactoferrin concentration in the eluate obtained during the purification process using a cation exchange resin in the control group 2, which was recorded as C2, and the volumes of the dialysate and the eluate were both recorded as V2.

[0032] According to the data obtained above, the recoveries of lactoferrin in the three groups of samples were calculated respectively. The results are shown in Table 1. The calculation method for the experimental group and the control group 1 is recovery rate (%) = (C1×V1-C2×V2) / (C1×V1)×100%; the calculation method for the control group 2 is recovery rate (%) = (C2×V2) / (C1×V1)×100%.

[0033] This example verifies that the extraction and purification effect of lactoferrin using the amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex of the present invention is better 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 with the control group 2.

[0034] The results of the experimental group and the control group 1 are compared, indicating that in the process of preparing amphoteric alkali lignin-sodium dodecylbenzene sulfonate in the present invention, the mass ratio of amphoteric alkali lignin and sodium dodecylbenzene sulfonate is 1:0.8, and the obtained amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex has a good extraction effect on lactoferrin, while when the mass ratio of amphoteric alkali lignin and sodium dodecylbenzene sulfonate is 1:0.4, the amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex has almost no extraction effect on lactoferrin.

[0035] This is because when the mass ratio of amphoteric alkali lignin and sodium dodecylbenzene sulfonate is 1:0.4, the surface of the obtained amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex is positively charged and cannot combine with the positively charged lactoferrin. However, when the mass ratio of amphoteric alkali lignin and sodium dodecylbenzene sulfonate is 1:0.8, the surface of the obtained amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex is negatively charged and can combine with the positively charged lactoferrin to separate and purify the lactoferrin.

[0036] Example 4 This example verifies the digestion of lactoferrin prepared by the present invention in simulated gastric juice and simulated intestinal juice through digestion simulation.

[0037] The simulated gastric fluid and the simulated intestinal fluid were prepared according to Table 2, and the pH of the simulated gastric fluid and the simulated intestinal fluid were adjusted to 3.0 and 7.4 with hydrochloric acid and sodium hydroxide, respectively.

[0038] 20 mg of lactoferrin prepared in Example 1 was added to 20 mL of simulated gastric fluid as an experimental group, and ordinary lactoferrin was used as a control for simulated digestion. Each sample was placed in a shaker and digested for 30 min, 60 min, 90 min, and 120 min at 37° C. and 100 r / min. After each stage of simulated digestion, sodium hydroxide solution was immediately added to destroy the shell structure of the microparticles and completely release lactoferrin. The amount of lactoferrin was measured and the retention rate of lactoferrin was calculated.

[0039] In addition, 20 mg of lactoferrin prepared in Example 1 was placed in 20 mL of simulated intestinal fluid and released by shaking on a shaker. 0.5 mL of supernatant was taken every 5 minutes to determine the content of lactoferrin therein. After each sampling, 0.5 mL of simulated intestinal fluid was added to maintain a constant solution volume. The cumulative release rate of lactoferrin during the release process was calculated based on the final amount of all dissolved lactoferrin.

[0040] like Figure 1 The figure shows the retention rate of lactoferrin in the simulated gastric juice digestion process. Figure 1It can be seen that after 30min, 60min, 90min, and 120min of simulated gastric juice digestion, the retention rates of lactoferrin were 96.43%, 87.52%, 83.07%, and 80.26%, respectively. This is because during the preparation of lactoferrin, most of the lactoferrin was coated inside the amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex, and only a small amount of lactoferrin was attached to the surface of the particles. The lactoferrin coated by the amphoteric alkali lignin-sodium dodecylbenzene sulfonate complex was basically not released when acidic simulated gastric juice was added.

[0041] like Figure 2 As shown, the cumulative release rate of lactoferrin during simulated intestinal digestion is shown. Figure 2 It can be seen that in the simulated intestinal fluid, the release rate of lactoferrin is very fast. Within 15 minutes, 90% of lactoferrin is released, and complete release of lactoferrin can be achieved within 20 minutes.

[0042] The specific implementation of the present invention described above does not constitute a limitation on the protection scope of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in 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, ultrasonicated at room temperature for 10 minutes, and then transferred to a dialysis bag and dialyzed in deionized water for 60 hours with a molecular weight cutoff of 100 Da. After the dialysis, the solid matter was separated and freeze-dried to obtain lactoferrin.

2. The method for preparing lactoferrin according to claim 1, characterized in that 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 it 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.

3. The method for preparing lactoferrin according to claim 2, characterized in that 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.

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

2.

5. The method for preparing lactoferrin according to claim 3, characterized in that: The molecular weight cut-off during dialysis was 1000 Da.

6. The method for preparing lactoferrin according to claim 3, characterized in that: The alkali lignin is obtained by fractional extraction with methanol.

7. 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.

8. 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.

9. The method for preparing lactoferrin according to claim 7, 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.

10. The method for preparing lactoferrin according to claim 6, characterized in that: The specific method of the methanol fractional extraction is: mixing lignin and methanol solution, and then magnetically stirring at a rotation speed of 300 rpm for 30 minutes to fully dissolve the lignin, filtering to obtain a lignin methanol solution, and then removing the methanol solution from the lignin methanol solution by rotary evaporation, volatilizing at room temperature for 2 hours, and then vacuum drying to obtain alkali lignin.

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