Preparation process of lactoferrin
By using amino hollow mesoporous silica-loaded chitosan and rennet in the lactoferrin preparation process, and combining specific adsorption technology of molecular imprinting resins, the problems of lactoferrin purity and cost in the prior art are solved. , high-purity and low-cost preparation of lactoferrin is achieved.
Patent Information
- Application Number
- CN202411345980.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-26
- Publication Date
- 2024-12-27
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
In the existing lactoferrin preparation process, casein removal methods are greatly affected by pH, resulting in limited product purity. The commonly used affinity column chromatography method is costly and has low specificity, making it difficult to achieve high-purity and low-cost milk. Ferritin preparation.
Using amino hollow mesoporous silica-loaded chitosan and rennet, skim milk was obtained by centrifugation, followed by specific adsorption and purification of lactoferrin using molecular imprinting resin, and whey was carried out by modified cation exchange resin. Isolation and purification to improve the purity of lactoferrin.
The preparation of high-purity lactoferrin is achieved, which reduces the preparation cost, avoids the risk of introducing impurities during casein removal, and improves the specificity and purity of the product.
Abstract
Description
Technical Field
[0001] The invention relates to the field of proteins, in particular to a preparation process of lactoferrin. Background Art
[0002] Lactoferrin is a non-heme iron-binding glycoprotein with multiple biological functions, playing an important role in clinical medicine, gene regulation, food and health care. In recent years, with the rapid development of the dairy industry, lactoferrin has been widely used in food, medicine, cosmetics and other products. Therefore, it is necessary to develop and improve the preparation process of lactoferrin and extract high-purity lactoferrin.
[0003] Lactoferrin is mostly derived from the milk of mammals, especially colostrum, where its content is the highest. However, colostrum contains a variety of substances, such as immunoglobulins, caseins, lactoferrin, etc., among which casein has the highest content, accounting for 80%. Therefore, in the preparation process of lactoferrin, casein must be removed first. The methods for removing casein are mainly isoelectric point method and enzymatic method. The isoelectric point method is greatly affected by pH when removing casein, resulting in limited purity of the final product. Although the casein content in the whey obtained after enzymatic hydrolysis is reduced, new free enzyme impurities will also be introduced into the whey.
[0004] After removing casein and collecting whey, it is necessary to further separate and purify lactoferrin in the whey. The separation and purification of lactoferrin include ion exchange chromatography, affinity column chromatography, gel chromatography, etc. Affinity column chromatography has high specificity and high purity, but the cost is also high. Ion exchange chromatography and gel chromatography generally have the problem of low specificity. It can be seen that in the preparation process of lactoferrin, a preparation process with higher purity, low cost and specificity is needed. Summary of the invention
[0005] The purpose of the present invention is to provide a preparation process of lactoferrin to solve the problems existing in the prior art.
[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:
[0007] A process for preparing lactoferrin comprises the following steps:
[0008] (1) Mixing amino hollow mesoporous silica and 1% glutaraldehyde aqueous solution in a mass ratio of 1:(3-4), stirring at room temperature for 20-30 minutes, filtering and washing 3-4 times with 0.01M, pH 7.4 phosphate buffer solution, stirring and mixing the washed amino hollow mesoporous silica, phosphate buffer solution, rennet and chitosan in a mass ratio of 1:(10-12):(0.5-0.7):(0.4-0.6) for 12-14 hours, filtering and washing 3-4 times with 0.01M, pH 7.4 phosphate buffer solution to obtain modified silica;
[0009] (2) adjusting the pH of skim milk to 6.0-6.2 with 1 mol / L hydrochloric acid solution, and keeping warm at 35°C for 30 minutes to obtain warm skim milk, mixing modified silica and pure water at a mass ratio of 1:(10-12), keeping warm at 35°C for 30 minutes to obtain a modified silica dispersion, mixing the modified silica dispersion and warm skim milk at a mass ratio of 1:(10-12), stirring and reacting at 50°C for 90-100 minutes, centrifuging at 4°C and 8000 r / min for 15 minutes, and collecting the supernatant to obtain whey;
[0010] (3) 4-vinylpyridine, lactoferrin and methanol were mixed, stirred at 30° C. for 20-30 min, pretreated resin, crosslinking agent and initiator were added, stirred and polymerized at 60° C., filtered and washed with a sodium hydroxide-ethanol mixture for 3-4 times, and washed with pure water for 3-4 times to obtain a molecularly imprinted resin;
[0011] (4) Mixing molecular imprinting resin and silver sulfate in a mass ratio of 1:(0.03-0.05), heating to 70° C., adding 98% concentrated sulfuric acid in an amount of 7-9 times the mass of the molecular imprinting resin dropwise at a rate of 2 mL / min, heating to 80° C. for reaction for 30-40 min, filtering with a glass sand funnel, immersing in an ice-water mixture, stirring for 20 min, filtering and washing with pure water for 6-8 times, and drying at 60° C. for 7-8 h to obtain a modified cation exchange resin;
[0012] (5) Activating the modified cation exchange resin to obtain an activated modified resin, mixing whey, activated modified resin and pure water in a mass ratio of 1: (0.5-0.6): (20-30), adjusting the pH to 6.8 with 1 M sodium hydroxide solution, stirring at 4° C. for 12 h at a stirring speed of 100-200 r / min, filtering and washing with pure water for 3-4 times, placing in a 5 wt % sodium chloride solution (50-60 times the mass of the activated modified resin) and stirring for 10 min to elute to obtain an eluate, ultrafiltering the eluate using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa, ultrafiltering 3-4 times, and freeze-drying to obtain lactoferrin.
[0013] As an optimization, the preparation process of the skimmed milk in step (2) is as follows: the bovine colostrum is skimmed using a high-speed refrigerated centrifuge at 4°C and 10,000 r / min for 30 minutes to remove the upper layer of fat and obtain skimmed milk; the bovine colostrum is collected within 72 hours after the dairy cow calves.
[0014] As an optimization, the mass ratio of 4-vinylpyridine, lactoferrin, methanol, pretreated resin, crosslinking agent and initiator in step (3) is 1:(0.2-0.3):(50-60):(5-6):(1-1.2):(0.01-0.02); the sodium hydroxide-ethanol mixed solution is prepared by 8wt% sodium hydroxide solution and anhydrous ethanol in a volume ratio of 1:1; the crosslinking agent is ethylene glycol dimethacrylate, and the initiator is dibenzoyl peroxide.
[0015] As an optimization, the preparation process of the pretreatment resin in step (3) is: the polystyrene resin is washed with pure water for 3-4 times, put into a Soxhlet extractor and extracted with anhydrous ethanol for 6 hours, and after the extraction, it is dried at 40°C for 6-8 hours to obtain the pretreatment resin. The polystyrene resin model is: NDA-1800 resin.
[0016] As an optimization, the activation step in step (5) is: immersing the modified cation exchange resin in pure water at 90-100° C. for 1-2 hours, and then immersing it in a 10% sodium chloride solution for 30-40 minutes to obtain an activated modified resin.
[0017] As an optimization, the particle size of the amino hollow mesoporous silica in step (1) is 100 nm; the deacetylation degree of the chitosan is ≥95%; and the chymosin activity is 890 IMCU / g.
[0018] As an optimization, the lactoferrin in step (3) is natural bovine lactoferrin, with a content of >98% and a brand of American Silma.
[0019] Compared with the prior art, the beneficial effects achieved by the present invention are:
[0020] When preparing lactoferrin, the present invention comprises the following steps: firstly, amino hollow mesoporous silica is used to load chitosan and rennet at the same time to obtain modified silica; bovine colostrum is used as a raw material, skim milk is obtained by centrifugation, casein in the skim milk is precipitated by the modified silica to obtain whey; then, lactoferrin is used as a template molecule, 4-vinylpyridine is used as a monomer, and polystyrene resin is used as a carrier, molecular imprinting resin is prepared under the action of a crosslinking agent and an initiator, the molecular imprinting resin is sulfonated to obtain a modified cation exchange resin, and the modified cation exchange resin is used to separate and purify the whey to obtain lactoferrin.
[0021] First, amino hollow mesoporous silica is used to load chitosan and rennet at the same time to obtain modified silica; bovine colostrum is used as a raw material, skim milk is obtained by centrifugation, and casein in the skim milk is precipitated using modified silica to obtain whey; rennet is a proteolytic enzyme that acts on casein to change its structure and lose its original stabilizing effect, thereby precipitating under the action of calcium ions; the amino group of chitosan is protonated under acidic conditions, so that the surface of chitosan is positively charged, and casein is negatively charged above the isoelectric point of 4.6-4.8. The positive charge on chitosan can attract casein and cooperate with rennet to precipitate casein. At the same time, chitosan has good antibacterial properties, and the possibility of microbial contamination is reduced during the operation of degreasing and removing casein from bovine colostrum; chitosan and rennet are fixed on the silica carrier, and will be removed together with casein during filtration, and no additional impurities will be introduced into the whey;
[0022] Then, using lactoferrin as a template molecule, 4-vinylpyridine as a monomer, and polystyrene resin as a carrier, a molecular imprinting resin is prepared under the action of a crosslinking agent and an initiator, the molecular imprinting resin is sulfonated to obtain a modified cation exchange resin, and then the modified cation exchange resin is used to separate and purify whey to obtain lactoferrin. Molecularly imprinted polymers are a type of polymer that uses a target molecule as a template and has specific selectivity. The molecular imprinting resin prepared using lactoferrin as a template molecule has holes that match lactoferrin, which can achieve specific adsorption of lactoferrin; the molecular imprinting resin is sulfonated to carry a strong acidic group on the resin, and the acidic group contained in the resin body after dissociation in water can bind to lactoferrin; the lactoferrin molecular imprinting and the acidic group on the modified cation exchange resin can synergistically adsorb lactoferrin, thereby improving the purity of lactoferrin. DETAILED DESCRIPTION
[0023] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.
[0024] The amino hollow mesoporous silica particles used in the following examples and comparative examples have a particle size of 100 nm and were purchased from Beijing Zhongke Keyou Nanotechnology Co., Ltd.; the chitosan deacetylation degree is ≥95%; the rennet activity is 890 IMCU / g and was purchased from Beijing Duoaite Biotechnology Co., Ltd.; the bovine colostrum is collected within 72 hours after calving; the lactoferrin is natural bovine lactoferrin with a content of >98% and a brand of American Silmar; the polystyrene resin model is NDA-1800 resin; the crosslinking agent is ethylene glycol dimethacrylate, and the initiator is dibenzoyl peroxide; the polystyrene resin model is NDA-1800 resin.
[0025] Embodiment 1:
[0026] A process for preparing lactoferrin comprises the following steps:
[0027] (1) mixing amino hollow mesoporous silica and 1% glutaraldehyde aqueous solution in a mass ratio of 1:3, stirring at room temperature for 20 minutes, filtering and washing 3-4 times with 0.01M, pH 7.4 phosphate buffer solution, stirring and mixing the washed amino hollow mesoporous silica, phosphate buffer solution, rennet and chitosan in a mass ratio of 1:10:0.5:0.4 for 14 hours, filtering and washing 4 times with 0.01M, pH 7.4 phosphate buffer solution to obtain modified silica;
[0028] (2) using a high-speed refrigerated centrifuge to defatted bovine colostrum at 4°C and 10,000 r / min for 30 min, removing the upper layer of fat to obtain skimmed milk; adjusting the pH of the skimmed milk to 6.2 using a 1 mol / L hydrochloric acid solution, and keeping it warm at 35°C for 30 min to obtain insulated skimmed milk, mixing modified silica and pure water at a mass ratio of 1:10, and keeping it warm at 35°C for 30 min to obtain a modified silica dispersion, mixing the modified silica dispersion and insulated skimmed milk at a mass ratio of 1:10, stirring and reacting at 50°C for 100 min, centrifuging at 4°C and 8,000 r / min for 15 min, and collecting the supernatant to obtain whey;
[0029] (3) The polystyrene resin was washed with pure water for 3 times, and extracted with anhydrous ethanol for 6 hours in a Soxhlet extractor. After the extraction, the resin was dried at 40° C. for 6-8 hours to obtain a pretreated resin. 4-vinylpyridine, lactoferrin, methanol, pretreated resin, crosslinking agent and initiator were weighed in a mass ratio of 1:0.2:50:5:1:0.01; 4-vinylpyridine, lactoferrin and methanol were mixed, stirred at 30° C. for 30 minutes, the pretreated resin, crosslinking agent and initiator were added, and the mixture was polymerized by stirring at 60° C., filtered and washed with a sodium hydroxide ethanol mixture for 3 times, wherein the sodium hydroxide ethanol mixture was prepared by 8% sodium hydroxide solution and anhydrous ethanol in a volume ratio of 1:1, and then washed with pure water for 3 times to obtain a molecularly imprinted resin;
[0030] (4) Molecularly imprinted resin and silver sulfate were mixed in a mass ratio of 1:0.03, heated to 70°C, 98% concentrated sulfuric acid (7 times the mass of the molecularly imprinted resin) was added dropwise at a rate of 2 mL / min, heated to 80°C for 40 min, filtered with a glass funnel, immersed in an ice-water mixture, stirred for 20 min, filtered, washed with pure water 6 times, and dried at 60°C for 8 h to obtain a modified cation exchange resin;
[0031] (5) The modified cation exchange resin was immersed in pure water at 100° C. and allowed to stand for 2 h, and then immersed in a 10% sodium chloride solution for 40 min to obtain an activated modified resin, whey, activated modified resin, and pure water were mixed at a mass ratio of 1:0.5:20, the pH was adjusted to 6.8 using a 1 M sodium hydroxide solution, and the mixture was stirred at 4° C. for 12 h at a stirring speed of 200 r / min, filtered, washed with pure water for 3 times, placed in a 5 wt % sodium chloride solution (50 times the mass of the activated modified resin) and stirred for 10 min to elute, to obtain an eluate, and the eluate was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa. After ultrafiltration 3 times, the mixture was freeze-dried to obtain lactoferrin.
[0032] Embodiment 2:
[0033] A process for preparing lactoferrin comprises the following steps:
[0034] (1) mixing amino hollow mesoporous silica and 1% glutaraldehyde aqueous solution in a mass ratio of 1:3.5, stirring at room temperature for 25 minutes, filtering and washing three times with 0.01M, pH 7.4 phosphate buffer solution, stirring and mixing the washed amino hollow mesoporous silica, phosphate buffer solution, rennet and chitosan in a mass ratio of 1:11:0.6:0.5 for 12-14 hours, filtering and washing 3-4 times with 0.01M, pH 7.4 phosphate buffer solution to obtain modified silica;
[0035] (2) using a high-speed refrigerated centrifuge to defatted bovine colostrum at 4°C and 10,000 r / min for 30 min, removing the upper layer of fat to obtain skimmed milk; adjusting the pH of the skimmed milk to 6.1 using a 1 mol / L hydrochloric acid solution, and keeping it warm at 35°C for 30 min to obtain warm skimmed milk, mixing modified silica and pure water at a mass ratio of 1:11, and keeping it warm at 35°C for 30 min to obtain a modified silica dispersion, mixing the modified silica dispersion and warm skimmed milk at a mass ratio of 1:11, stirring and reacting at 50°C for 95 min, centrifuging at 4°C and 8,000 r / min for 15 min, and collecting the supernatant to obtain whey;
[0036] (3) washing the polystyrene resin with pure water for 3-4 times, extracting it with anhydrous ethanol in a Soxhlet extractor for 6 hours, and drying it at 40° C. for 6-8 hours after the extraction to obtain a pretreated resin; weighing 4-vinylpyridine, lactoferrin, methanol, pretreated resin, crosslinking agent and initiator in a mass ratio of 1:0.25:55:5.5:1.1:0.015; mixing 4-vinylpyridine, lactoferrin and methanol, stirring at 30° C. for 20-30 minutes, adding the pretreated resin, crosslinking agent and initiator, stirring and polymerizing at 60° C., filtering and washing 3-4 times with a sodium hydroxide ethanol mixture, wherein the sodium hydroxide ethanol mixture is prepared by 8% sodium hydroxide solution and anhydrous ethanol in a volume ratio of 1:1, and then washing 3 times with pure water to obtain a molecularly imprinted resin;
[0037] (4) Molecularly imprinted resin and silver sulfate were mixed in a mass ratio of 1:0.04, heated to 70°C, 98% concentrated sulfuric acid in an amount of 8 times the mass of the molecularly imprinted resin was added dropwise at a rate of 2 mL / min, heated to 80°C for reaction for 35 min, filtered with a glass sand funnel, immersed in an ice-water mixture, stirred for 20 min, filtered and washed with pure water 7 times, and dried at 60°C for 7 h to obtain a modified cation exchange resin;
[0038] (5) The modified cation exchange resin is immersed in pure water at 90-100° C. and allowed to stand for 1-2 h, and then immersed in a 10 wt % sodium chloride solution for 30-40 min to obtain an activated modified resin, whey, activated modified resin, and pure water are mixed in a mass ratio of 1:(0.5-0.6):(20-30), the pH value is adjusted to 6.8 using a 1 M sodium hydroxide solution, and the mixture is stirred at 4° C. for 12 h at a stirring speed of 100-200 r / min, filtered, washed with pure water for 3-4 times, placed in a 5 wt % sodium chloride solution with a mass of 50-60 times that of the activated modified resin, and stirred for 10 min to elute to obtain an eluate, and the eluate is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa, and after ultrafiltration 3-4 times, freeze-dried to obtain lactoferrin.
[0039] Embodiment 3:
[0040] A process for preparing lactoferrin comprises the following steps:
[0041] (1) Mixing amino hollow mesoporous silica and 1% glutaraldehyde aqueous solution in a mass ratio of 1:4, stirring at room temperature for 20 minutes, filtering and washing three times with 0.01M, pH 7.4 phosphate buffer solution, stirring and mixing the washed amino hollow mesoporous silica, phosphate buffer solution, rennet and chitosan in a mass ratio of 1:12:0.7:0.6 for 12 hours, filtering and washing four times with 0.01M, pH 7.4 phosphate buffer solution to obtain modified silica;
[0042] (2) using a high-speed refrigerated centrifuge to defatted bovine colostrum at 4°C and 10,000 r / min for 30 min, removing the upper layer of fat to obtain skimmed milk; adjusting the pH of the skimmed milk to 6.0 using a 1 mol / L hydrochloric acid solution, and keeping it warm at 35°C for 30 min to obtain warm skimmed milk, mixing modified silica and pure water at a mass ratio of 1:12, and keeping it warm at 35°C for 30 min to obtain a modified silica dispersion, mixing the modified silica dispersion and warm skimmed milk at a mass ratio of 1:12, stirring and reacting at 50°C for 90 min, centrifuging at 4°C and 8,000 r / min for 15 min, and collecting the supernatant to obtain whey;
[0043] (3) The polystyrene resin was washed with pure water for 3 times, and extracted with anhydrous ethanol for 6 hours in a Soxhlet extractor. After the extraction, the resin was dried at 40° C. for 6-8 hours to obtain a pretreated resin. 4-vinylpyridine, lactoferrin, methanol, pretreated resin, crosslinking agent and initiator were weighed in a mass ratio of 1:0.3:-60:6:1.2:0.02; 4-vinylpyridine, lactoferrin and methanol were mixed, stirred at 30° C. for 20 minutes, and the pretreated resin, crosslinking agent and initiator were added. The mixture was polymerized by stirring at 60° C., filtered and washed with a sodium hydroxide ethanol mixture for 3 times. The sodium hydroxide ethanol mixture was prepared by 8% sodium hydroxide solution and anhydrous ethanol in a volume ratio of 1:1, and then washed with pure water for 4 times to obtain a molecularly imprinted resin;
[0044] (4) Molecularly imprinted resin and silver sulfate were mixed at a mass ratio of 1:0.05, heated to 70°C, 98% concentrated sulfuric acid in an amount of 9 times the mass of the molecularly imprinted resin was added dropwise at a rate of 2 mL / min, heated to 80°C for reaction for 30 min, filtered with a glass funnel, immersed in an ice-water mixture, stirred for 20 min, filtered, washed with pure water 6 times, and dried at 60°C for 7 h to obtain a modified cation exchange resin;
[0045] (5) The modified cation exchange resin was immersed in pure water at 90° C. and allowed to stand for 1 h, and then immersed in a 10% sodium chloride solution for 30 min to obtain an activated modified resin, whey, activated modified resin, and pure water were mixed at a mass ratio of 1:0.6:30, the pH was adjusted to 6.8 using a 1 M sodium hydroxide solution, stirred at 4° C. for 12 h at a stirring speed of 100 r / min, filtered and washed with pure water for 4 times, placed in a 5 wt % sodium chloride solution with a mass of 60 times that of the activated modified resin, stirred for 10 min to elute, and an eluate was obtained, and the eluate was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa. After ultrafiltration 3 times, the eluate was freeze-dried to obtain lactoferrin.
[0046] Comparative Example 1:
[0047] The difference between the preparation process of lactoferrin in comparative example 1 and embodiment 2 lies in step (3), wherein step (3) is modified as follows: washing a polystyrene resin with pure water for 3-4 times, extracting the polystyrene resin with anhydrous ethanol in a Soxhlet extractor for 6 hours, and drying the polystyrene resin at 40° C. for 6-8 hours after the extraction to obtain a pretreated resin; weighing 4-vinylpyridine, methanol, a pretreated resin, a crosslinking agent and an initiator in a mass ratio of 1:55:5.5:1.1:0.015; mixing 4-vinylpyridine, lactoferrin and methanol, stirring at 30° C. for 20-30 minutes, adding the pretreated resin, the crosslinking agent and the initiator, stirring and polymerizing at 60° C., filtering and washing the polystyrene resin with a sodium hydroxide ethanol mixture for 3-4 times, wherein the sodium hydroxide ethanol mixture is prepared by mixing 8% sodium hydroxide solution and anhydrous ethanol in a volume ratio of 1:1, and then washing the polystyrene resin with pure water for 3 times to obtain a molecularly imprinted resin.
[0048] Comparative Example 2:
[0049] The preparation process of lactoferrin in Comparative Example 2 differs from that in Example 2 in that step (1) is modified as follows: amino hollow mesoporous silica and 1% glutaraldehyde aqueous solution are mixed in a mass ratio of 1:4, stirred at room temperature for 20 min, filtered and washed 3 times with 0.01 M, pH 7.4 phosphate buffer solution, the washed amino hollow mesoporous silica, phosphate buffer solution, rennet and chitosan are stirred and mixed in a mass ratio of 1:12:0.7:0.6 for 12 h, filtered and washed 4 times with 0.01 M, pH 7.4 phosphate buffer solution to obtain modified silica.
[0050] Test example:
[0051] Test for the purity of lactoferrin:
[0052] Test method: The concentration C1 of lactoferrin in whey and the concentration C2 of lactoferrin in eluate were determined by ELISA method, the volume V1 of whey and the volume V2 of eluate were quantified, and the recovery rate of lactoferrin was calculated. The recovery rate was calculated by the formula: recovery rate (%) = (C2*V2) / (C1*V1)*100%.
[0053] The total protein concentration C3 in the eluate was determined by the BCA total protein concentration method, and the purity of lactoferrin was calculated based on the concentration of lactoferrin in the eluate in the ELISA method. The purity calculation formula is: purity (%) = C2 / C3*100%. The results are shown in Table 1.
[0054] Table 1
[0055] Recovery rate (%) purity(%) Example 1 97.65 92.4 Example 2 96.43 93.6 Example 3 97.26 92.1 Comparative Example 1 95.23 73.2 Comparative Example 2 95.79 84.7
[0056] From the comparison of the experimental data of Examples 1 to 3 and Comparative Examples 1 to 2 in Table 1, it can be found that the lactoferrin prepared by the present invention has good recovery rate and purity.
[0057] By comparison, the recovery rate and purity of Examples 1 to 3 are greater than those of Comparative Examples 1 to 2, indicating that, first, amino hollow mesoporous silica is used to simultaneously load chitosan and rennet to obtain modified silica; skim milk is obtained by centrifugation using bovine colostrum as a raw material, and casein in the skim milk is precipitated using modified silica to obtain whey; rennet is a proteolytic enzyme that acts on casein to cause structural changes in the casein, causing it to lose its original stabilizing effect, thereby causing precipitation under the action of calcium ions; the amino group of chitosan is used to load chitosan and rennet to obtain modified silica ... Protonation occurs under acidic conditions, making the surface of chitosan positively charged. Casein has a negative charge above the isoelectric point of 4.6-4.8. The positive charge on chitosan can attract casein and cooperate with chymosin to precipitate casein. At the same time, chitosan has good antibacterial properties. After defatting and removing casein from bovine colostrum, no additional sterilization operation is required, reducing the possibility of microbial contamination. Chitosan and chymosin are fixed on the silica carrier and will be removed together with casein during filtration, without introducing additional impurities into the whey.
[0058] Then, using lactoferrin as a template molecule, 4-vinylpyridine as a monomer, and polystyrene resin as a carrier, a molecular imprinting resin is prepared under the action of a crosslinking agent and an initiator, the molecular imprinting resin is sulfonated to obtain a modified cation exchange resin, and then the modified cation exchange resin is used to separate and purify whey to obtain lactoferrin. Molecularly imprinted polymers are a type of polymer that uses a target molecule as a template and has specific selectivity. The molecular imprinting resin prepared using lactoferrin as a template molecule has holes that match lactoferrin, which can achieve specific adsorption of lactoferrin; the molecular imprinting resin is sulfonated to carry a strong acidic group on the resin, and the acidic group contained in the resin body after dissociation in water can bind to lactoferrin; the lactoferrin molecular imprinting and the acidic group on the modified cation exchange resin can synergistically adsorb lactoferrin, thereby improving the purity of lactoferrin.
[0059] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. A process for preparing lactoferrin, characterized in that: The method comprises the following preparation steps: (1) Mixing amino hollow mesoporous silica and 1% glutaraldehyde aqueous solution in a mass ratio of 1:(3-4), stirring at room temperature for 20-30 minutes, filtering and washing 3-4 times with 0.01M, pH 7.4 phosphate buffer solution, stirring and mixing the washed amino hollow mesoporous silica, phosphate buffer solution, rennet and chitosan in a mass ratio of 1:(10-12):(0.5-0.7):(0.4-0.6) for 12-14 hours, filtering and washing 3-4 times with 0.01M, pH 7.4 phosphate buffer solution to obtain modified silica; (2) adjusting the pH of skim milk to 6.0-6.2 with 1 mol / L hydrochloric acid solution, and keeping warm at 35°C for 30 minutes to obtain warm skim milk, mixing modified silica and pure water at a mass ratio of 1:(10-12), keeping warm at 35°C for 30 minutes to obtain a modified silica dispersion, mixing the modified silica dispersion and warm skim milk at a mass ratio of 1:(10-12), stirring and reacting at 50°C for 90-100 minutes, centrifuging at 4°C and 8000 r / min for 15 minutes, and collecting the supernatant to obtain whey; (3) 4-vinylpyridine, lactoferrin and methanol were mixed, stirred at 30° C. for 20-30 min, pretreated resin, crosslinking agent and initiator were added, stirred and polymerized at 60° C., filtered and washed with a sodium hydroxide-ethanol mixture for 3-4 times, and washed with pure water for 3-4 times to obtain a molecularly imprinted resin; (4) Mixing molecular imprinting resin and silver sulfate in a mass ratio of 1:(0.03-0.05), heating to 70° C., adding 98% concentrated sulfuric acid in an amount of 7-9 times the mass of the molecular imprinting resin dropwise at a rate of 2 mL / min, heating to 80° C. for reaction for 30-40 min, filtering with a glass sand funnel, immersing in an ice-water mixture, stirring for 20 min, filtering and washing with pure water for 6-8 times, and drying at 60° C. for 7-8 h to obtain a modified cation exchange resin; (5) Activating the modified cation exchange resin to obtain an activated modified resin, mixing whey, activated modified resin and pure water in a mass ratio of 1: (0.5-0.6): (20-30), adjusting the pH to 6.8 with 1 M sodium hydroxide solution, stirring at 4° C. for 12 h at a stirring speed of 100-200 r / min, filtering and washing with pure water for 3-4 times, placing in a 5 wt % sodium chloride solution (50-60 times the mass of the activated modified resin) and stirring for 10 min to elute to obtain an eluate, ultrafiltering the eluate using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa, ultrafiltering 3-4 times, and freeze-drying to obtain lactoferrin.
2. A process for preparing lactoferrin according to claim 1, characterized in that: The preparation process of the skimmed milk in step (2) is as follows: skimming the bovine colostrum using a high-speed refrigerated centrifuge at 4°C and 10,000 r / min for 30 minutes to remove the upper layer of fat and obtain skimmed milk; the bovine colostrum is collected within 72 hours after calving.
3. A process for preparing lactoferrin according to claim 1, characterized in that: The mass ratio of 4-vinylpyridine, lactoferrin, methanol, pretreated resin, crosslinking agent and initiator in step (3) is 1:(0.2-0.3):(50-60):(5-6):(1-1.2):(0.01-0.02); the sodium hydroxide-ethanol mixed solution is prepared by 8wt% sodium hydroxide solution and anhydrous ethanol in a volume ratio of 1:1; the crosslinking agent is ethylene glycol dimethacrylate, and the initiator is dibenzoyl peroxide.
4. A process for preparing lactoferrin according to claim 1, characterized in that, The preparation process of the pretreated resin in step (3) is as follows: the polystyrene resin is washed with pure water for 3-4 times, put into a Soxhlet extractor and extracted with anhydrous ethanol for 6 hours, and after the extraction, it is dried at 40°C for 6-8 hours to obtain the pretreated resin. The polystyrene resin model is: NDA-1800 resin.
5. A process for preparing lactoferrin according to claim 1, characterized in that, The activation step in step (5) is as follows: immersing the modified cation exchange resin in pure water at 90-100° C. and leaving it to stand for 1-2 hours, and then immersing it in a 10% sodium chloride solution for 30-40 minutes to obtain an activated modified resin.
6. A process for preparing lactoferrin according to claim 1, characterized in that: In step (1), the particle size of the amino hollow mesoporous silica is 100 nm; the deacetylation degree of the chitosan is ≥95%; and the chymosin activity is 890 IMCU / g.
7. A process for preparing lactoferrin according to claim 1, characterized in that: The lactoferrin in step (3) is natural bovine lactoferrin, with a content of >98% and a brand of American Silma.