Process for extracting high-purity lactoferrin and lactoperoxidase from fresh milk

Abstract

The invention discloses a process for extracting high-purity lactoferrin and lactoperoxidase from fresh milk, which comprises the following steps: centrifugally degreasing the fresh milk to obtain skimmed milk; sterilizing and concentrating the obtained skimmed milk through a ceramic membrane to obtain sterilized concentrated milk liquid; adsorbing and eluting the sterilized concentrated milk liquid through cation exchange resin, and collecting eluent rich in lactoferrin and lactoperoxidase; desalting and concentrating the obtained eluent through a ceramic membrane to obtain secondary concentrated milk liquid; the obtained secondary concentrated milk liquid is introduced into a simulated moving bed chromatography system, a lactoferrin extracting solution is obtained from an extracting solution outlet of the simulated moving bed chromatography system, and a lactoperoxidase extracting solution is obtained from a raffinate outlet; carrying out desalination and concentration by using an ultrafiltration membrane, and carrying out spray drying to obtain a lactoferrin final product and a lactoperoxidase final product; the purity of lactoferrin in the obtained lactoferrin final product is 95% or above, and the purity of emulsified peroxidase in the obtained lactoperoxidase final product is 90% or above.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of dairy processing, and particularly relates to a process for extracting high-purity lactoferrin and lactoperoxidase from fresh cow milk. BACKGROUND

[0002] Lactoferrin and lactoperoxidase are important bioactive proteins in cow milk, which have multiple physiological functions such as antibacterial, antiviral, immunomodulatory and antioxidant, and are widely used in food, medicine and cosmetic industries. Currently, the common methods for separating these two proteins from cow milk include ion exchange chromatography, ultrafiltration, precipitation and conventional column chromatography. However, these methods have many limitations: for example, traditional ion exchange chromatography can adsorb lactoferrin and lactoperoxidase, but the separation efficiency is low and it is difficult to obtain high-purity products simultaneously; the selectivity of ultrafiltration and precipitation methods is poor, and impurities are easily introduced; and the conventional column chromatography has small processing capacity, discontinuous process and high cost, which is not suitable for industrial production.

[0003] Simulated moving bed chromatography (SMB) is a continuous chromatography technology, which realizes continuous separation of components by simulating the movement of the stationary phase, and has been widely used in the separation of sugars, chiral drugs and organic acids, but its application in the field of milk protein separation is less. In addition, the existing process can only extract one kind of protein, and cannot efficiently separate lactoferrin and lactoperoxidase simultaneously, resulting in resource waste and cost increase.

[0004] Therefore, there is an urgent need in the art for a continuous process that can efficiently and economically extract high-purity lactoferrin and lactoperoxidase from fresh cow milk simultaneously. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a process for extracting high-purity lactoferrin and lactoperoxidase from fresh cow milk, which addresses the shortcomings of the prior art.

[0006] To solve the above technical problems, the present application discloses a process for extracting high-purity lactoferrin and lactoperoxidase from fresh cow milk.

[0007] The process integrates technologies such as centrifugal degreasing, ceramic membrane filtration, cation exchange resin and simulated moving bed chromatography, to realize efficient simultaneous separation of the two proteins, with high product purity and good process continuity, suitable for large-scale production.

[0008] Specifically, the process method is as follows:

[0009] (1) Fresh cow milk is centrifuged to obtain skim milk;

[0010] (2) The skimmed milk obtained is subjected to sterilization and pre-concentration by a ceramic membrane filtration system to obtain sterilized concentrated milk liquid;

[0011] (3) The sterilized concentrated milk liquid obtained is passed through a cation exchange resin to adsorb lactoferrin and lactoperoxidase therein, and then eluted to collect eluate rich in lactoferrin and lactoperoxidase;

[0012] (4) The eluate obtained is subjected to desalination and concentration by a ceramic membrane to obtain secondary concentrated milk liquid;

[0013] (5) A simulated moving bed chromatography system is introduced, and the secondary concentrated milk liquid obtained is passed into the simulated moving bed chromatography system, wherein the simulated moving bed of the simulated moving bed chromatography system is composed of 8-24 identical chromatography columns, and is divided into four functional zones, each of which is composed of 2-6 chromatography columns connected in series, and the four zones are arranged and switched according to the liquid flow direction; the simulated moving bed chromatography system is provided with a feed inlet, an eluate inlet, an extract outlet and a raffinate outlet, and lactoferrin extract is obtained from the extract outlet and lactoperoxidase extract is obtained from the raffinate outlet;

[0014] (6) The lactoferrin extract and the lactoperoxidase extract obtained are respectively subjected to desalination and concentration by ultrafiltration membrane filtration to obtain lactoferrin concentrate and lactoperoxidase concentrate, respectively;

[0015] (7) The lactoferrin concentrate and the lactoperoxidase concentrate obtained are respectively subjected to spray drying to obtain lactoferrin and lactoperoxidase final products.

[0016] In step (1), the centrifugal skimming conditions are as follows: temperature 4-10℃, centrifugal force 3000-6000 g.

[0017] In step (2), the skimmed milk obtained is sterilized by a ceramic membrane, and the permeate obtained is concentrated by a ceramic membrane to obtain sterilized concentrated milk liquid. The pore size of the ceramic membrane used in the sterilization process is 1.0-1.8 μm, such as 1.4 μm, the operating temperature is 30-50℃, and the transmembrane pressure is 0.1-0.3 MPa; the pore size of the ceramic membrane used in the concentration process is 8-12 nm, such as 10 nm, the operating temperature is 30-50℃, the transmembrane pressure is 0.1-0.4 MPa, and the concentration of total protein in the sterilized concentrated milk liquid after concentration is 10%wt-15%wt.

[0018] In step (3), the sterilized concentrated milk liquid obtained is adjusted to pH 6.0-6.5 before being loaded, and then adsorbed and eluted by a cation exchange resin.

[0019] In step (3), the cation exchange resin uses cross-linked agarose as a matrix, with a resin particle size of 40-170 µm and an H ion exchange capacity of 0.08-0.30 mmol H ions. + / mL. In some embodiments, the preferred type of cation exchange resin is one of SP Sepharose 6FF, SP Sepharose Big Beads, CM Sepharose 6FF, CM Sephadex C-50, SP Sepharose Fast Flow, and Source 30S, but it is not limited to these six types. Cation exchange resins with equivalent separation efficiency are also within the scope of this invention.

[0020] In step (3), the treatment of the cation exchange resin includes equilibration, sample loading, and elution steps.

[0021] The equilibration solution used in the equilibration step is a 20-50 mM phosphate or citrate buffer solution with a pH of 6.0-6.5.

[0022] The sample loading volume is 15-25 BV, and the loading rate is 80-120 cm / h, such as 100 cm / h.

[0023] The elution solution used in the elution step is a 20-50 mM phosphate buffer solution with a pH of 7.0-7.5 containing 0.5-1.0 M NaCl.

[0024] The elution volume is 1-3 BV, such as 1.5 BV, and the elution rate is 30-50 cm / h, such as 40 cm / h.

[0025] In step (4), the ceramic membrane has a pore size of 8-12 nm, such as 10 nm, an operating temperature of 30-50℃, and a transmembrane pressure of 0.1-0.4 MPa; the total protein concentration of the secondary concentrated milk is above 1%wt.

[0026] In step (5), the stationary phase of the simulated moving bed chromatography system is a strong cation exchange resin, which uses polystyrene-divinylbenzene or cross-linked agarose as a matrix; when polystyrene-divinylbenzene is used as the matrix, the resin particle size is 25-35 µm, and the H ion exchange capacity is 0.15-0.30 mmol H ion. + / mL; when cross-linked agarose is used as the matrix, the resin particle size is 40-310 µm, and the H+ ion exchange capacity is 0.15-0.30 mmol H+. + / mL. In some embodiments, the preferred type of strong cation exchange resin is one of SP Sepharose 6FF, SP Sepharose Big Beads, and Source30S, but it is not limited to these three types. Cation exchange resins with equivalent separation efficiency are also within the scope of this invention.

[0027] In step (5), the eluent is a phosphate buffer with pH 6.2-6.8 and contains 0.15-0.35 M NaCl; the operating temperature of the simulated moving bed chromatography system is 10-25℃; and the switching time of the simulated moving bed chromatography system is 3-10 minutes.

[0028] In step (5), the flow rate ratios of the four functional zones (desorption zone I for strong adsorption components, key separation zone II, adsorption zone III for weak adsorption components, and resin regeneration zone IV) of the simulated moving bed chromatography system satisfy the following relationships: the flow rate ratio of zone I is 1.5-2.5; the flow rate ratio of zone II is 0.8-1.5; the flow rate ratio of zone III is 0.5-1.2; and the flow rate ratio of zone IV is 0.2-0.8.

[0029] In step (6), the molecular weight cutoff of the ultrafiltration membrane is 8-12 kDa, such as 10 kDa, the operating temperature of the ultrafiltration membrane is 35-45℃, such as 40℃, and the transmembrane pressure is 0.1-0.5 MPa, such as 0.3 MPa.

[0030] The present invention also provides a lactoferrin prepared by the above process, the purity of which is not less than 95%, the purity of emulsified peroxidase in the resulting lactoperoxidase final product is more than 90%, and the activity of the lactoperoxidase is more than 100 U / mg.

[0031] The beneficial effects of this invention are as follows:

[0032] (1) The present invention achieves sterilization and pre-concentration through ceramic membrane filtration, reducing the load of subsequent processing and improving efficiency.

[0033] (2) This invention utilizes cation exchange resin to selectively adsorb lactoferrin and lactoperoxidase, initially enriching the target proteins. Then, a simulated moving bed chromatography system is used for continuous separation, leveraging the difference in adsorption strength between the two proteins to achieve efficient separation with good process continuity. The entire process has high integration, mild operating conditions, avoids protein denaturation, and produces high-purity products, making it suitable for industrial applications.

[0034] (3) The process provided by the present invention first enriches two target proteins and then separates them using a simulated moving bed, which can significantly reduce the amount of elution solution used, making it more economical and environmentally friendly. Attached Figure Description

[0035] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0036] Figure 1 This is a schematic diagram of the simulated moving bed chromatography system of the present invention. Detailed Implementation

[0037] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0038] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0039] The parameters of the SP Sepharose Fast Flow cation exchange resin described in this invention are: 6% cross-linked agarose matrix, 45 µm-165 µm particle size, 0.18-0.25 mmol H₂O. + / mL Ion exchange capacity.

[0040] The parameters of the Source 30S strong cation exchange resin described in this invention are: polystyrene-divinylphenyl, 30 µm particle size, 0.18-0.25 mmol H₂O. + / mL ion exchange capacity.

[0041] The parameters of the CM Sepharose 6FF cation exchange resin described in this invention are: 6% cross-linked agarose matrix, 45 µm-165 µm, 0.10-0.15 mmol H₂O. + / mL ion exchange capacity.

[0042] The parameters of the SP Sepharose 6FF cation exchange resin described in this invention are: 6% cross-linked agarose matrix, 45 µm-165 µm particle size, 0.18-0.25 mmol H₂O. + / mL ion exchange capacity.

[0043] The parameters of the SP Sepharose Big Beads cation exchange resin described in this invention are: 6% cross-linked agarose matrix, 100 µm-300 µm particle size, 0.18-0.25 mmol H₂O. + / mL ion exchange capacity.

[0044] The flow rate ratio (m) mentioned in this invention is the ratio of the liquid phase flow rate to the apparent solid phase flow rate in each functional zone of the simulated moving bed.

[0045] In the following examples, the total protein concentration was determined using the conventional BCA method.

[0046] In the following examples, the total protein includes two proteins: lactoferrin and lactoperoxidase.

[0047] The purity of the lactoferrin final product described in the following examples was tested according to the national standard GB 5009.299-2024; the purity of the lactoperoxidase final product was tested according to the national standard GB 5009.299-2024, and the activity was tested according to the group standard T / CIFST 008-2022.

[0048] The activity of lactoperoxidase described in this invention is defined as follows: the amount of lactoperoxidase required to catalyze the oxidation of 1 μmol of substrate 3,3',5,5'-tetramethylbenzidine (TMB) per minute under the conditions of temperature 20 ℃~30 ℃ and pH 6.5~7.2 is expressed as 1 lactoperoxidase activity unit, i.e. 1U.

[0049] Example 1

[0050] (1) Take 100 L of fresh milk and centrifuge it at a temperature of 4℃ and a centrifugal force of 3000g to obtain skimmed milk.

[0051] (2) Skim milk is first sterilized by passing it through a ceramic membrane filtration system with a pore size of 1.4 μm (operating temperature 30℃, transmembrane pressure 0.1 MPa). The permeate is then concentrated by passing it through a ceramic membrane filtration system with a pore size of 10 nm (operating temperature 30℃, transmembrane pressure 0.3 MPa) until the total protein concentration in the concentrate reaches 10%. The concentrated milk is then adjusted to pH 6.0 with hydrochloric acid.

[0052] (3) Pack SP Sepharose Fast Flow cation exchange resin into the chromatographic column and equilibrate it with 20 mM, pH 6.0 phosphate buffer. Load the concentrated milk solution after pH adjustment (25 BV loading volume, 100 cm / h loading rate), and then elute with 20 mM, pH 7.0 phosphate buffer containing 0.5 M NaCl (40 cm / h elution volume, 3 BV elution volume), and collect the eluent.

[0053] (4) The eluent is desalted and concentrated using a ceramic membrane filtration system with a pore size of 10 nm (operating temperature 30℃, transmembrane pressure 0.3 MPa) to make the total protein concentration in the concentrate reach 1.0% to obtain secondary concentrated milk.

[0054] (5) Then, a simulated moving bed chromatography system (consisting of 8 columns connected in series, with Source 30S strong cation exchange resin as the stationary phase) is introduced, divided into four functional zones: Zone I, Zone II, Zone III, and Zone IV, each consisting of 2 columns connected in series. For example... Figure 1 As shown, the secondary concentrated milk was loaded through the inlet, the lactoperoxidase extract was collected at the outlet of zone III, the eluent was loaded through the inlet of zone I, and the lactoferrin extract was collected at the outlet of zone I. The eluent was a phosphate buffer solution with pH 6.2 and 0.15 M NaCl. The operating temperature was 10°C, and the column switching time was 3 minutes. The flow rate ratios for the four functional zones were set as follows: zone I 1.5, zone II 0.8, zone III 0.5, and zone IV 0.2.

[0055] (6) The lactoferrin extract and lactoperoxidase extract were desalted and concentrated by passing them through an ultrafiltration membrane (molecular weight cutoff 10 kDa) (operating temperature 40℃, transmembrane pressure 0.3 MPa) to obtain lactoferrin concentrate and lactoperoxidase concentrate, respectively.

[0056] (7) The lactoferrin concentrate and lactoperoxidase concentrate were spray-dried separately (inlet temperature 180℃, outlet temperature 80℃) to obtain the final lactoferrin product and the final lactoperoxidase product.

[0057] The purity of the lactoferrin final product was 95.5% as determined by HPLC; the purity of the lactoperoxidase final product was 90.2%, and the activity was 107 U / mg.

[0058] Example 2

[0059] (1) Take 200 L of fresh milk and centrifuge it at 10℃ and 6000g to obtain skimmed milk.

[0060] (2) Skim milk is first sterilized by passing it through a ceramic membrane filtration system with a pore size of 1.4 μm (operating temperature 40℃, transmembrane pressure 0.2 MPa). The permeate is then concentrated by passing it through a ceramic membrane filtration system with a pore size of 10 nm (operating temperature 40℃, transmembrane pressure 0.4 MPa) until the total protein concentration in the concentrate reaches 12%. The concentrated milk is then adjusted to pH 6.5 with citric acid.

[0061] (3) Pack CM Sepharose 6FF cation exchange resin into the chromatographic column and equilibrate it with 50 mM citrate buffer at pH 6.5. Load the concentrated milk solution after pH adjustment (20 BV loading volume, 100 cm / h loading rate), and then elute with 50 mM phosphate buffer at pH 7.5 containing 1.0 M NaCl (elution rate, 40 cm / h, elution volume, 1.5 BV), and collect the eluent.

[0062] (4) The eluent was desalted and concentrated using a ceramic membrane filtration system with a pore size of 10 nm (operating temperature 40℃, transmembrane pressure 0.2 MPa) to make the total protein concentration in the concentrate reach 1.5% to obtain secondary concentrated milk.

[0063] (5) Then, a simulated moving bed chromatography system (the system consists of 24 columns connected in series, with SP Sepharose Big Beads strong cation exchange resin as the stationary phase) is introduced, divided into four functional zones: Zone I, Zone II, Zone III, and Zone IV, each consisting of 6 columns connected in series. The secondary concentrated milk is loaded through the inlet, the lactoperoxidase extract is collected at the outlet of Zone III, the eluent is loaded through the inlet of Zone I, and the lactoferrin extract is collected at the outlet of Zone I. The eluent is a phosphate buffer solution with pH 6.8 containing 0.35 M NaCl, the operating temperature is 25°C, and the column switching time is 10 minutes. The flow rate ratios of the four functional zones are set as follows: Zone I 2.5, Zone II 1.5, Zone III 1.2, and Zone IV 0.8.

[0064] (6) The lactoferrin extract and lactoperoxidase extract were desalted and concentrated by passing them through an ultrafiltration membrane (molecular weight cutoff 10 kDa) (operating temperature 40℃, transmembrane pressure 0.3 MPa) to obtain lactoferrin concentrate and lactoperoxidase concentrate, respectively.

[0065] (7) The lactoferrin concentrate and lactoperoxidase concentrate were spray-dried separately (inlet temperature 180℃, outlet temperature 80℃) to obtain the final lactoferrin product and the final lactoperoxidase product.

[0066] The purity of the lactoferrin final product was 96.1% as determined by HPLC; the purity of the lactoperoxidase final product was 91.0%, and the activity was 103 U / mg.

[0067] Example 3

[0068] (1) Take 150 L of fresh milk and centrifuge it at 7℃ and 4500g to obtain skimmed milk.

[0069] (2) Skim milk is first sterilized by passing it through a ceramic membrane filtration system with a pore size of 1.4 μm (operating temperature 50℃, transmembrane pressure 0.3 MPa). The permeate is then concentrated by passing it through a ceramic membrane filtration system with a pore size of 10 nm (operating temperature 50℃, transmembrane pressure 0.4 MPa) until the total protein concentration in the concentrate reaches 15%. The concentrated milk is then adjusted to pH 6.3 with hydrochloric acid.

[0070] (3) Pack SP Sepharose Fast Flow cation exchange resin into the chromatographic column and equilibrate it with 35 mM, pH 6.3 phosphate buffer. Load the concentrated milk solution after pH adjustment (15 BV loading volume, 100 cm / h loading rate), and then elute with 35 mM, pH 7.3 phosphate buffer containing 0.75 M NaCl (40 cm / h elution volume, 2 BV elution rate). Collect the eluent.

[0071] (4) The eluent was desalted and concentrated using a ceramic membrane filtration system with a pore size of 10 nm (operating temperature 30℃, transmembrane pressure 0.3 MPa) to make the total protein concentration in the concentrate reach 1.2% to obtain secondary concentrated milk.

[0072] (5) Then, a simulated moving bed chromatography system (the system consists of 16 columns connected in series, with SP Sepharose 6FF strong cation exchange resin as the stationary phase) is introduced, divided into four functional zones: Zone I, Zone II, Zone III, and Zone IV, each consisting of 4 columns connected in series. The secondary concentrated milk is loaded through the inlet, the lactoperoxidase extract is collected at the outlet of Zone III, the eluent is loaded through the inlet of Zone I, and the lactoferrin extract is collected at the outlet of Zone I. The eluent is a phosphate buffer solution with pH 6.5 containing 0.25 M NaCl, the operating temperature is 18℃, and the column switching time is 6 minutes. The flow rate ratios of the four functional zones are set as follows: Zone I 2.0, Zone II 1.2, Zone III 0.8, and Zone IV 0.5.

[0073] (6) The lactoferrin extract and lactoperoxidase extract were desalted and concentrated by passing them through an ultrafiltration membrane (molecular weight cutoff 10 kDa) (operating temperature 40℃, transmembrane pressure 0.3 MPa) to obtain lactoferrin concentrate and lactoperoxidase concentrate, respectively.

[0074] (7) The lactoferrin concentrate and lactoperoxidase concentrate were spray-dried separately (inlet temperature 180℃, outlet temperature 80℃) to obtain the final lactoferrin product and the final lactoperoxidase product.

[0075] The purity of the lactoferrin final product was 95.8% as determined by HPLC; the purity of the lactoperoxidase final product was 90.5%, and the activity was 113 U / mg.

[0076] Example 4

[0077] (1) Take 120 L of fresh milk and centrifuge it at a temperature of 5℃ and a centrifugal force of 5000g to obtain skimmed milk.

[0078] (2) Skim milk is first sterilized by passing it through a ceramic membrane filtration system with a pore size of 1.4 μm (operating temperature 35℃, transmembrane pressure 0.15 MPa). The permeate is then concentrated by passing it through a ceramic membrane filtration system with a pore size of 10 nm (operating temperature 35℃, transmembrane pressure 0.2 MPa) until the total protein concentration in the concentrate reaches 13%. The concentrated milk is then adjusted to pH 6.2 with citric acid.

[0079] (3) Pack CM Sepharose 6FF cation exchange resin into the chromatographic column and equilibrate it with 25 mM citrate buffer at pH 6.2. Load the concentrated milk solution after pH adjustment (25 BV loading volume, 100 cm / h loading rate), and then elute with 25 mM phosphate buffer at pH 7.2 containing 0.6 M NaCl (elution rate, 40 cm / h, 2.5 BV elution volume), and collect the eluent.

[0080] (4) The eluent was desalted and concentrated using a ceramic membrane filtration system with a pore size of 10 nm (operating temperature of 40℃ and transmembrane pressure of 0.3 MPa) to achieve a total protein concentration of 1.1% in the concentrate, thus obtaining a secondary concentrated milk.

[0081] (5) Then, a simulated moving bed chromatography system (the system consists of 12 columns connected in series, with SP Sepharose 6FF strong cation exchange resin as the stationary phase) is introduced, divided into four functional zones: Zone I, Zone II, Zone III, and Zone IV, each consisting of 3 columns connected in series. The secondary concentrated milk is loaded through the inlet, the lactoperoxidase extract is collected at the outlet of Zone III, the eluent is loaded through the inlet of Zone I, and the lactoferrin extract is collected at the outlet of Zone I. The eluent is a phosphate buffer solution with pH 6.4 and 0.2 M NaCl, the operating temperature is 15℃, and the column switching time is 5 minutes. The flow rate ratios of the four functional zones are set as follows: Zone I 1.8, Zone II 1.0, Zone III 0.7, and Zone IV 0.4.

[0082] (6) The lactoferrin extract and lactoperoxidase extract were desalted and concentrated by passing them through an ultrafiltration membrane (molecular weight cutoff 10 kDa) (operating temperature 40℃, transmembrane pressure 0.3 MPa) to obtain lactoferrin concentrate and lactoperoxidase concentrate, respectively.

[0083] (7) The lactoferrin concentrate and lactoperoxidase concentrate were spray-dried separately (inlet temperature 180℃, outlet temperature 80℃) to obtain the final lactoferrin product and the final lactoperoxidase product.

[0084] The purity of the lactoferrin final product was 95.3% as determined by HPLC; the purity of the lactoperoxidase final product was 90.8%, and the activity was 102 U / mg.

[0085] Example 5

[0086] This embodiment aims to demonstrate that the process of separating lactoferrin and lactoperoxidase using simulated moving bed chromatography (SMB) has significant advantages over the traditional cation exchange resin sequential elution method in terms of product purity, yield, processing time, and cost.

[0087] Common pretreatment steps: Take 10 L of fresh milk and centrifuge it at 4℃ and 3000 g to obtain skim milk; first, pass the skim milk through a ceramic membrane filtration system with a pore size of 1.4 μm (operating temperature 30℃, transmembrane pressure 0.1 MPa) for sterilization, and then pass the permeate through a ceramic membrane filtration system with a pore size of 10 nm (operating temperature 30℃, transmembrane pressure 0.3 MPa) for concentration until the total protein concentration in the concentrate reaches 10%, to obtain about 3 L of concentrated milk;

[0088] Process A: Simulated moving bed chromatography:

[0089] ① Take 1.5 L of concentrated milk prepared in the common pretreatment step, adjust the pH to 6.0 with hydrochloric acid, and then load it onto an SPSepharose 6FF ion exchange resin column (column volume 100 mL, pre-equilibrated with 20 mM, pH 6.0 phosphate buffer) at a loading rate of 100 cm / h. Elute with elution buffer (20 mM, pH 7.0 phosphate buffer containing 0.75 M NaCl) at a elution rate of 40 cm / h and a elution volume of 300 mL to obtain approximately 300 mL of eluent containing lactoferrin and lactoperoxidase.

[0090] ② The above eluent was desalted and concentrated using a ceramic membrane filtration system with a pore size of 10 nm (operating temperature 30℃, transmembrane pressure 0.3 MPa) to make the total protein concentration in the concentrate reach 1.0%, resulting in approximately 100 mL of secondary concentrated milk.

[0091] ③ The secondary concentrated milk was introduced into a simulated moving bed chromatography system (the system consists of 8 columns connected in series, each with a volume of 10 mL, and the stationary phase is SP Sepharose 6FF strong cation exchange resin), divided into four functional zones: Zone I, Zone II, Zone III, and Zone IV, each zone consisting of 2 columns connected in series. For example... Figure 1 As shown, the secondary concentrated milk was loaded through the inlet, the lactoperoxidase extract was collected at the outlet of zone III, the eluent was loaded through the inlet of zone I, and the lactoferrin extract was collected at the outlet of zone I. The eluent was a phosphate buffer solution with pH 6.2 and 0.15 M NaCl, with a volume of 160 mL. The operating temperature was 10℃, and the column switching time was 3 minutes. The flow rate ratios for the four functional zones were set as follows: zone I 1.5, zone II 0.8, zone III 0.5, and zone IV 0.2.

[0092] Process B: Sequential elution with cationic resin

[0093] ① Take 1.5 L of concentrated milk prepared in the common pretreatment step, adjust the pH to 6.0 with hydrochloric acid, and then pass it through an SPSepharose 6FF ion exchange resin column (column volume 100 mL, pre-equilibrated with 20 mM, pH 6.0 phosphate buffer).

[0094] ② First, elute with low-salt elution buffer (20 mM phosphate buffer with 0.15M NaCl, pH 7.0) (elution rate 40 cm / h, elution volume 3 BV) and collect the lactoperoxidase fraction (about 300 mL).

[0095] ③ Elute with high-salt elution buffer (20 mM phosphate buffer with 0.75 M NaCl at pH 7.0) (elution rate of 40 cm / h, elution volume of 3 BV) and collect the lactoferrin fraction (approximately 300 mL).

[0096] Common post-processing steps: The eluents collected from processes A and B are desalted and concentrated by passing them through an ultrafiltration membrane (molecular weight cutoff of 10 kDa) (operating temperature of 40℃, transmembrane pressure of 0.3 MPa) to obtain lactoferrin concentrate and lactoperoxidase concentrate, respectively; then the lactoferrin concentrate and lactoperoxidase concentrate are spray-dried (inlet temperature of 180℃, outlet temperature of 80℃) to obtain lactoferrin final product and lactoperoxidase final product.

[0097] The comparison results of the two processes are shown in Table 1:

[0098] Table 1 Comparison of the two processes

[0099]

[0100] Note: a. The eluent consumption refers to the total amount of eluent used in steps ① (300 mL) and ③ (160 mL) of process A, and the total amount of eluent used in steps ② (300 mL) and ③ (300 mL) of process B.

[0101] In this embodiment, a simulated moving bed process (Process A) was used to achieve more efficient separation and reduce cross-contamination between lactoferrin and lactoperoxidase, resulting in a product with higher purity. In the sequential elution process (Process B), the elution steps may overlap, leading to a decrease in purity. Although the simulated moving bed process has a higher initial investment, the overall cost is lower in the long run due to improved yield and reduced solvent consumption.

[0102] The above embodiments demonstrate that the process of the present invention can stably obtain high-purity lactoferrin and lactoperoxidase under different conditions, with product purity reaching over 90%. Moreover, the process is continuous, efficient, and suitable for industrial production.

[0103] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A process for extracting high-purity lactoferrin and lactoperoxidase from fresh milk, characterized in that, Includes the following steps: (1) Fresh milk is centrifuged to remove fat, thus obtaining skim milk; (2) The obtained skim milk is sterilized and concentrated through a ceramic membrane to obtain sterilized concentrated milk; (3) The sterilized concentrated milk is adsorbed and eluted by cation exchange resin, and the eluent rich in lactoferrin and lactoperoxidase is collected. (4) The eluent obtained is desalted and concentrated by a ceramic membrane to obtain a secondary concentrated milk; (5) The obtained secondary concentrated milk is fed into a simulated moving bed chromatography system. The simulated moving bed chromatography system consists of 8-24 identical chromatographic columns and is divided into four functional zones. Each zone consists of 2-6 chromatographic columns connected in series. The four zones are arranged and switched according to the direction of liquid flow. The simulated moving bed chromatography system is equipped with a feed inlet, an eluent inlet, an extract outlet, and a retentate outlet. Lactoferrin extract is obtained from the extract outlet, and lactoperoxidase extract is obtained from the retentate outlet. (6) The obtained lactoferrin extract and lactoperoxidase extract were desalted and concentrated by ultrafiltration membrane to obtain lactoferrin concentrate and lactoperoxidase concentrate, respectively. (7) The lactoferrin concentrate and lactoperoxidase concentrate were spray-dried to obtain lactoferrin final product and lactoperoxidase final product, respectively. The purity of lactoferrin in the obtained lactoferrin final product is above 95%, and the purity of emulsified peroxidase in the obtained lactoperoxidase final product is above 90%.

2. The process according to claim 1, characterized in that, In step (1), the conditions for centrifugal degreasing are: temperature 4-10℃, centrifugal force 3000-6000g.

3. The process according to claim 1, characterized in that, In step (2), the obtained skim milk is sterilized by passing it through a ceramic membrane, and the obtained permeate is concentrated by passing it through a ceramic membrane to obtain sterilized concentrated milk.

4. The process according to claim 1 or 3, characterized in that, In step (2), the sterilization process uses a ceramic membrane with a pore size of 1.0-1.8 μm, an operating temperature of 30-50℃, and a transmembrane pressure of 0.1-0.3 MPa; the concentration process uses a ceramic membrane with a pore size of 8-12 nm, an operating temperature of 30-50℃, and a transmembrane pressure of 0.1-0.4 MPa; and the total protein concentration in the sterilized concentrated milk is 10%wt-15%wt.

5. The process according to claim 1, characterized in that, In step (3), the pH of the sterilized concentrated milk is adjusted to 6.0-6.5 and then adsorbed and eluted by cation exchange resin; the elution solution used in the elution is a 20-50 mM phosphate buffer solution with pH 7.0-7.5 containing 0.5-1.0M NaCl.

6. The process according to claim 1, characterized in that, In step (4), the ceramic membrane has a pore size of 8-12 nm, an operating temperature of 30-50℃, and a transmembrane pressure of 0.1-0.4 MPa; the total protein concentration of the secondary concentrated milk is above 1%wt.

7. The process according to claim 1, characterized in that, In step (5), the stationary phase of the chromatographic column is a strong cation exchange resin; the strong cation exchange resin uses polystyrene-divinylbenzene or cross-linked agarose as a matrix; when polystyrene-divinylbenzene is used as a matrix, the resin particle size is 25-35 µm, and the H ion exchange capacity is 0.15-0.30 mmol H ion exchange capacity. + / mL; when cross-linked agarose is used as the matrix, the resin particle size is 40-310 µm, and the H+ ion exchange capacity is 0.15-0.30 mmol H+. + / mL.

8. The process according to claim 1, characterized in that, In step (5), the eluent is a phosphate buffer with pH 6.2-6.8 and contains 0.15-0.35 M NaCl; the operating temperature of the simulated moving bed chromatography system is 10-25℃; and the switching time of the simulated moving bed chromatography system is 3-10 minutes.

9. The process according to claim 1, characterized in that, In step (5), the four functional zones are respectively zone I for desorption of strong adsorption components, zone II for key separation, zone III for adsorption of weak adsorption components, and zone IV for resin regeneration; the flow rate ratios of the four functional zones are respectively: zone I for 1.5-2.5; zone II for 0.8-1.5; zone III for 0.5-1.2; and zone IV for 0.2-0.

8.

10. The process according to claim 1, characterized in that, The activity of the lactoperoxidase is above 100 U / mg.

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