Method for preparing high-purity low-protein lactose by integrating expanded bed adsorption and crystallization

Abstract

The invention provides a high-purity low-protein lactose preparation method integrating expanded bed adsorption and crystallization. The invention provides a lactose preparation method which comprises the following steps: separating and purifying lactose from a lactose-containing mixture by using an extended bed adsorption system to obtain a lactose-containing solution, and concentrating and crystallizing to obtain lactose.

Description

Technical Field

[0001] This invention belongs to the field of deep processing and separation engineering technology of dairy products. More specifically, this invention relates to a method for preparing high-purity low-protein lactose by integrating extended bed adsorption and crystallization. Background Technology

[0002] Lactose, a major byproduct of the dairy industry, relies heavily on increasing its purity to extend its value chain. This is especially true in pharmaceutical excipients (particularly dry powder inhalers) and high-end infant formula, where the control of residual whey protein in lactose is extremely stringent. Existing lactose preparation methods are as follows: (1) Thermodynamic method (thermal precipitation method): This method denatures, aggregates, and precipitates major whey proteins such as β-lactoglobulin by heating at a specific pH (e.g., 90-95°C for 20 minutes). Although simple, this method has significant drawbacks: (A) Incomplete removal: It can only remove about 60-75% of the denatured proteins and cannot effectively remove α-lactalbumin and proteolytic fragments, which have better thermal stability. (B) Damage to product quality: High temperatures can easily trigger the Maillard reaction, leading to browning of the lactose solution, producing flavor compounds, and affecting the color and stability of the final product. (C) High energy consumption: Large-scale heating and subsequent cooling processes consume a large amount of energy.

[0003] (2) Physical separation method (membrane filtration): In industry, the combination of ultrafiltration (UF) and nanofiltration (NF) is commonly used. Ultrafiltration is used to retain most proteins, while nanofiltration is used to remove small molecule impurities. This method is currently the mainstream method for lactose preparation, but its limitations are: (A) Membrane fouling: Proteins and mineral salts are prone to deposit on the membrane surface, leading to a rapid decrease in membrane flux, requiring frequent chemical cleaning (CIP), shortening membrane life, and increasing operating costs. (B) Limited separation precision: Although the existing ultrafiltration process can retain large molecule proteins, it is difficult to remove some small molecule peptides and fine colloidal particles. These residual impurities will affect the formation of crystal nuclei or adsorb onto the crystal growth surface during subsequent crystallization, inhibiting the growth rate of lactose crystals and reducing the purity of the final product.

[0004] (3) Fine purification method (compacted bed chromatography): To obtain pharmaceutical-grade lactose, some lactose preparation methods add ion exchange or hydrophobic interaction chromatography after membrane filtration. The working mode is to pump the clarified lactose solution into a chromatographic column tightly packed with tiny resin particles (~50-150 µm). Disadvantages: (A) Stringent requirements for feed: This method requires extremely high clarity of the feed solution. Any particles, fat globules, or denatured protein aggregates that are not completely removed will quickly clog the column head sieve plate and the top of the packing bed, causing the column pressure to spike, the flow rate to drop, and ultimately forcing production to stop. (B) Lengthy and complex process chain: In order to protect the expensive chromatographic column, multiple pretreatment units such as diatomaceous earth filtration, plate and frame filtration, or microfiltration (MF) must be added upstream. This not only significantly increases the investment in equipment (CAPEX) and the floor space, but also makes the process cumbersome and reduces the material yield.

[0005] Therefore, there is an urgent need in this field to provide a new method for preparing lactose. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing high-purity, low-protein lactose by integrating extended bed adsorption and crystallization.

[0007] In a first aspect, the present invention provides a method for preparing lactose, the method comprising: separating and purifying lactose from a mixture containing lactose using an extended bed adsorption system to obtain a lactose-containing solution, and obtaining lactose by concentration and crystallization.

[0008] In one or more embodiments, the extended bed adsorption system includes an adsorbent, wherein the adsorbent comprises: a) A high-density material with a wet density ≥ 2.5 g / mL, wherein the high-density material is selected from one or more of stainless steel, tungsten carbide and zirconium dioxide; b) A hydrophilic porous polymer, wherein the hydrophilic porous polymer is selected from one or more of cross-linked agarose, cellulose, dextran, and polyvinyl alcohol (PVA); and, c) A ligand attached to a hydrophilic porous polymer, wherein the ligand comprises a hydrophobic group and an amine group.

[0009] In one or more embodiments, the ligand: (1) The hydrophobic group includes an aromatic group, a hydrophobic hydrocarbon group, and an amino acid side chain; preferably, the aromatic group includes a polycyclic ring and a heterocyclic ring, more preferably a benzene ring, a nitrogen-containing heterocyclic ring or a sulfur-containing heterocyclic ring, and / or, the hydrophobic hydrocarbon group is a hydrocarbon group with 4 or more carbon atoms, and / or, the amino acid side chain includes isoleucine or a branched alkane of leucine, or a sulfur-containing hydrophobic side chain of methionine; (2) The amino group includes: primary amino group, secondary amino group, tertiary amino group, quaternary ammonium group, preferably, the tertiary amino group includes guanidine group and pyridine tertiary amine.

[0010] In one or more embodiments, the ligand comprises: N-benzoyl-L-arginine, 4-mercaptoethylpyridine, alkylamine, diaminoalkane, phenylalkylamine, 2-amino-1-phenyl-1,3-propanediol, 2-mercapto-1-methylimidazolium, 2-mercaptobenzimidazole, 4-aminomethylpyridine, 2-aminomethylpyridine, tryptamine, histamine, 5-aminoindole, 4-(imidazol-1-yl)aniline, 2-mercaptoimidazolium, 5-aminobenzimidazole, N-(3-carboxypropionyl)aminodecylamine, N-pyromellitic acid-4-mercaptobutyric acid, 2-mercapto-5-sulfonic acid-benzimidazole, 2,5-dimercapto-thiadiazoline, 6-amino-4-yl-methyl-2 ... Hydroxy-2-naphthalenesulfonic acid, aminoalkyl carboxylic acid, tryptophan, p-aminohippuric acid, preferably the alkylamine includes pentanamine, hexylamine, heptanamine, octylamine, dodecylamine, hexadecamine, more preferably the ligand is N-benzoyl-L-arginine, 4-mercaptoethylpyridine or octylamine.

[0011] In one or more embodiments, the extended bed adsorption system includes one, two or more adsorption columns, in which the adsorbent is filled. Preferably, the adsorption column has a bottom distributor and a top outlet adapter. More preferably, the bottom distributor and / or the top outlet adapter both adopt a sieve structure or a filter structure. More preferably, when the lactose-containing mixture is pumped into the adsorption column at a flow rate of 200-600 cm / h, the stable bed height of the extended bed is 1.5-3.2 times that of the original settling bed, more preferably 1.5-3 times.

[0012] In one or more embodiments, the lactose concentration in the lactose-containing mixture is 15-30% (w / v), and preferably the lactose-containing mixture is whey or whey permeate.

[0013] In one or more embodiments, the lactose-containing solution: The protein content is less than or equal to 100 ppm, preferably less than or equal to 65 ppm, and / or, The protein removal rate is greater than or equal to 80%, preferably greater than or equal to 85%, more preferably greater than or equal to 90%, and / or, The lactose recovery rate is greater than or equal to 75%, preferably greater than or equal to 80%, and / or, The turbidity of the solution is less than or equal to 2 NTU, preferably less than or equal to 1.5 NTU.

[0014] A second aspect of the present invention provides lactose prepared using the preparation method described in any embodiment of the present invention.

[0015] A third aspect of the present invention provides an extended bed adsorption system for lactose separation and purification, the extended bed adsorption system comprising the adsorbent described in any embodiment of the present invention or the adsorption column described in any embodiment of the present invention.

[0016] In one or more embodiments, the method for lactose separation and purification is as described in any embodiment of the present invention.

[0017] A fourth aspect of the present invention provides the application of the extended bed adsorption system described herein in the separation and purification of lactose, the preparation of lactose, the improvement of lactose recovery rate, and the removal of proteins from lactose mixtures.

[0018] In one or more embodiments, the removal of proteins from the lactose mixture includes: reducing the protein concentration of the lactose mixture and / or increasing the protein removal rate of the lactose mixture.

[0019] In one or more embodiments, the proteins in the lactose mixture include β-lactoglobulin and α-lactalbumin.

[0020] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Detailed Implementation

[0021] Through in-depth research, the inventors discovered that using extended bed adsorption (EBA) for the separation and purification of lactose can efficiently remove β-lactoglobulin and α-lactalbumin from whey, controlling the protein residue in lactose to less than or equal to 65 ppm. The resulting lactose exhibits no browning, good thermal stability, and is clear and transparent after dissolution (turbidity less than or equal to 1.5 NTU). Furthermore, this method significantly improves lactose production efficiency: EBA purification results in almost no lactose loss, and the deep impurity removal significantly improves the kinetic environment for subsequent crystallization, reducing the impact of impurities on crystal growth. Consequently, within the same crystallization time, both the single-pass yield and crystal purity of lactose are significantly improved, resulting in a lactose recovery rate of over 80% in the finished product. In addition, the lactose preparation process using this EBA system exhibits good stability.

[0022] the term

[0023] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art as would be understood by those skilled in the art. Furthermore, unless otherwise stated, all methods, steps, techniques, and operations not specifically detailed herein can and have been performed in a manner known per se as would be understood by those skilled in the art.

[0024] In this document, the terms “contains,” “includes,” “containing,” and similar terms encompass the meanings of “basically composed of” and “composed of.” For example, when this document discloses “A contains B and C,” “A is basically composed of B and C” and “A is composed of B and C” should be considered as having been disclosed in this document.

[0025] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0026] The terms "above" and "below" both include the stated number.

[0027] Unless otherwise specified, percentages refer to weight percentages (wt%) and proportions refer to weight ratios (w / w).

[0028] In this document, when describing embodiments or examples, it should be understood that it is not intended to limit the invention to those embodiments or examples. Rather, all alternatives, modifications, and equivalents of the methods and materials described herein are covered within the scope defined by the claims.

[0029] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0030] The term "ppm" is an abbreviation for "Parts Per Million". In this document, unless otherwise specified, when describing the concentration of a component (such as protein) in a liquid mixture or solution, ppm refers to a unit of concentration, numerically equivalent to milligrams per liter (mg / L).

[0031] The term "NTU" is an abbreviation for "Nephelometric Turbidity Units," which are units of scattering turbidity used to characterize the turbidity of a solution.

[0032] The term "lactose" refers to a disaccharide composed of one molecule of glucose and one molecule of galactose linked by a β-1,4-glycosidic bond, with the chemical formula […]. Since lactose (which contains no water of crystallization) has a molecular weight of 342.30, it is classified as a small molecule sugar.

[0033] The term "lactose-containing mixture" refers to raw materials used in the production or preparation of lactose, typically a mixture containing lactose. The lactose-containing mixture may include whey or whey permeate. In some embodiments, the lactose concentration in the lactose-containing mixture may be 15-30% (w / v). In some specific embodiments, the lactose-containing mixture is a whey permeate.

[0034] The term "whey" refers to the mixture formed during the production of cheese or casein. It is usually a liquid and its main components include lactose, soluble proteins (such as β-lactoglobulin and α-lactalbumin), minerals (calcium, phosphorus, etc.), and trace amounts of fat.

[0035] The term "whey permeate" refers to a mixture formed during the lactose preparation process from whey, typically containing dry matter, lactose, minerals (or ash), proteins, and small amounts of other whey components. In some embodiments, whey permeate can be prepared from whey by membrane separation (e.g., ultrafiltration, microfiltration, etc.).

[0036] The terms "expanded bed adsorption," "extended bed," and "EBA" are used interchangeably, all referring to a method of adsorbing substances using fluidized particles. The principle of EBA is that special adsorbent particles, under the influence of an upward liquid flow, cause the bed to "fluidize" and expand, creating larger gaps between the particles. This allows solid particles to pass through without clogging, while the target molecules can still effectively bind to the adsorbent.

[0037] The term "adsorbent" refers to a solid-phase medium used in extended bed adsorption methods to specifically capture target molecules (such as proteins like β-lactoglobulin and α-lactalbumin) from complex mixtures (such as mixtures containing lactose).

[0038] Methods for preparing lactose and the lactose obtained

[0039] The present invention provides a method for preparing lactose, the method comprising: separating and purifying lactose from a mixture containing lactose using an extended bed adsorption system to obtain a lactose-containing solution (e.g., a flow-through solution), and obtaining lactose by concentration and crystallization.

[0040] The core advantage of this invention lies in utilizing an extended bed adsorption system to separate and purify lactose from a mixture containing lactose, achieving a one-step clarification and impurity removal process. Solid particles in the mixture flow directly through the interparticle channels with the liquid flow and exit from the top of the column without clogging; while proteins in the mixture that are difficult to remove by conventional methods (especially β-lactoglobulin and α-lactalbumin) diffuse into the adsorbent and are captured. Neutral lactose molecules do not interact with the ligands and flow out with the liquid flow, thus obtaining a high-purity lactose solution.

[0041] The extended bed adsorption system described in this invention comprises an adsorbent, the adsorbent comprising: a) A high-density material with a wet density ≥ 2.5 g / mL, wherein the high-density material is selected from one or more of stainless steel, tungsten carbide and zirconium dioxide; b) A hydrophilic porous polymer (e.g., said hydrophilic porous polymer coated on the outside of a high-density material), wherein said hydrophilic porous polymer is selected from one or more of cross-linked agarose, cellulose, dextran, and polyvinyl alcohol (PVA); and, c) A ligand attached to a hydrophilic porous polymer; wherein the ligand comprises a hydrophobic group and an amine group.

[0042] In this invention, high density generally refers to a wet density of the material ≥ 2.5 g / mL. "Wet density" refers to the density of the adsorbent particles in a moisture-containing state. This invention particularly emphasizes the importance of high density, preferably ≥ 2.6 g / mL, more preferably ≥ 2.8 g / mL or higher. In some embodiments, the wet density of the adsorbent is in the range of 2.5~3.5 g / mL, for example, 2.5 g / mL, 2.6 g / mL, 2.7 g / mL, 2.8 g / mL, 2.9 g / mL, 3.0 g / mL, 3.1 g / mL, 3.2 g / mL, 3.3 g / mL, 3.4 g / mL, 3.5 g / mL, or any combination thereof, more specifically, for example, 2.5~3.2 g / mL, 2.5~3 g / mL, 2.5~3 g / mL, etc. The high-density material can be a rigid material, such as, but not limited to, stainless steel, tungsten carbide, and zirconium dioxide. As the core framework of the adsorbent, it endows the adsorbent with the gravitational potential energy to maintain stable fluidization at high flow rates (≥500 cm / h) without being washed out.

[0043] In the adsorbent, the hydrophilic porous polymer is typically coated on the outside of the framework, and its main function is to provide a large specific surface area and abundant pore structure for protein molecules to enter and bind to ligands. In some embodiments, the average particle size of the adsorbent ranges from 100 to 300 µm, for example, 100 µm, 120 µm, 150 µm, 180 µm, 200 µm, 250 µm, 300 µm, or any combination thereof. In some embodiments, the hydrophilic porous polymer is selected from cross-linked agarose, cellulose, dextran, or polyvinyl alcohol (PVA). In some embodiments, the hydrophilic porous polymer is cross-linked agarose. In some specific embodiments, the weight percentage of the cross-linked agarose is 1 to 10%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or any combination thereof, more specifically, for example, 2 to 10%, 2 to 8%, 3 to 7%, or 4 to 6%.

[0044] In this invention, the ligand is typically a hybrid ligand. The hybrid mode refers to the simultaneous presence of hydrophobic activity (due to the hydrophobic group) and ion adsorption activity (due to the amine group). The ligand can be covalently bonded to the surface of a hydrophilic porous polymer. This "hybrid mode" allows the adsorbent to maintain a high adsorption capacity for proteins even in a high-salt environment without adjusting the conductivity of the feed solution (i.e., without dilution). In this invention, the "hydrophobic group" refers to a group capable of providing hydrophobic activity, such as aromatic groups (e.g., polycyclic rings, heterocycles, more specifically, benzene rings, nitrogen-containing heterocycles, sulfur-containing heterocycles, etc.), hydrophobic hydrocarbon groups (e.g., hydrocarbon groups with 4 or more carbon atoms), amino acid side chains (e.g., isoleucine, branched alkane structures of leucine, sulfur-containing hydrophobic side chains of methionine), etc. In this invention, the "amine group" refers to a nitrogen-containing organic functional group, including but not limited to: primary amine groups (-N...). The groups include secondary amines (-NH-), tertiary amines (e.g., guanidinyl, pyridine tertiary amine), and quaternary ammonium groups. It should be understood that the hydrophobic group and the amine group may be located on the same group (e.g., the pyridine ring) or on different groups. In some embodiments, the ligand may be, but is not limited to: N-benzoyl-L-arginine, 4-mercaptoethylpyridine, alkylamines (e.g., pentylamine, hexylamine, heptylamine, octylamine, dodecylamine, hexadecylamine), diaminoalkane, phenylalkylamine, 2-amino-1-phenyl-1,3-propanediol, 2-mercapto-1-methylimidazole, 2-mercaptobenzimidazole, 4-aminomethylpyridine, 2-aminomethylpyridine, tryptophan, histamine, 5-aminoindole, 4-(imidazol-1-yl)aniline, 2-mercaptoimidazole, 5-aminobenzimidazole, N-(3-carboxypropionyl)aminodecylamine, N-pyromellitic acid-aminodecylamine, 2-benzoylamino-4-mercaptobutyric acid, 2-mercapto-5-sulfonic acid-benzimidazole, 2,5-dimercapto-thiadiazo, 6-amino-4-hydroxy-2-naphthalenesulfonic acid, aminoalkylcarboxylic acid, tryptophan, and p-aminohippuric acid. In some specific embodiments, the ligand is selected from N-benzoyl-L-arginine, 4-mercaptoethylpyridine, or octylamine. In some embodiments, the density of the ligand is 50-200 µmol / mL. In some embodiments, the average particle size of the adsorbent is 100-300 µm, preferably 120-250 µm.

[0045] In some embodiments, the adsorbent comprises a) a high-density material with a wet density ≥ 2.5 g / mL, wherein the high-density material is selected from one or more of stainless steel, tungsten carbide, and zirconium dioxide (preferably the high-density material accounts for 60-90% of the total mass of the adsorbent, more preferably tungsten carbide, stainless steel, or zirconium dioxide with a density ≥ 2.5 g / mL); b) a hydrophilic porous polymer (e.g., the hydrophilic porous polymer coated on the high-density material), wherein the hydrophilic porous polymer is selected from one or more of cross-linked agarose, cellulose, dextran, and polyvinyl alcohol (PVA) (preferably cross-linked agarose, more preferably 4-6% cross-linked agarose); and c) a ligand composition attached to the hydrophilic porous polymer, wherein the ligand is selected from N-benzoyl-L-arginine, 4-mercaptoethylpyridine, or octylamine.

[0046] The adsorbent described in this invention can be prepared using conventional reverse-phase suspension thermal coagulation or emulsion-crosslinking methods. For example, a high-density material (such as tungsten carbide, stainless steel, or zirconium dioxide) can be dispersed in an aqueous solution containing a hydrophilic porous polymer (such as crosslinked agarose); this mixture is then poured into an oil phase containing an emulsifier and stirred to emulsify, forming droplets with a heavy core; the polymer is then gelled (cured) by cooling; after crosslinking and reinforcement, ligands are coupled to the surface of microspheres through a chemical reaction. In some embodiments, the adsorbent obtained after preparation is an adsorbent microsphere.

[0047] In some embodiments, the extended bed adsorption system includes an adsorption column in which the adsorbent is filled. It should be understood that the adsorbent (e.g., adsorbent microspheres) can be filled into the adsorption column of the extended bed adsorption system either all at once or in batches, typically using a wet packing method. After filling and settling, the adsorbent forms a dense bed at the bottom of the column, known as the initial settling bed. When operation begins, the feed solution is pumped from bottom to top. Under the balance of the drag force of the liquid flow and the gravity of the particles, the adsorbent particles separate from each other and become suspended, forming a stable extended bed (fluidized bed).

[0048] In some embodiments, when the high-density adsorbent described in this invention is used and the lactose-containing mixture is pumped in at a flow rate of 200-600 cm / h, the stable bed height of the extended bed is 1.5-3.0 times the height of the original settling bed. In some specific embodiments, when the high-density adsorbent described in this invention is used and the lactose-containing mixture is pumped in at a flow rate of 200-600 cm / h, the stable bed height of the extended bed is 1.5-3.2 times (preferably 1.5-3 times) the height of the original settling bed.

[0049] In some embodiments, the lactose-containing mixture may be a whey permeate. In some embodiments, the lactose concentration in the lactose-containing mixture may be 15-30% (w / v), preferably 15-25% (w / v). Excessively high concentrations of lactose (e.g., above 50% (w / v)) may lead to a significant increase in the viscosity of the feed solution, resulting in unstable bed expansion, easy channeling, hindered protein diffusion and mass transfer into the adsorbent, and decreased adsorption efficiency.

[0050] In some embodiments, the lactose-containing solution contains: The protein content is less than or equal to 100 ppm, preferably less than or equal to 90 ppm, 80 ppm, 70 ppm or 65 ppm, and / or, The protein removal rate is greater than or equal to 80%, preferably greater than or equal to 82%, 84%, 85%, 86% or 88%, more preferably greater than or equal to 90%, 91%, 92%, 93%, 94% or 95%, and / or, The lactose recovery rate is greater than or equal to 75%, preferably greater than or equal to 76%, 77%, 78%, 79%, or 80%, and / or, The turbidity of the solution is less than or equal to 2 NTU, preferably less than or equal to 1.9 NTU, 1.8 NTU, 1.7 NTU, 1.6 NTU or 1.5 NTU.

[0051] In some embodiments, the concentration causes the lactose-containing solution to become supersaturated. The solids content in the concentrated solution is typically controlled to be 55-65 wt% (e.g., 55 wt%, 60 wt%, 62 wt%, 65 wt%, or any combination thereof).

[0052] The concentration is carried out using the multi-effect vacuum evaporation method or MVR evaporation method known in the art, and the concentration temperature can be 60-75°C to avoid lactose caramelization.

[0053] In some implementations, the crystallization can employ a commonly used cooling crystallization method. This cooling crystallization method can be gradient cooling crystallization, for example, the first stage temperature is lowered to 45-50°C, with a residence time of 20-40 min, utilizing the rapid rotation rate at high temperature to promote nucleus stability and initial growth; the second stage temperature is lowered to 30-35°C, with a residence time of 20-40 min; and the third stage temperature is lowered to 10-15°C, with a residence time of 20-40 min, maximizing the reduction of lactose solubility through deep cooling. The total residence time for the entire crystallization process can be 90-120 min. This cooling crystallization method can be carried out using a three-stage tandem dynamic axial compression column (DBC) crystallizer.

[0054] In some embodiments, the method further includes the steps of separating and / or drying the crystallized lactose. The separation can be performed using methods known in the art, such as, but not limited to, centrifugation (e.g., horizontal spiral centrifugation) and vacuum filtration. The drying can be performed using methods known in the art, such as, but not limited to, fluidized bed drying. Typically, the dried lactose has a moisture content of 5.5 wt%, 5.0 wt%, 4.0 wt%, 3.0 wt%, 2.0 wt%, 1.0 wt%, or lower.

[0055] The preparation method of this invention for lactose preparation has higher efficiency: the extended bed adsorption system separates and purifies lactose from the lactose-containing mixture, effectively removing impurity proteins with almost no impact on the lactose content. The solution purified by the extended bed adsorption system exhibits a significantly improved crystallization yield, resulting in a lactose recovery rate of over 80.0% (e.g., 80.0%, 82.0%, 85.0%, or higher) in the finished lactose product.

[0056] The present invention also provides lactose prepared using the preparation method described in any embodiment of the present invention.

[0057] application

[0058] This invention provides the application of the extended bed adsorption system described in any embodiment of the invention in the separation and purification of lactose, the preparation of lactose, the improvement of lactose recovery rate, and the removal of proteins (especially β-lactoglobulin and α-lactalbumin) from lactose mixtures.

[0059] In some embodiments, the removal of proteins from the lactose mixture includes: reducing the protein concentration of the lactose mixture and / or increasing the protein removal rate of the lactose mixture.

[0060] The advantages of this invention include: (1) This invention discovers an extended bed (EBA) adsorbent suitable for the separation and preparation of lactose. This adsorbent can efficiently remove β-lactoglobulin and α-lactalbumin, and stably control the residual protein content in the finished lactose product to less than or equal to 65 ppm. This indicator is far superior to the current pharmacopoeia standards: the European Pharmacopoeia (Ph. Eur.) requires the absorbance (400 nm) of the lactose solution to be <0.04, and the protein limit test must meet the regulations; the United States Pharmacopeia (USP) has strict limits on "protein and light-absorbing impurities". Existing commercially available pharmaceutical lactose usually has a protein residue of 100-300 ppm, while the lactose prepared using the adsorbent of this invention has significantly improved purity, and has no browning, excellent thermal stability, and is clear and transparent after dissolution (turbidity ≤1.5 NTU).

[0061] (2) This invention provides an extended bed adsorption (EBA) system for preparing lactose. The resulting lactose exhibits no browning, good thermal stability, and is clear and transparent after dissolution. This method not only solves the "clogging" problem but also improves crystallization kinetics through deep impurity removal. Compared to traditional processes, this method eliminates trace proteins that inhibit crystal growth, increasing the single-pass lactose recovery rate by more than 10%, and achieving a final product lactose recovery rate of over 80%. Furthermore, the EBA system is insensitive to fluctuations in the turbidity of upstream feed (tolerating >50 NTU), eliminating the risk of downtime due to fluctuations in feed water quality and ensuring high continuity and stability in production.

[0062] (3) The extended bed adsorption system of the present invention simplifies the previous multiple filtration systems, eliminating the need for filtration consumables such as diatomaceous earth and filter cloth. Furthermore, the system can operate under low pressure and saves energy consumption from multiple material transfers and pumping, significantly reducing equipment investment costs, workshop floor space, and operating costs. In addition, the protein solution eluted using this extended bed adsorption system has a high concentration and can be used for feed protein recovery.

[0063] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0064] Experimental methods

[0065] 1. Preparation method of lactose

[0066] (1) Raw material pretreatment: The cheese whey is subjected to ultrafiltration (UF) treatment (molecular weight cutoff 10kDa) to remove most of the protein and fat, and increase the lactose concentration to 15-25% (w / v). The liquid at this time is called "whey permeate", which still contains 0.1-0.5% residual protein and a small amount of particulate matter, and appears slightly turbid (NTU: 5-50). Its pH is adjusted to 4.0-6.5. The ultrafiltration treatment was performed according to the method described in the reference "Concentration and purification of whey proteins by ultrafiltration" (Baldasso C, Barros TC, Tessaro IC. Concentration and purification of whey proteins by ultrafiltration. Desalination. 2011;278(1-3):381-386. doi:10.1016 / j.desal.2011.05.055) (specific parameters: molecular weight cutoff 10 kDa, transmembrane pressure 0.2-0.4 MPa, temperature 25-50℃).

[0067] (2) EBA processing

[0068] Equipment: The column is a high-density EBA-specific chromatography column (2.0 cm inner diameter, purchased from Cytiva) with both bottom distributor and top outlet adapter having a screen structure and equipped with a screen distributor.

[0069] Sample loading: The whey permeate obtained in (1) was pumped from the bottom of the EBA column at a linear flow rate of 200-600 cm / h.

[0070] Bed expansion: At this flow rate, high-density adsorbent particles are pushed upwards but not blown out of the column, forming a stable "fluidized bed" with a height expanded to 1.5-3.0 times the original settling bed height. Macroscopic flow channels are generated between the particles.

[0071] Separation mechanism: Particle passage: Solid particles in the feed liquid pass directly through the channels between the particles with the liquid flow and flow out from the top of the column without causing blockage.

[0072] Protein capture: Dissolved protein molecules enter the porous structure of the adsorbent through convection and diffusion, and are captured by binding with mixed-mode ligands with high affinity.

[0073] Lactose penetration: Neutral lactose molecules do not interact with ligands and flow smoothly out from the top of the column with the liquid flow.

[0074] Online monitoring: A UV-280nm detector is installed at the column top outlet to monitor the protein concentration in the effluent in real time. When the UV signal begins to rise, it indicates that the adsorbent is about to become saturated.

[0075] Adsorbent regeneration: After sample loading, perform the following operations in sequence: Elution: Elute the adsorbed proteins with 5 column volumes (CV) of 1.0 M NaCl + 20 mM phosphate buffer (pH 7.0); CIP cleaning: Cleaning and regeneration were performed using 3 column volumes (CV) of 0.5 M NaOH; Equilibration: Finally, reequilibrate with process buffer (20 mM citrate-sodium citrate buffer, pH 4.8) until the pH and conductivity of the effluent are stable, then it can be used for the next adsorption.

[0076] A dual-column EBA system is used in alternating operation: while one column is adsorbing, the other is regenerating (elution time 20-60 min). When column A is in the sample loading and adsorption phase, column B is sequentially eluting, regenerating, and rebalancing. When the online UV detector of column A shows protein breakthrough and the adsorbent is about to become saturated, the system automatically switches valves to introduce the feed solution into the ready column B, while simultaneously initiating regeneration of column A. This cycle repeats continuously, enabling 24-hour uninterrupted processing of upstream feed.

[0077] (3) Lactose Preparation: The liquid flowing out of the EBA is a highly clear pure lactose solution with extremely low protein content. This solution is concentrated to 55-65% solids content by an MVR evaporator and then enters a three-stage series dynamic axial compression column (DBC) crystallizer. A staged cooling strategy is adopted. The first stage temperature is reduced to 45-50℃, and the residence time is 20-40 min. The second stage temperature is reduced to 30-35℃, and the residence time is 20-40 min. The third stage temperature is reduced to 10-15℃, and the residence time is 20-40 min. Through deep cooling, the solubility of lactose is reduced to the greatest extent. The total residence time of the entire crystallization process is controlled at 90-120 min. Finally, the crystal slurry in the crystallizer is separated into solid and liquid by vacuum filtration. The separated wet crystals are sent to a fluidized bed for drying to obtain the finished lactose product.

[0078] 2. Preparation method of adsorbent

[0079] The reverse-phase suspension thermal coagulation method was used to disperse the framework material in an agarose aqueous solution, emulsify it into spheres, solidify and crosslink it, and obtain the adsorbent by coupling a mixed-mode ligand. The method was followed as described in (CN105713212B A method for preparing agarose crosslinked gel microspheres; Zhou, Q.-Z., Wang, L.-Y., Ma, G.-H., & Su, Z. (2007). Preparation of uniform-sized agarose beads by microporous membrane emulsification technique. Journal of Colloid and Interface Science, 311, 118–127.).

[0080] 3. Detection Method

[0081] (1) Methods for detecting protein concentration

[0082] The Bradford Coomassie Brilliant Blue method was used. Principle: When Coomassie Brilliant Blue G-250 dye binds to protein, the solution color changes from reddish-brown to blue, and the maximum absorption peak shifts from 465 nm to 595 nm. The absorbance is directly proportional to the protein concentration.

[0083] Procedure: Take 1 mL of the sample solution to be tested (pre-diluted according to concentration) and place it in a test tube. Add 5 mL of Bradford reagent (Coomassie Brilliant Blue G-250 solution) and vortex to mix. Incubate at room temperature for 5 minutes. Measure the absorbance at 595 nm using a UV-Vis spectrophotometer. Construct a standard curve using bovine serum albumin (BSA) as a standard and calculate the protein concentration in the sample.

[0084] (2) Methods for detecting protein removal rate

[0085] The formula is as follows, calculated based on the changes in protein concentration in the raw material solution and the flow-through solution: Protein removal rate (%) = [1 - (C)] out ×V out ) / (C in ×V in )]×100% C out : Protein concentration (mg / L) in the collected EBA flow-through solution; V out : The volume of liquid collected flowing through the vessel (L); C in Protein concentration (mg / L) of the sample loading solution (whey permeate); V in : Sample loading volume (L).

[0086] (3) Method for detecting moisture in lactose products

[0087] The Karl Fischer method was used to determine the moisture content of lactose.

[0088] The moisture content was determined using an automated Karl Fischer titrator equipped with a heated stirring table, with methanol-formamide (2:1) as the solvent. The sample mass was input, and the titration program was started. The instrument automatically titrated to the endpoint (potential jump point) and calculated the moisture content based on the volume of titrant consumed.

[0089] (4) Methods for detecting lactose concentration and purity

[0090] Quantitative analysis was performed using high performance liquid chromatography (HPLC) combined with a differential refractive index detector (RI).

[0091] Chromatographic column: ValueLab GP-NH2 / CN (250 mm × 4.6 mm) or similar product.

[0092] Detector: Differential refractive index detector (RI).

[0093] Mobile phase: acetonitrile:water = 70:30 (V / V), flow rate 1.0 mL / min, column temperature 30℃, detector temperature 30℃, injection volume 20 µL.

[0094] Quantitative method: Using the external standard method, a series of lactose standard solutions of different concentrations (e.g., 1.0–20.0 mg / mL) were prepared, and a standard curve was plotted (the lactose concentration was calculated from the peak area using HPLC, R0). 2 > 0.999).

[0095] (5) Method for detecting lactose recovery rate

[0096] The lactose recovery rate from raw materials to finished product was calculated using a combination of gravimetric method and HPLC purity correction.

[0097] Calculation formula: Lactose recovery rate (%) = [ M product × P product ×(1- W water )] / (C material ×V material )]×100% M product : The total mass (g) of the final dried lactose product obtained; P product The lactose purity (%) of the finished lactose was determined by HPLC. W water The moisture content (%) of the finished lactose was determined by the Karl Fischer method. C material The lactose concentration (g / L) in the sample feed solution (whey permeate) was determined by HPLC. V material : Total volume of the sample feed solution (L).

[0098] (6) Method for detecting the turbidity of the effluent

[0099] The determination was performed using a nephelometric turbidity meter (such as Hach 2100Q). Approximately 15 mL of the mixed eluent sample was placed in a sample vial, the vial walls were wiped clean, and the instrument was inserted for reading. Results are expressed as NTUs (Nephelometric Turbidity Units).

[0100] Example: Preparation and Screening of Adsorbents

[0101] Example 1: Formulation 1 of the EBA system adsorbent (standard high-density type)

[0102] This embodiment provides a composite adsorbent suitable for high-flow-rate extended bed adsorption (EBA) systems.

[0103] Framework: Tungsten carbide powder with an average particle size of 30 µm.

[0104] Matrix: 6% cross-linked agarose, coated on the outer layer of the backbone.

[0105] Agligand: N-benzoyl-L-arginine.

[0106] Particle size range: 100-300 µm (average particle size 180 µm).

[0107] Wet density: 2.8 g / mL.

[0108] Example 2: EBA System Adsorbent Formulation 2 (Lowest Density Type)

[0109] This embodiment provides an adsorbent with the lowest density parameters that meets the requirements for high flow rate operation.

[0110] Skeleton: 316 stainless steel powder.

[0111] Matrix: 6% cross-linked agarose.

[0112] Ligand: 4-Mercaptoethylpyridine (MEP).

[0113] Particle size range: 100-250 µm.

[0114] Wet density: 2.5 g / mL. This density is the critical lower limit for maintaining a flow rate of 500 cm / h without gel runoff.

[0115] Example 3: EBA System Adsorbent Formulation 3 (Ultra-High Density Type)

[0116] This embodiment provides an ultra-high density adsorbent suitable for high-viscosity liquids.

[0117] Framework: Tungsten carbide.

[0118] Matrix: cellulose.

[0119] Ligand: Octylamine.

[0120] Particle size range: 100-300 µm.

[0121] Wet density: 3.5 g / mL.

[0122] Large particle size and ultra-high density give the adsorbent extremely strong settling properties, making it suitable for processing concentrated whey with high viscosity.

[0123] Example 4: EBA System Adsorbent Formulation 4 (Low-Density Type)

[0124] This embodiment provides a conventional EBA adsorbent with insufficient density to verify the necessity of high density.

[0125] Skeleton: Composite skeleton containing quartz sand, with a quartz sand content of 25% (w / w).

[0126] Matrix: 4% cross-linked agarose.

[0127] Agligand: N-benzoyl-L-arginine.

[0128] Particle size range: 100-300 µm.

[0129] Wet density: 1.2 g / mL. This density is sufficient to maintain an expanded bed when processing low-viscosity buffer solutions, but a stable fluidized bed is not expected to form at high flow rates.

[0130] Example 5: EBA System Adsorbent Formulation 5 (Different Framework Types)

[0131] This embodiment provides an EBA adsorbent that uses zirconium dioxide microspheres as the framework material.

[0132] Framework: Zirconia microspheres.

[0133] Matrix: Polyvinyl alcohol (PVA).

[0134] Agligand: N-benzoyl-L-arginine.

[0135] Particle size range: 100-300 µm.

[0136] Wet density: 2.6 g / mL.

[0137] Example 6: EBA System Adsorbent Formulation 6 (Different Ligand Types)

[0138] This embodiment provides an EBA adsorbent using a non-mixed mode ligand.

[0139] Framework: Tungsten carbide.

[0140] Matrix: 6% cross-linked agarose.

[0141] Ligand: Trimethylammonium (non-mixed mode).

[0142] Particle size range: 100-300 µm.

[0143] Wet density: 2.8 g / mL.

[0144] This was used to verify the advantages of hybrid ligands in resisting salt interference.

[0145] Preparation Example 1: Lactose separation and purification using a medium-flow-rate EBA system and the adsorbent of Example 1

[0146] Raw materials: whey permeate (lactose 18% (w / v), protein concentration 3500 ppm, pH 5.8, turbidity 45 NTU).

[0147] EBA system: Column diameter 2.0 cm, settling bed height 20 cm, filled with the adsorbent of Example 1. The adsorbent framework is tungsten carbide powder with an average particle size of 30 µm. The matrix is ​​6% cross-linked agarose, coating the outer layer of the framework. The ligand is N-benzoyl-L-arginine. The particle size range is 100-300 µm (average particle size 180 µm). The wet density is 2.8 g / mL.

[0148] EBA system operating parameters: Sample loading flow rate 300 cm / h.

[0149] Results: The bed thickness within the column stably expanded to 40 cm (expansion factor 2.0). The system back pressure remained below 0.2 bar, with no clogging. After loading five bed volumes consecutively, the effluent UV detection signal showed no significant increase. Analysis of the collected effluent revealed a protein concentration reduced to 35 ppm, a protein removal rate of 99.0%, a lactose recovery rate of 82.5%, and a clear, transparent effluent with a turbidity of 0.9 NTU. These results demonstrate that the medium-flow-rate EBA system exhibits excellent performance and stable operation in the separation and purification of lactose.

[0150] Preparation Example 2: Lactose separation and purification using a low-flow-rate EBA system and the adsorbent of Example 1

[0151] Raw materials: whey permeate (lactose 18% (w / v), protein concentration 3500 ppm, pH 5.8, turbidity 45 NTU).

[0152] EBA system: Column diameter 2.0 cm, settling bed height 20 cm, filled with the adsorbent of Example 1. The adsorbent framework is tungsten carbide powder with an average particle size of 30 µm. The matrix is ​​6% cross-linked agarose, coating the outer layer of the framework. The ligand is N-benzoyl-L-arginine. The particle size range is 100-300 µm (average particle size 180 µm). The wet density is 2.8 g / mL.

[0153] EBA system operating parameters: The sample loading flow rate is reduced to 200 cm / h.

[0154] Results: The bed expanded stably to 30 cm (expansion factor 1.5). Due to the longer residence time, the proteins bound to the adsorbent more thoroughly. The protein concentration in the collected effluent was as low as 25 ppm, with a protein removal rate of 99.3%. However, the throughput was relatively low, the flow rate was slow, the output per unit time was low, and the relative dead volume loss was slightly high. The overall lactose recovery rate was 80.2%, and the effluent turbidity was 0.6 NTU.

[0155] Preparation Example 3: Lactose separation and purification using a high-flow-rate EBA system and the adsorbent of Example 1

[0156] Raw materials: whey permeate (lactose 18% (w / v), protein concentration 3500 ppm, pH 5.8, turbidity 45 NTU).

[0157] EBA system: Column diameter 2.0 cm, settling bed height 20 cm, filled with the adsorbent of Example 1. The adsorbent framework is tungsten carbide powder with an average particle size of 30 µm. The matrix is ​​6% cross-linked agarose, coating the outer layer of the framework. The ligand is N-benzoyl-L-arginine. The particle size range is 100-300 µm (average particle size 180 µm). The wet density is 2.8 g / mL.

[0158] EBA system operating parameters: Increase the sample loading flow rate to 500 cm / h.

[0159] Results: The bed stably expanded to 55 cm (expansion factor 2.75). The protein concentration in the effluent was 48 ppm, with a protein removal rate of 98.6%, a lactose recovery rate of 84.6%, and an effluent turbidity of 1.1 NTU. While protein residue was slightly increased, high-throughput continuous production reduced losses from intermittent operations, thus improving production efficiency. These results indicate that using a high-flow-rate EBA system for lactose separation and purification represents the optimal balance between reducing protein residue and maximizing production efficiency in lactose preparation.

[0160] Based on the results of preparation examples 1-3, a high-flow-rate EBA system was subsequently used to separate and purify lactose.

[0161] Preparation Example 4: Lactose separation and purification using a high-flow-rate EBA system and the adsorbent of Example 2

[0162] Raw materials: whey permeate (lactose 18% (w / v), protein concentration 3500 ppm, pH 5.8, turbidity 45 NTU).

[0163] EBA system: column diameter 2.0 cm, settling bed height 20 cm, filled with the adsorbent described in Example 2 (skeleton of 316 stainless steel, ligand of 4-mercaptoethylpyridine (MEP), density 2.5 g / mL).

[0164] Operating parameters: Sample loading flow rate 500 cm / h.

[0165] Results: The bed expanded stably to 62 cm (expansion factor 3.1). Due to the adsorbent density of 2.5 g / mL being slightly lower than 2.8 g / mL, the bed expansion rate was relatively large at the same high flow rate, approaching the column top adapter, requiring very careful operation.

[0166] The protein concentration in the flow-through was 55 ppm, the protein removal rate was 98.4%, the lactose recovery rate was 83.5%, and the turbidity of the effluent was 1.3 NTU. Although the stainless steel frame can also achieve EBA operation, due to its slightly lower density and excessive bed porosity, a small number of tiny impurity particles and proteins were allowed to penetrate, resulting in product indicators that were slightly inferior to those in Example 1, but still superior to the traditional process.

[0167] Preparation Example 5: Lactose separation and purification using a high-flow-rate EBA system and the adsorbent of Example 3.

[0168] Raw materials: whey permeate (lactose 18% (w / v), protein concentration 3500 ppm, pH 5.8, turbidity 45 NTU).

[0169] EBA system: column diameter 2.0 cm, settling bed height 20 cm, filled with the adsorbent described in Example 3 (skeleton is tungsten carbide, ligand is octylamine, particle size 100-300 µm).

[0170] Operating parameters: Sample loading flow rate 600 cm / h.

[0171] Results: The bed stably expanded to 44 cm (expansion factor 2.2). The large-diameter adsorbent particles settled rapidly, resulting in a very compact fluidized bed. The protein concentration in the flow-through was 65 ppm, with a protein removal rate of 98.1%, a lactose recovery rate of 84.3%, and an effluent turbidity of 1.5 NTU. Although the hydrodynamic performance was excellent (low pressure drop, no gel leakage), the larger adsorbent particle size lengthened the mass transfer path for protein diffusion into the microspheres. At an ultra-high flow rate of 600 cm / h, the dynamic adsorption loading decreased, resulting in a protein residue of 65 ppm, still significantly lower than the 100-300 ppm of traditional processes.

[0172] Preparation Example 6: Lactose separation and purification using a high-flow-rate EBA system and the adsorbent of Example 4

[0173] Raw materials: whey permeate (lactose 18% (w / v), protein concentration 3500 ppm, pH 5.8, turbidity 45 NTU).

[0174] EBA system: column diameter 2.0 cm, settling bed height 20 cm, filled with the adsorbent described in Example 4 (low-density agarose microspheres, density 1.2 g / mL).

[0175] Operating parameters: Try loading flow rate of 300 cm / h.

[0176] Result: The experiment failed. Less than 2 minutes after the feed pump was turned on, the bed rapidly expanded beyond the top adapter, and a large amount of adsorbent clogged the upper screen and leaked out with the flow-through liquid (gel leakage phenomenon), making it impossible to establish a stable fluidized bed. This fully demonstrates that a high-density skeleton (≥2.5 g / mL) is a key and necessary condition for the implementation of this invention at high flow rates.

[0177] Preparation Example 7: Lactose separation and purification using a high-flow-rate EBA system and the adsorbent of Example 5

[0178] Raw materials: whey permeate (lactose 18% (w / v), protein concentration 3500 ppm, pH 5.8, turbidity 45 NTU).

[0179] EBA system: column diameter 2.0 cm, settling bed height 20 cm, filled with the adsorbent described in Example 5 (skeletonium dioxide, density 2.6 g / mL).

[0180] Operating parameters: Sample loading flow rate 500 cm / h.

[0181] Results: The bed expanded stably to 58 cm (expansion factor 2.9), and the operation was stable. The protein concentration in the flow-through was 52 ppm, the protein removal rate was 98.5%, the lactose recovery rate was 83.1%, and the effluent turbidity was 1.2 NTU. The results were between those of Example 1 (tungsten skeleton) and Example 2 (stainless steel skeleton), indicating that as long as the skeleton density meets the requirements, high-density skeletons of different materials can achieve the separation and purification of lactose.

[0182] Preparation Example 8: Lactose separation and purification using a high-flow-rate EBA system and the adsorbent of Example 6

[0183] Raw materials: whey permeate (lactose 18% (w / v), protein concentration 3500 ppm, pH 5.8, turbidity 45 NTU).

[0184] EBA system: column diameter 2.0 cm, settling bed height 20 cm, filled with the adsorbent described in Example 6 (tungsten carbide framework, trimethylammonium ligand).

[0185] Operating parameters: Sample loading flow rate 500 cm / h.

[0186] Results: The hydrodynamic performance was consistent with that of Preparation Example 3 (expansion factor 2.75), but the impurity removal effect was extremely poor. The protein concentration in the flow-through was as high as 850 ppm, with a protein removal rate of only 75.7%, a lactose recovery rate of 72.0%, and an effluent turbidity of 5.0 NTU (milky white). Because the whey feedstock was not excessively diluted, its conductivity was high, and the adsorption capacity of ordinary ion exchange groups was strongly inhibited by competition from salt ions. This indicates that without using a mixed-mode ligand, it is difficult to achieve efficient impurity removal without diluting the feed solution.

[0187] Comparative Example 1: Using conventional compacted bed chromatography

[0188] System: 2.0 cm column diameter, 20 cm settling bed height, using conventional compacted bed strong anion exchange resin with Q (quaternary ammonium salt) as the ligand (trade name Q Sepharose Fast Flow, average particle size 90 μm, purchased from Cytiva).

[0189] Procedure: Pump the above-mentioned unfiltered raw material solution at a conventional flow rate of 150 cm / h.

[0190] Results: Less than 10 minutes after sample loading began, the column inlet pressure rapidly increased from 0.5 bar to over 3.0 bar, exceeding the system's upper limit, and the pump automatically shut down. Upon opening the column, it was found that the column head sieve plate and the packing surface were completely blocked by a layer of grayish-white precipitate. The experiment failed, proving that the compacted bed method cannot directly process this type of raw material.

[0191] Comparative Example 2: Using the traditional thermal precipitation method

[0192] Procedure: Heat the raw material liquid to 90-92°C with stirring, keep it at this temperature for 20 minutes, then cool and centrifuge to remove the precipitate.

[0193] Results: The protein concentration in the supernatant was still 1200 ppm, with a protein removal rate of only 65.7% and a lactose recovery rate of 60.6%. During centrifugation, a large amount of lactose mother liquor was carried in the precipitate, and the solution after centrifugation showed a distinct pale yellow color, indicating that the Maillard reaction had occurred. The product quality did not meet the standards.

[0194] Comparative Example 3: Membrane filtration method

[0195] Operation: A 10 kDa spiral wound ultrafiltration membrane module (polyethersulfone material) is used to perform cross-flow filtration of the feed liquid. The operating pressure is 3.0 bar and the concentration factor is 5 times.

[0196] Results: Although the 10 kDa ultrafiltration membrane retained most of the large protein molecules, a significant amount of small protein impurities remained in the permeate, resulting in a protein removal rate of 77.1%. After only one hour of operation, the membrane flux decreased from an initial 45 LMH to 25 LMH, a drop of 44%, indicating severe membrane pore blockage and concentration polarization. Due to the severe concentration polarization, lactose was significantly lost with the retentate, and subsequent crystallization was inhibited by residual impurities, resulting in a low single-pass yield; the lactose recovery rate was only 65.5%.

[0197] Comparative Example 4: EBA system using existing adsorbents

[0198] EBA system: column diameter 2.0 cm, settling bed height 20 cm, filled with commercially available low-density EBA adsorbent Streamline SP (purchased from Cytiva).

[0199] Operating parameters: Different flow rates were tested, and it was found that the adsorbent could only maintain stable fluidization at low flow rates (< 150 cm / h). Once the flow rate was increased to 200 cm / h, the bed expansion factor exceeded 3.5 times, and the adsorbent began to overflow from the top.

[0200] Results: Protein residue was 300 ppm, with a protein removal rate of 91.4%. Due to the lack of hydrophobic interactions, the adsorbent had a low loading capacity in high-salt whey, leading to premature protein penetration. Lactose recovery was 70.4%. The low flow rate and long production cycle made it easy for bacteria to grow and consume lactose. Frequent CIP cleaning was required to prevent contamination, resulting in increased physical losses.

[0201] In summary, this invention discloses a method for preparing high-purity, low-protein lactose by integrating extended bed adsorption and crystallization. The method includes: (1) taking whey or whey permeate and adjusting the pH to 4.0-6.5 as a pretreatment solution; (2) pumping the solution from bottom to top into an extended bed adsorption column packed with a high-density mixed-mode adsorbent, allowing the adsorbent bed to expand stably at a high flow rate of 200-600 cm / h, specifically adsorbing impurities (proteins) in the solution, while lactose flows out with the permeate; (3) collecting the permeate and concentrating, crystallizing, separating the solid and liquid phases, and drying to obtain the final product. The adsorbent has a wet density greater than or equal to 2.5 g / mL and a hydrophobic-charge-induced mixed-mode ligand, enabling it to directly treat high-turbidity solutions without clogging. This invention effectively solves the problems of long process flow, serious membrane fouling and low product purity in traditional processes. It can control the residual protein content in the finished lactose product to less than or equal to 65 ppm, the turbidity to less than or equal to 1.5 NTU, and the total lactose recovery rate to greater than or equal to 80%, thus achieving the efficient preparation of high-purity lactose.

[0202] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively 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 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. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. A method of making lactose, the method comprising: The lactose is separated and purified from a lactose-containing mixture by using an expanded bed adsorption system to obtain a lactose-containing solution, and the lactose is obtained by concentrating and crystallizing the lactose-containing solution.

2. The production method according to claim 1, wherein The expanded bed adsorption system comprises an adsorbent, wherein the adsorbent comprises: a) high density material with a wet density ≥ 2.5 g / mL, the high density material is selected from one or more of stainless steel, tungsten carbide and zirconium dioxide; b) hydrophilic porous polymer, wherein the hydrophilic porous polymer is selected from one or more of cross-linked agarose, cellulose, dextran and polyvinyl alcohol; and c) ligand connected to the hydrophilic porous polymer, wherein the ligand comprises a hydrophobic group and an amine group.

3. The production method according to claim 2, wherein The hydrophobic group in the ligand comprises an aromatic group, a hydrophobic hydrocarbon group, an amino acid side chain.

4. The production method according to claim 3, wherein The aromatic group comprises a polybasic ring and a heterocyclic ring.

5. The production method according to claim 4, wherein The aromatic group is a benzene ring, a nitrogen-containing heterocyclic ring or a sulfur-containing heterocyclic ring.

6. The production method according to claim 3, wherein The hydrophobic hydrocarbon group is a hydrocarbon group with more than 4 carbon atoms.

7. The production method according to claim 3, wherein The amino acid side chain comprises a branched alkane of isoleucine or leucine, or a sulfur-containing hydrophobic side chain of methionine.

8. The production method according to claim 2, wherein The amine group in the ligand comprises a primary amine group, a secondary amine group, a tertiary amine group, a quaternary ammonium group.

9. The production method according to claim 8, wherein The tertiary amine group comprises guanidino group, pyridine tertiary amine.

10. The production method according to claim 2, wherein The ligand comprises N-benzoyl-L-arginine, 4-mercaptoethylpyridine, alkylamine, diaminoalkane, phenylalkylamine, 2-amino-1-phenyl-1,3-propanediol, 2-mercapto-1-methylimidazole, 2-mercapto-benzimidazole, 4-aminomethylpyridine, 2-aminomethylpyridine, tryptamine, histamine, 5-aminoindole, 4-(imidazol-1-yl)aniline, 2-mercaptoimidazole, 5-amino-benzimidazole, N-(3-carboxypropionyl)aminodecane, N-mellitic acid aminodecane, 2-benzamido-4-mercaptobutyric acid, 2-mercapto-5-sulfonic acid benzimidazole, 2,5-dimercapto-thio-diazene, 6-amino-4-hydroxy-2-naphthalenesulfonic acid, aminoalkyl carboxylic acid, tryptophan, p-aminomandelic acid.

11. The production method according to claim 10, wherein The alkylamine comprises pentylamine, hexylamine, heptylamine, octylamine, dodecylamine, hexadecylamine.

12. The production method according to claim 10, wherein The ligand is N-benzoyl-L-arginine, 4-mercaptoethylpyridine or octylamine.

13. The production method according to claim 2, wherein The expanded bed adsorption system comprises one, two or more adsorption columns, and the adsorbent is filled in the adsorption columns.

14. The production method according to claim 13, wherein The adsorption column has a bottom distributor and a top outlet adapter.

15. The production method according to claim 14, wherein The bottom distributor and / or the top outlet adapter adopts a screen structure or a filter structure.

16. The production method according to claim 13, wherein When the lactose-containing mixture is pumped into the adsorption column at a flow rate of 200-600 cm / h, the stable bed height of the expanded bed is 1.5-3.2 times the original settled bed height.

17. The production method according to any one of claims 1 to 16, wherein The concentration of lactose in the lactose-containing mixture is 15-30% (w / v).

18. The production method according to claim 17, wherein The lactose-containing mixture is whey or whey permeate.

19. The production method according to any one of claims 1 to 16, wherein In the lactose-containing solution: the protein content is less than or equal to 100 ppm, and / or, the protein removal rate is greater than or equal to 80%, and / or, the lactose recovery rate is greater than or equal to 75%, and / or, the solution turbidity is less than or equal to 2 NTU.

20. The production method according to claim 19, wherein In the lactose-containing solution: the protein content is less than or equal to 65 ppm, and / or, a protein removal rate of greater than or equal to 85%, and / or, a lactose recovery rate of greater than or equal to 80%, and / or, a solution turbidity of less than or equal to 1.5 NTU.

21. Lactose prepared by the preparation method of any one of claims 1-20.

22. An expanded bed adsorption system for lactose separation and purification, characterized in that, The expanded bed adsorption system comprises the adsorbent of any one of claims 2-12 or the adsorption column of any one of claims 13-16.

23. The expanded bed adsorption system of claim 22, wherein, The method for separating and purifying lactose is as described in any one of claims 1-20.

24. Use of the expanded bed adsorption system of claim 22 or 23 in separating and purifying lactose, preparing lactose, improving lactose recovery rate, or removing protein in a lactose mixture.

25. The use of claim 24, wherein, The removing protein in the lactose mixture comprises reducing the protein concentration of the lactose mixture and / or improving the removal rate of protein in the lactose mixture.

26. The use of claim 25, wherein, The protein in the lactose mixture comprises beta-lactoglobulin and alpha-lactalbumin.