Preparation method of lactose based on asymmetric field flow separation

Abstract

The invention provides a lactose preparation method based on asymmetric field flow separation. The invention provides a method for separating and purifying lactose from a lactose-containing mixture, the method comprises the step of separating and purifying lactose from the lactose-containing mixture by utilizing asymmetric field flow separation, and in the asymmetric field flow separation, the flow velocity of transverse flow adopts an exponential decay mode, and the flow velocity of tangential flow adopts a constant flow mode. The invention also provides a preparation method of lactose crystals. The preparation method comprises the step of separating and purifying lactose from a lactose-containing mixture by using the method for separating and purifying lactose. The method effectively solves the problems of easy moisture absorption and low recovery rate of the product caused by difficult removal of the micro-crystallization inhibitor in the traditional membrane separation process, the purity of the obtained lactose product can reach 97% or above, the recovery rate of lactose is increased to 60% or above, and the method is suitable for industrial production of lactose.

Description

Technical Field

[0001] This invention belongs to the field of dairy product processing and separation and purification technology. More specifically, this invention relates to a method for preparing lactose based on asymmetric field flow separation. Background Technology

[0002] Currently, the industrial production of lactose generally employs membrane separation-based raw material purification processes. Among these, the combination of ultrafiltration and nanofiltration has become a common method for deproteinizing, decolorizing, and desalting whey permeate. This type of process can effectively remove large molecular proteins, colloids, and some inorganic salts from whey. Published Chinese patents include technical solutions for preparing refined lactose or desalted whey powder from whey through ultrafiltration, nanofiltration, and concentration (e.g., CN101491287B, CN102870952A, CN111919908A). In addition, ion exchange resins are also used for purifying sugar-containing mother liquors to further remove residual proteins and some pigment impurities, and they have a long-standing technical basis in the field of sugar solution treatment. For example, patent CN102482313B discloses a scheme for using ion exchange resins for the separation and purification of whey components. However, this method has certain drawbacks: the combined ultrafiltration and nanofiltration process relies solely on pore size sieving, which is ineffective for separating trace impurities (such as fine protein aggregates, Maillard reaction precursor colloids, calcium phosphate-protein complexes, etc.) with molecular weights between the ultrafiltration and nanofiltration cutoff ranges, or those with hydrodynamic radii significantly different from lactose but smaller molecular weights. Although these trace impurities are present in low concentrations, they have a strong crystal facet inhibition effect, severely inhibiting the growth of lactose crystals. This results in lactose product purity failing to exceed 97.0%, low recovery rates (typically only 40-50%), long crystallization cycles, and the product easily agglomerating due to the moisture absorption of impurities.

[0003] Therefore, there is an urgent need in this field to explore a novel method for preparing lactose that can precisely remove the aforementioned trace crystallization inhibitors in order to solve the above problems. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing lactose based on asymmetric field flow separation, so as to solve the problem of low lactose purity and recovery rate caused by incomplete removal of trace crystallization inhibitors in the prior art.

[0005] A first aspect of the present invention provides a method for separating and purifying lactose from a mixture containing lactose, the method comprising the step of separating and purifying lactose from the mixture containing lactose using asymmetric field flow separation; preferably, in the asymmetric field flow separation, the crossflow velocity adopts an exponential decay mode and the tangential flow velocity adopts a constant flow mode.

[0006] In one or more embodiments, the decay constant of the exponential decay mode is 0.05~0.10, preferably 0.07~0.08.

[0007] In one or more embodiments, in the exponential decay mode, the initial crossflow velocity is 1.0±0.3 mL / min, the time is 30±3 min, the decay constant is 0.07-0.08, and the final decayed crossflow velocity is 0.1±0.05 mL / min.

[0008] In one or more embodiments, the tangential flow rate is 6.0 ± 1.0 mL / min.

[0009] In one or more embodiments, the asymmetric field flow separation employs an open channel structure, the channel comprising an impermeable upper wall and a lower wall serving as an accumulation wall.

[0010] In one or more embodiments, the accumulation wall consists of a porous support and a filter membrane covering it.

[0011] In one or more embodiments, the filter membrane is a regenerated cellulose membrane with a molecular weight cutoff (MWCO) of 1 kDa to 10 kDa, more preferably 1 kDa to 5 kDa, and most preferably 1 kDa.

[0012] In one or more embodiments, the lactose-containing mixture includes whey, whey permeate, preferably whey permeate.

[0013] In one or more embodiments, the lactose-containing mixture contains more than 50% by dry weight of lactose, preferably 50-90%.

[0014] In one or more embodiments, the lactose-containing mixture is a lactose-containing mixture pretreated by microfiltration (MF) and / or ultrafiltration (UF).

[0015] In one or more embodiments, the microfiltration is performed using a membrane with a pore size of 0.4 to 0.5 μm, and / or the ultrafiltration is performed using a membrane with a molecular weight cutoff of 3 kDa to 10 kDa, more preferably using a 5 kDa polyethersulfone (PES) membrane.

[0016] In one or more embodiments, the method further includes the step of separating and purifying lactose from a lactose-containing mixture using nanofiltration.

[0017] In one or more embodiments, the nanofiltration percolation includes: separating and purifying the lactose-containing mixture using a nanofiltration membrane, and percolating with water or a buffer solution.

[0018] In one or more embodiments, the nanofiltration percolation further includes one or more conditions selected from the following: (1) The molecular weight cutoff of nanofiltration membranes is 150~300 Da; (2) Nanofiltration concentrates the lactose solution to a volume concentration factor (VCF) of 4 to 6, preferably 5.0 ± 0.1; (3) Percolation dilutes the lactose solution to a volume dilution factor (VDF) of 2 to 6, preferably 5.0 ± 0.1; (4) The pressure used for nanofiltration is 0.2~2.0 MPa, preferably 1.0~1.5 MPa; (5) Deionized water is used for percolation.

[0019] In one or more embodiments, the step of separating and purifying lactose from the lactose-containing mixture using asymmetric field flow separation precedes the step of separating and purifying lactose from the lactose-containing mixture using nanofiltration percolation.

[0020] A second aspect of the present invention provides a method for preparing lactose crystals, the method comprising the step of separating and purifying lactose from a mixture containing lactose using the method for separating and purifying lactose from a mixture containing lactose as described in any embodiment of the present invention.

[0021] In one or more embodiments, the preparation method includes the following steps: (1) Using the method for separating and purifying lactose from a mixture containing lactose as described in any embodiment of the present invention, lactose is separated and purified from the mixture containing lactose to obtain a purified lactose solution; (2) Concentrate the lactose purification solution described in (1) to a supersaturated state to obtain a lactose supersaturated solution; (3) Crystallize the lactose supersaturated solution described in (2) to obtain lactose crystals.

[0022] In one or more embodiments, in step (2), the concentration is to concentrate the lactose purification solution to a solid content of 50 wt% or more, and / or the concentration of lactose in the concentrated lactose supersaturated solution is 500 g / L or more.

[0023] In one or more embodiments, in step (3), the crystallization is induced by using nanocrystal seeds, preferably having a particle size D50 of 100-1000 nm, more preferably 100-500 nm, and most preferably 150-200 nm; and / or, the amount of nanocrystal seeds added is 0.01-0.5% (w / w), more preferably 0.05-0.25% (w / w), wherein the percentage is calculated relative to lactose solids.

[0024] In one or more embodiments, the preparation method further includes the step of separating and / or drying the lactose crystals described in (3); preferably, the separation is a solid-liquid separation, such as centrifugal separation or vacuum filtration separation, more preferably centrifugal separation, and / or, the drying is selected from any one or more of hot air drying, vacuum drying, airflow drying, static drying under negative pressure and fluidized bed drying, more preferably fluidized bed drying; preferably, the moisture content in the dried lactose microcrystals is 5.0 wt%~5.5 wt%.

[0025] In a third aspect, the present invention provides a lactose crystal, wherein the lactose crystal is prepared by the preparation method described in any embodiment of the present invention, preferably the lactose crystal is lactose monohydrate, more preferably the purity of the lactose crystal is ≥97%, and more preferably ≥99%.

[0026] A fourth aspect of the present invention provides applications selected from the following: (1) Application of asymmetric field flow separation in lactose separation and purification, preparation of lactose crystals, and / or improvement of lactose recovery rate, wherein the cross flow of the asymmetric field flow separation adopts an exponential decay mode and the tangential flow adopts a constant flow mode; (2) Application of asymmetric field flow separation and nanofiltration in lactose separation and purification, preparation of lactose crystals, and / or improvement of lactose recovery rate, wherein the cross flow of the asymmetric field flow separation adopts an exponential decay mode and the tangential flow adopts a constant flow mode.

[0027] In one or more embodiments, the method for separating and purifying lactose is as described in any embodiment of the present invention.

[0028] In one or more embodiments, the method for preparing the lactose crystals is as described in any embodiment of the present invention.

[0029] In one or more embodiments, the lactose crystals are lactose monohydrate.

[0030] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Detailed Implementation

[0031] Through in-depth research, the inventors discovered that applying the asymmetric field flow separation method, commonly used for the separation of biomacromolecules (such as proteins and viruses), to the separation and purification of lactose, by employing an exponentially decaying cross-flow field, achieves precise fractionation of components with minute differences in hydrodynamic radii. This is particularly effective for removing trace amounts of crystallization inhibitors (such as glycoprotein aggregates and residual milk proteins) in the 1 kDa to 100 kDa range, which are difficult to remove using traditional processes, demonstrating significant and stable removal efficiency. The inventors further optimized the membrane material and carrier liquid system in the asymmetric field flow separation method, preferably using a regenerated cellulose (RC) membrane in combination with a deionized water carrier liquid. This solves the problem of reduced recovery rates caused by the easy adsorption of lactose and proteins by traditional polyethersulfone (PES) membranes and avoids secondary contamination introduced by buffer salt carrier liquids. Furthermore, this invention uses nanoscale lactose seeds to induce crystallization, effectively avoiding interference from impurities on crystal lattice growth, reducing crystal defects and mother liquor encapsulation, thereby increasing lactose purity to over 97% and lactose recovery rate to over 60%.

[0032] the term

[0033] 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.

[0034] 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.

[0035] 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.

[0036] The terms "above" and "below" both include the stated number.

[0037] Unless otherwise specified, percentages refer to weight percentages and proportions refer to weight ratios in this article.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] The term "lactose crystals" refers to lactose in a crystalline structure. The term "lactose monohydrate" refers to a type of lactose crystal formed by the combination of one molecule of lactose and one molecule of water of crystallization.

[0042] The term "lactose-containing mixture" refers to a raw material 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 in the lactose-containing mixture has a dry weight percentage of 50% or more, 55% or more, 60% or more, 70% or more, or 75% or more, for example, 50-80%, 55-75%, or 60-75%. In some specific embodiments, the lactose-containing mixture is a whey permeate. In some embodiments, the lactose-containing mixture is a lactose-containing mixture pretreated by microfiltration (MF) and / or ultrafiltration (UF). In one or more embodiments, the microfiltration is performed using a membrane with a pore size of 0.4-0.5 μm, and / or the ultrafiltration is performed using a membrane with a molecular weight cutoff of 3 kDa to 10 kDa, preferably a 5 kDa polyethersulfone (PES) membrane.

[0043] 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.

[0044] The term "whey permeate" refers to a mixture formed during the lactose preparation process from whey, typically containing about 20% dry matter, of which 60% to 75% lactose, 15% to 25% minerals (or ash), 5% to 15% protein, and trace amounts of other whey components. In some embodiments, whey permeate can be prepared from whey by membrane separation (e.g., ultrafiltration, microfiltration, etc.).

[0045] The terms "asymmetric flow field-flow separation," "asymmetrical flow field-flow separation," and "AF4" are used interchangeably, all referring to a separation method based on fluid dynamics and diffusion behavior, which separates samples through asymmetric flow. The asymmetric flow typically includes vertical crossflow (crossflow) and laminar flow (tangential flow) within the channel.

[0046] The term "exponential decay" refers to the exponential decrease in velocity of crossflow or tangential flow over time in asymmetric flow separation methods. This can be expressed using the formula V(t) = V0 × e^(-t / t). -kt V(t) represents the exponential decay in the asymmetric field flow separation method, where V(t) represents the velocity of the exponentially decaying crossflow or tangential flow, V0 represents the initial crossflow or tangential flow velocity, k represents the decay constant, and t represents time.

[0047] The term "constant current" refers to the fact that the velocity of crossflow or tangential flow does not change over time in asymmetric field flow separation methods.

[0048] Methods for the isolation and purification of lactose

[0049] The present invention provides a method for separating and purifying lactose from a mixture containing lactose, the method comprising the step of separating and purifying lactose from the mixture containing lactose using asymmetric field flow separation; preferably, in the asymmetric field flow separation, the crossflow velocity adopts an exponential decay mode and the tangential flow velocity adopts a constant flow mode.

[0050] In some embodiments, the attenuation constant is 0.05~0.10, preferably 0.07~0.08, for example 0.077±0.002 or 0.077±0.001. In some specific embodiments, the initial crossflow velocity is 1.0±0.3 mL / min (preferably 1.0±0.2 mL / min, 1.0±0.1 mL / min or 1.0±0.05 mL / min), the time is 30±3 min (preferably 30±2 min or 30±1 min), the attenuation constant is 0.07-0.08, and the final attenuated crossflow velocity is 0.1±0.05 mL / min (preferably 0.1±0.04 mL / min, 0.1±0.03 mL / min, 0.1±0.02 mL / min or 0.1±0.01 mL / min).

[0051] In some embodiments, the tangential flow rate is 6.0 ± 1.0 mL / min (preferably 6.0 ± 0.5 mL / min, 6.0 ± 0.3 mL / min, 6.0 ± 0.2 mL / min or 6.0 ± 0.1 mL / min).

[0052] It should be understood that the crossflow velocity adopts an exponential decay mode, which can significantly broaden the separation window compared to the constant flow mode of crossflow: in the initial stage of separation, the high crossflow velocity can effectively suppress low molecular weight impurities; as the crossflow decays, the slightly larger molecules that were originally strongly compressed (such as casein micelle fragments and protein aggregates in the range of 1 kDa to 100 kDa) are gradually released but effectively separated from the lactose peak, thereby greatly improving the resolution of lactose crystallization inhibitors.

[0053] The asymmetric flow separation employs an open channel with an impermeable upper wall (such as polycarbonate or stainless steel) and a porous support and filter membrane forming an accumulation wall. In some embodiments, the filter membrane is a regenerated cellulose (RC) membrane with a molecular weight cutoff (MWCO) of 1 kDa to 10 kDa, most preferably a 1 kDa regenerated cellulose (RC) membrane. The term "Da" refers to the molecular weight cutoff, i.e., the molecular weight of molecules retained by the filter membrane. It should be understood that "a certain value + Da" indicates that the molecular weight cutoff is greater than or equal to that value; for example, a 1 kDa filter membrane means that when the filter membrane is used for filtration, the molecular weight of the molecules it retains is ≥1 kDa. In some preferred embodiments, the filter membrane is a regenerated cellulose (RC) membrane with a molecular weight cutoff of 1 kDa to 10 kDa, most preferably a 1 kDa regenerated cellulose (RC) membrane. In asymmetric flow separation methods, regenerated cellulose (RC) membranes are superior. RC membranes possess excellent hydrophilicity and electroneutrality, exhibiting low non-specific adsorption of lactose and residual milk proteins, while PES membranes readily adsorb proteins, causing contamination. Applying RC membranes for lactose separation can extend membrane life and significantly improve lactose purity and recovery rate.

[0054] In some embodiments, the method for separating and purifying lactose from a lactose-containing mixture further includes the step of separating and purifying lactose from the lactose-containing mixture using nanofiltration. In some embodiments, the nanofiltration includes: separating and purifying the lactose-containing mixture using a nanofiltration membrane and percolating with deionized water or a buffer solution. In some embodiments, the nanofiltration membrane has a molecular weight cutoff of 150-300 Da, for example, 150 Da, 160 Da, 180 Da, 200 Da, 220 Da, 240 Da, 260 Da, 280 Da, 300 Da, or any combination thereof. In some embodiments, nanofiltration concentrates the lactose solution to a volumetric concentration factor (VCF) of 4 to 6, such as 4.0 to 5.5, 4.2 to 5.2, or 4.5 to 5.0, more specifically, 5.0, and percolation dilutes the lactose solution to a volumetric dilution factor (VDF) of 2 to 6, such as 2.5 to 5.5, 3 to 5.2, or 4 to 5, more specifically, 5.0. In some embodiments, the pressure used for nanofiltration can be 0.2 to 2.0 MPa, such as 0.2 to 1.0 MPa, 0.5 to 1.5 MPa, or 1.0 to 2.0 MPa, more specifically, 1.0 to 1.5 MPa. In some preferred embodiments, percolation is performed using deionized water.

[0055] In some embodiments, the method for separating and purifying lactose from a lactose-containing mixture includes the steps of separating and purifying lactose from the lactose-containing mixture using asymmetric flow field separation and nanofiltration. In some embodiments, the step of separating and purifying lactose from the lactose-containing mixture using asymmetric flow field separation precedes the step of separating and purifying lactose from the lactose-containing mixture using nanofiltration. In some embodiments, the asymmetric flow field separation step and the nanofiltration step are performed sequentially. The nanofiltration step can remove salts and small molecule impurities from the lactose solution after asymmetric flow field separation, thereby obtaining a further purified lactose solution.

[0056] Preparation method of lactose crystals

[0057] This invention provides a method for preparing lactose crystals from a lactose-containing mixture, the method comprising the lactose separation and purification steps described in any embodiment of the invention. In some embodiments, the lactose separation and purification steps are steps of separating and purifying lactose from the lactose-containing mixture using asymmetric flow field separation and nanofiltration, preferably the asymmetric flow field separation step and the nanofiltration step are performed sequentially.

[0058] In some embodiments, the method for preparing lactose crystals from a lactose-containing mixture further includes a step of concentrating the purified lactose solution. In some embodiments, the concentration may bring the purified lactose solution to a supersaturated state. In some embodiments, the concentration to a supersaturated state can be determined by the solids content in the lactose solution; for example, the solids content in the concentrated supersaturated lactose solution may be above 50 wt%, above 55 wt%, or above 60 wt% (more specifically, 55-65 wt%, 56-64 wt%, 57-63 wt%, 58-62 wt%, or 59-61 wt%). In some embodiments, the lactose concentration in the concentrated supersaturated lactose solution may be above 500 g / L, above 520 g / L, or above 550 g / L, for example, 550-650 g / L. The concentration may be performed using concentration methods known in the art, such as, but not limited to, evaporation concentration. For example, the evaporation concentration temperature may be 65-75°C.

[0059] In some embodiments, the method for preparing lactose from a lactose-containing mixture further includes a crystallization step. In some embodiments, the crystallization can be performed by cooling a homogenized lactose-containing solution. It should be understood that the cooling crystallization can be carried out by various methods such as stirring cooling, crystallizer cooling, etc. In some embodiments, the crystallization can be induced using nanocrystal seeds. In some embodiments, the nanocrystal seeds refer to nanoscale seeds. The particle size D50 of the nanocrystal seeds can be 100~1000nm, for example 100~800nm, 150~500nm, 180~400nm, 100~300nm, or 120~250nm, preferably 150~200nm. In some embodiments, the amount of nanocrystal seeds added is 0.01~0.5% (w / w), 0.02~0.4% (w / w), 0.03~0.3% (w / w), or 0.04~0.25% (w / w), preferably 0.05~0.2% (w / w), where the percentages are calculated relative to the lactose solids. When nanocrystal seeds are added during the crystallization step to induce crystallization, the large specific surface area provided by the nanocrystal seeds allows for explosive nucleation of the lactose solution at relatively low supersaturation, avoiding the phenomenon of impurities being embedded in the crystal lattice at high supersaturation. This is beneficial for further improving the purity and recovery rate of lactose.

[0060] In some embodiments, the method for preparing lactose crystals from a lactose-containing mixture further includes the steps of separating and / or drying the crystallized lactose crystals. The separation can be a solid-liquid separation, such as, but not limited to, centrifugal separation. In some specific embodiments, the centrifugal separation can be performed using vacuum filtration or a horizontal screw centrifuge. The drying can be performed using methods known in the art, such as hot air drying, vacuum drying, airflow drying, static drying under negative pressure, fluidized bed drying, etc. In some specific embodiments, the drying is fluidized bed drying. In some embodiments, the moisture content in the dried lactose microcrystals is 5.0 wt% to 5.5 wt%.

[0061] lactose crystals

[0062] The present invention also provides lactose crystals prepared by the method for preparing lactose crystals from a lactose-containing mixture as described in any embodiment of the present invention. In some embodiments, the lactose crystals are lactose monohydrate.

[0063] In some embodiments, the lactose crystals prepared by the method of the present invention have a high purity, for example, a purity of 97% or higher, 97.5% or higher, 98% or higher, 98.5% or higher, 99% or higher.

[0064] In some embodiments, the recovery rate of lactose crystals prepared by the method of the present invention is high, for example, a recovery rate of 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, or higher.

[0065] application

[0066] This invention provides applications selected from the following: (1) Application of asymmetric field flow separation in lactose separation and purification, preparation of lactose crystals, and / or improvement of lactose recovery rate, wherein the cross flow of the asymmetric field flow separation adopts an exponential decay mode and the tangential flow adopts a constant flow mode; (2) Application of asymmetric field flow separation and nanofiltration in lactose separation and purification, preparation of lactose crystals, and / or improvement of lactose recovery rate, wherein the cross flow of the asymmetric field flow separation adopts an exponential decay mode and the tangential flow adopts a constant flow mode.

[0067] In some embodiments, the lactose crystals are lactose monohydrate.

[0068] The advantages of this invention include: (1) Improved separation accuracy: Existing membrane separation methods for separating lactose rely solely on pore size for sieving, making it difficult to separate impurities with similar molecular weights but different structures. This invention employs an asymmetric field flow separation (AF4) exponentially decaying flow field, based on the difference in Brownian diffusion coefficients for dynamic separation, removing impurities in the range of 1 kDa to 100 kDa, thus solving the problem of incomplete impurity removal leading to hindered lactose crystallization in traditional processes.

[0069] (2) Optimization of membrane material and carrier liquid: The membrane material commonly used in asymmetric field flow separation (AF4) systems is PES membrane, and salt buffer solution is usually used. In this invention, a hydrophilic RC membrane is specifically selected in combination with deionized water as carrier liquid to address the characteristics of lactose. This improvement overcomes the problem of low recovery rate caused by sample adsorption by PES membrane, while improving desalination efficiency and realizing the "greening" and "high recovery rate" of the process.

[0070] (3) Upgrade of seed induction strategy: Traditional crystallization often relies on natural nucleation or the addition of ordinary micron-sized seeds, which are easily affected by impurities, resulting in inconsistent crystal forms. This invention introduces nano-sized seeds into AF4-nanofiltration / percolation purification solution. By utilizing the large specific surface area and uniformity of the seeds, uniform growth is induced under low supersaturation, which significantly reduces mother liquor encapsulation, improves the purity of lactose products to over 97%, and achieves a recovery rate of over 60%, which is at the leading level in the industry.

[0071] 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.

[0072] Experimental Materials and Methods

[0073] The raw material is whey permeate from the cheese production process, with a volume of 1000mL, an initial lactose content of 2.5-4.5%, a protein residue of 0.4-0.9%, and a pH value of 6.4-6.8.

[0074] PES membrane: PES membrane produced by Synder Filtration, with a MWCO of 1 kDa.

[0075] Regenerated cellulose (RC) membrane: The regenerated cellulose membrane is manufactured by Microdyn-Nadir GmbH, Germany, with a MWCO of 1 kDa.

[0076] Polyethersulfone membrane: The polyethersulfone membrane is manufactured by Millipore, Inc., USA, with a MWCO of 5 kDa.

[0077] Spiral nanofiltration membrane with polyamide-polyethersulfone composite membrane: Spiral nanofiltration membrane with polyamide-polyethersulfone composite membrane produced by Veolia, France, with MWCO of 150-300 Da.

[0078] Asymmetric field flow separation was performed using an asymmetric field flow separation system manufactured by Wyatt Technology Corp.

[0079] Cooling crystallizer: A three-stage series continuous cooling crystallizer is built using the EasyMax automated crystallization workstation manufactured by Mettler Toledo of Switzerland, equipped with precision temperature control and stirring paddle.

[0080] The seed crystals were nanoscale seed crystals with a median diameter (D50) of 150-200 nm prepared by the applicant, and were prepared by ultrasound-assisted lactose antisolvent crystallization and wet media milling methods as described in the literature “Lactoseparticle engineering: Influence of ultrasound and anti-solvent on crystalhabit and particle size” (DOI:10.1016 / j.jcrysgro.2010.09.022) and “Production of sub-micron particles by wet comminution in stirred media mills” (DOI:10.1023 / B:JMSC.0000039214.12131.58).

[0081] Whey permeate pretreatment: First, microfiltration (pore size 0.45μm) is used to remove residual fat and suspended matter, and then spiral wound ultrafiltration (MWCO 5 kDa, polyethersulfone membrane) is used to initially remove protein, to obtain crude purified permeate (volume about 600mL-700mL, lactose concentration about 3.0%, protein residue 0.3-0.6%).

[0082] Detection methods: Lactose purity was determined by HPLC (Aminex HPX-87H column, differential refractive index detection); protein residue was determined by Lowry method or micro Kjeldahl method. Lactose recovery rate was calculated using a gravimetric method combined with HPLC purity correction, reflecting the lactose recovery rate from raw materials to finished product. Calculation formula: Lactose recovery rate (%) = [ M product × P product ×(1- W water )] / (Cmaterial ×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).

[0083] Example 1: AF4 standard exponential decay crossflow + crystallization stage with seed crystals

[0084] Take 1000 mL of whey permeate from the cheese production process, first pass it through microfiltration (pore size 0.45 μm) to remove residual fat and suspended matter, and then pass it through spiral wound membrane ultrafiltration (MWCO 5 kDa, polyethersulfone membrane) to preliminarily remove protein, to obtain crude purified permeate.

[0085] The crude purified permeate was pumped into the asymmetric field flow separation (AF4) system. The accumulation wall was a regenerated cellulose (RC) membrane (MWCO 1 kDa), and the carrier liquid was deionized water. The single injection volume was set to 5.0 mL (processing the feed solution through multiple injections), the injection flow rate was 1.0 mL / min, the focusing flow rate was 3.0 mL / min, and the focusing time was 10 min. During the elution stage, the channel flow rate (tangential flow rate) was fixed at 6.0 mL / min, and the crossflow adopted an exponential decay mode: the initial crossflow was 1.0 mL / min, which exponentially decayed to 0.1 mL / min within 30 min (the decay constant k≈0.077, determined by the instrument based on V(t)=V0). e -kt (Automatic control). The AF4 outlet is directly connected in series with a nanofiltration / percolation system (using a spiral wound nanofiltration membrane of MWCO 150-300 Da, made of polyamide-polyethersulfone composite membrane), which concentrates the solution to a volume concentration factor (VCF) of 5.0 under a pressure of 1.0-1.5 MPa. Then, pure water is introduced for constant volume percolation until the volume dilution factor (VDF) reaches 5.0 to remove impurities and small molecule minerals.

[0086] The above solution was heated and maintained at 70°C, then concentrated under reduced pressure to a solids content of 55 wt%-65 wt%, yielding a supersaturated lactose concentrate. The concentrate was pumped into a cooling crystallizer, with the temperature program controlled as follows: first stage temperature decreasing from 70°C to 45-50°C, residence time 20-40 min; second stage temperature decreasing from 45-50°C to 30-35°C, residence time 20-40 min; third stage temperature decreasing from 30-35°C to 10-15°C, residence time 20-40 min, with continuous crystallization at a stirring speed of 200 rpm. When the temperature reached 45°C, 0.20% (w / w, calculated relative to lactose solids) of nano-sized lactose seed crystals (particle size D50 of 150–200 nm) was added to the crystallizer inlet.

[0087] The crystallized slurry is subjected to solid-liquid separation (such as centrifugation or vacuum filtration), the mother liquor is partially recycled, and the wet crystals are dried in a fluidized bed to a moisture content of 5.0 wt%-5.5 wt%.

[0088] The final product was a dried α-lactose monohydrate powder sample, which was tested to have a purity of 99.8% and a recovery rate of 70.4%.

[0089] Example 2: The AF4 accumulation wall material is a polyethersulfone (PES) membrane.

[0090] Take 1000 mL of whey permeate from the cheese production process, first pass it through microfiltration (pore size 0.45 μm) to remove residual fat and suspended matter, and then pass it through spiral wound membrane ultrafiltration (MWCO 5 kDa, polyethersulfone membrane) to preliminarily remove protein, to obtain crude purified permeate.

[0091] The crude purified permeate was pumped into the asymmetric field flow separation (AF4) system, with a polyethersulfone (PES) membrane (MWCO 1 kDa) as the accumulation wall and deionized water as the carrier liquid. The single injection volume was set to 5.0 mL (processing the feed solution through multiple injections), the injection flow rate was 1.0 mL / min, the focusing flow rate was 3.0 mL / min, and the focusing time was 10 min. During the elution stage, the channel flow rate (tangential flow rate) was fixed at 6.0 mL / min, and the crossflow adopted an exponential decay mode: an initial crossflow of 1.0 mL / min, which exponentially decayed to 0.1 mL / min within 30 min (decay constant k≈0.077, determined by the instrument based on V(t)=V0). e -kt (Automatic control). The AF4 outlet is directly connected in series with a nanofiltration / percolation system (using a spiral wound nanofiltration membrane of MWCO 150-300 Da, made of polyamide-polyethersulfone composite membrane), which concentrates the solution to a volume concentration factor (VCF) of 5.0 under a pressure of 1.0-1.5 MPa. Then, pure water is introduced for constant volume percolation until the volume dilution factor (VDF) reaches 5.0 to remove impurities and small molecule minerals.

[0092] The above solution was heated and maintained at 70°C, then concentrated under reduced pressure to a solids content of 55 wt%-65 wt%, yielding a supersaturated lactose concentrate. The concentrate was pumped into a cooling crystallizer, with the temperature program controlled as follows: first stage temperature decreasing from 70°C to 45-50°C, residence time 20-40 min; second stage temperature decreasing from 45-50°C to 30-35°C, residence time 20-40 min; third stage temperature decreasing from 30-35°C to 10-15°C, residence time 20-40 min, with continuous crystallization at a stirring speed of 200 rpm. When the temperature reached 45°C, 0.20% (w / w, calculated relative to lactose solids) of nano-sized lactose seed crystals (particle size D50 of 150–200 nm) was added to the crystallizer inlet.

[0093] The crystallized slurry is subjected to solid-liquid separation (such as centrifugation or vacuum filtration), the mother liquor is partially recycled, and the wet crystals are dried in a fluidized bed to a moisture content of 5.0 wt%-5.5 wt%.

[0094] The final sample was a dried α-lactose monohydrate powder with a purity of 97.6% and a recovery rate of only 54.2%, indicating that the RC membrane has lower non-specific adsorption of lactose compared to the PES membrane, thus significantly improving the lactose recovery rate.

[0095] Example 3: AF4 Crossflow Constant Mode

[0096] Take 1000 mL of whey permeate from the cheese production process, first pass it through microfiltration (pore size 0.45 μm) to remove residual fat and suspended matter, and then pass it through spiral wound membrane ultrafiltration (MWCO 5 kDa, polyethersulfone membrane) to preliminarily remove protein, to obtain crude purified permeate.

[0097] The crude purified permeate was pumped into the asymmetric field flow separation (AF4) system, with a regenerated cellulose (RC) membrane (MWCO 1 kDa) as the accumulation wall and deionized water as the carrier liquid. The single injection volume was 5.0 mL (the entire feed solution was processed through multiple injections), the injection flow rate was 1.0 mL / min, the focusing flow rate was 3.0 mL / min, and the focusing time was 10 min. During the elution stage, the channel flow rate (tangential flow rate) was fixed at 6.0 mL / min, and the crossflow was in constant mode with a constant crossflow rate of 0.8 mL / min. The AF4 outlet was directly connected in series with a nanofiltration / percolation system (using a spiral wound nanofiltration membrane with a MWCO 150-300 Da polyamide-polyethersulfone composite membrane), concentrating the solution to a volume concentration factor (VCF) of 5.0 at 1.0-1.5 MPa. Then, pure water was introduced for constant-volume percolation until the volume dilution factor (VDF) reached 5.0 to remove impurities and small molecule minerals.

[0098] The above solution was heated and maintained at 70°C, then concentrated under reduced pressure to a solids content of 55 wt%-65 wt%, yielding a supersaturated lactose concentrate. The concentrate was pumped into a cooling crystallizer, with the temperature program controlled as follows: first stage temperature decreasing from 70°C to 45-50°C, residence time 20-40 min; second stage temperature decreasing from 45-50°C to 30-35°C, residence time 20-40 min; third stage temperature decreasing from 30-35°C to 10-15°C, residence time 20-40 min, with continuous crystallization at a stirring speed of 200 rpm. When the temperature reached 45°C, 0.20% (w / w, calculated relative to lactose solids) of nano-sized lactose seed crystals (particle size D50 of 150–200 nm) was added to the crystallizer inlet.

[0099] The crystallized slurry is subjected to solid-liquid separation (such as centrifugation or vacuum filtration), the mother liquor is partially recycled, and the wet crystals are dried in a fluidized bed to a moisture content of 5.0 wt%-5.5 wt%.

[0100] The final sample was a dried α-lactose monohydrate powder with a purity of 98.8% and a recovery rate of 66.4%. This indicates that the constant crossflow mode has low resolution in removing crystallization inhibitors within a specific molecular weight range, confirming the criticality of exponentially decaying crossflow for high-resolution removal of inhibitors.

[0101] Example 4: AF4 carrier fluid with added buffer salt solution

[0102] Take 1000 mL of whey permeate from the cheese production process, first pass it through microfiltration (pore size 0.45 μm) to remove residual fat and suspended matter, and then pass it through spiral wound membrane ultrafiltration (MWCO 5 kDa, polyethersulfone membrane) to preliminarily remove protein, to obtain crude purified permeate.

[0103] The crude purified permeate was pumped into the asymmetric field flow separation (AF4) system. The accumulation wall was a regenerated cellulose (RC) membrane (MWCO 1 kDa), and the carrier liquid was 0.01 M NaNO3 solution. The single injection volume was set to 5.0 mL (processing the feed solution through multiple injections), the injection flow rate was 1.0 mL / min, the focusing flow rate was 3.0 mL / min, and the focusing time was 10 min. During the elution stage, the channel flow rate (tangential flow rate) was fixed at 6.0 mL / min, and the crossflow adopted an exponential decay mode: the initial crossflow was 1.0 mL / min, which exponentially decayed to 0.1 mL / min within 30 min (the decay constant k≈0.077, determined by the instrument based on V(t)=V0). e -kt(Automatic control). The AF4 outlet is directly connected in series with a nanofiltration / percolation system (using a spiral wound nanofiltration membrane of MWCO 150-300 Da, made of polyamide-polyethersulfone composite membrane), which concentrates the solution to a volume concentration factor (VCF) of 5.0 under a pressure of 1.0-1.5 MPa. Then, pure water is introduced for constant volume percolation until the volume dilution factor (VDF) reaches 5.0 to remove impurities and small molecule minerals.

[0104] The above solution was heated and maintained at 70°C, then concentrated under reduced pressure to a solids content of 55 wt%-65 wt%, yielding a supersaturated lactose concentrate. The concentrate was pumped into a cooling crystallizer, with the temperature program controlled as follows: first stage temperature decreasing from 70°C to 45-50°C, residence time 20-40 min; second stage temperature decreasing from 45-50°C to 30-35°C, residence time 20-40 min; third stage temperature decreasing from 30-35°C to 10-15°C, residence time 20-40 min, with continuous crystallization at a stirring speed of 200 rpm. When the temperature reached 45°C, 0.20% (w / w, calculated relative to lactose solids) of nano-sized lactose seed crystals (particle size D50 of 150–200 nm) was added to the crystallizer inlet.

[0105] The crystallized slurry is subjected to solid-liquid separation (such as centrifugation or vacuum filtration), the mother liquor is partially recycled, and the wet crystals are dried in a fluidized bed to a moisture content of 5.0 wt%-5.5 wt%.

[0106] The final product was a dried α-lactose monohydrate powder sample with a purity of 98.1% and a recovery rate of 61.7%.

[0107] Example 5: Reducing the amount of seed crystals added during the crystallization stage

[0108] Take 1000 mL of whey permeate from the cheese production process, first pass it through microfiltration (pore size 0.45 μm) to remove residual fat and suspended matter, and then pass it through spiral wound membrane ultrafiltration (MWCO 5 kDa, polyethersulfone membrane) to preliminarily remove protein, to obtain crude purified permeate.

[0109] The crude purified permeate was pumped into the asymmetric field flow separation (AF4) system. The accumulation wall was a regenerated cellulose (RC) membrane (MWCO 1 kDa), and the carrier liquid was deionized water. The single injection volume was set to 5.0 mL (processing the feed solution through multiple injections), the injection flow rate was 1.0 mL / min, the focusing flow rate was 3.0 mL / min, and the focusing time was 10 min. During the elution stage, the channel flow rate (tangential flow rate) was fixed at 6.0 mL / min, and the crossflow adopted an exponential decay mode: the initial crossflow was 1.0 mL / min, which exponentially decayed to 0.1 mL / min within 30 min (the decay constant k≈0.077, determined by the instrument based on V(t)=V0). e-kt (Automatic control). The AF4 outlet is directly connected in series with a nanofiltration / percolation system (using a spiral wound nanofiltration membrane of MWCO 150-300 Da, made of polyamide-polyethersulfone composite membrane), which concentrates the solution to a volume concentration factor (VCF) of 5.0 under a pressure of 1.0-1.5 MPa. Then, pure water is introduced for constant volume percolation until the volume dilution factor (VDF) reaches 5.0 to remove impurities and small molecule minerals.

[0110] The above solution was heated and maintained at 70°C, then concentrated under reduced pressure to a solids content of 55 wt%-65 wt%, yielding a supersaturated lactose concentrate. The concentrate was pumped into a cooling crystallizer, with the temperature program controlled as follows: first stage temperature decreasing from 70°C to 45-50°C, residence time 20-40 min; second stage temperature decreasing from 45-50°C to 30-35°C, residence time 20-40 min; third stage temperature decreasing from 30-35°C to 10-15°C, residence time 20-40 min, with continuous crystallization at a stirring speed of 200 rpm. When the temperature reached 45°C, 0.05% (w / w, calculated relative to lactose solids) of nano-sized lactose seed crystals (particle size D50 of 150–200 nm) was added to the crystallizer inlet.

[0111] The crystallized slurry is subjected to solid-liquid separation (such as centrifugation or vacuum filtration), the mother liquor is partially recycled, and the wet crystals are dried in a fluidized bed to a moisture content of 5.0 wt%-5.5 wt%.

[0112] The final product was a dried α-lactose monohydrate powder sample, which was found to have a purity of 99.2% and a recovery rate of 64.8%.

[0113] Example 6: No seed crystals added during the crystallization stage

[0114] Take 1000 mL of whey permeate from the cheese production process, first pass it through microfiltration (pore size 0.45 μm) to remove residual fat and suspended matter, and then pass it through spiral wound membrane ultrafiltration (MWCO 5 kDa, polyethersulfone membrane) to preliminarily remove protein, to obtain crude purified permeate.

[0115] The crude purified permeate was pumped into the asymmetric field flow separation (AF4) system. The accumulation wall was a regenerated cellulose (RC) membrane (MWCO 1 kDa), and the carrier fluid was deionized water (without any buffer salts or additives). The single injection volume was set to 5.0 mL (processing the feed solution through multiple cyclic injections), the injection flow rate was 1.0 mL / min, the focusing flow rate was 3.0 mL / min, and the focusing time was 10 min. During the elution stage, the channel flow rate (tangential flow rate) was fixed at 6.0 mL / min, and the crossflow adopted an exponential decay mode: the initial crossflow was 1.0 mL / min, which exponentially decayed to 0.1 mL / min within 30 min (the decay constant k≈0.077, determined by the instrument based on V(t)=V0). e -kt (Automatic control). The AF4 outlet is directly connected in series with a nanofiltration / percolation system (using a spiral wound nanofiltration membrane of MWCO 150-300 Da, made of polyamide-polyethersulfone composite membrane), which concentrates the solution to a volume concentration factor (VCF) of 5.0 under a pressure of 1.0-1.5 MPa. Then, pure water is introduced for constant volume percolation until the volume dilution factor (VDF) is 5.0 to remove impurities and small molecule minerals.

[0116] The above solution was heated and maintained at 70°C, then concentrated under reduced pressure to a solids content of 55 wt%-65 wt%, yielding a supersaturated lactose concentrate. The concentrate was pumped into a cooling crystallizer, with the temperature program controlled as follows: first stage temperature decreasing from 70°C to 45-50°C, residence time 20-40 min; second stage temperature decreasing from 45-50°C to 30-35°C, residence time 20-40 min; third stage temperature decreasing from 30-35°C to 10-15°C, residence time 20-40 min, with continuous crystallization at a stirring speed of 200 rpm. No lactose seed crystals were added in this embodiment.

[0117] The crystallized slurry is subjected to solid-liquid separation (such as centrifugation or vacuum filtration), the mother liquor is partially recycled, and the wet crystals are dried in a fluidized bed to a moisture content of 5.0 wt%-5.5 wt%.

[0118] The final product was a dried α-lactose monohydrate powder with a purity of 99.0% and a recovery rate of 56.2%. This indicates that the absence of seed crystals led to uncontrollable nucleation, increased mother liquor encapsulation, and a significant reduction in the final product's recovery rate. The importance of adding seed crystals for further improving lactose purity and recovery rate is highlighted.

[0119] Example 7: Traditional process without AF4 separation

[0120] Take 1000 mL of whey permeate from the cheese production process, first pass it through microfiltration (pore size 0.45 μm) to remove residual fat and suspended matter, and then pass it through spiral wound membrane ultrafiltration (MWCO 5 kDa, polyethersulfone membrane) to preliminarily remove protein, to obtain crude purified permeate.

[0121] The crude purified permeate was directly pumped into a nanofiltration / percolation system (using a spiral wound nanofiltration membrane with a MWCO 150-300 Da polyamide-polyethersulfone composite membrane) without being treated by the asymmetric field flow separation (AF4) system. It was concentrated to a volume concentration factor (VCF) of 5.0 under a pressure of 1.0-1.5 MPa. Then, pure water was introduced for constant volume percolation until the volume dilution factor (VDF) reached 5.0 to remove impurities and small molecule minerals.

[0122] The above solution was heated and maintained at 70°C, then concentrated under reduced pressure to a solids content of 55 wt%-65 wt%, yielding a supersaturated lactose concentrate. The concentrate was pumped into a cooling crystallizer, with the temperature program controlled as follows: first stage temperature decreasing from 70°C to 45-50°C, residence time 20-40 min; second stage temperature decreasing from 45-50°C to 30-35°C, residence time 20-40 min; third stage temperature decreasing from 30-35°C to 10-15°C, residence time 20-40 min, with continuous crystallization at a stirring speed of 200 rpm. When the temperature reached 45°C, 0.20% (w / w, calculated relative to lactose solids) of nano-sized lactose seed crystals (particle size D50 of 150–200 nm) was added to the crystallizer inlet.

[0123] The crystallized slurry is subjected to solid-liquid separation (such as centrifugation or vacuum filtration), the mother liquor is partially recycled, and the wet crystals are dried in a fluidized bed to a moisture content of 5.0 wt%-5.5 wt%.

[0124] The final product was a dried α-lactose monohydrate powder sample with a purity of 95.5% and a recovery rate of 48.0%. The product had a slightly yellow appearance. Although this embodiment used a combination of microfiltration, ultrafiltration, nanofiltration, and nanocrystal seed crystallization, the lack of the precise separation step AF4 prevented the removal of trace impurities (such as fine protein aggregates and Maillard reaction precursor colloids) outside the pore size range of the microfiltration / ultrafiltration membranes. These impurities severely inhibited crystal growth and adsorbed onto the crystal surface, resulting in a product purity that could not reach above 97% and a significantly reduced recovery rate. This fully demonstrates the necessity and irreplaceability of the AF4 step in the high-purity lactose preparation process of this invention.

[0125] 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 for separating and purifying lactose from a mixture containing lactose, the method comprising the step of separating and purifying lactose from the mixture containing lactose using asymmetric field flow separation; Preferably, in the asymmetric field flow separation, the crossflow velocity adopts an exponential decay mode, and the tangential flow velocity adopts a constant flow mode.

2. The method as described in claim 1, characterized in that, The decay constant of the exponential decay mode is 0.05~0.10, preferably 0.07~0.08; more preferably, in the exponential decay mode, the initial crossflow velocity is 1.0±0.3 mL / min, the time is 30±3 min, the decay constant is 0.07~0.08, and the final decayed crossflow velocity is 0.1±0.05 mL / min; and / or, the tangential flow velocity is 6.0±1.0 mL / min.

3. The method as described in claim 1, characterized in that, The asymmetric field flow separation adopts an open channel structure, which includes an impermeable upper wall and a lower wall that serves as an accumulation wall; Preferably, the accumulation wall consists of a porous support and a filter membrane covering it; More preferably, the filter membrane is a regenerated cellulose membrane with a molecular weight cutoff of 1 kDa to 10 kDa, more preferably 1 kDa to 5 kDa, and most preferably 1 kDa.

4. The method according to any one of claims 1-3, characterized in that, The lactose-containing mixture includes whey, whey permeate, preferably whey permeate; and / or, in the lactose-containing mixture, the dry weight percentage of lactose is 50% or more, preferably 50-90%; Preferably, the lactose-containing mixture is a lactose-containing mixture pretreated by microfiltration and / or ultrafiltration; More preferably, the microfiltration is performed using a membrane with a pore size of 0.4~0.5μm, and / or the ultrafiltration is performed using a membrane with a molecular weight cutoff of 3 kDa~10 kDa, more preferably using a 5 kDa polyethersulfone membrane.

5. The method according to any one of claims 1-4, characterized in that, The method further includes the step of separating and purifying lactose from a mixture containing lactose using nanofiltration percolation; Preferably, the nanofiltration percolation includes: separating and purifying the lactose-containing mixture using a nanofiltration membrane, and percolating with water or a buffer solution; More preferably, the nanofiltration percolation further includes one or more conditions selected from the following: (1) The molecular weight cutoff of nanofiltration membranes is 150~300 Da; (2) Nanofiltration concentrates the lactose solution to a volume concentration factor of 4 to 6, preferably 5.0 ± 0.1; (3) Percolation dilutes the lactose solution to a volume dilution factor of 2 to 6, preferably 5.0 ± 0.1; (4) The pressure used for nanofiltration is 0.2~2.0 MPa, preferably 1.0~1.5 MPa; (5) Percolation is carried out using deionized water; More preferably, the step of separating and purifying lactose from the lactose-containing mixture using asymmetric field flow separation precedes the step of separating and purifying lactose from the lactose-containing mixture using nanofiltration percolation.

6. A method for preparing lactose crystals, characterized in that, The preparation method includes the step of separating and purifying lactose from a mixture containing lactose using the method described in any one of claims 1-5.

7. The preparation method according to claim 6, characterized in that, The preparation method includes the following steps: (1) Using the method of any one of claims 1-5, lactose is separated and purified from a mixture containing lactose to obtain a purified lactose solution; (2) Concentrate the lactose purification solution described in (1) to a supersaturated state to obtain a lactose supersaturated solution; (3) Crystallize the lactose supersaturated solution described in (2) to obtain lactose crystals.

8. The preparation method according to claim 7, characterized in that, In step (2), the concentration involves concentrating the lactose purification solution to a solid content of 50 wt% or more, and / or, the concentration of lactose in the concentrated lactose supersaturated solution is 500 g / L or more; and / or, In step (3), the crystallization is induced by using nanocrystal seeds, preferably with a particle size D50 of 100~1000 nm, more preferably 100~500 nm, and most preferably 150~200 nm; and / or, the amount of nanocrystal seeds added is 0.01~0.5% (w / w), more preferably 0.05~0.25% (w / w), wherein the percentage is calculated relative to lactose solids.

9. The preparation method according to any one of claims 6-8, characterized in that, The preparation method further includes the steps of separating and / or drying the lactose crystals described in (3); preferably, the separation is solid-liquid separation, centrifugal separation or vacuum filtration separation, more preferably centrifugal separation, and / or, the drying is selected from any one or more of hot air drying, vacuum drying, airflow drying, static drying under negative pressure and fluidized bed drying, more preferably fluidized bed drying; preferably, the moisture content in the dried lactose microcrystals is 5.0 wt%~5.5 wt%.

10. A type of lactose crystal, characterized in that, The lactose crystals are prepared by the preparation method according to any one of claims 6-9, preferably the lactose crystals are lactose monohydrate, more preferably the purity of the lactose crystals is ≥97%, and more preferably ≥99%.

11. Selected from the following applications: (1) Application of asymmetric field flow separation in lactose separation and purification, preparation of lactose crystals, and / or improvement of lactose recovery rate, wherein the cross flow of the asymmetric field flow separation adopts an exponential decay mode and the tangential flow adopts a constant flow mode; (2) Application of asymmetric field flow separation and nanofiltration in lactose separation and purification, preparation of lactose crystals, and / or improvement of lactose recovery rate, wherein the crossflow of the asymmetric field flow separation adopts an exponential decay mode and the tangential flow adopts a constant flow mode; Preferably, the method for separating and purifying lactose is as described in any one of claims 1-5, and / or the method for preparing lactose crystals is as described in any one of claims 6-9; and / or, the lactose crystals are lactose monohydrate.