Method for preparing and purifying cow milk oligosaccharide based on whey permeate

Abstract

The invention relates to the technical field of separation and enrichment of oligosaccharides, and particularly provides a preparation and purification method of cow milk oligosaccharides based on whey permeate, cow milk oligosaccharides are enriched by taking the whey permeate as a raw material, the reutilization of carbohydrates in the whey permeate is realized, and the economic value of the whey permeate is increased; the ceramic membrane and the nanofiltration spiral-wound membrane are combined, so that an efficient enrichment process is realized, lactose is removed thoroughly, the enriched oligosaccharide content is increased, and compared with a hydrolysis method for removing lactose, the problems of low efficiency, incomplete degradation and the like are avoided. Meanwhile, Z40-S filler is used, cow milk oligosaccharide and lactose in a sample can be well separated, the separation efficiency is high, the preparation scale is easy to enlarge, and industrial transformation can be achieved; in the preparation process, the cow milk oligosaccharide does not need to be derivatized, the original structure of the cow milk oligosaccharide is reserved, the purity is high, and an unexpected technical effect is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of separation and enrichment of oligosaccharides, and in particular to a method for preparing and purifying milk oligosaccharides based on whey permeate. Background Art

[0002] A growing body of research indicates that milk oligosaccharides play a crucial role in defending against enteric pathogens and maintaining intestinal microbial balance. Milk is rich in bioactive substances, such as antibodies, growth factors, and milk oligosaccharides. Among these, bovine milk oligosaccharides (BMOs) are expected to become a highly sought-after prebiotic product, exhibiting similar beneficial effects to human milk oligosaccharides (HMOs) in the intestines of infants and adults. BMOs are similar in composition to HMOs, consisting of monosaccharides such as glucose, galactose, N-acetylglucosamine, fucose, N-acetylneuraminic acid, and N-glycolylneuraminic acid, which is absent from the HMO structure. However, there is a significant difference in concentration between BMOs and HMOs (1-2 g / L and 12-14 g / L, respectively, in colostrum). Sialyl lactose (70%) predominates in bovine milk, primarily comprising 3'-SL and 6'-SL.

[0003] The increasing demand for milk oligosaccharides (HMOs) is driven by the difficulty in obtaining human milk, making it a commercially viable source. This has prompted the search for suitable sources of HMOs and the development of economically viable processes for their production. The main methods for preparing milk oligosaccharides include extraction from milk, chemical synthesis, enzymatic synthesis, and whole-cell fermentation using engineered strains. Each method has its advantages and limitations.

[0004] Whey permeate is a byproduct formed by ultrafiltration, which concentrates whey proteins from cheese whey leftover from the cheesemaking process. During the ultrafiltration process, whey proteins are retained by the membrane, while small molecules such as lactose and mineral salts permeate through the membrane, forming whey permeate. This permeate is light cream in color and has a slightly lower protein content and a higher lactose content (>80%) than standard whey. Global whey permeate production exceeds 200 million tons annually, and its full utilization is used to enrich milk oligosaccharides, thereby improving environmental and dairy processing-related issues. Current technologies for isolating oligosaccharides from whey permeate rely on a combination of lactose hydrolysis and membrane filtration. However, the high lactose concentration and low concentration of the target oligosaccharides make oligosaccharide separation challenging. Lactose hydrolysis also has drawbacks, such as the retention of monosaccharides in the hydrolyzate. After concentration by membrane filtration, the residual lactose and monosaccharide content in the hydrolyzate may be higher than the target oligosaccharides.

[0005] Therefore, it is of great significance to develop a method for preparing and purifying milk oligosaccharides based on whey permeate, which has high whey removal rate, high enrichment purity of milk oligosaccharides, few impurities and high preparation efficiency. Summary of the Invention

[0006] The purpose of the present invention is to address the problems existing in the prior art and provide a method for preparing and purifying milk oligosaccharides based on whey permeate, which has high whey removal rate, high milk oligosaccharide enrichment purity, few impurities and high preparation efficiency.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is: A method for preparing and purifying milk oligosaccharides based on whey permeate, comprising the following steps: Step 1: passing the bovine whey permeate aqueous solution through a ceramic membrane, washing with water, and collecting the permeate; Step 2: passing the permeate through a nanofiltration membrane and washing with water to obtain a cutoff liquid; Step 3: Pass the intercepted liquid through a Z40-S filler-filled preparation column, collect the column liquid, and perform reverse osmosis, freeze-drying, and vacuum drying to obtain the milk oligosaccharide component.

[0008] The present invention provides a method for preparing and purifying milk oligosaccharides based on whey permeate. This method uses whey permeate as a raw material to enrich milk oligosaccharides, enabling the reuse of carbohydrates in the whey permeate and increasing its economic value. Furthermore, the method combines ceramic membranes with nanofiltration membranes to achieve a highly efficient enrichment process, thoroughly removing lactose and increasing the oligosaccharide concentration. Compared with hydrolysis methods for lactose removal, this method avoids the problems of low efficiency and incomplete degradation. Furthermore, the present invention proposes the use of Z40-S filler (a charged ion exchange filler synthesized by bonding quaternary ammonium groups to silane groups, exhibiting extremely strong anion exchange capacity and particularly suitable for the exchange of weakly acidic compounds). This method effectively separates milk oligosaccharides from lactose in a sample, achieving high separation efficiency, facilitating scale-up of the preparation process, and enabling industrial conversion. During the preparation process, the milk oligosaccharides do not require derivatization, retaining their original structure and achieving high purity, achieving unexpected technical benefits.

[0009] Furthermore, Z40-S filler is a silica-based filler with strong anion exchange properties. Its high charge density and strong anion exchange capacity are achieved by bonding quaternary ammonium groups to silane groups. This makes it suitable for exchanging weakly acidic compounds, such as sialic acid derivatives in milk oligosaccharides. Since the target extract of this invention is acidic sugars, this filler demonstrates excellent separation efficiency. The filler structure is shown below.

[0010] .

[0011] Furthermore, in step 1, the bovine whey permeate aqueous solution is prepared by the following method: taking bovine whey permeate, adding it into 40-80°C water at a concentration of 30-60 mg / mL and dissolving it, standing it to precipitate, and then taking the supernatant and filtering it to obtain a filtrate.

[0012] Furthermore, in step 1, the pore size of the ceramic membrane is 400-600 nm. The main purpose of the ceramic membrane is to filter out macromolecular impurities and particulate matter in the aqueous solution of bovine whey permeate, including microorganisms, fat globules in whey, incompletely dissolved protein aggregates or precipitates, and some macromolecular proteins.

[0013] Furthermore, in step 2, the pore size of the nanofiltration roll membrane is 300-500D, and the pressure passing through the nanofiltration roll membrane is 0.2-0.25 MPa.

[0014] Furthermore, a Z40-S filler-packed preparative column was prepared using the following method: a 50DAC preparative column packed with Z40-S filler was used, the Z40-S filler was activated with methanol, the filler was then rinsed with formic acid water with a volume concentration of 3%-6%, and finally rinsed with pure water until neutral.

[0015] The present invention provides a method for preparing and purifying milk oligosaccharides based on whey permeate. This method uses whey permeate as a raw material to enrich milk oligosaccharides, thereby reusing carbohydrates in the whey permeate and increasing its economic value. Furthermore, the present invention combines a ceramic membrane with a nanofiltration roll membrane to achieve an efficient enrichment process, thoroughly removing lactose and increasing the oligosaccharide enrichment content. Compared with lactose removal by hydrolysis, this method avoids the problems of low efficiency and incomplete degradation. Furthermore, the present invention proposes the use of Z40-S filler (a charged ion exchange filler with extremely strong anion exchange capacity, suitable for exchanging weakly acidic compounds). This method can effectively separate milk oligosaccharides from lactose in a sample, achieves high separation efficiency, and is easy to scale up for industrial conversion. During the preparation process, the milk oligosaccharides do not require derivatization, retaining their original structure and achieving high purity, achieving unexpected technical results. DETAILED DESCRIPTION

[0016] The present invention will be described in detail below in conjunction with the solutions.

[0017] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the solutions and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0018] The embodiments of the present application are described in detail below. Examples of the embodiments are shown in the scheme, but unnecessary detailed descriptions may be omitted. For example, detailed descriptions of well-known matters and repeated descriptions of actually identical structures may be omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate understanding by those skilled in the art. In addition, the scheme and the following description are provided for those skilled in the art to fully understand the present application and are not intended to limit the subject matter described in the claims.

[0019] Unless otherwise defined, all technical and scientific terms used in this application have the same meanings as those commonly understood by technicians in the technical field to which this application belongs; the terms used in this application are only for the purpose of describing specific embodiments and are not intended to limit this application; unless otherwise specified, the numerical values ​​of the various parameters mentioned in this application can be measured using various measurement methods commonly used in the art (for example, they can be tested according to the methods given in the embodiments of this application).

[0020] The terms "include" and "have" in the description and claims of this application and any variations thereof are open expressions, that is, including the contents specified in this application but not excluding other contents.

[0021] In the description of this application, regardless of whether the word "about" or "approximately" is used, all numbers disclosed herein are approximate values. The value of each number may vary by less than 10% or by a reasonable difference considered by a person skilled in the art, such as 1%, 2%, 3%, 4% or 5%.

[0022] " range " disclosed in the present application is limited in the form of lower limit and upper limit, and given range is limited by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of special range. The scope limited in this way can be to include end value or not include end value, and can be arbitrarily combined, that is, any lower limit can form a range with any upper limit combination. For example, if the scope of 60-120 and 80-110 is listed for specific parameters, it is understood that the scope of 60-110 and 80-120 is also expected. In addition, if the minimum range value 1 and 2 are listed, and if the maximum range value 3,4 and 5 are listed, then the following range can all be expected: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In this application, unless otherwise specified, the numerical range " ab " represents the abbreviation of any real number combination between a and b, wherein a and b are all real numbers. For example, a numerical range of "0-5" indicates that all real numbers between "0-5" are listed herein, and "0-5" is simply an abbreviation for these numerical combinations. Furthermore, when a parameter is expressed as an integer ≥ 2, this is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0023] In the description of this application, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly specifying the quantity of the technical features indicated. "First feature" and "second feature" may include one or more of the features.

[0024] In the description of this application, "A and / or B" may include the case of A alone, the case of B alone, or any of the cases of A and B, where A and B are only used for example, and may be any technical feature connected by "and / or" in this application.

[0025] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0026] Unless otherwise specified, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0027] The present invention provides a method for preparing and purifying milk oligosaccharides based on whey permeate, comprising the following steps: Step 1: passing the bovine whey permeate aqueous solution through a ceramic membrane, washing with water, and collecting the permeate; Step 2: passing the permeate through a nanofiltration membrane and washing with water to obtain a cutoff liquid; Step 3: Pass the intercepted liquid through a Z40-S filler-filled preparation column, collect the column liquid, and perform reverse osmosis, freeze-drying, and vacuum drying to obtain the milk oligosaccharide component.

[0028] The invention uses whey permeate as a raw material to enrich milk oligosaccharides, thereby reusing the carbohydrates in the whey permeate and increasing its economic value. Furthermore, the invention combines a ceramic membrane with a nanofiltration roll membrane to achieve an efficient enrichment process, completely removing lactose and increasing the oligosaccharide enrichment content. Compared with the hydrolysis method for lactose removal, this method avoids the problems of low efficiency and incomplete degradation. Furthermore, the invention proposes the use of Z40-S filler (a charged ion exchange filler synthesized by bonding quaternary ammonium groups to silane groups, which has extremely strong anion exchange capacity and is suitable for exchanging weakly acidic compounds). This method can effectively separate milk oligosaccharides from lactose in the sample, with high separation efficiency, ease of scale-up, and industrial conversion. During the preparation process, the milk oligosaccharides do not require derivatization, retaining their original structure and high purity, achieving unexpected technical results.

[0029] In some examples, it was found that, in the technical solution protected by this application, only removing the ceramic membrane, only removing the nanofiltration spiral membrane, or only removing the Z40-S filler in the preparative column, while other conditions remained unchanged, significantly reduced the purity of milk oligosaccharides and the separation efficiency. Adjusting the order of membrane filtration also reduced the purification effect.

[0030] In some embodiments, in step 1, the bovine whey permeate aqueous solution is prepared by the following method: taking bovine whey permeate, dissolving it in 40-80°C water at a concentration of 30-60 mg / mL, standing it to precipitate, and then filtering the supernatant to obtain a filtrate.

[0031] In some embodiments, in step 1, the pore size of the ceramic membrane is 400-600 nm.

[0032] In some embodiments, in step 2, the pore size of the nanofiltration spiral membrane is 300-500D, and the pressure passing through the nanofiltration spiral membrane is 0.2-0.25 MPa. Specifically, the following steps are performed: 500 nm permeate is passed through a 300-500D spiral membrane (purchased from Nanjing Membrane Materials Industry Research Institute, model number NF300-500 98241). Top-wash with 20 L of water is performed four times, with 5 L of water added each time. The membrane pressure is controlled at 0.2-0.25 MPa (Note: This parameter is critical). 30% of the lactose can be removed, and the retentate is collected for later use (retentate volume 1 L, solids content 167 mg / mL).

[0033] The liquid chromatography-mass spectrometry analysis conditions of the intercepted liquid are as follows: loading 180 mL (30 g), water elution: 4 BV (the loading volume of the filler in the chromatographic column), elution with 5% formic acid water: 4 BV, and elution with 60% ethanol + 5% formic acid water: 4 BV.

[0034] Mobile phase A was acetonitrile, and mobile phase B was 10 mmol / L ammonium acetate (pH = 10.5); Chromatographic column: BEH Amide 1.7 μm, 2.1*150 mm (manufactured by Waters Corporation, USA, model: SKU: 186004802); Flow rate: 0.2 mL / min; Analysis method: 20~55%B, 30min.

[0035] Most of the acidic sugars are enriched in 1-2BV of acid water elution, and some are also enriched in pure water elution. The content in the remaining fractions is low, so the preparation can continue. At the same time, the preparation plan is adjusted: acid water elution is adjusted to 3BV, and 60% ethanol elution is adjusted to 2BV.

[0036] In some embodiments, a Z40-S filler-packed preparative column is prepared by the following method: a 50DAC preparative column is packed with Z40-S filler, the Z40-S filler is activated with methanol, the filler is then rinsed with formic acid water having a volume concentration of 3%-6%, and finally rinsed with pure water until neutral.

[0037] Specific embodiments are as follows: Example

[0038] Sample preparation: A 1.5 kg sample of bovine whey permeate was reconstituted in 50°C hot water at a solid-to-liquid ratio of 1:20, yielding 30 L of reconstituted solution, which was then allowed to stand overnight. When the supernatant was decanted, the bottom precipitate dispersed, and the decanted supernatant was subsequently filtered through filter paper. The filtered reconstituted supernatant was clarified using a 500 nm ceramic membrane and top-washed with 4 L of water, yielding 30 L of permeate (original (500 nm)) with a solids content of 43 mg / mL. The 30 L permeate was then nanofiltered using a 230333 nanofiltration membrane (purchased from Nanjing Membrane Materials Industry Research Institute, 230333 is the device model), yielding 30 L of clarified solution (230333-CQ1) and 2.5 L of retentate (230333-JL1). The content analysis is shown in Table 1.

[0039] 30 L of permeate was nanofiltered using nanofiltration membrane 230333 to obtain 30 L of clarified liquid (230333-CQ1) and 2.5 L of retentate (230333-JL1). The content analysis is shown in Table 1.

[0040] Table 1 230333-1 Acidic sugar content in actual sample (mg / ml) Lactose content in actual sample (mg / ml) Volume (L) Acidic sugar (g) Lactose (g) Original (500nm) 8.28 20.613 30 248.404 618.389 230333-CQ1 4.514 7.996 30 135.413 239.865 230333-JL1 20.584 102.612 2.5 51.459 256.531 According to the effect of membrane separation, acidic sugars with large molecular weight were retained by the spiral membrane. The content analysis of the samples after passing through the membrane showed that the yield of acidic sugars and lactose in the original sample after passing through the membrane was only 78.8%, which was a serious loss. Without considering the yield, the acidic sugar and lactose content in the permeate were compared and analyzed (assuming that the acidic sugars were permeated). Compared with the acidic sugar content in the original sample (28%), the acidic sugar content in the clarified liquid increased (36%). Example

[0041] Sample preparation: A 400 g sample of bovine whey permeate was dissolved in 8 L of 60°C hot water at a concentration of 50 mg / mL, allowed to stand for 4 h to separate out a precipitate, and then filtered with suction. The filtrate was passed through a 500 nm ceramic membrane and top washed with 20 L of water twice, each with 10 L of water. The 500 nm clarified solution was passed through a 300-500D (NF300-500 98241) spiral membrane and top washed with 20 L of water four times, each with 5 L of water. The membrane pressure was controlled at 0.2-0.25 MPa, which removed 30% of the lactose. The retained solution was then used for standby use (retained solution volume 1 L, solid content 167 mg / mL).

[0042] Z40-S preparative column packing: Take 300 g of Z40-S filler and load it into a 50DAC preparative column. Use 1.5 L of methanol to activate the Z40-S filler, then elute the filler with 1.5 L of 5% formic acid water, and finally elute with pure water until it becomes neutral.

[0043] Preparation plan: (1) Sample: 180 mL (30 g); (2) Water washing: 4BV; (3) 5% formic acid water elution: 4 BV; (4) 60% ethanol + 5% formic acid water elution: 4 BV; The target fractions are combined, subjected to membrane concentration treatment, freeze-dried, and dried under reduced pressure to obtain the acidic sugar fraction. Example

[0044] Process scale-up preparation: Dissolve 6 kg of bovine whey permeate in 120 L of 60°C hot water. Allow to stand overnight. Pass the supernatant through a 500 nm ceramic membrane. The clarified liquid from the 500 nm ceramic membrane is then passed through a 300-500D (NF300-500 98241) spiral membrane. The retentate (volume 11.2 L, solids content 186 mg / mL) is then used.

[0045] Preparation plan: (1) Sample volume: 800 mL (150 g); (2) Water washing: 4BV; (3) 5% formic acid water elution: 3BV; (4) 60% ethanol + 5% formic acid water elution: 2 BV; The target fractions are combined, subjected to membrane concentration treatment, freeze-dried, and dried under reduced pressure to obtain the acidic sugar fraction.

[0046] Using CAD detection: (1) Mobile phase A: 15 mmol / L ammonium acetate-acetonitrile; Mobile phase B: 15 mmol / L ammonium acetate (pH = 10.5).

[0047] (2) Chromatographic column: BEH Amide 1.7 μm, 2.1*150 mm, SN: 01743921818611.

[0048] (3) Flow rate: 0.2 mL / min.

[0049] (4) Analysis method: 20~40%B, 15min.

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing and purifying milk oligosaccharides based on whey permeate, characterized in that: The following steps are involved: Step 1: passing the bovine whey permeate aqueous solution through a ceramic membrane, washing with water, and collecting the permeate; Step 2: passing the permeate through a nanofiltration membrane and washing with water to obtain a cutoff liquid; Step 3: Pass the intercepted liquid through a Z40-S filler-filled preparation column, collect the column liquid, and perform reverse osmosis, freeze-drying, and vacuum drying to obtain the milk oligosaccharide component.

2. The method for preparing and purifying milk oligosaccharides based on whey permeate according to claim 1, characterized in that: In step 1, the bovine whey permeate aqueous solution is prepared by the following method: taking bovine whey permeate, adding it into 40-80°C water at a concentration of 30-60 mg / mL to dissolve it, standing it to precipitate, and then taking the supernatant and filtering it to obtain a filtrate.

3. The method for preparing and purifying milk oligosaccharides based on whey permeate according to claim 2, characterized in that: In step 1, the pore size of the ceramic membrane is 400-600 nm.

4. The method for preparing and purifying milk oligosaccharides based on whey permeate according to claim 1, characterized in that: In the step 2, the pore size of the nanofiltration roll membrane is 300-500D, and the pressure passing through the nanofiltration roll membrane is 0.2-0.25 MPa.

5. The method for preparing and purifying milk oligosaccharides based on whey permeate according to claim 1, characterized in that: The Z40-S filler-packed preparative column was prepared by the following method: a 50DAC preparative column was packed with Z40-S filler, the Z40-S filler was activated with methanol, the filler was then washed with formic acid water with a volume concentration of 3%-6%, and finally washed with pure water until neutral.