Method for separating milk oligosaccharide

Abstract

The invention provides a method for separating milk oligosaccharide, and belongs to the technical field of milk oligosaccharide separation. The invention aims to solve the technical problem that lactose and oligosaccharides are difficult to separate in order to improve the purity and production efficiency of milk oligosaccharides. The invention provides a method for separating milk oligosaccharides. The method comprises the following specific steps: step 1, filtering a waste liquid generated in preparation of whey liquid or whey eggs to obtain a filtrate; step 2, purifying the filtrate obtained in the step 1 by using a modified carbon filler column, and eluting by using an organic water solution to obtain an eluent; and 3, carrying out vacuum concentration on the eluent obtained in the step 2, and drying to obtain milk lactose oligosaccharide powder. The milk oligosaccharide is obtained by a non-chemical synthesis and synthetic biology method.

Description

Technical Field

[0001] The invention belongs to the technical field of separating milk oligosaccharides, and particularly relates to a method for separating milk oligosaccharides. Background Art

[0002] Milk oligosaccharides are a type of nutrient naturally present in milk, generally composed of 3-9 monosaccharides, including D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc), and sialic acid (Sia). Oligosaccharides naturally present in cow's milk are called bovine milk oligosaccharides (BMO), oligosaccharides naturally present in goat's milk are called caprine milk oligosaccharides (CMO), and milk oligosaccharides naturally present in human milk are called human milk oligosaccharides (HMOs, also known as human milk oligosaccharides). At present, there are more than 250 kinds of milk oligosaccharides with confirmed structures in breast milk. The main research objects are 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 3'-galactosyllactose (3'-Galactosyllactoses, 3'-GL), 4'-galactosyllactose (4'-Galactosyllactoses, 4'-GL) and 6'-galactosyllactose (6'-Galactosyllactoses, 6'-GL).

[0003] Studies have shown that milk oligosaccharides play an important role in improving intestinal flora microecology, maintaining intestinal barrier, regulating immunity, resisting pathogen infection and promoting neurodevelopment. Clinical intervention studies have shown that adding milk oligosaccharides can increase the proportion of bifidobacteria in the intestines of infants and young children, reduce the proportion of Escherichia coli, Streptococcus and toxin-producing Clostridium difficile, and the intestinal flora composition of infants and young children who consume foods containing milk oligosaccharides is more similar to that of breastfed children.

[0004] The production technology of milk oligosaccharides can be divided into three types: natural extraction, chemical synthesis and microbial synthesis. The product structure is exactly the same as HMOs in breast milk.

[0005] Natural extraction uses byproducts from cow and goat milk processing, and often uses membrane separation and chromatographic separation techniques. Nanofiltration and diafiltration are commonly used membrane separation methods. Since the molecular weight of lactose (342Da) is similar to the molecular weight of the main components of milk oligosaccharides (2'-FL, 3-FL, 3'-SL, 6'-SL) (487-633.Da), and the lactose content is very high, while the oligosaccharide content is very low, it is difficult and inefficient to separate, enrich and purify milk oligosaccharides using membrane technology. Exclusion chromatography and ion exchange chromatography are commonly used chromatographic separation methods. Exclusion chromatography uses the difference between the molecular weight of lactose and the molecular weight of milk oligosaccharides for separation. Due to the disadvantages of small sample load, slow separation, and expensive chromatographic column materials, exclusion chromatography is mostly used for the separation of milk oligosaccharides in scientific research. Ion exchange chromatography uses the difference in charge to separate lactose and acidic milk oligosaccharides. It is difficult to separate neutral milk oligosaccharides from lactose, resulting in a large loss of milk oligosaccharides. By modifying the ion exchange resin, the simulated moving bed chromatography (SMB) technology can be used to separate and purify milk oligosaccharides, but the equipment investment is large and the debugging is difficult. Due to the limitations of separation and extraction technology, it is difficult and costly to use the byproducts of cow and goat milk processing for natural extraction.

[0006] The chemical synthesis method has low efficiency, complex process, and food safety risks, so it has great market limitations and general development prospects. In 1999, ALY et al. used chemical methods for the first time to synthesize LNT and LNnT after 34 steps of reaction, but the yield was only 0.6%. SHERMAN et al. used propylene lactose and lactose as raw materials and introduced the glucosinolate method into the synthesis of LNT derivatives. Due to the many reaction steps of this synthetic strategy, the final yield was not ideal, only 8.6%. Although the chemical synthesis method has made great progress in the synthesis of HMOs, the use of a large amount of organic solvents, harsh reaction conditions, and lengthy reaction orders make its production cost expensive. The presence of toxic reagents also limits the application of chemically synthesized milk oligosaccharides in the food field.

[0007] Microbial synthesis technology based on bioengineered bacteria such as Escherichia coli and yeast is the main production form of commercial HMOs. In 2012, Hsu et al. used LNnT as a substrate and synthesized LNFP III using this method with a final yield of 49.6%. In 2015, Chen et al. started from lactoside and used a series of kinases, isomerases, uridyltransferases and glycosyltransferases to synthesize LNnT in a two-step method with a yield of 81%. Glycom, an American company, uses a modified strain (SCR6) of Escherichia coli K-12DH1 to ferment and produce 2'-FL. SCR6 was developed using recombinant DNA technology and added with sugar metabolism-related genes encoded by Klebsiella pneumoniae and Salmonella typhimurium, as well as 2'-FL biosynthesis-related genes encoded by Helicobacter pylori or Escherichia coli K-12DH1. On October 7, 2023, the National Health Commission approved two microbially synthesized milk oligosaccharides (2'-fucosyllactose and lactose-N-neotetraose) for use in infant formula, reconstituted milk powder (for children) and special medical infant food. Summary of the invention

[0008] The purpose of the present invention is to improve the purity and production efficiency of milk oligosaccharides and solve the technical problem of the difficulty in separating lactose and oligosaccharides.

[0009] The present invention provides a method for separating milk oligosaccharides, and the specific steps of the method are as follows:

[0010] Step 1: filtering the waste liquid generated by the production of whey liquid or whey protein to obtain a filtrate;

[0011] Step 2: After purifying the filtrate obtained in step 1 by using a modified carbon filler to fill a column, the filtrate is eluted with an aqueous solution of an organic solvent to obtain an eluate;

[0012] Step 3: The eluate obtained in step 2 is vacuum concentrated and then dried to obtain lactooligosaccharide powder.

[0013] It is further defined that the filtering method in step 1 is to separate the whey protein using an ultrafiltration membrane or a nanofiltration membrane, and the permeate is a solution containing lactose and milk oligosaccharides.

[0014] It is further defined that the method for obtaining the whey liquid in step 1 uses an ultrafiltration membrane with a maximum molecular weight cut-off of 5 KDa to process cheese.

[0015] It is further defined that the method for obtaining the whey protein liquid in step 1 is to filter the skim milk using a 100-3000 nm filter membrane, and the permeate is the whey protein liquid, and the skim milk is subjected to casein separation to obtain the whey protein liquid.

[0016] Further defined, casein is removed by acid precipitation, rennet method or microfiltration.

[0017] It is further defined that the purification method in step 2 is: using a modified carbon filler column to purify the obtained solution containing lactose and milk oligosaccharides.

[0018] Further defined, the elution method in step 2 is: loading 0.4-3BV at a flow rate of 0.3-1BV / h, using pure water to elute 2-3BV at a flow rate of 4-6BV / h for desalting, using 15% ethanol aqueous solution, 10% acetone aqueous solution, 10% ethanol aqueous solution or 50% methanol aqueous solution (V / V) at a flow rate of 4-6BV / h to elute 2-3BV to remove lactose, using 60% ethanol aqueous solution (V / V) at a flow rate of 6BV / h to elute 2BV.

[0019] It is further defined that the vacuum concentration in step 3 is: the obtained eluate rich in milk oligosaccharides is vacuum concentrated, the vacuum degree is -1 bar, the temperature gradient is 40-50°C, 40°C for 10 min, 42°C for 10 min, 44°C for 10 min, 46°C for 10 min, 48°C for 10 min, 50°C for 10 min, and finally concentrated to 40% of dry matter, and the product is concentrated while recovering ethanol to obtain a milk oligosaccharide concentrate.

[0020] It is further defined that the drying method in step 3 is as follows: the milk oligosaccharide concentrate is spray-dried with an inlet air temperature of 180° C. and an outlet air temperature of 95° C. to finally obtain milk oligosaccharide powder.

[0021] The present invention provides a milk oligosaccharide obtained by the above method.

[0022] Beneficial effects: The present invention provides a method for separating and purifying milk oligosaccharides from raw milk, whey, whey protein and whey protein production waste. Compared with the existing separation and purification methods, the method provided by the present invention has the following advantages:

[0023] 1. Compared with common separation fillers such as resins and agarose matrices, modified carbon fillers are used for separation, which has low purchase cost and extremely low waste cost in the later stage.

[0024] 2. Large volumes of samples (0.4-3BV) can be loaded without concentrating the raw materials. Molecular sieve fillers can only load samples of up to 0.1BV, and a certain concentration is required, otherwise the separation effect will be seriously reduced. Resin adsorption can be used for large volumes of samples. If resin adsorption is used, the separation process must use simulated moving bed technology. Too large a sample load affects the subsequent column separation, which limits the amount of sample loaded. In English literature, activated carbon is used to directly adsorb galacto-oligosaccharides (not the milk oligosaccharides purified by the present invention). The sample is loaded by placing the activated carbon in a solution of galacto-oligosaccharides and stirring for adsorption, which is very slow and labor-intensive.

[0025] 3. The equipment cost is low. Ordinary dairy companies can use the method provided by the present invention for production after simple transformation. Existing dairy processing and separation and purification equipment (disc separators, membrane concentration equipment, industrial medium-pressure chromatography pumps, industrial chromatography columns, spray drying, freeze drying machines) can be used. Disc separators remove fat from milk to reduce the impact of fat on subsequent extractions; membrane concentration equipment is a common production equipment, which can be used in the step of using membrane technology to separate whey protein and sugar as described in the present invention after a simple replacement of the membrane; industrial medium-pressure chromatography pumps and industrial chromatography columns are classic separation equipment and are the main equipment used to purify milk oligosaccharides and other impurities. Spray drying and freeze drying machines are classic drying equipment.

[0026] 4. The separation and purification speed is fast, and a complete purification process can be completed within 2 hours. For example, by using the classic three-column periodic counter-current chromatography, the purification process can be completed within 45 minutes.

[0027] This invention mainly involves the process of producing natural milk oligosaccharides from dairy processing waste (such as whey produced by cheese production, sugar-containing waste liquid produced by whey powder production, etc.), overcoming the problems described in previous literature regarding the use of membrane separation and chromatographic separation techniques, such as poor separation effect, slow speed, and large equipment investment.

[0028] The present invention uses modified carbon filler as a separation material and adopts a one-step column chromatography method for separation, so as to obtain a milk oligosaccharide mixture with at least 90% lactose removed. If the recovery rate is reduced, a lactose removal rate of more than 95% can be achieved. At present, most of the synthesized milk oligosaccharides on the market are separated and purified using simulated moving bed chromatography. This equipment is expensive and there are few manufacturers. A laboratory-level equipment is worth more than 800,000 yuan; the equipment debugging is complicated and requires professional engineers; using resin as a separation material, the subsequent recovery of the resin requires a special company to carry out, and the recovery cost is high. The one-step column chromatography equipment used in the present invention is cheap; the equipment is simple to operate, can be operated after simple training, and can be fully automated; the separation material used in the present invention is mainly a modified carbon filler, which can be used as fuel after recovery, and there is basically no recovery cost; the modified activated carbon separation material used in the present invention can be used in simulated moving bed chromatography for separation and purification, further improving the purity of the product.

[0029] The main process of the English literature is the adsorption of oligosaccharides by ordinary activated carbon, water elution, and elution with different concentrations of alcohol. It is an original separation method that requires a lot of manpower, consumes a lot of time, and cannot be automated. The residual lactose in the obtained product is relatively high. On this basis, optimization is carried out, and the chromatography column method is changed to the traditional activated carbon for separation. The prepared milk oligosaccharides contain more lactose, and the lactose removal rate is less than 70%. What is more serious is that the activated carbon has a strong adsorption capacity, and the adsorption is mostly dead adsorption, resulting in a large amount of milk oligosaccharide loss. Therefore, it is difficult to separate breast milk oligosaccharides from activated carbon. There are some research papers discussing the separation of milk oligosaccharides from waste products of dairy processing, which cannot be industrialized due to technical limitations or because the separation cost is too high. The early research object of milk oligosaccharides is milk oligosaccharides separated from breast milk, which is extremely expensive and sold by milligrams. The present invention is to extract milk oligosaccharides from milk and waste products produced by dairy products in an inexpensive way, which can be used for industrial production. There is literature on the use of activated carbon to separate functional sugars from soy whey. Soy whey is a term used to describe the wastewater produced by soybean processing, which refers to the whey of animal milk. The ingredients in soy whey are relatively simple, mainly glucose, xylose, sucrose raffinose, and stachyose. The structure of sugar is relatively simple. Oligosaccharides in milk are more complex sugars, and the separation difficulty will be higher. In addition, due to the strong adsorption capacity of ordinary activated carbon, a large number of target components are dead adsorbed, resulting in a low recovery rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a graph of the milk oligosaccharide analysis results of implementation 1, where the abscissa is the retention time and the ordinate is the response mv;

[0031] Figure 2 This is a graph of the analysis results of milk oligosaccharides in implementation 2, where the horizontal axis is the retention time and the vertical axis is the response mv;

[0032] Figure 3 This is a graph of the analysis results of milk oligosaccharides in implementation 3, where the horizontal axis is the retention time and the vertical axis is the response mv;

[0033] Figure 4 This is a graph of the milk oligosaccharide analysis results of implementation 4, where the abscissa is retention time and the ordinate is response mv. DETAILED DESCRIPTION

[0034] Preparation of modified carbon filler: Use scrapped industrial resin microspheres or newly made resin microspheres as starting microspheres, and obtain relatively clean microspheres through alkali washing, water washing and acid washing. Carbonization is carried out at 1000℃ under the protection of nitrogen:

[0035] 1. Pretreatment of resin microspheres: Take scrapped industrial resin microspheres and rinse them with tap water, add 3 times the volume of 1mol / L sodium hydroxide solution to clean and soak for 4-6 hours, rinse with deionized water until neutral, use 2 times the volume of 2mol / L hydrochloric acid aqueous solution, 1 times the volume of 1mol / L nitric acid aqueous solution and 1 times the volume of 3% hydrogen peroxide mixed solution to clean and soak for 4-6 hours, rinse with deionized water until neutral, add 3 times the volume of 0.2mol / L ferric chloride solution and 1 times the volume of 3% hydrogen peroxide solution to soak for 8 hours, and dry the pretreated resin microspheres or dry them at low temperature.

[0036] 2. Graphitization treatment: The dried pretreated resin microspheres were treated in a tubular furnace, heated to 800°C at 6°C / min under the protection of argon, heat treated for 3 hours after reaching the temperature, and naturally cooled to room temperature under the protection of argon to obtain modified carbon microspheres containing iron impurities.

[0037] 3. Cleaning and screening: Take out the modified carbon microspheres containing iron impurities, and use 2 volumes of deionized water to clean and soak for 6 hours. Take out the microspheres, and use 2 volumes of 4 mol / L hydrochloric acid aqueous solution and 1 volume of 2% hydrogen peroxide mixed solution to clean and soak for 12 hours. Take out the microspheres and wash them twice with deionized water. Take out the microspheres, add 2 volumes of 1 mol / L hydrochloric acid aqueous solution to clean and soak for 12 hours, and use deionized water to wash until neutral, take out the modified carbon microspheres and dry them or dry them at low temperature. Use a 40-mesh sieve to sieve the obtained modified carbon microspheres, and collect the sieve material, which is the modified carbon filler.

[0038] Description: The production process of food factories (such as water treatment, decolorization, purification and other processes) often produces a large amount of waste resins, which are usually handed over to environmental protection companies for recycling or resin manufacturers for recycling, which costs a lot of money. Most of these resins have a good spherical shape and uniform particle size, which are suitable as a source of carbon. Ferric chloride is a classic catalyst for reducing the temperature conditions of graphitization. Through the action of hydrogen peroxide, it can accelerate the reaction and increase the porosity of the microspheres. The graphitized microspheres are washed with hydrochloric acid to remove excess iron ions, and the addition of hydrogen peroxide can more fully remove impurities and improve the purity of the modified carbon microspheres. Screening is to obtain a relatively uniform modified carbon microsphere filler, which is beneficial to the subsequent separation and purification column filling and improves the resolution.

[0039] Example 1. A method for separating milk oligosaccharides

[0040] Raw milk is skimmed to obtain skimmed milk, and casein is separated from the skimmed milk to obtain whey protein solution. The method for separating casein is as follows: the raw milk is centrifuged and skimmed, and casein is removed by acid precipitation, rennet method or microfiltration method.

[0041] The method for separating whey protein is as follows: using an ultrafiltration membrane with a molecular weight cutoff of 1-5KDa to separate whey protein, using a nanofiltration membrane with a molecular weight cutoff greater than or equal to 1KDa to separate whey protein, and using repeated boiling or repeated freezing and thawing methods to separate whey protein.

[0042] 1. Use a disc separator to skim goat milk, and use a 100nm filter membrane to filter the skim milk. The permeate is whey protein liquid.

[0043] 2. Use an ultrafiltration membrane with a molecular weight cutoff of 3 KDa to separate whey protein, and the permeate is a solution containing lactose and milk oligosaccharides.

[0044] 3. Purify the obtained solution containing lactose and milk oligosaccharides using a modified carbon filler column, load 2BV at a flow rate of 1BV / h, use pure water to elute 2BV at a flow rate of 6BV / h for desalting, use 15% ethanol aqueous solution (V / V) to elute 3BV at a flow rate of 6BV / h to remove most of the lactose (90%-95% lactose), switch to 60% ethanol aqueous solution when the absorption at 200nm is 350AU, use 60% ethanol aqueous solution (V / V) to elute 2BV at a flow rate of 6BV / h.

[0045] 4. The 60% ethanol aqueous solution (V / V) eluent is a solution rich in milk oligosaccharides.

[0046] 5. The obtained solution rich in milk oligosaccharides is vacuum concentrated at a vacuum degree of -1 bar and a temperature gradient of 40-50°C, 40°C for 10 min, 42°C for 10 min, 44°C for 10 min, 46°C for 10 min, 48°C for 10 min, and 50°C for 10 min, and finally concentrated to 40% of dry matter. The product is concentrated while recovering ethanol to obtain a milk oligosaccharide concentrate.

[0047] 6. Freeze-dry the lactooligosaccharide concentrate at a pre-freezing temperature of -30°C, a pre-freezing time of 4 hours, a cold trap temperature of -50°C, and a vacuum degree of 1 Pa to finally obtain lactooligosaccharide powder.

[0048] After the sample was purified, the content of milk oligosaccharides was significantly increased, and the peak area of ​​milk oligosaccharides accounted for 89.33% of the total peak area. The final analysis results of milk oligosaccharides were shown in Figure 1 .

[0049] Example 2. A method for separating milk oligosaccharides

[0050] 1. The bovine whey obtained from cheese processing was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5 KDa. The permeate was a solution containing lactose and milk oligosaccharides.

[0051] 2. Use a modified carbon filler column to purify the obtained solution containing lactose and milk oligosaccharides, load 1BV at a flow rate of 0.5BV / h, use pure water to elute 3BV at a flow rate of 4BV / h, use 10% methanol aqueous solution (V / V) to elute 3BV at a flow rate of 4BV / h, and use 50% methanol aqueous solution (V / V) to elute 2BV at a flow rate of 4BV / h.

[0052] 4. The eluate of 50% methanol aqueous solution (V / V) is rich in milk oligosaccharides.

[0053] 5. The obtained solution rich in milk oligosaccharides is concentrated under vacuum at a vacuum degree of -1 bar and a temperature gradient of 40-50° C. The methanol is recovered and the product is concentrated to obtain a milk oligosaccharide concentrate.

[0054] 6. The milk oligosaccharide concentrate was spray dried with an inlet air temperature of 180°C and an outlet temperature of 95°C to finally obtain milk oligosaccharide powder.

[0055] After the sample was purified, the content of milk oligosaccharides was significantly increased, and the peak area of ​​milk oligosaccharides accounted for 91.38% of the total peak area. The final analysis results of milk oligosaccharides were shown in Figure 2 .

[0056] Example 3. A method for separating milk oligosaccharides

[0057] 1. Use a 100nm filter membrane to filter the nanofiltration permeate produced from goat whey protein concentrate (referring to the use of nanofiltration membrane to treat the whey solution obtained from dairy processing to remove part of the water, sugars and inorganic salts, etc., to increase the protein content in the whey solution, which is a routine operation for producing whey protein) to remove mechanical impurities and obtain a solution containing lactose and milk oligosaccharides.

[0058] 2. Use a modified carbon filler column to purify the obtained solution containing lactose and milk oligosaccharides, load 0.4BV at a flow rate of 0.3BV / h, use pure water to elute 3BV at a flow rate of 4BV / h, use 10% acetone aqueous solution (V / V) to elute 3BV at a flow rate of 4BV / h, and use 50% acetone aqueous solution (V / V) to elute 2BV at a flow rate of 4BV / h.

[0059] 4. The eluate of 50% acetone aqueous solution (V / V) is rich in milk oligosaccharides.

[0060] 5. The obtained eluate rich in milk oligosaccharides is concentrated under vacuum at a vacuum degree of -1 bar and a temperature gradient of 30-50° C. The acetone is recovered and the product is concentrated to obtain a milk oligosaccharide concentrate.

[0061] 6. Freeze-dry the lactooligosaccharide concentrate at a pre-freezing temperature of -30°C, a pre-freezing time of 4 hours, a cold trap temperature of -50°C, and a vacuum degree of 1 Pa to finally obtain lactooligosaccharide powder.

[0062] After the sample was purified, the content of milk oligosaccharides was significantly increased, and the peak area of ​​milk oligosaccharides accounted for 88.25% of the total peak area. The final analysis results of milk oligosaccharides were shown in Figure 3 .

[0063] Example 4. A method for separating milk oligosaccharides

[0064] 1. The waste liquid from the production of bovine colostrum (immunoglobulin) was filtered by 100nm microfiltration and 5KDa ultrafiltration to obtain a solution containing lactose and milk oligosaccharides from which mechanical impurities were removed.

[0065] 2. Use a modified carbon filler column to purify the obtained solution containing lactose and milk oligosaccharides, load 3BV at a flow rate of 0.4BV / h, use pure water to elute 2BV at a flow rate of 4BV / h, use 10% ethanol aqueous solution (V / V) to elute 3BV at a flow rate of 4BV / h, and use 60% ethanol aqueous solution (V / V) to elute 2BV at a flow rate of 4BV / h.

[0066] 4. The 60% ethanol aqueous solution (V / V) eluate is rich in milk oligosaccharides.

[0067] 5. The obtained eluate rich in milk oligosaccharides is concentrated under vacuum at a vacuum degree of -1 bar and a temperature gradient of 30-50° C. The ethanol is recovered and the product is concentrated to obtain a milk oligosaccharide concentrate.

[0068] 6. Freeze-dry the lactooligosaccharide concentrate at a pre-freezing temperature of -30°C, a pre-freezing time of 4 hours, a cold trap temperature of -50°C, and a vacuum degree of 1 Pa to finally obtain lactooligosaccharide powder.

[0069] After the sample was purified, the content of milk oligosaccharides was significantly increased, and the peak area of ​​milk oligosaccharides accounted for 92.56% of the total peak area. The final analysis results of milk oligosaccharides were shown in Figure 4 .

Claims

1. A method for separating milk oligosaccharides, characterized in that: The specific steps of the method are as follows: Step 1: filtering the waste liquid generated by the production of whey liquid or whey protein to obtain a filtrate; Step 2: After purifying the filtrate obtained in step 1 by using a modified carbon filler to fill a column, the filtrate is eluted with an aqueous solution of an organic solvent to obtain an eluate; Step 3: The eluate obtained in step 2 is vacuum concentrated and then dried to obtain lactooligosaccharide powder.

2. The method according to claim 1, characterized in that The filtration method in step 1 is to separate the whey protein using an ultrafiltration membrane or a nanofiltration membrane, and the permeate is a solution containing lactose and milk oligosaccharides.

3. The method according to claim 1, characterized in that The method for obtaining whey liquid in step 1 uses an ultrafiltration membrane with a maximum molecular weight cut-off of 5 KDa to process cheese.

4. The method according to claim 1, characterized in that In step 1, the method for obtaining the whey protein liquid is to filter the skim milk using a 100-3000 nm filter membrane, and the permeate is the whey protein liquid, and the skim milk is subjected to casein separation to obtain the whey protein liquid.

5. The method according to claim 4, characterized in that Casein is removed by acid precipitation, rennet method or microfiltration.

6. The method according to claim 1, characterized in that Purification method in step 2: purifying the obtained solution containing lactose and milk oligosaccharides using a modified carbon filler column.

7. The method according to claim 1, characterized in that The elution method in step 2 is as follows: load 0.4-3BV at a flow rate of 0.3-1BV / h, use pure water to elute 2-3BV at a flow rate of 4-6BV / h for desalting, use 15% ethanol aqueous solution, 10% acetone aqueous solution, 10% ethanol aqueous solution or 50% methanol aqueous solution (V / V) to elute 2-3BV at a flow rate of 4-6BV / h to remove lactose, and use 60% ethanol aqueous solution (V / V) to elute 2BV at a flow rate of 6BV / h.

8. The method according to claim 1, characterized in that Vacuum concentration in step 3: the obtained eluate rich in milk oligosaccharides is vacuum concentrated at a vacuum degree of -1 bar and a temperature gradient of 40-50°C, 40°C for 10 min, 42°C for 10 min, 44°C for 10 min, 46°C for 10 min, 48°C for 10 min, 50°C for 10 min, and finally concentrated to 40% of dry matter, and the product is concentrated while recovering ethanol to obtain a milk oligosaccharide concentrate.

9. The method according to claim 1, characterized in that: The drying method in step 3 is as follows: the milk oligosaccharide concentrate is spray dried with an inlet air temperature of 180° C. and an outlet air temperature of 95° C. to finally obtain milk oligosaccharide powder.

10. Milk oligosaccharides obtained by the method according to any one of claims 1 to 9.

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