CN118085121A - Method for increasing content of sialyllactose in milk oligosaccharide, oligosaccharide powder prepared by method and food - Google Patents
Method for increasing content of sialyllactose in milk oligosaccharide, oligosaccharide powder prepared by method and food Download PDFInfo
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- CN118085121A CN118085121A CN202410524903.3A CN202410524903A CN118085121A CN 118085121 A CN118085121 A CN 118085121A CN 202410524903 A CN202410524903 A CN 202410524903A CN 118085121 A CN118085121 A CN 118085121A
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- nanofiltration
- sialyllactose
- oligosaccharide
- trapped fluid
- concentration
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- 229920001542 oligosaccharide Polymers 0.000 title claims abstract description 58
- 150000002482 oligosaccharides Chemical class 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 44
- 239000000843 powder Substances 0.000 title claims abstract description 41
- OIZGSVFYNBZVIK-FHHHURIISA-N 3'-sialyllactose Chemical compound O1[C@@H]([C@H](O)[C@H](O)CO)[C@H](NC(=O)C)[C@@H](O)C[C@@]1(C(O)=O)O[C@@H]1[C@@H](O)[C@H](O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O)O[C@H](CO)[C@@H]1O OIZGSVFYNBZVIK-FHHHURIISA-N 0.000 title claims abstract description 13
- 235000013336 milk Nutrition 0.000 title claims abstract description 12
- 239000008267 milk Substances 0.000 title claims abstract description 12
- 210000004080 milk Anatomy 0.000 title claims abstract description 12
- 235000013305 food Nutrition 0.000 title claims abstract description 7
- 238000001728 nano-filtration Methods 0.000 claims abstract description 101
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 76
- 239000007788 liquid Substances 0.000 claims abstract description 47
- 239000003957 anion exchange resin Substances 0.000 claims abstract description 37
- 239000012530 fluid Substances 0.000 claims abstract description 36
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims abstract description 32
- 229910052801 chlorine Inorganic materials 0.000 claims abstract description 32
- 239000000460 chlorine Substances 0.000 claims abstract description 32
- 239000005862 Whey Substances 0.000 claims abstract description 30
- 102000007544 Whey Proteins Human genes 0.000 claims abstract description 30
- 108010046377 Whey Proteins Proteins 0.000 claims abstract description 30
- TYALNJQZQRNQNQ-UHFFFAOYSA-N #alpha;2,6-sialyllactose Natural products O1C(C(O)C(O)CO)C(NC(=O)C)C(O)CC1(C(O)=O)OCC1C(O)C(O)C(O)C(OC2C(C(O)C(O)OC2CO)O)O1 TYALNJQZQRNQNQ-UHFFFAOYSA-N 0.000 claims abstract description 29
- CILYIEBUXJIHCO-UHFFFAOYSA-N 102778-91-6 Natural products O1C(C(O)C(O)CO)C(NC(=O)C)C(O)CC1(C(O)=O)OC1C(O)C(OC2C(C(O)C(O)OC2CO)O)OC(CO)C1O CILYIEBUXJIHCO-UHFFFAOYSA-N 0.000 claims abstract description 29
- DVGKRPYUFRZAQW-UHFFFAOYSA-N 3 prime Natural products CC(=O)NC1OC(CC(O)C1C(O)C(O)CO)(OC2C(O)C(CO)OC(OC3C(O)C(O)C(O)OC3CO)C2O)C(=O)O DVGKRPYUFRZAQW-UHFFFAOYSA-N 0.000 claims abstract description 29
- CILYIEBUXJIHCO-UITFWXMXSA-N N-acetyl-alpha-neuraminyl-(2->3)-beta-D-galactosyl-(1->4)-beta-D-glucose Chemical compound O1[C@@H]([C@H](O)[C@H](O)CO)[C@H](NC(=O)C)[C@@H](O)C[C@@]1(C(O)=O)O[C@@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)O[C@@H]2CO)O)O[C@H](CO)[C@@H]1O CILYIEBUXJIHCO-UITFWXMXSA-N 0.000 claims abstract description 29
- OIZGSVFYNBZVIK-UHFFFAOYSA-N N-acetylneuraminosyl-D-lactose Natural products O1C(C(O)C(O)CO)C(NC(=O)C)C(O)CC1(C(O)=O)OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1O OIZGSVFYNBZVIK-UHFFFAOYSA-N 0.000 claims abstract description 29
- TYALNJQZQRNQNQ-JLYOMPFMSA-N alpha-Neup5Ac-(2->6)-beta-D-Galp-(1->4)-beta-D-Glcp Chemical compound O1[C@@H]([C@H](O)[C@H](O)CO)[C@H](NC(=O)C)[C@@H](O)C[C@@]1(C(O)=O)OC[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)O[C@@H]2CO)O)O1 TYALNJQZQRNQNQ-JLYOMPFMSA-N 0.000 claims abstract description 29
- 238000010612 desalination reaction Methods 0.000 claims abstract description 25
- 239000000945 filler Substances 0.000 claims abstract description 20
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims abstract description 16
- 239000008101 lactose Substances 0.000 claims abstract description 16
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 10
- 238000011033 desalting Methods 0.000 claims abstract description 8
- 238000001035 drying Methods 0.000 claims abstract description 6
- 238000013375 chromatographic separation Methods 0.000 claims abstract description 5
- 238000011097 chromatography purification Methods 0.000 claims abstract description 4
- 238000000108 ultra-filtration Methods 0.000 claims description 32
- 239000012528 membrane Substances 0.000 claims description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 239000003513 alkali Substances 0.000 claims description 15
- 238000010790 dilution Methods 0.000 claims description 13
- 239000012895 dilution Substances 0.000 claims description 13
- 239000012466 permeate Substances 0.000 claims description 12
- 239000006227 byproduct Substances 0.000 claims description 5
- 235000013351 cheese Nutrition 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 4
- 229910021641 deionized water Inorganic materials 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 3
- 235000020244 animal milk Nutrition 0.000 claims description 3
- 230000014759 maintenance of location Effects 0.000 claims description 3
- 238000001471 micro-filtration Methods 0.000 claims description 3
- 230000002829 reductive effect Effects 0.000 claims description 3
- 239000012465 retentate Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 2
- 239000002585 base Substances 0.000 claims 1
- 238000000926 separation method Methods 0.000 abstract description 12
- 235000013365 dairy product Nutrition 0.000 abstract description 2
- 239000012071 phase Substances 0.000 description 26
- 238000001514 detection method Methods 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- 235000020247 cow milk Nutrition 0.000 description 11
- 238000000909 electrodialysis Methods 0.000 description 9
- 239000011347 resin Substances 0.000 description 8
- 229920005989 resin Polymers 0.000 description 8
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 6
- 235000021255 galacto-oligosaccharides Nutrition 0.000 description 6
- 150000003271 galactooligosaccharides Chemical class 0.000 description 6
- 238000012856 packing Methods 0.000 description 5
- PXBFMLJZNCDSMP-UHFFFAOYSA-N 2-Aminobenzamide Chemical compound NC(=O)C1=CC=CC=C1N PXBFMLJZNCDSMP-UHFFFAOYSA-N 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 239000012614 Q-Sepharose Substances 0.000 description 3
- 235000013350 formula milk Nutrition 0.000 description 3
- QHXLIQMGIGEHJP-UHFFFAOYSA-N picoline - borane complex Substances [B].CC1=CC=CC=N1 QHXLIQMGIGEHJP-UHFFFAOYSA-N 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000002390 rotary evaporation Methods 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 235000020256 human milk Nutrition 0.000 description 2
- 210000004251 human milk Anatomy 0.000 description 2
- 239000008176 lyophilized powder Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- -1 BEH Amide Chemical class 0.000 description 1
- 229920002271 DEAE-Sepharose Polymers 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 235000013406 prebiotics Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000006268 reductive amination reaction Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Molecular Biology (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Sustainable Development (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Dairy Products (AREA)
Abstract
The invention discloses a method for improving sialyllactose content in milk oligosaccharide, oligosaccharide powder and food prepared by the method, belonging to the field of dairy product separation, comprising the following steps: sequentially ultrafiltering and nanofiltration of the whey liquid, separating and purifying the nanofiltration trapped fluid by chromatography, removing lactose-containing chromatography trapped fluid, collecting sialyllactose-containing chromatography trapped fluid, desalting, and drying to obtain oligosaccharide powder; wherein, in chromatographic separation and purification, chlorine type strong-alkaline anion exchange resin is adopted as a filler, and the mobile phase is HCl and/or acetic acid. The invention takes chlorine-type strong-alkalinity anion exchange resin as a filler, takes acetic acid or HCl as a mobile phase for separation and nanofiltration desalination to obtain oligosaccharide freeze-dried powder, wherein the content of 3 '-sialyllactose and 6' -sialyllactose in the oligosaccharide freeze-dried powder is more than 40 percent; compared with the prior art, the method improves.
Description
Technical Field
The invention relates to the technical field of dairy separation, in particular to a method for improving the content of sialyllactose in milk oligosaccharide, and oligosaccharide powder and food prepared by the method.
Background
The breast milk oligosaccharide has special biological activity function for the growth and development of infants, and is an indispensable component for the mother emulsification of infant formula milk powder. The natural resources of breast milk oligosaccharides are very limited, but the infant formula market is in great demand for them. It is possible to use cow milk oligosaccharides instead of cow milk oligosaccharides to be added to infant formulas. Whey powder is used as a byproduct generated in a plurality of production processes at present, has the characteristics of relatively low price, storage resistance and easy acquisition, and is suitable for being used as a raw material for producing cow milk oligosaccharide.
In the prior research of the applicant, on the basis of ultrafiltration and nanofiltration of the whey liquid, the obtained nanofiltration trapped fluid is subjected to chromatographic separation by using DEAE-sepharose or Q-sepharose as a filler and passing through a Superdex 30increase pre-packed column, and then is subjected to nanofiltration desalination or electrodialysis desalination and drying to obtain the cow milk oligosaccharide powder. The total mass percentage of the 3 '-sialyllactose and the 6' -sialyllactose in the cow milk oligosaccharide powder can reach 25 percent. However, the percentage of 3 '-sialyllactose and 6' -sialyllactose is still low, which is difficult to meet the demand, and a method for improving the content is needed.
Disclosure of Invention
Object of the Invention
In order to overcome the defects, the invention aims to provide a method for improving the content of sialyllactose in milk oligosaccharide, and oligosaccharide powder and food prepared by the method. The invention uses chlorine type strong alkaline anion exchange resin 1 or chlorine type strong alkaline anion exchange resin 2 as a filler, uses acetic acid or HCl as a mobile phase to separate and nanofiltration desalt, and the content of 3 '-sialyllactose and 6' -sialyllactose in the obtained oligosaccharide freeze-dried powder reaches more than 40%; compared with the prior art, the method improves.
Solution scheme
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
In a first aspect, the present invention provides a method for increasing sialyllactose content in milk oligosaccharides comprising the steps of: sequentially ultrafiltering and nanofiltration of the whey liquid, separating and purifying the nanofiltration trapped fluid by chromatography, removing lactose-containing chromatography trapped fluid, collecting sialyllactose-containing chromatography trapped fluid, desalting, and drying to obtain oligosaccharide powder;
wherein, in chromatographic separation and purification, chlorine type strong-alkaline anion exchange resin is adopted as a filler, and the mobile phase is HCl and/or acetic acid.
Further, the mesh number of the chlorine type strong-alkali anion exchange resin is 50-400, the water retention capacity is 39-80%, and the total exchange capacity is 0.6-1.2meq/mL.
Further, the mesh number of the chlorine type strong-alkali anion exchange resin is 50-400, the water retention capacity is 65% -80%, and the total exchange capacity is 0.6-0.7meq/mL.
Further, the chlorine-type strongly basic anion exchange resin is purchased from Shanghai' an de Biotechnology Co., ltd.
Further, in the mobile phase, the concentration of HCl is 0.05-0.5 mol/L; the concentration of acetic acid is 0.05-1 mol/L.
Further, the concentration of HCl is 0.15mol/L; the concentration of acetic acid is 1mol/L.
Further, the whey liquid is a byproduct whey liquid generated during cheese making, or a whey liquid obtained by redissolving desalted whey powder, or a permeate liquid prepared by microfiltration of defatted animal milk.
Further, ultrafiltering for at least three times, and concentrating the whey liquid to a concentration multiple of 2-7 times by ultrafiltration to obtain a first ultrafiltered permeate; adding water into the first ultrafiltration trapped fluid for dilution; ultrafiltering and concentrating again until the concentration multiple is 2-7 times to obtain a second ultrafiltrate; adding water into the ultrafiltration trapped fluid of the second time for dilution; ultrafiltering and concentrating again until the concentration multiple is 2-7 times to obtain a third ultrafiltrate; and so on.
Further, in the ultrafiltration step, the molecular weight cut-off of the ultrafiltration membrane is 5 kDa-30 kDa.
Further, the ultrafiltration conditions were: the temperature of the feed liquid is 20-45 ℃, the inlet pressure is 6-9 bar, and the outlet pressure is 5-8 bar; the membrane pressure difference is not more than 3ba.
Further, the nanofiltration times are at least three times, and ultrafiltration permeate is concentrated to be 2-13 times of concentration times through nanofiltration, so that first nanofiltration trapped fluid is obtained; adding water into the first nanofiltration trapped fluid for dilution; ultrafiltering and concentrating again until the concentration multiple is 2-13 times to obtain second nanofiltration trapped fluid; adding water into the second nanofiltration trapped fluid for dilution; carrying out nanofiltration and concentration again until the concentration multiple is 2-13 times to obtain a third nanofiltration trapped fluid; and so on.
Further, in the nanofiltration step, the molecular weight cut-off of the nanofiltration membrane is 100Da to 3000Da.
Further, the nanofiltration conditions are: the temperature of the feed liquid is 20-45 ℃, the inlet pressure is 16-20 bar, and the outlet pressure is 14-18 bar; the membrane pressure difference is not more than 3bar.
Further, the desalting step adopts nanofiltration for desalting.
Further, the nanofiltration desalination step comprises: separating the chromatographic collection liquid by nanofiltration, washing with deionized water until the conductivity is no longer reduced, and concentrating to the minimum circulation volume of the equipment to obtain nanofiltration concentrated retention liquid, wherein nanofiltration concentration is finished; optionally, in nanofiltration desalination, the molecular weight cut-off of the nanofiltration membrane is 100 Da-350 Da;
further, in nanofiltration desalination, the nanofiltration membrane is a nanofiltration acid-resistant membrane;
further, in nanofiltration desalination, the conditions are set as follows: the temperature of the feed liquid is 20-45 ℃, the inlet pressure is 10-15 bar, and the outlet pressure is 5-10 bar; the membrane pressure difference is not more than 3bar.
In a second aspect, there is provided an oligosaccharide powder prepared by the method of the first aspect;
The total mass percentage of the 3 '-sialyllactose and the 6' -sialyllactose in the oligosaccharide powder is not less than 40%.
In a third aspect, there is provided an oligosaccharide food or health product comprising an oligosaccharide powder obtainable by a process according to the first aspect or an oligosaccharide powder according to the second aspect.
Advantageous effects
The invention uses chlorine type strong alkaline anion exchange resin 1 or chlorine type strong alkaline anion exchange resin 2 as a filler, uses acetic acid or HCl as a mobile phase to separate and nanofiltration desalt, and the content of 3 '-sialyllactose and 6' -sialyllactose in the obtained oligosaccharide freeze-dried powder reaches more than 40%; compared with the prior art, the method improves.
Drawings
One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.
FIG. 1 is a separation graph of method a) according to example 3 of the present invention, wherein the chromatographic conditions are: in a chromatographic column of 1.5cm by 20cm, a chlorine type strongly basic anion exchange resin 2 is used as a filler, and HCl is used as a mobile phase. Wherein Lac is lactose, GOS is galactooligosaccharide, and SL is 3 '-sialyllactose and 6' -sialyllactose.
FIG. 2 is a separation graph of method b) of example 3 of the present invention, wherein the chromatographic conditions are: in a 10cm×50cm column, a chloride-type strongly basic anion exchange resin 1 was used as a packing, and HCl as a mobile phase. Wherein GOS is galacto-oligosaccharide and SL is 3 '-sialyllactose and 6' -sialyllactose.
FIG. 3 is a separation graph of method c) of example 3 of the present invention, wherein the chromatographic conditions are: in a chromatographic column of 1.5cm by 20cm, a chlorine type strongly basic anion exchange resin 1 is used as a filler, and acetic acid is used as a mobile phase. Wherein GOS is galacto-oligosaccharide and SL is 3 '-sialyllactose and 6' -sialyllactose.
FIG. 4 is a separation graph of method d) of example 3 of the present invention, wherein the chromatographic conditions are: in a 10cm×50cm column, a chlorine type strongly basic anion exchange resin 1 is used as a filler, and acetic acid is used as a mobile phase. Wherein Lac is lactose, GOS is galactooligosaccharide, and SL is 3 '-sialyllactose and 6' -sialyllactose.
FIG. 5 is a separation graph of method e) of example 3 according to the invention, wherein the chromatographic conditions are: in a 1.5 cm. Times.20 cm column, LKA98 resin was used as packing and acetic acid as mobile phase. Wherein Lac is lactose, GOS is galactooligosaccharide, and SL is 3 '-sialyllactose and 6' -sialyllactose.
FIG. 6 is a separation graph of method f) of example 3 of the present invention, wherein the chromatographic conditions are: in a 1.5 cm. Times.20 cm column, LKA20 resin was used as packing and acetic acid as mobile phase. Wherein Lac is lactose, GOS is galactooligosaccharide, and SL is 3 '-sialyllactose and 6' -sialyllactose.
FIG. 7 shows the results of UPLC detection of lyophilized powder of example 4 of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In addition, numerous specific details are set forth in the following description in order to provide a better illustration of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some embodiments, materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.
Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components.
The lactose of the invention is 1, 4-galactose glycoside glucose, the sialyllactose belongs to prebiotic oligosaccharide, and the oligosaccharide separated from the whey liquid is mainly sialyllactose.
The application provides a preparation method for separating and improving the content of 3 '-sialyllactose and 6' -sialyllactose in milk oligosaccharide powder from whey, and the adopted raw materials of the whey can have three sources: 1) The whey liquid is a byproduct whey liquid generated during cheese making; 2) Whey after re-dissolving desalted whey powder; 3) A permeate prepared by microfiltration of defatted animal milk (generally cow milk);
wherein, the byproduct whey liquid generated during cheese making or the whey liquid after re-dissolving the desalted whey powder can be directly or simply filtered and then directly ultrafiltered; generally, the whey liquid after re-dissolving the desalted whey powder is prepared by the desalted whey powder and water according to the mass ratio of 1:4-20.
In the following examples and comparative examples, the detection methods of lactose and cow's milk oligosaccharides were as follows:
the detection method of the cow milk oligosaccharide comprises the following steps: 100. Mu.L of skim milk was taken and diluted 10-fold. The diluted solution was centrifuged at 9600rpm at 4℃for 10min, and 100. Mu.L of the supernatant was collected. The supernatant was diluted 10 times, and after passing through a 0.22 μm nylon filter, 100. Mu.l of the supernatant was diluted five times to obtain a final diluted solution. Taking 100 mu L of the final dilution and the separated oligosaccharide sample, and respectively adding 50 mu L of 0.5 mol/L2-aminobenzamide (taking the 2-aminobenzamide as a derivative reagent) and 1 mol/L2-picoline borane complex (taking the 2-picoline borane complex as a sugar-labeled reductive amination reagent), wherein the volume ratio of the solvents used for the 2-aminobenzamide and the 2-picoline borane complex solution is 85:15 and acetic acid. And (3) after mixing uniformly, derivatizing for 2.5h at 65 ℃, taking out, cooling to room temperature, and then loading into a liquid phase vial for detection. Wherein the mobile phase is 100mmol/L ammonium formate solution (ph=4.5) and acetonitrile solution. The instrument is UltiMate 3000-high performance liquid chromatograph; the column was Waters UPLC BEH Amide (2.1 mm. Times.150 mm,1.7 μm) and the column temperature was set at 52 ℃. The excitation and emission wavelengths of the fluorescence detector were 250 and 425nm, respectively.
The invention is a further modification based on the applicant's prior application CN114539433a, and reference is made to CN114539433a for part of the methods, such as ultrafiltration, nanofiltration, detection, etc.
Example 1 ultrafiltration concentration
Ultrafiltration of the whey solution was performed at least three times:
Concentrating the whey liquid by ultrafiltration until the concentration multiple is 5 times to obtain a first ultrafiltration permeate;
Adding water into the first ultrafiltration trapped fluid for dilution, and concentrating until the concentration multiple is 5 times to obtain second ultrafiltration permeate;
adding water into the second ultrafiltration trapped fluid for dilution, and carrying out ultrafiltration concentration again until the concentration multiple is 5 times to obtain third ultrafiltration permeate; and so on.
The ultrafiltration times in this example were five times.
The molecular weight cut-off of the ultrafiltration membrane used in the embodiment is 5 kDa-30 kDa; optionally, the ultrafiltration membrane is a composite tubular organic membrane.
The ultrafiltration conditions in this example were: the temperature of the feed liquid is 20-45 ℃, the inlet pressure is 6-9 bar, and the outlet pressure is 5-8 bar; alternatively, the membrane pressure difference does not exceed 3bar.
EXAMPLE 2 nanofiltration concentration
The ultrafiltration permeate obtained in example 1 was concentrated by multiple nanofiltration:
concentrating the ultrafiltration permeate obtained in the example 1 to a concentration multiple of 2-13 times by nanofiltration to obtain a first nanofiltration trapped fluid;
Adding water into the first nanofiltration trapped fluid for dilution; carrying out nanofiltration and concentration again until the concentration multiple is 2-13 times to obtain second nanofiltration trapped fluid;
adding water into the second nanofiltration trapped fluid for dilution; carrying out nanofiltration and concentration again until the concentration multiple is 2-13 times to obtain a third nanofiltration trapped fluid; and so on.
The number of nanofiltration in this example was five.
The molecular weight cut-off of the nanofiltration membrane used in the embodiment is 100 Da-3000 Da; in the nanofiltration step, the nanofiltration membrane is a composite tubular organic membrane. The nanofiltration conditions are as follows: the temperature of the feed liquid is 20-45 ℃, the inlet pressure is 16-20 bar, and the outlet pressure is 14-18 bar; alternatively, the membrane pressure difference does not exceed 3bar.
Example 3 separation and purification
The separation and purification method comprises the following steps: the nanofiltration retentate of example 2 was subjected to chromatographic separation and purification, and lactose was eluted by deionized water and then purified, using a resin packing and purification method comprising the following steps:
a) The chlorine type strong-alkali anion exchange resin 2 is used as a filler (the parameters are: the mesh number is 50-400, the water retention capacity is 39% -48%, the total exchange capacity is 1.0-1.2 meq/mL), and the nanofiltration trapped fluid is loaded to a chromatographic column with the size of 1.5cm multiplied by 20 cm; the mobile phase is HCl with the flow rate of 0.05-0.5 mol/L and the flow rate of 0.5-7 mL/min, and 1-6 mL/pipe is collected. Preferably, the flow rate is 4.5mL/min, 3 mL/tube is collected, oligosaccharide and lactose detection is performed on each tube of the collected liquid, and the detection result is shown in FIG. 1.
B) The chlorine type strong-alkali anion exchange resin 1 is used as a filler (the parameters are: the mesh number is 50-400, the water retention capacity is 65% -80%, the total exchange capacity is 0.6-0.7meq/mL, the nanofiltration trapped fluid is loaded on a chromatographic column with the size of 10cm multiplied by 50cm, the mobile phase is 0.05-0.5 mol/L HCl, the flow rate is 20-70 mL/min, and 30-50 mL/tube is collected. Preferably, the flow rate is 35mL/min, 50 mL/tube is collected, oligosaccharide and lactose detection is performed on each tube of the collected liquid, and the detection result is shown in FIG. 2.
C) The chlorine type strong-alkali anion exchange resin 1 is used as a filler (the parameters are: the mesh number is 50-400, the water retention capacity is 65% -80%, the total exchange capacity is 0.6-0.7meq/mL, the nanofiltration trapped fluid is loaded on a chromatographic column with the size of 1.5cm multiplied by 20cm, the flowing phase is acetic acid with the flow rate of 0.05-1 mol/L, the flowing speed is 0.5-3.5 mL/min, and 1-5 mL/pipe is collected. Preferably at a flow rate of 1mL/min, 2 mL/tube was collected and oligosaccharide and lactose assays were performed on each tube of the collection as shown in FIG. 3.
D) The chlorine type strong-alkali anion exchange resin 1 is used as a filler (the parameters are: the mesh number is 50-400, the water retention capacity is 65% -80%, the total exchange capacity is 0.6-0.7meq/mL, the nanofiltration trapped fluid is loaded on a chromatographic column with the size of 10cm multiplied by 50cm, the flowing phase is acetic acid with the flow rate of 0.05-1 mol/L, the flowing speed is 30-60 mL/min, and 40-50 mL/pipe is collected. Preferably, the flow rate is 40mL/min, 50 mL/tube is collected, oligosaccharide and lactose detection is performed on each tube of the collected liquid, and the detection result is shown in FIG. 4.
E) And (3) taking LKA98 resin as a filler, loading the nanofiltration trapped fluid to a chromatographic column with the length of 1.5cm multiplied by 20cm, wherein the mobile phase is 0.05-1 mol/L acetic acid, the flow rate is 1-5 ml/min, and collecting 1-4 mL/tube. Preferably, the flow rate was 3mL/min, 2 mL/tube was collected, and oligosaccharide and lactose were detected for each tube of the collected solution, and the detection results were shown in FIG. 5.
F) And (3) taking LKA20 resin as a filler, loading the nanofiltration trapped fluid to a chromatographic column with the length of 1.5cm multiplied by 20cm, wherein the mobile phase is 0.05-1 mol/L acetic acid, the flow rate is 1-4 mL/min, and collecting 1-4 mL/tube. Preferably, the flow rate was 3mL/min, 2 mL/tube was collected, and oligosaccharide and lactose were detected for each tube of the collected solution, and the detection results were shown in FIG. 6.
The chlorine type strong-alkali anion exchange resin 1 and the chlorine type strong-alkali anion exchange resin 2 are purchased from Shanghai' an lan Biotechnology Co., ltd.
LKA98 and LKA20 resins were both purchased from eimeric health (china) biomedical limited; superdex30 increase pre-loaded chromatography column purchased from Situo; HCl and acetic acid were purchased from beijing chemical plant.
The results in FIGS. 1-6 show that 3 '-sialyllactose and 6' -sialyllactose cannot be separated by eluting with LKA98 resin and LKA20 resin as fillers and acetic acid as flowing phase 3 '-sialyllactose and 6' -sialyllactose; the chlorine type strong-alkali anion exchange resin 1 (or 2) is used as a filler, and HCl or acetic acid is used as flowing water to relatively separate 3 '-sialyllactose and 6' -sialyllactose, so that the 3 '-sialyllactose and the 6' -sialyllactose can be successfully separated.
Example 4, desalination:
Collecting chromatographic collection liquids of 3 '-sialyllactose and 6' -sialyllactose of the methods a) to d) in the example 3 respectively, combining the collection liquids in each method, wherein the chromatographic packing materials of a) to d) in the example 3 can effectively separate the cow's milk oligosaccharides, and collecting one or more chromatographic collection liquids can effectively separate the cow's milk oligosaccharides, wherein:
the purified collection 7L was separated using acetic acid as mobile phase (example 3, method d)) and was subjected to nanofiltration for salt removal.
The combined liquid separated with HCl as mobile phase (method b) of example 3) was split into two parts, one part was desalted by nanofiltration with an organic membrane separator and the other part was desalted by electrodialysis technique.
The nanofiltration desalination step comprises: separating 4.6L chromatographic collection liquid by nanofiltration, washing with deionized water until the conductivity is no longer reduced, concentrating to the minimum circulation volume of the equipment to obtain nanofiltration concentrated retention liquid, and ending nanofiltration concentration; in nanofiltration desalination, the molecular weight cut-off of the nanofiltration membrane is changed into 100Da to 350Da; in nanofiltration desalination, the nanofiltration membrane is a nanofiltration acid-resistant membrane; in nanofiltration desalination, the conditions were set as follows: the temperature of the feed liquid is 20-45 ℃, the inlet pressure is 10-15 bar, and the outlet pressure is 5-10 bar; alternatively, the membrane pressure difference does not exceed 3bar.
The electrodialysis step includes: 4.5L of chromatographic collection liquid is added into electrodialysis equipment for dialysis, and when the current is not changed any more, the desalination is finished. The conditions for electrodialysis are set as follows: adjusting voltage: 8V, material flow rate 50L/h, ultrapure water flow rate 50L/h, and polar liquid flow rate 45L/h.
And (3) performing rotary evaporation on the desalted liquid by using a Rongre-52A rotary evaporator, performing rotary evaporation on the desalted liquid to reach 10% of the original volume, and performing rotary evaporation on the desalted liquid by using a freeze dryer, and then performing freeze drying on the desalted liquid to obtain the freeze-dried powder. Firstly, placing the rotary steamed desalting solution on a sample shelf of a freeze dryer for prefreezing, setting a vacuum freeze drying temperature-rising program to be 20-2 h, 40-1 h and drying at 55 ℃ until the process is finished. The cold trap temperature is minus 50 ℃, the vacuum degree is 2.50-6.50 MPa, and the dry basis moisture content is lower than 5%.
The result of UPLC detection of the freeze-dried powder is shown in figure 7, wherein in nanofiltration desalination, the total content of 3 '-sialyllactose and 6' -sialyllactose in the freeze-dried powder after nanofiltration desalination of the separating liquid obtained by taking HCl or acetic acid as a mobile phase is not less than 40%; in electrodialysis desalination, the total content of 3 '-sialyllactose and 6' -sialyllactose in the lyophilized powder after the separation solution obtained by using HCl as a mobile phase is desalted by electrodialysis is about 28%.
The above results demonstrate that nanofiltration desalination is better.
Comparative example 1
The applicant changes the filling material from the Q-sepharose to the chlorine type strong alkaline anion exchange resin 1 or the chlorine type strong alkaline anion exchange resin 2 on the basis of the prior application CN114539433A, and the chlorine type strong alkaline anion exchange resin 1 can absorb more 3 '-sialyllactose and 6' -sialyllactose in the nanofiltration trapped liquid when the nanofiltration trapped liquid with the same amount is injected. The mobile phase of the prior application CN114539433A is modified to acetic acid or HCl by 0.1mol/L NaCl, so that more 3 '-sialyllactose and 6' -sialyllactose can be separated more efficiently. Specifically:
1.5g of the chlorine type strongly basic anion exchange resin 1, the chlorine type strongly basic anion exchange resin 2 and the Q-sepharose were taken out respectively, placed in 500mL glass conical flasks, 250mL nanofiltration retentate was added, and after shaking in a shaking incubator for 30, 60, 120, 180 minutes respectively under 120r/min conditions, 2mL of each was taken out of the supernatant, derivatization was performed and the 3 '-sialyllactose and 6' -sialyllactose contents were detected by UPLC to calculate sialyllactose loadings of the three fillers.
Through detection and calculation, the adsorption capacity of the Q-agarose gel is 2.96mg/g, the adsorption capacity of the chlorine type strong-alkali anion exchange resin 2 is 3.23mg/g, the adsorption capacity of the chlorine type strong-alkali anion exchange resin 1 is 8.11mg/g, and the adsorption capacity of the 3 '-sialyllactose and the 6' -sialyllactose of the chlorine type strong-alkali anion exchange resin 1 is the strongest through comparison.
The invention is preferably a chlorine type strong-alkali anion exchange resin 1, wherein the mesh number of the chlorine type strong-alkali anion exchange resin 1 is 50-400, the water retention capacity is 65-80%, and the total exchange capacity is 0.6-0.7meq/mL; and then nanofiltration or electrodialysis desalination is carried out, the molecular weight cut-off of nanofiltration membrane used for nanofiltration desalination is optimized to be 100 Da-350 Da, and the total content of 3 '-sialyllactose and 6' -sialyllactose in the finally obtained cow milk oligosaccharide powder is not less than 40%.
The invention uses chlorine type strong alkaline anion exchange resin 1 or chlorine type strong alkaline anion exchange resin 2 as a filler, uses acetic acid as a mobile phase to separate and nanofiltration desalt, and the content of 3 '-sialyllactose and 6' -sialyllactose in the obtained oligosaccharide freeze-dried powder reaches more than 42%; separating by taking HCl as a mobile phase, and carrying out nanofiltration desalination to obtain oligosaccharide freeze-dried powder, wherein the total content of 3 '-sialyllactose and 6' -sialyllactose reaches 41%; separating by taking HCl as a mobile phase, and carrying out electrodialysis desalination to obtain the oligosaccharide freeze-dried powder, wherein the total content of 3 '-sialyllactose and 6' -sialyllactose in the oligosaccharide freeze-dried powder reaches 28%. Compared with the prior art, the method improves.
The milk oligosaccharide powder obtained by the invention after drying is light yellow or milky yellow in color, lighter in flavor, smaller and uniform in particles, good in thermal stability and soluble in water. The technical scheme is used for solving the problem that the content of milk oligosaccharide in the existing milk oligosaccharide powder is low.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for increasing the sialyllactose content of milk oligosaccharides comprising the steps of: sequentially ultrafiltering and nanofiltration of the whey liquid, separating and purifying the nanofiltration trapped fluid by chromatography, removing lactose-containing chromatography trapped fluid, collecting sialyllactose-containing chromatography trapped fluid, desalting, and drying to obtain oligosaccharide powder;
wherein, in chromatographic separation and purification, chlorine type strong-alkaline anion exchange resin is adopted as a filler, and the mobile phase is HCl and/or acetic acid.
2. The method according to claim 1, wherein the mesh number of the chlorine-type strongly basic anion exchange resin is 50-400, the water retention capacity is 39% -80%, and the total exchange capacity is 0.6-1.2meq/mL;
Or the mesh number of the chlorine type strong-alkali anion exchange resin is 50-400, the water retention capacity is 65-80%, and the total exchange capacity is 0.6-0.7meq/mL;
or the chlorine-type strong base anion exchange resin is purchased from Shanghai Anbilde Biotechnology Co.
3. The method of claim 1, wherein the HCl concentration in the mobile phase is 0.05-0.5 mol/L; the concentration of the acetic acid is 0.05-1 mol/L;
or HCl concentration of 0.15mol/L; the concentration of acetic acid is 1mol/L.
4. The method according to claim 1, wherein the whey is a byproduct whey produced in cheese making, or a whey obtained by reconstitution of desalted whey powder, or a permeate obtained by microfiltration of defatted animal milk.
5. The method according to claim 1, wherein ultrafiltration is performed at least three times, and the first ultrafiltration permeate is obtained when the whey liquid is concentrated to a concentration multiple of 2-7 times by ultrafiltration; adding water into the first ultrafiltration trapped fluid for dilution; ultrafiltering and concentrating again until the concentration multiple is 2-7 times to obtain a second ultrafiltrate; adding water into the ultrafiltration trapped fluid of the second time for dilution; ultrafiltering and concentrating again until the concentration multiple is 2-7 times to obtain a third ultrafiltrate; and so on;
and/or, in the ultrafiltration step, the molecular weight cut-off of the ultrafiltration membrane is 5 kDa-30 kDa.
6. The method according to claim 1, wherein the nanofiltration is performed for at least three times, and the ultrafiltration permeate is concentrated by nanofiltration to a concentration factor of 2-13 to obtain a first nanofiltration retentate; adding water into the first nanofiltration trapped fluid for dilution; ultrafiltering and concentrating again until the concentration multiple is 2-13 times to obtain second nanofiltration trapped fluid; adding water into the second nanofiltration trapped fluid for dilution; carrying out nanofiltration and concentration again until the concentration multiple is 2-13 times to obtain a third nanofiltration trapped fluid; and so on;
and/or, in the nanofiltration step, the molecular weight cut-off of the nanofiltration membrane is 100 Da-3000 Da.
7. The method of any one of claims 1 to 6, wherein the desalting step is desalting by nanofiltration.
8. The method of claim 7, wherein the nanofiltration desalination step comprises: separating the chromatographic collection liquid by nanofiltration, washing with deionized water until the conductivity is no longer reduced, and concentrating to the minimum circulation volume of the equipment to obtain nanofiltration concentrated retention liquid, wherein nanofiltration concentration is finished;
And/or, in nanofiltration desalination, the molecular weight cut-off of the nanofiltration membrane is 100 Da-350 Da;
And/or, in nanofiltration desalination, the nanofiltration membrane adopted is a nanofiltration acid-resistant membrane;
And/or, in nanofiltration desalination, the conditions are set as follows: the temperature of the feed liquid is 20-45 ℃, the inlet pressure is 10-15 bar, and the outlet pressure is 5-10 bar; the membrane pressure difference is not more than 3bar.
9. An oligosaccharide powder prepared by the method of any one of claims 1 to 8; the total mass percentage of the 3 '-sialyllactose and the 6' -sialyllactose in the oligosaccharide powder is not less than 40%.
10. An oligosaccharide food or health product comprising an oligosaccharide powder obtainable by a process according to any one of claims 1 to 8 or an oligosaccharide powder according to claim 9.
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