CN115894578A - Preparation method of human milk oligosaccharide LNFP I reference substance - Google Patents
Preparation method of human milk oligosaccharide LNFP I reference substance Download PDFInfo
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Abstract
The invention discloses a preparation method of a human milk oligosaccharide LNFP I reference substance, which comprises the following steps: firstly, taking a human milk neutral sugar total sample as a raw material; dissolving the human milk neutral sugar total sample, and eluting by using a hydrophilic chromatographic filler and a gradient or isocratic mode by using water and an organic solvent as mobile phases to obtain an LNFP I crude product; then dissolving the LNFP I crude product, using a porous graphite carbon material as a chromatographic column stationary phase, using alkaline water and an organic solvent as mobile phases, and eluting by using a gradient or isocratic mode to obtain an LNFP I pure product; and finally, concentrating and freeze-drying the LNFP I pure product to obtain the human milk oligosaccharide LNFP I reference substance.
Description
Technical Field
The invention relates to a preparation method of oligosaccharide, in particular to a preparation method of a human milk oligosaccharide LNFP I reference substance.
Technical Field
Human milk oligosaccharides are a class of naturally occurring oligosaccharides present in human milk: plays an irreplaceable role in the early development of the digestive system of the infant, the perfection of the immune system after birth and the establishment of the ecological balance in vivo. The precondition for deep understanding of the biological and pharmacological activities of human milk oligosaccharides is to obtain human milk oligosaccharide samples, and therefore, the oligosaccharide preparation technology aiming at obtaining high purity human milk oligosaccharide reference products becomes one of the key steps in oligosaccharide research. However, since oligosaccharides are polar compounds, and contain different spatial configurations of monosaccharide structures, different glycosyl sequences, and different sugar substitution patterns, they can generate various structural species, thereby increasing the difficulty of separation.
The human milk oligosaccharide LNFP I is one of neutral sugar, and has a molecular formula of C 32 H 55 NO 25 The sequence is as follows: fuc alpha 1-2Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc, infant intestinal flora and the special structure of human milk oligosaccharide have a close inseparable relationship, and LNFP I can be used as a recognition marker of fucosylated human milk oligosaccharide. Many researches find that the components in the fucosylated human lactooligosaccharide can be effectively decomposed and utilized by bifidobacteria, and the bifidobacteria can regulate intestinal tracts, improve the metabolism of vitamins and enhance the function of liversIt can reduce the pH value of the intestinal tract by producing acetic acid and lactic acid through metabolism in the intestinal tract, inhibit the growth of saprophytic bacteria, maintain the microecological balance of the intestinal tract, reduce the infection risk of infants and resist inflammation caused by certain chronic diseases.
In recent years, the research on the preparation of human lactooligosaccharide has attracted much attention, and there are currently 3 main strategies for the separation and purification of human lactooligosaccharide: (1) Directly separating by adopting gel permeation chromatography or high-efficiency anion exchange chromatography according to the size of molecular weight or different charges; (2) After derivatization modification is carried out on the natural human milk oligosaccharide, separation is carried out by utilizing reverse phase chromatography or normal phase chromatography; (3) Synthesizing the breast milk oligosaccharide by a chemical method, an enzymatic method, a chemo-enzymatic method. Because of more isomers of human milk oligosaccharides, strategy 1 has lower selectivity, long time period and low preparation efficiency. Strategy 2, although it has some selectivity, the resulting oligosaccharide is in derivative form, destroying the original structure of native breast milk oligosaccharide. Strategy 3 chemical method faces to the electron and space obstruction of glycosyl donor, so that the glycosyl donor is difficult to form glycosidic bond, and simultaneously has the defects of byproducts generated by stereochemical racemization and the like in the reaction process, and the sources and active substrates of glycosyl transferase and glycosidase are limited to a certain extent in the enzymatic reaction.
Disclosure of Invention
The invention provides a preparation method of a human milk oligosaccharide LNFP I reference substance, which comprises the following steps:
s1, taking a human milk neutral sugar total sample as a raw material;
s2, mixing the human milk neutral sugar total sample with a first solvent, dissolving to obtain a first mixture, eluting by using a gradient or isocratic mode by using a hydrophilic chromatographic packing and a mobile phase of water and an organic solvent, and separating by using column chromatography to obtain an LNFP I crude product;
s3, mixing the crude LNFP I product with a second solvent, dissolving to obtain a second mixture, taking a porous graphite carbon material as a chromatographic column stationary phase, taking alkaline water and an organic solvent as mobile phases, eluting by using a gradient or isocratic mode, and separating by using column chromatography to obtain a pure LNFP I product;
and S4, concentrating and freeze-drying the LNFP I pure product to obtain the human milk oligosaccharide LNFP I control product.
Preferably, the first solvent in step S2 is a 40-60% methanol solution, and the second solvent in step S3 is an aqueous solution.
Preferably, the hydrophilic chromatographic filler in step S2 is a filler with cysteine polar groups bonded on the surface of silica gel, and the aqueous alkali solution in step S3 is 0.2% to 0.6% of aqueous ammonium hydroxide solution.
Preferably, the organic solvent in steps S2 and S3 is one or more of ethanol, methanol, and acetonitrile.
Preferably, the isocratic elution mode in step S2 is: the volume ratio of the aqueous solution in the mobile phase is 5-95 percent; or using a linear gradient approach: the volume concentration of the aqueous solution in the mobile phase changes from small to large, the initial volume concentration is 5-60%, and the final volume concentration is 40-95%; or the step gradient mode is as follows: more than 2 elution operations are randomly selected from 5 to 95 percent of the volume ratio of the aqueous solution in the mobile phase from small to large.
Preferably, the isocratic elution mode in step S3 is: the volume ratio of the organic solvent in the mobile phase is 5-95 percent; or using a linear gradient approach: the volume concentration of the organic solvent in the mobile phase changes from small to large, the initial volume concentration is 5-60%, and the final volume concentration is 10-95%; or the step gradient mode is as follows: more than 2 organic solvents are randomly selected from 5 to 95 percent of the volume ratio of the organic solvents in the mobile phase from small to large for elution.
Preferably, the chromatographic parameters in steps S2 and S3 are optimized as follows:
(1) the solid sample carrying amount is 0.01-15%;
(2) the flow rate is 0.1-2 times of column volume/min;
(3) the inner diameter of the chromatographic column is 4.6-500mm.
Preferably, the human milk neutral sugar total sample in the step S1 is prepared by the following steps:
s1, centrifuging a human milk sample at 0-10 ℃ for 5-150 min to remove upper-layer lipid to obtain a defatted breast milk sample, adding 1-5 times of volume of ethanol into the defatted breast milk sample, mixing uniformly, standing at 0-10 ℃ for 2-48 hours, centrifuging at 0-10 ℃ for 5-150 min, collecting upper-layer liquid, and evaporating to obtain a sample subjected to degreasing and protein removal;
s2, separating the degreased and deproteinized sample by using a silica gel surface-bonded aspartic acid polar group-bonded filler column chromatography, dissolving the degreased and deproteinized sample by using an ethanol aqueous solution with the volume concentration of 20-80%, wherein the mobile phase comprises 2-12 mM ammonium acetate buffer salt solution and an ethanol solution, the volume concentration of the buffer salt aqueous solution is changed from small to large, the initial volume concentration is 5-60%, the final volume concentration is 40-95%, collecting effluent liquid with the volume of 3-40 times of the column volume, and concentrating and freeze-drying the effluent liquid to obtain the neutral sugar total sample in the step S1.
The invention has the following advantages:
1. the solvent consumption is low, and the solvent in the process can be recycled;
2. the preparation conditions are simple, the sample does not need to be derivatized, and the original structure of the oligosaccharide is reserved;
3. the stationary phase of the chromatographic column used in the invention has good stability and is easy to be prepared in an enlarged way.
Drawings
FIG. 1 is an HPLC detection profile of an LNFP I control obtained in example 1 of the present invention;
FIG. 2 is an HPLC detection profile of an LNFP I control obtained in example 2 of the present invention;
FIG. 3 is an HPLC detection profile of an LNFP I control obtained in example 3 of the present invention;
FIG. 4 is an HPLC detection profile of LNFP I obtained in comparative example 1 of the present invention;
FIG. 5 is a schematic flow chart of the method of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to fig. 1 to 5 in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments, and based on the embodiments of the present invention, all other embodiments obtained by a person skilled in the art without making creative efforts belong to the protection scope of the present invention.
A method for preparing a human milk oligosaccharide LNFP I control as shown in fig. 5, said method comprising the steps of:
s1, taking a human milk neutral sugar total sample as a raw material;
s2, mixing the human milk neutral sugar total sample with a first solvent, dissolving to obtain a first mixture, eluting by using a hydrophilic chromatographic packing and a mobile phase of water and an organic solvent in a gradient or isocratic mode, and separating by using column chromatography to obtain a crude product LNFP I;
s3, mixing the crude LNFP I product with a second solvent, dissolving to obtain a second mixture, eluting by using a gradient or isocratic mode by using a porous graphite carbon material as a chromatographic column stationary phase and using alkaline water and an organic solvent as mobile phases, and separating by using column chromatography to obtain a pure LNFP I product;
and S4, concentrating and freeze-drying the LNFP I pure product to obtain the human milk oligosaccharide LNFP I reference substance.
Preferably, the first solvent in step S2 is a 40-60% methanol solution, and the second solvent in step S3 is an aqueous solution.
Preferably, the hydrophilic chromatographic filler in step S2 is a filler with cysteine polar groups bonded on the surface of silica gel, and the aqueous alkali solution in step S3 is 0.2% to 0.6% of aqueous ammonium hydroxide solution.
Preferably, the organic solvent in steps S2 and S3 is one or more of ethanol, methanol, and acetonitrile.
Preferably, the isocratic elution mode in step S2 is as follows: the volume ratio of the aqueous solution in the mobile phase is 5-95%; or using a linear gradient approach: the volume concentration of the aqueous solution in the mobile phase changes from small to large, the initial volume concentration is 5-60%, and the final volume concentration is 40-95%; or the step gradient mode is as follows: more than 2 elution operations are randomly selected from 5 to 95 percent of the volume ratio of the aqueous solution in the mobile phase from small to large.
Preferably, the isocratic elution mode in step S3 is: the volume ratio of the organic solvent in the mobile phase is 5-95 percent; or using a linear gradient approach: the volume concentration of the organic solvent in the mobile phase changes from small to large, the initial volume concentration is 5-60%, and the final volume concentration is 10-95%; or the step gradient mode is as follows: randomly selecting more than 2 from 5-95% of organic solvent in the mobile phase from small to large according to the volume ratio of the organic solvent in the mobile phase to carry out elution operation.
Preferably, the chromatographic parameters in steps S2 and S3 are optimized as follows:
(1) the solid sample carrying amount is 0.01-15%;
(2) the flow rate is 0.1-2 times of column volume/min;
(3) and the inner diameter of the chromatographic column is 4.6-500mm.
Preferably, the human milk neutral sugar total sample in the step S1 is prepared by the following steps:
s1, centrifuging a human milk sample at 0-10 ℃ for 5-150 min to remove upper-layer lipid to obtain a defatted breast milk sample, adding 1-5 times of ethanol in volume to the defatted breast milk sample, uniformly mixing, standing at 0-10 ℃ for 2-48 h, centrifuging at 0-10 ℃ for 5-150 min, collecting upper-layer liquid, and evaporating to dryness to obtain a defatted deproteinized sample;
s2, separating the degreased and deproteinized sample by using a silica gel surface-bonded aspartic acid polar group-bonded filler column chromatography, dissolving the degreased and deproteinized sample by using an ethanol aqueous solution with the volume concentration of 20-80%, wherein the mobile phase comprises 2-12 mM ammonium acetate buffer salt solution and an ethanol solution, the volume concentration of the buffer salt aqueous solution is changed from small to large, the initial volume concentration is 5-60%, the final volume concentration is 40-95%, collecting effluent liquid with the volume of 3-40 times of the column volume, and concentrating and freeze-drying the effluent liquid to obtain the neutral sugar total sample in the step S1.
Example 1:
(1) preparation of human milk neutral sugar total sample
1L of human milk was centrifuged at 4000rpm at 4 ℃ for 50min to remove upper lipids. And (3) taking the lower-layer water layer, adding 2 times of volume of absolute ethyl alcohol, uniformly mixing, standing at 4 ℃ for 12 hours, centrifuging at 4000rpm at 4 ℃ for 50min, collecting the upper-layer liquid, and evaporating to dryness to obtain the degreased protein-removed oligosaccharide sample. Dissolving a degreased protein-removed oligosaccharide sample by using a 30% ethanol water solution to prepare a solution with the concentration of 100mg/mL, wherein the sample size is 200mL, a filler with aspartic acid polar groups bonded on the surface of silica gel is adopted, and the inner diameter of a chromatographic column is 100mm; the flow rate is 240mL/min; the column temperature is 30 ℃; the mobile phase A was ethanol and B was 10mM ammonium acetate in water. Elution was performed under a linear gradient, the elution conditions being: the time is 60min, the initial volume concentration of B is 15 percent, the final volume concentration of B is 80 percent, all fractions within 20-60min are collected to obtain 10103.5mg of the oligosaccharide after protein and lactose removal by degreasing, and the oligosaccharide is confirmed to be a pure neutral human milk oligosaccharide mixture by chromatographic and mass spectrometric detection.
(2) Preparation of human milk oligosaccharide LNFP I reference substance
Weighing 500mg human milk neutral sugar, adding 10mL50% methanol solution, fully mixing and dissolving, using silica gel surface bonded cysteine polar group filler, and chromatographic column inner diameter 50mm; the sample injection amount is 4mL; the flow rate is 60mL/min; the mobile phase A is ethanol, the mobile phase B is methanol, the mobile phase C is water, and the elution conditions are as follows: time 50min, volume fraction of A55%, volume fraction of B10%, volume fraction of C35%. Collecting all fractions within 30-35min, concentrating, and lyophilizing to obtain crude LNFP I product.
Weighing 200mg of LNFP I crude product, adding 1mL of water, fully mixing and dissolving, and then using a graphitized carbon chromatographic column with the inner diameter of 10mm; the sample injection amount is 100 mu L; the flow rate is 3mL/min; the column temperature is 35 ℃; mobile phase a was acetonitrile and B was 0.4% ammonium hydroxide solution. Elution was performed under a linear gradient, the elution conditions being: the time is 20min: the initial volume concentration of A is 15%, the final volume concentration of A is 80%, all fractions within 12-18min are collected, and the pure LNFP I product is obtained after concentration and freeze-drying.
FIG. 1 is an HPLC detection profile of human milk oligosaccharide LNFP I control obtained in this example.
Detection conditions are as follows: liquid phase conditions: a chromatographic column: BEH Amide,2.1 × 150mm; the column temperature is 40 ℃; wavelength: 205nm; flow rate: 0.3mL/min; sample injection amount: 5 mul; mobile phase: acetonitrile (a), water (B); elution conditions: the elution time was 20min: the volume capacity of A was initially 70% and the volume capacity of A ended up to 30%.
The purity of the obtained human milk oligosaccharide LNFP I control product is more than 98 percent by HPLC analysis.
Example 2
(1) Preparation of human milk neutral sugar total sample
10L of human milk was centrifuged at 6000rpm at 4 ℃ for 80min to remove the upper lipid layer. And (3) taking the lower-layer water layer, adding 2 times of volume of absolute ethyl alcohol, uniformly mixing, standing at 4 ℃ for 12 hours, centrifuging at 6000rpm at 4 ℃ for 80min, collecting the upper-layer solution, and evaporating to dryness to obtain the degreased protein-removed oligosaccharide sample. Dissolving a degreased protein-removed oligosaccharide sample by using a 30% ethanol water solution to prepare a solution with the concentration of 100mg/mL, wherein the sample volume is 200mL, a filler with aspartic acid polar groups bonded on the surface of silica gel is adopted, and the inner diameter of a chromatographic column is 100mm; the flow rate is 240mL/min; the column temperature is 30 ℃; the mobile phase A is ethanol, and the mobile phase B is 10mM ammonium acetate aqueous solution. Elution was performed under a linear gradient, the elution conditions being: the time is 60min, the initial volume concentration of B is 15 percent, the final volume concentration of B is 80 percent, all fractions within 20-60min are collected, and 121.15g of the oligosaccharides with protein and lactose removed is obtained, and is confirmed to be a pure neutral human milk oligosaccharide mixture by chromatographic and mass spectrometric detection.
(2) Preparation of human milk oligosaccharide LNFP I reference substance
Weighing 5g of human milk neutral sugar, adding 100mL of 50% methanol solution, fully mixing and dissolving, and bonding a filler of cysteine polar groups on the surface of silica gel, wherein the inner diameter of a chromatographic column is 50mm; the sample injection amount is 20mL; the flow rate is 60mL/min; the mobile phase A is ethanol, the mobile phase B is methanol, the mobile phase C is water, and the elution is carried out under isocratic conditions, wherein the elution conditions are as follows: time 50min, volume fraction of A55%, volume fraction of B10%, volume fraction of C35%. Collecting all fractions within 30-35min, concentrating, and lyophilizing to obtain crude LNFP I product.
Weighing 2g of LNFP I crude product, adding 10mL of water, fully mixing and dissolving, and then using a graphitized carbon chromatographic column with the inner diameter of 10mm; the sample injection amount is 100 mu L; the flow rate is 3mL/min; the column temperature was 35 ℃; mobile phase a was acetonitrile and B was 0.4% ammonium hydroxide solution. Elution was performed under a linear gradient, the elution conditions being: the time is 20min: the initial volume concentration of A is 15%, the final volume concentration of A is 80%, all fractions within 12-18min are collected, and the pure LNFP I product is obtained after concentration and freeze-drying.
FIG. 2 is an HPLC detection profile of the human milk oligosaccharide LNFP I control obtained in this example.
Detection conditions are as follows: liquid phase conditions: a chromatographic column: BEH Amide, 2.1X 150mm; the column temperature is 40 ℃; wavelength: 205nm; flow rate: 0.3mL/min; sample introduction amount: 5 mul; mobile phase: acetonitrile (a), water (B); elution conditions: the elution time was 20min: the volume capacity of A was initially 70% and the volume capacity of A ended up to 30%.
The purity of the obtained human milk oligosaccharide LNFP I control product is more than 95 percent by HPLC analysis.
Example 3
(1) Preparation of human milk neutral sugar total sample
50L of human milk was centrifuged at 8000rpm at 4 ℃ for 100min to remove upper lipids. And (3) taking the lower-layer water layer, adding 2 times of volume of absolute ethyl alcohol, uniformly mixing, standing at 4 ℃ for 12 hours, centrifuging at 8000rpm at 4 ℃ for 100min, collecting the upper-layer solution, and evaporating to dryness to obtain the degreased protein-removed oligosaccharide sample. Dissolving a degreased protein-removed oligosaccharide sample by using a 30% ethanol water solution to prepare a solution with the concentration of 100mg/mL, wherein the sample volume is 200mL, a filler with aspartic acid polar groups bonded on the surface of silica gel is adopted, and the inner diameter of a chromatographic column is 100mm; the flow rate is 240mL/min; the column temperature is 30 ℃; the mobile phase A is ethanol, and the mobile phase B is 10mM ammonium acetate aqueous solution. Elution was performed under a linear gradient, the elution conditions being: the time is 60min, the initial volume concentration of B is 15 percent, the final volume concentration of B is 80 percent, all fractions within 20-60min are collected, and 503.5g of the oligosaccharides with protein and lactose removed is obtained, and the oligosaccharides are confirmed to be pure neutral human milk oligosaccharide mixture by chromatographic and mass spectrometric detection.
(2) Preparation of human milk oligosaccharide LNFP I reference substance
Weighing 10g of human milk neutral sugar, adding 200mL of 50% methanol solution, fully mixing and dissolving, and bonding a filler of cysteine polar groups on the surface of silica gel, wherein the inner diameter of a chromatographic column is 50mm; the sample injection amount is 20mL; the flow rate is 60mL/min; the mobile phase A is ethanol, the mobile phase B is methanol, the mobile phase C is water, and the elution conditions are as follows: 50min at 0, 55% of A, 10% of B and 35% of C, collecting all fractions within 30-35min, concentrating, and lyophilizing to obtain crude LNFP I product.
Weighing 5g of crude LNFP I product, adding 20mL of water, fully mixing and dissolving, and then using a graphitized carbon chromatographic column with the inner diameter of 10mm; the sample injection amount is 100 mu L; the flow rate is 3mL/min; the column temperature was 35 ℃; mobile phase a was acetonitrile and B was 0.4% ammonium hydroxide solution. Elution was performed under a linear gradient, the elution conditions being: the time is 20min: the initial volume concentration of A is 15%, the final volume concentration of A is 80%, all fractions within 12-18min are collected, and the pure LNFP I product is obtained after concentration and freeze-drying.
Fig. 3 is an HPLC detection profile of the human milk oligosaccharide LNFP I control obtained in this example.
Detection conditions are as follows: liquid phase conditions: and (3) chromatographic column: BEH Amide, 2.1X 150mm; the column temperature is 40 ℃; wavelength: 205nm; flow rate: 0.3mL/min; sample injection amount: 5 mul; mobile phase: acetonitrile (a), water (B); the elution time was 20min: the volume capacity of A was initially 70% and the volume capacity of A was 30% at the end.
The purity of the obtained human milk oligosaccharide LNFP I reference substance through HPLC analysis is more than 96 percent.
Comparative example 1:
(1) preparation of human milk neutral sugar total sample
5L of human milk was centrifuged at 6000rpm at 4 ℃ for 60min to remove the upper lipid layer. And (3) taking the lower-layer water layer, adding 2 times of volume of absolute ethyl alcohol, uniformly mixing, standing at 4 ℃ for 12 hours, centrifuging at 6000rpm at 4 ℃ for 60min, collecting the upper-layer liquid, and evaporating to dryness to obtain the degreased protein-removed oligosaccharide sample. Dissolving a degreased protein-removed oligosaccharide sample by using a 30% ethanol water solution to prepare a solution with the concentration of 100mg/mL, wherein the sample volume is 200mL, a filler with aspartic acid polar groups bonded on the surface of silica gel is adopted, and the inner diameter of a chromatographic column is 100mm; the flow rate is 240mL/min; the column temperature is 30 ℃; the mobile phase A was ethanol and B was 10mM ammonium acetate in water. Elution was performed under a linear gradient, the elution conditions being: the time is 60min, the initial volume concentration of B is 15 percent, the final volume concentration of B is 80 percent, all fractions within 20-60min are collected, and 60.55g of the oligosaccharides with protein and lactose removed is obtained, and the oligosaccharides are confirmed to be pure neutral human milk oligosaccharide mixture by chromatographic and mass spectrometric detection.
(2) Preparation of human milk oligosaccharide LNFP I reference substance
Weighing 500mg human milk neutral sugar, adding 10mL50% methanol solution, fully mixing and dissolving, using silica gel surface bonded cysteine polar group filler, and chromatographic column inner diameter 50mm; the sample injection amount is 4mL; the flow rate is 60mL/min; the mobile phase A was ethanol, B was methanol, C was water, and the elution was carried out under isocratic conditions for a washing time of 50min, the volume ratio of A was 55%, the volume ratio of B was 10%, and the volume ratio of C was 35% by volume. Collecting all fractions within 30-35min, concentrating, and lyophilizing to obtain LNFP I with purity of about 85%.
FIG. 4 is an HPLC detection profile of human milk oligosaccharide LNFP I obtained from this comparative example.
Therefore, a high-purity LNFP I reference substance is difficult to obtain by separating a human milk neutral sugar sample only by adopting a polar filler without using a graphitized carbon material, and further, the consumption of the solvent is low, and the solvent in the process can be recycled; the preparation condition is simple, and the sample does not need derivatization, so that the original structure of the oligosaccharide is reserved; particularly, the repeatability is good, and the chromatographic column stationary phase used in the invention has good stability and is easy to be prepared in an enlarged way.
It is to be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A method for preparing a human milk oligosaccharide LNFP I control, which is characterized in that the method comprises the following steps:
s1, taking a human milk neutral sugar total sample as a raw material;
s2, mixing the human milk neutral sugar total sample with a first solvent, dissolving to obtain a first mixture, eluting by using a gradient or isocratic mode by using a hydrophilic chromatographic packing and a mobile phase of water and an organic solvent, and separating by using column chromatography to obtain an LNFP I crude product;
s3, mixing the crude LNFP I product with a second solvent, dissolving to obtain a second mixture, taking a porous graphite carbon material as a chromatographic column stationary phase, taking alkaline water and an organic solvent as mobile phases, eluting by using a gradient or isocratic mode, and separating by using column chromatography to obtain a pure LNFP I product;
and S4, concentrating and freeze-drying the LNFP I pure product to obtain the human milk oligosaccharide LNFP I reference substance.
2. The method of claim 1, wherein: the first solvent in the step S2 is a 40-60% methanol solution, and the second solvent in the step S3 is an aqueous solution.
3. The method of claim 1, wherein: the hydrophilic chromatographic packing in the step S2 is a packing with cysteine polar groups bonded on the surface of silica gel, and the aqueous alkali solution in the step S3 is 0.2% -0.6% of ammonium hydroxide aqueous solution.
4. The method of claim 1, wherein: the organic solvent in the steps S2 and S3 is one or more than two of ethanol, methanol and acetonitrile.
5. The method of claim 1, wherein: the isocratic elution mode in the step S2 is as follows: the volume ratio of the aqueous solution in the mobile phase is 5-95%; or using a linear gradient approach: the volume concentration of the aqueous solution in the mobile phase changes from small to large, the initial volume concentration is 5-60%, and the final volume concentration is 40-95%; or the step gradient mode is as follows: randomly selecting more than 2 from 5-95% of the aqueous solution in the mobile phase from small to large according to the volume ratio of the aqueous solution in the mobile phase for elution.
6. The method of claim 1, wherein: the isocratic elution mode in the step S3 is as follows: the volume ratio of the organic solvent in the mobile phase is 5-95%; or using a linear gradient approach: the volume concentration of the organic solvent in the mobile phase changes from small to large, the initial volume concentration is 5-60%, and the final volume concentration is 10-95%; or the step gradient mode is as follows: randomly selecting more than 2 organic solvents from 5-95% according to the volume ratio of the organic solvents in the mobile phase from small to large for elution operation.
7. The method of claim 1, wherein: the chromatographic parameters in steps S2 and S3 are optimized as follows:
the solid sample carrying amount is 0.01-15%;
the flow rate is 0.1-2 times of the column volume/min;
the inner diameter of the chromatographic column is 4.6-500mm.
8. The method of claim 1, wherein: the human milk neutral sugar total sample in the step S1 is prepared by the following steps:
s1, centrifuging a human milk sample at 0 to 10 ℃ for 5 to 150min to remove an upper-layer lipid to obtain a defatted breast milk sample, adding 1 to 5 times of ethanol in volume into the defatted breast milk sample, mixing uniformly, standing at 0 to 10 ℃ for 2 to 48 hours, centrifuging at 0 to 10 ℃ for 5 to 150min, collecting an upper-layer solution, and evaporating to dryness to obtain a sample subjected to defatting and protein removal;
s2, separating the degreased and deproteinized sample by using a packed column chromatography of bonding aspartic acid polar groups on the surface of silica gel, dissolving the degreased and deproteinized sample by using an ethanol aqueous solution with the volume concentration of 20-80%, wherein the mobile phases are an ammonium acetate buffer salt solution with the volume concentration of 2-12mM and an ethanol solution, the volume concentration of the buffer salt aqueous solution is changed from small to large, the initial volume concentration is 5-60%, the final volume concentration is 40-95%, collecting an effluent liquid with the volume of 3-40 times of the column, and concentrating and freeze-drying the effluent liquid to obtain the neutral sugar total sample in the step S1.
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