CN116148397A

CN116148397A - Method for detecting oligosaccharide content of breast milk

Info

Publication number: CN116148397A
Application number: CN202310402569.XA
Authority: CN
Inventors: 王象欣; 高鹏; 解庆刚; 张影; 张永久; 蒋士龙; 崔东影; 陆思宇; 冷友斌
Original assignee: Feihe Gannan Dairy Products Co ltd; Feihe Harbin Dairy Co ltd; Feihe Longjiang Dairy Co ltd; Heilongjiang Feihe Dairy Co ltd Beijing Branch; Heilongjiang Feihe Dairy Co Ltd
Current assignee: Feihe Gannan Dairy Products Co ltd; Feihe Harbin Dairy Co ltd; Feihe Longjiang Dairy Co ltd; Heilongjiang Feihe Dairy Co ltd Beijing Branch; Heilongjiang Feihe Dairy Co Ltd
Priority date: 2023-04-14
Filing date: 2023-04-14
Publication date: 2023-05-23

Abstract

The invention belongs to the field of detection, and particularly relates to a method for detecting the content of breast milk oligosaccharide in a dairy product, which comprises the following steps: a step of pre-processing and a step of detecting, the step of pre-processing comprising: a step of removing protein and a step of enzymatic hydrolysis of galacto-oligosaccharide, and further comprises a step of adding an internal standard and a derivative reagent after the enzymatic hydrolysis of galacto-oligosaccharide, wherein the derivative reagent is procainamide; the detecting step includes: detecting the detection sample obtained by the pretreatment by a liquid chromatography-fluorescence analysis method; wherein the mobile phase in the liquid chromatograph comprises a polar solvent containing a chiral solvent.

Description

Method for detecting oligosaccharide content of breast milk

Technical Field

The invention belongs to the field of detection, and particularly relates to an analysis and detection technology for breast milk oligosaccharide substances in dairy products, in particular to dairy powder products.

Background

Breast milk is rich in various components for protecting neonate, such as immunoglobulin, congenital glycoprotein, antibody, bioactive peptide, glycolipid, free fatty acid, cytokine and chemotactic factor, oligosaccharide, etc. Wherein, breast milk oligosaccharide (Human milk oligosaccharides, HMOs) is a compound sugar, which is formed by connecting monosaccharides through glycosidic bonds and is divided into common oligosaccharide and functional oligosaccharide.

HMOs are abundant in breast milk, are the third largest component next to lactose and fat in breast milk, are 5-15 g/L in mature milk, are about 20 times higher than milk, and are 20-25 g/L in colostrum. The content and composition of HMOs vary widely among populations because the composition of HMOs is related to a variety of physiological parameters, including secretion and Lewis blood group status of the lactating mother. In order to accommodate the needs of growing children, oligosaccharides in milk also differ during the lactation phase, and HMOs content in colostrum is higher, decreases during lactation and is affected by mother's diet.

In addition, many of the biological functions of HMOs are related to their structure, e.g., 3' -sialyllactose (3 ' -SL) affects mouse colitis by selective intestinal bacterial colonization, whereas its isomer 6' -SL does not have this biological function. Also, studies show that the content of HMOs is different in different lactation period within 3 months, the increase of lactation period is accompanied by the change of the growth and development conditions of infants, and the change of the HMOs can be related to the change of the development conditions of infants, so that the change of the content of the HMOs and the influence factors thereof are significant for developing dairy products more similar to breast milk, especially infant formulas.

Although research shows that HMOs are difficult to digest and absorb by infants, the HMOs play an important role in healthy growth of infants, and have a plurality of important functions, including promoting growth of beneficial intestinal bacteria such as bifidobacteria, preventing adhesion of pathogenic bacteria in intestinal tracts, thereby reducing infection risk, maintaining intestinal microecological balance, promoting maturation of immune systems, participating in ganglioside and glycoprotein composition in brain tissues and the like.

HMOs are composed of 5 monosaccharide basic units, such as glucose, galactose, N-acetylglucosamine, fucose and N-acetylneuraminic acid, which usually contain a lactose core at the reducing end, and are prolonged together with the remaining 3 monosaccharide units by the action of several glycosyltransferases, and the number of monosaccharide compositions is generally 3 to 14. Depending on whether HMOs are sialylated, they can be classified into neutral HMOs (terminated with L-fucose) and acidic HMOs (terminated with sialic acid). Of these, 2'-fucosyllactose (2' -FL) and lacto-N-neotetraose (LNnT) have been currently approved by some countries or regions formally as additive for foods which can be added to infant formulas.

Further, since the composition of breast milk is complex, a large amount of lactose and milk fat in the components thereof are large interfering factors in the separation detection of HMOs. At present, the separation method for the HMOs mainly comprises High Performance Anion Exchange Chromatography (HPAEC), porous graphitized carbon chromatography (PGC), capillary Electrophoresis (CE) and High Performance Liquid Chromatography (HPLC), and is supplemented with ampere Pulse (PAD), ultraviolet (UV), fluorescence detector (FLD) or Mass Spectrum (MS) to achieve the purpose of separating and detecting the HMOs. Furthermore, because HMOs are themselves free of chromophores, the sensitivity on the detector is low, and derivatization reactions such as chromogenic active label labeling are often required, and commonly used labels are 2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), 2-aminoacridine ketone (AMAC), and the like.

In addition, although HPAEC has good separation capability on sugar, and PAD detector can achieve better sensitivity, the mobile phase (strong alkali environment) used by HPAEC cannot be used in tandem with mass spectrum, so that the application of HPAEC is limited from a certain aspect. CE has the advantages of low requirement on sample size, rapid and convenient operation, high resolution and the like, is mostly used for detecting sialylated HMOs, but one major disadvantage of CE is the existence of serious isomer co-elution phenomena such as 2' -FL, 3-FL and the like. In addition, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS) has a high sensitivity as compared with detection means such as fluorescence, but is widely used, but has a relatively high detection cost and a complicated pretreatment operation.

In the method for rapidly determining qualitative and quantitative content of breast milk oligosaccharide disclosed in cited document 1, the determination is carried out by ultra-high liquid chromatography and mass spectrometry after removing fat precipitate protein. Firstly, the method aims at the condition that the to-be-detected product is breast milk, the addition amount and the overall composition of HMOs in the breast milk and the HMOs in milk powder are different, and the interference of other components is large when the content of the HMOs in milk products such as milk powder is detected.

Citation 2 uses organic solvent as precipitant to precipitate protein, and adopts anion exchange chromatography to determine the content of breast milk oligosaccharide in milk after purification treatment, the pretreatment of the method is simple, but the types of breast milk oligosaccharide which can be detected are limited, and the method only comprises three types of 3'-SL, 6' -SL and LNT, which indicates that the disclosed detection method has limited separation effect on HMOs and cannot exclude other interference; in addition, the precipitation of proteins using organic solvents is time consuming and requires low temperatures, and the selection of ethanol and acetonitrile as precipitants may result in recrystallization of lactose or oligosaccharides, which may reduce the recovery of HMOs.

Reference 3 discloses a liquid chromatography-mass spectrometry detection method (external standard method) for HMOs, which utilizes sodium borohydride to perform reduction reaction on oligosaccharide, wherein the sodium borohydride can damage a hemiacetal structure of a reducing end of the oligosaccharide, reduce reducing end isocephalyl of the breast milk oligosaccharide into a diol structure with the identical structure, and reduce the complexity of the oligosaccharide structure and the chromatographic separation difficulty.

Citation literature:

citation 1: CN107192771B

Citation 2: CN112526022A

Citation 3: CN113686992A

Disclosure of Invention

Problems to be solved by the invention

Although the above prior art has conducted some studies on the detection of breast milk oligosaccharides in dairy products, it still cannot be completely adequate in terms of convenience, accuracy and economy of detection.

Based on the existing analysis means, the invention provides a detection method of high performance liquid chromatography-fluorescence analysis for the first time, the method improves the separation degree of various components by optimizing a pretreatment mode, and maximally eliminates the interference of other conventional added components in the dairy product, especially the frequently added galactooligosaccharide, thereby obtaining the detection method of the breast milk oligosaccharide content in the dairy product, which has the advantages of simple operation, low detection cost, good separation effect and high recovery rate.

In addition, in some preferred embodiments, a method for detecting HMOs by on-line solid phase extraction-liquid chromatography-fluorescence analysis is also provided by using a pretreatment column at the front end of the liquid chromatography column.

In addition, the quality control mode comprises the steps of adding a standard for recovery or selecting a sample added with the galactooligosaccharide and the breast milk oligosaccharide with known contents for sample reserving and retesting to obtain a stable detection range.

Solution for solving the problem

Through intensive studies by the inventors, it is considered that the above technical problems can be solved by the following scheme:

[1] a method for detecting the content of breast milk oligosaccharides in a dairy product, wherein the method comprises the following steps:

a step of pre-treatment and a step of detection,

the step of preprocessing comprises the following steps: a step of removing protein and a step of removing galactooligosaccharides, and further comprising a step of derivatizing treatment after the step of removing galactooligosaccharides, the step of derivatizing treatment being performed in the presence of a derivatizing agent comprising procainamine and an internal standard;

the detecting step includes: detecting the detection sample obtained in the pretreatment step by a liquid chromatography-fluorescence analysis method; wherein the mobile phase in the liquid chromatograph comprises a polar solvent containing a chiral solvent.

[2] The detection method according to [1], wherein in the step of removing protein, casein is removed by adjusting pH; the step of removing galacto-oligosaccharides is preceded by a step of removing fat.

[3] The detection method according to [1] or [2], wherein in the step of removing galactooligosaccharides, the galactooligosaccharides are hydrolyzed by adding an enzyme.

[4] The detection method according to [1] or [2], wherein in the step of pretreatment, the internal standard is selected from laminarin or mannotriose.

[5] The detection method according to [1] or [2], wherein the step of the derivatizing treatment further comprises a step of extraction after adding the derivatizing agent to obtain the detection sample.

[6] The detection method according to [5], wherein the step of extracting comprises performing centrifugation in the presence of a polar solvent to obtain a clarified liquid as the detection sample.

[7] The detection method according to [1] or [2], wherein in the step of detecting, a pretreatment column is further provided at the front end of a liquid chromatography column in the liquid chromatography, and the pretreatment column and the liquid chromatography column are connected through a multi-way valve.

[8] The detection method according to [1] or [2], wherein the polar solvent of the mobile phase in the liquid chromatography is one selected from the group consisting of nitrile solvents and alcohol solvents; the chiral solvent comprises a chiral alcohol solvent.

[9] The detection method according to [1] or [2], wherein the excitation wavelength in the fluorescence analysis method is 260 nm.+ -. 10nm, and the detection wavelength is 420 nm.+ -. 10nm.

[10] The test method according to [1] or [2], wherein the breast milk oligosaccharide comprises or is selected from one or more of 2' -fucosyllactose, 3' -sialyllactose,6' -sialyllactose, lactodifucosyltetrasaccharide, lactose-N-neotetraose, lactose-N-tetraose.

[11] The invention further provides application of the detection method in the detection of the dairy product, wherein the dairy product comprises infant formula milk powder, special medical formula milk powder, children formula milk powder and adult formula milk powder.

ADVANTAGEOUS EFFECTS OF INVENTION

Through implementation of the technical scheme, the invention can obtain the following technical effects:

the invention provides a method for detecting the content of breast milk oligosaccharide in a dairy product based on a liquid chromatography-fluorescence analysis method, which can avoid the influence of interference components in the dairy product on detection and improve the accuracy of detecting HMOs by optimizing a pretreatment mode, particularly removing conventionally added galactooligosaccharide components in the dairy product and selecting a derivative reagent.

Further, in the present invention, the chiral solvent is used in the mobile phase during liquid chromatography detection, thereby improving the degree of separation of various HMOs, and in particular, the degree of separation of lactose-N-neotetraose and lactose-N-tetraose can be improved.

In addition, in some preferred embodiments, the present invention can also provide an on-line solid phase extraction-liquid chromatography-fluorescence analysis (Online SPE-HPLC-FLD) method to detect HMOs in dairy products by providing a pretreatment column before the liquid chromatography column of liquid chromatography.

Therefore, the invention generally provides a method for detecting the content of breast milk oligosaccharide in the dairy product, which has the advantages of simple operation, low detection cost, good separation effect and high recovery rate.

Drawings

Fig. 1: on-line solid phase extraction column pipeline connection schematic diagram

Fig. 2: comparative example 1 HMOs Standard chromatogram

Fig. 3: comparative example 2 HMOs Standard chromatogram

Fig. 4: EXAMPLE 1 HMOs Standard chromatogram

Fig. 5: example 1 HMOs and GOS contrast chromatograms

Detailed Description

The following describes the present invention in detail. The following description of the technical features is based on the representative embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples. It should be noted that:

in the present specification, the numerical range indicated by "numerical values a to B" means a range including the end point value A, B.

In the present specification, the use of "substantially" or "substantially" means that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, more preferably 1%.

In the present specification, the meaning of "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

In this specification, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

In the present specification, the term "substantially" means that the deviation from the standard is 1% or less, preferably 0.5% or less.

In this specification, "column" has the same meaning as "chromatographic column".

Reference throughout this specification to "some specific/preferred embodiments," "other specific/preferred embodiments," "an embodiment," and so forth, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the elements may be combined in any suitable manner in the various embodiments.

The invention provides a method for detecting the content of breast milk oligosaccharide HMOs in milk or dairy products, which is mainly based on the following findings:

although a few detection methods have been developed for measuring the content of breast milk oligosaccharide in the prior art, various problems exist in production practice, for example, the detection cost is high when detection is performed by using liquid chromatography-mass spectrometry, and the detection signal is poor and some HMOs are not sufficiently separated when fluorescence detection is adopted. In addition, it was found that various additional components are present in a typical dairy product, and these components also have an influence on various detection results.

Accordingly, the present invention provides a method for detecting one or more breast milk oligosaccharides in a dairy product based on a number of detection practices. The influence of interfering components (such as galactooligosaccharides added in dairy products and the like) on detection accuracy can be avoided by optimizing a pretreatment method and selecting a derivatization reagent, and further, isomers in HMOs, especially lactose-N-neotetraose and lactose-N-tetraose isomers, can be effectively separated by using chiral solvents in a liquid chromatography mobile phase.

In addition, the front end of the liquid chromatographic column is provided with a pre-treatment column, so that the method for on-line solid phase extraction-high performance liquid chromatography-fluorescence analysis of the breast milk oligosaccharide in the dairy product can be further provided.

(Breast milk oligosaccharide)

HMOs typically consist of 3 to 14 monosaccharides. HMOs are composed of 5 monosaccharide basic units, such as glucose, galactose, N-acetylglucosamine, fucose and N-acetylneuraminic acid, which usually contain a lactose core at the reducing end, and are prolonged together with the remaining 3 monosaccharide units by the action of several glycosyltransferases, and the number of monosaccharide compositions is generally 3 to 14.

Depending on whether HMOs are sialylated, they can be classified into neutral HMOs (terminated with L-fucose) and acidic HMOs (terminated with sialic acid).

In the invention, the HMO targets are respectively:

2'-fucosyllactose (2' -FL);

3-fucosyllactose (3-FL);

3'-Sialyllactose (3' -SL);

6'-Sialyllactose (6' -SL);

lactose Disaccharide Fucosyltetrasaccharide (DFL);

lactose-N-tetraose (LNT);

lactose-N-neotetraose (LNnT);

wherein, the target substances except for 3'-SL and 6' -SL containing sialic acid are neutral HMOs.

The structure of each of the above breast milk oligosaccharides is as follows:

the detection methods provided below of the present invention may detect one or more of the above HMOs, and in some specific embodiments, 2, 4, or 7 species therein may be detected simultaneously.

(object of detection)

The measurement method of the present invention is mainly directed to a method for detecting the content of a specific component in a dairy product, and the dairy product to be detected is not particularly limited in principle, and may be, for example, a product derived from animal milk or various foods containing such animal milk. These dairy products may be in liquid form, solid form, or a solid-liquid mixture system, etc.

For animal milk, in some specific embodiments, it may be cow milk, sheep milk, horse milk, camel milk, or the like.

In some preferred embodiments of the present invention, the sample to be tested may be liquid milk, such as liquid cow milk, sheep milk, etc.; and can also be processed products of animal milk, such as dairy products, fermented milk, etc.

Further, the liquid milk may be liquid milk with or without degreasing, sterilization, etc., and typically may be a dairy product or the like for daily drinking; for milk powder products, it may include infant formula, adult milk, middle aged and elderly milk, formula for special medical uses, and the like.

The detection object of the present invention may be various foods containing the above-mentioned animal milk component. In some specific embodiments, these foods include infant complementary foods, functional dairy foods, and the like.

(pretreatment step)

In the present invention, the above-mentioned detection object is treated by a pretreatment step to be a detection sample that can be directly detected.

Specifically, the pretreatment step of the present invention mainly comprises a step of removing a protein component, a step of removing galactooligosaccharide, a step of derivatizing treatment, and optionally, one or more steps of adjusting a solid content and a step of removing fat.

Step of adjusting solid content

For different dairy products, it is desirable to prepare samples that can be tested, and therefore, in some embodiments, water is used to dilute the non-liquid or too high solids content test subjects, or, in other embodiments, too low solids content test subjects, by means of water removal, to raise their solids content to a suitable level.

There is no particular requirement for the specific manner of adjusting the solids content, and the test object may be mixed with water at ordinary temperature to form a substantially uniform system, and then subjected to the subsequent required treatment. And optionally grinding, stirring, etc. may be used to facilitate obtaining the substantially uniform system described above.

Step of fat removal

For the test object, since it may have a certain content of fat, for example, various kinds of powdered milk, liquid milk, and the like. Further, in order to reduce the influence of fat on the detection accuracy and the detection instrument, part or all of the fat may be removed in any desired case.

There is no particular limitation on the manner in which fat is removed, and in some particular embodiments, centrifugation may be used to remove fat. Further, in some preferred embodiments, 50 mass% or more, preferably 70 mass% or more, more preferably 90 mass% or more of the fat is separated and removed after the step of removing fat, based on the mass of the total fat in the subject.

Step of removing protein

As a necessary pretreatment step in the detection method of the present invention, it is necessary to remove protein components therein which may interfere with detection for the detection object to improve the effectiveness of detection and mitigate adverse effects on the detection instrument.

The step of removing proteins is not particularly limited in principle, and proteins in the subject may be at least partially removed, and in particular, part or all of casein may be removed by a method of removing proteins.

In some specific embodiments, the proteins therein, in particular casein, may be removed by means of pH adjustment. The method of adjusting the pH may be performed by adding an acidic component or a buffering component to the test object or an aqueous solution thereof. The acidic component may be various organic acids, for example, formic acid or acetic acid, and the buffering component may be a phosphoric acid-based buffering component or a phosphoric acid-salt composite buffering component. The pH value may be adjusted to a range of 4.2 to 4.6, preferably 4.3 to 4.5, from the viewpoint of sufficiently removing casein.

In other specific embodiments, the protein in the test subject may also be at least partially removed by one or more ultrafiltration treatments. The target component is removed by selection of a suitable ultrafiltration membrane.

Further, in some specific embodiments, 50 mass% or more, preferably 70 mass% or more, and more preferably 90 mass% or more of the protein in the subject is separated and removed by the step of removing the protein, based on the total protein content in the subject. In other preferred embodiments, 70 mass% or more, preferably 80 mass% or more, and more preferably 90 mass% or more of the casein component in the subject is separated and removed by the step of removing protein.

Step of removal of galactooligosaccharides

As described above, the presence of various additives, particularly galactooligosaccharides, which are often added to dairy products can be a significant interfering component in the detection of the content of HMOs, and therefore, it is necessary to remove galactooligosaccharides in the detection subject in pretreatment as an essential step in the detection method of the present invention.

As a means for removing galactooligosaccharides, there is basically no particular limitation, and in some embodiments of the present invention, an enzyme may be added to the protein-removed system to enzymatically decompose galactooligosaccharides therein, particularly to enzymatically decompose galactooligosaccharides as galactooligosaccharides.

Further, as the enzyme which can be used in the present invention, amyloglucosidase may be mentioned. The conditions for the enzymatic hydrolysis are not particularly limited as long as the enzymatic hydrolysis of galactooligosaccharides is sufficient.

Step of derivatization treatment

In the present invention, the test object may be subjected to derivatization after the step of removing galactooligosaccharide.

The derivatization treatment is mainly a group that modifies the target breast milk oligosaccharide in the test subject to impart fluorescence properties thereto. In some specific embodiments, the optical detectability of the galactooligosaccharide ingredient may be achieved by modifying the galactooligosaccharide with a derivatizing agent.

For the derivatizing agent, the present invention has found that the use of procainamide (see structure of formula 1 below) as the derivatizing agent having an optically active group is effective in modifying various breast milk oligosaccharides as compared with other conventional derivatizing agents and has good detectability when subjected to the fluorescent assay described below.

1 (1)

Further, in the derivatization treatment, an acidic component, a dehydrated component, a reducing component, a solvent, and the like may be added as needed in addition to the procainamide described above. The cleavage of the galacto-oligosaccharide end can be assisted with an acidic component, thereby liberating an aldehyde that can react with the primary amine groups of procainamide. The dehydration component mainly assists dehydration at the time of primary amine reaction of procainamide to form an aldimine structure, and examples of the dehydration component include glacial acetic acid. For the reducing component, mainly the aldimine structure described above is reduced to give the corresponding imine structure, and for the reducing component, one or more of borane compounds having reducing properties may be used. In addition, the solvent is not particularly limited, and one or more of polar solvents such as sulfones or amides may be used.

Furthermore, for the derivatization treatment of the present invention, it is typically a derivatization treatment performed in the presence of an internal standard to simultaneously impart optical/fluorescent detectability to the internal standard. For the internal standard useful in the present invention, it may typically be a saccharide, in some preferred embodiments, the internal standard may be laminarin or mannotriose, preferably the internal standard may be mannotriose. For the internal standard, it can be derivatized together with breast milk oligosaccharide in the same manner as described above.

Steps of other treatments

In the present invention, other pretreatment operations may be performed according to any need, in addition to the various treatment steps described above.

In some specific embodiments, a desalting treatment step may be included in addition to adjusting the solids content, removing proteins, galactooligosaccharides, fat components. The desalting method is not particularly limited, and for example, the desalting may be performed by a separate ultrafiltration step or simultaneously in the ultrafiltration treatment in the above-described step of removing proteins.

In other specific embodiments, after the derivatization treatment, to obtain a test sample suitable for detection, the method further comprises the step of extracting, wherein the step of extracting comprises centrifugation in the presence of a polar solvent to obtain a clarified liquid as a test sample that can be directly detected on-machine.

(step of detection)

The detection step of the present invention is mainly performed by using a combination of liquid chromatography and fluorescence detection device, wherein in some preferred embodiments, a line solid phase extraction-liquid chromatography-fluorescence analysis (Online SPE-HPLC-FLD) method is further obtained by adding a pretreatment column before the liquid chromatography column of liquid chromatography to detect HMOs in dairy products.

Liquid chromatography

The liquid chromatography used in the present invention may be High Performance Liquid Chromatography (HPLC) or ultra high performance liquid chromatography (UPLC).

In some specific embodiments, for chromatography columns in liquid chromatography, hydrophilic interaction chromatography (HILIC) may be used, typically glycosyl HILIC chromatography columns may be used. For such columns, it is possible to obtain them commercially, for example the XBridge type columns based on the ethylene bridge hybridization (BEH) particle technology offered by Waters corporation, which can meet the application requirements of UPLC (1.7 μm) and HPLC (2.5 μm and 3.5 μm). In some preferred embodiments, a Waters XBridge Glycan BEH Amide chromatographic column may be used.

Further, for the mobile phase of liquid chromatography (hereinafter also referred to as mobile phase a), a polar solvent containing a chiral solvent may be used from the viewpoint of effective separation of various kinds of breast milk oligosaccharides. The present invention has found that the use of chiral solvents allows good separation of difficult to separate isomers to improve the accuracy of the detection.

For chiral solvents, in some specific embodiments of the invention, chiral alcohol solvents may be used, and in some preferred embodiments, isopropyl alcohol may be used as the chiral solvent.

The polar solvent in the mobile phase a of the present invention is not particularly limited in principle, and for example, a nitrile solvent, an alcohol solvent, preferably, a nitrile solvent, more preferably, acetonitrile may be used.

Further, for the composition of mobile phase a, in some specific embodiments of the invention, the chiral solvent is used in an amount of 5 to 15% by volume, preferably 8 to 12% by volume, based on the total volume of both the chiral solvent and the polar solvent prior to mixing.

Furthermore, in some preferred embodiments of the present invention, from the viewpoint of improving the separation effect of the respective HMOs, an additional mobile phase (hereinafter also referred to as mobile phase B) may be used in the liquid chromatography, and the polarity or pH of the total mobile phase is adjusted by the use of mobile phase B to assist in improving the chromatographic separation effect. As a preferred mobile phase B, an aqueous solution of ammonium formate is used, wherein the concentration of ammonium formate may be 80 to 120mM, preferably 90 to 110mM.

Pretreatment column

In some preferred embodiments of the present invention, a pretreatment column may also be provided at the front end of the liquid chromatography column described above. By arranging the pretreatment column, the invention can provide an on-line solid phase extraction-high performance liquid chromatography-fluorescence analysis method.

As for the arrangement of the pretreatment column, on the one hand, as described above, it is possible to perform an extraction or purification function for the detection sample detected by the upper machine, and on the other hand, it is also possible to perform a protection function for the liquid chromatography column, that is, to protect the liquid chromatography column from the influence of the impurity components remaining in the detection sample.

Further, as for the kind of pretreatment column, in some specific embodiments, the same materials as the above-mentioned liquid chromatography column may be used, and in some preferred embodiments, the pretreatment column may differ from the liquid chromatography column only in length, for example, the pretreatment column has a length of 8% to 30%, preferably 10% to 20% of the length of the liquid chromatography column.

In addition, from the aspects of operation and good protection of the liquid chromatographic column, the pretreatment column and the liquid chromatographic column can be connected through a multi-way valve or the connection relationship can be switched. In some embodiments, the above-described connection or switching may be accomplished by a six-way valve.

In addition, as the mobile phase in operation of the pretreatment column, the same kind of mobile phase as in liquid chromatography can be used, but the composition (e.g., ratio of mobile phase a to mobile phase B) and flow rate of the mobile phase are allowed to be different.

Referring to a in fig. 1, after sample introduction, the pretreatment column is disconnected from the liquid chromatography column (i.e., the "analytical column"), at this time, the detection sample is extracted or purified on the pretreatment column under the action of the mobile phase, and the waste liquid flows out through the waste liquid outlet.

Referring to b in fig. 1, at a suitable retention time, the position of the six-way valve is switched such that the pretreatment column is connected in series with the liquid chromatography column, and the target detection substance is brought into the liquid chromatography column by the mobile phase for separation.

For the appropriate retention time or the time for which the six-way valve is switched, the retention time of the detection component on the pretreatment column can be pre-tested by the fluorescence detector depending on the condition of the mobile phase when the pretreatment column is in operation.

In addition, from the viewpoint of improving the operation efficiency, the flow rate and polarity of the mobile phase when the pretreatment column is operated are both greater than those when the liquid chromatography is operated.

Fluorescence analysis

The fluorescence analysis of the present invention is performed by a fluorescence detector, and the specific type and model of the fluorescence detector are not particularly limited, and the fluorescence detector can be obtained by commercial products.

In some specific embodiments, for fluorescence detectors, it may provide an excitation wavelength of 260 nm.+ -.10 nm, and a detection wavelength of 420 nm.+ -.10 nm.

Other auxiliary units

The auxiliary unit that can be used in the detection method of the present invention is not particularly limited. In some specific embodiments, these auxiliary units optionally may include:

the column temperature box is used for storing each chromatographic column and adjusting the temperature of each chromatographic column, for example, the temperature of the liquid chromatographic column can be controlled between 55 ℃ and 65 ℃ when the liquid chromatographic column works.

An automatic control device which can be programmed to switch the positions of the multi-way valves in the multi-way valve group at an appropriate time by programming or the like to realize the switching of the communication modes.

A liquid phase pump flow control device to adjust the rate at which the liquid phase pump supplies different mobile phases and mobile phases.

And the automatic sample injection device is used for realizing automatic sample injection detection.

(qualitative/quantitative analysis)

By the detection method described above according to the invention, qualitative and quantitative analysis of the dairy product can be provided by detecting the resulting standard curve by means of a standard.

Further, in the quantitative analysis, the standard curve can be corrected by means of the detected peak area for a standard sample of galactooligosaccharide of known content. Therefore, the actual content of HMO which is peaked at the same time can be calculated by only comparing the peak of the galactooligosaccharide raw material with the peak of the galactooligosaccharide raw material in each test.

Examples

The invention will be specifically illustrated by the following examples:

example 1:

example 1 was performed as follows.

(reagents and materials)

Unless otherwise specified, only analytically pure reagents were used.

Water, GB/T6682, first order;

acetonitrile (CH) ₃ CN): chromatographic purity;

glacial acetic acid (CH) ₃ COOH)；

Sodium acetate trihydrate (CH) ₃ COONa·3H ₂ O)；

Ammonia (NH) ₃ ·H ₂ O): the concentration is 25-30%;

formic acid (HCOOH): the purity is more than or equal to 99 percent;

dimethyl sulfoxide (DMSO, (CH) ₃ ) ₂ SO)： CAS No. 67-68-5；

2-methylpyridine borane complex: purity is more than or equal to 95%, CAS No. 3999-38-0;

procainamide (4-amino-N- (2-diethylenetriamine) benzamide): the purity is more than or equal to 98 percent;

amyloglucosidase (amyloglucosidase): ultra-high purity.

(preparation of reagents)

1) Sodium acetate buffer (0.1 m, ph=5.4):

1.36mg sodium acetate trihydrate is weighed into a 100mL volumetric flask, pH=5.4 is adjusted with glacial acetic acid, the volume is fixed with water, and the mixture can be stored for 1 week at 4 ℃.

2) Amyloglucosidase (Amyloglucosidase) solution (100 Units/mL):

and adding a proper amount of amyloglucosidase into a 50mL volumetric flask, adding sodium acetate buffer solution to a constant volume, and subpackaging into a 15 mL centrifuge tube, wherein the amyloglucosidase can be stored for 1 year at the temperature of minus 20 ℃.

3) Derivatizing reagent:

4.7 mL of DMSO and 2.0mL of glacial acetic acid are placed in a glass container according to a proportion, and are uniformly mixed by vortex to prepare a 30% glacial acetic acid+70% DMSO mixed solution. 571.2 mg procainamide and 642+ -5 mg of 2-methylpyridine borane are weighed into another glass container, 6mL of a 30% acetic acid+70% DMSO mixed solution is added, after vortex mixing is carried out, ultrasonic treatment is carried out for 10min until complete dissolution is carried out, and a 30% glacial acetic acid+70% DMSO mixed solution containing 0.35 mol/L procainamide and 1 mol/L2-methylpyridine borane is prepared.

4) Acetonitrile-water solution:

water 50mL and acetonitrile 150 mL are measured and mixed evenly to prepare acetonitrile (75 percent) -water (25 percent) solution.

5) 50% acetic acid solution:

taking 50% of water mL, 50% of glacial acetic acid mL, and mixing to obtain 50% acetic acid solution.

6) Mobile phase a (isopropanol/acetonitrile=10/90):

100mL isopropyl alcohol was added to a glass bottle containing 900 mL Acetonitrile (ACN) and mixed well by sonication.

7) Mobile phase B (100 mM ammonium formate, ph=4.4):

3.78mL of formic acid is added into 900 mL water, ammonia water is added to adjust the pH value to 4.4+/-0.02, the volume is fixed to 1L, and the mixture is uniformly mixed.

(Standard and internal standard)

The standards used were from China measuring institute and Carbosynth, the internal standard was from Megazyme, and the detailed information is shown in Table 1.

Table 1: standard substance information used

Standard and internal standard preparation:

1) Internal standard formulation (about 500. Mu.g/mL)

And (3) actual preparation: weighing 5mg internal standard into a 10 mL volumetric flask, and diluting ultrapure water to scale marks; the mixture is packaged in 1.5-mL to 2-mL screw cap centrifuge tubes, and can be stably stored for 12 weeks at-20+/-3 ℃.

2) Preparation of stock solution of series standard liquid

HMO control stock:

the exact purity and moisture content of the different HMO controls used for calibration should be known, precisely to the 3 significant digits. The concentration of the control solution was calculated using known purity and moisture content corrections. Ideally, the manufacturer (e.g., glycom) would provide the HMO control in the form of a sealed bottle, while providing the COA, indicating the purity and moisture content of the control provided. HMO control storage was performed according to the supplier's recommendations or it was stored in a desiccator at room temperature. If the supplier does not specify the moisture content of the control, the obtained control should be dried in a vacuum oven (vacuum, 60 ℃ C., dry overnight) or the moisture content of the control should be determined. The solution can be stored for one year at-18 ℃. The specific formulation is shown in table 2.

Table 2: preparation of 7 HMO stock solutions

Preparation of a standard curve:

according to Table 3, a mixed solution was prepared and scaled with water, using a 50mL volumetric flask for concentration level 1, a 10 mL volumetric flask for concentration level 2, and a 5mL volumetric flask for concentration levels 3-6. The corresponding concentrations ([ mu ] L/mL) of each test object prepared in Table 3 are shown in Table 4. The prepared solution can be stored at 4deg.C for 1 week, at-18deg.C for 1 year, and the number of freeze thawing cycles should not exceed 3.

The concentrations in tables 3 and 4 are used as references. The actual concentration of the calibration curve solution was calculated from the control purity using the exact concentration of each stock.

Table 3: preparation of 6-point calibration curves for 7 HMO mixtures

Table 4:7 HMO mixture working calibration curve solution concentrations (. Mu.g/mL) (assuming a control purity of 95%)

(instruments and devices)

Table 5: apparatus and device used in the examples

(sample pretreatment)

1) Sample preparation

Weighing 5.0000g of milk powder in a 50mL triangular flask, adding about 25mL of water at 40 ℃, then placing in a 70 ℃ water bath kettle, magnetically stirring for 20-25 min until the milk powder is completely dissolved, and standing to room temperature for later use. Ph=4.5 was adjusted with 50% acetic acid solution (4.2.5), transferred to a 50mL volumetric flask and filtered through constant volume filter paper.

2) Enzymolysis

Sucking 0.5mL sample filtrate or 0.5mL of working solution into a 2mL centrifuge tube, adding 0.5mL amyloglucosidase solution into the sample, and adding 0.5mL aqueous solution into the standard substance; then 0.5mL acetic acid buffer solution and 0.2 mL internal standard solution are added, vortex mixing is carried out, and incubation is carried out for 60+/-2 min under the condition of 60+/-2 ℃.

3) Derivatization

After 250. Mu.L of the derivative solution was added to a 2mL screw-capped centrifuge tube, 100. Mu.L of the sample extract or working solution was added and vortexed. Incubation was performed at 60.+ -. 2 ℃ for 60.+ -. 2min, after cooling, the lid was unscrewed in a fume hood, 1 mL acetonitrile-water solution was added, vortexed well, and the tube was centrifuged at 10000g for 5min, and the supernatant was then analyzed on a machine.

(detection)

In the detection step, a line solid phase extraction-liquid chromatography-fluorescence analysis (Online SPE-HPLC-FLD) method is obtained by adding a pretreatment column (connected with the liquid chromatography column through a multi-way valve) before the liquid chromatography column of the liquid chromatography so as to detect HMOs in a sample.

1) Liquid chromatography and fluorescence detection conditions

(1) Liquid chromatographic column: waters XBridge Glycan BEH Amide, 4.6X105 mm, 2.5 μm.

(2) Protective column: waters XBridge Glycan BEH Amide, 4.6X10. 20mm, 2.5 μm.

(3) Injector temperature: 5 ℃.

(4) Column temperature: 60 ℃.

(5) Sample injection amount: 10. mu L.

(6) Flow rate: 0.7 mL/min, on-line solid phase extraction 1.3 mL/min.

(7) Excitation wavelength: 260 nm.

(8) Emission wavelength: 420 nm.

2) Gradient elution

Table 6-1: gradient elution conditions

Table 6-2: multi-way valve switching program setting (on-line solid phase extraction column pipeline connection is as shown in figure 1)

2) Qualitative determination

The retention time of the chromatographic peak of the target compound in the sample after derivatization is compared with that of the chromatographic peak of the standard substance, and the variation range is within +/-2.5 percent.

3) Drawing of a Standard Curve

And under the condition of liquid chromatography, detecting the derived standard curve solution from low concentration to high concentration (the sample injection amount is 10 mu L), taking the ratio of the concentration of each target compound to the internal standard concentration as an abscissa, and taking the ratio of the peak area of the chromatographic peak of the target compound to the area of the internal standard chromatographic peak as an ordinate to obtain a standard curve regression equation, wherein the linear correlation coefficient is larger than 0.99.

4) Measurement of sample solution

Taking a sample to be detected after derivatization, sampling (the sampling amount is 10 mu L, and if the peak response is insufficient or saturated, the sampling amount is properly reduced or increased), calculating the concentration of each HMO in the test solution through a standard curve, and calculating the content of the HMO in the milk powder according to the following formula. The response value of the object to be tested in the test solution should be within the linear range of the standard curve, and should be re-measured after being diluted properly beyond the linear range.

Before each formulation experiment, weighing GOS raw materials (generally 500-800 mg, based on practice) according to the actual addition content of Galactooligosaccharide (GOS) in the formulation, dissolving in water and fixing volume in a 50mL volumetric flask, processing according to the steps of sample configuration and derivative treatment, and comparing GOS interference peaks according to the peak-to-peak time ratio of a standard substance, wherein the calibration peak area of each HMO in the sample is the area of each HMO peak minus the area of the GOS peak at the same time, and is the calibration peak area of each HMO peak.

5) Test data processing

The content X (mg/100 g) of each HMO in the sample was calculated according to the following formula:

wherein:

C-HMO concentration calculated according to a standard curve in micrograms per milliliter (μg/mL);

w is milk powder weighing (g);

v-constant volume (mL);

DF-dilution factor.

6) Precision of

The absolute difference between the two independent test results obtained under reproducible conditions is not more than 10% of the arithmetic mean.

Comparative example 1

Example 1 was repeated with additional procedures substituting 0.35 mol/L procainamide for 0.35 mol/L2-aminobenzoic acid (2-AA) as the derivatizing agent.

Comparative example 2

Mobile phase a using pure acetonitrile, other procedure repeat example 1.

FIG. 2 is a chromatogram of the HMOs standard of comparative example 1 showing that using 2-aminobenzoic acid (2-AA) as the derivatizing agent, a portion of the HMO did not peak while the isomer was not isolated. Fig. 3 is a chromatogram of the HMOs standard in comparative example 2 showing that LNT and LNnT isomers cannot be separated when pure acetonitrile is used for the mobile phase.

Industrial applicability

The method of the invention can be used industrially for detecting the content of breast milk oligosaccharide in dairy products.

Claims

1. A method for detecting the content of breast milk oligosaccharides in a dairy product, said method comprising the steps of:

a step of pre-treatment and a step of detection,

2. The method according to claim 1, wherein in the step of removing protein, casein is removed by adjusting pH; the step of removing galacto-oligosaccharides is preceded by a step of removing fat.

3. The method according to claim 1 or 2, characterized in that in the step of removing galactooligosaccharides, enzymes are added to hydrolyze the galactooligosaccharides.

4. The method according to claim 1 or 2, wherein in the step of pretreatment, the internal standard is selected from laminarin or mannotriose.

5. The method according to claim 1 or 2, wherein the step of derivatizing further comprises a step of extracting after adding the derivatizing agent to obtain the test sample.

6. The method according to claim 5, wherein the step of extracting comprises centrifuging in the presence of a polar solvent to obtain a clarified liquid as the detection sample.

7. The method according to claim 1 or 2, wherein in the step of detecting, a pretreatment column is further provided at a front end of a liquid chromatography column in the liquid chromatography, and the pretreatment column and the liquid chromatography column are connected by a multi-way valve.

8. The method according to claim 1 or 2, wherein the polar solvent of the mobile phase in the liquid chromatograph is one selected from the group consisting of nitrile solvents and alcohol solvents; the chiral solvent comprises a chiral alcohol solvent.

9. The method according to claim 1 or 2, wherein the excitation wavelength in the fluorescence analysis method is 260 nm.+ -. 10nm, and the detection wavelength is 420 nm.+ -. 10nm.

10. The method of claim 1 or 2, wherein the breast milk oligosaccharide comprises or is selected from one or more of 2' -fucosyllactose, 3' -sialyllactose,6' -sialyllactose, lactodifucosyltetrasaccharide, lacto-N-neotetraose, lacto-N-tetraose.

11. Use of the detection method according to any one of claims 1-10 in the detection of dairy products, including infant formula, special medical use formula, children formula, adult formula.