CN109270176B - Goat milk oligosaccharide determination method - Google Patents
Goat milk oligosaccharide determination method Download PDFInfo
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Abstract
The invention discloses a goat milk oligosaccharide determination method, which comprises the following steps: step one, degreasing and deproteinizing goat milk to obtain a goat milk oligosaccharide crude product; step two, removing lactose by using a Sephadex-G10 gel column to obtain a goat milk oligosaccharide sample; step three, utilizing carbon PacTMDetermining the lactose removal rate and the oligosaccharide retention rate of the sample by using a PA20 sugar separation column; and step four, optimizing liquid phase conditions and carrying out qualitative and quantitative analysis on the components and the structure of the product by adopting an ultra-high performance liquid phase Q-active Focus mass spectrum. The invention establishes a method for effectively separating and determining goat milk oligosaccharide for the first time, realizes effective removal of lactose by gel column chromatography on a goat milk sample with higher separation difficulty, realizes determination of isomer by optimization of liquid phase conditions, and identifies 49 oligosaccharides in goat milk by reasonable setting of a mass spectrometer.
Description
Technical Field
The invention relates to a method for separating and measuring goat milk oligosaccharide.
Background
Milk is a complex nutritional ingredient including fat, protein, carbohydrates, wherein milk also provides the nutritional needs of mammals. The lactose content in the carbohydrate is over 80% and the free lactooligosaccharide content is around 4% of the total sugars, so that the removal of lactose plays an important role in the analysis of lactooligosaccharides.
Studies have shown that milk oligosaccharides can balance gut microbiota to modulate the immune system, sialylated oligosaccharides can promote brain development, and serve as a source of galactose for the synthesis of galactocerebrosides, sialic acid for gangliosides in the grey brain matter, and glycoprotein production. It has been found that human milk contains more than 200 oligosaccharides, however, due to the differences in size, charge and structure, only low molecular weight human milk oligosaccharides have been synthesized chemically to date. Therefore, the search for alternative sources with biological activity similar to human milk oligosaccharides and their application in infant formulas has attracted attention. The goat milk is one of important milk base materials of infant formula powder, and researches show that the oligosaccharide content in the goat milk is about 0.25-0.30g/L, is about 8 times of that in cow milk, and is a potential breast milk oligosaccharide substitute. However, due to the limitation and technical barrier of the detection method, the research on the oligosaccharide structure in goat milk in China is laggard, so that the establishment of an effective goat milk oligosaccharide detection means has important significance for the research on goat milk oligosaccharides in China.
Based on these physiological functions, attention has been focused on the study of lactooligosaccharides. The domestic research on the structure of the goat milk oligosaccharide is only reported, and a goat milk oligosaccharide detection method is not provided. The separation and purification of oligosaccharides is the key to oligosaccharide detection, and its monitoring is very difficult due to the high complexity of its own composition, linkage, derivatization, micro-heterogeneity, etc. When the oligosaccharide in the goat milk is analyzed, some high-abundance oligosaccharides can cover detection signals of low-abundance oligosaccharides, so that the complete identification of the oligosaccharides is hindered, the oligosaccharide sample needs to be enriched and purified, the mixing of impurities is reduced, and the detection sensitivity is improved. On the other hand, the number of monosaccharide of the oligosaccharide in the goat milk is generally between 3 and 10, the molecular weight is similar, the operation difficulty is increased, and meanwhile, the retention time and the mass spectrogram of isomers are very similar, so that great difficulty is brought to the given character. Therefore, the selection of a proper method for separating and measuring the goat milk oligosaccharide is a very exploratory work.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
The invention also aims to provide a goat milk oligosaccharide determination method, which realizes effective removal of lactose which is an influence substance in oligosaccharide determination through gel column chromatography to obtain a goat milk oligosaccharide sample, then carries out quantitative analysis on lactose removal rate and oligosaccharide retention rate by utilizing high-efficiency anion exchange chromatography, identifies goat milk oligosaccharide by utilizing ultra-high performance liquid phase Q-active Focus mass spectrum, and realizes qualitative and quantitative analysis on goat milk oligosaccharide isomers by optimizing liquid phase conditions. By adopting the determination method, 49 goat milk oligosaccharides are finally identified, the structural composition of the 49 goat milk oligosaccharides is analyzed, the separation effect is good, the sample does not need to be derivatized, the original structure of the oligosaccharides is reserved, and in addition, the operation process is simple, the result is stable, and the repeatability is good.
To achieve these objects and other advantages in accordance with the present invention, there is provided a goat milk oligosaccharide assay method comprising the steps of:
step one, degreasing and deproteinizing goat milk, and extracting a goat milk oligosaccharide crude product;
secondly, removing lactose from the goat milk oligosaccharide crude product by using a Sephadex-G10 gel column to obtain a goat milk oligosaccharide sample;
step three, utilizing carbon PacTMDetermining the lactose removal rate and the oligosaccharide retention rate of the goat milk oligosaccharide sample by using a PA20 sugar separation column;
and step four, optimizing liquid phase conditions and performing qualitative and quantitative analysis on the components and the structure of the goat milk oligosaccharide sample by adopting an ultra-high performance liquid phase Q-active Focus mass spectrum.
Preferably, in the step one, the specific method for defatting and deproteinizing the goat milk comprises the following steps:
centrifuging goat milk for 15-30 min at the temperature of 3-8 ℃ and the rpm of 8000-10000, removing fat on the upper layer and a small amount of protein on the bottom, taking out the middle layer, adding absolute ethyl alcohol according to the volume ratio of 1: 2-1: 6, reacting for 2-12 h at the temperature of-80-4 ℃, centrifuging for 15-30 min at the temperature of 4000 Xg at the temperature of 3-8 ℃ after the reaction is finished, taking supernatant, and drying to obtain the goat milk oligosaccharide crude product.
Preferably, the drying is carried out by concentrating and drying with a vacuum concentrator.
Preferably, in the second step, the specific method for removing lactose comprises:
firstly, placing Sephadex-G10 gel in a beaker, soaking the gel in ultrapure water for 10-15 h, removing bubbles by a vacuum pump for 1-2 h, adding the gel into a chromatographic column, and prepressing the gel for 12h at the flow rate of 2.5-3 mL/min by using the ultrapure water;
and (3) loading the goat milk oligosaccharide crude product into a Sephadex-G10 gel column, taking ultrapure water as eluent, collecting column-passing liquid every 2min by using an automatic collector at the flow rate of 2-2.5 mL/min, detecting the column-passing liquid by using a phenol-sulfuric acid method, and placing the column-passing liquid in a refrigerator at the temperature of-80 ℃ for later use.
Preferably, in the third step, the specific method for measuring lactose removal rate and oligosaccharide retention rate is as follows:
adopting ultrapure water as a mobile phase A and 250mM NaOH as a mobile phase B, carrying out gradient elution on the goat milk oligosaccharide sample at the column temperature of 35 ℃ and the flow rate of 0.4mL/min, wherein the conditions of the gradient elution are as follows: 0-40.1min, 7.2% -100% A; 40.2-45min, 100% -100% A; 45.1-55min, 100% -7.2% A; the remaining liquid phase portion is mobile phase B. And detecting the lactose content in the sample by adopting a gold electrode pulse ampere detector, and collecting a collecting pipe containing the goat milk oligosaccharide.
Preferably, in the fourth step, the liquid phase conditions of the ultra-high performance liquid phase Q-active Focus mass spectrum are as follows:
adopting an ACQUITY UPLC amino chromatographic column, wherein the specification of the chromatographic column is 2.1mM multiplied by 100mM, the particle size of a column filler is 1.5-2 mu m, the column temperature is 30-40 ℃, the flow rate is 0.2-0.5 mL/min, a mobile phase A is acetonitrile, a mobile phase B is 10mM ammonium formate, and the gradient elution conditions are as follows: 0-20min, 95% -78% A; 20-35min, 78% -73% A; 35-38min, 73-62% A; 38-45min, 62% -50% A; 45-55min, 50% -95% A; 55-65min, 95% -95% A; the remainder of the liquid phase is mobile phase B.
Preferably, in the fourth step, the mass spectrum conditions of the ultra-high performance liquid phase Q-active Focus mass spectrum are as follows:
electrospray ion source: the spraying voltage of the positive ion mode and the spraying voltage of the negative ion mode are both 5-20 kV. The mass scanning range of the primary ions is 300-2000 m/z, and the column temperature is 30-40 ℃.
The invention at least comprises the following beneficial effects:
the method utilizes a Sephadex-G10 gel column to separate and purify the degreased and deproteinized goat milk, effectively removes influencing substances lactose, qualitatively and quantitatively analyzes oligosaccharide isomerides through optimization of liquid phase conditions, identifies 49 goat milk oligosaccharide components in the goat milk through reasonable setting of a mass spectrometer, and realizes analysis of the structures of the 49 goat milk oligosaccharides.
The determination method is simple and feasible, has stable result, and can be used for purification, separation and determination of the goat milk oligosaccharide.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a total ion flow diagram of a goat milk oligosaccharide sample in example 1 of the present invention.
FIG. 2 is a total ion flow diagram of isomers of Hex2Neu5Ac1 in a sample of goat milk oligosaccharides in example 1 of the present invention.
FIG. 3 is a total ion flow diagram of a goat milk oligosaccharide sample in example 2 of the present invention.
FIG. 4 is a total ion flow diagram of isomers of Hex2Neu5Ac1 in a sample of goat milk oligosaccharides in example 2 of the present invention.
FIG. 5 is a total ion flow diagram of a goat milk oligosaccharide sample in example 3 of the present invention.
FIG. 6 is a total ion flow diagram of isomers of Hex2Neu5Ac1 in a sample of goat milk oligosaccharides in example 3 of the present invention.
FIG. 7 is an ion chromatogram of a crude goat milk oligosaccharide in example 3 of the present invention.
FIG. 8 is an ion chromatogram of a goat milk oligosaccharide sample in example 3 of the present invention.
FIG. 9 is an ion chromatogram of a sample of goat milk oligosaccharide in comparative example 1.
FIG. 10 is an ion chromatogram of a sample of goat milk oligosaccharide in comparative example 2.
FIG. 11 is an ion chromatogram of a sample of goat milk oligosaccharide in comparative example 3.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The method for determining the goat milk oligosaccharide comprises the following steps:
step one, degreasing and deproteinizing goat milk, and extracting a goat milk oligosaccharide crude product;
secondly, removing lactose from the goat milk oligosaccharide crude product by using a Sephadex-G10 gel column to obtain a goat milk oligosaccharide sample;
step three, utilizing carbon PacTMDetermining the lactose removal rate and the oligosaccharide retention rate of the goat milk oligosaccharide sample by using a PA20 sugar separation column;
and step four, optimizing liquid phase conditions and performing qualitative and quantitative analysis on the components and the structure of the goat milk oligosaccharide sample by adopting an ultra-high performance liquid phase Q-active Focus mass spectrum.
In one embodiment, in the first step, the specific method for defatting and deproteinizing the goat milk comprises the following steps:
centrifuging goat milk for 15-30 min at the temperature of 3-8 ℃ and the rpm of 8000-10000, removing fat on the upper layer and a small amount of protein on the bottom, taking out the middle layer, adding absolute ethyl alcohol according to the volume ratio of 1: 2-1: 6, reacting for 2-12 h at the temperature of-80-4 ℃, centrifuging for 15-30 min at the temperature of 4000 Xg at the temperature of 3-8 ℃ after the reaction is finished, taking supernatant, and drying to obtain the goat milk oligosaccharide crude product.
In one embodiment, the drying is concentrated using a vacuum concentrator.
In one embodiment, in the second step, the specific method for removing lactose includes:
firstly, placing Sephadex-G10 gel in a beaker, soaking the gel in ultrapure water for 10-15 h, removing bubbles by a vacuum pump for 1-2 h, adding the gel into a chromatographic column, and prepressing the gel for 12h at the flow rate of 2.5-3 mL/min by using the ultrapure water;
and (3) loading the goat milk oligosaccharide crude product into a Sephadex-G10 gel column, taking ultrapure water as eluent, collecting column-passing liquid every 2min by using an automatic collector at the flow rate of 2-2.5 mL/min, detecting the column-passing liquid by using a phenol-sulfuric acid method, and placing the column-passing liquid in a refrigerator at the temperature of-80 ℃ for later use.
In one embodiment, the specific method for measuring lactose removal rate and oligosaccharide retention rate in step three is as follows:
adopting ultrapure water as a mobile phase A and 250mM NaOH as a mobile phase B, carrying out gradient elution on the goat milk oligosaccharide sample at the column temperature of 35 ℃ and the flow rate of 0.4mL/min, wherein the conditions of the gradient elution are as follows: 0-40.1min, 7.2% -100% A; 40.2-45min, 100% -100% A; 45.1-55min, 100% -7.2% A; the remaining liquid phase portion is mobile phase B. And detecting the lactose content in the sample by adopting a gold electrode pulse ampere detector, and collecting a collecting pipe containing the goat milk oligosaccharide.
In one embodiment, in the fourth step, the liquid phase conditions of the ultra-high performance liquid phase Q-active Focus mass spectrum are as follows:
adopting an ACQUITY UPLC amino chromatographic column, wherein the specification of the chromatographic column is 2.1mM multiplied by 100mM, the particle size of a column filler is 1.5-2 mu m, the column temperature is 30-40 ℃, the flow rate is 0.2-0.5 mL/min, a mobile phase A is acetonitrile, a mobile phase B is 10mM ammonium formate, and the gradient elution conditions are as follows: 0-20min, 95% -78% A; 20-35min, 78% -73% A; 35-38min, 73-62% A; 38-45min, 62% -50% A; 45-55min, 50% -95% A; 55-65min, 95% -95% A; the remainder of the liquid phase is mobile phase B.
In one embodiment, in the fourth step, the mass spectrum conditions of the ultra-high performance liquid phase Q-active Focus mass spectrum are as follows: electrospray ion source: the spraying voltage of the positive ion mode and the spraying voltage of the negative ion mode are both 5-20 kV. The mass scanning range of the primary ions is 300-2000 m/z, and the column temperature is 30-40 ℃.
Example 1
Step one, degreasing and deproteinizing goat milk. Centrifuging goat milk for 30min at 4 ℃ and 10000rpm, removing upper fat and a small amount of protein at the bottom, taking out the middle layer, adding absolute ethyl alcohol according to the volume ratio of 1:2, reacting for 4h at-20 ℃, centrifuging for 30min at 4000 Xg at 4 ℃ after the reaction is finished, taking supernatant, concentrating and drying by a vacuum concentrator, and obtaining the goat milk oligosaccharide crude product.
And step two, purifying the goat milk oligosaccharide crude product by using a Sephadex-G10 gel column to remove lactose. Firstly, putting Sephadex-G10 gel in a beaker, soaking the gel with ultrapure water for 12h, removing bubbles for 2h, then adding the gel into a chromatographic column with the specification of 3.6 multiplied by 60cm, and then pre-pressing the gel with ultrapure water for 12h at the flow rate of 2.5 mL/min. And (2) loading the goat milk oligosaccharide crude product into a Sephadex-G10 gel column, taking ultrapure water as eluent, enabling the flow rate to be 2.5mL/min, collecting column-passing liquid every 2min by using an automatic collector, detecting the column-passing liquid by using a phenol-sulfuric acid method, enabling the color to change when a 12 th tube is collected, and placing the column-passing liquid with the color changing after the 12 th tube in a refrigerator at-80 ℃ for later use.
Step three, utilizing carbon PacTMCarrying out quantitative analysis on the purified goat milk oligosaccharide sample by using a PA20 sugar separation column, using ultrapure water as a mobile phase A and 250mM NaOH as a mobile phase B, carrying out gradient elution on the purified goat milk oligosaccharide liquid at the column temperature of 35 ℃ and the flow rate of 0.4mL/min, wherein the conditions of the gradient elution are as follows: 0-40.1min, 7.2% -100% A; 40.2-45min, 100% -100% A; 45.1-55min, 100% -7.2% A; the remaining liquid phase portion is mobile phase B. And detecting the lactose content in the sample by adopting a gold electrode pulse ampere detector, and collecting a collecting pipe containing the goat milk oligosaccharide.
And step four, performing qualitative analysis on the components and the structure of the purified goat milk oligosaccharide sample by adopting an ultra-high performance liquid phase Q-active Focus mass spectrum.
The liquid phase conditions of the ultra-high performance liquid phase Q-active Focus mass spectrum are as follows: adopting an ACQUITY UPLC amino chromatographic column, wherein the specification of the chromatographic column is 2.1mM multiplied by 100mM, the particle diameter of a column packing is 1.7 mu m, the column temperature is 35 ℃, the flow rate is 0.2mL/min, the mobile phase A is acetonitrile, the mobile phase B is 10mM ammonium formate, and the gradient elution conditions are as follows: 0-18min, 95% -68% A; 18-30min, 68% -62% A; 30-38min, 62% -50% A; the remainder of the liquid phase is mobile phase B.
The mass spectrum conditions of the ultra-high performance liquid phase Q-active Focus mass spectrum are as follows: electrospray ion source: the spray voltage was 20kV for both positive and negative ion mode. The mass scanning range of the primary ions is 300-2000 m/z, and the column temperature is 35 ℃.
Example 2
Step one, degreasing and deproteinizing goat milk. Centrifuging goat milk for 30min at 4 ℃ and 10000rpm, removing upper fat and a small amount of protein at the bottom, taking out the middle layer, adding absolute ethyl alcohol according to the volume ratio of 1:2, reacting for 4h at-20 ℃, centrifuging for 30min at 4000 Xg at 4 ℃ after the reaction is finished, taking supernatant, concentrating and drying by a vacuum concentrator, and obtaining the goat milk oligosaccharide crude product.
And step two, purifying the goat milk oligosaccharide crude product by using a Sephadex-G10 gel column to remove lactose. Firstly, putting Sephadex-G10 gel in a beaker, soaking the gel with ultrapure water for 12h, removing bubbles for 2h, then adding the gel into a chromatographic column with the specification of 3.6 multiplied by 60cm, and then pre-pressing the gel with ultrapure water for 12h at the flow rate of 2.5 mL/min. And (2) loading the goat milk oligosaccharide crude product into a Sephadex-G10 gel column, taking ultrapure water as eluent, enabling the flow rate to be 2.5mL/min, collecting column-passing liquid every 2min by using an automatic collector, detecting the column-passing liquid by using a phenol-sulfuric acid method, enabling the color to change when a 12 th tube is collected, and placing the column-passing liquid with the color changing after the 12 th tube in a refrigerator at-80 ℃ for later use.
Step three, utilizing carbon PacTMCarrying out quantitative analysis on the purified goat milk oligosaccharide sample by using a PA20 sugar separation column, adopting ultrapure water as a mobile phase A and 250mM NaOH as a mobile phase B, controlling the column temperature at 35 ℃, and feeding the purified goat milk oligosaccharide liquid at the flow rate of 0.4mL/minLine gradient elution, conditions of gradient elution: 0-40.1min, 7.2% -100% A; 40.2-45min, 100% -100% A; 45.1-55min, 100% -7.2% A; the remaining liquid phase portion is mobile phase B. And detecting the lactose content in the sample by adopting a gold electrode pulse ampere detector, and collecting a collecting pipe containing the goat milk oligosaccharide.
And step four, performing qualitative analysis on the components and the structure of the purified goat milk oligosaccharide sample by adopting an ultra-high performance liquid phase Q-active Focus mass spectrum.
The liquid phase conditions of the ultra-high performance liquid phase Q-active Focus mass spectrum are as follows: adopting an ACQUITY UPLC amino chromatographic column, wherein the specification of the chromatographic column is 2.1mM multiplied by 100mM, the particle diameter of a column packing is 1.7 mu m, the column temperature is 35 ℃, the flow rate is 0.2mL/min, the mobile phase A is acetonitrile, the mobile phase B is 10mM ammonium formate, and the gradient elution conditions are as follows: 0-18min, 95% -74% A; 18-30min, 74% -69% A; 30-38min, 69% -62% A; 38-45min, 62% -50% A; the remainder of the liquid phase is mobile phase B.
The mass spectrum conditions of the ultra-high performance liquid phase Q-active Focus mass spectrum are as follows: electrospray ion source: the spray voltage was 20kV for both positive and negative ion mode. The mass scanning range of the primary ions is 300-2000 m/z, and the column temperature is 35 ℃.
Example 3
Step one, degreasing and deproteinizing goat milk. Centrifuging goat milk for 30min at 4 ℃ and 10000rpm, removing upper fat and a small amount of protein at the bottom, taking out the middle layer, adding absolute ethyl alcohol according to the volume ratio of 1:2, reacting for 4h at-20 ℃, centrifuging for 30min at 4000 Xg at 4 ℃ after the reaction is finished, taking supernatant, concentrating and drying by a vacuum concentrator, and obtaining the goat milk oligosaccharide crude product.
And step two, purifying the goat milk oligosaccharide crude product by using a Sephadex-G10 gel column to remove lactose. Firstly, putting Sephadex-G10 gel in a beaker, soaking the gel with ultrapure water for 12h, removing bubbles for 2h, then adding the gel into a chromatographic column with the specification of 3.6 multiplied by 60cm, and then pre-pressing the gel with ultrapure water for 12h at the flow rate of 2.5 mL/min. And (2) loading the goat milk oligosaccharide crude product into a Sephadex-G10 gel column, taking ultrapure water as eluent, enabling the flow rate to be 2.5mL/min, collecting column-passing liquid every 2min by using an automatic collector, detecting the column-passing liquid by using a phenol-sulfuric acid method, enabling the color to change when a 12 th tube is collected, and placing the column-passing liquid with the color changing after the 12 th tube in a refrigerator at-80 ℃ for later use.
Step three, utilizing carbon PacTMCarrying out quantitative analysis on the purified goat milk oligosaccharide sample by using a PA20 sugar separation column, using ultrapure water as a mobile phase A and 250mM NaOH as a mobile phase B, carrying out gradient elution on the purified goat milk oligosaccharide liquid at the column temperature of 35 ℃ and the flow rate of 0.4mL/min, wherein the conditions of the gradient elution are as follows: 0-40.1min, 7.2% -100% A; 40.2-45min, 100% -100% A; 45.1-55min, 100% -7.2% A; the remaining liquid phase portion is mobile phase B. And detecting the lactose content in the sample by adopting a gold electrode pulse ampere detector, and collecting a collecting pipe containing the goat milk oligosaccharide.
And step four, performing qualitative analysis on the components and the structure of the purified goat milk oligosaccharide sample by adopting an ultra-high performance liquid phase Q-active Focus mass spectrum.
The liquid phase conditions of the ultra-high performance liquid phase Q-active Focus mass spectrum are as follows: adopting an ACQUITY UPLC amino chromatographic column, wherein the specification of the chromatographic column is 2.1mM multiplied by 100mM, the particle diameter of a column packing is 1.7 mu m, the column temperature is 35 ℃, the flow rate is 0.2mL/min, the mobile phase A is acetonitrile, the mobile phase B is 10mM ammonium formate, and the gradient elution conditions are as follows: 0-20min, 95% -78% A; 20-35min, 78% -73% A; 35-38min, 73-62% A; 38-45min, 62% -50% A; the remainder of the liquid phase is mobile phase B.
The mass spectrum conditions of the ultra-high performance liquid phase Q-active Focus mass spectrum are as follows: electrospray ion source: the spray voltage was 20kV for both positive and negative ion mode. The mass scanning range of the primary ions is 300-2000 m/z, and the column temperature is 35 ℃.
Comparative example 1
In the second step, the crude goat milk oligosaccharide is purified and enriched by Sep-Pak C18 and graphitized column method, wherein, 5mL of methanol is used for balancing Sep-Pak C18 column, and 10mL (0.1% TFA) of ultrapure water solution is used for washing Sep-Pak C18And (3) a column. The crude goat milk oligosaccharide was dissolved in 400. mu.L (0.1% TFA) of ultra-pure water and loaded onto a Sep-Pak C18 column. Collecting eluate, and adding the eluate to Sep-Pak C18 column (this step)The procedure was repeated twice). Finally, washing the Sep-Pak C18 column with 3mL (0.1% TFA) ultrapure water solution, and collecting eluent, namely the purified goat milk oligosaccharide sample. The purified goat milk oligosaccharide sample was desalted using a graphitized column, which was first activated and equilibrated with 3 column volumes of 80% acetonitrile solution (20% solution containing 0.1% TFA) and ultrapure water, respectively. And (3) loading the eluent of the Sep-Pak C18 column, collecting the eluent (the flow rate is 0.5-1.0 mL/min), and repeating the step twice. The column was rinsed with 3 column volumes of ultrapure water to adsorb the oligosaccharides on the PGC packing at the bottom of the column for desalting. Finally, the oligosaccharides were eluted stepwise with 0.5mL of 10% acetonitrile (90% solution containing 0.1% TFA), 20% acetonitrile (80% solution containing 0.1% TFA), and 40% acetonitrile (60% solution containing 0.1% TFA) to obtain purified and enriched goat milk oligosaccharide samples. The other steps are the same as in example 3.
Comparative example 2
And secondly, purifying the goat milk oligosaccharide crude product by adopting an ultrafiltration membrane bag method, taking 50ml of the goat milk oligosaccharide crude product, and filtering and separating the goat milk oligosaccharide crude product by utilizing an ultrafiltration membrane with the molecular weight cutoff of 650Da for 4h, 5h and 6h respectively to obtain a filtrate which is a purified goat milk oligosaccharide sample. The other steps are the same as in example 3.
Comparative example 3
And secondly, purifying the goat milk oligosaccharide crude product by a dialysis method, adding the goat milk oligosaccharide crude product into a dialysis bag with the molecular weight cutoff of 1000Da, placing the dialysis bag in a refrigerator at 4 ℃ for 24 hours, changing water every 8 hours, and after changing water for 3 times, not increasing the solution in the dialysis bag any more to obtain a purified goat milk oligosaccharide sample. The other steps are the same as in example 3.
The following test results and analyses for each example and comparative example:
the total ion flow chart of the goat milk oligosaccharide sample after the high performance liquid chromatography separation in each example is shown in figures 1-3. The elution conditions of the liquid phase in example 1 were 0-18min, 95% -68% a; 18-30min, 68% -62% A; 30-38min, 62% -50% A, and the peaks between 18-22min are not separated from the graph 1, and the Hex2Neu5Ac1-1 and Hex2Neu5Ac1-2 isomers in the graph 2 are not separated under a positive and negative ion mode. The subsequent optimization of the high performance liquid chromatography condition is carried out according to the principle of high performance liquid chromatography separation and the characteristics of an Xbridge amino column, and the elution condition of the liquid phase in the embodiment 2 is optimized to be 0-18min and 95-74% A; 18-30min, 74% -69% A; 30-38min, 69% -62% A; 38-45min, 62% -50% A, and as can be seen from the combination of figure 3 and figure 4, the peaks with retention time between 20-27min in the total ion flow diagram are not separated, but it can be seen that the peaks with other retention times are obviously separated, and meanwhile, the peak areas of the intersecting Hex2Neu5Ac1-1 and Hex2Neu5Ac1-2 isomers are reduced, which indicates that the optimization effect is obvious. Further optimizing the liquid phase condition, wherein the elution condition of the liquid phase in the example 3 is optimized to be 0-20min, 95% -78% A; 20-35min, 78% -73% A; 35-38min, 73-62% A; 38-45min, 62% -50% of A, the chromatographic peaks in figure 5 are basically separated, and the isomers of Hex2Neu5Ac1-1 and Hex2Neu5Ac1-2 in figure 6 are also obviously separated and have better effect.
The Carbo Pac of the invention is utilizedTMPA20 sugar separation column was used to measure the crude and sample goat milk oligosaccharides obtained in example 3 and the sample goat milk oligosaccharides obtained in each comparative example, and the lactose content was quantitatively analyzed, the results are shown in Table 1:
TABLE 1 lactose removal Rate
As can be seen from Table 1, the result of purifying the goat milk oligosaccharide crude product to remove lactose by the method of comparative example 2 shows that the removal rate of lactose is the lowest, and is only 38.4%, which indicates that the determination method has low removal rate of lactose and poor separation effect. The results of the measurement methods of comparative example 1 and comparative example 3 show that the lactose content of the sample is low, the lactose removal rate is high, but the contents of lactose and lacto-oligosaccharide are both very low by combining the ion chromatogram 9 and fig. 11, which shows that although the lactose removal rate is high, the goat milk oligosaccharide is also greatly lost and the separation effect is poor by the measurement methods of comparative example 1 and comparative example 3. The determination method of the invention has high lactose removal rate, and the comparison of figure 8 shows that the method can separate lactose well, does not cause loss of goat milk oligosaccharide in the implementation process, has good separation effect and is convenient to operate.
The crude product of the goat milk oligosaccharide and the goat milk oligosaccharide sample purified in the embodiment are determined by ultra-high performance liquid phase Q-active Focus mass spectrometry under the optimized mobile phase conditions, the components and the structures of the goat milk oligosaccharide and the goat milk oligosaccharide are analyzed, and the determination results are shown in tables 2 and 3. Table 2 shows the mass spectrometry results of the crude goat milk oligosaccharides before purification, showing that 18 goat milk oligosaccharides can be detected from the crude goat milk oligosaccharides before purification, and table 3 shows the mass spectrometry results of the purified goat milk oligosaccharides, showing that 49 goat milk oligosaccharide structures can be detected by the method of the present invention.
From the results, the method for determining the goat milk oligosaccharide has the advantages of simple operation process and good separation effect, and can detect 49 goat milk oligosaccharide structures from goat milk.
TABLE 2 detection of goat milk oligosaccharide structure in goat milk oligosaccharide crude product
Note: + oligosaccharides detected in positive ion mode; oligosaccharides detected in negative ion mode
Table 3 sheep milk oligosaccharide structures detected in example 3
Note: + oligosaccharides detected in positive ion mode; oligosaccharides detected in negative ion mode
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (3)
1. The method for determining the goat milk oligosaccharide is characterized by comprising the following steps:
step one, degreasing and deproteinizing goat milk, and extracting a goat milk oligosaccharide crude product;
secondly, removing lactose from the goat milk oligosaccharide crude product by using a Sephadex-G10 gel column to obtain a goat milk oligosaccharide sample;
step three, utilizing carbon PacTMDetermining the lactose removal rate and the oligosaccharide retention rate of the goat milk oligosaccharide sample by using a PA20 sugar separation column;
optimizing liquid phase conditions and performing qualitative and quantitative analysis on the components and the structure of the goat milk oligosaccharide sample by adopting an ultra-high performance liquid phase Q-active Focus mass spectrum;
in the first step, the specific method for defatting and deproteinizing the goat milk comprises the following steps:
centrifuging goat milk for 15-30 min at 3-8 ℃ and 8000-10000 rpm, removing fat on the upper layer and a small amount of protein at the bottom, taking out the middle layer, adding absolute ethyl alcohol according to the volume ratio of 1: 2-1: 6, reacting for 2-12 h at-80-4 ℃, centrifuging for 15-30 min at 4000 Xg at 3-8 ℃ after the reaction is finished, taking supernatant, and drying to obtain a goat milk oligosaccharide crude product;
in the second step, the specific method for removing lactose comprises the following steps:
2.1) firstly placing Sephadex-G10 gel in a beaker, soaking the gel in ultrapure water for 10-15 h, removing bubbles by a vacuum pump for 1-2 h, adding the gel into a chromatographic column, and then prepressing the gel for 12h by using the ultrapure water at the flow rate of 2.5-3 mL/min;
2.2) loading the goat milk oligosaccharide crude product into a Sephadex-G10 gel column, taking ultrapure water as eluent, collecting column-passing liquid once every 2min by using an automatic collector, detecting the column-passing liquid by using a phenol-sulfuric acid method, and placing the column-passing liquid in a refrigerator at-80 ℃ for later use;
in the fourth step, the liquid phase conditions of the ultra-high performance liquid phase Q-active Focus mass spectrum are as follows:
adopting an ACQUITY UPLC amino chromatographic column, wherein the specification of the chromatographic column is 2.1mM multiplied by 100mM, the particle size of a column filler is 1.5-2 mu m, the column temperature is 30-40 ℃, the flow rate is 0.2-0.5 mL/min, a mobile phase A is acetonitrile, a mobile phase B is 10mM ammonium formate, and the gradient elution conditions are as follows: 0-20min, 95% -78% A; 20-35min, 78% -73% A; 35-38min, 73-62% A; 38-45min, 62% -50% A; 45-55min, 50% -95% A; 55-65min, 95% -95% A; the balance of the liquid phase is mobile phase B;
in the fourth step, the mass spectrum conditions of the ultra-high performance liquid phase Q-active Focus mass spectrum are as follows: electrospray ion source: the spraying voltage of the positive ion mode and the spraying voltage of the negative ion mode are both 5-20 kV, the mass scanning range of the primary ions is 300-2000 m/z, and the column temperature is 30-40 ℃.
2. The method for determining sheep milk oligosaccharide as claimed in claim 1, wherein the drying is carried out by vacuum concentration.
3. The method for determining goat milk oligosaccharide as claimed in claim 1, wherein in the third step, the specific method for determining lactose removal rate and oligosaccharide retention rate is as follows:
adopting ultrapure water as a mobile phase A and 250mM NaOH as a mobile phase B, carrying out gradient elution on the goat milk oligosaccharide sample at the column temperature of 35 ℃ and the flow rate of 0.4mL/min, wherein the conditions of the gradient elution are as follows: 0-40.1min, 7.2% -100% A; 40.2-45min, 100% -100% A; 45.1-55min, 100% -7.2% A; and the residual liquid phase part is a mobile phase B, a gold electrode pulse ampere detector is adopted to detect the lactose content in the sample, and a collecting pipe containing the goat milk oligosaccharide is collected.
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