CN109270176A - Sheep cream oligosaccharides measuring method - Google Patents
Sheep cream oligosaccharides measuring method Download PDFInfo
- Publication number
- CN109270176A CN109270176A CN201810962418.9A CN201810962418A CN109270176A CN 109270176 A CN109270176 A CN 109270176A CN 201810962418 A CN201810962418 A CN 201810962418A CN 109270176 A CN109270176 A CN 109270176A
- Authority
- CN
- China
- Prior art keywords
- sheep cream
- oligosaccharides
- sheep
- cream
- column
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Saccharide Compounds (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses a kind of sheep cream oligosaccharides measuring methods, comprising the following steps: Step 1: obtaining sheep cream oligosaccharides crude product to the degreasing of sheep cream, de- albumen；Step 2: removing lactose using Sephadex-G10 gel column, sheep cream oligosaccharide sample is obtained；Step 3: utilizing Carbo PacTMPA20 sugar splitter is measured the lactose removal rate and oligosaccharide retention rate of the sample；Step 4: optimizing liquid-phase condition and being carried out qualitative and quantitative analysis using ultra high efficiency liquid phase Q-Exactive Focus mass spectrum to the ingredient and structure of the product.The present invention establishes a kind of method efficiently separated and measure sheep cream oligosaccharides for the first time, to the biggish sheep milk sample product of separating difficulty, pass through gel filtration chromatography, realize effectively removing for lactose, the measurement that isomer is realized by the optimization of liquid-phase condition identifies 49 kinds of oligosaccharide in sheep cream by the reasonable set of mass spectrometer.
Description
Technical field
The present invention relates to a kind of separation of sheep cream oligosaccharides and measuring methods.
Background technique
Cream is that a kind of nutritional ingredient of complexity includes fat, protein, carbohydrate, and wherein it is dynamic to also provide lactation for cream
The nutritional need of object.The content of lactose is more than 80% in carbohydrate, and 4% that free newborn oligosaccharide content accounts for total reducing sugar is left
The right side, therefore the removal of lactose produces important role for the analysis of newborn oligosaccharides.
Research has shown that, newborn oligosaccharides can be with balance intestinal micropopulation to adjust immune system, and sialylated oligosaccharides can be with
Promote brain growth, and as the source of galactolipin for synthesizing galactocerebroside, sialic acid is for the nerve in ectocinerea
It saves glycosides rouge and glycoprotein generates.The study found that contain more than 200 oligosaccharide in human milk, however, due to size, charge and knot
The otherness of structure, so far, only the human milk oligosaccharides of low molecular weight can be by being chemically synthesized.Therefore, tool is found
There is the substitution source similar to human milk oligosaccharides bioactivity and is applied to the concern that infant formula has caused people.Sheep cream
It is one of the important newborn base-material of Infant Formula Enterprises, some researches show that oligosaccharide content is about 0.25-0.30g/L in sheep cream, is
8 times or so of cow's milk oligosaccharide content are the oligomeric sugar replacements of potential breast milk.But due to the limitation and art wall of detection method
It builds, the research of oligosaccharide structures more falls behind in China sheep cream, therefore establishes effective sheep cream oligosaccharide detection means to China
The research of sheep cream oligosaccharide is of great significance.
Based on these physiological functions, people are begun to focus in the research of newborn oligosaccharides.It is ground for sheep cream oligosaccharide structure the country
Study carefully and be rarely reported, and there is no the detection methods of a sheep cream oligosaccharides.The separation and purifying of oligosaccharide are the passes of oligomeric sugar detection
Key has its monitoring very big tired because of the high complexity of its own composition, connection, derivatization, microheterogeneity etc.
It is difficult.When analyzing the oligosaccharide in sheep cream, the oligosaccharide of some high abundances can cover the detection letter of low abundance oligosaccharide
Number, the complete identification of oligosaccharide is hindered, this just needs to carry out enriching and purifying operation to oligosaccharide sample, reduces the mixed of impurity
It closes, improves the sensitivity of detection.Since lactose content is higher in sheep cream, and the content of oligosaccharide is lower, separates low content ingredient
It is difficult, on the other hand, the monosaccharide number of oligosaccharides is generally between 3-10 in sheep cream, and molecular weight is close, even more increases
Operation difficulty, meanwhile, the retention time and mass spectrogram of isomer are again closely similar, to qualitative bring very big difficulty.Cause
This selects suitable method, and sheep cream oligosaccharides is separated and measured, is the very strong work of exploration.
Summary of the invention
It is an object of the invention to solve at least the above problems, and provide the advantages of at least will be described later.
It is a still further object of the present invention to provide a kind of sheep cream oligosaccharides measuring methods, by gel filtration chromatography, realize pair
Oligosaccharide determination influences substance lactose effectively removes, and sheep cream oligosaccharide sample is obtained, followed by high performance anion exchange chromatography
Quantitative analysis is carried out to lactose removal rate and oligosaccharide retention rate, using ultra high efficiency liquid phase Q-Exactive Focus mass spectrum to sheep
Newborn oligosaccharide is identified, is realized by the optimization to liquid-phase condition to the qualitative of sheep cream oligosaccharides isomer and quantitative point
Analysis.Using measuring method of the present invention, finally identifies 49 kinds of sheep cream oligosaccharides and realize to 49 kinds of sheep cream oligosaccharides knots
The analysis of structure composition, good separating effect, and sample is not necessarily to derivatization, remains the prototype structure of oligosaccharides, in addition operating process letter
It is single, it is as a result stable, it is reproducible.
In order to realize these purposes and other advantages according to the present invention, a kind of sheep cream oligosaccharides measuring method is provided, is wrapped
Include following steps:
Step 1: extracting sheep cream oligosaccharides crude product to the degreasing of sheep cream, de- albumen；
Step 2: removing lactose using Sephadex-G10 gel column to the sheep cream oligosaccharides crude product, sheep cream oligosaccharides sample is obtained
Product；
Step 3: utilizing Carbo PacTMPA20 sugar splitter is to the lactose removal rate of the sheep cream oligosaccharide sample and oligomeric
Sugared retention rate is measured；
Step 4: optimizing liquid-phase condition and using ultra high efficiency liquid phase Q-Exactive Focus mass spectrum to the sheep cream oligosaccharides
The ingredient and structure of sample carry out qualitative and quantitative analysis.
It is preferably, the degreasing of sheep cream, Deproteinated in the step 1 method particularly includes:
It takes sheep cream to be centrifuged 15~30min under the conditions of 3~8 DEG C, 8000~10000rpm, removes fat and the bottom on upper layer
Dehydrated alcohol is added in a small amount of protein, the ratio for taking out middle layer 1:2~1:6 by volume, and temperature is anti-at -80~4 DEG C
2~12h is answered, 4000 × g is centrifuged 15~30min at 3~8 DEG C after reaction, takes supernatant, and it is dry, obtain the sheep cream
Oligosaccharides crude product.
Preferably, described dry using vacuum concentration instrument concentrate drying.
Preferably, in the step 2, except the specific method of lactose includes:
First by Sephadex-G10 gel be placed in beaker with ultrapure water impregnate 10~15h, vacuum pump bubble removing 1~
It is added in chromatographic column after 2h, the specification of the chromatographic column is 1.6 × 60cm, the use of ultrapure water is then 2.5~3mL/ in flow velocity
Precompressed 12h under min；
The sheep cream oligosaccharides crude product is loaded in Sephadex-G10 gel column, uses ultrapure water for eluent, flow velocity
For 2~2.5mL/min, column liquid is crossed using the every 2min collection of automatic collector is primary, the column liquid of crossing is through phend-sulphuric acid
Detection, is placed in spare in -80 DEG C of refrigerators.
Preferably, which is characterized in that in the step 3, measure the specific side of lactose removal rate and oligosaccharide retention rate
Method are as follows:
Using ultrapure water as mobile phase A, 250mM NaOH is as Mobile phase B, and column temperature is 35 DEG C, with 0.4mL/min's
Flow velocity carries out gradient elution, the condition of gradient elution: 0-40.1min, 7.2%-100%A to the sheep cream oligosaccharide sample；
40.2-45min 100%-100%A；45.1-55min 100%-7.2%A；Remaining liquid phase part is Mobile phase B.Using gold
Electrode pulse ampere detector, lactose content in test sample, and collect the collecting pipe containing sheep cream oligosaccharides.
Preferably, in the step 4, the mass spectrographic liquid-phase condition of ultra high efficiency liquid phase Q-Exactive Focus are as follows:
Using ACQUITY UPLC amino chromatographic column, chromatographic column specification 2.1mm × 100mm, column packing partial size is 1.5~2 μ
M, 30~40 DEG C of column temperature, 0.2~0.5mL/min of flow velocity, mobile phase A is acetonitrile, and Mobile phase B is 10mM ammonium formate, gradient elution
Condition 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；Liquid phase remainder is Mobile phase B.
Preferably, in the step 4, the mass spectrographic Mass Spectrometry Conditions of ultra high efficiency liquid phase Q-Exactive Focus are as follows:
Electric spray ion source: positive ion mode and negative ion mode spray voltage are 5~20kV.The quality of primary ion
Scanning range 300--2000m/z, column temperature are 30~40 DEG C.
The present invention is include at least the following beneficial effects:
The present invention isolates and purifies degreasing and Deproteinated sheep cream using Sephadex-G10 gel column, effectively goes
It carries out qualitative and quantitative analysis except influence substance lactose, then by the optimization of liquid-phase condition to oligomeric sugar isomer, finally
By the reasonable set of mass spectrometer, 49 kinds of sheep cream oligosaccharide ingredients in sheep cream are identified, and are realized few to 49 kinds of sheep creams
The analysis of sugared structure.
Measuring method simple possible of the present invention, it is as a result stable, it can be used for the purifies and separates and measurement of sheep cream oligosaccharides.
Further advantage, target and feature of the invention will be partially reflected by the following instructions, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Detailed description of the invention
Fig. 1 is the total ion current figure of sheep cream oligosaccharide sample in the embodiment of the present invention 1.
Fig. 2 is the total ion current figure of the isomers of Hex2Neu5Ac1 in sheep cream oligosaccharide sample in the embodiment of the present invention 1.
Fig. 3 is the total ion current figure of sheep cream oligosaccharide sample in the embodiment of the present invention 2.
Fig. 4 is the total ion current figure of the isomers of Hex2Neu5Ac1 in sheep cream oligosaccharide sample in the embodiment of the present invention 2.
Fig. 5 is the total ion current figure of sheep cream oligosaccharide sample in the embodiment of the present invention 3.
Fig. 6 is the total ion current figure of the isomers of Hex2Neu5Ac1 in sheep cream oligosaccharide sample in the embodiment of the present invention 3.
Fig. 7 is the chromatography of ions figure of sheep cream oligosaccharides crude product in the embodiment of the present invention 3.
Fig. 8 is the chromatography of ions figure of sheep cream oligosaccharide sample in the embodiment of the present invention 3.
Fig. 9 is the chromatography of ions figure of sheep cream oligosaccharide sample in comparative example 1.
Figure 10 is the chromatography of ions figure of sheep cream oligosaccharide sample in comparative example 2.
Figure 11 is the chromatography of ions figure of sheep cream oligosaccharide sample in comparative example 3.
Specific embodiment
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art referring to specification text
Word can be implemented accordingly.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein are not precluded one or more
The presence or addition of a other elements or combinations thereof.
Sheep cream oligosaccharides measuring method of the present invention, comprising the following steps:
Step 1: extracting sheep cream oligosaccharides crude product to the degreasing of sheep cream, de- albumen；
Step 2: removing lactose using Sephadex-G10 gel column to the sheep cream oligosaccharides crude product, sheep cream oligosaccharides sample is obtained
Product；
Step 3: utilizing Carbo PacTMPA20 sugar splitter is to the lactose removal rate of the sheep cream oligosaccharide sample and oligomeric
Sugared retention rate is measured；
Step 4: optimizing liquid-phase condition and using ultra high efficiency liquid phase Q-Exactive Focus mass spectrum to the sheep cream oligosaccharides
The ingredient and structure of sample carry out qualitative and quantitative analysis.
It is in one embodiment, the degreasing of sheep cream, Deproteinated in the step 1 method particularly includes:
It takes sheep cream to be centrifuged 15~30min under the conditions of 3~8 DEG C, 8000~10000rpm, removes fat and the bottom on upper layer
Dehydrated alcohol is added in a small amount of protein, the ratio for taking out middle layer 1:2~1:6 by volume, and temperature is anti-at -80~4 DEG C
2~12h is answered, 4000 × g is centrifuged 15~30min at 3~8 DEG C after reaction, takes supernatant, and it is dry, obtain the sheep cream
Oligosaccharides crude product.
In one embodiment, described dry using vacuum concentration instrument concentrate drying.
In one embodiment, in the step 2, except the specific method of lactose includes:
First by Sephadex-G10 gel be placed in beaker with ultrapure water impregnate 10~15h, vacuum pump bubble removing 1~
It is added in chromatographic column after 2h, the specification of the chromatographic column is 1.6 × 60cm, the use of ultrapure water is then 2.5~3mL/ in flow velocity
Precompressed 12h under min；
The sheep cream oligosaccharides crude product is loaded in Sephadex-G10 gel column, uses ultrapure water for eluent, flow velocity
For 2~2.5mL/min, column liquid is crossed using the every 2min collection of automatic collector is primary, the column liquid of crossing is through phend-sulphuric acid
Detection, is placed in spare in -80 DEG C of refrigerators.
In one embodiment, which is characterized in that in the step 3, measure lactose removal rate and oligosaccharide retention rate
Method particularly includes:
Using ultrapure water as mobile phase A, 250mM NaOH is as Mobile phase B, and column temperature is 35 DEG C, with 0.4mL/min's
Flow velocity carries out gradient elution, the condition of gradient elution: 0-40.1min, 7.2%-100%A to the sheep cream oligosaccharide sample；
40.2-45min 100%-100%A；45.1-55min 100%-7.2%A；Remaining liquid phase part is Mobile phase B.Using gold
Electrode pulse ampere detector, lactose content in test sample, and collect the collecting pipe containing sheep cream oligosaccharides.
In one embodiment, in the step 4, the mass spectrographic liquid-phase condition of ultra high efficiency liquid phase Q-Exactive Focus
Are as follows:
Using ACQUITY UPLC amino chromatographic column, chromatographic column specification 2.1mm × 100mm, column packing partial size is 1.5~2 μ
M, 30~40 DEG C of column temperature, 0.2~0.5mL/min of flow velocity, mobile phase A is acetonitrile, and Mobile phase B is 10mM ammonium formate, gradient elution
Condition 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；Liquid phase remainder is Mobile phase B.
In one embodiment, in the step 4, the mass spectrographic Mass Spectrometry Conditions of ultra high efficiency liquid phase Q-Exactive Focus
Are as follows: electric spray ion source: positive ion mode and negative ion mode spray voltage are 5~20kV.The mass scanning of primary ion
Range 300--2000m/z, column temperature are 30~40 DEG C.
Embodiment 1
Step 1: to the degreasing of sheep cream, de- albumen.It takes sheep cream to be centrifuged 30min under the conditions of 4 DEG C, 10000rpm, removes upper layer
Fat and a small amount of protein in bottom, take out middle layer by volume 1:2 ratio be added dehydrated alcohol, temperature be -20 DEG C
Lower reaction 4h, 4000 × g is centrifuged 30min at 4 DEG C after reaction, takes supernatant, and vacuum concentration instrument is concentrated and dried, and obtains institute
State sheep cream oligosaccharides crude product.
Step 2: carrying out purifying except lactose using Sephadex-G10 gel column to the sheep cream oligosaccharides crude product.First will
Sephadex-G10 gel, which is placed in beaker, impregnates 12h, bubble removing 2h with ultrapure water, is then added in chromatographic column, the layer
The specification for analysing column is 3.6 × 60cm, the use of ultrapure water is then precompressed 12h under 2.5mL/min in flow velocity.By the sheep cream oligosaccharides
Crude product is loaded in Sephadex-G10 gel column, uses ultrapure water for eluent, flow velocity 2.5mL/min, is received using automatic
The every 2min collection of storage is primary to cross column liquid, crosses column liquid, the appearance when being collected into 12 pipe with phend-sulphuric acid detection is described
The later column liquid excessively for color change occur of 12nd pipe is placed in spare in -80 DEG C of refrigerators by color change.
Step 3: utilizing Carbo PacTMPA20 sugar splitter quantitatively divides the sheep cream oligosaccharide sample of the purifying
Analysis, using ultrapure water as mobile phase A, 250mM NaOH is as Mobile phase B, and column temperature is 35 DEG C, with the flow velocity pair of 0.4mL/min
The sheep cream oligosaccharides liquid of the purifying carries out gradient elution, the condition of gradient elution: 0-40.1min, 7.2%-100%A；
40.2-45min 100%-100%A；45.1-55min 100%-7.2%A；Remaining liquid phase part is Mobile phase B.Using gold
Electrode pulse ampere detector, lactose content in test sample, and collect the collecting pipe containing sheep cream oligosaccharides.
Step 4: using ultra high efficiency liquid phase Q-Exactive Focus mass spectrum to the sheep cream oligosaccharide sample of the purifying at
Divide and structure carries out qualitative analysis.
The mass spectrographic liquid-phase condition of the ultra high efficiency liquid phase Q-Exactive Focus are as follows: use ACQUITY UPLC amino color
Compose column, chromatographic column specification 2.1mm × 100mm, column packing partial size be 1.7 μm, 35 DEG C of column temperature, flow velocity 0.2mL/min, mobile phase A
For acetonitrile, Mobile phase B is 10mM ammonium formate, the condition of gradient elution are as follows: 0-18min, 95%-68%A；18-30min, 68%-
62%A；30-38min, 62%-50%A；Liquid phase remainder is Mobile phase B.
The mass spectrographic Mass Spectrometry Conditions of the ultra high efficiency liquid phase Q-Exactive Focus are as follows: electric spray ion source: cation mould
Formula and negative ion mode spray voltage are 20kV.The mass scan range 300--2000m/z of primary ion, column temperature are 35 DEG C.
Embodiment 2
Step 1: to the degreasing of sheep cream, de- albumen.It takes sheep cream to be centrifuged 30min under the conditions of 4 DEG C, 10000rpm, removes upper layer
Fat and a small amount of protein in bottom, take out middle layer by volume 1:2 ratio be added dehydrated alcohol, temperature be -20 DEG C
Lower reaction 4h, 4000 × g is centrifuged 30min at 4 DEG C after reaction, takes supernatant, and vacuum concentration instrument is concentrated and dried, and obtains institute
State sheep cream oligosaccharides crude product.
Step 2: carrying out purifying except lactose using Sephadex-G10 gel column to the sheep cream oligosaccharides crude product.First will
Sephadex-G10 gel, which is placed in beaker, impregnates 12h, bubble removing 2h with ultrapure water, is then added in chromatographic column, the layer
The specification for analysing column is 3.6 × 60cm, the use of ultrapure water is then precompressed 12h under 2.5mL/min in flow velocity.By the sheep cream oligosaccharides
Crude product is loaded in Sephadex-G10 gel column, uses ultrapure water for eluent, flow velocity 2.5mL/min, is received using automatic
The every 2min collection of storage is primary to cross column liquid, crosses column liquid, the appearance when being collected into 12 pipe with phend-sulphuric acid detection is described
The later column liquid excessively for color change occur of 12nd pipe is placed in spare in -80 DEG C of refrigerators by color change.
Step 3: utilizing Carbo PacTMPA20 sugar splitter quantitatively divides the sheep cream oligosaccharide sample of the purifying
Analysis, using ultrapure water as mobile phase A, 250mM NaOH is as Mobile phase B, and column temperature is 35 DEG C, with the flow velocity pair of 0.4mL/min
The sheep cream oligosaccharides liquid of the purifying carries out gradient elution, the condition of gradient elution: 0-40.1min, 7.2%-100%A；
40.2-45min 100%-100%A；45.1-55min 100%-7.2%A；Remaining liquid phase part is Mobile phase B.Using gold
Electrode pulse ampere detector, lactose content in test sample, and collect the collecting pipe containing sheep cream oligosaccharides.
Step 4: using ultra high efficiency liquid phase Q-Exactive Focus mass spectrum to the sheep cream oligosaccharide sample of the purifying at
Divide and structure carries out qualitative analysis.
The mass spectrographic liquid-phase condition of the ultra high efficiency liquid phase Q-Exactive Focus are as follows: use ACQUITY UPLC amino color
Compose column, chromatographic column specification 2.1mm × 100mm, column packing partial size be 1.7 μm, 35 DEG C of column temperature, flow velocity 0.2mL/min, mobile phase A
For acetonitrile, Mobile phase B is 10mM ammonium formate, the condition of gradient elution are as follows: 0-18min, 95%-74%A；18-30min, 74%-
69%A；30-38min, 69%-62%A；38-45min, 62%-50%A；Liquid phase remainder is Mobile phase B.
The mass spectrographic Mass Spectrometry Conditions of the ultra high efficiency liquid phase Q-Exactive Focus are as follows: electric spray ion source: cation mould
Formula and negative ion mode spray voltage are 20kV.The mass scan range 300--2000m/z of primary ion, column temperature are 35 DEG C.
Embodiment 3
Step 1: to the degreasing of sheep cream, de- albumen.It takes sheep cream to be centrifuged 30min under the conditions of 4 DEG C, 10000rpm, removes upper layer
Fat and a small amount of protein in bottom, take out middle layer by volume 1:2 ratio be added dehydrated alcohol, temperature be -20 DEG C
Lower reaction 4h, 4000 × g is centrifuged 30min at 4 DEG C after reaction, takes supernatant, and vacuum concentration instrument is concentrated and dried, and obtains institute
State sheep cream oligosaccharides crude product.
Step 2: carrying out purifying except lactose using Sephadex-G10 gel column to the sheep cream oligosaccharides crude product.First will
Sephadex-G10 gel, which is placed in beaker, impregnates 12h, bubble removing 2h with ultrapure water, is then added in chromatographic column, the layer
The specification for analysing column is 3.6 × 60cm, the use of ultrapure water is then precompressed 12h under 2.5mL/min in flow velocity.By the sheep cream oligosaccharides
Crude product is loaded in Sephadex-G10 gel column, uses ultrapure water for eluent, flow velocity 2.5mL/min, is received using automatic
The every 2min collection of storage is primary to cross column liquid, crosses column liquid, the appearance when being collected into 12 pipe with phend-sulphuric acid detection is described
The later column liquid excessively for color change occur of 12nd pipe is placed in spare in -80 DEG C of refrigerators by color change.
Step 3: utilizing Carbo PacTMPA20 sugar splitter quantitatively divides the sheep cream oligosaccharide sample of the purifying
Analysis, using ultrapure water as mobile phase A, 250mM NaOH is as Mobile phase B, and column temperature is 35 DEG C, with the flow velocity pair of 0.4mL/min
The sheep cream oligosaccharides liquid of the purifying carries out gradient elution, the condition of gradient elution: 0-40.1min, 7.2%-100%A；
40.2-45min 100%-100%A；45.1-55min 100%-7.2%A；Remaining liquid phase part is Mobile phase B.Using gold
Electrode pulse ampere detector, lactose content in test sample, and collect the collecting pipe containing sheep cream oligosaccharides.
Step 4: using ultra high efficiency liquid phase Q-Exactive Focus mass spectrum to the sheep cream oligosaccharide sample of the purifying at
Divide and structure carries out qualitative analysis.
The mass spectrographic liquid-phase condition of the ultra high efficiency liquid phase Q-Exactive Focus are as follows: use ACQUITY UPLC amino color
Compose column, chromatographic column specification 2.1mm × 100mm, column packing partial size be 1.7 μm, 35 DEG C of column temperature, flow velocity 0.2mL/min, mobile phase A
For acetonitrile, Mobile phase B is 10mM ammonium formate, the condition of gradient elution are as follows: 0-20min, 95%-78%A；20-35min, 78%-
73%A；35-38min, 73-62%A；38-45min, 62%-50%A；Liquid phase remainder is Mobile phase B.
The mass spectrographic Mass Spectrometry Conditions of the ultra high efficiency liquid phase Q-Exactive Focus are as follows: electric spray ion source: cation mould
Formula and negative ion mode spray voltage are 20kV.The mass scan range 300--2000m/z of primary ion, column temperature are 35 DEG C.
Comparative example 1
Second step is purified and is enriched with to sheep cream oligosaccharides crude product using Sep-Pak C18 and graphitization column method, is used first
Then the equilibrium methanol Sep-Pak C18 column of 5mL rinses Sep-Pak C with 10mL (0.1%TFA) ultra-pure water solution18Column.It will
Sheep cream oligosaccharides crude product is dissolved in 400 μ L (0.1%TFA) ultra-pure water solutions, is then added in Sep-Pak C18 column.Eluent is collected,
Eluent is added in Sep-Pak C18 column (this step is repeated twice) again.It is finally ultrapure water-soluble with 3m L (0.1%TFA)
Liquid rinses Sep-Pak C18 column, collects eluent, this eluent is the sheep cream oligosaccharide sample purified.Utilize graphitization column pair
The sheep cream oligosaccharide sample desalination of purifying, using the acetonitrile solution of 3 times of column volumes 80%, (20% contains the molten of 0.1%TFA first
Liquid) and ultrapure water activate and balance respectively graphitization column.By the eluent loading of Sep-Pak C18 column, eluent (flow velocity is collected
0.5~1.0mL/min), this step is repeated twice.With the ultrapure water pillar of 3 times of column volumes, purpose adsorption column bottom PGC is filled out
Oligosaccharides on material, in order to desalination.Finally use 10% acetonitrile solution of 0.5mL (90% solution containing 0.1%TFA), 20% second
Nitrile solution (80% solution containing 0.1%TFA), 40% acetonitrile solution (60% solution containing 0.1%TFA) by oligosaccharides substep
Elution, the sheep cream oligosaccharide sample after obtaining purification enrichment.Other steps are the same as embodiment 3.
Comparative example 2
Second step purifies sheep cream oligosaccharides crude product using ultrafiltration membrane bag method, and 50ml sheep cream oligosaccharides crude product is taken, using cutting
Staying molecular weight is that the ultrafiltration membrane of 650Da is filtered separation to it, and filtration time is respectively 4h, 5h, 6h, and it is pure for obtaining filtered fluid
The sheep cream oligosaccharide sample of change.Other steps are the same as embodiment 3.
Comparative example 3
Second step purifies sheep cream oligosaccharides crude product using dialysis, and sheep cream oligosaccharides crude product is added to molecular cut off
In the bag filter of 1000Da, to place 4 DEG C of refrigerators for 24 hours, a water is changed every 8h, after changing 3 water, solution no longer increases in bag filter
Add, obtains the sheep cream oligosaccharide sample of purifying.Other steps are the same as embodiment 3.
Below to the test result and analysis of each embodiment and comparative example:
Total ion current figure of the sheep cream oligosaccharide sample after high performance liquid chromatography separation is as shown in Figs. 1-3 in each embodiment.It is real
The elution requirement for applying liquid phase in example 1 is 0-18min, 95%-68%A；18-30min, 68%-62%A；30-38min, 62%-
50%A, the peak between 18-22min is there is no separating as shown in Figure 1, and Hex2Neu5Ac1-1 and Hex2Neu5Ac1-2 are different in Fig. 2
Structure body is also not separated by under negative ions mode.Referring to the principle of high performance liquid chromatography separation and the spy of XBridge nh 2 column
Point carries out the optimization of subsequent high-efficient liquid phase chromatogram condition, and the elution requirement of liquid phase is optimized for 0-18min in embodiment 2,95%-
74%A；18-30min, 74%-69%A；30-38min, 69%-62%A；38-45min, 62%-50%A, in conjunction with Fig. 3 and figure
4 it is found that peak of the retention time between 20-27min is not separated in total ion current figure, but can be seen that other retention times
Peak is obviously separated, while the peak area of Hex2Neu5Ac1-1 and Hex2Neu5Ac1-2 isomers intersection is reduced, and illustrates to optimize
Effect is obvious.Liquid-phase condition is advanced optimized, the elution requirement of liquid phase is optimized for 0-20min, 95%-78%A in embodiment 3；
20-35min, 78%-73%A；35-38min, 73-62%A；38-45min, 62%-50%A, Fig. 5 chromatographic peak are divided substantially
It opens, Hex2Neu5Ac1-1 and Hex2Neu5Ac1-2 isomers is also obviously separated in Fig. 6 and effect is preferable.
Utilize Carbo Pac of the present inventionTMPA20 sugar splitter is to sheep cream oligosaccharides crude product described in embodiment 3 and sheep cream
Sheep cream oligosaccharide sample described in oligosaccharide sample and each comparative example is measured, its lactose content of quantitative analysis, as a result such as 1 institute of table
Show:
The removal rate of 1 lactose of table
As shown in Table 1, purifying is carried out to sheep cream oligosaccharides crude product by 2 the method for comparative example and removes lactose, it is newborn as the result is shown
The removal rate of sugar is minimum, and only 38.4%, illustrate that the measuring method lactose removal rate is low, separating effect is bad.1 He of comparative example
Measuring method described in comparative example 3, sample lactose content is low as the result is shown, and lactose removal rate is high, but coupled ion chromatogram 9 and figure
Lactose and newborn oligosaccharide content are very low both known to 11, although illustrating that measuring method lactose described in comparative example 1 and comparative example 3 removes
Rate is high, but sheep cream oligosaccharides also has and is significantly lost, and separating effect is bad.Measuring method of the present invention, lactose removal
Rate is very high, it can separate well lactose known to comparison diagram 8, does not generate loss to sheep cream oligosaccharides again in implementation process, point
It is good from effect, and convenient for operation.
By the sheep cream oligosaccharide sample of the sheep cream oligosaccharides crude product and embodiment after purification mobile phase of the present invention optimized
Condition passes through ultra high efficiency liquid phase Q-Exactive Focus mass spectroscopy, the newborn oligosaccharide ingredient of both analyses and structure, measurement result
As shown in table 2, table 3.Table 2 is not mass spectral results of the sheep cream oligosaccharides crude product before purification, as the result is shown the not sheep before purification
The structure of 18 kinds of sheep cream oligosaccharides can be determined in newborn oligosaccharides crude product, table 3 is the mass spectral results of sheep cream oligosaccharide sample after purification, knot
Fruit, which shows, can detect that 49 kinds of sheep cream oligosaccharide structures through measuring method of the present invention.
It can be seen from the above result that sheep cream oligosaccharides measuring method of the present invention, operating process is simple, good separating effect, can
49 kinds of sheep cream oligosaccharide structures are detected from sheep cream.
Sheep cream oligosaccharide structure is detected in 2 sheep cream oligosaccharides crude product of table
Note: the oligosaccharide detected under+positive ion mode；The oligosaccharide detected under negative ion mode
The sheep cream oligosaccharide structure detected in 3 embodiment 3 of table
Note: the oligosaccharide detected under+positive ion mode；The oligosaccharide detected under negative ion mode
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and legend shown and described herein.
Claims (7)
1. sheep cream oligosaccharides measuring method, which comprises the following steps:
Step 1: extracting sheep cream oligosaccharides crude product to the degreasing of sheep cream, de- albumen；
Step 2: removing lactose using Sephadex-G10 gel column to the sheep cream oligosaccharides crude product, sheep cream oligosaccharide sample is obtained；
Step 3: utilizing Carbo PacTMPA20 sugar splitter protects the lactose removal rate and oligosaccharide of the sheep cream oligosaccharide sample
Rate is stayed to be measured；
Step 4: optimizing liquid-phase condition and using ultra high efficiency liquid phase Q-Exactive Focus mass spectrum to the sheep cream oligosaccharide sample
Ingredient and structure carry out qualitative and quantitative analysis.
2. sheep cream oligosaccharides measuring method as described in claim 1, which is characterized in that in the step 1, the degreasing of sheep cream, de- egg
White method particularly includes:
Sheep cream is taken to be centrifuged 15~30min under the conditions of 3~8 DEG C, 8000~10000rpm, fat and the bottom for removing upper layer are a small amount of
Protein, take out middle layer by volume 1:2~1:6 ratio be added dehydrated alcohol, temperature be -80~4 DEG C at reaction 2~
12h, 4000 × g is centrifuged 15~30min at 3~8 DEG C after reaction, takes supernatant, dry, and it is thick to obtain the sheep cream oligosaccharides
Product.
3. sheep cream oligosaccharides measuring method as claimed in claim 2, which is characterized in that described dry using vacuum concentration instrument concentration
It is dry.
4. sheep cream oligosaccharides measuring method as described in claim 1, which is characterized in that specific except lactose in the step 2
Method includes:
2.1) first by Sephadex-G10 gel be placed in beaker with ultrapure water impregnate 10~15h, vacuum pump bubble removing 1~
It is added in chromatographic column after 2h, the specification of the chromatographic column is 1.6 × 60cm, the use of ultrapure water is then 2.5~3mL/ in flow velocity
Precompressed 12h under min；
2.2) the sheep cream oligosaccharides crude product is loaded in Sephadex-G10 gel column, uses ultrapure water for eluent, flow velocity
For 2~2.5mL/min, column liquid is crossed using the every 2min collection of automatic collector is primary, the column liquid of crossing is through phend-sulphuric acid
Detection, is placed in spare in -80 DEG C of refrigerators.
5. sheep cream oligosaccharides measuring method as described in claim 1, which is characterized in that in the step 3, measure lactose removal
Rate and oligosaccharide retention rate method particularly includes:
Using ultrapure water as mobile phase A, 250mM NaOH is as Mobile phase B, and column temperature is 35 DEG C, with the flow velocity of 0.4mL/min
Gradient elution, the condition of gradient elution: 0-40.1min, 7.2%-100%A are carried out to the sheep cream oligosaccharide sample；40.2-
45min, 100%-100%A；45.1-55min 100%-7.2%A；Remaining liquid phase part is Mobile phase B.Using gold electrode arteries and veins
Ampere detector, lactose content in test sample are rushed, and collects the collecting pipe containing sheep cream oligosaccharides.
6. sheep cream oligosaccharides measuring method as described in claim 1, which is characterized in that in the step 4, ultra high efficiency liquid phase Q-
The mass spectrographic liquid-phase condition of Exactive Focus are as follows:
Using ACQUITY UPLC amino chromatographic column, chromatographic column specification 2.1mm × 100mm, column packing partial size is 1.5~2 μm, column
30~40 DEG C, 0.2~0.5mL/min of flow velocity of temperature, mobile phase A are acetonitrile, and Mobile phase B is 10mM ammonium formate, the item of gradient elution
Part 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；Liquid phase remainder is Mobile phase B.
7. sheep cream oligosaccharides measuring method as described in claim 1, which is characterized in that in the step 4, ultra high efficiency liquid phase Q-
The mass spectrographic Mass Spectrometry Conditions of Exactive Focus are as follows: electric spray ion source: positive ion mode and negative ion mode spray voltage are equal
For 5~20kV.The mass scan range 300--2000m/z of primary ion, column temperature are 30~40 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810962418.9A CN109270176B (en) | 2018-08-22 | 2018-08-22 | Goat milk oligosaccharide determination method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810962418.9A CN109270176B (en) | 2018-08-22 | 2018-08-22 | Goat milk oligosaccharide determination method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109270176A true CN109270176A (en) | 2019-01-25 |
| CN109270176B CN109270176B (en) | 2021-10-22 |
Family
ID=65154030
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810962418.9A Active CN109270176B (en) | 2018-08-22 | 2018-08-22 | Goat milk oligosaccharide determination method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109270176B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112526022A (en) * | 2020-11-27 | 2021-03-19 | 内蒙古伊利实业集团股份有限公司 | Method for detecting breast milk oligosaccharide in milk |
| CN114994214A (en) * | 2022-06-30 | 2022-09-02 | 北京三元食品股份有限公司 | Method for qualitatively detecting neutral oligosaccharide in breast milk |
| WO2023124706A1 (en) * | 2021-12-29 | 2023-07-06 | 北京三元食品股份有限公司 | Method for preparing milk oligosaccharide, oligosaccharide powder prepared using same, and food |
| WO2024001248A1 (en) * | 2022-06-30 | 2024-01-04 | 北京三元食品股份有限公司 | Method for qualitatively testing acidic oligosaccharides in breast milk |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090023171A1 (en) * | 2004-02-24 | 2009-01-22 | Christian Viskov | Method for determining specific groups constituting heparins or low molecular weight heparins |
| CN101613378A (en) * | 2009-07-17 | 2009-12-30 | 辽宁大学 | Acidic oligosaccharide and application thereof with fat-reducing and lipid-lowering effect |
| CN101817850A (en) * | 2010-04-19 | 2010-09-01 | 烟台大学 | Preparation method of single component of high-purity oligomate |
| CN102617746A (en) * | 2012-03-23 | 2012-08-01 | 山西大学 | Method for preparing multiple oligosaccharides by separating and purifying Chinese dates |
| JP5344787B2 (en) * | 2006-02-24 | 2013-11-20 | 独立行政法人科学技術振興機構 | Sulfated octasaccharide and decasaccharide derived from chondroitin sulfate of squid cartilage |
| CN104764847A (en) * | 2015-04-21 | 2015-07-08 | 福州大学 | Preparation method of oligosaccharide containing N-acetylated structure heparin |
| CN105949335A (en) * | 2015-12-31 | 2016-09-21 | 牡丹江友搏药业股份有限公司 | Oligosaccharide components with anti-platelet aggregation activity, and preparation method thereof |
| CN106248833A (en) * | 2016-09-26 | 2016-12-21 | 中国农业科学院农产品加工研究所 | Lac Bovis seu Bubali oligosaccharide assay method |
| CN106866749A (en) * | 2015-12-13 | 2017-06-20 | 中国科学院大连化学物理研究所 | A kind of preparation method of breast milk oligosaccharides |
| CN107192771A (en) * | 2017-05-04 | 2017-09-22 | 中国农业科学院农产品加工研究所 | The quantitative method of breast milk oligosaccharide fast qualitative |
| CN107621399A (en) * | 2016-07-14 | 2018-01-23 | 北京三元食品股份有限公司 | A kind of method of oligosaccharide in detection breast milk |
-
2018
- 2018-08-22 CN CN201810962418.9A patent/CN109270176B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090023171A1 (en) * | 2004-02-24 | 2009-01-22 | Christian Viskov | Method for determining specific groups constituting heparins or low molecular weight heparins |
| JP5344787B2 (en) * | 2006-02-24 | 2013-11-20 | 独立行政法人科学技術振興機構 | Sulfated octasaccharide and decasaccharide derived from chondroitin sulfate of squid cartilage |
| CN101613378A (en) * | 2009-07-17 | 2009-12-30 | 辽宁大学 | Acidic oligosaccharide and application thereof with fat-reducing and lipid-lowering effect |
| CN101817850A (en) * | 2010-04-19 | 2010-09-01 | 烟台大学 | Preparation method of single component of high-purity oligomate |
| CN102617746A (en) * | 2012-03-23 | 2012-08-01 | 山西大学 | Method for preparing multiple oligosaccharides by separating and purifying Chinese dates |
| CN104764847A (en) * | 2015-04-21 | 2015-07-08 | 福州大学 | Preparation method of oligosaccharide containing N-acetylated structure heparin |
| CN106866749A (en) * | 2015-12-13 | 2017-06-20 | 中国科学院大连化学物理研究所 | A kind of preparation method of breast milk oligosaccharides |
| CN105949335A (en) * | 2015-12-31 | 2016-09-21 | 牡丹江友搏药业股份有限公司 | Oligosaccharide components with anti-platelet aggregation activity, and preparation method thereof |
| CN107621399A (en) * | 2016-07-14 | 2018-01-23 | 北京三元食品股份有限公司 | A kind of method of oligosaccharide in detection breast milk |
| CN106248833A (en) * | 2016-09-26 | 2016-12-21 | 中国农业科学院农产品加工研究所 | Lac Bovis seu Bubali oligosaccharide assay method |
| CN107192771A (en) * | 2017-05-04 | 2017-09-22 | 中国农业科学院农产品加工研究所 | The quantitative method of breast milk oligosaccharide fast qualitative |
Non-Patent Citations (5)
| Title |
|---|
| CLEMENS KUNZ 等: "High-pH anion-exchange chromatography with pulsed amperometric detection and molar response factors of human milk oligosaccharides", 《JOURNAL OF CHROMATOGRAPHY B》 * |
| S. OBERMEIER 等: "Secretion of 13C-Labelled Oligosaccharides into Human Milk and Infant"s Urine after an Oral 13C-Galactose", 《ISOTOPES ENVIRON. HEALTH SHUTD.》 * |
| 刘世龙 等: "中性人乳寡糖的分离及结构鉴定", 《高等学校化学学报》 * |
| 李玉兰 等: "《生物化学实验》", 30 June 2008, 中国医药科技出版社 * |
| 渠婷: "金钗石斛低聚糖的分离、结构鉴定及其功能性研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112526022A (en) * | 2020-11-27 | 2021-03-19 | 内蒙古伊利实业集团股份有限公司 | Method for detecting breast milk oligosaccharide in milk |
| WO2023124706A1 (en) * | 2021-12-29 | 2023-07-06 | 北京三元食品股份有限公司 | Method for preparing milk oligosaccharide, oligosaccharide powder prepared using same, and food |
| CN114994214A (en) * | 2022-06-30 | 2022-09-02 | 北京三元食品股份有限公司 | Method for qualitatively detecting neutral oligosaccharide in breast milk |
| CN114994214B (en) * | 2022-06-30 | 2023-02-24 | 北京三元食品股份有限公司 | Method for qualitatively detecting neutral oligosaccharide in breast milk |
| WO2024001248A1 (en) * | 2022-06-30 | 2024-01-04 | 北京三元食品股份有限公司 | Method for qualitatively testing acidic oligosaccharides in breast milk |
| WO2024001249A1 (en) * | 2022-06-30 | 2024-01-04 | 北京三元食品股份有限公司 | Method for qualitatively detecting neutral oligosaccharides in breast milk |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109270176B (en) | 2021-10-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109270176A (en) | Sheep cream oligosaccharides measuring method | |
| Gerritsen et al. | On the determination of free amino acids in serum | |
| Saifer | Comparative study of various extraction methods for the quantitative determination of free amino acids from brain tissue | |
| CN110003323A (en) | A kind of method that double-aqueous phase system isolates and purifies protein | |
| CN106831596B (en) | A method of preparing erythrothioneine | |
| CN101290306A (en) | Milk and milk product tetracycline antibiotic residual quantity checking method | |
| JPS6254797B2 (en) | ||
| CN105092739A (en) | Method for measuring seven kinds of organic acid in rice wine by adopting solid-phase extraction-liquid-phase chromatogram method | |
| JP2019515664A (en) | Separation and purification method of mogroside V by subcritical water desorption technology | |
| CN102286589A (en) | Preparation method of turtle oligopeptide | |
| CN105601606B (en) | A kind of method for preparing high-purity nutgall catechin gallic acid ester GCG | |
| CN105566414B (en) | The method that four kinds of flavone glycosides are isolated and purified from waxberry flesh | |
| CN106188180A (en) | The isolation and purification method of tree peony anthocyanins isomer in a kind of black Fructus Lycii | |
| CN110343146A (en) | A kind of crab flavour mushroom flavor peptide and its preparation method and application | |
| CN107192775B (en) | A kind of measuring method of determining free amino acids in food content | |
| CN101480216A (en) | Method for extracting composite aminoacid from water melon or black seed melon | |
| CN101392016B (en) | Method for preparing high-purity soybean saponin A and B | |
| Boardman et al. | Isolation of two myoglobins from horse-heart extracts and the determination of the molecular weight of the main component | |
| CN103217498A (en) | Method for detecting dicyandiamide in milk powder with LC-MS (liquid chromatography/mass spectrometry) and sample preparation method | |
| CN101628930A (en) | Method for separating polypeptide with tris ligand and agarose medium | |
| CN109810014A (en) | A kind of two caffeoyl spermidine class compound selective enrichment methods in fructus lycii | |
| Trucksess et al. | High performance liquid chromatographic determination of aflatoxicol in milk, blood, and liver | |
| CN109212121A (en) | Red ginseng medicinal materials fingerprint and its construction method with formulation ingredients relevance | |
| CN111122722B (en) | Rapid determination of malic acid delta in fruit juice 13 Method of C value | |
| JPH07173182A (en) | Method for extracting and concentration milk-derived polar lipid consisting mainly of phespholipid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |