CN113899827A - Detection method of 3' -sialyllactose and application thereof - Google Patents
Detection method of 3' -sialyllactose and application thereof Download PDFInfo
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- CN113899827A CN113899827A CN202111157620.2A CN202111157620A CN113899827A CN 113899827 A CN113899827 A CN 113899827A CN 202111157620 A CN202111157620 A CN 202111157620A CN 113899827 A CN113899827 A CN 113899827A
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- 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
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- 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
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- 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
- G01N30/14—Preparation by elimination of some components
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- 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
- G01N2030/065—Preparation using different phases to separate parts of sample
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- 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
- G01N30/14—Preparation by elimination of some components
- G01N2030/146—Preparation by elimination of some components using membranes
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- General Physics & Mathematics (AREA)
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- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of biological detection, and particularly discloses a detection method of 3' -sialyllactose and application thereof. The detection method comprises the following steps: weighing a 3' -sialyllactose standard substance, dissolving by mobile phase ultrasonic wave, and preparing a standard working solution; establishing a standard curve by adopting high performance liquid chromatography for the standard working solution; heating the solution in the enzyme catalysis production in a boiling water bath, precipitating high molecular substances, centrifuging to obtain supernatant, diluting with mobile phase, filtering with disposable organic microporous filter membrane, separating different components of the filtered solution by high performance liquid chromatography, performing quantitative analysis by using a standard curve, and determining 3' -sialyllactose in the solution in real time. The invention improves the accuracy of the determination of the 3' -sialyllactose and has higher recovery rate by carrying out the technical means of heat treatment, ultracentrifugation, mobile phase dissolution, organic membrane filtration and the like on the solution in the enzyme catalysis production. The detection method is simple, quick, efficient and accurate.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a method for detecting 3' -sialyllactose in complex biological solution systems such as biological enzyme catalytic conversion solution and the like and application thereof.
Background
Human Milk Oligosaccharides (HMOs) are the 3 rd largest solid fraction next to lactose and fat in human breast milk, are not only of many kinds, but also have a high content, and in particular, in colostrum after delivery, have an oligosaccharide content far inferior to that of breast milk of other animals such as cow milk and goat milk. It has been proved that HMOs have effective prebiotic effect, can selectively stimulate the growth of beneficial bacteria in intestinal tract, indirectly inhibit the growth of harmful bacteria, and maintain the microecological balance of intestinal tract, thereby reducing the occurrence of digestive tract diseases to the maximum extent, further improving the health condition of host infants. The sialyloligosaccharide is an oligosaccharide compound containing one or a plurality of sialic acid molecules, is widely distributed in various biological tissues, and has important functions of biological information transmission and the like. Meanwhile, as one of the two most main oligosaccharide compositions in HMOs, the sialyloligosaccharide also shows unique physiological activity and superior market potential. There are two main forms of sialyloligosaccharides in HMOs: 3 '-SL and 6' -SL, which are prepared by dehydrating and condensing a molecule of sialic acid and a molecule of lactose catalyzed by alpha-2, 3 and alpha-2, 6 sialyl glycosyltransferase, respectively.
At present, the production process of sialyllactose is mainly based on biological enzymatic conversion, and the current method for detecting 3 ' -SL not only consumes long time, but also cannot quantify 3 ' -SL, so that a rapid and accurate 3 ' -SL quantitative detection method is urgently needed in the enzymatic synthesis process and the subsequent separation process.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for detecting 3' -sialyllactose in complex biological solution systems such as biological enzyme catalytic conversion solution and the like and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
in a first aspect, the present invention provides a method for detecting 3' -sialyllactose, comprising the steps of:
s1, weighing a 3 '-sialyllactose standard substance, dissolving the 3' -sialyllactose standard substance by using mobile phase ultrasonic, and preparing a standard working solution with the concentration of 0.1-1.0 g/L;
s2, establishing a standard curve by adopting high performance liquid chromatography to the standard working solution;
s3, heating the solution in the enzyme catalysis production in a boiling water bath to precipitate the high molecular substance, then centrifuging the precipitated solution, taking the supernatant, diluting the supernatant with a mobile phase, passing through a disposable organic microporous filter membrane, separating different components of the filtered solution by adopting high performance liquid chromatography, carrying out quantitative analysis by utilizing a standard curve, and measuring the 3' -sialyllactose in the solution in real time.
The detection method can be used for measuring the 3 '-sialyllactose produced by enzyme catalysis in real time and stabilizing the final yield of the 3' -sialyllactose, and has high accuracy for measuring the 3 '-sialyllactose and higher recovery rate of the obtained 3' -sialyllactose.
As a preferred embodiment of the detection method of 3' -sialyllactose, the high performance liquid chromatography adopts a TSKgel Amide-80 chromatographic column with the concentration of 4.6 multiplied by 250mm and 5 mu m, and ammonium formate solution with the concentration of 8-10 mmol/L and acetonitrile are used as mobile phases; the flow rate of the mobile phase is 0.9-1.1 ml/min; the column temperature is 50-60 ℃; the sample injection amount is 10-20 mu l; the absorption wavelength is 210 nm.
As a preferred embodiment of the method for detecting 3' -sialyllactose according to the present invention, the preparation step of the ammonium formate solution comprises: adding pure water into the ammonium formate crystal powder, preparing 8-10 mmol/L ammonium formate solution, ultrasonically mixing uniformly, then adding formic acid, and adjusting the pH value of the ammonium formate solution to 3.5-5.5, more preferably, adjusting the pH value of the ammonium formate solution to 4.0.
The inventor finds out through experiments that when the pH value of the ammonium formate solution is 3.5-5.5, the accuracy of 3 '-sialyllactose measurement is improved, and the recovery rate of 3' -sialyllactose is large.
The accuracy of the 3' -sialyllactose determination was highest and the recovery was greatest when the pH of the ammonium formate solution was 4.0. When the pH of the ammonium formate solution is outside the range of the present invention (e.g., 3.0, 6.0), the accuracy and recovery of 3 '-sialyllactose is reduced, indicating that the pH of the ammonium formate solution has a corresponding effect on the accuracy and recovery of the determination of 3' -sialyllactose.
In a preferred embodiment of the method for detecting 3' -sialyllactose according to the present invention, the volume ratio of the ammonium formate solution to acetonitrile is (2 to 8): (2-8).
When the volume ratio of the ammonium formate solution to the acetonitrile in the mobile phase is (2-8): (2-8), the accuracy and recovery rate of the 3' -sialyllactose measurement can be improved.
As a preferred embodiment of the method for detecting 3' -sialyllactose according to the present invention, the volume ratio of the ammonium formate solution to acetonitrile is 3: 7.
when the volume ratio of the ammonium formate solution to the acetonitrile is 3:7, the recovery rate of the 3' -sialyllactose can reach 98.65%, and the detection accuracy is higher.
In a preferred embodiment of the method for detecting 3' -sialyllactose according to the present invention, in step S3, the heating temperature is 70 to 100 ℃ and the heating time is 3 to 10 min.
According to the research of the inventor, when the heating temperature is 70-100 ℃ and the heating time is 3-10 min, the recovery rate of the 3 '-sialyllactose can be improved after the solution in the enzyme catalysis production is placed in a boiling water bath for heating, and the recovery rate of the 3' -sialyllactose can be maintained at 90-99.9%.
As a preferred embodiment of the method for detecting 3' -sialyllactose according to the present invention, the heating temperature is 100 ℃ and the heating time is 5 min.
When other conditions were not changed, the recovery rate of 3 '-sialyllactose increased with an increase in temperature by changing only the heating temperature, and the recovery rate of 3' -sialyllactose was the highest when the heating temperature was 100 ℃. When other conditions are not changed, the recovery rate of the 3 '-sialyllactose is increased along with the heating time only by changing the heating time, and the recovery rate of the 3' -sialyllactose is stable and is kept at about 99% after the heating time reaches 5 min.
In a preferred embodiment of the method for detecting 3' -sialyllactose according to the present invention, in the step S3, the centrifugation speed is 12000rpm, and the centrifugation time is 3 to 10 min.
When the centrifugation time of the solution is 3-10 min, the recovery rate of the 3 '-sialyllactose is basically stabilized to be more than 99%, and more preferably, the centrifugation time is 3-6 min, and the measured recovery rate of the 3' -sialyllactose is higher.
In a preferred embodiment of the method for detecting 3' -sialyllactose according to the present invention, in step S1, the concentrations of the standard working solutions are: 0.1g/L, 0.2g/L, 0.4g/L, 0.6g/L, 0.8g/L and 1.0 g/L.
In a second object, the present invention provides the use of the above assay for assessing the real-time yield and the final stable yield of enzymatically produced 3' -sialyllactose.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the accuracy of 3' -sialyllactose determination is improved by carrying out heat treatment, ultracentrifugation, mobile phase dissolution, organic membrane filtration and other technical means on the solution in enzyme catalysis production, and the recovery rate is higher; and establishing a standard curve on the basis of the standard substance by high performance liquid chromatography, detecting the 3 '-sialyllactose in the sample in the same way, and combining the standard curve to obtain the content of the 3' -sialyllactose. The detection method is simple, rapid, efficient and accurate, shortens the analysis time and enables the detection of the 3' -sialyllactose to be more accurate. The solution of the sample to be detected prepared by the method is stable in property, can be stored for 2-3 weeks in a closed environment at 4 ℃, and makes up for a short plate which cannot be stored for a long time in a biological system solution. The method is not only suitable for detecting the enzymatic production solution, but also can change the treatment parameters according to the actual conditions, thereby being suitable for systems such as post-treatment process solution, final product solution and the like, and having wide applicability.
Drawings
FIG. 1 is a high performance liquid chromatogram of a 3' -sialyllactose standard;
FIG. 2 is a standard working curve of a 3' -sialyllactose standard;
FIG. 3 is a high performance liquid chromatogram of the present invention for determining 3' -sialyllactose;
FIG. 4 is a graph I of the results of the effect of different heating temperatures on the recovery of 3' -sialyllactose;
FIG. 5 is a graph II of the results of the effect of different heating temperatures on the recovery of 3' -sialyllactose;
FIG. 6 is a graph I of the results of the effect of different heating times on the recovery of 3' -sialyllactose;
FIG. 7 is a graph II of the results of different heating times on the recovery of 3' -sialyllactose;
FIG. 8 is a graph I of the results of the effect of different high speed centrifugation times on the recovery of 3' -sialyllactose;
FIG. 9 is a graph II of the results of different high speed centrifugation time effects on recovery of 3' -sialyllactose;
FIG. 10 is a graph I of the results of the effect of different pH ammonium formate solutions on the recovery of 3' -sialyllactose;
FIG. 11 is a graph II of the results of the effect of ammonium formate solutions of different pH on the recovery of 3' -sialyllactose;
FIG. 12 is a graph I of the results of different volume ratios of ammonium formate solution and acetonitrile on the recovery of 3' -sialyllactose;
FIG. 13 is a graph II showing the results of different volume ratios of ammonium formate solution and acetonitrile on the recovery of 3' -sialyllactose.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
In the following examples and comparative examples, the experimental methods used were conventional ones unless otherwise specified, and the materials, reagents and the like used were commercially available ones unless otherwise specified.
Wherein the solution in the enzyme-catalyzed Production is derived from the literature "effective Production of 3' -Sialyllactose by simple grain Cell in One-dot Biosynthesis", which system comprises sialic acid, lactose, hexametaphosphate, CMPNa2 (disodium cytidine monophosphate), magnesium chloride hexahydrate, sodium hydroxide, E.coli BL21(DE3) DellacZ/pCSCP fermentation sludge and 50mM PBS, and the pH of the solution is 7.5.
Example 1 detection method of 3' -sialyllactose
A method for detecting 3' -sialyllactose comprises the following steps:
s1, taking ammonium formate crystal powder, adding pure water, preparing 10mmol/L ammonium formate solution, ultrasonically mixing uniformly, then adding formic acid, and adjusting the pH value of the ammonium formate solution to 4.0; mixing the prepared ammonium formate solution with acetonitrile in a ratio of 3:7, mixing in proportion, passing through a Jinteng organic microporous filter membrane (50 x 0.2um), and ultrasonically mixing uniformly to obtain a mobile phase;
s2, weighing a 3' -sialyllactose standard substance, dissolving the standard substance by mobile phase ultrasonic, and preparing standard working solutions with the concentrations of 0.1g/L, 0.2g/L, 0.4g/L, 0.6g/L, 0.8g/L and 1.0g/L respectively;
s3, adopting high performance liquid chromatography to the standard working solution, adopting an ultraviolet detector for measurement, drawing a standard working curve by taking the concentration as a horizontal coordinate and the chromatographic peak area of the measured object 3-SL as a vertical coordinate, and making a standard curve equation;
s4, heating 1-2 ml of solution in enzyme catalysis production in a boiling water bath at 100 ℃ for 5min to precipitate protein and other high molecular substances, centrifuging the precipitated solution at 12000rpm for 6min until the supernatant is completely separated from the bottom precipitate, taking the supernatant, diluting the supernatant with a mobile phase, filtering the supernatant with a disposable organic microporous filter membrane, separating different components of the filtered solution by adopting high performance liquid chromatography, and carrying out quantitative analysis by using a standard curve to determine the 3' -sialyllactose in the solution in real time.
The high performance liquid chromatography adopts a TSKgel Amide-80 chromatographic column with the thickness of 4.6 multiplied by 250mm and 5 mu m, and ammonium formate solution with the concentration of 10mmol/L and acetonitrile (the volume ratio is 3:7) are taken as mobile phases; the flow rate of the mobile phase is 1.0 ml/min; the column temperature is 50-60 ℃; the sample injection amount is 10-20 mu l; the absorption wavelength is 210 nm.
The high performance liquid chromatogram of the 3' -sialyllactose standard in example 1 is shown in fig. 1; the standard working curve of the 3' -sialyllactose standard is shown in fig. 2 (the regression equation is that y is 3285080x +25731.8, the linear correlation coefficient is more than 0.9999, and the linearity is good); the high performance liquid chromatogram for measuring 3' -sialyllactose in the present invention is shown in FIG. 3.
Example 2-7 detection method of 3' -sialyllactose
Example 2: compared with example 1, the difference is only that: the heating temperature in step S4 was 70 ℃ and the remaining parameters were the same as in example 1.
Example 3: compared with example 1, the difference is only that: the heating temperature in step S4 was 75 ℃ and the remaining parameters were the same as in example 1.
Example 4: compared with example 1, the difference is only that: the heating temperature in step S4 was 80 ℃ and the other parameters were the same as in example 1.
Example 5: compared with example 1, the difference is only that: the heating temperature in step S4 was 85 ℃ and the other parameters were the same as in example 1.
Example 6: compared with example 1, the difference is only that: the heating temperature in step S4 was 90 ℃ and the remaining parameters were the same as in example 1.
Example 7: compared with example 1, the difference is only that: the heating temperature in step S4 was 95 ℃ and the remaining parameters were the same as in example 1.
The recovery rates of 3' -sialyllactose determined by the detection methods of examples 1 to 7 are shown in FIGS. 4 to 5. When other conditions were not changed, the recovery of 3 '-sialyllactose could be improved by changing the heating temperature, and particularly, the recovery of 3' -sialyllactose was the highest (99.12%) when the heating temperature was 100 ℃. The recovery of 3' -sialyllactose was the lowest, 92.69%, when the heating temperature was 70 ℃. When the heating temperature is 80-100 ℃, the recovery rate of the 3' -sialyllactose is increased along with the increase of the temperature.
Examples 8 to 14, a method for detecting 3' -sialyllactose
Example 8: compared with example 1, the difference is only that: the heating time in step S4 was 3min, and the remaining parameters were the same as in example 1.
Example 9: compared with example 1, the difference is only that: the heating time in step S4 was 4min, and the remaining parameters were the same as in example 1.
Example 10: compared with example 1, the difference is only that: the heating time in step S4 was 6min, and the remaining parameters were the same as in example 1.
Example 11: compared with example 1, the difference is only that: the heating time in step S4 was 7min, and the remaining parameters were the same as in example 1.
Example 12: compared with example 1, the difference is only that: the heating time in step S4 was 8min, and the remaining parameters were the same as in example 1.
Example 13: compared with example 1, the difference is only that: the heating time in step S4 was 9min, and the remaining parameters were the same as in example 1.
Example 14: compared with example 1, the difference is only that: the heating time in step S4 was 10min, and the remaining parameters were the same as in example 1.
Comparative examples 1 to 2
Comparative example 1: compared with example 1, the difference is only that: the heating time of step S4 was 1min, and the remaining parameters were the same as in example 1.
Comparative example 2: compared with example 1, the difference is only that: the heating time of step S4 was 2min, and the remaining parameters were the same as in example 1.
The recovery rates of 3' -sialyllactose determined by the above-described assay methods of examples 8 to 14 and comparative examples 1 to 2 are shown in FIGS. 6 to 7. As a result: when other conditions are unchanged, the heating time is prolonged, the recovery rate of the 3 '-sialyllactose can be increased, when the heating time of the solution in the enzyme catalysis production reaches 5min, the recovery rate tends to be stable at a high value, and in order to save the experimental time, the recovery rate of the 3' -sialyllactose is better (99.12%) when the heating time is 5 min. On the other hand, when the heating time was 1min, the recovery rate of 3 '-sialyllactose was only 90.23%, and when the heating time was 2min, the recovery rate of 3' -sialyllactose was only 93.36%, and the recovery rate of 3 '-sialyllactose measured by the test method of comparative example 1-2 was inferior to that of 3' -sialyllactose measured by the test method of examples 8-14.
Example 15-17 method for detecting 3' -sialyllactose
Example 15: compared with example 1, the difference is only that: the centrifugation time in step S4 was 3min, and the remaining parameters were the same as in example 1.
Example 16: compared with example 1, the difference is only that: the centrifugation time in step S4 was 4min, and the remaining parameters were the same as in example 1.
Example 17: compared with example 1, the difference is only that: the centrifugation time in step S4 was 5min, and the remaining parameters were the same as in example 1.
Comparative examples 3 to 4
Comparative example 3: compared with example 1, the difference is only that: the centrifugation time in step S4 was 1min, and the remaining parameters were the same as in example 1.
Comparative example 4: compared with example 1, the difference is only that: the centrifugation time in step S4 was 2min, and the remaining parameters were the same as in example 1.
The recovery rates of 3' -sialyllactose determined by the above-described detection methods of examples 15 to 17 and comparative examples 3 to 4 are shown in FIGS. 8 to 9. As a result: the recovery of 3 '-sialyllactose was substantially stabilized at 99.0% or more when the high-speed centrifugation time was 3min or more, whereas the recovery of 3' -sialyllactose was 95.23% or 96.28% when the high-speed centrifugation time was 1min or 2min, and the recovery of 3 '-sialyllactose measured by the test method of comparative examples 3-4 was far inferior to that of 3' -sialyllactose measured in examples 15-17.
Examples 18 to 21, aDetection method of 3' -sialyllactose
Example 18: compared with example 1, the difference is only that: the pH of the ammonium formate solution in step S1 was 3.5, and the remaining parameters were the same as in example 1.
Example 19: compared with example 1, the difference is only that: the pH of the ammonium formate solution in step S1 was 4.5, and the remaining parameters were the same as in example 1.
Example 20: compared with example 1, the difference is only that: the pH of the ammonium formate solution in step S1 was 5.0, and the remaining parameters were the same as in example 1.
Example 21: compared with example 1, the difference is only that: the pH of the ammonium formate solution in step S1 was 5.5, and the remaining parameters were the same as in example 1.
Comparative example 5
Comparative example 5: compared with example 1, the difference is only that: the pH of the ammonium formate solution in step S1 was 3.0, and the remaining parameters were the same as in example 1.
The recovery rates of 3' -sialyllactose determined by the above-described detection methods of examples 18 to 21 and comparative example 5 are shown in FIGS. 10 to 11. The recovery of 3 '-sialyllactose was maximal (98.65%) when the pH of the ammonium formate solution was 4.0, and the accuracy of the measurement of 3' -sialyllactose was highest. Whereas when the pH of the ammonium formate solution is 3.0 and outside the pH range of the ammonium formate solution of the present invention, the recovery of 3' -sialyllactose is low, only 85.36%. When the pH of the ammonium formate solution was 6.0, the recovery of 3 ' -sialyllactose was also low, which is inferior to the recovery of 3 ' -sialyllactose determined in examples 18-21, further illustrating that the pH of the ammonium formate solution had a corresponding effect on the accuracy and recovery of the determination of 3 ' -sialyllactose.
Examples 22 to 27A method for detecting 3' -sialyllactose
Example 22: compared with example 1, the difference is only that: the volume ratio of ammonium formate solution to acetonitrile was 2:8, and the remaining parameters were the same as in example 1.
Example 23: compared with example 1, the difference is only that: the volume ratio of ammonium formate solution to acetonitrile was 4:6, and the remaining parameters were the same as in example 1.
Example 24: compared with example 1, the difference is only that: the volume ratio of ammonium formate solution to acetonitrile was 5:5, and the remaining parameters were the same as in example 1.
Example 25: compared with example 1, the difference is only that: the volume ratio of ammonium formate solution to acetonitrile was 6:4, and the remaining parameters were the same as in example 1.
Example 26: compared with example 1, the difference is only that: the volume ratio of ammonium formate solution to acetonitrile was 7:3, and the remaining parameters were the same as in example 1.
Example 27: compared with example 1, the difference is only that: the volume ratio of ammonium formate solution to acetonitrile was 8:2, and the remaining parameters were the same as in example 1.
Comparative examples 6 to 7
Comparative example 6: compared with example 1, the difference is only that: the volume ratio of ammonium formate solution to acetonitrile was 1:9, and the remaining parameters were the same as in example 1.
Comparative example 7: compared with example 1, the difference is only that: the volume ratio of ammonium formate solution to acetonitrile was 9:1, and the remaining parameters were the same as in example 1.
The recovery rates of 3' -sialyllactose determined by the above-described assay methods of examples 22 to 27 and comparative examples 6 to 7 are shown in FIGS. 12 to 13. When the volume ratio of the ammonium formate solution to the acetonitrile is 3:7, the recovery rate of the 3' -sialyllactose can reach 98.65%, and the detection accuracy is highest. When the volume ratio of ammonium formate solution to acetonitrile was 1:9 or 9:1, the measured recovery of 3' -sialyllactose was low, only 66.58% or 71.85%. It is shown that 3' -sialyllactose cannot be determined well and the accuracy is low when the volume ratio of ammonium formate solution to acetonitrile is outside the range of the present invention.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. The method for detecting 3' -sialyllactose is characterized by comprising the following steps:
s1, weighing a 3 '-sialyllactose standard substance, dissolving the 3' -sialyllactose standard substance by using mobile phase ultrasonic, and preparing a standard working solution with the concentration of 0.1-10 g/L;
s2, establishing a standard curve by adopting high performance liquid chromatography to the standard working solution;
s3, taking 1-2 ml of solution in the enzyme catalysis production, placing the solution in a boiling water bath for heating, precipitating high molecular substances, then centrifuging the precipitated solution, taking supernatant, diluting the supernatant with a mobile phase, filtering the supernatant with a disposable organic microporous filter membrane, separating different components of the filtered solution by adopting high performance liquid chromatography, carrying out quantitative analysis by utilizing a standard curve, and measuring the 3' -sialyllactose in the solution in real time.
2. The method for detecting 3' -sialyllactose as claimed in claim 1, wherein the high performance liquid chromatography uses a 4.6 x 250mm, 5 μm tskgel amide-80 column, and ammonium formate solution with a concentration of 8 to 10mmol/L and acetonitrile as mobile phases; the flow rate of the mobile phase is 0.9-1.1 ml/min; the column temperature is 50-60 ℃; the sample injection amount is 10-20 mu l; the absorption wavelength is 210 nm.
3. The method of detecting 3' -sialyllactose as claimed in claim 2, wherein the step of preparing the ammonium formate solution comprises: adding pure water into the ammonium formate crystal powder, preparing 8-10 mmol/L ammonium formate solution, ultrasonically mixing uniformly, then adding formic acid, and adjusting the pH value of the ammonium formate solution to 3.5-5.5, more preferably, adjusting the pH value of the ammonium formate solution to 4.0.
4. The method for detecting 3' -sialyllactose as claimed in claim 2, wherein the volume ratio of the ammonium formate solution to acetonitrile is (2 to 8): (2-8).
5. The method of detecting 3' -sialyllactose as claimed in claim 4 wherein the volume ratio of the ammonium formate solution to acetonitrile is from 3: 7.
6. the method for detecting 3' -sialyllactose according to claim 1, wherein the heating temperature is 70 to 100 ℃ and the heating time is 3 to 10min in step S3.
7. The method of detecting 3' -sialyllactose as claimed in claim 6, wherein the heating temperature is 100 ℃ and the heating time is 5 min.
8. The method for detecting 3' -sialyllactose according to claim 1, wherein in step S3, the centrifugation speed is 12000rpm and the centrifugation time is 3 to 10 min.
9. The method for detecting 3' -sialyllactose as claimed in claim 1, wherein in step S1, the concentrations of the standard working solutions are: 0.1g/L, 0.2g/L, 0.4g/L, 0.6g/L, 0.8g/L and 1.0 g/L.
10. Use of the assay of any one of claims 1 to 9 for assessing the real-time yield and the final stable yield of enzymatically produced 3' -sialyllactose.
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