CN112697926A - Method for detecting content of trehalose in sugar-saline beverage - Google Patents
Method for detecting content of trehalose in sugar-saline beverageDownload PDFInfo
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- CN112697926A CN112697926ACN202011617061.4ACN202011617061ACN112697926ACN 112697926 ACN112697926 ACN 112697926ACN 202011617061 ACN202011617061 ACN 202011617061ACN 112697926 ACN112697926 ACN 112697926A
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- HDTRYLNUVZCQOY-UHFFFAOYSA-Nα-D-glucopyranosyl-α-D-glucopyranosideNatural productsOC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1HDTRYLNUVZCQOY-UHFFFAOYSA-N0.000titleclaimsabstractdescription65
- HDTRYLNUVZCQOY-WSWWMNSNSA-NTrehaloseNatural productsO[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1HDTRYLNUVZCQOY-WSWWMNSNSA-N0.000titleclaimsabstractdescription65
- HDTRYLNUVZCQOY-LIZSDCNHSA-Nalpha,alpha-trehaloseChemical compoundO[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1HDTRYLNUVZCQOY-LIZSDCNHSA-N0.000titleclaimsabstractdescription65
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- RFSUNEUAIZKAJO-ARQDHWQXSA-NFructoseChemical compoundOC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1ORFSUNEUAIZKAJO-ARQDHWQXSA-N0.000claimsabstractdescription12
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- PKAUICCNAWQPAU-UHFFFAOYSA-N2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamineChemical compoundCNC.CC1=CC(Cl)=CC=C1OCC(O)=OPKAUICCNAWQPAU-UHFFFAOYSA-N0.000claimsabstractdescription6
- 239000007788liquidSubstances0.000claimsabstractdescription6
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- 238000006467substitution reactionMethods0.000description1
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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
- 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
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
Description
Technical Field
The invention belongs to the technical field of beverage detection, and particularly relates to a method for detecting the content of trehalose in a sugar-saline beverage.
Background
Trehalose is a non-reducing disaccharide having 2 glucose molecules bonded to each other via a hemiacetal hydroxyl group by an α -1,1 bond, and is widely found in plants, microorganisms and other organisms in nature. Many organisms can increase the content of self-trehalose through in vivo regulation under extreme conditions so as to resist the damage of the outside to the organisms. The trehalose can improve the stress resistance quality of the original species, and can be used as a protective additive in the drying process to keep the activity of biological products and prolong the storage life. At present, trehalose is widely used in food, cosmetics, medicines and the like. The analytical method of trehalose mainly comprises paper chromatography, thin layer chromatography, gas chromatography, high performance liquid chromatography and the like.
The existing detection method has the following problems and disadvantages:
in the existing trehalose analysis method, paper chromatography, thin layer chromatography and gas chromatography are complicated to operate, have large interference and have poorer accuracy and reproducibility than HPLC. The content of sodium salt contained in the sugar beverage by the HPLC method (1) is higher, and the separation degrees of a trehalose peak, a maltotriose peak and a glucose peak can not reach the required 1.3 when the sugar beverage is detected by a method in pharmacopoeia; (2) to remove interference from other peaks, the sugar brine beverage was diluted until the degree of separation was satisfactory, giving a theoretical amount of trehalose of 2mg/ml, at a concentration much lower than the 10mg/ml sample concentration indicated in the pharmacopoeia trehalose assay.
How to design a method for detecting the content of trehalose in a sugar-saline beverage, which effectively solves the problem that the interference of too high content of glucose in a prescription on a trehalose peak is too large by adjusting the concentration of a sample and changing the conditions of liquid chromatography, and meets the requirement of separation degree. The trehalose content in the beverage is effectively detected, and the interference in the detection process is reduced. This is a technical problem which is currently awaited to be solved in the art.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for detecting the content of trehalose in a sugar saline beverage, which effectively solves the problem that the interference of too high content of glucose in the existing prescription on a trehalose peak is too large, effectively detects the content of trehalose in the sugar beverage and reduces the interference in the detection process.
In order to solve the above problems, the object of the present invention is achieved by the following technical solutions:
a method for detecting the content of trehalose in a sugar-saline beverage is characterized by comprising the following steps:
step 1: diluting the test solution by 25 times: taking 10ml of sugar-saline beverage sample, and diluting to 250ml with water;
step 2: chromatographic conditions are as follows: adopting Bio-rad Aminex HPX-87℃, and a chromatographic column with the thickness of 300mm multiplied by 7.8mm and the column temperature of 77-87 ℃; water is used as a mobile phase, and the flow rate is 0.3 ml/min; a differential refraction detector with a detector temperature of 38 +/-5 ℃; taking appropriate amount of maltotriose, trehalose, anhydrous glucose and fructose, and preparing into solution containing 2mg, 40mg and 2mg of maltotriose, trehalose, anhydrous glucose and fructose respectively per 1ml with water as system applicability solution; precisely measuring 20 mu l, injecting into a liquid chromatograph, wherein the theoretical plate number is not less than 2000 calculated by a trehalose peak, and the separation degree of a maltotriose peak and the trehalose peak and the separation degree of the trehalose peak and an anhydrous glucose peak are both more than 1.3;
and step 3: control solution: 2mg/mL trehalose aqueous solution;
and 4, step 4: in the test solution, the relative standard deviation of the detection result of the trehalose content of the parallel sample is not more than 2.0%.
Preferably, in the step 2, the column temperature of the chromatographic column is 80 ℃, the flow rate of the mobile phase is 0.3ml/min, and the temperature of the detector is 40 ℃.
Compared with the prior art, the invention has the following advantages and positive effects:
(1) the content of trehalose in the sugar-saline beverage is effectively detected;
(2) the interference of glucose peak and maltotriose peak in the sugar saline beverage to the trehalose is reduced, and the separation degree is ensured to meet the requirement.
Drawings
FIG. 1 is a diagram showing the systematic applicability of the method for detecting the trehalose content in a sugar-containing aqueous beverage according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
Example 1:
(1) diluting the test solution by 25 times: a sample of sugar-saline beverage (10 ml) was taken and diluted to 250ml with water.
(2) Chromatographic conditions are as follows: adopting Bio-rad Aminex HPX-87C, and a chromatographic column with the thickness of 300mm multiplied by 7.8mm and the temperature of 77 ℃ for the chromatographic column; water is used as a mobile phase, and the flow rate is 0.3 ml/min; a differential refractive detector, a detector temperature of 35 ℃. Taking appropriate amount of maltotriose, trehalose, anhydrous glucose and fructose, and preparing into solution containing maltotriose, trehalose, anhydrous glucose and fructose 2mg, 40mg and 2mg per 1ml with water as system applicability solution; precisely measuring 20 μ l, injecting into liquid chromatograph, wherein the theoretical plate number is not less than 2000 calculated by trehalose peak, and the separation degree of maltotriose peak and trehalose peak and the separation degree of trehalose peak and anhydrous glucose peak are both greater than 1.3.
(3) Control solution: 2.052mg/mL and 2.034mg/mL trehalose in water.
The detection result shows that the trehalose content in the sugar saline beverage diluent sample is 1.940mg/ml which is calculated to be 96.9 percent of the marked amount in the formula; as shown in fig. 1, the system suitability meets the requirement, and the separation degree meets the requirement.
Example 2:
(1) diluting the test solution by 25 times: a sample of sugar-saline beverage (10 ml) was taken and diluted to 250ml with water.
(2) Chromatographic conditions are as follows: adopting Bio-rad Aminex HPX-87C, and a chromatographic column with the thickness of 300mm multiplied by 7.8mm and the temperature of 80 ℃; water is used as a mobile phase, and the flow rate is 0.3 ml/min; a differential refractive detector, detector temperature 40 ℃. Taking appropriate amount of maltotriose, trehalose, anhydrous glucose and fructose, and preparing into solution containing maltotriose, trehalose, anhydrous glucose and fructose 2mg, 40mg and 2mg per 1ml with water as system applicability solution; precisely measuring 20 μ l, injecting into liquid chromatograph, wherein the theoretical plate number is not less than 2000 calculated by trehalose peak, and the separation degree of maltotriose peak and trehalose peak and the separation degree of trehalose peak and anhydrous glucose peak are both greater than 1.3.
(3) Control solution: 1.987mg/mL and 2.044mg/mL trehalose in water.
The detection result shows that the trehalose content in the sugar saline beverage diluent sample is 1.955mg/ml, which is calculated to be 97.8% of the marked amount in the formula; the applicability of the system meets the requirement, and the separation degree meets the requirement.
Example 3:
(1) diluting the test solution by 25 times: a sample of sugar-saline beverage (10 ml) was taken and diluted to 250ml with water.
(2) Chromatographic conditions are as follows: adopting Bio-rad Aminex HPX-87℃, and a chromatographic column with the thickness of 300mm multiplied by 7.8mm and the temperature of 87 ℃; water is used as a mobile phase, and the flow rate is 0.3 ml/min; differential refractive detector, detector temperature 38 ℃. Taking appropriate amount of maltotriose, trehalose, anhydrous glucose and fructose, and preparing into solution containing maltotriose, trehalose, anhydrous glucose and fructose 2mg, 40mg and 2mg per 1ml with water as system applicability solution; precisely measuring 20 μ l, injecting into liquid chromatograph, wherein the theoretical plate number is not less than 2000 calculated by trehalose peak, and the separation degree of maltotriose peak and trehalose peak and the separation degree of trehalose peak and anhydrous glucose peak are both greater than 1.3.
(3) Control solution: 1.992mg/mL and 1.950mg/mL trehalose aqueous solution.
The detection result shows that the trehalose content in the sugar saline beverage diluent sample is 1.954mg/ml, which is calculated to be 97.7% of the marked amount in the formula; the applicability of the system meets the requirement, and the separation degree meets the requirement.
The above embodiments are only used for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; such modifications and substitutions do not depart from the spirit and scope of the present invention as set forth in the appended claims.
Claims (2)
1. A method for detecting the content of trehalose in a sugar-saline beverage is characterized by comprising the following steps:
step 1: diluting the test solution by 25 times: taking 10ml of sugar-saline beverage sample, and diluting to 250ml with water;
step 2: chromatographic conditions are as follows: adopting Bio-rad Aminex HPX-87℃, and a chromatographic column with the thickness of 300mm multiplied by 7.8mm and the column temperature of 77-87 ℃; water is used as a mobile phase, and the flow rate is 0.3 ml/min; a differential refraction detector with a detector temperature of 38 +/-5 ℃; taking appropriate amount of maltotriose, trehalose, anhydrous glucose and fructose, and preparing into solution containing 2mg, 40mg and 2mg of maltotriose, trehalose, anhydrous glucose and fructose respectively per 1ml with water as system applicability solution; precisely measuring 20 mu l, injecting into a liquid chromatograph, wherein the theoretical plate number is not less than 2000 calculated by a trehalose peak, and the separation degree of a maltotriose peak and the trehalose peak and the separation degree of the trehalose peak and an anhydrous glucose peak are both more than 1.3;
and step 3: control solution: 2mg/mL trehalose aqueous solution;
and 4, step 4: in the test solution, the relative standard deviation of the detection result of the trehalose content of the parallel sample is not more than 2.0%.
2. The method as claimed in claim 1, wherein the column temperature of the chromatographic column in step 2 is 80 ℃, the flow rate of the mobile phase is 0.3ml/min, and the temperature of the detector is 40 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011617061.4ACN112697926A (en) | 2020-12-31 | 2020-12-31 | Method for detecting content of trehalose in sugar-saline beverage |
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| CN202011617061.4ACN112697926A (en) | 2020-12-31 | 2020-12-31 | Method for detecting content of trehalose in sugar-saline beverage |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116223658A (en)* | 2022-12-30 | 2023-06-06 | 南京恩泰医药科技有限公司 | A kind of gluconate content assay method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11103885A (en)* | 1994-09-16 | 1999-04-20 | Nippon Shokuhin Kako Co Ltd | Method for producing saccharified solution containing trehalose |
| WO2008080093A2 (en)* | 2006-12-21 | 2008-07-03 | Verenium Corporation | Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them |
| WO2008100021A1 (en)* | 2007-02-15 | 2008-08-21 | Korea Advanced Institute Of Science And Technology | Genes which are related to trehalose biosynthesis and mass-production of trehalose using thereof |
| JP2012105677A (en)* | 2012-03-05 | 2012-06-07 | National Institute Of Agrobiological Sciences | Trehalose transporter gene and method for introducing trehalose in cell utilizing the same gene |
| CN109030656A (en)* | 2018-08-17 | 2018-12-18 | 华仁药业股份有限公司 | Detection method of the glucose in relation to substance in acetate Multiple electrolytes injection |
- 2020
- 2020-12-31CNCN202011617061.4Apatent/CN112697926A/enactivePending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11103885A (en)* | 1994-09-16 | 1999-04-20 | Nippon Shokuhin Kako Co Ltd | Method for producing saccharified solution containing trehalose |
| WO2008080093A2 (en)* | 2006-12-21 | 2008-07-03 | Verenium Corporation | Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them |
| WO2008100021A1 (en)* | 2007-02-15 | 2008-08-21 | Korea Advanced Institute Of Science And Technology | Genes which are related to trehalose biosynthesis and mass-production of trehalose using thereof |
| JP2012105677A (en)* | 2012-03-05 | 2012-06-07 | National Institute Of Agrobiological Sciences | Trehalose transporter gene and method for introducing trehalose in cell utilizing the same gene |
| CN109030656A (en)* | 2018-08-17 | 2018-12-18 | 华仁药业股份有限公司 | Detection method of the glucose in relation to substance in acetate Multiple electrolytes injection |
Non-Patent Citations (5)
| Title |
|---|
| ANDREA CP 等: "Differential synthesis of sucrose and trehalose in Euglena gracilis cells during growth and salt stress", 《PLANT SCIENCE》* |
| 吴辉 等: "果糖和麦芽糖作为有氧碳源对大肠杆菌NZN111两阶段发酵中厌氧发酵的影响", 《生物工程学报》* |
| 张曰辉 等: "高效液相色谱法检测奶糖中海藻糖等糖含量的研究", 《中国食品添加剂》* |
| 曾林燕 等: "3个品种黄精炮制前后小分子糖含量变化", 《中国实验方剂学杂志》* |
| 盛文胜 等: "不同蜂蜜中果糖·葡糖糖和蔗糖含量的测定", 《安徽农业科学》* |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116223658A (en)* | 2022-12-30 | 2023-06-06 | 南京恩泰医药科技有限公司 | A kind of gluconate content assay method |
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