Method for detecting reduced glutathione in yeast cells and yeast extracts

A technology of yeast extract and glutathione, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of low reliability, asymmetry, peak shape tailing of reduced glutathione, etc., to achieve Increase the number of theoretical plates, improve the chromatographic peak shape, and the effect of accurate and reliable detection

Inactive Publication Date: 2017-03-22
CHINA THREE GORGES UNIV +1
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  • Abstract
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Problems solved by technology

[0003] At present, the main detection method of reduced glutathione is DTNB derivatization-photometric method, because the sulfhydryl group is easy to cause false positive interference to this method, and the detection content is too high, so the reliability of this method is low, and it is not suitable for reduced glutathione Accurate Detection of Glycopeptide Content
Although it has been reported in the literature that the HPLC method is used to detect the content of reduced glutathione, the retention time of reduced glutathione in the chromatographic column is too short, and it is easy to overlap with other chemical components in yeast cells; The prototypical glutathione peak shape is tailed and asymmetrical; these seriously interfere with the determination of reduced glutathione

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  • Method for detecting reduced glutathione in yeast cells and yeast extracts
  • Method for detecting reduced glutathione in yeast cells and yeast extracts
  • Method for detecting reduced glutathione in yeast cells and yeast extracts

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Embodiment 1

[0026] Optimization of Analytical Conditions for High Performance Liquid Chromatography

[0027] The binary mobile phase is methanol-water phase (the water phase includes 1L deionized water, 2.2g / L sodium heptanesulfonate, 6.8g / L potassium dihydrogen phosphate, pH value is 4.5), methanol: the mixture of water phase The volume ratio is 8:92, and the standard analysis experiment is carried out on the chromatographic column Venusil XBP C18 column (5μm×10×250mm). Results The peak time of reduced glutathione on the chromatographic column was 2.5min, and the peak shape was seriously tailed. figure 1 is the HPLC chromatogram of the standard solution at pH 4.5 binary mobile phase. The chromatographic column is: Venusil XBP C18 column (5μm×10×250mm); the mobile phase is methanol-water binary mobile phase, wherein the water phase includes 1L deionized water, 2.2g / L sodium heptanesulfonate, 6.8g / L Potassium dihydrogen phosphate, pH 4.5. The mixing volume ratio of methanol:water phase ...

Embodiment 2

[0029] Influence of pH value of aqueous phase in binary mobile phase

[0030] Phosphoric acid was used to adjust the pH of the aqueous phase in the binary mobile phase to 4.5, 4.0, 3.5, 3.0, and 2.5 respectively. The mixed volume ratio of methanol: aqueous phase was 8:92. Carry out standard analysis experiments. The results showed that the pH value of the aqueous phase in the binary mobile phase had an important influence on the retention time of reduced glutathione on the column. The lower the pH value, the longer the retention time, and the sharper and more symmetrical the peak shape.

[0031] Phosphoric acid was used to adjust the pH of the aqueous phase in the binary mobile phase to 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, and 2.9, and the mixed volume ratio of methanol: aqueous phase was 8:92. 10×250mm) for standard analysis experiments. The results show that: when the pH of the aqueous phase is 2.5, the eluted reduced glutathione has a retention time of 16 minutes, and the peak ...

Embodiment 3

[0034] Production of reduced glutathione standard curve and regression equation

[0035] Take 80mg of reduced glutathione standard product, add deionized water to dissolve, dilute to 10mL, shake well to obtain reduced glutathione standard stock solution (8mg / mL); sequentially prepare 4mg / mL, 2mg / mL mL, 1mg / mL, 0.5mg / mL and 0.25mg / mL stock solution; HPLC measurement, record the peak area, take the concentration of reduced glutathione as the abscissa, and the corresponding peak area as the ordinate to perform linear regression, The linearity is: y=12506660x+905874, R 2 =0.9997 (wherein x is the reduced GSH concentration, and y is the corresponding peak area); the results show that the reduced glutathione mass concentration presents a good linear relationship between 0.25-4mg / mL; according to the signal-to-noise ratio S / N The minimum detectable concentration of reduced glutathione is 6.5 μg / mL.

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Abstract

The invention discloses a method for detecting reduced glutathione in yeast cells and yeast extracts. A high performance liquid chromatograph is used, and the reduced glutathione is qualitatively and quantitatively detected by an external standard method. The method adopts a methanol/water binary mobile phase (wherein sodium heptanesulfonate, potassium dihydrogen phosphate and phosphoric acid are added in the aqueous phase), the retention time of the reduced glutathione on a chromatographic column is regulated through adjustment of the pH value of a solution or adjustment of the ratio of two phases or cooperative adjustment, the number of theoretical tower plates is increased, the chromatographic peak type is improved, and detection of the content of the reduced glutathione in yeast is accurate and reliable. The method has the advantages of simple operation and high efficiency, provides a good reference for control of detection of glutathione in a process of yeast cell fermentation production of glutathione, and provides a method basis for increasing the quality standard of glutathione raw material.

Description

technical field [0001] The invention relates to a method for detecting reduced glutathione in yeast cells and yeast extracts, in particular to a method for measuring reduced glutathione in yeast fermentation products. Background technique [0002] Reduced glutathione (Glutathione, GSH), as an important pharmaceutical reagent, is widely used clinically, and the antioxidant properties of GSH make it favored as an additive in food and cosmetic processing industries. How to quickly, simply and efficiently detect the content of reduced glutathione is not only crucial to the quality control of the GSH production process by yeast fermentation, but also particularly critical to the quality standard control and improvement of GSH related products in the field of food and medicine. [0003] At present, the main detection method of reduced glutathione is DTNB derivatization-photometric method, because the sulfhydryl group is easy to cause false positive interference to this method, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 邓张双李知洪杜维力姚鹃刘秀继李啸陈良立邓艾平邹坤龚大春
Owner CHINA THREE GORGES UNIV
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