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Extraction and Quantification of Folic Acid in Corn Grain

A technology for grains and corns, applied in the field of plant folic acid extraction, can solve the problems of narrow linear range, low resolution, inability to distinguish folic acid, etc.

Active Publication Date: 2021-06-11
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, microbiological methods have the disadvantages of low resolution, narrow linear range, and inability to distinguish between different forms of folic acid

Method used

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  • Extraction and Quantification of Folic Acid in Corn Grain
  • Extraction and Quantification of Folic Acid in Corn Grain
  • Extraction and Quantification of Folic Acid in Corn Grain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1. Quantification of folic acid in corn kernels

[0061] 1. Preparation of standard solution

[0062] In addition to 5-F-THFGlu 1 and 13 C 5-F-THFGlu 1 Except, all standards were dissolved in methanol / water (1:1, v / v) containing 20 mM ammonium formate, 1% (w / v) L-ascorbic acid, and 0.5% (v / v) 2-mercaptoethanol middle. 5-F-THFGlu 1 and 13 C 5-F-THFGlu 1 Soluble in an aqueous solution containing 1% (w / v) L-ascorbic acid and 0.5% (v / v) 2-mercaptoethanol. All standard solutions have a concentration of 100 μg / mL and are stored at -80°C for future use. Use the extract to dilute.

[0063] 2. Method for extracting folic acid from corn kernels

[0064] Corn kernel freeze-dried powder : Fresh corn kernels are ground with MM 400 grinder (Retsch, Germany) under liquid nitrogen conditions and freeze-dried in ALPHA 1-4LDPlus freeze dryer (Christ, Germany) to obtain freeze-dried corn kernels, which are packed into 15ml or 50ml freeze-dried powder. Store in a tube at...

Embodiment 2

[0103] Embodiment 2. Optimization of folic acid extraction method

[0104] 1. Extraction solvent optimization

[0105] As the main solvent of the whole system, the extraction solution generally uses phosphate buffered saline (PBS) as the solvent. At the same time, mercaptoethanol and ascorbic acid were added as antioxidants. However, since phosphate is not volatile, a large amount of use will affect the ionization efficiency of the mass spectrometer and reduce the service life of the mass spectrometer. Therefore, we compared two extraction solvents, water and PBS, to assess the necessity of using phosphate buffered saline.

[0106] Taking corn kernels of GEMS 31 variety as a sample, the experiment was carried out according to the following method.

[0107] Extraction of folic acid from corn kernels: During extraction, 10-F-FA, THF, FA, 5, 10-CH=THF, 5-M-THFGlu 1-4 and 5-F-THFGlu 1-4 The standard substance is added in the freeze-dried powder of corn kernels with the extrac...

Embodiment 3

[0137] Example 3. Determination of single and polyglutamic acid folic acid content in the corn germination process

[0138] We compared the content changes of monoglutamic acid and polyglutamic acid folic acid in maize germ (GEMS 31) during germination. Embryo materials were collected on days 1-5 of germination, and folic acid in corn germs was extracted and detected by LC-MS / MS according to the method described in Example 1.

[0139] The result is as Figure 4 As shown, 5-M-THFGlu 1-4 , 5-F-THFGlu 1-4 , 5,10-CH=THF, 10-F-FA, FA and THF were detected in all samples. In the first 2 days, the content of folic acid was generally low, and the corn kernels were mainly in the stage of water absorption at this time. After the water absorption stage ended, the total content of 5-M-THF and 5-F-THF increased, and the corresponding distribution of mono- and polyglutamic acid folic acid also changed. 5-M-THFGlu 4 The proportion of the first 4 days remained at about 60%. From day 4 ...

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Abstract

The invention relates to plant folic acid extraction, in particular to a method for extracting and quantifying folic acid in corn grains. The method includes the following steps: take freeze-dried corn grain powder, add freshly prepared extract, vortex ultrasonically, shake at 100°C for 10 minutes, and cool rapidly on ice; add α-amylase, and perform enzymatic hydrolysis at 37°C with constant temperature shaking; Add protease, oscillate at 37°C for enzymatic hydrolysis; centrifuge, and take the supernatant; the formula of the extract is: every 100ml extract contains NaH 2 PO 4 249‑349 mg, Na 2 HPO 4 2H 2 O 659‑759mg, L‑ascorbic acid 1‑1.2g, 2‑mercaptoethanol 0.4‑1.0ml, pH 6.8‑7.0. The invention simultaneously accurately quantifies 12 kinds of folic acid with different mantissas and different oxidation states in corn grains, and provides technical support for dynamic analysis of folic acid metabolites.

Description

technical field [0001] The invention relates to plant folic acid extraction, in particular to a method for extracting and quantifying folic acid in corn grains. Background technique [0002] Folic acid is a class of water-soluble vitamins (VB9), including folic acid, tetrahydrofolate and their corresponding derivatives. As the core coenzyme factor of one-carbon metabolism, folic acid plays an important role in the one-carbon cycle and the synthesis of DNA, amino acids, proteins and other living substances. During the growth and development of animals and plants, folic acid with different mantissas and different oxidized forms has different biological activities and is crucial to the growth and development of animals and plants. Crops are the main source of folic acid for humans and animals, however, the folic acid content in crops is low, especially in cereals. Therefore, it has become a research hotspot to increase the folic acid level in plants through genetic engineerin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/04G01N30/06G01N30/34G01N30/72
CPCG01N30/02G01N30/04G01N30/06G01N30/34G01N30/72G01N2030/045G01N2030/062G01N2030/067
Inventor 韩莉妲万幸张春义
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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