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Extraction and quantitative methods for folic acid in corn grains

A technology for grain and corn, applied in the field of plant folic acid extraction, can solve the problems of narrow linear range, inability to distinguish folic acid, and low resolution, and achieve the effects of improving extraction efficiency, helping nutrition-oriented biological breeding, and ensuring stability

Active Publication Date: 2018-12-21
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 quantitative methods for folic acid in corn grains
  • Extraction and quantitative methods for folic acid in corn grains
  • Extraction and quantitative methods for folic acid in corn grains

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 extraction and quantitative methods for folic acid in corn grains. The extraction method comprises the following steps of takingcorn grain freeze-dried powder, adding a new extracting solution, carrying out ultrasonic vortex, oscillating at the temperature of 100 DEG C for 10 minutes, and rapidly cooling on ice; adding alpha-amylase and carrying out constant-temperature oscillating enzymolysis at 37 DEG C; adding protease and carrying out constant-temperature oscillating enzymolysis at 37 DEG C; and centrifuging and takingsupernate to obtain the folic acid, wherein a formula of the extracting solution is that every 100 ml of the extracting solution contains 249-349 mg of NaH2PO4, 659-759 mg of Na2HPO4.2H2O, 1-1.2 g ofL-ascorbic acid and 0.4-1.0 ml of 2-mercaptoethanol, and the pH is 6.8-7.0. According to the methods, 12 kinds of folic acid with different mantissa and oxidation states in the corn grains can be accurately quantified at the same time to provide technical support for dynamic analysis of the 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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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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