CN110879209A - Rapid detection method of gamma-aminobutyric acid - Google Patents
Rapid detection method of gamma-aminobutyric acid Download PDFInfo
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- CN110879209A CN110879209A CN201911247142.7A CN201911247142A CN110879209A CN 110879209 A CN110879209 A CN 110879209A CN 201911247142 A CN201911247142 A CN 201911247142A CN 110879209 A CN110879209 A CN 110879209A
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Abstract
The invention discloses a method for rapidly detecting gamma-aminobutyric acid (GABA), belonging to the technical field of biology. The Bertholt colorimetric method is optimized through screening comparison, a single-factor experiment and a response surface optimization experiment. The optimized Bertholt colorimetric method shortens the reaction time by 46 percent, and the distilled water is used for replacing 60 percent of ethanol solution to dilute the color developing solution, so that the detection efficiency is improved, and the detection cost is reduced. The optimized Bertholt colorimetric method for detecting the GABA content is faster, simpler and more accurate.
Description
Technical Field
The invention relates to a method for rapidly detecting gamma-aminobutyric acid (GABA).
Background
GABA is a natural non-protein amino acid and widely distributed in animals, plants and microorganisms. In mammals, GABA, an important inhibitory neurotransmitter, is involved in various metabolic activities and has various effects, so that many studies have been made in relation thereto. In the process of studying GABA, a suitable GABA detection method is selected firstly.
The GABA detection method comprises High Performance Liquid Chromatography (HPLC), Bertholt colorimetry, paper electrophoresis, paper chromatography and amino acid analyzer.
The GABA detection method specified in the national standard is a high performance liquid chromatography, the detection method mainly utilizes the o-phthalaldehyde and GABA to generate pre-column derivatization, and then utilizes ultraviolet reversed phase high performance liquid chromatography to determine the GABA concentration. The Bertholt colorimetric method is used for detecting the GABA content by utilizing the color reaction between phenol, sodium hypochlorite and free ammonia of GABA, has high detection sensitivity, can quickly detect a large number of GABA samples, and is simple and easy to implement; the accuracy of the result of measuring the GABA content by a paper electrophoresis method and a paper chromatography method is too low; the amino acid analyzer method has the same advantages and disadvantages as the HPLC method. Therefore, the Bertholt colorimetric method is the most convenient and rapid method for detecting GABA content and is easy to operate, the content of gamma-aminobutyric acid in germinated brown rice [ J ] is measured by using the Juan of the mineral forest, Cao, Song Jade, and the like through HPLC (high performance liquid chromatography) and the Bertholt colorimetric method in the food and feed industry, 2015(01): 61-65.) compares the advantages and the disadvantages of the HPLC method and the colorimetric method, and she finds that the separation degree and the repeatability of GABA are good, the precision is high, the result is accurate, but the colorimetric method is simple and rapid to operate and is suitable for measuring large batches of samples. The influence factor [ J ] of gamma-aminobutyric acid measured by a colorimetric method, Guangzhou chemical industry 2014,42(02): 78-80.) on the dosage of phenol and sodium hypochlorite is improved to a certain extent, but the problems of heating temperature, boiling water bath time, ice bath time and the like are lacked, so the improvement is incomplete, and the purpose of enabling the Bertholt colorimetric method to be safer, simpler and faster is not achieved. Zhao nations, etc. (Zhao nations, Wang Wei, colorimetric method for rapid determination of gamma-aminobutyric acid content [ J ]. food research and development 2015,36(10): 85-89.) try to establish a new method capable of rapidly determining GABA concentration, and the method utilizes the principle that glyoxylic acid, succinic acid, bromocresol green and free ammonia of GABA generate color reaction to prepare a color developing agent formed by mixing 0.5mg/mL glyoxylic acid, 4.0mg/mL succinic acid and 6.0 mg/mL bromocresol green, and can detect the GABA concentration at 617 nm. But the color developing agent used by the method has high toxicity and low detection precision.
The detection method comprises the following steps: the accuracy of detecting GABA content by paper electrophoresis and paper chromatography is too low; the detection efficiency of an amino acid analyzer method and a high performance liquid chromatography is low, and the detection period is long, so that the colorimetric method in daily experiments is the method most suitable for detecting the concentration of a large batch of GABA samples.
Disclosure of Invention
The invention aims to overcome the defect that the Bertholt colorimetric method is long in time consumption for detecting the GABA concentration, and provides a method which can be used for quickly detecting the GABA content and reducing the detection cost, and the method has higher goodness of fit with the detection result of an HPLC method.
When the method is used for detecting the GABA content, the relative standard deviation is within 3 percent, and compared with the detection result of the non-optimized Bertholt colorimetric method, the relative standard deviation is reduced by 1-2 percent, and the method is safe and reliable.
The invention relates to a method for rapidly detecting GABA, which comprises the following steps:
① drawing standard curve
Accurately preparing standard solutions of 0.02, 0.04, 0.06, 0.08, 0.10 and 0.12 mg/mL by using GABA standard products, putting 0.5 mL in an ice bath, adding 0.2 mL of buffer solution, 1 mL of phenol solution and 0.4 mL of sodium hypochlorite solution, fully oscillating, carrying out water bath reaction for 7 min, cooling for 9 min in the ice bath, adding 2 mL of distilled water, and measuring the absorbance value at 630 nm. And drawing a standard curve by taking the concentration as an abscissa and the absorbance as an ordinate.
The purity of the GABA standard substance is 99%;
the buffer solution is boric acid-borax buffer solution with pH of 9.0;
the phenol content of the phenol solution is 6%;
the sodium hypochlorite solution has a sodium hypochlorite content of more than 12.6%;
the ice bath temperature was 0 ℃;
the water bath temperature was 95 ℃.
② determining GABA content of liquid to be detected
Diluting the GABA sample solution to prepare a GABA solution to be detected, putting 0.5 mL into an ice bath, adding 0.2 mL of buffer solution, 1 mL of phenol solution and 0.4 mL of sodium hypochlorite solution, fully oscillating, carrying out water bath reaction for 7 min, cooling in the ice bath for 9 min, adding 2 mL of distilled water, measuring the absorbance value at 630 nm, and calculating the GABA content in the sample.
The buffer solution is boric acid-borax buffer solution with pH of 9.0;
the phenol content of the phenol solution is 6%;
the sodium hypochlorite solution has a sodium hypochlorite content of more than 12.6%;
the ice bath temperature was 0 ℃;
the water bath temperature was 95 ℃.
The method for measuring the GABA content simplifies the GABA measuring process, reduces the reaction temperature from boiling water to 95 ℃ compared with the prior method, replaces 60% ethanol solution with distilled water, reduces the total time of color reaction by 14 min, has smaller relative standard deviation of the final measuring result compared with the prior method, and has more consistent detecting result with the result of an HPLC method.
Compared with the prior art, the method for determining the GABA content has the following remarkable characteristics:
(1) the invention adopts a single-factor and response surface experiment to carry out an optimization experiment;
(2) the invention detects the GABA content, and compared with Zhao nationality, Li Xijuan, Shilin Juan (1. Zhao nationality, Wang Wei. colorimetric method for rapid determination of gamma-aminobutyric acid content [ J ]. food research and development, 2015,36(10): 85-89.2. Li Xijuan, Shanchunfeng, House Zedong. colorimetric method for determination of gamma-aminobutyric acid influence factor [ J ]. Guangzhou chemical industry 2014,42(02): 78-80.3. Shilin Juan, Cao Lei, Song Yu, etc. HPLC method and Bertholt colorimetric method for determination of gamma-aminobutyric acid content [ J ] in germinated brown rice, grain and feed industry, (2015 01): 61-65.), on the premise of ensuring detection precision, distilled water is used for replacing 60% ethanol solution to dilute chromogenic solution, thereby reducing detection cost.
(3) Compared with the original method, the color development reaction time of the invention is shortened by 14 min, 46% of the reaction time is saved, and the detection efficiency is improved.
(4) The relative deviation of the detection result of the invention is lower than 3%, and compared with the detection result of the non-optimized Bertholt colorimetric method, the relative standard deviation is reduced by 1-2%, and the result is more consistent with the result of an HPLC method.
Example 1:
① drawing standard curve
Accurately preparing standard solutions of 0.02, 0.04, 0.06, 0.08, 0.10 and 0.12 mg/mL by using GABA standard products, putting 0.5 mL in an ice bath, adding 0.2 mL of buffer solution, 1 mL of phenol solution and 0.4 mL of sodium hypochlorite solution, fully oscillating, carrying out water bath reaction for 7 min, cooling for 9 min in the ice bath, adding 2 mL of distilled water, and measuring the absorbance value at 630 nm. And drawing a standard curve by taking the concentration as an abscissa and the absorbance as an ordinate.
The purity of the GABA standard substance is 99%;
the buffer solution is boric acid-borax buffer solution with pH of 9.0;
the phenol content of the phenol solution is 6%;
the sodium hypochlorite solution has a sodium hypochlorite content of more than 12.6%;
the ice bath temperature was 0 ℃;
the water bath temperature was 95 ℃.
② determining GABA content of liquid to be detected
Diluting the GABA sample solution to prepare a GABA solution to be detected, putting 0.5 mL into an ice bath, adding 0.2 mL of buffer solution, 1 mL of phenol solution and 0.4 mL of sodium hypochlorite solution, fully oscillating, carrying out water bath reaction for 7 min, cooling in the ice bath for 9 min, adding 2 mL of distilled water, measuring the absorbance value at 630 nm, and calculating the GABA content in the sample.
The buffer solution is boric acid-borax buffer solution with pH of 9.0;
the phenol content of the phenol solution is 6%;
the sodium hypochlorite solution has a sodium hypochlorite content of more than 12.6%;
the ice bath temperature was 0 ℃;
the water bath temperature was 95 ℃.
By adopting the method to measure the GABA content, the total time of the color reaction is shortened by 14 min, and the relative standard deviation of the detection result is 2.48%.
Example 2:
① drawing standard curve
Accurately preparing standard solutions of 0.02, 0.04, 0.06, 0.08, 0.10 and 0.12 mg/mL by using GABA standard products, putting 0.5 mL in an ice bath, adding 0.2 mL of buffer solution, 1 mL of phenol solution and 0.4 mL of sodium hypochlorite solution, fully oscillating, carrying out water bath reaction for 7 min, cooling for 9 min in the ice bath, adding 2 mL of distilled water, and measuring the absorbance value at 630 nm. And drawing a standard curve by taking the concentration as an abscissa and the absorbance as an ordinate.
The purity of the GABA standard substance is 99%;
the buffer solution is boric acid-borax buffer solution with pH of 9.0;
the phenol content of the phenol solution is 6%;
the sodium hypochlorite solution has a sodium hypochlorite content of more than 12.6%;
the ice bath temperature was 0 ℃;
the water bath temperature was 95 ℃.
② determining GABA content of liquid to be detected
Diluting the GABA sample solution to prepare a GABA solution to be detected, putting 0.5 mL into an ice bath, adding 0.2 mL of buffer solution, 1 mL of phenol solution and 0.4 mL of sodium hypochlorite solution, fully oscillating, carrying out water bath reaction for 7 min, cooling in the ice bath for 9 min, adding 2 mL of distilled water, measuring the absorbance value at 630 nm, and calculating the GABA content in the sample.
The buffer solution is boric acid-borax buffer solution with pH of 9.0;
the phenol content of the phenol solution is 6%;
the sodium hypochlorite solution has a sodium hypochlorite content of more than 12.6%;
the ice bath temperature was 0 ℃;
the water bath temperature was 95 ℃.
By adopting the method to measure the GABA content, the total time of the color reaction is shortened by 14 min, and the relative standard deviation of the detection result is 2.88%.
Example 3:
① drawing standard curve
Accurately preparing standard solutions of 0.02, 0.04, 0.06, 0.08, 0.10 and 0.12 mg/mL by using GABA standard products, putting 0.5 mL in an ice bath, adding 0.2 mL of buffer solution, 1 mL of phenol solution and 0.4 mL of sodium hypochlorite solution, fully oscillating, carrying out water bath reaction for 7 min, cooling for 9 min in the ice bath, adding 2 mL of distilled water, and measuring the absorbance value at 630 nm. And drawing a standard curve by taking the concentration as an abscissa and the absorbance as an ordinate.
The purity of the GABA standard substance is 99%;
the buffer solution is boric acid-borax buffer solution with pH of 9.0;
the phenol content of the phenol solution is 6%;
the sodium hypochlorite solution has a sodium hypochlorite content of more than 12.6%;
the ice bath temperature was 0 ℃;
the water bath temperature was 95 ℃.
② determining GABA content of liquid to be detected
Diluting the GABA sample solution to prepare a GABA solution to be detected, putting 0.5 mL into an ice bath, adding 0.2 mL of buffer solution, 1 mL of phenol solution and 0.4 mL of sodium hypochlorite solution, fully oscillating, carrying out water bath reaction for 7 min, cooling in the ice bath for 9 min, adding 2 mL of distilled water, measuring the absorbance value at 630 nm, and calculating the GABA content in the sample.
The buffer solution is boric acid-borax buffer solution with pH of 9.0;
the phenol content of the phenol solution is 6%;
the sodium hypochlorite solution has a sodium hypochlorite content of more than 12.6%;
the ice bath temperature was 0 ℃;
the water bath temperature was 95 ℃.
By adopting the method to measure the GABA content, the total time of the color reaction is shortened by 14 min, and the relative standard deviation of the detection result is 2.57%. .
Claims (8)
1. A method for rapidly detecting gamma-aminobutyric acid comprises the following steps:
① drawing standard curve
Accurately preparing standard solutions of 0.02, 0.04, 0.06, 0.08, 0.10 and 0.12 mg/mL by using a gamma-aminobutyric acid (GABA) standard substance, putting 0.5 mL into an ice bath, adding 0.2 mL of buffer solution, 1 mL of phenol solution and 0.4 mL of sodium hypochlorite solution, fully oscillating, carrying out water bath reaction for 7 min, cooling for 9 min in the ice bath, adding 2 mL of distilled water, measuring an absorbance value at 630 nm, and drawing a standard curve by taking the concentration as an abscissa and the absorbance as an ordinate;
② determining GABA content of liquid to be detected
Diluting the GABA sample solution to prepare a GABA solution to be detected, putting 0.5 mL into an ice bath, adding 0.2 mL of buffer solution, 1 mL of phenol solution and 0.4 mL of sodium hypochlorite solution, fully oscillating, carrying out water bath reaction for 7 min, cooling in the ice bath for 9 min, adding 2 mL of distilled water, measuring the absorbance value at 630 nm, and calculating the GABA content in the sample.
2. The method for rapid detection of gamma-aminobutyric acid according to claim 1, wherein the purity of said GABA standard is 99%.
3. The method for rapidly detecting gamma-aminobutyric acid according to claim 1, wherein the buffer solution is a boric acid-borax buffer solution with pH of 9.0.
4. The method for rapidly detecting gamma-aminobutyric acid as claimed in claim 1, wherein the sodium hypochlorite solution contains sodium hypochlorite greater than 12.6%.
5. The method for rapidly detecting gamma-aminobutyric acid according to claim 1, wherein the phenol content of the phenol solution is 6%.
6. The method for rapidly detecting gamma-aminobutyric acid according to claim 1, wherein the water bath heating time is 7 min, and the temperature is 95 ℃.
7. The method for rapidly detecting gamma-aminobutyric acid as claimed in claim 1, wherein the ice bath cooling time is 9 min, and the temperature is 0 ℃.
8. The method for rapidly detecting gamma-aminobutyric acid according to claim 1, wherein the relative standard deviation of the detection result of the Bertholt colorimetric method is reduced by 1-2% compared with the detection result of the non-optimized Bertholt colorimetric method.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102388064A (en) * | 2009-03-27 | 2012-03-21 | 高裘企业公司 | Compositions and methods for screening and using compounds antagonizing spore-surface interactions |
| CN104663674A (en) * | 2007-10-23 | 2015-06-03 | 杜邦公司 | Fungicidal Mixtures |
| CN105925557A (en) * | 2016-06-13 | 2016-09-07 | 无锡布莱尼斯生物科技有限公司 | Method for gathering glutamate decarboxylase from Momordica grosvenori wastes to produce gamma-aminobutyric acid (GABA) |
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- 2019-12-09 CN CN201911247142.7A patent/CN110879209A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104663674A (en) * | 2007-10-23 | 2015-06-03 | 杜邦公司 | Fungicidal Mixtures |
| CN102388064A (en) * | 2009-03-27 | 2012-03-21 | 高裘企业公司 | Compositions and methods for screening and using compounds antagonizing spore-surface interactions |
| CN105925557A (en) * | 2016-06-13 | 2016-09-07 | 无锡布莱尼斯生物科技有限公司 | Method for gathering glutamate decarboxylase from Momordica grosvenori wastes to produce gamma-aminobutyric acid (GABA) |
Non-Patent Citations (2)
| Title |
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| 史小峰等: "传统发酵豆制品中γ-氨基丁酸的比色测定", 《中国调味品》 * |
| 黄柳舒等: "改良比色法测桑叶中γ-氨基丁酸含量及其热稳定性研究", 《食品科技》 * |
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