Method for enriching and extracting free glutamic acid from environmental water body and / or sediment

Abstract

The invention discloses a method for enriching and extracting free glutamic acid from an environmental water body and / or sediment. The method combines freeze drying, mechanical grinding, chemical extraction, cation exchange resin purification and low-temperature concentration. According to the method, the extraction efficiency, the yield and the purity of the free glutamic acid can be remarkably improved, sample background interference and glutamic acid degradation are avoided, and glutamic acid can be separated from other amino acids possibly existing in a sample, so that the purpose of enrichment extraction is achieved. Meanwhile, the method is easy and convenient to operate, efficient in extraction, capable of being used for on-site rapid treatment, wide in raw material source, low in cost and wide in application prospect in the field of environment analysis and ecological monitoring.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of environmental biochemistry, and in particular, to a method for enriching and extracting free glutamic acid from environmental water bodies and / or sediments. BACKGROUND

[0002] Glutamic acid is an amino acid with important biological functions, widely exists in nature, especially in the environment medium with high content of animals and plants and microorganisms. It is not only the basic unit of protein synthesis, but also plays a key role in energy metabolism, neurotransmitter conduction, nitrogen cycle and other life activities of organisms. In recent years, the research on glutamic acid as an environmental indicator substance has gradually attracted attention, especially in the fields of water body ecological environment and sediment biogeochemical cycle, its importance is increasingly prominent. In the process of water eutrophication and pollution, the change of glutamic acid content in sediment or sediment can reflect the microbial community activity, the biological transformation process of pollutants and the response degree of ecological system. Therefore, the concentration of free glutamic acid has been regarded as one of the important biomarkers for early identification of water eutrophication, ecological system health assessment and pollution source tracing.

[0003] The detection and monitoring of free glutamic acid has shown important value in many environmental and ecological restoration scenarios, such as water environment quality monitoring, through the change of glutamic acid level to monitor the nutritional status, pollution trend and ecological degradation degree of water body; ecological restoration effect evaluation, used to evaluate the improvement of nitrogen cycle and organic matter metabolism in water body or sediment system by plant remediation, microbial remediation and other technologies; pollution source identification and tracking, analyzing the distribution characteristics of glutamic acid in a specific area, which can help to locate organic pollution sources or evaluate exogenous material input; aquaculture and ecological management, glutamic acid as the product of the metabolic process of some aquatic organisms, its content can reflect the quality of bottom material and water stability, providing reference for scientific management of aquaculture environment.

[0004] The existing glutamic acid extraction methods are mostly derived from the analysis needs of food or biological samples, which generally rely on strong acid and strong base extraction, high temperature treatment or complex enzymatic steps. These conditions are not suitable for complex environmental samples, especially in complex environmental matrices such as water sediments, lake sediments and wetland soils. At present, the methods for extracting free glutamic acid from environmental samples (such as water and sediments) mainly include solvent extraction, solid phase extraction and ultrasonic assisted extraction. However, these methods generally have low extraction efficiency, insufficient purity, complicated operation steps and are sensitive to complex matrix interference, which limits their reliability and popularity in practical application. Especially in environmental samples with high diversity and complex impurity background, these traditional methods are difficult to meet the technical requirements of low detection limit, quantitative accuracy and batch analysis.

[0005] Therefore, developing a new method for enriching and extracting free glutamic acid suitable for complex environmental samples, with high extraction efficiency, high purity and good repeatability, has important significance for improving the accuracy of environmental monitoring data, expanding the application of amino acids in the field of ecological environment, and promoting the development of environmental science research and governance technology. SUMMARY

[0006] To overcome the problems of existing methods, such as glutamic acid degradation, increased sample background interference, low extraction efficiency, and inability to adapt to low concentration and complex environmental systems with multiple components, the present application provides a method for enriching and extracting free glutamic acid from environmental water and / or sediments.

[0007] The first object of the present application is to provide a method for enriching and extracting free glutamic acid from environmental water and / or sediments.

[0008] The present application claims the following: A method for enriching and extracting free glutamic acid from environmental water and / or sediments, comprising the following steps: S1. Freeze-drying the sample and mixing it with 0.1-1 M acid solution, shaking at 50-70℃ for 8-12 h, and then standing at 50-70℃ for 22-26 h; S2. Low-temperature centrifugal separation of the liquid, purification of the liquid using activated cation exchange resin, and then elution using ammonia solution, collecting the eluate to obtain free glutamic acid; Wherein, freeze-drying can remove water from the sample, enrich the target substance, and maintain the stability of free glutamic acid to prevent glutamic acid degradation; mixing with 0.1-1 M acid solution can improve the solubility of the target substance; shaking can promote the release of free glutamic acid in the sample.

[0009] Preferably, the environmental water includes aquaculture wastewater, seawater, sewage, and river and lake water.

[0010] Preferably, the sediments include aquaculture sediments, seawater sediments, sewage sediments, and river and lake sediments.

[0011] Preferably, in step S1, the sample is a sediment, and after freeze-drying the sample is mixed with 0.1-1 M acid solution, freeze-grinding is performed, and then shaking at 50-70℃ for 8-12 h and standing at 50-70℃ for 22-26 h. Wherein, freeze-grinding can ensure that the sediment and acid solution are in full contact, further improving the solubility of the target substance in the sediment sample.

[0012] Preferably, in step S1, the pressure of the freeze-drying is 20-25 Pa at -45 to -55℃.

[0013] More preferably, in step S1, the pressure of the freeze-drying is 23 Pa at -50℃.

[0014] Preferably, in step S1, the ratio of the sample to the acid solution is (1-2) g:10 mL.

[0015] More preferably, the ratio of the sample to the acid solution is 1 g:10 mL.

[0016] Preferably, in step S1, the sample is shaken at 60℃ for 10 h and then is left at 60℃ for 24 h.

[0017] Preferably, in step S1, the acid solution comprises a hydrochloric acid solution, a nitric acid solution or a sulfuric acid solution.

[0018] Preferably, in step S2, the low-temperature centrifugation is centrifugation at 2000-8000 rpm for 1-30 min at -30 to 25℃, which can simultaneously remove colloidal and lipid impurities.

[0019] More preferably, the low-temperature centrifugation is centrifugation at 6000 rpm for 5 min at 4℃.

[0020] Preferably, in step S2, the cation exchange resin comprises DOWEX-50WX8H + , DOWEX 50W-X8, DOWEX 88, DOWEX MONOSPHERE 99, DOWEX G-26, DOWEX 1X8, DOWEX SBR-2, DOWEX 650A or gel-type polystyrene sulfonic acid cation exchange resin.

[0021] More preferably, the cation exchange resin is DOWEX-50WX8H + cation exchange resin; after the supernatant is adsorbed by the activated cation exchange resin, impurities can be removed and quantitative adsorption of free glutamic acid can be completed.

[0022] Preferably, the mass ratio of the sample to the cation exchange resin is 1:(1-5).

[0023] More preferably, the mass ratio of the sample to the cation exchange resin is 1:1.

[0024] Preferably, in step S2, the activation method of the cation exchange resin is that the cation exchange resin is sequentially treated with an acid solution, washed with water, treated with a base solution, washed with water, treated with an acid solution and washed with water.

[0025] More preferably, the concentration of the acid solution and the base solution is 0.1-1 M.

[0026] Further preferably, the concentration of the acid solution and the base solution is 1 M.

[0027] Preferably, the concentration of the aqueous ammonia solution is 2-12 M.

[0028] More preferably, the concentration of the aqueous ammonia solution is 2 M.

[0029] Preferably, after the eluent is collected, the eluent is subjected to nitrogen blowing at 40-60℃; under this condition, the stability of free glutamic acid in the product can be maintained, and degradation reaction that may occur under high temperature conditions can be avoided.

[0030] Preferably, after the eluent is collected, the eluent is subjected to nitrogen blowing at 60℃.

[0031] Compared with the prior art, the present application has the following beneficial effects: The present application discloses a method for enriching and extracting free glutamic acid from environmental water bodies and / or sediments. The method of the present application can significantly improve the extraction efficiency, yield and purity of free glutamic acid, avoid sample background interference and glutamic acid degradation, and separate glutamic acid from other amino acids that may exist in the sample, thereby achieving the purpose of enrichment and extraction. At the same time, the method of the present application is simple to operate, efficient in extraction, can be used for on-site rapid processing, and has a wide application prospect in the field of environmental analysis and ecological monitoring due to its wide raw material sources and low cost. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Figure 1 is a flowchart of the process for enriching and extracting free glutamic acid.

[0033] Figure 2 Figure 2 is a GC-MS chromatogram of the amino acid product of Example 1.

[0034] Figure 3 Figure 3 is a GC-MS mass spectrum of the amino acid product of Example 1.

[0035] Figure 4 Figure 4 is a GC-MS chromatogram of the amino acid product of Example 2.

[0036] Figure 5 Figure 5 is a GC-MS chromatogram of the amino acid product of Comparative Example 2.

[0037] Figure 6 Figure 6 is a GC-MS chromatogram of the amino acid product of Comparative Example 3. DETAILED DESCRIPTION

[0038] The present application is further described below in connection with specific embodiments, which do not limit the present application in any manner. Unless otherwise specified, the reagents, methods and apparatus used in the following examples are conventional in the art.

[0039] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0040] Example 1: A method for enriching and extracting free glutamic acid from aquaculture water The present example provides a method for enriching and extracting free glutamic acid from aquaculture water Figure 1 , which specifically comprises the following steps: S1. Take an aquaculture water sample and place it in a beaker, and then place it in a freeze dryer (23 Pa, -50°C) to remove water and concentrate the target substance; S2. Take 1 g of the freeze-dried sample and place it in a 50 mL centrifuge tube, add 10 mL of 0.1 mol / L HCl solution to extract free glutamic acid, then place it in a 60°C constant temperature shaker (speed 140 rpm) for 10 h, and then transfer the mixed sample to a 60°C constant temperature incubator for 24 h; S3. Transfer the sample to a low-temperature centrifuge, centrifuge at 6000 rpm for 5 min at 4°C, and separate the supernatant. Take 1 g of DOWEX-50WX8H + cation exchange resin and sequentially activate it (soak for 2 min each time) in 1 mol / L HCl solution, ultrapure water, 1 mol / L NaOH solution, ultrapure water, 1 mol / L HCl solution, and ultrapure water to obtain activated cation exchange resin. The supernatant is passed through the activated cation exchange resin for free glutamic acid adsorption and concentration treatment, and then 10 mL of 2 mol / L ammonia solution is used to elute the glutamic acid on the cation exchange resin. The eluted mixed solution is placed in a 60°C constant temperature environment and blown dry with a gentle stream of nitrogen gas to obtain an amino acid powder.

[0041] Example 2: A method for enriching and extracting free glutamic acid from aquaculture sediments The present example provides a method for enriching and extracting free glutamic acid from aquaculture sediments Figure 1 , which specifically comprises the following steps: S1. Take an aquaculture sediment sample and place it in a beaker, and then place it in a freeze dryer (23 Pa, -50°C) to remove water and concentrate the target substance; S2. Take 1 g of freeze-dried sample and place it in a 50 mL centrifuge tube. Add 10 mL of 0.1 mol / L HCl solution to extract free glutamic acid, then place it in a frozen grinder. Set the frequency of the frozen grinder to 75 Hz, the temperature to -20°C, the running time to 30 s, the interval time to 15 s, and the cycle number to 3 times. After grinding, place the sample solution in a 60°C constant-temperature shaker (speed 140 rpm) and shake for 10 h. After mixing, transfer the sample to a 60°C constant-temperature incubator and stand for 24 h; S3. Transfer the sample to a low-temperature centrifuge and centrifuge at 6000 rpm for 5 min at 4°C to separate the supernatant. Take 1 g of DOWEX-50WX8H + cation exchange resin and sequentially activate it (soak for 2 min each time) in the following order: 1 mol / L HCl solution, ultrapure water, 1 mol / L NaOH solution, ultrapure water, 1 mol / L HCl solution, and ultrapure water to obtain the activated cation exchange resin. Pass the supernatant through the activated cation exchange resin for free glutamic acid adsorption and concentration treatment, then use 10 mL of 2 mol / L ammonia water solution to elute the glutamic acid on the cation exchange resin. Place the eluted mixed solution in a 60°C constant-temperature environment and blow dry it using a gentle nitrogen gas stream to obtain an amino acid powder.

[0042] Example 3. A method for enriching and extracting free glutamic acid from aquaculture water This example provides a method for enriching and extracting free glutamic acid from aquaculture water Figure 1 , which is performed according to Example 1, except that in step S2, 0.1 mol / L HCl solution is replaced with 0.1 mol / L nitric acid solution.

[0043] Example 4. A method for enriching and extracting free glutamic acid from aquaculture water This example provides a method for enriching and extracting free glutamic acid from aquaculture water, which is performed according to Example 1, except that in step S2, 1 g of freeze-dried sample is placed in a 50 mL centrifuge tube.

[0044] Example 5. A method for enriching and extracting free glutamic acid from aquaculture water This example provides a method for enriching and extracting free glutamic acid from aquaculture water Figure 1 , which is performed according to Example 1, except that in step S2, 0.1 mol / L HCl solution is replaced with 1 mol / L HCl solution.

[0045] Example 6. A method for enriching and extracting free glutamic acid from aquaculture water The present embodiment provides a method for enriching and extracting free glutamic acid from aquaculture water Figure 1 The enriching and extracting was performed according to Embodiment 1, except that in step S3, 1 g of DOWEX-50WX8H + The cation exchange resin was activated (soaked for 2 min each time) in the following order: 0.1 mol / L hydrochloric acid solution, ultrapure water, 0.1 mol / L NaOH solution, ultrapure water, 0.1 mol / L hydrochloric acid solution, and ultrapure water.

[0046] Embodiment 7: A method for enriching and extracting free glutamic acid from aquaculture water The present embodiment provides a method for enriching and extracting free glutamic acid from aquaculture water Figure 1 The enriching and extracting was performed according to Embodiment 1, except that in step S3, a gel-type polystyrene sulfonic acid cation exchange resin was used instead of DOWEX-50WX8H + cation exchange resin.

[0047] Embodiment 8: A method for enriching and extracting free glutamic acid from aquaculture water The present embodiment provides a method for enriching and extracting free glutamic acid from aquaculture water Figure 1 The enriching and extracting was performed according to Embodiment 1, except that in step S3, the mass of DOWEX-50WX8H + cation exchange resin was 5 g.

[0048] Embodiment 9: A method for enriching and extracting free glutamic acid from aquaculture water The present embodiment provides a method for enriching and extracting free glutamic acid from aquaculture water Figure 1 The enriching and extracting was performed according to Embodiment 1, except that in step S3, the concentration of the ammonia solution was 12 mol / L.

[0049] Embodiment 10: A method for enriching and extracting free glutamic acid from aquaculture water The present embodiment provides a method for enriching and extracting free glutamic acid from aquaculture water Figure 1 The enriching and extracting was performed according to Embodiment 1, except that in step S3, the mixed solution after elution was subjected to nitrogen blowing under constant temperature conditions at 40℃.

[0050] Comparative Example 1: A method for enriching and extracting free glutamic acid from aquaculture water The present comparative example provides a method for enriching and extracting free glutamic acid from aquaculture water Figure 1), enrichment extraction was carried out according to Example 1, except that in step S2, 0.01 mol / L HCl solution was used instead of 0.1 mol / L HCl solution.

[0051] Comparative Example 2 A method for enrichment extraction of free glutamic acid from cultured sediments This comparative example refers to the prior art (Extracting and desalting amino acids from soils and sediments: evaluation of methods, DOI: 10.1016 / 0038-0717(75)90074-7) and provides a method for enrichment extraction of free glutamic acid from cultured sediments, specifically comprising the following steps: S1. A sample of cultured sediments was taken into a beaker and placed in a freeze dryer (23 Pa, -50°C) to remove water and concentrate the target substance; S2. 3.21 g of the freeze-dried sample was weighed into a 15 mL digestion tube, 10 mL of 6 mol / L HCl solution was added, and hydrolysis was carried out at 110°C for 24 h; S3. After the hydrolysis treatment was completed, the sample solution was centrifuged at 6000 rpm for 5 min at room temperature, the supernatant was separated, and the supernatant was placed in a constant temperature oven at 60°C and blown dry with a gentle stream of nitrogen. The dried sample was redissolved in 10 mL of 0.1 mol / L HCl solution; S4. 1 g of DOWEX-50WX8H + The cation exchange resin was activated in the following order (soaked for 2 min each): 1 mol / L HCl solution, ultrapure water, 1 mol / L NaOH solution, ultrapure water, 1 mol / L HCl solution, ultrapure water, to obtain the activated cation exchange resin. The sample solution redissolved in step S3 was subjected to amino acid adsorption and concentration treatment by the cation exchange resin, and then 10 mL of 2 mol / L ammonia solution was used to elute the glutamic acid on the cation exchange resin. The eluted mixed solution was placed in a constant temperature oven at 60°C and blown dry with a gentle stream of nitrogen. Finally, the amino acid powder was obtained.

[0052] Comparative Example 3 A method for enrichment extraction of free glutamic acid from cultured water bodies This comparative example provides a method for enrichment extraction of free glutamic acid from cultured water bodies, which is carried out according to Comparative Example 2, except that the sample of cultured sediments is replaced by a sample of cultured water bodies.

[0053] Experimental Example 1 Gas chromatography-mass spectrometry (GC-MS) identification of amino acid products and quantitative analysis of free glutamic acid I. Experimental method 1. Respectively take 0.1 g of the amino acid powder prepared in Examples 1-10 and Comparative Examples 1-3, add 1 mL of acidified methanol solution (V 乙酰氯 :V 甲醇 =4:25, slowly prepare under ice bath), heat at 70°C for 1 h; 2. Terminate the reaction under ice bath condition, blow dry under 40°C constant temperature condition using gentle nitrogen flow. Add 250 μL dichloromethane, blow dry at room temperature, repeat this step once; 3. Add 1 mL mixed solution (V 乙酸酐 :V 吡啶 :V 丙酮 =1:2:5), and acylate under 60°C constant temperature condition for 10 min, then blow dry at room temperature; 4. Add 2 mL ethyl acetate and 1 mL saturated NaCl solution, vortex, extract the organic phase, remove the water phase; 5. Blow dry the organic phase at room temperature using gentle nitrogen flow. Add 1 mL dichloromethane, blow dry at room temperature, repeat this step once; 6. After blowing dry, add 500 μL of ethyl acetate and make up to volume, transfer to the sample bottle, and perform GC-MS detection on the machine.

[0054] II. Experimental results The free glutamic acid content prepared in Examples 1-10 and Comparative Example 1 is shown in Table 1. As can be seen from the results in Table 1, too low acid concentration in step S2 will result in low extraction efficiency and yield, and too high acid concentration can also result in hydrolysis of other amino acids in the sample, so the acid concentration needs to be controlled at 0.1-1 mol / L.

[0055] Table 1. Free glutamic acid content obtained by different enrichment extraction methods

[0056] The GC-MS chromatogram and mass spectrum of Example 1 are shown in Figure 2 and Figure 3 , the GC-MS chromatogram of Example 2 is shown in Figure 4 , the GC-MS chromatogram of Comparative Example 2 is shown in Figure 5 , and the GC-MS chromatogram of Comparative Example 3 is shown in Figure 6 . The results show that when using the methods of Comparative Examples 2 and 3 to extract amino acids in environmental water and sediments, only mixed amino acids can be extracted, and the glutamic acid signal cannot be highlighted, and the glutamic acid signal is easily covered; the methods of Examples 1 and 2 can highlight the signal peak of glutamic acid, which is convenient for further separation of glutamic acid.

[0057] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A method for the enrichment of the extraction of free glutamic acid from environmental waters and / or sediments, characterized in that, The method comprises the following steps: S1. After freeze-drying the sample, mix the sample with 0.1-1 M acid solution, shake at 50-70 DEG C for 8-12 h, and then stand at 50-70 DEG C for 22-26 h; S2. Low-temperature centrifugal separation of the liquid, purification of the liquid by using activated cation exchange resin, elution by using ammonia solution, and collection of the eluate to obtain free glutamic acid.

2. The method of claim 1, wherein, In step S1, the sample is a sediment, and after freeze-drying the sample, the sample is mixed with 0.1-1 M acid solution, freeze-grinded, then shaken at 50-70 DEG C for 8-12 h, and stood at 50-70 DEG C for 22-26 h.

3. The method of claim 1, wherein, In step S1, the sample and the acid solution are used in a ratio of (1-2) g:10 mL.

4. The method of claim 1, wherein, In step S1, the acid solution comprises a hydrochloric acid solution, a nitric acid solution or a sulfuric acid solution.

5. The method of claim 1, wherein, In step S2, the low-temperature centrifugal separation is at 2000-8000 rpm for 1-30 min at-30-25 DEG C.

6. The method of claim 1, wherein, In step S2, the cation exchange resin comprises DOWEX-50WX8H + DOWEX 50W-X8, DOWEX 88, DOWEX MONOSPHERE 99, DOWEX G-26, DOWEX 1X8, DOWEX SBR-2, DOWEX 650A or a gel type polystyrene sulfonic acid-based cation exchange resin.

7. The method of claim 1, wherein, The mass ratio of the sample to the cation exchange resin is 1:(1-5).

8. The method of claim 1, wherein, In step S2, the activation method of the cation exchange resin is: sequentially treating the cation exchange resin with an acid solution, washing with water, treating with an alkali solution, washing with water, treating with an acid solution and washing with water.

9. The method of claim 1, wherein, In step S2, the concentration of the ammonia solution is 2-12 M.

10. The method of claim 1, wherein, In step S2, after collecting the eluate, the eluate is subjected to nitrogen blowing at 40-60 DEG C.

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