Method for determining organic acid in reed root exudates based on ion chromatography and application

Abstract

The invention discloses a method for determining organic acid in reed root exudates based on ion chromatography and application, and belongs to the technical field of chemical analysis. According to the determination method, the ion chromatography is adopted, the type and the content of the organic acid in the reed root exudates cultured by various culture methods can be comprehensively analyzed, metabolite peaks are concise and visual, and the result is more accurate and credible. And the microbial inhibitor adopted when the reed root exudates are collected has no influence on the types and contents of components in the exudates, so that the credibility of the detection result is higher.

Description

TECHNICAL FIELD

[0001] The application relates to a method for determining organic acids in reed root exudates based on ion chromatography and application, and belongs to the technical field of chemical analysis. BACKGROUND

[0002] Root exudates not only affect the growth and development of plants, but also affect the rhizosphere micro-ecological environment, and exhibit various forms of allelopathy during the growth of plants. Therefore, research on the separation, purification and detection method of plant root exudates is of great significance to the rhizosphere micro-ecosystem and the growth and development of plants.

[0003] As a large number of microorganisms exist on the surface of plant roots, even if the plant roots are washed clean with sterile water, there is still a phenomenon that microorganisms degrade the root exudates in the hydroponic solution when the root exudates are collected in the hydroponic solution, so that the detected content of the root exudates is reduced, and errors are caused in accurately evaluating the quantity and type of the composition of the plant root exudates. Therefore, it is particularly crucial to reduce the degradation of the root exudates by microorganisms during the collection process when the plant root exudates are collected in the hydroponic solution.

[0004] Common low-molecular-weight organic acids in the root exudates of Gramineae plants include nine kinds of quinic acid, lactic acid, acetic acid, propionic acid, formic acid, malic acid, tartaric acid, oxalic acid and citric acid. The low-molecular-weight organic acids are usually quantitatively analyzed by high-performance liquid chromatography and ion chromatography. However, the current elution conditions cannot separate oxalic acid and an unknown impurity peak, which interferes with the analysis of the composition of the root exudates. SUMMARY

[0005] The purpose of the application is to provide a method for determining organic acids in reed root exudates based on ion chromatography and application, which can simultaneously determine nine kinds of low-molecular-weight organic acids in the reed root exudates.

[0006] The method for determining organic acids in reed root exudates based on ion chromatography comprises the following steps: (1) collecting the reed root exudates to be measured and concentrating and extracting the reed root exudates; (2) determining the extract by ion chromatography, wherein the eluent is NaOH or KOH, and the gradient elution conditions are as follows: 0-11 min, the KOH concentration is 1 mM; 11-24 min, the KOH concentration gradually increases from 1 mM to 15 mM; 24-37 min, the KOH concentration increases from 15 mM to 30 mM; 37-50 min, the KOH concentration again increases from 30 mM to 40 mM; and 51 min, the KOH concentration rapidly increases to 1 mM and is kept for 5 min; The organic acids tested included quinic acid, lactic acid, acetic acid, propionic acid, formic acid, malic acid, tartaric acid, oxalic acid, and citric acid.

[0007] Furthermore, the step of collecting the reed root secretions to be tested in step (1) is to obtain them by soaking the washed reed roots in sterile water containing NaN3.

[0008] Furthermore, the concentration of NaN3 is 0.1~0.25 g / L.

[0009] Furthermore, the concentration step described in step (1) includes filtering the collected reed root secretions through a membrane and then freeze-drying and reconstituted them.

[0010] Furthermore, the reed root secretions collected in step (1) are from the roots of hydroponically or soil-grown reeds.

[0011] Furthermore, the ion chromatography determination conditions described in step (2) also include an IonPacAS-11 analytical column, a column temperature of 25±2 ℃, and a flow rate of 1 mL·min. -1 .

[0012] The method described in this invention is applied to the analysis of the types and contents of organic acids in reed root exudates.

[0013] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: The determination method of this invention uses ion chromatography, which can comprehensively analyze the types and contents of organic acids in reed root exudates from various cultivation methods. The metabolite peaks are concise and intuitive, and the results are more accurate and reliable. Furthermore, the microbial inhibitors used when collecting reed root exudates do not affect the types and contents of components in the exudates, making the detection results even more reliable. Attached Figure Description

[0014] Figure 1 The effects of different microbial inhibitors on DOC in reed root exudates; Figure 2 This is an HPLC chromatogram of organic acid standards (final concentration of standard is 50 mg / L). Figure 3 This is a comparison of different gradient elution procedures (final concentration of standard is 0.5 mg / L); Figure 4 This is an ion chromatogram of 9 organic acid standards (final concentration of standards is 5 mg / L); the order is 1: quinic acid; 2: lactic acid; 3: acetic acid; 4: propionic acid; 5: formic acid; 6: malic acid; 7: tartaric acid; 8: oxalic acid; 9: citric acid; Figure 5 It refers to the content of organic acids in the root secretions of hydroponically grown reeds; Figure 6It refers to the content of organic acids in the root exudates of reeds under single and combined stresses of Cu and OTC. Detailed Implementation

[0015] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0016] Example 1

[0017] A method for determining organic acids in the root exudates of hydroponically grown reeds using ion chromatography, including methods for collecting, detecting, and analyzing the root exudates, with the specific operational steps as follows: (1) Cultivation of reed seedlings: Reed seedlings were cultivated using Hoagland nutrient solution. Six seedlings were transplanted into each pot, with a nutrient solution volume of 2.0 L. The seedlings were randomly arranged. The nutrient solution was aerated daily to ensure adequate oxygen supply to the plants, and 0.01 mol·L⁻¹ nutrient solution was used. -1 HCl or 0.01 mol·L -1 The pH of the nutrient solution was adjusted with NaOH to maintain it between 6.2 and 6.5, and the solution was changed 1-2 times per week. The reeds were cultivated in a glass greenhouse for one month, growing normally until they sprouted. Greenhouse conditions: temperature 25 ℃, light intensity 10000 Lux, light duration 16 h / 8 h (light / dark), humidity 75%.

[0018] In step (1), the Hoagland nutrient solution is prepared using ultrapure water as the solvent, with the pH adjusted to 6.2-6.5. The formula is shown in the table below: Table 1

[0019] (2) Collection of root exudates: Collect the reeds in the sprouting state obtained in step (1), and wash the roots of the reeds with sterile ultrapure water during the period of most vigorous root exudation from 13:00 to 17:00 in the afternoon. Then immerse the roots of the reeds in sterile water containing AgCl and NaN3 respectively and let them stand for 4 hours.

[0020] (3) Concentration and preservation of root exudates: The ultrapure water containing reed root exudates obtained in step (2) was filtered through a 0.45 μm filter membrane (MCE, Amicrom). The filtrate was the root exudate collection solution. 20 mL of the solution was used to determine the dissolved organic carbon (DOC) concentration using a total organic carbon analyzer (multi N / C +HT 1300, German). The amount of plant root exudates collected can be characterized by measuring the DOC concentration of the collection solution.

[0021] from Figure 1 It can be seen that the 0.1 g / L NaN3 treatment group ( Figure 1b) The DOC in the collected solution was significantly higher than that in the control group, while the 10 mg / L AgCl inhibitor treatment group ( Figure 1 a) Significantly lower than the control group. Further investigation into the optimal concentration of NaN3 yielded the following results: Figure 1 c. It can be seen that low concentration of NaN3 (0.01 g / L) cannot achieve complete antibacterial effect, while high concentration of NaN3 (0.25 g / L) will lead to a reduction in reed root exudates. Finally, it was found that 0.1 g / L of NaN3 is the optimal concentration. The remaining collected liquid was aliquoted into 30 mL tubes and immediately placed in a -20 ℃ freezer for pre-freezing. Then, it was placed in a freeze dryer (ChristALPHA 1-2 LD plus) and freeze-dried for 72 h. The resulting concentrated reed root exudate sample was stored in a -20 ℃ freezer and reconstituted for testing in the next step of analysis.

[0022] (4) Confirmation of the quantitative method for organic acids in root exudates: 1) The organic acid content in reed root exudates was analyzed using a high-performance liquid chromatograph (Perkin-Elmer Flexar, USA). Instrument conditions: Waters XBridge™ C 18 A reversed-phase column (250 mm × 4.6 mm, 5 μm) was used at a column temperature of 25 °C, with a mobile phase of 40 mmol·L⁻¹. -1 KH₂PO₄ solution (pH=2.6):methanol = 97:3 (v:v), flow rate 1.0 mL·min -1 The UV detector was used with a detection wavelength of 210 nm, and the injection volume was 10 μL. The sample was detected at a concentration of 50 mg·L⁻¹. -1 When using a mixed standard of organic acids (including quinic acid (KNS), lactic acid (RS), acetic acid (YS), propionic acid (BS), formic acid (JS), malic acid (PGS), tartaric acid (JSS), oxalic acid (CS), and citric acid (NMS), the peak elution patterns of each organic acid are shown in the table below. Figure 2 The oxalic acid peak eluted at 5.25 min, and was difficult to separate from an unknown confounding peak, which will affect the concentration of oxalic acid in root exudates (μg·L⁻¹). -1 Quantitative analysis of oxalic acid at orders of magnitude. Changing conditions such as the methanol content in the mobile phase and the injection volume did not significantly improve the separation of oxalic acid from other peaks.

[0023] 2) Low molecular weight organic acids in root exudates were quantitatively detected by ion chromatography.

[0024] The principle of ion chromatography for the simultaneous separation of multiple organic acids is achieved through gradient elution of the eluent (KOH / NaOH). First, it is necessary to determine the detection concentrations of each organic acid in the eluent. An ion chromatograph (Dionex™ ICS-5000 Starter Line IC) was used, with an IonPacAS-11 column (4 mm × 250 mm), a column temperature of 25 ± 2 ℃, an injection volume of 100 μL, and a conductivity detector. The detection cell temperature was 35 ℃, and the suppression current was 99 mA, mL·min. -1 The isocratic elution method using NaOH as the eluent was determined. Tests were conducted using a single standard solution of 1 mg / L for each sample. The results are as follows: five monoprotic organic acids (quinic acid, lactic acid, acetic acid, propionic acid, and formic acid) could be eluted and separated at a NaOH concentration of 1-2 mM; diprotic organic acids (malic acid, tartaric acid, and oxalic acid) could be eluted and separated at a NaOH concentration of 15-30 mM; while a triprotic acid (citric acid) required a NaOH concentration of 40 mM for elution.

[0025] Next, the gradient elution program was determined: Program 1: 0–15 min, KOH concentration 1 mM; 15–30 min, KOH concentration gradually increased from 1 mM to 20 mM; 30–45 min, KOH concentration increased again from 20 mM to 40 mM and held for 5 min. Program 2: 0–11 min, KOH concentration 1 mM; 11–24 min, KOH concentration gradually increased from 1 mM to 15 mM; 24–37 min, KOH concentration increased from 15 mM to 30 mM; 37–50 min, KOH concentration increased again from 30 mM to 40 mM; at 51 min, KOH concentration rapidly decreased to 1 mM and held for 5 min. The results showed that both elution programs could separate the nine organic acids, but when the organic acid concentration decreased to 0.5 mg·L⁻¹, the separation was significantly reduced. -1 At that time, under gradient elution program 1, the elution time of oxalic acid, tartaric acid and malic acid was prolonged and the conductivity detection signal was unstable, which affected their accurate quantification. Figure 3 -a); while gradient elution procedure 2 was used to detect 0.5 mg·L⁻¹. -1 Organic acids have a more stable baseline. Figure 3 -b) allows for better separation and accurate quantification of nine organic acids: quinic acid, lactic acid, acetic acid, propionic acid, formic acid, malic acid, tartaric acid, oxalic acid, and citric acid. Figure 4 ).

[0026] Preparation of standard organic acid solutions: Weigh 0.5 g (accurate to 0.0001 g) of each of the following: oxalic acid, citric acid, tartaric acid, succinic acid, malic acid, and quinic acid (all reagents are analytical grade). Dissolve them thoroughly in ultrapure water (18.2 MΩ·cm, Milli-Q) and bring the volume to 100 mL. The concentration of each organic acid is 5 g·L⁻¹. -1 This solution serves as a stock solution for each organic acid standard. Take 410 μL of formic acid (chromatographic grade), 500 μL of acetic acid and propionic acid (analytical grade), and 460 μL of lactic acid (analytical grade, 90%), and dilute to 100 mL with ultrapure water to prepare solutions with a concentration of 5 g·L⁻¹. -1 Standard stock solutions were prepared and stored at 4 °C. For testing, each organic acid stock solution was diluted to the required concentration with freshly prepared mobile phase; the concentration of a single standard solution was 1 mg·L⁻¹. -1 The concentration range of the mixed standard solution is 0.5~10 mg·L⁻¹. -1 The standard curve parameters for the nine organic acids are shown in Table 1, and the correlation coefficients (R²) are also shown. 2 The effective concentration ranges from 0.9901 to 0.9999, and the limit of detection (LOD) ranges from 7.37 to 89.9 μg·L⁻¹. -1 The limit of quantitation (LOQ) is 20.14–299.66 μg·L⁻¹. -1 .

[0027] Table 2

[0028] To further investigate the accuracy of ion chromatography in the detection of organic acids, 1 mg·L⁻¹ was prepared. -1 The standard solutions of organic acids were freeze-dried and concentrated according to the same processing steps. Five spiked blank samples were analyzed and determined. The spiked recovery rates of each organic acid are shown in Table 2. Except for acetic acid, propionic acid and formic acid, the recovery rates of the other six organic acids were all above 90% (C1: spiked measured value; C2: spiked theoretical value).

[0029] Table 3

[0030] (5) Actual detection of reed root exudates Ion chromatography was performed using gradient elution program 2 to detect root exudates from hydroponically grown reeds. The results are as follows: Figure 5 As shown, seven low-molecular-weight organic acids were detected, namely lactic acid, acetic acid, propionic acid, malic acid, tartaric acid, oxalic acid, and citric acid. The amount of lactic acid secreted was 0.0423 mg / g. -1 ·h -1DW, SD was 0.0021; acetic acid secretion was 0.0137 mg / g. -1 ·h -1 DW, SD was 0.0002; propionic acid secretion was 0.0131 mg / g. -1 ·h -1 DW, SD was 0.0042; malic acid secretion was 0.0432 mg / g. -1 ·h -1 DW, SD was 0.0005; tartaric acid secretion was 0.1032 mg / g. -1 ·h -1 DW, SD was 0.0039; oxalate secretion was 0.0063 mg / g. -1 ·h -1 DW, SD was 0.0001; citric acid secretion was 0.0100 mg / g. -1 ·h - 1 DW, SD is 0.0019.

[0031] Example 2 A method for determining organic acids in root exudates of reeds grown in root boxes using ion chromatography, including methods for collecting, detecting, and analyzing reed root exudates, with the specific operating steps as follows: (1) Cultivation of reeds: Reeds were cultivated in root boxes and subjected to OTC and heavy metal Cu for 90 consecutive days. 2+ Stress experiments.

[0032] (2) Collection of root exudates: Take the reeds cultured in step (1), perform destructive sampling of the root box, carefully separate the reed roots from the rhizosphere soil to obtain the complete root system, rinse repeatedly with ultrapure water and sterile water, avoiding damage to the reed roots during the process, and place the washed reeds in 1 L of sterile water (with 0.1 g·L⁻¹ added). -1 The reed roots were placed in a glass beaker containing NaN3, and the outer wall of the beaker was wrapped with tin foil to protect them from light. The beaker was left to stand for 4 hours to ensure that the reed roots were completely submerged in sterile water.

[0033] (3) Concentration and preservation of root exudates: The ultrapure water containing reed root exudates obtained in step (2) was filtered through a 0.45 μm filter membrane (MCE, Amicrom). The filtrate was the root exudate collection solution, which was immediately placed in a -20 ℃ freezer for pre-freezing, and then placed in a freeze dryer (ChristALPHA 1-2 LD plus) for freeze drying for 72 h. After freezing, it was taken out and stored in a -20 ℃ freezer for further analysis.

[0034] (4) Quantitative method for organic acids in root exudates: Nine organic acids (quinic acid, lactic acid, malic acid, tartaric acid, oxalic acid, and citric acid) were simultaneously separated by ion chromatography. The instrument conditions were: IonPacAS-11 (4 mm × 250 mm) column, column temperature 25 ± 2 ℃, injection volume 100 μL, conductivity detector (temperature 35℃), suppression current 99 mA, and flow rate 1 mL·min -1 Gradient elution with KOH eluent was performed. Quantitative analysis of organic acids in the root exudates revealed four organic acids in relatively high concentrations: malic acid, tartaric acid, oxalic acid, and citric acid. Results are as follows: Figure 5 As shown, the malic acid secretion levels in the CK, Cu, OTC, and OTC+Cu groups were 0.0140, 0.0046, 0.0060, and 0.0111 mg / g, respectively. -1 ·h - 1 DW, tartaric acid secretion was 0.0132, 0.0092, 0.0097 and 0.0214 mg·g. -1 ·h -1 DW, oxalate secretion was 0.0615, 0.0464, 0.0551 and 0.0809 mg·g. -1 ·h -1 DW, citric acid secretion was 0.1138, 0.0623, 0.11718, and 0.1131 mg / g. -1 ·h -1 DW; the total secretion of each organic acid showed the order of citric acid > oxalic acid > tartaric acid > malic acid; citric acid, with its strong detoxification ability, was secreted in higher amounts than other binary organic acids. From the perspectives of single and combined stress, under Cu single stress, the contents of all four organic acids were significantly reduced compared to the control (CK). P <0.05, malic acid and tartaric acid secretion levels were significantly lower in OTC single stress than in CK, while oxalic acid and citric acid secretion levels showed no significant difference from CK. P >0.05); Under combined Cu and OTC stress, the secretion of malic acid and citric acid was not significantly different from that of CK, while the secretion of tartaric acid and oxalic acid was significantly higher than that of CK. P <0.05).

[0035] In root exudates, the detoxification capacity of organic acids under stress is generally considered to be related to the relative positions of OH / COOH on the main carbon chain. Organic acids are classified into three categories: strong detoxifying acids (tribasic acids), mainly citric acid; moderately detoxifying acids (dibasic acids), mainly oxalic acid, malic acid, tartaric acid; and weakly detoxifying acids (monobasic acids), mainly formic acid, acetic acid. This can be understood as the production of more root exudates to resist stress when reed roots are under stress. However, during long-term stress, heavy metals and antibiotics may damage reed roots, which may explain the lower organic acid content under single stress compared to the control group. Under combined stress, oxytetracycline possesses multiple amino and hydroxyl functional groups that can interact with Cu... 2+ Metal ions undergo complexation reactions to form Cu-OTC complexes. Therefore, a complexation reaction exists between OTC and Cu under combined stress, which to some extent reduces the toxic effects of individual OTC and Cu stress on reeds. This allows the reed roots to actively respond to combined OTC and Cu stress by increasing the secretion of tartaric acid and oxalic acid. This is consistent with the trend of relative organic acid content changes under single and combined Cu and OTC stresses in this study.

Claims

1. A method for determining organic acids in reed root exudates based on ion chromatography, characterized in that, Includes the following steps: (1) Collect the root exudates of reeds to be tested and concentrate and extract them; (2) The extract was determined by ion chromatography. The eluent was NaOH or KOH. The gradient elution conditions were as follows: 0-11 min, KOH concentration was 1 mM; 11-24 min, KOH concentration gradually increased from 1 mM to 15 mM; 24-37 min, KOH concentration increased from 15 mM to 30 mM; 37-50 min, KOH concentration increased from 30 mM to 40 mM; at 51 min, KOH concentration rapidly decreased to 1 mM and was maintained for 5 min. The organic acids tested included quinic acid, lactic acid, acetic acid, propionic acid, formic acid, malic acid, tartaric acid, oxalic acid, and citric acid.

2. The method according to claim 1, characterized in that, The step of collecting the reed root secretions to be tested in step (1) is to obtain them by soaking the washed reed roots in sterile water containing NaN3.

3. The method according to claim 2, characterized in that, The concentration of NaN3 is 0.1~0.25 g / L.

4. The method according to claim 1, characterized in that, The concentration step described in step (1) includes filtering the collected reed root secretions through a membrane and then freeze-drying and reconstituted them.

5. The method according to claim 1, characterized in that, The reed root secretions collected in step (1) were from the roots of hydroponically or soil-grown reeds.

6. The method according to claim 1, characterized in that, The ion chromatography determination conditions described in step (2) also include an IonPacAS-11 analytical column, a column temperature of 25±2 ℃, and a flow rate of 1 mL·min. -1 .

7. The application of the method according to any one of claims 1 to 6 in analyzing the types and contents of organic acids in reed root exudates.

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