A method of using an apparatus for in situ collection of 2-acetyl-1-pyrroline synthesized by rice roots

Abstract

The application discloses a kind of 2-acetyl-1-pyrroline synthesized by in-situ collection rice root system and its use method in the field of plant biotechnology, the device includes hydroponic box, the upper portion of one side of hydroponic box is connected with first horizontal straight pipe, first horizontal straight pipe is communicated with first vertical pipe by first connecting elbow, first vertical pipe is connected with the water outlet end of water pump, the water inlet end of water pump is connected with liquid inlet by second horizontal straight pipe;The lower portion of the other side of hydroponic box is connected with third horizontal straight pipe, third horizontal straight pipe is communicated with second vertical pipe by second connecting elbow, and second vertical pipe is connected into collection bottle;Hydroponic box top is embedded with cultivation plate, and cultivation plate is provided with cultivation hole, and the junction of cultivation plate and the top of hydroponic box is also provided with sealing element for keeping airtight.The collecting device of the application is simple in structure and easy to build, and 2-acetyl-1-pyrroline synthesized by rice root system is successfully collected, solving the problem that 2-acetyl-1-pyrroline released by rice root system is easy to volatilize and difficult to store.

Description

Technical Field

[0001] This invention relates to the field of plant biotechnology, and in particular to a method for using a device for in-situ collection of 2-acetyl-1-pyrrolidine synthesized from rice roots. Background Technology

[0002] Fragrant rice, a rice variety with unique aromatic properties, is widely popular in the market and its high economic value has played a significant role in promoting agricultural economic development. Among the various volatile organic compounds that contribute to the aroma of fragrant rice, 2-acetyl-1-pyrrolidone is the most important aroma component, possessing a popcorn-like fragrance. Studies have shown that 2-acetyl-1-pyrrolidone is stored in rice plants by forming complexes with amylose, and is synthesized in large quantities during the grain-filling stage and translocated into the grains. 2-acetyl-1-pyrrolidone is highly volatile and is released in large quantities from the grains during rice cooking, becoming the main source of the rice's aroma.

[0003] Currently, methods for collecting 2-acetyl-1-pyrrololine can be categorized into simultaneous distillation-extraction (SDE) and headspace solid-phase microextraction (HSPME), depending on the collection materials and procedures. SDE involves simultaneously heating and condensing the organic and aqueous phases to extract 2-acetyl-1-pyrrololine into the organic solvent. HSPME uses fused silica fibers coated with a stationary phase to adsorb 2-acetyl-1-pyrrololine volatilized during heating. Both methods require gas chromatography-mass spectrometry (GC-MS) analysis to determine the specific concentration of 2-acetyl-1-pyrrololine collected. However, the application of these methods in rice mainly focuses on leaves and grains.

[0004] Rice roots are not only key organs for the plant to absorb water and mineral nutrients, but also participate in signal transduction and hormone regulation, responding to environmental changes. Studies have confirmed that the content of 2-acetyl-1-pyrrolidine precursors and the expression levels of genes related to 2-acetyl-1-pyrrolidine synthesis in roots are consistent with those in leaves, and rice roots themselves can synthesize 2-acetyl-1-pyrrolidine. However, roots contain little starch, and 2-acetyl-1-pyrrolidine is volatile and difficult to store in the roots. Therefore, the two methods mentioned above for collecting 2-acetyl-1-pyrrolidine are ineffective when applied to the collection of 2-AP from rice roots, often failing to quantify it due to detection limits below the instrument's detection limit. Currently, there is no systematic method for collecting 2-acetyl-1-pyrrolidine from roots, leaving research on the synthesis of 2-acetyl-1-pyrrolidine in roots unclear. Studying the synthesis of 2-acetyl-1-pyrroline in roots is a key part of revealing the aroma production mechanism of the whole plant and can provide strong scientific support for improving the aroma and quality of rice. Therefore, it is particularly important to establish a collection device for the release of 2-acetyl-1-pyrroline from rice roots. Summary of the Invention

[0005] The purpose of this invention is to overcome the deficiencies in the prior art and provide a method for using a device for in-situ collection of 2-acetyl-1-pyrrolline synthesized from rice roots, which avoids the volatilization of 2-acetyl-1-pyrrolline released from rice roots, facilitates storage, and has a simple structure and is easy to operate.

[0006] The objective of this invention is achieved as follows: an apparatus for in-situ collection of 2-acetyl-1-pyrrolline synthesized from rice roots, comprising a hydroponic box, wherein a first horizontal tube is connected to the upper part of one side of the hydroponic box, the first horizontal tube is connected to a first vertical tube via a first connecting bend, the first vertical tube is connected to the outlet of a water pump, and the inlet of the water pump is connected to an inlet via a second horizontal tube; a third horizontal tube is connected to the lower part of the other side of the hydroponic box, the third horizontal tube is connected to a second vertical tube via a second connecting bend, and the second vertical tube is connected to a collection bottle; a cultivation plate is fitted into the top of the hydroponic box, the cultivation plate has cultivation holes, the cultivation holes are provided with sponges for fixing experimental rice, and a sealing element is provided at the junction of the cultivation plate and the top of the hydroponic box to maintain a tight seal.

[0007] In operation, this invention ensures the airtightness of the entire device by setting a seal between the cultivation plate and the hydroponic box, thus confining the 2-acetyl-1-pyrrololine generated by the experimental rice roots within the device. Furthermore, the hydroponic box is strictly protected from light to prevent light from interfering with the synthesis of 2-acetyl-1-pyrrololine and the starch structure. Under sealed conditions, the 2-acetyl-1-pyrrololine released by the experimental rice roots complexes with starch and is stored in the rice nutrient solution. The collected rice nutrient solution is then tested to determine the content of 2-acetyl-1-pyrrololine.

[0008] Furthermore, the first horizontal pipe, the second horizontal pipe, the third horizontal pipe, the first vertical pipe, the second vertical pipe, the first connecting bend, and the second connecting bend are all made of polyvinyl chloride.

[0009] Furthermore, a first check valve is provided between the first horizontal pipe and the hydroponic box, a second check valve is provided between the third horizontal pipe and the hydroponic box, and a flow controller is provided between the second check valve and the second connecting bend.

[0010] Furthermore, the sponge is a hollow circular shape adapted to the cultivation holes.

[0011] Furthermore, the sealing element is a polyvinyl chloride film.

[0012] Furthermore, the hydroponic box is made of polypropylene and is filled with nutrient solution for rice growth.

[0013] A method for using an apparatus for in-situ collection of 2-acetyl-1-pyrrolidine synthesized from rice roots includes the following steps:

[0014] (1) Planting rice plants: Soaking and germinating rice seeds, planting the sprouting rice seeds evenly in a 96-well microplate, and placing them in a hydroponic box containing rice nutrient solution to cultivate until the three-leaf and one-heart stage.

[0015] (2) Preparation of rice nutrient solution: Add a small amount of deionized water to starch to form a suspension, pour it into boiling water and cook until clear and transparent, then cool in a water bath. Add N, P, K, Ca, Mg, SiO2, Mn, Mo, B, Zn, Cu, and Fe elements according to the rice nutrient solution formula determined by the International Rice Research Institute, and add deionized water until the starch concentration reaches 2 g / L. -1 Using 2.325 mol L -1 The pH of the nutrient solution was adjusted to 5.4 with sulfuric acid solution; high starch concentrations can form difficult-to-handle precipitates, gels, or uneven suspensions, while low starch concentrations can lead to insufficient collection of volatile gases. A starch concentration of 2 g / L is required. -1 This ensures that the 2-acetyl-1-pyrrolline synthesized by rice roots is stably complexed with starch;

[0016] (3) Transplanting rice plants: Transplant rice seedlings that have been cultivated to the three-leaf and one-heart stage into cultivation boards and fix the rice plants with hollow round sponges.

[0017] (4) Add rice nutrient solution: turn on the water pump and the first check valve, and transfer the rice nutrient solution from the inlet to the light-proof hydroponic box until the entire hydroponic box is filled.

[0018] (5) Sealing treatment: Close the first check valve and the second check valve, fit the cultivation plate on top of the hydroponic box, and tightly wrap the polyvinyl chloride film at the joint between the cultivation plate and the hydroponic box to ensure the airtightness of the device.

[0019] (6) Collect 2-acetyl-1-pyrroline: Place the completed collection device in a plant culture box or a suitable temperature environment for growth;

[0020] (7) Collect starch solution: Open the second check valve and flow controller, and collect 150 mL of starch solution into the collection bottle;

[0021] (8) Extraction of 2-acetyl-1-pyrrolline: The volatile organic compounds in the starch solution were concentrated using a simultaneous distillation-extraction apparatus and extracted into the organic phase dichloromethane;

[0022] (9) Determination of 2-acetyl-1-pyrrolidine: The content of 2-acetyl-1-pyrrolidine was obtained by gas chromatography-mass spectrometry.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] First, the collection device of this invention has a simple overall structure and is easy to assemble. It successfully collects 2-acetyl-1-pyrrololine synthesized by rice roots through an inlet, a sealed collection device, and an outlet collection device. Compared to existing technologies that obtain 2-acetyl-1-pyrrololine from rice plant leaves or grains, this invention solves the problems of easy volatilization and difficulty in storage of 2-acetyl-1-pyrrololine released from rice roots, filling a gap in the collection of 2-acetyl-1-pyrrololine synthesized by rice roots. Furthermore, the collection device of this invention allows for qualitative and quantitative measurement of 2-acetyl-1-pyrrololine synthesized by rice roots using gas chromatography-mass spectrometry. 2-acetyl-1-pyrrololine was detected in the nutrient solution of the fragrant rice variety Nanjing 9108, with a release rate of 6.295 ng / mL. -1 d -1 .

[0025] Secondly, the collection device of the present invention strictly protects the hydroponic box from light and seals it, which can prevent the starch structure from being broken due to light exposure, so that it still has the ability to complex with 2-acetyl-1-pyrrolline; the sealing treatment causes 2-acetyl-1-pyrrolline to accumulate in the hydroponic box, increasing the possibility of its complexation with starch. After 24 hours of collection, the presence of 2-acetyl-1-pyrrolline can be detected in the starch nutrient solution soaked in fragrant rice roots. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the device for in-situ collection of 2-acetyl-1-pyrrolidine synthesized from rice roots according to the present invention.

[0027] Figure 2 The image shows a gas chromatogram of starch nutrient solution collected by the device of this invention after GC-MS analysis.

[0028] Figure 3 The release rate of 2-acetyl-1-pyrrolidone was obtained by gas chromatography-mass spectrometry analysis of starch-containing nutrient solutions collected by the device of the present invention under different treatments.

[0029] The components include: 1. Liquid inlet; 2. Second horizontal pipe; 3. Water pump; 4. First vertical pipe; 5. First connecting bend; 6. First horizontal pipe; 7. First check valve; 8. Hydroponic box; 9. Cultivation plate; 10. Seal; 11. Experimental rice; 12. Sponge; 13. Rice nutrient solution; 14. Second check valve; 15. Third horizontal pipe; 16. Flow controller; 17. Second connecting bend; 18. Second vertical pipe; and 19. Collection bottle. Detailed Implementation Example 1

[0030] like Figure 1 The apparatus shown is for in-situ collection of 2-acetyl-1-pyrrolidine synthesized from rice roots. It includes a hydroponic box 8. A first horizontal tube 6 is connected to the upper part of one side of the hydroponic box 8. The first horizontal tube 6 is connected to a first vertical tube 4 via a first connecting bend 5. The first vertical tube 4 is connected to the outlet of a water pump 3. The inlet of the water pump 3 is connected to the inlet via a second horizontal tube 2. A third horizontal tube 15 is connected to the lower part of the other side of the hydroponic box 8. The third horizontal tube 15 is connected to a second vertical tube 18 via a second connecting bend 17. The second vertical tube 18 is connected to a collection bottle 19. A cultivation plate 9 is fitted into the top of the hydroponic box 8. The cultivation plate 9 has cultivation holes, and sponges 1 for fixing experimental rice 11 are placed inside the cultivation holes. 2. The sponge 12 is a hollow circular shape adapted to the cultivation hole. A polyvinyl chloride film sealant 10 is provided at the junction of the cultivation plate 9 and the top of the hydroponic box 8 to maintain a tight seal. The first horizontal pipe 6, the second horizontal pipe 2, the third horizontal pipe 15, the first vertical pipe 4, the second vertical pipe 18, the first connecting bend 5, and the second connecting bend 17 are all made of polyvinyl chloride. A first check valve 7 is provided between the first horizontal pipe 6 and the hydroponic box 8, and a second check valve 14 is provided between the third horizontal pipe 15 and the hydroponic box 8. A flow controller 16 is provided between the second check valve 14 and the second connecting bend 17. The hydroponic box 8 is made of polypropylene and is filled with rice nutrient solution 13 for rice growth. The nutrient solution 13 contains a concentration of 2 g / L. -1 High concentrations of starch can form difficult-to-handle precipitates, gels, or uneven suspensions, while low concentrations can lead to insufficient collection of volatile gases. A starch concentration of 2 g / L is also acceptable. -1 This ensures that the 2-acetyl-1-pyrrolline synthesized by rice roots is stably complexed with starch.

[0031] This device can successfully collect 2-acetyl-1-pyrrolidine released from rice roots, obtaining a starch nutrient solution with stable complexation of 2-acetyl-1-pyrrolidine, which facilitates further analysis and determination. Example 2

[0032] A method for using an apparatus for in-situ collection of 2-acetyl-1-pyrrolidine synthesized from rice roots includes the following steps:

[0033] (1) Planting rice plants: Soaking and germinating rice seeds, planting the sprouting rice seeds evenly in a 96-well microplate, and placing them in a hydroponic box 8 containing rice nutrient solution 13 to cultivate until the three-leaf and one-heart stage.

[0034] (2) Preparation of rice nutrient solution: Add a small amount of deionized water to starch to form a suspension, pour it into boiling water and cook until clear and transparent, then cool in a water bath. Add N, P, K, Ca, Mg, SiO2, Mn, Mo, B, Zn, Cu, and Fe elements according to the rice nutrient solution formula determined by the International Rice Research Institute, and add deionized water until the starch concentration reaches 2 g / L. -1 Using 2.325 mol L -1 The pH of the nutrient solution was adjusted to 5.4 using sulfuric acid solution;

[0035] (3) Transplanting rice plants: Transplant rice seedlings that have been cultivated to the three-leaf and one-heart stage into the cultivation board 9 and fix the rice plants with hollow circular sponges 12.

[0036] (4) Add rice nutrient solution: turn on the water pump 3 and the first check valve 7, and deliver the rice nutrient solution 13 from the inlet 1 to the light-proof hydroponic box 8 until the entire hydroponic box 8 is filled.

[0037] (5) Sealing treatment: Close the first check valve 7 and the second check valve 14, fit the cultivation plate 9 on top of the hydroponic box 8, and tightly wrap the polyvinyl chloride film at the joint between the cultivation plate 9 and the hydroponic box 8 to ensure the airtightness of the device.

[0038] (6) Collect 2-acetyl-1-pyrroline: Place the completed collection device in a plant culture box or a suitable temperature environment for growth;

[0039] (7) Collect starch solution: After culturing for 24 hours, open the second check valve 14 and the flow controller 16 to collect 150 mL of starch solution into the collection bottle 19;

[0040] (8) Extraction of 2-acetyl-1-pyrrolline: The volatile organic compounds in the starch solution were concentrated using a simultaneous distillation-extraction apparatus and extracted into the organic phase dichloromethane;

[0041] (9) Determination of 2-acetyl-1-pyrrolidine: The content of 2-acetyl-1-pyrrolidine was obtained by gas chromatography-mass spectrometry.

[0042] Specifically, rice roots were collected after soaking and growing, containing 2g L... -1 Starch in rice nutrient solution was analyzed by gas chromatography-mass spectrometry, and blank control group and experimental group were set up to collect and determine 2-acetyl-1-pyrrolidine.

[0043] After comparing the results, such as Figure 2 , 3 As shown, Figure 2 The gas chromatograms obtained after GC-MS analysis of the collected starch nutrient solutions are shown. 2-Acetyl-1-pyrrololine was not detected in the blank control group nutrient solution (no starch, no plants); 2-Acetyl-1-pyrrololine was not detected in the nutrient solution of experimental group one (with starch, no plants); 2-Acetyl-1-pyrrololine was not detected in the nutrient solution of experimental group two (with non-fragrant rice plants, with starch); and an absorption peak of 2-Acetyl-1-pyrrololine was detected in the nutrient solution of experimental group three (with fragrant rice plants, with starch).

[0044] Figure 3 The release rate of 2-acetyl-1-pyrrololine was determined by gas chromatography-mass spectrometry (GC-MS) of the collected starch-containing nutrient solutions under different treatments. In the blank control group (no starch, no plants), the 2-acetyl-1-pyrrololine content was almost zero; in experimental group one (with starch, no plants), the 2-acetyl-1-pyrrololine content was almost zero; in experimental group two (with non-aromatic rice plants, starch), the 2-acetyl-1-pyrrololine content was almost zero; and in experimental group three (with aromatic rice plants, starch), 2-acetyl-1-pyrrololine was detected, with a release rate of 6.295 ng / mL. -1 d -1 .

[0045] This invention is not limited to the above embodiments. Based on the technical solutions disclosed in this invention, those skilled in the art can make some substitutions and modifications to some of the technical features without creative effort, and all such substitutions and modifications are within the protection scope of this invention.

Claims

1. A method for using a device for in-situ collection of 2-acetyl-1-pyrrolline synthesized from rice roots, characterized in that: The device includes a hydroponic box. A first horizontal pipe is connected to the upper part of one side of the hydroponic box. The first horizontal pipe is connected to a first vertical pipe via a first connecting bend. The first vertical pipe is connected to the outlet of a water pump. The inlet of the water pump is connected to a liquid inlet via a second horizontal pipe. A third horizontal pipe is connected to the lower part of the other side of the hydroponic box. The third horizontal pipe is connected to a second vertical pipe via a second connecting bend. The second vertical pipe is connected to a collection bottle. A cultivation plate is fitted into the top of the hydroponic box. Cultivation holes are provided on the cultivation plate, and sponges for fixing experimental rice are placed inside the cultivation holes. A sealing element is also provided at the junction of the cultivation plate and the top of the hydroponic box to maintain a tight seal. The method of using this device includes: (1) Planting rice plants: Soaking and germinating rice seeds, planting the sprouting rice seeds evenly in a 96-well microplate, and placing them in a hydroponic box containing rice nutrient solution to cultivate until the three-leaf and one-heart stage. (2) Preparation of rice nutrient solution: Add a small amount of deionized water to starch to form a suspension, pour it into boiling water and cook until clear and transparent, then cool in a water bath. Add N, P, K, Ca, Mg, SiO2, Mn, Mo, B, Zn, Cu, and Fe elements according to the rice nutrient solution formula determined by the International Rice Research Institute, and add deionized water until the starch concentration reaches 2 g / L. -1 Using 2.325 mol L -1 The pH of the nutrient solution was adjusted to 5.4 using sulfuric acid solution; (3) Transplanting rice plants: Transplant rice seedlings that have been cultivated to the three-leaf and one-heart stage into cultivation boards and fix the rice plants with hollow round sponges. (4) Add rice nutrient solution: turn on the water pump and the first check valve, and transfer the rice nutrient solution from the inlet to the light-proof hydroponic box until the entire hydroponic box is filled. (5) Sealing treatment: Close the first check valve and the second check valve, fit the cultivation plate on top of the hydroponic box, and tightly wrap the polyvinyl chloride film at the joint between the cultivation plate and the hydroponic box to ensure the airtightness of the device. (6) Collect 2-acetyl-1-pyrroline: Place the completed collection device in a plant culture box or a suitable temperature environment for growth; (7) Collect starch solution: Open the second check valve and flow controller, and collect 150 mL of starch solution into the collection bottle; (8) Extraction of 2-acetyl-1-pyrrolline: The volatile organic compounds in the starch solution were concentrated using a simultaneous distillation-extraction apparatus and extracted into the organic phase dichloromethane; (9) Determination of 2-acetyl-1-pyrrolidine: The content of 2-acetyl-1-pyrrolidine was obtained by gas chromatography-mass spectrometry.

2. The method of using the device for in-situ collection of 2-acetyl-1-pyrrololine synthesized from rice roots according to claim 1, characterized in that: The first horizontal pipe, the second horizontal pipe, the third horizontal pipe, the first vertical pipe, the second vertical pipe, the first connecting bend, and the second connecting bend are all made of polyvinyl chloride.

3. The method of using the device for in-situ collection of 2-acetyl-1-pyrrololine synthesized from rice roots according to claim 2, characterized in that: A first check valve is provided between the first horizontal pipe and the hydroponic box, and a second check valve is provided between the third horizontal pipe and the hydroponic box. A flow controller is provided between the second check valve and the second connecting bend.

4. The method of using the device for in-situ collection of 2-acetyl-1-pyrrololine synthesized from rice roots according to claim 1, characterized in that: The sponge is a hollow circular shape adapted to the cultivation holes.

5. The method of using the device for in-situ collection of 2-acetyl-1-pyrrolidine synthesized from rice roots according to claim 1, characterized in that: The sealing element is a polyvinyl chloride film.

6. A method of using the device for in-situ collection of 2-acetyl-1-pyrrololine synthesized from rice roots according to any one of claims 1 to 5, characterized in that: The hydroponic box is made of polypropylene and is filled with nutrient solution for rice growth.

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