Evaluation method for gibberellin content based on water uptake of barley

Abstract

The present application provides a kind of based on the evaluation method of gibberellin content of barley water absorption rate, belongs to the field of analytical detection technology.The evaluation method of gibberellin content based on the water absorption rate of barley provided by the present application includes the following steps: determining the water absorption rate of barley at different time points in the germination process;Determine the gibberellin content of barley in the germination process;The measured water absorption rate of barley is regressed with gibberellin content, and the relationship curve is obtained to predict the gibberellin content in the germination process.The method can predict the gibberellin content in the germination process in advance by determining the water absorption rate of barley.It is simple to operate, does not need complex instrument, and the detection time is shortened, can satisfy the need of timely evaluation of gibberellin content in the germination process according to the detection result.

Description

Technical Field

[0001] This invention belongs to the field of analytical testing technology, and in particular relates to a method for evaluating gibberellin content based on barley water absorption rate. Background Technology

[0002] Barley is the main raw material for beer brewing. Through malting, the activity of various hydrolytic enzymes is activated, and during the saccharification process, the non-water-soluble substances in the malt are gradually hydrolyzed into small-molecule water-soluble nutrients, which are then used by yeast to ferment and produce alcohol and various flavor substances.

[0003] Plant hormones are trace organic compounds produced within plants that regulate (promote or inhibit) their own physiological processes. Plant hormones play a crucial regulatory role in plant growth and development, participating in the regulation of cell division, elongation, differentiation, germination, rooting, flowering, fruiting, sex determination, dormancy, and abscission. Gibberellin (GA) and abscisic acid (ABA) are the main hormones antagonistic to the regulation of barley germination. During barley processing, the conversion of starch to sugar is mainly catalyzed by α-amylase, β-amylase, and limiting dextrinase. Studies have shown that gibberellin induces an increase in α-amylase in the aleurone layer, while abscisic acid blocks this reaction. Therefore, the gibberellin content during barley processing is of great significance for seed germination and solubility.

[0004] Currently, the commonly used methods for determining gibberellin content in the wheat processing process include high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA). HPLC offers high accuracy for gibberellin determination, but the equipment is expensive and the testing costs are high. ELISA, on the other hand, has low sensitivity and relatively large inter-sample errors. Summary of the Invention

[0005] This invention provides a method for evaluating gibberellin content based on barley water absorption rate. This method can predict the gibberellin content during the germination process in advance by measuring the barley water absorption rate. It is simple to operate, requires no complex instruments, and shortens the detection time, meeting the need for timely evaluation of gibberellin content during germination based on detection results.

[0006] To achieve the above objectives, the present invention provides a method for evaluating gibberellin content based on barley water absorption rate, comprising the following steps:

[0007] The water absorption rate of barley at different time points during germination was determined.

[0008] Determining the gibberellin content in barley during germination;

[0009] Regression analysis was performed on the measured barley water absorption rate and gibberellin content to obtain the relationship curve, in order to predict the gibberellin content during the barley production process.

[0010] As a preferred option, the different time points are 0, 2, 4, and 8 hours.

[0011] As a preferred method, the water absorption rate of barley at different time points during germination was specifically determined as follows:

[0012] Take 5g of barley, add 20ml of sterile water, and incubate at 20-25℃. Remove the barley at different time points (0, 2, 4, 8h), wipe off the surface moisture with filter paper, weigh it (W), and calculate the water absorption rate.

[0013] Water absorption rate (%) = (W-5) / 5*100.

[0014] As a preferred method, the gibberellin content of barley during germination is specifically determined as follows:

[0015] Set up standard wells, sample wells and blank wells for gibberellin on the microplate. Add 50 μL of gibberellin standard of different concentrations to each standard well, add 50 μL of the sample to be tested to each sample well, and add 50 μL of sample diluent to each blank well.

[0016] Add 100 μL of horseradish peroxidase-labeled gibberellin antibody to each of the standard wells, sample wells and blank wells, cover with a membrane plate, gently shake to mix, and incubate at 37°C for 1 hour.

[0017] Shake off the liquid in the hole, fill each hole with washing solution, shake for 30 seconds, shake off the washing solution, pat dry with absorbent paper, and repeat the operation several times.

[0018] Add 50 μL each of hydrogen peroxide and tetramethylbenzidine to each well, gently shake to mix, and incubate at 37°C in the dark for 15 minutes.

[0019] Remove the microplate and quickly add 50 μL of 2M H2SO4 stop solution. Immediately after adding the stop solution, measure the OD value of each well at a wavelength of 450 nm.

[0020] Plot a linear regression curve of the standard with the concentration of the standard on the x-axis and the corresponding OD value on the y-axis, and calculate the concentration value of each sample according to the curve equation.

[0021] Preferably, the linear regression curve equation for the gibberellin is y = 0.0064x - 0.0679, R0 2 =0.9944.

[0022] As a preferred approach, regression analysis was performed on the measured barley water absorption rate and gibberellin content, including regression analysis on the 0-2 hour and / or 2-8 hour germination stages of barley.

[0023] As a preferred option, the gibberellin content of barley germinating for 0-2 hours is 5.36 * water absorption rate - 39.961.

[0024] When germination occurs 2-8 hours later, the gibberellin content is 11.64 * water absorption rate - 244.41.

[0025] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0026] The present invention provides a method for evaluating gibberellin content based on barley water absorption rate. Compared with existing detection technologies, this method can predict the gibberellin content during the germination process in advance by measuring the barley water absorption rate. This method is simple to operate, requires no complex instruments, and shortens the detection time, thus meeting the need for timely evaluation of gibberellin content during germination based on detection results. Attached Figure Description

[0027] Figure 1 Illustrations showing the water absorption rates of different barley varieties from 0 to 8 hours, provided by this invention.

[0028] Figure 2 A diagram illustrating the regression analysis equation for barley germination from 0 to 2 hours, provided in an embodiment of the present invention.

[0029] Figure 3 The regression analysis equation for barley germination 2-8 hours provided in this embodiment of the invention is illustrated. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1

[0032] Five different barley varieties were selected, and their water absorption rates were measured. The gibberellin content was then predicted based on regression equations. The results are shown in Table 1.

[0033] The water absorption rate of barley at different time points during germination was determined as follows:

[0034] Take 5g of barley, add 20ml of sterile water, and incubate at 20-25℃. Remove the barley at different time points (0, 2, 4, 8h), wipe off the surface moisture with filter paper, weigh it (W), and calculate the water absorption rate.

[0035] Water absorption rate (%) = (W-5) / 5*100.

[0036] The specific determination of gibberellin content in barley during germination (2 hours and 8 hours of germination) was as follows:

[0037] Grind an appropriate amount of barley in liquid nitrogen, weigh 0.1g of the ground sample, add 1mL of pre-cooled PBS solution, shake to mix, extract at 0-4℃ for 1-2h, centrifuge at 10000-12000rpm for 10-20min, and take the supernatant as the sample to be tested.

[0038] The required number of strips depends on the number of samples and standards. It is recommended to duplicate each standard and blank well. The number of samples should be determined based on the quantity; duplicates should be used whenever possible. Remove the required strips from the aluminum foil bag after equilibration at room temperature for 60 minutes. Before use, thoroughly mix all reagents. Avoid creating excessive foam in the liquid, as this can introduce air bubbles and cause errors during sample addition.

[0039] Set up standard wells, sample wells and blank wells for gibberellin on the microplate. Add 50 μL of gibberellin standard of different concentrations to each standard well, add 50 μL of the sample to be tested to each sample well, and add 50 μL of sample diluent to each blank well.

[0040] Add 100 μL of horseradish peroxidase-labeled gibberellin antibody to each of the standard wells, sample wells and blank wells, cover with a membrane plate, gently shake to mix, and incubate at 37°C for 1 hour.

[0041] Shake off the liquid in the hole, fill each hole with washing solution, shake for 30 seconds, shake off the washing solution, pat dry with absorbent paper, and repeat the operation several times.

[0042] Add 50 μL each of substrate A and B (hydrogen peroxide and tetramethylbenzidine) to each well, gently shake to mix, and incubate at 37°C in the dark for 15 minutes.

[0043] Remove the microplate and quickly add 50 μL of stop solution (2 M H2SO4). Immediately after adding the stop solution, measure the OD value of each well at a wavelength of 450 nm.

[0044] Plot a linear regression curve of the standard with the concentration of the standard on the x-axis and the corresponding OD value on the y-axis, and calculate the concentration value of each sample according to the curve equation.

[0045] The linear regression equation for gibberellin is y = 0.0064x - 0.0679, R0 2 =0.9944.

[0046] Water absorption rates of different barley varieties from 0 to 8 hours, such as Figure 1As shown, the first stage of water absorption is from 0 to 2 hours. During this time, all barley seeds are in a rapid water absorption phase, and gibberellin synthesis has begun slowly within the seed. The main factors affecting the water absorption rate at this stage are the seed coat and other components. The second stage is from 2 to 8 hours. During this stage, the water absorption rate of different barley seeds slows down compared to the first stage, entering a slow water absorption period. However, it is understandable that the water absorption rate of different barley varieties varies at different time points. External moisture is a crucial factor in seed germination, initiating germination and affecting gibberellin synthesis within the seed. Therefore... The non-germination process of barley The water absorption rate at the same time points (2 hours and 8 hours) and the gibberellin content of barley during the germination process (2 hours and 8 hours of germination) are also related. Correlation analysis was performed on the quantities to obtain the relationship curve. The details are as follows:

[0047] Germination time 0-2 hours: Gibberellin content (pmol / L) = 5.36 * water absorption rate - 39.961, R 2 =0.8284, such as Figure 2 As shown;

[0048] Germination time 2-8 hours: Gibberellin content (pmol / L) = 11.64 * water absorption rate - 244.41, R 2 =0.8047, as Figure 3 As shown.

[0049] Actual method for determining gibberellin content:

[0050] Germination experiments were conducted on six different barley varieties using traditional methods, and gibberellin content was determined using ELISA. The linear regression equation for gibberellin was y = 0.0064x - 0.0679, R0. 2 =0.9944, the test results are shown in Table 1 for the actual gibberellin content.

[0051] The specific measurement method is as follows:

[0052] (1) Take 100 grains of barley, place them in a petri dish containing double-layered filter paper, add 4 ml of sterile water, and place in a 16°C incubator in the dark. Take appropriate amounts of barley for testing at 2 hours and 8 hours.

[0053] (2) Take an appropriate amount of barley and grind it with liquid nitrogen. Weigh 0.1g of the ground sample, add 1mL of pre-cooled PBS solution, shake to mix, extract at 0-4℃ for 1-2h, centrifuge at 10000-12000rpm for 10-20min, and take the supernatant as the sample to be tested.

[0054] (3) Determine the required number of strips based on the number of samples to be tested plus the number of standards. It is recommended to make duplicates for each standard and blank well. The number of samples should be determined based on the quantity of samples. Make duplicates whenever possible. Take the required strips from the aluminum foil bag after equilibration at room temperature for 60 minutes. Before use, mix all reagents thoroughly. Do not allow the liquid to produce a large amount of foam, so as to avoid adding a large number of air bubbles during sample addition and causing errors in sample addition.

[0055] (4) Set up standard wells, sample wells and blank wells for gibberellin on the microplate. Add 50 μL of gibberellin standard of different concentrations to each standard well, add 50 μL of the sample to be tested to each sample well, and add 50 μL of sample diluent to each blank well.

[0056] (5) Add 100 μL of horseradish peroxidase-labeled gibberellin detection antibody to each of the standard wells, sample wells and blank wells, cover the membrane plate, gently shake to mix, and incubate at 37°C for 1 hour.

[0057] (6) Shake off the liquid in the hole, fill each hole with washing liquid, shake for 30 seconds, shake off the washing liquid, pat dry with absorbent paper, and repeat the operation several times.

[0058] (7) Add 50 μL each of substrate A and B (hydrogen peroxide and tetramethylbenzidine) to each well, gently shake to mix, and incubate at 37°C in the dark for 15 minutes.

[0059] (8) Take out the microplate and quickly add 50 μL of stop solution (2M H2SO4). After adding the stop solution, immediately measure the OD value of each well at a wavelength of 450 nm. Plot the linear regression curve of the standard with the standard concentration on the x-axis and the corresponding OD value on the y-axis. Calculate the concentration value of each sample according to the curve equation.

[0060] Table 1

[0061] Water absorption rate (%) after 2 hours 19.46 22.67 20.96 24.72 21.54 Predicting gibberellin content (pmol / L) 64.51 81.55 72.39 92.54 75.49 Actual gibberellin content (pmol / L) 62.48 70.77 72.12 98.11 79.20 8-hour water absorption rate (%) 30.97 35.59 30.67 36.44 31.83 Predicting gibberellin content (pmol / L) 116.08 169.86 112.59 179.75 126.09 Actual gibberellin content (pmol / L) 105.33 158.27 117.85 198.08 136.03

[0062] As shown in Table 1, the predicted gibberellin content of five barley varieties obtained using this method has an error of ≤15% compared to the actual gibberellin content determined by the traditional method, indicating that the evaluation method presented in this study is accurate. Furthermore, this evaluation method only requires analysis of the barley's water absorption rate at 2 and 8 hours, eliminating the need for large equipment and reducing the time required, whereas the traditional method requires germination experiments on the barley before determining the gibberellin content. Therefore, this method has significant advantages over the traditional approach.

Claims

1. A method for evaluating gibberellin content based on barley water absorption rate, characterized in that, Includes the following steps: The water absorption rate of barley at different time points during germination was determined. Determining the gibberellin content in barley during germination; Regression analysis was performed on the measured barley water absorption rate and gibberellin content to obtain the relationship curve, so as to predict the gibberellin content in the barley production process. Regression analysis was performed on the measured barley water absorption rate and gibberellin content, including regression analysis on the 0-2 hour germination stage and / or the 2-8 hour germination stage of barley.

2. The evaluation method according to claim 1, characterized in that, The different time points are 0, 2, 4, and 8 hours.

3. The evaluation method according to claim 1, characterized in that, The water absorption rate of barley at different time points during germination was specifically determined as follows: Take 5 g of barley, add 20 ml of sterile water, and incubate at 20-25℃. Remove the barley at different time points (0, 2, 4, and 8 hours), wipe off the surface moisture with filter paper, weigh it (W), and calculate the water absorption rate. Water absorption rate (%) = (W-5) / 5 100.

4. The evaluation method according to claim 1, characterized in that, The specific steps for determining the gibberellin content in barley during germination are as follows: Set up standard wells, sample wells and blank wells for gibberellin on the microplate. Add 50 μL of gibberellin standard of different concentrations to each standard well, add 50 μL of the sample to be tested to each sample well, and add 50 μL of sample diluent to each blank well. Add 100 μL of horseradish peroxidase-labeled gibberellin antibody to each of the standard wells, sample wells and blank wells, cover with a membrane plate, gently shake to mix, and incubate at 37°C for 1 hour. Shake off the liquid in the hole, fill each hole with washing solution, shake for 30 seconds, shake off the washing solution, pat dry with absorbent paper, and repeat the operation several times. Add 50 μL each of hydrogen peroxide and tetramethylbenzidine to each well, gently shake to mix, and incubate at 37°C in the dark for 15 minutes. Remove the microplate and quickly add 50 μL of 2M H2SO4 stop solution. Immediately after adding the stop solution, measure the OD value of each well at a wavelength of 450 nm. Plot a linear regression curve of the standard with the concentration of the standard on the x-axis and the corresponding OD value on the y-axis, and calculate the concentration value of each sample according to the curve equation.

5. The evaluation method according to claim 1, characterized in that, The gibberellin content of barley germinating for 0-2 hours was 5.36%. Water absorption rate -39.961, R 2 =0.8284; The gibberellin content was 11.64 g / L 2-8 hours after germination. Water absorption rate -244.41, R 2 =0.8047.

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