Correlation method for evaluating stress resistance of chitooligosaccharide
By using high-performance anion exchange chromatography and multiple regression model analysis, the systematic deficiencies in the evaluation of chitosan oligosaccharide stress resistance were addressed, enabling accurate assessment and optimized application under different adverse conditions, and supporting its promotion in agricultural production.
Patent Information
- Application Number
- CN202511931230.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-19
- Publication Date
- 2026-03-13
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Existing technologies lack systematic and comprehensive methods for evaluating the stress resistance of chitosan oligosaccharides, making it difficult to accurately assess their true effects on plants under different adverse conditions, and research is insufficient to support their widespread application in agricultural production.
High-performance anion exchange chromatography-integrated pulse amperometric detection was used for qualitative detection by ion chromatography. Combined with a multiple regression model, changes in physiological indicators were analyzed to classify the stress resistance level of chitosan oligosaccharides and provide optimized application schemes.
This study enabled a comprehensive and accurate assessment of the stress resistance of chitosan oligosaccharides, providing a scientific basis for their application, reducing costs, promoting their widespread use in agricultural production, and exploring their mechanism of action and long-term effects.
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Figure CN121656469A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant stress physiology technology, specifically a method for evaluating the stress resistance of chitosan oligosaccharides. Background Technology
[0002] In agricultural production, plants often face various abiotic stresses, such as low temperature, drought, and salt stress. These adverse conditions can severely affect plant growth, development, and yield, causing huge losses to agricultural production. Traditional methods for dealing with abiotic stresses mainly rely on chemical pesticides and irrigation, but these methods have many drawbacks. The large-scale use of chemical pesticides not only leads to environmental pollution but may also cause problems such as excessive pesticide residues in agricultural products, posing a potential threat to human health. Irrigation measures are greatly limited in arid or water-scarce areas, making it difficult to effectively solve the problem of drought stress in plants.
[0003] In recent years, chitosan oligosaccharides, as a natural bioactive substance, have gradually attracted researchers' attention due to their various biological functions, such as enhancing plant stress resistance and promoting plant growth. However, current research on the effects of chitosan oligosaccharides on improving plant stress resistance is mostly at the preliminary exploratory stage, lacking systematic and comprehensive evaluation methods. Existing studies tend to focus on the influence of single factors on the stress resistance effect of chitosan oligosaccharides, without comprehensively considering the interaction between multiple factors such as chitosan oligosaccharide concentration, degree of polymerization, and treatment time. This makes it difficult to accurately assess the true effect of chitosan oligosaccharides on plant stress resistance under different stress conditions. Furthermore, there is a lack of in-depth research on the mechanism of action and long-term effects of chitosan oligosaccharides on stress resistance, which to some extent limits the widespread application of chitosan oligosaccharides in agricultural production. Therefore, developing a scientific, systematic, and comprehensive method for evaluating the stress resistance effect of chitosan oligosaccharides is of significant practical importance. Summary of the Invention
[0004] The purpose of this invention is to provide a method for evaluating the stress resistance of chitosan oligosaccharides, so as to solve the problems mentioned in the background art.
[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for evaluating the stress resistance of chitosan oligosaccharides, comprising the following steps: S1. Qualitative detection by ion chromatography: High-performance anion exchange chromatography-integral pulse amperometric detection method was used to separate and detect chitosan oligosaccharide samples. The degree of polymerization of each component in the sample was calculated by comparing the retention time of the standard and the degree of polymerization-retention factor correlation equation. S2. Stress resistance treatment: The target plants were divided into a control group and a chitosan oligosaccharide treatment group. The treatment groups were sprayed with chitosan oligosaccharide solutions of different concentrations and degrees of polymerization, and stress conditions such as low temperature, drought and salt stress were set simultaneously. S3. Physiological index measurement: Plant samples are collected regularly to measure relative conductivity, malondialdehyde content, antioxidant enzyme activity and root vitality; S4. Data Correlation Analysis: Establish a multiple regression model of polymerization degree-concentration-physiological index changes to determine the key parameter combination of chitosan oligosaccharide's stress resistance effect; S5. Stress Resistance Level Classification: Based on the model output results, the stress resistance effect of chitosan oligosaccharide is classified into three levels: strong, medium and weak, and an optimized application scheme is output.
[0006] Preferably, in the ion chromatography qualitative detection step: The correlation equation is a linear equation: logk'=a·DP+c Where: k' is the retention factor, defined as t_R is the retention time of the sample component. is the dead time; a is a real parameter reflecting the slope of the effect of the degree of polymerization on the retention factor; c is a constant reflecting the basic retention characteristics of the chromatographic system; Parameters a and c were obtained by fitting the retention time-degree of polymerization data of the standard. The fitting methods included least squares or weighted least squares, and the goodness of fit R² ≥ 0.99.
[0007] Preferably, in the stress resistance treatment step: The stress conditions were set as follows: for low temperature stress, treatment at 4℃ for 24-72 hours, adjusted to 0-10℃ depending on the plant species; for drought stress, irrigation was stopped until the soil moisture content was ≤15%, and drought was simulated by polyethylene glycol at a concentration of 5-20%; for salt stress, irrigation was carried out with 100-200mM NaCl solution, using a mixed salt solution; foliar spraying or root irrigation was used, with spraying time in the early morning or evening to avoid high-temperature evaporation.
[0008] Preferably, in the physiological index measurement step: Relative conductivity determination: Take plant leaves or roots, rinse with deionized water, cut into 1cm segments, place in 20mL of deionized water, and extract by shaking at 25℃ for 24 hours. MDA content determination: The thiobarbituric acid method was used, and the extraction solution was 10% trichloroacetic acid; The reaction system was a 0.5% TBA solution in 20% TCA. After boiling in a water bath for 30 minutes and cooling, the absorbance at wavelengths of 532 nm and 600 nm was measured. The unit for MDA content is nmol / gFW.
[0009] Preferably, in the data correlation analysis step: Multiple regression model: Input variables include chitosan oligosaccharide concentration C (mg / L), degree of polymerization DP, and treatment time T (days); The output variables are the rate of change of relative conductivity ΔREC%, the rate of change of MDA content ΔMDA%, the rate of increase in antioxidant enzyme activity ΔSOD%, ΔPOD%, and ΔCAT%. The model expression is Where ε is the error term; Model validation: Cross-validation or independent test set validation was used, with a determination coefficient R² ≥ 0.85. The significance of the model was tested by analysis of variance, with a p-value < 0.05.
[0010] Preferably, the criteria for classifying stress resistance levels are as follows: High potency: ΔREC%≤30%, ΔMDA%≤25%, ΔSOD%≥40%, and at least two antioxidant enzyme activities increased by ≥30%; Medium-efficacy: 30% < ΔREC% ≤ 50%, 25% < ΔMDA% ≤ 40%, 20% ≤ ΔSOD% < 40%, and at least one antioxidant enzyme activity improvement rate ≥ 20%; Weak efficacy: ΔREC%>50%, ΔMDA%>40%, ΔSOD%<20%, and the rate of increase in antioxidant enzyme activity is <15%.
[0011] Preferably, the optimized application scheme includes: Concentration range: Adjusted according to the type of stress; Combination of aggregation degree: Low temperature stress: DP3-DP4 as the main component, supplemented by a small amount of DP2; Salt stress: DP2-DP3 are the main components, supplemented by DP5; Application frequency: For short-term stress, spray 1-2 times at intervals of 3-7 days; For prolonged stress, spray 3-5 times at 10-15 day intervals.
[0012] Preferably, the target plant includes: Food crops: rice, wheat, corn, soybeans; cash crops: rapeseed, cotton, tobacco, sugarcane; fruit crops: apples, citrus, grapes, strawberries; Treatment period: Seedling stage: 1-2 weeks after seed germination; Tillering stage: before jointing of grasses; Pre-flowering stage: 10-15 days before flower bud differentiation.
[0013] Preferably, the chitosan oligosaccharide stress resistance evaluation correlation method further includes stress resistance mechanism verification and long-term effect tracking: Verification of stress resistance mechanism: The expression levels of stress resistance-related genes were determined by real-time quantitative PCR; changes in cell ultrastructure were observed by transmission electron microscopy. Long-term effect tracking: Track the growth cycle of plants after treatment; evaluate the impact of chitosan oligosaccharides on soil microbial communities.
[0014] Preferably, the preparation steps of the chitosan oligosaccharide solution include: Dissolving: Add the chitosan oligosaccharide powder to deionized water and stir until completely dissolved; Sterilization: Sterilization by filtration; Stabilizer addition: Add 0.01-0.1% of Tween-20 or polyvinylpyrrolidone to improve solution stability.
[0015] The beneficial effects of this invention are as follows: 1. This invention establishes a multiple regression model of polymerization degree-concentration-physiological index changes, which comprehensively considers the interaction between multiple factors such as chitosan oligosaccharide concentration, polymerization degree, and treatment time. It can comprehensively and accurately evaluate the real effect of chitosan oligosaccharide on plant stress resistance under different adverse conditions, avoids the limitations of single-factor evaluation, and provides a more scientific and reliable basis for the application of chitosan oligosaccharide in agricultural production.
[0016] 2. In this invention, the stress resistance effect of chitosan oligosaccharides is classified into three levels: strong, medium, and weak. Specific optimized application schemes, including concentration ranges, degree of polymerization combinations, and application frequencies, are provided for different stress types and plant species. This helps to rationally select the application parameters of chitosan oligosaccharides according to different needs and conditions in actual agricultural production, thereby improving its application effect, reducing usage costs, and promoting the widespread application of chitosan oligosaccharides in agricultural production.
[0017] 3. This invention also includes steps for verifying the stress resistance mechanism and tracking long-term effects. By measuring the expression levels of stress resistance-related genes using real-time quantitative PCR and observing changes in cell ultrastructure using transmission electron microscopy, the mechanism by which chitosan oligosaccharides enhance plant stress resistance can be explored in depth, providing theoretical support for further optimization of the structure and function of chitosan oligosaccharides. Simultaneously, tracking the growth cycle of treated plants and assessing the impact of chitosan oligosaccharides on the soil microbial community can provide a comprehensive understanding of the long-term effects of chitosan oligosaccharides, ensuring their sustainable application in agricultural production. Attached Figure Description
[0018] Figure 1 This is a flowchart of the chitosan oligosaccharide stress resistance evaluation correlation method of the present invention. Detailed Implementation
[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.
[0020] like Figure 1 This invention provides a method for evaluating the stress resistance of chitosan oligosaccharides, which includes the following steps: S1. Qualitative detection by ion chromatography: High-performance anion exchange chromatography-integrated pulse amperometric detection (HPAEC-IPAD) was used to separate and detect chitosan oligosaccharide samples. The degree of polymerization (DP) of each component in the sample was calculated by comparing the retention time of the standard and the degree of polymerization-retention factor correlation equation. S2. Stress resistance treatment: The target plants were divided into a control group and a chitosan oligosaccharide treatment group. The treatment group was sprayed with chitosan oligosaccharide solution of different concentrations (5-500 mg / L) and degrees of polymerization (DP2-DP6), and stress conditions such as low temperature, drought and salt stress were set simultaneously. S3. Physiological index measurement: Plant samples were collected regularly to measure relative conductivity, malondialdehyde (MDA) content, antioxidant enzyme activity (SOD, POD, CAT) and root activity. S4. Data Correlation Analysis: Establish a multiple regression model of degree of polymerization (DP)-concentration-physiological index changes to determine the key parameter combination of chitosan oligosaccharide stress resistance effect; S5. Stress Resistance Level Classification: Based on the model output results, the stress resistance effect of chitosan oligosaccharide is classified into three levels: strong, medium and weak, and an optimized application scheme is output.
[0021] In the ion chromatography qualitative detection step: The correlation equation is a linear equation: logk'=a·DP+c Where: k' is the retention factor, defined as t_R is the retention time of the sample component. is the dead time; a is a real parameter reflecting the slope of the effect of the degree of polymerization on the retention factor; c is a constant reflecting the basic retention characteristics of the chromatographic system; Parameters a and c were obtained by fitting the retention time-polymerization degree data of the standards (DP2-DP6) using the least squares method or weighted least squares method, with a goodness of fit R² ≥ 0.99.
[0022] Among the stress resistance treatment steps: The stress conditions were set as follows: for low temperature stress, treatment at 4℃ for 24-72 hours, adjusted to 0-10℃ depending on the plant species; for drought stress, irrigation was stopped until the soil moisture content was ≤15%, and drought was simulated by polyethylene glycol (PEG6000) at a concentration of 5-20%; for salt stress, irrigation was carried out with 100-200mM NaCl solution, using a mixed salt solution (such as a 1:1 molar mixture of NaCl and Na2SO4); foliar spraying or root irrigation was used, with spraying in the early morning or evening to avoid high-temperature evaporation.
[0023] Among the steps for measuring physiological indicators: Relative conductivity determination: Take plant leaves or roots, rinse with deionized water, cut into 1cm segments, place in 20mL of deionized water, and extract by shaking at 25℃ for 24 hours. MDA content determination: The thiobarbituric acid (TBA) method was used, and the extraction solution was 10% trichloroacetic acid (TCA). The reaction system was a 0.5% TBA solution in 20% TCA. After boiling in a water bath for 30 minutes and cooling, the absorbance at wavelengths of 532 nm and 600 nm was measured. MDA content is expressed in nmol / gFW (fresh weight).
[0024] Among them, the data correlation analysis step includes: Multiple regression model: Input variables include chitosan oligosaccharide concentration (C, unit: mg / L), degree of polymerization (DP), and treatment time (T, unit: days); The output variables are the rate of change of relative conductivity (ΔREC%), the rate of change of MDA content (ΔMDA%), and the rate of increase in antioxidant enzyme activity (ΔSOD%, ΔPOD%, ΔCAT%). The model expression is Where ε is the error term; Model validation: Cross-validation or independent test set validation was used, with a determination coefficient R² ≥ 0.85. The significance of the model was tested by analysis of variance (ANOVA), with a p-value < 0.05.
[0025] The criteria for classifying stress resistance levels are as follows: High potency: ΔREC%≤30%, ΔMDA%≤25%, ΔSOD%≥40%, and at least two antioxidant enzyme activities increased by ≥30%; Medium-efficacy: 30% < ΔREC% ≤ 50%, 25% < ΔMDA% ≤ 40%, 20% ≤ ΔSOD% < 40%, and at least one antioxidant enzyme activity improvement rate ≥ 20%; Weak efficacy: ΔREC%>50%, ΔMDA%>40%, ΔSOD%<20%, and the rate of increase in antioxidant enzyme activity is <15%.
[0026] The optimized application solutions include: Concentration range: Adjusted according to the type of stress; Combination of aggregation degree: Low temperature stress: DP3-DP4 as the main component, supplemented by a small amount of DP2; Salt stress: DP2-DP3 are the main components, supplemented by DP5; Application frequency: For short-term stress (such as cold damage), spray 1-2 times at intervals of 3-7 days; For long-term stress (such as saline-alkali land), spray 3-5 times at 10-15 day intervals.
[0027] The target plants include: Food crops: rice, wheat, corn, soybeans; cash crops: rapeseed, cotton, tobacco, sugarcane; fruit crops: apples, citrus, grapes, strawberries; Treatment period: Seedling stage: 1-2 weeks after seed germination; Tillering stage: before jointing of grasses; Pre-flowering stage: 10-15 days before flower bud differentiation.
[0028] Among them, the correlation methods for evaluating the stress resistance of chitosan oligosaccharides also include verification of the stress resistance mechanism and long-term effect tracking: Verification of stress resistance mechanism: The expression levels of stress resistance-related genes (such as DREB, POD, and CAT) were determined by real-time quantitative PCR (qPCR); changes in cell ultrastructure were observed by transmission electron microscopy (TEM). Long-term effect tracking: Track the growth cycle of plants after treatment (e.g., yield, fruit setting rate); assess the impact of chitosan oligosaccharide on soil microbial community (e.g., 16S rRNA sequencing analysis).
[0029] The preparation steps for the chitosan oligosaccharide solution include: Dissolving: Add the chitosan oligosaccharide powder to deionized water and stir until completely dissolved; Sterilization: Sterilize by filtration (0.22μm filter membrane) or high temperature and high pressure sterilization (121℃, 15 minutes). Stabilizer addition: Add 0.01-0.1% of Tween-20 or polyvinylpyrrolidone (PVP) to improve solution stability.
[0030] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.
[0031] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A method for evaluating the stress resistance of chitosan oligosaccharides, characterized in that: Includes the following steps: S1. Qualitative detection by ion chromatography: High-performance anion exchange chromatography-integral pulse amperometric detection method was used to separate and detect chitosan oligosaccharide samples. The degree of polymerization of each component in the sample was calculated by comparing the retention time of the standard and the degree of polymerization-retention factor correlation equation. S2. Stress resistance treatment: The target plants were divided into a control group and a chitosan oligosaccharide treatment group. The treatment groups were sprayed with chitosan oligosaccharide solutions of different concentrations and degrees of polymerization, and low temperature, drought and salt stress conditions were set simultaneously. S3. Physiological index measurement: Plant samples are collected regularly to measure relative conductivity, malondialdehyde content, antioxidant enzyme activity and root vitality; S4. Data Correlation Analysis: Establish a multiple regression model of polymerization degree-concentration-physiological index changes to determine the key parameter combination of chitosan oligosaccharide's stress resistance effect; S5. Stress Resistance Level Classification: Based on the model output results, the stress resistance effect of chitosan oligosaccharide is classified into three levels: strong, medium and weak, and an optimized application scheme is output.
2. The chitosan oligosaccharide stress resistance evaluation correlation method according to claim 1, characterized in that: In the ion chromatography qualitative detection step: The correlation equation is a linear equation: logk'=a·DP+c Where: k' is the retention factor, defined as t_R is the retention time of the sample component. is the dead time; a is a real parameter reflecting the slope of the effect of the degree of polymerization on the retention factor; c is a constant reflecting the basic retention characteristics of the chromatographic system; Parameters a and c were obtained by fitting the retention time-degree of polymerization data of the standard. The fitting methods included least squares or weighted least squares, and the goodness of fit R² ≥ 0.
99.
3. The chitosan oligosaccharide stress resistance evaluation correlation method according to claim 1, characterized in that: In the stress resistance treatment step: The stress conditions were set as follows: for low temperature stress, treatment at 4℃ for 24-72 hours, adjusted to 0-10℃ depending on the plant species; for drought stress, irrigation was stopped until the soil moisture content was ≤15%, and drought was simulated by polyethylene glycol at a concentration of 5-20%; for salt stress, irrigation was carried out with 100-200mM NaCl solution, using a mixed salt solution; foliar spraying or root irrigation was used, with spraying time in the early morning or evening to avoid high-temperature evaporation.
4. The chitosan oligosaccharide stress resistance evaluation correlation method according to claim 1, characterized in that: In the physiological indicator measurement steps: Relative conductivity determination: Take plant leaves or roots, rinse with deionized water, cut into 1cm segments, place in 20mL of deionized water, and extract by shaking at 25℃ for 24 hours. MDA content determination: The thiobarbituric acid method was used, and the extraction solution was 10% trichloroacetic acid; The reaction system was a 0.5% TBA solution in 20% TCA. After boiling in a water bath for 30 minutes and cooling, the absorbance at wavelengths of 532 nm and 600 nm was measured. The unit for MDA content is nmol / gFW.
5. The chitosan oligosaccharide stress resistance evaluation correlation method according to claim 1, characterized in that: In the data correlation analysis step: Multiple regression model: Input variables include chitosan oligosaccharide concentration C (mg / L), degree of polymerization DP, and treatment time T (days); The output variables are the rate of change of relative conductivity ΔREC%, the rate of change of MDA content ΔMDA%, the rate of increase in antioxidant enzyme activity ΔSOD%, ΔPOD%, and ΔCAT%. The model expression is Where ε is the error term; Model validation: Cross-validation or independent test set validation was used, with a determination coefficient R² ≥ 0.
85. The significance of the model was tested by analysis of variance, with a p-value < 0.
05.
6. The chitosan oligosaccharide stress resistance evaluation correlation method according to claim 1, characterized in that: The criteria for classifying stress resistance levels are as follows: High potency: ΔREC%≤30%, ΔMDA%≤25%, ΔSOD%≥40%, and at least two antioxidant enzyme activities increased by ≥30%; Medium-efficacy: 30% < ΔREC% ≤ 50%, 25% < ΔMDA% ≤ 40%, 20% ≤ ΔSOD% < 40%, and at least one antioxidant enzyme activity improvement rate ≥ 20%; Weak efficacy: ΔREC%>50%, ΔMDA%>40%, ΔSOD%<20%, and the rate of increase in antioxidant enzyme activity is <15%.
7. The chitosan oligosaccharide stress resistance evaluation correlation method according to claim 1, characterized in that: The optimized application scheme includes: Concentration range: Adjusted according to the type of stress; Combination of aggregation degree: Low temperature stress: DP3-DP4 as the main component, supplemented by a small amount of DP2; Salt stress: DP2-DP3 are the main components, supplemented by DP5; Application frequency: For short-term stress, spray 1-2 times at intervals of 3-7 days; For prolonged stress, spray 3-5 times at 10-15 day intervals.
8. The chitosan oligosaccharide stress resistance evaluation correlation method according to claim 1, characterized in that: Target plants include: Food crops: rice, wheat, corn, soybeans; cash crops: rapeseed, cotton, tobacco, sugarcane; fruit crops: apples, citrus, grapes, strawberries; Treatment period: Seedling stage: 1-2 weeks after seed germination; Tillering stage: before jointing of grasses; Pre-flowering stage: 10-15 days before flower bud differentiation.
9. The chitosan oligosaccharide stress resistance evaluation correlation method according to claim 1, characterized in that: The method for evaluating the stress resistance of chitosan oligosaccharides also includes verification of the stress resistance mechanism and long-term effect tracking: Verification of stress resistance mechanism: The expression levels of stress resistance-related genes were determined by real-time quantitative PCR; changes in cell ultrastructure were observed by transmission electron microscopy. Long-term effect tracking: Track the growth cycle of plants after treatment; evaluate the impact of chitosan oligosaccharides on soil microbial communities.
10. The chitosan oligosaccharide stress resistance evaluation correlation method according to claim 1, characterized in that: The preparation steps of the chitosan oligosaccharide solution include: Dissolving: Add the chitosan oligosaccharide powder to deionized water and stir until completely dissolved; Sterilization: Sterilization by filtration; Stabilizer addition: Add 0.01-0.1% of Tween-20 or polyvinylpyrrolidone to improve solution stability.