Preparation method for high-acyl gellan oligosaccharide and use thereof in immune induction
The preparation of high-acyl gellan gum oligosaccharides via a complex oxidative hydrolysis method solves the production and environmental problems of existing plant immune inducers, achieving efficient and stable plant immune activation and disease control effects.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- QILU UNIVERSITY OF TECHNOLOGY (SHANDONG ACADEMY OF SCIENCES)
- Filing Date
- 2025-04-22
- Publication Date
- 2026-05-28
AI Technical Summary
Existing plant immune inducers have shortcomings such as complex production processes, unstable quality, high environmental risks, or insignificant effects. There is a lack of effective methods for the development and application of high-acyl gellan gum oligosaccharides in the field of plant immune inducement.
High-acyl gellan gum oligosaccharides were prepared using a complex oxidative hydrolysis method, which included hydrolysis with trifluoroacetic acid and hydrogen peroxide under specific conditions, combined with methanol solution treatment and purification using a 5 kDa filter membrane to obtain oligosaccharides with high purity and specific degree of polymerization.
It significantly activates the plant immune system, enhances plant disease resistance, reduces disease occurrence, increases crop yield and quality, and is environmentally friendly with no environmental risks.
Smart Images
Figure CN2025090323_28052026_PF_FP_ABST
Abstract
Description
A method for preparing high-acyl gellan gum oligosaccharides and their application in immune induction. Technical Field
[0001] This invention belongs to the field of preparation and application of oligosaccharide active substances, and specifically relates to a method for preparing high-acyl gellan gum oligosaccharides and their application in immune induction. Background Technology
[0002] In agricultural production, plant diseases have always been a significant factor affecting crop yield and quality. To effectively control plant diseases and reduce the use of chemical pesticides, strategies that utilize the plant's own immune system to enhance its disease resistance have received widespread attention. Plant immune inducers, as substances capable of inducing and activating the plant's own immune system, play a crucial role.
[0003] Currently, there are various types of plant immune inducers, including microbial inducers (such as Bacillus, Trichoderma, and other microorganisms and their metabolites), chemically synthesized inducers (such as some salicylic acid analogs), and some inducers derived from natural products. However, these existing immune inducers have some limitations to varying degrees.
[0004] As for microbial inhibitors, their production process often relies on complex microbial fermentation and culture conditions, requiring strict production environment control. Furthermore, the quality stability of different batches is difficult to control precisely, leading to fluctuations in effectiveness during practical applications. Moreover, microorganisms themselves have a certain activity cycle, and storage and transportation conditions are relatively demanding, increasing the cost and difficulty of use.
[0005] While chemically synthesized inhibitors can ensure product quality consistency through relatively precise chemical synthesis processes, some chemically synthesized substances may pose potential environmental residue risks, which can easily have adverse effects on the ecological environment such as soil and water bodies. Furthermore, long-term use may lead to drug resistance in pathogens, affecting the effectiveness of their continued use.
[0006] For some natural product-derived inducers, on the one hand, there are problems such as limited raw material sources, complex extraction processes, and high costs; on the other hand, their effect of inducing plant immune activation may not be significant or their range of action may be narrow, making it difficult to meet the wide range of needs for plant immune enhancement in various crops under different growth environments.
[0007] Meanwhile, high-acyl gellan gum, as a natural polysaccharide with unique physicochemical properties, has been widely used in food, cosmetics, and other fields. However, there are few reports on the development and application of its oligosaccharide form in plant immune induction. Existing methods for preparing oligosaccharides from polysaccharides mostly suffer from drawbacks such as difficulty in controlling the hydrolysis process, a wide distribution of the degree of polymerization of the product, and difficulty in accurately obtaining oligosaccharides with a specific degree of polymerization and high activity. This limits the potential advantages of high-acyl gellan gum oligosaccharides in the field of plant immune induction.
[0008] Therefore, there is an urgent need to develop an efficient, stable, and environmentally friendly method for preparing high-acyl gellan gum oligosaccharides and to clarify its application effect in plant immune induction, so as to fill the gaps in existing plant immune inducing agents and provide a new and effective means for the prevention and control of plant diseases in agricultural production. Summary of the Invention
[0009] To address the above-mentioned technical problems, this invention proposes a method for preparing high-acyl gellan gum oligosaccharides and demonstrates its excellent application effect in inducing and activating the plant's own immune system and improving plant disease resistance. It is expected to bring new breakthroughs and developments to the field of plant immune induction and resistance.
[0010] A method for preparing high-acyl gellan gum oligosaccharides mainly employs a complex oxidative hydrolysis method, comprising the following steps:
[0011] (1) Select food-grade high acyl gellan gum with a purity ≥99%, dissolve it in pure water at a solid-liquid ratio of 0.5-5:20, add 0.5-5% trifluoroacetic acid and 0.5-1% hydrogen peroxide, stir evenly, raise the temperature to 70-90℃, keep it for 1-2 hours, and carry out heating hydrolysis treatment.
[0012] (2) After the hydrolysis product obtained in (1) is concentrated to 1 / 4-1 / 10 of the original volume by pressure evaporation at no higher than 60°C, methanol solution is added according to the ratio of concentrate:methanol = 1:3-1:5, and then evaporated to dryness under reduced pressure at 60°C to remove residual trifluoroacetic acid and hydrogen peroxide.
[0013] (3) The product after evaporation is dissolved in pure water at a mass ratio of 1:9. Then, it is purified by using a filter membrane with a molecular weight cutoff of 5 kDa. The permeate is collected and then evaporated under reduced pressure at 60°C to obtain high acyl gellan gum oligosaccharides with a degree of polymerization of 4-10.
[0014] The application of the high-acyl gellan gum oligosaccharide obtained through the above preparation steps in immune induction is also a key aspect protected by this invention.
[0015] The present invention has the following advantages and effects compared with the prior art:
[0016] (1) The preparation method of the present invention can efficiently and stably prepare high-purity, high-acyl gellan gum oligosaccharides with a specific degree of polymerization of 4-10;
[0017] (2) The prepared high-acyl gellan gum oligosaccharide can be prepared into a solution and sprayed on Arabidopsis leaves. It can significantly activate the Arabidopsis salicylic acid pathway, increase the level of reactive oxygen species in its tissues, and increase the enzyme activity of phenylalanine transaminase and catalase in the tissues. It can also significantly activate the plant immune system of Arabidopsis, resist the invasion of pathogenic fungus Pseudomonas fluorescens Pst DC3000, and reduce the plant disease index.
[0018] (3) The high acyl gellan gum oligosaccharide prepared by the present invention has shown significant effects in inducing plant immunity and resistance, which helps to reduce the occurrence of plant diseases and improve crop yield and quality. Attached Figure Description
[0019] Figure 1 shows the TLC spectra before and after degradation in Example 1;
[0020] Figure 2 shows the infrared spectra of gellan gum before and after degradation in Example 1;
[0021] Figure 3 shows the LC-MS of the gellan gum oligosaccharide mixture in Example 1;
[0022] Figure 4 shows the disease index analysis of each group after infection by pathogen DC3000 in Example 4;
[0023] Figure 5 shows the gene expression in Arabidopsis leaves after treatment with gellan gum oligosaccharide in Example 4 (Figure A: relative expression level of gene PDF1.2; Figure B: relative expression level of gene PR1).
[0024] Figure 6 shows the detection of reactive oxygen species using laser confocal microscopy in Example 4;
[0025] Figure 7 shows the changes in the defensive enzymes in Arabidopsis thaliana after spraying with gellan gum oligosaccharide in Example 4.
[0026] Figure 8 shows the specific implementation method of the salicylic acid and jasmonic acid content in Arabidopsis thaliana after infection in Example 4. Detailed Implementation
[0027] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments. Example 1
[0028] Food-grade high-acyl gellan gum with a purity of 99.5% was selected. A certain amount of the high-acyl gellan gum was dissolved in pure water at a solid-liquid ratio of 1:20 to obtain a high-acyl gellan gum solution.
[0029] Add 2% trifluoroacetic acid and 0.8% hydrogen peroxide to the above solution. After stirring thoroughly with a magnetic stirrer, transfer the solution to a reactor equipped with heating and stirring functions, heat to 80°C, and hydrolyze for 1.5 hours. After hydrolysis, transfer the hydrolysis product to a rotary evaporator and concentrate it under pressure at 50°C until it is concentrated to 1 / 6 of its original volume. Then, slowly add methanol solution at a ratio of 1:4 (concentrate:methanol), stir thoroughly, and evaporate again under reduced pressure at 60°C using a rotary evaporator to remove residual trifluoroacetic acid and hydrogen peroxide. Add pure water to the evaporated product at a mass ratio of 1:9, dissolve thoroughly with ultrasonic assistance, and then purify by passing through a filter membrane with a molecular weight cutoff of 5 kDa. Collect the permeate, and finally place the permeate in a vacuum drying oven and evaporate under reduced pressure at 60°C to obtain the high-acyl gellan gum oligosaccharide product.
[0030] As shown in Figures 1-3, the high-acyl gellan gum oligosaccharide products obtained in this embodiment were characterized by thin-layer chromatography, infrared spectroscopy, and liquid chromatography-mass spectrometry, respectively. Example 2
[0031] Food-grade high-acyl gellan gum with a purity of 99.2% was selected. An appropriate amount of the high-acyl gellan gum was accurately weighed and dissolved in pure water at a solid-liquid ratio of 0.5:20 (g / mL) to prepare a high-acyl gellan gum solution.
[0032] Add 0.5% trifluoroacetic acid and 0.5% hydrogen peroxide to the above solution. After thorough mixing with a mechanical stirrer, transfer the solution to a three-necked flask equipped with temperature control and stirring. Heat to 70°C and hydrolyze for 2 hours. After hydrolysis, transfer the hydrolysis product to a vacuum concentration apparatus and concentrate under pressure at 40°C until it is reduced to 1 / 10 of its original volume. Then, carefully add methanol solution at a ratio of 1:3 (concentrate:methanol), stir well, and evaporate to dryness under reduced pressure at 60°C using a vacuum distillation apparatus to remove residual trifluoroacetic acid and hydrogen peroxide. Add pure water to the evaporated product at a mass ratio of 1:9 and dissolve thoroughly with the aid of a magnetic stirrer. Then, purify using a filter membrane with a molecular weight cutoff of 5 kDa. Collect the permeate and finally place it in a vacuum drying oven and evaporate to dryness under reduced pressure at 60°C to obtain the high-acyl gellan gum oligosaccharide product. Example 3
[0033] Food-grade high-acyl gellan gum was selected, and its purity reached 99.8% after professional testing. A certain amount of the high-acyl gellan gum was measured and dissolved in pure water at a solid-liquid ratio of 5:20 (g / mL) to form a high-acyl gellan gum solution.
[0034] Add 5% trifluoroacetic acid and 1% hydrogen peroxide to the above solution. After thorough mixing with a vigorous stirrer, transfer the solution to a reflux evaporator and heat to 90°C for hydrolysis for 1 hour. After hydrolysis, transfer the hydrolysis product to a rotary evaporator and concentrate it under pressure at 55°C until it is reduced to 1 / 4 of its original volume. Then, slowly add methanol solution at a ratio of 1:5 (concentrate:methanol), stir well, and evaporate again under reduced pressure at 60°C using a rotary evaporator to remove residual trifluoroacetic acid and hydrogen peroxide. Add pure water to the evaporated product at a mass ratio of 1:9 and dissolve it thoroughly with the aid of a stirrer. Then, purify the product using a filter membrane with a molecular weight cutoff of 5 kDa, collect the permeate, and finally place the permeate in a vacuum drying oven and evaporate it under reduced pressure at 60°C to obtain the high-acyl gellan gum oligosaccharide product. Example 4
[0035] Several healthy Arabidopsis thaliana plants with uniform growth were selected and divided into a blank control group and experimental groups with different doses. The treatment methods are as follows:
[0036] Blank control group: No additional treatment was given to the Arabidopsis plants. They were maintained with normal routine care, and suitable light, temperature and humidity conditions were kept. The blank control group was sprayed with distilled water in the same way as the experimental group for oligosaccharide spraying.
[0037] Different dosage experimental groups: The high-acyl gellan gum oligosaccharide product prepared in Example 1 was used to prepare oligosaccharide solutions of 50, 100, 200, and 500 μg / mL. The oligosaccharide solutions were sprayed onto the leaves of Arabidopsis thaliana using a small spray bottle, with 1.5 mL of oligosaccharide solution being sprayed evenly on each plant.
[0038] Twenty-four hours later, Arabidopsis thaliana plants sprayed with oligosaccharides were collected for enzyme activity testing. Five samples were collected from each treatment group, and the experiment was repeated three times.
[0039] Twenty-four hours after treatment, the levels of reactive oxygen species (ROS) (Figure 6), transcription level of PR1 (a key gene in the salicylic acid pathway) (Figure 5), expression level of PDF1.2 (a key gene in the jasmonic acid pathway) (Figure 5), activity levels of defense-related enzymes such as CAT / PAL (Figure 7), malondialdehyde (Figure 7), and content of salicylic acid and jasmonic acid in the plants (Figure 8) were measured in each group of Arabidopsis thaliana plants, and their disease index was analyzed.
[0040] Regarding the disease index, different concentrations of gellan gum oligosaccharide were sprayed on Arabidopsis thaliana three days before Pst DC3000 infection. Within the concentration range of 50 μg / mL to 500 μg / mL, 100 μg / mL gellan gum oligosaccharide showed the best effect, as shown in Figure 4 (left). The yellowing area and number of yellowed leaves were significantly less than those of Arabidopsis thaliana directly infected with Pst DC3000. As shown in Figure 4 (right), the disease index of Arabidopsis thaliana directly injected with Pst DC3000 was 92.4%, the disease index of Arabidopsis thaliana injected with Pst DC3000 after spraying with gellan gum oligosaccharide was 81.2%, and the disease index of Arabidopsis thaliana injected with Pst DC3000 after spraying with 100 μg / mL oligosaccharide was 80.5%. Compared with the control group, the disease index of Arabidopsis thaliana after spraying with gellan gum oligosaccharide decreased by 11.9%.
[0041] After spraying with gellan gum oligosaccharide, the salicylic acid pathway gene PR1 in Arabidopsis thaliana did not change, while the gene PDF1.2, which is responsible for regulating the jasmonic acid pathway, was upregulated by 9.08 times. Therefore, this paper speculates that gellan gum oligosaccharide regulates immunity through the jasmonic acid pathway of plant immunity, thereby resisting the invasion of pathogens.
[0042] Under excitation light, green fluorescence was clearly visible in the upper epidermis of Arabidopsis treated with gellan gum oligosaccharide, while no green fluorescence was observed in the upper epidermis of Arabidopsis treated with water, indicating that gellan gum oligosaccharide induces a large amount of reactive oxygen species to be produced in the upper epidermis of Arabidopsis.
[0043] Twenty-four hours after spraying oligosaccharides, the enzyme activity of CAT also increased, with a significant increase of 22.16% compared to the control, and a significant increase of 33.6% in PAL enzyme activity.
[0044] Spraying oligosaccharides did not significantly alter the salicylic acid and jasmonic acid levels in Arabidopsis thaliana. However, injecting DC3000 alone significantly increased the salicylic acid content, consistent with the results of Q-PCR detection of changes in gene PR1. Spraying oligosaccharides three days prior, followed by DC3000 injection, resulted in a significant change in jasmonic acid content in Arabidopsis thaliana, reaching 4.78 times that of the control group. This is consistent with the results of Q-PCR detection of the resistance gene PDF1.2, further demonstrating that gellan gum oligosaccharides regulate plant immunity by activating the jasmonic acid pathway in plants.
[0045] In summary, the high-acyl gellan gum oligosaccharide preparation method provided by this invention utilizes food-grade, high-purity high-acyl gellan gum and employs a composite oxidative hydrolysis method to prepare high-acyl gellan gum oligosaccharides that can effectively activate the plant's immune system and reduce disease index.
[0046] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.
Claims
1. A method for preparing high-acyl gellan gum oligosaccharides, characterized in that, The main steps include the following: (1) Dissolve high acyl gellan gum in pure water with a solid-liquid ratio of 0.5-5:20 to obtain a high acyl gellan gum solution; (2) Add 0.5-5% trifluoroacetic acid and 0.5-1% hydrogen peroxide to the high acyl gellan gum solution obtained in (1), stir evenly, and then heat to 70-90℃ for hydrolysis for 1.5-2 hours; (3) The hydrolyzed material is rotary evaporated at 40-55℃ and concentrated under reduced pressure to 1 / 10-1 / 4 of the original volume. Then, methanol is slowly added according to the volume ratio of concentrate to methanol = 1:3-5, and rotary evaporated again. (4) Add pure water to the material obtained in (3) at a mass ratio of 1:9 to dissolve it, filter it, and evaporate the obtained filtrate under reduced pressure at 60°C to obtain the high acyl gellan gum oligosaccharide product.
2. The method for preparing a high-acyl gellan gum oligosaccharide as described in claim 1, characterized in that, (1) The medium-high acyl gellan gum is food grade with a purity of ≥99%.
3. The method for preparing a high-acyl gellan gum oligosaccharide as described in claim 1, characterized in that, (1) The solid-liquid ratio of medium-high acyl gellan gum to pure water is 1:
20.
4. The method for preparing a high-acyl gellan gum oligosaccharide as described in claim 1, characterized in that, (2) Add 2% trifluoroacetic acid and 0.8% hydrogen peroxide, stir evenly and heat to 80°C, and hydrolyze for 1.5 hours.
5. The method for preparing a high-acyl gellan gum oligosaccharide as described in claim 1, characterized in that, (3) The hydrolyzed material is rotary evaporated at 50°C and concentrated under reduced pressure to 1 / 6 of the original volume. Then, methanol is slowly added according to the volume ratio of concentrate to methanol = 1:4, and rotary evaporated again.
6. The method for preparing a high-acyl gellan gum oligosaccharide as described in claim 1, characterized in that, (4) uses a filter membrane with a molecular weight cutoff of 5 kDa for filtration.
7. The application of the high-acyl gellan gum oligosaccharide prepared by the preparation method described in claim 1 in the field of immune induction.
8. The application as described in claim 7, characterized in that, The high-acyl gellan gum oligosaccharide was prepared into a solution and applied by spraying.
9. The application as described in claim 8, characterized in that, The concentration of the solution is 50 μg / mL to 500 μg / mL.