Biotransformation preparation method of curdlan oligosaccharide and application of curdlan oligosaccharide in preparation of immune induced resistance products

Abstract

The invention belongs to the field of preparation and application of oligosaccharide active substances, and provides a biotransformation preparation method of curdlan oligosaccharide and application of the curdlan oligosaccharide in preparation of immune induced resistance products. According to the preparation method, a composite oxidation hydrolysis technology is adopted, food-grade and high-purity raw materials are selected, and the curdlan oligosaccharide with the polymerization degree of 4-10 is successfully prepared through the steps of reagent addition in a specific proportion, strict hydrolysis condition control, concentration, impurity removal, membrane filtration purification and the like. After the oligosaccharide is prepared into a solution, the solution is sprayed on Arabidopsis thaliana leaves, the salicylic acid signal channel of Arabidopsis thaliana can be obviously activated, and the activity of related enzymes in the Arabidopsis thaliana can be improved, so that the immune system of plants is activated, the invasion of pathogenic bacteria is effectively resisted, and the disease index is reduced. Therefore, the curdlan oligosaccharide is an efficient plant immune resistance inducer material. The invention not only provides a new effective substance for the field of plant immune induced resistance, but also develops a corresponding preparation method, and shows a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of preparation and application of oligosaccharide active substances, and specifically relates to a biotransformation preparation method of guar gum oligosaccharide and its application in the preparation of immune-inducing products. Background Technology

[0002] In agricultural production, plant diseases seriously affect crop yield and quality. Therefore, strategies that rely on the plant's own immune system to resist diseases have attracted much attention, and plant immune inducers play a key role in this process.

[0003] Each existing immune inducer has its limitations. Specifically, the production of microbial inducers relies on complex fermentation conditions and has strict environmental requirements, making batch-to-batch quality inconsistency difficult. Furthermore, their storage and transportation conditions are quite demanding, resulting in high costs. While chemically synthesized inducers are relatively stable in quality, they pose a risk of environmental residues and can easily lead to drug resistance in pathogens. Naturally derived inducers, on the other hand, face challenges such as limited raw materials, complex extraction processes, and high costs. In addition, their immune activation effects may be less than ideal, or their range of action may be relatively narrow.

[0004] Currant has wide applications in the food and cosmetics industries; however, the development and application of its oligosaccharide form in plant immune induction is extremely limited. This is because the conversion of polysaccharides into oligosaccharides presents drawbacks such as difficulty in precisely controlling hydrolysis and a wide range of product polymerization degrees. These problems restrict the potential advantages of currant oligosaccharides in the field of plant immune induction.

[0005] Therefore, in order to fill the gaps in existing immune inducers and help control agricultural diseases, it is urgent to develop an efficient, stable and environmentally friendly method for preparing gellan gum oligosaccharides and to clarify its specific effects in application. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a biotransformation preparation method for gellan gum oligosaccharides and its application in immune induction. This preparation method, through optimizing process parameters at each step and screening suitable bacterial strains, efficiently and stably prepares gellan gum oligosaccharides and explores their application in immune induction, thereby overcoming the shortcomings of existing technologies.

[0007] The technical solution of this invention is: a method for preparing gellan gum oligosaccharides, mainly employing a complex oxidative hydrolysis method, comprising the following steps: A method for preparing gellan gum oligosaccharides by biotransformation mainly includes the following steps: (1) Dissolve guar gum in pure water with a solid-liquid ratio of 0.5-3:20 to obtain a guar gum solution; (2) Trichoderma harzianum strains were cultured in liquid culture medium induced by kerogenase hydrolase. Trichoderma harzianum AB-17-1 was cultured at 28℃ for 24 hours, centrifuged at 10000rpm for 10 minutes, and the supernatant was collected as crude enzyme solution. (3) Add the crude enzyme solution obtained in (2) to the gellan gum solution obtained in (1), adjust the pH to 4.0-6.0, stir evenly, and then heat to 40-55℃ for enzymatic hydrolysis for 1.5-2 hours; (4) After enzymatic hydrolysis, the material is rotary evaporated at 40-55℃ and concentrated under reduced pressure to 1 / 10-1 / 4 of the original volume. Then, ethanol is slowly added at a volume ratio of 1:3-5 for concentrate:ethanol. The precipitate is collected and dissolved in pure water at a mass ratio of 1:9. The steps of adding ethanol, collecting precipitate, and dissolving in pure water are repeated three times. (5) Filter the material obtained in (4), and evaporate the permeate under reduced pressure at 60°C to obtain the guar gum oligosaccharide product.

[0008] Preferably, in (1), the kerogen gum is food grade with a purity of ≥99%; the solid-liquid ratio of kerogen gum to pure water is 1:20.

[0009] Preferably, by mass fraction, the formulation of the kerogenase-induced liquid culture medium in (2) is as follows: The ingredients are: 0.1-0.5% kerogen gum, 0.1-0.5% sodium nitrate, 0.5-3% dipotassium hydrogen phosphate, 0.1-0.5% potassium chloride, 0.1-0.5% magnesium sulfate, and the balance being pure water. Furthermore, the formulation of the liquid culture medium for inducing keratogel hydrolase in (2) is as follows: The ingredients are: 0.3% kerogen gum, 0.5% sodium nitrate, 0.5% dipotassium hydrogen phosphate, 0.1% potassium chloride, 0.2% magnesium sulfate, and the remainder is pure water.

[0010] Preferably, the mass fraction of the crude enzyme solution added in (3) is 5-10%.

[0011] Furthermore, in (3), the crude enzyme solution added has a mass fraction of 5%, the pH is adjusted to 5.0, the temperature is raised to 45°C, and the enzyme is hydrolyzed for 2 hours.

[0012] Preferably, in (4), the enzymatically hydrolyzed material is rotary evaporated at 50°C, concentrated under reduced pressure to 1 / 5 of the original volume, and then ethanol is slowly added at a volume ratio of concentrate to ethanol = 1:4. The precipitate is collected and then dissolved in pure water at a mass ratio of 1:9. The steps of adding ethanol, collecting precipitate, and dissolving in pure water are repeated three times.

[0013] Preferably, in (5), a filter membrane with a molecular weight cutoff of 5 kDa is used for filtration.

[0014] The application of the guar gum oligosaccharide obtained through the above preparation steps in the preparation of immune-inducing products is also a key protected aspect of this invention.

[0015] Preferably, the guar gum oligosaccharide is prepared into a solution with a concentration of 25 μg / mL to 200 μg / mL for spraying.

[0016] The present invention has the following advantages and effects compared with the prior art: (1) By means of the preparation method provided by the present invention, guar gum oligosaccharides can be prepared in an efficient and stable manner, and the obtained guar gum oligosaccharides have the characteristics of high purity, and their specific degree of polymerization is in the range of 4-10. (2) The oligosaccharide obtained by the present invention is prepared into a solution and then sprayed on Arabidopsis leaves. It can significantly activate the salicylic acid pathway of Arabidopsis, thereby increasing the level of reactive oxygen species in Arabidopsis tissues. At the same time, the enzyme activities of phenylalanine transaminase and catalase in the tissues will also be increased accordingly. It can also significantly activate the plant immune system of Arabidopsis to resist the invasion of pathogenic fungus Pseudomonas fluorescens Pst DC3000, thereby reducing the disease index of the plant. (3) The ketoran oligosaccharide prepared by the present invention has shown a very significant effect in inducing plant immunity and resistance, which can reduce the occurrence of plant diseases to a certain extent and has a positive effect on improving the yield and quality of crops. Attached Figure Description

[0017] Figure 1 enzyme-producing strain Trichoderma harzianum Colony morphology, hyphal morphology, and phylogenetic identification results of A-B17-1; Figure 2 TLC chromatograms of gellan gum oligosaccharides (① glucose + lactose control ② gellan gum oligosaccharides). Figure 3 The infrared absorption spectra of guar gum and its oligosaccharides are shown. Figure 4 The results of ESI-MS characterization of the degree of polymerization of guar gum oligosaccharides; Figure 5 Changes in disease index induced by Pst DC3000 after Arabidopsis thaliana pretreatment with ketoran oligosaccharide; Figure 6 Analysis of Pst DC3000 growth in leaves of Arabidopsis thaliana after pretreatment with ketoran oligosaccharides; Figure 7 Changes in the expression levels of salicylic acid resistance genes in Arabidopsis thaliana after pretreatment with ketorum oligosaccharides; Figure 8Changes in the expression levels of resistance genes via the jasmonic acid pathway in Arabidopsis thaliana after pretreatment with guar gum oligosaccharides; Figure 9 Analysis of ROS production levels in Arabidopsis thaliana leaves pretreated with ketorum oligosaccharides; Figure 10 The changes in the activity of defensive enzymes in Arabidopsis thaliana after pretreatment with guar gum oligosaccharides were investigated. Detailed Implementation

[0018] 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.

[0019] Example 1 Accurately weigh 1g of food-grade gellan gum (purity ≥99%) and add it to 20mL of pure water at a solid-liquid ratio of 1:20. Use a magnetic stirrer to stir thoroughly at room temperature until the gellan gum is completely dissolved to obtain a gellan gum solution for later use.

[0020] A liquid culture medium for inducing curdlan hydrolysate enzymes was prepared with the following formula: curdlan 0.3%, sodium nitrate 0.5%, dipotassium hydrogen phosphate 0.5%, potassium chloride 0.1%, magnesium sulfate 0.2%, with the remainder added to 100% with pure water. The self-selected *Trichoderma harzianum* strain was then used. Trichoderma harzianum A-B17-1 was inoculated into an Erlenmeyer flask containing 100 mL of the culture medium and incubated for 24 hours in a shaker at 28°C and 180 rpm.

[0021] After the culture is completed, the culture medium is transferred to a centrifuge tube and centrifuged at 10,000 rpm for 10 minutes. After centrifugation, the upper fermentation supernatant is carefully collected, which is the crude enzyme solution, used for subsequent enzymatic hydrolysis steps.

[0022] Take 10 mL of the obtained gellan gum solution and add 5% (i.e., 0.5 mL) of the crude enzyme solution from the fermentation supernatant of Trichoderma harzianum. Slowly adjust the pH of the solution to 5.0 using dilute hydrochloric acid solution. After stirring evenly with a magnetic stirrer, transfer the solution to a constant temperature water bath and heat it to 45°C. Continue enzymatic hydrolysis at this temperature for 2 hours to ensure that the enzymatic hydrolysis is complete.

[0023] The enzymatically hydrolyzed material was transferred to a rotary evaporator, and the temperature was set to 50°C for rotary evaporation to concentrate the material to 1 / 5 of its original volume under reduced pressure. Then, ethanol was slowly added to the concentrate at a volume ratio of 1:4 (concentrate:ethanol) while stirring. Once a precipitate formed, it was collected by filtering with filter paper. The precipitate was then dissolved by adding pure water at a mass ratio of 1:9. This process of adding ethanol to precipitate and dissolving in pure water was repeated three times to remove impurities from the material.

[0024] The material after the above treatment is filtered through a filter membrane with a molecular weight cutoff of 5 kDa, the permeate is collected, and the permeate is transferred to an evaporator and evaporated under reduced pressure at 60°C to obtain the guar gum oligosaccharide product.

[0025] The Trichoderma harzianum strain used in this invention Trichoderma harzianum The colony morphology, hyphal morphology, and phylogenetic identification results of A-B17-1 are as follows: Figure 1 As shown; The gellan gum oligosaccharides obtained in this embodiment were analyzed and characterized using thin-layer chromatography, infrared spectroscopy, and electrospray ionization mass spectrometry, respectively. The results are shown in the figure. Figures 2-4 .

[0026] Example 2

[0027] Weigh 1.5g of food-grade gellan gum (purity ≥99%) and add it to 20mL of pure water at a solid-liquid ratio of 1.5:20. Dissolve the gellan gum completely by ultrasonic-assisted stirring (ultrasonic power 200W, ultrasonic time 10 minutes) to obtain a gellan gum solution for later use.

[0028] Prepare a liquid culture medium for inducing curdlan hydrolysate enzymes with the following formula: curdlan 0.4%, sodium nitrate 0.3%, dipotassium hydrogen phosphate 0.8%, potassium chloride 0.2%, magnesium sulfate 0.3%, and pure water to 100%. Introduce the *Trichoderma harzianum* strain... Trichoderma harzianum A-B17-1 was inoculated into an Erlenmeyer flask containing 150 mL of the culture medium and incubated for 24 hours in a shaker at 30°C and 200 rpm.

[0029] After the culture is completed, centrifugation is performed at 10,000 rpm for 10 minutes, and the upper fermentation supernatant is collected as crude enzyme solution.

[0030] Take 12 mL of gellan gum solution, add 8% (i.e., 0.96 mL) of crude enzyme solution, adjust the pH value to 5.5 with sodium hydroxide solution, stir well, and then place the solution in a constant temperature water bath and heat it to 50℃ for 1.8 hours for enzymatic hydrolysis.

[0031] The enzymatically hydrolyzed material was placed in a rotary evaporator and concentrated under reduced pressure to 1 / 4 of its original volume. Then, ethanol was added at a volume ratio of concentrate to ethanol of 1:3.5. After the precipitate was collected, it was dissolved in pure water at a mass ratio of 1:9. This step was repeated three times to remove impurities.

[0032] The processed material was filtered through a 5 kDa filter membrane, the permeate was collected, and then the permeate was evaporated under reduced pressure at 60°C to obtain the gellan gum oligosaccharide product.

[0033] Example 3

[0034] Measure 0.8g of food-grade glucon (purity ≥99%) and mix it with 20mL of pure water at a solid-liquid ratio of 0.8:20. Stir the mixture at 300rpm for 30 minutes using a mechanical stirrer to ensure complete dissolution of the glucon and obtain a glucon solution for later use.

[0035] A liquid culture medium for inducing gellan gum hydrolysis was prepared, comprising: 0.2% gellan gum, 0.4% sodium nitrate, 1.2% dipotassium hydrogen phosphate, 0.3% potassium chloride, 0.4% magnesium sulfate, and the remainder being purified water. The *Trichoderma harzianum* strain was then introduced into the medium. Trichoderma harzianum A-B17-1 was inoculated into a culture flask containing 80 mL of the culture medium and incubated for 24 hours in a shaker at 26°C and 160 rpm.

[0036] After the culture is completed, the culture medium is transferred to centrifuge tubes and centrifuged at 10,000 rpm for 10 minutes. The fermentation supernatant is collected as crude enzyme solution.

[0037] Take 8 mL of gellan gum solution, add 6% (i.e. 0.48 mL) of crude enzyme solution, adjust the pH value to 4.5 with dilute hydrochloric acid, stir evenly, and heat to 42℃. Enzymatically hydrolyze at this temperature for 2 hours.

[0038] The enzymatically hydrolyzed material was transferred to a rotary evaporator and concentrated under reduced pressure to 1 / 3 of its original volume. Then, ethanol was added at a volume ratio of concentrate to ethanol of 1:4.5. The precipitate was collected and dissolved in pure water at a mass ratio of 1:9. This process was repeated three times to wash away impurities.

[0039] The processed material was filtered through a 5 kDa filter membrane, the permeate was collected, and the permeate was evaporated under reduced pressure at 60°C to obtain the guar gum oligosaccharide product, which was labeled as sample 3.

[0040] Example 4

[0041] 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: 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.

[0042] Different dosage experimental groups: The oligosaccharide product of guar gum prepared in Example 1 was used to prepare oligosaccharide solutions of 25, 50, 100 and 200 μg / mL. The oligosaccharide solutions were sprayed on the leaves of Arabidopsis thaliana with a small spray bottle, and 1.5 mL of oligosaccharide solution was sprayed evenly on each plant.

[0043] Twenty-four hours later, Arabidopsis thaliana plants sprayed with oligosaccharide solution were collected for enzyme activity detection. Five samples were collected from each treatment group, and the experiment was repeated three times.

[0044] Twenty-four hours after treatment, the disease index of Arabidopsis thaliana plants in each group was measured. Figure 5 ), the growth of PstDC3000 in plant leaves ( Figure 6 ), transcriptional levels of key genes in the salicylic acid pathway ( Figure 7 ), transcriptional levels of key genes in the jasmonic acid pathway ( Figure 8 ), leaf ROS production level ( Figure 9 ), changes in the activity levels of defense enzymes in Arabidopsis thaliana ( Figure 10 ).

[0045] Regarding the disease index, different concentrations of guar gum oligosaccharide were sprayed on Arabidopsis thaliana three days before Pst DC3000 was used to infect the plant. The guar gum oligosaccharide treatment group with a concentration of 50 mg / L showed the best effect, with the disease index decreasing by 41.2%, which was 25.83%, 29.16%, and 27.3% higher than that of 25 mg / L, 100 mg / L, and 200 mg / L, respectively.

[0046] After oligosaccharide pretreatment, the immune system in Arabidopsis thaliana was activated. When Pst DC3000 infected the plant, Arabidopsis thaliana was able to promptly initiate a strong defense response, thereby inhibiting the proliferation of Pst DC3000 within the plant. The amount of Pst DC3000 growth inside Arabidopsis thaliana leaves can, to some extent, reflect the degree of activation of the plant's defense capabilities. Compared with the untreated group, pretreatment with 50 mg / L guar gum oligosaccharides reduced the growth of Pst DC3000 in diseased leaves by 77.84%, which was 17.54%, 32.62%, and 36.00% higher than that with 25 mg / L, 100 mg / L, and 200 mg / L, respectively.

[0047] After treating Arabidopsis thaliana with glucon oligosaccharides for 24 h, the expression of salicylic acid pathway resistance marker genes pr1, pr2, and pr5 was upregulated by 10.93-fold, 6.97-fold, and 8.1-fold, respectively. However, the expression of jasmonic acid pathway resistance marker genes pdf1.2 and vsp did not show significant changes. This indicates that glucon oligosaccharide pretreatment may activate the plant immunity of Arabidopsis thaliana through the salicylic acid pathway to resist the invasion of Pst DC3000.

[0048] Fluorescence microscopy revealed no fluorescence around the stomata on the lower epidermis of Arabidopsis leaves in the water-treated group, while significant ROS production was observed in the glucon oligosaccharide-treated group. This indicates that pretreatment with glucon oligosaccharides can induce ROS production in Arabidopsis leaves, and ROS may activate downstream defense responses in Arabidopsis through MAPK cascade signaling.

[0049] After treating Arabidopsis thaliana with gluconolan oligosaccharides for 24 h, the changes in the activities of four defensive enzymes—POD, SOD, CAT, and PAL—were investigated. Oligosaccharide treatment increased POD activity by 35.20%, SOD by 59.01%, CAT by 23.80%, and PAL by 41.18%. These results indicate that, on the one hand, gluconolan oligosaccharides may enhance lignin synthesis in Arabidopsis thaliana through PAL and POD, thereby strengthening its disease resistance; on the other hand, the increased SOD and CAT activities may alleviate oxidative stress in Arabidopsis thaliana, protecting plant cells from oxidative damage.

[0050] In summary, the method for preparing glucon oligosaccharides provided by this invention utilizes food-grade, high-purity glucon and employs a compound oxidative hydrolysis method to prepare glucon oligosaccharides that can effectively activate the plant's immune system and reduce disease index.

[0051] 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 gellan gum oligosaccharides through biotransformation, characterized in that, The main steps include the following: (1) Dissolve guar gum in pure water with a solid-liquid ratio of 0.5-3:20 to obtain a guar gum solution; (2) Trichoderma harzianum strains were cultured in liquid culture medium induced by kerogenase hydrolase. Trichoderma harzianum AB-17-1 was cultured at 28℃ for 24 hours, centrifuged at 10000rpm for 10 minutes, and the supernatant was collected as crude enzyme solution. (3) Add the crude enzyme solution obtained in (2) to the gellan gum solution obtained in (1), adjust the pH to 4.0-6.0, stir evenly, and then heat to 40-55℃ for enzymatic hydrolysis for 1.5-2 hours; (4) After enzymatic hydrolysis, the material is rotary evaporated at 40-55℃ and concentrated under reduced pressure to 1 / 10-1 / 4 of the original volume. Then, ethanol is slowly added at a volume ratio of 1:3-5 for concentrate:ethanol. The precipitate is collected and dissolved in pure water at a mass ratio of 1:

9. The steps of adding ethanol, collecting precipitate, and dissolving in pure water are repeated three times. (5) Filter the material obtained in (4), and evaporate the permeate under reduced pressure at 60°C to obtain the guar gum oligosaccharide product.

2. The method for preparing gellan gum oligosaccharides by biotransformation as described in claim 1, characterized in that, (1) The guar gum is food grade with a purity of ≥99%; the solid-liquid ratio of guar gum to pure water is 1:

20.

3. The method for preparing gellan gum oligosaccharides by biotransformation as described in claim 1, characterized in that, The formulation of the liquid culture medium for inducing keratogalactosidase in (2) by mass fraction is as follows: The ingredients are: 0.1-0.5% kerogen gum, 0.1-0.5% sodium nitrate, 0.5-3% dipotassium hydrogen phosphate, 0.1-0.5% potassium chloride, 0.1-0.5% magnesium sulfate, and the balance is pure water.

4. The method for preparing gellan gum oligosaccharides by biotransformation as described in claim 3, characterized in that, The formulation of the liquid culture medium for inducing keratogalactosidase in (2) by mass fraction is as follows: The ingredients are: 0.3% kerogen gum, 0.5% sodium nitrate, 0.5% dipotassium hydrogen phosphate, 0.1% potassium chloride, 0.2% magnesium sulfate, and the remainder is pure water.

5. The method for preparing gellan gum oligosaccharides by biotransformation as described in claim 1, characterized in that, (3) The mass fraction of the crude enzyme solution added is 5-10%.

6. The method for preparing gellan gum oligosaccharides by biotransformation as described in claim 1, characterized in that, (3) The crude enzyme solution added has a mass fraction of 5%, the pH is adjusted to 5.0, the temperature is raised to 45℃, and the enzyme is hydrolyzed for 2 hours.

7. The method for preparing gellan gum oligosaccharides by biotransformation as described in claim 1, characterized in that, (4) The enzymatically hydrolyzed material is rotary evaporated at 50°C and concentrated under reduced pressure to 1 / 5 of the original volume. Then, ethanol is slowly added at a volume ratio of 1:4 for concentrate to ethanol. The precipitate is collected and dissolved in pure water at a mass ratio of 1:

9. The steps of adding ethanol, collecting precipitate, and dissolving in pure water are repeated three times.

8. The method for preparing gellan gum oligosaccharides by biotransformation as described in claim 1, characterized in that, (5) uses a filter membrane with a molecular weight cutoff of 5 kDa for filtration.

9. The application of the gellan gum oligosaccharide prepared by the preparation method described in claim 1 in the preparation of immune-inducing products.

10. The application as described in claim 9, characterized in that, The oligosaccharide of guar gum is prepared into a solution with a concentration of 25 μg / mL-200 μg / mL for spraying.

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