Application of paenibacillus kribbensis space mutant strain exopolysaccharide in preparation of polysaccharide nano-selenium

Abstract

The invention discloses an application of paenibacillus kribbensis space mutant strain exopolysaccharide in preparation of polysaccharide nano-selenium. The invention provides polysaccharide nano-selenium, which is prepared by mixing paenibacillus kribbensis exopolysaccharide, inorganic selenium and a reducing agent and reacting, has nano-scale particle size, and has higher yield and purity and better stability than nano-selenium prepared from conventional microbial strains. Meanwhile, the polysaccharide nano-selenium has a good antagonistic effect on various plant pathogenic fungi and can be used for preventing and treating various plant diseases; when being applied to tobacco production, the tobacco growth can be obviously promoted, and more effective methods are provided for promoting the growth of crops and increasing the yield. The preparation method of the polysaccharide nano-selenium provided by the invention is simple, low in cost, high in product purity and environment-friendly, and provides more efficient and rapid synthesis preparation ways for biosynthesis of the polysaccharide nano-selenium.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosynthetic nanomaterials, and more specifically relates to the use of an exopolysaccharide of a space-faring mutant strain of Paenibacillus cribenbergii in the preparation of polysaccharide nano-selenium. Background Art

[0002] Selenium is an essential trace element for plant growth, serving both nutritional enhancement and disease prevention. Nanoselenium, with its small particle size (100-1000 nm), high bioactivity, and low toxicity, offers unique value in agriculture. It can be applied to the leaves or roots to increase crop selenium content, meeting the demand for selenium-rich agricultural products, and inhibit plant pathogens through the nanoscale effect, making it a research hotspot for green agricultural inputs. However, traditional nanoselenium preparation methods (such as chemical reduction and physical methods) rely on harsh conditions (high temperature, strong acid and alkali), resulting in product aggregation, poor stability, insufficient biocompatibility, and environmental pollution. Direct application of inorganic selenium (such as sodium selenite) is easily fixed in the soil, resulting in low conversion efficiency and high ecological risks. Therefore, green and efficient biosynthesis technologies are urgently needed. Plant diseases (such as pepper anthracnose, tea root rot, and wheat stem rot) are common in agricultural production. Traditional chemical pesticides have led to resistance and residue problems, creating an urgent need for new biocontrol agents. The antibacterial activity of nanoselenium is related to its ability to induce oxidative stress and damage cell walls in pathogens. For example, existing research has shown that the Paenibacillus cribensis strain △PS04-2 can be used to produce nanoselenium for the prevention and treatment of various plant diseases. However, due to its single-action nature, nanoselenium is susceptible to environmental influences, resulting in poor stability, aggregation, and oxidation.

[0003] Polysaccharide-Selenium Nanoparticles (P-SeNPs) are nano-scale elemental selenium (Se) prepared by chemical, physical or biological methods using natural polysaccharides (such as chitosan, maitake mushroom polysaccharide, tiger milk ganoderma polysaccharide, etc.) as stabilizers or carriers. 0 ) complex. Existing disclosed biological methods for preparing polysaccharide nanoselenium include preparing polysaccharide nanoselenium through extracellular polysaccharides of bacterial strains, such as Rhizobium caragana exopolysaccharide-SeNPs, Grifola frondosa polysaccharide (GFPs)-SeNPs, Ganoderma lucidum polysaccharide (LRP)-SeNPs, and Agaricus rutaecarpa polysaccharide-SeNPs. Polysaccharide nanoselenium has great potential in the fields of medicine, food, and agriculture due to its low toxicity, high efficiency, and versatility. However, there are still relatively few disclosed polysaccharide nanoselenium. Therefore, in response to the bottlenecks of traditional nanoselenium preparation and the needs of green agricultural development, developing more efficient and environmentally friendly polysaccharide nanoselenium synthesis methods and clarifying its application potential in plant disease prevention and control and crop growth promotion are of great significance for promoting the industrialization of selenium-enriched microbial preparations, reducing dependence on chemical pesticides, and increasing the selenium content of crops. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the problems of instability, yield and purity of nano-selenium synthesized by traditional microorganisms, and to provide an application of an extracellular polysaccharide of a space mutant strain of Paenibacillus kriebensis in the preparation of polysaccharide nano-selenium.

[0005] The first object of the present invention is to provide polysaccharide nano-selenium.

[0006] The second object of the present invention is to provide a method for preparing polysaccharide nano-selenium.

[0007] The third object of the present invention is to provide applications of polysaccharide nano-selenium.

[0008] The third object of the present invention is to provide a space mutant strain of Paenibacillus criebensis ΔPS04-17 and the use of its exopolysaccharide.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The present invention provides a polysaccharide nano-selenium, which is prepared from Paenibacillus cribbensis ( Paenibacillus kribbensis ) It is prepared by mixing extracellular polysaccharides with inorganic selenium and a reducing agent.

[0010] The present invention uses a space-age mutant of Paenibacillus cribensis, ΔPS04-17, to ferment, purify, and refine extracellular polysaccharides. The resulting polysaccharide nano-selenium, after reaction with organic selenium and a reducing agent, has a nanometer-scale particle size and exhibits superior conversion efficiency, stability, yield, and purity compared to nano-selenium produced directly from the ΔPS04-17 strain. It also avoids agglomeration, flocculation, and compatibility. The polysaccharide nano-selenium provided by the present invention significantly outperforms nano-selenium produced from biological strains, exhibiting greater stability and yield, and provides additional methods and sources for the biosynthesis of polysaccharide nano-selenium.

[0011] The preparation principle of the polysaccharide nano-selenium provided by the present invention is: inorganic selenium and a reducing agent undergo a reduction reaction, and extracellular polysaccharide is used as a template material for modification to obtain polysaccharide nano-selenium.

[0012] Preferably, the Bacillus kriebensis is the space mutant strain ΔPS04-17 of Bacillus kriebensis, which has been deposited in the Guangdong Provincial Microbial Culture Collection Center on March 10, 2022, with the deposit number: GDMCC NO: 62233.

[0013] The invention provides a method for preparing Paenibacillus criebensis exopolysaccharide. The method comprises the following steps: inoculating Paenibacillus criebensis into a fermentation medium, obtaining a fermentation liquid after fermentation, and purifying, removing protein, dialyzing and freeze-drying the fermentation liquid to obtain the exopolysaccharide.

[0014] Preferably, the purification employs alcohol precipitation method, and the protein removal employs Sevage method.

[0015] Preferably, the preparation method of the exopolysaccharide is as follows: inoculating Paenibacillus chibensis into a fermentation medium, culturing, and obtaining a fermentation liquor of △PS04-17; taking the fermentation milk-white bacterial liquid, heating in a water bath, then diluting with distilled water, cooling, centrifuging, and discarding the bacterial precipitate; slowly adding anhydrous ethanol to the supernatant on ice, stirring, and storing in a refrigerator, so that white flocculent polysaccharide is precipitated and gathered into white block polysaccharide; redissolving the precipitated polysaccharide in distilled water, dialyzing after removing the protein, and freeze-drying to obtain the exopolysaccharide of △PS04-17.

[0016] Preferably, the fermentation medium employs modified Czapek medium, and the specific formula is as follows: sodium nitrate 1-3 g / L, potassium phosphate dibasic 0.5-1.5 g / L, potassium chloride 0.4-0.6 g / L, magnesium sulfate 0.4-0.6 g / L, ferrous sulfate 0.005-0.02 g / L, and sucrose 25-35 g / L, and the pH value is 7.0-7.2.

[0017] More preferably, the formula of the modified Czapek medium is as follows: sodium nitrate 1.5 g / L, potassium phosphate monobasic 1.0 g / L, potassium chloride 0.5 g / L, magnesium sulfate 0.5 g / L, ferrous sulfate 0.01 g / L, and sucrose 30.0 g / L, and the pH value is 7.0-7.2.

[0018] As a more specific embodiment, the present application provides a specific preparation method of the exopolysaccharide of Paenibacillus chibensis: (1) inoculating Paenibacillus chibensis space mutant △PS04-17 bacterial liquid into modified Czapek medium at an amount of 1%, and culturing at 28°C in a constant-temperature shaker at 150 rpm / min for 4 days to obtain a fermentation liquor of △PS04-17.

[0019] (2) taking 50 mL of the milk-white bacterial liquid of △PS04-17 after fermentation, heating in a water bath at 100°C for 30 min, diluting twice with distilled water, cooling, and centrifuging at 8000 r / min for 15 min, at this time, there is a layer of bacterial precipitate at the bottom of the centrifuge tube, and the supernatant is transparent milk-white liquid, and the bacterial precipitate is discarded.

[0020] (3) collecting the supernatant into a pre-cooled beaker, slowly adding 3 times the volume of anhydrous ethanol to the supernatant on ice, stirring clockwise with a glass rod, and storing in a refrigerator for 12-24 h, so that white flocculent polysaccharide is precipitated and gathered into white block polysaccharide.

[0021] (4) The precipitated polysaccharide was collected in a 50 mL centrifuge tube and redissolved in distilled water. A chloroform: n-butanol = 5:1 (V:V) binary system was prepared and added to the polysaccharide extract at a ratio of 1:5 (V solvent: V fermentation broth). The mixture was mixed and vigorously shaken. After standing and stratification, the solvent layer and the protein at the interface with the aqueous layer were removed. The organic solvent was then added in the above ratio until no denatured protein was produced at the interface. The resulting aqueous solution was dialyzed for 24 h and freeze-dried to obtain the purified ΔPS04-17 exopolysaccharide.

[0022] The present invention provides a method for preparing polysaccharide nano-selenium. The method comprises the following steps: taking extracellular polysaccharide, adding deionized water to dissolve the polysaccharide, and obtaining a ΔPS04-17 extracellular polysaccharide solution; adding an organic selenium solution and a reducing agent solution to the extracellular polysaccharide solution; subjecting the obtained mixed solution to ultrasonic mixing in a water bath at a certain temperature for a certain period of time, and obtaining the ΔPS04-17 extracellular polysaccharide nano-selenium solution; collecting the reaction solution, centrifuging it, discarding the supernatant, washing the precipitate, and purifying it to obtain the polysaccharide nano-selenium.

[0023] Preferably, the organic selenium is sodium selenite with a concentration of 0.1-2 mol / L.

[0024] Preferably, the reducing agent is ascorbic acid with a concentration of 1 to 5 mol / L.

[0025] More preferably, 2 mol / L ascorbic acid and 0.5 mol / L sodium selenite solution are used.

[0026] At the same time, studies have shown that polysaccharide nano-selenium has a good antagonistic effect on a variety of plant pathogenic fungi, and can be used to prevent and control a variety of plant diseases such as pepper anthracnose, tea tree root rot, wheat stem base rot, gray mold, etc. It can be used in the development and application of biocontrol agents, selenium-rich preparations and fertilizers; and the use of polysaccharide nano-selenium in tobacco production can significantly promote the growth of tobacco, providing more effective methods for promoting crop growth and increasing yields.

[0027] The present invention provides application of polysaccharide nano-selenium in inhibiting plant pathogens or in preventing and treating plant diseases caused by pathogens.

[0028] Preferably, the pathogen is Colletotrichum capsicum ( Colletotrichum fructicola ), Fusarium oxysporum ( Fusarium cugenangense ), Pseudomonas graminearum ( Fusarium pesudograminearum )、Botrytis cinerea( Botrytis cinerea ) one or more.

[0029] Preferably, the plant diseases are one or more of pepper anthracnose, tea tree root rot, wheat stem rot, and gray mold.

[0030] The present invention provides the application of the polysaccharide nano-selenium in promoting tobacco growth.

[0031] The present invention provides the use of the polysaccharide nano-selenium in the preparation of antibacterial products or growth-promoting products.

[0032] The present invention provides a product containing the polysaccharide nano-selenium.

[0033] The Paenibacillus cribensis ΔPS04-17 exopolysaccharide provided by the present invention is prepared from the Paenibacillus cribensis ΔPS04-17 strain and can convert inorganic selenium into nano-selenium. The reaction conditions are mild and the operation is simple, providing more efficient and rapid synthesis pathways for improving the conversion efficiency of nano-selenium and the biosynthesis of polysaccharide nano-selenium. Furthermore, the exopolysaccharide of the ΔPS04-17 strain provided by the present invention can quickly complete the conversion of inorganic selenium in a relatively short period of time, with a conversion efficiency far exceeding that of the ΔPS04-17 strain for inorganic selenium conversion, a high conversion rate, and a shorter conversion time, providing more conversion raw materials for polysaccharide nano-selenium biosynthesis strains.

[0034] Therefore, the present invention also provides the use of the space mutant strain ΔPS04-17 of Paenibacillus kriebensis and its extracellular polysaccharide in converting inorganic selenium into polysaccharide nano-selenium or in preparing polysaccharide nano-selenium.

[0035] The present invention has the following beneficial effects: The present invention provides a polysaccharide nano-selenium, which is prepared by reacting the exopolysaccharide of the space mutant ΔPS04-17 strain of Paenibacillus kriebensis with inorganic selenium and a reducing agent. This polysaccharide nano-selenium has better conversion efficiency, stability, yield, and purity than nano-selenium produced directly from the ΔPS04-17 strain, and does not undergo agglomeration, flocculation, or compatibility reactions. Furthermore, this polysaccharide nano-selenium exhibits excellent antagonism against various plant pathogenic fungi, making it suitable for the prevention and treatment of various plant diseases and the development of more biocontrol agents, selenium-enriched preparations, and fertilizers. The polysaccharide nano-selenium can be used in tobacco production to significantly promote tobacco growth, providing a more effective method for increasing crop growth and yield.

[0036] The extracellular polysaccharide of the △PS04-17 strain provided by the present invention can quickly complete the conversion of inorganic selenium in a relatively short period of time, and its conversion efficiency far exceeds the conversion of inorganic selenium by the △PS04-17 strain, with a high conversion rate and a shorter conversion time, providing more conversion raw materials for the biosynthesis strains of polysaccharide nano-selenium. The polysaccharide nano-selenium prepared by the present invention is significantly better than the nano-selenium prepared by biological strains, has better stability and yield, and provides more methods and sources for the biological synthesis of polysaccharide nano-selenium. In addition, the method for preparing nano-selenium from the extracellular polysaccharide of the △PS04-17 strain provided by the present invention has low equipment requirements, simple process, low cost, high product purity, and is environmentally friendly, providing more efficient and rapid ways to synthesize biological nano-selenium for the biosynthesis of nano-selenium. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The exopolysaccharide of the space mutant △PS04-17 of Paenibacillus kriebensis (A is the crude exopolysaccharide precipitated by adding three volumes of anhydrous ethanol to the supernatant of the centrifuged △PS04-17 fermentation broth; B is the form of the crude exopolysaccharide of △PS04-17 after deproteinization by the Sevage method, dialysis, and freeze-drying to constant weight).

[0038] Figure 2 It is a △PS04-17 extracellular polysaccharide nano-selenium solution.

[0039] Figure 3 This is the SEM scanning electron microscopy observation of △PS04-17 extracellular polysaccharide.

[0040] Figure 4 This is the SEM scanning electron microscope observation image of polysaccharide nanoselenium particles synthesized from △PS04-17 extracellular polysaccharide.

[0041] Figure 5 This is the EDS analysis diagram of the elemental composition of △PS04-17 polysaccharide nanoselenium particles and △PS04-17 nanoselenium particles (A in the figure is △PS04-17 refined extracellular polysaccharide nanoselenium; B is △PS04-17 nanoselenium).

[0042] Figure 6 This is a diagram of the atomic percentage of elements contained in △PS04-17 polysaccharide nanoselenium particles and △PS04-17 nanoselenium particles (A in the figure is △PS04-17 extracellular polysaccharide nanoselenium; B is △PS04-17 nanoselenium).

[0043] Figure 7 This is the standard curve for the determination of selenium content in precipitation samples.

[0044] Figure 8 This is a curve of selenium content in the precipitate within 12 hours of the reaction between △PS04-17 extracellular polysaccharide nanoselenium solution and △PS04-17 nanoselenium solution (A in the figure is △PS04-17 extracellular polysaccharide nanoselenium solution; B is △PS04-17 nanoselenium solution).

[0045] Figure 9 The figure shows the external morphology comparison of the △PS04-17 extracellular polysaccharide nanoselenium solution and the △PS04-17 nanoselenium solution after being in a water bath at high temperature (60°C) for 4 hours (A in the figure is the △PS04-17 extracellular polysaccharide nanoselenium solution; B is the △PS04-17 nanoselenium solution).

[0046] Figure 10 This is the control effect of the control group on plant pathogenic fungi (A in the figure is the pathogen of tea root rot; B is the pathogen of wheat stem base rot; C is the pathogen of gray mold).

[0047] Figure 11 This is the control effect of △PS04-17 polysaccharide nano-selenium on plant pathogenic fungi (in the figure, A is the pathogen of tea root rot; B is the pathogen of wheat stem base rot; C is the pathogen of gray mold).

[0048] Figure 12 This is a diagram showing the growth-promoting effect of △PS04-17 polysaccharide nano-selenium on tobacco (A in the figure is the control group, sprayed with clean water; B is the treatment group, sprayed with △PS04-17 refined polysaccharide synthetic polysaccharide nano-selenium solution).

[0049] Figure 13 These are the experimental results of promoting tobacco growth by △PS04-17 polysaccharide nano-selenium (A in the figure is the control group, sprayed with clean water; B is the treatment group, sprayed with △PS04-17 refined polysaccharide and synthetic polysaccharide nano-selenium solution).

[0050] Figure 14 The lesion area statistics of the control effect of pepper anthracnose (A in the figure is the control group inoculated with pathogens only; B is the treatment group sprayed with △PS04-17 polysaccharide nano-selenium solution first and then inoculated with pathogens). DETAILED DESCRIPTION

[0051] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0052] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0053] The Paenibacillus sp. used in the examples Paenibacillus kribbensis ) The space-induced mutant strain △PS04-17 is the preliminary research result of the applicant's research team. The △PS04-17 strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on March 17, 2022, with the deposit number: GDMCC NO: 62233, and is recorded in the prior art: CN114480222 A.

[0054] Example 1 Preparation of purified exopolysaccharide from Paenibacillus cribenbergii ΔPS04-17 (1) According to the modified Czapek medium formula (sodium nitrate 1.5 g / L, potassium dihydrogen phosphate 1 g / L, potassium chloride 0.5 g / L, magnesium sulfate 0.5 g / L, ferrous sulfate 0.01 g / L, sucrose 30.0 g / L, pH 7.0-7.2), 500 mL of modified Czapek medium was prepared as the fermentation medium, and then the space mutant of Paenibacillus kriebensis △PS04-17 was inoculated at 1% of the medium. The culture was fermented at 28°C in a constant temperature shaker at 150 rpm / min for 4 days to obtain △PS04-17 fermentation liquid.

[0055] (2) Take 50 mL of fermented △PS04-17 milky white bacterial liquid, heat it in a 100℃ water bath for 30 min, dilute it twice with distilled water, cool it, and centrifuge it at 8000 r / min for 15 min. At this time, there will be a layer of bacterial sediment at the bottom of the centrifuge tube, and the supernatant will be a transparent milky white liquid. Discard the bacterial sediment.

[0056] (3) Collect the supernatant into a pre-cooled beaker, slowly add 3 times the volume of anhydrous ethanol on ice, and stir clockwise with a glass rod while adding. After refrigeration for 12 to 24 hours, white flocculent polysaccharides will precipitate and aggregate into white block polysaccharides ( Figure 1 A).

[0057] (4) The precipitated polysaccharide was collected in a 50 mL centrifuge tube and redissolved in distilled water. A chloroform: n-butanol = 5:1 (V:V) binary system was prepared and added to the polysaccharide extract at a ratio of 1:5 (V solvent: V fermentation broth). The mixture was mixed and shaken vigorously. After standing and stratification, the solvent layer and the protein at the junction with the water layer were removed. The organic solvent in the above ratio was continued to be added until no denatured protein was produced at the junction. The obtained aqueous phase solution was dialyzed for 24 h and freeze-dried to obtain the refined △PS04-17 extracellular polysaccharide ( Figure 1 B).

[0058] Example 2 Determination of purified extracellular polysaccharide from △PS04-17 (1) △PS04-17 refined polysaccharide solution: The ΔPS04-17 refined extracellular polysaccharide solid prepared in Example 1 was dissolved in 50 mL of water and stirred to obtain a refined polysaccharide solution, which was a transparent white liquid with low viscosity.

[0059] (2) Draw the glucose standard curve using the phenol-sulfuric acid method: ① Accurately weigh 100 mg of analytical grade glucose dried to constant weight at 105°C into a 100 mL volumetric flask and dissolve it in distilled water to make up to volume to obtain a 1 mg / mL standard glucose solution.

[0060] ②Take phenol 5 g, put in 100 mL volumetric flask, add distilled water to the mark, shake well, then put in brown reagent bottle and refrigerate, get 5 % phenol solution.

[0061] ③Take 0, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL of glucose standard solution into 15 mL centrifuge tube respectively, add distilled water to 1.0 mL. Add 1.0 mL 5 % phenol solution into the test solution, then quickly add 5.0 mL concentrated sulfuric acid vertically, stand at room temperature for 10 min. Then put in boiling water bath for 20 min, the orange color of the mixture gradually deepens with the increase of glucose concentration.

[0062] Then take 100 μL of the reaction solution of different concentrations of standard glucose into 96-well plate, 4 replicates for each concentration, adjust zero with blank solution, measure the absorbance of the solution at wavelength of 490 nm, get the absorbance of the reaction solution of different concentrations of glucose and take the average value, then plot the standard curve of glucose with glucose mass concentration as abscissa and absorbance value as ordinate.

[0063] (3) Use phenol-sulfuric acid method to determine the polysaccharide content of △PS04-17 refined polysaccharide solution: Dilute the △PS04-17 refined polysaccharide solution by 50 times, take 1 mL of the diluted refined polysaccharide solution into 15 mL, add 1.0 mL of 5 % phenol solution, then quickly add 5.0 mL of concentrated sulfuric acid vertically, stand at room temperature for 10 min. Then put in boiling water bath for 20 min. Take 100 μL and add into 96-well plate, 4 replicates for each concentration, adjust zero with blank solution, measure the absorbance of the solution at wavelength of 490 nm, calculate the total sugar content of △PS04-17 refined polysaccharide solution according to the standard curve of glucose.

[0064] The results show that after the reaction of standard glucose solution with concentrations of 0 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL and 1000 μg / mL with phenol and sulfuric acid, the average absorbance of the solution at wavelength of 490 nm is 0.13, 0.86, 1.59, 2.34, 3.07 and 3.76 respectively, and the standard curve of glucose is calculated as y = 0.0036x + 0.1348, R 2= 0.9998, indicating that the standard curve exhibits good linearity. The average absorbance at 490 nm of the 50-fold diluted ΔPS04-17 purified polysaccharide solution after reaction with phenol and sulfuric acid solution was 2.00. Based on the standard curve, the polysaccharide content in the 50-fold diluted ΔPS04-17 purified polysaccharide solution was 518.11 μg / mL, resulting in a polysaccharide content of 25.91 mg / mL in the purified ΔPS04-17 exopolysaccharide solution.

[0065] Example 3 Preparation of PS04-17 Extracellular Polysaccharide Nanoselenium The △PS04-17 exopolysaccharide prepared in Example 1 was dissolved in sterile water at a concentration of 10 g / L, and 100 mL was used. 100 mL of 2 mol / L ascorbic acid (VC) solution and 100 mL of 0.5 mol / L sodium selenite (Na2SeO3) solution were prepared separately and added to 100 mL of the △PS04-17 exopolysaccharide solution. The volume was made up to 1 L with deionized water, shaken well, and heated in a 50°C constant temperature water bath for 4 h to obtain a red, transparent Paenibacillus Cribensis △PS04-17 exopolysaccharide nanoselenium solution. Figure 2 As shown, the selenium content in the obtained △PS04-17 extracellular polysaccharide nanoselenium solution is 4 g / L (the determination method is described later).

[0066] Example 4 △PS04-17 extracellular polysaccharide conversion effect on sodium selenite The concentrations of △PS04-17 extracellular polysaccharide solution (10 g / L) and VC solution (100 mL 2 mol / L) were kept constant, and solutions with different sodium selenite (Na2SeO3) concentrations of 0.5, 1, 2, 3, and 4 mol / L were prepared. The solution without sodium selenite was used as the control treatment and mixed with the polysaccharide and VC solutions for reaction. Three replicates were set up for each treatment, and the volume of each bottle was 150 mL. After heating and culturing in a 50℃ water bath for 4 h, the fermentation broth treated with polysaccharide nanoselenium was centrifuged at 12000 rpm for 5 min, and the supernatant was collected and the sodium selenite content was determined by Agilent 5800 inductively coupled plasma optical emission spectrometry.

[0067] The test results are shown in Table 1, which show that the content of inorganic selenium in the total selenium of all fermentation systems treated with different sodium selenite concentrations is less than 1%, indicating that more than 99% of selenium salts are converted into elemental selenium and polysaccharide organic selenium.

[0068] Table 1 Conversion rate of △PS04-17 extracellular polysaccharide to different sodium selenite concentrations

[0069] Example 5 Characterization and Analysis of Extracellular Polysaccharide Nanoselenium in △PS04-17 Preparation of △PS04-17 nano-selenium solution: △PS04-17 strain was activated and prepared with a concentration of 1×10 6 cfu / mL seed solution was inoculated into Czapek culture medium containing selenium salt at a concentration of 0.5 mol / L at a 1% inoculum rate to obtain the △PS04-17 nano-selenium solution prepared by direct fermentation of the △PS04-17 strain.

[0070] Samples of the △PS04-17 exopolysaccharide solution, the △PS04-17 nanoselenium solution, and the △PS04-17 exopolysaccharide nanoselenium solution were centrifuged at 12,000 rpm for 10 minutes. The supernatant was discarded, and the pellet was fixed in 2.5% glutaraldehyde solution at 4°C overnight. The samples were removed from the glutaraldehyde and dehydrated in a gradient of 50%, 70%, and 100% ethanol, each replacement for approximately 15 minutes. The samples were then removed and dried in a supercritical dryer. After drying for approximately one hour, the samples were removed, mounted on a sample stage, gold-sprayed, and tested. Images and photographs were taken using a Hitachi SU8020 field-emission scanning electron microscope. The elemental composition of the nanoselenium particles was analyzed by scanning electron microscopy and EDS.

[0071] Scanning electron microscopy images of the blank control group Figure 3 As shown in FIG, in the △PS04-17 extracellular polysaccharide solution without adding selenite, no nano-selenium synthesis was observed. Figure 4 As shown, spherical nano-selenium is produced, and the arrows in the figure indicate nano-selenium particles; the particle size of the nano-selenium particles is measured to be 50~400nm.

[0072] Further, the elemental selenium samples extracted from the △PS04-17 nano-selenium solution and the △PS04-17 extracellular polysaccharide nano-selenium solution were analyzed by energy dispersive spectrometer (EDS), and EDS point scanning was performed on selenium particles with a particle size of about 100 nm. The obtained elemental energy spectrum of the nano-selenium particles is shown in the figure below. Figure 5 As shown, the figure shows the △PS04-17 nano-selenium solution ( Figure 5 A) and △PS04-17 extracellular polysaccharide nanoselenium solution ( Figure 5 B) Se characteristic peak, confirming that the synthesized granular material is nano-selenium.

[0073] The atomic percentages of elements in the nano-selenium particles prepared from △PS04-17 and the polysaccharide nano-selenium particles prepared from the extracellular polysaccharide of △PS04-17 were statistically analyzed. Figure 6As shown, it can be seen that the element detected by the polysaccharide nano selenium particles in group A is selenium (Se) with a weight percentage of 6.63% and an atomic percentage of 1.12%; the element detected by the nano selenium particles in group B is selenium (Se) with a weight percentage of 3.38% and an atomic percentage of 0.56%, it can be seen that the yield of nano selenium prepared by the exopolysaccharide of △PS04-17 is higher and the purity is better.

[0074] Example 6 Determination of selenium content in nano selenium solution and stability test 1. Determination of nano selenium yield The concentration of the exopolysaccharide solution of △PS04-17 (10 g / L) and the VC solution (100 mL 2 mol / L) was kept unchanged, and a solution of sodium selenite (Na2SeO3) with a concentration of 0.5 mol / L was prepared and mixed with the polysaccharide and VC solution for the reaction. Meanwhile, the △PS04-17 strain was activated to prepare a seed solution with a concentration of 1×10 6 cfu / mL, and inoculated into a Czapek's culture solution containing selenium salt with a concentration of 0.5 mol / L at an inoculation amount of 1%, to obtain a nano selenium solution prepared from the fermentation broth of the strain.

[0075] That is, the exopolysaccharide nano selenium solution of △PS04-17 and the nano selenium solution of △PS04-17 were prepared with a concentration of sodium selenite (Na2SeO3) of 0.5 mol / L, respectively, and 3 replicates were set for each treatment, and the liquid volume of each bottle was 150 mL; under the conditions of 28 ℃ and shaking speed of 8000 r / min, the fermentation liquid containing selenium was cultured for 12 h, then a proper amount of the fermentation liquid containing selenium was centrifuged at 12000 r / min for 5 min, and placed in a 4 ℃ refrigerator for 24 h to make the solution stratify obviously, then the red nano selenium precipitate was discarded, and the precipitate was washed with 10 mL chloroform, 10 mL ethanol and 10 mL sterile water in turn, finally the nano selenium particles were resuspended in 15 mL ultrapure water, placed in a-80 ℃ ultra-low temperature refrigerator for freezing for 12 h, and vacuum freeze-dried for 36 h, to obtain purified nano selenium particles.

[0076] The determination of selenium content in the precipitate sample was carried out by drawing a standard curve, with the culture time as the abscissa and the nano selenium yield as the ordinate, to draw a curve graph, as shown in Figure 7 The curve equation is y = 17.189x - 0.001, R 2 = 0.9995, and the standard curve has a good linear relationship.

[0077] Subsequently, the nano-selenium precipitate sample of the culture was washed with 1 mL of 0.1 mol / L Tris-HCl (pH 8.0), washed twice with 2 mL of 1 mol / L NaCl, and 2 mL of 1 mol / L Na2S was added to the precipitate, mixed, and reacted for 1 h. The absorbance of the reaction solution at 500 nm was measured, and the content and yield of nano-selenium were calculated using the following formula:

[0078] The results are as follows Figure 8 As shown, the △PS04-17 strain can also synthesize nanoselenium after fermentation. With increasing incubation time, the nanoselenium yields in both the △PS04-17 exopolysaccharide nanoselenium solution and the △PS04-17 nanoselenium solution precipitate gradually increased. Comparing the nanoselenium yields between the two groups revealed that the △PS04-17 exopolysaccharide nanoselenium solution rapidly converted almost all of the inorganic selenium in the solution within 12 hours, achieving a yield close to 100% (curve A). However, the △PS04-17 nanoselenium solution remained in a vigorous reaction phase, with the nanoselenium yield continuing to rise (curve B). This indicates that the △PS04-17 exopolysaccharide has the ability to convert inorganic selenium faster than the △PS04-17 strain, achieving the same conversion efficiency in just 4 hours as the △PS04-17 strain did in 12 hours. This conversion efficiency far exceeds that of direct conversion of inorganic selenium by the △PS04-17 strain.

[0079] 2. Stability After completing the selenium content determination test in the precipitated samples, the two test solutions were incubated at 28°C and shaker speed of 8000 r / min for 2 days to ensure complete reaction. Appropriate amounts of the two nano-selenium solutions were simultaneously placed in a 60°C water bath for 4 hours, and the external morphologies of the solutions were observed for comparison.

[0080] The results are as follows Figure 9 As shown in the figure, after 4 h of reaction in a 60 ℃ water bath, the △PS04-17 extracellular polysaccharide nano-selenium solution showed no significant change and was in the form of red nano-selenium ( Figure 9 A); However, under high temperature conditions, the nano-selenium particles in the △PS04-17 nano-selenium solution matured due to the lack of polysaccharide as a carrier template, and underwent reactions such as agglomeration, flocculation, and compatibility. The particle size increased significantly, and the solution turned from red to black, indicating that the product was unstable ( Figure 9 B). This indicates that nano-selenium prepared from △PS04-17 extracellular polysaccharide has better stability.

[0081] Example 7 △ PS04-17 extracellular polysaccharide nano-selenium prevention and treatment effect on pathogens The control effect of △PS04-17 extracellular polysaccharide nano-selenium on a variety of common plant fungal disease pathogens was determined. The pathogen was tea tree root rot pathogen (Fusarium oxysporum Fusarium cugenangense ), wheat stem rot pathogen (Pseudofus graminearum Fusarium pesudograminearum ), gray mold pathogen (Botrytis cinerea Botrytis cinerea ).

[0082] Inhibitory activity against pathogenic fungi: Using the stand-off plate culture method, a 5 mm thick cake of the corresponding pathogenic fungi was inoculated in the center of a PDA plate. A Paenibacillus cribensis ΔPS04-17 exopolysaccharide nanoselenium solution was prepared using the same method as in Example 2. Using a pipette, 20 μL of the polysaccharide nanoselenium solution was dripped onto four spots 2.5 cm from the cake. A culture plate inoculated with only the pathogenic fungi served as a control. The plates were incubated at 28°C. After 5 days of stand-off culture, colony diameters were measured and the inhibition rate was calculated.

[0083] Inhibition rate = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100%.

[0084] The results showed that compared with the control group, Figure 10 As shown in Figure 2, the extracellular polysaccharide nanoselenium from △PS04-17 has a good inhibitory effect on the growth of three pathogenic fungi: tea root rot, wheat stem base rot, and gray mold. Figure 11 As shown, the antibacterial rates were 68%, 52% and 83% respectively.

[0085] Example 8 △ PS04-17 extracellular polysaccharide nano-selenium growth-promoting effect on tobacco A potted experiment was conducted to evaluate the growth-promoting effect of a ΔPS04-17 exopolysaccharide nano-selenium solution on tobacco plants. The Paenibacillus cribensis ΔPS04-17 exopolysaccharide nano-selenium solution was prepared using the same method as in Example 2. Tobacco potted plants of uniform growth and size were selected, and their initial plant heights were recorded for grouping. The experiment was divided into two groups: control group A (foliar spraying only with 50 mL of sterile water) and treatment group B (foliar spraying with 50 mL of a 100-fold diluted 4 g / L purified ΔPS04-17 exopolysaccharide nano-selenium solution). Each group of plants was sprayed with an appropriate amount of water every seven days. After one month of incubation, plant growth was observed and data recorded.

[0086] The results are as follows Figure 12 As shown in the figure, the growth and cotyledon number of tobacco plants treated with △PS04-17 extracellular polysaccharide nano-selenium solution were better than those of the control group; the height of the plants after treatment changed as shown in the figure. Figure 13As shown in the results, the tobacco plant height in the control group A increased from 15.8 cm to 22.9 cm, while the tobacco plant height in the treatment group B increased from 15.4 cm to 27.5 cm, indicating that the application of △PS04-17 extracellular polysaccharide nano-selenium solution can promote the growth of tobacco plants and has a certain growth-promoting effect.

[0087] Example 9 △ PS04-17 extracellular polysaccharide nano-selenium control effect on pepper anthracnose A nanoselenium solution of Paenibacillus cribensis ΔPS04-17 exopolysaccharide was prepared using the same method as in Example 2. Uniformly sized chili peppers were selected, cleaned, disinfected with 75% alcohol, and air-dried for later use. The pepper fruits were pierced with a sterile toothpick. The treatment group was first sprayed thoroughly and evenly with the ΔPS04-17 exopolysaccharide nanoselenium solution and then air-dried. Fresh pepper anthracnose bacteria ( Colletotrichum fructicola ) fungus blocks (8 mm in diameter) were covered with sterile cotton and kept moist with sterile water. They were then stored in an incubator at 28°C and 85% humidity. Pepper fruits sprayed with sterile water served as controls. Each treatment was replicated three times, with six fruits per replicate. The development of pepper lesions was recorded by measuring the area of ​​lesions on the pepper fruits, and the relative inhibition rate was calculated.

[0088] The results are as follows Figure 14 As shown in the figure, the average area of ​​lesions in the control group was 4.38 cm 2 The average lesion area of ​​the treatment group was 0.16 cm 2 , indicating that △PS04-17 refined extracellular polysaccharide nano-selenium has a good preventive and control effect on pepper anthracnose, with a prevention efficiency of 96.3%.

[0089] In summary, the present invention provides an exopolysaccharide nanoselenium, which is prepared by reacting the exopolysaccharide of the space mutant △PS04-17 strain of Paenibacillus kriebensis with inorganic selenium and a reducing agent. Using the exopolysaccharide of the △PS04-17 strain to prepare polysaccharide nanoselenium has a better conversion efficiency to inorganic selenium (sodium selenite) than that prepared by the △PS04-17 strain, and can reduce toxic sodium selenite in a short time to obtain polysaccharide nanoselenium with nanometer-sized particles. Furthermore, the polysaccharide nanoselenium prepared by using the exopolysaccharide of the △PS04-17 strain has a higher yield and purity than that prepared by the △PS04-17 strain, and also has better stability. Furthermore, the polysaccharide nanoselenium synthesized by the exopolysaccharide of the △PS04-17 strain has a strong antagonistic effect against various plant pathogenic fungi, can be used to prevent and control a variety of plant diseases, and has great development and application potential in selenium-enriched biocontrol agents and fertilizers. Its use in tobacco production can significantly promote tobacco growth, providing a more effective method for improving crop growth and yield.

[0090] The polysaccharide nano-selenium prepared from the extracellular polysaccharide of the △PS04-17 strain provided by the present invention has a preparation method with low equipment requirements, simple process, low cost, high product purity, and is environmentally friendly, providing a more efficient and rapid way to synthesize biological nano-selenium for the biosynthesis of nano-selenium.

[0091] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A polysaccharide nano-selenium, characterized in that: The polysaccharide nanoselenium is prepared from Paenibacillus kriebensis ( Paenibacillus kribbensis ) It is prepared by mixing extracellular polysaccharides with inorganic selenium and a reducing agent.

2. The polysaccharide nanoselenium according to claim 1, characterized in that The preparation method of the Paenibacillus criebensis exopolysaccharide comprises the following steps: inoculating Paenibacillus criebensis into a fermentation medium, obtaining a fermentation liquid after fermentation, and purifying, removing protein, dialysis, and freeze-drying the fermentation liquid to obtain the exopolysaccharide.

3. The polysaccharide nano-selenium according to claim 1, characterized in that The inorganic selenium is sodium selenite, and the reducing agent is ascorbic acid.

4. The method for preparing polysaccharide nano-selenium according to claim 3, characterized in that: Add 1-5 mol / L ascorbic acid solution and 0.1-2 mol / L sodium selenite solution to the extracellular polysaccharide solution of Paenibacillus kriebensis, shake well and heat to react for 2-4 hours to obtain a red, transparent polysaccharide nano-selenium solution. Centrifuge, discard the supernatant, wash the precipitate, and purify to obtain polysaccharide nano-selenium.

5. The use of the polysaccharide nano-selenium according to claim 1 in inhibiting plant pathogens or in preventing and treating plant diseases caused by pathogens, characterized in that: The pathogen is pepper fruit anthracnose ( Colletotrichum fructicola ), Fusarium oxysporum ( Fusarium cugenangense ), Pseudomonas graminearum ( Fusarium pesudograminearum )、Botrytis cinerea( Botrytis cinerea ) one or more.

6. The application according to claim 5, characterized in that The plant diseases are one or more of pepper anthracnose, tea tree root rot, wheat stem base rot, and gray mold.

7. Use of the polysaccharide nano-selenium according to claim 1 in promoting tobacco growth.

8. Use of the polysaccharide nano-selenium according to claim 1 in the preparation of antibacterial products or growth-promoting products.

9. A product, characterized in that Containing the polysaccharide nano-selenium according to any one of claims 1 to 3.

10. Use of a space mutant strain of Paenibacillus kriebensis ΔPS04-17 and its exopolysaccharide in converting inorganic selenium into polysaccharide nano-selenium or in preparing polysaccharide nano-selenium, characterized in that: The space mutant strain ΔPS04-17 of Paenibacillus kribensis was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 10, 2022, with the deposit number: GDMCC NO: 62233.

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