Membrane material containing extracellular polysaccharide of refrigerated bacillus, growth promoter and preparation method thereof

Abstract

The present invention relates to a membrane material and growth promoter containing Bacillus frostbite exopolysaccharide and a preparation method. The bacterial exopolysaccharide is mixed in 100mL phosphate buffer at a mass volume ratio of 1% to 3% in a pH=6.5, and a polysaccharide solution is obtained after sufficient stirring and dissolution; the membrane material is obtained after activation, cross-linking, and functional addition. The present invention is the first to utilize the fermentation product of Bacillus frostbite PG‑2 for development and utilization, and the fermentation product is deeply analyzed to obtain the molecular marker specific sequence EpsG of the bacterial exopolysaccharide gel. Through the functional analysis of the product, the present invention provides a rice-based soil improver, rice growth promoter, and rice slow-release fertilizer for the first time. Its effect has been confirmed by experiments.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation product production, in particular to a growth-promoting agent containing extracellular polysaccharide of Refrigerium glacialis and a preparation method thereof. Background Art

[0002] Exopolysaccharides are natural macromolecular polymers produced by microorganisms. Many studies have shown that polysaccharides have a variety of biological activities, such as antioxidant, antibacterial, antitumor, and immunomodulatory functions. The marine environment has rich bacterial resources, and it is feasible to find strains with development potential in them. Bacterial exopolysaccharides are secreted by bacteria of various genera. They are polymers composed of carbohydrates that can help cells adhere, adapt to the environment, and tolerate stress. They have a certain effect on bacterial survival, adaptation, and continuous colonization. When exopolysaccharides are part of the cell wall components, they can provide structural integrity and protection for bacteria and contribute to the formation of biofilms. Bacterial exopolysaccharides have excellent biological activities, such as immunomodulation, prebiotic effects, anticancer, antioxidant, antidiabetic, antiviral, and anti-gastritis properties. Bacterial exopolysaccharides have broad commercial application potential in the pharmaceutical and food industries. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a growth promoter containing extracellular polysaccharide of Bacillus frostii and a preparation method thereof.

[0004] The technical solution adopted by the present invention to solve its technical problem is:

[0005] A rice growth promoter containing polysaccharides secreted by microorganisms, containing polysaccharides obtained by fermentation of Gelidibacter sp. PG-2, wherein the Gelidibacter sp. is PG-2, the classification name is Gelidibacter sp., the preservation number is CGMCC No. 30528, the preservation date is May 8, 2024, and the preservation unit is General Microbiology Center of China National Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0006] Moreover, the microbial culture medium is LB-Z medium: 25 g / L lactose, 15 g / L NaCl, 10 g / L peptone, 5 g / L yeast extract, pH 7.0±0.2.

[0007] A polysaccharide-containing sustained-release film material, the preparation steps are as follows:

[0008] (1) The bacterial extracellular polysaccharide is mixed in 100 mL of phosphate buffer at a mass volume ratio of 1% to 3% in a pH = 6.5, and the polysaccharide solution is obtained after being fully stirred and dissolved; the bacteria is: polysaccharide obtained by fermentation of Gelidibacter sp. PG-2, the Gelidibacter sp. is PG-2, the classification name is: Gelidibacter sp., the preservation number is: CGMCC No. 30528, the preservation date is: May 8, 2024, the preservation unit is: General Microbiology Center of China National Microbiological Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0009] (2) Activation treatment: adding activators to the polysaccharide solution in sequence and stirring to evenly activate the solution, wherein the weight ratio of activator to polysaccharide is 1:1;

[0010] (3) Cross-linking: The mass ratio of the polysaccharide solution treated with the activator to the cross-linking agent is 10:1-1.5, the pH is 4, and after stirring and mixing, the mixture is treated at 55-65° C. for 45 minutes, and the pH is adjusted to 7 to obtain a polysaccharide solution treated with a cross-linking agent;

[0011] (4) Functional addition: The functional additive is mixed into water in a mass ratio of 1:30-100 to the cross-linking agent treated polysaccharide solution and stirred evenly. The mixture is boiled in a boiling water bath and then cooled to 60°C. The mixture is then mixed with the cross-linking agent treated polysaccharide solution in a volume ratio of 1:1 and stirred at 55-65°C for 45 minutes to obtain a sustained-release membrane material.

[0012] Moreover, the activator is Tween 20, and the weight ratio of the activator-containing polysaccharide solution to the activator is 1:1.5.

[0013] Moreover, the cross-linking agent is a combination of one or more (or more than two) of sodium citrate, glutaraldehyde, divinylbenzene, glycidyl methacrylate, and sodium trimetaphosphate.

[0014] Moreover, the functional additive is one or both of carboxymethyl starch and cationic starch.

[0015] Moreover, the phosphate buffer solution is a solution of disodium hydrogen phosphate and potassium dihydrogen phosphate mixed in a certain proportion, the concentrations of the disodium hydrogen phosphate and potassium dihydrogen phosphate solutions are both 0.05-0.08 mol / L, and the pH is adjusted to 5-7.

[0016] A rice growth promoter contains the above-mentioned polysaccharide sustained-release film material.

[0017] Moreover, the Gelidibacter sp. is PG-2, the classification name is: Gelidibacter sp., the preservation number is: CGMCC No.30528, the preservation date is: May 8, 2024, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0018] Moreover, the Gelidibacter sp. is PG-2, the classification name is: Gelidibacter sp., the preservation number is: CGMCC No.30528, the preservation date is: May 8, 2024, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0019] A polysaccharide containing microorganism secretion, characterized in that the expression sequence is EpsG, see sequence 1.

[0020] The advantages and positive effects achieved by the present invention are:

[0021] The present invention is the first to develop and utilize the fermentation product of Bacillus cyrogenus PG-2, and conducts in-depth analysis of the fermentation product to obtain the specific molecular marker sequence EpsG of bacterial extracellular polysaccharide gel. Through functional analysis of the product, the present invention provides a rice-based soil improver, rice growth promoter, and rice slow-release fertilizer for the first time. The effect has been confirmed by experiments.

[0022] The original strain used in the present invention is a strain isolated from the seabed mud of Bohai Sea. It has good salt tolerance and can show good secretion characteristics under 4°C. The strain has a significant low temperature resistance effect and is especially suitable for cultivation conditions in the north.

[0023] The present invention conducts field experiments on the slow-release fertilizer coated with the slow-release film, and finds that the slow-release fertilizer has an effect on the water retention performance of the soil, the slow release of nitrogen, and the water holding rate of the soil, and can significantly improve the soil performance, which is beneficial to increasing plant yield.

[0024] The present invention conducts rice planting experiments on the slow-release fertilizer coated with the slow-release film, and finds that the slow-release fertilizer has a significant effect on increasing the height and weight of rice roots and stems, and has a good effect on maintaining the fertility of the rhizosphere soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The effect of different fertilizer addition amounts on soil water holding capacity.

[0026] Figure 2 The water retention performance of PG-2 extracellular polysaccharide gel water-retaining slow-release fertilizer (3% w / w) in soil.

[0027] Figure 3PG-2 extracellular polysaccharide gel water-retaining slow-release fertilizer (3% w / w) slow-releases nitrogen in the soil.

[0028] Figure 4 It is the available phosphorus in rhizosphere soil after PG-2 polysaccharide gelation.

[0029] Figure 5 This is the nitrate nitrogen in the rhizosphere soil after PG-2 polysaccharide gelation.

[0030] Figure 6 Dry weight of rice roots after PG-2 polysaccharide gelation.

[0031] Figure 7 Dry weight of rice stem after PG-2 polysaccharide gelation.

[0032] Figure 8 The rice stem thickness after PG-2 polysaccharide gelation.

[0033] Fig. 9 Fresh weight roots of rice after PG-2 polysaccharide gelation.

[0034] Fig.10 Fresh weight of rice stem after PG-2 polysaccharide gelation.

[0035] Fig.11 The height of rice plants after PG-2 polysaccharide gelation. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0037] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0038] The Gelidibacter sp. used in the present invention is PG-2, with a classification name of Gelidibacter sp., a preservation number of CGMCC No. 30528, a preservation date of May 8, 2024, and a preservation unit of the General Microbiology Center of China National Microbiological Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0039] The recommended culture medium is LB-Z medium: 25g / L lactose, 15g / L NaCl, 10g / L peptone, 5g / L yeast extract, pH 7.0±0.2.

[0040] The method for producing extracellular polysaccharides using the above-mentioned alkali-resistant cold bacillus comprises the following steps: inoculating the strain PG-2 in the above-mentioned LB-Z culture medium at an inoculum concentration of 2-5% by volume and culturing and fermenting in a constant temperature oscillator at 4-10°C and 200rpm for 3-5 days, placing the fermentation liquid in a centrifuge tube, removing the bacteria, collecting the fermentation liquid, and then separating the polysaccharide. After measurement, the polysaccharide content is 530-840 mg / L. If the polysaccharide concentration is increased, the method of increasing the lactose concentration by 25 g / L can be selected. It is determined by HPLC to be a heteropolysaccharide, which is composed of mannose, glucosamine, glucuronic acid, galactose, aminogalactose, glucose, galactose and fucose, and the molecular weight is 492,094 Da.

[0041] The present invention obtains the specific molecular marker sequence EpsG (sequence 1) of bacterial extracellular polysaccharide gel by analyzing the polysaccharide.

[0042] The extracellular polysaccharide can be obtained by using existing technology, such as precipitating polysaccharides by trichloroacetic acid method to obtain a polysaccharide mixture. The recommended polysaccharide precipitation method is: centrifuge the fermentation broth for 20 minutes at 10,000×g and 4°C, concentrate the supernatant 100 times by vacuum distillation rotary instrument, add 10% trichloroacetic acid to the cell-free supernatant, keep it at 4°C overnight, and centrifuge it at 10,000×g and 4°C for 15 minutes to remove protein; then, mix the supernatant with 4 times the volume of pre-cooled ethanol for precipitation, keep it at 4°C for 12 hours, collect the polysaccharide precipitate after centrifugation at 10,000×g for 15 minutes at 4°C, place the obtained EPS in a dialysis bag with a cutoff of 14,000Da, dialyze it in deionized water for 2 days to remove impurities, freeze the polysaccharide solution in the dialysis bag for 12 hours, and then obtain a dry polysaccharide sample by freeze drying.

[0043] The present invention can directly use the fermentation liquid with the bacterial cells removed when preparing the growth promoter or slow-release fertilizer in the later stage, thus avoiding the step of polysaccharide enrichment and saving costs.

[0044] The method for preparing the polysaccharide-containing sustained-release film provided by the present invention comprises the following steps:

[0045] (1) 1-3 g of bacterial extracellular polysaccharide is mixed in 100 ml of 100 mL of phosphate buffer solution at a mass volume ratio of 1%-3% at pH=6.5, and the polysaccharide solution is obtained after sufficient stirring and dissolution. The phosphate buffer solution is a solution of disodium hydrogen phosphate and potassium dihydrogen phosphate mixed in a certain proportion, the concentrations of the disodium hydrogen phosphate and potassium dihydrogen phosphate solutions are both 0.05-0.08 mol / L, and the pH is adjusted to 5-7.

[0046] (2) Activation: 2-6 g of an activator is sequentially added to the polysaccharide solution, wherein the activator is Tween 20, and the weight ratio of the polysaccharide to the activator is 1:2;

[0047] (3) Cross-linking: The mass ratio of the polysaccharide solution treated with the activator to 10-15 g of the cross-linking agent is 10:1-1.5, the pH value is 4, and after stirring and mixing, the mixture is treated at 55-65° C. for 45 min, and the pH is adjusted to 7 to obtain a polysaccharide solution treated with the cross-linking agent;

[0048] The cross-linking agent is one or more (or more than two) of sodium citrate, glutaraldehyde, divinylbenzene, glycidyl methacrylate, and sodium trimetaphosphate;

[0049] (4) Functional addition: The mass ratio of 1.1-3.7 g of functional additive to about 110 g of cross-linking agent polysaccharide solution is 1:30-100. The functional additive is added into water and stirred evenly. After boiling in a boiling water bath, it is cooled to 60°C. Then, it is mixed with the cross-linking agent treated polysaccharide solution in a volume ratio of 1:1. The mixture is stirred at 55-65°C for 45 minutes to obtain a sustained-release membrane material.

[0050] The functional additive is one or both of carboxymethyl starch and cationic starch;

[0051] The method for preparing a fertilizer containing a slow-release film provided by the present invention comprises the following steps:

[0052] The granular fertilizer is placed in a coating machine, preheated to 55-65° C., and the film material of the slow-release fertilizer is sprayed on the surface of the granular fertilizer. After cooling, a gel structure is formed, and a coating is formed on the surface of the granular fertilizer. The coating is repeated multiple times to obtain a coated slow-release fertilizer based on PG-2 extracellular polysaccharide gel, wherein the film material of the slow-release fertilizer accounts for 5-10% of the weight of the coated slow-release fertilizer based on PG-2 extracellular polysaccharide gel;

[0053] Sustained release effect test

[0054] like Figure 1 The effect of different fertilizer addition amounts on soil water holding capacity is shown in Figure 1 As shown in the figure, the maximum water holding rate of the soil without adding PG-2 polysaccharide water-retaining slow-release fertilizer is 28%, while after adding 1%, 2%, 3%, 4% and 5% slow-release fertilizer respectively, the maximum water holding rate of the soil is 29.7%, 31.0%, 33.6%, 33.9% and 27.8%.

[0055] like Figure 2 As shown, the soil persistence rates of the soil without PG-2 polysaccharide water-retaining slow-release fertilizer were 28%, 19.2%, 6.8% and 1.4% after 1d, 3d, 7d and 15d, respectively; while the soil persistence rates of the soil with 3% slow-release fertilizer were 33.6%, 28.8%, 23.8% and 14% after 1d, 3d, 7d and 15d, respectively.

[0056] like Figure 3 The figure shows the change of nitrogen release rate in soil of PG-2 polysaccharide slow-release fertilizer over time. It can be seen from the figure that the release rate of nitrogen in the fertilizer is 5.7%, 9.7%, 19.1% and 29.5% respectively after being buried in the soil for 1, 3, 7 and 15 days. Note: For urea granules that are not coated, they can release 98% of their nitrogen within 12 hours.

[0057] Growth promoting effect test

[0058] Universal plastic flower pots (8 cm in diameter, 6.5 cm in bottom diameter, 11 cm in height) were used for soil culture of rice. 3% (w / w) (6 g) of ordinary fertilizer, ordinary fertilizer + polysaccharide, control slow-release fertilizer, and PG-2 polysaccharide water-retaining slow-release fertilizer were buried. After 1d, 3d, 7d, and 15d, the changes in rhizosphere soil available phosphorus and nitrate nitrogen, rice dry weight of roots and stems, rice stem diameter, rice fresh weight of roots and stems, and rice plant height were measured. Note: During the experiment, three different points in the flower pots were randomly selected for sampling. About 10 g of soil was mixed, air-dried, and sieved to test the content of soil available phosphorus and nitrate nitrogen; the dry and fresh weight, stem diameter, and plant height of rice roots and stems were measured during the experiment.

[0059] like Figures 4 to 11 As shown, the slow-release fertilizer provided by the present invention can promote the growth of different parts of rice. Compared with the control slow-release fertilizer, the available phosphorus and nitrate nitrogen in the soil increased by 15.3% and 19.7% respectively after applying the polysaccharide slow-release fertilizer for 15 days. In addition, after applying the polysaccharide slow-release fertilizer for 15 days, the dry weight of rice roots, dry weight of stems, stem diameter, fresh weight of roots and fresh weight of stems increased by 21.7%, 21.8%, 17.3%, 19.9% ​​and 18.3% respectively; while there was no significant change in plant height.

[0060] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A rice growth-promoting agent containing polysaccharides secreted by microorganisms, characterized in that: Contains polysaccharides obtained by fermentation of Gelidibacter PG-2, wherein the Gelidibacter is PG-2, the classification name is Gelidibacter sp., the preservation number is CGMCC No.30528, the preservation date is May 8, 2024, and the preservation unit is General Microbiology Center of China National Committee for the Preservation of Microorganisms, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

2. The rice growth-promoting agent containing polysaccharides secreted by microorganisms according to claim 1, characterized in that: The microbial culture medium is LB-Z medium: 25 g / L lactose, 15 g / L NaCl, 10 g / L peptone, 5 g / L yeast extract, pH 7.0±0.

2.

3. A polysaccharide-containing sustained-release film material, characterized in that: The preparation steps are as follows: (1) The bacterial extracellular polysaccharide is mixed in 100 mL of phosphate buffer at a mass volume ratio of 1% to 3% in a pH = 6.5, and the polysaccharide solution is obtained after being fully stirred and dissolved; the bacteria is: polysaccharide obtained by fermentation of Gelidibacter sp. PG-2, the Gelidibacter sp. is PG-2, the classification name is: Gelidibacter sp., the preservation number is: CGMCC No. 30528, the preservation date is: May 8, 2024, the preservation unit is: General Microbiology Center of China National Microbiological Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. (2) Activation treatment: adding activators to the polysaccharide solution in sequence and stirring to evenly activate the solution, wherein the weight ratio of activator to polysaccharide is 1:1; (3) Cross-linking: The mass ratio of the polysaccharide solution treated with the activator to the cross-linking agent is 10:1-1.5, the pH is 4, and after stirring and mixing, the mixture is treated at 55-65° C. for 45 minutes, and the pH is adjusted to 7 to obtain a polysaccharide solution treated with a cross-linking agent; (4) Functional addition: The functional additive is mixed into water in a mass ratio of 1:30-100 to the cross-linking agent treated polysaccharide solution and stirred evenly. The mixture is boiled in a boiling water bath and then cooled to 60°C. The mixture is then mixed with the cross-linking agent treated polysaccharide solution in a volume ratio of 1:1 and stirred at 55-65°C for 45 minutes to obtain a sustained-release membrane material.

4. The polysaccharide sustained-release film according to claim 3, characterized in that: The activator is Tween 20, and the weight ratio of the activator-containing polysaccharide solution to the activator is 1:1.

5.

5. The polysaccharide sustained-release film according to claim 3, characterized in that: The cross-linking agent is one or a combination of two or more of sodium citrate, glutaraldehyde, divinylbenzene, glycidyl methacrylate, and sodium trimetaphosphate; and the functional additive is one or two of carboxymethyl starch and cationic starch.

6. The polysaccharide sustained-release film according to claim 3, characterized in that: The phosphate buffer solution is a solution of disodium hydrogen phosphate and potassium dihydrogen phosphate mixed in a certain proportion, the concentrations of the disodium hydrogen phosphate and potassium dihydrogen phosphate solutions are both 0.05-0.08 mol / L, and the pH is adjusted to 5-7.

7. A rice growth promoter, characterized in that: Contains the polysaccharide sustained-release film material according to claim 3.

8. The use of polysaccharide fermented from Bacillus cyrtonema PG-2 as a rice growth promoter, characterized in that: The Gelidibacter sp. is PG-2, the classification name is: Gelidibacter sp., the preservation number is: CGMCC No.30528, the preservation date is: May 8, 2024, the preservation unit is: General Microbiology Center of China National Microbiological Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

9. The use of polysaccharide fermented from Bacillus cyrogenus PG-2 as a soil conditioner, characterized in that: The Gelidibacter sp. is PG-2, the classification name is: Gelidibacter sp., the preservation number is: CGMCC No.30528, the preservation date is: May 8, 2024, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

10. A polysaccharide containing microorganism secretion, characterized in that: The expressed sequence is EpsG, see sequence 1.

Images