KR100864399B1 - A method for capsulating useful agricultural culture using alginate shell bead having improved drought resistance viability of useful agricultural culture - Google Patents

Abstract

A method for encapsulating useful agricultural microorganisms by using alginate shell beads is provided to improve drought resistance of agricultural microorganisms, reduce costs for encapsulation by replacing expensive cationic poly-L-lysine by inexpensive and environment-friendly chitosan, and enhance survival ability of agricultural microorganisms at room temperature. A method for encapsulating useful agricultural microorganisms by using alginate shell beads comprises the steps of: dissolving alginate in NaOH solution, and regulating pH to 7.0-7.5 to prepare alginate solution; floating Serratia plymuthica A21-4 or Pseudomonas fluorescens B16 as a useful agricultural microorganism in the alginate solution; adding dropwise of the microorganism-floating alginate solution into 0.1M of CaCl2 solution by using a low pressure LC pump to form spherical beads; agitating and washing the spherical beads; firstly coating the washed beads with chitosan; secondly coating the chitosan-coated beads with silica solution; and washing the coated beads to obtain alginate/chitosan-based hydrogel with diameter of 3-5 mm, wherein the overall reaction temperature is 4 deg. C. A number of colony of the Serratia plymuthica A21-4 is 3x10^9 CFU/ml and a number of colony of the Pseudomonas fluorescens B16 is 2x10^9 CFU/ml.

Description

A method for capsulating useful agricultural culture using alginate shell bead having improved drought resistance viability of useful agricultural culture}

1 is a schematic diagram of preparation of alginate beads by a multilayer process;

2 is a view showing a manufacturing process of alginate shell beads,

3 is a diagram illustrating a stabilization process of alginate beads by type of multiple cations,

4 is a diagram illustrating electron microscopic cross-sectional structure of alginate beads, the left (poly-L-lysine) and the middle (polyethyleneimine) parts are alginate bead structures not loaded with bacteria, and the right (using chitosan) part is S plymuthica A21-4 is loaded,

5 is an apparatus for producing hundreds of μM to 1 mm alginate shell microbeads, which has a structure of an injection system powered by compressed air;

Figure 6 is a microstructure of (A) swelling microscopy structure of the alginate bead prepared by the capsule prepared by the compressed air injection system again by the CaCl ₂ / chitosan / silicate treatment method, and (B) after lyophilization SEM photo (500 μm),

Figure 7 Freeze-dried S. plymuthica Differential scanning thermal analysis traces according to the moisture content of the loaded chitosan-based beads,

8 and 9 are lyophilized S. plymuthica Melt enthalpy per gram of ice obtained from DSC melt endotherm (melt endotherm) with water content of loaded chitosan based beads.

The present invention relates to a method for encapsulating agricultural useful microorganisms using alginate shell beads to improve the cold-resistance viability of useful agricultural microorganisms, and more particularly, to inexpensive and environmentally friendly poly-L-lysine used for alginate gel stabilization. The present invention relates to the provision of a hydrogel that has not only been replaced with phosphorus chitosan but also improved the cold resistance of useful agricultural microorganisms.

This is because, in formulating microorganisms, most of the cases in which the shelf life is short and cannot be commercialized cannot be hydrated. The most suitable material to compensate for this weakness is hydrogel.

In particular, microorganisms intended to be used as biopesticides must maintain density while living on the surface of plants for a long time. The absolute condition for the survival of such microorganisms is water. The use of useful microorganisms can be maximized if microorganisms are included in the hydrogel network with water of activity to be able to act, and can be discharged as necessary to maintain the plant's root zone.

On the other hand, the encapsulation technique is to enclose or coat the solid particles or liquid with a film of a high molecular material to be sealed at a controlled rate under specific conditions. Hydrophilic polymers that can be used in hydrogels to encapsulate microorganisms have been developed in many chemical forms. In particular, the development of water-soluble polymers (mainly polysaccharides and some polyelectrolytes, etc.) is thought to be almost endless.

Among them, the most used and studied material for encapsulation so far is alginate, a natural polymer material. Alginate is a polymer of β-D-mannuronic acid and α-L-guluronic acid, produced mainly by algae or bacteria.

Uronic acids that make up alginate are negatively charged because they have a carboxyl group at their ends. Therefore, when bivalent cations such as Mg ²⁺ , Ca ²⁺ , Sr ²⁺ , and Ba ²⁺ are added, physical crosslinking is formed to form a hydrogel as shown in FIG. 1.

In particular, Ca ^{+ 2,} so that crosslinking by cationic best form Ca ^{2 +} / studies on the enzymes and micro-encapsulation of the bacteria of the alginate gel have been reported most frequently.

It is important to stabilize the gel is such as Ca ^{2 +} / alginate gel is formed by physical cross-linking. To this end, stabilization of gels using various multiple cations such as multiple cationic poly-L-lysine has been attempted.

In addition, Ca ^{2 +} / results of the alginate gel and a complex of multiple cation in one step the silica coating to enhance with a sol-gel process that has been used since the late 1970's have also been published a lot. The presence of such silicas has been found to enhance the stability of alginate capsules and significantly affect bacterial fixation.

The use of useful microorganisms encapsulated in biological processes in the natural environment, such as bioremediation, has been reported to have many advantages over free cell suspension. In particular, encapsulation of bacteria into hydrogels can reduce biological and abiotic stresses by providing a number of advantages, including protecting the bacteria from the harmful effects of dangerous compounds and increasing survival and metabolism.

However, Ca ^{2 +} / Once you've loaded alginate beads of useful microorganisms in gel capsules made by a multi-cation and silica processing stored at room temperature in a wet state structure is most easily broken or useful microbes can not survive long-term. Lyophilization methods are commonly used to stabilize pharmaceuticals, microorganisms, and many food and biological products. Lyophilized samples are characterized to minimize degradation reactions stored at room temperature for long periods of time and to maintain proper physical, chemical and biological stability.

Whether useful microorganisms survive the long-term storage after lyophilizing the encapsulated hydrogel is determined by the condition and structural properties of the water in the hydrogel. In general, differential scanning calorimetry (DSC) is used to investigate the characteristics of these intrinsic water structures. DSC measures the difference in the flow of heat generated from a sample by applying the same hourly heat to the sample and an inert reference material, and measures the state and structural properties of the water by measuring the change in heat capacity generated from a phase change (from ice to water). It can be seen.

The application of hydrogel has been applied mainly to the field of medicine. In addition, mass application of hydrated gels (mainly in the form of organic polymers) may be used as an adjuvant for reinforcing the structure of concrete, or added to foods to preserve the viscosity of foods and preserve the shape of processed foods. In recent years, the use of polysaccharide hydrogels in the decomposition of oil and the production of materials through the immobilization of bacteria or activated sludge.

However, although the application to agriculture is relatively small in both domestic and foreign countries, many applications are expected in the future in terms of preventing environmental pollution, protecting water resources, improving crop productivity, and minimizing damage caused by a single crop.

On the other hand, in order to increase the structural stability of the negatively charged alginate beads, cationic poly-L-lysine is used at present, but since the poly-L-lysine is too expensive, it is inexpensive and excellent in performance to replace poly-L-lysine. There is also a focus on developing other multications.

Accordingly, one object of the present invention is to provide beads in an environmentally friendly manner by utilizing the physicochemical properties of the structural properties of natural polymers without chemical crosslinking.

Another object of the present invention is to provide a method for encapsulating agricultural useful microorganisms using the obtained beads.

It is another object of the present invention to provide a practically useful microorganism-supported hydrogel material to increase the productivity of crops. Another object is to provide a practically useful microorganism-supported hydrogel material to increase the productivity of crops.

According to the first aspect of the present invention,

Suspending the agriculturally useful microorganisms in alginate;

The step of cross-linking was suspended alginate Ca ^{2 +} and the physical form of spherical beads;

Primary coating the spherical beads with chitosan; And

Provided is a method of encapsulating microorganisms useful for agriculture using an alginate shell bead comprising a secondary coating with silica.

According to the second aspect of the present invention,

An alginate / chitosan based hydrogel having improved sweat resistance of 3 to 5 mm in diameter obtained by the first aspect is provided.

According to the third aspect of the present invention,

An alginate / chitosan based hydrogel having improved sweat resistance of 400 μm to 1 mm in diameter obtained by the first aspect is provided.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

In the present invention, Serratia useful agricultural microorganisms useful for the prevention of pepper disease using natural polymer polysaccharides Try the encapsulation of plymuthica A21-4 and Psudomonas fluorescence B16 and microbial supported practically useful to increase the productivity of crops provide a hydrogel material.

To this end, starting with the natural polymer is alginate and by physical cross-linking, multi-layer process, and the silica coating with Ca ^{2 +} manufacturing the alginate shell beads.

Looking at these shell beads manufacturing method in more detail as follows.

First, agricultural useful bacteria are suspended in alginate. The agricultural useful bacteria include, but are not limited to, Serratia plymuthica A21-4 or Psudomonas It is preferable to use pepper blight control bacteria consisting of fluorescens B16.

Serratia plymuthica A21-4 is more preferably cultured in NR medium (Nutrient rich medium) containing 1% yeast extract, 1.5% nutrient broth and 0.2% ammonium sulfate to 3 × 10 ⁹ CFU / ml colony. , Psudomonas fluorescens B16 is more preferably used in culture to the colony number 2 × 10 ⁹ CFU / ml in NR medium.

A was floated alginate is cross-linked Ca ^{2 +} and the physical form of spherical beads. Specifically, alginic acid was dissolved in NaOH solution and adjusted to pH 7.0-7.5 to prepare alginate solution, and then the alginate solution was converted into CaCl ₂ with LC low pressure pump. It is slowly dropped into the solution to form spherical beads.

The obtained spherical beads are stirred and washed with water, and chitosan is added as a multi-cation coating material and stirred to first coat. The coated beads are then reacted with silica for secondary coating and washing with water. At this time, the alginate gel formed does not show any particular difference in moisture content according to the preparation temperature, but since the alginate gel prepared at 4 ° C. is more mechanically stable, the total reaction temperature for making the alginate gel is performed within a temperature range of 4 ° C. It is preferable.

In this way, alginate / chitosan based hydrogels having a diameter of 3 to 5 mm can be produced. The prepared hydrogel is preferably lyophilized or dried to enhance the viability of the microorganisms in the gel.

As the diameter of the beads thus prepared is about 3 to 5 mm, there is no problem in the hydrogel formulation used in the pot, but it may be difficult to adhere to the plant because the size is too large for packaging such as foliar spraying. There is a need to make the bead size to a few tens to hundreds of microns, and the method for producing such a small diameter hydrogel is as follows.

That is, spherical beads are made by mixing the above-mentioned useful microorganisms in a barium chloride solution and then spraying them into a solution in which alginate is dissolved using an air driven spray system. The syringe pump speed of the air injector should be 18-18.6 ml / h and the air pressure should be 4.5-5.5 LPM AIR. At this time, the smaller the velocity and the higher the pressure, the smaller the size of the spray droplets, but smaller than the pump speed can reduce the useful microorganisms suspended and can be made into a non-uniform capsule.

The hundreds of micron size capsules thus obtained were replaced with CaCl ₂ Slowly drop into solution to form spherical beads, stir and wash, add chitosan and stir to coat primary.

The coated beads may be reacted with silica for secondary coating, and washed with water to prepare alginate / chitosan based hydrogels of 400 μm to 1 mm in diameter.

At this time, the total reaction temperature is preferably carried out in the temperature range of 4 ℃ for the same reason as when preparing the alginate / chitosan-based hydrogel having a diameter of 3 to 5 mm.

In addition, the obtained hydrogel is preferably lyophilized or dried to enhance the viability of the microorganisms in the gel.

On the other hand, modeling and optimization experiments for the above process was carried out through the following examples, and the biophysical properties related to the viability of bacteria of alginate beads were identified through moisture content, electron microscopy, DSC analysis, and the like. The result was the following:

First, agricultural useful microorganisms Serratia plymuthica A21-4 and Pseudomonas for encapsulation of fluorescens B16 lie in alginate cross-linked -Ca ^{2 +} / multi-layer forming cation / silica coating process it made the 3 ~ 5 ㎜ alginate beads shell blank. In particular, we have successfully developed a process for replacing poly-L-lysine, which is currently used for stabilizing alginate gels in the literature, with inexpensive (16,000 times) and environmentally friendly chitosan.

Second, S. plymuthica A21-4 encapsulated 3 to 5 mm alginate beads were generally round in shape, highly porous inside, and most of the spaces were interconnected. The excellent supporting ability of alginate beads against S. plymuthica A21-4 bacteria is also associated with the layered structure in these beads (see Figures 4 and 6).

Third, the prepared chitosan-based S. plymuthica After lyophilization of the A21-4 alginate gel beads, 3 months at room temperature, the bacteria show a very high survival rate, demonstrating that the alginate gel matrix provides a good protective film of Serratia bacteria (see FIGS. 7-9).

Fourth, as a result of DSC experiments for analyzing the state and structural properties of the intrinsic water of the lyophilized alginate gel beads, it was confirmed that about 1.5 times as much as the bound water "bound water". These binding waters are thought to play a very important role in the survival of S. plymuthica A21-4 during the freezing process because of the presence of alginate gels (FIGS. 7-9).

<Example>

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. The following examples are intended to illustrate the invention and are not intended to limit the invention thereto.

< 3 to 5 mm for chitosan-coated based microencapsulation Modeling and Optimization Experiments for Making Alginate Shell Beads >

Comparative example

As shown in Figure 2, Coradin et al., The alginic acid by (Chem. Commun., 2001, 2496-2497) Ca 2 + crosslinked / polyacrylic -L- lysine layer formed / silica coating / citrate derived alginate removed The encapsulation of bacteria was attempted by the hollow capsule formation method.

However, the process is less stable in subsequent processes such as lyophilization.

Accordingly, the following example was carried out by focusing on substituting the original citrate process of Coradin and replacing the layer forming material with cheap chitosan.

Example  1-Manufacture

Alginate solution was prepared by dissolving alginic acid (Sigma) in a concentration of 1.5 wt / v% in 0.06 M NaOH solution to pH 7.5. The alginate solution was obtained in a 10 ㎖ LC low-pressure pump (Gilson M312) using a yellow tip 200 ㎕ 0.1 M CaCl ₂ Slow dropping into 100 ml of solution formed spherical beads. It was stirred for 1 h at 4 ° C. and then washed twice with 0.05 M Tris-HCl buffer (pH 7.2).

Then, as shown in FIG. 3, 0.01 wt / v% poly-L-lysine or 0.1 wt / v% polyethyleneamine (PEI) or 0.01 wt / v% chitosan, respectively, was added as a multi-cation coating material and stirred for 30 minutes. The beads were first coated.

In order to improve the mechanical properties of the obtained alginate / polycationic capsule, the capsules were reacted with 0.01 M sodium silicate solution for 2 hours, and the capsules were coated again, and washed twice with 0.05 M Tris-HCl buffer (pH 7.2).

< Example  2- CaCl ₂  By concentration and reaction time Alginate Bead  Water content measurement>

One) Ca ^{2 +} Concentration and Ca ^{2 +} Moisture content of capsule according to reaction time of

In alginate gel Ca ^{2 +} has physical cross-linking agent cross-linked to the change in the relative concentrations of exemplary bar which is expected to adjust the cross-link density, in this embodiment, measuring the moisture of the capsules made in accordance with the Ca ^{2 +} concentration because the role as (physical crosslinks) It was checked whether the density was adjusted.

In order to measure the moisture content of the capsule prepared in Example 1, the filter paper (Advantec) cut to a suitable size in a vacuum oven and dried for 24 hours, weighed, and weighed and dried by placing the capsule made at 4 ℃ on the filter paper The weight of the previous capsule (Wt (wet)) was measured. After completely drying the filter paper with the capsules in the vacuum oven for 24 hours, the weight of the capsule after drying (Wt (dry)) is weighed and then the capsule weight after drying from the weight of the capsule before drying, as shown in Equation 1 below. Then, the moisture content of the capsule was measured.

[{Wt (wet)-Wt (dry)} / Wt (wet)] X 100 = capsule moisture%

Ca ^{2 +} concentration and the capsules to the water content (%) The results of capsules obtained by the production temperature are summarized in Table 1.

Room temperature (25 ℃) Primary Secondary 0.05 M CaCl ₂ 94.9% 96.3% 0.1 M CaCl ₂ 98.9% 97.3% 0.15 M CaCl ₂ 96.0% 98.3% 0.2 M CaCl ₂ 97.2% 97.3% Primary Secondary 0.1 M CaCl ₂ 97.2% 98.6% 0.2 M CaCl ₂ 98.7% 97.6%

4 ℃ Primary Secondary 0.1 M CaCl ₂ 97.2% 98.6% 0.2 M CaCl ₂ 98.7% 97.6%

As shown in Table 1a and 1b, the alginate gel did not show any particular difference depending on the Ca ²⁺ concentration and capsule preparation temperature within the margin of error. However, since gels prepared at room temperature are easily broken down by touching them, gels prepared at 4 ° C are considered to be more stable mechanically, and therefore, gels prepared at 4 ° C may be more stable. Moisture content (%) experiment was performed, the results are summarized in Table 2 below.

4 ℃ 1hr 0.1 M CaCl ₂ 92.0% 0.2 M CaCl ₂ 98.2% 0.4 M CaCl ₂ 95.9%

4 ℃ 0.1 M CaCl ₂ 1hr 94.4% 2hr 92.0% 3hr 93.2% 4hr 96.2%

As shown in Table 2, the alginate gel did not show a special difference depending on the Ca ²⁺ concentration and the reaction time within the error range, and did not show a significant difference in the mechanical strength, etc. in the Ca ²⁺ concentration and time optimization. , 0.1 M CaCl ₂ , 1 hour was determined as the optimum reaction conditions.

Example 3 Encapsulation of Useful Bacteria and Measurement of the Number of Bacteria

As useful bacteria, the following two strains were selected as the bacteriostatic bacterium.

1) Strains: Serratia plymuthica  A21-4

After incubating the bacteria for 30 hours at 30 ° C. in nutrient-rich NR medium, the bacteria (3 × 10 ⁹ CFU / ml) recovered by centrifugation (8,700 g, 30 min) were suspended in alginate solution to form the beads. Loaded into capsules.

2) Strains: Pseudomonas fluorescens  B16

After incubating the bacteria at 30 ° C. for 12 hours in nutrient rich NR medium, the cells (2 × 10 ⁹ CFU / ml) recovered by centrifugation (8,700 g, 30 min) were used to load the capsules. However, since it contains a substance such as EPS, it was washed twice with PBS buffer to purely separate bacteria and used.

3) S. plymuthica  Due to various physical factors of bacteria Survival rate  Research

Serratia Prior to loading and lyophilizing plymuthica A21-4 bacteria in alginate hydrogel beads, the survival rates of the relevant physicochemical stress factors were examined.

In other words, the effect on the survival rate at the loading concentration of alginate itself and how long the cultured bacteria themselves can survive under lyophilized conditions.

① S. plymuthica  Wow Alginate  solution( pH  7.5) 1: 1 When floating  Surviving population (control, Alginate  Influence by the polymer itself)

400 μl of S. plymuthica NR culture was harvested and suspended in 400 μl of alginate solution (pH 7.5) at room temperature for 1 hour and then spreaded to determine the number of colonies. The number of colonies obtained was 3.0 × 10 ⁸ to 8.0 × 10 ⁹ CFU / ml, with no significant effect as expected.

② S. plymuthica  The number of the surviving population of the culture medium itself by lyophilization (control population)

After culturing S. plymuthica in NR medium for 12 hours, the recovered culture was first frozen in a general freezer (-20 ° C) for about 5 hours and then frozen in Deep Freezer (-70 ° C) for 24 hours. Frozen samples were lyophilized (Eyla freeze drier, 24 h to 48 h) in an Eyla freeze dryer (FD-1000, GCD-051X pump). For reference, all subsequent lyophilization experiments were performed under the same conditions.

Lyophilized bacteria were placed in PBS buffer for 24 hours and then spreaded to determine the number of colonies. The surviving population of lyophilized S. plymuthica ranged from 1 × 10 ² to 1 × 10 ³ CFU / ml, indicating that most of the bacteria died from severe East Sea damage.

4) S. plymuthica Loading  For multiple cations pH  optimization

The alginate polymer has a carboxyl group in the side chain, and the dissolved aqueous solution has a pH of about 4. However, the optimal growth pH of Serratia plymuthica A21-4 was 7. This was performed to optimize the alginate gel production according to the pH.

At this time, poly-L-lysine, an alginate gel stabilizer mainly used in the encapsulation of animal bacteria, was considered to be too expensive to be used as an agricultural material, and polyethyleneimine (3000) was used as an alternative cationic polymer of poly-L-lysine. Cheaper times) and chitosan (16,000 times cheaper) were selected first.

S. plymuthica loaded alginates were prepared using the three types of multiple cations described above and compared with various properties.

First, 400 μl of S. plymuthica NR culture was harvested and suspended in 400 μl of pH 4.0 alginate solution and pH 7.5 alginate solution (pH adjusted with 10 N NaOH), respectively. This was added to 4 ml of 0.1 M CaCl ₂ solution by LC low pressure pump (Gilson M312) using 200 μl yellow tip, stirred for 1 hour, washed with 0.05 M Tris-HCl buffer (pH 7.2), and 0.01% poly 2 ml of -L-lysine or 0.1 wt / v% polyethyleneimine or 0.01 wt / v% chitosan, respectively, was allowed to react for 30 minutes.

Water was washed twice with Tris-HCl buffer (pH 7.2), 2 ml of 0.01 M sodium silicate solution was reacted for 2 hours, and washed twice with Tris-HCl buffer (pH 7.2).

The number of colonies was measured by mechanically crushing and spreading the beads in order to measure the viability of the bacteria according to the time of lyophilization of the finished beads and the time left at room temperature (0 h, 24 h, 48 h, 72 h, 120 h). The viability results of S. plymuthica cells in alginate capsules according to pH and multiple cationic species are shown in Table 3 below.

Alginate / poly-L-lysine / silicate 0 hr 24 hr 48 hr 72 hr 96 hr 120 hr pH 4 1.47 × 10 ⁷ CFU / ml 5.49 × 10 ⁷ CFU / mL 2.01 × 10 ⁷ CFU / mL 1.54 × 10 ⁷ CFU / mL 2.37 × 10 ⁷ CFU / mL 3.24 × 10 ⁷ CFU / mL pH 7.5 7.98 × 10 ⁸ CFU / mL 1.84 × 10 ⁸ CFU / mL 4.52 × 10 ⁸ CFU / mL 2.69 × 10 ⁸ CFU / mL 3.71 × 10 ⁸ CFU / mL 1.38 × 10 ⁸ CFU / mL

Alginate / Polyethyleneimine / Silicate 0 hr 24 hr 48 hr 72 hr 96 hr 120 hr pH 4 2.53 × 10 ⁷ CFU / mL 4.3 × 10 ⁷ CFU / mL 1.18 × 10 ⁷ CFU / mL 2.11 × 10 ⁷ CFU / mL 1.07 × 10 ⁷ CFU / mL 1.53 × 10 ⁷ CFU / mL pH 7.5 2.53 × 10 ⁸ CFU / mL 1.75 × 10 ⁸ CFU / mL 3.24 × 10 ⁸ CFU / mL 2.07 × 10 ⁸ CFU / mL 2.82 × 10 ⁸ CFU / mL 1.09 × 10 ⁸ CFU / ml

Alginate / chitosan / silicate 0 hr 24 hr 48 hr 72 hr 96 hr 120 hr pH 4 1.82 × 10 ⁷ CFU / mL 5.46 × 10 ⁷ CFU / mL 1.79 × 10 ⁷ CFU / mL 1.2 × 10 ⁷ CFU / mL 2.3 × 10 ⁷ CFU / mL 1.60 × 10 ⁷ CFU / mL pH 7.5 3.68 × 10 ⁸ CFU / mL 1.3 2 × 10 ⁸ CFU / mL 1.83 × 10 ⁸ CFU / mL 3.25 × 10 ⁸ CFU / mL 1.09 × 10 ⁸ CFU / mL 1.67 × 10 ⁸ CFU / mL

As shown in the table, the survival rate of S. plymuthica cells was the highest at pH 7 for all three multiple cations. In addition, it was confirmed that the cheapest chitosan showed better results in viability and structural stability than the other two cations. In particular, it was concluded that the biodegradable chitosan was economical and environmentally friendly as the stabilizer of the alginate gel because polyethyleneimine synthetic material was difficult to decompose.

5) S. plymuthica cell of Alginate At the bead Intelligence  Research

Chitosan-based S. plymuthica alginate-gel beads prepared in paragraph 4) were washed twice with Tris-HCl buffer (pH 7.2) and then soaked in fresh Tris-HCl buffer (pH 7.2) for 2 days ( Volume ratio = wet gel 30: buffer 70) and then spreading to measure the number of cells supported by the gel beads in the buffer soaked state.

Each surviving population in the buffer layer and beads was measured by the ability of the beads to support the S. plymuthica cells when the alginate beads were immersed in Tris-HCl buffer (pH7.2) for 48 h, and summarized in Table 4 below.

S. plymuthica Buffer layer 5.00 × 10 ⁵ ~ 2.38 × 10 ⁶ Within the bead 4.14 × 10 ⁸

As shown in Table 4, even if the wet gel is soaked in the buffer for two days, the number of bacteria in the alginate beads does not change significantly within 48 hours. From this, it can be seen that the supporting capacity of the alginate gel against S. plymuthica bacteria. It can also be inferred that the release of bacteria from alginate beads occurs very slowly.

On the other hand, Gram-negative S. plymuthica is also assumed to adhere to the inner layer of the alginate layer, which is a layered structure in the beads made in this study, it was also confirmed in FIG.

6) S. plymuthica Wow P. fluorescens Relative encapsulation and survival

In this example, S. plymuthica A21-4 and P. fluorescens B16 were selected as useful bacteria.

That is, as described above, alginate beads are prepared using the three kinds of multi-cations as described above, and the two bacteria are left in the air at room temperature, and then left for a time (0 hr, 24 hr, 48 hr, 72 hr, In order to measure the survival rate according to 120 hr), the capsules were placed in sterile distilled water for 24 hours, then crushed and spreaded to determine the number of colonies, and the relative encapsulation and survival rates of S. plymuthica and P. fluorescens according to multiple cations The results are summarized in Table 5 below.

Alginate / poly-L-lysine / silicate Elapsed time 0.1 M CaCl ₂ S. plymythica P. fluorescens 0 hr 7.98 × 10 ⁸ CFU / mL 4.56 × 10 ⁶ CFU / mL 24 hr 2.84 × 10 ⁸ CFU / mL 2.04 × 10 ⁶ CFU / mL 48 hr 4.52 × 10 ⁸ CFU / mL 6.29 × 10 ⁶ CFU / mL 72 hr 2.69 × 10 ⁸ CFU / mL - 96 hr 3.71 × 10 ⁸ CFU / mL - 120 hr 1.38 × 10 ⁸ CFU / mL -

Alginate / Polyethyleneimine / Silicate Elapsed time 0.1 M CaCl ₂ S. plymythica P. fluorescens 0 hr 2.53 × 10 ⁸ CFU / mL 3.42 × 10 ⁶ CFU / mL 24 hr 1.75 × 10 ⁸ CFU / mL 1.03 × 10 ⁶ CFU / ml 48 hr 3.24 × 10 ⁸ CFU / mL 2.78 × 10 ⁶ CFU / mL 72 hr 2.07 × 10 ⁸ CFU / mL - 96 hr 2.82 × 10 ⁸ CFU / mL - 120 hr 1.09 × 10 ⁸ CFU / ml -

Alginate / chitosan / silicate Elapsed time 0.1 M CaCl ₂ S. plymythica P. fluorescens 0 hr 3.68 × 10 ⁸ CFU / mL 2.07 × 10 ⁶ CFU / mL 24 hr 1.32 × 10 ⁸ CFU / mL 1.28 × 10 ⁶ CFU / mL 48 hr 1.83 × 10 ⁸ CFU / mL 2.62 × 10 ⁶ CFU / mL 72 hr 3.25 × 10 ⁸ CFU / mL - 96 hr 1.09 × 10 ⁸ CFU / mL - 120 hr 1.67 × 10 ⁸ CFU / mL -

As shown in Table 5, it can be seen that the loading rate and survival time between the two Gram-negative bacteria are very different. In other words, S. plymuthica showed no significant change in viability within 120 hours, whereas P. fluorescens did not survive after 72 hours. From this, it was confirmed that P. fluorescens of two bacteria is more cold-resistant.

Therefore, the preparation of encapsulated alginate gel through lyophilization of the present invention and crop seedling and field studies were limited to S. plymuthica A21-4.

7) Encapsulated  3 months later to survive S. plymuthica  A21-4 population

After freeze-drying the chitosan-based S. plymuthica alginate gel beads prepared in Experiment 6) for 3 months in a Falcon tube, the capsules were placed in sterile distilled water for 24 hours and then crushed and spreaded to determine the number of colonies. Was measured.

As a ^{result, 1.0 × 10 5 ~ 1.1 ×} 10 7 CFU / ml of that, good protective film of some tteoleojigin compared to the initial 10 ^8, but that the bacteria after 3 months represents the relatively very high survival rate seen alginate gel matrix is Serratia bacteria ( to provide protection during storage.

8) S. plymuthica  A21-4 encapsulated  3 to 5 mm Alginate Bead  Structural properties

Gel beads prepared by LC low pressure pump injection using a 200 μl yellow tip were lyophilized and gold coated to observe morphology under an electron microscope. For cross-sectional photography, the lyophilized sample was coagulated again with liquid nitrogen, cut with a knife, and SEM observation was performed.

As shown in FIG. 4, the structural properties of the alginate beads are generally rounded (˜0.5 mm diameter), and the interior is quite porous and most of the spaces appear to be interconnected. The overall spherical structure was not significantly different according to the multiple cations used, but the internal space structure was found to be very different depending on the type of multiple cations used and whether the bacteria were loaded.

Among them, the case of using chitosan multiple cations loaded with bacteria was the least structurally dense. It is believed that this was caused by flocculation of the bacteria in a 1.5 wt / v% solution of alginate polymer, which caused some aggregation during bead formation, which led to many voids during the lyophilization process.

< Example  Hundreds of μm ~ 1 by 4-chitosan-coated based microencapsulation mm Alginate  Shell Microbeads  Manufacturing (using compressed air powered injection system)>

   The beads made by the 200 μl yellow tip / low pressure pump method described above have a size of about 3 to 5 mm, which is not a problem in the hydrogel formulation used in the pot, but it is too large for packaging such as foliar spraying and thus it may be difficult to adhere to the plant. have.

Therefore, since the bead size needs to be reduced to a level of several tens to hundreds of microns, a method of making alginate beads having a relatively small size up to several hundred microns using an air injector of alginate solution using compressed air will be described later.

One) Hundreds of microns Serratia Loaded Alginate Bead  Manufacture and characteristics

As shown in FIG. 8, 9 g of PVA and 1.5 g of barium chloride were dissolved in 100 ml of DW to sterilize 5.5 g of 4 ml of alginic acid mixed with PBS buffer and S. plymuthica in a 2: 1: 1 ratio to 1 liter of DW. 150 ml of melted solution was flowed at a syringe pump speed of 18 to 18.6 ml / h, and spherical beads were dropped to 4.5 to 5.5 LPM AIR (smaller flow rates and higher air pressures). .

However, the capsules were not immediately aggregated by washing with sodium chloride (9%) solution, but the obtained bacterial loaded beads were found to entangle with each other between the beads during the lyophilization process to reduce the volume, and also most of the bacteria were killed.

Therefore, in order to structurally stabilize the micron beads and increase the survival rate of bacteria, an attempt was made to stabilize the alginate beads by adding CaCl ₂ / chitosan / silicate method to the process.

That is, 0.1 M CaCl ₂ to several hundred micron sized capsules obtained in 8 Spherical beads were formed by slowly dropping into 100 ml of solution. It was stirred at 4 ° C. for 1 hour and washed twice with 0.05 M Tris-HCl buffer (pH 7.2).

Then, 0.01 wt / v% chitosan was added thereto and stirred for 30 minutes to coat the beads with chitosan.

As before, in order to improve the mechanical properties of the alginate / chitosan capsules, the capsules were reacted with 0.01 M sodium silicate solution for 2 hours, and the capsules were coated with silicate once more and washed twice with 0.05 M Tris-HCl buffer (pH 7.2).

Micron Alginate Bead  characteristic

In order to measure the number of viable cells, capsules made by an air spray system / additional surface treatment process were placed in sterile distilled water for 24 hours, and then crushed and spreaded to determine the number of colonies.

① Number of surviving population: 2.75 × 10 ⁸ CFU / ml

② Bead Size: 400 ㎛ ~ 1 ㎜

Encapsulation rate made with an air injection system The beads coated again according to the original method were observed after Coomassie blue staining under a microscope and the results are shown in FIG. 6A.

As can be seen from the microscopic observation, it can be seen that the structure is spherical and the size is about 400 μm in the swollen state. However, it can be seen from Fig. 6b that corresponds to an electron micrograph after freeze-drying the alginate beads, which is a small leaf type having a diameter of ˜500 μm.

The peculiar thing is that the spherical diameter in the swelling state is also ~ 500 ㎛, which shows that the swelling shape occurs only in a specific direction.

However, the electron microscopic cross-section of gel beads made by LC low pressure pump injection method using 200 μl yellow tip consists of several layers, but the structure of gel beads made by air injection system is assumed to be unoccupied shell type beads. It is also possible that the beads made with the air injection system required an additional stabilization process to reflect the shell-type structure.

< Example  5- Alginate  Physicochemical Study on the Structural Properties of Water in Gels>

On the other hand, Figure 6 relates to a physicochemical study (Differential Scanning Calorimetry study) on the structural properties of the water in the alginate gel. That is, the bacteria consist of about 60 to 70wt% of water. In addition, the material used as the encapsulation matrix of bacteria is also Ca ²⁺ cross-linked alginate hydrogel, the water content of 95 ~ 99%. Therefore, the condition and structural properties of water determine the life and death of bacterial bacteria.

Therefore, the effects of the intrinsic water structure on the viability of bacteria were investigated using differential scanning thermoanalysis (ice melting point) technique.

1) Lyophilized S. plymuthica Moisture Content in Loaded Capsules wt %

   Place the lyophilized about 10 mg capsules in the e-tube into the e-tube and dried in a vacuum oven at 50 ° C. for 24 and 48 hr, and weighed to determine the moisture wt% content of the lyophilized capsules. It was measured based on.

[{Wt (freeze-dried) -Wt (vacuum-dried)} / Wt (freeze-dried)]] X 100 = moisture content wt% in capsule

The results obtained are summarized in Table 6 below.

Residual water content wt% in lyophilized alginate beads 24 h 48 h Moisture Content in Alginate Beads  10wt% 10wt%

2) lyophilized S. plymuthica Differential Scanning Calorimetry (DSC) Study of Loaded Chitosan-based Beads

Lyophilized 0.2 to 0.3 mg S. plymuthica The loaded beads were placed in a DSC sample pan, and then 0.2 μl, 0.4 μl, 0.6 μl, 0.8 μl, 1 μl, 2 μl, and 3 μl of distilled water were placed in each pan, immediately sealed, and used for experiment after standing at room temperature of 24 hr. It was.

At this time, only bead of relatively constant weight was selected by 0.2 ~ 0.3 mg experiment.

A sealed empty pan was placed in the reference cell of the DSC (Model DSC Q10) instrument, and the sample pan was placed on the sample cell, left at a temperature of -60 ° C, and tested at a scan rate of 10 ° C / min. Onset T and Tm (peak temperature) at which ice begins to melt were determined using the instrument's standard software.

Swelling 0.2 μL of water in 0.2 mg beads showed no melting endothermic temperature by ice. However, increasing the amount of water to 0.4 [mu] l shows the melting endotherm at around -10 [deg.] C. That is, considering the weight of the bead about 0.2 mg, about 1.5 times the weight ratio of the bead may be referred to as water that does not exhibit a melting endotherm, that is, bound water (see FIG. 7).

It can be seen that 10% of the weight loss obtained from the vacuum drying of the above lyophilized beads is thus bound water.

If 10% of the water in the freeze-dried gel is all bound water, it can be seen that "bound water", which is 1.5 times the weight of the beads during the last drying process, is also removed. Since the binding water is due to the presence of alginate gel, it is thought that this binding water played an important role in the survival of S. plymuthica during the freezing process.

For reference, Figure 8 is lyophilized S. plymuthica Graph showing melt enthalpy per gram of ice obtained from DSC melting endothermic temperature with water content of loaded chitosan-based beads, where enthalpy per gram of Y-axis contained 0.2 mg of beads. Where the X-axis meets S. plymuthica The amount of bound water where the loaded chitosan-based beads showed no melting was 0.34 g.

9 is lyophilized S. plymuthica Graph showing melt enthalpy per gram of ice obtained from DSC melt endothermic temperature with water content of loaded chitosan-based beads, where enthalpy per gram of Y-axis is recalculated from the enthalpy value of FIG. The enthalpy shows a parabolic curve, but extrapolation with the X-axis showed 0.33 g, which is almost the same as in FIG. 8.

According to the present invention, it is possible to replace poly-L-lysine, which is currently used for alginate gel stabilization, with low-cost (16,000 times) and environmentally friendly chitosan, as well as excellent carrying capacity against S. plymuthica A21-4 bacteria. After 3 months at room temperature, bacteria showed very high survival rates.

Claims (16)

A method of encapsulating agricultural useful microorganisms using alginate shell beads, Dissolving alginic acid in NaOH solution and adjusting to pH 7.0-7.5 to prepare an alginate solution; Suspending the pepper blight control bacterium of Serratia plymuthica A21-4 or Psudomonas fluorescens B16 as a useful agricultural microorganism in the alginate solution; Slowly dropping the suspended alginate solution into 0.1 M CaCl ₂ solution with LC low pressure pump to form spherical beads; Stirring and washing the obtained spherical beads; Adding chitosan to the washed beads and stirring to first coat; Second coating the coated beads with a silica solution; And Washing the obtained coating to obtain an alginate / chitosan based hydrogel having a diameter of 3 to 5 mm; Including, The whole process is carried out at 4 ℃ temperature encapsulation method of agricultural useful microorganisms using alginate shell beads to improve the cold resistance viability of useful agricultural microorganisms delete The method of claim 1, wherein the Serratia plymuthica A21-4 method characterized by using the culture to be 3 × 10 ⁹ CFU / ml culture medium colonies from NR The method of claim 1, wherein the Psudomonas fluorescens B16 is used in a culture of NR medium so that the number of colonies 2 × 10 ⁹ CFU / ml delete delete delete delete delete The method of claim 1, wherein the obtained hydrogel is lyophilized or dried to enhance the viability of microorganisms in the gel. delete delete A method of encapsulating agricultural useful microorganisms using alginate shell beads, As a useful microorganism for farming, Serratia plymuthica A21-4 or Pseudomonas fluorescens B16 was mixed with barium chloride solution, and then alginate was dissolved in an air sprayer using a syringe pump speed of 18-18.6 ml / h and Spraying under conditions of 4.5 to 5.5 LPM AIR; Slowly dropping the obtained hundred micron size capsule into 0.1 M CaCl ₂ solution for 1 hour to form spherical beads; Stirring and washing the obtained beads: Adding chitosan to the washed beads and stirring to first coat; Second coating the coated beads with a silica solution; And Washing the obtained beads to obtain an alginate / chitosan based hydrogel having a diameter of 400 μm to 1 mm; The whole process is carried out at 4 ℃ temperature encapsulation method of agricultural useful microorganisms using alginate shell beads to improve the cold resistance viability of useful agricultural microorganisms, characterized in that The method of claim 13 wherein the Serratia plymuthica A21-4 method characterized by using the culture to be 3 × 10 ⁹ CFU / ml culture medium colonies from NR The method according to claim 13, wherein the Psudomonas fluorescens B16 is cultured in NR medium so as to have a colony number of 2 × 10 ⁹ CFU / ml. The method of claim 13, wherein the obtained hydrogel is lyophilized or dried to enhance the viability of microorganisms in the gel.

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