JP5397942B2 - Satch-decomposing microbe-encapsulating microcapsules, lawn conservation method using the same, and method for producing the microcapsules - Google Patents

Description

  TECHNICAL FIELD The present invention relates to a satch-decomposing bacteria-encapsulating microcapsule suitable for decomposing and removing a satch deposited on a lawn, a method for preserving a lawn using the microcapsule, and a method for producing the microcapsule.

  Generally, lawn mowers are regularly used to keep the lawn height in the green of golf courses, stadiums such as soccer, baseball and horse races, and lawns such as gardens. Accumulate in between to produce a thatch. This thatch is a pile of turfgrass, dead leaves, stems, roots, etc., but as the accumulation increases, it becomes difficult for air and water to penetrate into the soil and the roots of turfgrass. Fertilizers and pesticides are absorbed by the satch and become less effective, and toxic gas is easily generated due to the propagation and decay of pathogenic bacteria and pests, causing turfgrass growth failure and death. Therefore, it is necessary to remove it before the accumulation of thatch increases as a care for keeping the grassland healthy.

  Conventionally, as a means for removing the satch, a method of scraping the satch with a blade of a self-propelled or towed machine called vertical mower on a large lawn such as a golf course or a stadium has been adopted. In a grassy field, it is normal to scrape the satch manually using a rake or rake (Non-patent Document 1). However, either the mechanical work or the manual work is very troublesome for the satch removal work, and there is a concern that the work may damage the turf grass.

http://www.takii.co.jp/green/howto/howto5.html (2009/9/11) "Turf, greening, green manure | Encyclopedia of lawn. Takii seedlings"

  In light of the above-mentioned circumstances, the present inventors have conducted experimental research on the possibility of means for decomposing and reducing the volume of a satch using microorganisms. As a result, there are microorganisms that exhibit high activity as satch-decomposing bacteria, It has been found that microorganisms can be easily propagated by general culturing means and can be sufficiently used on an industrial scale. However, the method of directly spraying such a sorghum-degrading bacterium on turf land is affected by severe natural conditions such as sunlight, temperature change, and rainfall. It was difficult to exhibit a stable thatch decomposition action over a long period of time.

  In view of this, the inventors of the present invention have made extensive studies on application to that-degrading bacteria, focusing on the immobilization of microorganisms by microcapsules in the course of further experimental research. As a result, porous microcapsules made of environmentally degradable polymers can stably hold satch-degrading bacteria and efficiently establish them on lawn, and protect satch-degrading bacteria from environmental changes. In addition to being able to do so, the environmentally degradable polymer of the microcapsule is used as a feed, which suppresses the inactivation of the satch-degrading bacteria and ensures its high satch-degrading ability for a long period of time without impacting the environment on the sprayed lawn. It has been found that the present invention can be exhibited, and the present invention has been made.

Thatch-decomposing bacteria-encapsulating microcapsules according to the invention of claim 1 are porous and hollow lumens comprising an environmentally degradable polymer mainly composed of poly-ε-caprolactone having an average molecular weight of 10,000 to 500,000. It is assumed that the inner portion of the microcapsule having the inner capsule is filled with the inner aqueous phase containing the satch-degrading bacteria .

  As a preferred embodiment of the microcapsules containing that-degrading bacterium of claim 1, the invention of claim 2 is that the bacterium that belongs to the genus Bacillus subtilis, and the invention of claim 3 is that the average particle size is 10 to 3, Each of them is specified to be in the range of 000 μm.

The lawn conservation method according to the invention of claim 4 is characterized in that the satch-decomposing bacteria-encapsulating microcapsules according to any one of the above 1 to 3 are sprayed on the lawn.

The method for producing microcapsules containing that-degrading bacteria according to the invention of claim 5 is characterized in that the environmentally degradable wall material polymer mainly composed of poly-ε-caprolactone having an average molecular weight of 10,000 to 500,000 is mainly composed of dichloromethane. low boiling point organic organic phase the solvent formed by dissolving the by admixing alginate aqueous solution containing thatch degrading bacteria, liquid droplets of the alginate aqueous solution containing thatch decomposing bacteria as the inner aqueous phase in the organic phase After preparing an S / O emulsion in which the S / O emulsion is dispersed, the S / O emulsion is added and mixed in the aqueous phase and stirred for a predetermined time, whereby the droplets of the S / O emulsion are dispersed in the outer aqueous phase. S / O / W emulsion prepared, and then the organic solvent in the organic phase is removed by heating or / and drying in liquid under reduced pressure to gel the wall material polymer. Ri, is characterized in that to produce microcapsules inner aqueous phase is filled comprising thatch-degrading bacteria to the cavity shape of the lumen of the microcapsules porous consisting of environmentally degradable polymers.

The satch-degrading bacteria-encapsulating microcapsules according to the invention of claim 1 are such that the satch-degrading bacteria are stably supported on the microcapsules and can be efficiently fixed by spraying on the lawn . Since the hollow microcapsule has a hollow inner cavity filled with an internal aqueous phase containing satch-degrading bacteria, it is included even when exposed to severe natural conditions such as sunlight, temperature changes, and rainfall. The satch-degrading bacteria are protected from environmental changes, and in addition, the environmentally degradable polymer that forms the microcapsules serves as a feed for the satch-degrading bacteria. The amount of decrease due to release from the capsule is always reliably replenished, and the poly-ε-caprolactone used as the environmentally degradable polymer is excellent in biodegradability and biocompatibility, and is particularly well compatible with that degradation bacteria. Satch-degrading bacteria remain highly active without metabolic stress. Therefore, in the lawn where the microcapsules are sprayed, the satch-degrading bacteria are stably released over a long period of time, and the high satch-degrading ability is continuously exhibited. Air and water do not penetrate easily, and fertilizers and pesticides that have been spread are not absorbed by the satch, making it difficult for them to work, and the generation of toxic gases due to the propagation and decay of pathogenic bacteria and pests is prevented. Is maintained in a healthy state for a long time, and the microcapsules themselves are degraded over time in the natural environment and eventually disappear. Nor.

  According to the invention of claim 2, the satch-degrading bacterium encapsulated in the microcapsule exhibits a high satch-degrading action, is hardly inactivated in a microcapsule-encapsulated state, is relatively easy to culture and can be obtained at low cost. There is an advantage.

   According to the invention of claim 3, since the average particle size of the microcapsules containing such saccharolytic bacteria is in a specific range, it is difficult to be washed away by rainfall or flooding after being sprayed on lawn, and the vegetation status and soil status of turfgrass There is no change.

According to the lawn land preservation method according to the invention of claim 4 , since the satch-decomposing bacteria-encapsulating microcapsules are sprayed on the lawn ground, the satch-degrading bacteria are stably released over a long period of time and have a high thatch decomposition ability. persistently demonstrated, or become air and water is less likely to penetrate to the soil or turf grass-roots deposited thick thatch, scatter the fertilizer and pesticides is not able to or less likely effectiveness is absorbed by the thatch, In addition, the generation of toxic gas due to the propagation and decay of pathogenic bacteria and pests is prevented, so that turfgrass is maintained in a healthy state for a long period of time, and the environmental burden caused by the sprayed microcapsules does not occur.

According to the method for producing a microcapsule containing a satch-decomposing bacterium according to the invention of claim 5, the satch-degrading bacterium is contained in the lumen of a porous microcapsule made of an environmentally degradable polymer mainly composed of poly-ε-caprolactone. As a microcapsule filled with an inner aqueous phase, it is possible to efficiently and easily mass-produce microcapsules that can stably exhibit high thatch degradation ability over a long period of time.

The relationship between the culture time of the satch-degrading bacteria, the number of bacteria in the medium and the pH in the medium is shown, (a) is a correlation characteristic diagram of the first culture, and (b) is a correlation characteristic diagram of the second culture. It is a correlation characteristic figure of the shaking time and cellulose degradation rate in the activity evaluation test of a satch degradation bacterium. It is a scanning electron microscope photograph figure which shows the Satch decomposition | disassembly microbe inclusion microcapsule prepared in Examples 1-3 of this invention, and its cross section. It is a scanning electron micrograph figure which shows the gel bead obtained by the preparation test of the alginate-chitosan gel bead in Example 4 of this invention. It is a scanning electron micrograph figure which shows the cross section of the alginic acid chitosan gel bead which included the satch decomposition | disassembly microbe prepared in the same Example 4, its cross section, and the microbe alginate-chitosan gel bead obtained by the said preparation test.

  Thatch-decomposing bacteria-encapsulating microcapsules of the present invention are those in which porous decomposing bacteria are encapsulated in porous microcapsules made of an environmentally degradable polymer. It is released to the end and exhibits a high thatch decomposition ability. Therefore, in turfland sprayed with microcapsules containing that sachet-degrading bacteria, the satch accumulates thickly, making it difficult for air and water to penetrate into the soil and the roots of turfgrass. As a result, the generation of toxic gases due to the propagation and decay of pathogenic bacteria and pests is prevented, so that turfgrass is stably maintained for a long time in a healthy state. The microcapsule itself is an environmentally degradable polymer that constitutes a feed for the satch-degrading bacteria and contributes to preventing the deactivation of the satch-degrading bacteria. Since it disappears, there is no environmental impact on the sprayed turf land and its surroundings.

  The satch-degrading bacterium used in the present invention is not particularly limited as long as it has a satch-degrading ability, but Bacillus subtilis genus known as so-called Bacillus subtilis is preferable. That is, the genus Bacillus subtilis has an advantage that it has a high ability of degrading that batch and is not easily inactivated in a microcapsule-encapsulated state, and is relatively easy to culture and available at low cost.

  The environmentally degradable polymer constituting the porous microcapsule includes so-called biodegradable polymer that is decomposed by microorganisms in the soil into water and carbon dioxide, and so-called biodegradable polymer that degrades over time in natural environment with sunlight, water, etc. A biodegradable polymer is suitable for use as a feed for a satch-degrading bacterium, although it contains a disintegrating polymer component. This biodegradable polymer is divided into chemical synthesis systems, natural product systems, and microbial systems in terms of production method, and any of them may be used. Specific examples include polycaprolactone, polylactic acid, polyglycolic acid, polybutylene succinate, Aliphatic polyesters such as polyethylene succinate, polyvinyl alcohol, polyphosphoric acid, polyamino acids, chitosan, chitin, cellulose, starch, cellulose acetate, bacterial cellulose, polysaccharides such as curdlan and pullulan, biopolyester, and composites thereof Etc.

  The size of the satch-decomposing bacteria-encapsulating microcapsules is preferably in the range of 10 to 3,000 μm as the average particle size. If it is too small, it is easy to be washed away by rainfall or flooding. There are concerns that change the situation.

  There are no particular restrictions on the form of the porous microcapsule, but as a preferred specific example thereof, there is one or a plurality of hollow lumens in the porous meat part, and a satch-degrading bacterium in the lumen part. A core-shell form filled with an inner aqueous phase containing, and a rough sponge-like surface with a skin-like surface, and an inner aqueous phase containing a satch-degrading bacterium filled in the sponge-like voids Gel bead form. In particular, in the former core-shell form, the satch-degrading bacteria grow actively in the lumen without being affected by environmental changes, and the decrease due to the release outside the microcapsule is always replenished reliably, and thus high thatch degradation There is an advantage that the ability can be stably exhibited over a longer period of time, and a high microcapsule recovery rate can be obtained.

  In order to produce the above-mentioned core-shell microcapsules, first, an environmentally degradable protective polymer aqueous solution containing satch-degrading bacteria is added to an organic phase O in which an environmentally degradable wall material polymer is dissolved in a low-boiling organic solvent. By adding and mixing, an S / O emulsion in which droplets of a protective polymer aqueous solution containing satch-decomposing bacteria are dispersed as an inner aqueous phase S in the organic phase O is prepared. Then, the S / O emulsion is added and mixed in the aqueous phase and stirred for a predetermined time to prepare an S / O / W emulsion in which droplets of the S / O emulsion are dispersed in the outer aqueous phase W. . Next, drying in liquid by heating or / and decompression is performed, and the organic solvent in the organic phase O of the S / O / W emulsion is removed to gel the wall material polymer, thereby comprising an environmentally degradable polymer. A microcapsule in which an inner water phase S containing a satch-degrading bacterium is filled in a lumen of the porous microcapsule is generated.

  Here, as the low-boiling organic solvent of the organic phase O, a nonpolar solvent having a boiling point of 85 ° C. or lower is preferable for drying in liquid by heating and / or reduced pressure, for example, dichloromethane (boiling point 40 ° C.), chloroform (61 ° C.), ethyl acetate (77 ° C.), 1.2-dichloroethane (83.5 ° C.), and the like are suitable. It is recommended to use alone or in combination with others as the subject. That is, since dichloromethane has a particularly low boiling point, gelation of the wall material polymer by in-liquid drying proceeds efficiently, and the temperature and pressure conditions required for in-liquid drying are eased and energy consumption can be reduced.

  The environmentally degradable polymer used for the organic phase O wall material polymer is not particularly limited, but a polymer mainly composed of poly-ε-caprolactone is recommended. This is because, by using poly-ε-caprolactone as a wall material polymer, microcapsules excellent in recovery rate and physical strength can be produced, and in addition, compatibility between poly-ε-caprolactone and that-degrading bacteria is particularly good. This is because the activity of the satch-degrading bacterium persists at a high level by using this as a diet, so that a better satch-degrading ability is exhibited. In addition, as this poly-epsilon-caprolactone, that whose average molecular weight is 10,000-500,000 is suitable.

  In addition, in the organic phase O, an appropriate emulsion stabilizer is preferably blended for preparing an S / O emulsion by adding the inner aqueous phase S. Examples of such emulsion stabilizers include a spanning surfactant such as sorbitan monooleate, and various surfactants, water-soluble resins, water-soluble polysaccharides and the like generally used for emulsion preparation.

  The environmentally degradable protective polymer used in the inner aqueous phase S may be any water-soluble polymer that does not interfere with the activity and growth of that-satch-degrading bacteria. For example, a water-soluble polymer such as alginate, κ-carrageenan, chitosan, etc. Examples thereof include saccharides and polyvinyl alcohol, but an alginate such as sodium alginate is particularly recommended because of its good compatibility with that degradation bacteria. The concentration of the aqueous solution in the case of using this sodium alginate is preferably about 0.5 to 10% by mass, and if it is too high, the dispersion stability of the S / O emulsion is lowered and aggregation tends to occur. In addition, polypeptone, a yeast extract, magnesium sulfate, etc. can be suitably mix | blended in the protective agent polymer aqueous solution as a nutrient source of a satch-degrading bacterium.

  When the S / O emulsion is prepared by adding and mixing the inner aqueous phase S in the organic phase O, it is recommended to stir under ice cooling in order to protect the satch-degrading bacteria. Moreover, it is desirable that the aqueous phase used for the outer aqueous phase W of the S / O / W emulsion contains a dispersion stabilizer. The dispersion stabilizer is not particularly limited, and any of those generally used for emulsion preparation can be used. From the viewpoint that an excellent emulsion effect can be obtained by using a high molecular weight emulsion stabilizer. Are particularly preferred. When this gelatin is used, the concentration in the outer aqueous phase is preferably about 0.1 to 10% by mass. Furthermore, after drying in the liquid, a proteolytic enzyme such as papain may be added for the purpose of degrading the high molecular weight gelatin to a low molecular weight and improving the filtration characteristics of the prepared microcapsules and the outer aqueous phase.

  In the above-mentioned drying in the liquid, in order to volatilize and remove the low boiling point organic solvent in the organic phase, one or both of heating and depressurization is performed with stirring, but it is recommended to simultaneously perform heating and depressurization in terms of processing efficiency. . In the heating, the temperature of the emulsion may be raised stepwise or continuously over several hours, but the highest temperature may be lower than the boiling point of the low boiling organic solvent. Further, in the reduced pressure, the pressure of the atmosphere in contact with the liquid level of the emulsion may be reduced stepwise or continuously over several hours, but the maximum reduced pressure may be about a fraction of the atmospheric pressure. Thus, when the organic solvent having a low boiling point in the organic phase is mainly dichloromethane, the maximum ultimate temperature is about 30 ° C. and the maximum pressure reduction is about 400 hPa in the liquid drying in which heating and decompression are performed simultaneously. In addition, it is good for the stirring speed in this liquid drying to be about 50-500 rpm.

  On the other hand, the microcapsules in the form of gel beads are not particularly limited, but the alginic acid-chitosan gel beads are excellent in the supportability and habitability of that-satch-degrading bacteria, and in addition to being able to exhibit high thatch-degrading ability stably over a long period of time, gel beads Is recommended because it is easy to prepare.

  In order to produce a porous microcapsule composed of alginate-chitosan gel beads encapsulating this satch-degrading bacterium, an aqueous solution containing an acid component for dissolving chitosan in water and calcium chloride as a gelling agent for alginic acid is used. The alginate-chitosan gel beads encapsulating the satch-degrading bacteria are produced by adding dropwise the alginate aqueous solution containing the satch-degrading bacteria to the continuous phase as a dispersed phase to freeze-dry the gel beads obtained by filtration and washing. And collect it.

  In the above continuous phase in this production method, the pH is increased by adding an alkali such as sodium hydroxide in order to increase the activity of the satch-degrading bacterium and promote the formation of a composite film by electrostatic interaction between alginic acid and chitosan. It is desirable to adjust to a weak acidity of about 5 to 5.5. In addition, when the alginate aqueous solution containing the satch-decomposing bacteria in the dispersed phase is added dropwise to this continuous phase, it is desirable to set the liquid temperature at a moderate temperature of 1 to 20 ° C. in order to protect the satch-degrading bacteria.

In addition, as chitosan, although commercially available chitosan aqueous solution can be used, a difference arises in the property of the alginate-chitosan gel bead produced by the viscosity. By the way, the viscosity and pH of three kinds of trade names chitosan 5, chitosan 50, and chitosan 300 manufactured by Wako Pure Chemical Industries, Ltd., which are typical commercial products of chitosan aqueous solutions, are different as follows.
Viscosity (mPa · s) pH (10g / l water immersion, 25 ° C)
Chitosan 5 ... 0-10 ... 8.0-100.0
Chitosan 50 ... 10 to 100 ... 7.0 to 9.0
Chitosan 300 ... 100-500 ... 6.0-8.0
Thus, alginic acid-chitosan gel beads can be prepared by using any of these three types of chitosan. However, when the pH of the continuous phase is increased by adding the above-mentioned alkali, the formation of gel beads is caused by chitosan 50 and chitosan 300. At the same time, it is easy to cause agglomeration in part, but the chitosan 5 does not agglomerate even when the pH is raised to 5.4. Therefore, it is recommended to use a chitosan aqueous solution having a viscosity of 10 mPa · s or less in order to increase the activity of the satch-degrading bacteria by adjusting the pH to slightly acidic.

[Culture of that-degrading bacteria]
Bacillus su-btilis NBRC13719 as a satch-degrading bacterium in sterilized 702 medium (1 g polypeptone and 0.2 g Yeast exract and 0.1 g MgSO ₄ .7H ₂ O dissolved in 100 ml distilled water, pH 7.0) In a incubator, the cells were cultured for a predetermined number of days with a shaker at a temperature of 30 ° C. and a stirring speed of 170 rpm, and 0.1 ml of the 702 medium after the culture was collected, and 0.9 wt% physiological saline was added. The mixture was mixed with 9 ml, and 0.1 ml of the mixed solution was collected and mixed with 0.9 ml of the same physiological saline, so that the 702 medium was diluted to 100 million volume times. 802 agar medium (polypeptone 1g of distilled water 100 ml, Yeast the Extract of 0.2g, MgSO ₄ · 7H ₂ O of 0.1 g, agar 1.5g dissolved, pH 7.0) was added to in And allowed to stand at 30 ° C. in Yubeta, 1 day, 2 days, and counting the colonies of thatch degrading bacteria were formed after 3 days with measuring the number of viable bacteria, the pH was measured in the media by a pH meter. This culture test was performed twice under the same conditions in order to confirm reproducibility. As a result, the relationship between the culture time in 702 medium (days = day), the number of viable bacteria (CFU / ml) in the 802 agar medium and the pH in the medium on each count day from 1st to 3rd In the test of Fig. 1 (a), the second test was as shown in Fig. 1 (b).

  As shown in FIGS. 1 (a) and 1 (b), the viable count of that-degrading bacteria reaches a peak after 2 to 3 days of culture, and the pH in the medium tends to increase as the culture time increases. Soon, it was confirmed that it became substantially constant at a pH of less than 9.

[Activity evaluation of that-degrading bacteria]
A sample solution in which 0.25 g of cellulose was dissolved in 10 ml of 0.9% by weight physiological saline was placed in a test tube and sterilized, and the above-described culture test (the number of days of culture for the first test, agar) was added to the sample solution. 0.0967 g of the Satch-degrading bacterium obtained in 3 days after leaving the medium) was added and mixed with a wet weight, and the test tube was sealed and sealed at 30 ° C. and 150 rpm for a predetermined time (0 day, 0.5 day, 1 day). 2 days, 3 days, 4 days, 5 days) After shaking, the collected liquid was filtered and freeze-dried, and the dry weight was measured. Then, the difference in dry weight after 0 days of shaking and n days after shaking was taken as the weight loss of cellulose, and the cellulose degradation rate by each shaking time was determined, and the results shown in FIG. 2 were obtained. From FIG. 2, the cellulose degradation rate increased with shaking time (days), and the cellulose degradation rate reached nearly 50% in 4 days of shaking. It is clear that this satch-degrading bacterium has a high satch-degrading ability. is there.

Example 1
<Preparation of S / O emulsion>
An organic phase O is prepared by adding and mixing 5 g of poly-ε-caprolactone having an average molecular weight of 10,000 and 0.1% by weight of sorbitan monooleate in 50 ml of dichloromethane, while a 1% by weight aqueous solution of sodium alginate (viscosity of 80 to 120 cp), 1 g of the above cultured Bacillus su-btilis NBRC13719 as a wet-degrading bacterium was added and mixed in a wet weight to prepare an inner aqueous phase S, and this inner aqueous phase S was added to the organic phase O and cooled with ice. A S / O emulsion was prepared by stirring with a homogenizer at a stirring speed of 6,000 rpm for 10 minutes.

<Preparation of S / O / W emulsion>
200 ml of 0.5% by weight gelatin aqueous solution is accommodated as an outer aqueous phase W in a closed stirring tank equipped with an air supply / exhaust port and a water jacket, and the total amount of the prepared S / O emulsion is added to the stirring tank. The S / O / W emulsion in which the droplets of the S / O emulsion are dispersed in the outer water phase W is obtained by stirring for 10 minutes at a stirring speed of 150 rpm while maintaining the liquid temperature at 15 ° C. by water cooling. Prepared.

<Drying in liquid and collecting microcapsules>
About the prepared S / O / W emulsion, while maintaining a stirring speed of 150 rpm, by vacuum suction from the exhaust port and circulation of warm water to the water jacket, in the first stage, the liquid temperature is 25 ° C. and the atmospheric pressure is 600 hPa for 2 hours. In the second stage, the liquid temperature is 30 ° C. and the atmospheric pressure is 600 hPa for 3 hours. In the third stage, the liquid temperature is 30 ° C. and the atmospheric pressure is 500 hPa for 4 hours. In the fourth stage, the liquid temperature is 30 ° C. and the atmospheric pressure is 400 hPa for 5 hours. After performing the step-in-liquid drying treatment, papain is added and mixed at a rate of 0.5 g / L with respect to the outer aqueous phase, and the produced Satch-degrading bacteria-encapsulated microcapsules are separated by filtration and washed with distilled water. And recovered.

Examples 2 and 3
The organic phase O poly-ε-caprolactone was used in Example 2 except that 5 g of an average molecular weight of 40,000 was used in Example 2, and 5 g of an average molecular weight of 70,000 to 100,000 was used in Example 3. In the same manner as that, satch-degrading bacteria-encapsulating microcapsules were prepared and collected.

Average droplet diameter of S / O emulsion (S / O-EM) and W / S / O emulsion (W / S / O-EM) in Examples 1 to 3 above, average of obtained microcapsules (MC) The particle size, recovery amount and recovery rate are shown in the following Table 1 together with the average molecular weight of the poly-ε-caprolactone (PCL) used. Moreover, the scanning electron microscope photograph figure of the whole microcapsule obtained in each Example and a cross section is shown as number (1)-(3) corresponding to an Example number in FIG. In Table 1, the average droplet size of the emulsion was obtained from a stereomicrograph, and the average particle size of the microcapsule was obtained from a scanning electron micrograph, and 50 samples were actually measured to obtain an average value. The microcapsule recovery rate (%) was calculated as (microcapsule dry weight × 100) / (Na alginate mass + poly-ε-caprolactone mass + sorbitan monooleate mass).

As shown in Table 1, in any of Examples 1 to 3, a high microcapsule recovery rate of 85% or more was obtained. Further, as compared with Examples 1 to 3, the larger the average molecular weight of the poly-ε-caprolactone used in the organic phase O, the larger the average droplet size of the W / S / O emulsion and the average particle size of the microcapsules. However, it can be seen that there is almost no difference due to the average molecular weight of ε-caprolactone in the average droplet diameter and the microcapsule recovery rate of the S / O emulsion. On the other hand, from the electron micrograph of FIG. 3 , the Satch-degrading bacteria-encapsulating microcapsules obtained in Examples 1 to 3 are all in the form of a core shell having a plurality of hollow lumens in the porous meat part. I understand that.

Example 4
<Preparation test of alginic acid-chitosan gel beads>
Three kinds of chitosan according to the above-mentioned chitosan standards were used, respectively, and 0.417 g of hydrochloric acid corresponding to 0.1% by weight and 0.1% of 0.14% hydrochloric acid were added to 150 ml of 4 g / L chitosan aqueous solution in a closed stirring tank equipped with a water jacket. A 0.5M equivalent calcium chloride 0.833g was added and mixed to prepare a continuous phase, and a 1M aqueous sodium solution was added to adjust the pH. The continuous phase was maintained at a liquid temperature of 4 ° C by water cooling, while the dispersed phase was As a result, 20 ml of a 1.8 w / v% sodium alginate aqueous solution (viscosity 500 to 600 cp) is dropped by a syringe, and gently stirred at 120 rpm for 30 minutes to produce alginate-chitosan gel beads, followed by filtration and washing. Gel beads were lyophilized and collected.

Table 2 shows the amount and rate of recovery of the gel beads and the gel bead generation status together with the viscosity and continuous phase pH of the chitosan used. FIG. 4 shows scanning electron micrographs of gel beads obtained under various preparation conditions with different chitosan species and continuous phase pH. The recovery rate was calculated as [weight of recovered gel beads (including aggregates) × 100] / (Na alginate mass + chitosan mass). The gel bead production status was evaluated in the following five stages.
◎ ・ ・ ・ The best production situation and good condition after drying.
○: Good gel beads are generated.
Δ: Gel beads are formed, but aggregation is partially observed.
▲ ・ ・ ・ Gel beads are formed, but soft and brittle.
X: No gel beads are formed.

  As shown in the electron micrograph of FIG. 4, gel beads are generated even when the pH of the continuous phase is set to either 2.2 or 5.4 using three types of chitosan. Moreover, as shown in Table 2, there is almost no difference in the amount of gel beads recovered and the recovery rate due to the difference in chitosan species and continuous phase pH. However, when the pH of the continuous phase is adjusted with an aqueous sodium solution using chitosan 50,300, the gel beads are partially agglomerated together with the formation of gel beads. On the other hand, when chitosan 5 is used, the above aggregation does not occur even when the pH of the continuous phase is increased to 5.4. From this result, chitosan 5 was used in the preparation of the next that decomposed bacteria encapsulating alginic acid-chitosan gel beads.

<Preparation of that-decomposing bacteria-containing alginic acid-chitosan gel beads>
Similar to the preparation test of the alginate-chitosan gel beads, except that chitosan 5 was used as chitosan and 0.65 g of the cultured Bacillus su-btilis NBRC13719 was added as a satch-degrading bacterium to the dispersed phase sodium alginate aqueous solution in a wet weight. In this way, satch-decomposing bacteria-encapsulating alginic acid-chitosan gel beads were prepared.

Table 3 shows the recovery amount and recovery rate of the obtained gel-degrading bacteria containing gel beads together with the continuous phase pH. FIG. 5 shows scanning electron micrographs (s) (a) and (b) of the Satch-degrading bacteria-encapsulating gel beads obtained when the continuous phase pH is 2.2 and 5.4, and the continuous phase pH is 2.2. Scanning electron micrograph (c) of the cross-section of the obtained Satch-degrading bacteria-encapsulating gel beads, and scanning electron of the cross-section of the germ-free gel beads prepared using chitosan 5 in the preparation test under the condition of continuous phase pH 2.2 A micrograph (d) is shown. The recovery rate was calculated as [weight of recovered gel beads (including aggregates) × 100] / (Na alginate mass + chitosan mass + bacterial weight).

  From the electron micrograph of FIG. 5, the prepared satch-degrading bacterium-encapsulated alginic acid-chitosan gel beads have a skin-like surface and the entire interior is a rough sponge-like shape, and the satch-degrading bacterium is present in the sponge-like void portion. It can be seen that the gel is in the form of gel beads filled with an inner aqueous phase containing. In addition, from the results in Table 2, the recovery rate of that-degrading bacteria-encapsulating gel beads is slightly lower than the recovery rate of germ-free gel beads (see Table 1). This shows that the dispersibility with respect to the continuous phase tends to be reduced because the aqueous sodium alginate solution in the dispersed phase contains a satch-degrading bacterium.

Reference Example Three types of chitosan according to the standard of chitosan described above were used, and a dispersed phase was prepared by adding and mixing 1 v / v% acetic acid to 10 ml of a 1 w / w% concentration chitosan aqueous solution, and equipped with a water jacket. 100 ml of a 4 w / w concentration polyphosphoric acid aqueous solution was placed in a closed stirring tank, pH was adjusted by adding a 1 M aqueous sodium chloride solution, and the continuous phase was maintained at a liquid temperature of 4 ° C. by water cooling. A dispersed phase chitosan aqueous solution was dropped by a syringe, and the mixture was gently stirred at 120 rpm for 30 minutes to produce polyphosphate-chitosan gel beads. Then, the gel beads obtained by filtration and washing were freeze-dried and collected.

Table 4 shows the amount and rate of recovery of the gel beads and the state of gel bead formation together with the viscosity and continuous phase pH of the chitosan used. The recovery rate (%) was calculated as [weight of recovered gel beads (including aggregates) × 100] / (polyphosphoric acid mass + chitosan mass). Moreover, the gel bead production | generation condition was evaluated in the same five steps as the case of the said alginate-chitosan gel bead.

  As shown in Table 4, gel beads are produced when the pH of the continuous phase is adjusted to 5.4 using chitosan 50,300, but continuous phase is also obtained when chitosan 5 is used or chitosan 50,300 is used. When the pH is not adjusted (pH 2.2), good gel beads cannot be produced. Therefore, under the condition that the pH of the continuous phase is adjusted to 5.4 using chitosan 50,300, a Satch-degrading bacterium is added to and mixed with the dispersed phase chitosan aqueous solution, and polyphosphate-chitosan gel beads are prepared in the same manner as described above. However, it was difficult to disperse the disperse phase in the continuous phase, and it was not possible to prepare a satchel-degrading bacteria-encapsulated gel bead.

Claims (5)

A satch-degrading bacterium is placed in the lumen of a microcapsule having a porous and hollow lumen made of an environmentally degradable polymer mainly composed of poly-ε-caprolactone having an average molecular weight of 10,000 to 500,000. Thatch-decomposing bacteria-encapsulated microcapsules filled with the inner aqueous phase .   The satch-decomposing bacteria-encapsulating microcapsules according to claim 1, wherein the satch-decomposing bacterium is a genus Bacillus subtilis.   The satch-degrading bacteria-encapsulating microcapsules according to claim 1 or 2, wherein the average particle size is in the range of 10 to 3,000 µm. A method for preserving a lawn, comprising spraying the microcapsules containing that-degrading bacteria according to any one of claims 1 to 3 onto a lawn. In an organic phase in which an environmentally degradable wall material polymer mainly composed of poly-ε-caprolactone having an average molecular weight of 10,000 to 500,000 is dissolved in a low boiling point organic solvent mainly composed of dichloromethane, by admixing alginate aqueous solution containing, after the droplets of alginate aqueous solution containing thatch degrading bacteria to prepare a S / O emulsion was dispersed as an internal aqueous phase in the organic phase, the S / O emulsion An S / O / W emulsion in which droplets of the S / O emulsion are dispersed in the outer aqueous phase is prepared by adding and mixing in the aqueous phase and stirring for a predetermined time, and then the organic solvent in the organic phase It was removed in-liquid drying by heating and / or reduced pressure by gelling the wall material polymer, a porous consisting of environmentally degradable polymers of the microcapsules cavity shaped Thatch decomposing bacteria-encapsulating microcapsule production method, characterized in that to produce the microcapsules inner aqueous phase is filled comprising thatch-degrading bacteria to the cavity.