Environmentally responsive <i>Paecilomyces lilacinus </i>microbead and preparation method thereof

Abstract

An environmentally responsive Paecilomyces lilacinus microbead and its preparation method and application are provided. The Paecilomyces lilacinus microbead includes a capsule core and a capsule wall; wherein, in the capsule core, Paecilomyces lilacinus spore powder, a vegetable oil, glucose, peptone, a cellulose nanofiber, sodium citrate, and a surfactant are combined to form an emulsifiable capsule core; and in the capsule wall, chitosan, gelatin, polyvinyl alcohol, glycerol, and water are combined to form a water-responsive shell. The environmentally responsive Paecilomyces lilacinus microbeads can stimulate the dissolution of polyvinyl alcohol in the shell according to the moisture content in the soil, so that the water in the soil flows into the microbeads. The cellulose nanofibers in the microbeads absorb water and expand, blocking the holes in the shell, resulting in the inability of spores to flow out.

Claims

1. An environmentally responsive Paecilomyces lilacinus microbead, comprising a capsule core and a capsule wall; wherein, in the capsule core, Paecilomyces lilacinus spore powder, a vegetable oil, glucose, peptone, a cellulose nanofiber, sodium citrate, and a surfactant are combined to form an emulsifiable capsule core; and in the capsule wall, chitosan, gelatin, polyvinyl alcohol, glycerol, and water are combined to form a water-responsive shell.

2. The environmentally responsive Paecilomyces Lilacinus microbead of claim 1, wherein parts by weight of compositions of the capsule core are as follows: 20 to 30 parts of the Paecilomyces Lilacinus spore powder, 40 to 60 parts of the vegetable oil, 2 to 3 parts of the sodium citrate, 1 to 1.5 parts of the surfactant, 1 to 1.5 parts of the glucose, 0.5 to 1 part of the peptone, and 3 to 5 parts of the cellulose nanofiber.

3. The environmentally responsive Paecilomyces Lilacinus microbead of claim 1, wherein parts by weight of compositions of the capsule wall are as follows: 1.5 to 2 parts of the chitosan, 8 to 10 parts of the gelatin, 3 to 5 parts of the polyvinyl alcohol, 6 to 10 parts of the glycerol, and 60 to 100 parts of the water.

4. The environmentally responsive Paecilomyces Lilacinus microbead of claim 1, wherein a mass ratio of the capsule core to the capsule wall is (4-5):(2-3).

5. The environmentally responsive Paecilomyces Lilacinus microbead of claim 1, wherein the vegetable oil is one selected from the group consisting of corn oil, soybean oil and grapeseed oil.

6. The environmentally responsive Paecilomyces Lilacinus microbead of claim 1, wherein the surfactant is an anionic emulsifier or a nonionic emulsifier.

7. The environmentally responsive Paecilomyces Lilacinus microbead of claim 1, wherein the surfactant is a mixture of alkylphenol polyoxyethylene ether, fatty alcohol polyoxyethylene ether and sodium lauryl sulfate, and a mass ratio of the alkylphenol polyoxyethylene ether, the fatty alcohol polyoxyethylene ether and the sodium lauryl sulfate is 2:2:1.

8. A preparation method of the environmentally responsive Paecilomyces Lilacinus microbead of claim 1, comprising the following steps: (1) preparation of a core material solution: adding and mixing the Paecilomyces lilacinus spore powder, the vegetable oil and the sodium citrate to obtain a first mixture, and then adding the surfactant into the first mixture to form an emulsifiable concentrate, and then adding the glucose, the peptone and the cellulose nanofiber into the emulsifiable concentrate to obtain the core material solution; (2) preparation of a wall material solution: mixing and heating the chitosan, the gelatin, the polyvinyl alcohol, the glycerol and the water at 95-100° C. to obtain a second mixture, and performing a heat preservation on the second mixture at 95-96° C. to obtain the wall material solution; (3) injecting the core material solution and the wall material solution obtained in step (1) and (2) into a dripping pill machine respectively, adjusting a dripping speed to control a ratio of the core material solution and the wall material solution, and dripping the core material solution and the wall material solution into liquid paraffin to obtain a primary microbead; (4) removing the liquid paraffin on a surface of the primary microbead obtained in step (3) to obtain a treated primary microbead, and performing a drying treatment on the treated primary microbead in a roller under a constant temperature and constant humidity to obtain the environmentally responsive Paecilomyces lilacinus microbead.

9. The preparation method of claim 8, wherein the vegetable oil is one selected from the group consisting of corn oil, soybean oil and rapeseed oil.

10. The preparation method of claim 8, wherein the surfactant is an anionic emulsifier or a nonionic emulsifier.

11. The preparation method of claim 10, wherein the surfactant is a mixture of alkylphenol polyoxyethylene ether, fatty alcohol polyoxyethylene ether and sodium lauryl sulfate, and a mass ratio of the alkylphenol polyoxyethylene ether, the fatty alcohol polyoxyethylene ether and the sodium lauryl sulfate is 2:2:1.

12. The preparation method of claim 8, wherein the ratio of the core material solution to the wall material solution in step (3) is (4-5):(2-3).

13. The preparation method of claim 8, wherein after the drying treatment, a mass percent of the capsule core to the environmentally responsive Paecilomyces lilacinus microbead is 85% and a mass percent of the capsule wall to the environmentally responsive Paecilomyces lilacinus microbead is 15%.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Schematic diagram of structure and function for the product of the invention;

(2) FIG. 2: Schematic diagram of sodium citrate cross-linked chitosan and gelatin in the product of the invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(3) In order to make the purpose, technical solution and advantages of this patent clearer, the following is a further detailed description of this patent in combination with the particular embodiment. It is understood that the particular embodiments described herein are solely for the purpose of interpreting the patent and are not intended to limit the invention.

(4) The structure of the environmentally responsive microbead is shown in FIG. 1 and FIG. 2, and the following is further explained in combination with specific embodiments.

Embodiment 1: A Water-Responsive Paecilomyces lilacinus Microbead

(5) (1) Preparation of core material solution: 250 g of Paecilomyces lilacinus spore powder, 600 g of corn oil, and 20 g of sodium citrate were added into a beaker and stirred at room temperature for 30 min to mix well, followed by 10 g of the mixture of surfactants dodecylphenol polyoxyethylene ether, polyoxyethylene lauryl ether, and sodium lauryl sulfate (mass ratio 2:2:1); after the formation of EC, 15 g of glucose, 10 g of peptone, and 40 g of cellulose nanofibers were successively added and stirred at room temperature for 30 min to obtain the core material solution.

(6) (2) Preparation of wall material solution: 1.5 g of chitosan, 10 g of gelatin, 4 g of polyvinyl alcohol (the degree of polymerization of polyvinyl alcohol is 500-600), and 8 g of glycerol were added to 80 mL of distilled water, heated to 95-100° C., mixed well, and kept at 95-96° C. to obtain the wall material solution.

(7) (3) Inject the core material and wall material solution obtained in step (1) and (2) into the dripping pill machine, respectively; the dripping speed of the capsule core and the capsule wall is 40 ml/min and 30 ml/min respectively; drip into the condensate liquid paraffin, remove the condensate on the microbead surface, dry in a 35% constant humidity roller at constant temperature 25° C. to obtain the water-responsive Paecilomyces lilacinus microbeads with core material and wall material accounting for 85% and 15% of microbead mass, respectively.

Comparative Example 1: A Water-Responsive Paecilomyces lilacinus Microbead (Cellulose Nanofiber Deletion Set

(8) (1) Preparation of core material solution: 250 g of Paecilomyces lilacinus spore powder, 600 g of corn oil, and 20 g of sodium citrate were added into a beaker and stirred at room temperature for 30 min to mix well, followed by 10 g of a mixture of surfactants dodecylphenol polyoxyethylene ether, polyoxyethylene lauryl ether, and sodium lauryl sulfate (mass ratio 2:2:1); after the formation of EC, 15 g of glucose and 10 g of peptone were successively added and stirred at room temperature for 30 min to obtain the core material solution.

(9) (2) Preparation of wall material solution: 1.5 g of chitosan, 10 g of gelatin, 4 g of polyvinyl alcohol (the degree of polymerization of polyvinyl alcohol is 500-600), and 8 g of glycerol were added to 80 mL of distilled water, heated to 95-100° C., mixed well, and kept at 95-96° C. to obtain the wall material solution.

(10) (3) Inject the core material and wall material solution obtained in step (1) and (2) into the dripping pill machine, respectively; the dripping speed of the capsule core and the capsule wall is 40 ml/min and 30 ml/min respectively; drip into the condensate liquid paraffin, remove the condensate on the microbead surface, dry in 35% constant humidity roller at a constant temperature 25° C. to obtain the water-responsive Paecilomyces lilacinus microbeads with core material and wall material accounting for 85% and 15% of microbead mass, respectively.

Comparative Example 2: A Water-Responsive Paecilomyces lilacinus Microbead (Polyvinyl Alcohol Missing Set

(11) (1) Preparation of core material solution: 250 g of Paecilomyces lilacinus spore powder, 600 g of corn oil, and 20 g of sodium citrate were added into a beaker and stirred at room temperature for 30 min to mix well, followed by 10 g of a mixture of surfactants dodecylphenol polyoxyethylene ether, polyoxyethylene lauryl ether, and sodium lauryl sulfate (mass ratio 2:2:1); after the formation of EC, 15 g of glucose, 10 g of peptone, and 40 g of cellulose nanofibers were successively added and stirred at room temperature for 30 min to obtain the core material solution.

(12) (2) Preparation of wall material solution: 1.5 g of chitosan, 10 g of gelatin, and 8 g of glycerol were added to 80 mL of distilled water, heated to 95-100° C., mixed well, and kept at 95-96° C. to obtain wall material solution.

(13) (3) Inject the core material and wall material solution obtained in step (1) and (2) into the dripping pill machine, respectively; the dripping speed of the capsule core and the capsule wall is 40 ml/min and 30 ml/min respectively; drip into the condensate liquid paraffin, remove the condensate on the microbead surface, dry in a 35% constant humidity roller at a constant temperature 25° C. to obtain 85% and 15% of microbead mass for core material and wall material, respectively.

Comparative Example 3: A Water-Responsive Paecilomyces lilacinus Microbead (Surfactant Missing Set

(14) (1) Preparation of core material solution: 250 g of Paecilomyces lilacinus spore powder, 600 g of corn oil, and 20 g of sodium citrate were added into a beaker and stirred at room temperature for 30 min and mixed well, followed by 15 g of glucose, 10 g of peptone, and 40 g of cellulose nanofibers successively and stirred at room temperature for 30 min to obtain the core material solution.

(15) (2) Preparation of wall material solution: 1.5 g of chitosan, 10 g of gelatin, 4 g of polyvinyl alcohol (the degree of polymerization of polyvinyl alcohol is 500-600), and 8 g of glycerol were added to 80 mL of distilled water, heated to 95-100° C., mixed well, and kept at 95-96° C. to obtain the wall material solution.

(16) (3) Inject the core material and wall material solution obtained in step (1) and (2) into the dripping pill machine, respectively; the dripping speed of the capsule core and the capsule wall is 40 ml/min and 30 ml/min respectively; drip into the condensate liquid paraffin, remove the condensate on the microbead surface, dry in 35% constant humidity roller at a constant temperature 25° C. to obtain 85% and 15% of microbead mass for core material and wall material, respectively.

(17) Case Effect Test:

(18) (1) Microbead Change and Viable Bacteria Release in Water Environment

(19) Take 30 g of microbeads prepared in Embodiment 1 and Comparative Examples 1-3, divide them into three portions, place them in a culture dish containing a small amount of sterile water, place them at 28° C. for 5 days, observe the changes in the internal and external structure of microbeads every day, and determine the number of bacterial strains in the microbeads, the result is Table 1.

(20) TABLE-US-00001 TABLE 1 Strain number in microbeads Internal variation External variation (cfu/g) Dayl Embodiment 1 Moisture increased Small amount of water 2.4 × 10.sup.7 adsorption on microbead shell Comparative Moisture increased Small amount of water 2.2 × 10.sup.6 Example 1 adsorption on microbead shell Comparative No significant change No significant change 2.4 × 10.sup.7 Example 2 Comparative Moisture increased Small amount of water 2.3 × 10.sup.7 Example 3 adsorption on microbead shell Day2 Embodiment Moisture increased Small amount of water 5.6 × 10.sup.7 1 adsorption on microbead shell Comparative EC reduced Small amount of water 4.4 × 10.sup.5 Example 1 adsorption on microbead shell Comparative No significant change No significant change 2.6 × 10.sup.7 Example 2 Comparative Moisture increased Small amount of water 2.2 × 10.sup.7 Example 3 Adsorption on microbead shell Day3 Embodiment Moisture increased, trace Small amount of water 6.5 × 10.sup.8 1 hyphal growth adsorption on microbead shell Comparative Small amount of hyphal Small amount of water 3.3 × 10.sup.3 Example 1 growth with reduced EC adsorption on microbead shell Comparative No significant change No significant change 2.1 × 10.sup.7 Example 2 Comparative Moisture increased, no Small amount of water 2.3 × 10.sup.7 Example 3 hyphal growth adsorption on microbead shell Day4 Embodiment Moisture increased, A small amount of 1.4 × 10.sup.9 1 heavy hyphal growth hyphal in the microbead shell and thinning of the shell layer Comparative Small amount of hyphal Small amount of water 5.8 × 10.sup.2 Example 1 growth with reduced EC adsorption on microbead shell Comparative No significant change No significant change 2.3 × 10.sup.7 Example 2 Comparative Moisture increased, no Small amount of water 2.3 × 10.sup.7 Example 3 hyphal growth adsorption on microbead shell Day5 Embodiment Moisture increased, Small amount of 4.8 × 10.sup.9 1 heavy hyphal growth hyphal in microbead shell, shell layer breaks Comparative Extensive hyphal growth Small amount of water 4.4 × 10.sup.2 Example 1 with reduced EC adsorption on microbead shell Comparative No significant change No significant change 2.5 × 10.sup.7 Example 2 Comparative Moisture increased, no Small amount of water 2.1 × 10.sup.7 Example 3 hyphal growth adsorption on microbead shell

(21) The test results are shown in Table 1, and the environmentally responsive Paecilomyces lilacinus microbeads prepared according to the method described in Embodiment 1 can adjust the release rate and amount of entrapped viable bacteria according to the moisture content. When polyvinyl alcohol is added into the shell, the polyvinyl alcohol in the shell is dissolved due to the water in the microbead shell, so that the external water enters the microbead and promotes the germination of bacteria cell. When there is no polyvinyl alcohol in the shell but a small amount of moisture, the external moisture cannot enter the microbeads. When cellulose nanofibers are added to the core material, cellulose nanofibers absorb water and adhere to the wall, so that the internal EC encapsulated strains cannot leak, and the strains grow and propagate inside, so as to achieve the effect of slow release. When the EC core is used (vegetable oil and surfactant were added at the same time), the strain can grow and propagate normally. When the capsule core is only a non-EC phase, the strain is wrapped inside the oil phase and cannot contact the external environment for the time being, so it cannot propagate normally for the time being, resulting in that the strain cannot function in a short time.

(22) (2) Effect of Storage Time on Strain Viability:

(23) The samples prepared in Embodiment 1 and Comparative Examples 1-3 were sealed and stored at room temperature for 0-180 days. After storage, take 15 g of the sample, dilute it in 100 g of normal saline, add 0.5 wt % Tween 80, and place it in a conical flask. Shake it with glass microbeads (spread flat at the bottom) in a shaker (200 rpm) for 45 min. Then, the strains were diluted with normal saline according to the activity and number of strains. The dilutions were spread on Rose Bengal medium, incubated in a 28° C. incubator for 48 h, and counted. As shown in Table 2, the results showed that the strain activity of the product prepared in Embodiment 1 remained well during storage, indicating that the microbeads prepared by this method could better protect the bacteria cell during storage. For the product prepared with Comparative Example 3, there will be trace loss during storage, due to the use of non-EC type oil-phase as the core for Comparative Example 3, there will be loss in the presence of non-EC type oil-phase in the spores.

(24) TABLE-US-00002 TABLE 2 Storage Comparative Comparative Comparative Time Embodiment 1 Example 1 Example 2 Example 3 (days) (cfu/g) (cfu/g) (cfu/g) (cfu/g) 0 2.4 × 10.sup.7 2.5 × 10.sup.7 2.3 × 10.sup.7 2.1 × 10.sup.7 30 2.2 × 10.sup.7 1.7 × 10.sup.7 2.1 × 10.sup.7 1.5 × 10.sup.7 60 2.1 × 10.sup.7 1.5 × 10.sup.7 1.5 × 10.sup.7 1.3 × 10.sup.7 90 1.9 × 10.sup.7 1.4 × 10.sup.7 1.2 × 10.sup.7 1.1 × 10.sup.7 120 1.8 × 10.sup.7 1.3 × 10.sup.7 1.1 × 10.sup.7 9.8 × 10.sup.6 180 1.5 × 10.sup.7 1.1 × 10.sup.7   1 × 10.sup.7 9.6 × 10.sup.6

(25) (3) Field Tests

(26) Fertilizer efficacy tests were conducted on the control effect of root-knot nematodes, and data collection and analysis were performed on the test samples. A total of four treatments (Treatment I: Embodiment 1; Treatment II: Comparative Example 1; Treatment III: Comparative Example 2; Treatment IV: Comparative Example 3) and an untreated control were designed in the experiment. The plants tested are cucumbers. Before transplanting, 5 g of prepared samples were added to each soil pit, and other cultivation measures were uniformly and normally operated to ensure a consistent plant growth environment. After 30 days of colonization, the yield was counted, and at the late harvest stage, the roots were excavated to investigate the root knot index and evaluate the control effect. The control effect is shown in Table 3, and obviously Embodiment 1 can effectively control root-knot nematodes and improve cucumber yield.

(27) Grading criteria for root-knot nematodes:

(28) Grade 0: Healthy roots, no root knots

(29) Grade 1; few root knots, “root knot %”<25%

(30) Grade 2: Moderate number of root-knots, “root knot %”≈25%-50%

(31) Grade 3: High number of root-knots, “root knot %”≈50%-75%

(32) Grade 4: Too many root-knots, “root knot %”>75%
Root-knot index=Σ(each grade of disease index×Corresponding number of diseased strain/Investigated strain number)×100%
Prevention and control effect=(1−Root-knot index in treatment area/Root-knot index in control area)×100%

(33) TABLE-US-00003 TABLE 3 Root-knot Prevention and Yield index control effect (%) increase (%) Treatment I 19 75.64 25.6 Treatment II 38 51.28 16.8 Treatment III 53 32.05  6.9 Treatment IV 41 47.43 10.4 Control 78 — —

Embodiment 2: A Water-Responsive Paecilomyces lilacinus Microbead

(34) (1) Preparation of core material solution: 200 g of Paecilomyces lilacinus spore powder, 400 g of soybean oil, and 25 g of sodium citrate were added into a beaker and stirred at room temperature for 30 min to mix well, followed by 12 g of a mixture of surfactant octylphenol polyoxyethylene ether and sodium alkyl sulfate (mass ratio 2:1); after the formation of EC, 10 g of glucose, 5 g of peptone, and 30 g of cellulose nanofibers were successively added and stirred at room temperature for 30 min to obtain the core material solution.

(35) (2) Preparation of wall material solution: 1.5 g of chitosan, 8 g of gelatin, 3 g of polyvinyl alcohol (the degree of polymerization of polyvinyl alcohol is 500-600), and 6 g of glycerol were added to 60 mL of distilled water, heated to 95-100° C., mixed well, and kept at 95-96° C. to obtain the wall material solution.

(36) (3) Inject the core material and wall material solution obtained in step (1) and (2) into the dripping pill machine, respectively; the dripping speed of capsule core is 40 ml/min, the dripping speed of capsule wall is 20 ml/min; drip into the condensate liquid paraffin, remove the condensate on the microbead surface, dry in a 35% constant humidity roller at constant temperature 25° C. to obtain the water-responsive Paecilomyces lilacinus microbeads with core material and wall material accounting for 80% and 20% of microbead mass, respectively.

Embodiment 3: A Water-Responsive Paecilomyces lilacinus Microbead

(37) (1) Preparation of core solution: 300 g of Paecilomyces lilacinus spore powder, 500 g of rapeseed oil, and 30 g of sodium citrate were added to a beaker and stirred at room temperature for 30 min to mix well, followed by 12 g of a mixture of surfactants castor oil polyoxyethylene ether, styrenyl phenol polyoxyethylene ether, and calcium dodecyl benzene sulfonate (mass ratio 3:1:1); after the formation of EC, 12 g of glucose, 8 g of peptone, and 50 g of cellulose nanofibers were successively added and stirred at room temperature for 30 min to obtain the core solution.

(38) (2) Preparation of wall material solution: 2.0 g of chitosan, 10 g of gelatin, 5 g of polyvinyl alcohol (the degree of polymerization of polyvinyl alcohol is 500-600), and 10 g of glycerol were added to 100 mL of distilled water, heated to 95-100° C., mixed well, and kept at 95-96° C. to obtain the wall material solution.

(39) (3) Inject the core material and wall material solution obtained in step (1) and (2) into the dripping pill machine, respectively; the dripping speed of capsule core is 50 ml/min, the dripping speed of capsule wall is 30 ml/min; drip into the condensate liquid paraffin, remove the condensate on the microbead surface, dry in a 35% constant humidity roller at constant temperature 25° C. to obtain the water-responsive Paecilomyces lilacinus microbeads with core material and wall material accounting for 90% and 10% of microbead mass, respectively.

(40) The embodiments above represent only a few embodiments of the invention, the detailed and specific description of which cannot be construed as a limitation of the scope of the patent. It should be noted that, for the general technicians in this field, without separating from the conception of this patent, the aforesaid modes of execution can also make several deformations, combinations and improvements, which are all within the scope of protection of this patent. Therefore, the scope of protection of this patent shall be subject to the claims.