Dried biological compositions and methods thereof

Abstract

The present disclosure relates to a dried biological composition comprising at least one silica, a mixture of polysaccharides and glycoproteins, at least one microorganism, at least one sugar or sugar alcohol, and a resin. The dried biological composition is produced by the steps of (a) mixing at least one silica, a mixture of polysaccharides and glycoproteins, at least one microorganism, and at least one sugar or sugar alcohol in an aqueous solution, (b) spray drying the mixture of (a), and (c) coating the spray-dried mixture (b) with a compound comprising a resin in a fluidized bed. The composition is intended for agricultural applications such as foliar application or seed treatment.

Description

[Technical field]

[0001] FIELD OF THE DISCLOSURE This disclosure relates generally to dried biological compositions and methods of making and using same.

[0002] Background technology US Patent No. 8,409,822 (Trevino et al.) discloses a composition for dry delivery of microorganisms, comprising precipitated silica granules having a porous structure and microorganisms loaded throughout the pores of the precipitated silica granules, the composition functioning to allow the growth of the microorganisms in the pores of the precipitated silica granules. Also, US Patent No. 9,296,989 (Trevino et al.) relates to a composition for dry delivery of live cells, comprising an inert carrier substrate having pores, live cells loaded into the pores of the inert carrier substrate, and a surface layer disposed on the outer surface of the inert carrier substrate loaded with the live cells, the surface layer being permeable to molecules that aid in the cell growth of the live cells, such that the composition functions to allow increased growth of the live cells in the inert carrier substrate compared to other compositions in which the surface layer is absent. Although the composition of Trevino et al. is disclosed as "dry", it is not actually dried, as it is disclosed that the pores of the precipitated silica granules are substantially filled with a liquid containing live microorganisms. Trevino et al.'s silica acts as an absorbent and is loaded with 25-75% live microorganisms. At this loading level, the loaded silica is defined as free-flowing and dry to the touch. The use of these compositions is relatively limited because the concentration of microorganisms and the water content in the silica are not optimized, and the microorganisms can still respire, which can result in rapid loss of activity.

[0003] Various protective agents, such as sulfoxides, alcohols, monosaccharides, polysaccharides, amino acids, peptides, glycoproteins, and other additives, have been used to protect microorganisms from dehydration damage. U.S. Pat. No. 5,360,607 (Eyal et al.) discloses an improved stable dried pellet biopesticide composition comprising an inert carrier capable of supporting fungal growth and promoting conidial sporulation, and an entomogenous fungal biomass prepared by submerged fermentation of an isolate of the fungus Paecilomyces fumosoroeus. However, this method uses alginate to encapsulate natural pellets, which are subject to variations, particularly in the moisture content (e.g., water activity (WA)) that microorganisms depend on to survive and respire. w ) level).

[0004] WO 2020 / 104612 describes a dried biological composition comprising (1) a substrate and (2) a microorganism loaded on a surface of the substrate, the composition having a total moisture content of about 0.01% to about 15% by weight.

[0005] International Publication No. 2012 / 118795 2 describes a seed coating composition comprising a seed and a specific layer coating.

[0006] CN101069499 describes a method for treating seeds, which includes a film-forming agent, a colorant and an oxygen-enriched porous inorganic material.

[0007] The prior art does not address the problem of maintaining the stability of microorganisms while stored in a dried form. It is therefore an object of the present invention to provide a highly concentrated, dry, stable form of microorganisms during storage.

[0008] summary This object is achieved by a dried biological composition comprising at least one silica, a mixture of polysaccharides and glycoproteins, at least one microorganism, at least one sugar or sugar alcohol, and at least one resin.

[0009] The silica may be fumed silica or precipitated silica, preferably precipitated silica. The precipitated silica is preferably 10 ml 2 / g~550m 2 / g, more preferably 200m 2 / g~550m 2 / g, more preferably 480m 2 / g~520m 2 The precipitated silica has a BET surface area of ​​1000 μm / g. The precipitated silica has a particle size d50 of preferably 5 μm to 200 μm, more preferably 30 μm to 100 μm, and even more preferably 40 μm to 60 μm. The precipitated silica has a total water content of preferably 1% to 15% by weight, more preferably 5% to 10% by weight, and even more preferably 6% to 8% by weight. The precipitated silica has a dose of oil (DOA) of preferably 10 mL / 100 g to 500 mL / 100 g, more preferably 200 mL / 100 g to 400 mL / 100 g, and even more preferably 280 mL / 100 g to 300 mL / 100 g.

[0010] The dried biological composition is preferably 0.1 ml 2 / g~5m 2 / g, more preferably 0.5m 2 / g~2m 2 / g, more preferably 0.5m 2 / g~1m 2 The dried biological composition has a BET surface area of ​​0.1 to 0.6, more preferably 0.15 to 0.5, and even more preferably 0.2 to 0.4. The dried biological composition has a particle size d50 of preferably 5 μm to 700 μm, more preferably 300 μm to 600 μm, and even more preferably 450 μm to 550 μm. The dried biological composition has a total water content of preferably 0% to 15% by weight, more preferably 2% to 10% by weight, and even more preferably 3% to 6% by weight. The dried biological composition has a water activity of preferably 0.1 to 0.6, more preferably 0.15 to 0.5, and even more preferably 0.2 to 0.4.

[0011] The mixture of polysaccharides and glycoproteins may be gum arabic. Gum arabic may have a concentration of 1% to 30% by weight, preferably 5% to 15% by weight, in the dried biological composition. Gum arabic may act as a protectant and provide an advantage in terms of viability during spray drying.

[0012] The microorganisms were Bacillus subtilis QST713, Pasteuria usage, Beauveria bassiana, Coniothyrium minitans, Chondrostereum purpureum, Paecilomyces lilacinus, Aschersonia aleyrodis, Beauveria brongniartii, Hirsutella thompsonii, Isaria fumosorosea, Isaria sp., Lecanicillium longisporum, and longisporum, Lecanicillium muscarium, Lecanicillium sp., Metarhizium anisopliae, Metarhizium anisopliae var.acridum, Nomuraea rileyi, Sporothrix insectorum, Cydia pomonella GV, Phytophthora palmivora, Lagenidium giganteum, Bacillus thuringiensis, Pseudomonas protegens, Bradyrhizobium, Mycorrhiza, Clonostachys rosea, Saccharomyces cerevisiae, Pichia pastoris, Aspergillus niger niger, Aspergillus oryzae, or Hansenula, Bacillus spp. and Lactobacillus spp., or any combination thereof, preferably selected from the group consisting of Bacillus thuringiensis, Pseudomonas protegens, Bradyrhizobium, Mycorrhiza, Clonostachys rosea, and any combination thereof, more preferably Pseudomonas protegens.

[0013] The sugar may be trehalose, saccharose, a disaccharide or a polysaccharide, preferably isomaltulose or palatinose. The sugar alcohol may be isomalt.

[0014] The resin may be shellac.

[0015] The dried biological composition preferably comprises at least one precipitated silica, a mixture of polysaccharides and glycoproteins, at least one microorganism, a sugar or sugar alcohol, and shellac.

[0016] The dried biological composition more preferably comprises at least one of precipitated silica, gum arabic, at least one microorganism, isomalt and shellac.

[0017] The dried biological composition most preferably comprises at least one of precipitated silica, gum arabic, Pseudomonas protegens, isomalt and shellac.

[0018] The dried biological composition may include a plasticizer, preferably polyethylene glycol (PEG) or glycerin, more preferably PEG400.

[0019] The dried biological composition preferably comprises 5% to 15% by weight of silica, 5% to 15% by weight of a mixture of polysaccharides and glycoproteins, 2% to 8% by weight of microorganisms, 20% to 30% by weight of sugars and sugar alcohols, and 30% to 60% by weight of resin.

[0020] The dried biological composition preferably comprises 5% to 15% by weight of precipitated silica, 5% to 15% by weight of gum arabic, 2% to 8% by weight of microorganisms, 20% to 30% by weight of sugars and sugar alcohols, and 30% to 60% by weight of resins.

[0021] The dried biological composition preferably comprises 5% to 15% by weight of precipitated silica, 5% to 15% by weight of gum arabic, 2% to 8% by weight of Pseudomonas protegens, 20% to 30% by weight of isomalt, and 30% to 60% by weight of shellac.

[0022] The dried biological composition may have a core-shell structure, where the core comprises silica, a mixture of polysaccharides and glycoproteins, microorganisms and sugars or sugar alcohols, and the shell comprises a resin, preferably shellac.

[0023] The dried biological composition may contain 30% to 60% by weight of a resin, preferably shellac.

[0024] The dried biological composition preferably comprises 5% to 15% by weight of precipitated silica, 5% to 15% by weight of gum arabic, 2% to 8% by weight of Pseudomonas protegens, 20% to 30% by weight of isomalt, and 30% to 60% by weight of shellac, and the composition has a core-shell structure, the core comprising precipitated silica, gum arabic, Pseudomonas protegens and isomalt, and the shell comprising shellac.

[0025] The method according to the invention comprises the steps of: (a) mixing at least one silica, a mixture of polysaccharides and glycoproteins, at least one microorganism, and at least one sugar or sugar alcohol in an aqueous solution; (b) spray drying the mixture of (a); (c) coating the spray-dried mixture (b) with a resin-containing compound in a fluidized bed; Includes.

[0026] The mixing step (a) may be carried out with at least one silica, a mixture of polysaccharides and glycoproteins, at least one microorganism and at least one sugar or sugar alcohol in an aqueous solution.

[0027] The aqueous solution in step (a) may be a saline solution.

[0028] The aqueous solution of step (a) (i) mixing a mixture of polysaccharides and glycoproteins, such as gum arabic, with a sugar or sugar alcohol, such as isomalt, and with silica, preferably precipitated silica, such as Sipernat® 50; (ii) centrifuging the microorganisms and suspending the residue after centrifugation in saline or Luria Broth (Luria-Miller Bertani Broth); (iii) After the suspension is complete, the two solutions (i) and (ii) are mixed for about 30 to 60 minutes. It may be produced by

[0029] The spray drying process (b) may be carried out at a spray dryer inlet temperature of 76° C. to 80° C. The outlet temperature may be 45° C. to 60° C. The two-fluid nozzle using air or inert gas was not pressurized in the system. The entire process may be carried out under a nitrogen atmosphere.

[0030] Atomization of the mixture may be carried out in co-current with hot drying gas from the top of the spray dryer.

[0031] In the fluidized bed coating process (c), the process gas may be nitrogen with a maximum bed temperature of 40°C to 50°C, preferably 40°C. The resin may be an aqueous resin solution. The aqueous resin solution may comprise polyethylene glycol or glycerin. The aqueous resin solution may be sprayed into the fluidized bed. The aqueous resin solution may be a shellac solution, preferably an ammonium salt shellac solution. The ammonium salt shellac solution may comprise 25% by weight of pure shellac. The ammonium salt shellac solution may comprise PEG400 (e.g. 5% by weight based on dry shellac) as a plasticizer.

[0032] The dried biological compositions of the present invention can be used in agricultural applications, such as seed treatment or foliar spray, as well as food and feed applications.

[0033] The final dried biological composition has improved storage stability.

[0034] Working Example Particle size (d50) Particle size measurement of the substrate is performed on a HORIBA Laser Scattering Dry Particle Size Distribution Analyzer LA-950 according to the angle of scattered laser light according to ISO 13220.

[0035] Total water content Moisture measurements of dried substrates or microbial powders are performed with a Satorius IR Moisture Balance. An amount of approximately 0.3 g of sample powder is weighed out and the sample is heated to a temperature of 105° C. and held on an aluminum plate until constant mass is reached.

[0036] water activity The water activity of the sample (a w ) is measured up to 22°C. w ) is measured by placing the sample in a defined water activity environment and tracked using a measurement device. The humidity sensor is based on a capacitive polymer moisture sensing element consisting of a hygroscopic dielectric material placed between a pair of electrodes. The sensor uses a plastic or polymer as the dielectric material. Gaseous water molecules can pass through the sensor. As the humidity increases the material reacts and the geometry of the sensor determines the value of the capacitance which is related to the amount of water molecules present. Thus the humidity, relative humidity, is tracked over time in the chamber and therefore in the sample to follow the equilibrium between the sample and the gaseous environment.

[0037] BET surface area and DOA The BET surface area of ​​substrates (e.g., silica) is measured on a Micromeritics TriStar 3020 instrument by the BET nitrogen adsorption method of Brunaur et al. (J. Am. Chem. Soc., 60, 309 (1938)), which is known in the art for granular materials, e.g., silica and silicate materials. Nitrogen adsorption-desorption isotherms were collected at 77 K. 50-100 mg of powder samples are degassed at 105 °C for 2 h before measurement. The Barrett-Joyner-Halenda (BJH) model is used to calculate the BET surface area. DOA adsorption measurements are performed using ISO 19246.

[0038] Colony forming units (CFU) The concentration of the microorganisms is determined by plate counting using a serial dilution technique. The product powder containing the microorganisms is stirred in sterile water in the presence of Triton X-100 surfactant to mobilize the microorganisms from the carrier. The resulting suspension of individual microorganisms is serially diluted several times, 10-fold each time. Each time, the diluted sample is plated on a sterile agar plate and incubated. After several days, the microorganisms present are visible as dots on the agar. When the dilution is sufficient to reduce the number on the plate to a countable amount, the number of colonies is counted and multiplied by the dilution factor to determine the population in the original sample.

[0039] Storage Test The storage test is carried out at 25°C. The storage test is carried out in a thermostatic chamber with a salinity that defines a water activity environment in the ambient air with a relative humidity of 40% (sodium iodide), corresponding to a water activity of 0.4. Uncoated samples from the spray drying process as a reference and coated samples from the fluidized bed coating process are added in 1 g portions to the chamber in open Falcon tubes. Over time, the relative humidity in the closed atmosphere becomes identical to the relative humidity in the open sample tube for each gram of sample. Thus, the cells are in equilibrium with the relative humidity of the ambient air. Over a period of time (several weeks), samples are taken at regular intervals from the thermostatic chamber. The samples are then analyzed by the described CFU method.

[0040] The physical properties of the substrates were measured using the analytical methods described above and are summarized below.

[0041] Example 1 (Comparative) In one flask, 6390 g of saline is placed. This is solution (i). 655 g of gum arabic is weighed out and stirred with a propeller mixer until the gum arabic is completely dissolved. Then, 1829 g of isomalt (Risumalt®), a sugar alcohol, is added to the solution. Once completely dissolved, 770 g of Sipernat® 50 is added to the liquid and mixed until evenly dispersed. Sipernat® 50 is a 500 ml solution of BET 500 ml. 2 / g, d50 50 μm and total moisture content ≦7% by weight.

[0042] The bacterial biomass of Pseudomonas protegens Migula, Pf-5 is harvested from an overnight culture in a shake flask by centrifugation at 12000 rpm for 10 min. The harvested biomass cell pellet containing the microorganisms is transferred to a second flask. 1600 g of these microorganisms are resuspended in 1600 g of fresh Luria Broth (Luria-Miller Bertani Broth) medium using a propeller mixer. This solution is called solution (ii).

[0043] Solution (i) is added to solution (ii) and stirred for an additional 60 minutes. This solution is called solution (iii).

[0044] Solution (iii) is spray-dried using a GEA Niro Minor (MM-100) spray dryer. The inlet temperature is adjusted to 80° C. with a flow rate of 18 g / min to 20 g / min. The corresponding outlet temperature is 50° C. to 53° C. The dryer is used in co-current flow and the particles sprayed using a two-fluid nozzle are dried within a few seconds in the machine. The atomization and drying gas is nitrogen. The resulting particles are collected in a cyclone from which the product is stored refrigerated at 4° C.

[0045] Example 2 (Example of the present invention) 2.4 kg of the spray-dried product of Example 1 are stored in a refrigerator at 4° C. until the next day, when they are used in a fluid bed called Procell LabSystems manufactured by Glatt. In one run, 350 g of the spray-dried product of Example 1 are provided in the fluid bed machine. The 30 ml of the fluid bed machine used for the fluidization of the powders is 3 With 1475 g of ammonium shellac solution (SSB® AQUAGOLD from Stoever GmbH&Co.KG Bremen) preheated to 80° C. / h, 1475 g of ammonium shellac solution (SSB® AQUAGOLD from Stoever GmbH&Co.KG Bremen) are sprayed onto the fluidized powder bed using a nozzle pressure of 1.5 bar. The aqueous ammonium-shellac solution has a concentration of 25% by weight of dry shellac in water. A plasticizer is added to this solution at a concentration of 5% by weight depending on the weight of dry shellac used. The plasticizer is PEG400. The drying time is 140 min.

[0046] The bed temperature is about 40° C. The shellac forms a shell around the spray dried particles, coating them accordingly. The coated powder contains shellac on its outside.

[0047] Using the above or similar methods, the BET surface area and average particle size of the products immediately after processing were measured and are summarized in Table 1.

[0048] [Table 1]

[0049] Using the above or a similar method, the total moisture content and water activity (a w ) levels were measured and are summarized in Table 2.

[0050] [Table 2]

[0051] Storage water activity (a wThe effects of storage temperature (°C), moisture content on initial CFU, and CFU after 1 to 3 weeks are summarized in Table 3.

[0052] [Table 3]

[0053] As can be seen from Table 1 above, the blockage of open pores by the addition of a shellac coating reduces the BET surface area.

[0054] At 40% RH, the uncoated powder (Example 1) shows lower stability compared to the shellac coated powder (Example 2) under identical storage conditions (Table 3). After 3 weeks, the coated material has a LOG loss of 3.09 and the uncoated material has a LOG loss of 7.46.

[0055] It can therefore be concluded that shellac coating significantly reduces the mortality rate of viable cells during storage compared to uncoated material and prevents cell death.

Claims

1. A dried biological composition comprising at least one silica, a mixture of polysaccharides and glycoproteins, at least one sugar or sugar alcohol, at least one microorganism, and at least one resin, wherein the composition has a core-shell structure, the core comprising the silica, the mixture of polysaccharides and glycoproteins, the microorganism, and the sugar or sugar alcohol, and the shell comprising the resin.

2. 10. The dried biological composition of claim 1, wherein the silica is precipitated silica.

3. Precipitated silica is 10m 2 / g~550m 2 3. The dried biological composition of claim 2, having a BET surface area of 1000 nm / g.

4. The microorganisms include Bacillus subtilis QST713, Pasteuria usagae, Beauveria bassiana, Coniothyrium minitans, Chondrostereum purpureum, Paecilomyces lilacinus, Aschersonia aleyrodis, Beauveria brongniartii, Hirsutella thompsonii, Isaria fumosorosea, Isaria sp., Lecanicillium longisporum, and the like. longisporum, Lecanicillium muscarium, Lecanicillium sp., Metarhizium anisopliae, Metarhizium anisopliae var. acridumacridum, Nomuraea rileyi, Sporothrix insectorum, Cydia pomonella GV, Phytophthora palmivora, Lagenidium giganteum, Bacillus thuringiensis, Pseudomonas protegens, Bradyrhizobium, Mycorrhiza, Clonostachys rosea, Saccharomyces cerevisiae, Pichia pastoris, Aspergillus niger niger, Aspergillus oryzae, or Hansenula, Bacillus spp. and Lactobacillus spp., or any combination thereof.

5. 5. The dried biological composition of claim 4, wherein the microorganism is Pseudomonas protegens.

6. 10. The dried biological composition of claim 1, wherein the resin is shellac.

7. 2. The dried biological composition of claim 1, wherein the sugar or sugar alcohol is trehalose, sucrose or a polysaccharide, or isomalt.

8. 10. The dried biological composition of claim 1, comprising precipitated silica, gum arabic, Pseudomonas protegens, isomalt, and shellac.

9. 10. The dried biological composition of claim 1, comprising 30% to 60% by weight of resin.

10. 10. The dried biological composition of claim 1, comprising 5% to 15% by weight silica, 5% to 15% by weight mixture of polysaccharides and glycoproteins, 2% to 8% by weight microorganisms, 30% to 60% by weight resin, and 20% to 30% by weight sugars and sugar alcohols.

11. below, (a) mixing at least one silica, a mixture of polysaccharides and glycoproteins, at least one microorganism, and at least one sugar or sugar alcohol in an aqueous solution; (b) spray drying the mixture of (a); (c) coating the spray-dried mixture (b) with a resin-containing compound in a fluidized bed 10. A method for producing the dried biological composition of claim 1, comprising:

12. 12. The method for producing a dried biological composition of claim 11, wherein saline is added in step (a).

13. 12. The method for producing a dried biological composition of claim 11, wherein glycerin or polyethylene glycol is added in step (c).

14. 10. Use of the dried biological composition of claim 1 for seed formulation or foliar spray application.