WO2019232410A1

WO2019232410A1 - Spray drying composition and related methods

Info

Publication number: WO2019232410A1
Application number: PCT/US2019/034960
Authority: WO
Inventors: Ted W. DEISENROTH; Toan Van PHO; Grit BAIER; Rute da Conceicao TAVARES ANDRE; Rebecca Schmitt
Original assignee: Basf Corporation
Priority date: 2018-05-31
Filing date: 2019-05-31
Publication date: 2019-12-05

Abstract

A method for encapsulating an agent is provided. The method includes the steps of: flowing a first fluid through a first path, the first fluid including one or more agent and a cross-linkable biopolymer; flowing a second fluid through a second path; flowing a gas through a third path; and combining the first fluid, second fluid and gas to encapsulate the agent in a cross-linked matrix. A spray-dried composition, seed coating composition and method for preparing a coated seed are also provided.

Description

SPRAY DRYING COMPOSITION AND RELATED METHODS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.

62/678,653, filed May 31 , 2018, the disclosure of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.

REFERENCE TO SEQUENCE LISTING, A TABLE, OR A COMPUTER PROGRAM LISTING

COMPACT DISC APPENDIX

[0003] Not applicable.

BACKGROUND

[0004] Incorporation of beneficial microorganisms (bacteria, fungi, virus) or other active agents in applicable food, agricultural, pharmaceutical or cosmetic formulations is challenging due to the harsh processing and chemical conditions used in industrial processes. Such conditions can destroy any beneficial microorganisms or render other agents inactive.

[0005] Microencapsulation by spray-drying is a well-known methodology used to protect the sensitive microorganisms or other active agents from external stress factors by entrapping the active material within a protective matrix, which is essentially inert to the material being encapsulated. Encapsulation materials include biologically available matrix compounds such as hydrogels, biopolymers and polysaccharides that can provide a controlled release of the bioactive and protection against environmental stress. Alginate is a well-known encapsulation matrix which can be cross-linked in the presence of divalent ions such as calcium. Various approaches to encapsulation by spray-drying are the subject of, for example, U.S. Patent No. 9,700,519; WO2017/044473; WO2013/096883; Strobel et ai,“Industrially-Scalable

Microencapsulation of Plant Beneficial Bacteria in Dry Cross-Linked Alginate Matrix”, Industrial Biotechnology , Vol. 14, No. 3 (2018), pp. 138-147; Santa-Maria et ai,“Microencapsulation of bioactives in cross-linked alginate matrices by spray drying”, Journal of Microencapsulation, Vol. 29, Issue 3 (2012), pp. 286-295; and Strobel et al.,“In situ cross-linking of alginate during spray drying to microencapsulate lipids in powder”, Food Hydrocolloids, Vol. 58 (2016), pp. 141-149.

[0006] The survivability of beneficial microorganisms or the activity of active agents during the spray-drying process is usually low due to the harsh temperature and shear to which the cells are exposed. Thus, encapsulation can be difficult to upscale and further difficult to incorporate in the end application. In addition, the shelf-life of such formulations often do not meet field or use requirements. [0007] Furthermore, encapsulation using cross-linking based on pH drop can potentially cause safety issues by the release of harmful gases that lower the yield of viable microbial cells in microbial formulations or result in deactivating other active agents. Accordingly, there remains a need for stable formulations comprising beneficial microbes or other active agents with higher cross-linking matrices and methods of making them that provide improved bacterial yield and survivability.

BRIEF SUMMARY

[0008] According to one aspect, a method for encapsulating an agent is provided. The method includes the steps of: flowing a first fluid through a first path, the first fluid including one or more agent and a cross-linkable biopolymer; flowing a second fluid through a second path; flowing a gas through a third path; and combining the first fluid, second fluid and gas to encapsulate the agent in a cross-linked matrix. According to one embodiment, the second fluid includes at least one cross-linking component comprising a multivalent cation. According to one embodiment, the multivalent cation is Ca⁺², Ba⁺², Cr⁺², Cu⁺², Fe⁺², Mg⁺², Zn⁺². According to one embodiment, the first fluid further includes at least one cross-linking component that is an acid soluble multivalent cation and the second fluid includes an acid that is succinic acid, adipic acid, acrylic acid, glutaric acid, ascorbic acid, gallic acid, caffeic acid, and any combination thereof. According to one embodiment, the first fluid further comprises at least one amphiphilic polymer. According to one embodiment, the at least one amphiphilic polymer is a poloxamer.

[0009] According to one embodiment, the agent is one or more bacteria, protein, or enzyme. According to one embodiment, the bacteria is gram negative. According to one embodiment, the gram negative bacteria is a Bradyrhizobium or Rhizobium.

[0010] According to one embodiment, the method further comprises fermenting the bacteria in a fermentation medium prior to encapsulation, wherein the fermentation medium prior to fermentation comprises trehalose. According to one embodiment, the fermentation medium prior to fermentation comprises about 5% m/v trehalose. According to one

embodiment, the method further comprises adding trehalose to the fermentation medium after fermentation and prior to encapsulation. According to one embodiment, the fermentation medium prior to fermentation comprises about 2% m/v trehalose, and wherein about 8% m/v trehalose is added to the fermentation medium after fermentation and prior to encapsulation.

[001 1] According to one embodiment, the gas is air, nitrogen, or a mixture thereof.

According to one embodiment, the cross-linkable biopolymer includes an alginate. According to one embodiment, the method further includes the step of desiccating the encapsulated agent. [0012] According to another aspect, an encapsulated agent produced by the methods disclosed herein are also provided. A spray-dried composition produced by the methods disclosed herein is also provided.

[0013] According to another aspect, a spray-dried composition is provided. The spray- dried composition may be produced by any of the methods provided herein. The spray-dried composition includes a biopolymer encapsulating agent such as one or more bacteria. The bacteria exhibits a concentration of at least 1 ^c10⁸ cfu/g after spray-drying. According to one embodiment, the bacteria exhibit a concentration of at least 1 ^c10⁸ cfu/g for at least 30 days of storage, at least 60 days of storage, at least 90 days of storage, at least 120 days of storage, at least 150 days of storage, or at least 180 days of storage. According to one embodiment, the bacteria are nitrogen fixing bacteria. According to one embodiment, the bacteria are gram negative bacteria. According to one embodiment, the bacteria are Rhizobium spp. or

Bradyrhizobium spp. According to one embodiment, the initial water activity (a_w) of the composition is between about 0.05 to about 0.15. According to one embodiment, the biopolymer encapsulating the bacteria is a cross-linked biopolymer. According to one embodiment, the biopolymer includes alginate. According to one embodiment, the spray-dried composition further includes at least one amphiphilic polymer. According to one embodiment, the at least one amphiphilic polymer is a poloxamer. According to one embodiment, the composition further includes at least one sugar. According to one embodiment, the sugar is trehalose, sucrose, lactose, raffinose, or a mixture thereof.

[0014] According to another aspect, a seed coating composition is provided. The seed coating composition includes a spray-dried composition as provided herein that includes at least one biopolymer encapsulated agent. The seed coating also includes water. According to one embodiment, the encapsulated agent is one or more bacteria, protein, or enzyme. According to one embodiment, the bacteria is gram negative. According to one embodiment, the gram negative bacteria of the spray-dried composition may be Rhizobium spp., Bradyrhizobium spp., or a combination thereof. According to one embodiment, the biopolymer includes an alginate. According to one embodiment, the seed coating composition is applied to a soybean seed.

[0015] According to another aspect, a method for preparing a coated seed is provided.

The method includes the steps of: preparing a seed coating composition, the seed coating composition including at least one biopolymer encapsulated agent; applying the seed coating composition to a surface of the seed to prepare a coated seed; and drying the coated seed. According to one embodiment, the encapsulated agent is one or more bacteria, protein, or enzyme. According to one embodiment, the bacteria is gram negative. The gram negative bacteria of the spray-dried composition may be Rhizobium spp., Bradyrhizobium spp., or a combination thereof. According to one embodiment, the biopolymer includes an alginate. According to one embodiment, the seed coating composition is applied to a soybean seed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is schematic of a tri-channel nozzle of a spray dryer according to one embodiment.

[0017] Figure 2 depicts the bacterial survivability results for the comparison of samples prepared according to Example 1 and Example 2 with Hydagen558P alginate and B. japonicum 532c and stored at 28 °C.

[0018] Figure 3 depicts the bacterial survivability results for the comparison of samples prepared according to Example 1 and Example 2 with Sigma-Aldrich alginate and B. japonicum 532c and stored at 28 °C.

[0019] Figure 4 depicts the bacterial survivability results for the comparison of samples prepared according to Example 1 and Example 2 with Hydagen558P alginate and SEMIA 5079/5080 and stored at 28 °C.

[0020] Figure 5 depicts the bacterial survivability results for the comparison of samples prepared according to Example 1 and Example 2 with Sigma-Aldrich Alginate and SEMIA 5079/5080 and stored at 28 °C.

DETAILED DESCRIPTION

[0021] The present disclosure will now be described more fully hereinafter with reference to exemplary embodiments and aspects thereof. All embodiments and aspects can be combined in any way or combination. These exemplary embodiments are described so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. Indeed, the present disclosure may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. The following definitions are meant to clarify, but not limit, the terms defined. If a particular term used herein is not specifically defined, such term should not be considered indefinite. Rather, terms are used within their accepted meanings.

[0022] As used in the specification, and in the appended claims, the singular forms“a”,

“an”,“the”, include plural referents unless the context clearly dictates otherwise. As used in the specification, and in the appended claims, the words "optional" or "optionally" mean that the subsequently described event or circumstance can or cannot occur.

[0023] As used herein the term“agent” refers to any chemical or biological component

(or a combination thereof), the application of which causes or provide a beneficial and/or useful effect in agriculture including, but not limited to, agriculturally beneficial microorganisms, biostimulants, nutrients, pesticides (e.g. acaricides, fungicides, herbicides, insecticides, and nematicides), plant signal molecules, enzymes and proteins.

[0024] As used herein the term“agriculturally beneficial microorganisms” refers to microorganisms having at least one agriculturally beneficial property (e.g., the ability to fix nitrogen, the ability to solubilize phosphate and/or the ability to produce an agriculturally beneficial agent, such as a plant signal molecule) such as, for example, bacteria.

[0025] As used herein the term“biostimulant” refers to an agent or combination of agents the application of which enhances one or more metabolic and/or physiological processes of a plant or plant part (e.g., carbohydrate biosynthesis, ion uptake, nucleic acid uptake, nutrient delivery, photosynthesis and/or respiration).

[0026] As used herein the term“biopolymer” refers to any molecule that polymerizes with multivalent ions and has at least one polymerizable moiety that cross-links in the presence of a multivalent ion and includes alginates, pectin, carrageenan, chitosan, gelatin, cellulose, starch, polyglutamic acid, whey proteins, casein, or any combination thereof. This term includes both monomers and polymers.

[0027] As used herein the term“cross-linking component” refers to a multivalent cation that facilitates cross-linking of the biopolymer.

[0028] As used herein the term“colony forming units”,“cfu”, or“CFU” refers to microbial cell/spore capable of propagating on or in a substrate (e.g., a soil) when conditions (e.g., temperature, moisture, nutrient availability, pH, etc.) are favorable for microbial growth.

[0029] As used herein the term“multivalent cation” refers to any cation with more than one electron in its valence shell, including but is not limited to: Ba⁺², Ca⁺², Cr⁺², Cu⁺², Fe⁺², Fe⁺³, Mg⁺², Pb⁺², Pb⁺⁴, Sn⁺², Sn⁺⁴ and Zn⁺².

[0030] As used herein the terms“yield” or“initial survivability” refer to the percentage of colony forming units surviving after the spray-drying method as provided herein. The yield is calculated based on the following formula: CFU_out / CFU_n x 100.

[0031] As used herein the term“survivability” refers to the colony forming units (CFU) at a point in time after formation of the spray-dried composition as provided herein. [0032] As used herein the term“water activity” or“a_w” refers to the partial vapor pressure of water in a substance divided by the standard state partial vapor pressure of water.

[0033] The present disclosure provides for both compositions and methods that provide an agent encapsulated within a cross-linked matrix. The methods provided herein eliminate producing harmful side products such as, for example, ammonia or ammonia gas. The encapsulated agents prepared according the methods provided herein exhibit increased survivability when stored at elevated temperatures (28-32°C).

[0034] According to one embodiment, a method is provided for making a spray-dried composition comprising an agent encapsulated in a cross-linked matrix. According to a particular embodiment, the method utilizes a tri-channel nozzle spray-drying system as depicted in FIG. 1. Two channels serve as a first path and second path for a first fluid and second fluid, respectively. A third channel serves as a path for at least one gas. According to one embodiment, the first fluid path passes through a first path via an internal channel nozzle.

According to one embodiment, the second fluid passes through a second path via an external channel nozzle. The first and second fluids react which each other during spraying. While not being bound to a particular theory, it is believed that physical separation of the first fluid and the second fluid prevents pre-mature cross-linking and that upon combining the fluids, the biopolymer or agent from the first fluid reacts with the cross linking component from the second fluid during spray-drying to form an encapsulated, highly cross-linked agent. Thus, the physical separation of the two fluids provides a better encapsulation efficiency.

[0035] According to one embodiment, the agent may be one or more chemical or biological agent including but not limited to agriculturally beneficial microorganisms (e.g., bacteria), biostimulants, nutrients, pesticides (e.g. acaricides, fungicides, herbicides, insecticides, and nematicides), plant signal molecules, enzymes and proteins.

[0036] According to a particular embodiment, the agent is a bacteria. The bacteria may be any type of bacteria that may be encapsulated in biopolymer. The bacteria may be gram positive or gram negative bacteria. Suitable bacteria include, but is not limited to, Azospirillum brasilense INTA Az-39, Bacillus amyloliquefaciens D747, Bacillus amyloliquefaciens NRRL B- 50349, Bacillus amyloliquefaciens TJ1000, Bacillus amyloliquefaciens FZB24, Bacillus amyloliquefaciens FZB42, Bacillus amyloliquefaciens 1 N937a, Bacillus amyloliquefaciens IT- 45, Bacillus amyloliquefaciens T J 1000, Bacillus amyloliquefaciens MB1600, Bacillus

amyloliquefaciens BS27 (deposited as NRRL B-5015), Bacillus amyloliquefaciens BS2084 (deposited as NRRL B-50013), Bacillus amyloliquefaciens 15AP4 (deposited as ATCC PTA- 6507), Bacillus amyloliquefaciens 3AP4 (deposited as ATCC PTA-6506), Bacillus amyloliquefaciens LSSA01 (deposited as NRRL B-50104), Bacillus amylollquefaclens ABP278 (deposited as NRRL B-50634), Bacillus amyloliquefaciens 1013 (deposited as NRRL B-50509), Bacillus amyloliquefaciens 918 (deposited as NRRL B-50508), Bacillus amyloliquefaciens 22CP1 (deposited as ATCC PTA-6508) and Bacillus amyloliquefaciens BS18 (deposited as NRRL B-50633), Bacillus cereus 1-1562, Bacillus firmus 1-1582, Bacillus lichenformis BA842 (deposited as NRRL B-50516), Bacillus lichenformis BL21 (deposited as NRRL B-50134), Bacillus mycoides NRRL B-21664, Bacillus pumilus NRRL B-21662, Bacillus pumilus NRRL B- 30087, Bacillus pumilus ATCC 55608, Bacillus pumilus ATCC 55609, Bacillus pumilus GB34, Bacillus pumilus KFP9F, Bacillus pumilus QST 2808, Bacillus subtilis ATCC 55078, Bacillus subtilis ATCC 55079, Bacillus subtilis MB1 600, Bacillus subtilis NRRL B-21661 , Bacillus subtilis NRRL B-21665, Bacillus subtilis CX-9060, Bacillus subtilis GB03, Bacillus subtilis GB07,

Bacillus subtilis QST-713, Bacillus subtilis FZB24, Bacillus subtilis D747, Bacillus subtilis 3BP5 (deposited as NRRL B-50510), Bacillus thuhngiensis ATCC 13367, Bacillus thuhngiensis GC- 91 , Bacillus thuhngiensis NRRL B-21619, Bacillus thuhngiensis ABTS-1857, Bacillus thuhngiensis SAN 401 1 , Bacillus thuhngiensis ABG-6305, Bacillus thuhngiensis ABG-6346, Bacillus thuhngiensis AM65-52, Bacillus thuhngiensis SA-12, Bacillus thuhngiensis SB4,

Bacillus thuhngiensis ABTS-351 , Bacillus thuhngiensis HD-1 , Bacillus thuhngiensis EG 2348, Bacillus thuhngiensis EG 7826, Bacillus thuhngiensis EG 7841 , Bacillus thuhngiensis DSM 2803, Bacillus thuhngiensis NB-125, Bacillus thuhngiensis NB-176, Bradyrhizobium elkanii SEMIA 501 , Bradyrhizobium elkanii SEMIA 587, Bradyrhizobium elkanii SEMIA 5019,

Bradyrhizobium japonicum NRRL B-50586 (also deposited as NRRL B-59565), Bradyrhizobium japonicum NRRL B-50587 (also deposited as NRRL B-59566), Bradyrhizobium japonicum NRRL B-50588 (also deposited as NRRL B-59567), Bradyrhizobium japonicum NRRL B-50589 (also deposited as NRRL B-59568), Bradyrhizobium japonicum NRRL B-50590 (also deposited as 40 NRRL B-59569), Bradyrhizobium japonicum NRRL B-50591 (also deposited as NRRL B- 59570), Bradyrhizobium japonicum NRRL B-50592 (also deposited as NRRL B-59571), Bradyrhizobium japonicum NRRL B-50593 (also deposited as NRRL B-59572), Bradyrhizobium japonicum NRRL B-50594 (also deposited as NRRL B-50493), Bradyrhizobium japonicum NRRL B-50608, Bradyrhizobium japonicum NRRL B-50609, Bradyrhizobium japonicum NRRL B-50610, Bradyrhizobium japonicum NRRL B-50611 , Bradyrhizobium japonicum NRRL B- 50612, Bradyrhizobium japonicum NRRL B-50726, Bradyrhizobium japonicum NRRL B-50727, Bradyrhizobium japonicum NRRL B-50728, Bradyrhizobium japonicum NRRL B-50729, Bradyrhizobium japonicum NRRL B-50730, Bradyrhizobium japonicum SEMIA 566,

Bradyrhizobium japonicum SEMIA 5079, Bradyrhizobium diazoefficiens SEMIA 5080, Bradyrhizobium japonicum USDA 6, Bradyrhizobium japonicum USDA 110, Bradyrhizobium japonicum USDA 122, Bradyrhizobium japonicum USDA 123, Bradyrhizobium japonicum USDA 127, Bradyrhizobium japonicum USDA 129, Bradyrhizobium japonicum USDA 532C,

Pseudomonas jessenii PS06, Rhizobium leguminosarum S012A-2 (IDAC 080305-01), Sinorhizobium fredii CCBAU1 14, Sinorhizobium fredii USDA 205 and combinations thereof, as well as microorganisms having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to any of the aforementioned strains. According to a particular embodiment, the agent is a gram negative bacteria. According to a further embodiment, the agent is a

Bradyrhizobium sp. or Rhizobium sp.

[0037] In another embodiment, where the agent is a bacteria, the method of

encapsulating a bacterial agent in a cross-linked biopolymer results in a percentage yield of encapsulated bacteria that is typically greater than about 20% according to the following formula:

CFUout / CFUin x 100.

According to a particular embodiment, the percentage yield of encapsulated bacteria is typically greater than about 40%.

[0038] The biopolymer in the first fluid may be any molecule that polymerizes with multivalent ions and has at least one polymerizable moiety that cross-links in the presence of a multivalent ion and includes, but is not limited to, alginates, pectin, carrageenan, chitosan, gelatin, cellulose, starch, polygalacturonates, collagen, latex, polyglutamic acid, soy, whey proteins, casein, agarose, xanthan gum, maltodextrin, gallan gum or any combination thereof. According to a particular embodiment, the biopolymer includes an alginate (e.g., A1 112 from Sigma-Aldrich^® or Hydagen 558P from BASF^®). The biopolymer and agent may be combined in the first fluid prior to flowing through a first path. Biopolymers may further be combined with other things such as sugars, for example trehalose, proteins or other polymers, both

biopolymers and synthetic polymers.

[0039] The at least one cross-linking component as disclosed herein may include a multivalent cation. Suitable multivalent cations include different salts of multivalent cations such as Ca⁺², Ba⁺², Cr⁺², Cu⁺², Fe⁺², Mg⁺², Zn⁺². The multivalent ions should be capable of cross- linking. In one embodiment the multivalent cation is water soluble, such as for example, calcium chloride. In another embodiment the multivalent cation is acid soluble. Suitable salts of acid soluble multivalent ions include dicalcium phosphate, calcium carbonate and calcium oxalate. The acid soluble multivalent cation should be soluble at a low pH. [0040] The at least one organic acid as disclosed herein may include oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, or any combination thereof. The organic acid may be titrated to a slightly acidic pH by the addition of a base. According to a particular embodiment, the organic acid may exhibit a pH of typically about 5.0 to about 7.0.

[0041] According to a particular embodiment, the first fluid typically includes at least one agent that is combined with or suspended with at least one biopolymer. According to such an embodiment, the second fluid includes the cross-linking component.

[0042] According to another embodiment, the first fluid typically includes at least one agent that is combined with or suspended with at least one biopolymer and at least one cross- linking component. According to such an embodiment, the second fluid includes an organic acid. According to some embodiments, the first fluid and the second fluid are free of citric acid.

[0043] The method of encapsulating an agent in a cross-linked biopolymer includes the steps of flowing the first fluid through a first path and flowing the second fluid through a second path. The method of encapsulating an agent in a cross-linked biopolymer further includes the step of flowing a gas through a third path. The gas may include any inert gas acceptable as a carrier/drying gas. According to particular embodiment, the gas includes air, nitrogen or a mixture thereof.

[0044] The method of encapsulating an agent in a cross-linked biopolymer further includes the step of combining the first fluid, second fluid, and gas to encapsulate the agent in the biopolymer. Such a step may be carried out at any appropriate conditions to produce an agent that is encapsulated in the biopolymer. According to one embodiment, the inlet temperature is typically from about 100°C to about 160°C. According to one embodiment, the inlet temperature is typically from about 120°C to about 140°C. According to one embodiment, the inlet temperature is typically about 130°C. According to one embodiment, the outlet temperature is typically from about 40°C to about 80°C. According to one embodiment, the outlet temperature is typically from about 50°C to about 70°C. According to one embodiment, the outlet temperature is typically about 60°C. According to one embodiment, cross linking between the cross-linking component and the biopolymer is limited prior to combining the fluid and gas.

[0045] The method of encapsulating an agent in a cross-linked biopolymer may further include the step of introducing at least one polymer to the first fluid prior to combining with the second fluid and gas. The polymer may a biopolymer or a synthetic polymer and may be added to increase survivability of the bacteria in the resulting spray-dried composition in the presence of limited oxygen and humidity. Exemplary polymers include ethylene oxide/polyethylene oxide tri-block copolymers. The polymer may be added to increase survivability of a bacterial agent in the resulting spray-dried composition in the presence of limited oxygen and humidity. The polymer may be an amphiphilic polymer or poloxamer (e.g., poloxamer 188 available from Sigma Aldrich^®). The polymer may be added at a concentration of typically from about 0.05% to about 2.0% based on the weight of polymer per volume of fluid.

[0046] According to one embodiment, the spray-dried composition further includes a sugar. In a further aspect, the sugars is trehalose, sucrose, lactose, raffinose and a mixture thereof. Sugars may derivatized or functionalized. In another embodiment, the composition further includes a polysaccharide. Exemplary polysaccharides include but are not limited to maltodextrin and starch. In another embodiment, the composition includes mixtures of sugars and polysaccharides.

[0047] The method of encapsulating an agent in a cross-linked biopolymer results in a spray-dried composition that is collected and stored. According to one embodiment, the spray- dried composition is a powder that is collected in a cyclone and collection chamber of a spray drying system.

[0048] The method of encapsulating an agent in a cross-linked biopolymer may further include the step of desiccating the spray-dried composition. According to one embodiment, the spray-dried composition is protected from light and conditioned in a desiccator at ambient temperature for typically about 5-9 days. According to one embodiment, the spray-dried composition is protected from light and conditioned in a desiccator at ambient temperature from typically about 7 days. According to one embodiment, the spray-dried composition attains a water activity of typically about 0.5 or less upon desiccation. According to one embodiment, the spray-dried composition attains a water activity (a_w) of typically about 0.1 or less upon desiccation. The spray-dried composition may be stored in a sterile container until use.

[0049] The spray-dried composition may be formulated to be in the form of a powder.

The composition may be stored at a temperature between about 4° to about 40° C and a relative humidity of between about 1 % to about 80%. Powders may be further formulated as an application to plant parts. Exemplary plant parts include any part of a plant, including cells, and tissues derived from plants and may specifically refer to any of plant components or organs (e.g., leaves, stems roots etc.), plant tissues, plant cells and seeds. Examples of plant parts, include, but are not limited to, anthers, embryos, flowers, fruits, fruit bodies, leaves, ovules, pollen, rhizomes, roots, seeds, shoots, stems and tubers, as well as scions, rootstocks protoplasts, and calli. [0050] A seed coating composition is also provided. The seed coating composition includes a spray-dried composition as provided herein that includes at least one biopolymer encapsulated agent. The seed coating also includes water. According to one embodiment, the encapsulated agent is one or more bacteria, protein, or enzyme. According to one embodiment, the bacteria is gram negative. According to one embodiment, the gram negative bacteria of the spray-dried composition may be Rhizobium spp., Bradyrhizobium spp., or a combination thereof. Preferred applications include seed coatings for application to soybean seeds.

[0051] A method for preparing a coated seed is also provided. The method includes the steps of: preparing a seed coating composition, the seed coating composition including at least one biopolymer encapsulated agent; applying the seed coating composition to a surface of the seed to prepare a coated seed; and drying the coated seed. According to one embodiment, the encapsulated agent is one or more bacteria, protein, or enzyme. According to one embodiment, the bacteria is gram negative. The gram negative bacteria of the spray-dried composition may be Rhizobium spp., Bradyrhizobium spp., or a combination thereof. According to one embodiment, the biopolymer is an alginate.

[0052] As a person skilled in the art will readily appreciate, the above description is meant as an illustration of implementation of the principles this disclosure. This description is not intended to limit the scope or application of this system in that the system is susceptible to modification, variation and change, without departing from the spirit of this disclosure, as defined in the following claims.

EXAMPLE 1 (M2)

[0053] Preparation of fluids

[0054] Bacteria stocks: Bradyrhizobium spp. were supplied via batch fermentation as follows: a 2L PETG seed shake flask containing 500mL of a generic medium such as YMB was used. The shake flask was sterile inoculated via a glycerol stock or interchangeably a slant media wash or agar plate scrape. The flask was placed in an incubator at temperatures between 26-32°C. The flask was shaken at medium speed for 4-7 days. A stainless steel fermenter containing 20L generic Rhizobia media was inoculated. The fermentation was run in batch mode with low agitation and aeration for 14 days or until after steady state was reached. Media was aseptically harvested and filled into sterilized plastic bladders at 4 °C until use.

Bradyrhizobium japonicum strain 532c was obtained from a generic Rhizobia media e.g.

containing complex raw materials, a nitrogen and carbon source, salts, vitamins and trace elements as well as a small amount of antifoam with pH between 5.5 and 7.5. The media also contained 50g/L trehalose. Bradyrhizobium japonicum SEMIA 5079 was obtained from a generic Rhizobia media e.g. containing complex raw materials, a nitrogen source, carbon sources, salts, vitamins and trace elements as well as a small amount of antifoam with pH between 5.5 and 7.5. Bradyrhizobium diazoefficiens SEMIA 5080 was obtained from a generic Rhizobia media e.g. containing complex raw materials, a nitrogen source, carbon sources, salts, vitamins and trace elements as well as a small amount of antifoam with pH between 5.5 and 7.5.

[0055] Two batches were evaluated and fluids were prepared for each batch using the method described below:

1) B. japonicum 532c

2) B. japonicum SEMIA 5079 / B. diazoefficiens SEMIA 5080

3) Fermentation broths with or without added polysaccharides were tested.

[0056] Fluid 1 (Samples 1-3, 6-8, 11-13, 15-171: A sodium-alginate (0.5 to 2.5 wt%,

Hydagen^® 558P BASF^® or 2% to 4% Alginate from Sigma-Aldrich, Germany, Prod. Nr.

W201502) was dissolved in 100 ml_ double distilled water. The mixture was stirred for several hours (or overnight) at room temperature until homogenous. Formulation additives (sugars, polymers) were added at this point, if required. The mixture was sterile filtered before adding the microorganisms. One-hundred ml_ of bacteria culture were centrifuged (7000 rpm /15 min /

4°C) and re-suspended in 50 ml_ of the Na-alginate solution.

[0057] Fluid 2 (Samples 2, 3, 7, 8, 12, 13, 16, 17): A solution of CaCI₂ (0.1 to 0.4 wt%) was prepared with double distilled water and sterile filtered before use.

EXAMPLE 2 (M3)

[0058] Preparation of fluids

[0059] Bradyrhizobium spp. were obtained by the same method described in Example 1.

[0060] Fluid 1 (Samples 4, 5, 9, 10, 14, 18): A solution with sodium-alginate (0.5 to 2.5 wt %, Hydagen^® 558P from BASF^® or 2% to 4% Alginate from Sigma-Aldrich, Germany, Prod.

Nr. W201502) and 0.3 to 0.5 wt % CaHP0₄ was prepared in sterile filtered double distilled water. The solution was stirred overnight at room temperature until homogenous. Formulation additives (sugars, polymers) were added at this point, if required. One-hundred mL of bacteria culture were centrifuged (7000 rpm /15 min / 4 °C) and re-suspended in 50 mL of the Na- alginate/CaHP0₄ solution.

[0061] Fluid 2 (Samples 4, 5, 9, 10, 14, 18): A succinic acid solution (1.25 to 2 wt.%) was prepared in double distilled water. The pH of the solution was adjusted to 5.5 with an aqueous solution of NaOH. The solution was sterile filtered before use.

EXAMPLE 3 [0062] Modified fermentation

[0063] The fermentation process of Examples 1 and 2 were further modified to include trehalose in the fermentation media. Two modified fermentation runs were made, the first included 5% m/v trehalose added to the media before fermentation. The second run included 2% m/v trehalose added to the media before fermentation and 8% m/v trehalose added to the media after fermentation, resulting in a total 10% m/v trehalose amount in the media.

EXAMPLE 4

[0064] Spray drying process for Examples 1 and 2

[0065] Table 1 contains compositions of formulations of Samples 1-18 to be spray-dried.

TABLE 1

[0066] Spray drying was using a Buchi Labortechnik Mini Sprayer B-290 equipped with a three fluid nozzle as shown in FIG.1. The first fluid is dosed through the central channel and the second fluid is dosed through one of the side channels. The third channel supplies the drying air.

[0067] The inner channel is supplied with the alginate containing feedstock (Fluid 1) through a peristaltic pump at a rate of 1.8 ml/min. The second feedstock (Fluid 2) is supplied over a Cori-Flow M13 (Bronkhorst Cori-Tech) pump at a rate of 3.5 ml/min. The drying air used is nitrogen. The drying parameters are set as follows: Inlet temperature 110 -130 °C, aspirator 50%, Q-flow 55 mm and drying gas supplied at a rate of 25 Nm³/h. The outlet temperature is around 58-61 °C.

[0068] Spray dried material was stored in aluminum foil bags with Mitsubishi Gas

Chemical RP-3A moisture and oxygen absorbers. These RP-3A absorbers decreased the oxygen level below 0.1 % and lowered the a_w to approximately 0.05 to 0.1. Water activity (a_w) was measured using a Rotronic HygroPalm HP23-AW-A portable water activity analyzer. The samples were then stored in an incubator held at 28 °C.

[0069] Bacteria grown by the modified fermentation methods of Example 3 were then subsequently spray-dried according to the methods described for Examples 1 and 2.

EXAMPLE 5

[0070] Survivability studies

[0071] The compositions of Examples 1 and 2 (all batches) were then tested at pre determined intervals to determine bacterial survivability. A 0.05g of powder sample is weighed out in a conical tube and mixed with 20 mL of Peptone buffer containing Tween 80 and vortexed for 10 minutes. Samples were aseptically removed from the conical tubes for use with a 96-well plate used to prepare 1 to 10 serial dilutions using sterile Peptone Buffer with Tween 80.

Sample was pipetted on the surface of Congo Red Yeast Mannitol Agar (CRYMA) spot plates to create 10pL spots per dilution. Each spot plate offers 6 boxes for 6 dilutions and thus provides a larger range to count the colonies at optimal dilution. Samples are absorbed into the agar for 10-15 minutes and incubated for 6-8 days at 28°C. After incubation of plates, visible colonies are counted. Statistically accurate counts for spot plates range in 3 to 30 colonies per dilution square. Results are calculated in cfu/mL sample according to the following formula: average cfu per dilution square / 10 pL x reciprocal of dilution factor x1000 pL/mL = cfu/mL.

[0072] Initial percent (%) survivability after spray drying was calculated as Total CFUout /

Total CFUin. Total CFUout was calculated as CFU/mL of rehydrated sample ^* 20 mL (which is the volume of Peptone buffer used to rehydrate) / mass of powder rehydrated (which is typically 0.05 g) * mass of powder collected. Total CFUin was calculated as CFU/mL * total volume of the solution that the pre-spray drying aliquot was taken from. For Example 1 and 2, the pre-spray drying aliquot was typically the bacteria + succinic acid resuspension, with a volume of 50 mL. Both percent yield/initial survivability and storage conditions were tested.

[0073] At pre-determined intervals, samples of the powders were rehydrated to measure the survivability in CFU/g. CFU/g was calculated as CFU/mL of the rehydrated sample * 20 mL (which is the volume of Peptone buffer used to rehydrate) / mass of powder rehydrated (which is typically 0.05 g).

[0074] Seed coating process for Examples 1 and 2

[0075] For samples prepared according to Examples 1 and 2, seed coating

compositions were prepared via the following process. Each sample were first rehydrated in the appropriate volume of Peptone buffer with Tween 80 to prepare a solution of approximately 1x10¹⁰ CFU/mL. In a sterile microcentrifuge tube, the seed coating composition was prepared by combining 0.4 mL of the rehydrated sample, 0.4 mL of the commercially used extender, and 1.2 mL of water. Optionally, if the extender was not used, then an additional 0.4 mL of water was added. The microcentrifuge tube was vortexed for approximately 1 minute to combine the ingredients. 50 g of soybean seeds were placed into a plastic jar. Using a pipette, 0.163 mL of the seed coating composition was applied onto the seeds. The plastic jar was capped, placed into a FlackTek SpeedMixer™ DAC 150 FVZ-K, and mixed at 800 rpm for 10 seconds. Once coated, the seeds were poured into resealable foil or plastic bags. The bags were left open in a biosafety cabinet at ambient laboratory conditions for the coated seeds to dry for about 1 hour.

[0076] Alternatively, for larger scale seed treatments, 500 g of soybean seeds were placed into a resealable plastic bag. Using a pipette, 1.63 mL of the seed coating composition was applied onto the seeds. The plastic bag was sealed and shaken for approximately 1 minute. The bags were unsealed and left open in a biosafety cabinet at ambient laboratory conditions for the coated seeds to dry for about 1 hour.

EXAMPLE 6

[0077] Results

[0078] Table 3 and Figures 2 and 3 show results from testing with B. japonicum 532c.

TABLE 3

[0079] Table 4 and Figures 4 and 5 show results from testing with B. japonicum SEMI A

5079 / B. diazoefficiens SEMI A 5080.

TABLE 3

Claims

CLAIMS We claim:

1. A method for encapsulating an agent comprising:

a. flowing a first fluid through a first path, the first fluid comprising one or more agent and a cross-linkable biopolymer;

b. flowing a second fluid through a second path;

c. flowing a gas through a third path; and

d. combining the first fluid, second fluid and gas to encapsulate the agent in a cross-linked matrix.

2. The method of claim 1 , wherein the second fluid comprises at least one cross-linking component comprising a soluble multivalent cation.

3. The method of claim 2, wherein the multivalent cation is Ca⁺², Ba⁺², Cr⁺², Cu⁺², Fe⁺², Mg⁺², Zn⁺².

4. The method of claim 1 , wherein the first fluid further comprises at least one cross-linking component comprising an acid soluble multivalent cation and the second fluid comprises an acid selected from the group consisting of succinic acid, adipic acid, acrylic acid, glutaric acid, ascorbic acid, gallic acid, caffeic acid, and any combination thereof.

5. The method of claim 1 , wherein the first fluid further comprises at least one amphiphilic polymer.

6. The method of claim 5, wherein the at least one amphiphilic polymer is a poloxamer.

7. The method of claim 1 , wherein the agent is one or more bacteria, protein, or enzyme.

8. The method of claim 7, wherein the bacteria is gram negative.

9. The method of claim 8, wherein the gram negative bacteria is a Bradyrhizobium or

Rhizobium.

10. The method of claim 7, further comprising fermenting the bacteria in a fermentation medium prior to encapsulation, wherein the fermentation medium prior to fermentation comprises trehalose.

11. The method of claim 10, wherein the fermentation medium prior to fermentation comprises about 5% m/v trehalose.

12. The method of claim 10, further comprising adding trehalose to the fermentation medium after fermentation and prior to encapsulation.

13. The method of claim 12, wherein the fermentation medium prior to fermentation comprises about 2% m/v trehalose, and wherein about 8% m/v trehalose is added to the fermentation medium after fermentation and prior to encapsulation.

14. The method of claim 1 , wherein the gas is air, nitrogen, or a mixture thereof.

15. The method of claim 1 , wherein the cross-linkable biopolymer comprises an alginate.

16. The method of claim 1 , further comprising the step of desiccating the encapsulated agent.

17. An encapsulated agent produced by the method of claim 1.

18. A spray-dried composition produced the method of claim 1.

19. A spray-dried composition comprising:

a biopolymer encapsulating bacteria, the bacteria having a concentration of at least 1 x10⁸ cfu/g after spray-drying.

20. The spray-dried composition of claim 19, wherein the bacteria have a concentration of at least 1 x 10⁸ cfu/g for at least 30 days of storage.

21. The spray-dried composition of claim 19, wherein the bacteria have a concentration of at least 1 x 10⁸ cfu/g for at least 60 days of storage.

22. The spray-dried composition of claim 19, wherein the bacteria have a concentration of at least 1 x 10⁸ cfu/g for at least 90 days of storage.

23. The spray-dried composition of claim 19, wherein the bacteria have a concentration of at least 1 10⁸ cfu/g for at least 120 days of storage.

24. The spray-dried composition of claim 19, wherein the bacteria have a concentration of at least 1 10⁸ cfu/g for at least 150 days of storage.

25. The spray-dried composition of claim 19, wherein the bacteria have a concentration of at least 1 10⁸ cfu/g for at least 180 days of storage.

26. The spray-dried composition of claim 19, wherein the bacteria are nitrogen fixing bacteria.

27. The spray-dried composition of claim 19, wherein the bacteria are gram-negative bacteria.

28. The spray-dried composition of claim 27, wherein the bacteria are selected from

Rhizobium spp. and Bradyrhizobium spp.

29. The spray-dried composition of claim 19, wherein the initial water activity (a_w) of the composition is between about 0.05 to about 0.15.

30. The spray-dried composition of claim 19, wherein the biopolymer encapsulating the bacteria is a cross-linked biopolymer.

31. The spray-dried composition of claim 19, wherein the biopolymer comprises an alginate.

32. The spray-dried composition of claim 19, further comprising at least one amphiphilic polymer.

33. The spray-dried composition of claim 32, wherein the at least one amphiphilic polymer is a poloxamer.

34. The spray-dried composition of claim 19, further comprising a sugar, polysaccharide or mixture thereof.

35. The spray-dried composition of claim 34, wherein the sugar is selected from trehalose, sucrose, lactose, raffinose and a mixture thereof.

36. A seed coating composition comprising a water and a spray-dried composition produced by the method of claim 1.

37. A seed coating composition comprising:

a spray-dried composition comprising at least one biopolymer encapsulated agent; and water.

38. The seed coating composition of claim 37, wherein the agent is one or more bacteria, protein, or enzyme.

39. The seed coating composition of claim 37, wherein the bacteria is gram negative.

40. The seed coating composition of claim 39, wherein the gram negative bacteria are selected from Rhizobium spp. and Bradyrhizobium spp.

41. The seed coating composition of claim 37, wherein the biopolymer comprises an alginate.

42. The seed coating composition of claim 37, wherein the seed is a soybean seed.

43. A method for preparing a coated seed comprising:

a. preparing a seed coating composition, the seed coating composition comprising at least one biopolymer encapsulated agent;

b. applying the seed coating composition to a surface of the seed to prepare a coated seed; and

c. drying the coated seed.

44. The method of claim 43, wherein the agent is one or more bacteria, protein, enzyme.

45. The method of claim 43, wherein the bacteria is gram negative.

46. The method of claim 45, wherein the gram negative bacteria are selected from

Rhizobium spp. and Bradyrhizobium spp.

47. The method of claim 43, wherein the biopolymer comprises an alginate.

48. The method of claim 43, wherein the seed is a soybean seed.