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WO2019232416A1 - Composition de séchage par atomisation et procédés associés - Google Patents

Composition de séchage par atomisation et procédés associés Download PDF

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Publication number
WO2019232416A1
WO2019232416A1 PCT/US2019/034970 US2019034970W WO2019232416A1 WO 2019232416 A1 WO2019232416 A1 WO 2019232416A1 US 2019034970 W US2019034970 W US 2019034970W WO 2019232416 A1 WO2019232416 A1 WO 2019232416A1
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Prior art keywords
bacteria
spray
dried composition
acid
biopolymer
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PCT/US2019/034970
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English (en)
Inventor
Ted W. DEISENROTH
Toan Van PHO
Michael Patrick BENVENUTO
R.D. Piran Cargeeg
Grit BAIER
Rute Da Conceicao TAVARES ANDRE
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BASF Corp
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BASF Corp
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Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms

Definitions

  • beneficial microorganisms as alternatives to chemical pesticides and synthetic fertilizers in agricultural production is an area of increasing interest.
  • Application of beneficial microorganisms to seeds is an efficient mechanism for placement of microbial inoculants into soil where the microorganisms will be well positioned to colonize seedling roots and protect against disease and pests.
  • Microbial inoculants are thus used in the agricultural industry as a means for promoting plant health.
  • Inoculant compositions comprising plant promoting bacteria or nitrogen fixing bacteria are well known and a commonly used biofertilizer.
  • Rhizobacteria are commonly applied as inoculants and include nitrogen-fixers and phosphate- solubilizers which enhance the availability of the macronutrients nitrogen and phosphorus to the host plant.
  • Rhizobium The most commonly applied rhizobacteria are Rhizobium and closely related genera. Rhizobium are nitrogen-fixing bacteria that form symbiotic associations within nodules on the roots of legumes. Such behavior increases host nitrogen nutrition and is important to the cultivation of soybeans, chickpeas and many other leguminous crops.
  • PGP plant growth promotion
  • Bacterial inoculants are, however, only effective when, after application, the microorganisms are readily able to survive and thrive in soil conditions.
  • Application of bacterial inoculant formulations and the soil environment itself subjects these inoculants to a variety of stresses, including temperature, mechanical, light, oxidative and osmotic stress, all of which impact the survivability of the bioactive.
  • a limitation to the use of bacterial inoculants is a low organism survival rate.
  • Encapsulation by spray-drying is a well-known methodology used to improve bacterial inoculation formulations by entrapping the bioactive 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.
  • Various approaches to encapsulation by spray-drying are the subject of, for example, U.S. Patent No.
  • a method for encapsulating a bacteria includes the steps of: flowing a fluid through a first path, the fluid including the bacteria, a cross-linking component and a biopolymer; flowing a gas through a second path; and combining the fluid and gas to encapsulate the bacteria in the biopolymer to form a spray-dried composition.
  • the percentage yield of encapsulated bacteria is greater than 20% according to the following formula: CFUout / CFUin x 100.
  • the cross-linking component includes an acid soluble multivalent cation.
  • the multivalent cation is Ca +2 , Ba +2 , Cr +2 , Cu +2 , Fe +2 ,
  • the bacteria is gram negative.
  • the gram negative bacteria is a Bradyrhizobium.
  • the gas flowed through the second path is air, nitrogen, or a mixture thereof.
  • the biopolymer is an alginate.
  • the bacteria is suspended in ammonium succinate, sodium alginate, or a combination thereof prior to flowing the fluid through the first path.
  • the fluid further includes at least one amphiphilic polymer.
  • the at least one amphiphilic polymer is a poloxamer.
  • the method further includes the step of desiccating the spray-dried composition.
  • the method further comprises fermenting the bacteria in a fermentation medium prior to encapsulation, wherein the fermentation medium prior to fermentation comprises trehalose.
  • the fermentation medium prior to fermentation comprises about 5% m/v trehalose.
  • the method further comprises adding trehalose to the fermentation medium after fermentation and prior to encapsulation.
  • 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.
  • a method for encapsulating an agent is provided.
  • the method includes the steps of: flowing a fluid through a first path, the fluid including the agent and biopolymer; flowing a gas through a second path; and combining the fluid and gas to encapsulate the agent in the biopolymer to form a spray-dried composition.
  • the percentage yield of encapsulated bacteria is greater than 20% according to the following formula:
  • the agent is one or more bacteria, protein, or enzyme.
  • the bacteria is gram negative.
  • the gram negative bacteria is a Bradyrhizobium.
  • the biopolymer is an alginate.
  • the agent is suspended in ammonium succinate, sodium alginate, or a combination thereof prior to flowing the fluid through the first path.
  • the gas flowing through the second path is air, nitrogen, or a mixture thereof.
  • the fluid further includes at least one amphiphilic polymer.
  • the at least one amphiphilic polymer is a poloxamer.
  • the method further includes the step of desiccating the spray-dried composition and optionally removing oxygen.
  • 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 bacteria. The bacteria exhibits a
  • the bacteria exhibit a concentration of at least 1 10 8 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.
  • the bacteria are nitrogen fixing bacteria.
  • the bacteria are gram-negative bacteria.
  • the bacteria are Rhizobium spp. or Bradyrhizobium spp.
  • the initial water activity (a w ) of the composition is between about 0.05 to about 0.15.
  • the biopolymer encapsulating the bacteria is a cross-linked biopolymer.
  • the biopolymer includes alginate.
  • the spray-dried composition further includes at least one amphiphilic polymer.
  • the at least one amphiphilic polymer is a poloxamer.
  • the composition further includes at least one sugar.
  • the sugar is trehalose, sucrose, lactose, raffinose, or a mixture thereof.
  • a 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.
  • the encapsulated agent is one or more bacteria, protein, or enzyme.
  • the bacteria is gram negative.
  • the gram negative bacteria of the spray-dried composition may be Rhizobium spp. , Bradyrhizobium spp., or a combination thereof.
  • the biopolymer is an alginate.
  • the seed coating composition is applied to a soybean seed.
  • 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.
  • the encapsulated agent is one or more bacteria, protein, or enzyme.
  • the bacteria is gram negative.
  • the gram negative bacteria of the spray-dried composition may be Rhizobium spp. , Bradyrhizobium spp., or a combination thereof.
  • the biopolymer is an alginate.
  • the seed coating composition is applied to a soybean seed.
  • Figure 1 is schematic of a dual-channel nozzle of a spray dryer according to one embodiment.
  • Figure 2 depicts the bacterial survivability results for the comparison of compositions prepared by Example 1 and Example 2.
  • Figure 3 depicts the bacterial survivability results for the comparison of compositions prepared by Example 1 with and without polysaccharides.
  • Figure 4 depicts the bacterial survivability results for the comparison of compositions prepared by Example 1 with and without polymer.
  • Figure 5 depicts the bacterial survivability results for the comparison of samples prepared according to Example 1 and Example 2 and stored at 28 °C.
  • agent refers to any chemical or biological component
  • 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.
  • 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).
  • biopolymer refers to any molecule that polymerizes with multivalent ions and has at least one polymerizable moiety that may cross-link in the presence of a multivalent ion and includes, but is not limited to, alginates, pectin, carrageenan, chitosan, gelatin, cellulose, starch, polyglutamic acid, whey proteins, casein, agarose, xanthan gum, maltodextrin, gallan gum or any combination thereof. This term includes both monomers and polymers.
  • cross-linking component refers to a multivalent cation that facilitates cross-linking of the biopolymer.
  • colony forming units 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.
  • a substrate e.g., a soil
  • conditions e.g., temperature, moisture, nutrient availability, pH, etc.
  • 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 , CC 2 , Cu +2 , Fe +2 , Fe +3 , Mg +2 , Pb +2 , Pb +4 , Sn +2 , Sn +4 and Zn +2 .
  • 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: CFUout / CFUin x 100.
  • 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.
  • 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.
  • the present disclosure describes both compositions and methods that provide an agent or bacteria encapsulated within a biopolymer.
  • the methods as provided herein produce a spray-dried composition that exhibit enhanced yield and survivability of the encapsulated agent or bacteria and increased shelf life at elevated temperatures.
  • the biopolymer is cross-linked with a multivalent cation.
  • the matrix is not cross-linked.
  • the encapsulated agents prepared according the methods provided herein exhibit increased spray-drying yield as well as survivability when stored at elevated temperatures (28-32°C).
  • a method for encapsulating a bacteria in a cross-linked biopolymer.
  • the method utilizes a dual path or dual-channel nozzle spray-drying system as depicted in FIG. 1.
  • Suitable spray drying systems include the Buchi B-290 which is equipped with a dual-channel nozzle.
  • the spray dryer feed line, pump and nozzle may be sterilized with ethanol prior to use.
  • the method of encapsulating bacteria in a cross-linked biopolymer includes the step of flowing a fluid through a first path or channel in the nozzle.
  • the fluid includes at least one bacteria strain, at least one cross-linking component and at least one biopolymer.
  • the biopolymer in the 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.
  • the biopolymer includes an alginate (e.g., A11 12 from Sigma-Aldrich ® or HYDAGEN ® 558P from BASF ® ).
  • alginate e.g., A11 12 from Sigma-Aldrich ® or HYDAGEN ® 558P from BASF ®
  • Biopolymers may further be combined with other molecules such as sugars, for example trehalose, proteins or other polymers, both biopolymers and synthetic polymers.
  • the at least one cross-linking component in the fluid may include an acid soluble form of a multivalent cation.
  • Suitable multivalent cations include different salts of acid soluble multivalent cations such as Ca +2 , Ba +2 , Cr 12 , Cu +2 , Fe +2 , Mg +2 , Zn +2 .
  • the multivalent ions should be capable of cross-linking.
  • Suitable salts of multivalent ions include dicalcium phosphate, calcium carbonate, calcium oxalate and other insoluble calcium salts.
  • the multivalent cation should be soluble at a reduced pH.
  • the spray-dried composition further includes a sugar.
  • the sugar is trehalose, sucrose, lactose, raffinose and a mixture thereof. Sugars may be derivatized or functionalized.
  • the spray- dried composition further includes a polysaccharide. Exemplary polysaccharides include, but are not limited to, maltodextrin and starch.
  • the spray-dried composition may further include a combination of sugar and polysaccharides.
  • the bacteria in the fluid may be centrifuged into a pellet and re-suspended or combined with an organic acid prior to flowing the fluid through a first path.
  • Suitable organic acids include oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid or sebacic acid.
  • the organic acid may be titrated to a slightly acidic pH by the addition of a base.
  • the organic acid may exhibit a pH of typically about 5.0 to about 7.0
  • the fluid is free of citric acid.
  • the organic acid is sterile prior to being utilized to re-suspend the bacteria.
  • the bacteria in the fluid may be centrifuged into a pellet and suspended or combined with a combination of organic acid and biopolymer.
  • the bacterial pellet is suspended in an ammonium salt of a dicarboxylic acid, sodium alginate, or a combination thereof prior to flowing the fluid through the first path.
  • the dicarboxylic acid may be succinic acid.
  • the bacterial pellet is suspended in ammonium succinate, sodium alginate, or a combination thereof prior to flowing the fluid through the first path.
  • the fluid includes typically a 1 : 1 mixture of biopolymer and a bacteria/organic acid mixture.
  • Such an embodiment also includes a cross- linking component that is stirred or agitated in the fluid to keep the cross-linking component dispersed in the fluid prior to flowing the fluid through a first path.
  • the method of encapsulating a bacteria strain in a cross-linked biopolymer further includes the step of flowing a gas through a second path or channel in the nozzle.
  • the gas may include any inert gas acceptable as a carrier/drying gas.
  • the gas includes air, nitrogen or a mixture thereof.
  • the method of encapsulating a bacteria strain in a cross-linked biopolymer further includes the step of combining the fluid and gas to encapsulate the bacteria in the biopolymer. Such a step may be carried out at any appropriate conditions to produce a bacteria strain that is encapsulated in the biopolymer.
  • the inlet temperature is typically from about 100°C to about 160°C.
  • the inlet temperature is typically from about 120°C to about 140°C.
  • the inlet temperature is typically about 130°C.
  • the outlet temperature is typically from about 40°C to about 80°C.
  • the outlet temperature is typically from about 50°C to about 70°C.
  • the outlet temperature is typically about 60°C.
  • cross linking between the cross-linking component and the biopolymer is limited prior to combining the fluid and gas.
  • the method of encapsulating a bacteria strain in a cross-linked biopolymer may further include the step of introducing at least one polymer to the fluid prior to combining the 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 triblock copolymers.
  • 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 0.01 % to about 2% based on the weight of polymer per volume of fluid.
  • the method of encapsulating a bacteria strain in a cross-linked biopolymer results in a spray-dried composition that is collected and stored.
  • the spray-dried composition is a powder that is collected in a cyclone and collection chamber of a spray-drying system.
  • the method of encapsulating a bacteria in a cross-linked biopolymer may further include the step of desiccating the spray-dried composition and optionally removing oxygen.
  • the spray-dried composition is protected from light and conditioned in a desiccator at ambient temperature from typically about 5-9 days.
  • the spray-dried composition is protected from light and conditioned in a desiccator at ambient temperature from typically about 7 days.
  • the spray-dried composition attains a water activity of typically about 0.5 or less upon desiccation.
  • the spray-dried composition attains a water activity of typically about 0.1 or less upon desiccation.
  • the spray-dried composition may be stored in a sterile container until use.
  • the bacteria encapsulated in a cross-linked biopolymer according to the method as provided herein may be any type of bacteria that may be encapsulated in a biopolymer.
  • the bacteria may be gram positive or gram negative bacteria.
  • Suitable bacteria include, but are not limited to, Azospirillum brasilense INTA Az-39, Bacillus amyloliquefaciens D747, Bacillus amyloliquefaciens NRRL B-50349, Bacillus amyloliquefaciens TJ1000, 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
  • the gram negative bacteria is a Bradyrhizobium sp.
  • the percentage yield of encapsulated bacteria is typically greater than about 20% according to the following formula:
  • the percentage yield of encapsulated bacteria is typically greater than about 40%. According to a particular embodiment, the percentage yield of encapsulated bacteria is typically greater than about 60%.
  • a spray-dried composition produced by the aforementioned method is also provided.
  • the spray-dried composition includes biopolymer encapsulating bacteria, the bacteria having a concentration of at least 1x10 8 cfu/g after spray drying.
  • the bacteria have a concentration of at least
  • 1x10 8 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, at least 180 days of storage, at least 210 days of storage, at least 240 days of storage, at least 270 days of storage, at least 300 days of storage, at least 330 days of storage or at least 360 days of storage.
  • a method for encapsulating an agent in a biopolymer that is not cross-linked.
  • the method utilizes a dual-channel or dual path nozzle spray-drying system as depicted in FIG. 1.
  • Suitable spray drying systems include the Buchi B-290 which is equipped with a dual-channel nozzle.
  • the spray dryer feed line, pump and nozzle may be sterilized with ethanol prior to use.
  • the method of encapsulating an agent in a biopolymer includes the step of flowing a fluid through a first path or channel in the nozzle.
  • the fluid includes at least one agent and at least one biopolymer.
  • the biopolymer in the fluid may be an alginate, 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.
  • the biopolymer includes an alginate (e.g., A1 112 from Sigma-Aldrich ® or HYDAGEN 558P from BASF ® ).
  • alginate e.g., A1 112 from Sigma-Aldrich ® or HYDAGEN 558P from BASF ®
  • Biopolymers may further be combined with other molecules such as sugars, for example trehalose, proteins or polymers, either biopolymers or synthetic polymers.
  • the fluid includes a 1 :1 mixture of biopolymer and an agent/organic acid mixture.
  • the resulting mixture is fed directly into the first path as provided herein.
  • Suitable organic acids include oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid or sebacic acid.
  • the organic acid may be titrated to a slightly acidic pH by the addition of a base.
  • the organic acid may exhibit a pH of typically about 5.0 to about 7.0.
  • the fluid is free of citric acid.
  • the organic acid is sterile prior to being utilized to re-suspend the agent.
  • the agent in the fluid may be centrifuged into a pellet and suspended or combined with a combination of organic acid and biopolymer.
  • the agent pellet is suspended in an ammonium salt of a dicarboxylic acid, sodium alginate, or a combination thereof prior to flowing the fluid through the first path.
  • the dicarboxylic acid may be succinic acid.
  • the agent pellet is suspended in ammonium succinate, sodium alginate, or a combination thereof prior to flowing the fluid through the first path.
  • the method of encapsulating an agent in a biopolymer further includes the step of flowing a gas through a second path or channel in the nozzle.
  • the gas may include any inert gas acceptable as a carrier/drying gas.
  • the gas includes air, nitrogen or a mixture thereof.
  • the method of encapsulating an agent in a biopolymer further includes the step of combining the 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.
  • the inlet temperature is typically from about 100°C to about 160°C.
  • the inlet temperature is typically from about 120°C to about 140°C.
  • the inlet temperature is typically about 130°C.
  • the outlet temperature is typically from about 40°C to about 80°C.
  • the outlet temperature is typically from about 50°C to about 70°C.
  • the outlet temperature is typically about 60°C.
  • the method of encapsulating an agent in a biopolymer may further include the step of introducing at least one polymer to the fluid prior to combining the fluid and gas.
  • the polymer may a biopolymer or a synthetic polymer and may be added to increase survivability of the agent 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 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% based on the weight of polymer per volume of fluid.
  • the method of encapsulating an agent in a biopolymer forms a spray-dried composition that is collected and stored.
  • the spray-dried composition is a powder that is collected in a cyclone and collection chamber.
  • the method of encapsulating an agent may further include the step of desiccating the spray-dried composition and optionally removing oxygen.
  • the spray-dried composition is protected from light and conditioned in a desiccator at ambient temperature from typically about 5-9 days.
  • the spray-dried composition is protected from light and conditioned in a desiccator at ambient temperature from typically about 7 days.
  • the spray-dried composition attains a water activity of typically about 0.5 or less upon desiccation.
  • the spray-dried composition attains a water activity of typically about 0.1 or less upon desiccation.
  • the spray-dried composition may be stored in a sterile container until use.
  • the agent encapsulated in a biopolymer that is not cross-linked according to the methods provided herein may be 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 (e.g., bacteria), biostimulants, nutrients, pesticides (e.g. acaricides, fungicides, herbicides, insecticides, and nematicides), plant signal molecules, enzymes and proteins.
  • the agent is a bacteria.
  • the bacteria may be any type of bacteria that may be encapsulated in a biopolymer.
  • the bacteria may be gram positive or gram negative bacteria.
  • Suitable bacteria include, but are 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 1T-45, Bacillus amyloliquefaciens TJ1000, 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 P
  • amyloliquefaciens 1013 (deposited as NRRL B-50509), Bacillus amyloliquefaciens 918
  • 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,
  • the gram negative bacteria is a Bradyrhizobium sp.
  • the percentage yield of encapsulated bacteria is typically greater than about 20% according to the following formula:
  • the percentage yield of encapsulated bacteria is typically greater than about 40%. According to a particular embodiment, the percentage yield of encapsulated bacteria is typically greater than about 60%.
  • a spray-dried composition is also provided.
  • the spray-dried composition includes biopolymer encapsulating bacteria, the bacteria having a concentration of at least 1x10 8 cfu/g after spray-drying.
  • the bacteria have a concentration of at least
  • 1x10 8 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, at least 180 days of storage, at least 210 days of storage, at least 240 days of storage, at least 270 days of storage, at least 300 days of storage, at least 330 days of storage or at least 360 days of storage.
  • the bacteria include any type of plant growth promoting bacteria including nitrogen fixing bacteria, phosphate solubilizers, bacteria impacting ACC deaminase activity and the production of siderophores and phytohormones.
  • the bacteria is a nitrogen fixing bacteria.
  • the initial water activity (a w ) of the composition is typically between about 0.01 to about 0.4. According to one embodiment, the initial water activity (a w ) of the composition is typically between about 0.03 to about 0.3. According to one embodiment, the initial water activity (a w ) of the composition is typically between about 0.04 to about 0.20. According to one embodiment, the initial water activity (a w ) of the composition is typically between about 0.05 to about 0.15.
  • the spray-dried composition includes at least one amphiphilic polymer.
  • the at least one amphiphilic polymer is a poloxamer.
  • Exemplary polymers include ethylene oxide/polyethylene oxide tri-block
  • the spray-dried composition further includes a sugar.
  • the sugar is trehalose, sucrose, lactose, raffinose and a mixture thereof. Sugars may derivatized or functionalized.
  • the spray- dried composition further includes a polysaccharide. Exemplary polysaccharides include, but are not limited to, maltodextrin and starch. In another embodiment the spray-dried composition may further include a combination of sugar and polysaccharides.
  • 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 and applied 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, calli and the like.
  • 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.
  • the encapsulated agent is one or more bacteria, protein, or enzyme.
  • the bacteria is gram negative.
  • 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.
  • a method for preparing a coated seed 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.
  • the encapsulated agent is one or more bacteria, protein, or enzyme.
  • the bacteria is gram negative.
  • the gram negative bacteria of the spray-dried composition may be Rhizobium spp. , Bradyrhizobium spp., or a combination thereof.
  • the biopolymer is an alginate.
  • 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.
  • an alginate feedstock was prepared by slowly adding sodium alginate (4 wt%, low viscosity A1 1 12 alginate supplied by Sigma-Aldrich ® ) to a stirred suspension of 0.5 wt% CaHP0 4 in water. The mixture was stirred overnight at room temperature and then sterilized via an autoclave (45 min liquid cycle at 121 °C) the next day.
  • a second feedstock of succinic acid was prepared by dissolving succinic acid (2 wt%) in water, titrating the solution to pH 5.6 with NH 4 OH (aq) , and sterilizing the solution via filtration through a 0.2 pm filter. If required, formulation additives, such as sugars and polymers, were added to the succinic acid solution and dissolved prior to sterile filtration. If the formulation additives were not fully soluble in water, then a separate mixture of the additives in water was prepared and sterilized via an autoclave (45 min liquid cycle at 121 °C).
  • Example 1 b For Samples 25 and 26 prepared according to Example 1 b, a second sodium alginate (HYDAGEN ® 558P, BASF) was also used with Example 1. In this case, a lower percentage of alginate was utilized due to the high viscosity of the dissolved HYDAGEN ® alginate.
  • the HYDAGEN ® alginate feedstock was prepared by slowly adding HYDAGEN ® 558P alginate (2 to 2.5 wt%) to a stirred suspension of 0.25 to 0.313 wt% CaHP0 in water. The mixture was stirred overnight at room temperature and then sterilized via an autoclave (45 min liquid cycle at 121 °C) the next day.
  • a second feedstock of succinic acid was prepared by dissolving succinic acid (1 to 1.25 wt%) in water, titrating the solution to pH 5.6 with NH 4 OH (aq) , and sterilizing the solution via filtration through a 0.2 pm filter. If required, formulation additives, such as sugars and polymers, were added to the succinic acid solution and dissolved prior to sterile filtration. If the formulation additives were not fully soluble in water, then a separate mixture of the additives in water was prepared and sterilized via an autoclave (45 min liquid cycle at 121 °C).
  • the bacteria were prepared by centrifuging 100 ml. of the bladder (typically ⁇ 1x10 10 CFU/mL) in conical tubes at 4700 rpm for 10 min. The supernatant was decanted, and the cell pellet was re-suspended in 50 ml. of the succinic acid solution.
  • Bradyrhizobium spp. were obtained by the same method described in Example 1 above and feedstocks were prepared for each batch of Bradyrhizobium.
  • a sodium alginate feedstock was prepared by slowly adding sodium alginate (4 wt%, low viscosity A1 1 12 alginate from Sigma-Aldrich ® ) to water. The solution was stirred overnight at room temperature and then sterilized via an autoclave (45 min liquid cycle at 121 °C) the next day.
  • a second feedstock of succinic acid was prepared by dissolving succinic acid (2 wt%) in water, titrating the solution to pH 5.6 with NH 4 OH (aq) , and sterilizing the solution via filtration through a 0.2 pm filter. If required, formulation additives, such as sugars and polymers, were added to the succinic acid solution and dissolved prior to sterile filtration. If the formulation additives were not fully soluble in water, then a separate mixture of the additives in water was prepared and sterilized via an autoclave (45 min liquid cycle at 121 °C).
  • the bacteria were prepared by centrifuging 100 mL of the bladder (typically
  • Example 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 (Samples 1-19 and 25-29).
  • 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.
  • Table 1 contains compositions for spray-drying of Samples 1-34.
  • Formulations were spray dried using a Buchi Labortechnik Mini Sprayer B-290 equipped with a two fluid nozzle.
  • the spray dryer was sterilized by running 70% ethanol through the feed line, pump, and nozzle without compressed gas or heat. The ethanol was left to sit for 3 minutes, and then sterile water was run through to purge the apparatus. The collected ethanol and water was discarded. The exposed end of the feed tube was then left to sit in a beaker of 70% ethanol to maintain sterility.
  • the spray dryer was heated to an inlet temperature of 130°C with an aspirator rate of 100%. Twenty to 30 ml. of water was then pumped through the feed tube to lower the outlet temperature to ⁇ 60°C.
  • HygroPalm HP23-AW-A portable water activity analyzer After the drying period was over, the samples were then transferred to a desiccator with additional silica gel desiccant in an incubator held at 32 °C. Control samples were prepared by storing the spray dried powder in capped conical tubes under the same temperature conditions.
  • spray dried material was stored in conical tubes with gas- permeable seals (Thomson AirOtopTM Enhanced seals) to allow further drying to occur while maintaining sterility.
  • the conical tubes were stored in aluminum foil bags with Mitsubishi Gas Chemical RP-3A moisture and oxygen absorbers.
  • the RP-3A absorbers decreased the oxygen level below 0.1 % and lowered the a w to approximately 0.05 to 0.1.
  • the a w was measured using a Rotronic HygroPalm HP23-AW-A portable water activity analyzer.
  • the foil bags were stored in BD GasPakTM EZ incubation containers containing additional RP-3A packets.
  • the samples were stored at room temperature (23 °C), 4 °C, or gradually increasing temperatures from 4 °C to 23 °C, for time periods ranging from ⁇ 7 to 30 days.
  • the BD containers with the samples were then transferred to an incubator held at 28 °C.
  • Control samples were prepared by storing the spray dried powder in capped conical tubes under the same temperature conditions.
  • RP-3A moisture and oxygen absorbers Chemical RP-3A moisture and oxygen absorbers.
  • the RP-3A absorbers kept the oxygen level below 0.1 % and the a w to approximately 0.05 to 0.1.
  • the foil bags were stored in BD GasPakTM EZ incubation containers containing additional RP-3A packets.
  • the BD containers with the samples were stored in an incubator held at 28 °C. Control samples were prepared by storing the spray dried powder in capped conical tubes under the same temperature conditions.
  • compositions of Examples 1 and 2 were then tested 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.
  • Total CFUin 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 CFU in 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.
  • 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).
  • 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 1x10 10 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.
  • 0.163 mL of the seed coating composition was applied onto the seeds.
  • the plastic jar was capped, placed into a FlackTek SpeedMixerTM DAC 150 FVZ-K, and mixed at 800 rpm for 10 seconds.
  • 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.
  • FIG. 2 summarizes the results for Samples 1 , 2, 3, 19, 27, 35, and 36 stored in an incubator held at 32 °C. Both Examples 1 and 2 resulted in at least 1x10 8 CFU/g after 60 days of storage with the desiccated samples (designated as“desic.”) as compared to the control samples, which were not desiccated (designated as“control”). Thus the compositions resulting from the methods described in had an enhanced bacterial survivability. Futhermore, the compositions made by Examples 1 and 2 demonstrated enhanced bacterial survivability compared to the reference compositions (Samples 35 and 36) made by Example 7.
  • Example 1 The compositions of Example 1 were then evaluated by testing the difference between those samples with (Samples 7 and 25) and without (Samples 8 and 26)
  • Example 1 The compositions of Example 1 (Samples 1-3 and 10-15) and survivability was measured as described above. These samples were stored in a desiccator in an incubator held at 32 °C. The results are summarized in FIG.4. The samples that include polymer show an improved survivability between 40 and 80 days over samples without polymer. Thus initial survivability was enhanced with the use of poloxamer.
  • FIG. 5 summarizes the results for Samples 20-24, 29, and 31-34 made by Examples 1 and 2.
  • the samples were stored with RP-3A moisture and oxygen absorbers in an incubator held at 28 °C.
  • the samples exhibited high survivability of at least 1x10 8 CFU/g up to about 140 days of storage, especially with the addition of a protein or sugar.
  • a reference sample denoted as“Alginate” was prepared via the following procedure.
  • a sodium alginate feedstock was prepared by slowly adding sodium alginate (4 wt%, low viscosity A1 1 12 alginate from Sigma-Aldrich®) to water. The solution was stirred overnight at room temperature and then sterilized via an autoclave (45 min liquid cycle at 121 °C) the next day.
  • the bacteria were prepared by centrifuging 100 mL of the bladder (typically ⁇ 1x10 10 CFU/mL) in conical tubes at 4700 rpm for 10 min. The supernatant was decanted, and the cell pellet was re-suspended in 50 mL of the alginate feedstock. The formulation was then spray dried according to the standard process for Examples 1 and 2.
  • a second reference sample denoted as“Sigma + CaHP04” was prepared via the following procedure.
  • An alginate feedstock was prepared by slowly adding sodium alginate (4 wt%, low viscosity A1 1 12 alginate supplied by Sigma-Aldrich®) to a stirred suspension of 0.5 wt% CaHP04 in water. The mixture was stirred overnight at room temperature and then sterilized via an autoclave (45 min liquid cycle at 121 °C) the next day.
  • the bacteria were prepared by centrifuging 100 mL of the bladder (typically ⁇ 1x10 10 CFU/mL) in conical tubes at 4700 rpm for 10 min. The supernatant was decanted, and the cell pellet was re-suspended in 50 mL of the alginate feedstock. The formulation was then spray dried according to the standard process for Examples 1 and 2.

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Abstract

La présente invention concerne à la fois des compositions et des procédés qui fournissent un agent encapsulé à l'intérieur d'un biopolymère. Les procédés selon l'invention produisent une composition de séchage par atomisation qui présente un rendement et une capacité de survie améliorés de l'agent encapsulé et une durée de conservation accrue à des températures élevées.
PCT/US2019/034970 2018-05-31 2019-05-31 Composition de séchage par atomisation et procédés associés Ceased WO2019232416A1 (fr)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030138936A1 (en) * 2000-05-02 2003-07-24 Taiji Mizuguchi Spray-dried microbial cells
US20090238885A1 (en) * 2006-05-22 2009-09-24 Nizo Food Research B.V. Protein encapsulated particles
US20100189767A1 (en) * 2006-09-19 2010-07-29 Eyal Shimoni Probiotic compositions and methods of making same
US20100266727A1 (en) * 2007-11-07 2010-10-21 Encoate Holdings Limited Stabilisation of dried biological material
US20130323362A1 (en) * 2010-12-06 2013-12-05 Degama Berrier Ltd. Composition and method for improving stability and extending shelf life of probiotic bacteria and food products thereof
US20140348815A1 (en) * 2011-12-23 2014-11-27 The Regents Of The University Of California Spray dry method for encapsulation of biological moieties and chemicals in polymers cross-linked by multivalent ions for controlled release applications
WO2017044545A1 (fr) * 2015-09-11 2017-03-16 Novozymes Bioag A/S Compositions d'inoculant stables et procédés de production de ces compositions
WO2017191093A1 (fr) * 2016-05-02 2017-11-09 Lacto Research Sprl Procede d'enrobage de microorganismes, poudre desdits microorganismes enrobes et composition pharmaceutique, nutraceutique, cosmetique, alimentaire ou sanitaire la comprenant

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030138936A1 (en) * 2000-05-02 2003-07-24 Taiji Mizuguchi Spray-dried microbial cells
US20090238885A1 (en) * 2006-05-22 2009-09-24 Nizo Food Research B.V. Protein encapsulated particles
US20100189767A1 (en) * 2006-09-19 2010-07-29 Eyal Shimoni Probiotic compositions and methods of making same
US20100266727A1 (en) * 2007-11-07 2010-10-21 Encoate Holdings Limited Stabilisation of dried biological material
US20130323362A1 (en) * 2010-12-06 2013-12-05 Degama Berrier Ltd. Composition and method for improving stability and extending shelf life of probiotic bacteria and food products thereof
US20140348815A1 (en) * 2011-12-23 2014-11-27 The Regents Of The University Of California Spray dry method for encapsulation of biological moieties and chemicals in polymers cross-linked by multivalent ions for controlled release applications
WO2017044545A1 (fr) * 2015-09-11 2017-03-16 Novozymes Bioag A/S Compositions d'inoculant stables et procédés de production de ces compositions
WO2017191093A1 (fr) * 2016-05-02 2017-11-09 Lacto Research Sprl Procede d'enrobage de microorganismes, poudre desdits microorganismes enrobes et composition pharmaceutique, nutraceutique, cosmetique, alimentaire ou sanitaire la comprenant

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