Formulations containing biological agents and methods of use

Abstract

Formulations containing a biological agent and processed fibroin that exhibit enhanced stability and survivability of the biological agent, their methods of preparation as well as methods of treating a seed are provided. The formulations optionally include additives, particularly additives that, when combined with processed fibroin, provide excellent stability and survivability of biological agents including microbes.

Description

FORMULATIONS CONTAINING BIOLOGICAL AGENTS AND METHODS OF USEFIELD

[0001] The present disclosure relates to formulations containing biological agents, including formulations for agricultural use, and methods of using such compositions. More specifically, the present disclosure relates to formulations containing biological agents that exhibit enhanced stability and survivability, their methods of preparation as well as methods of treating a surface such as a seed.BACKGROUND

[0002] Formulations containing biological agents, such as microbes, are used in a variety of industries such as agriculture, animal health and nutrition, medicine and healthcare, consumer health, food and beverage production, waste and environmental remediation, construction, household and professional cleaning, and industrial applications and manufacturing, for example. In agriculture, microbes are a sustainable option to provide increased agricultural productivity, protection against pathogens and tolerance to stress, for example. In medicine, human health, consumer health and / or animal health, microbes can be used as prebiotics, probiotics and postbiotics to improve gastrointestinal health, skin health, oral health, and vaginal health. In the food and beverage industry, microbes can be used for fermentation as well as food production. In industrial manufacturing, microbes can be used to create biofuel, reduce or sequester environmental pollutants, clean up waste spills, as well as for general streamlining of manufacturing processes. In construction, microbes can be used for concrete stabilization and soil stabilization. Industrial applications include bioenergy such as batteries. Biological agents are generally formulated into delivery systems that can preserve the efficacy of the agents as well as deliver them to their site of action.

[0003] An important agricultural application of biological agents is in seed treatment. Seed treatment is the process of treating seeds with active ingredients to support the germination and / or health and growth of a wide variety of crops. Typical examples include the application of pesticides, such as fungicides, insecticides, nematicides and plant growth regulators, seed colorants and coatings as well as other active ingredients like fertilizers. Agriculturallyacceptable formulations may include chemical active ingredients, biological active ingredients, or a combination of the two. Typically, compositions based on biological active ingredients are susceptible to environmental stress, impacting their survival, viability and performance, and typically have a short shelf life. Therefore, there is a need for formulations including agriculturally acceptable formulations comprising biological active agents that exhibit enhanced stability and survivability.SUMMARY

[0004] In an aspect, described herein are formulations comprising effective amounts of a biological agent and processed fibroin. In an aspect, the formulation exhibits enhanced stability of the biological agent compared to a biological agent formulation without processed fibroin under equivalent environmental conditions. Exemplary biological agents include bacterial species, fungal species, algal species, viral species, protozoal species, extracts and extracellular vesicles from the foregoing, and combinations thereof. The formulations optionally additionally include additives such as stabilizing additives. In a specific aspect, the formulation is coated on a seed.

[0005] In another aspect, a method of preparing a formulation is described. The method comprises a) providing an effective amount of a biological agent, and b) combining the biological agent with an effective amount of processed fibroin, and optionally at least one additive.

[0006] In another aspect, a method of protecting a seed is provided. The method of protecting seed comprises (a) providing an agriculturally acceptable formulation, the formulation comprising an effective amount of a biological agent and processed fibroin; and (b) applying the formulation to the seed in an agriculturally effective amount.

[0007] This summary has been provided so that the nature of the disclosure may be understood quickly. A more complete understanding of the disclosure can be obtained by reference to the following detailed description of the preferred embodiments thereof concerning the attached drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The foregoing features and other features of the present disclosure will now be described with reference to the drawings of a preferred embodiment. The illustrated embodiments are intended to illustrate, but not to limit the disclosure. The drawings include the following Figures:

[0009] Fig. 1 illustrates the impact of processed fibroin, both with and without glycerol additives, on Rhizobium leguminosarum on seed stabilization.

[0010] Fig. 2 illustrates the impact of processed fibroin, both with and without trehalose additives, on Bradyrhizobium japonicum on seed stabilization.

[0011] Fig. 3 illustrates the impact of processed fibroin, both with or without the addition of trehalose and sorbitol additives, on Pink Pigmented Facultative Methylotroph (PPFM) on seed stabilization at 20°C.

[0012] Fig. 4 illustrates the impact of processed fibroin, both with or without the addition of trehalose and sorbitol additives, on a Pink Pigmented Facultative Methylotroph (PPFM) on seed stabilization at 30° C.

[0013] Fig. 5 illustrates the impact of processed fibroin % on B.firmus in-pack survival at 30°C.

[0014] Fig. 6 illustrates the impact of processed fibroin % on B.firmus on-seed survival at 30°C / 80% RH.

[0015] Fig. 7 illustrates the impact of processed fibroin on Rhizobium leguminosarum on seed stabilization.

[0016] Fig. 8 illustrates the impact of processed fibroin on Pseudomonas mosselii on seed stabilization.DETAILED DESCRIPTION

[0017] 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.

[0018] 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.

[0019] As used herein, the term “biological agents” refers to living microorganisms such as bacteria, fungi, algae, viruses, and protozoa, as well as extracts and / or extracellular vesicles from plants, bacteria, yeasts, and algae. Biological agents also include nucleic acid therapeutics such as DNA, RNA, miRNA, RNAi, and anti-sense RNA as well as polypeptides including antibodies.

[0020] In an aspect, the biological agent is an “agriculturally beneficial microorganism”. As used herein, the term “agriculturally beneficial microorganisms” refers to microorganisms having at least one agriculturally beneficial property (e.g., but not restricted to) the ability to fix nitrogen, the ability to solubilize nutrients such as phosphate or potassium and / or the ability to produce an agriculturally beneficial agent, such as a phytohormones, plant signal molecules, and / or biopesticidal actives / inducers) such as, for example, bacteria. Agriculturally beneficial microorganisms have been well-described in the art and are not limited to the agriculturally beneficial microorganisms exemplified herein.

[0021] As used herein, the term “colony forming units”, “cfu”, or “CFU” is a measurement of the number of viable microbial cells in a sample. CFU includes microbial cell / spores capable of propagating in a host, on or in a substrate (e.g., on a root or in soil) when conditions (e.g., temperature, moisture, nutrient availability, pH, etc.) are favorable for microbial growth.

[0022] As used herein, the term “additives” refers to any substance included in a composition with an active agent or primary component, often, but not limited to, serving as a carrier, diluent, or vehicle for the active agent or primary component. In some embodiments, additives may be compounds or compositions approved for use by the US Food and Drug Administration (FDA). In some embodiments, additives may increase stability of the formulation and / or stability of one or more other formulation components such as the stability of the biological agent.

[0023] As used herein, the terms “yield” or “initial survivability” refer to the percentage of colony forming units surviving after the application of a formulation as provided herein, such as application to a seed. The yield is calculated based on the following formula:CFU0Utx 100CFUin

[0024] As used herein, the term “survivability” refers to viable colony forming units (CFU) at a point in time after preparation of a formulation, after administration of a formulation, or after application of a formulation to a surface. An exemplary surface is a propagule surface, such as a seed.

[0025] As used herein, an “effective amount” is an amount of a substance which produces a desired effect. In the case of a biological, an effective amount, e.g., an agriculturally effective amount, is an amount sufficient to provide the desired activity such as fungicidal activity, plant health activity, biostimulant activity, plant regulation activity and the like. In the case of processed fibroin, an effective amount is an amount that provides stabilization or survivability to a biological agent as described herein.

[0026] As used herein, the term On-Seed Survival, or “OSS” refers to the quantified viable target microbial propagule(s) population recovered from the seed surface following the treatment of the seed with the aforementioned biological agent. The microbial propagules are assessed from the target treated seed (e.g. soybean, corn, etc.) using microbial enumeration techniques at selected time points from treated seeds which have been stored under prescribed environmental conditions (temperature and relative humidity). OSS is reported as cfu / seed.

[0027] As used herein, the term In-Pack Stability (Survival), or “IPS” refers to the quantified viable target microbial propagule(s) population recovered from the formulation. The microbial propagules are assessed from the formulation using microbial enumeration techniques at selected time points stored under prescribed environmental conditions (temperature). IPS is reported as cfu / mL.

[0028] As used herein, “processed fibroin” is fibroin which has been purified from silk fibers, e.g., silk cocoons, through a process typically including removal of additional proteins such as sericin. Processed fibroin is composed of amino acids, particularly glycine, alanine, and serine. Processed fibroin preparations can have different molecular weights depending on the processing conditions employed.

[0029] In an aspect, as used herein, high molecular weight processed fibroin (“high MW SF”) is defined as processed fibroin having a molecular weight of greater than 10 kDa, such as greater than 10 kDa to about 500 kDa .(e.g., 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa,50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, 100 kDa, 125 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa), as determined by ultra high pressure liquid chromatography-size exclusion chromatography (UPLC-SEC) using a calibration curve of standards IgG (150 kDa), BSA (66.4 kDa), Myoglobin (17 kDa), and Uracil (112 Da). In an aspect, high MW SF has a molecular weight of greater than 10 kDa to 150 kDa as determined by ultra high pressure liquid chromatography-size exclusion chromatography (UPLC-SEC) using a calibration curve of standards IgG (150 kDa), BSA (66.4 kDa), Myoglobin (17 kDa), and Uracil (112 Da).

[0030] In an aspect, low molecular weight processed fibroin (“low MW SF”) is defined as processed fibroin having a molecular weight of less than or equal to than 10 kDa, such as greater than 0.5 kDa to 10 kDa (e.g., 0.5 kDa, 0.75 kDa, 1 kDa, 1.5 kDa, 2 kDa, 2.5 kDa, 3, kDa, 4 kDa, 4.5 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, and the like), as determined by ultra high pressure liquid chromatography-size exclusion chromatography (UPLC-SEC) using a calibration curve of standards IgG (150 kDa), BSA (66.4 kDa), Myoglobin (17 kDa), and Uracil (112 Da). In a specific aspect, Low MW SF has a molecular weight of about 0.5 kDa to about 2 kDa as determined by ultra high pressure liquid chromatography-size exclusion chromatography (UPLC-SEC) using a calibration curve of standards IgG (150 kDa), BSA (66.4 kDa), Myoglobin (17 kDa), and Uracil (112 Da).

[0031] Formulations containing a biological agent

[0032] Described herein are formulations including a biological agent.

[0033] Formulations including biological agents face an inherent challenge: biological agents typically have a limited shelf life, and living biological agents also have limited survivability once administered or exposed to environmental conditions. The longevity of these compositions is influenced by several factors, including the specific biological agents used (such as bacteria, fungi, or other microorganisms), the product formulation, storage conditions, and the inclusion of preservatives and / or stabilizers. These biological agent-containing formulations include living active ingredients that may comprise microbes like fungi and bacteria, as well as extracts from plants, bacteria, yeasts, and algae. While all organisms have potential applications, some are more widely and effectively utilized than others. Typically, spore-forming organisms demonstrate greater survival resilience and durability. For instance, Bacillus strains used as biological agents for seed treatment are known for their high efficacy, and good shelf life, andare thus frequently used in practice. Organisms that do not have a spore phase are more challenging to stabilize than those that produce resilient spores and / or vegetative cells. Rhizobiaceae bacteria, which do not have a spore phase, can encounter stability limitations; nonetheless, they are widely utilized due to their agronomically beneficial symbiotic relationship with legumes, which facilitates nitrogen fixation - an important aspect of economic, agricultural, and environmental sustainability. Additionally, a robust and effective formulation is crucial for the success of all organisms, especially for Rhizobiaceae members and other gram-negative bacteria or non-spore-forming microorganisms.

[0034] To address the stability concerns associated with such biological agents, the present disclosure describes the use of processed fibroin in combination with biological agents including agriculturally beneficial organisms and / or chemicals to provide a formulation. The resulting formulation provides stability to the biological agents in the composition, thereby enhancing shelf-life stability and / or improving or maintaining post-administration or in-field performance of the compositions.

[0035] In one embodiment, a formulation is intended for agricultural applications and the methods for utilizing this formulation are described. In an embodiment, the agriculturally acceptable composition comprises agriculturally beneficial microorganisms.

[0036] In one embodiment, the agriculturally acceptable formulation comprises an effective amount of an agriculturally beneficial microorganism along with processed fibroin. In one embodiment, an agriculturally acceptable formulation comprises an effective amount of an agriculturally beneficial microorganism, an effective amount of processed fibroin, and at least one additive. In one embodiment, a method of preparing and using the agriculturally acceptable formulation is also provided.

[0037] Biological Agents

[0038] Exemplary biological agents include bacterial species, fungal species, algal species, protozoan species, and combinations thereof. In an embodiment, the biological agent is in a vegetative cell , spore or other living or attenuated cell form, or in the form of an extract or vesicle, or material from natural or synthetic fermentation or bioreaction of natural or genetically modified microorganisms, or of natural compounds. Biological agents also include nucleic acids and (poly)peptides.

[0039] Exemplary bacterial species include gram-negative bacteria, gram-positive bacteria, spore-forming bacteria, and non-spore-forming bacteria.

[0040] Exemplary fungal species include fungi belonging to the phyla; Glomeromycota, Ascomycota, Basidiomycota, and / or Zygomycota.

[0041] In an embodiment, the biological agent is an agricultural biological agent, which can be a microorganism with fungicidal activity, a microorganism with insecticidal activity, a microorganism with nematicidal activity, a microorganism with plant health activity (also known to as biofertilizer, a microorganism with biostimulant or Plant Growth Promotor (PGP or PGPR) activity), a microorganism with plant regulation activity, or a combination thereof. In one embodiment, a combination of such microorganisms may be employed.

[0042] Microorganisms: Exemplary agriculturally beneficial microorganisms include Acremonium spp, Ampelomyces quisqualis, Aspergillus flavus, Aureobasidium pullulans, Bacillus altitudinis, B. amyloliquefaciens, B. amyloliquefaciens ssp. plantarum (also referred to as B. velezensis), B. cereus, B. idriensis, B. licheniformis, B. megaterium (also referenced as Priestia megaterium), B. methylotrophicus, B. mojavensis, B. mycoides, B. pumilus, B. solisalsi (B. solisilvae), B. subtilis, B. subtilis var. amyloliquefaciens, B. velezensis, Burkholderia cepacia, Candida oleophila, C. saitoana, Cladosporium spp, Cladosporium cladosporioides, Clavibacter michiganensis, Clonostachys rosea f catenulate (also named Gliocladium catenulatum), Coleosporium spp, Coniothyrium minitans, Cryptococcus albidus, C flavescens, Dilophospora alopecuri, Erisiphe spp, Erwinia gerundensis, Flavobacterium spp, Fusarium oxysporum, Geobacillus spp, Gliocladium roseum, G. virens, Hypocrea spp, Lysobacter spp, Lysobacter antibioticus, L. enzymogenes, Metschnikowia fructicola, Microbacterium trichothecenolyticum, Microcystis, Microdochium dimerum, Microsphaeropsis ochracea, Muscodor albus, Nomura spp, Paenibacillus spp, Paenibacillus alvei, P. epiphyticus, P. polymyxa, Pantoea vagans, P. agglomerans, Peronospora spp, Phlebiopsis gigantea, Pleospora spp, Pseudomonas spp, P. aeruginosa, P. chlororaphis, P. fluorescens, P. proradix, P. putida, P. mosselii, Pseudozyma spp, Pythium oligandrum, Saccharomyces cerevisiae, Simplicillium lanosoniveum, Sphaerodes mycoparasitica, Streptomyces spp, S. griseoviridis, S. lydicus, S. microflavus, S. venezuelae, S. violaceusniger, Talaromyces flavus, Trichoderma asperelloides, T. asperellum, T. atroviride, T. fertile, T. gamsii, T. harmatum, T. harzianum, T. polysporum, T. stromaticum, T. virens (FKA Gliocladium virens), T. viride, Typhula phacorrhiza, Ulocladium oudemansii, Verticillium albo-atrum (FKA V. dahliae) ; Agrobacterium radiobacter, Bacillus cereus, B.firmus, B. thuringiensis, B. thuringiensis ssp. aizawai, B. t. ssp. israelensis, B. t. ssp. galleriae, B. t. ssp. kurstaki, B. t. ssp. tenebrionis, Beauveria bassiana, B. brongniartii, Burkholderia spp., Chromobacterium subtsugae, Cordyceps spp, Cydia pomonella granulovirus (CpGV), Cryptophlebia leucotreta granulovirus (CrleGV), Dactylella spp, Entomophthora spp, Helicoverpa armigera nucleopolyhedrovirus (HearNPV), Helicoverpa zea nucleopolyhedrovirus (HzNPV), Helicoverpa zea single capsid nucleopolyhedrovirus (HzSNPV), Hirsutella spp, Isaria fumosorosea, Lecanicillium lecanii (FKA Verticillium lecanii), L. longisporum (FKA L lecanii),L. muscarium, Lysinibacillus sphaericus (FKA Bacillus sphaericus), Metarhizium anisopliae, M. anisopliae var. anisopliae, M. anisopliae var. acridum, Microsporidium, Nomuraea rileyi, Paecilomyces fumosoroseus, Paenibacillus popilliae, Pasteuria spp., P. nishizawae, P. penetrans, P. ramosa, P. thomea, P. usgae, P. califomica, Purpur eocillium lilacinum (FKA Paecilomyces lilacinus), Spodoptera littoralis nucleopolyhedrovirus (SpliNPV), Streptomyces avidinni, Tsukamurella paurometabola, Xenorhabdus spp, Yersinia entomophaga, Wolbachia pipientis, Zoophtora radicans, Metarhizium robertsii; Actinomycetes spp, Agrobacterium spp, Alcaligenes, Anabaena spp, Aphanizomenon spp, Arthrobacter spp, A. globiformis, Aureobacterium, Azoarcus spp, Azospirillum amazonense, A. brasilense, A. lipoferum, A. irakense, A. halopraeferens, Azotobacter spp, A. vinelandii, Azorhizobium spp, Bacillus idriensis, B. simplex, Bradyrhizobium spp., B. elkanii, B. japonicum, B. liaoningense, B. lupini, Brevibacillus spp, Burkholderia vietnamiensis, Chaetomium globosum, Clostridium spp, Comamonas spp, Corynebacterium spp, Delftia acidovorans, Enterobacter spp, E. cloacae, E. aerogenes, Enterococcus spp, Frankia alni, Gigaspora spp, Glomus spp, G. aggregatum, G. clarum, G. deserticola, G. etunicatum, Glomus spp intraradices (also references as G intraradicalis), G. mosseae, Gluconacetobacter diazotrophicus (FKA Acetobacter diazotrophicus), Herbaspirillum spp, Hydrogenophaga spp, Klebsiella spp, K. variicola, K. oxytoca, Kosakonia spp, K. sacchari, Lactobacillus spp, Mesorhizobium spp., Methanotrophic bacteria (Pink Pigmented Facultative Methylotrophs or PPFM), Methylobacterium spp, M. aminovorans, M. fujisawaense, M. mesophilicum, M. nodulans, M. oryzae, M. phyllosphaerae,M. radiotolerans, M. rhodesianum, M. symbioticum, M. thiocyanatum, Methylotrophicus extorquens (also referenced as Methylobacterium extorquens), Microbacterium oxydans, Micrococcus spp, Mortierella spp, Myrothecium verrucaria, Nostoc spp, Pantoea spp,Penicillium spp, Penicillium bilaiae (also referenced as P bilaii), P. brevicompactum, P. canescens, P. expansum, P. fellatanum, P. gaestrivorus, P. glabrum, P.jessenii, P. peoriae, P. radicum, P. raistrickii, P. steckii, P. vermiculatum, Photorhabdus spp, Phyllobacterium spp, Rhizobium leguminosarum bv. phaseoli, R. I. bv. trifolii, R. I. bv. viciae (also referenced as Rd. bv. viceae), R. tropici, Rhizopus spp, Rhizopogon spp, R. amylopogon, Scleroderma cepa, Serratia spp, S. marcescens, Sinorhizobium meliloti., Sphingobacterium spp, Stenotrophomonas spp, Streptomyces galbus, Thiobacillus spp, or Bacillus tequilensis, Suillus spp.

[0043] Exemplary microorganisms for use in probiotic applications, for example, include yeast (e.g., Saccharomyces boulardii), gram-negative bacteria (e.g., E. coli Nissle, Akkermansia muciniphila, Prevotella bryantii, etc.), gram-positive bacteria (e.g., Bifidobacterium animalis (including subspecies lactis), Bifidobacterium longum (including subspecies infantis), Bifidobacterium bifidum, Bifidobacterium pseudoIongum, Bifidobacterium thermophilum, Bifidobacterium breve, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus heiveticus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus deibrueckii (including subspecies bulgaricus), Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus gasseri, Lactobacillus brevis (including subspecies coagulans), Bacillus cereus, Bacillus subtilis (including var. Natto), Bacillus polyfermenticus, Bacillus clausii, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus, Faecalibacterium prausnitzii, Streptococcus thermophilus, Brevibacillus brevis, Lactococcus lactis, Leuconostoc mesenteroides, Enterococcus faecium, Enterococcus faecalis, Enterococcus durans, Clostridium butyricum, Sporolactobacillus inulinus, Sporolactobacillus vineae, Pediococcus acidilactici, Pediococcus pentosaceus, etc.), and the like.

[0044] Prebiotics and / or postbiotics can be used alone or in combination with probiotics. The term “prebiotic” as used herein includes compounds that stimulate the growth and or activity of bacteria and include, but are not limited to, inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS), a-gluco-oligosaccharides, a low gas producing prebiotic and combinations thereof, and the metabolic modifying component may comprise chromium. The term “postbiotic” as used herein, unless otherwise specified, refers to non-viable bacterial products or metabolic by products from the probiotic organism. Postbiotics can be sourced from the fermentation products of metabolites of Lactobacillus and Bifidobacterium, for example. Postbiotics can be found in fermented fruits and vegetables, chocolate, and tea or coffee.

[0045] Microbes used in healthcare applications include, but are not limited to, Streptococcus pyogenes (Acute Pharyngitis, Rheumatic Fever), Streptococcus pneumoniae (Pneumonia, Otitis Media), Prevotella intermedia, Porphymoronas gingivalis, Fusobacterium nucleatum (Periodontal disease / Gingivitis, Halitosis), Streptococcus agalactiae (Neonatal Sepsis and Meningitis), Solobacterium moore, Porphyromonas endodontalis, Fusobacterium nucleatum, Eubacterium sulci, Parvimonas micra, Eubacterium saburreum, Atopobium parvulum, Micromonas micros (Halitosis), Turicella otitidis, Alloiococcus otitidis, Moraxella catarrhalis, Actinomyces viscosus (Root Surface Caries), Streptococcus mutans, Streptococcus sobrinus (Tooth Decay, Dental Caries), Prevotella intermedia (Ulcerative gingivitis), Fusobacterium nucleatum, A. actinomycetemcomitans, Porphyromonas gingivalis, Bacteroides intermedins, Actinomyces naeslundii (Periodontitis), Streptococcus intermedins (Abscess Formation), Streptococcus agalactiae (Sepsis), Bacteroides species, Prevotella species, Bifidobacterium, Faecalibacterium, Lachnospiraceae (fecal-derived bacteria that are then used in fecal bacterial transplants), and the like.

[0046] Microbes are used in concrete applications, (e.g., “self-healing concrete”) including, but not limited to, Bacillus sphaericus, Bacillus subtilis, Bacillus cereus, Sporosarcinia pasteurii, and fungi.

[0047] Microbes used in the dairy industry include, but are not limited to, Lacticaseibacillus rhamnosus, Ligilactobacillus salivarius, Lactiplantibacillus plantarum, Lactobacillus helveticus, Lactococcus spp. , Leuconostoc spp. , and the like.

[0048] Microbes used in battery applications include, but are not limited to, Geobacter species, Shewanwella species, Synechocystis species, and Thiobacillus thioparus.

[0049] Microbes used in aquaculture include species used as probiotics as well as species used for food, such as Bacillus species, lactic acid bacteria, Vibrio species, Aeoromonas species, Saccharomyces cerevisiae, Aspergillus species , and Fusarium venenatum.

[0050] Microbial-based cleaning products can include Bacillus species, Lactobacillus species, Saccharomyces cerevisiae, Bifidobacteria and Rhodopseudomonas . Bioremediation of spills and toxins may require combinations of microorganisms (e.g. bacteria and algae), each of which require stabilization.

[0051] The biological agent can also be an engineered microorganism (e.g., bacteria such as E. coli and Gluconacetobacter xylinus, or yeast such as Saccharomyces cerevisiae) engineered toproduce chemicals in “microbial cell factories” that are important in industrial applications, e.g., bacterially derived cellulose.

[0052] Processed Fibroin

[0053] In one embodiment, the composition of the present disclosure comprises processed fibroin, which can be sourced from a silk producer or other sources as described herein. Silk fibroin, or simply fibroin, is the primary structural protein found in silk produced by silkworms, particularly the Bombyx mori species. This protein is known for its exceptional mechanical properties, biocompatibility, and biodegradability, making it a valuable material in various applications, particularly in the fields of biomaterials, tissue engineering, and drug delivery. When sourced from a silk producer such as Bombyx mori, for example, fibroin is obtained from silk fibers after the removal of the outer sericin, another protein that acts as a protective coating.

[0054] Processed fibroin, is composed of amino acids, particularly glycine, alanine, and serine, and, under certain processing conditions, has a unique crystalline structure of antiparallel beta sheets that contributes to its strength and stability. Processed fibroin can be obtained from silk fibers through various methods, including aqueous extraction, solvent dissolution, and electrospinning. These methods aim to produce fibroin in different forms, such as films, hydrogels, scaffolds, or fibers, depending on the intended application. Due to its natural origin, processed fibroin is highly biocompatible.

[0055] Besides natural silk-producing organisms like silkworms, silk and / or silk fibroin can also be sourced from genetically modified organisms (GMOs), recombinant silk fibroin produced through DNA technology, and fully synthetic silk fibroin created via peptide synthesis. Different species and production methods yield silk fibroin with varying physical and chemical properties, and blends of silk fibroin from multiple sources can offer unique, enhanced characteristics. GMOs and recombinant techniques allow for tailored silk fibroin properties, while synthetic silk fibroin enables precise control over composition without relying on biological silk producers. As used herein, processed fibroin refers to fibroin sourced from any of the above- identified sources.

[0056] The unique characteristics of silk fibroin make it suitable for diverse applications in textiles, medicine, and agriculture. Polymer strength and flexibility have supported classical uses of silk fibroin in textiles and materials, while silk fibroin biocompatibility has gained attention more recently for applications in the fields of medicine.

[0057] The inventors have discovered that combining processed fibroin with biological agents in a formulated composition results in a stable formulation with an extended shelf life and extended survivability of the biological agent. This increased longevity is attributed to the enhanced survivability and stability of the biological agents when paired with processed fibroin. Additionally, the inventors discovered that specific characteristics of processed fibroin, defined, for example, by particular molecular weight and purity specifications, when combined with a biological agent, produce stable formulations that exhibit enhanced survivability and stability such as for agricultural applications.

[0058] The processing conditions, the molecular weight (kDa), and the purity of the processed fibroin alter the stability of the biological agents in the formulations. The degumming conditions, dissolution conditions, purification conditions, and further processing such as hydrolysis, can affect the properties of processed fibroin including molecular weight, the ability to form stable, concentrated solutions, and the level of both organic and inorganic impurities. The molecular weight of processed fibroin can affect its properties, influencing factors such as application, processing, and compounding characteristics. Additionally, the purity of the processed fibroin can ensure that the biological agents in the agriculturally acceptable formulation remain stable and active for an extended period, thereby enhancing the composition's shelf life. For example, residual chaotropic agents, including but not limited to lithium, bromide, and calcium, used during the dissolution of degummed silk fibers can be minimized by specific processing techniques to improve application of processed fibroin. These particular impurities could have especially negative impacts on the survival of the biological agent in close contact with the fibroin, and could negatively impact the local environment wherever the formulation is deposited, for example the soil, the gut, or in a food or beverage product. Additionally, by eliminating these inorganic impurities using specific processing techniques, the ingredient is easier to formulate for any use case. For example, by removing more calcium, it is possible to formulate in phosphate-buffered solutions without the possibility of precipitating calcium phosphate, increasing its applicability across all fields.

[0059] Molecular weight / polydispersity and Purity of the Processed Fibroin

[0060] The various molecular weight characteristics include the number average molecular weight (MN), average molecular weight (MW), viscosity average molecular weight (MV), Z average molecular weight (MZ), and Z+l average molecular weight (MZ+1). Peak averagemolecular weight (MP) may also be determined. Another parameter which is frequently used when describing polymers is the polydispersity index (PDI). This parameter gives an indication of how broad a range of molecular weights is in the sample. The PDI is defined as:PDI = —A MNPDI equal to 1.0 is a monodisperse polymer while a PDI greater than 1.0 is a poly disperse polymer. The broader the molecular weight range, the higher the PDI value. Unless indicated otherwise, molecular weight as used herein is number average molecular weight.

[0061] In an aspect, the processed fibroin has a number average molecular weight of about 500 kDa, 450 kDa, 400 kDa, 350 kDa, 300 kDa, 250 kDa, 200 kDa, 150 kDa, less than 100 kDa, less than 90 kDa, less than 80 kDa, less than 70 kDa, less than 60 kDa, less than 50 kDa, less than 40 kDa, less than 30 kDa, less than 20 kDa, less than 10 kDa, less than 5 kDa, less than 2 kDa, etc., such as 0.5 to 80 kDa, 0.5 to 50 kDa, 0.5 to 45 kDa, 30 to 55 kDa, 30 to 40 kDa, 45 to 55 kDa, 0.5 to 2 kDa, or 0.5 to 5 kDa, 2 to 80 kDa, 2 to 50 kDa, 2 to 45 kDa, 2 to 5 kDa, or 2 to 10 kDa as measured by size exclusion chromatography (SEC), specifically by high-performance liquid chromatography (HPLC)-SEC. In one embodiment, the processed fibroin has a number average molecular weight of as low as 0.5 kDa, or 2 kDa or 2.5 kDa and as high as about 150 kDa, as measured by size exclusion chromatography (SEC). In another aspect, the processed fibroin has a weight average molecular weight of 10 kDa or less, such as 2 to 10 kDa or 0.5 to 10 kDa as determined by dynamic light scattering (DLS), i.e., SEC with multi-angle light scattering (SEC- MALS). Additionally, in an embodiment, the processed fibroin has a poly dispersity index of less than 1.5, 1.4, 1.3, 1.2, or 1.1 as determined by dynamic light scattering.

[0062] In SEC, also called gel filtration or gel permeation chromatography, molecules are separated based on their size as they pass through a column packed with porous beads. In UPLC- SEC, a sample is first run through UPLC, then through a column packed with porous particles. Combining UPLC and SEC can improve speed, resolution and automation of the process. SEC and UPLC-SEC, however, do not provide absolute molecular weights, so the elution times of the samples must be compared to known standards to determine the molecular weight of a sample. Typically, a calibration curve is created with known standards to determine molecular weight. The standard used to determine molecular weight herein is a Waters BEH200 Protein Standard Mix (Thyroglobulin (660 kDa), IgG (150 kDa), BSA (66.4 kDa), Myoglobin (16.7 kDa), and Uracil (112 Da)).

[0063] As described herein, processed fibroin with a number average molecular weight of 0.5 to 500 kDa, for example, can be determined by UPLC-SEC using a calibration curve of standards IgG (150 kDa), BSA (66.4 kDa), Myoglobin (17 kDa), and Uracil (112 Da).

[0064] In another aspect, processed fibroin with a number average molecular weight of less than 55 kDa, or less than 40 kDa, can be measured by UPLC-SEC using a calibration curve of standards IgG (150 kDa), BSA (66.4 kDa), Myoglobin (17 kDa), and Uracil (112 Da).

[0065] As described herein, processed fibroin with a number average molecular weight of about 10 kDa or less, such as 0.5 to 10 kDa, can be determined by UPLC-SEC using a calibration curve of standards IgG (150 kDa), BSA (66.4 kDa), Myoglobin (17 kDa), and Uracil (112 Da)).

[0066] While UPLC-SEC and the Waters BEH200 Protein Standard Mix are used to determine number average molecular weights herein, other methods may be used to determine molecular weights, with the caveat that the reported molecular weight of a sample can vary with the methods used to determine molecular weight. For example, SEC-MALS provides absolute molecular weight measurements without relying on standards. The absolute molecular weight determined by SEC-MALS may be different from that determined by UPLC-SEC and the Waters BEH200 Protein Standard Mix.

[0067] In addition to molecular weight, the purity of processed fibroin can influence its stabilizing properties within the composition. The purity of the processed fibroin is inversely proportional to the amount of unwanted protein and inorganic impurities present in the processed fibroin. The purity can be assessed by measuring the percentage of silk fibroin in relation to the unwanted proteins and the amount of inorganic impurities present.

[0068] There are several methods available for producing processed fibroin with the desired molecular weight and purity. The molecular weight and purity levels of the processed fibroin can vary with each method.

[0069] In some processes, preparing processed fibroin involves extracting it from silkworm cocoons by removing the sericin (degumming) through heating in an alkaline solution, then dissolving the fibroin in a concentrated chaotropic salt solution such as lithium bromide, followed by dialysis to purify the fibroin solution. The purified solution can then be further processed into various forms like solutions, powders, films, sponges, or fibers depending on the desired application.

[0070] Silk fibers from silkworm moth (Bombyx mori) cocoons include two main components, sericin (usually present in a range of 20-30% by weight ) and silk fibroin (usually present in a range of 70-80% by weight). Structurally, silk fibroin forms the center of the silk fibers and sericin acts as the gum coating the fibers. After degumming, the silk fibers are dissolved in an aqueous solution containing chaotropic agents like lithium bromide or calcium chloride, followed by dilution and filtration to remove particulates.

[0071] In an aspect, processed fibroin is produced by providing raw silk (e.g., cocoons or unpurified silk such as silk yarn), the raw silk comprising fibers containing silk fibroin and sericin. First, the raw silk is degummed in a salt solution, specifically a sodium carbonate solution with a sodium carbonate concentration of 0.02 to 1.0 M sodium carbonate, such as 0.02 M, 0.05 M, 0.1 M, 0.2 M, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 M, at a temperature of about 60° to about 100°C, specifically 80° to 90°, such as 80°, 85° or 90°, and for a time of greater than 60 minutes to about 480 minutes. In a preferred aspect, degumming is performed in 0.5 M sodium carbonate at 85°C for 240 to 360 minutes. In an aspect, degumming provides degummed silk fibers having a sericin concentration of 0-0.5 wt%.

[0072] After the degumming, the silk fibroin fibers are further processed by dissolving them, preferably in an aqueous solution comprising a chaotropic agent. Exemplary chaotropic agents include lithium bromide, lithium chloride, calcium chloride, ethanol, guanidinium chloride, and urea. Dissolving preferably includes using 5M to 13M lithium bromide for 1 hour to overnight at 50°C to 100°C to provide dissolved silk fibers, or dissolving the degummed silk fibers using a mixture of calcium chloride, ethanol, and water in a molar ratio of 1:2:8, respectively, for 1 hour to overnight at 50°C to 100°C to provide dissolved silk fibers.

[0073] After the silk fibroin fibers are dissolved, they can be diluted prior to further purification. In an aspect, the dissolved silk fibers are diluted in water to provide a concentration of 5 to 20% w / v silk fibroin fibers. Optionally the diluted fibroin solution is filtered through a polypropylene, polyethersulfone, nylon, or cellulose, diatomaceous earth, perlite depth prefilter to remove particulates and provide a clarified silk fiber solution.

[0074] In an aspect, the aqueous silk fibroin solution having a concentration of greater than or equal to 5% w / v silk fibroin is then purified by exchanging the chaotropic salt from the silk fibroin solution at a pH below the isoelectric point of the silk fibroin, wherein the pH is between2 and 5, replacing the chaotropic salt with a buffer comprising 10 to 300 mM of a second salt, or a combination thereof, to prepare the purified silk fibroin.

[0075] In an aspect, the diluted silk fibroin fibers are then purified by tangential flow filtration (TFF) using, for example, continuous diafiltration by tangential flow filtration (TFF). Diafiltration is the fractionation process that washes smaller molecules through a membrane and leaves larger molecules in the retentate without significantly changing concentration. It can be used to remove salts or exchange buffers. It can remove ethanol or other small solvents or additives.

[0076] In continuous diafiltration, the diafiltration solution (water, buffer, or a salt solution) is added to the sample feed reservoir at the same rate as filtrate is generated. In this way the volume in the sample reservoir remains constant, but the small molecules (e.g., salts) that can freely permeate through the membrane are washed away in the filtrate (also called the permeate). Using salt removal as an example, each additional diafiltration volume (DV; also referred to herein as a diavolume) reduces the salt concentration further as the salt ions are removed in the filtrate. (A diafiltration volume is the volume of sample before the diafiltration solution is added.) Anything that isn’t filtered out is the “retentate”. In the present case, the retentate includes the majority of the silk fibroin.

[0077] In the process described herein, in one method, the “sample”, also called the retentate, which includes the silk fibroin, is pH adjusted down to pH 2 to 5, for example, from its original pH of 8.5-9.

[0078] In an aspect, exchanging salt ions from the aqueous silk fibroin solution is by continuous diafiltration by tangential flow filtration (TFF) with a 5 kDa to 10 kDa molecular weight cut-off membrane by a process comprising providing a reduced pH retentate and filtering with at least three diafiltration volumes with a replacement feed of water, wherein the reduced pH retentate is a retentate comprising the silk fibroin and having a pH of 2 to 5. In an aspect, prior to providing the reduced pH retentate, the method comprises filtering at least 3 diafiltration volumes, preferably at least 5 diafiltration volumes, with a water replacement feed.

[0079] In another aspect, exchanging salt ions from the aqueous silk fibroin solution is by dialysis against the buffer having a pH of 2-5, wherein a pH of 2-5 is maintained through at least a portion of the dialysis procedure, preferably through the entire dialysis procedure.

[0080] US Patent No. 12,024,538 describes methods for reducing impurities in silk fibroin preparations and is incorporated herein by reference. Silk fibroin is produced from raw silk, which undergoes a degumming process in a sodium carbonate solution (0.05 to 1 M) at elevated temperatures (60-90°C) for 60 to 480 minutes, resulting in degummed silk fibers with minimal sericin content. Unlike prior art processes that use lower concentrations of sodium carbonate, this method using higher concentrations of sodium carbonate for degumming produces silk fibroin suitable for further processing, allowing for higher concentrations in subsequent steps. After degumming, the silk fibers are dissolved in an aqueous solution containing chaotropic agents like lithium bromide (e.g., lithium bromide (LiBr) at concentrations between 5M to 13M) or calcium chloride, followed by dilution and filtration to remove particulates. The dissolved silk fibroin solution is then purified by exchanging chaotropic salts at a pH between 2 and 5, using methods like tangential flow filtration (TFF) or dialysis. This purification process helps in removing smaller molecules while retaining the silk fibroin. By using the TFF / Dialysis methods, the purified silk fibroin preparation comprises 10 to 600 ppm lithium per mg silk, or 10 to 600 ppm bromine per mass of silk fibroin. The resulting silk fibroin preparation can be further adjusted to a neutral pH (7-9) and may be stored at concentrations ranging from 5% to 20% w / v. The average MW is controlled by both the initial degumming step (more sodium carbonate, higher incubation temperature, and longer incubation time all lead to a lower average MW product), and by controlled hydrolysis of the purified silk fibroin solution in water after TFF. For example, by incubating 15% silk fibroin with 0.1 M sodium hydroxide at 80°C for 24 hours, one can lower the MW, and then pH adjust back to 7-8 using concentrated HC1. Overall, the method produces a high-quality silk fibroin solution suitable for various applications, addressing limitations found in prior silk fibroin purification techniques.

[0081] In one embodiment, the processed fibroin is prepared using the method described in US Patent 12,024,538. It is to be understood that the processed fibroin may be prepared by any of the known processes available. While the embodiment here describes a specific method of producing processed fibroin, it will be understood by those skilled in the art that various other methods may also be used to create the processed fibroin of desired characteristics. For purposes of this disclosure, the desired molecular weight and purity of the fibroin play an integral part in stabilizing the biological agent, thereby increasing the stability of the resultant formulation. In one embodiment, the processed fibroin having a molecular weight of as low as 0.5 kDa and ashigh as 150 kDa, as measured by size exclusion chromatography (SEC) is used in the agriculturally acceptable formulation. Residual chaotropic salts used to prepare process fibroin can affect the purity of the processed fibroin. For example, in one embodiment, the processed fibroin may be prepared by a process comprising dissolving degummed silk fibroin fibers in 5 M to 13 M LiBr, and the processed silk fibroin comprises 10 to 600 mg, specifically 10 to 400, 10 to 300, 10 to 100 or 10 to 50 mg lithium per kg silk fibroin as determined by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS), and / or 10 to 600 mg, specifically 10 to 400, 10 to 300, 10 to 100 or 10 to 50 mg bromine per kg silk fibroin as determined by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) is used in the formulation.

[0082] In another aspect, the processed fibroin may be prepared by a process comprising dissolving degummed silk fibroin fibers in a chaotropic agent comprising calcium chloride, ethanol, and water in a molar ratio of 1:2:8, respectively, and the purified silk fibroin comprises 10 to 500 mg calcium per kg fibroin as determined by Inductively Coupled Plasma - Mass Spectrometry (ICP-MS).

[0083] In an aspect, a processed fibroin preparation prepared by the foregoing methods can be further modified to reduce the molecular weight. For example, hydrolyzing a processed fibroin solution having an initial molecular weight of 40 to 50 kDa overnight in either acidic or basic conditions and at elevated temperature can provide processed fibroin having a weight average molecular weight of less than 10 kDa. This can include incubation in 0.05 - 5 M NaOH solution, specifically 0.1 M, or in 0.05 - 5 M HC1 solution, specifically 0.25 M, at 30 - 95°C, specifically at 80°C, for 6 - 96 hours, specifically for 24 hours. The average molecular weights of processed fibroin from these processes can range from 0.5 kDa to 10 kDa, for example.

[0084] Additives

[0085] In one embodiment, the processed fibroin and the biological agent may additionally comprise one or more additives. In one embodiment, the one or more additives may include one or more of sucrose, maltodextrin, lactose, phosphate salts, sodium chloride, potassium phosphate monobasic, potassium phosphate dibasic, sodium phosphate dibasic, sodium phosphate monobasic, polysorbate 80, phosphate buffer, phosphate buffered saline, sodium hydroxide, glycerol, sorbitol, mannitol, ribose, riboflavin, inositol, xylitol, xylose, lactose USP, Starch 1500, microcrystalline cellulose, potassium chloride, sodium borate, boric acid, sodium borate decahydrate, magnesium chloride hexahydrate, calcium chloride dihydrate, sodium hydroxide,Avicel®, dibasic calcium phosphate dehydrate, tartaric acid, citric acid, fumaric acid, succinic acid, malic acid, hydrochloric acid, polyvinylpyrrolidone, copolymers of vinylpyrrolidone and vinyl acetate, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethyl cellulose, polyvinyl alcohol, polyethylene glycol, acacia, trehalose, and sodium carboxymethylcellulose. One or more of the additives may include phosphate buffer. One or more of the additives may include phosphate buffered saline. One or more of the additives may include sucrose. One or more of the additives may include trehalose. One or more of the additives may include sorbitol. One or more of the additives may include Xylitol. One or more of the additives may include mannitol. The additives may include boric acid, sodium borate decahydrate, sodium chloride, potassium chloride, magnesium chloride hexahydrate, calcium chloride dihydrate, sodium hydroxide, and hydrochloric acid. The processed silk may include at least one additive selected from one or more members of the group consisting of sorbitol, triethylamine, 2-pyrrolidone, alpha-cyclodextrin, benzyl alcohol, beta-cyclodextrin, dimethyl sulfoxide, dimethylacetamide (DMA), dimethylformamide, ethanol, gamma-cyclodextrin, glycerol, glycerol formal, hydroxypropyl beta-cyclodextrin, kolliphor® 124, kolliphor® 181, kolliphor® 188, kolliphor® 407, kolliphor® EL (cremophor EL), Cremophor® RH 40, cremophor® RH 60, dalpha- tocopherol, PEG 1000 succinate, polysorbate 20, polysorbate 80, solutol HS 15, sorbitan monooleate, poloxamer-407, poloxamer-188, Labrafil® M-1944CS, Labrafil® M-2125CS, Labrasol®, Gelucire® 44 / 14, Softigen® 767, mono- and di-fatty acid esters of PEG 300, PEG 400, or PEG 1750, kolliphor® RH60, N-methyl-2-pyrrolidone, castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, medium chain triglycerides of coconut oil, medium chain triglycerides of palm seed oil, beeswax, d-alpha-tocopherol, oleic acid, medium-chain mono-glycerides, medium-chain di-glycerides, alpha-cyclodextrin, betacyclodextrin, hydroxypropyl-beta-cyclodextrin, sulfobutylether-beta-cyclodextrin, hydrogenated soy phosphatidylcholine, distearoylphosphatidylglycerol, L-alphadimyristoylphosphatidylcholine, L- alpha-dimyristoylphosphatidylglycerol, PEG 300, PEG 300 caprylic / capric glycerides (Softigen 767), PEG 300 linoleic glycerides (Labrafil® M-2125CS), PEG 300 oleic glycerides (Labrafil M-1944CS), PEG 400, PEG 400 caprylic / capric glycerides (Labrasol®), polyoxyl 40 stearate (PEG 1750 monosterate ), polyoxyl 8 stearate (PEG 400 monosterate ), polysorbate 20, polysorbate 80, polyvinyl pyrrolidone, propylene carbonate, propylene glycol, solutol HS15,sorbitan monooleate (Span™ 20), sulfobutylether-beta-cyclodextrin, transcutol, triacetin, 1- dodecylazacyclo-heptan-2- one, caprolactam, castor oil, cottonseed oil, ethyl acetate, medium chain triglycerides, methyl acetate, oleic acid, safflower oil, sesame oil, soybean oil, tetrahydrofuran, glycerin, and PEG 4kDa.

[0086] Additives may be added to the formulations to improve the properties of the formulation such as the physical properties of the formulation including processibility. In an aspect, the additive may be a stabilizer, which generally refers to an additive which improves the stability, e.g., the shelf stability of the formulation. In another aspect, the additive is a stabilizing additive, which is used herein for additive which improve the stability of the biological agent in the formulations. Certain additives may have stabilizer and stabilizing additive properties.

[0087] In an aspect, the stabilizer or stabilizing additive is a long chain polysaccharide, including, but not limited to, hyaluronic acid, alginate, xanthan gum, gum arabic, pullulan, corn starch, cellulose, hemicellulose, carboxymethylcellulose, pectin, inulin, glucagon, dextran, cyclodextrin, lignin, chitin, chitosan, methylan, and the like, and combinations thereof.

[0088] In another aspect, the stabilizer or stabilizing additive is a protein such as collagen, gelatin, bovine serum albumin, elastin, keratin, sericin, whey protein, beta-lactoglobulin, alphalactalbumin, an immunoglobulin, lactoferrin, lactoperoxidase, and the like, and combinations thereof.

[0089] In an aspect, the stabilizer or stabilizing additive, is a disaccharide or a sugar alcohol. Exemplary disaccharides include lactose, sucrose, maltose, trehalose, and the like, specifically trehalose. Exemplary sugar alcohols include glycerol, ethylene glycol, sorbitol, threitol, arabitol, ribitol, ribose, xylose, xylitol, erythritol, galactitol, fucitol, iditol, tagatose, altritol, inositol, volemitol, isomalt, mailitol, malotriiol, lactitol, ployglycitol, maltotetratiol, and mannitol, specifically sorbitol.

[0090] In another aspect, the additive is an additive that promotes fibroin gel formation, such as alcohols including methanol, ethanol, n-propanol, n-butanol, 1,3 -propanediol and glycerol.

[0091] In an aspect, a powdered, e.g. spray dried, silk fibroin / glycerol combination can be dissolved in water and incubated at room temperature for a period of time to form a gel. A processed fibroin gel can be formed in solution or in situ as a coating on a substrate, for example, a seed coating. In an aspect, in a processed fibroin gel, the weight ratio of processed fibroin togelling additive, e.g., glycerol, can be 0.1 to 100, or 0.1 to 50, 0.2 to 50, 002 to 10, 0.5 to 10, or 0.5 to 5.

[0092] The formulations may comprise a stabilizing additive for the biological agent. As used herein, a stabilizing additive may improve stability of a biological agent when used on its own, or in combination with silk fibroin. Advantageously, when a stabilizing additive is combined with silk fibroin, the resulting biological agent stability due to the combination may be greater than either the silk fibroin alone or the stabilizing additive alone at the same concentration. In an aspect, a certain level of biological agent stability can be achieved with lower amounts of silk fibroin when used in combination with a stabilizing additive as compared to each of the silk fibroin or stabilizing additive alone. For example, a combination of 1% w / v sorbitol and 2% w / v low MW SF may provide comparable or better stability compared to 4% w / v low MW SF with no additive. Similarly, a combination of 2% w / v low MW SF and 1-5% w / v sorbitol may increase stability to levels not observed with sorbitol alone.

[0093] The combination of silk fibroin with a stabilizing additive produces unexpected and surprising effects on the biological agent’s stability. In an aspect, the stabilizing effect of silk fibroin and a stabilizing additive is less than additive, that is, the total stability may be less than the sum of the stability determined for each agent individually, but still greater than either agent alone at a given concentration.

[0094] In another aspect, the stabilizing effect of silk fibroin and a stabilizing additive is additive, that is, the total stability is about the sum of the stability determined for each agent individually at a given concentration.

[0095] And in yet another aspect, the stabilizing effect of silk fibroin and a stabilizing additive is synergistic, that is, the total stability may be significantly greater than the sum of the stability determined for each agent individually at a given concentration. In an aspect, synergy can be determined using the Colby method which is well-known in the art.

[0096] In an aspect, the ratio of the weight of the stabilizing additive to silk fibroin is 1 :20 to 20: 1, such as 1:0.4, 1:0.5, 1 :0.8, 1 :1, 1:2, 1: 3, 1:4, 1:5, 1 :6, 1:7; 1:8, 1:9, 1 :10, and any ratio in between.

[0097] Process for preparing the formulation

[0098] In one embodiment, a process for preparing the formulation comprising a biological agent, processed fibroin, and optionally an additive is provided. The processed fibroin may beprovided as a solution with desired viscosity or as spray-dried powder. Additives may optionally be added to either the solution or the powder. The biological agent may then be suspended in the solution or powder comprising the processed fibroin to manufacture the formulation. The disclosure includes both liquid and solid formulations.

[0099] One format for the processed fibroin is a solution of silk fibroin in water. Processed fibroin with a molecular weight of about 0.5 to 150 kDa, for example, provides solutions with manageable viscosities even with protein concentrations exceeding 15% (w / v) and as high as 50% (w / v). A processed fibroin solution is the simplest starting point for a variety of different uses (hydrogels, films, solid casting, etc.) As is known in the art, even in the absence of additives, fibroin solutions can form hydrogels. For the methods described herein, both solution and hydrogel formulations of processed fibroin may be used. In the case of a hydrogel formation, the hydrogel may be shaken prior to application. While the appearance of the processed fibroin may no longer be a gel after shaking, many of the properties of the gel may be maintained.

[0100] In yet another embodiment, processed fibroin can be provided in the form of a spray- dried powder. A fibroin solution of desired molecular weight of desired quantity is spray-dried using traditional methods. The fibroin solution in water can be sprayed dried to achieve a fibroin powder. Using processed fibroin prepared by the methods described herein, spray drying provides a shelf-stable powder that is completely reconstitutable in water up to concentrations of >20% (w / v). In one embodiment, a 15% (w / v) fibroin solution is spray-dried using a Buchi Mini lab-scale spray dryer. Spray-dried fibroin powder may be used to provide solid formulations, or dissolved or suspended in water to provide liquid formulations.

[0101] Additionally, additives may optionally be included in the spray drying process. In one of the examples described herein for the biological agent Rhizobium leguminosarum bv. viceae, a solution of 15% (w / v) silk fibroin and 4.5% (v / v) glycerol in water was spray-dried, which resulted in a powder that was roughly 23% glycerol by mass. The gelling agent, e.g., glycerol, can be 0.01 to 50% by weight of the final dried powder. This method produces a powder that is also readily reconstitutable in water, but due to the presence of glycerol (an additive that contributes to silk fibroin beta-sheet formation through hydrogen bonding), the solution eventually gels after reconstituting. Further, this method of reproducible gelling (which can be tuned with the concentration of powder added, temperature, and time) can provide a formulation achieving the elevated stability. Additionally, this powder, when left at room temperature, mayeventually form beta-sheets at the microscopic level, making the powder itself not reconstitutable, which may have use for other methods. Beta-sheet formation may be mitigated by vacuum packing the resultant spray-dried formulation and / or the use of anti-desiccants.

[0102] In an embodiment, the processed fibroin comprises 0.01 to 50% w / v (such as 0.01-30% w / v, 1-30% w / v, 0.1-12% w / v, 1-10 % w / v, and all ranges in between) of a formulation in liquid form. The processed fibroin comprises 0.01 to 99.99% w / w of a formulation in solid form (such as 50 to 99.9% w / w, 75 to99% w / w, 85 to 99 % w / w, and all ranges in between). Optionally an additive may be added to the processed fibroin. The additive may be selected from any of the additive listed above such as one or more of sugars, polysaccharides, sugar alcohols, or nucleating promoters (e.g. fine particulate matter - separately or as part of the target material delivery) to enhance conformational change (e.g., to the Silk II 0 sheet motif) of the protein, forming an amphiphilic structure further enabling protection, both pre and post application. Thickness (impacting microbial protection and release characteristics) can be modified using variable application volumes, fibroin of selected concentrations and / or molecular weight. In one embodiment, the additives may comprise up to 99% by w / w or w / v of the formulation, such as 0.1 to 99% w / w or w / v. In one embodiment, such as for a coated seed composition, the biological agent may be added in an amount of l-lxl0e9 cfu / seed, preferably lxl0e2-lxl0e7 cfu / seed.

[0103] Under equivalent environmental conditions, the formulation with processed fibroin and optional additives exhibits at least 10% to 50,000%, 50% to 10,000%, or 100% to 5,000% or higher enhanced stability of the biological agents. Specific examples of increases in on-seed stability can be 46%, 50%, 63%, 73%, 76%, 80%, 86%, 93%, 96%, 103%, 104%, 119%, 122%, 153%, 443%, 449%, 518%, 524%, 669%, 1,088%, 1,141%, 1264%, 3,300%, 23,900%. Examples of enhanced in-pack stability can be at least 10% to 50,000%. Examples of increased in pack stability can be 10,406%, 24,276% and 27,428%.

[0104] Studies indicate that the processed fibroin provides increased pre- and post- application stability of the biological cells to a wide range of micro-organisms under elevated temperature conditions. The gram-positive bacterium - Bacillus firmus responds to the sequential addition of fibroin (up to the maximum 3% evaluated in these studies). Both in-pack and on-seed stability are increased with all fibroin concentrations evaluated over the 12-month evaluation period under the elevated storage temperatures.Seed Treatment

[0105] In a particularly preferred embodiment, the agriculturally acceptable formulation is utilized as a seed treatment formulation. According to one aspect of the present disclosure, the seeds are coated, such as, substantially uniformly coated with one or more layers of the formulations disclosed herein using conventional methods of mixing, spraying, or a combination thereof, through the use of treatment application equipment that is specifically designed and manufactured to accurately, safely, and efficiently apply seed treatment products to seeds. Such equipment uses various types of coating technology such as rotary coaters, drum coaters, fluidized bed techniques, spouted beds, rotary mists, or a combination thereof. Liquid seed treatments such as those of the present disclosure can be applied via either a spinning “atomizer” disk, or a spray nozzle which evenly distributes the seed treatment onto the seed as it moves though the spray pattern. Alternatively, a simple application tube can dispense the seed treatment directly onto the seed in the seed treatment equipment, allowing the seed movement and flow action to fully coat the seed. Preferably, the seed is then mixed or tumbled for an additional period of time to achieve additional treatment distribution and dried.

[0106] The seeds may be coated via a batch or continuous coating process. In a continuous coating embodiment, continuous flow equipment simultaneously meters both the seed flow and the seed treatment products. A slide gate, cone and orifice, seed wheel, or weighing device (belt or diverter) regulates seed flow. Once the seed flow rate through treating equipment is determined, the flow rate of the seed treatment is calibrated to the seed flow rate in order to deliver the desired dose to the seed as it flows through the seed treating equipment. Additionally, a computer system may monitor the seed input to the coating machine, thereby maintaining a constant flow of the appropriate amount of seed.

[0107] In a batch coating embodiment, batch treating equipment weighs out a prescribed amount of seed and places the seed into a closed treating chamber or bowl where the corresponding dose of seed treatment is then applied. This batch is then dumped out of the treating chamber in preparation for the treatment of the next batch. With computer control systems, this batch process is automated enabling it to continuously repeat the batch treating process.

[0108] In either embodiment, the seed coating machinery can optionally be operated by a programmable logic controller that allows various equipment to be started and stopped withoutemployee intervention. The components of this system are commercially available through several sources such as Gustafson Equipment of Shakopee, Minn.

[0109] A variety of additives known to one of ordinary skill such as adhesives or binders may be added to the agriculturally acceptable formulation. Exemplary binders include those composed of an adhesive polymer that may be natural or synthetic and is without phytotoxic effect on the seed to be coated. At least one colorant may also be added. Any of a variety of colorants may be employed, including organic chromophores classified as nitroso, nitro, azo, including monoazo, bisazo and polyazo, diphenylmethane, triarylmethane, xanthene, methine, acridine, thiazole, thiazine, indamine, indophenol, azine, oxazine, anthraquinone and phthalocyanine. Other additives that can be added include trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc. A polymer or other dust agent can be applied to retain the treatment on the seed surface.

[0110] In one embodiment, the seed coating formulation of the present disclosure may contain at least one filler which is an organic or inorganic, natural or synthetic component with which the active components are combined to facilitate its application onto the seed. Exemplary fillers are inert solids such as clays, natural or synthetic silicates, silica, resins, waxes, solid fertilizers (for example, ammonium salts), natural soil minerals, such as kaolins, clays, talc, lime, quartz, attapulgite, montmorillonite, bentonite or diatomaceous earths, or synthetic minerals, such as silica, alumina or silicates, in particular aluminum or magnesium silicates.

[0111] The present disclosure also provides a method of protecting a seed comprising providing the agriculturally acceptable formulation comprising effective amounts of a biological agent and processed silk fibroin, and applying the formulation in an effective amount, that is, an agriculturally effective amount. In another embodiment, the seed treatment formulation may be applied to seed in combination with a pesticide (such as a fungicide, insecticide, nematicide), a seed coating, a seed colorant, and / or other seed applied treatments.

[0112] Any crop seed can be treated in accordance with the disclosure. This includes genetically modified crops, non-genetically modified crops and combinations thereof. Crop seeds that can be treated in accordance with the present disclosure include such crops as fruits and vegetables. In one embodiment, without limitation, the crop seeds that may be coated include soybean, wheat, sunflower, barley, rice, rapeseed / canola, sugar beet, sunflower, tuber / rhizome producing crops, tomato, bean, carrot, tobacco, vegetable, fruit, grass turf, and flower seeds. Preferably, soybean,corn, canola, oil seed rape, rice, legume and pulse crops, cereals or cotton seeds are coated with the present formulations.

[0113] Having disclosed the subject matter of the present invention, it should be apparent that many modifications, substitutions and variations of the present invention are possible in light thereof. It is to be understood that the present invention can be practiced other than as specifically described. Such modifications, substitutions and variations are intended to be within the scope of the present application.Stabilization of Biological Agents

[0114] Processed fibroin, including but not limited to processed fibroin having a molecular weight of about 10 kDa or higher (measured using the UPLC-SEC method described herein), can have conformational changes which enhance the ability of fibroin to stabilize biological agents. Without being held to theory, silk fibroin beta-sheet structures seem to have benefits across multiple coating formulations with regard to the survival of biological agents both in-pack (as a packaged liquid formulation) and on-seed (dried onto the seed surface). These benefits extend to multiple types of biological agents, including but not limited to gram-positive bacteria, gramnegative bacteria, and spore-forming bacteria. And while these examples demonstrate extended survival using silk fibroin in agricultural coating formulations, the effects seen have broad applicability anywhere these or other biological agents are utilized. The effect could be related to controllable aspects of the silk fibroin, including but not limited to silk fibroin concentration, purity level, molecular weight, pH, format, beta-sheet percentage, time to gelation, and chemical modification or cross-linking. Other processes could be used to supplement and control how silk fibroin influences biological agent survival, including but not limited to shaking, heating, cooling, freezing, stirring, combining with other formulants, and controlling in what order formulants are added. By controlling these and other process variables, silk fibroin can both by itself and in combination with other formulants provide a meaningful and unexpected stability benefit for numerous biological agents across many industries. In an aspect, the processed fibroin, with or without additives, stabilizes (e.g., improves survival) the biological agent in the formulation. Without being held to theory, low molecular weight SF may undergo conformational changes (beta sheet formations) with or without the presence of certain excipients.

[0115] In an aspect, when the biological agent is a spore forming bacterium, processed fibroin alone can provide significant stabilization, both in solution (e.g., in-pack) and on a surface (e.g., on a seed). As shown in Example 5, over time at 30°C, a processed fibroin solution containing suspended pore-forming bacteria forms a gel. Unexpectedly, these formulations with processed fibroin alone can provide over a 10,000%, a 20,000% or even a 24,000% improvement in survival of a spore forming bacterium. Optionally, the addition of a gel-inducing agent (also called a beta-sheet inducing agent, or an excipient) such as glycerol can accelerate gel formation at lower temperatures.

[0116] Also, when the biological agent is a spore forming bacterium, the processed fibroin alone can improve the on-surface (e.g., on-seed) survival of the spore forming bacteria. As shown in Example 6, processed fibroin alone can provide greater than a 40%, 70% or 100% improvement in survival of a spore forming bacterium coated on a surface.

[0117] In another aspect, when the bacteria is a non-spore forming bacterium, such as a nonspore forming gram negative or gram-positive bacterium, processed fibroin alone can provide a 50% or more improvement in on-surface (e.g., on-seed) survival of the non-spore forming bacteria. This is shown in Example 1. Also shown in Example 1 is that the addition of glycerol to the formulation can enhance stabilization, providing a more than 1000% improvement in survival of the non-spore forming bacteria. Without being held to theory, it is believed that in some processed fibroin preparations, the addition of glycerol aids in beta sheet formation which improves hydrogel network formation. Hydrogel formation may protect the microbe, for example a bacterium or a fungi, from physical stress, sudden dehydration, and other physicochemical destabilization effects to pH, osmolarity, or surface charge.

[0118] In an aspect, when the biological agent is a non-spore forming, environmentally unstable bacterium, adding a gel-inducing agent to the formulation prior to application to a surface (also called pre-gelling) can improve the survival of the bacteria. Environmental conditions include temperature, water, salinity and chemicals. Pre-gelling can include combining processed fibroin, a sugar alcohol such as glycerol, and the bacteria and allowing the formulation to form a hydrogel (by visual observation) prior to coating onto a surface (e.g., a seed). It is predicted that the pre-gelled composition will protect the sensitive biological agents during all steps of the coating process, from application to drying and storage.

[0119] In another aspect, when the bacterium is a non-spore forming bacterium, such as a nonspore forming gram negative or gram-positive bacterium, processed fibroin with additives such as a disaccharide sugar (e.g., trehalose) or a polyhydroxy alcohol (e.g., sorbitol) can provide a 50%, 100% or more improvement in on-surface (e.g., on-seed) survival of the non-spore forming bacteria. For example, as shown in Example 2, the addition of processed fibroin to trehalose can increase non-spore forming bacteria survival by 2-fold or more. Also, the addition of processed fibroin to sorbitol can increase non-spore forming bacteria survival by 100-fold or more. Further, as shown in Example 3, the addition of processed fibroin to trehalose can increase non-spore forming bacteria survival by 10-fold or more. The addition of processed fibroin to sorbitol can also increase non-spore forming bacteria survival by 100-fold or more. Yet further, as shown in Example 3, the addition of processed fibroin to trehalose can increase non-spore forming bacteria survival by 35 -fold or more or even 250-fold or more. The addition of processed fibroin to sorbitol can also increase non-spore forming bacteria survival by 70-fold or more.

[0120] In an aspect, when the bacterium is a non-spore forming bacterium, the ratio of fibroin to disaccharide sugar (e.g., trehalose) is 1 :1 to 1:5, specifically 1 :2 to 1:3. In another aspect, when the bacteria is a non-spore forming bacteria, the ratio of fibroin to poly hydroxy alcohol (e.g., sorbitol) is 1 :1 to 1:5, specifically 1:2 to 1:4.

[0121] In another aspect, processed fibroin with low molecular weight, for example as low as 10 kDa, 9 kDa, 8 kDa, 7 kDa, 6 kDa, 5 kDa, 4 kDa, 3 kDa, 2.5 kDa, 2 kDa, 1 kDa, 0.5 kDa (or lower) stabilizes biological agents as described herein.Methods, Examples 1-6

[0122] In these Examples, the number average molecular weight of the processed fibroin was determined using a Waters Acquity H-Class UPLC equipped with a Waters BEH 200A 1.7 mm, 4.8 x 300 cm UPLC SEC column. Sample temperature was maintained at 4°C. An isocratic flow at 0.3 mL / min of mobile phase 100 mM Tris-HCl with 400 mM anhydrous sodium perchlorate, pH 8.0 was used to elute silk fibroin (SF) from the column. Mobile phase was prepared by dissolving 12.1 g of Tris base and 48.1 g of sodium perchlorate in 800 mL of DI water. The pH was adjusted to 8.0 with 6N HC1. DI water was added to a final volume of IL. The solution was sterile filtered through a 0.2 pm poly ethersulfone membrane filter. Samples were diluted to 1% SF in mobile phase and 2 pL injections were used. SF elution was monitored at 280 nm and 220 nm. The average molecular weight was calculated using a standard curve prepared from a WatersBEH 200A Protein SEC Standard Mix (IgG (150 kDa), BSA (66.4 kDa), Myoglobin (17 kDa), and Uracil (112 Da).

[0123] In Examples 1-6, processed fibroin (either 40-70 kDa MW fibroin (e.g., 51.3 kDa by UPLC-SEC, Waters BEH200 Protein Standard Mix) or 25-45 kDa MW fibroin (e.g., 40.5 kDa by UPLC-SEC, Waters BEH200 Protein Standard Mix)) was mixed at differing concentrations with and without additives at identified concentrations to prepare agricultural formulations. The processed fibroin in the aqueous formulation was between 1-30% w / v, while the additives were between 0.01-30% w / v. In one aspect the additives could be 0.05-5%, 0.1-5%, 1-10%, 5-15%, 10-20%, 10-25%, 10-30% w / v. The composition in the form of a suspension of the biological agent in the fibroin / additive solution was then applied to seeds to create treatment samples. The treatment samples were stored under differing environmental conditions to assess biological stabilization over time (assessed as cfu / mL or cfu / seed using generally recognized standard protocols for those ordinarily skilled in the art of microbial determination). In some cases, the formulation was applied to the target seed at the stated rate, with the seed being mixed with the treatment to provide an even seed coverage. Treated seeds were then subsampled to be stored under differing environmental conditions, with viable cfu / seed being assessed over time using generally recognized standard protocols for those ordinarily skilled in the art of microbial determination. Fibroin and non-fibroin controls were also included in the experimental design.

[0124] Processed fibroin was introduced into the following formulations using a wide variety of different formats and chemical and physical configurations. Importantly, extended stability was observed using multiple different starting and ending points for the processed fibroin configuration. These include, but are not limited to, processed fibroin solutions, spray-dried powders, hydrogels, chemically cross-linked hydrogels, films, freeze-dried powders, and solid constructs. The results are shown in Fig. 1-6 and Tables 1-6.Example 1 : Stabilization of Rhizobium leguminosarum bv. viceae with processed fibroin with and without glycerol

[0125] In this example, the impact of processed fibroin, both with or without glycerol additive, on the stability and viability on Rhizobium leguminosarum bv. viceae at 15°C on pea seeds was assessed. The control was Rhizobium leguminosarum bv. viceae with no fibroin or glycerol.Rhizobium leguminosarum bv. viceae is an agriculturally important bacterium which can be unstable under environmental conditions.

[0126] As is well-known in the art, fibroin solutions can form a 3 -dimensional network structure having the properties of a gel. In an aspect, a fibroin gel can be formed by physical crosslinking in which the coils or a-helices of fibroin undergo a transition to a P-sheet network, forming a hydrated 3 -dimensional network. These physical hydrogels can be formed through self-assembly, ultrasound, shear force, electric field, heat, pH change, organic solvent, and / or surface treatment with a surfactant. Alcohols such as methanol, ethanol and glycerol, for example, can stimulate fibroin gel formation. Without being held to theory, it is believed that in some cases these physical silk fibroin hydrogels can stabilize added biological agents.

[0127] In another aspect, a fibroin gel can be formed by chemical crosslinking in which covalent bonds are formed between fibroin amino acid sidechains.

[0128] Fibroin hydrogels, including those with enzymatic, irradiation, and photo treatment mediated cross-linking could all provide a unique structural environment that contributes to extended survival of added biological agents. This would be particularly useful if hydrogen bonding negatively impacted the biological agent, or if the conditions that would induce the physical cross-linked network were deleterious to extended viability.

[0129] In the first preparation method, processed fibroin was introduced by itself to determine its effect on stabilization. In this method, a processed fibroin spray-dried powder was dissolved in an aqueous Rhizobium leguminosarum by. viceae suspension to provide a liquid formulation. After the powder was fully dissolved, the coating formulation was applied to and immediately dried on seeds. In a second preparation method, a processed fibroin / glycerol combination spray- dried powder (for example, 15% w / v processed fibroin / 4.5% w / v glycerol) was dissolved in an aqueous suspension comprising Rhizobium leguminosarum bv. viceae to form a coating formulation, and then the coating formulation was applied to and immediately dried on seeds. The theory was that spray drying the processed fibroin and glycerol would accelerate gel formation once the powder was added to the Rhizobium leguminosarum bv. viceae suspension. Without being held to theory, it is believed that during this drying process, as water evaporates and the local concentration of silk fibroin and glycerol increases, a hydrogel is formed on the seed surface. In a third preparation method, a processed fibroin / glycerol combination powder was dissolved in an aqueous Rhizobium leguminosarum bv. viceae suspension form a liquidformulation and the formulation was allowed to gel (e.g., form beta-sheets) before being introduced to the seeds. This pre-gelled formulation was used to coat seeds. It is believed that this “pre-gelled” formulation showed superior OSS, indicating that protecting this fragile active with the beta-sheet structures may be important even from the initial application / drying step. While not done in this experiment, the pre-gelled formulation can be shaken such that it does not re-form as a cohesive gel, however, it is believed that the shaken gel does maintain the stabilization enhancement performance. This indicates that it is possible for the hydrogel formulation to be broken apart, perhaps to facilitate ease of use, while still maintaining its benefits. The data in Fig. 1 and Table 1 is for method 1 (3% w / v processed fibroin / 0.9% w / v), method 2 (3% w / v processed fibroin / 23% glycerol powder, not pre-gelled) and method 3 (3% w / v processed fibroin / 23% glycerol powder, pre-gelled).

[0130] Fig. 1 and Table 1 provide the OSS data for Rhizobium leguminosarum bv. viceae on seed stabilization.Table 1: Rhizobium leguminosarum bv. viceae OSS data, 18-day assessment on pea seeds

[0131] As shown in Fig. 1 and Table 1, while processed fibroin by itself provided a 50% increase in survival compared to control, processed fibroin with glycerol without pre-gelling provided about a 100% improvement in survival, and glycerol with pre-gelling provided greater than a 1000% improvement in survival. Without being held to theory, it is believed that the glycerol additive facilitates the formation of a beta-sheet crystalline structure by encouraging hydrogen bonding within the processed fibroin. These beta-sheet structures are predicted to form a hydrogel network around the biological agent, protecting it from physical stress, sudden dehydration, and other physicochemical destabilization effects on pH, osmolarity, or surfacecharge. For this example, it appears that the protective beta-sheet structures form during the drying step on the surface of the seed, suggesting that the biological agent’s instability may arise during drying and the associated water loss. Protection of this biological agent during drying is thus predicted to improve seed stabilization.Example 2: Stabilization of Bradyrhizobium japonicum with processed fibroin with trehalose or sorbitol

[0132] Trehalose, a disaccharide sugar, and sorbitol, a polyhydroxy alcohol, have been used to stabilize microbes from environmental conditions. Fig. 2 and Table 2 illustrate the impact of the addition of processed fibroin to trehalose and sorbitol additives, on the stability and viability of an agriculturally acceptable formulation comprising B. japonicum at a temperature of 20°C on soybean seeds. The processed fibroin spray-dried powder, trehalose or sorbitol and B. japonicum were mixed in water to form a suspension of the B. japonicum in the fibroin / additive.

[0133] As shown in Fig. 2 and Table 2, compared to trehalose or sorbitol alone, the addition of 5% w / v processed fibroin improved 12 week survival by over 100%. Trehalose alone increased survival by about 60%, while sorbitol alone had no effect on Bradyrhizobium japonicum survival.Table 2: Bradyrhizobium japonicum OSS data, 12 week assessment on soybean seeds

[0134] The combination of trehalose or sorbitol with processed fibroin and Bradyrhizobium japonicum provided an unexpected increase in survival. Of note, in this example, processed fibroin alone had little effect on stabilization (data not shown). Without being held to theory, the improvement with the combination of processed fibroin and disaccharide sugar or sugar alcohol could be related to hydration retention, bonding interactions, structure formation, and / or astructural interaction between the processed fibroin, the additives, and the cell walls of the biological active.Example 3: Stabilization of Methylorubrum extorquens with processed fibroin with trehalose or sorbitol at 20°C on corn seeds

[0135] Fig. 3 and Table 3 illustrate the impact of the addition of processed fibroin to trehalose or sorbitol additives on the stability and viability of PPFM (Methylorubrum extorquens also known as Methylobacterium extorquens) at 20°C on corn seeds. The processed fibroin, trehalose or sorbitol and PPFM were mixed in water to form a suspension of PPFM in the fibroin / additive.

[0136] As shown in Fig. 3 and Table 3, compared to trehalose or sorbitol alone, the addition of 5% or 7% w / v processed fibroin generally improved 12 week survival by over 100% to over 1000%. Trehalose alone increased survival by about 120%, while sorbitol alone had no effect on Methylobacterium extorquens.Table 3: Methylobacterium extorquens: OSS data, 12 week assessment on corn seeds

[0137] As with Bradyrhizobium japonicum, the combination of trehalose or sorbitol with processed fibroin and Methylobacterium extorquens provided an unexpected increase in survival. Of note, in this example, processed fibroin alone had little effect on stabilization (data not shown). Without being held to theory, the improvement with the combination of processed fibroin and disaccharide sugar or sugar alcohol could be related to hydration retention, bonding interactions, structure formation, and / or a structural interaction between the silk fibroin, the additives, and the cell walls of the active. Noticeably, in this example there was an increase insurvival with an increase in processed fibroin present in combination with the trehalose and sorbitol. Without being held to theory, this indicates that the stabilization effect provided by these processed fibroin preparations is proportional to the amount of processed fibroin, and perhaps also depends on the strength of the fibroin network as affected by the presence of disaccharide sugar or sugar alcohol. The unexpected improvement in survival from 5% to 7% w / v processed fibroin in the presence of 15% w / v trehalose indicates that there may be a sharp cutoff for the most improved survival effect. Additionally, processed fibroin concentrations greater than 7% w / v may continue to show additionally improved beneficial effects.Example 4: Stabilization of Methylorubrum extorquens with processed fibroin with trehalose or sorbitol at 30°C on corn seeds

[0138] Fig. 4 and Table 4 illustrate the addition of processed fibroin to trehalose and sorbitol additives on the stability and viability of PPFM (at 30°C on corn seeds). The processed fibroin, trehalose or sorbitol and PPFM were mixed in water to form a suspension of PPFM in fibroin / additive.

[0139] As shown in Fig. 4 and Table 4, compared to trehalose or sorbitol alone, the addition of 5% or 7% w / v processed fibroin generally provided an increase in 4 week survival of over 70% with sorbitol, and over 3000% with trehalose. Trehalose alone increased survival by about 93%, while sorbitol alone had no effect on PPFM.Table 4: Methylobacterium extorquens: OSS data, 4 week assessment on corn seeds

[0140] Similar to the data at 20°C, the combination of trehalose or sorbitol with processed fibroin and Methylobacterium extorquens provided an unexpected increase in survival at 30°C.Example 5: In-pack stabilization of Bacillus firmus with processed fibroin at 30° C

[0141] Fig. 5 and Table 5 illustrate the impact of processed fibroin on the In-Pack stability and viability of B. firmus (30°C Storage Temp). Different percentage processed fibroin solutions were thoroughly mixed with Bacillus firmus with the resultant mixture stored at 30°C for the assessment timeframe. These samples formed a hydrogel while standing. The stability in the as- formed hydrogel was tested over time.

[0142] The cfu / mL of B. firmus was assessed over time, with very clear positive impacts (e.g., more than 10,000% improvement in survival) of all three fibroin treatments (1%, 2% and 3% w / v) tested. In general, formulations with a higher concentration of processed fibroin are expected to lead to a higher percentage of beta-sheet structures. Without being held to theory, this could explain the dose dependence in this data, where a greater concentration of processed fibroin leads to a more structured and denser beta-sheet crystal network (e.g., a hydrogel), and therefore provides more protection and a superior stability outcome.Table 5: In-pack stability of Bacillus firmus with processed fibroinExample 6: Stabilization of B. firmus with processed fibroin at 30° C on seeds

[0143] Fig. 6 and Table 6 illustrate the impact of processed fibroin on the stability and viability of B. firmus on seeds (seed stored at 30° C / 80% RH). At a 12 month assessment, the survival increased about 50% or more when B. firmus was formulated with processed fibroin.Table 6: Stabilization of B. firmus with processed fibroin at 30° C on seeds

[0144] Compared to the data in Table 5, similar trends are observed in Table 6 but without the same magnitude of effect. This could be for numerous reasons. Without being held to theory, these on seed samples were held in a much higher relative humidity, which will impact stability endpoints. Additionally, it is possible that the benefit the silk fibroin beta-sheet network provides is more pronounced in bulk format.Methods, Examples 7-8

[0145] A low number average molecular weight processed silk fibroin having an average molecular weight of about 2 kDa as determined by UPLC-SEC using a calibration curve of standards IgG (150 kDa), BSA (66.4 kDa), Myoglobin (17 kDa), and Uracil (112 Da) was prepared. To prepare the 2 kDa MW SF, processed silk fibroin having a molecular weight of 30 to 60 kDa was prepared (degummed for 4 or 6 hours in 0.5 M sodium carbonate, solubilized in LiBr and purified using TFF as described herein). A 15% w / v solution of the processed silk fibroin was hydrolyzed in 0.1 or 0.25 M NaOH for between 0.5 and 24 hours at 80°C. Then 0.1 or 0.25 M HC1 was added to neutralize the solution, followed by filtration through a 0.22 pm filter to remove particulates. The 2 kDa MW SF was then spray dried to form a powder.

[0146] Example 7: In vitro stability with 2 kDa MW SF

[0147] An in vitro assay was developed in which bacteria (e.g., Rhizobium leguminosarum bv. viceae) were mixed with processed fibroin (referred to below as SF) solutions- 100 pL of SF / bacteria solution was pipetted into each well of a 96-well microplate, and the entire plate was dried (16 hours in a fume hood). The plate was then incubated at 22°C and ambient humidity for 2 weeks before being reconstituted in distilled water to dissolve the film and release bacteria. The % survival of bacteria was determined by LIVE / DEAD™ BacLight™ (L7012 kit) fluorescence assay using the manufacturer’s protocol. A 100% dead standard was made by treating Rhizobium leguminosarum bv. viceae with 70% isopropyl alcohol for 1 hour and rinsing with saline. A 100% live standard was made by rinsing Rhizobium leguminosarum bv. viceae with saline. Standards were made using 90: 10, 50:50, and 10:90 live:dead solutions.

[0148] Stabilization with silk fibroin of molecular weight between 2 kDa and 50 kDa was compared in the in vitro assay. The data is provided in Table 7.31Table 7: Stabilization of Rhizobium leguminosarum bv. viceae with processed silk fibroin in vitro, listed as % bacteria survival compared to fresh control after 14 days

[0149] Unexpectedly, when added at the same concentration, in the in vitro assay, the 2 kDa MW SF alone provided improved stabilization of Rhizobium leguminosarum bv. viceae compared to the 40 or 50 kDa MW SF alone treatments.

[0150] In order to determine the effect of processed fibroin having lower molecular weight (kDa), e.g., 2 kDa, on bacterial survival, the in vitro study was repeated using a wider range of silk fibroin “SF” concentrations. The samples in this study also included appropriate blanks for each condition, where everything except the bacteria was added to the well and then treated the same from then on. It was observed that different concentrations of SF would impact the fluorescence intensity in different ways, so it was helpful to make sure that each sample was adjusted to its own individual blank. The data in Table 8 is blank-adjusted.Table 8: Stabilization of Rhizobium leguminosarum bv. viceae with 2 kDa MW SF in vitro

[0151] Table 8 demonstrates the effectiveness of the 2 kDa MW SF treatment on bacterial survival in vitro and also shows an unexpected dose response. The survival trendline is nearly linear from 0% to 10% SF (R-squared is 0.9592). The benefit peaks at 10% SF in this in vitro experiment. Above 10% SF, the stabilization effect begins to decrease again, although it is much higher than the control. This indicates that there may be an optimal concentration of SF with low MW SF (e.g. 2 kDa MW) for this stabilizing effect on Rhizobium leguminosarum bv. viceae. Without being held to theory, it is believed that the interactions between processed fibroin and microbes at the cellular level may be sensitive to the amount of processed fibroin in the surrounding environment in both excess and absence.

[0152] The effect of additives on the 2 kDa MW SF stabilization was also studied in the in vitro assay as shown in Table 9. In column 2, % survival was adjusted for background fluorescence by the same methods described above. Column 3 includes unadjusted values for comparison.Table 9: Stabilization of Rhizobium leguminosarum bv. viceae with 2 kDa MW SF in vitro

[0153] Under the experimental conditions, the addition of trehalose or sucrose at the tested w / v concentrations to 4% 2 kDa MW SF does not appear to improve stabilization of Rhizobium leguminosarum bv. viceae. This surprising result leads to the hypothesis that lower MW SF may stabilize microbes in a different manner as compared to the higher molecular weight SF, replacing or complementing otherwise known stability extending additives.Example 8: On seed stabilization of Rhizobium leguminosarum bv. viceae

[0154] On seed-stabilization experiments were performed as above, comparing the performance of silk fibroin of different molecular weights. 2 kDa MW SF spray-dried powder was dissolved in an aqueous Rhizobium leguminosarum by. viceae suspension to provide a liquid formulation.After the powder was fully dissolved, the coating formulation was applied to and immediately dried on seeds

[0155] In Fig. 7, the data is plotted as % survival compared to control for 0, 7, 14, 21, 28, 35 and 42 days. Table 10 also lists the values for day 21 and day 42. Four different batches of 10% 2 kDa MW SF (shown as Batch A-D), all around the same kDa MW value were prepared in the same way. The data compares % survival on treated seeds as compared to control seeds at each timepoint. At each timepoint, all 2 kDa MW groups are superior to the control. The protection from drying before the time 0 measurement is particularly noticeable. Without being held to theory, it is believed that the effectiveness of lower MW SF (e.g., 2 kDa MW) may derive from a surface interaction with the bacteria itself, or from another physicochemical reaction in the microenvironment of the cells. The stabilization effect of lower MW SF is observed through at least 21 days, confirming the in vitro findings and suggesting that stability can be achieved in unique way(s) using processed fibroin of different MW and with optional additives.Table 10: Stabilization of Rhizobium leguminosarum bv. viceae with 2 kDa MW SF on-seed at 21 days and 42 daysExample 9- Stabilization of Rhizobium leguminosarum bv. viceae with low MW SF and additives in vitro

[0156] To continue to investigate the relationship between bacterial survival and the presence of different additives and stabilizers, an in vitro screen of Rhizobium leguminosarum bv. viceae stability was completed. In this study, 4% w / v silk fibroin at 1.92 kDa was combined with 1% w / v of a stabilizer where the stabilizer was selected from gum arabic, gellan gum, cellulose, oleicacid, lignosulfonate, maltodextrin, chitosan, sodium alginate, carboxymethyl cellulose (CMC), inulin, gelatin, trehalose, glycerol, sorbitol, polyvinyl alcohol (PVA), riboflavin, corn starch, or sucrose. Table 11 lists the percentage survival for samples that could be blank-adjusted.

[0157] Table 11 : Stabilization of Rhizobium leguminosarum bv. viceae with low MW SF and additives in vitro

[0158] The data demonstrates that certain additives can impact the survival of the Rhizobium leguminosarum bv. viceae positively when used in combination with a lower amount of low MW SF. Without being held to theory, the negative values are believed to be due to complete bacteria death as well as some interference from the riboflavin (which has a strong orange coloration). It is believed that combining low MW SF with certain additives could increase stability of microbes in the in vitro assay and in on seed stability. It is to be understood that the additives similar to the ones listed in Table 11 could also provide similar results as the one listed in Table 11 and the list of additives is not comprehensive.

[0159] To investigate this relationship further, an expanded screen was completed. In this study, 2% or 4% w / v silk fibroin at 1.88 kDa was combined with either 1%, 2%, or 5% w / v of either gum arabic, polyvinyl alcohol (PVA), glycerol, or sorbitol. These samples were dried and left for one week at room temperature, then treated as described above. Table 12 lists the percentsurvival for samples that could be blank adjusted. Tables 8 -10 highlight stabilization of Rhizobium leguminosarum bv. viceae with low MW SF in vitro without additives.Table 12: Stabilization of Rhizobium leguminosarum bv. viceae with low MW SF and additives in vitro

[0160] A comparison of Tables 1-11 show even though the absolute numbers for percentage survival may vary depending on the additive or the other variables, however, the trends in stabilization are reproducible.

[0161] The data above demonstrate that different additives may have different impacts on viability in combination with low MW SF. It is clear though that a combination of the low MW SF and additives provide better stability as compared to additives alone. In general, low MW SF exhibited improved Rhizobium leguminosarum bv. viceae stabilization when combined with additives, and did not show reduced viability from the values achieved with the additives alone. In particular, the benefit seen in the sorbitol containing groups demonstrated a clear and unexpected increase in Rhizobium leguminosarum bv. viceae viability beyond values seen with both sorbitol and 2% or 4% w / v low MW SF on their own. Without being held to theory, there may be some beneficial combinations of low MW SF and sorbitol, among other additives, that could lead to an increase in survival, while using reduced amounts of SF and / or additive, which could provide a manufacturing, supply chain, application and commercial advantage.Example 10: On seed stabilization of the gram-negative bacteria - Pseudomonas mosselii

[0162] On seed-stabilization experiments were performed as above in order to determine the performance of low kDa silk fibroin and identify the titration level (% w / v) impact. 2.604 kDa MW SF spray-dried powder was dissolved in an aqueous Pseudomonas mosselii suspension to provide a liquid formulation, resulting in a 0% w / v (control), 6% w / v, 10% w / v and 14% w / v titration level of 2.604 kDa MW fibroin. After the spray-dried powder was fully dissolved for each of the treatments, the coating formulation was applied to and allowed to dry on the soybean seed. The treated seed were stored at 10°C for the duration of the study.

[0163] In Fig. 8 the impact of this low kDa fibroin on on-seed stability with Pseudomonas mosselii is shown. In Table 13, the data is tabulated as recovered viable cfu / seed for 0 (TO), 3 (T3), 6 (T6), and 9 (T9) weeks after treatment. At each timepoint, all 2.604 kDa MW groups are superior to the control. The protection from drying before the time 0 measurement is noticeable. Without being held to theory, it is believed that the effectiveness of lower MW SF (e.g., 2.604 kDa MW) may derive from a surface interaction with the bacteria itself, or from another physicochemical reaction in the microenvironment of the cells. The stabilization effect of lower MW SF is observed through at least 9 weeks, with an optimal concentration identified from the rate range tested of 10% w / v (248% increase in survival vs the control).Table 13: On-Seed Stabilization of Pseudomonas mosselii with a range of concentrations of2.604 kDa MW SF at Time Zero (TO), 3 Weeks (T3), 6 Weeks (T6), and 9 Weeks (T9)

[0164] This data again confirms the in vitro findings being relatable to a very different bacterial genus - suggesting that stability can be achieved in unique ways using different MW kDa processed fibroin. It is to be understood that this and the other examples are illustrative only. Variations in concentrations or other parameters may be made without departing from the scope of the disclosure, and such variations are expected to yield substantially similar results.

[0165] Although the present disclosure has been described with reference to specific embodiments, these embodiments are illustrative only and not limiting. Many other applications and embodiments of the present disclosure will be apparent in light of this disclosure and the following claims.

Claims

CLAIMS1. A formulation comprising effective amounts of a biological agent and processed fibroin, wherein the formulation exhibits enhanced stability of the biological agent compared to a biological agent formulation without processed fibroin under equivalent environmental conditions.

2. The formulation of claim 1, wherein the formulation additionally comprises at least one additive.

3. The formulation of claim 1, wherein the biological agent is selected from a bacterial species, a fungal species, an algal species, a viral species, a protozoal species, an extract or vesicle from the foregoing, a nucleic acid, a polypeptide, or a combination thereof.

4. The formulation of claim 1, wherein the biological agent is a microorganism selected from gram-positive bacteria, gram-negative bacteria, spore-forming bacteria, non-spore forming bacteria, and fungi belonging to the phyla Glomeromycota, Ascomycota, Basidiomycota, and / or Zygomycota.

5. The formulation of claim 1, wherein the biological agent is a microorganism with fungicidal activity, a microorganism with insecticidal activity, a microorganism with nematicidal activity, a microorganism with plant health and / or plant growth regulation activity, or a combination thereof.

6. The formulation of claim 1, wherein the formulation exhibits at least 10% to 50000% or more enhanced stability of the biological agent compared to the biological agent formulation without processed fibroin under equivalent environmental conditions.

7. The formulation of claim 1, wherein the processed fibroin has a molecular weight of from 0.5 to 500 kDa as measured by ultra high pressure liquid chromatography-size exclusion chromatography (UPLC-SEC) using a calibration curve of standards IgG (150 kDa), BSA (66.4 kDa), Myoglobin (17 kDa), and Uracil (112 Da).

8. The formulation of claim 7, wherein the processed fibroin has a molecular weight of less than 55 kDa, or less than 40 kDa, as measured by UPLC-SEC using a calibration curve of standards IgG (150 kDa), BSA (66.4 kDa), Myoglobin (17 kDa), and Uracil (112 Da).

9. The formulation of claim 8, wherein the processed fibroin has a molecular weight of from 30 to 80 kDa as measured by size exclusion chromatography (UPLC-SEC) as measured byUPLC-SEC using a calibration curve of standards IgG (150 kDa), BSA (66.4 kDa), Myoglobin (17 kDa), and Uracil (112 Da).

10. The formulation of claim 1, wherein the processed fibroin has a weight average molecular weight of 10 kDa or less as determined by UPLC-SEC using a calibration curve of standards IgG (150 kDa), BSA (66.4 kDa), Myoglobin (17 kDa), and Uracil (112 Da).

11. The formulation of claim 10, wherein the processed fibroin has a weight average molecular weight of about 0.5 to about 2 kDa as determined by UPLC-SEC using a calibration curve of standards IgG (150 kDa), BSA (66.4 kDa), Myoglobin (17 kDa), and Uracil (112 Da).

12. The formulation of claim 1, wherein the formulation is a liquid formulation and the processed fibroin is present in the formulation in an amount of 0.01 to 50% w / v fibroin.

13. The formulation of claim 1, wherein the formulation is a liquid formulation and the processed fibroin is present in the formulation in an amount of 0.1% to 12% w / v fibroin.

14. The formulation of claim 1, wherein the formulation is a solid formulation and the processed fibroin is present in the formulation in an amount of 0.01 to 99.9% w / w fibroin.

15. The formulation of claim 1, wherein the formulation is a solid formulation and the processed fibroin is present in the formulation in an amount of 50 to 99.9% w / w fibroin.

16. The formulation of claim 1, wherein the processed fibroin has a purity of 10 to 600 mg of lithium per kg of fibroin.

17. The formulation of claim 1, wherein the processed fibroin has a purity of 10 to 600 ppm bromine per mass of fibroin.

18. The formulation of claim 1, wherein the formulation is coated on a seed, and wherein the biological agent is a microorganism present in an amount of 1 - 1x10e9 cfu / seed, preferably lxl0e2 - lxl0e7 cfu / seed.

19. The formulation of claim 2, wherein the additive is a stabilizing additive selected from gum arabic, gellan gum, cellulose, oleic acid, lignosulfonate, maltodextrin, chitosan, sodium alginate, carboxymethyl cellulose (CMC), inulin, gelatin, trehalose, glycerol, sorbitol, polyvinyl alcohol (PVA), riboflavin, corn starch, sucrose, and combinations thereof.

20. The formulation of claim 2, wherein the additive is added in the amount of 0.01-30% w / v .

21. The formulation of claim 2, wherein the additive is present in the amount of 0.05-5% w / v, 0.1-5% w / v, 1-10% w / v, 5-15% w / v, 10-20% w / v, 10-25% w / v, 10-30% w / v.

22. The formulation of claim 1 , wherein the formulation is coated on a surface.

23. The formulation of claim 1, wherein the processed fibroin is prepared from silk fibroin by: preparing an aqueous silk fibroin solution having a concentration of greater than or equal to5% w / v silk fibroin from the silk fibroin preparation, wherein the silk fibroin preparation and aqueous solution comprise a chaotropic salt; and exchanging the chaotropic salt from the aqueous silk fibroin solution by continuous diafiltration by tangential flow filtration (TFF) to prepare purified silk fibroin, wherein TFF is done with a solution having a pH below the isoelectric point of the silk fibroin, wherein the pH is between 2 and 5 and the pH is maintained between 2 and 5 during at least a portion of the TFF diafiltration; wherein the chaotropic salt is LiBr, wherein the silk fibroin preparation was prepared by a process comprising dissolving degummed silk fibroin fibers in 5 M to 13 M LiBr, wherein the purified silk fibroin comprises 10 to 600 mg lithium per kg silk fibroin as determined by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS), and wherein the only solvent in the method of purifying is water.

24. A method of preparing a formulation comprising: a) providing an effective amount of a biological agent; b) combining the biological agent with an effective amount of processed fibroin, and optionally at least one additive.

25. The method of claim 24, wherein in step (b) the biological agent is suspended in a solution of the processed fibroin.

26. The method of claim 24, wherein in step (b) the biological agent is combined with the processed fibroin by spray drying the effective amount of the processed fibroin on the biological agent.

27. The method of claim 24, wherein the biological agent is a microorganism selected from gram-positive bacteria, gram-negative bacteria, spore-forming bacteria, non-spore forming bacteria, and fungi belonging to the phyla Glomeromycota, Ascomycota, Basidiomycota, and / or Zygomycota.

28. A method of protecting a seed comprising:(a) providing an agriculturally acceptable formulation, the agriculturally acceptable formulation comprising an effective amount of a biological agent and processed fibroin; and(b) applying the agriculturally acceptable formulation to the seed in an agriculturally effective amount.

29. The method of claim 28, wherein the biological agent is an agriculturally beneficial microorganism.

30. The method of claim 28, wherein the formulation additionally comprises at least one additive.

31. The method of claim 30, wherein the additive is selected from gum arabic, gellan gum, cellulose, oleic acid, lignosulfonate, maltodextrin, chitosan, sodium alginate, carboxymethyl cellulose (CMC), inulin, gelatin, trehalose, glycerol, sorbitol, polyvinyl alcohol (PVA), riboflavin, corn starch, sucrose, and combinations thereof.

32. The method of claim 31, wherein the agriculturally acceptable formulation is a liquid formulation and the additive is present in the amount of 0.01-30% w / v.

33. The method of claim 28, wherein the method additionally comprises applying a pesticide, a seed coating, a seed colorant, seed applied treatment, or a combination thereof.

34. The method of claim 28, wherein the biological agent is a microorganism selected from gram-positive bacteria, gram-negative bacteria, spore-forming bacteria, non-spore forming bacteria, and fungi belonging to the phyla; Glomeromycota, Ascomycota, Basidiomycota, and / or Zygomycota..

35. The formulation of claim 1, wherein the microorganism is selected from Rhizobium (including R. leguminosarum - symbiovars), Bradyrhizobium (including B. japonicum, B. elkanii and B. arachis), Beauveria bassiana, Methylobacterium (including Methylorubrum extorquens), Pseudomonas including P. mosselii, and Bacillus including B. firmus, B. thuringiensis, B. subtilis, B. amyloliquefaciens) .

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