The present invention relates to a method for encapsulation of microparticles (e.g., fungal conidia), involving (i) suspending microparticles in an aqueous solution containing at least one sugar to form an aqueous suspension wherein the concentration of said sugar is about 0.1% to about 10% w/v, and (ii) spray drying said aqueous suspension with a spray dryer, wherein the inlet temperature of said spray dryer is about 40° C. to about 140° C. and the outlet temperature of said spray dryer is about 20° C. to about 80° C.
Trichoderma spp. are able to control root and foliar pathogens of plants through mycoparasitism, nutrient competition and rhizosphere competence, enzymes, induced defense responses in plants, and metabolism of germination stimulants (Howell, C. R., Plant Disease, 87: 4-10 (2003)). Recent studies by Advanced Biological Marketing, Inc. (ABM) in Van Wert, Ohio and Cornell University showed that Trichoderma strains may induce changes in the micro floral composition on roots, enhance nutrient uptake, stabilize soil nutrients, promote root development, and increase root hair formation (Harman, G. E., Phytopathology, 96: 190-194 (2006)). Trichoderma spp. produces three kinds of propagules (or structures that can give rise to a new individual organism): hyphae, chlamydospores, and conidia (Papavizas, G. C., Annual Review of Phytopathology, 23: 23-54 (1985)). Biomass that contains hyphae as the main propagules of Trichoderma spp. cannot withstand drying and loses viability during dehydration. Conidia can be produced abundantly in a short period of time, and therefore have been used as the active ingredient in most Trichoderma spp. based products. Efforts have been made to produce chlamydospores and desiccation tolerant conidia from liquid fermentation (Harman, G. E., et al., Biological Control, 1:23-28 (1991); Jin, X., et al., Biological Control, 1: 237-243 (1991); Jin, X., et al., Biological Control, 7: 267-274 (1996); Eyal, J., et al., Journal of Industrial Microbiology & Biotechnology, 19:163-168 (1997)). Companies engaged in the development of Trichoderma conidia based products are usually small and lack the financial support to build up large liquid fermentation facilities. Trichoderma conidia are currently produced by two-phase solid fermentation systems. Inoculum is produced by liquid culturing and then transferred to a solid matrix for aerial conidial production by solid fermentation. Aerial conidia of T. harzianum are hydrophilic in nature and it is difficult to separate them from the solid substrate by sieving. Washing conidia off the solid fermentation substrate and then centrifuging the suspension has become the choice for harvesting. Conidia produced by either liquid fermentation or two-phase solid fermentation must be dried to prevent spoilage by microbial contamination and to induce dormancy for formulation development and shelf-life (Jin, X., et al., Principles in the development of biological control systems employing Trichoderma species against soil-born plant pathogenic fungi, In: Leatham, G. F. (Ed.), “Frontiers in industrial mycology”, 1992, Chapman & Hall, Inc., New York, N.Y., pp. 174-195). Drying the conidial pastes of Trichoderma spp. in large scale production remains a major hurdle because conidia lose viability during the drying process at elevated temperatures.
There are three drying technologies employed most often by industry: fluid bed-, freeze-, and spray-drying. Freeze drying has been used to preserve microorganisms for decades and is the preferred method for keeping culture collections worldwide (Morgan, C. A., et al., Journal of Microbiological Methods, 66: 183-193 (2006)). The disadvantages of freeze-drying are cross contamination, viability loss, and high costs of processing. Furthermore, our experience showed that it was difficult to break freeze dried cakes of Trichoderma conidia, and the conidia lost viability because of the heat generated by milling. Fluid bed drying is usually used to process relatively large particles for glomeration and particle coating. A contact-sorption drying method was developed to dry Penicillium bilaii in a fluidized bed dryer (Tadayyon, A., and G. A. Hill, Journal of Chemical Technology and Biotechnology, 68: 277-282 (1997)). Fungal spores in a liquid suspension were injected into the dryer using an air-shear atomizer from the top of the dryer. Instant skim milk powder was fluidized as a protecting agent, moisture sorbent, and carrier. The resulting product survived well in refrigeration for 3 months, but only survived several days under room temperatures. Spray-drying is mostly used in the dairy industry because of the relatively low cost compared to freeze-drying (Morgan et al., 2006). Spray-drying was used to process several fungal conidia, including conidia of Trichoderma harzianum, without success (Larena, I., et al., Journal of Applied Microbiology, 94: 508-514 (2003); Guijarro, B., et al., Biocontrol Science and Technology, 16: 257-269 (2006); personal communication with Dan Custis and Gary Harman, 2009). Comparison of drying methods were conducted to study the effects of freeze-, spray-, and fluid bed-drying on conidia viability of Epicoccum nigrum and Penicillium frequentans, biological control agents against Monilinia spp. that cause peach brown rot disease, and P. oxalium for the control of Fusarium wilt of tomatoes (Larena, I., et al., Journal of Applied Microbiology, 94: 508-514 (2003); Larena, I., et al., Journal of Phytopathology, 151: 600-606 (2003); Guijarro, B., et al., Biocontrol Science and Technology, 16: 257-269 (2006)). Spray drying resulted in considerable loss of conidial viability. Comparison of drying technologies was also conducted in drying conidia of entomopathogenic fungi Beauveria brongniartii and Metarhizium anisopliae (Horaczek, A., and H. Viernstein, Biological Control, 31: 65-71 (2004)). Spray drying caused severe damage to the conidia and resulted in low viability and prolonged germination.
Microencapsulation can enhance the activity of some biological control agents and protect them from adverse conditions in preparing bioherbicide inocula (Winder, R. S., et al., Biocontrol Science and Technology, 13:155-169 (2003)). Attempts have been made in microencapsulation of nuclear polyhedrosis virus, Bacillus thurigenisis, and hydrophobic aerial conidia of Metarhizium anisopliae and Beauveria bassiana, which are used in biological control of insects (U.S. Pat. No. 4,948,586; Horaczek and Viernstein, 2004; Liu, C. P., and S. D. Liu, Journal of Microencapsulation, 26: 377-384 (2009); Liu, C. P., and S. D. Liu, Drying Technology, 27: 747-753 (2009)). An encapsulation method was also developed for either ascospores or conidia of potential biological control agents, including Talaromyces flavus, Gliocladium virens, Pennicillum oxalicum, and Trichoderma viride (Fravel, D. R., et al., Phytopathology, 75: 774-777 (1985)). Conidia were encapsulated in an alginate-clay matrix and were dried at room temperature. New systems of encapsulating Trichoderma spp. have been developed and applied to several Trichoderma species and strains (Cho, C. F., and W. C. Lee, Journal of Bioscience and Bioengineering, 87: 822-824 (1999); Mafia, R. G., et al., Fitopatologia brasileira, 28: 101-105 (2003); El-Katatny, M. H., et al., Food Technology and Biotechnology, 41: 219-225 (2003)). Trichoderma mycelium and conidia were encapsulated in pellets containing gluten, wheat bran, rice husk, oak bran, maize meal, or chitin as nutrient base. However, the density of conidia in the alginate pellets is low, and the shelf life is not enough to support commercialization of Trichoderma based products. Furthermore, alginate encapsulation is difficult to scale up because it is labor intensive, and air drying pellets at room temperatures takes a long time, requires a large space, and easily gets contaminated. An encapsulation technology was developed using water insoluble absorbents to remove the water gently from the conidial biomass at room temperature and to encapsulate the conidia (U.S. Patent Application Publication No. PCT/US2006/034744 (2006)). The water insoluble, water-absorbent substances can be any organic or inorganic material capable of removing moisture gently from the suspension of Trichoderma conidia. The disadvantage is that the final product contains 80-90% of water insoluble, water-absorbent substances which result in blocked pipelines in field applications (Custis, 2007, personal communication). It is ideal that Trichoderma formulations should contain 5×109 cfu/g to be effective in a variety of applications (U.S. Patent Application Publication No. PCT/US2006/034744 (2006)). Conidial density or quantity in the dried formulation of Trichoderma is critical for field application because higher conidial density in a formulation should result in better efficacy. To achieve this conidial density in any formulation, efforts must be made to provide technical powders that contain at least 90% of pure conidia.
We have developed a method for the microencapsulation of conidia (e.g., T. harzianum) through spray-drying which significantly improved the survival percentages of conidia and resulted in a high concentration of viable conidia in the final product after drying.