JPWO2019067379A5 - - Google Patents

Description

In one embodiment, the preparation of a liquid microbial-based product increases the concentration of microorganisms to 1 billion propagules per milliliter, optionally adding additives, preservatives and/or pH modifiers. further comprising adding The "off-the-shelf" liquid product is filled into containers (eg, one-gallon containers), sealed, and labeled for a variety of uses, including commercial settings.

In one embodiment, the preparation of a liquid microbial-based product increases the concentration of microorganisms to 1 billion propagules per milliliter, optionally adding additives, preservatives and/or pH modifiers. further comprising adding The "off-the-shelf" liquid product is filled into containers (eg, one-gallon containers), sealed, and labeled for a variety of uses, including commercial settings.

After the culture process is finished, the temperature of the incubator is raised to about 40° C., dry air is replenished, moist air is exhausted, and drying is performed for about 3-4 days. The dried microbial-based product is ground, milled, and micronized to the desired particle size. Propagule concentration should be at least 1×10 ⁹ conidia per gram of dry product, with 1×10 ¹⁰ , 1×10 ¹¹ , 1×10 ¹² and even 1×10 ¹³ propagules per gram. do.

Example 3 - Conidiogenesis in Cultures Conidia are harvested from biologically pure cultures of Trichoderma harzianum grown in reactors. The nutrient medium composition consisted of glucose (30 g/L), yeast extract (2.8 g/L), _{KH2PO4 (} 1.0 g/L), _MgSO4 . _7H2O (0.5 g/L), KCl (0.5 g/L), _FeSO4 . _7H2O (0.01 g/L), _ZnSO4 . _7H2O (0.01 g/L), _CuSO4 . Contains _5H2O (0.005 g/L). The initial pH of the culture is 5.5 and the temperature is 25-28°C. The culture volume is approximately 100 gallons. After 5 days of culture, the yield exceeds about 5×10 ⁸ to 5×10 ⁹ conidia per milliliter of culture.

Yield from one tray is about 652.60 grams before drying and processing. After completion of the culture treatment, the temperature of the incubator is increased to about 40° C., dry air is replenished, moist air is exhausted, and drying is performed for about 3 to about 4 days. After drying and thorough milling, over 4 pounds of Trichoderma product is produced per tray. The dried microbial-based product is ground, milled and micronized to the desired particle size and mixed with dry diatomaceous earth and commercial compost to redistribute residual moisture and standardize the final product. Propagule concentrations should not be less than about 1×10 ⁶ , preferably less than 1×10 ⁹ conidia per gram of dry product.

Claims (19)

A method of producing a liquid form of a Trichoderma-based product and a solid state of a Trichoderma-based product, comprising:
(a) preparing an alginate agar bead inoculum from a Trichoderma sp. culture;
(b) culturing said alginate agar bead inoculum in a nutrient broth culture medium in a reactor to a desired scaled-up microbial density in said alginate agar bead inoculum , said desired scaled-up microbial density being said at least 1×10 ⁹ conidia per gram of alginate agar bead inoculum ;
(c) harvesting said alginate agar bead inoculum from said nutrient broth culture medium;
(d) using said cultured alginate agar bead inoculum to inoculate a submerged fermentation reactor to prepare said liquid form Trichoderma-based product, and using said cultured alginate agar bead inoculum to prepare a solid state fermentation reactor; to prepare said solid state Trichoderma-based product;
A method that includes steps . 2. The method of claim 1, wherein said Trichoderma microorganism is selected from the group consisting of Trichoderma reesei , Trichoderma harzianum , Trichoderma viride and Trichoderma hamatum . The step (a) includes mixing a 5% homogeneous seed culture slurry of the seed culture with sterile liquid nutrient medium, 1% agar , and 2% sodium alginate to form an inoculum solution, and a drip shower device. depositing droplets of said inoculum solution into a mixing vessel containing 1% calcium chloride, said droplets comprising alginate agar beads comprising nutrient components and microbial particles embedded in alginate agar masses using 2. The method of claim 1, forming an inoculum. The step (b) collects the alginate agar bead inoculum and a sufficient volume of the nutrient broth culture medium to disperse a high concentration of Trichoderma mycelium within and throughout the surface of the alginate agar bead inoculum. 2. The method of claim 1, comprising culturing in a reactor comprising 2. The method according to claim 1, wherein said step (b) is carried out under continuous aeration agitation at a temperature of about 28° C. to about 30° C. and a pH of about 5.0 to about 6.5 for about 1 to about 10 days. described method. After said step (c) and before said step (d), treating said alginate agar bead inoculum for storage, said treating comprising collecting said alginate agar bead inoculum from said reactor and removing said alginate . 2. The method of claim 1, comprising placing the agar bead inoculum in a cryopreservation solution comprising about 50% glycerol and water. 7. The method of claim 6 , wherein said cryopreservation solution is in a container selected from the group consisting of flasks, tubes, vials and dishes. 8. The method of claim 7 , wherein the container contains up to 50 alginate agar bead inoculants . The method of claim 7 , wherein the container is placed in a freezer at a temperature of -80°C to -10°C. The method of claim 7 , wherein the container is placed in a refrigerator at a temperature of -10°C to 4°C. In step (d), preparing the liquid form of the Trichoderma -based product comprises: about 2. The method of claim 1, comprising culturing said alginate agar bead inoculum at a temperature of 28°C to about 30°C and a pH of about 5.0 to about 6.5. In step (d), preparing the liquid form of the Trichoderma-based product comprises increasing the concentration of the Trichoderma to at least 1×10 ⁹ per milliliter propagule with the nutrient broth culture medium; 12. The method of claim 11 , optionally comprising adding preservatives, additives and/or pH modifiers. In step (d), preparing the solid state Trichoderma-based product comprises mixing the harvested alginate agar bead inoculum with a nutrient broth medium and a solid or semi-solid substrate to form a mixture. , spreading said mixture on a heated proof tray and culturing said mixture in said tray in an incubator at 30° C. for 3-10 days with constant aeration of ambient air. 14. The method of claim 13 , wherein said incubator is a proofing oven. 14. The method of claim 13 , wherein the substrate is vermiculite and the ratio of vermiculite to alginate agar bead inoculum in the mixture ranges from 3:1 to 4:1. 14. The method of claim 13 , wherein said substrate is a food product selected from the group consisting of corn flour, rice, beans and pasta. increasing the temperature of the incubator to about 40° C., replenishing dry air, evacuating moist air, and drying the trays for about 3 to about 4 days to produce a dry product; 14. The method of claim 13 , further comprising grinding the material with a grinder to a desired particle size and mixing the ground dry product with dry diatomaceous earth and commercial compost. 18. The method of claim 17 , further comprising packing the dry product mixed with diatomaceous earth and commercial compost into commercial sealed bags. 19. The method of claim 18 , wherein the concentration of Trichoderma in the dry product after mixing with the diatomaceous earth and commercial compost is 1 x 10< ⁶ > or more conidia per gram.