WO1995025163A1 - Procedes de production de spores fongiques et compositions contenant ces dernieres - Google Patents

Procedes de production de spores fongiques et compositions contenant ces dernieres Download PDF

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Publication number
WO1995025163A1
WO1995025163A1 PCT/CA1995/000094 CA9500094W WO9525163A1 WO 1995025163 A1 WO1995025163 A1 WO 1995025163A1 CA 9500094 W CA9500094 W CA 9500094W WO 9525163 A1 WO9525163 A1 WO 9525163A1
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primary
phase
spores
phase fermentation
fermentation
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PCT/CA1995/000094
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English (en)
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Robert Duncan Carmichael
Catherine Brigetta Kuiack
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Philom Bios Inc.
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Priority to AU17039/95A priority Critical patent/AU1703995A/en
Publication of WO1995025163A1 publication Critical patent/WO1995025163A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N3/00—Spore forming or isolating processes

Definitions

  • Types of biological input products which can be formulated with the fungal spore active ingredients include plant fertility products such as fertilizers and supplements, and pest control products such as mycoherbicides, mycoinsecticides and mycofungicides.
  • the prior art teaches that the final products of the various single-phase liquid fermentation processes, i.e., a mixture of mycelia and spores referred to as fungal propagules, are not separated but rather, are used directly in selected applications or are incorporated into various compositions. Analyses of the prior art suggest that the disclosed processes are typically expensive, provide low productivities with poor stability of the final products and consequently, have poor commercial potential and limited application.
  • Walker, U.S. Patent 4,419,120, and Tabachnik, U.S. Patent 4,837,155 teach that processes disclosedly previously for the production of fungal propagules are laboratory processes and do not produce sufficient amounts of material in large-enough volumes in short-enough time to be commercially viable.
  • Walker, U.S. Patent 4,419,120, and Tabachnik, U.S. Patent 4,837,155 teach individually and separately, that economic large-scale production of fungal propagules is based on single-phase liquid fermentation processes which may, but not necessarily consist of multiple stages. Fungal propagules produced by previously disclosed processes tend to be comprised of mixtures containing mycelia and spores, and tend to be very unstable and have very short shelf lives.
  • This invention relates to the discovery of a process which enables the production of very large quantities of fungal spores in submerged liquid fermentations.
  • This process comprises the simultaneous manipulation and maintenance of three key components of fermentations i.e., (a) the carbon: nitrogen ratios of the media, (b) the growth-rate-limiting concentrations of the nutrient components of liquid fermentation media and/or adjustment of the physical fermentation parameters such as aeration or agitation so that the fungal growth rates are affected, and (c) the addition or deletion of sporulation inhibiting or inducing agents into the media, such that two distinct phases of fungal growth occur, i.e., first the production of mycelial biomass and then, sporulation and spore production.
  • These three fermentation components may be manipulated individually or in combination at each stage of the fermentation process so that the onset and duration of the two fungal growth phases are precisely controlled through out the duration of the fermentation process.
  • This process comprises preparing a fungal inoculum stock which is used to inoculate a fermenter vessel containing a nutrient-balanced liquid medium with a low carbon:nitrogen nutrient ratio.
  • a fermentation is conducted in this vessel to produce primarily mycelial biomass. If more biomass is desired, then the mycelial culture produced in the first fermentation can be serially transferred to one or more larger fermenters containing the low carbon:nitrogen ratio liquid medium.
  • the mycelial biomass is transferred to a final fermenter vessel containing a liquid medium with an altered carbon:nitrogen nutrient ratio, said carbon:nitrogen ratio altered such that further mycelial production is limited, but such that spore production is stimulated and optimized.
  • a further fermentation is conducted in the final fermenter to produce the desired spores.
  • the final product consisting of mycelial biomass and spores, is removed from the vessel and the spores are separated from the mycelial biomass.
  • the resulting spore suspension is concentrated into a slurry.
  • the spore slurry can be used as an active ingredient for compositions comprised of liquids, powders or granules. Alternatively, the spore slurry can be dried prior to its use as an active ingredient in compositions.
  • the fermentation medium used during the first phase of fungal growth i.e., production of mycelial biomass
  • the low carbon:nitrogen ratio medium may also contain sporulation-suppressing medium components to further minimize sporulation during the phase of mycelial growth and development .
  • the fermentation medium used during the second phase of fungal growth i.e., sporulation, has an altered carbon:nitrogen ratio relative to the medium used for mycelial growth, and does not contain any sporulation- inhibiting medium components but rather, may contain sporulation inducing medium components.
  • the spores produced during the final fermentation are preferably separated from the mycelial biomass by screening, straining, or sieving.
  • the resulting spore suspension is preferably concentrated into a slurry by centrifugation or filtration, for subsequent processing.
  • the spore slurry may be then packaged in a water- and gas-impermeable container, or added as an active ingredient into commercially useful compositions.
  • the spores may be stabilized by the addition of a stabilizing agent to the spore slurry prior to packaging or use as an active ingredient in compositions. For optimum stability of commercial compositions, it is preferable to add a stabilizing agent to the spore slurry prior to preparing the compositions.
  • the spore slurry may be dried, preferably by spray-drying, freeze-drying, or air-drying.
  • the spores are preferably stabilized prior to drying by the addition of a stabilizing agent to the spore slurry or alternatively, by adjusting the relative humidity of the dried spores to a constant, preferably in a range between 12% to 33% moisture content.
  • a stabilizing agent it is preferable to add to the spore slurry prior to drying, or to adjust the relative humidity of the dried spores to a constant in the range of 12% to 33% moisture content.
  • compositions which are comprised of fungal spores as the active ingredients in the form of spore slurries, and carriers thereof.
  • compositions containing concentrated spore slurries, and compositions containing dried, stabilized spores are preferably packaged in sealed water- and gas-impermeable containers, and preferably stored frozen.
  • compositions comprised of spore slurries and powders, and spore slurries and granules, are preferably packaged in sealed water-impermeable containers, and can be stored at temperatures in the range of -85C to 25C.
  • single-phase fermentation refers to fermentation processes in which fungal propagules consisting of mixtures of mycelia and spores, ' are produced by growing the cultures in a liquid medium until the nutrient components are exhausted such that no further growth occurs.
  • This term refers to single-phase fermentations which are harvested after one complete growth period in a fermenter vessel, and also to cultures which are produced by expansion through serial transfers from a nutrient-exhausted vessel into a larger vessel containing fresh liquid medium.
  • two-phase fermentation process refers to all fermentation processes wherein the manipulation of the carbon:nitrogen ratio and the growth- rate-limiting nutrient concentration of the fermentation medium enables precise control over the onset and duration of mycelial biomass growth and development, and over the stimulation and optimization of sporulation.
  • primary-phase fermentation refers to the production of primarily mycelial biomass in a fermenter vessel containing a nutrient-balanced low carbon:nitrogen ratio liquid medium such that carbon sources are completely depleted from the medium before the nitrogen sources are exhausted.
  • multi-stage refers to serial transfers of mycelial biomass produced during primary-phase fermentation, to successively larger fermenter vessels, so that large quantities of mycelial biomass are produced.
  • final-phase fermentation refers to the production of large quantities of spores in a fermenter vessels containing a nutrient-balanced liquid medium containing a balanced or high carbon:nitrogen ratio, such that the nitrogen sources are depleted at the same rate or more quickly, than the carbon sources.
  • C:N ratios are used to define and describe the nutrient availability in fermentation media, and are typically referred to as "high”, “low”, or “balanced”. For example, growth of a fungal culture in a fermentation medium with a “high” C:N ratio will result in a residual level of carbon remaining in the medium after the nitrogen sources have been completely exhausted. In a “low” C:N ratio medium, excess nitrogen will remain in the medium after the microbial culture has completely depleted the carbon sources. In a “balanced” C:N ratio, both carbon and nitrogen sources are depleted simultaneously.
  • the growth rate of a microbial culture in a fermentation medium is primarily determined by nutrient concentration. When all required nutrients are available in excess, fungal growth occurs at a maximal rate. However, when the supply of an essential nutrient (e.g., carbon) is limiting fungal growth to less than the maximal rate, that nutrient, e.g., carbon, is referred to as the growth-rate-limiting nutrient.
  • the primary carbon sources in fermentation media are from sugars or other carbohydrates .
  • the primary carbon sources typically do not contain nitrogen.
  • Nitrogen sources are typically supplied as "defined” compounds comprised of nitrate or ammonium ions, organic compounds such as amino acids, or complex materials such as yeast extract. Although, some of the primary nitrogen sources may contain carbon, the C:N ratio of the nitrogen compounds is typically very low.
  • Cgm Colletotrichum gloeosporioides f.sp. malvae
  • the medium is C:N balanced when the ratio of sucrose:yeast extract is 6:1 (mass:mass) .
  • the growth rate of Cgm will be at a maximum when the concentrations of sucrose and yeast extract are at the 6:1 ratio, e.g., 10 g/L sucrose and 1.67 g/L yeast extract.
  • a C:N ratio less than 6:1 (e.g., 5.5:1) will result in the medium defined as a "low C:N medium” for Cgm, even if 55 g/L sucrose and 10 g/L yeast extract are supplied, because residual nitrogen would remain in the medium after the carbon is depleted.
  • a C:N ratio greater than 6:1 e.g., 6.5:1 would be considered a "high” C:N medium for Cgm.
  • a liquid fermentation medium for Fusarium heterosporum is considered “balanced” , when the C:N ratio is 14:1., i.e., 10.5 g/L glucose and 0.75 g/L yeast extract .
  • a key feature of our invention is the precise control during the fermentation process, over the physiological change of the fungal culture from mycelial biomass production to spore production. It should be noted that in order for fermentation production of fungal spores to be commercially viable, a number of transfers of the mycelial biomass, while it is in a state of vegetative growth, to successively larger fermenter vessels is required in order to achieve the necessary volume of mycelial biomass needed to produce the target spore yields in the final fermentation step.
  • a low C:N ratio fermentation medium is preferentially used to produce large quantities of mycelial biomass.
  • sporulation will be suppressed by the residual nitrogen remaining in the exhausted low C:N ratio medium.
  • Fungal sporulation processes are triggered by transferring the mycelial biomass into a fermenter vessel containing an enriched liquid medium with a high C:N ratio to ensure that high levels of carbon are available for further enabling sporulation to continue after the nitrogen sources are exhausted.
  • a further discovery according to this invention is that it is also possible to further increase spore yields in the final-phase fermentation by raising the C:N ratio in the last stage of the primary- phase fermentation.
  • a sporulation inhibitor must be included with the high C:N ratio medium in this step to enable maximal mycelial biomass yields while minimizing sporulation.
  • the large-scale production of fungal spores includes a multi-staged two-phase liquid fermentation process.
  • the first phase i.e., the primary phase
  • the mycelial biomass and any spores produced during the primary-phase fermentations are not recovered, but are used to inoculate the next stage of primary-phase fermentation.
  • the final phase of production i.e., final-phase fermentation, is where the spores to be used as the active ingredient of commercial compositions, are produced and subsequently harvested.
  • the production process is preferably initiated by using a fungal spore stock to inoculate agar plates, shake flasks or fermenter vessels as appropriate.
  • Fungal spore stocks are produced on a suitable solid agar medium such as sucrose-yeast-extract agar (SYE) or potato dextrose agar (PDA) .
  • SYE sucrose-yeast-extract agar
  • PDA potato dextrose agar
  • the spores are harvested by flooding the surfaces of the agar medium with sterile distilled water and gently agitating the culture.
  • the resulting spore suspension is quickly removed from the agar medium, suspended in a sterile glycerol solution and either used immediately, or stored at -40C to -85C.
  • Fungal inoculum stocks may also be produced from fungal cultures grown on suitable agar media by excising part or all of the culture, which may contain mycelia or mycelia plus spores, from the media, and then homogenizing the excised culture in sterile liquid medium.
  • the second type of fungal inoculum stock i.e., prepared from mycelia or mycelia plus spores, is used immediately after preparation.
  • the primary-phase fermentation is initiated by inoculating an appropriate fungal stock into a liquid medium which is nutrient-balanced but contains a low C:N ratio.
  • a low C:N ratio medium for C ⁇ lletotrichum sp . such as Colletotrichum gloeosporioides f.sp. alvae ( Cgm) could contain, but is not restricted to, sucrose (10 g/L) , yeast extract (5 g/L) , mono- potassium phosphate (5 g/L) , ammonium sulfate (10 g/L) with the pH adjusted to 6.
  • the ammonium sulfate will inhibit sporulation by Cgm mycelia if the fermentation in any primary-phase vessel is allowed to continue after the carbon sources are exhausted.
  • An example of a low C:N ratio primary-phase fermenter medium for Penicillium sp . such as Penicillium bilaii may contain, but is not restricted to, glucose (20 g/L) , tryptone (10 g/L) , malt extract (10 g/L) , KH 2 P0 4 (5 g/L) with the pH adjusted to 6.
  • the primary-phase fermentations are performed under controlled conditions which may include, but are not restricted to, airflow between 0.9 - 30 1pm, agitation between 50 - 800 rp , temperature maintained between 18 - 35C, pH maintained at 6.0 + . 0.5, back-pressure maintained between 0 - 0.5 bar, dissolved oxygen levels maintained above 50%.
  • Culture foaming can be controlled by the addition of sterilized commercial anti-foaming agent (e.g., 10% Sigma Emulsion A) .
  • Mycelial biomass production during primary-phase fermentation can be greatly increased by incorporating multiple stages of primary-phase fermentation.
  • the mycelial biomass produced during the first primary-phase fermentation (i.e., stage 1) , is aseptically transferred into a stage-2 fermenter vessel containing the low C:N ratio liquid medium, with the stage-2 vessel being 10 times larger than the stage-1 vessel.
  • stage 1 the mycelial biomass produced during the stage-2 primary- phase fermentation
  • stage-3 the stage-3 vessel being 10 times larger than the stage-2 vessel.
  • the primary-phase fermentations are continued until sufficient mycelial biomass has been generated to inoculate a final-phase fermenter vessel which is typically 10 times larger than the immediately preceding primary-phase vessel.
  • the time-period required for completion of primary-phase fermentation will range between 2 - 14 days, depending on the number of transfers to larger vessels required to generate the preferred colume of mycelial biomass required to transfer into the final-phase fermenter vessel.
  • a final-phase fermentation is initiated by inoculation of the final-phase liquid medium with the preferred volume of primary-phase culture.
  • the final- phase medium has an altered C:N ratio relative to the primary-phase medium and does not contain sporulation- suppressing compounds, such that sporulation and spore production are optimized.
  • a final-phase medium for Colletotrichum sp may contain, but is not restricted to, sucrose (5-30 g/L) , yeast extract (0-5 g/L) , mono- potassium phosphate (1-5 g/L) , tryptic soy broth (0 - 30 g/L) .
  • An example of a final-phase medium for Penicillium sp An example of a final-phase medium for Penicillium sp .
  • Antibiotic compounds may be added to the primary- phase and final-phase media to ensure that the biological purity of the fungal cultures is maintained during the fermentation process.
  • Penstrep Sigma cat. no. P0906
  • the media contains 50 UI/mL penicillin and 0.05 mg/mL streptomycin.
  • the final-phase fermentations are performed under controlled conditions for airflow, agitation, temperature, pH, back-pressure, and dissolved oxygen which may include, but are not restricted to, airflow between 0.9 - 40 1pm, agitation between 50 - 800 rpm, temperature maintained between 10 - 35C, pH maintained between 4 - 7, back ⁇ pressure maintained between 0 - 0.5 bar, dissolved oxygen levels maintained above 50%.
  • Culture foaming can be controlled by the addition of sterilized commercial anti- foaming agent (e.g., 10% Sigma Emulsion A) .
  • the final product of the final-phase fermentation consists of mycelial biomass and spores, and typically occurs within 60 - 72 hours, with spore yields in the range of 2 X 10 7 to 5 X 10 8 spores/mL of culture.
  • the spores are separated from the sieving, straining, or screening.
  • the final fermentation product consisting of mycelia and spores may be homogenized, then centrifuged to remove the mycelial debris from the spores .
  • the resulting spore suspension is concentrated into a slurry by centrifugation or filtration.
  • the concentrated spore slurry may be directly incorporated into liquid, powder or granular compositions which are then packaged in water- and gas-impermeable containers.
  • the spore slurry may be stabilized by the addition of a stabilizing compound such as glycerol, prior to incorporation into compositions which are then packaged in water- and gas-impermeable containers.
  • the spore slurry may also be dried, preferably using a process such as spray-drying, freeze-drying or air-drying, and then incorporated into liquid, powder or granular compositions which are then packaged in water- and gas-impermeable containers.
  • Dried spores may be stabilized by adjusting their final moisture content to a constant in the range of 12 - 33%, and incorporated into liquid, wettable powder or granular compositions which are then packaged in water- and gas-impermeable containers.
  • the following example is of a small-scale, three- stage, two-phase fermentation process for the production of Colletotrichum gloeosporioides f.sp. malvae [ Cgm) spores.
  • stage-1 primary- phase mycelial biomass cultures were pooled and transferred into a vessel containing 16 L of the Cgm primary-phase fermentation medium and grown under controlled conditions (stage 2) .
  • stage-2 primary-phase mycelial biomass culture was transferred into a vessel containing 170 L of Cgm final- phase fermenter medium which consisted of sucrose (30 g/L) , yeast extract (5 g/L) , mono-potassium phosphate (5 g/L) , with the pH adjusted to 6 (stage 1) , and then grown under controlled conditions (stage 3) .
  • the titre of spores produced was 3 X 10 7 spores/mL.
  • the spores produced were separated from the mycelial biomass by sieving.
  • the resulting spore suspension was concentrated by centrifugation.
  • the resulting concentrated spore slurry was air-dried in trays.
  • the viability of the dried spores was 2.0 X 10 9 cfu/g (i.e., colony-forming units per gram of product) .
  • a Cgm production inoculum stock culture was plated onto SYE agar and grown at room temperature for 6 days. Spores produced .were suspended in sterile culture medium and transferred into 5 vessels, each containing 150 mL of Cgm primary-phase fermentation medium which consisted of sucrose (10 g/L) , yeast extract (5 g/L) , mono-potassium phosphate (5 g/L) , ammonium sulfate (10 g/L) with the pH adjusted to 6 (stage 1) . After 3-days' growth in the stage-1 vessels, the stage-1 primary-phase Cgm mycelial biomass cultures were pooled and transferred into a vessel containing 16 L of the primary-phase fermentation medium and then grown under controlled conditions (stage 2) . After 2-days' growth in the stage-2 vessel, the Cgm mycelial biomass was transferred into a vessel containing 170 L of the primary-phase fermentation medium and then grown under controlled conditions (stage 3) . (B) Final-phase fermentation:
  • stage-3 primary-phase Cgm mycelial biomass culture was transferred into a vessel containing 1000 L of Cgm final- phase fermenter medium which consisted of sucrose (30 g/L) , yeast extract (5 g/L) , mono-potassium phosphate (5 g/L) , with the pH adjusted to 6 and then grown under controlled conditions (stage 4) .
  • Cgm final- phase fermenter medium which consisted of sucrose (30 g/L) , yeast extract (5 g/L) , mono-potassium phosphate (5 g/L)
  • stage 4 the titre of Cgm spores produced was 2.2 X 10 7 spores/ml.
  • the following example is a laboratory model of a large-scale, three-stage two-phase fermentation process for the production of Penicillium bilaii (Pb) spores.
  • a frozen Pb production inoculum stock culture (1 X10 10 spores) was thawed at room temperature, and then inoculated into a vessel containing 10 L of " Pb primary- phase medium #1" which consisted of glucose (20 g/L) , tryptone (10 g/L) , malt extract (10 g/L) , KH 2 P0 4 (5 g/L) with the pH adjusted to 6 with NaOH, and then grown under controlled conditions (stage 1) which consisted of airflow at 2 1pm for 24 hours and then adjusted to 10 1pm for 24 hours, agitation at 600 rpm, temperature maintained at 25C, pH maintained at 6.0 ⁇ 0.1 with 2N H 2 S0 4 or 2N NaOH as required.
  • stage 2 which consisted of sucrose (10 g/L) , mono-sodium glutamate (6 g/L) , MgS0 4 .7H 2 0 (2 g/L) , KH 2 P0 4 (1 g/L) with the pH adjusted to 6, and then grown under controlled conditions (stage 2) which consisted of airflow 10 1pm, agitation at 600 rpm, temperature maintained at 25C, pH maintained at 6.0 + . 0.1 with 2N H 2 S0 4 or 2N NaOH as required. Culture foaming was controlled by the constant addition of a 10% Sigma
  • Emulsion A anti-foam agent The C:N ratio of this medium was 13.6:1.
  • stage 3 After a 24-hour fermentation in the stage-2 vessel, the pH of the fermenter culture was lowered to 4.5 after which, the mycelial biomass was transferred into a vessel containing 9 L of final-phase medium which consisted of sucrose (18 g/L), mono-sodium glutamate (1 g/L) , MgS0 4 .7H 2 0 (0.2 g/L) , KH 2 P0 4 (0.5 g/L) , Ca (N0 3 ) 2 .4H 2 0 (8.9 g/L) with the pH adjusted to 4.5 with NaOH, and then grown under controlled conditions (stage 3) which consisted of airflow
  • Pb primary-phase medium #1 which consisted of glucose (20 g/L) , casein peptone (10 g/L) , malt extract (10 g/L) , KH 2 P0 4 (2.5 g/L) , plus 110 mL of a trace elements solution consisting of boric acid (4 g/L) , cupric sulfate (0.4 g/L) , ferric chloride (2 g/L), manganous chloride (0.4 g/L) , sodium molybdate (0.4 g/L), zinc chloride (4.4 g/L) , and P-2000 anti-foam agent (0.005%) with the pH maintained between 5.8 - 6.0 with NaOH and H 2 S0 4 .
  • stage 1 The primary-phase culture was then grown under controlled conditions (stage 1) which consisted of airflow at 14 1pm for 24 hours and then adjusted to 130 1pm for 16 hours, agitation at 400 rpm, temperature maintained at 25C, pH between 4.5 and 6.0 with 2N H 2 S0 4 or 2N NaOH as required.
  • stage 1 The C:N ratio of the primary-phase medium was 3:1.
  • the entire 220- L of mycelial biomass was transferred into a 3,000-L stage-2 primary-phase fermentation vessel containing 2,200 L of " Pb primary-phase medium #2" which consisted of sucrose (10 g/L), mono-sodium glutamate (6 g/L) , MgS0 4 .7H 2 0 (2 g/L) , KH 2 P0 4 (1 g/L) plus 2 L of the trace elements solution and P-2000 antifoam agent (0.0005%) with the pH maintained between 5.8 and 6.0 with NaOH or H 2 S0 4 .
  • Pb primary-phase medium #2 consisted of sucrose (10 g/L), mono-sodium glutamate (6 g/L) , MgS0 4 .7H 2 0 (2 g/L) , KH 2 P0 4 (1 g/L) plus 2 L of the trace elements solution and P-2000 antifoam agent (0.0005%) with the pH maintained between 5.8 and 6.0 with NaOH or H 2 S0 4 .
  • the second-stage primary-phase culture grown under controlled conditions which consisted of airflow 1.3 1pm, agitation at 200 rpm, temperature maintained at 25C, pH maintained at 6.0 ⁇ 0.1 with 2N H 2 S0 4 or 2N NaOH as required.
  • the C:N ratio of the second Pb primary-phase fermentation medium was 13.6 :1.
  • stage 3 The final-phase culture was then grown under controlled conditions (stage 3) which consisted of airflow 11 1pm for 60 hours after which is was reduced to 5 1pm for 12 hours, agitation at 100 rpm, temperature maintained at 25C, pH maintained at 4.7 ⁇ 0.1 with 2N H 2 S0 4 or 2N NaOH as required.
  • the C:N ratio of the final-phase medium was 7:1.
  • the titre of Pb spores produced was 2.1 X 10 8 spores/ml.
  • Penicillium bilaii ( Pb) spores produced in EXAMPLE 4 were separated from the mycelial biomass by passing the final-fermentation product through a 48" vibratory screen. The resulting spore suspension was concentrated into a slurry by continuous centrifugation. The concentrated spore slurry was collected in 55-gal drums. Prior to drying, the spore slurry was stabilized by the addition of sucrose (1 g/L) and mon-sodium glutamate (1 g/L) into the drums and well-mixed. Then, the stabilized slurry was pumped from the 55-gal drums into a feed line which sprayed the spore slurry into 7.5-foot spray drier.
  • the inlet temperature was maintained in a range of 128C - 132C, while the outlet temperature was maintained between 48C to 52C.
  • Spray-dried spores were collected in double-walled plastic bags.
  • the viability of the Pb spores after drying, was 5.2 X 10 8 ( ⁇ 2.98X10 8 ) cfu/g (i.e., colony-forming-units per gram) of dried spores .
  • the dried, stabilized spores were packaged and sealed in plastic-lined aluminum foil pouches that were water- and gas-impermeable. After three-months of storage at -20C, the viability of the Pb spores packaged in the aluminum foil pouches was 2.4 X 10 8 cfu/g.
  • Penicillium bilaii ( Pb) spores produced with the large-scale two-phase fermentation process outlined in EXAMPLE 4 were separated from the mycelial biomass by passing the final-fermentation product through a 48" vibratory screen. The resulting spore suspension was concentrated into a slurry by continuous centrifugation. The concentrated spore slurry was collected in 55-gal drums.
  • the concentrated Pb spore slurry was diluted with sterile distilled water and then stabilized by the addition of glycerol (final glycerol concentration in the composition was 10% v/v) .
  • a green food dyestuff was also added to the composition (13% w/v) .
  • the liquid spore concentrate composition was then packaged and sealed in plastic-lined aluminum foil pouches. After 1 week of storage at -20C, the Pb titre in the liquid composition was 1.89 X 10 9 ( ⁇ 1.4X10 8 ) . After 3 months of storage at -20C, the Pb titre in the liquid composition «as 1.33 X 10 9 (+ 2.1X10 7 ) .
  • EXAMPLE 7 EXAMPLE 7:
  • the Pb titre in the inoculated peat was 8.21 X 10 8 cfu/g (colony-forming-units per gram) .
  • the Pb titre was 8.41 X 10 8 cfu/g, and 8.35 X 10 8 cfu/g after 3 months of storage.
  • Penicillium bilaii ( Pb) concentrated spore slurry produced with the large-scale two-phase fermentation process outlined in EXAMPLE 4 was used to prepare a granular composition.
  • the PJ -coated calcined clay granules were dispensed into plastic bags (1 kg/bag) , tightly sealed, and then stored at 20C.
  • the moisture content of the Pb- coated granules was 17%.
  • the titre of Pb on the clay granules was determined immediately after coating, and subsequently at 4, 8 and 22 weeks. The data are recorded in Table 1.
  • Table 1 Effects of storage on the titre of PJ -coated calcined clay granules .

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Abstract

Procédés permettant d'effectuer un processus de fermentation fongique en deux phases à plusieurs étages utilisé pour produire de grandes quantités de spores fongiques pouvant servir de principes actifs dans des compositions du type commercial. La première phase du processus de fermentation stimule de préférence la croissance de mycélium fongique et on peut considérablement augmenter le volume de la biomasse mycélienne produite pendant cette phase en transférant successivement en série la biomasse mycélienne dans des cuves plus grandes. La deuxième phase c'est-à-dire la phase finale du processus de fermentation stimule de préférence la sporulation fongique et la production de spores. Les spores fongiques produits selon le procédé de cette invention peuvent être traités sous forme de pâtes concentrées ou de poudres déshydratées. Les compositions du type commercial pouvant être préparées avec ces produits de spores fongiques comme principes actifs, comprennent des poudres, des liquides et des granulés mouillables.
PCT/CA1995/000094 1994-03-15 1995-02-24 Procedes de production de spores fongiques et compositions contenant ces dernieres WO1995025163A1 (fr)

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WO1999024555A2 (fr) * 1997-11-10 1999-05-20 Dschida William J A Production de parois cellulaires fongiques et utilisation de celles-ci en tant que matiere premiere destinee a des textiles
WO2014124120A1 (fr) * 2013-02-06 2014-08-14 Envera, Llc Mélanges séchés de composés germinatifs et de spores
WO2015130911A1 (fr) * 2014-02-27 2015-09-03 Envera, Llc Mélanges séchés de composés germinatifs pour spores fongiques
CN107557407A (zh) * 2017-09-26 2018-01-09 华南理工大学 一种调控裂褶菌发酵产物裂褶多糖分子量的方法
US9932543B2 (en) 2014-08-06 2018-04-03 Envera, Llc Bacterial spore compositions for industrial uses
WO2019140093A1 (fr) * 2018-01-15 2019-07-18 Locus Ip Company, Llc Production submergée aérobie à grande échelle de champignons
US11172669B2 (en) 2016-11-16 2021-11-16 Locus Agriculture Ip Company, Llc Materials and methods for the control of nematodes
US11286456B2 (en) 2017-09-28 2022-03-29 Locus Agriculture Ip Company, Llc Large scale production of liquid and solid trichoderma products
US11324224B2 (en) 2017-07-27 2022-05-10 Locus Agriculture Ip Company, Llc Efficient production of Pichia yeasts and their use for enhancing plant and animal health
US11377585B2 (en) 2019-06-20 2022-07-05 Locus Ip Company, Llc Co-cultivation of a myxobacterium and acinetobacter for enhanced production of emulsan
US11414640B2 (en) 2017-10-31 2022-08-16 Locus Ip Company, Llc Matrix fermentation systems and methods for producing microbe-based products
US11447430B2 (en) 2018-05-08 2022-09-20 Locus Agriculture Ip Company, Llc Microbe-based products for enhancing plant root and immune health
US11479749B2 (en) 2017-04-07 2022-10-25 Locus Ip Company, Llc Production and cryopreservation of high concentration inocula
US11758924B2 (en) 2019-04-12 2023-09-19 Locus Solutions Ipco, Llc Pasture treatments for enhanced carbon sequestration and reduction in livestock-produced greenhouse gas emissions

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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999024555A2 (fr) * 1997-11-10 1999-05-20 Dschida William J A Production de parois cellulaires fongiques et utilisation de celles-ci en tant que matiere premiere destinee a des textiles
WO1999024555A3 (fr) * 1997-11-10 1999-07-22 William J A Dschida Production de parois cellulaires fongiques et utilisation de celles-ci en tant que matiere premiere destinee a des textiles
EA034962B1 (ru) * 2013-02-06 2020-04-13 Инвира Элайси, Ллс Высушенные смеси со способствующими прорастанию спор соединениями
WO2014124120A1 (fr) * 2013-02-06 2014-08-14 Envera, Llc Mélanges séchés de composés germinatifs et de spores
KR20150133699A (ko) * 2013-02-06 2015-11-30 엔베라, 엘엘씨 건조된 포자 발아성 화합물 혼합물
US9447376B2 (en) 2013-02-06 2016-09-20 Envera, Llc Dried spore germinative compound mixtures
KR102302522B1 (ko) 2013-02-06 2021-09-14 엔베라 엘아이씨, 엘엘씨 건조된 포자 발아성 화합물 혼합물
US10308909B2 (en) 2013-02-06 2019-06-04 Envera Lic, Llc Dried spore germinative compound mixtures
WO2015130911A1 (fr) * 2014-02-27 2015-09-03 Envera, Llc Mélanges séchés de composés germinatifs pour spores fongiques
US9932543B2 (en) 2014-08-06 2018-04-03 Envera, Llc Bacterial spore compositions for industrial uses
US11172669B2 (en) 2016-11-16 2021-11-16 Locus Agriculture Ip Company, Llc Materials and methods for the control of nematodes
US11825827B2 (en) 2016-11-16 2023-11-28 Locus Solutions Ipco, Llc Materials and methods for the control of nematodes
US11479749B2 (en) 2017-04-07 2022-10-25 Locus Ip Company, Llc Production and cryopreservation of high concentration inocula
US11324224B2 (en) 2017-07-27 2022-05-10 Locus Agriculture Ip Company, Llc Efficient production of Pichia yeasts and their use for enhancing plant and animal health
CN107557407A (zh) * 2017-09-26 2018-01-09 华南理工大学 一种调控裂褶菌发酵产物裂褶多糖分子量的方法
US11286456B2 (en) 2017-09-28 2022-03-29 Locus Agriculture Ip Company, Llc Large scale production of liquid and solid trichoderma products
US11414640B2 (en) 2017-10-31 2022-08-16 Locus Ip Company, Llc Matrix fermentation systems and methods for producing microbe-based products
WO2019140093A1 (fr) * 2018-01-15 2019-07-18 Locus Ip Company, Llc Production submergée aérobie à grande échelle de champignons
US11412740B2 (en) 2018-01-15 2022-08-16 Locus Ip Company, Llc Large-scale aerobic submerged production of fungi
US11447430B2 (en) 2018-05-08 2022-09-20 Locus Agriculture Ip Company, Llc Microbe-based products for enhancing plant root and immune health
US11758924B2 (en) 2019-04-12 2023-09-19 Locus Solutions Ipco, Llc Pasture treatments for enhanced carbon sequestration and reduction in livestock-produced greenhouse gas emissions
US11377585B2 (en) 2019-06-20 2022-07-05 Locus Ip Company, Llc Co-cultivation of a myxobacterium and acinetobacter for enhanced production of emulsan

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