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WO2025114547A1 - Amélioration du rendement de la fermentation par supplémentation des milieux - Google Patents

Amélioration du rendement de la fermentation par supplémentation des milieux Download PDF

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Publication number
WO2025114547A1
WO2025114547A1 PCT/EP2024/084124 EP2024084124W WO2025114547A1 WO 2025114547 A1 WO2025114547 A1 WO 2025114547A1 EP 2024084124 W EP2024084124 W EP 2024084124W WO 2025114547 A1 WO2025114547 A1 WO 2025114547A1
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Prior art keywords
bacillus
lipopeptides
glutamate
production
increased
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Akram ABI
Patricia Cuevas DOMINGUEZ
Cesar FONSECA
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Chr Hansen AS
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Chr Hansen AS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

Definitions

  • the present disclosure relates to methods withing the field of improving yields in fermentation processes by supplementation of fermentation media.
  • bacteria of Bacillus sp. genus have received much attention due to the wide variety of antibiotic compounds they produce, their long shelf life, their fast growth in culture, and their ability to colonize leaf surfaces [1 , 2, 3, 4],
  • Bacillus subtilis such as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus mycoides, Bacillus circulans, Bacillus megaterium, Bacillus pumilus, Bacillus mojavensis, and Bacillus thuringiensis show antimicrobial activity.
  • the antimicrobial activity of these bacteria is due to their ability to produce lipopeptides e.g. of the surfactin, iturin, and fengycin families, which differ in the amino acid sequence and the branching of the fatty acid chain.
  • surfactins exhibit high antibacterial activity, whereas iturins and fengycins are recognized for their antifungal activity [4],
  • B. subtilis and B. amyloliquefaciens to control various diseasecausing microorganisms in a wide variety of crops, including fruit and vegetable crops such as blackberry, grape, raspberry, strawberry, tomato, cucumber, black pepper, orange, melon, apple, peach, custard apple, banana, papaya, mango, and kiwi.
  • EP231 1936 discloses a B. subtilis strain KS1 (NITE BP-569) as a biological control agent to counteract several phytopathogenic microorganisms in vine crops.
  • WO 98/21968 discloses an antibiotic produced by B. subtilis A0153 (ATCC 55614) effective against bacterial and fungal infections and also as method for protecting plants that comprises the application of these antibiotic compounds.
  • WO9850422 disclose antibiotic compounds produced by the B. subtilis strain AQ713 (equivalent to strain QST713, deposited as NRRL B-21661) and its mutants which exhibit insecticidal, nematidical, antifungal, and antibacterial activity.
  • US201 1/0318386 describes methods for inducing systemic resistance against various pathogens through the use of biological controllers of the Bacillus genus, specifically of the isolated B. mojavensis 203-7 and isolated B. mycoides species.
  • ES 2345969 describes a phytostrengthener for application on banana and plantain pseudostems, which includes B. subtilis, Trichoderma viride, and B. megaterium var phosphaticum.
  • WO14178032 discloses a process for increasing the production of biomass of microorganisms of the Bacillus genus, including Bacillus subtilis EA-CB0015 and Bacillus amyloliquefaciens EA- CB0959.
  • the biomass obtained by the process can be separated from the culture medium using conventional methods of centrifugation or microfiltration, whereas the active metabolites can be obtained by extraction with solvents, precipitation, adsorption, or chromatography.
  • the amount of biomass of microorganisms of Bacillus sp. obtained can range between 3.0 and 20.0 g/L.
  • fermentation processes are expensive and time-consuming to setup and therefore maximizing the output from the fermentation processes is key in ensuring the profitability of the materials produced in the fermentation processes and the development of optimized fermentation strategies are highly desired.
  • the present disclosure relates to methods for increasing production of one or more lipopeptides in a fermentation process.
  • the present disclosure relates to a composition
  • a composition comprising a fermentation broth or one or more lipopeptides produced according to the methods disclosed herein.
  • the present disclosure relates to the use of a fermentation broth, or a composition as disclosed herein for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen.
  • the present disclosure relates to a method for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen comprising administering a fermentation broth, or a composition produced by the methods disclosed herein, to the plant.
  • the present disclosure relates to methods for increasing lipopeptide production in fermentations comprising a Bacillus spp.
  • This increased production is achieved by providing a Bacilllus spp., providing a suitable fermentation medium, supplementing the fermentation medium with glutamate, and initiating and running a fermentation process by introducing the provided Bacillus spp. into the supplemented fermentation medium.
  • the present disclosure relates to a method for increasing production of one or more lipopeptides in a fermentation process comprising, the method comprising the steps a) providing a bacillus spp. b) providing a suitable fermentation medium c) providing a supplemented fermentation medium by supplementing the suitable fermentation medium with glutamate d) initiating a fermentation process by introduction of the bacillus spp. provided in step a) into the supplemented fermentation medium provided in step c), and e) running the fermentation process, thereby providing a fermentation broth with an increased content of one or more lipopeptides.
  • the one or more lipopeptides is selected as one or more of fengycin and iturin.
  • the supplementation of the fermentation medium with aspartate relates to supplementing a fermentation medium with 3-30 g/L glutamate, 5-25 g/L glutamate, 7-20 g/L glutamate, 10-15 g/L glutamate, 7-15 g/L glutamate, 10-20 g/L glutamate, 5-15 g/L glutamate, 5-13 g/L glutamate, 7-13 g/L glutamate, 9-11 g/L glutamate, 5 g/L glutamate, 6 g/L glutamate, 7 g/L glutamate, 8 g/L glutamate, 9 g/L glutamate, 10 g/L glutamate, 11 g/L glutamate, 12 g/L glutamate or 13 g/L glutamate, 14 g/L glutamate, or 15 g/L glutamate.
  • step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with at least 3 g/L glutamate, at least 4 g/L glutamate, at least 5 g/L glutamate, 6 g/L glutamate, at least 7 g/L glutamate, at least 8 g/L glutamate, at least 9 g/L glutamate, at leastl 0 g/L glutamate, up to 15 g/L glutamate, up to 18 g/L glutamate, up to 20 g/L glutamate, up to 22 g/L glutamate, up to 24 g/L glutamate, up to 26 g/L glutamate, up to 28 g/L glutamate, or up to 30 g/L glutamate.
  • step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with at least 3 g/L glutamate.
  • step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with at least 5 g/L glutamate.
  • step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with at least 7 g/L glutamate. In one or more exemplary embodiments, step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with at least 8 g/L glutamate.
  • step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with at least 9 g/L glutamate.
  • step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with at least 10 g/L glutamate.
  • step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with 3-30 g/L glutamate, 5-25 g/L glutamate, 7-20 g/L glutamate, 10-15 g/L glutamate, 7-15 g/L glutamate, 10-20 g/L glutamate, 5-15 g/L glutamate, 5-13 g/L glutamate, 7-13 g/L glutamate, 9-11 g/L glutamate, 5 g/L glutamate, 6 g/L glutamate, 7 g/L glutamate, 8 g/L glutamate, 9 g/L glutamate, 10 g/L glutamate, 1 1 g/L glutamate, 12 g/L glutamate or 13 g/L glutamate, 14 g/L glutamate, or 15 g/L glutamate.
  • step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with 7-15 g/L glutamate, I Q- 20 g/L glutamate, 5-15 g/L glutamate, 7-13 g/L glutamate, or 9-11 g/L glutamate.
  • step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with 10-20 g/L glutamate.
  • step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with 7-15 g/L glutamate.
  • step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with 5-15 g/L glutamate.
  • step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with 5-13 g/L glutamate.
  • step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with 7-13 g/L glutamate.
  • step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with 7-10 g/L glutamate.
  • step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with 10-15 g/L glutamate.
  • the method disclosed herein comprises an additional step, step f) that relates to the recovery of the one or more lipopeptides from the fermentation broth.
  • the method as disclosed herein further comprises a recovery step f) for recovering the one or more lipopeptides from the fermentation broth.
  • the recovery step f) for recovering one or more lipopeptides from the fermentation broth comprises a centrifugation or filtration step.
  • the method disclosed herein comprises an additional step, step g) that relates to the purification of the one or more lipopeptides from the fermentation broth.
  • the method as disclosed herein further comprises a purification step g) for providing one or more purified lipopeptides.
  • the method as disclosed herein comprises both a recovery step f) and a purification step g).
  • an increased production of one or more lipopeptides of the method a production of one or more lipopeptides that is increased by 5-30%, 5-25%, 8-22%, 10-20%, 12-18%, 12-16%, 12-14% or 14-16%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by 8- 18%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by 10- 16%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by 12- 14%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by at least 5%, 6, % 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by at least 8%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by at least 9%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by at least 10%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by up to 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by up to 12%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by up to 14%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by up to 16%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by up to 18%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by up to 20%. In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by up to 22%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by up to 24%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by up to 26%.
  • the one or more lipopeptides is selected from the group consisting of fengycin and iturin.
  • the one or more lipopeptides is selected as fengycin and iturin.
  • the one or more lipopeptides is selected as fengycin.
  • an increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by 5-30%, 5-25%, 8- 22%, 10-20%, 12-18%, 12-16%, 12-14% or 14-16%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by 8-18%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by 10-16%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by 12-14%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by at least 5%, 6, % 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by at least 8%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by at least 9%. In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by at least 10%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by up to 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by up to 12%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by up to 14%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by up to 16%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by up to 18%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by up to 20%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by up to 22%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by up to 24%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by up to 26%.
  • the one or more lipopeptides is selected as iturin.
  • an increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by 4-10%, 5-12%, 5-14%, 5-30%, 5-25%, 8-22%, 10-20%, 12-18%, 12-16%, 12-14% or 14-16%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by 4-10%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by 5-12%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by 5-14%. In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by at least 5%, 6, % 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by at least 5%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by at least 6%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by at least 7%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by at least 8%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by at least 9%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by at least 10%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by up to 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by up to 12%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by up to 14%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by up to 16%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by up to 18%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by up to 20%. In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by up to 22%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by up to 24%.
  • the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by up to 26%.
  • the present disclosure also relates to compositions with an increased content of one or more lipopeptides as produced according to the method as disclosed herein.
  • the present disclosure relates to a composition comprising a fermentation broth produced according to the method as disclosed herein, a composition comprising one or more lipopeptides recovered in step f) of the method as disclosed herein, or a composition comprising the purified one or more lipopeptides obtained in step g) of the method as disclosed herein.
  • the present disclosure relates to a composition comprising a fermentation broth produced according to the method as disclosed herein, a composition comprising fengycin and/or iturin recovered in step f) of the method as disclosed herein, or a composition comprising the purified fengycin and/or iturin obtained in step g) of the method as disclosed herein.
  • the present disclosure relates to the use of a fermentation broth as produced by the method as disclosed herein, the use of the one or more lipopeptides as produced by the method as disclosed herein, the use of a composition comprising a fermentation broth produced according to the method as disclosed herein, the use of a composition comprising one or more lipopeptides recovered in step f) of the method as disclosed herein, and/or the use of a composition comprising the purified one or more lipopeptides obtained in step g) of the method for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen.
  • the present disclosure relates to the use of a fermentation broth as produced by the method as disclosed herein, the use of the fengycin and/or iturin as produced by the method as disclosed herein, the use of a composition comprising a fermentation broth produced according to the method as disclosed herein, the use of a composition comprising the fengycin and/or iturin recovered in step f) of the method as disclosed herein, and/or the use of a composition comprising the purified fengycin and/or iturin obtained in step g) of the method for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen.
  • the fermentation broth, the compositions and/or the one or more lipopeptides produced according to the method can also be used in a method for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen.
  • the fermentation broth, the compositions, or the one or more lipopeptides produced according to the method are used in a method for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen comprising administering one or more of the fermentation broth, the compositions, or the one or more lipopeptides produced according to the method to the plant.
  • the fermentation broth, the compositions, or the fengycin and/or iturin produced according to the method are used in a method for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen comprising administering one or more of the fermentation broth, the compositions, or the fengycin and/or iturin produced according to the method to the plant.
  • the recovery step f) and the purification step g) of the method as disclosed herein comprises one or more of the following steps
  • an adsorption step comprising adsorption of one or more lipopeptides onto an adsorption column
  • an adsorption step comprising adsorption of one or more lipopeptides onto a geopolymer
  • Geopolymers that are useful in the methods of the present disclosure comprises kieselguhr, diatomite, diatomaceous earth, Kaolin (Chinese clay), bentonite, talcum, volcanic ash, volcanic rock, clay, perlite, lignin, drilling mud, diatomic earth and synthetic silica.
  • the purification step g) is performed on the material recovered in step f) of the methods as disclosed herein.
  • the pH is adjusted to between pH 4.5 and 8.0 prior to adsorption onto a geopolymer or an adsorption column.
  • Adjustment of pH in step f) or step g) comprises adjusting the pH to 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or adjusting the pH to within the ranges 4.5-8.0 pH, 5.0-7.0 pH or 5.5-7.0.
  • a bacteria belonging to the Bacillus spp. in the context of the present disclosure is any bacteria that belongs within the Bacillus genus.
  • the Bacillus genus is well defined in the prior art and the selection and identification of bacterial species belonging to the Bacillus genus is a routine task for the person skilled in the art.
  • a bacteria belonging to the Bacillus spp. is any bacteria that belongs to the Bacillus genus.
  • the Bacillus spp. as used in the method as disclosed herein is a bacteria belonging to a species selected from the list consisting of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus mycoides, Bacillus circulans, Bacillus megaterium, Bacillus pumilus, Bacillus mojavensis, Bacillus thuringiensis, Bacillus simplex, Bacillus safensis, Bacillus atrophaeous, Bacillus methylotrophicus, Bacillus siamensis, Bacillus vallismortis, and Bacillus tequilensis.
  • the Bacillus spp. as used in the method as disclosed herein is a bacteria belonging to the Bacillus amyloliquefaciens species.
  • the Bacillus spp. as used in the method M1 is a bacteria selected from the list consisting of Bacillus amyloliquefaciens subsp. amyloliquefaciens, Bacillus amyloliquefaciens subsp. plantarum, B. amyloliquefaciens strain with the DSMZ accession number DSM 25840, B. amyloliquefaciens strain with the DSMZ accession number DSM 27032, B. amyloliquefaciens strain with the DSMZ accession number DSM 27033 and B. amyloliquefaciens strain with the DSMZ accession number DSM 34003.
  • the Bacillus spp. as used in the method M1 is a Bacillus amyloliquefaciens strain selected from the list consisting of DSM 25840, DSM 27032, DSM 27033 and DSM 34003.
  • the Bacillus spp. as used in the method M1 is a Bacillus amyloliquefaciens strain selected from the list consisting of DSM 25840, DSM 27032 and DSM 27033. In one or more exemplary embodiments of the present disclosure, the Bacillus spp. as used in the method M1 is Bacillus amyloliquefaciens strain DSM 34003.
  • the strain Bacillus amyloliquefaciens was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 03 April
  • the strain Bacillus amyloliquefaciens was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 21 March
  • the strain Bacillus amyloliquefaciens was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 21 March 2013, under the accession No. DSM 27033.
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen
  • the strain Bacillus amyloliquefaciens was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 24 August 2021 , under the accession No. DSM 34003.
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen
  • Fig. 1 shows lipopeptide concentrations and bioactivity measured in terms of ID50 (The half maximal inhibitory dilution of the fermentates) in end-of-fermentation fermentates from cultures of Bacillus amyloliquefaciens DSM 34003 incubated in BioLector Pro, at 1500 rpm, 33°C, pH 7.8-8.0, for 40 hours, in 0.8 mL LC medium supplemented with glutamate and/or aspartate as depicted in table 1 .
  • ID50 The half maximal inhibitory dilution of the fermentates
  • Table 1 Glutamate and/or aspartate supplementation in the variants of LC medium in the experiments conducted with Bacillus amyloliquefaciens DSM 34003 in BioLector Pro, at 33°C, pH 7.8-8.0 for 40 h.
  • Example 1 Lipopeptide yield of Bacillus amyloliquefaciens cultured in supplemented LC medium
  • the lipopeptide contents of the fermentate from each of the cultures described in example 1 culture was also analyzed and quantified and the iturin and fengycin contents are shown in figure 1 .
  • the supplementation of LC medium with glutamate clearly increases the production of lipopeptides while aspartate supplementation results in a lower production of lipopeptides.
  • Supplementation with 7.0 and 10 g/L glutamate yielded 410 and 400 mg/L iturin compared to 380 and 370 mg/L in cultures grown in supplemented with 0 and 3.4 g/L glutamate (LC-varO and LC medium) representing an increase in iturin yield of at least 5% and up to 11%, while the iturin yield in cultures supplemented with only aspartate were much lower at around 260-310 mg/L iturin (LC-var3, LC-var4 and LC-var5).
  • Supplementation with 7.0 and 10 g/L glutamate yielded 280 and 290 mg/L fengycin compared to 240/250 mg/L in cultures grown in supplemented with 0 and 3.4 g/L glutamate (LC-varO and LC-medium) which is an increase of at least 10% and up to an increase of 17%, while the fengycin yield in cultures supplemented with only aspartate were much lower at around 160-200 mg/L fengycin (LC-var3, LC-var4 and LC-var5).
  • ID50 The half maximal inhibitory dilution of biological samples
  • the inhibitory potency was assessed by filling microtiter plates with 200 pl Fusarium Graminearum conidia media mixture in a total concentration of 5.0x10 5 F. graminearum conidia and supplementing this mixture with 50 pl sample. Following mixing of media and samples, the microtiter plates are transferred to an incubator or robotic incubator and OD measurements at OD620 is performed for each sample every 20 hours for 120 hours, measuring the increase in OD overtime. Antibiotics were added to prevent the outgrowth of Bacillus (spores and/or vegetative cells) in the samples over the course of the experiment.
  • a dose-response curve with OD/increase in OD is plotted using data from the dilution series for each fermentate at a specific time interval.
  • the slope of the dose-response curve for the fermentate dilution series is normalized against the averaged slope of the uninhibited sample (negative control/isopropanol only) and multiplied by 100.
  • a sigmoidal model is fitted onto the logistic relationship between the inhibition and no inhibition state.
  • the half maximal inhibitory dilution (ID50) of the sample fermentate is defined in Equation 1.
  • Equation 1 Sigmoidal formula describing the dose-response of F. graminearum growth inhibition at different dilutions.
  • A is the origin of the function, also the ID50;
  • B describes the dilution factor;
  • C stands for the slope of the function.
  • the fit of the sigmoidal curve to the data is controlled by visual inspection and poor fits are considered as outlier and excluded.
  • IC50 half maximal inhibitory concentration
  • Equation 2 Sigmoidal formula describing the dose-response of F. graminearum growth inhibition at different dilutions.
  • A is the origin of the function, also the ID50;
  • B describes the dilution factor;
  • C stands for the slope of the function.
  • the fit of the sigmoidal curve to the data is controlled by visual inspection and poor fits are considered as outlier and excluded.
  • the deviations between calculated responses from the samples and the model are minimized by a script to optimize the model fit.
  • Model fitting is constrained to minimize false interpretations caused by outliers. Therefore, the variables A, B and C are >0; while A is ⁇ 500; B is ⁇ 1 and C is ⁇ 2.
  • the ID50 values are calculated for each fermentate dilution series and a comparison of these ID50 values are represented by the curve shown in figure 1 .
  • the ID50 of fermentates from Bacillus amyloliquefaciens cultured in LC medium supplemented with glutamate in concentrations of 7 and 10 g/L (LC-var1 and LC-var2) is greater than when the LC medium is supplemented with 0 or 3.4 (LC-varO and LC medium) and much greater than when LC medium is supplemented with aspartate (LC-var3-6).
  • a method for increasing production of one or more lipopeptides in a fermentation process comprising, the method comprising the steps a) providing a bacillus spp. b) providing a suitable fermentation medium c) providing a supplemented fermentation medium by supplementing the suitable fermentation medium with glutamate d) initiating a fermentation process by introduction of the bacillus spp. provided in step a) into the supplemented fermentation medium provided in step c), and e) running the fermentation process, thereby providing a fermentation broth with an increased content of one or more lipopeptides.
  • the suitable fermentation medium is supplemented with up to 15 g/L glutamate, up to 18 g/L glutamate, up to 20 g/L glutamate, up to 22 g/L glutamate, up to 24 g/L glutamate, up to 26 g/L glutamate, up to 28 g/L glutamate, or up to 30 g/L.
  • recovery step f) comprises a centrifugation step or filtration step.
  • Bacillus spp. is selected as one from the list consisting of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus mycoides, Bacillus circulans, Bacillus megaterium, Bacillus pumilus, Bacillus mojavensis, and Bacillus thurigiensis, Bacillus simplex, Bacillus safensis, Bacillus atrophaeous, Bacillus methylotrophicus, Bacillus siamensis, Bacillus vallismortis, Bacillus tequilensis.
  • Bacillus spp. is Bacillus amyloliquefaciens.
  • Bacillus spp. is a Bacillus amyloliquefaciens strain selected from the list consisting of Bacillus amyloliquefaciens subsp. amyloliquefaciens, Bacillus amyloliquefaciens subsp. plantarum, B. amyloliquefaciens strain with the DSMZ accession number DSM 25840, B. amyloliquefaciens strain with the DSMZ accession number DSM 27032, B. amyloliquefaciens strain with the DSMZ accession number DSM 27033, and B. amyloliquefaciens strain with the DSMZ accession number DSM 34003.
  • Bacillus spp. is B. amyloliquefaciens strain with the DSMZ accession number DSM 34003.
  • a composition comprising a fermentation broth produced according to the method of any one of the preceding items, a composition comprising the one or more lipopeptides recovered in step f) according to the method of any one of the preceding items, or a composition comprising the purified one or more lipopeptides obtained in step g) according to the method of any one of the preceding items.
  • Use of a fermentation broth produced by a method according to any one of the items 1-75 or a composition according to item 76 for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen.
  • the use according to item 77, wherein the infection in a plant is caused by a fungal pathogen.
  • a method for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen comprising administering a fermentation broth produced by the method according to any one of items 1-75 or a composition according to item 76 to the plant.
  • T Stein Bacillus subtilis antibiotics: structures, syntheses and specific functions, Molecular Microbiology, vol. 56, pp. 854-857, 2005.

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Abstract

La présente invention concerne des procédés permettant d'améliorer le processus de fermentation pour produire les lipopeptides antifongiques fengycine et iturine par supplémentation du milieu de fermentation en glutamate à l'aide de Bacillus amyloliquefaciens.
PCT/EP2024/084124 2023-11-30 2024-11-29 Amélioration du rendement de la fermentation par supplémentation des milieux Pending WO2025114547A1 (fr)

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