Improving fermentation output by media supplementation

Abstract

The present disclosure relates to methods for improving the output of fermentation processes by supplementation of the fermentation medium.

Description

[0001] TITLE: Improving fermentation output by media supplementation

[0002] The present disclosure relates to methods withing the field of improving yields in fermentation processes by supplementation of fermentation media.

[0003] BACKGROUND

[0004] Among the microorganisms used for biological control, 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],

[0005] In particular, certain species of Bacillus such as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus mycoides, Bacillus circulans, Bacillus megaterium, Bacillus pumilus, Bacillus mojavensis, and Bacillus thuringiensis show antimicrobial activity.

[0006] 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],

[0007] The prior art describes the use of 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.

[0008] WO9850422, WO9909819, and W00029426 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.

[0009] 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. In turn, 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. In a preferred embodiment of WO14178032, the amount of biomass of microorganisms of Bacillus sp. obtained can range between 3.0 and 20.0 g / L.

[0010] Bacteria belonging to the Bacillus species are used in many different applications including use in animal feeds and in methods to improve the nutritional status of animals. For example WO2013153159 discloses the use of bacillus spores for use in animal feeds whereas W02004 / 095939 discloses the use of Bacillus spores in aquatic animal feeds and both

[0011] WO2010 / 070005 and US2003 / 0124104 disclose probiotic use of bacillus spores, and in particular use of bacillus subtilis spores in probiotics.

[0012] However, 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.

[0013] SUMMARY

[0014] In a broadest aspect, the present disclosure relates to methods for improving the sporulation ratio of Bacillus spp. in a fermentation broth produced in a fermentation process by supplementation of a fermentation medium with aspartate.

[0015] In one aspect, the present disclosure relates to a composition comprising the fermentation broth produced according to the methods disclosed herein or spores recovered and / or purified from a fermentation broth produced according to the methods disclosed herein.

[0016] In another aspect, the present disclosure relates to the use of a fermentation broth or composition produced according to the methods disclosed herein as for controlling an infection in a plant caused by a fungal or bacterial pathogen.

[0017] In another aspect, the present disclosure relates to the use of a fermentation broth or composition produced according to the methods disclosed herein as a probiotic.

[0018] In another aspect, the present disclosure relates to the use of a fermentation broth or composition produced according to the methods disclosed herein in an animal feed.

[0019] In another aspect, the present disclosure relates to the use of a fermentation broth or composition produced according to the methods disclosed herein in an aquatic animal feed. In another aspect, the present disclosure relates to the use of a fermentation broth or composition produced according to the methods disclosed herein as a nematocidal or antimicrobial agent.

[0020] In another aspect, the present disclosure relates to the use of a fermentation broth or composition produced according to the methods disclosed herein as an antibacterial and / or antifungal agent.

[0021] In another aspect, the present disclosure relates to the use of a fermentation broth or composition produced according to the methods disclosed herein in a method for improving the nutritional status of an animal or aquatic animal.

[0022] In another aspect, the present disclosure relates to the use of a fermentation broth or composition produced according to the methods disclosed herein in a method for controlling, preventing ameliorating an infection in a plant that is caused by a fungal or bacterial pathogen.

[0023] DETAILED DESCRIPTION

[0024] It has been surprisingly shown herein that supplementing a fermentation medium with aspartate (aspartic acid) resulted in increased spore yield as well as sporulation ratio when bacteria belonging to the species Bacillus amyloliquefaciens were cultivated in the aspartate supplemented fermentation medium (see example 1 and figure 1).

[0025] As shown in figure 1 , Bacillus amyloliquefaciens cultures in fermentation media supplemented with 7 g / L (LC-var4) and 10 g / L (LC-var5) aspartate (table 1) showed a respective increase in sporulation ratio to about 86% and 89% respectively compared to a sporulation ratio of 79% in unsupplemented fermentation media (LC-varO) and about 83% in fermentation media supplemented with 3.4 g / L glutamate (LC / LC medium) while supplementation of the fermentation medium with higher amounts of glutamate resulted in lower sporulation ratios. Simultaneously, the spore yield was increased to about 1.1x1 O10(1.1 E+10) and 1.3x1 O10(1.3E+10) in LC-var-4 and LC- var5 compared to about 6x109in LC-varO and about 8x109(8E+10) in LC-varO.

[0026] Because it is the spores of Bacillus amyloliquefaciens (and in more general terms spores of Bacillus spp.) that are of interest and not vegetative cells in terms of providing an antimicrobial protective effect, increasing the sporulation ratio is a very important parameter as it determines how cost / effective the fermentation process is in terms of output per resource / sugar input.

[0027] Therefore, the present disclosure relates to the provision of methods for improving the sporulation ratio and / or spore yield in fermentations comprising Bacillus spp.

[0028] A method M1 for improving sporulation ratio in Bacillus fermentations

[0029] It was surprisingly found in the present disclosure that supplementation of a fermentation medium with aspartate increases the sporulation ration of Bacillus amyloliquefaciens. This effect is considered likely to also hold true for other member-species of the Bacillus spp. genus. This improved sporulation ratio 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.

[0030] In particular, the present disclosure relates to a method M1 for increasing the sporulation ratio and / or spore yield of a Bacillus spp. in a fermentation broth, the method comprising a1) providing a bacillus spp. b1) providing a suitable fermentation medium c1) supplementing the suitable fermentation medium with aspartate, thereby forming a supplemented fermentation medium d1) initiating a fermentation process by introduction of the bacillus spp. provided in step a1) into the supplemented fermentation medium provided in step c1), and e1) running the fermentation process. thereby providing a fermentation broth with an increased sporulation ratio of bacillus spp. and / or an increased Bacillus spp. spore yield.

[0031] In one or more exemplary embodiments of the present disclosure, the method M1 relates to a method for providing a fermentation broth with an increased sporulation ratio of a Bacillus spp..

[0032] The key to achieving an increased sporulation ratio lies in the supplementation medium with aspartate. Methods for producing and supplementing fermentation media with desirable additional nutrients are commonly described in the prior art. Accordingly, the use of such methods for obtaining a supplemented fermentation medium is considered merely a routine task for the person skilled in the art.

[0033] In the present context the supplementation of the fermentation medium with aspartate relates to supplementing a fermentation medium with 3-30 g / L aspartate, 5-25 g / L aspartate, 7-20 g / L aspartate, 10-15 g / L aspartate, 7-15 g / L aspartate, 10-20 g / L aspartate, 5-15 g / L aspartate, 5-13 g / L, 7-13 g / L aspartate, 9-11 g / L aspartate, 5 g / L aspartate, 6 g / L aspartate, 7 g / L aspartate, 8 g / L aspartate, 9 g / L aspartate, 10 g / L aspartate, 11 g / L aspartate, 12 g / L aspartate or 13 g / L aspartate,

[0034] 14 g / L aspartate, or 15 g / L aspartate.

[0035] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein relates to supplementing the suitable fermentation medium provided in step b1) with at least 3 g / L aspartate, at least 4 g / L aspartate, at least 5 g / L aspartate, 6 g / L aspartate, at least 7 g / L aspartate, at least 8 g / L aspartate, at least 9 g / L aspartate, at least 10 g / L aspartate, up to 15 g / L aspartate, up to 18 g / L aspartate, up to 20 g / L aspartate, up to 22 g / L aspartate, up to 24 g / L aspartate, up to 26 g / L aspartate, up to 28 g / L aspartate, or up to 30 g / L aspartate.

[0036] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein relates to supplementing the suitable fermentation medium provided in step b1) with at least 3 g / L aspartate.

[0037] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein relates to supplementing the suitable fermentation medium provided in step b1) with at least 5 g / L aspartate.

[0038] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein relates to supplementing the suitable fermentation medium provided in step b1) with at least 7 g / L aspartate.

[0039] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein relates to supplementing the suitable fermentation medium provided in step b1) with at least 8 g / L aspartate.

[0040] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein relates to supplementing the suitable fermentation medium provided in step b1) with at least 9 g / L aspartate.

[0041] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein relates to supplementing the suitable fermentation medium provided in step b1) with at least 10 g / L aspartate.

[0042] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein relates to supplementing the suitable fermentation medium provided in step b1) with 3-30 g / L aspartate, 5-25 g / L aspartate, 7-20 g / L aspartate, 10-15 g / L aspartate, 7-15 g / L aspartate, 10-20 g / L aspartate, 5-

[0043] 15 g / L aspartate, 5-13 g / L, 7-13 g / L aspartate, 9-11 g / L aspartate, 5 g / L aspartate, 6 g / L aspartate, 7 g / L aspartate, 8 g / L aspartate, 9 g / L aspartate, 10 g / L aspartate, 11 g / L aspartate, 12 g / L aspartate or 13 g / L aspartate, 14 g / L aspartate, or 15 g / L aspartate.

[0044] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein relates to supplementing the suitable fermentation medium provided in step b1) with 7-15 g / L aspartate, IQ- 20 g / L aspartate, 5-15 g / L aspartate, 7-13 g / L aspartate, or 9-11 g / L aspartate. In one or more exemplary embodiments, step c1) of the method M1 disclosed herein relates to supplementing the suitable fermentation medium provided in step b1) with 10-20 g / L aspartate.

[0045] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein relates to supplementing the suitable fermentation medium provided in step b1) with 7-15 g / L aspartate.

[0046] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein relates to supplementing the suitable fermentation medium provided in step b1) with 5-15 g / L aspartate.

[0047] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein relates to supplementing the suitable fermentation medium provided in step b1) with 7-13 g / L aspartate.

[0048] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein relates to supplementing the suitable fermentation medium provided in step b1) with 7-10 g / L aspartate.

[0049] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein relates to supplementing the suitable fermentation medium provided in step b1) with 10-15 g / L aspartate.

[0050] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein relates to supplementing the suitable fermentation medium provided in step b1) with 7-15 g / L aspartate.

[0051] In the context of the present disclosure, the sporulation ratio is calculated as the number of Bacillus spp. spores divided by the sum of Bacillus spp. spores and Bacillus spp. vegetative cells in a fermentation broth.

[0052] In one or more exemplary embodiments, the increased sporulation ratio of method M1 is a sporulation ratio of at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89% or at least 90%.

[0053] In one or more exemplary embodiments, the increased sporulation ratio of method M1 is a sporulation ratio of at least 80%.

[0054] In one or more exemplary embodiments, the increased sporulation ratio of method M1 is a sporulation ratio of at least 81%.

[0055] In one or more exemplary embodiments, the increased sporulation ratio of method M1 is a sporulation ratio of at least 82%.

[0056] In one or more exemplary embodiments, the increased sporulation ratio of method M1 is a sporulation ratio of at least 83%.

[0057] In one or more exemplary embodiments, the increased sporulation ratio of method M1 is a sporulation ratio of at least 84%.

[0058] In one or more exemplary embodiments, the increased sporulation ratio of method M1 is a sporulation ratio of at least 85%. In one or more exemplary embodiments, the increased sporulation ratio of method M1 is a sporulation ratio of at least 86%.

[0059] In one or more exemplary embodiments, the increased sporulation ratio of method M1 is a sporulation ratio of at least 87%.

[0060] In one or more exemplary embodiments, the increased sporulation ratio of method M1 is a sporulation ratio of at least 88%.

[0061] In one or more exemplary embodiments, the increased sporulation ratio of method M1 is a sporulation ratio of at least 89%.

[0062] In one or more exemplary embodiments, the increased sporulation ratio of method M1 is a sporulation ratio that falls within the range of 80-95%, 80-90%, 80-85%, 81-88%, 82-85, 80-87%, 82-87%, 82-90%, 83-88%, 85-95%, 85-90%, 85-88%, 87-89%, 87-95%, 87-93% or 87-90%.

[0063] In one or more exemplary embodiments, the increased sporulation ratio of method M1 is a sporulation ratio that falls within the range of 80-85%.

[0064] In one or more exemplary embodiments, the increased sporulation ratio of method M1 is a sporulation ratio that falls within the range of 82-87%.

[0065] In one or more exemplary embodiments, the increased sporulation ratio of method M1 is a sporulation ratio that falls within the range of 83-88.

[0066] In one or more exemplary embodiments, the increased sporulation ratio of method M1 is a sporulation ratio that falls within the range of 85-89%.

[0067] In one or more exemplary embodiments, the increased sporulation ratio of method M1 is a sporulation ratio that falls within the range of 80-90%.

[0068] Methods for the recovery of Bacillus spores from a fermentation process or from a fermentation medium are well known in the prior art, and the choice of a suitable method of recovery is considered a routine choice that the skilled person will make in accordance with circumstances. In the present context it is generally contemplated that the spores are recovered alone from the fermentation medium during or at the end of fermentation.

[0069] Accordingly, in some cases it is desired to separate the Bacillus spp. spores from the fermentation broth. In these cases, the method M1 disclosed herein comprises an additional step, step f1) that relates to the recovery of the Bacillus spp. spores from the fermentation broth.

[0070] In one or more exemplary embodiments, the method M1 as defined herein, further comprises a recovery step f1) for recovering Bacillus spp. spores from the fermentation broth.

[0071] In one or more exemplary embodiments, the recovery step f1) for recovering Bacillus spp. spores from the fermentation broth comprises a centrifugation or filtration step. In some cases, it is desired to purify the Bacillus spp. spores produced according to the method disclosed herein. In these instances, the method M1 disclosed herein comprises an additional step, step g) that relates to the purification of the Bacillus spp. spores from the fermentation broth.

[0072] In one or more exemplary embodiments, the method M1 as defined herein, further comprises a purification step g1) for providing purified Bacillus spp. spores.

[0073] In some cases, it is desired to both include a recovery step and a purification step in the method M1 as disclosed herein. Thus, in one or more exemplary embodiments the method M1 as disclosed herein comprises both a recovery step f1) and a purification step g1).

[0074] In one or more exemplary embodiments, the present disclosure relates to a composition comprising Bacillus spp. spores produced according to the method M1 .

[0075] In one or more exemplary embodiments, the present disclosure relates to a composition comprising a fermentation broth produced according to the method M1 , a composition comprising Bacillus spp. spores recovered in step f1) of the method M1 and / or a composition comprising the purified Bacillus spp. spores produced according to step g1) of the method M1.

[0076] In one or more exemplary embodiments, the present disclosure relates to the use of a composition produced by the method M1 , a fermentation broth produced by the method M1 , Bacillus spp. spores recovered in step f1) of the method M1 and / or purified spores provided in step g1) of the method M1 for controlling an infection in a plant caused by a fungal or bacterial pathogen.

[0077] In one or more exemplary embodiments, 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 composition produced by the method M1 , a fermentation broth produced by the method M1 , Bacillus spp. spores recovered in step f1) of the method M1 and / or purified spores provided in step g1) of the method M1 to the plant.

[0078] In one or more exemplary embodiments, the present disclosure relates to the use of a composition produced by the method M1 , a fermentation broth produced by the method M1 , Bacillus spp. spores recovered in step f1) of the method M1 and / or purified spores provided in step g1) of the method M1 for controlling an infection in a plant caused by a fungal pathogen.

[0079] In one or more exemplary embodiments, the present disclosure relates to the use of a composition produced by the method M1 for controlling an infection in a plant caused by a bacterial pathogen.

[0080] In one or more exemplary embodiments, the present disclosure relates to the use of a composition produced by the method M1 , a fermentation broth produced by the method M1 , Bacillus spp. spores recovered in step f1) of the method M1 and / or purified spores provided in step g1) of the method M1 as a probiotic. In one or more exemplary embodiments, the present disclosure relates to the use of a composition produced by the method M1 , a fermentation broth produced by the method M1 , Bacillus spp. spores recovered in step f1) of the method M1 and / or purified spores provided in step g1) of the method M1 in an animal feed.

[0081] In one or more exemplary embodiments, the present disclosure relates to a method for improving health and nutritional status of an animal comprising administering an animal feed comprising a composition produced by the method M1 , a fermentation broth produced by the method M1 , Bacillus spp. spores recovered in step f1) of the method M1 and / or purified spores provided in step g1) of the method M1 to the animal.

[0082] In one or more exemplary embodiments, the present disclosure relates to the use of a composition produced by the method M1 , a fermentation broth produced by the method M1 , Bacillus spp. spores recovered in step f1) of the method M1 and / or purified spores provided in step g1) of the method M1 in an aquatic animal feed.

[0083] In one or more exemplary embodiments, the present disclosure relates to a method for improving health and nutritional status of an aquatic animal comprising administering an aquatic animal feed comprising a composition produced by the method M1 , a fermentation broth produced by the method M1 , Bacillus spp. spores recovered in step f1) of the method M1 and / or purified spores provided in step g1) of the method M1 to the aquatic animal.

[0084] In one or more exemplary embodiments, the present disclosure relates to the use of a composition produced by the method M1 , a fermentation broth produced by the method M1 , Bacillus spp. spores recovered in step f1) of the method M1 and / or purified spores provided in step g1) of the method M1 as a nematocidal or antimicrobial agent.

[0085] In one or more exemplary embodiments, the present disclosure relates to the use of a composition produced by the method M1 , a fermentation broth produced by the method M1 , Bacillus spp. spores recovered in step f1) of the method M1 and / or purified spores provided in step g1) of the method M1 as an antibacterial and / or antifungal agent.

[0086] In one or more exemplary embodiments, the present disclosure relates to the use of a composition produced by the method M1 , a fermentation broth produced by the method M1 , Bacillus spp. spores recovered in step f1) of the method M1 and / or purified spores provided in step g1) of the method M1 as an antibacterial agent.

[0087] In one or more exemplary embodiments, the present disclosure relates to the use of a composition produced by the method M1 , a fermentation broth produced by the method M1 , Bacillus spp. spores recovered in step f1) of the method M1 and / or purified spores provided in step g1) of the method M1 as an antifungal agent. Recovery and purification steps

[0088] The recovery step f1) and the purification step g1) of the method M1 as defined herein comprises one or more of the following steps

[0089] 1) a filtration step,

[0090] 2) a centrifucation step,

[0091] 3) a pH adjustment step

[0092] 4) an adsorption step comprising adsorption of one or more lipopeptides onto an adsorption column,

[0093] 5) an adsorption step comprising adsorption of one or more lipopeptides onto a geopolymer,

[0094] 6) a chromatography step

[0095] 7) a CaCh flocculation step

[0096] 8) a solvent extraction step

[0097] 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.

[0098] In one or more embodiments of the present disclosure, the purification step g1) is performed on the material recovered in step f1) of the methods as disclosed herein.

[0099] In one or more exemplary embodiments, the pH is adjusted to between pH 4.5 and 8.0 prior to adsorption onto a geopolymer or an adsorption column.

[0100] Adjustment of pH in step f1) or step g 1 ) 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.

[0101] Bacillus spp.

[0102] 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.

[0103] Thus, in one or more exemplary embodiments of the present disclosure, a bacteria belonging to the Bacillus spp. as used in the method M1 is any bacteria that belongs to the Bacillus genus. In one or more exemplary embodiments of the present disclosure, the Bacillus spp. as used in the method M1 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.

[0104] In one or more exemplary embodiments of the present disclosure, the Bacillus spp. as used in the method M1 is a bacteria belonging to the Bacillus amyloliquefaciens species.

[0105] In one or more exemplary embodiments of the present disclosure, 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.

[0106] In one or more exemplary embodiments of the present disclosure, 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.

[0107] In one or more exemplary embodiments of the present disclosure, 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.

[0108] 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.

[0109] Deposits and expert solution

[0110] The applicant requests that a sample of the deposited micro-organisms stated below may only be made available to an expert, until the date on which the patent is granted.

[0111] The strain Bacillus amyloliquefaciens was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 03 April 2012, under the accession No. DSM 25840.

[0112] 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 27032. 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.

[0113] 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.

[0114] The deposits were made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

[0115] BRIEF DESCRIPTION OF THE FIGURES

[0116] Figure 1

[0117] Fig.1 shows spore counts, cell counts and sporulation ratio of end-of-fermentatation fermentates from cultures of Bacillus amyloliquefaciens DSM 34003 incubated in BioLector Pro, at 1500 rpm, 33°C, pH7.8-8.0, for 40 hours, in 0.8 mL LC medium supplemented with glutamate and / or aspartate as depicted in table 1 .

[0118] EXAMPLES

[0119] Materials and methods

[0120] The experiments described herein were performed with Bacillus amyloliquefaciens DSM 34003 in BioLector Pro, flower-type plates (MF32C-BOH2), at 1500 rpm, 33°C, pH 7.8-8.0, for 40 h, in 0.8 mL LC medium. The LC-medium was supplemented with a concentration corresponding to 0-10 g / L glutamate and / or aspartate in the LC-medium as described in table 1 .

[0121] Table 1 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.

[0122] Example 1 - Sporulation ratio and spore yield of Bacillus amyloliquefaciens cultured in supplemented LC medium

[0123] Bacillus amyloliquefaciens cultures were incubated for 40 hours in flower type plates 0.8 mL LC medium supplemented with monosodium glutamate and / or aspartate (aspartic acid) as described under materials and methods. After 40 hours, the Bacillus spore counts and cell counts in the cultures were measured using flow cytometry.

[0124] As shown in figure 1 Bacillus amyloliquefaciens cultures in fermentation media supplemented with aspartate in a concentration of 7 (LC-var4) and 10 (LC-var5) g / L showed a respective increase in sporulation ratio to about 86% and 89% respectively (see figure 1) compared to a sporulation ratio of 79% in unsupplemented fermentation media (LC-varO) and about 83% in fermentation media supplemented with 3.4 g / L glutamate (LC / LC medium). Simultaneously, the spore yield was increased to about 1.1x1010(1.1 E+10) and 1.3x1 O10(1.3E+10) in LC-var-4 and LC-var5 compared to about 6x109in LC-varO and LC var1 and about 8x109(8E+9) in LC medium, LC-var2 and LC- var3 indicating a spore yield increase of about 60% in LC medium supplemented with aspartate in a concentration of 7 and 10 g / L compared to unsupplemented LC medium.

[0125] Interestingly, supplementing LC medium with 7.0 and 10.0 g / L glutamate did not have a beneficial effect on spore yield which remained comparable to levels obtained with LC medium. However, supplementing LC medium with 7.0 and 10.0 aspartate provided a clear increase in the sporulation ratio and yield when compared to LC medium and LC medium supplemented with glutamate. Supplementing LC medium with both 5.0 g / L aspartate and glutamate slightly increased the sporulation ratio and increased spore yield when compared to LC medium or LC supplemented with glutamate, although the spore yield did not reach the levels the cultures supplemented with higher amounts of aspartate.

[0126] Example 1 shows that aspartate supplementation of the fermentation medium increases B. amyloliquefaciens DSM 34003 spore concentration, yield and ratio, with spore counts over 1 E10 and a sporulation ratio above 80%. The aspartate supplementation to the production media may significantly increase the spore yield of and therefore contribute to productivity improvement in terms of reducing production costs of products containing this spore product. Therefore, this approach can be applied to all production processes of other Bacillus spp. where spores that are commercially relevant.

[0127] It is interesting to note that supplementation with glutamate did not result in the same increase in sporulation ratio, thus indicating supplementation with either one of the two amino acids result in opposite responses in Bacillus cultures (e.g. aspartate increasing sporulation and glutamate decreasing sporulation). This suggests that the increased sporulation ratio seen herein is not simply a response to a general nutritional boost caused by the addition of an amino acid to a fermentation medium, but rather a specific metabolic response by the Bacillus cultures to the addition of aspartate.

[0128] ITEMS

[0129] 1 . A method M1 for increasing sporulation ratio and / or spore yield of Bacillus spp. in a fermentation broth, the method comprising a1) providing a bacillus spp. b1) providing a suitable fermentation medium c1) supplementing the suitable fermentation medium with aspartate, thereby forming a supplemented fermentation medium d1) initiating a fermentation process by introduction of the bacillus spp. provided in step a1) into the supplemented fermentation medium provided in step c1), and e1) running the fermentation process. thereby providing a fermentation broth with an increased sporulation ratio of Bacillus spp. and / or an increased Bacillus spp. spore yield.

[0130] 2. The method M1 according to item 1 , for increasing the sporulation ratio of Bacillus spp. and increasing the Bacillus spp. spore yield in a fermentation broth.

[0131] 3. The method M1 according to any one of items 1-2, for increasing the sporulation ratio of Bacillus spp. in a fermentation broth.

[0132] 4. The method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with at least 3 g / L aspartate, at least 4 g / L aspartate, at least 5 g / L aspartate, 6 g / L aspartate, at least 7 g / L aspartate, at least 8 g / L aspartate, at least 9 g / L aspartate, or at least 10 g / L aspartate. The method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with at least 3 g / L aspartate. The method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with at least 5 g / L aspartate. The method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with at least 7 g / L aspartate. The method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with at least 10 g / L aspartate. The method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with up to 15 g / L aspartate, up to 18 g / L aspartate, up to 20 g / L aspartate, up to 22 g / L aspartate, up to 24 g / L aspartate, up to 26 g / L aspartate, up to 28 g / L aspartate, or up to 30 g / L. The method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 3-30 g / L aspartate, 5-25 g / L aspartate, 7-20 g / L aspartate, 10-15 g / L aspartate, 7-15 g / L aspartate, 10-20 g / L aspartate, 5-15 g / L aspartate, 5-13 g / L aspartate, 7-13 g / L aspartate, 9-11 g / L aspartate, 5 g / L aspartate, 6 g / L aspartate, 7 g / L aspartate, 8 g / L aspartate, 9 g / L aspartate, 10 g / L aspartate, 11 g / L aspartate, 12 g / L aspartate or 13 g / L aspartate, 14 g / L aspartate, or 15 g / L aspartate. The method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 7-15 g / L aspartate, 10-20 g / L aspartate, 5-15 g / L aspartate, 5-13 g / L aspartate, 7-13 g / L aspartate, or 9-11 g / L aspartate. The method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 7-15 g / L aspartate. The method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 10-20 g / L aspartate. 14. The method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 5-15 g / L aspartate.

[0133] 15. The method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 5-13 g / L aspartate.

[0134] 16. The method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 7-13 g / L aspartate.

[0135] 17. The method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 7-10 g / L aspartate.

[0136] 18. The method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 10-15 g / L aspartate.

[0137] 19. The method M1 according to any one of the preceding items, wherein the sporulation ratio is calculated as the number of bacillus spores divided by number of vegetative bacillus cells + the number bacillus spores.

[0138] 20. The method M1 according to any one of the preceding items, wherein the increased sporulation ratio is a sporulation ratio of at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89% or at least 90%.

[0139] 21. The method M1 according to any one of the preceding items, wherein the increased sporulation ratio is at least 80%.

[0140] 22. The method M1 according to any one of the preceding items, wherein the increased sporulation ratio is at least 81%.

[0141] 23. The method M1 according to any one of the preceding items, wherein the increased sporulation ratio is at least 82%.

[0142] 24. The method M1 according to any one of the preceding items, wherein the increased sporulation ratio is at least 85%. 25. The method M1 according to any one of the preceding items, wherein the increased sporulation ratio is about 89-90%.

[0143] 26. The method M1 according to any one of the preceding items, wherein the increased sporulation ratio falls within the range of 80-95%, 80-90%, 80-85%, 81-88%, 82-85, 80- 87%, 82-87%, 82-90%, 83-88%, 85-95%, 85-90%, 85-88%, 87-89%, 87-95%, 87-93% or 87-90%.

[0144] 27. The method M1 according to any one of the preceding items, wherein the increased sporulation ratio falls within the range of 80-85%.

[0145] 28. The method M1 according to any one of the preceding items, wherein the increased sporulation ratio falls within the range of 82-87%.

[0146] 29. The method M1 according to any one of the preceding items, wherein the increased sporulation ratio falls within the range of 83-88.

[0147] 30. The method M1 according to any one of the preceding items, wherein the increased sporulation ratio falls within the range of 80-90%.

[0148] 31. The method M1 according to any one of the preceding items, the method further comprising a recovery step f1), for recovering Bacillus spp. spores from the fermentation broth.

[0149] 32. The method M1 according to item 31 , wherein the recovery step f1) comprises a centrifugation step or filtration step.

[0150] 33. The method M1 according to any one of the preceding items, the method further comprising a purification step g1) for providing purified Bacillus spp. spores.

[0151] 34. The method M1 according to any one of the preceding items, wherein the 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. 35. The method M1 according to any one of the preceding items, wherein the Bacillus spp. is Bacillus amyloliquefaciens.

[0152] 36. The method M1 according to any one of the preceding items, wherein the 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.

[0153] 37. The method M1 according to any one of the preceding items, wherein the Bacillus spp. is B. amyloliquefaciens strain DSM 34003.

[0154] 38. A composition comprising a fermentation broth produced according to the method M1 of any one of the preceding items, a composition comprising the Bacillus spp. spores recovered in step f1) of the method M1 according to any one of items 27-32, or a composition comprising the purified Bacillus spp. spores obtained in step g1) of the method M1 according to any one of items 29-32.

[0155] 39. Use of a fermentation broth produced according to the method of any one of items 1-37 or a composition according to item 38 for controlling an infection in a plant caused by a fungal or bacterial pathogen.

[0156] 40. Use of a fermentation broth produced according to the method of any one of items 1-37 or a composition according to item 38 as a probiotic.

[0157] 41. Use of a fermentation broth produced according to the method of 1-37 or a composition according to item 38 in an animal feed.

[0158] 42. Use of a fermentation broth produced according to the method of any one of items 1-37 or a composition according to item 38 in an aquatic animal feed.

[0159] 43. Use of a fermentation broth produced according to the method of any one of items 1-37 or a composition according to item 38 as a nematocidal or antimicrobial agent. 44. Use of a fermentation broth produced according to the method of any one of items 1-37 or a composition according to item 38 as an antibacterial and / or antifungal agent. 45. A method for improving health and nutritional status of an animal comprising administering an animal feed comprising a fermentation broth produced according to the method of any one of items 1-37 or a composition according to item 38 to an animal.

[0160] 46. A method for improving health and nutritional status of an aquatic animal comprising administering an animal feed a fermentation broth produced according to the method of any one of items 1-37 or a composition according to item 38 to an aquatic animal.

[0161] 47. A method for controlling, preventing or ameliorating an infection in a plant caused by a fungal or bacterial pathogen comprising a fermentation broth produced according to the method of any one of items 1-37 or a composition according to item 38 to the plant.

[0162] References

[0163] 1 . M. Shoda: Bacterial Control of Plant Disease, Journal of Bioscience and Bioengineering, pp. 515-521 , 200.

[0164] 2. H. P. Bais, R. Fall and .J M. Vivanco: Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas s y r i n g e is facilitated by biofilm formation and surfactin production, Plant Physiology, vol. 134, pp. 307-319, 2004.

[0165] 3. T Stein: Bacillus subtilis antibiotics: structures, syntheses and specific functions, Molecular Microbiology, vol. 56, pp. 854-857, 2005.

[0166] 4. M. Ongena and P. Jacques: Bacillus lipopeptides: versatile weapons for plant disease biocontrol, Applied Microbiology and Biotechnology, vol. 16, No. 3, pp. 115-125, 2008.

Claims

CLAIMS1 . A method M1 for increasing sporulation ratio and / or spore yield of Bacillus spp. in a fermentation broth, the method comprising a1) providing a bacillus spp. b1) providing a suitable fermentation medium c1) supplementing the suitable fermentation medium with aspartate, thereby forming a supplemented fermentation medium d1) initiating a fermentation process by introduction of the bacillus spp. provided in step a1) into the supplemented fermentation medium provided in step c1), and e1) running the fermentation process. thereby providing a fermentation broth with an increased sporulation ratio of Bacillus spp. and / or an increased Bacillus spp. spore yield.

2. The method M1 according to claim 1 , for increasing the sporulation ratio of Bacillus spp. in a fermentation broth.

3. The method M1 according to any one of the preceding claims, wherein the suitable fermentation medium is supplemented with at least 5 g / L aspartate.

4. The method M1 according to any one of the preceding claims, wherein the suitable fermentation medium is supplemented with at least 7 g / L aspartate.

5. The method M1 according to any one of the preceding claims, wherein the suitable fermentation medium is supplemented with at least 10 g / L aspartate.

6. The method M1 according to any one of the preceding claims, wherein the suitable fermentation medium is supplemented with 5-13 g / L aspartate.

7. The method M1 according to any one of the preceding claims, wherein the increased sporulation ratio is at least 80%.

8. The method M1 according to any one of the preceding claims, wherein the increased sporulation ratio falls within the range of 80-90%.

9. The method M1 according to any one of the preceding claims, the method further comprising a recovery step f1), for recovering Bacillus spp. spores from the fermentation broth.

10. The method M1 according to any one of the preceding claims, the method further comprising a purification step g1 , for providing purified Bacillus spp. spores.11 . The method M1 according to any one of the preceding claims, wherein the 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.

12. A composition comprising a fermentation broth produced according to the method M1 of any one of the preceding claims, a composition comprising the Bacillus spp. spores recovered in step f1) of the method M1 according to any one of claims 9-11 , or a composition comprising the purified Bacillus spp. spores obtained in step g1) of the method M1 according to any one of claims 10-11.

13. Use of a fermentation broth produced according to the method of any one of claims 1-11 or a composition according to claim 12 as a probiotic.

14. Use of a fermentation broth produced according to the method of 1-11 or a composition according to claim 12 in an animal feed or in an aquatic animal feed.

15. Use of a fermentation broth produced according to the method of any one of claims 1-11 or a composition according to claim 12 as an antimicrobial, nematocidal, antibacterial or antifungal agent.

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