Improving fermentation output by media supplementation

Abstract

The present disclosure relates to methods for improving the output of a fermentation process by supplementing the fermentation medium.

Description

[0001] This disclosure relates to methods in the field of improving fermentation process yield by supplementing fermentation medium. Background Technology

[0002] Among microorganisms used for biocontrol, Bacillus species have attracted much attention due to the variety of antibiotic compounds they produce, their long shelf life, their rapid growth in cultures, and their ability to colonize leaf surfaces [1, 2, 3, 4].

[0003] In particular, certain Bacillus species, such as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus mycoides, Bacillus circulans, Bacillus megaterium, Bacillus pumilus, Bacillus mojavensis, and Bacillus thuringiensis, have exhibited antimicrobial activity.

[0004] The antimicrobial activity of these bacteria is due to their ability to produce lipopeptides such as surfactants, itursin, and fenestrogens, which differ in their amino acid sequences and fatty acid chain branching. Surfactants exhibit high antibacterial activity, while itursin and fenestrogens are known for their antifungal activity[4].

[0005] The prior art describes the use of Bacillus subtilis and Bacillus amyloliquefaciens for controlling various pathogenic microorganisms in a wide variety of crops, including fruit and vegetable crops such as blackberries, grapes, raspberries, strawberries, tomatoes, cucumbers, black pepper, oranges, melons, apples, peaches, custard apples, bananas, papayas, mangoes, and kiwifruit. EP 2311936 discloses a Bacillus subtilis strain KS1 (NITE BP-569) as a biocontrol agent against several plant pathogenic microorganisms in vine crops. WO 98 / 21968 discloses an antibiotic produced by Bacillus subtilis A0153 (ATCC 55614) effective against bacterial and fungal infections; and also discloses methods for protecting plants, including the application of these antibiotic compounds.

[0006] WO 9850422, WO 9909819 and W00029426 disclose antibiotic compounds produced by Bacillus subtilis strain AQ713 (equivalent to strain QST713, deposited as NRRL B-21661) and its mutants, which exhibit insecticidal, nematode-killing, antifungal and antibacterial activities.

[0007] US 2011 / 0318386 describes a method for inducing systemic resistance against various pathogens using biocontrol agents of the genus Bacillus, particularly isolated Bacillus mojaveniformis 203-7 and isolated species of Bacillus mycosis. Furthermore, ES 2345969 describes a plant tonic for application to plantain pseudostems of bananas and plantains, comprising Bacillus subtilis, Trichoderma viride, and B. megaterium var. phosphaticum.

[0008] WO 14178032 discloses a process for improving biomass production from Bacillus microorganisms, including Bacillus subtilis EA-CB0015 and Bacillus amyloliquefaciens EA-CB0959. The biomass obtained by this process can be separated from the culture medium using conventional centrifugation or microfiltration methods, while active metabolites can be obtained by solvent extraction, precipitation, adsorption, or chromatography. In a preferred embodiment of WO 14178032, the amount of biomass obtained from the Bacillus species can range from 3.0 to 20.0 g / L.

[0009] Bacteria belonging to the genus Bacillus are used in many different applications, including in animal feed and in methods for improving the nutritional status of animals. For example, WO 2013153159 discloses the use of Bacillus spores in animal feed, while WO 2004 / 095939 discloses the use of Bacillus spores in aquatic animal feed, and both WO 2010 / 070005 and US 2003 / 0124104 disclose the probiotic use of Bacillus spores, and in particular the use of Bacillus subtilis spores in probiotics.

[0010] However, establishing fermentation processes is expensive and time-consuming, so maximizing the output of fermentation processes is key to ensuring the profitability of the materials produced in fermentation processes, and the development of optimized fermentation strategies is highly anticipated. Summary of the Invention

[0011] In its broadest sense, this disclosure relates to a method for improving the spore formation rate of Bacillus species in the fermentation broth produced during a fermentation process by supplementing the fermentation medium with aspartic acid.

[0012] In one aspect, this disclosure relates to a composition comprising fermentation broth produced according to the methods disclosed herein or spores recovered and / or purified from fermentation broth produced according to the methods disclosed herein.

[0013] On the other hand, this disclosure relates to the use of fermentation broths or compositions produced according to the methods disclosed herein for the prevention and treatment of infections in plants caused by fungal or bacterial pathogens.

[0014] On the other hand, this disclosure relates to the use of fermentation broth or compositions produced according to the methods disclosed herein as probiotics.

[0015] On the other hand, this disclosure relates to the use of fermentation broths or compositions produced according to the methods disclosed herein in animal feed.

[0016] On the other hand, this disclosure relates to the use of fermentation broths or compositions produced according to the methods disclosed herein in aquatic animal feed.

[0017] On the other hand, this disclosure relates to the use of fermentation broth or compositions produced according to the methods disclosed herein as nematicides or antimicrobial agents.

[0018] On the other hand, this disclosure relates to the use of fermentation broth or compositions produced according to the methods disclosed herein as antibacterial and / or antifungal agents.

[0019] On the other hand, this disclosure relates to the use of fermentation broth or compositions produced according to the methods disclosed herein in methods for improving the nutritional status of animals or aquatic animals.

[0020] On the other hand, this disclosure relates to the use of fermentation broth or compositions produced according to the methods disclosed herein in methods for preventing, controlling, or improving infections in plants caused by fungal or bacterial pathogens. Detailed Implementation

[0021] This article unexpectedly shows that when bacteria belonging to the Bacillus amyloliquefaciens species are cultured in an aspartic acid-supplemented fermentation medium, the addition of aspartic acid (aspartic acid) to the fermentation medium leads to an increase in spore yield and spore formation rate (see Example 1 and...). Figure 1 ).

[0022] like Figure 1The results showed that Bacillus amyloliquefaciens cultures supplemented with 7 g / L (LC-var4) and 10 g / L (LC-var5) aspartic acid (Table 1) exhibited spore formation rates increased to approximately 86% and 89%, respectively. In contrast, the spore formation rate was 79% in the unsupplemented fermentation medium (LC-var0) and approximately 83% in the fermentation medium supplemented with 3.4 g / L glutamate (LC / LC medium), with higher glutamate supplementation leading to lower spore formation rates. Simultaneously, spore yields increased to approximately 1.1 × 10⁻⁶ in both LC-var4 and LC-var5. 10 (1.1E+10) and 1.3 × 10 10 (1.3E+10), in comparison, the spore yield in LC-var0 is approximately 6 × 10⁻⁶. 9 Furthermore, the spore yield in LC-var0 is approximately 8 × 10⁻⁶. 9 (8E+10).

[0023] Since the spores of Bacillus amyloliquefaciens (and more broadly, spores of Bacillus species) are of interest in providing antimicrobial protection rather than vegetative cells, increasing the spore formation rate is a very important parameter because it determines the cost / benefit of the fermentation process in terms of output per unit of resource / sugar input.

[0024] Therefore, this disclosure relates to providing methods for improving spore formation rate and / or spore yield in fermentation containing Bacillus species.

[0025] Method M1 for improving spore formation rate in Bacillus fermentation In this disclosure, it was unexpectedly found that supplementing the fermentation medium with aspartic acid increased the sporulation rate of *Bacillus amyloliquefaciens*. This effect is considered likely to also apply to other members of the *Bacillus* genus.

[0026] This improved spore formation rate is achieved by providing a Bacillus species, providing a suitable fermentation medium, supplementing the fermentation medium with glutamate, and initiating and operating the fermentation process by introducing the provided Bacillus species into the supplemented fermentation medium.

[0027] In particular, this disclosure relates to a method M1 for increasing the spore formation rate and / or spore yield of Bacillus species in fermentation broth, the method comprising: a1) Provide species of the genus Bacillus. b1) Provide a suitable fermentation medium, c1) Supplementing a suitable fermentation medium with aspartic acid to form a supplemented fermentation medium. d1) The fermentation process is initiated by introducing the Bacillus species provided in step a1) into the supplemented fermentation medium provided in step c1); and e1) Run the fermentation process, This provides a fermentation broth with increased spore formation rate and / or increased spore yield of Bacillus species.

[0028] In one or more exemplary embodiments disclosed herein, method M1 relates to a method for providing a fermentation broth having an increased spore formation rate of Bacillus species.

[0029] The key to achieving an improved spore formation rate lies in supplementing the culture medium with aspartic acid. Methods for producing fermentation media and supplementing them with desired additional nutrients are generally described in the prior art. Therefore, obtaining a supplemented fermentation medium using such methods is considered routine for those skilled in the art.

[0030] In the context of this invention, supplementing the fermentation medium with aspartic acid involves supplementing the fermentation medium with 3-30 g / L aspartic acid, 5-25 g / L aspartic acid, 7-20 g / L aspartic acid, 10-15 g / L aspartic acid, 7-15 g / L aspartic acid, 10-20 g / L aspartic acid, 5-15 g / L aspartic acid, 5-13 g / L aspartic acid, 7-13 g / L aspartic acid, 9-11 g / L aspartic acid, 5 g / L aspartic acid, 6 g / L aspartic acid, 7 g / L aspartic acid, 8 g / L aspartic acid, 9 g / L aspartic acid, 10 g / L aspartic acid, 11 g / L aspartic acid, 12 g / L aspartic acid, or 13 g / L aspartic acid, 14 g / L aspartic acid, or 15 g / L aspartic acid.

[0031] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein involves supplementing the suitable fermentation medium provided in step b1) with at least 3 g / L aspartic acid, at least 4 g / L aspartic acid, at least 5 g / L aspartic acid, 6 g / L aspartic acid, at least 7 g / L aspartic acid, at least 8 g / L aspartic acid, at least 9 g / L aspartic acid, at least 10 g / L aspartic acid, up to 15 g / L aspartic acid, up to 18 g / L aspartic acid, up to 20 g / L aspartic acid, up to 22 g / L aspartic acid, up to 24 g / L aspartic acid, up to 26 g / L aspartic acid, up to 28 g / L aspartic acid, or up to 30 g / L aspartic acid.

[0032] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein involves supplementing the suitable fermentation medium provided in step b1) with at least 3 g / L aspartic acid.

[0033] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein involves supplementing the suitable fermentation medium provided in step b1) with at least 5 g / L aspartic acid.

[0034] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein involves supplementing the suitable fermentation medium provided in step b1) with at least 7 g / L aspartic acid.

[0035] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein involves supplementing the suitable fermentation medium provided in step b1) with at least 8 g / L of aspartic acid.

[0036] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein involves supplementing the suitable fermentation medium provided in step b1) with at least 9 g / L aspartic acid.

[0037] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein involves supplementing the suitable fermentation medium provided in step b1) with at least 10 g / L aspartic acid.

[0038] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein involves supplementing the suitable fermentation medium provided in step b1) with 3-30 g / L aspartic acid, 5-25 g / L aspartic acid, 7-20 g / L aspartic acid, 10-15 g / L aspartic acid, 7-15 g / L aspartic acid, 10-20 g / L aspartic acid, 5-15 g / L aspartic acid, 5-13 g / L aspartic acid, 7-13 g / L aspartic acid, 9-11 g / L aspartic acid, 5 g / L aspartic acid, 6 g / L aspartic acid, 7 g / L aspartic acid, 8 g / L aspartic acid, 9 g / L aspartic acid, 10 g / L aspartic acid, 11 g / L aspartic acid, 12 g / L aspartic acid, or 13 g / L aspartic acid, 14 g / L aspartic acid, or 15 g / L aspartic acid.

[0039] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein involves supplementing the suitable fermentation medium provided in step b1) with 7-15 g / L aspartic acid, 10-20 g / L aspartic acid, 5-15 g / L aspartic acid, 7-13 g / L aspartic acid, or 9-11 g / L aspartic acid.

[0040] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein involves supplementing the suitable fermentation medium provided in step b1) with 10-20 g / L aspartic acid.

[0041] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein involves supplementing 7-15 g / L of aspartic acid into a suitable fermentation medium provided in step b1).

[0042] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein involves supplementing 5-15 g / L of aspartic acid into a suitable fermentation medium provided in step b1).

[0043] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein involves supplementing 7-13 g / L of aspartic acid into a suitable fermentation medium provided in step b1).

[0044] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein involves supplementing 7-10 g / L of aspartic acid into a suitable fermentation medium provided in step b1).

[0045] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein involves supplementing the suitable fermentation medium provided in step b1) with 10-15 g / L aspartic acid.

[0046] In one or more exemplary embodiments, step c1) of the method M1 disclosed herein involves supplementing 7-15 g / L of aspartic acid into a suitable fermentation medium provided in step b1).

[0047] In the context of this disclosure, the spore formation rate is calculated as the number of Bacillus species spores in the fermentation broth divided by the sum of Bacillus species spores and Bacillus species vegetative cells.

[0048] In one or more exemplary embodiments, the increased spore formation rate of method M1 is 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%.

[0049] In one or more exemplary embodiments, the improved spore formation rate of method M1 is at least 80%.

[0050] In one or more exemplary embodiments, the improved spore formation rate of method M1 is at least 81%.

[0051] In one or more exemplary embodiments, the improved spore formation rate of method M1 is at least 82%.

[0052] In one or more exemplary embodiments, the improved spore formation rate of method M1 is at least 83%.

[0053] In one or more exemplary embodiments, the improved spore formation rate of method M1 is at least 84%.

[0054] In one or more exemplary embodiments, the improved spore formation rate of method M1 is at least 85%.

[0055] In one or more exemplary embodiments, the improved spore formation rate of method M1 is at least 86%.

[0056] In one or more exemplary embodiments, the improved spore formation rate of method M1 is at least 87%.

[0057] In one or more exemplary embodiments, the improved spore formation rate of method M1 is at least 88%.

[0058] In one or more exemplary embodiments, the improved spore formation rate of method M1 is at least 89%.

[0059] In one or more exemplary embodiments, the improved spore formation rate of method M1 is a spore formation rate falling 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%.

[0060] In one or more exemplary embodiments, the improved spore formation rate of method M1 is a spore formation rate falling within the range of 80%-85%.

[0061] In one or more exemplary embodiments, the improved spore formation rate of method M1 is a spore formation rate falling within the range of 82%-87%.

[0062] In one or more exemplary embodiments, the improved spore formation rate of method M1 is a spore formation rate falling within the range of 83%-88%.

[0063] In one or more exemplary embodiments, the improved spore formation rate of method M1 is a spore formation rate falling within the range of 85%-89%.

[0064] In one or more exemplary embodiments, the improved spore formation rate of method M1 is a spore formation rate falling within the range of 80%-90%.

[0065] Methods for recovering Bacillus spores from fermentation processes or fermentation media are well known in the art, and the selection of a suitable recovery method is considered a conventional choice that a person skilled in the art would make based on the circumstances. In the context of this invention, the separate recovery of spores from the fermentation medium during or at the end of fermentation is generally considered.

[0066] Therefore, in some cases, it is desirable to separate Bacillus spores from the fermentation broth. In these cases, the method M1 disclosed herein includes an additional step, namely step f1, involving the recovery of Bacillus spores from the fermentation broth.

[0067] In one or more exemplary embodiments, method M1 as defined herein further includes a recovery step f1 for recovering Bacillus spores from the fermentation broth.

[0068] In one or more exemplary embodiments, the recovery step f1) for recovering Bacillus spores from the fermentation broth includes a centrifugation or filtration step.

[0069] In some cases, it is desirable to purify Bacillus spores produced according to the methods disclosed herein. In these cases, the method M1 disclosed herein includes an additional step, namely step g, involving the purification of Bacillus spores from the fermentation broth.

[0070] In one or more exemplary embodiments, method M1 as defined herein further includes a purification step g1 for providing purified Bacillus spores.

[0071] In some cases, it is desirable to include both a recovery step and a purification step in a method M1 as disclosed herein. Therefore, in one or more exemplary embodiments, a method M1 as disclosed herein includes both a recovery step f1) and a purification step g1).

[0072] In one or more exemplary embodiments, this disclosure relates to a composition comprising spores of a Bacillus species produced according to method M1.

[0073] In one or more exemplary embodiments, this disclosure relates to a composition comprising a fermentation broth produced according to method M1, a composition comprising Bacillus spores recovered in step f1) of method M1, and / or a composition comprising purified Bacillus spores produced according to step g1) of method M1.

[0074] In one or more exemplary embodiments, this disclosure relates to the use of compositions produced by method M1, fermentation broth produced by method M1, Bacillus spores recovered in step f1) of method M1, and / or purified spores provided in step g1) of method M1 for the prevention and control of infections in plants caused by fungal or bacterial pathogens.

[0075] In one or more exemplary embodiments, this disclosure relates to a method for preventing, controlling, or improving infections in plants caused by fungal or bacterial pathogens, the method comprising applying to the plant a composition produced by method M1, a fermentation broth produced by method M1, Bacillus spores recovered in step f1) of method M1, and / or purified spores provided in step g1) of method M1.

[0076] In one or more exemplary embodiments, this disclosure relates to the use of compositions produced by method M1, fermentation broth produced by method M1, Bacillus spores recovered in step f1) of method M1, and / or purified spores provided in step g1) of method M1 for the prevention and control of infections caused by fungal pathogens in plants.

[0077] In one or more exemplary embodiments, this disclosure relates to the use of a composition produced by method M1 for the prevention and treatment of infections in plants caused by bacterial pathogens.

[0078] In one or more exemplary embodiments, this disclosure relates to the use of compositions produced by method M1, fermentation broths produced by method M1, Bacillus spores recovered in step f1) of method M1, and / or purified spores provided in step g1) of method M1 as probiotics.

[0079] In one or more exemplary embodiments, this disclosure relates to the use of compositions produced by method M1, fermentation broths produced by method M1, Bacillus spores recovered in step f1) of method M1, and / or purified spores provided in step g1) of method M1 in animal feed.

[0080] In one or more exemplary embodiments, this disclosure relates to a method for improving the health and nutritional status of an animal, the method comprising administering animal feed to the animal comprising a composition produced by method M1, a fermentation broth produced by method M1, Bacillus spores recovered in step f1) of method M1, and / or purified spores provided in step g1) of method M1.

[0081] In one or more exemplary embodiments, this disclosure relates to the use of compositions produced by method M1, fermentation broths produced by method M1, Bacillus spores recovered in step f1) of method M1, and / or purified spores provided in step g1) of method M1 in aquatic animal feed.

[0082] In one or more exemplary embodiments, this disclosure relates to a method for improving the health and nutritional status of an aquatic animal, the method comprising administering an aquatic animal feed to the aquatic animal comprising a composition produced by method M1, a fermentation broth produced by method M1, Bacillus spores recovered in step f1) of method M1, and / or purified spores provided in step g1) of method M1.

[0083] In one or more exemplary embodiments, this disclosure relates to the use of compositions produced by method M1, fermentation broth produced by method M1, Bacillus spores recovered in step f1) of method M1, and / or purified spores provided in step g1) of method M1 as nematicides or antimicrobial agents.

[0084] In one or more exemplary embodiments, this disclosure relates to the use of compositions produced by method M1, fermentation broth produced by method M1, Bacillus spores recovered in step f1) of method M1, and / or purified spores provided in step g1) of method M1 as antibacterial and / or antifungal agents.

[0085] In one or more exemplary embodiments, this disclosure relates to the use of compositions produced by method M1, fermentation broth produced by method M1, Bacillus spores recovered in step f1) of method M1, and / or purified spores provided in step g1) of method M1 as antibacterial agents.

[0086] In one or more exemplary embodiments, this disclosure relates to the use of compositions produced by method M1, fermentation broth produced by method M1, Bacillus spores recovered in step f1) of method M1, and / or purified spores provided in step g1) of method M1 as antifungal agents.

[0087] Recovery and purification steps The recovery step f1) and purification step g1) of method M1 as defined herein include one or more of the following steps: 1) Filtering steps, 2) Centrifugation step, 3) pH adjustment steps, 4) An adsorption step, which includes adsorbing one or more lipopeptides onto an adsorption column. 5) An adsorption step, which includes adsorbing one or more lipopeptides onto the geopolymer. 6) Chromatographic steps, 7) CaCl2 flocculation step, 8) Solvent extraction step.

[0088] Geopolymers that can be used in the methods disclosed herein include kieselguhr, diatomite, diatomaceous earth, kaolin (Chinese clay), bentonite, talc, volcanic ash, volcanic rock, clay, perlite, lignin, drilling mud, diatomic earth, and synthetic silica.

[0089] In one or more embodiments of this disclosure, the material recovered in step f1) of the method disclosed herein is subjected to a purification step g1).

[0090] In one or more exemplary embodiments, the pH is adjusted to between pH 4.5 and 8.0 before adsorption onto the geopolymer or adsorption column.

[0091] The pH adjustment in step f1) or step g1) includes adjusting the pH to 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, or 7.5, or adjusting the pH within the range of 4.5-8.0, 5.0-7.0, or 5.5-7.0.

[0092] Bacillus species In the context of this disclosure, bacteria belonging to the genus *Bacillus* are any bacteria within the genus *Bacillus*. The genus *Bacillus* is well-defined in the prior art, and the selection and identification of bacterial species belonging to the genus *Bacillus* is a routine task for those skilled in the art.

[0093] Therefore, in one or more exemplary embodiments disclosed herein, the bacteria belonging to the genus Bacillus used, such as in method M1, are any bacteria belonging to the genus Bacillus.

[0094] In one or more exemplary embodiments disclosed herein, the Bacillus species used in method M1 are bacteria belonging to a list of species selected from the following: Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus mycosis fungoides, Bacillus circulans, Bacillus megaterium, Bacillus pumilus, Bacillus mojaveii, Bacillus thuringiensis, Bacillus simplex, Bacillus safensis, Bacillus atrophaeous, Bacillus methylotrophicus, Bacillus siamensis, Bacillus vallismortis, and Bacillus tequilensis.

[0095] In one or more exemplary embodiments disclosed herein, the Bacillus species used in method M1 is a bacterium belonging to the Bacillus amyloliquefaciens species.

[0096] In one or more exemplary embodiments disclosed herein, the Bacillus species used in method M1 may be selected from the list consisting of: Bacillus amyloliquefaciens subsp. amyloliquefaciens, Bacillus amyloliquefaciens subsp. plantarum, Bacillus amyloliquefaciens strain with DSMZ accession number DSM 25840, Bacillus amyloliquefaciens strain with DSMZ accession number DSM 27032, Bacillus amyloliquefaciens strain with DSMZ accession number DSM 27033, and Bacillus amyloliquefaciens strain with DSMZ accession number DSM 34003.

[0097] In one or more exemplary embodiments disclosed herein, the Bacillus species used in method M1 are selected from the list of Bacillus amyloliquefaciens strains consisting of: DSM 25840, DSM 27032, DSM 27033 and DSM34003.

[0098] In one or more exemplary embodiments disclosed herein, the Bacillus species used in method M1 are selected from the list of Bacillus amyloliquefaciens strains consisting of DSM 25840, DSM 27032 and DSM 27033.

[0099] In one or more exemplary embodiments disclosed herein, the Bacillus species used, such as in method M1, is Bacillus amyloliquefaciens strain DSM 34003.

[0100] Preservation and expert solutions The applicant requests that, prior to the date of patent grant, the deposited microbial samples described below be provided only to experts.

[0101] The strain *Bacillus amyloliquefaciens* was deposited on April 3, 2012, with accession number DSM 25840 at the German Center for Microbiology and Cell Culture (DSMZ) GmbH (Inhoffenstr. 7B, D-38124Braunschweig, Germany).

[0102] The strain *Bacillus amyloliquefaciens* was deposited on March 21, 2013, with accession number DSM 27032 at the German Center for Microbial and Cell Culture Collection (DSMZ) GmbH (7B Inhofenstrasse, Braunschweig, Germany, D-38124).

[0103] The strain *Bacillus amyloliquefaciens* was deposited on March 21, 2013, with accession number DSM 27033 at the German Center for Microbial and Cell Culture Collection (DSMZ) GmbH (7B Inhofenstrasse, Braunschweig, Germany, D-38124).

[0104] The strain *Bacillus amyloliquefaciens* was deposited on August 24, 2021, with accession number DSM 34003 at the German Center for Microbial and Cell Culture Collection (DSMZ) GmbH (7B Inhofenstrasse, Braunschweig, Germany, D-38124).

[0105] The deposit was made in accordance with the Budapest Treaty on the international recognition of the deposit of microorganisms for patent proceedings purposes. Attached Figure Description

[0106] Figure 1 Figure 1 The spore count, cell count, and spore formation rate of the fermentation product are shown at the end of fermentation of Bacillus amyloliquefaciens DSM 34003 culture in BioLector Pro at 1500 rpm, 33 °C, pH 7.8–8.0 in 0.8 mL of LC medium supplemented with glutamic acid and / or aspartic acid (as depicted in Table 1).

[0107] Example Materials and Methods The *Bacillus amyloliquefaciens* DSM 34003 strain used in this study was subjected to 40 h of BioLector Pro flower-type plates (MF32C-BOH2) at 1500 rpm, 33°C, and pH 7.8–8.0 in 0.8 mL LC medium. The concentrations of glutamate and / or aspartic acid supplemented to the LC medium corresponded to 0–10 g / L in the LC medium as described in Table 1.

[0108] Table 1 Table 1: Glutamic acid and / or aspartic acid supplementation in LC medium variants during an experiment using Bacillus amyloliquefaciens DSM 34003 in BioLector Pro at 33°C and pH 7.8–8.0 for 40 h.

[0109] Example 1 – Spore formation rate and spore yield of Bacillus amyloliquefaciens cultured in supplemented LC medium Bacillus amyloliquefaciens cultures were incubated for 40 hours in 0.8 mL of LC medium supplemented with monosodium glutamate and / or aspartic acid (aspartic acid) in a cloverleaf plate, as described in Materials and Methods. After 40 hours, the spore and cell counts of Bacillus spp. in the cultures were measured by flow cytometry.

[0110] like Figure 1 The results showed that Bacillus amyloliquefaciens cultures supplemented with aspartic acid at concentrations of 7 (LC-var4) and 10 (LC-var5) g / L exhibited sporulation rates that increased to approximately 86% and 89%, respectively (see [link to original text]). Figure 1 In comparison, the spore formation rate was 79% in the unsupplemented fermentation medium (LC-var0) and approximately 83% in the fermentation medium supplemented with 3.4 g / L glutamic acid (LC / LC medium). Meanwhile, spore yields in LC-var-4 and LC-var-5 increased to approximately 1.1 × 10⁻⁶. 10 (1.1E+10) and 1.3 × 10 10 (1.3E+10), in comparison, the spore yield in LC-var0 and LC var1 is approximately 6 × 10⁻⁶. 9 The spore yield in LC medium, LC-var2, and LC-var3 was approximately 8 × 10⁻⁶. 9(8E+9) indicates that spore production was increased by approximately 60% in LC media supplemented with 7 and 10 g / L aspartic acid compared to unsupplemented LC media.

[0111] Interestingly, supplementing LC medium with 7.0 and 10.0 g / L glutamic acid had no beneficial effect on spore yield, remaining comparable to levels obtained with LC medium alone. However, supplementing LC medium with 7.0 and 10.0 g / L aspartic acid provided a significant increase in spore formation rate and yield compared to both LC medium and LC medium supplemented with glutamic acid. When compared to LC medium or LC medium supplemented with glutamic acid, supplementing LC medium with 5.0 g / L aspartic acid and glutamic acid slightly increased both spore formation rate and spore yield, but the spore yield did not reach the level of cultures supplemented with higher amounts of aspartic acid.

[0112] Example 1 demonstrates that aspartic acid supplementation in the fermentation medium improves the spore concentration, yield, and ratio of *Bacillus amyloliquefaciens* DSM 34003, with spore counts exceeding 1E10 and spore formation rates exceeding 80%. Supplementing the production medium with aspartic acid significantly increases spore yield and thus contributes to productivity improvements in reducing the production costs of products containing this spore product. Therefore, this method can be applied to all production processes of other *Bacillus* species where spores are commercially relevant.

[0113] Interestingly, it was noted that glutamate supplementation did not lead to the same increase in sporulation rate, thus indicating that supplementation of either of the two amino acids produces opposite responses in Bacillus cultures (e.g., aspartic acid increases sporulation, while glutamate decreases it). This suggests that the increased sporulation rate observed in this study is not merely a response to a general nutritional enhancement caused by the addition of amino acids to the fermentation medium, but rather a specific metabolic response of Bacillus cultures to the addition of aspartic acid.

[0114] project 1. A method M1 for increasing the spore formation rate and / or spore yield of Bacillus species in fermentation broth, the method comprising: a1) Provide species of the genus Bacillus. b1) Provide a suitable fermentation medium, c1) Supplement the suitable fermentation medium with aspartic acid to form a supplemented fermentation medium. d1) The fermentation process is initiated by introducing the Bacillus species provided in step a1) into the supplemented fermentation medium provided in step c1); and e1) Run the fermentation process described above. This provides a fermentation broth with increased spore formation rate and / or increased spore yield of Bacillus species.

[0115] 2. The method M1 according to Project 1, wherein the method is used to increase the spore formation rate of the Bacillus species in the fermentation broth and to increase the spore yield of the Bacillus species.

[0116] 3. Method M1 according to any one of items 1-2, the method being used to increase the spore formation rate of the Bacillus species in the fermentation broth.

[0117] 4. Method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with at least 3 g / L aspartic acid, at least 4 g / L aspartic acid, at least 5 g / L aspartic acid, 6 g / L aspartic acid, at least 7 g / L aspartic acid, at least 8 g / L aspartic acid, at least 9 g / L aspartic acid, or at least 10 g / L aspartic acid.

[0118] 5. Method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with at least 3 g / L aspartic acid.

[0119] 6. Method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with at least 5 g / L aspartic acid.

[0120] 7. Method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with at least 7 g / L aspartic acid.

[0121] 8. Method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with at least 10 g / L aspartic acid.

[0122] 9. Method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with up to 15 g / L aspartic acid, up to 18 g / L aspartic acid, up to 20 g / L aspartic acid, up to 22 g / L aspartic acid, up to 24 g / L aspartic acid, up to 26 g / L aspartic acid, up to 28 g / L aspartic acid, or up to 30 g / L aspartic acid.

[0123] 10. Method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 3-30 g / L aspartic acid, 5-25 g / L aspartic acid, 7-20 g / L aspartic acid, 10-15 g / L aspartic acid, 7-15 g / L aspartic acid, 10-20 g / L aspartic acid, 5-15 g / L aspartic acid, 5-13 g / L aspartic acid, 7-13 g / L aspartic acid, 9-11 g / L aspartic acid, 5 g / L aspartic acid, 6 g / L aspartic acid, 7 g / L aspartic acid, 8 g / L aspartic acid, 9 g / L aspartic acid, 10 g / L aspartic acid, 11 g / L aspartic acid, 12 g / L aspartic acid, or 13 g / L aspartic acid, 14 g / L aspartic acid, or 15 g / L aspartic acid.

[0124] 11. Method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 7-15 g / L aspartic acid, 10-20 g / L aspartic acid, 5-15 g / L aspartic acid, 5-13 g / L aspartic acid, 7-13 g / L aspartic acid, or 9-11 g / L aspartic acid.

[0125] 12. Method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 7-15 g / L aspartic acid.

[0126] 13. Method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 10-20 g / L aspartic acid.

[0127] 14. Method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 5-15 g / L aspartic acid.

[0128] 15. Method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 5-13 g / L aspartic acid.

[0129] 16. Method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 7-13 g / L aspartic acid.

[0130] 17. Method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 7-10 g / L aspartic acid.

[0131] 18. Method M1 according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 10-15 g / L aspartic acid.

[0132] 19. Method M1 according to any one of the preceding items, wherein the spore formation rate is calculated as the number of Bacillus spores divided by the number of Bacillus cells + the number of Bacillus spores.

[0133] 20. The method M1 according to any one of the preceding items, wherein the improved spore formation rate is 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%.

[0134] 21. The method M1 according to any one of the preceding items, wherein the increased spore formation rate is at least 80%.

[0135] 22. The method M1 according to any one of the preceding items, wherein the increased spore formation rate is at least 81%.

[0136] 23. The method M1 according to any one of the foregoing items, wherein the increased spore formation rate is at least 82%.

[0137] 24. The method M1 according to any one of the preceding items, wherein the improved spore formation rate is at least 85%.

[0138] 25. The method M1 according to any one of the preceding items, wherein the increased spore formation rate is about 89%-90%.

[0139] 26. The method M1 according to any one of the preceding items, wherein the increased spore formation rate 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%.

[0140] 27. The method M1 according to any one of the preceding items, wherein the increased spore formation rate falls within the range of 80%-85%.

[0141] 28. Method M1 according to any one of the preceding items, wherein the increased spore formation rate falls within the range of 82%-87%.

[0142] 29. The method M1 according to any one of the preceding items, wherein the increased spore formation rate falls within the range of 83%-88%.

[0143] 30. The method M1 according to any one of the preceding items, wherein the increased spore formation rate falls within the range of 80%-90%.

[0144] 31. The method M1 according to any one of the foregoing items, the method further comprising a recovery step f1 for recovering Bacillus spores from the fermentation broth.

[0145] 32. The method M1 according to item 31, wherein the recovery step f1) includes a centrifugation step or a filtration step.

[0146] 33. The method M1 according to any one of the foregoing items, the method further comprising a purification step g1 for providing purified Bacillus spores.

[0147] 34. Method M1 according to any one of the preceding items, wherein the Bacillus species is selected from one of the following list: Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus mycosis fungoides, Bacillus circumflex, Bacillus megaterium, Bacillus pumilus, Bacillus mojaveii, Bacillus thuringiensis, Bacillus simplex, Bacillus safortus, Bacillus atrophus, Bacillus methyltrophicus, Bacillus sicca, Bacillus oryzae, and Bacillus tekirae.

[0148] 35. Method M1 according to any one of the preceding items, wherein the Bacillus species is Bacillus amyloliquefaciens.

[0149] 36. Method M1 according to any one of the preceding items, wherein the Bacillus species is selected from the list of Bacillus amyloliquefaciens strains consisting of: Bacillus amyloliquefaciens subsp. amyloliquefaciens, Bacillus amyloliquefaciens plantarum, Bacillus amyloliquefaciens strain with DSMZ accession number DSM 25840, Bacillus amyloliquefaciens strain with DSMZ accession number DSM 27032, Bacillus amyloliquefaciens strain with DSMZ accession number DSM 27033, and Bacillus amyloliquefaciens strain with DSMZ accession number DSM34003.

[0150] 37. Method M1 according to any one of the preceding items, wherein the Bacillus species is Bacillus amyloliquefaciens strain DSM 34003.

[0151] 38. A composition comprising fermentation broth produced by method M1 according to any one of the preceding items, a composition comprising the spores of the Bacillus species recovered in step f1) of method M1 according to any one of items 27-32, or a composition comprising the purified spores of the Bacillus species obtained in step g1) of method M1 according to any one of items 29-32.

[0152] 39. Use of the fermentation broth produced by any one of items 1-37 or the composition described in item 38 for the prevention and treatment of infections in plants caused by fungal or bacterial pathogens.

[0153] 40. Use of the fermentation broth produced by any one of items 1-37 or the composition described in item 38 as a probiotic.

[0154] 41. Use of the fermentation broth produced by any one of items 1-37 or the composition described in item 38 in animal feed.

[0155] 42. Use of the fermentation broth produced by any one of items 1-37 or the composition described in item 38 in aquatic animal feed.

[0156] 43. Use of the fermentation broth produced by any one of items 1-37 or the composition described in item 38 as a nematicide or antimicrobial agent.

[0157] 44. Use of fermentation broth produced by any one of items 1-37 or composition according to item 38 as an antibacterial and / or antifungal agent.

[0158] 45. A method for improving the health and nutritional status of an animal, the method comprising administering to the animal an animal feed comprising a fermentation broth produced by any one of items 1-37 or a composition according to item 38.

[0159] 46. ​​A method for improving the health and nutritional status of aquatic animals, the method comprising administering to the aquatic animals an animal feed comprising a fermentation broth produced by any one of items 1-37 or a composition according to item 38.

[0160] 47. A method for preventing, controlling, or improving infections in plants caused by fungal or bacterial pathogens, the method comprising applying to the plant a fermentation broth produced by any one of items 1-37 or a composition according to item 38. References 1. M. Shoda: Bacterial Control of Plant Disease, Journal of Bioscience and Bioengineering, pp. 515-521, 2000. 2. H. P. Bais, R. Fall and J M. Vivanco: Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringe is facilitated by biofilm formation and surfactin production, Plant Physiology, Vol. 134, pp. 307-319, 2004. 3. T Stein: Bacillus subtilis antibiotics: structures, syntheses and specific functions, Molecular Microbiology, Vol. 56, pp. 854-857, 2005. 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

1. A method M1 for increasing the spore formation rate and / or spore yield of Bacillus species in fermentation broth, the method comprising: a1) Provide species of the genus Bacillus. b1) Provide a suitable fermentation medium, c1) Supplement the suitable fermentation medium with aspartic acid to form a supplemented fermentation medium. d1) The fermentation process is initiated by introducing the Bacillus species provided in step a1) into the supplemented fermentation medium provided in step c1); and e1) Run the fermentation process described above. This provides a fermentation broth with increased spore formation rate and / or increased spore yield of Bacillus species.

2. The method M1 according to claim 1, wherein the method is used to increase the spore formation rate of the Bacillus species in the 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 aspartic acid.

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 aspartic acid.

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 aspartic acid.

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

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

8. The method M1 according to any one of the preceding claims, wherein the increased spore formation rate 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 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 spores.

11. The method M1 according to any one of the preceding claims, wherein the Bacillus species is selected from one of the following list: Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus mycosis fungoides, Bacillus circumflex, Bacillus megaterium, Bacillus pumilus, Bacillus mojaveii, Bacillus thuringiensis, Bacillus simplex, Bacillus safortus, Bacillus atrophus, Bacillus methyltrophicus, Bacillus sicca, Bacillus oryzae, and Bacillus tekirae.

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

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

14. Use of the fermentation broth produced by the method according to any one of claims 1-11 or the composition according to claim 12 in animal feed or aquatic animal feed.

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

Images