Methods for stabilizing liquid bacterial endophytic spore compositions

Abstract

The present invention relates to a method for stabilizing a liquid bacterial endophytic spore composition. The invention also relates to a multifunctional penetration preservation strategy for the suspension of the liquid bacillus endophytic spores.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 424,643, filed on November 11, 2022, the contents of which are incorporated herein by reference in their entirety. Field of the Invention

[0003] The present invention relates to a method for stabilizing liquid bacterial endospore compositions. The present invention also relates to a versatile osmotic preservation strategy for liquid Bacillus endospore suspensions. Background of the Invention

[0005] Endospores are metabolically dormant, persistent, non-reproductive cells formed by bacteria of the phylum Bacillota (basal synonym Firmicutes) in response to nutrient restriction or other adverse environmental conditions. Endospore-forming genera include Bacillus, Brevibacillus, Lysinibacillus, and Paenibacillus, among others. Endospores are resistant to a variety of physical and chemical stressors that would kill vegetative bacterial cells, including extreme temperatures, chemical disinfectants, desiccation, and ultraviolet radiation. Due to this resilience, endospores (particularly those formed by members of the genus Bacillus) are popular inclusions in microbial biostimulants and biofertilizer formulations. Liquid endospore suspensions may be considered commercially superior to freeze-dried and spray-dried powders because they are easier to integrate into unit operations in fertilizer coating facilities and grower management practices. However, known concentrated liquid endospore suspensions face several challenges that affect product stability. These suspensions often contain high concentrations of biomolecules left over from the fermentation process (JPGorsuch, Z. Jones, Heliyon 6 (2020) e03419), which can support the growth of heterotrophic spoilage organisms in the absence of aggressive antimicrobial preservation packaging (Setlow, P., J. Appl. Microbiol. 126, 348-358, 2018). Although endospores are more resistant to chemical stressors than vegetative bacterial cells, many endospores may be unstable to concentrated preservative levels with sufficient contact time (Setlow, P., J. Bacteriol. 196, 1297-1305, 2014). Therefore, a preservative system that prevents the proliferation of spoilage organisms without compromising the viability of the endospores formulated in the product is essential. Additionally, Bacillus endospores may be susceptible to non-nutritional germination, where biomolecules such as peptidoglycan or dipicolinate trigger germination responses that deprive endospores of characteristic metabolic dormancy and associated resistance to environmental and chemical stressors (Setlow, P., J. Bacteriol. 196, 1297-1305, 2014). Because such biomolecules can be present in concentrated industrial endospore suspensions (JPGorsuch, Z. Jones, Heliyon 6 (2020) e03419), preservative systems that mitigate the non-nutritional germination responses of unstable endospores are also essential for product stability. Such systems can work chemically (by directly inactivating non-nutritional germination compounds) or biologically (by managing the non-nutritional germination responses of endospores).

[0006] Products such as Crop Liquid TM A 1:1:1:1 blend of four Bacillus species, Bacillus amyliliquefaciens (BA), Bacillus subtilis (BS), Bacillus pumilus (BP), and Bacillus licheniformis (BL), experiences spoilage from environmental microorganisms and loss of endospore viability over time under stress storage conditions. While advanced chemical preservative packaging adequately protects the product from spoilage from environmental contaminants, they also have a negative impact on endospore stability. Therefore, there is a need in the art for a preservative system that can mitigate the spoilage threat posed by cold-tolerant environmental contaminants while also improving stability outcomes for labile Bacillus endospores. SUMMARY OF THE INVENTION

[0008] Described herein are compositions and methods for extending the storage time and reducing spoilage of liquid endospore suspensions. The present disclosure provides an optimized preservative system for liquid endospore suspensions that creates a physical and chemical environment within the suspension that antagonizes spoilage organisms while supporting endospore dormancy and stability. This, in turn, allows the product to be shipped and stored for extended periods of time without special handling requirements to inhibit or delay the growth of environmental spoilage organisms such as bacteria, yeasts, fungi, viruses, and / or pests.

[0009] Thus, in one aspect, the present disclosure provides a composition comprising a liquid endospore suspension and at least one penetrating preservative.

[0010] In some embodiments, the liquid endospore suspension is an aqueous suspension.

[0011] In some embodiments, the liquid endospore suspension comprises at least one endospore-forming Bacillus.

[0012] In some embodiments, the at least one endospore-forming Bacillus is selected from the group consisting of Bacillus, Brevibacillus, Paenibacillus, and Lysinibacillus.

[0013] In some embodiments, the Bacillus species is selected from the group consisting of B. amyloliquefaciens (BA), B. subtilis (BS), B. pumilus (BP), B. licheniformis (BL), B. thuringiensis, B. velezensis, B. endophyticus, and Priestia megaterium (Bacillus megaterium).

[0014] In some embodiments, at least one osmotic preservative is an osmotic pressure regulator. Additionally, in some embodiments, the osmotic pressure regulator reduces water energy and / or induces preservation of endospores.

[0015] In some embodiments, at least one osmotic preservative is a chloride salt. Additionally, in some embodiments, at least one osmotic preservative is selected from the group consisting of sodium chloride, calcium chloride, potassium chloride, magnesium chloride, and any other chloride salt.

[0016] In some embodiments, the composition comprises at least about 10.0 wt % of at least one osmotic preservative.

[0017] In some embodiments, the liquid endospore suspension is stable at 40°C for at least 180 days.

[0018] Another aspect of the present disclosure provides a method of inhibiting the proliferation of at least one environmental spoilage organism in a liquid endospore suspension, wherein the method comprises using at least one penetrating preservative.

[0019] In some embodiments, the at least one environmental spoilage organism is selected from a bacterial contaminant, a yeast contaminant, or a fungal contaminant. Additionally, in some embodiments, the bacterial contaminant is selected from the group consisting of Pseudomonas, Citrobacter, Klebsiella, Shigella, Enterobacter, and Serratia species.

[0020] In some embodiments, the at least one endospore-forming Bacillus is selected from the group consisting of Bacillus, Brevibacillus, Paenibacillus, and Lysinibacillus. In some embodiments, the at least one endospore-forming Bacillus comprises a species from a genus selected from the group consisting of Bacillus, Brevibacillus, Paenibacillus, and Lysinibacillus.

[0021] In some embodiments, the Bacillus species is selected from the group consisting of Bacillus amyloliquefaciens (BA), Bacillus subtilis (BS), Bacillus pumilus (BP), Bacillus licheniformis (BL), Bacillus thuringiensis, Bacillus velezensis, Bacillus endophyticus, and Priesteria megaterium (synonym Bacillus megaterium).

[0022] In some embodiments, at least one osmotic preservative is an osmotic pressure regulator. Additionally, in some embodiments, the osmotic pressure regulator reduces water energy and / or induces preservation of endospores.

[0023] In some embodiments, the at least one osmotic preservative is selected from the group consisting of sodium chloride, calcium chloride, potassium chloride, magnesium chloride, and any other chloride salt.

[0024] In some embodiments, the liquid endospore suspension is stable at 40°C for at least 180 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Shown are spoilage organisms recovered from samples of commercially available endospore suspensions protected with standard chemical preservatives and exposed to standard unit operations in a customer facility, necessitating the inclusion of advanced antimicrobial inhibitor systems in future product iterations.

[0027] Figure 2 The stability of a model endospore blend under stress conditions in the presence of standard chemical preservatives is shown. BA: Bacillus amyloliquefaciens; BS: Bacillus subtilis; BP: Bacillus pumilus; BL: Bacillus licheniformis.

[0028] Figure 3 The use of advanced chemical preservation packaging to mitigate the spoilage of endospore suspensions by environmental microorganisms is shown. The success of the chemical preservation strategy was evaluated using a customized preservative efficacy test.

[0029] Figure 4 The stability of a model endospore blend under stress conditions in the presence of advanced chemical preservatives is shown. Individual species subpopulations within the blend were evaluated using a custom spread plate assay, with morphological scoring allowing each population to be reported individually from the individual blends tested.

[0030] Figure 5 Shown are morphological scores of APC plates (a), allowing assessment of populations of individual species BA (b), BS (c), BP (d) and BL (e) in mixed species suspensions.

[0031] Figure 6 The stability of BA endospores in various penetrating preservative systems is shown.

[0032] Figure 7 The stability of BS endospores in various penetrating preservative systems is shown.

[0033] Figure 8 The stability of BP endospores in various penetrating preservative systems is shown.

[0034] Figure 9 The stability of BL endospores in various penetrating preservative systems is shown. Detailed Description of the Invention

[0036] The present disclosure relates to an optimized preservative system for liquid endospore suspensions (i.e., aqueous endospore suspensions) that creates a physical and chemical environment within the suspension that antagonizes spoilage organisms while supporting endospore dormancy and stability. Advantages of the preservative system for liquid endospore suspensions include, but are not limited to, mitigating the threat of spoilage from cold-tolerant environmental contaminants while also improving stability outcomes for labile Bacillus endospores. The compositions of the present disclosure can extend storage time, reduce spoilage risk, and support endospore dormancy and stability in liquid endospore suspensions.

[0037] One aspect of the present disclosure relates to a composition comprising a liquid endospore suspension and at least one penetrating preservative. A related aspect of the present disclosure relates to a composition comprising an aqueous endospore suspension and at least one penetrating preservative. Notably, the preservative system comprising at least one penetrating preservative creates a physical and chemical environment within the liquid endospore suspension that antagonizes spoilage organisms while supporting endospore dormancy and stability.

[0038] In some embodiments, the liquid endospore suspension comprises at least one endospore-forming bacillus. In some embodiments, at least one endospore-forming bacillus is from the genus Bacillus, brevis, Paenibacillus, lysinibacillus or another Bacillus. In some embodiments, at least one endospore-forming bacillus is a species within the genus Bacillus, brevis, Paenibacillus or lysinibacillus. In some embodiments, the bacillus species is bacillus amyloliquefaciens (BA), bacillus subtilis (BS), bacillus pumilus (BP), bacillus licheniformis (BL), bacillus thuringiensis, bacillus velez, endophytic bacillus, Priesteria megaterium (base synonym bacillus megaterium) or another bacillus species. In some embodiments, the bacillus species is bacillus amyloliquefaciens (BA), bacillus subtilis (BS), bacillus pumilus (BP) or bacillus licheniformis (BL).

[0039] In some embodiments, the liquid endospore suspension comprises one or more of the bacillus phyla that form endospores. In some embodiments, one or more of the bacillus phyla that form endospores are species from the genus Bacillus, Brevibacillus, Paenibacillus, Lysinibacillus, another Bacillus genus, or any combination thereof. In some embodiments, one or more of the bacillus phyla that form endospores are species from the genus Bacillus, Brevibacillus, Paenibacillus, or Lysinibacillus. In some embodiments, the bacillus species is bacillus amyloliquefaciens (BA), bacillus subtilis (BS), bacillus pumilus (BP), bacillus licheniformis (BL), bacillus thuringiensis, bacillus velez, endophytic bacillus, Priesteria megaterium (base synonym bacillus megaterium), another bacillus species, or any combination thereof. In some embodiments, the Bacillus species is Bacillus amyloliquefaciens (BA), Bacillus subtilis (BS), Bacillus pumilus (BP), or Bacillus licheniformis (BL), or a combination thereof. In some embodiments, Bacillus amyloliquefaciens (BA), Bacillus subtilis (BS), Bacillus pumilus (BP), and Bacillus licheniformis (BL) spores are present in equal concentrations in the liquid endospore suspension as determined by colony forming units (CFU). In some embodiments, Bacillus amyloliquefaciens (BA), Bacillus subtilis (BS), Bacillus pumilus (BP), and Bacillus licheniformis (BL) spores are not present in equal concentrations in the liquid endospore suspension as determined by CFU.

[0040] Bacillus species such as Bacillus, Brevibacillus, Paenibacillus, and Lysinibacillus are known to those of ordinary skill in the art. Such species are available from recognized depository centers. Exemplary depository information for bacterial species that can be used in the liquid endospore suspensions of the present disclosure is provided in Table A below.

[0041] Table A. Deposit numbers for exemplary bacterial species

[0042]

[0043] *American Type Culture Collection (atcc.org)

[0044] **The Chinese General Microbiological Culture Collection Center (cgmcc.net)

[0045] ***JCM online strain catalog (RIKEN BRC Microbe Division, jcm.brc.riken.jp)

[0046] In some embodiments, the liquid endospore suspension has about 1×10 6 to about 1×10 12 Colony forming units / mL (CFU / mL), for example 1×10 6 to about 1×10 11 CFU / mL, about 1×10 8 to 1×10 12 CFU / mL or approximately 1 × 10 9 to 1×10 12 CFU / mL of microorganisms. In some embodiments, the liquid endospores have a concentration of about 1×10 7 to about 1×10 11 CFU / mL of microorganisms. In some embodiments, the liquid endospores have a concentration of about 1×10 8 to about 1×10 10 CFU / mL of microorganisms. In some embodiments, the liquid endospores have a concentration of about 5×10 8 to about 1×10 10 CFU / mL of microorganisms. In some embodiments, the liquid endospores have a concentration of about 5×10 8 to about 5×10 9 The concentration of microorganisms in CFU / mL.

[0047] In some embodiments, the at least one osmotic preservative is an osmotic pressure regulator. In some embodiments, the at least one osmotic pressure regulator is a combination of osmotic pressure regulators. In some embodiments, the osmotic pressure regulator increases the osmotic pressure within the liquid endospore suspension. In some embodiments, the osmotic pressure regulator decreases the osmotic pressure within the liquid endospore suspension. In some embodiments, the osmotic pressure regulator reduces water energy and / or induces preservation of endospores within the liquid endospore suspension.

[0048] In some embodiments, the preservative system can include at least one osmotic preservative, at least two osmotic preservatives, at least three osmotic preservatives, or four or more osmotic preservatives. In some embodiments, the preservative system can include at least one osmotic pressure regulator, at least two osmotic pressure regulators, at least three osmotic pressure regulators, or four or more osmotic pressure regulators. Alternatively, in some embodiments, the preservative system can be a chemical and / or physical preservative system.

[0049] In some embodiments, the at least one osmotic preservative comprises a salt or a combination of salts. In some embodiments, the at least one osmotic preservative comprises one or more chloride salts. In some embodiments, the at least one osmotic preservative comprises sodium chloride, calcium chloride, potassium chloride, magnesium chloride, or any combination thereof. In some embodiments, the osmotic preservative increases the osmotic pressure within the liquid endospore suspension. In some embodiments, the osmotic preservative decreases the osmotic pressure within the liquid endospore suspension. In some embodiments, the osmotic preservative reduces water energy and / or induces preservation of endospores within the liquid endospore suspension.

[0050] In some embodiments, the preservative system can include at least one penetrating preservative, at least two penetrating preservatives, at least three penetrating preservatives, or four or more penetrating preservatives. In some embodiments, the preservative system can include at least one salt, at least two salts, at least three salts, or four or more salts. Alternatively, in some embodiments, the preservative system can be a chemical and / or physical preservative system.

[0051] In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative can be stable at about 20° C. to about 40° C. for at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 1.5 years, or at least 2 years. In some embodiments, a stable aqueous microbial composition can be stable at about 20° C. to about 40° C. for about 1 month to about 2 years, about 2 months to about 1.5 years, about 3 months to about 1 year, about 3 months to about 8 months, about 6 months to about 3 years, for example, about 6 months to about 2 years, about 6 months to about 1.5 years, about 6 months to about 1 year, about 1 year to about 3 years, about 1 to 2 years, or about 1 to 1.5 years. In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative can be stable at about 20° C. to about 40° C. for about 3 months, about 6 months, about 9 months, about 1 year, about 1.5 years, or about 2 years. For example, a composition comprising a liquid endospore suspension and at least one penetrating preservative can be stable at about 40°C for about 6 months (ie, 180 days).

[0052] In some embodiments, the composition comprising a liquid endospore suspension and at least one penetrating preservative can be stable at room temperature for at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 1.5 years, or at least 2 years. In some embodiments, the composition comprising a liquid endospore suspension and at least one penetrating preservative can be stable at room temperature for about 1 month to about 2 years, about 2 months to about 1.5 years, about 3 months to about 1 year, about 3 months to about 8 months, about 6 months to about 3 years, such as about 6 months to about 2 years, about 6 months to about 1.5 years, about 6 months to about 1 year, about 1 year to about 3 years, about 1 to 2 years, or about 1 to 1.5 years. For example, the composition comprising a liquid endospore suspension and at least one penetrating preservative can be stable at room temperature for about 3 months, about 6 months, about 9 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, or about 3 years.

[0053] In some embodiments, the composition comprising the liquid endospore suspension and at least one osmotic preservative can remain biostable for at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 1.5 years, or at least 2 years at about 20° C. to about 40° C. In some embodiments, the stable aqueous microbial composition can remain biostable for about 6 months to about 3 years, e.g., about 1 month to about 2 years, about 2 months to about 1.5 years, about 3 months to about 1 year, about 3 months to about 8 months, about 6 months to about 2 years, about 6 months to about 1.5 years, about 6 months to about 1 year, about 1 year to about 3 years, about 1 to 2 years, or about 1 to 1.5 years at about 20° C. to about 40° C. In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative can remain biostable for about 3 months, about 6 months, about 9 months, about 1 year, about 1.5 years, or about 2 years at about 20° C. to about 40° C. For example, a composition comprising a liquid endospore suspension and at least one penetrating preservative can remain biostable for about 6 months (i.e., 180 days) at about 40° C.

[0054] In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative can remain biostable at room temperature for at least 3 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 1.5 years, or at least 2 years. In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative can remain biostable at room temperature for about 6 months to about 3 years, such as about 6 months to about 2 years, about 6 months to about 1.5 years, about 6 months to about 1 year, about 1 year to about 3 years, about 1 to 2 years, or about 1 to 1.5 years. For example, a composition comprising a liquid endospore suspension and at least one penetrating preservative can remain biostable at room temperature for about 3 months, about 6 months, about 9 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, or about 3 years.

[0055] In some embodiments, the composition comprising a liquid endospore suspension and at least one osmotic preservative can remain biologically stable under various humidity storage conditions for the aforementioned time periods. In some embodiments, the humidity is ambient humidity. In some embodiments, the humidity is from about 10% to about 90%, for example, from about 10% to about 80%, from about 10% to about 75%, from about 10% to about 70%, from about 10% to about 65%, from about 10% to about 60%, from about 20% to about 90%, from about 20% to about 80%, from about 20% to about 75%, from about 20% to about 70%, from about 20% to about 65%, from about 20% to about 60%, from about 30% to about 90%, from about 30% to about 80%, from about 30% to about 75%, from about 30% to about 70%, from about 30% to about 65%, or from about 30% to about 60%. In some embodiments, the humidity is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75%. In some embodiments, the humidity is up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, or up to 75%.

[0056] In some embodiments, the composition comprising a liquid endospore suspension and at least one penetrating preservative can comprise from about 0.01 wt% (weight percent) to 15.0 wt% of the penetrating preservative, for example, from about 0.01 wt% to 10.0 wt%, from about 0.01 wt% to 5.0 wt%, from about 0.01 wt% to 3.0 wt%, from about 0.01 wt% to 1.0 wt%, from about 0.1 wt% to 15.0 wt%, from about 0.1 wt% to 10.0 wt%, from about 0.1 wt% to 5.0 wt%. %, about 0.1 wt% to 3.0 wt%, about 0.1 wt% to 1.0 wt%, about 0.5 wt% to 15.0 wt%, about 0.5 wt% to 10.0 wt%, about 0.5 wt% to 5.0 wt%, about 0.5 wt% to 3.0 wt%, about 0.5 wt% to 1.0 wt%, about 1.0 wt% to 15.0 wt%, about 1.0 wt% to 10.0 wt%, about 1.0 wt% to 5.0 wt%, or about 1.0 wt% to 3.0 wt% of an osmotic preservative. In some embodiments, the composition can comprise at least about 0.01 wt%, at least about 0.05 wt%, at least about 0.1 wt%, at least about 0.5 wt%, at least about 1.0 wt%, at least about 2.0 wt%, at least about 3.0 wt%, at least about 5.0 wt%, at least about 10.0 wt%, at least about 15.0 wt%, at least about 20.0 wt%, at least about 25.0 wt%, at least about 30.0 wt%, at least about 35.0 wt%, at least about 40.0 wt%, at least about 45.0 wt%, or at least about 50.0 wt% of an osmotic preservative. In some embodiments, a composition comprising a liquid endospore suspension and at least one osmotic preservative can comprise between about 0.01 wt% and 20.0 wt% of an osmotic preservative. In some embodiments, a composition comprising a liquid endospore suspension and at least one osmotic preservative can comprise between about 1 wt% and 20.0 wt% of an osmotic preservative. In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative may comprise about 5.0 wt% to 20.0 wt% of the penetrating preservative. In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative may comprise about 5.0 wt% to 10.0 wt% of the penetrating preservative. In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative may comprise about 0.01 wt% to 10.0 wt% of the penetrating preservative. In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative may comprise about 0.1 wt% to 5.0 wt% of the penetrating preservative. In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative may comprise about 0.1 wt% to 1.0 wt% of the penetrating preservative.In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative may comprise about 0.5 wt% to 1 wt% of the penetrating preservative. In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative may comprise about 0.5 wt% to 2.0 wt% of the penetrating preservative. In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative comprises about 1.0 wt% of the penetrating preservative. In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative comprises about 5.0 wt% of the penetrating preservative. In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative comprises about 10.0 wt% of the penetrating preservative. In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative comprises about 15.0 wt% of the penetrating preservative. In some embodiments, the composition comprising a liquid endospore suspension and at least one osmotic preservative comprises about 20.0 wt% of the osmotic preservative. In some embodiments, the at least one osmotic preservative comprises sodium chloride, calcium chloride, potassium chloride, magnesium chloride, or any combination thereof.

[0057] In some embodiments, the composition comprising a liquid endospore suspension and at least one osmotic preservative can comprise about 5 g of osmotic preservative per 1 L of composition, about 6 g of osmotic preservative per 1 L of composition, about 7 g of osmotic preservative per 1 L of composition, about 8 g of osmotic preservative per 1 L of composition, about 9 g of osmotic preservative per 1 L of composition, about 10 g of osmotic preservative per 1 L of composition, about 11 g of osmotic preservative per 1 L of composition, about 12 g of osmotic preservative per 1 L of composition, about 13 g of osmotic preservative per 1 L of composition, about 14 g of osmotic preservative per 1 L of composition, about 15 g of osmotic preservative per 1 L of composition, about 16 g of osmotic preservative per 1 L of composition, about 18 g of osmotic preservative per 1 L of composition, about 20 g of osmotic preservative per 1 L of composition, about 21 g of osmotic preservative per 1 L of composition, about 22 g of osmotic preservative per 1 L of composition, about 23 g of osmotic preservative per 1 L of composition, about 24 g of osmotic preservative per 1 L of composition, about 25 g of osmotic preservative per 1 L of composition, about 26 g of osmotic preservative per 1 L of composition, about 27 g of osmotic preservative per 1 L of composition, about 28 g of osmotic preservative per 1 L of composition, about 29 g of osmotic preservative per 1 L of composition, about 30 g of osmotic preservative per 1 L of composition, about 31 g of osmotic g osmotic preservative / 1L composition, about 30 g osmotic preservative / 1L composition, about 40 g osmotic preservative / 1L composition, about 50 g osmotic preservative / 1L composition, about 60 g osmotic preservative / 1L composition, about 70 g osmotic preservative / 1L composition, about 80 g osmotic preservative / 1L composition, about 90 g osmotic preservative / 1L composition, about 100 g osmotic preservative / 1L composition, about 110 g osmotic preservative / 1L composition, about 120 g osmotic preservative / 1L composition, about 130 g osmotic preservative / 1L composition, about 140 g osmotic preservative / 1L composition, about 150 g osmotic preservative / 1L composition or about 200 g osmotic preservative / 1L composition. In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative may comprise about 50 g of penetrating preservative per 1 L. In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative may comprise about 100 g of penetrating preservative per 1 L. In some embodiments, a composition comprising a liquid endospore suspension and at least one penetrating preservative may comprise about 150 g of penetrating preservative per 1 L. In some embodiments, the at least one penetrating preservative comprises sodium chloride, calcium chloride, potassium chloride, magnesium chloride, or any combination thereof.

[0058] In some embodiments, the composition comprising the liquid endospore suspension comprises one or more additional preservatives (i.e., in addition to the osmotic preservatives described herein), buffers, suspending agents, surfactants, or combinations thereof. Exemplary additional preservatives, buffers, suspending agents, and surfactants are described in WO 2021 / 022128, the contents of which are incorporated herein by reference in their entirety.

[0059] In some embodiments, the composition comprising the liquid endospore suspension may comprise one or more additional preservatives, i.e., one or more additional preservatives in addition to the penetrating preservatives described herein. The composition comprising the liquid endospore suspension may comprise one preservative, two preservatives, three preservatives, or more, including a penetrating preservative.

[0060] In some embodiments, a composition comprising a liquid endospore suspension can comprise from about 0.01 wt% to 15.0 wt% of one or more additional preservatives, such as from about 0.01 wt% to 10.0 wt%, from about 0.01 wt% to 5.0 wt%, from about 0.01 wt% to 3.0 wt%, from about 0.01 wt% to 1.0 wt%, from about 0.1 wt% to 15.0 wt%, from about 0.1 wt% to 10.0 wt%, from about 0.1 wt% to 5.0 wt%, from about 0.1 wt% to 3.0 wt%, from about 0.01 wt% to 1.0 wt%, from about 0.1 wt% to 15.0 wt%, from about 0.1 wt% to 10.0 wt%, from about 0.1 wt% to 5.0 wt%, from about 0.1 wt% to 3.0 wt%. % to 3.0 wt %, about 0.1 wt % to 1.0 wt %, about 0.5 wt % to 15.0 wt %, about 0.5 wt % to 10.0 wt %, about 0.5 wt % to 5.0 wt %, about 0.5 wt % to 3.0 wt %, about 0.5 wt % to 1.0 wt %, about 1.0 wt % to 15.0 wt %, about 1.0 wt % to 10.0 wt %, about 1.0 wt % to 5.0 wt %, or about 1.0 wt % to 3.0 wt % of one or more additional preservatives.

[0061] The one or more additional preservatives may be selected from modified isothiazolinyl compounds, parabens, modified quaternary amines, modified ureas, glycerol derivatives, 2-bromo-2-nitro-1,3-propanediol, natural oils, organic acids having a molecular weight of no more than 200 and at least one pKa greater than 4.2, and combinations thereof.

[0062] Examples of modified isothiazolinone compounds include, but are not limited to, 1,2-benzisothiazolin-3-one, methylisothiazolinone, methylchloroisothiazolinone, benzisothiazolinone, and combinations thereof.

[0063] Examples of esters of parahydroxybenzoic acid include, but are not limited to, methylparaben, ethylparaben, propylparaben, and combinations thereof.

[0064] Examples of modified quaternary amines include, but are not limited to, benzethonium chloride or cetylpyridinium chloride, and combinations thereof.

[0065] Examples of modified ureas include, but are not limited to, diazolidinyl urea, imidazolidinyl urea, and combinations thereof.

[0066] Examples of glycerol derivatives include, but are not limited to, ethylhexylglycerin.

[0067] Examples of natural oils include, but are not limited to, grapefruit seed extract, tea tree oil, thyme oil, lemongrass oil, oregano oil, rosemary oil, lavender oil, and combinations thereof.

[0068] Examples of organic acids include, but are not limited to, acetic acid, citric acid, ascorbic acid, sorbic acid, propionic acid, butyric acid, oxalic acid, succinic acid, malic acid, tartaric acid, fumaric acid, aconitic acid, dipicolinic acid, amino acids, and combinations thereof. Examples of amino acids include, but are not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0069] In some embodiments, the one or more additional preservatives comprise calcium sulfate. In some embodiments, the composition comprising the liquid endospore suspension comprises from about 0.01 wt% to about 5 wt% calcium sulfate. In some embodiments, the composition comprising the liquid endospore suspension comprises from about 0.1 wt% to about 1 wt% calcium sulfate. In some embodiments, the composition comprising the liquid endospore suspension comprises from about 0.2 wt% to about 1.5 wt% calcium sulfate. In some embodiments, the composition comprising the liquid endospore suspension comprises about 0.25 wt% calcium sulfate.

[0070] In some embodiments, the one or more additional preservatives include 1,2-benzisothiazolin-3-one. In some embodiments, the composition comprising the liquid endospore suspension comprises about 0.01 wt% to about 2.0 wt%, about 0.01 wt% to about 1.5 wt%, about 0.01 wt% to about 1.0 wt%, or about 0.05 wt% to about 1.0 wt% of 1,2-benzisothiazolin-3-one.

[0071] In some embodiments, the composition comprising the liquid endospore suspension may comprise about 0.01 wt% to about 0.2 wt% of a modified benzisothiazoline, a paraben, a modified urea, or a mixture thereof, about 0.05 wt% to about 1 wt% of a low molecular weight organic acid, and / or about 0.1 wt% to about 10 wt% of an inorganic salt. In some embodiments, the preservation solution or stable aqueous microbial composition may comprise about 0.01 wt% to about 0.1 wt% of 1,2-benzisothiazolin-3-one, about 0.1 wt% to about 1 wt% of sorbic acid, and about 0.1 wt% to 10 wt% of sodium chloride.

[0072] The composition comprising a liquid endospore suspension can comprise from about 0.01 wt% to 15.0 wt% of a suspending agent, for example, from about 0.01 wt% to 10.0 wt%, from about 0.01 wt% to 5.0 wt%, from about 0.01 wt% to 3.0 wt%, from about 0.01 wt% to 1.0 wt%, from about 0.1 wt% to 15.0 wt%, from about 0.1 wt% to 10.0 wt%, from about 0.1 wt% to 5.0 wt%, from about 0.1 wt% to 3.0 wt%, from about 0.01 wt% to 1.0 wt%, from about 0.1 wt% to 15.0 wt%, from about 0.1 wt% to 10.0 wt%, from about 0.1 wt% to 5.0 wt%, from about 0.1 wt% to 3.0 wt%. .0wt%, about 0.1wt% to 0.5wt%, about 0.1wt% to 0.4wt%, about 0.5wt% to 15.0wt%, about 0.5wt% to 10.0wt%, about 0.5wt% to 5.0wt%, about 0.5wt% to 3.0wt%, about 0.5wt% to 1.0wt%, about 1.0wt% to 15.0wt%, about 1.0wt% to 10.0wt%, about 1.0wt% to 5.0wt% or about 1.0wt% to 3.0wt% of a suspending agent.

[0073] Compositions comprising liquid endospore suspensions can include one, two, three, or more suspending agents. Suspending agents are particularly useful for suspending the microorganisms in the aqueous composition, thereby providing desired physical stability. Without wishing to be bound by theory, suspending agents may alter the viscosity of the aqueous composition, thereby preventing the microbial species from settling to the bottom of the container.

[0074] Suspending agent can be polymer, surfactant or its combination. In some embodiments, suspending agent is polymer. The example of polymer can include but not limited to xanthan gum, guar gum, gum arabic, carboxymethyl cellulose, sodium polyacrylate, polyethylene glycol, ethylene oxide-propylene oxide (EO-PO) block copolymer, modified starch, modified polyacrylate, modified methyl methacrylate, polyethyleneimine, sodium polyaspartate, poly-gamma-glutamic acid or its combination. In some embodiments, suspending agent is a blend of xanthan gum and gum arabic (for example, Solagum TM AX). In some embodiments, the storage solution or stable aqueous microbial composition may comprise about 0.1 wt% to 1.0 wt% polymer.

[0075] In some embodiments, the suspending agent comprises a rheology modifier. A rheology modifier is a substance that changes the rheological properties of a material, i.e., a substance added to a formulation to change the viscosity. Suitable rheology modifiers include, but are not limited to, commercially available rheology modifiers including urea and modified forms of urea, such as -410. Other rheology modifiers are known to those of ordinary skill in the art.

[0076] Examples of surfactants include, but are not limited to, primary alkyl alcohol ethoxylates, secondary alkyl alcohol ethoxylates, primary alkyl alcohol propoxylates, secondary alkyl alcohol propoxylates, and combinations thereof. In some embodiments, the surfactant has a cloud point of about 30° C. to about 80° C. and a hydrophilic-lipophilic balance of about 5 to about 15. In some embodiments, the surfactant is a C-13 branched primary alcohol having an average ethoxylation of 5 to 10, for example, 5, 6, 7, 8, 9, or 10.

[0077] In some embodiments, the surfactant includes a fully saturated ethoxylated primary branched C13 alcohol such as Synperonic TM 13 / 7 or Synperonic TM 13 / 6. In some embodiments, the surfactant comprises an alcohol ethoxylate, such as Ecosurf TM In some embodiments, the surfactant comprises a mixture of 58.0% to 62.0% D-pyranose, oligodecyloctyl glycoside, and 38.0% to 42.0% water, and contains less than 2% decanol and less than 1.0% octanol, such as Triton TM CG110. In some embodiments, the surfactant includes a secondary polyether polyol such as Tergitol TM L-62. In some embodiments, the surfactant includes a secondary alcohol ethoxylate, such as Tergitol TM 15-S-12. In some embodiments, the surfactant includes a nonionic alkyl EO / PO copolymer, such as Tergitol TM XDLW.

[0078] In some embodiments, the liquid endospore suspension can comprise about 0.1 wt% to 10 wt% surfactant, such as about 0.1 wt% to 5 wt%, 0.1 wt% to 2 wt%, 0.1 wt% to 1 wt%, 0.5 wt% to 1 wt%, or 0.5 wt% to 2 wt% surfactant.

[0079] In some embodiments, suspending agents may include xanthan gum, gum arabic, and alcohol ethoxylates.

[0080] The composition comprising the liquid endospore suspension can comprise from about 0.01 wt% to 15.0 wt% of a buffering agent, for example, from about 0.01 wt% to 10.0 wt%, from about 0.01 wt% to 5.0 wt%, from about 0.01 wt% to 3.0 wt%, from about 0.01 wt% to 1.0 wt%, from about 0.1 wt% to 15.0 wt%, from about 0.1 wt% to 10.0 wt%, from about 0.1 wt% to 5.0 wt%, from about 0.1 wt% to 3.0 wt%. %.0 wt %, about 0.1 wt % to 1.0 wt %, about 0.5 wt % to 15.0 wt %, about 0.5 wt % to 10.0 wt %, about 0.5 wt % to 5.0 wt %, about 0.5 wt % to 3.0 wt %, about 0.5 wt % to 1.0 wt %, about 1.0 wt % to 15.0 wt %, about 1.0 wt % to 10.0 wt %, about 1.0 wt % to 5.0 wt %, or about 1.0 wt % to 3.0 wt % of a buffer.

[0081] The buffer can be in an amount sufficient to maintain the pH of the composition comprising the liquid endospore suspension at greater than 4.2, e.g., greater than 4.5, greater than 5.0, greater than 5.5, greater than 6.0, greater than 6.5, or greater than 7.0. In some embodiments, the buffering agent can be in an amount sufficient to maintain the pH of the composition comprising a liquid endospore suspension in the range of about 4.3 to about 12.0, e.g., about 4.3 to about 11.0, about 4.3 to about 10.0, about 4.3 to about 9.5, about 4.3 to about 9.0, about 4.3 to about 8.5, about 5.0 to about 12.0, about 5.0 to about 11.0, about 5.0 to about 10.0, about 5.0 to about 9.5, about 5.0 to about 9.0, about 5.0 to about 8.5, about 5.5 to about 12.0, about 5.5 to about 11.0, about 5.5 to about 10.0, about 5.5 to about 9.5, about 5.5 to about 9.0, or about 5.5 to about 8.5.

[0082] The pH of the composition comprising the liquid endospore suspension can be selected to maximize the shelf life of spores and / or colonies stored in the aqueous composition for extended periods of time. A pH of less than about 4 can adversely affect the stability of certain bacterial spores at high temperatures. For example, Bacillus spores can demineralize at low pH and lose their resistance to high temperature storage. Therefore, a pH greater than about 4.2, about 4.5, about 5.0, about 5.5, or about 6.0 is desirable for improving the biostability of Bacillus spores. In some embodiments, the pH of the composition comprising the liquid endospore suspension is greater than about 6.0.

[0083] In some aspects, the present disclosure relates to methods of using the compositions of the present disclosure for inhibiting the proliferation of at least one environmental spoilage organism. The compositions of the present disclosure can antagonize spoilage organisms while supporting endospore dormancy and stability.

[0084] Another aspect of the present disclosure relates to a method of inhibiting the proliferation of at least one environmental spoilage organism in a liquid endospore suspension, wherein the method comprises using at least one penetrating preservative.

[0085] In some embodiments, the methods comprise inhibiting the proliferation of at least one environmental spoilage organism in a composition comprising a liquid endospore suspension by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, the proliferation of at least one environmental spoilage organism in a liquid endospore suspension is inhibited by about 10% to about 95%, e.g., about 10% to about 85%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 20% to about 95%, about 20% to about 85%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 30% to about 95%, about 30% to about 85%, about 30% to about 75%, about 30% to about 70%, about 30% to about 65%, or about 30% to about 60%. In some embodiments, the inhibition of proliferation of at least one environmental spoilage organism in a liquid endospore suspension is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the inhibition of proliferation of at least one environmental spoilage organism is measured relative to the proliferation of the at least one environmental spoilage organism in an otherwise equivalent composition comprising a liquid endospore suspension that does not contain the at least one penetrating preservative.

[0086] In some embodiments, the at least one environmental spoilage organism includes, but is not limited to, a bacterial contaminant, a yeast contaminant, or a fungal contaminant. In some embodiments, the at least one environmental spoilage organism comprises a bacterial contaminant, a yeast contaminant, a fungal contaminant, or any combination thereof. In some embodiments, the bacterial contaminant comprises a Pseudomonas species, a Citrobacter species, a Klebsiella species, a Shigella species, an Enterobacter species, a Serratia species, or any combination thereof.

[0087] In some embodiments, the method comprises inhibiting the proliferation of at least one environmental spoilage organism in a composition comprising a liquid endospore suspension comprising endospore-forming Bacillus species. In some embodiments, the endospore-forming Bacillus species is Bacillus species, Brevibacillus species, Paenibacillus species, Lysinibacillus species, or any combination thereof.

[0088] In some embodiments, the method comprises inhibiting the proliferation of at least one environmental spoilage organism in a composition comprising a liquid endospore suspension comprising a Bacillus species. In some embodiments, the Bacillus species comprises Bacillus amyloliquefaciens (BA), Bacillus subtilis (BS), Bacillus pumilus (BP), Bacillus licheniformis (BL), Bacillus thuringiensis, Bacillus velezensis, Bacillus endophyticus, Priesteria megaterium (synonym Bacillus megaterium), another Bacillus species, or any combination thereof.

[0089] In some embodiments, the aforementioned methods include the use of at least one osmotic preservative. In some embodiments, the at least one osmotic preservative is an osmotic pressure regulator. In some embodiments, the at least one osmotic pressure regulator is a combination of osmotic pressure regulators. In some embodiments, the osmotic pressure regulator increases the osmotic pressure within the liquid endospore suspension. In some embodiments, the osmotic pressure regulator decreases the osmotic pressure within the liquid endospore suspension. In some embodiments, the osmotic pressure regulator reduces water energy and / or induces preservation of endospores within the liquid endospore suspension.

[0090] In some embodiments, the aforementioned method includes the use of a preservative system. In some embodiments, the preservative system can include at least one osmotic preservative, at least two osmotic preservatives, at least three osmotic preservatives, or four or more osmotic preservatives. In some embodiments, the preservative system can include at least one osmotic pressure regulator, at least two osmotic pressure regulators, at least three osmotic pressure regulators, or four or more osmotic pressure regulators. Alternatively, in some embodiments, the preservative system can be a chemical and / or physical preservative system.

[0091] In some embodiments, the at least one osmotic preservative is a salt or a combination of salts. In some embodiments, the at least one osmotic preservative is one or more chloride salts. In some embodiments, the at least one osmotic preservative is sodium chloride, calcium chloride, potassium chloride, magnesium chloride, or any combination thereof. In some embodiments, the osmotic preservative increases the osmotic pressure within the liquid endospore suspension. In some embodiments, the osmotic preservative decreases the osmotic pressure within the liquid endospore suspension. In some embodiments, the osmotic preservative reduces water energy and / or induces preservation of endospores within the liquid endospore suspension.

[0092] In some embodiments, the preservative system can include at least one penetrating preservative, at least two penetrating preservatives, at least three penetrating preservatives, or four or more penetrating preservatives. In some embodiments, the preservative system can include at least one salt, at least two salts, at least three salts, or four or more salts. Alternatively, in some embodiments, the preservative system can be a chemical and / or physical preservative system.

[0093] When used in agronomic applications, the compositions of the present disclosure can provide a number of desirable characteristics related to reduced spoilage and endospore stability, including, but not limited to, antagonism of spoilage organisms and support of endospore dormancy and stability.

[0094] Although the present teaching has been described in conjunction with various embodiments and examples, it is not intended that the present teaching be limited to such embodiments or examples. On the contrary, as those skilled in the art will recognize, the present teaching encompasses various alternatives, modifications and equivalents.

[0095] Although various inventive embodiments have been described and illustrated herein, it will be readily apparent to those skilled in the art that various other devices and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein may be contemplated, and each of these variations and / or modifications is considered to be within the scope of the inventive embodiments described herein. More generally, it will be readily understood by those skilled in the art that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend on the specific application or applications for which the inventive teachings are used. Those skilled in the art will recognize many equivalents to the specific inventive embodiments described herein. Therefore, it will be understood that the foregoing embodiments are presented only by way of example, and within the scope of the appended claims and their equivalents, inventive embodiments may be practiced in a manner different from that specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, product, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits and / or methods is included within the inventive scope of the present disclosure if such features, systems, articles, materials, kits and / or methods are not mutually inconsistent.

[0096] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0097] Unless expressly indicated to the contrary, the indefinite articles “a” and “an” as used herein in the specification and claims should be understood to mean “at least one.” Any ranges recited herein are inclusive.

[0098] As used throughout this specification and claims, the terms "substantially," "approximately," and "about" generally mean plus or minus 10% of the stated value, eg, about 100 would include 90 to 110.

[0099] The phrase "and / or" as used herein in the specification and in the claims should be understood to mean "one or both" of the elements so connected (i.e., elements that are present jointly in some cases and separately in other cases). Multiple elements listed using "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so connected. In addition to the elements specifically indicated by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically indicated. Thus, as a non-limiting example, in one embodiment, reference to "A and / or B" when used in conjunction with open language (such as "comprising") may refer to only A (optionally including elements other than B); in another embodiment, to only B (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); and so on.

[0100] As used in the specification and claims herein, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, that is, including at least one of a plurality of elements or a list of elements, but also including more than one, and optionally including additional unlisted items. Only terms that clearly indicate the contrary, such as "only one" or "exactly one", or "consisting of..." when used in the claims, will refer to the inclusion of exactly one element of a plurality of elements or a list of elements. Generally speaking, when preceded by an exclusive term such as "any one", "one of...", "only one of..." or "exactly one of...", the term "or" as used herein should only be interpreted to indicate an exclusive alternative (i.e., "one or the other, but not both"). "Substantially consisting of..." when used in the claims should have its ordinary meaning as used in the field of patent law.

[0101] As used herein in the specification and in the claims, the phrase "at least one" when referring to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each element specifically listed in the list of elements and not excluding any combination of elements in the list of elements. This definition also allows that elements other than the elements specifically identified in the list of elements to which the phrase "at least one" refers may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B," or equivalently, "at least one of A and / or B") may, in one embodiment, mean at least one (optionally including more than one) A, but no B (and optionally including elements other than B); in another embodiment, mean at least one (optionally including more than one) B, but no A (and optionally including elements other than A); in yet another embodiment, mean at least one (optionally including more than one) A and at least one (optionally including more than one) B (and optionally including other elements); and so on.

[0102] As used herein, "wt%" means percent by weight.

[0103] As used herein, a "preservative system" refers to one or more penetrating preservatives or at least one penetrating preservative. Furthermore, the preservative system prevents the proliferation of spoilage organisms in a liquid endospore suspension without compromising the viability of the endospores in the product formulation. The proliferation of spoilage organisms can be measured using methods known in the art. For example, in the pharmaceutical and personal care industries, USP 41 / 51 requires a two-order-of-magnitude reduction in the challenge microbial population for a preservative to be considered effective.

[0104] In the claims and the foregoing description, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "including," and the like are to be construed as open-ended, meaning including but not limited to. As provided in Section 2111.03 of the U.S. Patent Office Manual of Patent Examining Procedures, only the transitional phrases "consisting of" and "consisting essentially of" are to be closed or semi-closed transitional phrases, respectively.

[0105] As used herein, the term "biostable" refers to an aqueous microbial composition whose microbial titer does not change by more than 25% (e.g., no more than 20%, no more than 15%, or no more than 10%) after storage at room temperature for a period of time, e.g., at least 6 months. In some embodiments, the term "biostable" refers to a microbial titer that remains at or above the relevant label claim.

[0106] Unless otherwise stated, the claims should not be construed as limited to the described order or elements. It should be understood that various changes in form and details may be made by one skilled in the art without departing from the spirit and scope of the appended claims. Protection is claimed for all embodiments and equivalents thereof that fall within the spirit and scope of the appended claims.

[0107] Although methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, illustrative methods and materials are now described. Other features, objects and advantages of the present invention will be apparent from the specification, as well as from the claims. In this specification and the appended claims, the singular also includes the plural, unless the context clearly indicates otherwise. Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. All patents and publications cited in this disclosure are incorporated herein by reference in their entirety. Example

[0108] The present disclosure is further illustrated by the following examples and synthesis examples, and the following examples and synthesis examples are not to be construed as limiting the scope or spirit of the present disclosure to the specific procedures described herein. It should be understood that providing examples is to illustrate certain embodiments, and is thereby not intended to limit the scope of the present disclosure. It should also be understood that, without departing from the spirit of the present disclosure and / or the scope of the appended claims, a variety of other embodiments, modifications thereof, and equivalents thereof that can be imagined by those skilled in the art can be taken.

[0109] Example 1. Preparation of Bacillus endospores

[0110] The microorganisms of the present disclosure can be grown using standard submerged liquid fermentation methods known in the art.

[0111] The independent starter culture (starter culture) of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens and Bacillus pumilus is grown according to the following general scheme, and is adjusted according to the needs of every kind of organism: 2 grams of nutrient broth (Nutrient Broth), 2 grams of AmberFerm (yeast extract) and 4 grams of maltodextrin are added in 250mL Erlenmeyer flask (Erlenmeyer flask).Add 100mL distilled deionized water, and stir flask until all dry ingredients dissolve.The flask is covered and placed in the autoclave operated at 121 ℃ and 15psi for 30 minutes.After cooling, inoculate flask with one of 1mL pure microorganism strain.The flask is sealed and placed on orbital shaker at 30 ℃.Allow culture to grow 3-5 days.This procedure (or parallel operation) is repeated for every kind of organism.

[0112] Larger culture is prepared by adding 18 grams of nutrient broth, 18 grams of AmberFerm and 36 grams of maltodextrins into a 1 liter flask with 900 mL of distilled deionized water. The flask is sealed and sterilized as above. After cooling, 100 mL of microbial culture medium from a 250 mL Erlenmeyer flask is added. The 1 liter flask is sealed, placed on an orbital shaker, and allowed to grow again for 3-5 days at 30 ℃.

[0113] During the final growth phase before introduction into the fermentor, the culture from the 1-liter flask was aseptically transferred to a sterilized 6-liter container and fermented at 30°C with aeration until reaching stationary phase. The contents of each 6-liter culture flask were transferred to a separate fermentor, which was also filled with sterilized growth medium composed of 1 part yeast extract and 2 parts dextrose. The separate fermentors were operated aerobically at pH 7.0 and the optimal temperature for each species.

[0114] When the cell density reaches 10 11 After the fermentation was complete, the fermentor was heat-shocked at >80°C to induce spore formation. Spores were recovered from the liquid fermentation medium by filtration and then isolated at approximately 10 11 The titer of CFU / mL was resuspended in aqueous medium.Each stock aqueous suspension of pure Bacillus spores was refrigerated (~4°C).

[0115] With a value greater than about 40×10 10 A Bacillus concentrate at a final titer of CFU / mL was prepared by mixing separate stock aqueous suspensions of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus pumilus into an aqueous culture medium stabilized with 0.25 wt% calcium sulfate.

[0116] Example 2. Stability of Liquid Bacillus Endospore Suspension

[0117] In one experiment, multiple bacteria were isolated from samples of contaminated products, and the culturable microbial communities differed in species composition (JPGorsuch, Z. Jones, Heliyon 6 (2020) e03419). MacConkey agar plates were prepared from serial dilutions of two separate contaminated product samples from southern Vietnam, showing qualitative differences in colony morphology between their dominant cultured species. Figure 1As shown in Figure 2, members of the genus Bacillus are subject to instability mechanisms such as spoilage by environmental microorganisms. Without wishing to be bound by theory, the results from the experiments described above, i.e., the recovery of spoilage organisms from samples of commercially available endospore suspensions protected with standard chemical preservatives and exposed to standard unit operations at a customer's facility, support the need to include advanced antimicrobial inhibitor systems in future endospore suspension products to eliminate or reduce the amount of spoilage organisms.

[0118] In a second experiment, the stability of a blend of four industrially relevant Bacillus species (Bacillus amyloliquefaciens (BA), Bacillus subtilis (BS), Bacillus pumilus (BP), and Bacillus licheniformis (BL)) under stress conditions was determined in the presence of standard chemical preservatives including, but not limited to, Proxel TM and Bioban TM Subpopulations of individual species within blends were assessed using a custom spread plate assay with morphological scoring (Gorsuch, J., Journal of Microbiological Methods 164, 105682, 2019) that allows for separate reporting of each population from a single tested blend. Figure 2 As shown in Figure 1, one Bacillus species, Bacillus subtilis (BS), experiences instability mechanisms such as loss of endospore viability over time under stress storage conditions. Without wishing to be bound by theory, results from the experiments described above show that after 100 days of storage, while BA, BP, and BL retain viability, BS rapidly loses viability and drops below the detection limit of the assay.

[0119] In the third experiment, the effects of blends of four industrially relevant Bacillus species (Bacillus amyloliquefaciens (BA), Bacillus subtilis (BS), Bacillus pumilus (BP), and Bacillus licheniformis (BL)) with advanced chemical preservation packaging ( Figure 3 The stability of the formula 1-6). Figure 3 As shown in , stability was assessed by determining the ability of the blends to mitigate environmental microbial spoilage using a customized preservative efficacy test (JPGorsuch, Z. Jones, Heliyon 6 (2020) e03419). Figure 4 As shown in , the subpopulations of individual species within the blends were assessed using a custom spread plate assay with morphological scoring (Gorsuch, J., Journal of Microbiological Methods 164, 105682, 2019), which allows for separate reporting of each population from a single tested blend. Although advanced chemical preservative packaging adequately protects the product from spoilage by environmental contaminants ( Figure 3 ), but they also had a negative impact on the endospore stability of three of the four Bacillus species (BA, BS, and BP) over time ( Figure 4 ). Thus, over time, the product failed to meet its label claims. Without wishing to be bound by theory, the results from the experiments described above suggest a need for a preservative system that can mitigate the spoilage threat posed by cold-tolerant environmental contaminants while also improving stability results for labile Bacillus endospores.

[0120] Example 3. Stability of Liquid Bacillus Endospore Suspension and Osmotic Preservative System

[0121] Osmotic preservative strategies based on salts such as sodium chloride (NaCl) and potassium chloride (KCl) are used in the food production industry to protect the substrate of interest from microbial spoilage (Pizarro, J., Revista Argentina de Microbiología, Vol. 50, No. 1, pp. 56-61). The mechanism of action of these salt-based systems is understood to be disruption of cell structure and function (Gandhi A, J Dairy Sci. 2014, 97(10):5939-51). However, Bacillus endospores are naturally dehydrated (Setlow, P., 2014, Bacteriol. 196, 1297-1305) and are therefore not subject to this type of cell disruption. Additionally, in spores of some Bacillus species, sodium chloride (NaCl) is known to inhibit both vegetative and non-vegetative germination responses (Nagler K, Appl Environ Microbiol. 2014; 80(4):1314-1321). There are no permeable preservation packages available that produce both effects (prevent product spoilage and stabilize endospore viability of otherwise labile species).

[0122] In one experiment, prototype endospore suspensions were screened to identify osmotic pressure additives that produced the set of parameters discussed above (Table 1). Each prototype was a mixed species assemblage consisting of equal concentrations of BA, BS, BP, and BL endospores. Each species represented a testable subpopulation within the entire assemblage, and each population was individually assessed over time using a custom diffusion plate assay, with morphological scoring allowing each population to be reported individually from a single test mixture.

[0123] A range of industrially relevant solutes (sodium chloride, magnesium chloride, potassium chloride and calcium chloride, respectively) were screened for their ability to prevent product spoilage and mitigate endospore instability. Samples were stored under stressful environmental storage conditions (constant 40°C). Preservative efficacy was assessed at 0, 30, 59 and 90 days of storage (sampling in progress) using a published PET assay (JPGorsuch, Z.Jones, Heliyon 6 (2020) e03419). Results were compared to a positive control comprising the advanced chemical preservation packaging described above. Individual endospore populations were assessed at 0, 14, 30, 60, 90, 120 and 150 days of storage (sampling in progress) using a published plate count assay that includes morphological scoring of individual species within a microbial collection ( Figure 5 ), allowing each population within the collection to be counted separately over time.

[0124] Table 1 - Composition of prototype endospore suspension.

[0125]

[0126] The ability (Table 2) of the proliferation of various permeation preservative packaging to inhibit environmental spoilage organisms was examined using a customized PET assay. The values ​​shown represent the change multiple of the colonies of bacterial contaminants and yeast contaminants relative to their initial inoculation levels when the assay began. Without wishing to be bound by theory, any negative result is considered to be a pass, with larger negative values ​​indicating a more aggressive microbicidal effect. Without wishing to be further bound by theory, the results of the above-described experiments show that NaCl and CaCl tend to have less aggressive effects than KCl and MgCl, indicating a slightly weaker antimicrobial effect.

[0127] Table 2 - Customized PET assay examining inhibition of proliferation of environmental spoilage organisms.

[0128]

[0129] The results of the stability study of Bacillus endospores collected from the mixture described in Table 1 are presented in a separate figure ( Figure 6-Figure 9 Each individual species represented a testable subpopulation in the mixture via morphological scoring. In all treatments except the treatment preserved with CaCl2, the BA population ( Figure 6 ) stability and historical data ( Figure 1 ) compared favorably. The stability of the BS population in the test blend (Table 1) Figure 7 )—historically unstable under these storage conditions—exhibited the same instability profile only in treatments stored with NaCl ( Figure 1), while treatments preserved with KCl, MgCl2, and CaCl2, respectively, showed positive stability profiles not previously observed for this species. In all treatments, the BP and BL populations (respectively Figure 8 and Figure 9 ) stability and historical data ( Figure 1 ) is more favorable than

[0130] As discussed above, the optimal preservative system for liquid endospore suspensions creates a physical and chemical environment within the suspension that antagonizes spoilage organisms while supporting endospore dormancy and stability. PET assay data (Table 2) show that MgCl2 and KCl have the greatest antimicrobial activity against field-relevant spoilage organisms in the tested Bacillus collection. Endospore stability data ( Figure 6 ) showed that endospore stability of all Bacillus species was preserved only in formulations preserved with MgCl and KCl, as assessed by in vitro culturability. Without wishing to be bound by theory, the results indicate that these materials create a physical and chemical environment that meets the criteria listed above (i.e., antagonizing spoilage organisms while supporting endospore dormancy and stability).

[0131] Without wishing to be bound further by theory, the results suggest that it is possible that the mechanisms behind spoilage prevention may differ from those behind endospore stabilization, resulting in a set of parallel parameters that must be met to produce an optimal endospore suspension. For example, antimicrobial efficacy may be mediated by the water activity or osmotic pressure of the solution, or by the interaction of specific cations with the cells of the spoilage organisms. Endospore stabilization, on the other hand, may be mediated by the water activity or osmotic pressure of the solution, by the interaction of specific cations with the endospore structure, or by chemical fixation of non-vegetative germinates in the suspension supernatant.

[0132] Without wishing to be bound by theory, the results show that a composition comprising endospore-forming Bacillus and a penetrating preservative system creates a physical and chemical environment within a liquid Bacillus formulation that simultaneously favors endospore stability under stress conditions and antagonizes environmental spoilage organisms.

[0133] Equivalent plan

[0134] Although the present invention has been described in conjunction with the specific embodiment set forth above, many substitutions, modifications and other variations of the present invention will be apparent to those skilled in the art. All such substitutions, modifications and variations are intended to fall within the spirit and scope of the present invention.

Claims

1. A composition comprising a liquid endospore suspension and at least one penetrating preservative.

2. The composition of claim 1, wherein the liquid endospore suspension is an aqueous suspension.

3. The composition of claim 1, wherein the liquid endospore suspension comprises at least one endospore-forming Bacillus species.

4. The composition of claim 1, wherein the at least one endospore-forming Bacillus is selected from the group consisting of Bacillus, Brevibacillus, Paenibacillus, and Lysinibacillus.

5. The composition of claim 1 , wherein the Bacillus species is selected from the group consisting of B. amyloliquefaciens (BA), B. subtilis (BS), B. pumilus (BP), B. licheniformis (BL), B. thuringiensis, B. velezensis, B. endophyticus, and Priestia megaterium (Bacillus megaterium).

6. The composition of claim 1, wherein the at least one osmotic preservative is an osmotic pressure regulator.

7. The composition of claim 6, wherein the osmotic pressure regulating agent reduces water energy and / or induces preservation of the endospores.

8. The composition of claim 1, wherein the at least one osmotic preservative is a chloride salt.

9. The composition according to claim 8, wherein the at least one osmotic preservative is selected from sodium chloride, calcium chloride, potassium chloride, magnesium chloride and any other chloride salt.

10. The composition of claim 1, wherein the composition comprises at least about 10.0 wt% of the at least one osmotic preservative.

11. The composition of claim 1 , wherein the liquid endospore suspension is stable at 40°C for at least 180 days.

12. A method of inhibiting the proliferation of at least one environmental spoilage organism in a liquid endospore suspension, wherein the method comprises using at least one penetrating preservative.

13. The method of claim 12, wherein the at least one environmental spoilage organism is selected from the group consisting of bacterial contaminants, yeast contaminants, and fungal contaminants.

14. The method of claim 13, wherein the bacterial contaminant is selected from the group consisting of Pseudomonas species, Citrobacter species, Klebsiella species, Shigella species, Enterobacter species, and Serratia species.

15. The method of claim 12, wherein the at least one endospore-forming Bacillus is selected from the group consisting of Bacillus species, Brevibacillus species, Paenibacillus species, and Lysinibacillus species.

16. The method of claim 12, wherein the Bacillus species is selected from the group consisting of Bacillus amyloliquefaciens (BA), Bacillus subtilis (BS), Bacillus pumilus (BP), Bacillus licheniformis (BL), Bacillus thuringiensis, Bacillus velezensis, Bacillus endophyticus, and Priesteria megaterium (synonymous with Bacillus megaterium).

17. The method of claim 12, wherein the at least one osmotic preservative is an osmotic pressure regulator.

18. The method of claim 17, wherein the osmotic pressure regulating agent reduces water energy and / or induces preservation of the endospores.

19. The method of claim 12, wherein the at least one osmotic preservative is selected from the group consisting of sodium chloride, calcium chloride, potassium chloride, magnesium chloride, and any other chloride salt.

20. The method of claim 12, wherein the liquid endospore suspension is stable at 40°C for at least 180 days.

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