Mycosubtilin in baked products

Abstract

This invention relates to a method for preserving baked goods, comprising providing dough containing antifungalin or a functional derivative thereof, shaping the dough, and baking the shaped dough to obtain a baked goods, and relates to dough containing antifungalin or a functional derivative thereof, premixes, and baked goods.

Description

Technical Field

[0001] This invention relates to baking products, dough for preparing baking products, and methods for preserving baking products. Background Technology

[0002] Microbial spoilage of baked goods caused by mold, yeast, and bacteria is a serious problem, leading to significant food waste. Fungal growth can cause sensory defects in baked goods, ranging from visual degradation to noticeable changes in smell, taste, or texture, and can also have negative health effects through mycotoxins produced by some molds. To address this economic and safety issue, the baking industry has been working to identify processing methods that can make bread safe and extend its shelf life. Physical methods and chemical preservatives have long been used. However, public authorities are encouraging the food industry to limit the use of chemical preservatives and develop natural methods for food preservation. This is accompanied by a strong societal demand for "clean label" foods, as consumers seek more natural, less processed, and safer products.

[0003] While natural preservatives have been quite successful in replacing chemical preservatives in various types of food and beverages, finding effective natural preservatives for baked goods, especially bread, remains a challenge. This is related to factors such as the complex matrix of dough / bread, the shear forces during mixing, the intense heat treatment during baking, and the lack of flavoring compounds that can mask the potential sensory effects of preservatives.

[0004] Furthermore, overcoming post-baking contamination of baked goods (primarily fungal and yeast contamination) requires effective bakery design to minimize contamination, isolation of low- and high-risk areas, efficient air handling systems and maintenance, and intensive cleaning programs. However, due to the lack of proper hygiene conditions and design in most bakeries, many facilities suffer from post-baking contamination, leading to increased pressure on the preservation of baked goods.

[0005] Various natural preservatives have been explored for preserving baked goods, including plant extracts (Rizzello et al., 2015, Appl. Environ. Microbiol., 81:4195-4206; Wei et al., 2009, J. Food Sci., 74: M177-M184), polyphenols (Juanying Ou: Advances in food and Nutrition Research, Vol 98, 2021, pp207-252), essential oils (Debonne et al., 2019, Food microbiology, 83, 9-17), antimicrobial peptides (Thery et al., 2016, Innovative Food Science and Emerging Technologies, 38, 160-168), and antifungal yeasts (Coda et al., 2013, Food Microbiology, 33: (243-251), etc. However, many of these compounds failed to effectively prevent mold growth, mainly due to their interaction with dough / bread matrix, lack of reasonable resistance to heat and shear mixing forces, oxidation and volatilization of active ingredients, and strong sensory effects (Debonne et al., 2019, Food Microbiology, 83, 9-17; https: / / doi.org / 10.1016 / bs.afnr.2021.02.009).

[0006] Therefore, there is a need for a natural preservative for baked goods that has comparable or better activity against molds, yeasts, and / or bacteria that commonly contribute to the spoilage of baked goods, especially bread, compared to chemical preservatives.

[0007] Biosurfactants are known to possess many interesting properties, including antimicrobial properties. Among various biosurfactants, lipopeptides produced by Bacillus spp. have been extensively evaluated in vitro for their effectiveness against food spoilage microorganisms such as Rhizopus stolonifer, Paecilomyces variotii, and Byssochlamys fulva (Kourmentza et al., Frontiers in Microbiology, 2021, Vol 11, Article 561060), and plant pathogens such as Zymoseptoria tritici (Mejri, S. et al., Environ Sci PollutRes, 25, 29822-29833) and Fusarium oxysporum (Mihalache, G. et al., *Environmental Science & Pollution Research*, 25, 29784-29793 (2018) and *Venturia inaequalis* (Desmyttere et al., *Frontiers in Microbiology*, 22 October 2019, Vol. 10, https: / / doi.org / 10.3389 / fmicb.2019.02327). However, the application of lipopeptides in the preservation of baked goods has not been reported.

[0008] The inventors recognized that the lipopeptide biosurfactant antimycin has a similar ability to preserve baked goods at a similar level compared to typical chemical preservatives used (such as calcium propionate). Therefore, this invention relates to a method for preserving baked goods, comprising providing dough containing antimycin or a functional derivative thereof, shaping the dough, and baking the shaped dough to obtain the baked goods. Summary of the Invention

[0009] On one hand, the present invention provides a method for preserving baked goods, comprising providing dough containing antifungal agents or functional derivatives thereof, shaping the dough and baking the shaped dough to obtain baked goods.

[0010] Preferably, the dough contains an antifungal agent, and preferably the antifungal agent comprises one or more isomers of the antifungal agent selected from iso-C15, anteiso-C15, iso-C16, n-C16, anteiso-C17, iso-C17, iso-C18, and n-C18. Particularly good results have been achieved with the C17 isomer of the antifungal agent or a mixture of antifungal agents, wherein one or more C17 isomers are the predominant isomer.

[0011] Antifungalin is preferably produced by bacteria of the genus Bacillus, preferably selected from Bacillus subtilis, Bacillus thuringiensis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, and Bacillus mohair, more preferably Bacillus subtilis. Based on flour weight (FWB), the antifungalin or its functional derivative is preferably added in an amount of at least 0.1 wt.%, preferably at least 0.3 wt.%, more preferably about 0.1 wt.% to about 1 wt.% FWB. Regarding the amount of antifungalin or its functional derivative, based on the total weight of the product prepared using the antifungalin, the antifungalin or its functional derivative is generally added in an amount of at least 0.02 wt.%, preferably at least 0.06 wt.%, more preferably at least 0.2 wt.%, and most preferably at least 0.3 wt.% based on the weight of the product. Preferably, the amount is at most about 1 wt.% based on the weight of the product, more preferably at most about 0.8 wt.% based on the weight of the product, and particularly at most about 0.6 wt.% based on the weight of the product.

[0012] If chemical preservatives are present, the dough further preferably contains up to 0.3 wt.% of chemical preservatives based on the weight of the flour, more preferably up to 0.05 wt.% of chemical preservatives, even more preferably up to 0.01 wt.% of chemical preservatives, and particularly substantially no chemical preservatives. In terms of the amount of chemical additives based on the total weight of the product, the amount is advantageously in the range of 0-0.3 wt.%, preferably in the range of 0-0.1 wt.%, more preferably up to 0.03 wt.%, and even more preferably up to 0.006 wt.%.

[0013] The present invention also relates to a baking product, preferably a baking product obtainable by the method according to the present invention, which contains antifungalin or a functional derivative thereof.

[0014] The baked product contains antifungal subtilisin, preferably wherein the antifungal subtilisin comprises one or more isomers of antifungal subtilisin selected from iso-C15, anteiso-C15, iso-C16, n-C16, anteiso-C17, iso-C17, iso-C18 and n-C18.

[0015] The baked goods are preferably free of chemical preservatives, and more preferably, the baked goods are clean label products.

[0016] The baked products according to the invention are preferably selected from bread, such as bread rolls, bagels, rolls, bread rolls, croissants, baguettes, pretzels, custard rolls, muffins and flatbreads; dessert cakes, such as butter cakes, sponge cakes, chiffon cakes, Genoa cakes, muffins, chocolate cakes, cheesecakes and angel food cakes; pastries, pies, biscuits, flatbreads and pizzas, preferably wherein the baked products are bread, more preferably bread rolls.

[0017] According to the present invention, the baked product advantageously has an extended shelf life compared to comparable baked products that use the same weight of chemical preservatives, preferably calcium propionate instead of one or more antifungal agents, but are otherwise identical.

[0018] During the accelerated shelf life test (ASLT), when stored at about 21°C, the baked products preferably have an accelerated shelf life of at least 96 hours, preferably at least 120 hours, more preferably at least 144 hours, and especially at least 168 hours.

[0019] In another aspect, the present invention provides dough or premix for preparing baked goods, the dough or premix comprising antimycin or a functional derivative thereof, the antimycin preferably comprising one or more isomers of antimycin selected from iso-C15, anteiso-C15, iso-C16, n-C16, anteiso-C17, iso-C17, iso-C18 and n-C18.

[0020] Based on the weight of flour, the dough or premix according to the invention for preparing baked products preferably contains at least 0.1 wt.% antifungal and / or up to 0.1 wt.% chemical preservatives, more preferably substantially free of chemical preservatives.

[0021] This invention also relates to the use of antimicrobial agents, specifically antimicrobial agents, in baked goods. The invention further relates to the use of antimicrobial agents, specifically antimicrobial agents, against one or more of the following: *Penicillium paneum*, *Penicillium roqueforti*, *Rhizopus spp.* (especially *Rhizopus stolonifer*), *Aspergillus ruber*, and *Hyphopichia spp.* (especially *Hyphopichia burtonii*). The antimicrobial use according to the invention is particularly suitable for beverages or foods, preferably baked goods, especially bread. In baked goods, especially bread, antimicrobial agents can be further specifically used as antimicrobial agents against *A. niger*. The use according to the invention (as an antimicrobial agent or preservative) is generally in vitro (non-medical). Attached Figure Description

[0022] Figure 1 Chemical structures of antifungal subtilisin anteiso-C17 and antifungal subtilisin iso-C17.

[0023] Figure 2A Accelerated shelf-life test (ASLT) of the antimicrobial effects of antifungal subtilisin on Aspergillus niger and Penicillium liquefaction: a snapshot of bread inoculated with ±100 spores of each mold at 6 days and 16 hours.

[0024] Figure 2B Accelerated shelf-life test (ASLT) of the antimicrobial effect of antifungal subtilisin on Aspergillus niger and Penicillium liquefaction: Time to appearance (TOA) of mold in control bread (left column: data on Penicillium liquefaction and Aspergillus niger), bread with added antifungal subtilisin (added at 0.3% flour weight (FWB), middle column), and bread with added calcium propionate (0.3% FWB, right column).

[0025] Figure 3 The effects of adding 0.1 wt.% and 0.3 wt.% antifungal agents on water activity and pH compared to control bread (without preservatives) and bread containing calcium propionate (0.3% FWB).

[0026] Figure 4 The effect of adding antifungal agent (0.3% FWB) on dough properties compared to control bread (no preservatives) and bread containing calcium propionate (0.3% FWB) when using a DoughLAB mixer (PerkinElmer). Detailed Implementation

[0027] Unless otherwise stated, as used herein, the term "or" means "and / or". Unless otherwise stated, as used herein, the terms "a" or "an" mean "at least one / an".

[0028] The term “substantially” is generally used herein to indicate that a product has the specified general characteristic or function. When referring to a quantifiable characteristic, this term is particularly used to indicate that the quantifiable characteristic is greater than 90%, more particularly greater than 95%, and even more particularly greater than 98% of the maximum value of the characteristic. The term “substantially free” is generally used herein to mean that a substance is absent (below the detection limit achievable by analytical techniques available at the effective application date), or is present in such a low amount that the substance does not significantly affect the properties of a product substantially free of the substance, or is present in such a low amount (trace) that the substance does not need to be labeled on packaged products substantially free of the substance. In practice, quantitatively, based on the total weight of the product in which the substance is present, a product is generally considered substantially free of the substance if the content of the substance is 0–0.1 wt.%, particularly 0–0.01 wt.%, more particularly 0–0.005 wt.%.

[0029] As those skilled in the art will understand, the term "about" regarding values ​​generally includes a range around that value. In particular, the range is from at least 10% below the value to at least 10% above the value, and more specifically, from 5% below the value to 5% above the value. When a "noun" (e.g., compound, additive, etc.) is mentioned in the singular, it is intended to include the plural unless otherwise stated.

[0030] "Mycosubtilin" is a natural cyclic lipopeptide with a peptide sequence of L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Ser-L-Asn and a saturated β-amino fatty acid fused to an N-terminal Asn residue on a carboxylic acid. Several isomers of mycosubtilin have been described, depending on the length of the fatty acid carbon chain and whether the fatty acid is straight or branched. The term mycosubtilin covers any naturally occurring isomer of mycosubtilin, either in isolated form or as a mixture of isomers, including mycosubtilin iso-C15, mycosubtilin anteiso-C15, mycosubtilin iso-C16, mycosubtilin n-C16, mycosubtilin anteiso-C17, mycosubtilin iso-C17, mycosubtilin iso-C18, and mycosubtilin n-C18. The structures of antifungal subtilisin anteiso-C17 and antifungal subtilisin iso-C17 are in Figure 1 As shown in the image.

[0031] As used herein, the term "functional derivative" refers to a baked product containing an antimicrobial subtilisin derivative with significant antimicrobial activity, thereby extending the shelf life of the baked product compared to a control baked product that lacks antimicrobial subtilisin but is otherwise identical. Functional activity can be determined using, for example, the accelerated baking test described in Example 2 below.

[0032] As used herein, the term "dough" refers to a mixture of dough ingredients in an amount suitable for preparing a baked product. The dough ingredients used depend on the baked product being prepared. For example, typical dough ingredients for bread preparation include flour, water, salt, and yeast. Sweet baked products often also contain one or more sweeteners, such as sugar or sugar substitutes, such as aspartame or honey. Typically, dough is kneadable or pourable at 25°C and atmospheric pressure. After the dough has been heated for a sufficient time, a baked product is obtained, which is a solid food product that is no longer kneadable or pourable.

[0033] Although the antimicrobial activity of many natural compounds against fungi involved in the spoilage of baked goods has been described in vitro, most of these compounds failed to extend the shelf life of baked goods in baking tests. While the exact mechanisms remain unknown, the inventors anticipate that many natural antimicrobial compounds will be inactivated or inhibited by common dough ingredients, for example, due to the binding of the antimicrobial compounds to one or more of the dough ingredients.

[0034] The inventors discovered that baked goods containing antimicrobial subtilisin as a natural preservative exhibited a longer shelf life compared to control baked goods (containing no preservatives) and reference baked goods (containing calcium propionate as a chemical preservative). This is surprising because other natural compounds known to exhibit antimicrobial activity, including natural compounds derived from the same class of lipopeptides and / or structurally similar compounds, have failed to extend the shelf life of baked goods.

[0035] Antifungalin is a natural lipopeptide composed of a cyclic heptapeptide and a saturated β-amino fatty acid moiety with a length of fourteen to eighteen carbon atoms. It belongs to the itucrin family, which also includes itucrin A and itucrin A2. L Iridin C, Bacillus D, Bacillus F, Bacillus L and Bacillus L C (Guez et al., 2022. Metabolites, 12, 107).

[0036] Antimycin can exist as one or more different isomers or mixtures thereof. The isomerism of antimycin is determined by the branching of the carbon chain of the fatty acid moiety of antimycin.

[0037] The fatty acid portion of the antifungal subtilisin is preferably methyl-branched. When the fatty acid is a methyl-branched fatty acid, the branch point can be located on the penultimate carbon atom, resulting in an isomethyl-branched fatty acid, or the branch point can be located on the penultimate carbon atom, resulting in an anteiso methyl-branched fatty acid.

[0038] Furthermore, the fatty acid preferably comprises about fourteen to about eighteen carbon atoms, more preferably sixteen or seventeen carbon atoms.

[0039] Preferably, the antifungal subtilisin includes one or more isomers selected from antifungal subtilisin iso-C15, antifungal subtilisin anteiso-C15, antifungal subtilisin iso-C16, antifungal subtilisin n-C16, antifungal subtilisin anteiso-C17, antifungal subtilisin iso-C17, antifungal subtilisin iso-C18, and antifungal subtilisin n-C18.

[0040] On the one hand, the antimycobacterial isomer is an isolated antimycobacterial isomer, preferably an isolated antimycobacterial isomer selected from antimycobacterial iso-C15, antimycobacterial anteiso-C15, antimycobacterial iso-C16, antimycobacterial n-C16, antimycobacterial anteiso-C17, antimycobacterial iso-C17, antimycobacterial iso-C18, and antimycobacterial n-C18.

[0041] The isomer can be isolated from a mixture of isomers or can be prepared synthetically, for example using a suitable chemical-enzymatic synthesis scheme.

[0042] The isomers can be separated from the mixture of isomers using any suitable method known in the art, such as high-performance liquid chromatography (HPLC).

[0043] On the other hand, the antifungal subtilisin preferably comprises a mixture of antifungal subtilisin isomers, more preferably a mixture of two or more isomers of antifungal subtilisin.

[0044] More preferably, the mixture of at least two or more antifungal subtilisin isomers comprises at least anteiso-C15 and anteiso-C17 antifungal subtilisin, at least anteiso-C15 and iso-C18 antifungal subtilisin, at least anteiso-C15 and n-C18 antifungal subtilisin, at least anteiso-C15 and iso-C15 antifungal subtilisin, at least anteiso-C15 and iso-C16 antifungal subtilisin, at least anteiso-C15 and n- C16 antifungal blight inhibitor, at least anteiso-C15 and iso-C17 antifungal blight inhibitor, at least anteiso-C17 and iso-C18 antifungal blight inhibitor, at least anteiso-C17 and n-C18 antifungal blight inhibitor, at least anteiso-C17 and iso-C15 antifungal blight inhibitor, at least anteiso-C17 and iso-C16 antifungal blight inhibitor, at least anteiso-C17 and n-C16 antifungal blight inhibitor, at least anteiso-C17 antifungal blight inhibitor. Substances and ISO-C17 antifungal substances, at least ISO-C18 antifungal substances and n-C18 antifungal substances, at least ISO-C18 antifungal substances and ISO-C15 antifungal substances, at least ISO-C18 antifungal substances and ISO-C16 antifungal substances, at least ISO-C18 antifungal substances and n-C16 antifungal substances, at least ISO-C18 antifungal substances and ISO-C17 antifungal substances, at least n-C18 antifungal substances and ISO-C15 antifungal substances, at least n-C18 antifungal substances and ISO-C16 antifungal substances, at least n- C18 antibacterial blight inhibitor and n-C16 antibacterial blight inhibitor, at least n-C18 antibacterial blight inhibitor and iso-C17 antibacterial blight inhibitor, at least iso-C15 antibacterial blight inhibitor and iso-C16 antibacterial blight inhibitor, at least iso-C15 antibacterial blight inhibitor and n-C16 antibacterial blight inhibitor, at least iso-C15 antibacterial blight inhibitor and iso-C17 antibacterial blight inhibitor, at least iso-C16 antibacterial blight inhibitor and n-C16 antibacterial blight inhibitor, at least iso-C16 antibacterial blight inhibitor and iso-C17 antibacterial blight inhibitor, or at least n-C16 antibacterial blight inhibitor and iso-C17 antibacterial blight inhibitor.

[0045] Alternatively or additionally, the antimycin may comprise a mixture of three or more antimycin isomers.

[0046] More preferably, the mixture of at least three or more antifungal bacillus isomers comprises at least iso-C18 antifungal bacillus, n-C18 antifungal bacillus, and iso-C15 antifungal bacillus; at least iso-C18 antifungal bacillus, n-C18 antifungal bacillus, and anteiso-C15 antifungal bacillus; at least iso-C18 antifungal bacillus, n-C18 antifungal bacillus, and iso-C16 antifungal bacillus; at least iso-C18 antifungal bacillus, n-C18 antifungal bacillus, and n-C16 antifungal bacillus; at least iso-C18 antifungal bacillus, n-C18 antifungal bacillus, and iso-C17 antifungal bacillus; at least ISO-C18 antibacterial bacillus, n-C18 antibacterial bacillus, and anteiso-C17 antibacterial bacillus, at least ISO-C18 antibacterial bacillus, ISO-C15 antibacterial bacillus, and anteiso-C15 antibacterial bacillus, at least ISO-C18 antibacterial bacillus, ISO-C15 antibacterial bacillus, and ISO-C16 antibacterial bacillus, at least ISO-C18 antibacterial bacillus, ISO-C15 antibacterial bacillus, and n-C16 antibacterial bacillus, at least ISO-C18 antibacterial bacillus, ISO-C15 antibacterial bacillus, and ISO-C17 antibacterial bacillus, at least ISO-C18 antibacterial bacillus, ISO-C15 Antifungal blight inhibitors and anteiso-C17 antifungal blight inhibitors, at least iso-C18 antifungal blight inhibitors, anteiso-C15 antifungal blight inhibitors and iso-C16 antifungal blight inhibitors, at least iso-C18 antifungal blight inhibitors, anteiso-C15 antifungal blight inhibitors and n-C16 antifungal blight inhibitors, at least iso-C18 antifungal blight inhibitors, anteiso-C15 antifungal blight inhibitors and iso-C17 antifungal blight inhibitors, at least iso-C18 antifungal blight inhibitors, anteiso-C15 antifungal blight inhibitors and anteiso-C17 antifungal blight inhibitors, at least iso-C18 antifungal blight inhibitors, iso-C16 antifungal blight inhibitors Subtilisin and n-C16 antifungal subtilisin, at least iso-C18 antifungal subtilisin, iso-C16 antifungal subtilisin and iso-C17 antifungal subtilisin, at least iso-C18 antifungal subtilisin, iso-C16 antifungal subtilisin and anteiso-C17 antifungal subtilisin, at least iso-C18 antifungal subtilisin, n-C16 antifungal subtilisin and iso-C17 antifungal subtilisin, at least iso-C18 antifungal subtilisin, n-C16 antifungal subtilisin and anteiso-C17 antifungal subtilisin, at least iso-C18 antifungal subtilisin, iso-C17 antifungal subtilisin and anteiso-C17 antifungal subtilisinAt least n-C18 anti-bacterial mycotoxin, iso-C15 anti-bacterial mycotoxin, and anteiso-C15 anti-bacterial mycotoxin; at least n-C18 anti-bacterial mycotoxin, iso-C15 anti-bacterial mycotoxin, and iso-C16 anti-bacterial mycotoxin; at least n-C18 anti-bacterial mycotoxin, iso-C15 anti-bacterial mycotoxin, and n-C16 anti-bacterial mycotoxin; at least n-C18 anti-bacterial mycotoxin, iso-C15 anti-bacterial mycotoxin, and iso-C17 anti-bacterial mycotoxin; at least n-C18 anti-bacterial mycotoxin, iso-C15 anti-bacterial mycotoxin, and anteiso-C17 anti-bacterial mycotoxin; at least n-C18 anti-bacterial mycotoxin and anteiso-C15 anti-bacterial mycotoxin. and iso-C16 antifungal, at least n-C18 antifungal, anteiso-C15 antifungal and n-C16 antifungal, at least n-C18 antifungal, anteiso-C15 antifungal and iso-C17 antifungal, at least n-C18 antifungal, anteiso-C15 antifungal and anteiso-C17 antifungal, at least n-C18 antifungal, iso-C16 antifungal and n-C16 antifungal, at least n-C18 antifungal, iso-C16 antifungal and iso-C17 antifungal, at least n-C18 antifungal At least n-C18 anti-fungal, at least n-C16 anti-fungal, at least n-C16 anti-fungal, at least n-C17 anti-fungal, at least n-C18 anti-fungal, at least n-C16 anti-fungal, at least n-C17 anti-fungal, at least n-C18 anti-fungal, at least n-C17 anti-fungal, at least n-C17 anti-fungal, at least n-C17 anti-fungal, at least n-C15 anti-fungal, at least n-C16 anti-fungal, at least n-C15 ... And n-C16 antifungal agent, at least iso-C15 antifungal agent, anteiso-C15 antifungal agent and iso-C17 antifungal agent, at least iso-C15 antifungal agent, anteiso-C15 antifungal agent and anteiso-C17 antifungal agent, at least iso-C15 antifungal agent, iso-C16 antifungal agent and n-C16 antifungal agent, at least iso-C15 antifungal agent, iso-C16 antifungal agent and iso-C17 antifungal agent, at least iso-C15 antifungal agent, iso-C16 antifungal agent and anteiso-C17 antifungal agent.At least iso-C15 anti-bacterial mycotoxin, n-C16 anti-bacterial mycotoxin, and iso-C17 anti-bacterial mycotoxin; at least iso-C15 anti-bacterial mycotoxin, iso-C16 anti-bacterial mycotoxin, and anteiso-C17 anti-bacterial mycotoxin; at least iso-C15 anti-bacterial mycotoxin, iso-C17 anti-bacterial mycotoxin, and anteiso-C17 anti-bacterial mycotoxin; at least anteiso-C15 anti-bacterial mycotoxin, iso-C16 anti-bacterial mycotoxin, and n-C16 anti-bacterial mycotoxin; at least anteiso-C15 anti-bacterial mycotoxin, iso-C16 anti-bacterial mycotoxin, and iso-C17 anti-bacterial mycotoxin; at least anteiso-C15 anti-bacterial mycotoxin, iso-C16 anti-bacterial mycotoxin, and anteiso-C17 anti-bacterial mycotoxin. At least anteiso-C15 antibacterial blight inhibitor, n-C16 antibacterial blight inhibitor, and iso-C17 antibacterial blight inhibitor; at least anteiso-C15 antibacterial blight inhibitor, n-C16 antibacterial blight inhibitor, and anteiso-C17 antibacterial blight inhibitor; at least anteiso-C15 antibacterial blight inhibitor, iso-C17 antibacterial blight inhibitor, and anteiso-C17 antibacterial blight inhibitor; at least iso-C16 antibacterial blight inhibitor, n-C16 antibacterial blight inhibitor, and iso-C17 antibacterial blight inhibitor; at least iso-C16 antibacterial blight inhibitor, n-C16 antibacterial blight inhibitor, and anteiso-C17 antibacterial blight inhibitor; or at least n-C16 antibacterial blight inhibitor, iso-C17 antibacterial blight inhibitor, and anteiso-C17 antibacterial blight inhibitor.

[0047] Alternatively or additionally, the antifungalin comprises a mixture of four or more antifungalin isomers.

[0048] More preferably, the mixture of at least four or more antifungal bacillus isomers includes at least iso-C18 antifungal bacillus, n-C18 antifungal bacillus, iso-C15 antifungal bacillus, and anteiso-C15 antifungal bacillus; at least iso-C18 antifungal bacillus, n-C18 antifungal bacillus, iso-C15 antifungal bacillus, and iso-C16 antifungal bacillus; at least iso-C18 antifungal bacillus, n-C18 antifungal bacillus, iso-C15 antifungal bacillus, and n-C16 antifungal bacillus; at least iso-C18 antifungal bacillus, n-C18 antifungal bacillus, and iso-C15 antifungal bacillus. Bacillus subtilis and iso-C17 antibacterial bacillus, at least iso-C18 antibacterial bacillus, n-C18 antibacterial bacillus, iso-C15 antibacterial bacillus and anteiso-C17 antibacterial bacillus, at least iso-C18 antibacterial bacillus, n-C18 antibacterial bacillus, anteiso-C15 antibacterial bacillus and iso-C16 antibacterial bacillus, at least iso-C18 antibacterial bacillus, n-C18 antibacterial bacillus, anteiso-C15 antibacterial bacillus and n-C16 antibacterial bacillus, at least iso-C18 antibacterial bacillus, n-C18 antibacterial bacillus, anteiso-C15 antibacterial bacillus Antimicrobial and iso-C17 antimicrobial, at least iso-C18 antimicrobial, n-C18 antimicrobial, anteiso-C15 antimicrobial and anteiso-C17 antimicrobial, at least iso-C18 antimicrobial, n-C18 antimicrobial, iso-C16 antimicrobial and n-C16 antimicrobial, at least iso-C18 antimicrobial, n-C18 antimicrobial, iso-C16 antimicrobial and iso-C17 antimicrobial, at least iso-C18 antimicrobial, n-C18 antimicrobial, iso-C16 antimicrobial and anteiso so-C17 antibacterial bactericide, at least iso-C18 antibacterial bactericide, n-C18 antibacterial bactericide, n-C16 antibacterial bactericide and iso-C17 antibacterial bactericide, at least iso-C18 antibacterial bactericide, n-C18 antibacterial bactericide, iso-C16 antibacterial bactericide and anteiso-C17 antibacterial bactericide, at least iso-C18 antibacterial bactericide, n-C18 antibacterial bactericide, n-C16 antibacterial bactericide and iso-C17 antibacterial bactericide, at least iso-C18 antibacterial bactericide, n-C16 antibacterial bactericide and anteiso-C17 antibacterial bactericide,At least iso-C18 anti-bacterial bactericide, n-C18 anti-bacterial bactericide, iso-C17 anti-bacterial bactericide, and anteiso-C17 anti-bacterial bactericide; at least iso-C18 anti-bacterial bactericide, iso-C15 anti-bacterial bactericide, anteiso-C15 anti-bacterial bactericide, and iso-C16 anti-bacterial bactericide; at least iso-C18 anti-bacterial bactericide, iso-C15 anti-bacterial bactericide, anteiso-C15 anti-bacterial bactericide, and n-C16 anti-bacterial bactericide; at least iso-C18 anti-bacterial bactericide, iso-C15 anti-bacterial bactericide, anteiso-C15 anti-bacterial bactericide, and iso-C17 anti-bacterial bactericide. At least iso-C18 anti-bacterial bacteriocin, iso-C15 anti-bacterial bacteriocin, anteiso-C15 anti-bacterial bacteriocin, and anteiso-C17 anti-bacterial bacteriocin; at least iso-C18 anti-bacterial bacteriocin, iso-C15 anti-bacterial bacteriocin, iso-C16 anti-bacterial bacteriocin, and n-C16 anti-bacterial bacteriocin; at least iso-C18 anti-bacterial bacteriocin, iso-C15 anti-bacterial bacteriocin, iso-C16 anti-bacterial bacteriocin, and iso-C17 anti-bacterial bacteriocin; at least iso-C18 anti-bacterial bacteriocin, iso-C15 anti-bacterial bacteriocin, iso-C16 anti-bacterial bacteriocin, and anteiso-C17 anti-bacterial bacteriocin. At least iso-C18 antifungal, anteiso-C15 antifungal, iso-C16 antifungal, and n-C16 antifungal; at least iso-C18 antifungal, iso-C15 antifungal, iso-C16 antifungal, and iso-C17 antifungal; at least iso-C18 antifungal, iso-C15 antifungal, iso-C16 antifungal, and anteiso-C17 antifungal; at least iso-C18 antifungal, iso-C15 antifungal, iso-C16 antifungal, and iso-C17 antifungal; at least... ISO-C18 antibacterial bactericide, ISO-C15 antibacterial bactericide, n-C16 antibacterial bactericide, and anteiso-C17 antibacterial bactericide, at least ISO-C18 antibacterial bactericide, ISO-C15 antibacterial bactericide, ISO-C17 antibacterial bactericide, and anteiso-C17 antibacterial bactericide, at least ISO-C18 antibacterial bactericide, anteiso-C15 antibacterial bactericide, ISO-C16 antibacterial bactericide, and n-C16 antibacterial bactericide, at least ISO-C18 antibacterial bactericide, anteiso-C15 antibacterial bactericide, ISO-C16 antibacterial bactericide, and ISO-C17 antibacterial bactericide,At least iso-C18 antifungal bacillus, anteiso-C15 antifungal bacillus, iso-C16 antifungal bacillus, and anteiso-C17 antifungal bacillus; at least iso-C18 antifungal bacillus, iso-C16 antifungal bacillus, n-C16 antifungal bacillus, and iso-C17 antifungal bacillus; at least iso-C18 antifungal bacillus, iso-C16 antifungal bacillus, n-C16 antifungal bacillus, and anteiso-C17 antifungal bacillus; at least n-C18 antifungal bacillus, iso-C15 antifungal bacillus, anteiso-C15 antifungal bacillus, and iso-C16 antifungal bacillus. Subtilisin, at least n-C18 antifungal subtilisin, iso-C15 antifungal subtilisin, anteiso-C15 antifungal subtilisin and n-C16 antifungal subtilisin, at least n-C18 antifungal subtilisin, iso-C15 antifungal subtilisin, anteiso-C15 antifungal subtilisin and iso-C17 antifungal subtilisin, at least n-C18 antifungal subtilisin, iso-C15 antifungal subtilisin, anteiso-C15 antifungal subtilisin and anteiso-C17 antifungal subtilisin, at least n-C18 antifungal subtilisin, iso-C15 antifungal subtilisin, iso-C16 antifungal subtilisin and n-C16 antifungal subtilisin At least n-C18 anti-bacterial mycotoxin, iso-C15 anti-bacterial mycotoxin, iso-C16 anti-bacterial mycotoxin, and iso-C17 anti-bacterial mycotoxin; at least n-C18 anti-bacterial mycotoxin, iso-C15 anti-bacterial mycotoxin, iso-C16 anti-bacterial mycotoxin, and anteiso-C17 anti-bacterial mycotoxin; at least n-C18 anti-bacterial mycotoxin, iso-C15 anti-bacterial mycotoxin, iso-C16 anti-bacterial mycotoxin, and anteiso-C17 anti-bacterial mycotoxin; at least n- C18 antifungal agent, iso-C15 antifungal agent, iso-C17 antifungal agent, and anteiso-C17 antifungal agent, at least n-C18 antifungal agent, anteiso-C15 antifungal agent, iso-C16 antifungal agent, and n-C16 antifungal agent, at least n-C18 antifungal agent, anteiso-C15 antifungal agent, iso-C16 antifungal agent, and iso-C17 antifungal agent, at least n-C18 antifungal agent, anteiso-C15 antifungal agent, iso-C16 antifungal agent, and anteiso-C17 antifungal agent,At least n-C18 antibacterial blight inhibitor, anteiso-C15 antibacterial blight inhibitor, n-C16 antibacterial blight inhibitor, and iso-C17 antibacterial blight inhibitor; at least n-C18 antibacterial blight inhibitor, anteiso-C15 antibacterial blight inhibitor, n-C16 antibacterial blight inhibitor, and anteiso-C17 antibacterial blight inhibitor; at least n-C18 antibacterial blight inhibitor, iso-C16 antibacterial blight inhibitor, n-C16 antibacterial blight inhibitor, and iso-C17 antibacterial blight inhibitor; at least n- C18 antibacterial blight inhibitor, iso-C16 antibacterial blight inhibitor, n-C16 antibacterial blight inhibitor, and anteiso-C17 antibacterial blight inhibitor, at least n-C18 antibacterial blight inhibitor, n-C16 antibacterial blight inhibitor, iso-C17 antibacterial blight inhibitor, and anteiso-C17 antibacterial blight inhibitor, at least n-C18 antibacterial blight inhibitor, n-C16 antibacterial blight inhibitor, iso-C17 antibacterial blight inhibitor, and anteiso-C17 antibacterial blight inhibitor, at least iso-C15 antibacterial blight inhibitor, anteiso-C15 antibacterial blight inhibitor, iso-C16 antibacterial blight inhibitor, and n-C16 antibacterial blight inhibitor, at least iso-C15 antibacterial blight inhibitor... Antibacterial agents, including anteiso-C15, iso-C16, and iso-C17, with at least iso-C15, anteiso-C15, iso-C16, and iso-C17, and at least iso-C15, anteiso-C15, n-C16, and iso-C17. At least iso-C15 antifungal, anteiso-C15 antifungal, iso-C17 antifungal, and anteiso-C17 antifungal; at least iso-C15 antifungal, iso-C16 antifungal, n-C16 antifungal, and iso-C17 antifungal; at least iso-C15 antifungal, iso-C16 antifungal, n-C16 antifungal, and anteiso-C17 antifungal; at least iso-C15 antifungal, n-C16 antifungal, iso-C17 antifungal, and anteiso-C17 antifungal.At least anteiso-C15 antibacterial blight inhibitor, iso-C16 antibacterial blight inhibitor, n-C16 antibacterial blight inhibitor, and iso-C17 antibacterial blight inhibitor.

[0049] Alternatively or additionally, the antifungal subtilisin comprises a mixture of five or more antifungal subtilisin isomers.

[0050] More preferably, the mixture of at least five or more antibacterial subtilisin isomers includes at least antibacterial subtilisin iso-C18, antibacterial subtilisin n-C18, antibacterial subtilisin iso-C15, antibacterial subtilisin anteiso-C15 and iso-C16, at least antibacterial subtilisin iso-C18, antibacterial subtilisin n-C18, antibacterial subtilisin iso-C15, antibacterial subtilisin anteiso-C15 and n-C16, and at least antibacterial subtilisin iso-C18, antibacterial subtilisin n-C18, antibacterial subtilisin iso-C15, antibacterial subtilisin anteiso-C15 and iso-C17. Antibacterial blight inhibitors, at least antibacterial blight inhibitor iso-C18, antibacterial blight inhibitor n-C18, antibacterial blight inhibitor iso-C15, antibacterial blight inhibitor anteiso-C15 and anteiso-C17; at least antibacterial blight inhibitor iso-C18, antibacterial blight inhibitor n-C18, iso-C15 antibacterial blight inhibitor, iso-C16 antibacterial blight inhibitor and n-C16 antibacterial blight inhibitor; at least antibacterial blight inhibitor iso-C18, antibacterial blight inhibitor n-C18, iso-C15 antibacterial blight inhibitor, iso-C16 antibacterial blight inhibitor and iso-C17 antibacterial blight inhibitor; at least antibacterial blight inhibitor iso-C18, antibacterial blight inhibitor... At least the following anti-mycobacterium compounds are present: n-C18, iso-C15, iso-C16, and anteiso-C17. Antibacterial agents and anteiso-C17 antibacterial agents, at least antibacterial agents iso-C18, n-C18, anteiso-C15, iso-C16, and n-C16 antibacterial agents, at least antibacterial agents iso-C18, n-C18, anteiso-C15, n-C16, and iso-C17, at least antibacterial agents iso-C18, n-C18, anteiso-C15, n-C16, and anteiso-C17.At least antibacterial bactericides ISO-C18, n-C18, anteiso-C15, ISO-C17, and anteiso-C17; at least antibacterial bactericides ISO-C18, ISO-C15, anteiso-C15, ISO-C16, and n-C16; at least antibacterial bactericides ISO-C18, ISO-C15, anteiso-C15, ISO-C16, and ISO-C17; at least antibacterial bactericides ISO-C18, antibacterial bactericides... Antifungal agents ISO-C15, anteiso-C15, ISO-C16, and anteiso-C17, and at least antifungal agents ISO-C18, ISO-C15, anteiso-C15, n-C16, and ISO-C17, and at least antifungal agents ISO-C18, ISO-C15, anteiso-C15, n-C16, and anteiso-C17, and at least antifungal agents ISO-C18, anteiso-C15, n-C16, and anteiso-C17, and at least antifungal agents ISO-C18, anteiso-C15, and i so-C16 antibacterial blight inhibitor, n-C16 antibacterial blight inhibitor, and iso-C17 antibacterial blight inhibitor, at least antibacterial blight inhibitor iso-C18, antibacterial blight inhibitor anteiso-C15, iso-C16 antibacterial blight inhibitor, n-C16 antibacterial blight inhibitor, and anteiso-C17 antibacterial blight inhibitor, at least antibacterial blight inhibitor iso-C18, antibacterial blight inhibitor anteiso-C15, iso-C16 antibacterial blight inhibitor, iso-C17 antibacterial blight inhibitor, and anteiso-C17 antibacterial blight inhibitor, at least antibacterial blight inhibitor iso-C18, antibacterial blight inhibitor anteiso-C15, n-C16 antibacterial blight inhibitor, iso- C17 antibacterial and anteiso-C17 antibacterial, at least antibacterial iso-C18, iso-C16 antibacterial, n-C16 antibacterial, iso-C17 antibacterial and anteiso-C17 antibacterial, at least antibacterial n-C18, antibacterial iso-C15, antibacterial anteiso-C15, iso-C16 antibacterial and n-C16 antibacterial, at least antibacterial n-C18, antibacterial iso-C15, antibacterial anteiso-C15, iso-C16 antibacterial and iso-C17 antibacterial.At least antibacterial bactericides n-C18, iso-C15, anteiso-C15, iso-C16, and anteiso-C17; at least antibacterial bactericides n-C18, iso-C15, anteiso-C15, n-C16, and iso-C17; at least antibacterial bactericides n-C18, iso-C15, anteiso-C15, and n-C16. Anteiso-C17 antibacterial blight inhibitor, at least antibacterial blight inhibitor n-C18, antibacterial blight inhibitor iso-C15, antibacterial blight inhibitor anteiso-C15, iso-C17 antibacterial blight inhibitor and anteiso-C17 antibacterial blight inhibitor, at least antibacterial blight inhibitor n-C18, antibacterial blight inhibitor anteiso-C15, iso-C16 antibacterial blight inhibitor, n-C16 antibacterial blight inhibitor and iso-C17 antibacterial blight inhibitor, at least antibacterial blight inhibitor n-C18, antibacterial blight inhibitor anteiso-C15, iso-C16 antibacterial blight inhibitor Antibacterial agents, n-C16 antibacterial agent and anteiso-C17 antibacterial agent, at least antibacterial agent n-C18, antibacterial agent anteiso-C15, iso-C16 antibacterial agent, iso-C17 antibacterial agent and anteiso-C17 antibacterial agent, at least antibacterial agent n-C18, iso-C16 antibacterial agent, n-C16 antibacterial agent, iso-C17 antibacterial agent and anteiso-C17 antibacterial agent, at least iso-C15 antibacterial agent, anteiso o-C15 antifungal agent, iso-C16 antifungal agent, n-C16 antifungal agent, and iso-C17 antifungal agent, at least iso-C15 antifungal agent, iso-C16 antifungal agent, n-C16 antifungal agent, and anteiso-C17 antifungal agent, at least anteiso-C15 antifungal agent, iso-C16 antifungal agent, n-C16 antifungal agent, iso-C17 antifungal agent, and anteiso-C17 antifungal agent.

[0051] Alternatively or additionally, the antifungal subtilisin comprises a mixture of six or more antifungal subtilisin isomers.

[0052] More preferably, the mixture of at least six or more antifungal subtilisin isomers includes at least iso-C18 antifungal subtilisin, n-C18 antifungal subtilisin, iso-C15 antifungal subtilisin, anteiso-C15 antifungal subtilisin, iso-C16 antifungal subtilisin, and n-C16 antifungal subtilisin; at least iso-C18 antifungal subtilisin, n-C18 antifungal subtilisin, iso-C15 antifungal subtilisin, anteiso-C15 antifungal subtilisin, iso-C16 antifungal subtilisin, and iso-C17 antifungal subtilisin; at least iso-C18 antifungal subtilisin, n-C18 antifungal subtilisin, iso-C15 antifungal subtilisin, and a... Nteiso-C15 antibacterial blight inhibitor, iso-C16 antibacterial blight inhibitor, and anteiso-C17 antibacterial blight inhibitor, at least iso-C18 antibacterial blight inhibitor, n-C18 antibacterial blight inhibitor, iso-C15 antibacterial blight inhibitor, anteiso-C15 antibacterial blight inhibitor, n-C16 antibacterial blight inhibitor, and iso-C17 antibacterial blight inhibitor, at least iso-C18 antibacterial blight inhibitor, n-C18 antibacterial blight inhibitor, iso-C15 antibacterial blight inhibitor, anteiso-C15 antibacterial blight inhibitor, n-C16 antibacterial blight inhibitor, and anteiso-C17 antibacterial blight inhibitor, at least iso-C18 antibacterial blight inhibitor, n-C18 antibacterial blight inhibitor. Mycobacterium tumefaciens, iso-C15 mycobacterium tumefaciens, anteiso-C15 mycobacterium tumefaciens, iso-C17 mycobacterium tumefaciens and anteiso-C17 mycobacterium tumefaciens, at least iso-C18 mycobacterium tumefaciens, n-C18 mycobacterium tumefaciens, iso-C15 mycobacterium tumefaciens, n-C16 mycobacterium tumefaciens and iso-C17 mycobacterium tumefaciens, at least iso-C18 mycobacterium tumefaciens, n-C18 mycobacterium tumefaciens, iso-C15 mycobacterium tumefaciens, iso-C16 mycobacterium tumefaciens, iso-C17 mycobacterium tumefaciens and anteiso-C17 mycobacterium tumefaciens, at least iso-C18 mycobacterium tumefaciens, n-C18 Antimycin, anteiso-C15 antimycin, iso-C16 antimycin, n-C16 antimycin, and anteiso-C17 antimycin, at least iso-C18 antimycin, n-C18 antimycin, anteiso-C15 antimycin, iso-C16 antimycin, n-C16 antimycin, and iso-C17 antimycin, at least iso-C18 antimycin, n-C18 antimycin, anteiso-C15 antimycin, iso-C16 antimycin, iso-C17 antimycin, and anteiso-C17 antimycin.At least iso-C18 antifungal, iso-C15 antifungal, anteiso-C15 antifungal, iso-C16 antifungal, n-C16 antifungal, and iso-C17 antifungal; at least iso-C18 antifungal, iso-C15 antifungal, anteiso-C15 antifungal, iso-C16 antifungal, n-C16 antifungal, and anteiso-C17 antifungal; at least iso-C18 antifungal, iso-C15... Antifungal agents, including anteiso-C15 antifungal agent, iso-C16 antifungal agent, iso-C17 antifungal agent, and anteiso-C17 antifungal agent, with at least iso-C18 antifungal agent, iso-C15 antifungal agent, iso-C16 antifungal agent, n-C16 antifungal agent, iso-C17 antifungal agent, and anteiso-C17 antifungal agent, with at least iso-C18 antifungal agent, anteiso-C15 antifungal agent, and iso-C16 antifungal agent. Bacillus subtilis, n-C16 antibacterial bacillus, iso-C17 antibacterial bacillus and anteiso-C17 antibacterial bacillus, at least n-C18 antibacterial bacillus, iso-C15 antibacterial bacillus, anteiso-C15 antibacterial bacillus, iso-C16 antibacterial bacillus, n-C16 antibacterial bacillus and iso-C17 antibacterial bacillus, at least n-C18 antibacterial bacillus, iso-C15 antibacterial bacillus, anteiso-C15 antibacterial bacillus, iso-C16 antibacterial bacillus, n-C16 antibacterial bacillus The text appears to contain a series of seemingly unrelated characters and symbols, possibly a corrupted or incomplete sentence. A direct translation wouldn't be meaningful without further context or clarification.

[0053] Alternatively or additionally, the antifungalin comprises a mixture of seven or more antifungalin isomers.

[0054] More preferably, the mixture of at least seven or more antifungal subtilisin isomers includes iso-C18 antifungal subtilisin, n-C18 antifungal subtilisin, iso-C15 antifungal subtilisin, anteiso-C15 antifungal subtilisin, iso-C16 antifungal subtilisin, n-C16 antifungal subtilisin, and iso-C17 antifungal subtilisin, with at least iso-C18 antifungal subtilisin, n-C18 antifungal subtilisin, iso-C15 antifungal subtilisin, anteiso-C15 antifungal subtilisin, iso-C16 antifungal subtilisin, n-C16 antifungal subtilisin, and... Anteiso-C17 antibacterial blight inhibitor, at least iso-C18 antibacterial blight inhibitor, n-C18 antibacterial blight inhibitor, iso-C15 antibacterial blight inhibitor, anteiso-C15 antibacterial blight inhibitor, iso-C16 antibacterial blight inhibitor, iso-C17 antibacterial blight inhibitor, and anteiso-C17 antibacterial blight inhibitor, at least iso-C18 antibacterial blight inhibitor, n-C18 antibacterial blight inhibitor, iso-C15 antibacterial blight inhibitor, anteiso-C15 antibacterial blight inhibitor, iso-C16 antibacterial blight inhibitor, iso-C17 antibacterial blight inhibitor, and ant eiso-C17 antibacterial bactericide, at least iso-C18 antibacterial bactericide, n-C18 antibacterial bactericide, iso-C15 antibacterial bactericide, iso-C16 antibacterial bactericide, n-C16 antibacterial bactericide, iso-C17 antibacterial bactericide and anteiso-C17 antibacterial bactericide, at least iso-C18 antibacterial bactericide, n-C18 antibacterial bactericide, anteiso-C15 antibacterial bactericide, iso-C16 antibacterial bactericide, n-C16 antibacterial bactericide, iso-C17 antibacterial bactericide and anteiso-C17 antibacterial bactericide. The herbicides include at least iso-C18 antifungal blight inhibitor, iso-C15 antifungal blight inhibitor, anteiso-C15 antifungal blight inhibitor, iso-C16 antifungal blight inhibitor, n-C16 antifungal blight inhibitor, iso-C17 antifungal blight inhibitor, and anteiso-C17 antifungal blight inhibitor, or at least n-C18 antifungal blight inhibitor, iso-C15 antifungal blight inhibitor, anteiso-C15 antifungal blight inhibitor, iso-C16 antifungal blight inhibitor, n-C16 antifungal blight inhibitor, iso-C17 antifungal blight inhibitor, and anteiso-C17 antifungal blight inhibitor.

[0055] Alternatively or additionally, the antifungal subtilisin comprises a mixture of eight antifungal subtilisin isomers, preferably the mixture of the eight antifungal subtilisin isomers comprising at least iso-C15 antifungal subtilisin, anteiso-C15 antifungal subtilisin, iso-C16 antifungal subtilisin, n-C16 antifungal subtilisin, iso-C17 antifungal subtilisin, anteiso-C17 antifungal subtilisin, iso-C18 antifungal subtilisin, and n-C18 antifungal subtilisin.

[0056] Particularly good results have been obtained with mixtures of isomers with the antimycin C17 isomer and / or the antimycin C16 isomer as the main components. Such mixtures typically have an antimycin C17 content ranging from 35 wt.% to 80 wt.% based on the total antimycin isomers and / or an antimycin C16 content ranging from 6 wt.% to 50 wt.% based on the total weight of the antimycin isomers.

[0057] Preferably, the content of the antimycin C17 isomer is at least about 40 wt.% based on the total antimycin, more preferably based on the total antimycin isomer being in the range of about 40 wt.% to about 60 wt.%, and even more preferably, the content of the antimycin C17 isomer is in the range of 45 wt.% to 55 wt.% based on the total weight of the antimycin isomer, such as about 50 wt.% based on the total weight of the antimycin isomer.

[0058] Typically, based on the total weight of antimycin, in a mixture of antimycin isomers including the antimycin C16 isomer, the antimycin C16 isomer comprises at least 5 wt.%. Preferably, based on the total weight of antimycin, the antimycin C16 content ranges from about 20 wt.% to about 55 wt.%, more preferably based on the total weight of the antimycin isomers, ranging from about 25 wt.% to about 50 wt.%, and even more preferably based on the total weight of the antimycin isomers, ranging from about 35 wt.% to about 42 wt.%.

[0059] In an advantageous embodiment, in addition to the antimycobacterial C17 isomer and / or antimycobacterial C16 isomer, the isomer mixture also includes the antimycobacterial C15 isomer and / or antimycobacterial C18 isomer. In the mixture having the C17 and / or C16 isomers as the main isomers, the content of the antimycobacterial C15 isomer is typically less than 15 wt.%, particularly at most 10 wt.%, and more particularly in the range of 3 wt.% to 9 wt.%, for example, about 4 wt.% to about 6 wt.%, based on the total weight of the antimycobacterial isomers.

[0060] In a mixture having C17 and / or C16 isomers as the main isomers, the content of the antimycin C18 isomer is generally less than 25 wt.% based on the total weight of the antimycin isomers, preferably based on the total weight of the antimycin isomers being in the range of about 2 wt.% to about 20 wt.%, and more particularly based on the total weight of the antimycin isomers being in the range of about 4 wt.% to about 15 wt.%.

[0061] Specific examples of useful antifungal compound mixtures are disclosed in Kourmentza et al., Frontiers in Microbiology, Vol 11 (January 2021), article 561060, the content of which on antifungal compound mixtures is incorporated herein by reference. Preferred examples described therein are MI, M-II, M-III, and M-IV, with MI and M-IV being particularly preferred.

[0062] Antifungal subtilisin isomers can exist in any suitable weight ratio in a mixture of isomers.

[0063] Preferred mixtures of antimycin isomers include antimycin iso-C16, antimycin n-C16, antimycin anteiso-C17, and antimycin iso-C17, in a weight ratio preferably of about 26 (antimycin iso-C16): 1 (antimycin iso-C16): 2 (antimycin n-C16): 45 (antimycin anteiso-C17): 23 (antimycin iso-C17).

[0064] The antifungalin can be produced by suitable host cells or prepared by chemical synthesis. If the antifungalin is produced by suitable host cells, a mixture of antifungalin isomers is usually obtained.

[0065] Preferably, the antimycobacterial agent is produced by a suitable host cell (e.g., a Bacillus cell). Methods for producing antimycobacterial agents are well described in the art, for example, Guez et al., 2021, *Fronts in Bioengineering and Biotechnology*, 9, 678469; Kourmentza et al., *Frontiers in Microbiology*, Vol. 11 (January 2021), article 561060; or Dussert et al., *Frontiers in Microbiology*, Vol. 13 (June 2022), article 914713.

[0066] In principle, the antifungal subtilisin can be produced by any Bacillus species with a suitable mechanism for synthesizing it, preferably selected from Bacillus subtilis, Bacillus thuringiensis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, and Bacillus mohaiweii, and more preferably Bacillus subtilis.

[0067] More preferably, the antifungal subtilisin is produced by a microorganism selected from the following: Bacillus subtilis ATCC 6633, Bacillus subtilis ATCC 21332, Bacillus subtilis ATCC 9943, Bacillus subtilis BBG21, Bacillus subtilis BBG100, Bacillus subtilis BBG116, Bacillus subtilis BBG125, Bacillus subtilis BBG131, Bacillus subtilis BBG133, Bacillus subtilis BV12I37, Bacillus subtilis S499, Bacillus subtilis LBS1, Bacillus licheniformis BAS50, Bacillus licheniformis ATC 14580, and Bacillus thuringiensis BBG300.

[0068] More preferably, the antifungal subtilisin is produced by Bacillus subtilis strain ATCC6633, Bacillus subtilis strain BBG100, Bacillus subtilis strain BBG133, Bacillus subtilis BV12I37, or Bacillus subtilis LBS1. Particularly good results have been achieved using the antifungal subtilisin isomer produced by Bacillus subtilis LBS1 (see also Kourmentza et al. (2021) and Dussert et al. (2022)).

[0069] Further examples of strains that can be used to produce anti-substantiin are *Bacillus subtilis* BLIP2 (DOI: 10.3389 / fmicb.2020.561060); *Bacillus subtilis* BBG125 (DOI: 10.3389 / fbioe.2021.815337); *Bacillus subtilis* BBG116 (DOI: 10.1007 / s11356-017-9162-7); *Bacillus subtilis* Z15 (DOI: 10.1371 / journal.pone.0269861); *Bacillus subtilis* 370 (Journal of Clinical Investigation, 28.5 (1949): 924-926); *Bacillus subtilis* RFB112 (DOI: 10.1016 / j.seppur.2012.11.017).

[0070] Alternatively or additionally, the antimycin may be produced by genetically modified host cells, wherein the gene driving the expression of the antimycin has been stably transfected or upregulated. Using genetically modified host cells can advantageously allow for the production of antimycin with higher purity or yield.

[0071] The antifungalin produced by suitable host cells is typically isolated from the host cells. In other words, the antifungalin is preferably isolated at least partially from the fermentation mixture. Furthermore, prior to use in dough, baked goods, or methods according to the invention, the produced antifungalin is preferably optionally partially or substantially purified to remove one or more impurities from the fermentation mixture. For example, substantial purification would include isolating bacteria from the fermentation culture by microfiltration and / or centrifugation, etc., and cell-free fermentation cultures are typically concentrated by membrane filtration and / or one or more other steps (such as precipitation, solvent extraction, etc.) to produce antifungalin of higher purity.

[0072] The antimycin can also be purified using any suitable method known in the art, such as high performance liquid chromatography, for example using the methods described by Guez et al. (2021, Frontiers in Bioengineering and Biotechnology, 9, 678469), Kourmentza et al. (2021) or Dussart et al. (2022).

[0073] In the implementation scheme, the purity of the (purified) antifungal subtilisin is at least 50%, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 86%, even more preferably at least 87%, even more preferably at least 88%, such as at least 89%, even more preferably at least 90%, even more preferably at least 91%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and most preferably at least 99% purity or 100% purity.

[0074] Typical impurities present in fermentation mixtures containing antimycotic subtilisin obtained from suitable host cells include other excretory compounds such as proteins and residual components from the fermentation medium.

[0075] Alternatively, the antifungalin produced (and isolated from) by suitable host cells can be used in baked goods, dough, or methods according to the invention without substantial purification, i.e., as part of a fermentation mixture. Using the fermentation mixture directly in dough or baked goods reduces the steps involved in preparing the antifungalin, thereby reducing complexity and saving time and cost.

[0076] Alternatively or additionally, functional derivatives of antifungal subtilisin can be used in dough, premixes, or baking products or methods according to the present invention.

[0077] Preferably, the functional derivative is an antifungalin analogue.

[0078] Preferably, compared to a control baked product that is substantially preservative-free but otherwise identical, the functional derivative has the ability to increase the shelf life of the baked product against fungi by at least 5%. More preferably, as determined by the accelerated shelf life test described in Example 2, compared to the control, the functional derivative has the ability to increase the shelf life of the baked product by at least 7%, more preferably at least 10%, even more preferably at least 12%, even more preferably at least 15%, even more preferably at least 20%, even more preferably at least 25%, even more preferably at least 30%, even more preferably at least 35%, even more preferably at least 40%, even more preferably at least 45%, even more preferably at least 50%, even more preferably at least 55%, even more preferably at least 60%, even more preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 90%, and most preferably 100%.

[0079] The functional derivatives of the antifungal subtilisin may include, for example, modified peptide sequences, modified fatty acids, or combinations thereof.

[0080] For example, in functional derivatives, one or more amino acids in the antimycin peptide sequence may be replaced by one or more other natural or non-natural amino acids. Typically, the substituted amino acid is replaced by an amino acid with similar physicochemical properties (also referred to in the art as a "conservative mutation" or "conservative substitution"). Alternatively or additionally, the amino acid may be substituted with an enantiomer of the same amino acid.

[0081] Therefore, the functional derivative is preferably a cyclic lipopeptide having a β-amino fatty acid fused to an N-terminal amino acid residue on a carboxylic acid. More preferably, the cyclic lipopeptide has a peptide sequence of L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Ser-L-Asn, or, as determined on the full length of the anti-mycobacterial lipopeptide, has at least 57% sequence identity, preferably at least 71%, and more preferably at least 85% sequence identity, compared to the peptide sequence L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Ser-L-Asn.

[0082] Alternatively or additionally, in the functional derivative, the carbon chain of the fatty acid moiety can be shorter or longer than the fatty acid chain of the antifungalin. In principle, the carbon chain of the fatty acid moiety of the functional derivative can be of any length. Typically, the carbon chain of the fatty acid moiety comprises about six to about twenty-two carbon atoms.

[0083] Preferably, the carbon chain of the fatty acid may include about eight to about twenty carbon atoms, particularly about twelve to about nineteen carbon atoms, and most preferably about fourteen to about eighteen carbon atoms.

[0084] Most preferably, the functional derivative is a cyclic lipoheptapeptide having a peptide sequence of L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Ser-L-Asn, or a peptide having at least 57%, preferably at least 71%, more preferably at least 85% sequence identity with the peptide sequence L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Ser-L-Asn as determined over the full length of the antimycin lipoheptapeptide, and a β-amino fatty acid fused to a terminal amino group of an N-terminal amino acid residue on a carboxylic acid, wherein the carbon chain of the fatty acid has about fourteen to about eighteen carbon atoms, provided that the functional derivative is not the same as the antimycin.

[0085] Based on the weight of flour, the antifungal agent or its functional derivative is preferably present in the baked goods in an amount of at least 0.1 wt.%, more preferably at least 0.2 wt.%, more preferably at least 0.3 wt.%, even more preferably at least 0.4 wt.%, even more preferably at least 0.5 wt.%, and most preferably at least 1 wt.%. At such concentrations of antifungal agent, good antimicrobial properties are obtained in the baked goods without adversely affecting their structure and texture.

[0086] Preferably, based on the weight of flour, the amount of the antifungal subtilisin or its functional derivative is from about 0.1 wt.% to about 1 wt.%, more preferably from about 0.2 wt.% to about 0.8 wt.%, and even more preferably from about 0.3 wt.% to about 0.5 wt.%.

[0087] Alternatively or additionally, based on the dry weight of the baked product, the antifungal agent or its functional derivative is preferably present in an amount of at least 0.06 wt.%, more preferably at most 0.1 wt.%, even more preferably at least 0.2 wt.%, and most preferably at least 0.3 wt.% based on the dry weight of the baked product.

[0088] Alternatively or additionally, the amount of the antifungal subtilisin or its functional derivative thereof is preferably from about 0.06 wt.% to about 1 wt.% based on the dry weight of the baked product, more preferably from about 0.2 wt.% to about 0.6 wt.%, and even more preferably from about 0.3 wt.% to about 0.5 wt.% based on the dry weight of the baked product.

[0089] The advantage of using antifungal agents like subtilisin or its functional derivatives to reduce spoilage in baked goods is that it allows for a reduction in the amount of chemical preservatives. Common chemical preservatives include calcium propionate, sodium propionate, organic propionates, potassium sorbate, and sorbic acid.

[0090] Therefore, the present invention preferably relates to dough (preparation method), premix, or baked product, which, based on the weight of flour, contains at most 0.3 wt.%, more preferably at most 0.2 wt.%, even more preferably at most 0.1 wt.%, even more preferably at most 0.05 wt.%, even more preferably at most 0.01 wt.%, and particularly substantially free of chemical preservatives. Alternatively or additionally, the present invention preferably relates to dough (preparation method), premix, or baked product, which, based on the dry weight of the baked product, contains at most 0.3 wt.%, preferably at most 0.1 wt.%, more preferably at most 0.05 wt.%, even more preferably at most 0.01 wt.%, and particularly substantially free of chemical preservatives.

[0091] Baked goods that are substantially free of chemical preservatives are preferred clean-label baked goods. "Clean label" is a term used in this field to refer to food that is substantially free of chemical additives, such as chemical preservatives.

[0092] Therefore, the dough, premixes and baked products according to the present invention are preferably substantially free of chemical additives.

[0093] The dough can be prepared in a manner known per se, provided that antimycin or a functional derivative thereof is incorporated therein. Preferably, the dough is prepared by mixing antimycin or a functional derivative thereof with one or more other typical dough ingredients to form a dough in which antimycin is dispersed. As used herein, the term "dispersed therein" means that the antimycin is distributed throughout the dough, and preferably, the antimycin or a functional derivative thereof is uniformly distributed throughout the dough. This means that the concentration of antimycin is substantially the same in two randomly extracted samples of the dough or baked product (e.g., a sample taken from the core region of the dough or baked product, i.e., at least one centimeter from the surface or edge, and a sample taken from the surface of the dough or baked product).

[0094] As used in this article, a premix refers to a mixture of the dry dough components of dough. Premixes can be used to prepare dough, i.e., by mixing the premix with the liquid portion of the dough (usually water and / or oil).

[0095] Mixing can be achieved using any suitable method known in the art, such as by using a mechanical mixer or by kneading the dough by hand. The dough ingredients can be added in any particular order. For example, all the dough ingredients can be added at once, or a portion of the dough ingredients can be mixed first, followed by the remaining dough ingredients.

[0096] Typical dough ingredients used to prepare dough, premixes, or baked products according to the invention include starch (preferably provided by flour), water, fat, and salt.

[0097] Preferably, the dough, premix, or baked product contains starch, more preferably cereal starch. Preferably, the cereal starch is selected from wheat starch, corn starch, and rice starch.

[0098] Alternatively or additionally, the dough or baked product comprises flour, preferably selected from wheat flour, spelt flour, corn flour, oat flour, barley flour, rye flour, sorghum flour, buckwheat flour, millet flour, black wheat flour, amaranth flour, tamarisk flour, rice flour, quinoa flour, tapioca starch, potato flour, chickpea flour, coconut flour, almond flour, cassava flour, arrowroot flour, and combinations thereof. More preferably, the dough, premix, or baked product comprises wheat flour, spelt flour, rye flour, corn flour, rice flour, or combinations thereof.

[0099] The dough or baked product further preferably contains fat. The fat can be a solid fat (such as shortening, butter, or margarine) or a liquid fat (vegetable oil), which is typically liquid at a temperature of about 25°C and atmospheric pressure. Vegetable oils generally contain lower levels of saturated fatty acids, which is considered beneficial from a health perspective.

[0100] Preferably, the dough or baked product contains vegetable oil, more preferably selected from sunflower oil, olive oil, walnut oil, canola oil, rapeseed oil, peanut oil, coconut oil, sesame oil, grapeseed oil, and avocado oil.

[0101] It has been found that the antimicrobial activity of many natural preservatives is substantially neutralized in the presence of fats (especially vegetable oils) and / or starch. However, studies have shown that antimicrobial subtilisin or its functional derivatives are advantageously able to extend the shelf life of baked goods containing vegetable oils and / or starches, in which other natural preservatives known in the art have essentially lost all antimicrobial activity. Therefore, antimicrobial subtilisin or its functional derivatives are advantageously suited as natural preservatives in baked goods containing vegetable oils and / or starches.

[0102] The dough, premix, or baked product may further contain one or more leavening agents to provide a leavening effect, resulting in a product with a larger volume. Preferably, the leavening agent is yeast, more preferably baker's yeast.

[0103] Baking powder and fermentation acids, such as calcium monophosphate (MCP), sodium aluminum phosphate (SALP), sodium aluminum sulfate (SAS), and sodium acid pyrophosphate (SAPP), can also be used.

[0104] Dough or baked goods may further contain sweeteners such as sugar, aspartame, stevia, or honey. Sweeteners are commonly used in the preparation of sweet baked goods such as dessert cakes, pastries, pies, or cookies.

[0105] The dough or baked goods may further contain water. The water may be provided as is, such as using tap water or mineral water, or it may be provided from another source, such as in the form of (plant-based) emulsions.

[0106] Dough, premixes, or baked products may optionally contain one or more other typical dough ingredients, such as emulsifiers (e.g., lecithin); thickeners (e.g., gum); salt; ascorbic acid, ammonium sulfate, flavorings, etc. If the dough, premixes, or baked products contain one or more of these optional ingredients, they are preferably natural ingredients, such as sunflower or soy lecithin or natural flavorings.

[0107] The amount of dough component present in dough, premix, or baked goods depends on the type of baked goods being prepared. Those skilled in the art can select the amount of dough component based on baking common sense and the information provided herein.

[0108] Based on experience, the baked goods preferably contain at least 40 wt.% flour, more preferably at least 50 wt.%, even more preferably at least 70 wt.%, even more preferably at least 80 wt.%, even more preferably at least 85 wt.%, and most preferably at least 90 wt.% flour, based on the total dry weight of the baked goods. Typically, the baked goods contain about 40 wt.% to about 98 wt.%, more preferably about 70 wt.% to about 95 wt.% flour.

[0109] Based on experience, and considering the total weight of the baked goods, the baked goods preferably contain at least 0.1 wt.% fat, more preferably at least 0.3 wt.%, and even more preferably at least 1 wt.%. Preferably, the baked goods contain about 0.1 wt.% to about 30 wt.%, more preferably about 0.2 wt.% to about 20 wt.%, and most preferably about 0.3 wt.% to about 15 wt.% fat (preferably vegetable oil).

[0110] In the method according to the invention, dough can be shaped and baked to obtain a baked product. Depending on the type of baked product and the type of dough ingredients used, any suitable method known in the art can be used to shape and bake the baked product.

[0111] The appropriate method for shaping dough usually depends on the dough's consistency, such as its firmness and viscosity. For firm dough, it can often be shaped mechanically or manually and subsequently baked without the need for a device to hold its shape. For example, croissants, pizzas, etc., typically do not require molds.

[0112] More fluid doughs (sometimes referred to as "batter" in the industry) typically require sturdy devices, such as robust molds, to hold their shape. Additionally, molds may be needed to maintain a specific shape if one is desired. Some cakes (such as buttercakes) and pies may require devices like molds to hold their shape before or during baking.

[0113] Suitable baking conditions can be selected to bake the baked products according to the invention. The baked products can be heated by dry heat (e.g., in an oven or on a hot plate), by steam, or by hot oil (e.g., in a skillet (frying) or deep fryer). However, dry heat is preferred for heating the baked products, and heating in an oven is preferred.

[0114] Those skilled in the art can select appropriate forming and baking conditions based on common general knowledge and the information provided herein.

[0115] In principle, antifungal agents such as subtilisin or its functional derivatives have the ability to reduce spoilage in virtually all types of baked goods. According to the invention, baked goods are preferably selected from breads, such as loaves, bagels, rolls, bread rolls, croissants, baguettes, pretzels, bacon rolls, muffins, and flatbreads; desserts and cakes, such as butter cakes, sponge cakes, chiffon cakes, Genoa cakes, muffins, chocolate cakes, cheesecakes, and angel food cakes; pastries, pies, biscuits, flatbreads, and pizzas. Particularly good results have been achieved with breads, especially loaves.

[0116] According to the present invention, antifungalin or its functional derivatives are capable of preventing spoilage of the baked goods. Without wishing to be bound by any theory, the inventors believe that antifungalin or its functional derivatives exhibit antimicrobial activity against typical microorganisms involved in the spoilage of baked goods, thereby acting as a natural preservative for baked goods.

[0117] Key microorganisms known to be involved in the spoilage of baked goods, including bread, are *Aspergillus niger* and *Penicillium paneum*, but other microorganisms are also known to play a role, such as *Penicillium roqueforti*, *Monascus ruber*, *Rhizopus stolonifer*, *Pichia burtonii*, and *Saccharomyces cerevisiae*. Most microorganisms involved in the spoilage of baked goods are fungi, but yeasts and bacteria can also be involved, although usually to a lesser extent.

[0118] Therefore, the present invention relates to a baked product (a method for preserving baked products), which has an extended shelf life compared to a control baked product.

[0119] The control baked product is a baked product of the same type. For example, if the shelf life of a loaf of bread is to be determined, then the control baked product is also a loaf of bread.

[0120] The control baked product was prepared from the same dough, provided that the antifungal agent or its functional derivative was not present, or was replaced by an equal weight of a chemical preservative (preferably calcium propionate).

[0121] Baked products according to the invention preferably have a shelf life of at least 48 hours, preferably at least 72 hours, more preferably at least 96 hours, even more preferably at least 120 hours, even more preferably at least 144 hours, and particularly at least 168 hours, which can be determined by the accelerated shelf life test as described in Example 2. In an embodiment, as determined by the accelerated shelf life test as described in Example 2, baked products according to the invention have a shelf life of up to 336 hours, particularly up to 312 hours, more particularly up to 288 hours, even more particularly up to 264 hours, even more particularly up to 240 hours, even more particularly up to 216 hours, or most particularly up to 192 hours. Preferably, the shelf life is between about 48 hours and about 336 hours, more preferably between about 72 hours and about 312 hours, even more preferably between about 96 hours and about 288 hours, even more preferably between about 120 hours and about 264 hours, even more preferably between about 144 hours and about 240 hours, and particularly between about 168 hours and about 24 hours.

[0122] Advantageously, accelerated shelf-life challenge tests offer increased sensitivity, reproducibility, and statistical power. In accelerated shelf-life testing, a test sample of the baked product to be evaluated is inoculated with a predetermined number of cells / spores of known spoilage microorganisms, typically Aspergillus niger spores and / or Penicillium thuringiensis spores. To improve accuracy, a batch of freeze-dried fungal spores is usually used in the test to ensure that spore viability and quantity are constant, thus enabling comparative results.

[0123] The shelf life of baked goods is assessed by monitoring the presence of microorganisms (preferably fungi) through photographic imaging and visual inspection of photographs and baked goods. Accelerated shelf life is defined as the time, in hours, from inoculation of the baked goods with the microorganism to be assessed until spoilage of the baked goods becomes visible to the naked eye (e.g., due to the formation of fungal colonies).

[0124] The present invention also relates to the use of antifungal subtilisin as an antimicrobial agent against one or more of the following: Penicillium tumefaciens, Penicillium loudi, Rhizopus (especially Rhizopus thuringiensis), Monascus purpureus, and Pichia pastoris (especially Pichia pastoris Burton).

[0125] For the purposes of clarity and concise description, certain features are described herein as part of the same or separate embodiments; however, it should be understood that the scope of the invention may include embodiments having a combination of all or some of the described features.

[0126] The present invention is illustrated by the following embodiments.

[0127] Example

[0128] Example 1: In vitro antifungal activity of various lipopeptides

[0129] The antifungal susceptibility of six different lipopeptide compositions to Aspergillus niger D-02906, Penicillium tumefaciens CBS 302.97, Penicillium loudichotomum CBS 174.87, Rhizopus spp. CBS 819.97, Monascus purpureus D-061178, Pichia pastoris C-00349, and Saccharomyces cerevisiae NCYC 77 was evaluated using a broth microdilution assay in 96-well plates.

[0130] The lipopeptides were supplied by Lipofabrik (France). Antimycin was produced by Bacillus subtilis LBS1, surfactant by Bacillus subtilis BLIP2, phytosalicylate by Bs2504, and a mixture of antimycin / surfactant was produced by Bacillus subtilis BLIP2. Kourmentza et al. (2021) outlined the details of lipopeptide production and purification. Antifungal susceptibility testing was performed in 96-well plates using the microdilution method, as described in Espinel-Ingroff & Canton 2007A (Antifungal susceptibility testing of filamentous fungi, edited by R. Schwalbe, L. Steele-Moore, and C. Goodwin; Antimicrobial susceptibility testing protocols, Boca Raton, FL: Taylor & Francis, pp 209-241); and in Espinel-Ingroff & Canton, 2007B (Antifungal susceptibility testing of yeasts, edited by R. Schwalbe, L. Steele-Moore & AC. Goodwin; Antimicrobial susceptibility testing protocols, Boca Raton, FL: Taylor & Francis, pp 209-241). Taylore & Francis, pp. 173-207, used this to determine the minimum inhibitory concentration (MIC) of lipopeptides for each strain.

[0131] The growth medium used in the microdilution assay was a semi-solid yeast extract sucrose (YES) medium containing 20 g / L yeast extract, 150 g / L sucrose, 0.5 g / L magnesium sulfate, and 1.25 g / L agar, adjusted to a water activity of 0.97 with glycerol and to pH 5.6 with 0.1 M citric acid and 0.1 M trisodium citrate (Medina et al., 2012, *Fungal Biology* 116(1):161-169). For the sensitivity test of *Saccharomyces cerevisiae*, YES broth was used without agar to avoid the formation of single colonies in the wells.

[0132] Filamentous fungal cultures were grown in PDA at 25°C for 7 days (Aspergillus niger, Penicillium chrysogenum, and Penicillium loudi), 10 days (Monascus purpureus), or 3 days (Rhizopus spp.). Spore suspensions were prepared in 0.85% sterile saline solution, and the OD530 nm of the suspension was adjusted to produce 0.4 × 10⁻⁶ spores. 6 -5×10 6 CFU / mL.

[0133] Yeast was cultured for 2 days at 25°C on Sabouraud dextrose agar (SAB) (Pichia pastoris Burton's) or at 30°C on malt extract agar (MEA) (Saccharomyces cerevisiae). Colonies were collected and suspended in 0.85% sterile saline solution, and the turbidity of the suspension was adjusted to produce 1×10⁻⁶ cells / mL. 6 -5×10 6 CFU / mL. The spore and cell suspension was further diluted in culture medium to prepare a working inoculum suspension.

[0134] The tested lipopeptides were antimycin 89% purity, surfactant >80% purity, antimycin / surfactant 80:20 (42% purity), antimycin / surfactant 80:20 (34% purity), and antimycin / surfactant 80:20 (23% purity). They were dissolved in dimethyl sulfoxide (DMSO) and further diluted in growth medium to obtain a final test concentration range of 0.5-64 mg / L.

[0135] Inoculate the wells of a 96-well plate with 100 μL of working inoculum and 100 μL of lipopeptide dilution in culture medium, so that each well contains 0.4 × 10⁻⁶ μL of lipopeptide. 4 -5×10 4 CFU / mL, and the final DMSO content in the wells was 1 vol.%.

[0136] Each test plate contained a sterile control well with 200 µL of culture medium and a growth control well without antifungal agents, containing 100 µL of culture medium and 100 µL of inoculum. The plates were then sealed with paraffin film and incubated at 25°C for 72 hours, except for Rhizopus spp. plates which were incubated for 48 hours and Saccharomyces cerevisiae plates which were incubated at 30°C.

[0137] The minimum inhibitory concentration (MIC) is defined as the lowest concentration at which the tested lipopeptide prevents any discernible growth after the incubation period. Each test consists of three technical replicates, and is repeated three times on different days and using different cultures (biological replicates). MIC results are reported as a range containing multiple MIC measurements, or as the reported value when all measurements show only one MIC.

[0138] The broth microdilution assay allows for the determination of the MIC values ​​of lipopeptides for each test fungal strain (Table 1).

[0139] Table 1: Minimum Inhibitory Concentration (MIC) values ​​of various yeasts and molds involved in the spoilage of baked goods (in mg lipopeptide / L broth).

[0140]

[0141] As shown in Table 1, antimycin exhibited good in vitro antifungal activity, while surfactant showed no antifungal activity at concentrations up to 64 mg / L. Even at the lowest purity (23%), the antimycin / surfactant 80:20 ratio showed better antimicrobial activity than surfactant alone.

[0142] Example 2: In vivo antifungal activity of various lipopeptides

[0143] The antifungal activity of three different lipopeptide compositions against the fungi *Aspergillus niger* and *Penicillium liquefaction* in bread was determined. Different types of bread were prepared: a control without any preservatives, a reference containing 0.3% calcium propionate based on flour weight (FWB), and bread containing lipopeptides comprising 0.1% and 0.3% FWB of antifungin (>99% purity), 0.1% and 0.3% FWB of surfactant (80% purity), 0.1%, 0.3%, and 0.6% FWB of fengycin (64% purity), and 0.1% FWB of antifungin / surfactant 80:20 (42% purity). Bread dough was prepared by mixing the dough ingredients in Table 2 at 120 rpm for 5 minutes using a DoughLAB mixer (PerkinElmer). The dough was then proofed in a 40°C, humidity-free proofing cabinet (GS MIWE) for 70 minutes. Divide the dough into multiple portions (8 × 50 g), round them, and bake at 200°C for 15 minutes without steam. Of the resulting bread, six loaves were used to prepare two 6-well plates for ASLT, and two more for pH and aw measurements. The 6-well plates were prepared and ASLT was performed as follows: After baking, the bread was cooled in a HEPA filter chamber for 90 minutes and sliced. Using a cookie cutter (3.2 cm in diameter), three random breadcrumbs (from the inside of the bread) were cut from each loaf and placed in a sterile 6-well plate (2 loaves × 3 slices). Two 6-well plates were prepared for each bread type. Three aliquots (20 μL) of bread slices in each 6-well plate were inoculated with ±100 spores of *Aspergillus niger* or *Penicillium liquefactionum*. Therefore, a total of 18 spots (6 loaves × 3 aliquots) of each mold were inoculated per experiment. Freeze-dried spores were used in all experiments. The inoculated bread was incubated at room temperature of approximately 21°C until visible mold appeared. The appearance of mold was imaged by photographic photography. Visually inspect the images, and TOA is defined as the time (in hours) for mold to appear after mold inoculation.

[0144] Table 2: Bread Recipes

[0145]

[0146] The results are as follows Figure 1 As shown in Figure 2 and Table 3.

[0147] Table 3: Time of appearance (TOA) of Aspergillus niger / Penicillium liquefaction in bread containing biosurfactants compared to control bread; the times shown are the times when the fungus appeared later (+) or earlier (-) compared to the control (no preservative) and the reference bread containing 0.3 wt.% calcium propionate but otherwise identical, in hours; wt.% is based on flour weight.

[0148]

[0149] From Table 3 and Figure 1 As shown in Figure 2, compared to the control without preservatives, the presence of 0.3 wt.% antifungal subtilisin delayed bread spoilage by more than 66 hours (after which the experiment was stopped) and 41.16 hours, respectively, based on flour weight, for Aspergillus niger and Penicillium liquefaction. Furthermore, compared to a comparable reference bread containing 0.3 wt.% calcium propionate but otherwise identical, the presence of Aspergillus niger and Penicillium liquefaction was delayed by more than 31.67 hours and 23.16 hours, respectively. This indicates that antifungal subtilisin is a promising natural preservative for baked goods, exhibiting superior preservative capabilities compared to equivalent amounts of commonly used chemical preservatives.

[0150] It is noteworthy that, compared to bread containing calcium propionate, an antifungal content of 0.3 wt.% is required based on flour weight to delay fungal appearance. This relatively high level of antifungal required here, compared to in vitro results (Example 1), suggests that the antifungal is partially inhibited or inactivated in the bread.

[0151] Interestingly, although in vitro experiments demonstrated that the lipopeptide mixture of antifungal agents (subtilisin / surfactant) had antifungal activity against Aspergillus niger and Penicillium viniferum, the mixture did not delay the appearance of fungi in bread at all.

[0152] Furthermore, compared to the control, the presence of 0.1 wt.% of cymoxanil only slightly delayed the appearance of Aspergillus niger and Penicillium tumefaciens, but did not delay their appearance compared to the calcium propionate-based reference.

[0153] Example 3: In vitro antifungal activity of antifungal subtilisin in the presence of various common ingredients in baked goods

[0154] To assess whether the various components present in the bread formulation affect the antifungal activity of antifungal subtilisin against Aspergillus niger and Penicillium tumefaciens, as described above (Example 1), an in vitro antifungal susceptibility test was performed in the presence of the bread components to detect changes in MIC.

[0155] Different formulations of YES medium were prepared as follows to test different concentrations of each component: 1.25% and 2.5% wheat starch, 0.15% and 0.3% sunflower oil (according to Gutierrez et al., 2008, International Journal of Food Microbiology, 124(1), 921-97, containing 0.1% sterile filtered Tween 80 to promote mixing and stabilize the emulsion), 0.36% and 0.72% salt (NaCl), 0.25% and 0.5% ascorbic acid, 0.0125% and 0.025% amylase, and 0.00625% and 0.0125% xylanase.

[0156] In YES medium with and without bread ingredients, 100% purity antifungalin was tested with two-fold dilutions, within a final concentration range of 0.0625–8 mg / L. The MIC was defined as the lowest concentration at which the tested lipopeptide prevented any identifiable growth after 72 hours. Each test consisted of three technical replicates, repeated three times on different days and using different cultures (biological replicates). MIC results were reported as a range containing multiple MIC measurements, or as the reported value if all measurements showed only one MIC.

[0157] The MIC (micronaire value) of 100% pure anti-mycotic agent (μg / L culture medium) was determined using the method described in Example 1 in the presence of starch, sunflower seed oil, sodium chloride, ascorbic acid, amylase, and xylanase. The results are shown in Table 4.

[0158] Table 4: Effects of antifungal activity of antifungal agents in bread ingredients against Aspergillus niger and Penicillium viniferum

[0159]

[0160] As shown in Table 4, the MIC of anti-mycobacterium increased approximately 4-8 times in the presence of sunflower seed oil and approximately 8-16 times in the presence of wheat starch. No increase in MIC was observed in the presence of NaCl, ascorbic acid, and amylase. Clearly, at the tested concentrations, xylanase can increase the MIC of anti-mycobacterium by up to 2-fold.

[0161] The results showed that sunflower seed oil and starch, even at concentrations lower than those present in bread (using the above formulation), inhibited the antifungal activity of antimycin. This explains why a relatively high concentration of antimycin is required in bread as shown in Example 2, compared to in vitro in Example 1.

[0162] Example 4: Determination of water activity and pH of bread containing antifungal subtilisin

[0163] The water activity and pH of bread containing antifungal agents prepared according to the method of Example 2 were determined as follows, and compared with a control (without preservatives) and a reference bread containing 0.3 wt.% calcium propionate. To measure the pH of the bread, 10 g of bread was placed in 90 ml of water (10% w / v) and suspended using a stirrer. The pH of the resulting bread suspension was measured using a calibrated portable pH meter (Accumet AB150, Fischer Scientific). Water activity (α) was measured using an Aqualab3 (METER Group). wTo do this, fill the sample cup with breadcrumbs that cover the bottom but do not exceed half full. When not actively reading the sample, store it in a sealed package or in a sample cup with the sample cup lid on. Load the sample into the AquaLab3 and record the data. When equilibrium is reached (within 5 minutes or less), the AquaLab3 will report the reading.

[0164] The results are as follows Figure 3 As shown.

[0165] from Figure 3 The above figure shows that the pH of the bread according to the invention is between 5.3 and 5.5. This is comparable to the control and slightly lower than the bread containing 0.3 wt.% calcium propionate.

[0166] Similarly, the water activity was between approximately 0.94 and 0.96, which is comparable to both the control and reference breads.

[0167] Therefore, it was concluded that, compared with the control and the reference bread containing calcium propionate (3% FWB), the pH and water activity of the bread according to the invention did not change significantly.

[0168] Example 5: Dough properties of bread according to the present invention compared with control and reference bread

[0169] The flour water absorption, dough formation time, and kneading resistance of the dough according to the present invention were determined using a DoughLAB mixer (PerkinElmer) and compared with a control (no preservatives) and a reference (0.3 wt.% calcium propionate) as follows. The DoughLAB measured the kneading resistance, which was monitored as a torque value and plotted as a time curve. Figure 4 To do this, place all the ingredients listed in Table 2 except for water into the mixing bowl of the DoughLAB mixer, with the water added automatically by the mixer. Mix the dough at 120 rpm for 5 minutes.

[0170] The results are shown in Table 5 and Figure 4 As shown.

[0171] Table 5: Dough properties of the dough according to the present invention compared with control (no preservative) and reference dough containing 0.3 wt.% calcium propionate.

[0172]

[0173] From Table 5 and Figure 4 It can be concluded that the presence of antifungal subtilisin did not significantly affect the dough properties.

Claims

1. A method for preserving a baked product, comprising providing dough containing antifungal agents or functional derivatives thereof, shaping the dough, and baking the shaped dough to obtain a baked product.

2. The method according to claim 1, wherein the dough comprises antifungal subtilisin, preferably wherein the antifungal subtilisin comprises one or more antifungal subtilisin isomers selected from antifungal subtilisin C17 isomer, antifungal subtilisin C16 isomer, antifungal subtilisin C15 isomer, and antifungal subtilisin C18 isomer, preferably the antifungal subtilisin isomers selected from anteiso-C17, iso-C17, iso-C15, anteiso-C15, iso-C16, n-C16, iso-C18, and n-C18.

3. The method according to claim 1 or 2, wherein the antifungal subtilisin is produced by bacteria from the genus Bacillus, preferably Bacillus subtilis.

4. The method according to any one of the preceding claims, wherein the antifungal subtilisin or its functional derivative is added in an amount of at least 0.06 wt.% based on the dry weight of the baked product, preferably at least 0.1 wt.% based on the dry weight of the baked product, more preferably at least 0.2 wt.%.

5. The method according to any one of the preceding claims, wherein the amount of chemical preservative is in the range of 0 wt.% to 0.3 wt.% based on the total dry weight of the baked product, preferably up to 0.03 wt.%, more preferably up to 0.006 wt.%.

6. The method according to any one of the preceding claims, wherein the dough further comprises starch and / or vegetable oil, particularly sunflower seed oil.

7. A baking product, preferably obtainable by the method according to any one of claims 1 to 6, wherein the baking product comprises antifungal agent or a functional derivative thereof.

8. The baked product according to claim 7, wherein the baked product comprises antifungal subtilisin, preferably wherein the antifungal subtilisin comprises one or more antifungal subtilisin isomers selected from the antifungal subtilisin C17 isomer, antifungal subtilisin C16 isomer, antifungal subtilisin C15 isomer, and antifungal subtilisin C18 isomer, preferably the antifungal subtilisin isomers selected from anteiso-C17, iso-C17, iso-C15, anteiso-C15, iso-C16, n-C16, iso-C18, and n-C18.

9. The baking product according to claim 7 or 8, wherein, based on the dry weight of the baking product, the baking product contains up to 0.1 wt.% of chemical preservatives, more preferably the baking product is substantially free of chemical preservatives, and even more preferably the baking product is a clean label baking product.

10. The baked product according to any one of claims 7 to 9, wherein the baked product is selected from bread, such as bread rolls, bagels, rolls, bread rolls, croissants, baguettes, pretzels, custard rolls, muffins and flatbreads; dessert cakes, such as butter cakes, sponge cakes, chiffon cakes, Genoa cakes, muffins, chocolate cakes, cheesecakes and angel food cakes; pastries, pies, biscuits, flatbreads and pizzas, preferably wherein the baked product is bread, more preferably bread rolls.

11. The baked product according to any one of claims 7 to 10, wherein the baked product has an extended shelf life compared with a comparable baked product having an equal weight of chemical preservatives, preferably calcium propionate instead of one or more antifungal agents, but otherwise identical.

12. The baked product according to any one of claims 7 to 11, wherein, as determined by accelerated shelf life testing (ASLT), the baked product has an accelerated shelf life of at least 96 hours, preferably at least 120 hours, more preferably at least 144 hours, and particularly at least 168 hours when stored at about 25°C.

13. A dough or premix for preparing baked goods, said dough or premix comprising antimycin or a functional derivative thereof, preferably the antimycin comprising one or more antimycin isomers selected from the antimycin C17 isomer, antimycin C16 isomer, antimycin C15 isomer, and antimycin C18 isomer, preferably the antimycin isomer is selected from anteiso-C17, iso-C17, iso-C15, anteiso-C15, iso-C16, n-C16, iso-C18, and n-C18.

14. The dough or premix for preparing baked goods according to claim 13, wherein, based on the dry weight of the baked goods, the dough contains at least 0.06 wt.% antifungal and / or at most 0.1 wt.% chemical preservatives, more preferably substantially free of chemical preservatives.

15. Use of antifungal subtilisin as a preservative in baked goods.

16. Antimicrobial agents are used in food or beverages against one or more of the following: Penicillium paneum, Penicillium roqueforti, Rhizopus spp., especially Rhizopus stolonifer, Monascus ruber, and Pichia spp., especially H. burtonii.

17. The use of antifungal subtilisin as an antimicrobial agent in bread against Aspergillus niger.

18. The use according to any one of claims 15 to 17, wherein at least one antibacterial subtilisin selected from the group consisting of antibacterial subtilisin C17 isomer, antibacterial subtilisin C16 isomer, antibacterial subtilisin C15 isomer and antibacterial subtilisin C18 isomer, preferably selected from anteiso-C17, iso-C17, iso-C15, anteiso-C15, iso-C16, n-C16, iso-C18 and n-C18.

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