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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/52—Propionic acid; Butyric acids
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  • C12P7/00—Preparation of oxygen-containing organic compounds
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  • C12P7/56—Lactic acid
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  • C12N2500/00—Specific components of cell culture medium
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  • C12N2500/36—Lipids
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• the present invention pertains to a method for suppressing the growth of gram-positive bacteria in culturing gram-negative bacteria, moulds and yeasts.
• the present invention relates to antibacterial agents which comprise a lactylate, a glycerol ester or a mixture thereof and to the use of such agents in fermenter cultures.
• bacterial control agents include, for example, (natural) antibiotics and sulphite at a concentration which does not affect the viability of the yeast but which prevents outgrowth of contaminating bacteria. Details of such agents are disclosed in: Ruckle et al., Hop acids as natural anti-bacterials can efficiently replace antibiotics in ethanol production, International Sugar Journal 108: 139-147 (2006); and, Limayem et al., Alternative antimicrobial compounds to control potential Lactobacillus contamination in bioethanol fermentations, Journal of Environmental Science and Health, Part B 46(8): 709-714, (2011).
• contaminating gram positive bacteria are also pathogenic bacteria such as: Enterococcus, Clostridium, Listeria, Staphylococcus, various Bacillus species, and Streptococcus.
• Contaminants are undesirable because they can seriously compromise the optical purity of the lactic acid and they can produce other fermentation products and thereby lower the fermentation yield. There are therefore good reasons to suppress the outgrowth of these micro-organisms.
• lactylates and / or glycerol esters of medium chain fatty acids specifically inhibits the outgrowth of gram-positive bacteria when culturing gram-negative bacteria or moulds and yeasts.
• the producer microorganisms Upon adding an effective amount of a lactylate, a glycerol ester or a mixture thereof, the producer microorganisms continue to grow in a fermenter but the growth of contaminating gram-positive bacteria is prevented or suppressed. This ensures that the fermentation process is not disturbed by outgrowth of unwanted microorganisms, unwanted side-products and inefficient use of energy sources. It furthermore diminishes the required additive amount of bacterial contaminating control agents such as antibiotics.
• a lactylate refers to a compound having an acyl group from the fatty acid attached to one (monolactylates) or several lactic acid molecules (dilactylates etc.) and a proton (H+) or another cation attached to the terminal carboxylate.
• the fatty acid moiety consists typically of a hydrocarbon chain attached to a carboxyl group at the end.
• the hydrocarbon chain can contain different numbers of carbon atoms, and the bonds between the carbon atoms can be saturated or unsaturated.
• Lactylates are known surfactants. These surfactants are made by reacting R-lactic acid or S-lactic acid or any mixture of these two with a fatty acid and concurrently neutralizing with a base. Lactylates are well known in the food industry and are used in personal care applications to improve skin feel, skin softness and moisturization, and to reduce tackiness during wet to dry transition after product application. It is known that certain lactylates possess antimicrobial properties. US2007/010856 (Cohen et al.) describes sutures treated with inter alia a lactylate as an antimicrobial compound. US2006/062832 (Lopes) describes a wipe composition comprising a lactylate as an antimicrobial compound.
• WO2004/037177 (Eveready Battery Inc.) describes an antibacterial shaving foam or gel formulation which contains lactylate as an antimicrobial compound.
• WO01/06877 (Rhodia) describes the use of lactylates in combination with hop acids in food; the hop acids have activity against gram positive bacteria and lactylates are proposed as one possible adjunct ingredient amongst a very broad range of food-grade emulsifiers (page 6). The specific use of lactylates is not exemplified and the fact that lactylates have an individual activity against gram positive bacteria is not recognised in this reference.
• WO 2004/107877 Patent Application Laida BV
• an antimicrobial composition comprising a mixture of lactic acid or a derivative thereof and an inorganic acid.
• the composition is described as an antimicrobial in general.
• Salmonella and Escherichia coli is specified. While lactylates are mentioned as possible lactic acid derivatives, their use is not further elucidated.
• a fermentation medium comprising:
• an antimicrobial agent selected from:
• R represents a C4-C18 acyl group, the acyl group having an alkyl or alkenyl chain which may be branched or unbranched;
• Ri , R2 and R3 each independently represent H or a C4-C18 acyl group, the acyl group having an alkyl or alkenyl chain which may be branched or unbranched, with the proviso that at least one of Ri , R2 or R3 is H and at least one of Ri , R2, or R3 is an acyl group;
• M represents a proton (H + ) or a counter-cation chosen from the group Li, Na, K, Ca, Mg, Zn, Fe(ll), Cu, Mn, Ag, ammonium or substituted ammonium having one or more (C1-4)alkyl optionally substituted with one or more hydroxy;
• a is an integer of from 1 to 3;
• b is 1 or 2 equaling the valency of M.
• the term "mixture of such compounds” as used herein denotes that the antibacterial agent may contain: two or more compounds which conform to Formula 1 ; two or more compounds which conform to Formula 2; or, mixtures of compounds as defined in Formula 1 and Formula 2.
• the fermentation medium may comprise the antibacterial agent in an amount of from 0.001 to 0.5 wt. %, preferably from 0.025 to 0.5 wt.%, based on the total weight of the medium.
• the fermentation medium may comprise the antibacterial agent at a concentration of from 0.1 to 1000 mg/L, preferably from 0.5 to 500 mg/L and more preferably of from 1 to 100 mg/L of fermentation medium.
• the fermentation medium may further comprise an inoculant comprising a culture of gram-negative bacteria, moulds or yeasts.
• the antibacterial agent may be added to the medium prior to, after or together with that culture of gram-negative bacteria, moulds or yeasts.
• the fermentation medium is obtained by: contacting the substrate for microbial growth with said antibacterial agent for a period of from 1 minute to 48 hours, preferably of from 1 to 24 hours, before inoculation of the fermentation medium with the fermenting micro- organism.
• the fermentation medium may be obtained by adding said antibacterial agent to an aqueous, recycled process stream that is a stream which is derived from a product of the fermentation but which is recycled as an input to the fermentation medium for inter alia water balance.
• an inoculant for a fermentation medium comprising:
• an antibacterial agent selected from:
• R represents a C4-C18 acyl group, the acyl group having an alkyl or alkenyl chain which may be branched or unbranched;
• Ri , R2 and R3 each independently represent H or a C4-C18 acyl group, the acyl group having an alkyl or alkenyl chain which may be branched or unbranched, with the proviso that at least one of Ri , R2 or R3 is H and at least one of Ri , R2, or R3 is an acyl group;
• M represents a proton (H + ) or a counter-cation chosen from the group Li, Na, K, Ca, Mg, Zn, Fe(ll), Cu, Mn, Ag, ammonium or substituted ammonium having one or more (C1-4)alkyl optionally substituted with one or more hydroxy;
• a is an integer of from 1 to 3;
• b is 1 or 2 equaling the valency of M.
• This second aspect of the invention prevents the contamination of the inoculant with gram-positive bacteria.
• the anti-bacterial agent Upon inoculating the fermentation medium, the anti-bacterial agent becomes dispersed in that medium where it can continue to suppress or prevent the growth of gram-positive bacteria which might be introduced as contaminants of the medium.
• the present invention also provides for the use of an antibacterial agent for suppressing the growth of gram-positive contaminating bacteria in culturing gram-negative bacteria, moulds or yeasts, said antibacterial agent being selected from: a lactylate in accordance with Formula 1 , as defined hereinabove; a glycerol ester in accordance with Formula 2, as defined hereinabove; or, a mixture of such compounds.
• a method for preventing or reducing microbial infections caused by gram-positive bacteria in a fermenter culture of gram- negative bacteria comprising adding to the culture an effective amount of an antimicrobial agent selected from: a lactylate in accordance with Formula 1 , as defined hereinabove; a glycerol ester in accordance with Formula 2, as defined above; and, a mixture of such compounds.
• an antimicrobial agent selected from: a lactylate in accordance with Formula 1 , as defined hereinabove; a glycerol ester in accordance with Formula 2, as defined above; and, a mixture of such compounds.
• a method for obtaining a fermentation product comprising the steps of: providing a fermentation medium; and, introducing into said medium an inoculant comprising a culture of gram-negative bacteria, moulds or yeasts.
• microbial growth refers to an increase in the number and / or metabolic activity of microbial cells, including bacteria, molds, fungi, and algae.
• the term "fermentation medium” as used herein means a three phase (solid-liquid-gas) system which is retained within the fermentation vessel.
• the liquid phase contains water, dissolved nutrients, dissolved substrates for microbial growth and dissolved metabolites; the source of the water is not limited and includes, in particular, process waters, such as backset and/or thin stillage, scrubber water, evaporator condensate or distillate, side stripper water from distillation, or other fermentation product plant process water.
• the solid phase comprises individual cells, pellets, insoluble substrates for microbial growth and precipitated metabolic products.
• substrate refers to any substance or compound that is converted or meant to be converted into another compound by the action of an enzyme.
• substrate is intended to encompass not only compounds that provide a carbon source suitable for use as a starting material, such as any biomass derived carbohydrate, but also intermediate metabolites used in a metabolic pathway associated with a microorganism.
• the fermentation medium may typically comprise, as a substrate, one or more fermentable carbohydrates, such as sugars.
• the fermentation medium including fermentation substrate and other raw materials used in the fermentation process of the invention may be processed - by milling, liquefaction, saccharification or the like - prior to or simultaneously with the fermentation process.
• the fermentation medium can refer to the medium before the fermenting micro-organism is added, such as, the medium in or resulting from liquefaction and/or saccharification, as well as the medium which comprises the fermenting organism, such as, the medium used in simultaneous saccharification and fermentation (SSF) or one-step fermentation processes.
• SSF simultaneous saccharification and fermentation
• fermentation vesset means the vessel in which the fermentation reaction is carried out.
• fermentation vessel may be used interchangeably with fermentation vessel.
• inoculant means the original source of a complex microbial community which is intended to be added to the fermentation vessel but which does not limit the final composition of the microbial community; the final composition is determined by the operating conditions and productivity of the fermentation vessel.
• the inoculant is typically formed by propagation of the desired microorganism(s) in a suitable propagation tank, which tank is much smaller than the fermentation vessel.
• the inoculant typically includes a culture of one or more "production strains" of microorganisms which may have been adapted by natural selection or by biotechnological means to produce the fermentation product of interest.
• An exemplary, but non-limiting, inoculant contains a culture of the yeast Saccharomyces cerevisiae for the production of ethanol.
• the term "culture” is used in a manner known in the art to denote the propagation of microbial organisms, in particular production strains, in a predetermined culture media conducive to their growth.
• the term “fermenter culture” refers to a propagation of one or more productions strains of microorganisms present within said fermentation medium or within an inoculant suitable for said fermentation medium.
• gram-negative bacteria which may be cultured and utilized in the present invention as the production strains.
• Exemplary, but non-limiting, gram-negative bacteria include: Escherichia coli; Acinetobacter; Bordetella; Brucella; Campylobacter; Cyanobacteria; Enterobacter; Erwinia; Franciscella; Helicobacter; Klebsiella; Legionella; Moraxella; Neisseria; Pantooea; Pasteureiiaceae, especially bacteria of the Actinobacillus genus, Hemophilus genus and
• the fermenter culture or fermentation medium comprises one or more gram negative bacterium selected from the group consisting of: Escherichia coli; Pseudomonas species; and, Pasteureiiaceae species.
• moulds which might be mentioned are moulds of the genera: Aspergillus, in particular Aspergillus oryzae, Aspergillus terreus and Aspergillus niger, Rhizopus, for instance Rhizopus oligosporus and Rhizopus oryzae; Fusarium, such as Fusarium oxysporum; Mucor, such as Mucor racemosus; Cladosporium, such as Cladosporium herbarum; Penicillum, such as Penicillum expansum; and, Trichoderma, such as Trichoderma harzanium.
• Aspergillus in particular Aspergillus oryzae, Aspergillus terreus and Aspergillus niger, Rhizopus, for instance Rhizopus oligosporus and Rhizopus oryzae
• Fusarium such as Fusarium oxysporum
• Mucor such as Mucor racemosus
• Cladosporium such as Cladosporium herbarum
• Penicillum such
• yeast genera which may be employed in the present invention include: Brettanomyces; Candida; Dekkera; Pichia; and, Saccharomyces.
• the carbohydrates that a specific microorganism can ferment are either commonly known to the person of ordinary skill in the art or are easily accessible in the published, background literature.
• common carbohydrates fermentable by lactic acid producing microorganisms include but are not limited to: Cs sugars such as arabinose, xylose and ribose; C6 sugars such as glucose, fructose, galactose, rhamnose and mannose; and, C12 sugars such as sucrose, maltose and isomaltose.
• the fermentable carbohydrates are primarily derived from starch-based or sugar-based feedstocks.
• feedstocks include, but are not limited to: corn, wheat, triticale, barley, cassava, rye, graded starch stream rendered from the aforementioned feedstocks, sugar cane, sugar beet, molasses, rice straw, potato waste, wood waste, switch grass, pine and other wood derivatives, municipal waste, food waste and beverage (alcoholic and non-alcoholic) industry waste.
• An exemplary, but non-limiting, feedstock for the fermentation by Saccharomyces cerevisiae is molasses.
• the content of fermentable carbohydrates in biomass may be determined by methods known in the art.
• a particularly instructive disclosure is Milne et al., Sourcebook of Methods of Analysis for Biomass Conversion and Biomass Conversion Processes. SERI/SP-220-3548. Golden, CO: Solar Energy Research Institute, February 1990.
• fermentation products which may be produced by gram negative bacteria, moulds and yeasts, such as the aforementioned microorganisms, there might be mentioned: ethanol; 1 ,3-propanediol; glycerol; butanol; 1 ,4-butanediol; arabitol; xylitol; sorbitol; mannitol; acetoin; acetic acid; propionic acid; 3-hydroxy propionic acid; lactic acid; succinic acid; furandicarboxylic acid; fumaric acid; malic acid; adipic acid; citric acid; aconitic acid; glutamic acid; itaconic acid; levulinic acid; glutaric acid; aspartic acid; malonic acid; glycine; serine; threonine; lysine; isoprene; and, polyhydroxybutyrate.
• the fermentation medium which comprises the antibacterial agent as defined in the present invention may be for the production of: ethanol; 1 ,3- propanediol; glycerol; butanol; 1 ,4-butanediol; arabitol; xylitol; sorbitol; mannitol; acetic acid; propionic acid; 3-hydroxy propionic acid; lactic acid; succinic acid; 2,5-furandicarboxylic acid; fumaric acid; malic acid; adipic acid; citric acid; aconitic acid; glutamic acid; itaconic acid; levulinic acid; glutaric acid; aspartic acid; malonic acid; and, mixtures thereof.
• the antibacterial agent has been added to a fermentation medium for the production of: 1 ,4-butanediol; propionic acid; 3-hydroxypropionic acid; lactic acid; succinic acid; 2,5-furandicarboxylic acid; fumaric acid; malic acid; or itaconic acid. And it is most preferred to use said antibacterial agent in a fermentation medium for the production of: propionic acid; lactic acid; succinic acid; 1 ,4-butanediol; or, 2,5-furandicarboxylic acid.
• the present invention also encompasses: the dimerization of that lactic acid to obtain lactide; the synthesis of polylactic acid by polycondensation of that lactic acid; and, the synthesis of polylactic acid by polymerization of lactide obtained from that lactic acid.
• the desired fermentation product(s) mentioned herein and obtainable by fermentation of a fermentable substrate with a suitable microorganism will be produced as a component of a composition which typically further comprises: traces of the fermentable substrate; other substances produced by the microorganism; and, traces of the microorganism itself such as cellular debris and / or cellular components.
• the term "fermentation product” is intended to encompass both the crude product and the product after it has subjected to clarification, purification and / or concentration.
• Exemplary, but non-limiting methods of purification include one or more of: filtration, including micro- and ultrafiltration; distillation; (re-)crystallization; extraction; chemical treatment, such as acidification; ion exchange; activated carbon treatment; and, electrodialysis.
• Gram-positive bacteria are stained dark blue or violet by gram staining, mainly due to a high amount of peptidoglycan in their cell wall.
• the present invention is concerned with suppressing or preventing the growth of such bacteria in fermenter cultures.
• the invention is particularly concerned with suppressing or preventing the growth of Gram positive bacteria comprising: Enterococci; Clostridium, in particular Clostridium perfringens and Clostridium pasteurianum; Listeria, in particular Listeria monocytogenes and Listeria innocua; Staphylococcus, in particular Staphylococcus aureus; various Bacillus species, in particular Bacillus anthracis, Bacillus cereus and Bacillis subtilis; and, Streptococcus.
• acyl group means a functional group derived from removing a hydroxyl group from a carboxylic acid and which has the formula R x CO- wherein R x is an alkyl group or alkenyl group that is attached to the CO group with a single bond. One or more unsaturated bonds may be present in the alkenyl group.
• the total number of carbon atoms in the acyl chain thus is the number carbon atoms in the R x chain + 1.
• (C4-C18)acyl means an acyl group having 4-18 carbon atoms.
• a glycerol ester bearing only one C4-C18 acyl group side-chain is termed a (C4-
• C18)glycerol mono-ester a glycerol ester that bears two C4-C18 acyl groups is termed a (C4- C15)glycerol di-ester.
• Mixtures of said esters may be termed (C4-C18)glycerol mono/di-esters or (C4- C18)glycerol mono/di.
• acyl groups include: C6 acyl groups such as iso-hexanoyl groups; C8 acyl groups such as iso-octanoyl groups; C10 acyl groups such as decanoyl groups; C12 acyl groups such as lauryl (dodecanoyl); C14 acyl groups such myristyl (tetradecanoyl) groups; C16 acyl groups such as cetyl and palmityl (hexadecanoyl) groups; and, C18 acyl groups such as octadecanoyl.
• the compounds defined in Formula 1 and Formula 2 above are used for suppression of the growth of gram-positive contaminating bacteria in culturing gram-negative bacteria, moulds or yeasts through the addition of one or more of these compounds to the culture media. In suppressing the growth of gram-positive contaminating bacterial, it is possible to improve production of the desired fermentation product.
• the present invention does not preclude the addition, as the antibacterial agent, of two or more compounds which conform to Formula 1 or two or more compounds which conform to Formula 2. Moreover, mixtures of compounds as defined in Formula 1 and Formula 2 may also be used.
• the total amount of antibacterial agent administered to the fermentation medium, inoculum or fermenter culture is such that it is effective to suppress, prevent or reduce contaminations caused by gram-positive bacteria.
• the exact amount of antibacterial agent will depend on a number of factors such as the particular agent used, the production strain being cultured, the type of medium and the energy source. In most embodiments however, the fermentation medium or other culture medium will contain from 0.001 to 0.5 wt. %, preferably from 0.025 to 0.5 wt.%, and more preferably from 0.1 to 0.5 wt.% based on the total weight of the medium, of the defined antibacterial agent.
• the fermentation medium may comprise at least one adjunct antimicrobial ingredient which has efficacy against gram positive bacteria but which does not substantially effect gram negative bacteria.
• adjunct ingredients may be added directly to the fermentation medium as an exogenous ingredient. Additionally or alternatively, the adjunct ingredients may be included in an inoculant for the fermentation medium.
• the fermentation medium, inoculant or fermenter culture may comprises up to 1 wt.%, based on the total weight of the medium, of an adjunct antimicrobial agent selected from the group consisting of: lysozyme; nisin; pediocin; ⁇ -Polylysine; Protamin; Hop beta acids; rosin acids; pimelic acid; benzoic acid; p-hydroxybenzoic acid; salicylic acid; cinnamic acid; citric acid; saturated fatty acids with a chain length of from 8 to 16 carbon atoms; sugar esters of saturated fatty acids with a chain length of from 8 to 16 carbon atoms; and, mixtures thereof.
• the fermentation medium may comprise up to 2000 mg/L of said adjunct antimicrobial agent.
• lactylates employed in the present invention possess a structure according to Formula 1 herein- below: (R-(0-CH(CH 3 )-CO) a 0) b M (Formula 1);
• R represent a C4-C18 acyl group, preferably a C8 to C14 acyl group, more preferably a C10-C14 acyl group and most preferably a C12-C14 acyl group.
• M represents a proton (H + ) or a counter-cation chosen from the group Li, Na, K, Ca, Mg, Zn, Fe(ll), Cu, Mn, Ag, ammonium or substituted ammonium having one or more (C1-4)alkyl optionally substituted with one or more hydroxyl groups.
• M is selected from the group consisting of Na, K, Ca and Mg. More preferably, M is Na.
• the group (0-CH(CH3)-CO)0 represents a lactyl radical of either the R or S configuration (as defined in Section E in the 1979 edition of the lUPAC Nomenclature of Organic Chemistry) derived from R- or S-lactic acid.
• the group might also represent a mixture of such stereo-isomeric configurations.
• the value of b is equal to the valency of M. "b” thereby attains the value of 1 if M is a proton (H + ) or a monovalent cation such as Na, K, Ag, ammonium (NH 4 ) or substituted ammonium, "b” attains the value of 2 where M is a divalent cation such as Ca, Mg, Zn, Mn, Fe(ll) or Cu.
• Exemplary lactylates of Formula 1 which find utility as anti-bacterial agents in the present invention include but are not limited to: dodecanoyl-lactylate (C12-lactylate); tetradecanoyl-lactylate (C14- lactylate); hexadecanoyl-lactylate (C16-lactylate); octadecanoyl lactylate (C18:0-lactylate) and, octadec-9-enoyl-lactylate (C18:1 -lactylate).
• glycerol esters suitable for use as antibacterial agents in the present invention are defined in Formula 2: CH2OR1-CHOR2-CH2OR3 (Formula 2)
• Ri, R2 and R3 each independently represent H or a C4-C18 acyl group with the proviso that at least one of Ri , R2 or R3 is H and at least one of Ri , R2, or R3 is an acyl group.
• one or two of Ri, R2 or R3 are C6-C14 acyl groups and the remaining R n groups are H.
• one or two of Ri , R2 and R3 are C8 acyl groups and the remaining R n groups are H.
• the present invention does not preclude the use of a mixture of glycerol mono- and di- esters within the antibacterial agent.
• good results have been obtained from the use of (C8)glycerol mono/di-esters.
• Examples AMCET 200C C8-glycerol mono- and diester mixture, purchased from Corbion Caravan, Lenexa, Kansas, U.S.
• AMCET 3400E A mixture of decanoyl-lactylate (C10-lactylate) and dodecanoyl-lactylate (C12- lactylate, purchased from Corbion Caravan, Lenexa, Kansas, U.S.A.
• AMCET 4530E A mixture of dodecanoyl-lactylate (C12-lactylate) and tetradecanoyl-lactylate (C14- lactylate), purchased from Corbion Caravan, Lenexa, Kansas, U.S.A. ATCC: American Type Culture Collection, Manassas, Virginia, U.S.A.
• Bioscreen C A culture system available from Oy Growth Curves Ab Ltd, Helsinki, Finland. The Bioscreen C kinetically measures the development of turbidity (growth) by vertical photometry in up to 200 wells simultaneously.
• EMPLEX C18-lactylate, purchased from Corbion Caravan, Lenexa, Kansas, U.S.
• MIC Minimal Inhibitory Concentration, as measured in an optical density test, is the lowest concentration at which the increase in absorbance of a culture did not exceed the threshold value, which was defined as the average increase in absorbance value of the blanks plus three times the standard deviation.
• Olacta Octadecenoyl-lactylate (C18:1-lactylate) purchased from Corbion Caravan, Lenexa, Kansas, U.S.A.
• Pationic 122A A mixture of two lactylates, specifically sodium decanoyl lactylate (sodium caproyl lactylate) and sodium dodecanoyl lactylate (sodim lauroyl lactylate) in a 1.3: 1 mol ratio, purchased from Corbion Caravan, Lenexa, Kansas, U.S.A.
• Example 1 Effect of tetradecanoyl-lactylate (C14-lactylate) on mixed cultures of Escherichia coli and Clostridium pasteurianum To determine whether tetradecanoyl-lactylate (C14-lactylate) could prevent Clostridium pasteurianum
• JEG2 (NCCB 100154, NCCB: Netherlands Culture Collection of Bacteria, Utrecht, Netherlands) from growing in a culture of a bioengineered homolactic R-lactic acid producing Escherichia coli TG128 (NRRL B-30962.NRRL: Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research, Peoria, Illinois, U.S.A.) three different fermentations were set up and carried out simultaneously. These fermentations were:
• Fermenter 1 Escherichia coli TG128 pure culture fermentation; ⁇ ) Fermenter 2. Escherichia coli TG128 mixed with Clostridium pasteurianum JEG2; iii) Fermenter 3. Escherichia coli TG128 mixed with Clostridium pasteurianum JEG2 with 0.05% (w/v) tetradecanoyl-lactylate (Ci 4 -lactylate: Corbion Caravan, Lenexa, Kansas, U.S.A.) addition.
• Fermenter 1 , 2 and 3 received 3.5 I sterile growth medium of the following composition: 3.25 I demineralised water, 385 g glucose monohydrate, 12.25 g di-ammonium phosphate, 17.75 g di-potassium hydrogen phosphate, 12.25 g potassium dihydrogen phosphate, 3.5 ml of a 1 M solution of betaine-hydrochloride, 5.25 ml of a 1 M solution of MgS0 4 (magnesium sulphate), 3.5 ml of a 1 M solution of CaCh (calcium chloride) and 5.25 ml of a trace metal solution.
• the trace element solution contains per liter: 1.6 g FeC (iron(lll)-chloride), 0.2 g C0CI2.6H2O (cobalt-chloride), 0.1 g CuCI 2 (copper-chloride), 0.2 g ZnCI 2 .4H 2 0 (zinc-chloride) 0.2 g NaMo0 4 (sodium-molybdate), H3BO3 (boric acid) and 10 ml 37 % (w/w) HCI (hydrochloric acid). Fermentor 3 received 0.05 % (w/v) tetradecanoyl-lactylate (Ci 4 -lactylate).
• All three fermenters were equipped with a pH probe.
• the pH of the fermentation was controlled at a value of 6.5 by the addition of a slurry of Ca(OH)2 in demineralised water.
• the concentration of the Ca(OH)2 slurry was approximately 220 g/l.
• the temperature of the fermentors was kept constant at a value of 37°C.
• Fermenter 2 started to produce a large volume of foam and a putrid smell.
• Microscopic examination of the culture broth revealed the presence of large numbers of endospore bearing cells. This phenomenon is seen when Clostridium pasteurianum JEG2 is growing unrestricted.
• Fermenter 3 which was also inoculated with a mixed culture of Escherichia coli TG 128 and Clostridium pasteurianum JEG2 but which also received 0.05 % (w/v) tetradecanoyl-lactylate (Ci 4 -lactylate) produced no foam or putrid smell.
• microscopic examination of the culture broth taken from Fermenter 3 showed that it contained no endospore bearing cells.
• Fermenter 3 was in every respect similar to the performance of Fermenter 1 which was inoculated with a pure culture of Escherichia coli TG128. Moreover chemical analysis of fermentation broth (Table 1) showed that there is no difference in impurity profile between the Escherichia coli TG128 standard fermentation (Fermenter 1) and the Escherichia coli I Clostridium pasteurianum JEG2 mixed culture with tetradacanoyl-lactylate (Fermenter 3). The percent enantiomeric excess of the lactate produced in Fermenters 1 and 3 is close to 100 and only a small amount of S-lactate was detected in Fermenter 3, probably introduced by saponification of the lactylate ester.
• the percent enantiomeric excess in Fermenter 2 was considerably lower due to the unrestricted growth of Clostridium pasteurianum JEG2. Furthermore, the total amount of lactic acid produced in Fermenter 2 was also considerably lower. Percent enantiomeric excess is defined as: ((R- S)/(R+S))*100 and where R and S represent the respective fractions of enantiomers in the R- and S- lactate containing fermentation broth.
• Example 2 Effect of mixtures of decanoyl-lactylate (C10-lactylate) and dodecanoyl-lactylate (C12- lactylate) or mixtures of dodecanoyl-lactylate (C12-lactylate) and tetradecanoyl- lactylate (C14-lactylate) on mixed cultures of Escherichia coli and Clostridium pasteurianum
• lactylates as defined in Formula 1 and glycerol esters as defined in Formula 2 were tested against Clostridium perfringens ATCC 13124 in a Bioscreen C culture system.
• the optical density of the cultures was automatically measured at fixed time intervals at 420 - 580 nm using a wide band filter.
• the growth rate of the test organisms was determined at 30°C.
• the Bioscreen was placed inside an anaerobic cabinet equipped with a type M-12 oxygen sensor (In Vivo2 400 hypoxia workstation, Biotrace International Pic, Bridgend, United Kingdom).
• the oxygen tension was regulated at 0 % oxygen using a Ruskinn gas mixer module (Biotrace International Pic).
• Brain heart infusion broth was prepared with varying amounts of different lactylates and glycerol esters as indicated in Table 2 herein below.
• Tetradecanoic acid (Myristic acid), Sodium tetradecyl sulfate (Sodium myristyl sulfate) were purchased from Sigma-Aldrich.
• the pH of the media was adjusted to 6.0 with 9 M sulphuric acid using a Handylab pH 12 pH meter equipped with a Blueline 16 pH (micro) probe (No. 285129163). All media were sterilised by filtration using 0.45 ⁇ cellulose acetate filters (Minisart syringefilter, sterile and non-pyrogenic, no. 16555, Sartorius, Gottingen, Germany) (9). 300 ⁇ of each medium was transferred to a panel of a sterile Bioscreen Honeycombe 100 well plate (Thermo electron Oy, Vantaa, Finland). Completed well plates were stored at -30°C until further use.
• Clostridium perfringens ATCC 13124 were prepared in screw-capped tubes (100 x 16 mm) containing 10 ml brain heart infusion broth (Oxoid CM225, Basingstoke, United Kingdom) for 24 hours at 30°C.
• Table 3 shows the MIC values of the lactylates, glycerol esters, tetradecanoic acid (myristic acid) and sodium tetradecyl sulfate (sodium myristyl sulfate) for Clostridium perfringens ATCC 13124 in brain heart infusion broth. In the parentheses the number of repeats is given. Table 3. MIC values of different fatty acid derivatives for Clostridium perfringens ATCC 13124 in brain heart infusion broth. (The number of repeats is provided in brackets.)
• lactylates and glycerol esters are capable of suppressing the growth of Clostridium perfringens ATCC 13124.
• the pH of the media was adjusted to 6.0 with 9 M sulphuric acid using a Handylab pH 12 pH meter equipped with a Blueline 16 pH (micro) probe (no. 285129163). All media were sterilised by filtration using 0.45 ⁇ cellulose acetate filters (Minisart syringefilter, sterile and non-pyrogenic, no. 16555, Sartorius, Gottingen, Germany). 300 ⁇ of each medium was transferred to a panel of a sterile Bioscreen Honeycombe 100 well plate (Thermo electron Oy, Vantaa, Finland). Completed well plates were stored at 4°C until further use. Well plates were inoculated with 3 ⁇ of the appropriate test culture using a sterile Hamilton repeating dispenser (Hamilton, Bonaduz, Switzerland.
• Liquid seed cultures were prepared of the following cultures were used in this study:
• Escherichia coli serotype 0157:H7 (ATCC 700728);
• Salmonella enterica ATCC 13076, S. Enteritidis
• Salmonella enterica ATCC 13311 , S. Typhimurium
• Salmonella enterica JAVA (NCTC 8458, NCTC: National Collection of Type Cultures, Porton Down, Salisbury, United Kingdom);
• Bacillus cereus (ATCC 11778);
• the data which is summarised in Tables 4 and 5 herein below, shows that Gram-positive bacteria are more susceptible to these compounds than Gram-negatives species.
• the data in Table 5 also shows that AMCET 200C is active against a much wider range of organisms than the lactylates and it encompasses also the Gram negative bacteria.
• the effective concentration of AMCET 200C (C8- glycerol mono/di) against gram-negative bacteria is 0.5 - 1 % (w/w).
• the concentration range tested was: 0 - 1 % (w/w). Within Table 5, a "+" sign indicates inhibition, NT: not tested. Gram positive organisms are Listeria, Bacillus, Staphylococcus and Lactobacillus.
• Example 5 Ethanol fermentation with Saccharomyces cerevisiae This Example documents the effect of a low concentration of a lactylate blend within an ethanol fermentation with Saccharomyces cerevisiae, said fermentation running on cane-sugar molasses and being deliberately contaminated with a mixed culture of Lactobacillus species. Cultures and culture conditions
• Saccharomyces cerevisiae MUCL30115 was obtained from the Mycotheque de I'Universite Catholique de Louvain (BCCM/MUCL, Louvain-la-Neuve, Belgium) and pre-cultured in a yeast-peptone-glucose broth (YPG).
• the YPG-broth contained, per litre of demineralized water: 40 g glucose monohydrate; 10 g BactoTM Peptone (Becton, Dickinson and Company, Sparks, Maryland, USA); and, 5 g BactoTM Yeast extract (Becton, Dickinson and Company, Sparks, Maryland, USA).
• the pH of the medium was adjusted to 6.0 - 7.0 with 1 N HCI. Cultures were incubated in shake flasks at room temperature.
• Lactobacillus brevis LMG11438 was obtained from the Laboratorium voor Microbiologie, Universiteit Gent (BCCM/LMG, Gent, Belgium). Lactobacillus fermentum AR748 and Lactobacillus fructivorans AR742 were obtained from Corbion Purac B.V., Gorinchem, The Netherlands. All strains were pre- cultured on MRS-broth (de Man et al (1960J A medium for the cultivation of lactobacilli. J. Appl. Bacteriology 23(1 ): 130-135) and incubated at 30°C in stationary screw capped flasks. A mixed culture was prepared by mixing equal volumes of the three Lactobacillus cultures.
• Saccharomyces culture both these cultures also received 10 ml of the mixed Lactobacillus culture.
• 9-10 vol.% of inoculum was removed from each fermenter (A, B) and respectively transferred to further fermenters ( ⁇ ', ⁇ ') containing fresh medium: the fermentation in the primary fermenters (A, B) was allowed to proceed.
• ⁇ ', ⁇ ' further fermenters
• L(+) lactic acid, D(-) lactic acid and residual glucose were determined using enzymic procedures. Specifically and each in accordance with the given manufacturer's protocol: glucose was assayed using the K-Gluc kit available from Megazyme International; D-lactic acid was assayed using the K-Date kit available from Megazyme International; and, L-Lactic acid was assayed using the L- Date kit available from Megazyme International.
• Table 6 below indicates the determined amounts of L(+) lactic acid, D(-) lactic acid and ethanol in the fermentations which have been contaminated with the mixed culture of Lactobacillus species.
• those fermenting cultures of Saccharomyces cerevisiae which also contain Pationic 122A there is shown to be a significant lowering of the standing concentrations of L(+) and, in particular, D(-) lactic acid as compared to cultures which do not contain the lactylate blend.
• the ethanol concentration of cultures of Saccharomyces cerevisiae which contain Pationic 122A is significantly elevated over those cultures in which the lactylate blend in absent.
• the positive effects of Pationic 122A could be maintained for at least 6 - 8 consecutive transfers.

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