FATTY ACID ESTERS AGAINST INFECTIONS IN FERMENTATIONS

Abstract

An antibacterial agent suppresses the growth of gram-positive contaminating bacteria. The antibacterial agent is selected from: a lactylate in accordance with the general formula (R(OCH(CH.sub.3)CO).sub.aO).sub.bM; a glycerol ester in accordance with the general formula CH.sub.2OR.sub.1CHOR.sub.2CH.sub.2OR.sub.3; and, mixtures thereof. In the general formulae: R represents a C4-C18 acyl group, R.sub.1, R.sub.2 and R.sub.3 are each independently selected from 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 R.sub.1, R.sub.2 or R.sub.3 is H and at least one of R.sub.1, R.sub.2, or R.sub.3 is an acyl group; M represents a proton (H.sup.+) or a counter-cation chosen from the group Li, Na, K, Ca, Mg, Zn, Fe(II), Cu, Mn, Ag, ammonium or substituted ammonium having one or more (C1-4)alkyl optionally substituted with one or more hydroxyl.

Claims

1. A fermentation medium comprising: a substrate for microbial growth; and, as an exogenous, added ingredient, an antimicrobial agent selected from: i) a lactylate in accordance with Formula 1,
(R(OCH(CH.sub.3)CO).sub.aO).sub.bM(Formula 1); ii) a glycerol ester in accordance with Formula 2,
CH.sub.2OR.sub.1CHOR.sub.2CH.sub.2OR.sub.3(Formula 2); and, iii) a mixture of such compounds, wherein: R represents a C4-C18 acyl group, the acyl group having an alkyl or alkenyl chain which may be branched or unbranched; R.sub.1, R.sub.2 and R.sub.3 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 R.sub.1, R.sub.2 or R.sub.3 is H and at least one of R.sub.1, R.sub.2, or R.sub.3 is an acyl group; M represents a proton (H.sup.+) or a counter-cation chosen from the group Li, Na, K, Ca, Mg, Zn, Fe(II), 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; and, b is 1 or 2 equaling the valency of M.

2. The fermentation medium according to claim 1 further comprising: an inoculant comprising a culture of gram-negative bacteria, moulds or yeasts.

3. The fermentation medium according to claim 1, wherein the antibacterial agent is the lactylate according to Formula 1 or the salt thereof.

4. The fermentation medium according to claim 3, wherein R represents an acyl group with a straight or branched chain consisting of 4 to 18 carbon atoms.

5. The fermentation medium according to claim 4, wherein R represents an acyl group with a straight or branched chain consisting of 12 to 14 carbon atoms.

6. The fermentation medium according to claim 5, wherein a in Formula 1 is 1.

7. The fermentation medium according to claim 1, wherein the antibacterial agent is a glycerol ester according of Formula 2.

8. The fermentation medium according to claim 7, wherein one or two of R.sub.1, R.sub.2 and R.sub.3 are acyl groups with 8 carbon atoms and the other(s) is (are) H.

9. The fermentation medium according to claim 1, wherein the amount of the antibacterial agent is from 0.001 to 0.5 weight %, based on the total weight of the medium.

10. The fermentation medium according to claim 9, wherein the amount of the antibacterial agent is between 0.025 and 0.5 weight %, based on the total weight of the medium.

11. The fermentation medium according to claim 1 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.

12. The fermentation medium according to claim 1 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.

13. An inoculant for a fermentation medium comprising: a culture of gram-negative bacteria, moulds or yeasts; and, an antibacterial agent selected from: i) a lactylate in accordance with Formula 1,
(R(OCH(CH.sub.3)CO).sub.aO).sub.bM(Formula 1); ii) a glycerol ester in accordance with Formula 2,
CH.sub.2OR.sub.1CHOR.sub.2CH.sub.2OR.sub.3(Formula 2); and, iii) a mixture of such compounds, wherein: R represents a C4-C18 acyl group, the acyl group having an alkyl or alkenyl chain which may be branched or unbranched; R.sub.1, R.sub.2 and R.sub.3 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 R.sub.1, R.sub.2 or R.sub.3 is H and at least one of R.sub.1, R.sub.2, or R.sub.3 is an acyl group; M represents a proton (H.sup.+) or a counter-cation chosen from the group Li, Na, K, Ca, Mg, Zn, Fe(II), 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; and, b is 1 or 2 equaling the valency of M.

14. (canceled)

15. 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,
(R(OCH(CH.sub.3)CO).sub.aO).sub.bM(Formula 1); a glycerol ester in accordance with Formula 2,
CH.sub.2OR.sub.1CHOR.sub.2CH.sub.2OR.sub.3(Formula 2); and, a mixture of such compounds wherein: R represents a C4-C18 acyl group, the acyl group having an alkyl or alkenyl chain which may be branched or unbranched; R.sub.1, R.sub.2 and R.sub.3 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 R.sub.1, R.sub.2 or R.sub.3 is H and at least one of R.sub.1, R.sub.2, or R.sub.3 is an acyl group; M represents a proton (H.sup.+) or a counter-cation chosen from the group Li, Na, K, Ca, Mg, Zn, Fe(II), 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; and, b is 1 or 2 equaling the valency of M.

16. A method for obtaining a fermentation product, said method 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, said fermentation medium comprises: a substrate for microbial growth; and, as an exogenous, added ingredient, an antimicrobial agent selected from: i) a lactylate in accordance with Formula 1,
(R(OCH(CH.sub.3)CO).sub.aO).sub.bM(Formula 1); ii) a glycerol ester in accordance with Formula 2,
CH.sub.2OR.sub.1CHOR.sub.2CH.sub.2OR.sub.3(Formula 2); and, iii) a mixture of such compounds, wherein: R represents a C4-C18 acyl group, the acyl group having an alkyl or alkenyl chain which may be branched or unbranched; R.sub.1, R.sub.2 and R.sub.3 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 R.sub.1, R.sub.2 or R.sub.3 is H and at least one of R.sub.1, R.sub.2, or R.sub.3 is an acyl group; M represents a proton (H.sup.+) or a counter-cation chosen from the group Li, Na, K, Ca, Mg, Zn, Fe(II), 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; and, b is 1 or 2 equaling the valency of M.

17. The method according to claim 16, wherein said inoculant comprises gram-negative bacteria selected from the group consisting of: Escherichia coli; Acinetobacter; Bordetella; Brucella; Campylobacter; Cyanobacteria; Enterobacter; Erwinia; Franciscella; Helicobacter; Klebsiella; Legionella; Moraxella; Neisseria; Pantooea; Pasteurellaceae; Pseudomonas; Proteus; Salmonella; Selenomonadales; Serratia; Shigella; Treponema; Vibrio; Yersinia; Zynomonas; and, combinations thereof.

18. The method according to claim 17, wherein said inoculant comprises one or more gram-negative bacterium selected from the group consisting of: Escherichia coli; Pseudomonas species; and, Pasteurellaceae species.

19. The method according to claim 16, wherein said inoculant comprises one or more moulds selected from the genera Aspergillus and Rhizopus.

20. The method according to claim 16, wherein said inoculant comprises one or more yeasts selected from the genera: Brettanomyces; Candida; Dekkera; Pichia; and, Saccharomyces.

21. The method according to claim 16 for obtaining a fermentation product selected from the group consisting 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.

22. The method according to claim 16 for obtaining a fermentation product selected from: propionic acid; lactic acid; succinic acid; 1,4-butanediol; and, 2,5-furandicarboxylic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0064] Broadly, 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.

[0065] 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.

[0066] 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.

[0067] In addition to the aforementioned antibacterial agent based on lactylates and/or glycerol ester, 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. Such 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.

[0068] In an embodiment, 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. In an alternative expression, the fermentation medium may comprise up to 2000 mg/L of said adjunct antimicrobial agent.

Formula 1

[0069] The lactylates employed in the present invention possess a structure according to Formula 1 herein-below:

(R(OCH(CH.sub.3)CO).sub.aO).sub.bM(Formula 1);

[0070] 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.

[0071] M represents a proton (H.sup.+) or a counter-cation chosen from the group Li, Na, K, Ca, Mg, Zn, Fe(II), Cu, Mn, Ag, ammonium or substituted ammonium having one or more (C1-4)alkyl optionally substituted with one or more hydroxyl groups. Preferably M is selected from the group consisting of Na, K, Ca and Mg. More preferably, M is Na.

[0072] The group (OCH(CH.sub.3)CO)O represents a lactyl radical of either the R or S configuration (as defined in Section E in the 1979 edition of the IUPAC Nomenclature of Organic Chemistry) derived from R- or S-lactic acid. The group might also represent a mixture of such stereo-isomeric configurations.

[0073] The value of b is equal to the valency of M. b thereby attains the value of 1 if M is a proton (H.sup.+) or a monovalent cation such as Na, K, Ag, ammonium (NH.sub.4) or substituted ammonium. b attains the value of 2 where M is a divalent cation such as Ca, Mg, Zn, Mn, Fe(II) or Cu.

[0074] The value of a may be from 1 to 3, with 1 being preferred. Lactylates in which a is 1 are termed monolactylates; compounds where a is 2 are termed dilactylates; and, compounds where a is 3 are termed trilactylates. Monolactylates (a=1) are preferred for use herein. However, it is noted that where a monolactylate is included in the antimicrobial agent, this does not preclude the presence of trace amounts of dilactylates and trilactylates therein; the higher order species can arise in the course of synthesizing the monolactylate.

[0075] 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).

[0076] Methods of synthesis of such lactylates are known in the art. Mention may be made of: U.S. Pat. No. 3,883,669 (Tsen et al.); U.S. Pat. No. 4,146,548 (Forsythe); Elliger, A convenient preparation of pure stearoyl-2-lactylic acid, Journal of Agricultural and Food Chemistry 27: 527 (1979); and, WO 2012/036693 (Caravan Ingredients Inc.). Further, crude lactylates obtained in such synthetic methods may be purified by conventional methods, including but not limited to: filtration; centrifugation; distillation; crystallization; extraction; and, chromatography.

Formula 2

[0077] The glycerol esters suitable for use as antibacterial agents in the present invention are defined in Formula 2:

CH.sub.2OR.sub.1CHOR.sub.2CH.sub.2OR.sub.3(Formula 2)

[0078] R.sub.1, R.sub.2 and R.sub.3 each independently represent H or a C4-C18 acyl group with the proviso that at least one of R.sub.1, R.sub.2 or R.sub.3 is H and at least one of R.sub.1, R.sub.2, or R.sub.3 is an acyl group.

[0079] In a first embodiment, one or two of R.sub.1, R.sub.2 or R.sub.3 are C6-C14 acyl groups and the remaining R.sub.n groups are H. Preferably, one or two of R.sub.1, R.sub.2 and R.sub.3 are C8 acyl groups and the remaining R.sub.n groups are H.

[0080] It is noted that the present invention does not preclude the use of a mixture of glycerol mono- and di-esters within the antibacterial agent. For example, good results have been obtained from the use of (C8)glycerol mono/di-esters.

[0081] Methods of synthesis of such mono- and di-esters are known in the art. For example, the commercial synthesis of C4-C18 esters of glycerol is typically carried out by two different routes: direct esterification of the fatty acid with the glycerol (glycerolysis), catalyzed by a homogeneous acid, such as sulphuric or sulfonic acids; or, by transesterification of triglycerides and polyalcohol catalyzed by alkaline hydroxides like NaOH, KOH or Ca(OH).sub.2 and sodium salts of low molecular weight alcohols, such as methanol. Reference may also be made to: Mostafa et al. Production of mono-, di-, and triglycerides from waste fatty acids through esterification with glycerol Advances in Bioscience and Biotechnology, 2013, 4, 900-907; Hyun et al. A single step non-catalytic esterification of palm fatty acid distillate (PFAD) for biodiesel production. Fuel, 93, 373-380 (2012). Further, crude glycerol esters obtained in such synthetic methods may be purified by conventional methods, including but not limited to: filtration; centrifugation; distillation; crystallization; extraction; and, chromatography.

[0082] The invention is further illustrated by the following examples, which show the inventive merits of this invention, without the invention being limited thereto or thereby.

EXAMPLES

[0083] AMCET 200C: C8-glycerol mono- and diester mixture, purchased from Corbion Caravan, Lenexa, Kans., U.S.

[0084] AMCET 3400E: A mixture of decanoyl-lactylate (C10-lactylate) and dodecanoyl-lactylate (C12-lactylate, purchased from Corbion Caravan, Lenexa, Kans., U.S.A.

[0085] AMCET 4530E: A mixture of dodecanoyl-lactylate (C12-lactylate) and tetradecanoyl-lactylate (C14-lactylate), purchased from Corbion Caravan, Lenexa, Kans., U.S.A.

[0086] ATCC: American Type Culture Collection, Manassas, Va., U.S.A.

[0087] 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.

[0088] EMPLEX: C18-lactylate, purchased from Corbion Caravan, Lenexa, Kans., U.S.

[0089] 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.

[0090] Olacta: Octadecenoyl-lactylate (C18:1-lactylate) purchased from Corbion Caravan, Lenexa, Kans., U.S.A.

[0091] Pationic 122A: A mixture of two lactylates, specifically sodium decanoyl lactylate (sodium caproyl lactylate) and sodium dodecanoyl lactylate (sodium lauroyl lactylate) in a 1.3:1 mol ratio, purchased from Corbion Caravan, Lenexa, Kans., U.S.A.

Example 1: Effect of Tetradecanoyl-Lactylate (C14-Lactylate) on Mixed Cultures of Escherichia coli and Clostridium pasteurianum

[0092] 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, Ill., U.S.A.) three different fermentations were set up and carried out simultaneously. These fermentations were: [0093] i) Fermenter 1: Escherichia coli TG128 pure culture fermentation; [0094] ii) Fermenter 2. Escherichia coli TG128 mixed with Clostridium pasteurianum JEG2; [0095] iii) Fermenter 3. Escherichia coli TG128 mixed with Clostridium pasteurianum JEG2 with 0.05% (w/v) tetradecanoyl-lactylate (C14-lactylate: Corbion Caravan, Lenexa, Kans., U.S.A.) addition.

[0096] All three fermentations were carried out in sterile 7 litre fermenters. Fermenter 1, 2 and 3 received 3.5 l sterile growth medium of the following composition: 3.25 l 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 MgSO.sub.4 (magnesium sulphate), 3.5 ml of a 1 M solution of CaCl.sub.2) (calcium chloride) and 5.25 ml of a trace metal solution. The trace element solution contains per liter: 1.6 g FeCl.sub.3(iron(111)-chloride), 0.2 g CoCl.sub.2.6H.sub.2O (cobalt-chloride), 0.1 g CuCl.sub.2 (copper-chloride), 0.2 g ZnCl.sub.2.4H.sub.2O (zinc-chloride) 0.2 g NaMoO.sub.4 (sodium-molybdate), H.sub.3BO.sub.3 (boric acid) and 10 ml 37% (w/w) HCl (hydrochloric acid). Fermentor 3 received 0.05% (w/v) tetradecanoyl-lactylate (C14-lactylate).

[0097] 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).sub.2 in demineralised water. The concentration of the Ca(OH).sub.2 slurry was approximately 220 g/l. The temperature of the fermentors was kept constant at a value of 37 C.

[0098] Each fermenter (1, 2 and 3) was inoculated with 80 ml of an actively growing overnight culture of Escherichia coli TG128. Fermenters 2 and 3 were also inoculated with 1 ml of a culture of Clostridium pasteurianum JEG2 growing on brain heart infusion broth. Depending on the progress of the fermentations the fermenter cultures were operated for 25-35 hours after which they were analysed. The results of the (chemical) analyses are summarized in Table 1 herein below:

TABLE-US-00001 TABLE 1 Fermenter 3 Escherichia Fermenter 2 coli TG128 + Escherichia Clostridium coli TG128 + pasteurianum Fermenter 1 Costridium NCCB 100154 + Escherichia pasteurianum Tetradecanoyl- Component (g/l) coli TG128 NCCB 100154 lactylate R-lactate 82.6 28.2 81.8 S-lactate 0 2 0.3 % enantiomeric 100 86.8 99.3 excess: (R S)/ (R + S) Ethanol 0.07 0.4 0.08 glucose 1.9 14.4 2.3 Formic acid <0.2 2 <0.2 Acetic acid 0.2 2.3 0.3 Propionic acid <0.1 <0.1 <0.1 Butyric acid <0.1 6.6 0.1 Pyruvic acid <0.1 <0.1 <0.1 2-hydroxy butyric <0.1 <0.1 <0.1 acid Glycolic acid <0.5 <0.5 <0.5 Oxalic acid <0.2 <0.2 <0.2 Sorbic acid <0.1 <0.1 <0.1 Fumaric acid <0.2 <0.2 <0.2 Succinic acid 0.1 <0.1 <0.1 Benzoic acid <0.3 <0.3 <0.3 Maleic acid <0.2 <0.2 <0.2 Malic acid <0.5 <0.5 <0.5 Citric acid <0.5 <0.5 <0.5

[0099] 12 Hours after inoculation it was observed that 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.

[0100] Fermenter 3 which was also inoculated with a mixed culture of Escherichia coli TG128 and Clostridium pasteurianum JEG2 but which also received 0.05% (w/v) tetradecanoyl-lactylate (C.sub.14-lactylate) produced no foam or putrid smell. Moreover, microscopic examination of the culture broth taken from Fermenter 3 showed that it contained no endospore bearing cells.

[0101] The performance of 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/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.

[0102] The percent enantiomeric excess in Fermenter 2 on the other hand 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: ((RS)/(R+S))*100 and where R and S represent the respective fractions of enantiomers in the R- and S-lactate containing fermentation broth.

[0103] Exactly the same results were obtained when Fermenter 3 was fortified with 0.025% (w/v) tetradecanoyl-lactylate (C14-lactylate) instead of 0.05% (w/v).

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

[0104] In an identical experimental set up as described in Example 1, the efficacy of 0.05% (w/v) AMCET 3400E and 0.05% (w/v) AMCET 4530E to suppress the growth of Clostridium pasteurianum JEG2 in a culture of Escherichia coli TG128 was tested.

[0105] The performance of Fermenter 3 with either AMCET 3400E or AMCET 4530E 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 showed that there is no difference in impurity profile between the Escherichia coli TG128 standard fermentation (Fermenter 1) and the Escherichia coli/Clostridium pasteurianum JEG2 mixed culture with AMCET 3400E or AMCET 4530E (Fermenter 3). The percent enantiomeric excess of the lactate produced in Fermenter 1 and 3 is close to 100 for AMCET 3400E and AMCET 4530E.

Example 3: In Vitro Tests of Lactylates Against Clostridium perfringens

[0106] The efficacy of lactylates as defined in Formula 1 and glycerol esters as defined in Formula 2 to inhibit growth was tested against Clostridium perfringens ATCC 13124 in a Bioscreen C culture system.

[0107] 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. In order to assure low oxygen conditions the Bioscreen was placed inside an anaerobic cabinet equipped with a type M-12 oxygen sensor (In Vivo.sub.2 400 hypoxia workstation, Biotrace International Plc, Bridgend, United Kingdom). The oxygen tension was regulated at 0% oxygen using a Ruskinn gas mixer module (Biotrace International Plc).

[0108] Brain heart infusion broth was prepared with varying amounts of different lactylates and glycerol esters as indicated in Table 2 herein below.

[0109] The following compounds were tested: Octanoyl-lactylate (C8-lactylate), Decanoyl-lactylate (C10-lactylate), Dodecanoyl-lactylate (C12-lactylate), Tetradecanoyl-lactylate (C14-lactylate), Hexadecanoyl-lactylate (C16-lactylate), Olacta (octadecenoyl-lactylate, C18:1-lactylate), AMCET 3400E, AMCET 4530E, C8-glycerol mono/di, C10-glycerol mono/di, C12-glycerol mono/di, C14-glycerol mono/di, Tetradecanoic acid (Myristic acid) and Sodium tetradecyl sulfate (Sodium myristyl sulfate).

TABLE-US-00002 TABLE 2 Concentration Concentration Species Range % (w/v) Step size % (w/v) Octanoyl-lactylate 0-0.5 0.1 (C8-lactylate) Decanoyl-lactylate 0-0.1 0.02 (C10-lactylate) Dodecanoyl-lactylate 0-0.01 0.002 (C12-lactylate) Tetradecanoyl-lactylate 0-0.01 0.001 (C14-lactylate) Hexadecanoyl-lactylate 0-0.01 0.002 (C16-lactylate) Olacta (Octadecenoyl- 0-0.1 0.02 lactylate, C18:1- lactylate) AMCET 3400E 0-0.01 0.001 (C10/C12- lactylate) AMCET 4530E 0-0.01 0.001 (C12/C14- lactylate) C8- glycerol mono/di 0-0.5 0.05 C10- glycerol mono/di 0-0.1 0.01 C12- glycerol mono/di 0-0.01 0.001 C14- glycerol mono/di 0-0.01 0.001 Tetradecanoic acid 0-0.01 0.001 (Myristic acid) Sodium tetradecyl sulfate 0-0.01 0.001 (Sodium myristyl sulfate) Tetradecanoic acid (Myristic acid), Sodium tetradecyl sulfate (Sodium myristyl sulfate) were purchased from Sigma-Aldrich.

[0110] 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 m cellulose acetate filters (Minisart syringefilter, sterile and non-pyrogenic, no. 16555, Sartorius, Gttingen, Germany) (9). 300 l 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. Well plates were inoculated with 3 l seed culture using a sterile Hamilton repeating dispenser (Hamilton, Bonaduz, Switzerland). Liquid seed cultures of Clostridium perfringens ATCC 13124 were prepared in screw-capped tubes (10016 mm) containing 10 ml brain heart infusion broth (Oxoid CM225, Basingstoke, United Kingdom) for 24 hours at 30 C.

[0111] 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-US-00003 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.) MIC value Species % (w/v) Octanoyl-lactylate (C8-lactylate) 0.05 (2) Decanoyl-lactylate (C10-lactylate) 0.04 (2) Dodecanoyl-lactylate (C12-lactylate) 0.002 (2) Tetradecanoyl-lactylate (C14-lactylate) 0.001 (2) Hexadecanoyl-lactylate (C16-lactylate) 0.002 (2) Olacta (Octadecenoyl-lactylate, C18:1- lactylate) 0.02 (2) AMCET 3400E (C10/C12- lactylate) 0.02 (3) AMCET 4530E (C12/C14- lactylate) 0.001 (3) C8- glycerol mono/di 0.1 (3) 0.2 (4) C10- glycerol mono/di 0.02 (2) 0.04 (2) C12- glycerol mono/di >0.01 (2) C14- glycerol mono/di >0.01 (4) Tetradecanoic acid (Myristic acid) >0.01 (3) Sodium tetradecyl sulfate (Sodium myristyl sulfate) 0.001 (3)

[0112] It appears that even at very low concentration, lactylates and glycerol esters are capable of suppressing the growth of Clostridium perfringens ATCC 13124.

Example 4: Antimicrobial Properties of Some Lactylates and C8-Glycerol Mono/Di-Esters

[0113] The efficacy of a selected number of different lactylates as defined in formula 1 and C8-glycerol mono/di ester (Caprylic mono/di) as defined in formula 2 to inhibit growth was tested against a selection of gram-positive and gram-negative bacteria 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.

[0114] Brain heart infusion broth was prepared with varying amounts of lactylates or C8-glycerol mono/di ester (Caprylic mono/di). The following compounds were tested: AMCET 3400E; AMCET 4530E; EMPLEX; and, AMCET 200C.

[0115] 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 m cellulose acetate filters (Minisart syringefilter, sterile and non-pyrogenic, no. 16555, Sartorius, Gttingen, Germany). 300 l 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 l of the appropriate test culture using a sterile Hamilton repeating dispenser (Hamilton, Bonaduz, Switzerland.

[0116] Liquid seed cultures were prepared of the following cultures were used in this study: [0117] Escherichia coli serotype 0157:H.sub.7 (ATCC 700728); [0118] Escherichia coli (ATCC 8739); [0119] Staphylococcus aureus (ATCC 6538P); [0120] Listeria monocytogenes (F2399); [0121] Listeria monocytogenes (ATCC 7644); [0122] Listeria monocytogenes NFPA 83 (Seman, D. L., A. C. Borger, et al. (2002) Journal of Food Protection 65(4): 651-658); [0123] Listeria monocytogenes LCDC 861 (Seman, D. L., A. C. Borger, et al. (2002) Journal of Food Protection 65(4): 651-658); [0124] Listeria innocua (ATCC 33090); [0125] Listeria innocua TNO strain (TNO, Zeist, The Netherlands); [0126] Salmonella enterica (ATCC 13076, S. Enteritidis); [0127] Salmonella enterica (ATCC 13311, S. Typhimurium); [0128] Salmonella enterica JAVA (NCTC 8458, NCTC: National Collection of Type Cultures, Porton Down, Salisbury, United Kingdom); [0129] Lactobacillus sakei (DSMZ 20017, DSMZ: Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany); [0130] Lactobacillus plantarum (DSMZ 20174); [0131] Lactobacillus curvatus (DSMZ 20019); [0132] Bacillus cereus (ATCC 11778); [0133] Pseudomonas lundensis (LMG 13517, LMG: Belgian Coordinated Collections of Microorganisms/LMG Bacteria Collection, Gent, Belgium); and, [0134] Pseudomonas fragi (LMG 2191).

[0135] All cultures were transferred daily in screw capped tubes (10016 mm) containing 10 ml brain heart infusion broth (Oxoid CM0225, Basingstoke, UK). Lactobacillus species were transferred in MRS broth (Oxoid CM0359). All cultures were incubated at 30 C. and without agitation.

[0136] We have studied the effects of different concentrations of AMCET 3400E, AMCET 4530E, EMPLEX and AMCET 200C.

[0137] 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).

TABLE-US-00004 TABLE 4 Effect of AMCET 3400E, AMCET 4530E, EMPLEX and AMCET 200C on different Gram positive and Gram negative bacteria. AMCET AMCET AMCET 3400E 4530E 200C (C8- (C10/C12- (C12/C14- glycerol Strain lactylate) lactylate) Emplex mono/di) Listeria + (+) + (+) () () monocytogenes LCDC 861 Listeria + (+) + (+) () () monocytogenes NFPA 83 Listeria + (+) + (+) () () monocytogenes ATCC 7644 Bacillus cereus + (+) + (+) () () ATCC 11778 Staphylococcus aureus + (+) + (+) () () ATCC 6538P Lactobacillus plantarum + () + () () () DSM 20174 Lactobacillus curvatus + () + () () () DSM 20019 Lactobacillus sakei + (+) + (+) () () DSM 20017 Escherichia coli () () () () ATCC 8739 Escherichia coli () () () () O157:H7 ATCC 700728 Salmonella enterica () () () () ATCC 13311 Salmonella enterica () () () () ATCC 13076 Salmonella enterica () () () () JAVA strain Pseudomonas lundensis () () () () LMG 13517 Pseudomonas fragi () () () () LMG 2191

[0138] Two concentration ranges were tested: 0-0.1% (w/w) and 0-0.01% (w/w). Within Table 4, the results for the 0-0.01% concentration range are shown in the brackets. A + sign indicates inhibition. Gram positive organisms are Listeria, Bacillus, Staphylococcus and Lactobacillus.

TABLE-US-00005 TABLE 5 Effect of AMCET 3400E, AMCET 4530E, EMPLEX and AMCET 200C on different Gram positive and Gram negative bacteria. AMCET AMCET AMCET 3400E 4530E 200C (C8- (C10/C12- (C12/C14- glycerol Strain lactylate) lactylate) Emplex mono/di) Listeria + + + + monocytogenes F2399 Listeria + + + + monocytogenes LCDC 861 Listeria + + + + monocytogenes NFPA 83 Listeria + + + + monocytogenes ATCC 7644 Listeria innocua + + + + ATCC 33090 Listeria innocua + + + + TNO strain Bacillus cereus + + + + ATCC 11778 Staphylococcus aureus + + + + ATCC 6538P Lactobacillus curvatus NT NT NT + DSM 20019 Lactobacillus sakei NT NT NT + DSM 20017 Escherichia coli NT NT NT + ATCC 8739 Escherichia coli + O157:H7 ATCC 700728 Salmonella enterica + ATCC 13311 Salmonella enterica + ATCC 13076 Salmonella enterica + JAVA strain Pseudomonas lundensis NT NT NT LMG 13517 Pseudomonas fragi NT NT NT LMG 2191

[0139] 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

[0140] 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

[0141] Saccharomyces cerevisiae MUCL30115 was obtained from the Mycotheque de l'Universit 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 Bacto Peptone (Becton, Dickinson and Company, Sparks, Md., USA); and, 5 g Bacto Yeast extract (Becton, Dickinson and Company, Sparks, Md., USA). The pH of the medium was adjusted to 6.0-7.0 with 1N HCl. Cultures were incubated in shake flasks at room temperature.

[0142] 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 (1960) 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.

[0143] All fermentation experiments were carried out in 3 litre jacketed glass fermenters containing 0.5 litres of a liquid medium having the following composition: 50 g cane-sugar molasses (85 Brix); and, 450 ml demineralized water. The temperature of each fermentation was controlled at 30 C. using a circulating water-bath and the pH was controlled at 5.5 with 1N NaOH.

[0144] Two primary fermenters (A, B) were each inoculated with 50 ml of the actively fermenting Saccharomyces culture: both these cultures also received 10 ml of the mixed Lactobacillus culture. To one of these fermenters (A) was added a 0.5 ml of a solution containing 10% (w/w) Pationic 122A in order to study the effect of lactylates thereon.

[0145] After 24 hours of fermentation, 9-10 vol. % of inoculum was removed from each fermenter (A, B) and respectively transferred to further fermenters (A, B) containing fresh medium: the fermentation in the primary fermenters (A, B) was allowed to proceed. Using this back slopping technique, six to eight further transfers of inoculum were performed, each after 24 hours of fermentation in the source fermenter.

Analytical Methods

[0146] The amounts of 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.

[0147] Organic acids and ethanol were determined by Gas Chromatographic analysis.

Results

[0148] 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. In 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. Moreover, 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.

TABLE-US-00006 TABLE 6 Transfer Pationic 122A L(+) Lactic D() Lactic Ethanol No. Present acid (g/l) acid (g/l) (% w/w) Molasses Fermentation with Saccharomyces cerevisiae contaminated with LAB mixed culture 2 No 1.40 6.80 1.20 3 No 1.18 6.50 1.30 4 No 1.28 6.79 1.60 5 No 1.16 3.15 1.30 6 No 1.42 3.31 1.60 Average 1.29 5.31 1.40 Molasses Fermentation with Saccharomyces cerevisiae contaminated with LAB mixed culture and in presence of Pationic 122A 2 Yes 0.15 2.10 1.30 3 Yes 0.16 0.90 2.10 4 Yes 0.15 0.98 2.20 5 Yes 0.21 0.97 2.20 6 Yes 0.23 1.33 1.80 Average 0.18 1.25 1.92

[0149] It will be apparent to those skilled in the art, upon consideration of the specification, that various modifications can be made in the disclosed embodiments without departing from the scope of the invention. It is therefore intended that the embodiments and examples be considered illustrative only, with the true scope of the invention being indicated by the following claims.