FORMULATION SYSTEMS FOR ANTIMICROBIAL GLYCOLIPIDS

Abstract

The invention relates to a composition comprising one or more antimicrobial glycolipids and one or more formulation stabilizers. The invention also relates to methods of preparing the compositions and their application in water containing food, beverage, cosmetic, home care and medical products.

Claims

1. A composition comprising: (i) a glycolipid component comprising: at least one antimicrobial glycolipid according to general formula (I) ##STR00010## wherein m is 3 to 5; n is 2 to 5; o is 0 or 1; and p ss 3 to 17; with the proviso that the sum m+n+o+p is not less than 14; and R is a carbohydrate moiety bound via one of its carbon atoms to the binding oxygen; and/or a physiologically acceptable salt thereof; and/or an ester thereof: in open chain form, wherein any of the hydroxyl groups of general formula (I) including any of the hydroxyl groups of the carbohydrate moiety R is intermolecularly esterified with a carboxylic acid; and/or in form of a lactone intramolecularly formed between the terminal carboxylic acid group of general formula (I) with any of the hydroxyl groups of general formula (I) including any of the hydroxyl groups of the carbohydrate moiety R; and (ii) a formulation component comprising at least one formulation stabilizer; wherein the relative weight ratio of the formulation component to the glycolipid component is 100:1 to 1:2, based on the total weight of all antimicrobial glycolipids according to general formula (II) in the composition and based on the total weight of all formulation stabilizers in the composition.

2. The composition according to claim 1, wherein the at least one antimicrobial glycolipid according to general formula (I) is an ester in open chain form, wherein the carboxylic acid is a C.sub.3-C.sub.10-alkanoic acid.

3. The composition according to claim 1 wherein the formulation component comprises a formulation stabilizer selected from cyclodextrins.

4. The composition according to claim 3, wherein the cyclodextrin is selected from alpha-cyclodextrin, beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin and methyl-beta-cyclodextrin.

5.-57. (canceled)

58. The composition according to claim 4, wherein the relative weight ratio of the cyclodextrin to the glycolipid component is 5:1 to 2.5:1

59. The composition according to claim 1, wherein the formulation component comprises a formulation stabilizer selected from polysorbates.

60. The composition according to claim 59, wherein the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, polysorbate 120, and a mixture of any of the foregoing.

61. The composition according to claim 60, wherein the relative weight ratio of the polysorbate to the glycolipid component is 20:1 to 8:1.

62. The composition according to claim 1, wherein R is a moiety of the subformula ##STR00011## wherein the rings A, B and C are monosaccharide moieties, each independently from the others, with 5 or 6 ring members, wherein one or more of the hydroxyl groups may be acylated.

63. The composition according to claim 62, wherein the rings A and B are xylopyranose moieties and the ring G is a glucopyranose moiety.

64. The composition according to claim 1, wherein the at least one antimicrobial glycolipid is a glycolipid according to general formula (II) ##STR00012## wherein s is 1 or 2; t is 8 or 7; R.sup.1 means H or OH; R.sup.2 means H or -C.sub.1-C.sub.6-alkyl; and R.sup.3, R.sup.4, R.sup.5, R.sup.6 and R.sup.7, independently of one another, mean H or C(O)C.sub.1-C.sub.6-alkyl.

65. The composition according to claim 64, wherein at least one of R.sup.3, R.sup.4, R.sup.5, R.sup.6 and R.sup.7 means C(O)C.sub.1-C.sub.6-alkyl.

66. The composition according to claim 1, wherein the at least one antimicrobial glycolipid is selected from compounds (II-A) to (II-D), ##STR00013## ##STR00014## physiologically acceptable salts thereof, and mixtures thereof.

67. The composition according to claim 66, wherein the at least one antimicrobial glycolipid is compound (II-A) or a physiologically acceptable salts thereof.

68. The composition according to claim 1, wherein the glycolipid component comprises a mixture of more than one antimicrobial glycolipid according to general formula (I).

69. The composition according to claim 64, wherein the glycolipid component comprises a mixture of more than on(c) antimicrobial glycolipid according to general formula (II).

70. The composition according to claim 69, wherein the glycolipid component comprises a mixture of at least a first, a second, and a third antimicrobial glycolipid according to general formula (II), wherein: the relative weight content of the first antimicrobial glycolipid according to general formula (II) is 30 to 50 wt.-%; the relative weight content of the second antimicrobial glycolipid according to general formula (II) is 20 to 50 wt.-%; the relative weight content of the third antimicrobial glycolipid according to general formula (II) is 5 to 10 wt.-%, relative to the total weight of all antimicrobial glycolipids that are comprised in the glycolipid component.

71. The composition according to claim 69, wherein the glycolipid component comprises a mixture of at least a first, a second, and a third antimicrobial glycolipid according to general formula (II), wherein: the first antimicrobial glycolipid has a nominal molecular weight of 970 Da; the second antimicrobial glycolipid has a nominal molecular weight of 1012 Da; and the third antimicrobial glycolipid has a nominal molecular weight of 1054 Da.

72. The composition according to claim 66, wherein the polysorbate is polysorbate 80.

73. An orally consumable water based product comprising the composition according to claim 1.

Description

EXAMPLES

Preparation of Compositions

[0119] A glycolipid mixture with the following weight distribution was used:

TABLE-US-00017 Glycolipid Nominal molecular weight [Da] * ~886 0.4% ~928 4.6% ~954 5.8% ~970 (e.g., either 2x acetyl or 1x isovaleryl) 41.9% ~1012 (e.g. 2x acetyl and 1x isovaleryl) 32.3% ~1054 7.7% Other glucolipids 7.3% * relative wt. % of all glycolipids in sample.

[0120] The total glycolipid content in sample was 95 wt. % of dry mass.

Example 1: Water Based Stock Compositions of alpha-cyclodextrin and a Mixture of Antimicrobial Glycolipids

[0121] Alpha-cyclodextrin (a-CD) and a mixture of antimicrobial glycolipids according to general formula (I) were poured into a glass vessel as solid materials as to generate a volume of 3 mL stock composition in relative quantities as outlined in the table below:

TABLE-US-00018 Constituents 1-1 1-2 1-3 1-4 1-5 Alpha-cyclodextrin 10 g/L 50 g/L 50 g/L 50 g/L 50 g/L Mixture of 4 g/L 6.7 g/L 5 g/L 12.5 g/L 10 g/L antimicrobial glycolipids

[0122] Thus, for 3 mL of stock composition e.g. 1-1, 30 mg alpha-cyclodextrin and 12 mg mixture of antimicrobial glycolipids were employed. Subsequently, 3 mL demineralized water were added as to generate four stock compositions with different relative concentrations as indicated. The resultant stock, compositions were intensively stirred with a magnetic stirrer at room temperature for 30 min. to yield white slightly turbid compositions ready for use.

Example 2: Solid Powder Mixtures of alpha-cyclodextrin and a Mixture of Antimicrobial Glycolipids

[0123] 1 g of alpha-cyclodextrin powder was poured into two 60 mL glass containers each. Subsequently, a mixture of antimicrobial glycolipids according to general formula (I) (milled with mesh size 0.5 mm) was added in amounts of 1 g and 0.4 g, respectively, as to yield the mass distribution outlined in the table below:

TABLE-US-00019 Constituents 2-1 2-2 Alpha-cyclodextrin 71.4 wt.-% 50 wt.-% Mixture of antimicrobial glycolipids 28.6 wt.-% 50 wt.-%

[0124] The glass container was sealed and continuously shaken for 10 min, at room temperature yielding an optically homogeneous mixture of the two powders.

Example 3: Water Based Stock Compositions of Polysorbate 80 and a Mixture of Antimicrobial Glycolipids

[0125] 4 g (4 ml) polysorbate 80 (PS80) were poured into three 60 mL glass containers each and mixed with 40 mL demineralized water. The compositions were intensively stirred with a magnetic stirrer at room temperature for 30 min. In three separate containers 0.4 g, 1 g and 1.6 g mixture of antimicrobial glycolipids according to general formula (I) were poured and subsequently given into the three polysorbate 80 solutions each. The stock compositions had the following concentrations:

TABLE-US-00020 Constituent 3-1 3-2 3-3 Polysorbate 80 80 g/L 80 g/L 80 g/L Mixture of antimicrobial glycolipids 8 g/L 20 g/L 32 g/L

[0126] The separate containers were washed with 23 mL demineralized water and this wash water was also poured into the three combined polysorbate 80 solutions each. The volume of each composition was adjusted to 50 mL by addition of water. The three glass containers were sealed, shaken and subsequently intensively stirred using a magnet stirrer at highest speed to yield a ready for use clear ivory coloured stock composition.

Performance of Compositions

Example 4: Screening for Suitable Formulation Enhancing Agents Used in Combination with Mixture of Antimicrobial Glycolipids

[0127] As to identify formulation stabilizer a screening was conducted in which additives typically used in food, cosmetic or medical applications were combined with a mixture of antimicrobial glycolipids in a water based formulation. A rather high concentration of 1000 mg/l for the formulation enhancing agents was used as to not miss potential effects whereas the mixture of antimicrobial glycolipids was applied in typical use concentrations, i.e. 5, 10 and 25 g/ml.

[0128] A total number of 36 additives were investigated as listed: Xanthan, Guar gum, Pektin, Polyvinylpolypyrrolidone, Glucuronolacton, beta-Cyclodextrin, Sorbitan monostearate, Pektin, Sorbitan monolaurate, Polysorbat 80, Glycocholat, myo-Inositol, Polyethylenglycol, Gum arabic, Locust bean gum, Agar, Alpha-Cyclodextrin, -Carrageen, -Carrageen, Konjac Gum, Tara Gum, Lecithin (from eggs), Lecithin (from soy beans), EDTA, Polyvinylpyrrolidone, Saccharoseacetatisobutyrate (SAIB), Methylcellulose, Hydroxypropylcellulose, Glycerol ester of wood rosin, Carboxymethylcellulose, Sodium alginate, Traganth, Polysorbat 20, Polysorbat 60, Lyso-Lecithin.

[0129] Portions of about 50 mL each for two beverages. Gerolsteiner Orangenlimonade (turbid orange lemonade, de-gassed; cloudy beverage) and REWE Apfelsaft (clear apple juice, sterile filtered; clear beverage) as well as for 10 mM citrate buffer is water (pH 3.0), were mixed each with aliquots of a mixture of antimicrobial glycolipids and the respective additives as to establish for each individually screened additive three combinations of concentrations (1000 g/ml (with few exceptions as listed below) additive with 5, 10 and 25 g/ml mixture of antimicrobial glycolipids, respectively); three controls with neither adding a mixture of antimicrobial glycolipids nor additive were run in parallel.

[0130] The necessary aliquots of a mixture of antimicrobial glycolipids were transferred from an aqueous stock solution containing 1 mg/ml mixture of antimicrobial glycolipids into the nine test solutions prepared for each additive.

[0131] The necessary aliquots of additives were transferred directly into the 50 mL portions.

[0132] For measurement of the turbidity the turbidity infrared device AL250T-IR from AQUALYTIC was used. It was measured at three time points: day 0, 3, 7. All test samples were stored and handled at room temperature.

[0133] A qualitative overview on all additives tested for formulation stabilising effects in using a mixture of antimicrobial glycolipids in three water based product formulation, citrate buffer pH 3, orange lemonade and apple juice is given in the table below. In addition, the influence of the additives on the MIC of a mixture of antimicrobial glycolipids is described qualitatively. Also the effect of the additive itself on the water based products applied is described independent form the influence and interdependencies with a mixture of antimicrobial glycolipids.

TABLE-US-00021 10 mM Cloudy Conc. of citrate orange Clear additive Effect of additive itself on water buffer lemon- apple Effect Component (mg/ml) based product (pH 3) ade juice on MIC Guar gum 1 nd nd n n Pektin (from 1 Increase of turbidity ic ic nd n apple) Glucuronolacton 1 n n n n Beta- 1 ic n ic lw Cyclodextrin Sorbitan 0.1 Weakly soluble dc dc n sw monostearate Pektin (from 1 Increase of turbidity ic n ic n citrus) Sorbitan 1 nd nd nd n monolaurate Polysorbate 80 1 Increase of turbidity ic ic ic lw Glycocholat 1 not compatible with orange n n n n lemonade myo-Inositol 1 dc n dc n Polyethylenglycol 1 n n n n Gum arabic 0.125 ic n nd nd Gum arabic from 1 dc n n n acacia tree Locust bean gum Not soluble nd Agar 1 nd n nd n alpha- 1 ic ic ic lw Cyclodextrin -Carrageen 1 Increase of turbidity dc n n n -Carrageen 1 Precipitation n n n n Konjac Gum 0.5 Not completely dissolved n n dc n Tara Gum 0.5 Not completely dissolved dc n dc n Lecithin (from 1 Increase of turbidity, lipid dc ic dc nd eggs) precipitate and film at surface Lecithin (from 1 Increase of turbidity dc ic dc w soy) EDTA 1 dc n n Impr. Pektin variant 3 1 n n n n Polyvinyl- 1 dc n n n pyrrolidone k12 Polyvinyl- 1 dc n dc n pyrrolidone k30 Polyvinyl- 1 dc n n n pyrrolidone k90 Saccharose- 0.2 dc n n n acetateisobutyrat Methyl cellulose 1 n n n n Hydroxypropyl 1 dc n n n cellulose Glycerol ester of 0.2 nd n n n wood rosin Carboxymethyl 1 n n n n cellulose Sodium alginate 1 n n n n Xanthan 1 Increase of turbidity and viscosity ic ic ic n Xanthan variant 1 Increase of turbidity and viscosity ic ic ic n 2 Xanthan variant 1 Increase of turbidity and viscosity ic ic ic n 3 Traganth 1 n n n n Polysorbate 80 1 Not compatible with apple juice. ic ic ic lw Clear citrus lemonade used instead Polysorbate 20 1 Not compatible with apple juice. nd ic ic lw Clear citrus lemonade used instead Polysorbate 60 1 Not compatible with apple juice. nd ic ic lw Clear citrus lemonade used instead Lyso-Lecithin 1 Strong increase of turbidity nd nd ic w Legend: n = neutral, w = weaker, lw = little weaker, sw = strongly weaker, dc = decrease in compatibility, ic = increase in compatibility, nd = not clear or not determined, impr = improved;

[0134] Since xanthan and pektin additive applied (as bolded is the above list) displayed promising formulation stabilizing effects in this initial testing at rather high concentrations, testing was repeated and effect observed for a longer period of time, i.e. >=14 days, under same conditions as described above. All experiments done and reported in duplicate. Samples were compared against control (blank without addition of antimicrobial glycolipids or xanthan) It revealed that the compatibility improving effect of these additives fades out over time as illustrated for xanthan in the table below:

TABLE-US-00022 25 g/ml of mixture of antimicrobial glycolipids in Orange Lemonade time control control Xanthan 1 mg/ml Xanthan 1 mg/ml [d] Results of the visual control 0 Turbid, no particles, Turbid, no particles, Same as control Same as control no precipitate no precipitate 7 Turbid, no particles, Turbid, no particles, Same as control Turbid, particles, no precipitate no precipitate some precipitation 14* Turbid, no particles, Turbid, no particles, Turbid, particles, Turbid, particles, no precipitate no precipitate strong precipitate strong precipitate 25 g/ml of mixture of antimicrobial glycolipids in Apple Juice time control control Xanthan 1 mg/ml Xanthan 1 mg/ml [d] Results of the visual control 0 clear, no particles, clear, no particles, Weakly turbid, no Weakly turbid, no no precipitate no precipitate particles, no precipitate particles, no precipitate 7 clear, no particles, clear, no particles, Weakly turbid, no Weakly turbid, no no precipitate no precipitate particles, no precipitate particles, no precipitate 14* clear, no particles, clear, no particles, Clear with Clear with no precipitate no precipitate precipitation precipitation *not further observed beyond day 14 since compatibility was disturbed already after 14 days.

[0135] Improvement of compatibility by xanthan and pektin was also investigated at lower concentrations, i.e. 5, 10. 50, 100, 250, 500 and 750 g/ml. For pektin at least 500 g/ml were required and for Xanthan at least 250 g/ml to observe the desired effect in the said water based products as used here.

[0136] It can be concluded from the above experimental data that xanthans and pektin can be used to improve the formulation stabilization of a mixture of antimicrobial glycolipids in water based product formulations for a limited period of time less than 7 days. In case stability is required for a longer period of time, then other additives, in particular cyclodextrins and polysorbates are to be used.

Example 5: Application of Combinations of alpha-cyclodextrin and Mixture of Antimicrobial Glycolipids

[0137] As to confirm the formulation stabilizer properties of alpha-cyclodextrin for the use of a mixture of antimicrobial glycolipids in water based products, in particular beverages, combinations of both components at different concentrations were investigated in two commercially available beverages which lack compatibility using a mixture of antimicrobial glycolipids alone, i.e. in the absence of alpha-cyclodextrin.

[0138] Portions of about 50 mL each for the two beverages, Gerolstetner Orangenlimonade (turbid orange lemonade, de-gassed; cloudy beverage) and REWE Apfelsaft (clear apple juice, sterile filtered; clear beverage), were mixed with aliquots of a mixture of antimicrobial glycolipids and alpha-cyclodextrin as to establish the depicted nine combinations of concentrations (4-2 to 4-4 and 4-6 to 4-11, respectively); two control solutions with neither adding a mixture of antimicrobial glycolipids nor alpha-cyclodextrin were run in parallel (4-1 and 4-5, respectively).

[0139] The necessary aliquots of a mixture of antimicrobial glycolipids were transferred from an aqueous stock solution containing 1 mg/ml mixture of antimicrobial glycolipids into the two beverages, Gerolsteiner Orangenlimonade and REWE Apfelsaft, respectively.

[0140] The necessary aliquots of alpha-cyclodextrin were transferred from art aqueous stock solution containing 10 mg/ml of alpha-cyclodextin into the two beverages, Gerolsteiner Orangenlimonade and REWE Apfelsaft, respectively.

[0141] For measurement of the turbidity the turbidity infrared device AL250T-IR from AQUALYTIC was used. It was measured at three time points; day 0, 3, 7. All test samples were stored and handled at room temperature.

a) Orange Lemonade

[0142]

TABLE-US-00023 25 g/ml of mixture of antimicrobial glycolipids 4-1 4-2 4-3 4-4 control alpha-cyclodextrin alpha-cyclodextrin alpha-cyclodextrin 10 g/ml 100 g/ml 500 g/ml

[0143] Results of the turbidity measurements are summarized in the tables below:

TABLE-US-00024 25 g/ml of mixture of antimicrobial glycolipids 4-1 4-2 4-3 4-4 Time [d]/turbidity control 10 g/ml 100 g/ml 500 g/ml 0 346 NTU 339 NTU 345 NTU 331 NTU 3 299 NTU 263 NTU 293 NTU 298 NTU 7 242 NTU nd 232 NTU 253 NTU nd = not determined

[0144] Results of the visual control are summarized in the tables below:

TABLE-US-00025 25 g/ml of mixture of antimicrobial glycolipids Time 4-1 4-2 4-3 4-4 [d] control 10 g/ml 100 g/ml 500 g/ml 0 Turbid, no particles, no Same as control Same as control Same as precipitate control 3 Turbid, no particles, Strong turbidity and particles, Same as control Same as small precipitate precipitation control 7 Turbid, no particles, nd Strong turbidity and particles, Same as small precipitate precipitation control

b) Apple Juice

[0145]

TABLE-US-00026 10 g/ml of mixture of antimicrobial glycolipids (clear solution) 4-5 4-6 4-7 4-8 4-9 4-10 4-11 control alpha- alpha- alpha- alpha- alpha- alpha- cyclodextrin cyclodextrin cyclodextrin cyclodextrin cyclodextrin cyclodextrin l0 g/ml 25 g/ml 50 g/ml 75 g/ml 100 g/ml 500 g/ml

[0146] Results of the turbidity measurements are summarized in the sables below:

TABLE-US-00027 10 g/ml of mixture of antimicrobial glycolipids Time [d]/ 4-5 4-6 4-7 4-8 4-9 4-10 4-11 turbidity control 10 g/ml 25 g/ml 50 g/ml 75 g/ml 100 g/ml 500 g/ml 0 0.47 NTU 1.55 NTU 0.68 NTU 0.63 NTU 0.71 NTU 0.70 NTU 0.76 NTU 3 0.49 NTU 1.14 NTU 0.81 NTU 0.67 NTU 0.61 NTU 0.79 NTU 0.79 NTU 7 0.46 NTU nd 0.72 NTU nd nd 0.82 NTU nd nd = not determined

[0147] Results of the visual control are summarized in the tables below:

TABLE-US-00028 10 g/ml of mixture of antimicrobial glycolipids 4-5 4-6 4-7 4-8 4-9 4-10 4-11 Time [d] control 10 g/ml 25 g/ml 50 g/ml 75 g/ml 100 g/ml 500 g/ml 0 clear, no Same as Same as Same as Same as Same as Same as particles, control control control control control control not precipitate 3 clear, no Precipitation Same as Same as Same as Same as Same as particles, control control control control control not precipitate 7 clear, no nd Clear, no nd nd Same as nd particles, particles, control not precipitate precipitation

[0148] It can be concluded from the above experimental data that the 10 g/ml solution of a mixture of antimicrobial glycolipids in the clear beverage Apple Juice can safely be stabilized by combining with 100 g/ml alpha-cyclodextrin whereas amounts of 10-25 g/ml alpha-cyclodextrin obviously are not yet sufficient due to the observed precipitation. As demonstrated in Example 9, the plain 10 g/ml solution of a mixture of antimicrobial glycolipids in the clear beverage Apple Juice displayed precipitation already after 3 days. For the 25 g/ml solution of mixture of antimicrobial glycolipids in the cloudy beverage Orange Lemonade safe stabilization can be achieved by adding 500 g/ml alpha-cyclodextrin whereas amounts of 100 g/mi alpha-cyclodextrin obviously are not yet sufficient due to the observed increase in turbidity and precipitation after 7 days. As demonstrated in Example 9, the plain 25 g/ml solution of a mixture of antimicrobial glycolipids in the cloudy beverage Orange Lemonade displayed precipitation already after 3 days.

Example 6: Temperature Dependency of Application of Combinations of alpha-cyclodextrin and a Mixture of Antimicrobial Glycolipids

[0149] Applying a similar protocol as outlined under Example 5, the temperature dependency of the effect on compatibility was observed for alpha-cyclodextrin in combination with a mixture of antimicrobial glycolipids for the clear beverage apple juice. However, for the concentration of 10 g/ml of mixture of antimicrobial glycolipids three combination with alpha-cyclodextrin, 25 (6-1, 6-2,6-9, 6-10, 6-17, 6-18), 75 (6-3, 6-4, 6-11, 6-12, 6-19, 6-20) and 100 g/ml (6-5, 6-6, 6-13, 6-14, 6-21, 6-22) and for the concentration of 25 g/ml of mixture of antimicrobial glycolipids only 100 g/ml alpha-cyclodextrin (6-7, 6-8, 6-1, 6,15, 6,-16, 6-23, 6-24) was used all with two test points, respectively. Besides room temperature, also 6 C. and 40 C. were applied; in addition the time period of observation was extended to 14 days, yielding the following results:

TABLE-US-00029 6 C. 10 g/ml of mixture of antimicrobial glycolipids time 6-1 6-2 6-3 6-4, 6-5, 6-6, [d] control 25 g/ml 25 g/ml 75 g/ml 75 g/ml 100 g/ml 100 g/ml 0 clear, no Slightly Slightly Same as Same as Same as Same as particles, not turbid, no turbid, no control control control control precipitate particles, particles, not precipitate not precipitate 7 clear, no Same as Same as Same as Same as Same as Same as particles, not control control control control control control precipitate 14 clear, no Same as Same as Same as Same as Same as Same as particles, not control control control control control control precipitate 21 clear, no clear, no clear, no Same as Same as Same as Same as particles, not particles, particles, control control control control precipitate precipitate precipitate 28 clear, no Same as Same as Same as Same as particles, not control control control control precipitate

TABLE-US-00030 time 6 C. 25 g/ml of mixture of antimicrobial glycolipids [d] control 6-7, 100 g/ml 6-8, 100 g/ml 0 clear, no Slightly turbid, no Slightly turbid, no particles, not particles, not precipitate particles, not precipitate precipitate 7 clear, no Slightly turbid, no Slightly turbid, no particles, not particles, not precipitate particles, not precipitate precipitate 14 clear, no Slightly turbid, no Slightly turbid, no particles, not particles, not precipitate particles, not precipitate precipitate 21 clear, no clear, no particles, clear, no particles, particles, not precipitate precipitate precipitate

TABLE-US-00031 20 C. 10 g/ml of mixture of antimicrobial glycolipids time 6-9, 6-10, 6-11, 6-12, 6-13, 6-14, [d] control 25 g/ml 25 g/ml 75 g/ml 75 g/ml 100 g/ml 100 g/ml 0 clear, no Slightly Slightly Same as Same as Same as Same as particles, not turbid, no turbid, no control control control control precipitate particles, particles, not precipitate not precipitate 7 clear, no Slightly Slightly Same as Same as Same as Same as particles, not turbid, no turbid, no control control control control precipitate particles, particles, not precipitate not precipitate 14 clear, no nd nd Same as Same as Same as Same as particles, not control control control control precipitate 21 clear, no Slightly Slightly clear, slight clear, slight Same as Same as particles, not turbid, no turbid, no precipitate precipitate control control precipitate particles, particles, precipitate precipitate contaminated clear, slight clear, slight Same as Same as precipitate precipitate control control

TABLE-US-00032 time 20 C. 25 g/ml of mixture of antimicrobial glycolipids [d] control 6-15, 100 g/ml 6-16, 100 g/ml 0 clear, no Slightly turbid, no Slightly turbid, no particles, not particles, not precipitate particles, not precipitate precipitate 7 clear, no Same as control Same as control particles, not precipitate 14 clear, no Slightly turbid, no Slightly turbid, no particles, not particles, precipitate particles, precipitate precipitate 21 clear, no Slightly turbid, no Slightly turbid, no particles, not particles, precipitate particles, precipitate precipitate

TABLE-US-00033 40 C. 10 g/ml of mixture of antimicrobial glycolipids time 6-17, 6-18, 6-19, 6-20, 6-21, 6-22, [d] control 25 g/ml 25 g/ml 75 g/ml 75 g/ml 100 g/ml 100 g/ml 0 clear, no Slightly Slightly Same as Same as Same as Same as particles, not turbid, no turbid, no control control control control precipitate particles, particles, not precipitate not precipitate 7 clear, no Slightly Slightly Same as Same as Same as Same as particles, not turbid, no turbid, no control control control control precipitate particles, particles, not precipitate not precipitate 14 clear, no Slightly Slightly clear, no clear, no Same as Same as particles, not turbid, no turbid, no particles, particles, control control precipitate particles, particles, slight slight slight slight precipitate precipitate precipitate precipitate 21 clear, no Slightly Slightly clear, no clear, no Same as Same as particles, not turbid, no turbid, no particles, particles, control control precipitate particles, particles, slight slight slight slight precipitate precipitate precipitate precipitate 28 clear, no clear, no clear, no Same as Same as particles, not particles, particles, control control precipitate slight slight precipitate precipitate

TABLE-US-00034 time 40 C. 25 g/ml of mixture of antimicrobial glycolipids [d] control 6-23, 100 g/ml 6-24, 100 g/ml 0 clear, no Slightly turbid, no Slightly turbid, no particles, not particles, not precipitate particles, not precipitate precipitate 7 clear, no Slightly turbid, no Slightly turbid, no particles, not particles, not precipitate particles, not precipitate precipitate 14 clear, no Particles, precipitate Particles, precipitate particles, not precipitate 21 clear, no Particles, precipitate Particles, precipitate particles, not precipitate

[0150] Is can be concluded from the above experimental data that the improvement of compatibility by adding alpha-cyclodextrin is temperature dependent. Surprisingly, improvement of compatibility is stronger at lower temperature in the order of 4 C.>room temperature>40 C.; lack of compatibility of 10 g/ml mixture of antimicrobial glycolipids with apple juice can successfully avoided by adding only 75 g/ml at 4 C. whereas at 40 C. 100 g/ml are needed. This is in opposite to what is normally expected since solubility is typically increased at higher temperature. This confirms that the improvement of compatibility is not a simple solubility enhancement but an unexpected finding in opposite to the normal expectation of a person trained in the field. Also the improvement of compatibility, applying alpha-cyclodextrin, is long lasting >28 days using 10 g/ml mixture of antimicrobial glycolipids. This is substantially different to pektin and xanthan, depicted in Experiment 4 where the compatibility improving effect faded out already after 7 to 14 days.

Example 7: Application of Combinations of beta-cyclodextrin and a Mixture of Antimicrobial Glycolipids

[0151] As to confirm the compatibility improving properties of beta-cyclodextrin for the use of a mixture of antimicrobial glycolipids in water based products, in particular beverages, combinations of both components at different concentrations were investigated in two commercially available beverages which lack compatibility using a mixture of antimicrobial glycolipids alone, i.e. in the absence of beta-cyclodextrin.

[0152] Portions of about 50 mL each for the two beverages, Gerolsteiner Orangenlimonade (orange lemonade, de-gassed, cloudy beverage) and REWE Apfelsaft (apple juice, sterile filtered, clear beverage), were mixed with aliquots of a mixture of antimicrobial glycolipids and beta-cyclodextrin, using volumes as indicated in the table below, as to establish the ten combinations of concentrations; two control solutions wish neither adding a mixture of antimicrobial glycolipids nor beta-cyclodextrin, was run in parallel.

[0153] The necessary aliquots of a mixture of antimicrobial glycolipids were transferred from an aqueous stock solution containing 1 mg/ml mixture of antimicrobial glycolipids in the two beverages, Gerolsteiner Orangenlimonade and REWE Apfelsaft, respectively.

[0154] The necessary aliquots of alpha-cyclodextrin were transferred from an aqueous stock solution containing 10 mg/ml mixture of alpha-cyclodextrin in the two beverages, Gerolsteiner Orangenlimonade and REWE Apfelsaft, respectively.

[0155] For measurement of the turbidity the turbidity infrared device AL250T-IR from AQUALYTIC was used. It was measured at three time points: day 0, 3, 7. All test samples were stored and handled at room temperature.

a) Orange Lemonade

[0156]

TABLE-US-00035 25 g/ml of mixture of antimicrobial glycolipids 5-3 5-4 5-5 5-2 100 g/ml 250 g/ml 500 g/ml 5-6 5-1 50 g/ml beta- beta- beta- beta- 1 mg/ml beta- Combined liquids control cyclodextrin cyclodextrin cyclodextrin cyclodextrin cyclodextrin v Lemonade [ml] 50 49.25 49 48.25 47 44.5 v beta-cyclodextrin 0 0.25 0.5 1.25 2.5 5 Stock [ml] v mixture of 0 1.25 1.25 1.25 1.25 1.25 antimicrobial glycolipids Stock [ml]

[0157] Results of the turbidity measurements are summarized in the tables below:

TABLE-US-00036 25 g/ml mixture of antimicrobial glycolipids in 5-5 5-6 time 5-1 5-2 5-3 5-4 500 1000 [d] control 50 g/ml 100 g/ml 250 g/ml g/ml g/ml 0 346 NTU 342 NTU 341 NTU 339 NTU 320 298 NTU NTU 7 260 NTU 212 NTU 214 NTU 219 NTU 217 220 NTU NTU

[0158] Results of the visual control are summarized in the tables below:

TABLE-US-00037 25 g/ml mixture of antimicrobial glycolipids time 5-1 5-2 5-3 5-4 5-5 5-6 [d] control 50 g/ml 100 g/ml 250 g/ml 500 g/ml 1000 g/ml 0 Turbid, no Same as Same as Same as Same as Same as particles, not control control control control control precipitate 7 Turbid, no Little more Little more Little more Little more Little more particles, not precipitation precipitation precipitation precipitation precipitation precipitate

b) Apple Juice

[0159]

TABLE-US-00038 10 g/ml of mixture of antimicrobial glycolipids (clear solution) 5-9 5-10 5-11 5-8 100 g/ml 250 g/ml 500 g/ml 5-12 5-7 50 g/ml beta- beta- beta- beta- 1 mg/ml beta- Combined liquids control cyclodextrin cyclodextrin cyclodextrin cyclodextrin cyclodextrin v apple juice [ml] 50 49.25 49 48.25 47 44.5 v beta-cyclodextrin 0 0.25 0.5 1.25 2.5 5 Stock [ml] v mixture of 0 0.5 0.5 0.5 0.5 0.5 antimicrobial glycolipids Stock [ml]

[0160] Results of the turbidity measurements are summarized in the tables below:

TABLE-US-00039 10 g/ml of mixture of antimicrobial glycolipids (clear solution) 5-12 time 5-7 5-8 5-9 5-10 5-11 1000 [d] control 50 g/ml 100 g/ml 250 g/ml 500 g/ml g/ml 0 0.43 NTU 1.09 NTU 0.98 NTU 0.72 NTU 0.69 NTU 0.72 NTU 7 0.45 NTU 0.52 NTU 0.54 NTU nd nd nd

[0161] Results of the visual control are summarized in the tables below:

TABLE-US-00040 10 g/ml of mixture of antimicrobial glycolipids (clear solution) time 5-7 5-8 5-9 5-10 5-11 5-12 [d] control 50 g/ml 100 g/ml 250 g/ml 500 g/ml 1000 g/ml 0 clear, no particles, Same as control Same as control Same as Same as Same as not precipitate control control control 7 clear, no particles, Little more Little more nd nd nd not precipitate precipitation precipitation nd = not determined

[0162] It can be concluded from the above experimental data that the 10 g/ml solution of a mixture of antimicrobial glycolipids in the clear beverage Apple Juice can fairly be stabilized by combining with 50 g/ml beta-cyclodextrin. As demonstrated in Example 7, the plain 10 g/ml solution of a mixture of antimicrobial glycolipids in the clear beverage Apple Juice displayed precipitation already after 3 days. For the 25 g/ml solution of mixture of antimicrobial glycolipids in the cloudy beverage Orange Lemonade reasonable safe stabilization can be achieved by adding 50 g/ml beta-cyclodextrin. As demonstrated in Example 7, the plain 25 g/ml solution of a mixture of antimicrobial glycolipids in the cloudy beverage Orange Lemonade displayed precipitation already after 3 days.

Example 8: Application of Stock Solutions from Example 3 in Water Based Products

[0163] 2400 ml of two beverages, Gerolsteiner Orangenlimonade (orange lemonade, de-gassed, cloudy beverage) and Kastell Zitronensprudel (citrus lemonade, sterile filtered, clear beverage), each were fitted into a 500 mL Erlenmeyer flask. Subsequently, a defined volume, as shown in the table below, of the stock solution, as generated under Example 3, was poured into the beverages and stirred for 5 min, at room temperature:

TABLE-US-00041 Beverage citrus lemonade orange lemonade 6-1 6-2 6-3 6-4 PS80-1 PS80-2 PS80-1 PS80-2 Stock solution 3-1 3-2 3-1 3-2 (see Example 3) v Beverage 400 ml 400 ml 400 ml 400 ml v Stock 0.5 ml 0.5 ml 1.25 ml 1.25 ml solution c Polysorbate 100 g/ml 100 g/ml 250 g/ml 250 g/ml 80 c mixture 10 g/ml 25 g/ml 25 g/ml 100 g/ml antimicrobial glycolipids

[0164] 250 mL portion of the four solutions in the Erlenmeyer flasks were poured into clear and sterile polystyrol conical tubes and sealed for investigation of stability and compatibility of such beverages at three different temperatures (6/20/40 C.) each, resulting in four tubes per temperature and beverage. Such 24 tubes were stored for seven days and observed at day 0, 3 and 7 by measurement of turbidity as well as by optical inspection. A control solution without adding any components was run in parallel.

[0165] The results are depicted in the following tables; without additives means that neither stock solution, nor polysorbate 80 or a mixture of antimicrobial glycolipids was added.

a) Citrus Lemonade

[0166] Results of the turbidity measurements at 6 C., 20 C. and 40 C. are summarized in the tables below:

TABLE-US-00042 6 C. Polysorbate 80 0.1 g/L 6-1 6-2 time [d] control 10 g/ml 25 g/ml 0 0.58 NTU 0.56 NTU 0.57 NTU 0.61 NTU 0.63 NTU 3 0.63 NTU 0.64 NTU 0.62 NTU 0.64 NTU 0.68 NTU 7 0.60 NTU 0.62 NTU 0.62 NTU 0.64 NTU 0.64 NTU

TABLE-US-00043 20 C. Polysorbate 80 0.1 g/L 6-1 6-2 time [d] control 10 g/ml 25 g/ml 0 0.57 NTU 0.59 NTU 0.53 NTU 0.64 NTU 0.65 NTU 3 0.60 NTU 0.59 NTU 0.61 NTU 0.54 NTU 0.53 NTU 7 0.58 NTU 0.60 NTU 0.60 NTU 0.61 NTU 0.61 NTU

TABLE-US-00044 40 C. Polysorbate 80 0.1 g/L 6-1 6-2 time [d] control 10 g/ml 25 g/ml 0 0.58 NTU 0.59 NTU 0.58 NTU 0.64 NTU 0.66 NTU 3 0.58 NTU 0.58 NTU 0.58 NTU 0.58 NTU 0.59 NTU 7 0.62 NTU 0.63 NTU 0.60 NTU 0.64 NTU 0.60 NTU

[0167] Results of the visual control at 6 C., 20 C. and 40 C. are summarized in the tables below:

TABLE-US-00045 6 C. Polysorbate 80 0.1 g/L Time 6-1 6-2 [d] control 10 g/ml 25 g/ml 0 Clear solution, no Same as Same as Same as Same as particles, no control control control control precipitates 3 Clear solution, no Same as Same as Same as Same as particles, no control control control control precipitates 7 Clear solution, no Same as Same as Same as Same as particles, no control control control control precipitates

TABLE-US-00046 20 C. Polysorbate 80 0.1 g/L 6-1 6-2 Time [d] control 10 g/ml 25 g/ml 0 Clear solution, Same as Same as Same as Same as no particles, no control control control control precipitates 3 Clear solution, Same as Same as Same as Same as no particles, no control control control control precipitates 7 Clear solution, Same as Same as Same as Same as no particles, no control control control control precipitates

TABLE-US-00047 40 C. Polysorbate 80 0.1 g/L 6-1 6-2 Time [d] control 10 g/ml 25 g/ml 0 Clear solution, Same as Same as Same as Same as no particles, control control control control no precipitates 3 Clear solution, Same as Same as Same as Same as no particles, control control control control no precipitates 7 Clear solution, Same as Same as Same as Same as no particles, control control control control no precipitates

b) Orange Lemonade

[0168] Results of the turbidity measurements at 6 C., 20 C. and 40 C. are summarized in the tables below:

TABLE-US-00048 6 C. Polysorbate 80 0.25 g/L 6-3 6-4 time [d] Control* 25 g/ml 100 g/ml 0 324 NTU 280 NTU 278 NTU 221 NTU 219 NTU 3 293 NTU 257 NTU 261 NTU 205 NTU 204 NTU 7 266 NTU 245 NTU 242 NTU 201 NTU 199 NTU

TABLE-US-00049 20 C. Polysorbate 80 0.25 g/L 6-3 6-4 time [d] control 25 g/ml 100 g/ml 0 324 NTU 281 NTU 274 NTU 229 NTU 221 NTU 3 273 NTU 223 NTU 228 NTU 183 NTU 185 NTU 7 260 NTU 204 NTU 204 NTU 64 NTU 66 NTU

TABLE-US-00050 40 C. Polysorbate 80 0.25 g/L 6-3 6-4 time [d] control 25 g/ml 100 g/ml 0 310 NTU 280 NTU 277 NTU 226 NTU 216 NTU 3 254 NTU 174 NTU 175 NTU 126 NTU 101 NTU 7 244 NTU 163 NTU 168 NTU 95 NTU 94 NTU

[0169] Results of the visual control at 6 C., 20 C. and 40 C. are summarized in the tables below:

TABLE-US-00051 6 C. Polysorbate 80 0.25 g/L Time 6-3 6-4 [d] control 25 g/ml 100 g/ml 0 Clear solution, no Same as control Same as control Same as control Same as control particles, no precipitates 3 Clear solution, no Slightly turbid, no Slightly turbid, no Trub, keine Trub, keine particles, weak particles, weak particles, weak Partikel, kein Partikel, kein precipitation precipitate precipitate Bodensatz Bodensatz 7 Clear solution, no Same as control Same as control Same as control Same as control particles, weak precipitation

TABLE-US-00052 20 C. Polysorbate 80 0.25 g/L Time 6-3 6-4 [d] control 25 g/ml 100 g/ml 0 Clear solution, no Same as control Same as control Same as control Same as control particles, no precipitates 3 Clear solution, no Slightly turbid, no Slightly turbid, no Turbid, no Turbid, no particles, weak particles, weak particles, weak particles, no particles, no precipitation precipitate precipitate precipitate precipitate 7 Clear solution, no Same as control Same as control separation of separation of particles, weak liquid phases, liquid phases, precipitation particles particles

TABLE-US-00053 40 C. Polysorbate 80 0.25 g/L 6-3 6-4 Time [d] control 25 g/ml 100 g/ml 0 Clear solution, no Same as control Same as control Same as control Same as control particles, no precipitates 3 Clear solution, no Same as control Same as control Clear solution, Clear solution, particles, weak particles, strong particles, strong precipitation precipitation precipitation 7 Clear solution, no Same as control Same as control Clear solution, Clear solution, particles, weak particles, strong particles, strong precipitation precipitation precipitation *control means that neither polysorbate 80 nor a mixture of antimicrobial glycolipids was added

[0170] It can be concluded from the above experimental data that the 10 g/ml and 25 g/ml solution of a mixture of antimicrobial glycolipids in the clear beverage Citrus Lemonade can safely be stabilized by combination with 100 g/ml Polysorbate 80; this stability could be even confirmed for three different storage temperatures. As demonstrated in Example 7, the plain 10 g/ml solution of a mixture of antimicrobial glycolipids in the clear beverage Citrus Lemonade displayed precipitation already after 3 days. For the 25 g/ml solution of mixture of antimicrobial glycolipids in the cloudy beverage Orange Lemonade safe stabilisation can be achieved by adding 250 g/ml Polysorbate 80 whereas the amount of 250 g/ml Polysorbate 80 combined with 100 g/ml of mixture of antimicrobial glycolipids obviously is not sufficient due to the observed increase precipitation and reduced turbidity after 7 days. As demonstrated in Example 7, the plain 25 g/ml solution of a mixture of antimicrobial glycolipids in the cloudy beverage Orange Lemonade displayed precipitation already after 3 days.

Example 9: Compatibility Experiments of a Mixture of Antimicrobial Glycolipids without Applying any Formulation Stabilizer

[0171] For comparison a mixture of antimicrobial glycolipids was applied to the beverages used in Examples 4-8 without applying any of the formulation stabilizers, i.e. polysorbates and cyclodextrins.

[0172] Portions of about 50 mL each for the three beverages, Gerolsteiner Orangenlimonade (orange lemonade, de-gassed, cloudy beverage), Kastell Zitronensprudel (citrus lemonade, sterile filtered, clear beverage), and REWE Apfelsaft (clear apple juice, sterile filtered, clear beverage), were mixed with aliquots of a mixture of antimicrobial glycolipids as to establish the nine test solutions; three control solutions without adding a mixture of antimicrobial glycolipids were ran in parallel:

TABLE-US-00054 7-1 7-2 7-3 7-4 control mixture of antimicrobial mixture of mixture of glycolipids antimicrobial antimicrobial 5 g/ml glycolipids glycolipids 10 g/ml 25 g/ml

[0173] Test sample preparation and experimental processing was identical as outlined in Examples 4 and 5 and visual observation revealed the following results:

[0174] REWE Apfelsaft (apple juice, sterile filtered):

TABLE-US-00055 mixture of antimicrobial glycolipids [g/ml] 7-11 7-21 7-31 7-41 time [d] control 5 g/ml 10 g/ml 25 g/ml 0 Clear, no particle, no Same as control Slight turbidity Slight turbidity precipitate 3 Clear, no particle, no Same as control Clear but slight Clear but strong precipitate precipitation precipitation 7 Clear, no particle, no Same as control Clear but slight Clear but strong precipitate precipitation precipitation 14 Clear, no particle, no Same as control precipitate

[0175] Gerolsteiner Orangenlimonade (orange lemonade, de-gassed):

TABLE-US-00056 mixture of antimicrobial glycolipids [g/ml] 7-12 7-22 7-32 7-42 time [d] control 5 g/ml 10 g/ml 25 g/ml 0 Turbid, no particles, no Same as control Same as control Less turbid precipitation 3 Turbid, no particles, no Same as control Same as control Clear, precipitation precipitation 7 Turbid, no particles, no Same as control Same as control Clear, precipitation precipitation 14 Turbid, no particles, no Same as control Same as control precipitation

[0176] Kastell Zitronensprudel (citrus lemonade, sterile filtered):

TABLE-US-00057 mixture of antimicrobial glycolipids [g/ml] 7-13 7-23 7-33 7-43 time [d] control 5 g/ml 10 g/ml 25 g/ml 0 clear, no particles, no Same as control Same as control Same as control precipitation 3 clear, no particles, no Same as control Slight precipitate Slight precipitate precipitation 7 clear, no particles, no Same as control Same as control precipitation precipitation 14 clear, no particles, no Same as control Same as control precipitation

[0177] It can be concluded from the above experimental data that the compatibility of a mixture of antimicrobial glycolipids dissolved in the clear beverage Citrus Lemonade and Apple juice, as used here, is limited to a concentration of 10 g/ml whereas at concentration of 25 g/ml precipitation can be observed already after 3 becoming prominent after 7 days. Compatibility of a mixture of antimicrobial glycolipids dissolved in the cloudy beverage Orange Lemonade, as used here, is limited to an even lower concentration of 5 g/ml whereas at a concentration of 10 g/ml precipitation can be observed already after 3 days. In case stability is given after 7 days it stays stable even after 14 days.

Example 10: Comparison of Minimum Inhibitory Concentrations (MICs) of a Mixture Antimicrobial Glycolipids Alone or in Combination with Formulation Stabilisers

[0178] The table below lists the MIC values determined for a mixture antimicrobial glycolipids alone or in combination with formulation stabilizers for two spoiling organisms: Saccharomyces cerevisiae MUCL 53497 and Aspergillus niger ATCC 16404:

TABLE-US-00058 S. cerevisiae A. niger inventive inventive MIC [g/mL] of glycolipids in in Concentration combination comparative combination comparative of formulation with without with without stabilizer formulation formulation formulation formulation Formulation stabilizer [g/mL] stabilizer stabilizer stabilizer stabilizer alpha-Cyclodextrin 25 25 12.5 12.5 3.1 50 12.5 6.3 3.1 3.1 100 25 12.5 3.1 3.1 250 50 50 3.1 3.1 500 100 50 12.5 3.1 beta-Cyclodextrin 100 12.5 6.3 3.1 3.1 250 25 25 12.5 3.1 500 25 12.5 3.1 3.1 Methyl-beta- 50 12.5 6.3 3.1 3.1 cyclodextrin 100 25 12.5 3.1 3.1 250 50 25 3.1 3.1 Hydroxypropyl-beta- 250 25 12.5 6.3 3.1 cyclodextrin 1000 50 50 6.3 3.1 100 12.5 3.1 6.3 1.6 250 25 3.1 12.5 1.6 Polysorbate 20 100 6.3 3.1 6.3 12.5 250 25 3.1 12.5 12.5 Polysorbate 60 75 12.5 3.1 6.3 12.5 100 12.5 3.1 6.3 12.5 Polysorbate 80 50 50 12.5 12.5 3.1 100 12.5 6.3 3.1 1.6 250 25 12.5 3.1 1.6 500 100 25 12.5 3.1

[0179] Combinations were carried out using different concentrations of formulation stabilizers, as indicated. MIC values were determined by inoculation with 110E5 CFU/mL of the corresponding micro-organism, subsequent incubation in SDB medium at 28 C. for 48 h and visual inspection of microbial growth. The lowest concentration without detectable microbial growth was considered as MIC. All determinations were done in duplicate.

[0180] It can be concluded from the above experimental data that a mixture antimicrobial glycolipids retains its antimicrobial efficacy, as demonstrated above by the MIC values against the yeast and mold strain even in combination with cyclodextrins and polysorbates, as listed. However, the MIC value depends on the concentration of cyclodextrins and polysorbates used, i.e. the higher the concentrations of cyclodextrins and polysorbates are the higher the MIC values were measured. Beyond a concentration level of 500 g/ml, cyclodextrins or polysorbates, the mixture antimicrobial glycolipids becomes ineffective.

Example 11: Application of Combinations of methyl-beta-cyclodextrin and a Mixture of Antimicrobial Glycolipids

[0181] As to confirm the formulation stabilization properties of methyl-beta-cyclodextrin for the use of a mixture of antimicrobial glycolipids in water based products, in particular beverages, combinations of both components at different concentrations were investigated in two commercially available beverages which lack compatibility using a mixture of antimicrobial glycolipids alone, i.e. in the absence of methyl-beta-cyclodextrin.

[0182] Two beverages, Gerolsteiner Orangenlimonade (turbid orange lemonade, de-gassed; cloudy beverage) and REWE Apfelsaft (clear apple juice, sterile filtered; clear beverage), were used and test solutions prepared as described for Example 5 applying a stock solution of 50 mg/ml methyl-beta-cyclodextrin as well as a 5 mg/ml stock solution of a mixture of antimicrobial glycolipids both in sterile water, respectively.

[0183] For measurement of the turbidity the turbidity infrared device AL250T-IR from AQUALYTIC was used. It was measured at five time points: day 0, 3, 7, 14 and 28. All test samples were stored and handled at room temperature.

[0184] Results on compatibility for Orange Lemonade and Apple Juice:

TABLE-US-00059 Beverage Apple Juice mixture of antimicrobial 0 10 10 10 10 10 glycolipids [g/mL] methyl-beta-cyclodextrin 0 50 75 100 150 250 [g/mL] methyl-beta-cyclodextrin 5 7.5 10 15 25 vs. mixture of antimicrobial glycolipids time [d] turbidity [NTU] 0 0.67 1.71 1.21 1.05 0.92 0.88 28 0.72 0.86 1.50 1.77 1.78 1.80 Visual observation 0 clear, no weakly turbid, no Same as Same as Same as Same as particles, not particles, no control control control control precipitate precipitate 7 clear, no weakly turbid, no Same as Same as Same as Same as particles, not particles, no control control control control precipitate precipitate 14 clear, no weakly turbid, no Same as Same as Same as Same as particles, not panicles, no control control control control precipitate precipitate 21 clear, no weakly turbid, no Same as Same as Same as Same as particles, not particles, no control control control control precipitate precipitate 28 clear, no Small precipitate Same as Same as Same as Same as particles, not control control control control precipitate

TABLE-US-00060 Beveragae Orange Lemonade mixture of antimicrobial 0 25 25 25 25 25 glycolipids [g/mL] methyl-beta-cyclodextrin 0 100 250 375 500 1000 [g/mL] methyl-beta-cyclodextrin 4 10 15 20 40 vs. mixture of antimicrobial glycolipids time [d] turbidity [NTU] 0 274 283 270 269 258 244 28 194 145 161 173 189 185 Visual observation 0 Turbid, no Same as Same as Same as Same as Same as particles, no control control control control control precipitate 7 Turbid, no precipitate Same as Same as Same as Same as particles, no control control control control precipitate 14 Turbid, no precipitate Cloudy, small Small Same as Same as particles, no precipitate precipitate control control precipitate 21 Turbid, no precipitate Cloudy, small Small Same as Same as particles, no precipitate precipitate control control precipitate 28 Turbid, no precipitate Cloudy, small Small Same as Same as particles, no precipitate precipitate control control precipitate

[0185] It can be concluded from the above experimental data that the 10 g/ml solution of a mixture of antimicrobial glycolipids in the clear beverage Apple Juice can safely be stabilized for 28 d by combining with 75 g/ml methyl-beta-cyclodextrin whereas amounts of 50 g/ml methyl-beta-cyclodextrin obviously are not yet sufficient due to the observed small precipitation. As demonstrated in Example 9, the plain 10 g/ml solution of a mixture of antimicrobial glycolipids in the clear beverage Apple Juice displayed precipitation already after 3 days. For the 25 g/ml solution of mixture of antimicrobial glycolipids in the cloudy beverage Orange Lemonade safe stabilization can be achieved for 28 d by adding 500 g/ml methyl-beta-cyclodextrin whereas amounts of 250 g/ml methyl-beta-cyclodextrin obviously are not yet sufficient due to the observed increase in turbidity and precipitation after 14 days. As demonstrated in Example 9, the plain 25 g/ml solution of a mixture of antimicrobial glycolipids in the cloudy beverage Orange Lemonade displayed precipitation already after 3 days.

Example 12: Application of Combinations of hydroxypropyl-beta-cyclodextrin and a Mixture of Antimicrobial Glycolipids

[0186] As to confirm the compatibility improving properties of hydroxypropyl-beta-cyclodextrin for the use of a mixture of antimicrobial glycolipids in water based products, in particular beverages, combinations of both components at different concentrations were investigated in two commercially available beverages which lack compatibility using a mixture of antimicrobial glycolipids alone, i.e. in the absence of hydroxypropyl-beta-cyclodextrin.

[0187] Two beverages, Gerolsteiner Orangenlimonade (turbid orange lemonade, de-gassed; cloudy beverage) and REWE Apfelsaft (clear apple juice, sterile filtered; clear beverage), were used and test solutions prepared as described for Example 5 applying a stock solution of 5 g/ml hydroxypropyl-beta-cyclodextrin as well as a 5 g/ml stock solution of a mixture of antimicrobial glycolipids both in sterile water, respectively.

[0188] For measurement of the turbidity the turbidity infrared device AL250T-IR from AQUALYTIC was used. It was measured at five time points: day 0, 3, 7, 14 and 28. All test samples were stored and handled at room temperature.

[0189] Results on compatibility for Orange Lemonade and Apple Juice:

TABLE-US-00061 Beverage Apple Juice mixture of 0 10 10 10 10 10 antimicrobial glycolipids [g/mL] hydroxypropyl-beta- 0 50 75 100 150 250 cyclodextrin [g/mL] hydroxypropyl-beta- 5 7.5 10 15 25 cyclodextrin vs. mixture of antimicrobial glycolipids time [d] turbidity [NTU] 0 0.68 2.03 1.55 1.86 1.49 1.09 28 0.75 0.76 0.8 0.81 0.93 1.86 Visual observation 0 clear, no Weakly turbid, Weakly turbid, Weakly turbid, Same as Same as particles, not no particles, no particles, no particles, control control precipitate not precipitate not precipitate not precipitate 7 clear, no particles Small particles Thin particles Same as Same as particles, not control control precipitate 14 clear, no particles Small particles Thin particles Same as Same as particles, not control control precipitate 21 clear, no particles Small particles This particles Same as Same as particles, not control control precipitate 28 clear, no particles Small particles Thin particles Same as Same as particles, not control control precipitate

TABLE-US-00062 Beverage Orange Lemonade mixture of antimicrobial 0 25 25 25 25 25 glycolipids [g/mL] hydroxypropyl-beta- 0 100 250 375 500 1000 cyclodextrin [g/mL] hydroxypropyl-beta- 4 10 15 20 40 cyclodextrin vs. mixture of antimicrobial glycolipids time [d] turbidity [NTU] 0 276 272 272 257 261 249 28 197 148 148 152 158 176 Visual observation 0 Turbid, no Same as Same as Same as Same as Same as particles, no control control control control control precipitate 7 Turbid, no precipitate precipitate precipitate Small Same as particles, no precipitate control precipitate 14 Turbid, no precipitate precipitate precipitate Small Same as particles, no precipitate control precipitate 21 Turbid, no precipitate precipitate precipitate Small Same as particles, no precipitate control precipitate 28 Turbid, no precipitate precipitate precipitate Small Very little particles, no precipitate precipitation precipitate

[0190] It can be concluded from the above experimental data that the 10 g/ml solution of a mixture of antimicrobial glycolipids in the clear beverage Apple Juice can safely be stabilized for 28 d by combining with 150 g/ml hydroxypropyl-beta-cyclodextrin whereas amounts of 100 g/ml hydroxypropyl-beta-cyclodextrin obviously are not yet sufficient due to the observed small precipitation. As demonstrated in Example 9, the plain 10 g/ml solution of a mixture of antimicrobial glycolipids in the clear beverage Apple Juice displayed precipitation already after 3 days. For the 25 g/ml solution of mixture of antimicrobial glycolipids in the cloudy beverage Orange Lemonade safe stabilization can be achieved for 28 d by adding 1000 g/ml hydroxypropyl-beta-cyclodextrin whereas amounts of 500 g/ml hydroxypropyl-beta-cyclodextrin obviously are not yet sufficient due to the observed increase in turbidity and precipitation after 7 days. As demonstrated in Example 9, the plain 25 g/ml solution of a mixture of antimicrobial glycolipids in the cloudy beverage Orange Lemonade displayed precipitation already after 3 days.

Example 13: Preserving Challenge Tests of a Mixture of Antimicrobial Glycolipids in Combination with Formulation Stabiliser in Selected Beverages

Case 1: Volvic Juicy Sommerfrchte (Fruit Drink 10% Juice)

[0191] a) Initial experiments showed that addition of a mixture of antimicrobial glycolipids according w the invention (in the following also abbreviated as AGL) into this fruit drink leads to slight incompatibilities regarding the visual appearance of the beverage.

TABLE-US-00063 c (AGL) [g/ml] Time: 0 (control) 5 10 25 Visual appearance 1 h weakly turbid, no same as control same as control same as control particles, no sediment 28 d clear, no particles, similar as control, similar as control, cloudy particles, thin sediment slightly more more sediment predominantly at the sediment at the bottom Turbidity [NTU] 1 h 6.11 7.68 8.70 11.40 28 d 0.60 0.57 0.49 0.49

[0192] The observed incompatibility effects were further increased when the experiment was repeated at 6 C. (i.e. refrigerator conditions).

[0193] b) However, when the fruit drink containing 5 or 10 g/ml AGL was challenged by adding certain food-spoiling microorganisms, it was shown that the tested AGL concentrations safely prevent spoilage of the beverage. No viable microorganisms were found in the beverage compositions after 28 day inoculation period at room temperature.

[0194] The preserving challenge test was carried out as follows:

[0195] The beverage was spoiled with a mixture of three yeasts or three molds. Yeast mixture: Saccharomyces cerevisiae, Zygosacchammyces rouxii, Zygosacchoromyces bailii. Mold mixture: Aspergillus niger, Byssachlamys nivea, Pencillium roqueforti. The mixture of glycolipids was added to the beverage in different concentrations, and afterward it was inoculated with either the yeast or the mold mixture with a concentration of 100 colony forming units (cfu) per ml for either of the mixtures. Incubation was done for 28 h days at room temperature, using sterile centrifuge tubes (50 ml) closed with a screw lid and filled with 40 ml beverage as vessel. The tubes were inspected visually on regular basis in order to assess physical compatibility as well as microbial growth. After 28 days, microbial growth was quantified by colony count on agar plates incubated for 72 h with 100 ml of each beverage sample.

[0196] c) In order to overcome the limited compatibility of AGL in this fruit drink, certain amounts of alpha-cyclodextrin (a-CD) as formulation stabilizer were added as to achieve a formulation exhibiting both physicochemical and antimicrobial stability.

[0197] Test parameters are given in the table below. The test for compatibility was combined with a preserving challenge test as described in the preceding paragraph (section b).

TABLE-US-00064 c (-CD) 0 (control) 35 70 100 [g/ml] c (AGL) 7 7 10 [g/ml] Time: Visual appearance 1 h weakly turbid, no Same Same Same particles, no sediment as control as control as control 28 d Clear, no particles, Same Same Same thin sediment as control as control as control

[0198] While the non-preserved control was completely spoiled with yeasts and molds after seven days at room temperature, no microbial spoilage was found in the formulations containing AGL and a-CD during the complete test period of 28 days. Colony count confirmed that no microbial growth occurred in these preparations.

[0199] Compatibility (i.e. absence of any visual differences between formulations containing AGL and the original beverage without AGL) was confirmed under refrigerator conditions (6 C.) for 28 days. No visual difference to control was observed.

[0200] Thus, the combination of AGL with a-CD as formulation stabilizer allows a safe preservation of the beverage against microbial spoilage.

Case 2: Schweppes Indian Tonic Water (Carbonated Soft Drink)

[0201] a) Initial experiments showed that addition of AGL into this carbonated soft drink leads to slight incompatibilities regarding the visual appearance of the beverage. In particular, the turbidity of the beverage increases slightly with the AGL concentration.

TABLE-US-00065 c (AGL) [g/ml] Time: 0 (control) 5 10 25 Visual appearance 1 h clear, no particles, no slightly turbid, no slightly turbid, no slightly turbid, no sediment particles, no sediment particles, no sediment particles, no sediment 28 d clear, no particles, no slightly turbid, no similar as control, cloudy particles, sediment particles, no sediment more sediment predominantly at the at the bottom Turbidity [NTU] 1 h 0.66 1.89 3.06 5.94 28 d 0.55 0.49 0.47 0.48

[0202] At refrigerator conditions (6 C.), compatibility was found to be worse due to particle formation.

[0203] b) A preserving challenge test (same method and conditions as for Case 1: Volvic Juicy Sommerfrchte) showed that all tested AGL concentrations (5, 10, 25 g/ml) safely prevented spoilage of the beverage. Despite the visual appearance changed as described in section a), no microbial growth occurred. Without addition of AGL, spoilage of the soft drink occurred after incubation for 14 d at room temperature.

[0204] c) Addition of a-CD as formulation stabilizer stabilized the beverage formulation and maintained the antimicrobial activity of the AGL. This was confirmed by repeating the preserving challenge test using concentrations as listed in the following table.

TABLE-US-00066 c (-CD) 0 (control) 35 70 50 100 [g/ml] c (AGL) 7 7 10 10 [g/ml] Time: Visual appearance 1 h clear, no particles, no Same as Same as Same as Same as sediment control control control control 28 d clear, no paricles, no Same as Same as Same as Same as sediment control control control control

[0205] While the non-preserved control was spoiled with yeasts and molds after 14 days at room temperature, no microbial growth was found in the formulations containing AGL and a-CD during the complete test period of 28 days. Colony count confirmed that no microbial growth had occurred in these preparations.

[0206] Compatibility (i.e. absence of any visual differences between formulations containing AGL and the original beverage without AGL) was confirmed under refrigerator conditions (6 C.) for 28 days. No visual difference to control was observed.

[0207] Thus, the combination of AGL with a-CD as formulation stabilizer allows a safe preservation of the beverage against microbial spoilage.

Case 3: Clear Apple Juice

[0208] a,b) Initial experiments showed that addition of 5 g/ml AGL (or higher concentrations) into clear apple juice safely protected against microbial growth in a challenge test as described before (Case 1, section b). However, addition of AGL to apple juice also provoked formation of thin sediment (at 5 g/ml AGL) or cloudy particles (at 5 and 10 g/ml AGL).

[0209] c) Addition of a-CD as formulation stabilizer stabilized the beverage formulation and maintained the antimicrobial activity of AGL. This was confirmed by repeating the preserving challenge test using concentrations as listed in the following table.

TABLE-US-00067 c (-CD) 0 (control) 70 100 [g/ml] c (AGL) 7 10 [g/ml] Time: Visual appearance 1 h clear, no particles, no Same as control Same as control sediment 28 d clear, no particles, Same as control Same as control very thin sediment

[0210] While the non-preserved control was spoiled with yeasts and molds after 2 days at room temperature, no microbial growth was found in the formulations containing AGL and a-CD during the test period of 28 days. Colony count confirmed that no microbial growth had occurred in the preparations.

[0211] Compatibility (i.e. absence of any visual differences between formulations containing AGL and the original beverage without AGL) was also con finned under refrigerator conditions (6 C.) for 28 days. No visual difference to control was obtained.

[0212] Thus, the combination of AGL with a-CD as formulation stabilizer formulation stabilizer allows a safe preservation of the beverage against microbial spoilage.

Comparison of alpha-cyclodextrin with Polysorbate 60:

[0213] The preferred formulation stabilizers polysorbates and cyclodextrins have been further investigated towards their practicability in application in beverages as well as their reliability in use. The results are compiled its the following table:

TABLE-US-00068 formulation formulation stabilizing compatibility with water stabilizer handling solubility in water effect* based products** alpha- Powder, easy to weigh Very well soluble in +++ Very good, no negative cyclodextrin and dose water at ambient interaction observed among temperature 148 different beverages tested polysorbate Thick liquid, quite Dissolution requires +++ Not reliable: turbidity and 60 demanding to strong stirring and precipitation observed for precisely weigh and heating >50 C. certain beverages dose *indicates the effect when applied to stabilize mixtures of antimicrobial glycolipids in a beverage, provided that the formulation stabilizer is compatible with the beverage when applied w/o mixtures of antimicrobial glycolipids **indicates whether the formulation stabilizer is compatible when applied to the beverage w/o mixtures of antimicrobial glycolipids

[0214] Although polysorbates demonstrate a good formulation stabilizing effect of water bases products when preserved with Glycolipids, polysorbates do have certain disadvantages in handling and solubility. In addition the reliability to be used in beverages is limited due to the observed limitation in compatibility. Therefore, the cyclodextrins, in particular alpha-cyclodextrin, appear superior as a broadly applicable and reliable formulation stabilizer for preservation of water based products with mixtures of antimicrobial glycolipids.