NOVEL BACTERIAL FERMENT OF LACTOBACILLUS SPECIES

Abstract

A bacterial ferment obtained by a method including the following steps: (a) culturing Lactobacillus species in at least one growth medium for at least 24 hours; (b) separating the mixture via centrifugation; (c) adding lactic acid to the supernatant obtained from step b) in an amount sufficient to lower the pH value of the supernatant; (d) stabilizing the resulting mixture with the addition of at least one preservative and/or at least one multifunctional; and optionally (e) filtering the mixture to remove any final precipitation.

Claims

1. A bacterial ferment obtained by a method comprising of the following steps: (a) culturing Lactobacillus species in at least one growth medium for at least 24 hours; (b) separating the mixture via centrifugation to obtain a supernatant; (c) adding lactic acid to the supernatant obtained from step b) in an amount sufficient to lower the pH value of the supernatant to obtain a resulting mixture; (d) stabilizing the resulting mixture by adding at least one preservative and/or at least one multifunctional; and optionally (e) filtering the mixture to remove any final precipitation.

2. A method for producing a bacterial ferment, comprising of the following steps: (a) culturing Lactobacillus species in at least one growth medium for at least 24 hours; (b) separating the mixture via centrifugation to obtain a supernatant; (c) adding lactic acid to the supernatant obtained from step b) in an amount sufficient to lower the pH value of the supernatant to obtain a resulting mixture; (d) stabilizing the resulting mixture with the addition of at least one preservative and/or at least one multifunctional; and optionally (e) filtering the mixture to remove any final precipitation.

3. The method of claim 2, wherein the growth medium comprises a blend of yeast extract, yeast peptone, sodium acetate, dextrose, sucrose, ammonium citrate, potassium phosphate, ascorbic acid, L-cysteine, magnesium sulfate, tween-80 and/or water.

4. The method of claim 2, further comprising combining the mixture with at least one another mixture of a different Lactobacillus species prepared by a process comprising: culturing the different Lactobacillus species in at least one growth medium for at least 24 hours; separating the mixture via centrifugation to obtain a supernactant; adding lactic acid to the supernatant in an amount sufficient to lower the pH value of the supernatant to obtain a resulting mixture; stabilizing the resulting mixture with the addition of at least one preservative and/or at least one multifunctional; and optionally filtering the mixture to remove any final precipitation.

5. The method of claim 2, wherein the at least one multifunctional is a polyol selected from the group consisting of 1,2-pentanediol, 1,2-hexanediol, 1,2-heptanediol, 1,2-octanediol, and mixtures thereof.

6. The method of claim 2, wherein Lactobacillus species is selected from the group consisting of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, and Lactobacillus salivarius.

7. The method of claim 2, wherein said lactic acid is added in an amount of from about 2 to about 10 wt.-percent, based on the total amount of the mixture.

8. A method comprising topically administering the bacterial ferment according to claim 1 to skin to improve the appearance of the skin and/or to prevent body odour.

9. A non-therapeutical method for strengthening a skin barrier function, and/or reducing transepidermal water loss, and/or inducing an expression of filaggrin, and/or increasing components related to a natural moisturizing factor, the method comprising topically administering the bacterial ferment according to claim 1.

10. A method comprising topically applying the bacterial fermemt according to claim 1 to treat and/or to prevent a skin condition.

11. The method according to claim 10, wherein the skin condition is selected from the group consisting of loss of skin barrier function, inflammatory skin conditions, growth of pathogenic microorganisms, and combinations thereof.

12. The method according to claim 10, wherein the skin condition is selected from the group consisting of atopic dermatitis, microbial infection, dry skin, itchy skin, sensitive skin, atopic skin, skin inflammation, microbial dysbiosis, rosacea, psoriasis, rash, and acne.

13. The method according to claim 10, wherein topically applying the bacterial ferment comprises (a) providing anti-inflammatory activity, and/or (b) inhibiting pathogenic microorganisms, and/or (c) maintaining and/or establishing and/or restoring a healthy state of a skin microbiome, and/or (d) improving an immune response of skin.

14. A composition comprising the bacterial ferment according to claim 1, wherein the composition is a pharmaceutical composition or a cosmetic composition.

15. The composition according to claim 14, wherein the composition is selected from the group consisting of oil in water emulsion, water in oil emulsion, crème, lotion, and ointment.

16. A product comprising the bacterial ferment according to claim 1, wherein the product is a pharmaceutical product or a cosmetic product.

17. The composition according to claim 15, wherein the composition is selected from the group consisting of oil in water emulsion, water in oil emulsion, crème, lotion, and ointment.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0160] FIG. 1 shows a flow scheme according to the invention.

[0161] FIG. 2 shows the viability vs. lactic acid addition of the bacterial ferment.

[0162] FIG. 3 shows the pH reflex response and time.

DETAILED DESCRIPTION

[0163] The following examples are intended to illustrate the present invention without limiting the invention. Unless indicated otherwise all amounts, parts and percentages are based on the weight and the total amount or on the total weight and the total amount of the preparations.

Processes and Characteristics of Lactic Acid Bacteria Ferments

EXAMPLES 1

[0164] Lactobacillus plantarum was inoculated from a 1 ml frozen seed stock and grown under sterile, aseptic conditions at 37 centigrade in 1liter growth medium, then transferred to 400-liter intermediate growth medium after 24 hours and then transferred to a 5000 liter culture vessel after a subsequent phase of growth at 24 hours. The bacterial growth medium consisted of a blend of yeast extract, yeast peptone, sodium acetate, dextrose, sucrose, ammonium citrate, potassium phosphate, ascorbic acid, L-cysteine, magnesium sulfate, tween-80 and water. 5000 litres of a light brown, pungent smelling, growth media containing Lactobacillus plantarum that had just reached the stationary growth phase was separated by centrifugation in a sterile disc stack centrifuge. The supernatant from the centrifugation step was depleted of bacteria, however it still contained 3,500,000 lactobacilli per ml. Sodium benzoate was added during the collection process to a final concentration of 0.5%. Then, 70 liters of 88% aqueous lactic acid was mixed into to the supernatant and the mixture allowed to incubate at room temperature for 48 hours. The ferment was then blended with ferment derived from Lactobacillus casei prepared with the same process of centrifugation, addition of sodium benzoate and pH shock to produce a mixture of materials from different probiotic species. This mixture was then passed through an activated carbon filter, this carbon treated mixture was then passed through a catalytic carbon filter and the resulting treated mixture collected in a stilling tank. After 8 hours an aqueous mixture of 2% pentylene glycol was added to the mixture in the stilling tank and mixed then 30% ammonium hydroxide is added in a stepwise fashion until the pH reached 4.6. The mixture was centrifuged through a high g-force clarifying centrifuge to remove any final precipitation. The decanted supernatant was filtered through a 0.2 micron filter before being filled into 25L screw cap containers.

EXAMPLES 2

[0165] Lactobacillus gasseri was inoculated from a 1 ml frozen seed stock and grown under sterile, aseptic conditions at 37 centigrade in 1liter growth medium, then transferred to 400 liter intermediate growth medium after 24 hours and then transferred to a 5000 liter culture vessel after a subsequent phase of growth at 24 hours. The bacterial growth medium consisted of a blend of yeast extract, yeast peptone, sodium acetate, dextrose, sucrose, ammonium citrate, potassium phosphate, ascorbic acid, L-cysteine, magnesium sulfate, tween-80 and water. 5000 liters of a dark brown, pungent smelling, growth media containing Lactobacillus gasseri that had just reached the stationary growth phase was separated by centrifugation in a sterile disc stack centrifuge. The supernatant from the centrifugation step was depleted of bacteria, however it still contained 1,500,000 lactobacilli per ml. Sodium benzoate was added during the collection process to a final concentration of 0.5%. Then, 70 liters of 88% aqueous lactic acid was mixed into to the supernatant and the mixture allowed to incubate at room temperature for 48 hours. This mixture was then passed through an activated carbon filter, this carbon treated mixture was then passed through a catalytic carbon filter. To further decolor the material was passed through an additional filter containing granulated manganese dioxide to remove excess tannins and the resulting treated mixture collected in a stilling tank. After 8 hours an aqueous mixture of 2% pentylene glycol was added to the mixture in the stilling tank and mixed then 30% ammonium hydroxide is added in a stepwise fashion until the pH reached 4.6. After a stilling period of 48 hours the clarified mixture was centrifuged through a high g-force clarifying centrifuge and the resulting supernatant clarified by diatomaceous earth filtration to remove any final precipitation. The decanted supernatant was filtered through a 0.2 micron filter before being filled into 25L screw cap containers.

EXAMPLES 3

[0166] The following bacterial strains were prepared under sterile conditions and grown individually each at a scale of 5000-liter fermentation: Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermenturn, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei and Lactobacillus salivarius. Each strain was harvested individually by sterile centrifugation and the supernatant was treated with 0.5% sodium benzoate and ‘shocked’ with 5-8% lactic acid addition, followed by incubation at room temperature for 24 hours. The treated culture supernatant mixtures were then blended together to reach a specific colour hue specification of yellow. This mixture was then passed through an activated carbon filter, this carbon treated mixture was then passed through a catalytic carbon filter. An aqueous mixture of pentylene glycol was added to the mixture to reach a final concentration of 2% in the stilling tank and an ammonium hydroxide stock was added in a stepwise fashion until the pH reached 4.6. After a stilling period of 48 hours the clarified mixture was clarified by diatomaceous earth filtration to remove any final precipitation. The decanted supernatant was filtered through a 0.2 micron filter before being filled into 25L screw cap containers.

EXAMPLES 4 to 6, Comparison Examples C1 to C2

[0167] The following bacterial strains were prepared under sterile conditions and grown individually each at a scale of 5000 litre fermentation: Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermenturn, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei and Lactobacillus salivarius. Each strain was harvested individually by sterile centrifugetion and the supernatant was treated with 0.5% sodium benzoate and treated with 3-5% lactic acid addition, followed by passing through a 20 kV electric field. The treated culture supernatant mixtures were then blended together to reach a specific color hue specification of yellow. This mixture was then passed through an activated carbon filter, this carbon treated mixture was then passed through a catalytic carbon filter. An aqueous mixture of pentylene glycol was added to the mixture to reach a final concentration of 2% in the stilling tank and an ammonium hydroxide stock was added in a stepwise fashion until the pH reached 4.6. After a stilling period of 48 hours the clarified mixture was clarified by diatomaceous earth filtration to remove any final precipitation. The decanted supernatant was filtered through a 0.2 micron filter before being filled into 25L screw cap containers.

Modulation of Filaggrin Levels in Skin Explants

[0168] Filaggrins play a key role in the cornification process of the human epidermis by cross-linking keratins. Additionally, they can incorporate into the lipid envelope of keratinocytes, supporting its role as skin barrier to prevent moisture loss. Filaggrin can therefore be used as a marker for skin barrier function and terminal keratinocyte differentiation in the epidermis.

[0169] To test for potential modulation of filaggrin levels by the bacterial ferment according to the invention (), abdominal skin samples (from plastic surgery) from a female donor (born in 1993) were used after obtaining informed consent. The skin samples were characterized in terms of phototype and classified as “Light” (ITA° =43°). Skin samples were cut into pieces of approx. 8×3 mm (diameter x thickness) and placed on a perforated stainless-steel ring. Cultivation was carried out at an air-liquid interface using modified William's E medium. Six skin samples were used per treatment.

[0170] Test samples were applied topically to the tissue explants by first cleaning the skin surface with a cotton pad and subsequently applying 4 μl of test sample on top of the biopsies. Finally, the explant was covered with a delivery membrane (6 mm diameter). Application was performed daily from D0 to D5 (day 0 to day 5) of the organ culture.

[0171] On D6 of the experiment, the skin explants were harvested, fixated and sectioned (2 sections per explant) prior to immuno-staining with a specific antibody (Rabbit polyclonal anti-filaggrin, Santa Cruz, cat# sc30229). Detection was carried out using Dako REAL™ Detection System, Alkaline Phosphatase/RED, Rabbit/Mouse (Agilent, cat# K5005). The images of the slides were acquired and the signal was quantified by estimating the intensity and distribution of the pink/red staining within the epidermis using ImageJ (NIH, USA). Data were normalized to the dimension of the analysed skin surface (expressed in pixels).

[0172] In the experiment, the performance of the bacterial ferment according to the invention was evaluated vs PBS as vehicle control, as well as a sample of the culture broth prior to fermentation with Lactobacilli (Raw), a sample of raw medium containing the preservative system employed in the bacterial ferment according to the invention (2% Hydrolite-5), as well as a competitor benchmark product. Data for these treatments are summarized in Table 1

TABLE-US-00007 TABLE 1 Semi-quantitative evaluation of fillagrin modulation by the samples indicated in the table. Values shown are averages from 12 sections ± standard deviation. Statistical significance was tested using a t-test. Average % Change Filaggrin vs Vehicle p- Ex. Sample Score SD Control value 0 PBS Vehicle 2.34 0.92 0 1 0 Raw Medium 1.71 0.6 −27 0.66 Control 0 Preservative 2.47 0.67 1 1 Medium-Control 4 Bacterial ferment 5.29 1.54 126 <0.01 according to the invention C1 Competitor 2.27 1.42 −3 0.62 Benchmark

[0173] Skin samples treated with the bacterial ferment according to the invention (Example 4) exhibited a clear and significant increase in fillaggrin levels (+126% vs vehicle control), whereas the controls and the competitor benchmark lowered filaggrin levels in skin explants or did not modulate them at all.

Modulation of Interleukin-8 Levels

[0174] Interleukin-8 (IL8) is an important intermediate signalling molecule in the response of tissues to insults, resulting in an inflammation reaction. As the phenomenon of irritated skin is tightly correlated to mild inflammatory conditions, levels of IL8 are being routinely used as biomarkers for irritation. IL8 is secreted by keratinocytes in the epidermis upon treatment with stresses such as UV light, chemical compounds or bacterial infection. IL8 is a chemoattractant which recruits other inflammatory cell types upon secretion.

[0175] To assess the effect of the bacterial ferment according to the invention on the secretion levels of IL8, reconstituted 3D skin models (EpiDerm™, MatTek) were used. After a short acclimation phase (daily medium changes), the models were switched to hydrocortisone-free medium, and test compounds were added topically to the reconstituted models. 50 μM dexamethasone was used as positive control, PBS served as negative control in the experiment. After 72h incubation at 37° C. 5% CO2, the 3D models were washed with PBS and subsequently, an inflammatory response was stimulated by addition of 1 μg/ml Ma in the medium for approx. 24h. Supernatants were collected after this phase and the IL8 concentration in these supernatants was determined using an ELISA kit (Human IL8/CXCL1 DuoSet ELISA, R&D Systems). To assess potential toxic effects of the test compounds on the 3D models, a standard MTT assay was carried out to assess viability of the 3D models at the end of the experiment. The results are shown in Table 2.

TABLE-US-00008 TABLE 2 IL8 concentrations in the supernatants of MatTek EpiDerm ™ 3D reconstituted skin models after treatment with test compounds and subsequent stimulation with 1 μg/ml IL1a. Values shown are averages from triplicates ± SEM. P-value is based on a T-test. Viability was determined using a standard MTT assay, values are normalized to stimulated control. IL-8 Viability concentration (% vs [pg/ml] ± p- stimulated Ex. Sample SEM value control) Unstimulated Control 149 ± 7.88 <0.01 105 Stimulated Control 1552 ± 89.96 1 100 50 μM Dexamethasone 1225 ± 18.61 0.011 90 Raw Media Control  2172 ± 127.57 <0.01 114 Preservative Media 1466 ± 63.9  0.385 126 Control 5 Bacterial ferment  954 ± 61.45 <0.01 128 according to the invention C1 Competitor Benchmark 1475 ± 38.55 0.385 105

[0176] The bacterial ferment according to the invention exhibits a clear soothing effect by statistically significantly reducing the IL8 concentration in the supernatant by 39% compared to the stimulated control. Interestingly, the Raw Media Control (Culture broth prior to Lactobacillus fermentation) shows a pronounced irritating effect on the 3 models, whereas the Preservative Control as well as the Competitor Benchmark product both exhibit no modulation of IL8 concentration.

Modulation of Skin Moisture

[0177] The ability of the bacterial ferment according to the invention to influence and improve skin moisture was assessed in a clinical trial with water as the placebo. The study was conducted over a period of two weeks in a randomized, comparative design using blinded test samples. 22 female study subjects (age range 26-65 weeks) were selected in adherence to the Helsinki declaration. Subjects with skin conditions, as well as pregnant and lactating women were excluded from the study. The subjects applied the test samples twice daily on the volar forearms after thorough washing of the forearms with 10% sodium lauryl ether sulphate, 4% NaCI in water.

[0178] Skin moisture was assessed doing corneometer measurements (using a CM 825 corneometer probe connected to a MDD4 (Multidisplay Device, Courage+Khazaka electronic GmbH)) in the treated area at the beginning (day 0) and the end (day 13) of the application period. The measurement principle is based on a non-invasive capacity determination of the skin, which is quantitatively correlated to the water content.

[0179] Results are displayed as the means over subjects in difference of corneometer units (.Math.d13-d0) ±standard abbreviation. Statistical significance of difference of treatments was calculated vs untreated control using ANOVA (Tukey test). The results are shown in Table 3.

TABLE-US-00009 TABLE 3 Results skin moisture Ex. Sample Means of C.U. ANOVA p-value Untreated −0.1 0.807 — Placebo 1.6 0.741 0.43 6 Bacterial ferment 3.0 0.676 0.029 according to the invention

[0180] As shown in Table 3, the bacterial ferment according to the invention causes a significant increase in C.U., and thus in skin moisture content, over the 13d study duration.

EXAMPLES 7

[0181] Reduction of Lactobacillus Viability by Treatment with Lactic Acid

[0182] To determine efficacy of the lactic acid treatment for reduction of Lactobacillus viability, a series of concentrations of lactic acid was added to Lactobacillus cultures. After 12h incubation at 37° C. with stirring, pH of the samples was measured and Lactobacillus viability was determined by plating of serial dilutions for samples treated with lactic acid. The results are shown in Table 4:

TABLE-US-00010 TABLE 4 Effect of addition of various amounts of lactic acid on pH of the broth and bacterial count after incubation at 37° C. for 12 h Lactic Acid (% (w/v) pH after 12 hrs Lactobacillus count (log cfu/ml) 0 7.14 9.90 2 4.56 7.54 2.8 5.56 7.08 3 4.17 5.45 3.4 4.56 4.72 3.6 4.08 5.09 4 3.94 3.52 4.4 3.88 3.60 4.8 3.83 3.00 5 3.75 1.00 5.4 3.78 1.38 6 3.65 1.95 6.8 3.56 1.70

[0183] As shown in Table 4, addition of lactic acid had a drastic effect on the viability of Lactobacillus cultures. Bacterial counts dropped rapidly with increasing amounts of lactic acid; the optimal reduction of Lactobacillus count was reached in a concentration range of ca. 5% (w/v) lactic acid.

Formulation Examples

[0184] In the following, the invention is illustrated by the following formulation examples 1 to 15. The amounts are indicated as % by weight for all formulations.

TABLE-US-00011 TABLE 1 Deodorant Spray with ACH Ingredients EU INCI Amount Tri ethylcitrate Triethyl citrate 3.0 Solubilizer ® PEG-40 Hydrogenated Castor Oil, 1.0 Trideceth-9, Propylene Glycol, Aqua Ethanol 96% Ethanol 30.0 Ethylhexylglycerin Ethylhexylglycerin 0.3 SymDeo ® B125 2-Methyl 5-Cycloheylpentanol 0.25 SymClariol ® Decylene Glycol 0.25 Frescolat ® ML Menthyl Lactate 0.6 Tego ® Cosmo P Polyglyceryl-3 Caprylate 0.5 813MB Locron ® L (50%) Aqua, Aluminum Chlorohydrate 25.0 Perfume oil PO1, Parfum 0.5 PO2, PO3, PO4, or PO5 Water, demin. Aqua Add 100 Bacterial ferment Lactobacillus Ferment, Ammonium 5.0 according to the Lactate, Lactic Acid, Pentylene invention Glycol

TABLE-US-00012 TABLE 2 Deodorant Roll-on Ingredients EU INCI Amount Aqua/Water Aqua Add 100 Bacterial ferment Lactobacillus Ferment, Ammonium 5.0 according to the Lactate, Lactic Acid, Pentylene Glycol invention Hydrolite ® CG Caprylyl Glycol 0.3 SymSave ® H Hydroxyacetophenone 0.5 EDTA NA2 Disodium EDTA 0.1 Dracorin ® GOC Glyceryl oleate citrate; Caprylic/ 4.0 Capric triglyceride Dragoxat ® 89 Ethyl hexyl isononanoate 5.0 SymMollient ® S Cetearyl Nonanoate 1.0 green Genuvisco ® CG Carrageenan 0.5 131 Potassium Sorbate Potassium Sorbate 0.5 Farnesol Farnesol 0.3 Perfume oil PO1, Perfume 1.0 PO2, PO3, PO4, or PO5 SymGuard ® CD Phenylpropanol, o-Cymen-5-ol, 0.3 Decylene Glycol Sodium Hydroxide, Aqua; Sodium hydroxide 0.2 50% solution

TABLE-US-00013 TABLE 3 Flash SOS Face Gelee Ingredients EU INCI Amount Dracorin ® GOC Glyeryl Oleate Citrate, Caprylic/ 0.6 Capric Triglyceride SymMollient ® PDCC Propanediol Dicaprylate/Caprate 6.0 Dragoxat ® 89 Ethylhexyl Isononanoate 5.0 PCL-Liquid ® 100 Cetearyl Ethylhexanoate 1.5 Frescolat ® X-Cool Menthyl Ethylamido Oxalate 1.0 Sym Relief ® 100 Bisabolol, Zingiber Officinale Root 0.1 Extract Sym Decanox ™ HA Caprylic/Capric Triglyceride, 0.8 Hydroxymethoxyphenyl Decanone Alpha Tocopherol DL Tocopherol 0.1 Carbopol ® Ultrez 20 Acrylates/C10-30 Alkyl Acrylate 0.4 Polymer Crosspolymer Aqua/Water Aqua Add 100 Glycerin Glycerin 3.0 SymMollient ® W/S Trideceth-9, PEG-5 Isononanoate, 1.0 Aqua SymDiol ® 68 1,2-Hexanediol, Caprylyl Glycol 0.5 SymSave ® H Hydroxyacetophenone 0.5 Bacterial ferment Lactobacillus Ferment, Ammonium 20.0 according to the Lactate, Lactic Acid, Pentylene Glycol invention Aloe Vera Gel Aloe Barbadensis Leaf Juice 1.5 Concentrate Sodium Hydroxide Aqua, Sodium hydroxide 0.7 10% solution Perfume oil PO1, PO2, Parfum 0.5 PO3, PO4, or PO5 Color 0.03

TABLE-US-00014 TABLE 4 Intimate Sensi-Gel Ingredients EU INCI Amount Aqua/Water Aqua Add 100 Sodium Benzoate Sodium benzoate 0.3 Keltrol ® CG-T Xanthan Gum 0.2 Cyamopsis Cyamopsis Tetragonolobus Gum 0.1 Tetragonolobus Gum Glycerin Glycerin 4.0 Bacterial ferment Lactobacillus Ferment, Ammonium 5.0 according to the Lactate, Lactic Acid, Pentylene invention Glycol Tego ® Betain F50 Cocamidopropyl betaine 14.0 Solubilizer PEG-40 Hydrogenated castor oil, 3.0 Trideceth-9, Propylene Glycol, Aqua Hydrolite ® 6 1,2 Hexanediol 1.0 SymGuard ® CD Phenylpropanol, o-cymen-5-ol, 0.3 Decylene Glycol Sym Relief ® 100 Bisabolol, Zingiber officinale root 0.1 extract Perfume oil PO1, PO2, Parfum 0.2 PO3, PO4, or PO5 Frescolat ® X-cool Menthyl ethylamido oxalate 0.4 Plantacare ® 2000 UP Decyl Glucoside 2.0 Lactic acid 90% Nat. Lactic Acid, Aqua 0.3

TABLE-US-00015 TABLE 5 Cleansing Micellar Gel Ingredients INCI Amount Water, distilled Water (Aqua) Ad 100 Carbopol ® ETD2050 Carbomer 0.7 Polymer Bacterial ferment Lactobacillus Ferment, Ammonium 10.0 according to the Lactate, Lactic Acid, Pentylene invention Glycol SymOcide ® PS Phenoxyethanol, 1.0 Decylene glycol,1,2-hexanediol Sodium Hydroxide 10% Aqua, Sodium Hydroxide 3.28 solution SymMollient ® W/S Trideceth-9, PEG-5 Isononanoate, 1.0 Aqua SymSol ® PF-3 Aqua, Pentylene glycol, Sodium 3.0 lauryl sulfoacetate, Sodium oleoyl sarcosinate, Sodium chloride, Sodium oleate Perfume oil PO1; PO2; Parfum 1.0 PO3; PO4 or PO5 SymVital ® AR Zingiber officinale (ginger) root 0.2 extract Extrapone ® Seaweed Aqua, Butylene glycol, Fucus 1.0 vesiculosus extract Actipone ® Dandelion Glycerin 1.0 Juice (Organic) GW Taraxacum officinale juice Aqua Colour I Colour 0.35 Colour II Colour 0.20 Caprylhydroxamic Acid Caprylhydroxamic Acid 0.2 Phenylpropanol Phenylpropanol 0.2

TABLE-US-00016 TABLE 6 Aloe Shower Cream Ingredients EU INCI Amount Water demin. Water Add 100 Glycerin 85% Glycerin, Water 3.0 Bacterial ferment Lactobacillus Ferment, Ammonium 5.0 according to the Lactate, Lactic Acid, Pentylene Glycol invention Dermosoft ® 1388 Glycerin, Water, Sodium Levulinate, 3.5 ECO Sodium Anisate Genuvisco ® CG-131 Carrageenan 0.8 Keltrol ® CG-SFT Xanthan Gum 0.3 Amisoft ® CS-22 Sodium Cocoyl Glutamate, Disodium 20.0 Cocoyl Glutamate, Water Plantacare ® 2000 UP Decyl Glucoside 7.0 Aloe Vera Gel Aloe Barbadensis Leaf Juice 1.5 Concentrate SymOleo ® Vita 7 Glycine Soja (Soybean) Oil, 5.0 Gossypium Herbaceum (Cotton) Seed Oil, Mangifera Indica (Mango) Seed Butter, Olea Europaea (Olive) Fruit Oil, Persea Gratissima (Avocado) Oil, Prunus Amygdalus Dulcis (Sweet Almond) Oil, Theobroma Cacao (Cocoa) Seed Butter Citric acid solution Aqua, citric acid 6.7 10% Perfume oil PO1, PO2, Parfum 0.4 PO3, PO4, or PO5 Cosmopheres ® Mannitol, Microcrystalline Cellulose, 2.0 GYAM-M Aloe Barbadensis Leaf Juice Powder, Chromium Oxides greens, Iron Oxides

TABLE-US-00017 TABLE 7 Fresh Hair shampoo Ingredients EU INCI Amount Aqua Aqua Add 100 EDTA B Powder Disodium EDTA 0.1 Nativacare ™ 5600 Zea Mays (Corn Starch) 2.0 Rheocare ® XGN Xanthan Gum 0.5 Texapon ® NSO UP Sodium Laureth sulfate 35.0 Dehyton ® K Cocamidopropyl betaine 8.0 Plantacare ® 2000 UP Decyl Glucoside 4.0 Perfume oil PO1, PO2, Parfum 1.0 PO3, PO4, or PO5 SymSave ® H Hydroxyacetophenone 0.5 Hydrolite ® 6 1,2-Hexanediol 1.0 SymClariol ® Decylene Glycol 0.3 Frescolat ® ML cryst Menthyl Lactate 1.0 SymCalmin ® Pentylene Glycol, Butylene Glycol, 0.2 Hydroxyphenyl propamidobenzoic acid Crinipan ® AD Climbazole 0.2 Propylene Glycol Propylene Glycol 2.0 Bacterial ferment Lactobacillus Ferment, Ammonium 5.0 according to the Lactate, Lactic Acid, Pentylene invention Glycol SymHair ® Shield Pentylene Glycol, Aqua, Glycerin, 1.0 Triticum vulgare bran extract, 1,2- Hexanediol, Capryly glycol Sodium Hydroxide Aqua, Sodium hydroxide 2.2 10% solution

TABLE-US-00018 TABLE 8 Hair conditioner with UV protection Ingredients INCI Amount Renex ™ PEG 6000 PEG-150 2.5 Hair Conditioner Base Cetyl alcohol, behentrimonium 3.0 chloride, Triticum Vulgare (Wheat) bran extract, linoleic acid PCL-Solid Stearyl heptanoate, stearyl caprylate 0.5 Dowsil ™ 5200 Laurylmethicone copolyol 0.5 Natrosol ™ 250 HR Hydroxyethylcellulose 0.5 Benzophenone-4 Benzophenone-4 1.0 Neo Heliopan ® AP Disodiumphenyldibenz-imidazole 1.0 tetrasulphonate Amino methyl propanol Amino methyl propanol 2.0 Xiameter ® 949 Amodimethicone, cetrimonium 2.0 MEM-0949 Emulsion chloride, trideceth-12 Perfume oil PO1, PO2, Parfum 0.8 PO3, PO4, or PO5 Hydrolite ® 6 1,2-Hexanediol 0.5 Bacterial ferment Lactobacillus Ferment, Ammonium 5.0 according to the Lactate, Lactic Acid, Pentylene invention Glycol Water Water (Aqua) Add 100

TABLE-US-00019 TABLE 9 Low Cost-Refreshing Body Lotion Ingredients EU INCI Amount Aqua/Water Aqua Add 100 Bacterial ferment Lactobacillus Ferment, Ammonium 10.0 according to the Lactate, Lactic Acid, Pentylene Glycol invention Extrapone ® Aqua, Propylene Glycol, Butylene Glycol, 1.0 Camomile Chamomilla recutita, (Matricaria) Flower extract, Bisabolol Amaze ® XT Dehydroxanthan Gum 0.7 Avicel ® PC591 Cellulose, Cellulose Gum 0.3 Caprylic/Capric Caprylic/Capric Triglyceride 5.0 Triglyceride Dragoxat ® 89 Ethylhexyl Isononanoate 3.0 Dracorin ® GOC Glyceryl Oleate Citrate, CaprylicCapric 0.3 Triglyceride Ethanol 96% Alcohol denat. 5.0 Frescolat ® ML Menthyl Lactate 0.3 Perfume oil PO1, Parfum 0.2 PO2, PO3, PO4, or PO5 Sodium Hydroxide Aqua, Sodium hydroxide 0.1 10% solution SymOcide PS Phenoxyethanol, Decylene Glycol, 1,2 1.0 Hexanediol

TABLE-US-00020 TABLE 10 Anti-aging Face Cream, soft touch Ingredients EU INCI Amount Aqua/Water Aqua Add 100 Glycerin 99.5% Glycerin 2.0 Dragosine ® Carnosine 0.1 Kelcogel ® CG-HA Gellan Gum 0.1 Keltrol ® CG-SFT Xanthan Gum 0.4 Dracorin ® CE Glyceryl Stearate Citrate 2.0 Lanette ® O Cetearyl Alcohol 5.0 SymMollient ® Propanediol Dicaprylate/ 3.0 PDCC Caprate SymMollient ® S Cetearyl Nonanoate 2.0 green Plantoil Triglyceride Caprylic/Capric Triglyceride 8.0 Dragoxat ® 89 Ethylhexyl Isononanoate 2.0 SymFinity ® 1298 Echinacea Purpurea Extract 0.1 LIFTONIN XPERT Water, Mannan, Pentylene 2.0 Eco Glycol, Citric Acid Bacterial ferment Lactobacillus Ferment, 20.0 according to the Ammonium Lactate, Lactic invention Acid, Pentylene Glycol Perfume oil PO1, Parfum 0.3 PO2, PO3, PO4, or PO5

TABLE-US-00021 TABLE 11 Erythema Cotton Cream Raw material/Ingredients INCI Amount Dehymuls ® PGPH Polyglyceryl-2 5.25 Dipolyhydroxystearate Tegosoft ® MM Myristyl Myristate 1.00 Cacao Butter Theobroma Cacao Seed Butter 0.50 Caprylic/Capric Caprylic/Capric Triglyceride 5.00 Triglyceride Alpha Tocopherol DL Tocopherol 0.10 Jojoba Oil Simmondsia Chinensis Seed Oil 10.00 Bentone Gel ® GTCCV Caprylic/Capric Triglyceride, 2.50 Stearalkonium Hectorite, Propylene Carbonate Sweet Almond Oil Prunus Amygdalu Dulcis (Sweet 10.00 (Organic) Almond) Oil Cutina ® HR Hydrogeated Castor Oil 2.50 Zinc Oxide Zinc Oxide 1.20 Cetiol ® SB 45 Butyrospermum Parkii Butter 0.50 Sym Decanox ™ HA Caprylic/Capric Triglyceride, 1.00 Hydroxymethoxyphenyl Decanone Aqua/Water Aqua Add 100 Magnesium Sulfate Magnesium Sulphate Hepta 0.50 Heptahydrate Hydrate Glycerin Glycerin 5.00 SymOcide ® PS Phenoxyethanol, Decylene Glycol, 1.00 1,2 Hexanediol Hydrolite ® 5 green Pentylene Glycol 1.00 Bacterial ferment Lactobacillus Ferment, Ammonium 5.00 according to the Lactate, Lactic Acid, Pentylene invention Glycol SymGlucan ® Aqua, Glycerin, 1,2-Hexanediol, 5.00 Caprylyl Glycol, Beta-Glucan Extrapone ® Cotton Glycerin, Aqua, PEG-40 1.00 Milk P Hydrogenated Castor Oil, Trideceth-9, Nonfat Dry Milk (Sine Adipe Lac), Gossypium Herbaceum Seed oil, 1,2 Hexanediol, Caprylyl Glycol DragoCalm ® Aqua, Glycerin, Avena Sativa 1.00 Kernel Extract Perfume oil PO1; PO2; Parfum 0.05 PO3; PO4; or PO5

TABLE-US-00022 TABLE 12 Natural Night Cream Raw material/Ingredients INCI Amount Deionised Water Aqua Add 100 Glycerin 99.5% Glycerin 2.0 Dermofeel PA-3 Sodium Phytate; Aqua; Alcohol 0.1 Dermosoft 1388 eco Glycerin, Aqua, Sodium 3.0 Levulinate, Sodium Anisate Veegum Ultra Granules Magnesium Aluminium Silicate 0.5 Keltrol CG-RD Xanthan Gum 0.5 Dermofeel SL Sodium Stearoyl Lactylate 2.0 Dermofeel PS Polyglyceryl-3 Stearate 3.0 Dermosoft decalact MB Sodium Caproyl/Lauroyl Lactylate 0.8 Phytosqualan, veg. grade Squalane 4.0 Cegosoft SBE Butyrosperum Parkii Butter; 2.0 Tocopherol Cutina GMS Glyceryl Stearate 2.0 Lanette O Cetearyl Alcohol 1.0 Sunflower oil Helianthus Annuus Seed Oil 4.0 Dermofeel sensolv Isoamyl Laurate 5.0 Bacterial ferment Lactobacillus Ferment, Ammonium 20.0 according to the Lactate, Lactic Acid, Pentylene invention Glycol Perfume oil PO1; PO2; Parfum 0.3 PO3; PO4; or PO5

TABLE-US-00023 TABLE 13 Anti-Wrinkle Day Emulsion exp. SPF 15 Raw material/Ingredients INCI Amount Emulsiphos ® Potassium cetyl phosphate, 2.5 Hydrogenated palm glycerides Neo Heliopan ® 303 Octocrylene 2.0 Neo Heliopan ® OS Ethylhexyl salicylate 5.0 Isoadipate Diisopropyl adipate 2.0 SymMollient ® S green Cetearyl nonanoate 2.0 Caprylic/Capric Caprylic/capric triglyceride 3.0 Triglyceride Dragoxat ® 89 Ethylhexyl isononanoate 1.0 PCL-Liquid ® 100 Cetearyl ethyl hexanoate 3.0 SymUrban ® Benzylidene 0.3 dimethoxydimethylindanone SymDecanox ™ HA Caprylic/capric triglyceride 2.0 Hydroxymethoxyphenyl decanone Edeta ® BD Disodium edta 0.1 Keltrol ® CG-SFT Xanthan gum 0.2 Cosmedia ® SP Sodium polyacrylate 0.4 Aqua/Water Aqua Add 100 Glycerin Glycerin 3.0 Neo Heliopan ® Hydro Phenylbenzimidazole sulfonic 2.0 acid Biotive ® L-Arginine Arginine 1.0 Sodium Hydroxide 10% Aqua, Sodium hydroxide 1.2 solution Dragosine ® Carnosine 0.2 SymSave ® H Hydroxyacetophenone 0.5 SymDiol ® 68 ( 1,2-hexanediol, Caprylyl glycol 0.3 Phenoxyethanol Phenoxyethanol 0.2 Bacterial ferment Lactobacillus Ferment, 10.0 according to the Ammonium Lactate, Lactic Acid, invention Pentylene Glycol SymLift ® Aqua, Trehalose, Glycerin, 5.0 Pentylene glycol, Beta-glucan, Hordeum vulgare seed extract, Sodium hyaluronate, 1,2- hexanediol, Caprylyl glycol Macadamia Oil (refined) Macadamia integrifolia seed oil 0.2 Ethanol 96% Alcohol denat. 3.0 Sensocel ® 10 Cellulose 1.0 Sipernat ® 11 PC Hydrated silica 1.0 Perfume oil PO1; PO2; Parfum 0.4 PO3; PO4; or PO5

TABLE-US-00024 TABLE 14 Perfect Hand with exp. SPF 15, UVA/UVB Balanced Raw material/Ingredients INCI Amount Emulsiphos ® Potassium cetyl phosphate 1.5 Hydrogenated palm glycerides Lanette ® O Cetearyl alcohol 1.0 Tegosoft ® MM Myristyl myristate 0.5 Neo Heliopan ® OS Ethylhexyl salicylate 5.0 Edeta ® BD Disodium EDTA 0.1 Dragoxat ® 89 Ethylhexyl isononanoate 2.0 Isoadipate Diisopropyl adipate 6.0 PCL-Liquid ® 100 Cetearyl ethylhexanoate 3.0 Corapan ® TQ Diethylhexyl 2,6-naphthalate 2.0 Cetiol ® SB 45 Butyrospermum parkii butter 1.5 SymBright ™ 2036 Sclareolide 0.2 SymDecanox ™ HA Caprylic/capric triglyceride, 1.0 Hydroxymethoxyphenyl decanone SymCare ® O Hexyldecanol, Pentylene glycol, 2.0 4-t-butylcyclohexanol, Bisabolol, Cetylhydroxyproline palmitamide, Hydroxyphenyl propamidobenzoic acid, Stearic acid, Brassica campestris (rapeseed) sterols, Zingiber officinale (ginger) root extract Genuvisco ® Chondrus crispus powder 0.4 Carrageenan CG-131 Keltrol ® CG-SFT Xanthan gum 0.2 Aqua/Water Aqua Add 100 Neo Heliopan ® Hydro Phenylbenzimidazole sulfonic 2.5 acid Biotive ® L-Arginine Arginine 1.0 Sodium Hydroxide 10% Aqua 1.3 solution Sodium hydroxide Glycerin Glycerin 2.0 SymSave ® H Hydroxyacetophenone 0.5 SymDiol ® 68 1,2-hexanediol, Caprylyl glycol 0.5 RonaFlair ® Flawless Silica Ci 77892, Ci 77491 1.5 Tego ® Feel C10 Cellulose 1.5 Bacterial ferment Lactobacillus Ferment, 10.0 according to the Ammonium Lactate, Lactic invention Acid, Pentylene Glycol Perfume oil PO1; PO2; Parfum 0.5 PO3; PO4; or PO5