<i>Lactobacillus plantarum </i>for skin care
12128129 · 2024-10-29
Assignee
Inventors
- Manuel PESARO (Holzminden, DE)
- Dominik Stuhlmann (Holzminden, DE)
- Lisa GARBE (Holzminden, DE)
- Gerhard Schmaus (Höxter-Bosseborn, DE)
- Kerstin HOLMGREN (Helsingborg, SE)
- Niklas Larsson (Lund, SE)
Cpc classification
A61P29/00
HUMAN NECESSITIES
A61K8/99
HUMAN NECESSITIES
A61K9/14
HUMAN NECESSITIES
International classification
A61K8/99
HUMAN NECESSITIES
A61K9/14
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
Abstract
The present invention relates to microorganisms for use in the treatment and/or prevention of skin conditions by topical application. In particular, the microorganisms are for use in the treatment and/or prevention of loss of skin barrier function, inflammatory conditions and/or growth of pathogenic microorganism. The invention further relates to pharmaceutical or cosmetic compositions or products comprising the microorganisms Provided is also a cosmetic use of the microorganisms or compositions for application on the skin and a cosmetic method, in particular to improve the appearance of the skin.
Claims
1. A method for improving the appearance of non-mucosal skin comprising topically applying to the non-mucosal skin a topical composition comprising: (a) attenuated or dead Lactobacillus plantarum HEAL 19 (DSM 15313) having an intact physical structure, wherein the Lactobacillus plantarum HEAL 19 (DSM 15313) have been heat-treated at a temperature of 60 to 121 C. for 1 second to 120 minutes; (b) one or more carriers, excipients, further active ingredients, or a combination thereof; (c) one or more preservatives; and (d) water; wherein the method improves the appearance of the non-mucosal skin by reducing or preventing skin irritation, dry skin, rash, acne, or skin aging.
2. The method of claim 1, wherein the method treats or improves a skin condition.
3. The method of claim 2, wherein the skin condition is loss of skin barrier function, an inflammatory skin condition, growth of a pathogenic microorganism, or a combination thereof.
4. The method of claim 2, wherein the skin condition is atopic dermatitis, a microbial infection, dry skin, itchy skin, sensitive skin, inflammation of the skin, rosacea, psoriasis, rash, acne, or a combination thereof.
5. The method of claim 1, wherein the Lactobacillus plantarum HEAL 19 (DSM 15313), is applied to the skin in a pharmaceutical or cosmetic composition, the pharmaceutical or cosmetic composition comprising: (a) 0.01 to 5% dry weight, based on a total weight of the pharmaceutical or cosmetic composition, of the Lactobacillus plantarum HEAL 19 (DSM 15313) in an amount sufficient for improving the appearance of the skin by reducing or preventing skin irritation, dry skin, rash, acne, or skin aging; and (b) one or more carriers, excipients, further active ingredients, or a combination thereof, wherein the pharmaceutical or cosmetic composition comprises at least 10{circumflex over ()}8 cells of the Lactobacillus plantarum HEAL 19 (DSM 15313) per gram of the pharmaceutical or cosmetic composition.
6. The method of claim 5, wherein the Lactobacillus plantarum HEAL 19 (DSM 15313) have been subjected to freeze-drying, spray-drying, or granulating.
7. The method of claim 6, wherein the Lactobacillus plantarum HEAL 19 (DSM 15313) have been freeze-dried, spray-dried, or granulated with a carrier selected from inulin, starch, gummi arabicum, whey protein, skim milk powder, maltodextrin, or a combination thereof.
8. The method of claim 5, wherein the pharmaceutical or cosmetic composition is in the form of an oil-in-water or water-in-oil emulsion, an ointment, a crme, a lotion, or a gel.
9. The method of claim 8, wherein the method treats dry skin and comprises applying the pharmaceutical or cosmetic composition to dry skin.
10. The method of claim 8, wherein the method treats atopic dermatitis and comprises applying the pharmaceutical or cosmetic composition to skin suffering from atopic dermatitis.
11. The method of claim 8, wherein the method treats a microbial infection and comprises applying the pharmaceutical or cosmetic composition to skin suffering from a microbial infection.
12. The method of claim 8, wherein the method treats itchy or sensitive skin and comprises applying the pharmaceutical or cosmetic composition to the itchy or sensitive skin.
13. The method of claim 8, wherein the method treats inflammation of the skin and comprises applying the pharmaceutical or cosmetic composition to inflamed skin.
14. The method of claim 8, wherein the method treats rosacea, psoriasis, rash, a combination thereof and comprises applying the pharmaceutical or cosmetic composition to skin suffering from rosacea, psoriasis, rash, or a combination thereof.
15. A method for treating non-mucosal skin comprising topically applying a pharmaceutical or cosmetic composition to non-mucosal skin suffering a skin condition selected from atopic dermatitis, a microbial infection, dryness, itchiness, inflammation, rosacea, psoriasis, rash, acne, or a combination thereof, the pharmaceutical or cosmetic composition comprising: (a) 0.01 to 5% dry weight, based on a total weight of the pharmaceutical or cosmetic composition, of non-viable Lactobacillus plantarum HEAL 19 (DSM 15313) having an intact physical structure; and one or more further carriers, excipients, further active ingredients, or a (b) combination thereof; wherein the pharmaceutical or cosmetic composition comprises at least 10{circumflex over ()}8 cells of the non-viable Lactobacillus plantarum HEAL 19 (DSM 15313) per gram of the pharmaceutical or cosmetic composition, and is in the form of an oil-in-water or water-in-oil emulsion, an ointment, a crme, a lotion, or a gel.
16. The method of claim 1, wherein the topical composition further comprises Lactobacillus plantarum HEAL 99 (DSM 15316).
17. The method of claim 16, wherein the Lactobacillus plantarum HEAL 19 (DSM 15313) and Lactobacillus plantarum HEAL 99 (DSM 15316) have been subjected to freeze-drying, spray-drying, or granulating with a carrier selected from inulin, starch, gummi arabicum, whey protein, skim milk powder, maltodextrin, or a combination thereof, and wherein the Lactobacillus plantarum HEAL 19 (DSM 15313) and Lactobacillus plantarum HEAL 99 (DSM 15316), and the carrier, are in a weight ratio of 1:9 to 3.7.
Description
SHORT DESCRIPTION OF THE FIGURES
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(18) The invention is further illustrated by the examples below, which are not to be understood as limiting for the scope.
(19) The following experimental procedures were used in the examples:
(20) Preparation of Heat-Treated Lactobacilli Suspensions
(21) The growth of the L. plantarum strains HEAL 19 and HEAL 99 can be obtained in various culture media. The carbon source can be glucose or starch. Meat extract, yeast extract, peptone or protease peptone (vegetable) etc. can be used as nitrogen source. The pH of the culture medium should be about 6. The culture temperature is variable, but preferably 37 C. Culture duration may be about 8 to 24 h. Shaking or aerated shaking can be added.
(22) After cultivation, a washing process can be added. It can be done as follows. Cells can first be collected by centrifugation, supernatant decanted and cell pellets suspended with liquid PBS, cell culture medium or purified water. The described procedure can be repeated if needed.
(23) Heat-treatment of the Lactobacilli suspension can be done before or after a washing procedure. It can be achieved by ultrasound, UV-irradiation or heat, whereas heat-treatment is preferable. It can be performed with temperatures from 60 C. to 121 C. For temperatures above 100 C., an autoclave may be used. Heat-treatment durations can range from 1 s to 120 min, such as from 20 s to 120 min. Heat-treatment between 70 and 100 C. for 20 s to 15 min using an industrial pasteurizer is preferable. Optional further processing for the heat-treated Lactobacilli can include freeze-drying, spray-drying, granulating etc. This can serve the purpose of improving applicability and/or stability of the product itself.
(24) The final product can be a powder, granulate, suspension or solution and is defined to be applied in cosmetic compositions in a concentration of 0.001 to 10% w/v. This relates to a concentration of 110.sup.7-110.sup.11 cells/mL cosmetic formulation. More preferably, the final product is applied in a concentration of 0.01 to 5% w/v, and even more preferably, in 0.02 to 1% w/v, such as from, 0.1 to 1% w/v.
(25) Effects of Lactobacillus Strains on AMP Gene Expression in Keratinocytes Grown as Mono-Layer
(26) AMP inductions were measured with real time PCR analysis using Taq-Man Array fast 96-well plates.
(27) HaCaT keratinocytes were cultivated in EpiLife medium, cascade Biologics, Gibco, incl. HKGS, Gibco, life technologies. Test substances were diluted in equal medium and added in a volume of 2 mL per well on 6-well plates. All samples are tested on 100% confluent cells incubated at 37 C., 5% CO2. After 24 h incubation time all samples were removed and cells washed twice with Dulbecco's Phosphate Buffered Saline with Ca and Mg, Capricorn. RNA isolation was performed using RNeasy Mini Kit, Qiagen. The procedure has been done as describes in the RNeasy Mini Handbook.
(28) Cells were lysed and precipitated with ethanol, the homogenate was washed three times using the spin column and the mRNA was eluated with purified water. Depending on the sample the procedure was slightly adjusted.
(29) RNA concentration was measured using CuvetteG 1.0 and BioPhotometer, Eppendorf by measuring the absorption at 260 nm. Control values, like E260/280 or E260/230 were calculated simultaneously. Transcription was done with a minimum of 0.5-1 g RNA per sample.
(30) Reverse transcription was done using high capacity RNA-to-cDNA Kit, Applied Biosystems. The procedure was based on the provided protocol. All samples were incubated in a PCR Thermocycler, Biometra running 60 min at 37 C. for reverse transcription, followed by 5 min at 95 C. (enzyme inactivation) and holding temperature at 4 C. in reaction room.
(31) Quantitative Real-Time PCR was done using StepOne Plus Fast Real Time PCR Instrument, Applied Biosystems. Initial steps of the RT-PCR included a first heating phase holding for 20 s at 95 C., followed by 40 cycles of cDNA denaturation for 3 s at 95 C. and annealing/elongation at 60 C. for 30 s.
(32) Analysis was done by 2-ACT Methodin detail: 1. Standardization of Ct values with reference-gen (HPRT)
C.sub.Tvalue.sub.gene=C.sub.Tvalue.sub.geneC.sub.Tvalue.sub.Referencegene (HPRT) 2. Subtraction of ACT-value of control sample (untreated) and ACT-value (treated)
C.sub.Tvalue.sub.gene=C.sub.Tvalue.sub.treatedC.sub.Tvalue.sub.control 3. Calculation of RQ-Ratio (relative quantification-value)
RQvalue=2.sup.CT
(33) A RQ-Value of >2 was defined as relevant induction of a gene.
(34) Anti-Inflammatory Effects of Lactobacillus plantarum HEAL 19 and HEAL 99
(35) HaCaT keratinocytes were cultivated in 96-well plates using RPMI-A, medium sterile filtered with L-Glutamine, Capricorn, supplemented with 10% fetal calf serum (FCS), Capricorn. The plates were incubated at 37 C. and 5% CO2. After 24 hours starvation with cell culture medium (RPMI, 1% FCS) the cells were washed once. The anti-inflammatory substances/inactivated bacteria suspensions were suspended in starvation medium, applied and incubated for 24/48 h with parameters described above. Dexamethasone in a concentration of 10 M was used as positive control. To remove bacterial cells after pre-incubation all wells were washed with cell culture medium three times. IL-1alpha, Gibco, life technologies was dissolved in H.sub.2Odest, diluted in starvation medium and pipetted to wells, preferably at a concentration of 30 ng/mL. Alternatively, a heat inactivated Staphylococcus aureus (DSM799) suspension was used as pro-inflammatory agent in a concentration of 0.1 mg/mL-1 mg/mL dry mass. After 8 h incubation the supernatants were separated. All samples were stored at 80 C. until further analyses.
(36) IL-8 concentrations were measured with Human CXCL8/IL-8, DuoSet ELISA, Development Systems, R&D Systems. The experimental procedure was performed as described in the provided protocol.
(37) After calculating means of the duplicate/triplicate values and subtraction of the average zero standard optical density the calculation was done using four parameter logistic curve-fit for creating standard curves.
(38) Induction of Hyaluronic Acid
(39) Human epidermal keratinocyte progenitors (HEPKp) generated 3d human skin models were cultivated and systemically treated with test substances. Briefly, defined concentrations of test substances were added to cell culture medium and each skin model was treated in cell culture dishes for up to 9 days at 37 C. and 5% CO.sub.2. After the treatment, collected supernatants were stored at 80 C. until further analyses. Viability of the cells was controlled by MTT standard method.
(40) Hyaluronic acid concentrations were measured by TECOmedical hyaluronic acid plus ELISA. The experimental procedure was performed as described in the provided protocol.
(41) Antimicrobial Effects of the Lactobacillus Strains
(42) Growth curves of various bacteria were measured photometrically at 620 nm.
(43) Inhibition of Staphylococcus aureus:
(44) 96-well MTP were prepared according to DIN58940-8 with autoclaved CASO medium (MERCK 1.05459) and test substances dissolved in DMSO or distilled water. The relevant wells were inoculated with S. aureus (DSM799/ATCC6538) using stock cultures maintained in 10% glycerin at 20 C. An inoculum of 1-610.sup.6 cfu was added per well. Test substances were analyzed using three or four replicate wells during incubation at 37 C. for 16 hours. Growth curves were determined in each well via absorption at 620 nm using a Sunrise Photometer (Tecan, Austria) and Magellan Software. A concentration of 125 ppm farnesol and 4 ppm triclosan was used as positive control for growth inhibition, both pre-dissolved in DMSO (Merck 802912).
(45) For calculation, averages of detected absorptions were calculated and normalized with blank values. These OD values were plotted in function of time for graphical presentation.
(46) Induction of Moisturizing Factor Filaggrin in 3D Skin Models
(47) Three dimensional skin models derived from human epidermal keratinocyte progenitors (HEPKp) and were systemically treated with test substances in CnT-PR-3D medium for 4 to 9 days. The filaggrin release was detected in the media by ELISA, Enzyme-linked Immunosorbent Assay Kit for filaggrin, Cloud-Clone Corp.
(48) Barrier Improving Efficacy on SDS Damaged 3D Skin Models for Dry Skin
(49) For this purpose an in vitro study was performed on a 3D epidermis model system for dry skin. The model system consisted of 14 day-old, mature epidermis cell cultures which had a disturbed epidermal skin barrier due to SDS treatment. Disturbance of the epidermal barrier was measured as loss of intercellular lipid lamellae. The skin repair performance was determined by transmission electron microscopy (TEM) after treatment with L. plantarum HEAL 19 formulated in a cosmetic emulsion or placebo emulsion and then further cultivated to observe reformation of intercellular lipid lamellae.
(50) Ex Vivo Studies
(51) Ex vivo trials were done by Cutech Srl, Italy. Effects of described Lactobacillus strains on various parameters were analyzed in ex vivo skin models. Human skin from abdominal plastic surgery was used. Applications of heat inactivated bacteria suspensions were done systemically or topically, in formulation or solved in cell culture medium Tape stripping may be used. Concentrations and incubation times may vary.
(52) All data were processed in terms of mean, standard deviation and standard error of mean (SEM) for each treatment.
(53) Epidermal Filaggrin, Involucrin and Cytokeratin 14
(54) For analyses, the models were immune-stained with the selected antibody filaggrin rabbit polyclonal [H-300], involucrin mouse monoclonal (SY5) and/or cytokeratin 14 (CK14) rabbit monoclonal (EPR17350). The amount of the antigen present in each slide was evaluated by estimating the intensity and the distribution of red dye within the epidermis. The obtained data were normalized for the length of the basal lamina.
(55) Skin Barrier Integrity (Rhodamine B)
(56) Ex vivo skin models have been treated topically with the L. plantarum strains before the application of the diesel particulate (i.e. 1650b). All skin samples have been incubated at 35 C., 5% CO2 and environmental humidity. At the end of the experimental phase the skin samples have been harvested stained with Rhodaminde B, cryo-fixed and cut at the cryostat for consequent image acquisition and analysis. The analysis of Rhodamine B fluorescence has been performed within two sections of the epidermis area for each skin model. Images have been analyzed by evaluating the fluorescence through Image-J application (NIH, USA).
(57) Anti-Inflammatory Effects
(58) Above described ex vivo human skin models have been used to verify the anti-inflammatory potential of the L. plantarum strains. The models were treatment with a L. plantarum HEAL 19 cosmetic emulsion followed by the application of the external noxious agent and pollutant 1650b, simulating air pollution. At a selected endpoint, the organ cell culture medium has been withdrawn from wells and analyzed for IL-8 using Deluxe set Human IL-8 of Biolegend, Inc.
(59) In Vivo Studies
(60) In vivo trials were done at Kosmoscience Cincia & Tecnologia Cosmtica Ltda, Brazil. They assessed relevant skin parameters after application of a cosmetic o/w formulation containing L. plantarum HEAL 19 lyophilisate.
(61) The o/w emulsion had the following composition:
(62) TABLE-US-00001 Placebo Active Phase Ingredients INCI % % A Dracorin GOC Glyceryl Oleate Citrate, 2 2 Caprylic/Capric/ Triglyceride LanetteO Cetearyl Alcohol 3 3 PCL liquid 100 Cetearyl Ethylhexanoate 2.5 2.5 Dragoxat 89 Ethylhexyl Isononanoate 2.2 2.2 Xiameter 200 Fluid Dimethicone 0.3 0.3 350 CS B Carbopol EDT 2020 Aerylates/C10-30 alkyl 0.2 0.2 acrylate Crosspolymer Keltrol CG Xanthan Gum 0.2 0.2 C Water ad 100 ad 100 SymSave H Hydroxyacetophenone 0.5 0.5 2-Phenoxyethanol Phenoxyethanol 0.5 0.5 EDTA disodium edta 0.1 0.1 D NaOH 10% 0.2 0.2 E L. plantarum 1.0 HEAL19 Total = 100.00 100.00 pH 5.3 5.3
(63) Twenty two subjects with extra dry skin participated in the study. Topical application of the emulsion was done twice-daily on the inner forearm. Measurements took place at day 0, 7, 14 and 21.
(64) Skin Hydration by Corneometry
(65) The measurement of capacitance was performed using a Corneometer 825 probe coupled to a Multi Probe Adapter, MPA 5 (CKeletronics, Germany). Variation of the Capacitance and the skin hydration provided by the L. plantarum HEAL 19 formulation in relation to the placebo was calculated.
(66) Effectiveness of the Skin Barrier by Evaporimetry
(67) Transepidermal water loss (TEWL) was measured using a Tewameter 300 probe coupled to a Multi Probe Adapter, MPA 5 (CKeletronics, Germany). Variation of the TEWL values and the cutaneous barrier fortification provided by the L. plantarum HEAL 19 formulation in relation to the placebo was calculated. Fortification of the skin barrier can be observed as decrease in the TEWL value.
(68) The following results demonstrate the efficiency of the present invention. Single experiments should illustrate but not limit effects of the invention.
Example 1: AMP Gene Expression in HaCa Keratinocytes
(69) L. plantarum HEAL 19 induces the release of a wide range of antimicrobial peptides in human keratinocytes. All these peptides together represent a complex defense system of the human skin. Defensins for instance, that are able to kill Gram positive bacteria, were upregulated. In sum, the upregulated peptides are effective against a broad spectrum of potentially pathogenic Gram positive and Gram negative bacteria, fungi, eukaryotic parasites and/or viruses.
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(71) Results demonstrate that important AMPs like -Defensin 1, 3 or 4 and/or Psoriasin and/or Calprotectin are induced by the treatment with L. plantarum HEAL 19. This treatment results in higher expression of AMPs in human skin, which relates to a stronger first defense line in human skin and thereby supports the skin barrier. Furthermore, the induced expression of -Defensin or Cathelicidin can balance the AMP fingerprint in diseases such as in atopic dermatitis. Thus, the skin microbiome can be restored, maintained and/or improved by treatment of L. plantarum HEAL 19.
(72) The inductions of AMPs by L. plantarum HEAL 19 are concentration dependent. In
(73) Similar effects can be observed using L. plantarum HEAL 99 when HaCaT keratinocytes are treated.
(74) In epidermal 3D human skin models, it has been shown that various AMP coding genes are similarly induced (data not shown).
Example 2: Anti-Inflammatory Effects of Lactobacillus Strains (In Vitro/Ex Vivo)
(75) Skin diseases, lesions or irritations mostly correlate with an inflammation of affected skin areas. It was shown that besides the described beneficial effects of the Lactobacillus strains, their topical application results in an anti-inflammatory effect. In this connection, the Lactobacillus strains support skin health by a further mechanism.
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(77) It has been shown that similar anti-inflammatory effect of L. plantarum HEAL 19 can be seen in ex vivo human skin models. As shown in
(78) Inflammation in skin can not only be induced by pro-inflammatory cytokines but also by bacteria, specifically bacteria pathogenic to the skin, by cell to cell contact.
(79) S. aureus is frequently found in eczemous skin lesions of AD patients and is often responsible for aggravating the disease status and/or encouraging inflammation. The treatment with L. plantarum strains on skin results in lower inflammation, it concomitantly reduces redness and the skin is soothed and calmed.
Example 3: Antimicrobial Effect of Lactobacillus plantarum HEAL 19 and HEAL 99
(80) It is known that S. aureus is consistently found in eczemous skin lesions of AD patients for instance and seems to be important and responsible for aggravating the disease status. Potential infections of the skin can be prevented by inhibiting growth of pathogens, like S. aureus by application of the L. plantarum strains on skin.
(81) Growth curves of S. aureus were created by measuring optical density during 16 h incubation.
Example 4: Barrier Strengthening
(82) A: Induction of Involucrin and Cytokeratin 14 (CDK14) in Ex Vivo Studies
(83) Involucrin is a protein providing structural support to the skin cells and thereby allows the cell to resist invasion by micro-organisms. Cytokeratin 14 is usually found as a heterodimer and forms the cytoskeleton of epithelial cells. Both marker proteins increased by topical treatment using various concentrations of L. plantarum HEAL 19 in ex vivo regenerated human skin models, as shown in
(84) It is hereby demonstrated, that topical treatment of skin with the described invention directly leads to a strengthening of skins barrier function.
(85) B: Skin Barrier Integrity
(86) The invention shows a protective activity on the skin barrier by the reduction of rhodamine B penetration in ex vivo models after treatment with L. plantarum HEAL 19 and stimulation with diesel particulate (1650b). The evaluation of skin morphology allows determining whether a compound affects the structure of the treated skin samples. To perform this evaluation, the skin sections have been stained with rhodamine B staining. Following the staining for each skin sample the integrity of the epidermis has been evaluated.
(87) C: Barrier Improving Efficacy
(88) The intercellular lipid lamellae in the intercellular space of the stratum corneum, used as a quality measure of the epidermal barrier, were analyzed and quantitatively evaluated. The topical treatment with 0.5% L. plantarum HEAL 19 in a skin care formulation leads to an increased repair performance of the 3D epidermal models system for dry skin (
Example 5: Hyaluronic Acid Induction in 3D Skin Models
(89) Hyaluronic acid (HYA) plays a series of important roles in skin. It is necessary in skin to maintain epidermal barrier function and the structure of the stratum corneum. Furthermore, it plays an important role in immobilizing water in tissues, in tissue repair or in influencing cell differentiation and proliferation (Weindl et al 2004). All this can be realized with the treatment of the described L. plantarum strains.
(90)
Example 6: Hydration of Skin
(91) A: Filaggrin Release from 3D Skin Models
(92) Increased release of the protein filaggrin into the medium could be detected after 3 to 9 days incubation.
(93) Topical application of the described invention leads to filaggrin upregulation in human skin and subsequently to a strengthening of the skin barrier and positive effects on skin moisturization.
(94) B: Filaggrin Induction Ex Vivo
(95) The induction of filaggrin due to the topical treatment with the invention was confirmed in studies using ex vivo human skin models.
(96) Similar concentration dependent effects were observed in in vitro experiments investigating HEPKp 3D human skin models (data not shown).
Example 7: In Vivo Data
(97) To confirm effects observed in model systems, an in vivo study was performed with a panel of subjects suffering from extra dry skin. Heat-treated L. plantarum HEAL 19 was incorporated into a cosmetic cream and applied on inner forearms of panelists. Effects on skin moisture retention capacity (capacitance) and transepidermal water loss (TEWL) were monitored after 0, 7, 14, and 21 days in comparison to untreated and placebo-treated areas.
(98) The in vivo study confirmed effects suggested by molecular biological investigations: capacitance steadily increased over treatment time and effects were significantly stronger than in placebo treatments at all time points. TEWL decreased significantly in the treatments Lactobacilli-containing cream, whereas the placebo had no effect.
(99) These results demonstrate the capacity of the invented heat-treated Lactobacilli strains to increase moisturization and barrier strength of the skin leading to significantly improvement of skin health.
(100)
Example 8: Growth Curves for L. plantarum Spp
(101) A 96-well MTP was prepared with autoclaved MRS medium (Oxoid CM0359). Three replicate wells were inoculated with each L. plantarum spp. strain, using stock cultures maintained in 10% glycerin at 20 C. An inoculum of OD=0.1 was added per well and the 96-well MTP incubated at 37 C. for 16 hours. Growth curves were determined in each well via absorption at 620 nm using a Sunrise Photometer (Tecan, Austria) and Magelan Software. Saline (0.9% Sodium chloride solution) was used as negative growth control.
(102) For calculation, averages of detected absorptions were calculated and plotted in function of time for graphical presentation. The tested strains as well as the obtained growth curves can be seen in
Formulation Examples 1 to 15
(103) Formulations (compositions) comprising Lactobacilli according to the invention having skin soothing and barrier strengthening effects: 1. Skin lightening day cream o/w 2: Skin-soothing lotion 3: After sun balm, itch reducing 4: Calming body spray 5: Sunscreen lotion (o/w, broadband protection) 6: w/o night cream 7: Scalp soothing Anti dandruff shampoo 8: Self-tanning cream 9: Anti itch barrier repair cream 10: Antiperspirant/deodorant roll-on 11: Emulsion with UV-A/B-broadband protection 12: Sun spray with UV-A/B-broadband protection with low oil content 13: Skin-lightening balm with UV-A/UV-B protection 14: Scalp soothing hair conditioner with UV-B/UV-A protection, rinse off 15: Anti-itch hair conditioner, leave on
(104) TABLE-US-00002 Raw Material % BY WEIGHT/FORMULATION EXAMPLE Name/INCI INCI 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Lactobacillus 0.1 0.2 0.3 0.05 0.2 0.5 0.4 0.02 0.6 0.1 0.3 0.01 1 0.1 0.15 plantarum HEAL19 (pasteurized) Lactobacillus 0.05 0.1 0.1 0.02 0.1 0.1 0.1 0.05 0.2 0.1 0.05 0.02 0.2 0.3 0.15 plantarum HEAL99 (pasteurized) Abil 350 Dimethicone 0.5 2.0 1.0 0.5 0.5 0.3 0.1 Allantoin Allantoin 0.2 0.1 0.25 Aloe Vera Water (Aqua). 3.0 3.0 0.45 Gel Aloe Concentrate Barbadensis 10/1 * Leaf Juice Alpinia Leaf Alpinia 1.0 0.5 Extract Blend Officinarum Leaf Extract. Alpinia conchigera Leaf Extract. Alpinia Blepharocalyx Leaf Extract Alugel 34 TH Aluminium 1.0 Stearate Arbutin -Arbutin 0.2 ()-alpha Bisabolol 0.15 0.2 0.1 Bisabolol Butylene Butylene 5.0 3.0 3.0 Glycol Glycol Carbopol Carbomer 0.1 0.2 0.2 Ultrez-10 Ceramide BIO* Cetylhydroxy- 0.1 0.2 0.5 proline Palmitamide Cetiol OE Dicaprylyl Ether 4.0 Cetiol SB 45 Butyrospermum 1.0 Parkii (Shea Butter) Citric Acid Citric Acid 0.4 0.3 0.3 10% sol. Comperlan Cocamide MEA 0.5 100 Crinipan AD Climbazole 0.5 Curcuma Curcuma 0.5 Extract Xanthorrhiza Root Extract Curcuma Curcuma Longa 1.5 Root Extract (Turmeric) Root Extract Dehyquart ACA Cetrimonium 0.2 0.5 Chloride Dehyquart SP Quaternium-52 0.5 4.0 Dihydroxy- Dihydroxy- 5.0 acetone acetone Dow Corning Cyclohexasilox- 2.0 246 Fluid ane and Cyclopentasilox- ane Dow Corning Cyclomethicone 0.5 345 Fluid D-Panthenol Panthenol 1.0 Dracorin Glyceryl 5.0 5.0 1.5 1.0 1.0 CE* Stearate Citrate Dracorin Glyceryl Oleate 2.0 GOC* Citrate. Caprylic/Capric Triglyceride Drago-Beta- Water (Aqua). 0.3 Glucan* Butylene Glycol. Glycerin. Avena Sativa (Oat). Kernel Extract Dragoderm* Glycerin. 2.0 Triticum Vulgare (Wheat) Gluten. Water (Aqua) DragoCalm Water (Aqua), 1.0 0.8 Glycerin, Avena Sativa (Oat) Kernel Extract Drago-Oat- Water (Aqua). 1.0 2.0 Active* Butylene Glycol. Avena Sativa (Oat) Kernel Extract Dragosan Sorbitan 6.0 W/O P* Isostearate. Hydrogenated Castor Oil. Ceresin. Beeswax (Cera Alba) Dragosantol Bisabolol 0.3 0.1 0.3 0.2 0.1 0.1 100* Dragoxat 89 * Ethylhexyl 2.0 0.1 Ethylisononan- oate EDETA BD Disodium EDTA 0.1 0.1 0.1 0.1 Emulsiphos * Potassium Cetyl 2.0 1.5 2.0 1.5 0.1 Phosphate. Hydrogenated Palm Glycerides Ethanol 96% Ethanol 2.0 30.0 13.0 5.0 Euxyl K702 Dehydroacetic 0.5 Acid. Benzoic Acid. Phenoxyethanol. Polyaminopropyl Biguanide. Ethylhexyl- glycerin Euxyl K712 Sodium 0.2 0.3 Benzoate. Potassium Sorbate Extrapone Glycerin. Water 0.2 0.7 Green Tea (Aqua). Camellia GW * Sinensis Leaf Extract Extrapone Glycerin. Water 0.3 0.5 Rosemary (Aqua). GW* Rosmarinus officinalis (Rosemary) Leaf Extract Extrapone Propylene 1.0 Witch Hazel Glycol. Distillate Hamamelis colourless* Virginiana (Witch Hazel) Water. Water (Aqua). Hamamelis Virginiana (Witch Hazel) Extract Farnesol* Farnesol 0.5 Fragrance Fragrance 0.3 Rose* Fragrance Fragrance 0.3 0.3 0.3 0.2 0.4 0.4 0.5 0.3 0.3 1.0 0.5 0.4 0.5 0.1 WHITE * FrescolatMGA* Menthone 0.5 0.3 Glycerol Acetal FrescolatML Menthyl Lactate 0.8 0.2 cryst.* FrescolatX- Menthyl 1.0 COOL* Ethylamido Oxalate Genapol Sodium Laureth 37.0 LRO liquid Sulfate Glycerol 85% Glycerin 3.0 2.0 4.0 4.7 2.0 1.5 3.0 Glyceryl Glyceryl 2.0 2.0 2.0 4.0 Stearate Stearate Hydrolite-5 * Pentylene Glycol 5.0 3.5 Hydroviton Water. Glycerin. 1.0 4.5 24* Sodium Lactate. TEA Lactate. Serine. Lactic Acid. Urea. Sorbitol. Sodium Chloride. Lauryl Diethylenedi- aminoglycine. Lauryl Aminopropyl- glycine. Allantoin Hydroviton Water. 1.0 1.0 PLUS* Pentylene Glycol. Glycerin. Fructose. Urea. Citric Acid. Sodium Hydroxide. Maltose. Sodium PCA. Sodium Chloride. Sodium Lactate. Trehalose. Allantoin. Sodium hyaluronate. Glucose Irgasan DP 300 Triclosan 0.3 Isoadipate* Diisopropyl 1.0 1.0 Adipate Isodragol* Triisononanoin 2.0 3.0 1.0 Isopropyl Isopropyl 4.0 4.0 Palmitate Palmitate Karion F Sorbitol 2.0 Keltrol RD Xanthan Gum 0.2 0.1 0.2 0.3 0.2 Kojic acid Kojic Acid 1.0 0.5 0.2 0.3 Lanette 16 Cetyl Alcohol 1.0 1.0 1.2 Lanette E Sodium Cetearyl 0.7 Sulfate Lanette O Cetearyl Alcohol 3.0 1.0 2.0 Lara Care A- Galactoarabinan 0.3 2.5 1.5 200 Magnesium Magnesium 0.7 Chloride Chloride Merquat 550 Polyquaternium- 0.5 7 NaOH 10% Sodium 0.3 sol. Hydroxide Naringin 4.5.7- 0.5 2.0 Trihydroxyflavon e7-O- Neohesperidoside Natrosol 250 Hydroxyethyl- 0.3 HHR cellulose Neo Butyl Methoxy- 1.0 Heliopan dibenzoyl- 357* methane Neo Disodium Phenyl 10 22.0 1.5 Heliopan Dibenzimidazole AP * Tetrasulfonate (10% as sodium salt) Neo Ethylhexyl 5.0 3.0 25.0 Heliopan Methoxy- AV* cinnamate Neo Isoamyl p- 5.0 Heliopan Methoxy- E1000* cinnamate Neo Homosalate 5.0 5.0 Heliopan HMS* Neo Phenylbenz- 6.7 Heliopan imidazole Hydro* Sulfonic Acid (15% as sodium salt) Neo 4-Methylbenzyl- 1.5 33.3 10.0 Heliopan idene Camphor MBC * Neo Ethylhexyl 5.0 2.0 Heliopan Salicylate OS* Neo PCL Trideceth-9. wssl. N * PEG-5 Ethylhexanoate. Water Neutral Oil Caprylic/Capric 6.0 4.0 2.0 6.0 10.0 2.0 1.0 Triglyceride Oxynex 2004 BHT 0.1 Paraffin Oil Mineral Oil 4.0 PCL Liquid Cetearyl 3.0 5.0 7.0 12.0 3.0 3.0 0.6 0.3 100* Ethylhexoate PCL Solid * Stearyl 2.0 3.0 Heptanoate. Stearyl Caprylate Pemulen TR-2 Acrylates/C10- 0.3 0.2 30 Alkyl Acrylate Crosspolymer Polymer JR Polyquaternium- 0.1 400 10 Propylene Propylene 5.0 0.8 0.8 0.8 Glycol Glycol Retinyl Retinyl 0.2 Palmitate in Palmitate Oil Sepigel 305 Polyacrylamide. 1.0 C13-14 Isoparaffin. Laureth-7 Sodium Sodium 2.0 1.0 Ascorbyl Ascorbyl Phosphate Phosphate Sodium Sodium 0.5 Benzoate Benzoate Sodium Sodium 1.0 Chloride Chloride Sodium Sodium 0.3 0.6 0.4 2.8 Hydroxide Hydroxide (10% sol.) Solubilizer PEG-40 2.0 2.2 611674* Hydrogenated Castor Oil. Trideceth-9. Water (Aqua) Sun Flower Oil Helianthus 5.0 Annuus (Sunflower) Seed Oil Sweet Prunus dulcis 5.0 Almond Oil SymCalmin Pentylene 1.0 1.0 Glycol. Butylene Glycol. Hydroxyphenyl Propamidobenzoic Acid SymDeo 2-Methyl 5- 0.5 B125* Cyclohexyl- pentanol SymDeo Dimethyl 0.5 MPP* Phenylbutanol Symdiol68* 1.2-Hexanediol. 0.5 Caprylylglycol. Symdiol68T * 1.2-Hexanediol. 0.5 Caprylylglycol. Tropolone SymGlucan Aqua, Glycerin, 5 1,2-Hexandiol, Caprylyl Glycol, Beta-Glucan SymMatrix* Maltodextrin. 0.1 0.3 1.0 Rubus Fruticosus (Blackberry) Leaf Extract SymMollient Cetearyl 1.5 S * Nonanoate SymMollient Trideceth-9, 0.5 2.0 W/S PEG-5 Isononanoate, Water (Aqua) SymOcide Phenoxyethanol, 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.2 1.2 PH Hydroxy- acetophenone, Caprylyl Glycol, Aqua SymOcideP Phenoxyethanol. 1.0 S * Decylene Glycol. 1.2 Hexanediol Polyoxyethylene Laureth-9 0.5 1.0 (9) Lauryl Ether SymRelief Bisabolol. 0.1 100* Zingiber Officinale (Ginger) Root Extract SymRelief S Bisabolol, 0.2 Hydroxy- methoxyphenyl Decanone SymRepair Hexyldecanol. 2.0 100* Bisabolol. Cetyl- hydroxyproline Palmitamide. Stearic Acid. Brassica Campestris (Rapeseed) Sterols SymSitive Pentylene 1.5 0.5 1609 * Glycol. 4-t- Butylcyclohexanol SymSolPF3 * Water. 1.5 Pentylene Glycol. Sodium Lauryl Sulfoacetate. Sodium Oleoyl Sarcosinate. Sodium Chloride. Disodium Sulfoacetate. Sodium Oleate. Sodium Sulfate SymVital Zingiber 0.1 0.1 AgeRepair* Officinale (Ginger) Root Extract SymWhite377* Phenylethyl 0.5 0.5 1.0 Resorcinol Tego Betain Cocamidopropyl 6.0 1.0 1.0 L7 Betaine Tegosoft PC Polyglyceryl 3- 0.3 31 Caprate Tegosoft TN C12-15 Alkyl 5.0 5.0 Benzoate Texapon Sodium Laureth 4.0 NSO BZ Sulfate Tocopherol Tocopheryl 0.5 0.5 3.0 0.3 0.5 Acetate Acetate Triethanolamine. Triethanolamine 0.5 0.5 99% Water. Water (Aqua) ad 100 demineralized Zedoaria Curcuma 2.0 1.5 Leaf Extract Zedoaria Leaf Extract Zirkonal L Aluminium 37.0 450 Zirconium Pentachloro- hydrate (40% aqueous solution)
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