LACTOBACILLUS PLANTARUM CNCM I-4026 PREPARATIONS AND SKIN HEALTH

Abstract

The present invention relates generally to compositions with a health benefit. In particular, the invention relates to bacterial preparations of Lactobacillus plantarum CNCM I-4026 (NCC 2936). An aspect of the invention is a composition for use in the treatment or prevention of skin infections comprising a bacterial preparation of Lactobacillus plantarum CNCM I-4026. A further aspect is a cosmetic composition comprising a bacterial preparation of Lactobacillus plantarum CNCM I-4026.

Claims

1. A method for the treatment or prevention of skin infections comprising administering a bacterial preparation of Lactobacillus plantarum CNCM I-4026 to an individual in need of same.

2. A method according to claim 1 wherein the bacterial preparation is applied topically.

3. A method according to claim 1 wherein the bacterial preparation is a fermentation broth of Lactobacillus plantarum CNCM I-4026 or an extract thereof.

4. A method according to claim 3 wherein the Lactobacillus plantarum CNCM I-4026 have been at least partially removed from the fermentation broth.

5. A method according to claim 1 wherein the Lactobacillus plantarum CNCM I-4026 have been rendered non-viable.

6. A method according to claim 1 wherein the bacterial preparation is the supernatant of a fermentation broth of Lactobacillus plantarum CNCM I-4026 after centrifugation.

7. A method according to claim 1 wherein the skin infections are selected from the group consisting of boils, carbuncles, styes, impetigo, cellulitis, acne vulgaris and combinations of these.

8. A method according to claim 1 wherein the skin infections are staphylococcal infections.

9. A method according to claim 1 wherein the skin infections are methicillin-resistant Staphylococcus aureus infections.

10. A method according to claim 1 wherein the composition is applied at a dosage of between 1 and 200 mg of the bacterial preparation of Lactobacillus plantarum CNCM I-4026 on a dry basis per 100 cm.sup.2 of skin, 1 to 4 times per day.

11. A composition for use method according to claim 1 wherein the composition is administered to a subject suffering from burns, cuts, abrasions, pressure ulcers or combinations of these.

12. A method according to claim 1 wherein the composition is administered to a subject suffering from atopic dermatitis.

13. A method according to claim 1 wherein the composition further comprises a compound with antimicrobial properties selected from the group consisting of tea tree oil, eugenol, thymol, carvacrol, cinnamaldehyde, allyl isothiocyanate, lauric acid, palmitoleic acid, linoleic acid, acetic acid, propionic acid, butyric acid and combinations of these.

14. A method according to claim 1 wherein the composition is a pet care product.

15. A cosmetic composition comprising a bacterial preparation of Lactobacillus plantarum CNCM I-4026 wherein the composition is selected from the group consisting of cleansing, protective or care creams, milks or lotions for the face, for the hands, for the feet, for the major anatomical folds or for the body; makeup products; skincare gels or foams; shampoos, soaps; bath compositions; deodorant compositions, aftershave gels or lotions; and depilatory creams.

Description

EXAMPLES

Example 1: Lactobacillus plantarum NCC2936 Antimicrobial Activity

[0037] A vial of L. plantarum CNCM I-4026 was reactivated in 10 ml MRS broth (Difco) and incubated overnight at 30° C. in sterile anaerobic conditions (1st culture). A second culture was made by inoculating 10m1 of MRS broth with 2% of the previous culture (2nd culture). An inoculum was then prepared in 250 ml of MRS broth, inoculated at 2% with the 2nd culture. The final fermentation was performed in a 1 litre bioreactor containing MRS broth. Following the fermentation, the culture was centrifuged for 20 minutes at 3300×g (gravities) in sterile 1 L plastic bottles. The resulting pellet was discarded and the supernatant was filtered through a 0.2 μm filter. The filtered supernatant was frozen at −40° C. in a sealed aluminium bag and freeze-dried using a LyoBeta 35 instrument. The resulting powder was stored at 4° C. and rehydrated in the appropriate media for the inhibition assays.

[0038] Strains of typical microorganisms were collected from a variety of sources. In general, the selected strains were environmental isolates from food products and clinical sources. The strains included both clinical and food isolates of Staphylococcus aureus, together with an isolate of Staphylococus epidermidis. To perform the inhibition assays, cultures were grown overnight using appropriate media. Overnight cultures were diluted to achieve a starting inoculum of between 10.sup.3 and 10.sup.4 CFU/ml in normal growth media or media adjusted to pH 5.5. The freeze-dried filtered supernatant described above was rehydrated at 5% (w/v) in the growth media to form an antimicrobial stock solution. In standard multiwell plates, 20 μl of this stock antimicrobial solution was added to the diluted culture to give a final volume of 200 μl per well and a final concentration of 0.5% of the freeze dried material. The plate was loaded in a Bioscreen C Microbiological Analyser (Labsystems Helsinki, Finland) and incubated at 30° C. or 37° C. with shaking for 24-48 hours. The Optical Density (OD) was recorded every 15 minutes at 600 nm.

[0039] A delay or failure to record the Time To Detection (TTD) for each culture in the presence of the antimicrobial compared to the control was taken as the measure of inhibition. The TTD was determined when the OD.sub.600 reading increased by 0.2 above the background for the media plus the antimicrobial. A delay was recorded when a slower TTD relative to the control was observed and this difference could vary considerably but generally required a minimum change of 2 hours. All assays were performed in triplicate. The results are presented in tables 1 and 2 below.

[0040] The results show that the bacterial preparation of Lactobacillus plantarum CNCM I-4026 has surprisingly wide-ranging antimicrobial properties; inhibiting or delaying the growth of many microorganisms associated with poor health and in particular Staphylococcus aureus, Staphylococus epidermidis and Escherichia coli which may cause or complicate skin infections. The bacterial preparation of Lactobacillus plantarum CNCM I-4026 is shown to inhibit or delay growth of both Gram-positive and Gram-negative bacteria.

TABLE-US-00001 TABLE 1 Inhibition assay results for NCC2936 supernatant-Food safety/spoilage microorganisms. Food safety/spoilage microorganism Gram Positive Gram Negative Bacillus cereus I Escherichia coli I Bacillus licheniformis I Pseudomonas fluorescens I Lactobacillus plantarum X Salmonella enteriditis D Enterococcus faecium I Enterobacter cloacae D Staphylococcus aureus (food source) I Listeria monocytogenes I I = No TTD determined, X = Growth detected and D = Delay in TTD.

TABLE-US-00002 TABLE 2 Inhibition assay results for NCC2936 supernatant-Respiratory/oral microorganisms. Respiratory/oral microorganism Gram Positive Gram Negative Streptococcus pneumoniae I Haemophilus influenzae I Streptococcus pseudopneumoniae I Haemophilus parainfluenzae I Streptococcus mitis I Pseudomonas aeruginosa I Streptococcus oralis I Staphylococcus aureus (clinical isolate) I Staphylococus epidermidis I I = No TTD determined, X = Growth detected and D = Delay in TTD.

Example 2: Comparison of Lactobacillus plantarum Strains

[0041] Three plantarum strains were compared for their antimicrobial activity; L. plantarum CNCM I-4026, L. plantarum ATCC 8014 and L. plantarum WCFS1. L. plantarum ATCC 8014 has been shown to display antimicrobial properties [B. W. Lash et al., Food Microbiology 22, 199-204 (2005)] [N. A. I. Ismatul et al., Asian Journal of Biochemical and Pharmaceutical Research, 3, 90-98 (2012)], as has L. plantarum WCFS1 [M. H. J. Sturme et al., Microbiology, 153, 3939-3947 (2007)].

[0042] The L. plantarum strains were prepared and assayed in the same manner as Example 1 against three microorganisms Staphylococcus aureus, Enterococcus faecium and Escherichia coli. The OD.sub.600 readings after 72 hours are presented in table 3. None of the cultures with L. plantarum strains reached an OD.sub.600 reading of above 0.2 after 72 hours (the level for TTD in Example 1), but the OD.sub.600 readings for L. plantarum CNCM I-4026 were significantly lower than those of the other L. plantarum strains ATCC 8014 and WCFS1. This demonstrates that L. plantarum CNCM I-4026 is particularly effective in limiting the growth of microorganisms including Staphylococcus aureus, an important cause of skin infections.

TABLE-US-00003 TABLE 3 OD.sub.600 inhibition assay results for different L. plantarum strains after 72 hours MRS (Unfermented NCC 2936 WCFS1 ATCC 8014 broth) Control Staphylococcus aureus 0.08 0.12 0.12 1.5 1.4 Enterococcus faecium 0.09 0.14 0.14 0.97 0.5 Escherichia coli 0.06 0.15 0.14 0.9 0.94

Example 3: Oil-In-Water Emulsion Skin Cream Comprising a Bacterial Preparation of Lactobacillus plantarum CNCM I-4026

[0043] A sample of the freeze-dried supernatant of L. plantarum CNCM I-4026 from Example 1 is used to formulate a skin cream as follows:

[0044] Heat oil phase containing (in weight % overall):

[0045] Almond Oil 5%

[0046] Triglyceride 5%

[0047] Cetyl alcohol 2%

[0048] Stearic acid 2%

[0049] then blend with water phase containing:

[0050] Water 80%

[0051] 2% freeze-dried Lactobacillus plantarum NCC2936 supernatant

[0052] Glycerin 2.5%

[0053] EDTA 0.5%

[0054] and finally add 1% paraben mix.