WO2018134745A1

WO2018134745A1 - A eutrophicating product for cosmetic use and a relative use

Info

Publication number: WO2018134745A1
Application number: PCT/IB2018/050286
Authority: WO
Inventors: Alberto RODOLFI; Elisabetta CASELLI
Original assignee: Copma S.C. A R.L.
Priority date: 2017-01-19
Filing date: 2018-01-17
Publication date: 2018-07-26
Also published as: IT201700005552A1

Abstract

The disclosed eutrophicating product for cosmetic use, comprises: a base compound having a nourishing and/or emollient and/or hydrating and/or moisturising and/or protective action; spores of probiotic bacteria mixed with the base compound it further comprises bacteriophage elements mixed with the base compound and having bactericide activity on predetermined undesired bacterial species present, the bacteriophage elements, in cooperation with the spores of probiotic bacteria, being able to carry out a combined and synergic action against damaging micro-organisms. Also disclosed is the use of a eutrophicating product for treatment and protection of the skin and adnexa thereof.

Description

A EUTROPHICATING PRODUCT FOR COSMETIC USE AND A RELATIVE USE

DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention relates to the technical sector relating to cosmetic treatments for topical use, in the field of eutrophicating products having a nourishing and/or emollient and/or hydrating and/or moisturising and/or protective action.

There is at present a progressively growing demand for products for eutrophicating products for treatment and protection of the skin and its adnexa (hair, nails) based on natural, non-allergenic and biological formulations.

DESCRIPTION OF THE PRIOR ART

Many of the products at present on the market for the treatment of the skin are based on the presence of chemical and/or pharmacological additives, which have the aim of preventing the onset of infections/inflammation, or of facilitating the restoring of the skin functions following the development of infections/inflammation of the skin and the adnexa.

It is known that the skin and its adnexa are colonised by numerous microorganisms (especially bacteria and fungi), which in their entirety form the cutaneous commensal microbiota.

However, the presence of some types of micro-organisms on the skin can constitute a risk for development of infections or inflammations.

As the presence of benign non-pathogenic micro-organisms effectively opposes colonisation by potentially pathogenic micro-organisms, the Applicant notes that there already exist detergent products on the free market, which contain probiotic bacteria of the Bacillus genus, with the aim of limiting the use of chemical disinfectants and facilitating a benign colonisation of the skin, so that the skin can combat pathogenic micro-organisms and render their population difficult.

These products exploit a "biocontrol" approach based on the fact that the probiotic micro-organisms (non-pathogens) can colonise the skin and the adnexa, competing with the proliferation of other species that are potentially risky for the health of individuals.

The bacteria of the Bacillus genus represent a vast group of spore-forming Gram-positive bacteria, ubiquitous in nature and also present in the human intestine.

The spores formed by the Bacillus bacteria can survive for very long times, because of their resistance to desiccation, heat and many chemical substances, and are therefore ideal for probiotic applications in the field of cosmetics.

It is also noteworthy that from the point of view of safety, the Bacillus species present in the products used are considered low/null risk for pathogens (non- pathogenic).

The presence of the probiotics further prevents re-colonisation by contaminant micro-organisms, thus stably maintaining hygiene where the probiotics are applied.

As this is a system based on the biological mechanism of competitive antagonism between probiotics and contaminant microbes, it can however be necessary to make prolonged use thereof in order to reach the stable destruction of the potentially harmful microbial load.

SUMMARY OF THE INVENTION

An aim of the present invention is to obviate the above drawbacks by providing a eutrophicating product for cosmetic use able to act rapidly and effectively against any potentially-dangerous microbial species, including those of the multi-resistant type.

The above-indicated aims are obtained by a cosmetic product realised according to claim 1 , and a use of the product according to claim 9.

In particular embodiments, the product for cosmetic use comprises the following compounds, in combination:

• the base compound can be in the solid, liquid or gaseous state;the base compound can comprise: Cocoa butter, Butyrospermum Parkit Butter,

Caprylyl Glycol, Carnauba, Cera Alba, Ceresin, Cetearyl Alcohol, Cetearyl Glucoside, Cetearyl Isononanoate, Ethylhexylmethoxycinnamate, Glyceril stearate citrate, Glycerin, Isopropryl Myristate, Lanolin, Panthenol, Paraffinum Liquidum, PEG-7 hydrogenated castor oil, Phenoxyethanol, Silicones, Sodium Carbomer, Sodium Carboxymethyl Betaglucan, Tocopheryl Acetate, Xanthana gum;

• the probiotic bacteria spores are mixed with the base compound at a concentration comprised between 10²-10⁹ spores/ml;

• the bacteriophage elements are mixed with the base compound at a concentration comprised between 10³-10⁹ PFU/ml;

• the probiotic bacteria are of the Bacillus genus (preferably though not exclusively of the species Bacillus subtilis, Bacillus megaterium and Bacillus pumilus);

• the bacteriophage elements comprise bacteriophages of the family Caudovirales and/or of the family Microviridae and/or of the family

Leviviridae and/or of the family Inoviridae and/or of the family Tectiviridae and/or of the family Corticoviridae;

The characteristics of the invention are specified in the following in which some preferred, but not exclusive, embodiments are described. BRIEF DESCRIPTION OF THE DRAWINGS:

Figures 1-3 are graphs relating to the reduction of the presence, over time (months), respectively of Staphylococcus aureus (Gram-positive bacterium), Enterobacteriaceae spp. (Gram-negative bacteria) and Candida albicans (fungus), on inert non-porous surfaces treated with product containing probiotic bacteria.

Figures 4-6 are graphs relating to the reduction of the presence, over time (hours), respectively of Staphylococcus aureus, Enterobacteriaceae spp. and Candida albicans, on inert non-porous surfaces treated with product containing bacteriophages.

Figures 7-9 are graphs relating to the reduction of the presence, over time (weeks), respectively of Staphylococcus aureus, Enterobacteriaceae spp. and Candida albicans, on inert non-porous surfaces treated with product containing probiotic bacteria and bacteriophages.

Figure 10 is a graph relating to the reduction of the presence, over time (days), respectively of Staphylococcus aureus on inert non-porous surfaces treated with product simultaneously containing probiotic bacteria and bacteriophages, in which the synergic action can be appreciated with respect to those treated with only probiotic bacteria or only bacteriophages.

Figure 1 1 is a graph representing the reduction, after 24 hours, of the presence of Staphylococcus aureus on pig skin, treated with a eutrophicating product for cosmetic use containing only probiotics (PRO), only bacteriophages (BP) or a combination of probiotics and bacteriophages (PRO+BP). The proposed eutrophicating product for cosmetic use innovatively comprises:

- a base compound having an emollient and/or hydrating and/or moisturising and/or protective action;

- spores of probiotic bacteria mixed with the base compound;

- bacteriophage elements mixed with the base compound and having bactericide activity on predetermined undesired bacterial species present, the bacteriophage elements, in cooperation with the spores of probiotic bacteria, being able to carry out a combined and synergic action against damaging micro-organisms. The invention also relates to a use of a eutrophicating product for cosmetic use, for treatment and protection of the skin and its adnexa.

The base compound can comprise one or more of the following: Cocoa butter, Butyrospermum Parkit Butter, Caprylyl Glycol, Carnauba, Cera Alba, Ceresin, Cetearyl Alcohol, Cetearyl Glucoside, Cetearyl Isononanoate, Ethylhexylmethoxycinnamate, Glyceril stearate citrate, Glycerin, Isopropryl Myristate, Lanolin, Panthenol, Paraffinum Liquidum, PEG-7 hydrogenated castor oil, Phenoxyethanol, Silicones, Sodium Carbomer, Sodium Carboxymethyl Betaglucan, Tocopheryl Acetate, Xanthana gum. The probiotic bacteria spores are mixed with the base compound at a concentration comprised between 10²-10⁹ spores/ml.

The bacteriophage elements specifically directed against bacteria potentially able to cause infection/inflammation of the skin or adnexa can be present in the base compound at a concentration comprised between 10³-10⁹ spore/ml. By way of non-limiting example, the probiotic bacteria can be of the Bacillus genus, including the species Bacillus subtilis, Bacillus megaterium and Bacillus pumilus.

This does not exclude the possibility that further probiotic bacteria can be used, as a function of the undesired bacterial species to be contrasted. The bacteriophage element can advantageously be selected, alternatively or in combination, according to the bacterial species to be contrasted, from among the following families: Caudovirales, Microviridae, Leviviridae, Inoviridae, Tectiviridae, Corticoviridae.

As is known, the bacteriophage elements of the Caudovirales family also comprise the Myoviridae, Siphoviridae, Podoviridae families.

The eutrophicating product of the invention for cosmetic use, in the preferred embodiments as indicated in the foregoing, is particularly suitable for cleansing of the skin and the skin adnexa. It can be produced in the solid state and/or the paste state (for example for soaps, ointments, creams, gels etc.) or in the liquid state (for example lotions, milks, water-consistency liquids etc.) or in the gaseous state (for example for sprays etc.). The action of the bacteriophages is specific, as specific types of lithic bacteriophages kill specific types of bacteria, and therefore perform a potentially very effective and specific action in the direct battle against undesired bacteria.

It is further to be evidenced how the use of specific bacteriophages prevents the risk of damage to the probiotic Bacillus, which can therefore be kept intact and effective in their activity.

The use of bacteriophages in products for cosmetic use is particularly indicated as they are rather stable in differing temperature conditions, pH and salinity of the environment, and can therefore be added to base compounds without suffering in terms of activity.

As the Applicant is not at present empowered to carry out laboratory testing on animals or human beings, it has used alternative methods to carry out experimental testing on inert surfaces.

These laboratory tests give evidence of the synergic effect deriving from the combined use of probiotic bacteria and bacteriophage elements with respect to the single use of probiotic bacteria or bacteriophage elements.

Test n° 1 (use only of probiotic bacteria)

The activity of PCHS detergents containing probiotics has been amply evaluated on inert surfaces, where they have shown themselves to be able to destroy the microbial load by about 90% more with respect to traditional eutrophicating products, with daily use for about one month.

After a period of two months destruction, the contaminant load is stably low regarding both the bacteria and the fungi [Caselli et ai, 2016]. Figures 1 -3 illustrate the antimicrobial action (bacteria and fungi) of a detergent product based only on probiotics.

The trend of contamination by Staphylococcus aureus (taken as an example of Gram positive bacteria), Enterobacteriaceae spp. (as an example of Gram negative bacteria) and Candida albicans (as an example of fungi), was measured on the field, with application of Rodac plates of specific media for the indicated bacteria and fungi species.

Test n° 2 (use only of bacteriophages)

Figure 4-6 illustrate the anti-bacterial action of the bacteriophages on an inert surface contaminated by specific target bacteria.

The trend of contamination by Staphylococcus aureus (taken as an example of Gram positive bacteria), Pseudomonas aeruginosa (as an example of Gram negative bacteria) and Candida albicans (as an example of fungi), was measured in vitro, on experimental models of inert surfaces having an area of 24 cm².

The surfaces were contaminated with a known quantity of micro-organism (10² CFU/24 cm²) and then treated by application of growing concentrations of specific bacteriophages (respectively 10³, 10⁴, 10⁵ PFU), therefore in ratios of 10:1 , 100:1 and 1000:1 with the seeded target bacteria. For the fungi a combination of the bacteriophages used against the Gram positive and Gram negative bacteria was used.

The residual contamination was evaluated after 1 , 3, 6 hours by application of Rodac plates of specific medium for the indicated bacterial and fungi species.

The results are expressed as mean values ± S.D. of samples in duplicate in three independent experiments.

It can be noted that already after one hour, and at the lower concentration, the bacteriophages are able to remove more than 90% of the bacterial cells against which they are specifically directed. The treatment with only bacteriophages has the limitation of being directed only against the bacteria specifically recognised by the bacteriophages, and therefore in a case of contamination by bacterial species different from those against which the bacteriophages are specifically directed, the surface treated remains contaminated by the species not attacked by the bacteriophages, as the bacteriophages are by definition specific for a bacterial species, and therefore not able to attack other bacterial species, nor are they able to attack species of fungi.

These limitations of the bacteriophages are overcome by the addition of the probiotics, which instead have a slower action, but generalised and independent of the bacterial or fungi species present.

Test n° 3 (combined use of probiotic bacteria / bacteriophages)

Figures 7-9 illustrate the effect of the combined and synergic antimicrobial action obtained with the contemporary presence of probiotic bacteria and bacteriophages on surfaces contaminated by the specific microbes illustrated.

The trend of contamination by Staphylococcus aureus (taken as an example of Gram positive bacteria), Pseudomonas aeruginosa (as an example of Gram negative bacteria) and Candida albicans (as an example of fungi), was measured in vitro, on experimental models of inert surfaces of non-porous and sterile material having an area of 24 cm².

The surfaces were contaminated with a known quantity of micro-organism (10³ CFU/24 cm²) and then treated by application of a solution containing probiotic bacteria (10³ CFU/ml) and specific bacteriophages (10⁴ PFU/ml).

For the fungi a combination of the bacteriophages used against the Gram positive and Gram negative bacteria was used.

The residual contamination was evaluated after 1 , 2, 3 and 4 hours by application of Rodac plates of specific medium for the indicated bacterial and fungi species. The results are expressed as mean values ± S.D. of samples in duplicate in three independent experiments. Comparison of tests 1-3

Figure 10 illustrates the direct comparison between the antimicrobial action of the no. 3 treatments on the contamination by Staphyococcus aureus, measured in vitro on experimental models of inert surfaces of 24 cm². The surfaces were contaminated with a known quantity of micro-organism (10³ CFU/24 cm²) and then treated by application of a solution containing probiotic bacteria growing concentrations of (10³ CFU/ml), only specific bacteriophages (10⁴ PFU/ml), or the combination of probiotics and bacteriophages at the indicated concentrations. The residual contamination was evaluated after 1 , 2, 3 and 7 hours by application of Rodac plates of specific medium for Staphyococcus aureus.

The results are expressed as mean values ± S.D. of samples in duplicate in three independent experiments. From the experimental tests carried out, it can easily be noted how the contemporaneous presence of Bacillus probiotics and anti-pathogen bacteriophages significantly increases the effectiveness of the destruction of the quantity on inert surfaces, thanks to the synergic action carried out against the various damaging micro-organisms.

On the one hand the bacteriophages destroy the contaminant load extremely rapidly, but have a limited action over time and do not succeed in preventing re-contamination, nor can they attack bacterial species that are different from those specifically recognised.

On the other hand the probiotics reduce the contaminant load slowly but constantly, thanks to the competitive mechanisms, inhibiting recontamination and with an action that is independent of the microbial species present. The contemporaneous presence of probiotics and bacteriophages guarantees the rapidity of the specific antimicrobial action and contemporaneous inhibition of recontamination, ensuring destruction of the pathogen that is rapid and stable over time.

The applicant believes, however, that by virtue of the nature of composition of a eutrophicating product (creams, ointments), characterised by the presence of high concentrations of oily base substances, it is not at all out of the question for a mixture of probiotics and bacteriophages to act synergically on an organic surface that is irregular and porous such as the skin surface. Bacteriophages, in fact, due to their characteristic structure, might remain blocked internally of the oily matrix without being able to reach the surface of the target bacteria. Further, the same base compound might function as the source of carbon for the growth of the damaging microbial species.

Experiments were therefore carried out that evidence (for the first time) the ability of probiotics and bacteriophages to act together and synergically in the decontamination of an irregular, organic and porous surface such as the skin, even when suspended in an oily matrix.

Not being able to use human skin or voluntary subjects (as it is not ethically possible to artificially contaminate a subject then to verify the decontaminating ability of a product) in vitro tests were carried out on pig skin. Pig skin has relative characteristics (organic constitution, porosity, irregularity of the surface) that make it alike human skin. The test was carried out as described in the following.

Staphylococcus aureus was chosen as the reference microorganism for the tests carried out, as Staphylococci are part of the human cutaneous microbiota.

Flaps of pig skin, with fur removed, were frozen at -20% and irradiated with UV rays to eliminate any original contamination.

The flaps treated in this way were then cut into identical fragments of 2x2 cm per side (4 cm² surface).

Each flap was contaminated with 10³ CFU of S. aureus ATCC 25923, and then the flaps were treated with 0.1 ml of the following compounds, containing a eutrophicating base with the absence or presence of probiotic elements and/or bacteriophages (in triplicate). The results have been included in figure 1 1 , in which: 1. CTR(-): indicates negative control; no treatment

2. CTRb: indicates base control; treatment with only eutrophicating base

3. CTR(+): indicates positive control; treatment with eutrophicating base to which a high-level disinfectant has been added 4. PRO: indicates treatment with eutrophicating base containing probiotics (10⁵ CFU/ml)

5. BP: indicates treatment with eutrophicating base containing bacteriophages (10⁷ CFU/ml)

6. PRO+BP: indicates treatment with eutrophicating base containing probiotics and bacteriophages (respectively 10⁵ CFU/ml and 10⁷ PFU/ml).

The treatment was carried out by the spreading of the flaps of skin on the surface, up to complete absorption.

The treat skin flaps were left to dry and then deposited on Baird-Parker agar medium plates, which enables growth and enumeration of the S. aureus residue CFU.

Following a 24 h incubation at 37°C, the CFU were counted.

The results are reported in the graph of figure 11 , expressed as a mean value ± SD referred to samples in triplicate in 3 independent experiments.

From the experimental testing carried out, reported in figure 11 , it can clearly be observed that the single presence of probiotic Bacillus or bacteriophage anti-S. aureus is translated into a decontaminating ability of the eutrophicating product relative to the pathogen bacterium. However, the contemporary presence of probiotics and bacteriophages leads to the elimination of 92% of this bacterium, significantly higher than what is obtained with single use of probiotics or bacteriophages, and comparable to the effectiveness obtained with a high-level disinfectant, without however having the damaging features thereof with respect to the skin. In a comparison of figure 1 1 with figure 10, it emerges that the effect after 24 hours in relation to the eutrophicating product for cosmetic use with the effect obtained with the solution is greater than that obtained with the solution used in test 3 comprising probiotic bacteria/bacteriophages. In fact, in figure 11 , in the case of the eutrophicating product PRO+BT, the contamination after only 24 hours was much lower than the eutrophicating base containing BP bacteriophages. Instead, in figure 10, after 24 hours on inert surfaces the datum relative to the sole combination of probiotics and bacteriophages coincides approximately to that of the solution containing only bacteriophages. Therefore with the eutrophicating cosmetic product according to the invention a synergic effect is obtained that is greater in shorter times (24 hours).

Note that this is particularly important as the skin and its adnexa are typically treated periodically every day and thus it is particularly advantageous that the eutrophicating product according to the invention is effective in the period between one treatment and another. The realising of a eutrophicating product for cosmetic use containing at the same time Bacillus probiotics and anti- pathogen bacteriophages has the obvious advantage, with respect to traditional products, of ensuring and extremely rapid and immediate destruction of the contaminant bacterial load. This is thanks to the combined action performed by the bacteriophages which are able, in a few hours, to kill the target bacteria, and by the probiotic bacteria, which are able to persistently colonise the cutaneous surface, replacing the pathogens.

The presence of the bacteriophages thus represents an empowering of the anti-bacterial action of the product bases on only probiotic Bacillus as it facilitates and increases the action of the probiotics, which are also active on fungi.

This guarantees an immediate sanitising effect during the initial step of the cosmetic treatment, enabling the probiotic bacteria to maintain a stably low presence over time of potentially pathogenic bacteria. The addition of specific bacteriophage elements to the base compound mixed with spores of probiotic Bacillus advantageously enables using the product in a directed way, responding to specific needs and/or situations of particular interest, such as for example the destruction of one or more specific bacterial species that are particularly prevalent and/or damaging.

The presence in the product of the invention of a greater number of families of bacteriophage families (Caudovirales, Microviridae, Leviviridae, Inoviridae, Tectiviridae and Corticoviridae) enables the product itself to have a bactericide activity on a broader group of treatable bacterial species. The predetermined families of bacteriophage elements can be present in the product that is the object of the invention, both singly and in combination, so as to include the possibility of diversified treatments according to the specific need.

From the above it is clear that the eutrophicating product for cosmetic use of the invention, having a nourishing and/or emollient and/or hydrating and/or moisturising and/or protective action, is able to act in a particularly rapid and effective way against any microbial species, including those of the multi- resistant type (superbugs). The product is particularly effective for cosmetic treatment of the skin and skin adnexa.

Claims

1) A eutrophicating product for cosmetic use, comprising: a base compound having a nourishing and/or emollient and/or hydrating and/or moisturising and/or protective action; spores of probiotic bacteria mixed with the base compound; characterised in that it further comprises bacteriophage elements mixed in the base compound and having a bactericide action on predetermined undesired and present bacterial species, the bacteriophage elements, in cooperation with the spores of probiotic bacteria, being able to carry out a combined and synergic action against damaging micro-organisms. 2) The product of claim 1 , characterised in that the base compound can be in the solid and/or paste, liquid or gaseous state.

3) The product of claim 1 or 2, characterised in that the base compound comprises one or more of following elements: Cocoa butter, Butyrospermum Parkit Butter, Caprylyl Glycol, Carnauba, Cera Alba, Ceresin, Cetearyl Alcohol, Cetearyl Glucoside, Cetearyl Isononanoate, Ethylhexylmethoxycinnamate, Glyceril stearate citrate, Glycerin, Isopropryl Myristate, Lanolin, Panthenol, Paraffinum Liquidum, PEG-7 hydrogenated castor oil, Phenoxyethanol, Silicones, Sodium Carbomer, Sodium Carboxymethyl Betaglucan, Tocopheryl Acetate, Xanthana gum. 4) The product of one of claims from 1 to 3, characterised in that the spores of probiotic bacteria are mixed with the base compound at a concentration comprised between 10²-10⁹ spores/ml.

5) The product of one of claims from 1 to 4, characterised in that the bacteriophage elements are mixed with the base compound at a concentration comprised between 10³-10⁹ PFU/ml.

6) The product of one of claims from 1 to 5, characterised in that the probiotic bacteria are of the Bacillus genus.

7) The product of claim 6, characterised in that the probiotic bacteria are of the Bacillus subtilis, Bacillus megaterium and Bacillus pumilus species. 8) The product of one of claims from 1 to 7, characterised in that the bacteriophage elements comprise bacteriophages of the Caudovirales family and/or of the Microviridae family and/or of the Leviviridae family and/or of the Inoviridae family and/or of the Tectiviridae family and/or of the Corticoviridae family.

9) Use of a eutrophicating product for cosmetic use, according to any one of the preceding claims from 1 to 8 for treatment and protection of the skin and adnexa thereof.