ITRM20110406A1 - PLANTARICINE AND BIOMASS INCLUDING PLANTARS FOR USE IN THE COSMETIC AND MEDICAL FIELD. - Google Patents

Description

Insoles and biomass including insoles for use in the cosmetic and medical fields

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The present invention relates to insoles and biomass including insoles for use in the cosmetic and medical fields. In particular, the present invention relates to the use of plantaricin A, N or K or their mixtures or to a biomass containing one or more of the plantaricins mentioned above possibly in association with the lactic bacteria used for their preparation and their uses in stimulation angiogenesis and, therefore, an adequate blood supply of fundamental importance in normal cell growth and hair growth processes.

The hair follicle undergoes cycles of transformations passing from a quiescent phase (telogen) to a growth phase (anagen) with a rapid proliferation of follicular keratinocytes and the lengthening of the hair shaft followed by a regression phase (catagen) which leads to the involution of the follicle. These cyclical changes result in a remodeling of the epithelial and dermal components.

It is known that during the anagen phase of hair growth there is a phase of development of the vascular network, probably to increase the inflow of nutritional elements that fully support the needs of rapid cell division. On the contrary, during the telogen phase, hair loss is accompanied by a disappearance of blood vessels at the level of the dermal papilla and the bulb in general.

The expression of vascular endothelial growth factor (VEGF) and its receptors is related to the formation of capillaries.

The presence of VEGF in the hair bulb is necessary for the induction and maintenance of effective angiogenesis in the life cycle of the hair. In particular, the expression of VEGF in the human follicle varies during the hair cycle, in particular: during the anagen phase VEGF is strongly expressed, while it decreases during the catagen and telogen phases.

In in vitro experiments it was further shown that VEGF is an autocrine growth factor for dermal papilla cells. The formation of new blood vessels that occurs through the expression of VEGF is a regulated mechanism. The expression of VEGF is in fact induced in cells treated with interleukin-6, interleukin-1beta, TGF-beta.

It is also known that pharmacological substances, such as Minoxidil, are active on the vascularization through the induction of VEGF and through this mechanism they exert their effectiveness in situations of alopecia.

To date, therefore, the methods used to restore the function of the follicle in situations of hair loss that are based on the stimulation of VEGF are known, in fact there are substances (peptides) of vegetable origin that mimic the action of this growth factor enough to be defined growth factor like.

It has recently been observed that bacteria are able to release and perceive signal molecules as a response to changes in the surrounding environment, including changes in their cell density and / or in the number of cells of other microbial species that populate the same ecosystem. (Sturme et al., 2007. Making sense of quorum sensing in lactobacilli: a special focus on Lactobacillus plantarum WCFA1. Microbilogy 153: 3939-3947). In lactic bacteria, these responses, which occur according to a `` quorum sensing '' (QS) mechanism, include signal molecules called type 2 self-inductors (AI-2, mainly derivatives of furanones), synthesized through the activity LuxS enzyme (Miller and Bassler, 2003. LuxS quorum sensing: more than just a numbers game. Curr Opin Microbiol 6: 191-197), or signal molecules called pheromone peptides or self-inducing peptides (AIP) (Nakajama et al., 2001. Gelatinase biosynthesis-activating pheromone: a peptide lactone that mediates a quorum sensing in Enterococcus faecalis. Mol Microbiol 41: 145-154). It has recently been shown that the L. plantarum WCFS1 genome contains a large number of genes that code for AIP peptides, together with other genes that code for other functions involved in the mechanisms of â € œquorum sensingâ € (Sturme et al ., 2007.

Making sense of quorum sensing in lactobacilli: a special focus on Lactobacillus plantarum WCFA1. Microbilogy 153: 3939-3947). Some studies have shown that the system responsible for the synthesis of pheromone peptides of the plantaricin type is involved in the mechanisms of intra-species cellular communication. In this case the pheromone peptide is used as a tool to measure the cell density of the species synthesizing the molecule (Diep et al., 1994. The gene encoding plantaricin A, a bacteriocin from Lactobacillus plantarum C11, is located on the same transcription unit as an agr-like regulatory system. Appl Environ Microbiol 60: 160-166). Other studies have also shown that plantaricin-type pheromone peptides may be involved in inter-species cell communication mechanisms. In particular, the presence of competing microorganisms can activate the regulatory system that is involved in the mechanisms of microbial antagonism (Maldonado et al., 2004. Production of plantaricin NC8 by Lactobacillus plantarum NC8 is induced in the presence of different types of Gram- positive bacteria. Arch Microbiol 181: 8-16). In the presence of high cell densities of other microbial species, the pheromone peptide favors a cascade series of phosphorylation reactions involving complex metabolic regulation phenomena, which culminate in the synthesis of signal molecules which in the specific case act as antimicrobial compounds of the type bacteriocin (e.g. plantaricin types A, K and N) (Hauge et al., 1998. Plantaricin A is an ampkiphilic alpha-helical bacteriocin-like pheromone which exerts antimicrobial and pheromone activities through different mechanisms. Biochemistry 37: 16026-16032) .

Although the mechanism of cellular communication between prokaryotic and eukaryotic cells has been partially demonstrated, the literature on the interactions between signal molecules involved in the `` quorum sensing '' mechanisms of bacteria (e.g. peptide pheromones) and cells is very limited. of the intestinal mucosa of man. The only example is represented by the pentapeptide CSF, synthesized by the probiotic microorganism Bacillus subtilis, as a signal molecule involved in the phenomena of competence and sporulation (Fujija et al., 2007. The Bacillus subtilis quorum-sensing molecule CSF contributes to intestinal homeostasis via OCTN2, a host cell membrane transporter. Cell Host Microbe 1: 299-308). It has been shown that this pentapeptide is able to cause the induction of p38 MAP kinase, kinase B (Akt) and cryotolerance, favoring the prevention of oxidative damage in the intestine and thus reinforcing the barrier functions . In the current state of knowledge, no publication or patent has also considered the effect of signal molecules, involved in the mechanisms of cellular communication between bacteria, on the hair follicle.

The inventors of the present invention have now found that plantaricin, in particular planatricin A, has a positive effect on the stimulation of VEGF, therefore, on angiogenesis, by keratinocytes.

The plantaricins can be of synthetic origin or produced by microbiological processes. The use of biomass obtained through microbiological procedures and containing plantaricins is also advantageous. The preparation of biomass containing plantaricins can advantageously take place through the co-cultivation of two lactic bacteria: L. plantarum DC400 (deposited at the DSMZ on 21 December 2009 with N. DSM 23213) and L. rossiae DPPMA174 (deposited at the DSMZ in date 21 December 2009 with DSM No. 23214). The cultivation of L. plantarum DSM 23213 in conditions of co-culture with L. rossiae DSM 23214 is able to activate the synthesis of pheromone peptides of the plantaricin type (in particular plantaricin A) obtaining concentrations of approx.

50 times higher than those observed in the presence of the mono-culture of L. plantarum DC400 (DSM 23213). It has also been shown that the co-cultivation of L. plantarum DC400 (DSM 23213) with other species of lactic bacteria, also isolated from â € œnatural yeastâ €, is not able to stimulate the synthesis of pheromone peptides as in the case of the association with L. rossiae. Another important aspect of the above-mentioned procedure is that the synthesis of plantaricin A is possible not only on culture media usually used for the cultivation of lactic bacteria in the laboratory, but also on grape must, whey and aqueous extracts of fruit and vegetable products. This coculture process for obtaining plantaricins is the subject of patent applications IT RM2010A000004 and PCT / IT2011 / 000003 in which the synthesis of biomass containing plantaricins is reported, in particular plantaricin type A (PlnA) as a response mechanism to the co-cultivation of the two lactic bacteria L. plantarum and L. rossiae that populate the same food ecosystem, such as the â € œnatural yeastâ € used for the production of leavened products. The patent applications describe a standardized and optimized biotechnological protocol that provides for the co-cultivation of the two bacteria in CDM (Chemically Defined Medium), WFH (Wheat Flour Hydrolyzate) (Gobbetti, 1998. The sourdough microflora: interactions of lactic acid bacteria and yeasts . Trends Food Sci Technol 9: 267-274), grape must (diluted with 1% soluble carbohydrates, added 0.5% maltose and 0.5% yeast extract, pH 5.6 ), whey (added 0.5% maltose and 0.5% yeast extract, pH 5.6) or aqueous extracts of fruit and vegetables (added 0.5% maltose and 0.5% yeast extract, pH 5.6) for 18 - 24 h at 30 -37 ° C. In particular, before co-cultivation, lactic acid bacteria are cultured for 24 h, collected by centrifugation (10,000 x g for 15 min at 4 ° C), washed twice in 50 mM phosphate buffer, pH 7.0 and resuspended in water. at the cell density of 9.0 log cfu / ml, inoculated (4%, for each species) under co-culture conditions on one of the above mentioned culture media.

At the end of the cultivation, the cells may or may not be removed from the culture broth by centrifugation, thus subjecting the supernatant to a dehydration process by drying or lyophilization.

Growing L. plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214) in coculture conditions on any of the previously described substrates, synthesis of plantaricin A was observed at a concentration between 2.5 and 4.0 µg / mL. Under mono-culture conditions, the concentration of plantaricin A synthesized by L. plantarum DC400 (DSM 23213) was in the order of approx. 0.06 µg / mL. In conditions of co-culture with other species of lactic bacteria (eg Pediococcus pentosaceus, Lactobacillus pentosus, Lactobacillus brevis, Lactobacillus rossiae, Lactobacillus rhamnosus) the synthesis of plantaricin A was decidedly lower. In conditions of co-cultivation with L. rossiae DPPMA174 (DSM 23214), the synthesis of other pheromone peptides, such as plantaricin type K and N, was also observed, although at lower concentrations than plantaricin A, and precisely in the range of 0.02-0.06 µg / ml.

In an in vitro biological evaluation on keratinocytes, incubation with different concentrations 0.1, 1 and 10 µg / ml of plantaricin A, both of synthetic origin and contained in the biomass described above, conducted for different contact times, resulted in stimulating VEGF .

The inventors of the present invention have also found that the biomass obtained by the process described above and containing one or more plantaricins, possibly in association with the lactic bacteria L. plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214), has greater effectiveness in promoting angiogenesis at the bulb level compared to the use of plantaricin alone.

Therefore, the specific object of the present invention is a biomass comprising one or more plantaricins A, N or K, preferably plantaricin A, said biomass being obtained by co-culture of Lactobacillus plantarum DSM 23213 and Lactobacillus rossiae DSM 23214, or one or more plantaricins selected from plantaricin A, K or N, preferably plantaricin A, called biomass and said one or more plantaricins for use as a cosmetic for the treatment of hair and skin appendages such as eyelashes and eyebrows.

A further object of the present invention is a biomass comprising one or more plantaricins A, N or K, preferably plantaricin A, said biomass being obtained by co-culture of Lactobacillus plantarum DSM 23213 and Lactobacillus rossiae DSM 23214, or one or more plantaricins selected from plantaricin A, K or N, preferably plantaricin A, called biomass and said one or more plantaricins for use, cosmetic or pharmaceutical, in the treatment or prevention of hair loss.

The present invention also relates to a biomass comprising one or more plantaricins A, N or K, preferably plantaricin A, said biomass being obtained by co-culture of Lactobacillus plantarum DSM 23213 and Lactobacillus rossiae DSM 23214, or one or more plantaricins selected from plantaricin A , K or N, preferably plantaricin A, called biomass and called one or more plantaricins for use, cosmetic or pharmaceutical, in the treatment or prevention of conditions of alteration of the scalp, such as for example alopecia induced by chemotherapy, induced alopecia from radiotherapy, aerated alopecia, androgenetic alopecia, telogen effluvium, or skin appendages such as eyelashes and eyebrows.

The biomass according to the present invention or one or more plantaricins are able, in fact, to improve the health of the hair, to strengthen the hair follicle, to increase vascularization by improving the supply of nutrients to the cells.

The biomass according to the present invention can be prepared by a process which includes or consists of the following steps:

a) propagate in culture the two lactic bacteria Lactobacillus plantarum DSM 23213 and Lactobacillus rossiae DSM 23214;

b) co-inoculate a substrate selected from the group consisting of CDM, WFH, grape must, whey or extracts of fruit and vegetable products, with an aqueous suspension of the lactic bacteria defined in step a);

c) incubate; and eventually,

d) centrifuge the culture broth to remove the lactic bacteria cells.

In particular, the suspension of phase a) can have a cell density equal to approx. 9.0 Log cfu / ml for each of the two species and can be added to the substrate in a percentage ranging from 1 to 4% with respect to the volume of the substrate.

The incubation is carried out at a temperature from 30 to 37 ° C, preferably 30 ° C, for 18 - 24 h, preferably 18 h. Centrifugation is carried out at 10,000 x g for 15 min at 4 ° C. The biomass preparation process can further comprise a step e) of dehydration of the supernatant obtained in step d) by drying or lyophilization.

A further object of the present invention is a pharmaceutical or cosmetic composition comprising or consisting of a biomass comprising one or more plantaricins A, N or K, of the biomass being obtained by co-culture of Lactobacillus plantarum DSM 23213 and Lactobacillus rossiae DSM 23214, or one or more plantaricins chosen from plantaricin A, K or N, for the uses defined above.

The present invention will now be described by way of illustration, but not of limitation, according to its preferred embodiments, with particular reference to the figures of the attached drawings, in which: Figure 1 (A-D) shows the graphs of the VEGF values obtained in human keratinocytes NCTC2544 from Elisa assay experiments in human keratinocytes NCTC2544. NCTC2544 were treated with culture medium only (positive control); Synthetic PlnA 10 µg / ml; biomass containing PlnA from co-culture of Lactobacillus plantarum DC400 and Lactobacillus rossiae DPPMA174, containing PlnA at a concentration of 10 µg / ml; or hyaluronic acid at a concentration of 200 µg / ml. The analyzes were performed after incubation at 37 ° C, 5% CO2 for 4 (Fig. 1A), 8 (Fig. 1B), 16 (Fig. 1C) and 24h (Fig. 1D).

EXAMPLE 1: Study on the effects of plantaricin A and biomass including plataricin on cell proliferation of keratinocytes MATERIALS AND METHODS

Cell cultures

The line used is a cell line of human keratinocytes NCTC 2544 (Perry V.P. et al., 1957) kept in culture in sterile flasks, incubated at 37 ° C in a humid atmosphere with 5% CO2 in culture medium MEM (Minimum Essential Medium ) added with 10% fetal bovine serum (FBS), 2mM L-glutamine, 1% non-essential amino acids, in the presence of 1% penicillin and streptomycin. Cells grow in vitro by adhering to the surface of the culture plates, in monolayer.

Compounds tested

â € ¢ Biomass including plantaricins, in particular Plantaricin A, from co-culture of L. plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214); â € ¢ Synthetic Plantaricin A (NeoMPS PolyPeptide laboratories S.A.-Strasbourg, France);

â € ¢ Positive control (untreated cells);

â € ¢ Commercial control (hyaluronic acid);

PHASE 1

1.1 Thawing of the cell culture

The procedure involves the thawing of the cell line, stored in cryovials at -80 ° C, the subsequent elimination of the exhausted medium and DMSO by centrifugation and the resuspension of the cells in complete culture medium.

1.2 Cell line propagation

The immortalized line of human keratinocytes NCTC 2544 (15) is used, maintained in culture in sterile flasks, incubated at 37 ° C in a humid atmosphere with 5% CO2 in culture medium MEM (Minimum Essential Medium) added with 10% of fetal bovine serum (FBS), 2mM L-glutamine, 1% non-essential amino acids, in the presence of 1% penicillin and streptomycin.

The 1: 3 split of the cell line is carried out every 2 days upon reaching the monolayer by washing with 1X PBS (phosphate buffer without Ca <2+ and> Mg <2+>) and detachment of the cells with a trypsin-EDTA solution. 37 ° C for 2 minutes.

STEP 2

2.1 Cytotoxicity test by MTT assay (for the determination of the non-cytotoxic concentrations of biomasa to be used)

The assay is used to evaluate the decrease in cell viability using a chromogenic oxidizing agent (MTT bromide) corresponding to a polycyclic system (C18H16BrN5S) equipped with a tetrazole ring that can be easily reduced by mitochondrial dehydrogenases or other electronic transport systems. , forming - by opening the tetrazole ring - a nitrogenous chromogenic compound called formazan, whose characteristic functional group is R <1> NHâˆ'N = CR <2> âˆ'N = NR <3>.

The immortalized line of human keratinocytes NCTC 2544 is used, maintained in culture in sterile flasks, incubated at 37 ° C in a humid atmosphere with 5% CO2 in culture medium MEM (Minimum Essential Medium) added with 10% of fetal bovine serum (FBS) , 2mM L-glutamine, 1% non-essential amino acids, in the presence of 1% penicillin and streptomycin.

The procedure includes:

- Transfer of cells in the exponential growth phase into 96-well plates;

- Overnight incubation at 37 ° C;

- Replacement of the culture medium with 100 µl of the substances to be tested;

- Incubation at 37 ° C, 5% CO2 for 24 / 48h / 72h;

- At the end of the incubation time, aspirate the medium and wash with PBS;

- Dilution of the previously prepared MTT stock solution in complete medium without phenol red (10 µl of stock solution in 100 µl of complete medium without phenol red);

- Application of MTT to cells (100µl) and incubation for 3h at 37 ° C;

- At the end of the incubation, elimination of the MTT from the wells and extraction of the formazan by resuspending the cells in 100 µl of DMSO solution;

- Spectrophotometric reading at 570 and 630nm;

STEP 3

Analysis of the cellular response to damage by evaluating the expression levels of VEGF-A by Elisa assay.

The immortalized line of human keratinocytes NCTC 2544 is used, maintained in culture in flasks, incubated at 37 ° C in a humid atmosphere with 5% CO2 in culture medium MEM (Minimum Essential Medium) added with 10% of fetal bovine serum (FBS) , 2mM glutamine, 1% non-essential amino acids, in the presence of 1% penicillin and streptomycin. The experiment initially requires the development of a monolayer of confluent cells in 12-well plates.

After reaching confluence, the cells were exposed to treatment with the compounds to be tested for 4, 8, 16, 24h. The compounds to be tested were diluted in complete culture medium to obtain the following treatment concentrations: PlnA from co-culture 0.1-1-10Î1⁄4g / ml; Synthetic PlnA 0.1-1-10Î1⁄4g / ml; hyaluronic acid (from a commercial solution of Connettivina, Fidia S.p.A.) 200Î1⁄4g / ml. Cells incubated in culture medium alone were used as a positive control.

At the end of each incubation time, the culture supernatants were collected and subjected to Elisa assay for VEGF-A.

ELISA is the acronym of Enzyme-Linked Immunoabsorbent Assay, it is a technique for the quantification of proteins that manages to combine the specificity of an antibody with the sensitivity of a simple enzymatic assay, using antibodies or antigens conjugated with enzymes. The 96-well plates used for the assay are pre-layered plates with the specific antibody for the protein to be tested (VEGF-A).

The standards and samples are subsequently added to the wells together with a solution of biotinylated antibody specific for the protein to be tested and a mixture of avidin conjugated with horseradish oxidase (HRP). After incubation a TMB substrate solution (3,3 ', 5,5' tetramethylbenzidine) will be added to each well.

Only the wells containing the protein of interest will exhibit a color change.

The enzyme-substrate reaction ends with the addition of a solution of sulfuric acid and the color change will be measured through a spectrophotometer at a wavelength of 450 nm ± 2 nm.

The protein concentration in the samples was then determined by comparing the O.D. of the samples compared to the standard curve.

RESULTS

Table 1: MTT assay

Control and Compounds Tested Vitality Concentrations (pg / mL)

After 24h of After 48h of treatment treatment Negative control * 100pg / ml 111.295 ± 0 .165 104 .483 ± 0.4; 200pg / ml 115.239 ± 0 .171 121 .738 ± 0.2 400pg / ml 107.767 ± 0 .086 84 .848 ± 0.0 PlnA (biomass) 0 .Olpg / ml 103.380 ± 0 .000 131 .860 ± 0.3; 0.lpg / ml 95 .850 ± 0.317 128 .819 ± 0.3 lpg / ml 98 .970 ± 0.074 117.653 ± 0.3 10pg / ml 89 .360 ± 0.306 136 .660 ± 0.0 50pg / ml 2 .030 ± 0.010 1 .497 ± 0.0 100pg / ml 2 .200 ± 0.005 2 .685 ± 0.0 PlnA 0 .Olpg / ml 105.730 ± 0 .116 138 .014 ± 0.3 (synthesis); 0.lpg / ml 99 .200 ± 0.206 133 .595 ± 0.4 lpg / ml 106.810 ± 0 .014 116 .679 ± 0.6 10pg / ml 113.350 ± 0 .143 106 .629 ± 0.4 50pg / ml 122.560 ± 0 .132 67.736 ± 1 .3 100pg / ml 94 .360 ± 0.014 82.537 ± 0 .9 * Hyaluronic acid in spray formulation (Fidia Farmaceutici)

Toxicity was tested by MTT assay for both synthetic Plantaricin (PlnA) and co-culture biomass on NCTC 2544 keratinocytes.

Compared to the control (untreated cells), incubation with synthetic PlnA from 0.01 to 50 µg / ml significantly increases (p <0.05) cell proliferation (Table 1), while exposure to 100 µg / ml causes a significant decrease (p <0.05) in cell proliferation.

The greatest effect of synthesis PlnA is found after 72h.

After 48 and 72h of incubation, exposure to coculture biomass containing PlnA from 0.01 to 10 µg / ml determines a significant increase (p <0.05) in cell proliferation. No significant increase was found after 24 h of incubation. A marked decrease in cell proliferation is observed with incubations of co-culture biomass containing PlnA greater than or equal to 50 µg / ml.

Hyaluronic acid at 100 and 200 µg / ml increases cell proliferation. After 48 and 72h incubation with 400 µg / ml hyaluronic acid, cell proliferation is inhibited.

Compared to synthetic PlnA and co-culture biomass, hyaluronic acid is less effective in increasing cell proliferation.

Figure 1 A-D shows that VEGF expression is markedly induced both by treatment with synthetic PlnA, but above all by co-culture biomass containing PlnA.

Even hyaluronic acid, used as a commercial negative control stimulates the synthesis of the VEGF gene but the effect is lower than that produced by Plantaricin A.

In particular, for all the treatment times considered, the 10 µg / ml coculture PlnA causes the highest increase in VEGF. In particular, at 10 µg / ml the induction by the coculture PlnA is always greater than the same quantity of synthetic PlnA.

This study therefore demonstrated how the pheromone peptide PlnA, which is commonly involved in the quorum-sensing mechanisms of lactic bacteria, is positively perceived by human keratinocytes.

EXAMPLE 2: TOPICAL LOTION

Component (chemical name / INCI) ...... Quantity w / w (%) Denatured alcohol. ................... 13 - 18 Mannitol ........................... .. 0.5 - 2 Betain ............................... 0.05 - 0.5 PEG - 40 Hydrogenated castor oil ..... 0.1 - 0.5 VP / VA Copolymer ...................... 0.01 - 0.05 Parfum .. ............................. 0.05 - 0.2 Biomass containing Plantaricin A ... 0.001 - 0.8 Aqua. ................................ to taste 100

EXAMPLE 3: STRENGTHENING LONGING MASCARA

Component (chemical name / INCI) ...... Quantity w / w (%) Biomass containing Plantaricin A ... 0.001 - 0.80 Isopropyl alcohol ................ .... 5 - 10

Cera alba / beeswax .................... 5 - 10 Stearic acid ..................... .... 3.0 - 5.0 Tricontanyl PVP ...................... 3.0 - 5.0 Dimethicone ... ....................... 3.0 - 5.0 Ceteareth - 12 ............... ........ 1.0 - 3.0 Mica ................................ 1.0 â € “3.0 Carnauba Wax / Carnauba Wax ........... 1.0 â €“ 3.0 Glyceryl stearate ............. ....... 1.0 - 3.0 Silica ............................... 1, 0 - 3.0 Ceteth - 20 .......................... 1.0 - 3.0 Polyvinyl alcohol ... ................. 1.0 - 3.0 Phenoxyethanol ....................... 0.5 â € “1.0 Parfum ............................... 0.1 - 0.7 Panthenol. ........................... 0.1 - 1.0 Propylene glycol .............. ....... 0.1 - 1.0 Disodium stearoyl glutamate .......... 0.1 - 1.0 Ammonium hydroxide .............. ..... 0.01 - 1.0 Propylparaben ........................ 0.05 - 0.19 Diazolidinyl Urea .... ................ 0.05 - 0.5 Hydrolyzed keratin ................... 0.01 - 1, 0 Disodium EDTA ........................ 0.02 - 0.10 Quaternium - 15 .............. ........ 0.01 - 0.2 Aluminum hydroxide .................. 0.01 - 1.0 C.I. 77007 / Ultramarines .............. q.b

THERE. 77491, 77492, 77499 / Iron Oxides. q.s.

THERE. 77891 / Titanium dioxide .......... to taste

Aqua ................................. to taste 100 g EXAMPLE 4: SHAMPOO

Component (chemical name / INCI) ....... Quantity w / w (%) Disodium Laureth Sulfosuccinate ...... 1 - 5 Magnesium Laureth Sulfate ............ 5 - 9

PEG - 7 Glyceryl Cocoate ............. 0.50 - 1.0 Cocamide MIPA ....................... . 0,5 - 2,0 Peg - 200 Hydrogenated Glyceryl Palmate 0,5 - 2,0 Polyquaternium - 10 .................. 0,1 - 0,5 Tetrasodium EDTA ..................... 0.05 - 0.20 Sodium Lauroyl Sarcosinate ........... 1 - 4 Tetrasodium EDTA .. ................... 0.05 - 0.20 BHA ....................... ........... 05 - 0.015 Potassium Undecylenoyl Wheat Protein. 0.50 - 1 Laureth - 4 .......................... 0.01 - 0.80 Parfum ........ ....................... 0.10 - 0.80 Glycol Distearate .................. .. 0.50 - 1.0 Laureth - 7 .......................... 0.50 - 0.80 Sodium Cocoamphoacetate ... ........... 0.05 - 3.0 Cocamidopropyl Betaine ............... 0.01 - 2 Sodium Laureth Sulfate ....... ........ 0.01 - 3 Sodium Hydroxymethylglycinate ........ 0.20 - 0.45 Biomass containing Plantaricin A ... 0.005 - 1.0 Benzoic acid ........ ................. 0.5 - 0.10 Sodium hydroxyde ..................... to taste

Citric acid .......................... to taste

Aqua ................................. to taste 100

EXAMPLE 5: ANDROGE-NETICA ALOPECIA TREATMENT LOTION

Component (chemical name / INCI) ....... Quantity w / v (%)

Hydroxypropyltrimonium hyaluronathe .. 0.005 - 0.50 Polyurethane - 26 .................... 0.004 - 4 Lecithin .............. ............... 0.005 - 5 Alcohol denat ........................ 15 - 20

PEG - 40 Hydrogenated Castor Oil ..... 0,5 - 2 Octadecyl di-t-butyl-4-hydroxyhydrocinnamate 0,02 0,05

Parfum ............................... 0,10 - 0,25 Helianthus annuus seed oil ....... .... 0.001 - 0.01 Lactic acid .......................... to taste at pH 4.9 Biomass containing Plantaricin A ... 0.001 - 0.50 Aqua ................................ . to taste 100 ml EXAMPLE 6: TRICHOLOGICAL SERUM

Component (chemical name / INCI) ....... Quantity w / v (%) Hydroxypropyltrimonium Hyaluronathe .. 0.005 - 0.50 Polyurethane - 26 ................. ... 0.004 - 4 Lecithin ............................. 0.005 - 5 Denatured alcohol ......... .......... 15 - 20 Hydrogenated Castor oil .............. 0.50 - 0.9 Octadecyl Di-t-butyl-4-hydroxyhydrocinnamate ... .................................. 0.01 - 0.05 Parfum ........ ....................... 0.01 - 0.05 Ethoxydiglycol ................... .... 0.05 - 1.0 Hydroxypropyl guar .................... 0.1 - 0.8 Lactic acid ........ ................... q.s. to pH = 5 Biomass containing Plantaricin A ... 0.001 - 0.50 Aqua ................................. q.s. 100 ml EXAMPLE 7: CONDITIONING BALM

Component (chemical name / INCI) ....... Quantity w / w (%) Behentrimonium Methosulfate .......... 0.5 - 3.0 Panthenol ......... ................... 0.5 - 3.0 Cetearyl Alcohol ..................... 0 , 5 - 4.0 Palmitic acid ........................ 0.5 - 4.0 Myrystic acid ......... ............... 0.5 - 4.0 Hydrolyzed Wheat Protein ............. 0.05 - 1.0 Cetrimonium Chloride ... .............. 1.0 - 3.0 Penthylene glycol .................... 5.0 Phenoxyethanol ... .................... 0.5 - 1.0 Parfum ...................... ......... 0.1 - 0.3 Biomass containing Plantaricin A ... 0.01 - 0.50 Aqua ................... .............. to taste 100 g

Claims (10)

CLAIMS 1) Biomass comprising one or more plantaricins A, N or K, said biomass being obtained by coculture of Lactobacillus plantarum DSM 23213 and Lactobacillus rossiae DSM 23214, or one or more plantaricins chosen from plantaricin A, K or N, said biomass and called a or more plantaricins for use as a cosmetic for the treatment of hair and skin appendages such as eyelashes and eyebrows. 2) Biomass comprising one or more plantaricins A, N or K, said biomass being obtained by coculture of Lactobacillus plantarum DSM 23213 and Lactobacillus rossiae DSM 23214, or one or more plantaricins chosen from plantaricin A, K or N, said biomass and called a or more orthotics for use in the treatment or prevention of hair loss. 3) Biomass comprising one or more plantaricins A, N or K, said biomass being obtained by coculture of Lactobacillus plantarum DSM 23213 and Lactobacillus rossiae DSM 23214 or one or more plantaricins chosen from plantaricin A, K or N, called biomass and called one or more plantaricins for use in the treatment or prevention of altered states of the scalp or skin appendages such as eyelashes and eyebrows. 4) Biomass or plantaricins for the use according to claim 3, in which the states of alteration of the scalp are selected from the group consisting of alopecia induced by chemotherapy, alopecia induced by radiotherapy, alopecia aerata, androgenetic alopecia, telogen effluvium. 5) Biomass for use according to each of claims 1 to 4, wherein said biomass is prepared by means of a process which comprises or consists of the following steps: a) propagate in culture the two lactic bacteria Lactobacillus plantarum DSM 23213 and Lactobacillus rossiae DSM 23214; b) co-inoculate a substrate selected from the group consisting of CDM, WFH, grape must, whey or extracts of fruit and vegetable products, with an aqueous suspension of the lactic bacteria defined in step a); c) incubate; and eventually, d) centrifuge the culture broth to remove the lactic bacteria cells. 6) Biomass for the use according to claim 5, wherein, the suspension of phase a) has a cell density equal to approx. 9.0 Log cfu / ml for each of the two species and is added to the substrate in a percentage ranging from 1 to 4% with respect to the volume of the substrate. 7) Biomass for the use according to any one of claims 5-6 wherein the incubation is carried out at a temperature from 30 to 37 ° C, preferably 30 ° C, for 18 - 24 h, preferably 18 h. 8) Biomass for the use according to each of claims 5-7, wherein the centrifugation is carried out at 10,000 x g for 15 min at 4 ° C. 9) Biomass for use according to any one of claims 5-8, wherein the process further comprises a step e) of dehydration of the supernatant obtained in step d) by drying or lyophilization. 10) Pharmaceutical or cosmetic composition comprising or consisting of a biomass comprising one or more plantaricins A, N or K, of the biomass being obtained by co-culture of Lactobacillus plantarum DSM 23213 and Lactobacillus rossiae DSM 23214, or one or more plantaricins selected from plantaricin A, K or N, for use as defined in any one of claims 1 to 9.