ITRM20110330A1 - PROCEDURE FOR THE PREPARATION OF A BIOMASS INCLUDING PLANTARICINA AND ITS USES IN MEDICAL FIELD. - Google Patents

Description

PROCEDURE FOR PREPARING A BIOMASS

INCLUDING INSOLE AND ITS USES IN THE MEDICAL FIELD

The present invention relates to a process for preparing a biomass comprising plantaricin and its uses in the medical field. In particular, the present invention relates to a process for the preparation of plantaricin A, N or K or their mixtures or of a biomass containing one or more of the plantaricins mentioned above in association with the lactic bacteria used for their preparation and their uses in the stimulation of barrier functions of intestinal cells or keratinocytes of the human epidermis.

It is known that the epidermis and the intestinal wall perform a barrier function against the external environment and the harmful agents it contains. The barrier function of the epidermis and intestinal wall can be weakened by various factors. One of the conditions that most frequently involve alterations in the barrier function of the skin is exposure to various environmental factors. In normal conditions, however, the skin is able to adapt to any exogenous insults through an adaptation that guarantees the achievement of a "steady state" and a sufficient degree of tolerance. The continuous aggression to the skin by detergents and chemical substances with an irritating action (organic solvents, soaps, detergent solutions) can, for example, damage not only the lipid component of the surface film of the skin, but also the intercellular component of the stratum corneum, compromising its its barrier capabilities.

This results in mild dermatitis characterized by an increase in transepidermal water loss, slight desquamation and loss of elasticity of the stratum corneum, which may eventually result in the formation of small, continuously superficial solutions. This mild irritation, often subclinical, triggers reparative processes which, through an increase in lipid synthesis and a stimulation of the proliferative activity of the basal keratinocytes, allow the achievement of a new equilibrium and the restoration of the barrier function.

When the skin barrier is no longer able to adequately carry out its defensive functions, the risk of inflammatory skin diseases arising, triggered by the release of cytokines with consequent production on site of phlogogenic mediators and free radicals increases. The latter, in addition to causing direct damage to DNA and proteins through an oxidation mechanism, can cause peroxidation of the lipids of the cell membranes of epidermal cells.

The epidermis does not contain blood vessels, however it retains a small amount of water, which is essential for the physiological balance and the very integrity of the horny tissue. The water flow, coming from the capillaries of the dermis, passes the dermal-epidermal junction and slips between the rows of corneocytes, reaching levels similar to that of the generality of the tissues, estimated at around 60-70% in the deep areas (compact horn) , and 10-35% in the more superficial regions (corneum disjoint). The fact that, even in its outermost part, in contact with the air, the epidermis is able to maintain a reserve of moisture is based on two particular functions: the barrier activity that prevents the evaporation of fluids, and the overall hydrophilic power of the stratum corneum (water holding capacity), determined by the presence of highly hygroscopic particles. Experiments based on the progressive stripping of very thin corneocytic layers of the disjoint horny layer, and of the hydrolipidic film that covers it, show that the barrier function is not greatly affected, while it is gradually more compromised with the removal of the deeper layers of the underlying layer. compact corneum, of which the cemented wall structure is well known, with corneocyte bricks made up of clusters of keratin, compacted by filaggrin, and coated with a rigid protein envelope. Electron microscopy explained that the corneocytes are welded by modified desmosomal formations (corneosomes) and are engulfed by a lipid glue known as intercorneocyte cement, which creates a flexible and nearly impenetrable barrier film. Stressful situations that can acutely or chronically involve the skin barrier and in which the organism puts in place a series of homeostatic mechanisms that allow the epidermis to monitor its efficiency and restore it when it fails regardless of its nature of acute injurious noxa. The skin reacts to chronic skin stress induced by prolonged exposure to low levels of humidity in the air, typical of arid climates, with compensatory adaptive phenomena that cause an increase in the proliferation of basal cells and the consequent increase in the thickness of the layer horny and of the skin as a whole. The production and exocytosis of Odland's bodies and inter-corneocytic lipids (which maintain their normal composition) also increase in order to allow the epidermis a better defense of its own water resources. However, the lack of humidity causes a phase change in the liquid-lipid stratification of the intercorneocyte cement, which undergoes crystallization. This leads to less plasticity and rigidity to the tractions induced by the muscle-joint movements and to the extrinsic dynamic stresses, which easily cause micro-injuries. Another aspect of the coin of similar adaptive responses is given by hyperkeratosis: the increase in the proliferative layer is not counterbalanced by a parallel increase in the rhythm of exfoliation of the corneocytes which, instead of falling apart, remain conglobated in voluminous corneous clusters and they detach only in the form of coarse flakes. Furthermore, chronic environmental stresses activate a cytokine cascade that assumes the histological aspects of inflammation, with hypertrophy and degranulation of mast cells in the dermis. This may serve to explain the exacerbation of inflammatory skin diseases that can be observed in the winter season. Each acute insult brought to the barrier induces a cytokine response on the part of the affected skin cells, the effects of which are reflected on the keratinocytes themselves and on the underlying dermis, determining important morphological and functional consequences aimed at restoring the barrier function, the texture of the epidermal surface, and above all to liven up the activities of the fibroblasts at the base of the improvement of the compactness and turgor of the dermis.

To date, the methods used to restore the barrier function consist in the use of restorative and water-regulating substances that are able to obviate the poor skin defense capacity due to the alteration of the barrier function. These substances are compounds with a rehydrating and restructuring action. Many dermocosmetic products allow skin rehydration and, with their restorative function, promote skin regeneration. However, they act through a simple cosmetic "masking" of a rough surface, and do not have a real therapeutic effect, which should instead take place through a repairing and regenerating action aimed at restoring the morphofunctional integrity of the skin.

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 human epidermis.

The authors of the present invention have now found that plantaricin, in particular planatricin A, has a positive effect on the barrier functions of the keratinocytes of the human epidermis as well as of intestinal cells.

Studies concerning plantaricin preparation processes using mono-cultures (intraspecies) of bacteria are known. However, the cultivation of the microorganisms of interest takes place on complex and very expensive culture media to be used at industrial level in the preparation of signal molecules for therapeutic purposes.

The authors of the present invention have now perfected a process for the preparation of plantaricin by using a co-culture of two specific lactic bacteria capable of obtaining a much higher yield than those obtained by the known technique. In particular, the cultivation of L. plantarum DC400 (filed with the DSMZ on 21 December 2009 with No. DSM 23213) in conditions of co-culture with L. rossia and DPPMA174 (filed with the DSMZ on 21 December 2009 with No. DSM 23214; initially L. DPPMA174 was deposited with the DSMZ as L. sanfranciscensis DPPMA174, however on 31 May 2011 the correct taxonomic designation of lactobacillus was communicated to the DSMZ) is able to activate the synthesis of peptides pheromone of the plantaricin type (in particular plantaricin A) obtaining concentrations 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 process according to the present invention 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 extracts. aqueous fruit and vegetable products.

At the current state of knowledge, no publication or patent has reported the synthesis of plantaricin type A (PlnA) as a response mechanism to the co-cultivation of two lactic bacteria (e.g. L. plantarum and L. sanfranciscensis) that populate the same food ecosystem. , such as the â € œnatural yeastâ € used for the production of bakery leavened products. In the literature, an increase in plantaricin A synthesis has also never been observed in the conditions of co-culture (inter-species) compared to the conditions of mono-culture (intra-species). Furthermore, as mentioned above, the known processes use complex and very expensive cultivation media.

Furthermore, it was surprisingly found that the biomass obtained by the process of the invention and containing one or more plantaricins in association with the lactic bacteria L. plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214) has a greater efficacy in strengthening the barrier function at the level of the epidermis or intestinal wall compared to the use of plantaricin alone.

The lactic bacteria according to the present invention belong to the Lactobacillus genus and have been isolated from â € œnatural yeastâ € for the production of breads typical of Southern Italy.

A biotechnological protocol has been standardized and optimized which 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 milk (added 0.5% maltose and 0.5% yeast extract, pH 5.6) or aqueous extracts of fruit and vegetable products (added 0.5% maltose and 0.5% extract yeast, pH 5.6) for 18 - 24 h at 30 - 37 ° C. 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.

A diagram of the biotechnological protocol for the formulation of the plantaricin A-based preparation is described below.

Propagation of lactic acid bacteria cultures at 30 ° C for 24 h, washing, suspension in water at a cell density of 9.0 log cfu / ml and co-inoculation (1-4%) of the culture medium (CDM, WFH, must of € ™ grapes, whey, aqueous extracts of fruit and vegetables)

Cultivation at 30 - 37 ° C for 18 - 24 h

Cell removal by centrifugation

Dehydration of the supernatant by drying or lyophilization

Formulation of the preparation for application in dermo-cosmetics

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 cocultivation conditions 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 one of the possible formulations, the application of 2.5 µg / ml of plantaricin A was found to stimulate the barrier functions as demonstrated using a reconstituted epidermis model (SkinEthic®) and the Transepithelial Electric Resistence (TEER) assay. . Similar results were obtained on cells of intestinal origin Caco-2 / TC7 capable of reproducing the intestinal mucosa.

As demonstrated by complementary analyzes using microbiological, chromatographic techniques and in vitro and ex vivo cell culture assays, the coculture of L. plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214), not used in previous studies, according to the present invention allows: (i) the synthesis of signal molecules involved in inter-species cellular communication mechanisms at a concentration not detectable in the presence of other associations between lactic bacteria of the same ecosystem; (ii) the synthesis of plantaricin A, and other plantaricins (K and N), also starting from low-cost substrates; and (iii) a protective effect by strengthening the barrier functions at the level of epidermis and intestinal cells, demonstrating how signal molecules synthesized by prokaryotic cells are also perceived by eukaryotic cells.

Therefore, the specific object of the present invention is a biotechnological process for the synthesis of a biomass comprising or consisting of at least one plantaricin selected from plantaricin A, K or N, preferably A, or their mixtures and the lactic bacteria Lactobacillus plantarum DSM 23213 and Lactobacillus rossiae DSM 23214 or for the preparation of one or more plantaricins selected from plantaricin A, K or N, preferably A, or their mixtures, said process comprising or consisting 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 is added to the substrate in a percentage ranging from 1 to 4% with respect to the volume of the substrate. The incubation phase can be carried out at a temperature from 30 to 37 ° C, preferably 30 ° C, for 18 - 24 h, preferably 18 h, while the centrifugation can be carried out at 10,000 x g for 15 min at 4 ° C

The process according to the invention 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 biomass obtainable or obtained by the process as defined above, comprising or consisting of at least one plantaricin selected from plantaricin A, K or N, preferably A, or their mixtures and the lactic bacteria Lactobacillus plantarum DSM 23213 and Lactobacillus rossiae DSM 23214.

The present invention further relates to a pharmaceutical or cosmetic composition comprising or consisting of biomass as defined above, as an active ingredient, in association with one or more pharmaceutically acceptable excipients and / or adjuvants.

A particular aspect of the present invention also relates to the use of the biomass or composition as defined above for the preparation of a medicament to reinforce the barrier function at the level of the epidermis or intestinal wall or for the healing of wounds.

A further object of the present invention is the lactic bacterium Lactobacillus plantarum DSM 23213 or Lactobacillus rossiae DSM 23214 or their mixture.

Furthermore, the present invention relates to the use of one or more plantaricins selected from plantaricin A, K or N, preferably A, for the preparation of a medicament to reinforce the barrier function at the level of the epidermis or for the preparation of a medicament. for wound healing.

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.

Figure 1a shows the Multidimensional HPLC (MDLC) analysis coupled with electrospary-ionisation (ESI) ion trap MS (nano-ESI / MS-MS) conducted on the cell-free supernatant of the broth-culture obtained by cocultivation of Lactobacillus plantarum DC400 (DSM 23213) and Lactobacillus rossiae DPPMA174 (DSM 23214). Figure 1b shows the chromatogram obtained from the real chromatogram of Figure 1a based on the specific acquisition time with m / z ratios relative to plantaricin A. Figure 1c shows a species-relevant MS / MS spectrum based on the m / z ratio of 1493.7 observed in the chromatogram of Figure 1a.

Figure 2 shows the concentration of plantaricin A synthesized from the mono-culture of Lactobacillus plantarum DC400 (DC400), L. plantarum DPPMA 20 (DPPMA20), Lactobacillus pentosus 12H5 (12H5), Lactobacillus rossiae DPPMA174 (DPPMA174) and Peduscoccus £ 2XA pentace ) and from the co-cultures of L. plantarum DC400 with L. plantarum DPPMA20 (DC400-DPPMA20), L. pentosus 12H5 (DC400-12H5); L. rossiae DPPMA174 (DC400-DPPMA174) or P. pentosaceus 2XA3 (DC400-2XA3). The data are the average of three experiments conducted in triplicate.

Figure 3 shows the growth kinetics of Lactobacillus rossiae DPPMA174. Mono-culture (â—); coculture with Lactobacillus plantarum DC400 (â— ‹); monoculture with purified plantaricin A (2.5 µg / ml) (∠†); and mono-culture with chemically synthesized plantaricin A (2.5 µg / ml) (â – ²). Purified plantaricin A corresponds to that synthesized in the co-culture between L. plantarum DC400 and L. rossiae DPPMA174. The data are the average of three experiments conducted in triplicate.

Figure 4 shows portions of the gel related to the two-dimensional electrophoretic analysis of the proteins expressed by Lactobacillus rossiae DPPMA174 after 18 h of cultivation. Panel A, mono-culture; panel B, coculture with Lactobacillus plantarum DC400; and panel C, mono-culture in the presence of purified plantaricin A (2.5 µg / ml). The numbers marked with ovals or triangles refer to proteins that have shown an increase or decrease in the level of expression during cultivation with L. plantarum DC400 or with purified plantaricin A. The numbers marked with diamonds or double triangles refer to proteins that have shown an increase or decrease in the level of expression only during co-cultivation with L. plantarum DC400.

Figure 5 shows the Transepithelial Electric Resistance (TEER) (Ohms x cm <2>) of reconstituted epidermis (SkinEthic®) after exposure for 0 and 24 h with PBS buffer, plantaricin A (2.5 µg / ml) or biomass containing 2 , 5 µg / ml of plantaricin A. Data are the average of three experiments conducted in triplicate.

Figure 6 shows the viability of Caco2 / TC7 cells measured as Neutral Red uptake after 24, 48 and 72 h incubation with purified plantaricin A (2.5 µg / ml) from Lactobacillus plantarum DC400 (DC400) mono-culture or from co-culture with Lactobacillus rossiae DPPMA174 (DC400-DPPMA174). The fraction purified from the mono-culture of L. rossiae DPPMA174 which eluted under the same chromatographic conditions as plantaricin A, was used as a negative control (DPPMA174). Another negative control is represented by the DMEM culture medium (DMEM). Chemically synthesized plantaricin A (2.5 µg / ml) was used as a positive control (PlnA). The data are the average of three experiments conducted in triplicate. The asterisk indicates significant differences (P <0.01) with respect to the negative control. Figure 7 shows the viability of Caco2 / TC7 cells measured as Neutral Red uptake after 24, 48 and 72 h incubation with interferon γ (IFN-γ) (1000 U / ml) alone and with IFN-γ plus purified plantaricin A (2.5 µg / ml) (IFN-γ DC400-DPPMA174). The purified plantaricin comes from the co-culture between Lactobacillus plantarum DC400 and Lactobacillus rossiae DPPMA174. DMEM culture medium was used as a negative control (DMEM). Chemically synthesized plantaricin A (2.5 µg / ml) together with IFN-γ was used as a positive control (PlnA). The data are the average of three experiments conducted in triplicate. The asterisk indicates significant differences (P <0.01) with respect to the negative control.

Figure 8 shows the Transepithelial Electric Resistence (TEER) (Ohms x cm <2>) of Caco2 / TC7 cells after 24 and 48 h of incubation. The incubation was carried out with: plantaricin A (2.5 µg / ml) purified from Lactobacillus plantarum DC400 (DC400) monoculture; plantaricin A (2.5 µg / ml) purified from the co-culture between L. plantarum DC400 and Lactobacillus rossiae DPPMA174 (DC400-DPPMA174); chemically synthesized plantaricin A (2.5 µg / ml) (PlnA); interferon γ (IFN-γ) (1000 U / ml) and plantaricin A purified from the coculture between L. plantarum DC400 and L. rossiae DPPMA174 (IFN-γ DC400-DPPMA174); and IFN-γ and chemically synthesized plantaricin A (PlnA IFN-γ). DMEM culture medium (DMEM) was used as a negative control. The data are the average of three experiments conducted in triplicate. The asterisk indicates significant differences (P <0.01) with respect to the negative control. Figure 9 shows the results of the wound healing assay on keratinocytes treated with control, plantaricin A or biomass containing plantaricin A. Figure 10 shows the distance between the margins of a wound treated with control, plantaricin A or biomass containing plantaricin A.

Figure 11 shows the percentage increase in healing of the wound treated with the control, plantaricin A or biomass containing plantaricin A. culture (a) and synthetic Plantaricin (b), at the different time intervals considered. The application of the cut on the cellular monolayers was done by using a 200 ml tip. The cells were treated with co-culture and synthesis Plantaricin independently and at concentrations of 0.1, 1 and 10µg / ml.

Figure 13 shows the percentage of cells migrated through the cutting area after treatment with plantaricin from co-culture (0.1, 1 and 10µg / ml). The migration percentages were determined by measuring the free area between the two cutting lines created after 0, 4, 8, 12, 24, 48 and 72h hours from the initial cut. Bars represent means ± S.E.M. of two independent experiments each conducted in triplicate.

Figure 14 shows the percentage of cells migrated through the cutting area compared to the initial time after treatment with synthetic Plantaricin (0.1, 1 and 10µg / ml). The migration percentages were determined by measuring the free area between the two cutting lines created after 0, 4, 8, 12, 24, 48 and 72h hours from the initial cut. Bars represent means ± S.E.M. of two independent experiments each conducted in triplicate.

Figure 15 shows the percentage of free area compared to the initial cutting area after treatment of the NCTC2544 human keratinocyte monolayer with co-culture Plantaricin (0.1, 1 and 10µg / ml). the percentages were determined by measuring the free area between the two cutting lines with respect to the initial free cutting area after 0, 4, 8, 12, 24, 48 and 72h hours from the initial cut The bars represent the means ± S.E.M. of two independent experiments each conducted in triplicate.

Figure 16 shows the percentage of free area compared to the initial cutting area after treatment of the NCTC2544 human keratinocyte monolayer with synthetic Plantaricin (0.1, 1 and 10µg / ml). the percentages were determined by measuring the free area between the two cutting lines with respect to the initial free cutting area after 0, 4, 8, 12, 24, 48 and 72h hours from the initial cut The bars represent the means ± S.E.M. of two independent experiments each conducted in triplicate.

Figure 17 shows the effect of coculture plantaricin (0.1, 1 and 10µg / ml), synthetic plantaricin (0.1, 1 and 10µg / ml) and connectivin (200µg / ml) on expression of TGFβ1. in a monolayer of human keratinocytes NCTC 2544, etched to create a cut with a tip of 200 µl.

Example 1: Synthesis and purification of plantaricin-type pheromone peptides and study of the effects on epidermis and Caco-2 cells.

L. plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214) belonging to the Collection of Crops of the Department of Plant Protection and Applied Microbiology of the University of Bari, previously isolated from â € œnatural yeastâ €, are been propagated at 30 ° C for 24 h in modified MRS (mMRS), containing, in addition to the normal ingredients, 5% maltose and 10% yeast water - final pH 5.6.

Cells cultured for 24 h, harvested 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 a cell density of 9.0 log cfu / ml are been inoculated (4%, for each species) under mono-culture or co-culture conditions on WFH culture medium (Gobbetti, 1998. The sourdough microflora: interactions of lactic acid bacteria and yeasts. Trends Food Sci Technol 9: 267-274 ). The same procedure was applied and the same results, described later, were obtained using grape must (diluted to 1% of soluble carbohydrates, added of 0.5% of maltose and 0.5% of extract yeast, pH 5.6), whey (see previous additions) or aqueous extracts of fruit and vegetable products (see previous additions) as substrates for cultivation. The incubation was carried out for 18 h at 30 ° C. After cell removal by centrifugation (10,000 x g for 10 min at 4 ° C), the supernatants of the mono- and co-cultures were added with trifluoroacetic acid (0.05%) and centrifuged at 10,000 x g for 10 min. The supernatant was filtered through filters with porosity equal to 0.22 µm. The HPLC analysis was carried out using the AKTA Purifier system (GE Healthcare) with a detector operating at 214 nm and using a C18 XTerra reverse phase column (Waters, Mildford). A mixture of water, 2-propanol and trifluoroacetic acid (0.05%) was used as the mobile phase. All fractions containing plantaricin type A were analyzed by multidimensional chromatograph (MDLC) coupled with an ESI-ion trap MS mass spectrometer. The conditions of analysis for the identification and quantification of plantaricins A, K and N are those described in the publication Di Cagno et al. (Di Cagno et al., 2010. Quorum sensing in sourdough Lactobacillus plantarum DC400 (DSM 23213): induction of plantaricin A (PlnA) under co-cultivation with other lactic acid bacteria and effect of PlnA on bacterial and Caco-2 cells. Proteomics in press).

(2) Growth kinetics of Lactobacillus rossiae DPPMA174 (DSM 23214)

The data relating to the growth kinetics of L. rossiae DPPMA174 (DSM 23214) were elaborated by means of the Gompertz equation, subsequently modified (Zwietering et al., 1990. Modeling of bacterial growth curve. Appl Environ Microbiol 56: 1875- 1881). Cell counting was performed by plating on SDB culture medium at 30 ° C for 48 h. Cell viability and the number of damaged and / or dead cells were determined using the LIVE / DEAD bacLight Bacterial Viability kit (Molecular Probes, INc., Cambridge).

(3) Two-dimensional electrophoretic analysis and protein identification

The two-dimensional electrophoretic analysis of the cytoplasmic proteins of L. rossiae DPPMA174 (DSM 23214) cultured in mono- and co-culture was carried out using the immobiline-polyacrylamide system (De Angelis et al., 2005. Biochim. Biophys. Acta . 1762: 80-93). Four gels for each condition were analyzed and the data were normalized according to the procedure proposed by Bini et al. (Bini et al., 1997. Protein expression profiles in human breast ductal carcinoma and histologically normal tissue. Electrophoresis. 18: 2831-2841).

Protein identification was carried out by LC-ESI-MS / MS analysis and comparison of the sequences obtained on different databases (National Center for Biotechnology Information, Bethesda, MD, USA; ProFound, http: //www.prowl. rockefeller.edu/cgibin/ProFound).

(4) Assays on reconstituted epidermis and measurement of TEER (Transepithelial Electric Resistance)

The reconstituted human epidermis SkinEthic® (Reconstructed Human Epidermis) consists of normal keratinocytes of the human epidermis in multilayer form. It is a completely differentiated epidermis after cultivation of human keratinocytes in a chemically defined medium (MCDM 153), without the addition of fetal bovine serum, on an inert support of porous polycarbonate at the air-liquid interface for 17 days. At this stage of development, the morphological analysis shows a multi-layered vital epidermis and a stratum corneum formed by more than ten compact cell layers. SkinEthic® reconstituted human epidermis was used according to the methods previously described (Di Cagno et al., 2009. Synthesis of γ-amino butyric acid (GABA) by Lactobacillus plantarum DSMZ19463: functional grape must beverage and dermatological application. Biotechnol Microbiol DOI: 10.1007 / s00253-009-23704).

TEER measurement was performed by Millicell-ERS Volthommeter (Millipore, Billerica, MA). The measurement was expressed in Ohms x cm <2> (Di Cagno et al., 2010. Quorum sensing in sourdough Lactobacillus plantarum DC400: induction of plantaricin A (PlnA) under co-cultivation with other lactic acid bacteria and effect of PlnA on bacterial and Caco-2 cells. Proteomics in press).

(5) Caco-2 / TC7 cell assays

Caco-2 cells (clone TC7) of human origin were cultured in Dulbecco's medium (DMEM), supplemented with fetal bovine serum (10%), non-essential amino acids (1%), gentamicin / streptomycin (50 µg / ml), glutamine (2 mM) and 4-2-hydroxyethyl-1-piperazinylethanesulfonic acid (1%) (Di Cagno et al., 2010. Quorum sensing in sourdough Lactobacillus plantarum DC400: induction of plantaricin A (PlnA) under co-cultivation with other lactic acid bacteria and effect of PlnA on bacterial and Caco-2 cells. Proteomics in press). Cell viability was determined by Neutral Red dye absorption assay (Balls et al., 1987. Approaches to validation alternative methods in toxicology. In: Goldber A.M. (Ed). N.Y. Academic Press pp. 45-58). After treatment for 24 -72 h with the different preparations, the cells were washed with PBS buffer and incubated for 4 h at 37 ° C with a solution of Neutral Red (33 mg / l). Subsequently, the cells were again washed with PBS buffer and treated with lysing solution (50% ethanol in water containing 1% acetic acid). Plate reading was performed with a Novapath plate reader (Biorad, Hercules, CA) Di Cagno et al., 2010. Quorum sensing in sourdough Lactobacillus plantarum DC400: induction of plantaricin A (PlnA) under co-cultivation with other lactic acid bacteria and effect of PlnA on bacterial and Caco-2 cells. Proteomics in press).

For TEER measurement, Caco-2 / TC7 cells were inoculated (7.5 x 10 <4> cells / ml) in a microplate container with 24 cells and a polyethylene filter (porosity equal to 0.4 µm) . Before treatment, the cells were incubated for 21 days at 37 ° C. The treatments with the different preparations were carried out for 18, 24 and 48 h. The integrity of the cell layer was therefore determined by measuring the TEER.

Results

(1) Synthesis and purification of plantaricin-type pheromone peptides

After 18 h of development in WFH culture medium, the mono-culture of L. plantarum DC400 (DSM 23213) reached a cell density of 9.27 ± 0.18 log cfu / ml. The values of µmax and Î »were, respectively, of the order of approx. 0.27 log cfu / ml / h and 3.77 h. The cell density of lactic bacteria varied from 9.0 ± 0.05 (L. brevis CR13) to 9.43 ± 0.31 log cfu / ml (L. plantarum DPPMA20). The µmax values varied from 0.11 ± 0.05 (L. pentosus 12H5) to 0.15 ± 0.04 log cfu / ml / h (L. plantarum DPPMA20), as well as the value of Î »Ã ̈ ranged from 0.37 ± 0.07 (P. pentosaceus 2XA3) to 4.20 ± 0.36 h (L. rossiae DPPMA174 (DSM 23214)). In comparison with mono-culture, the cell density of L. plantarum DC400 (DSM 23213) did not change significantly (P> 0.05) (9.06 ± 0.34 - 9.28 ± 0.42 log cfu / ml) when the microorganism has been grown in co-culture with the other lactic bacteria. Cell yield of L. plantarum DPPMA20, Lactobacillus paralimentarius 8D, L. pentosus 12H5, Lactobacillus reuteri and Weissella cibaria 10XA16 was also not affected by co-culture. Conversely, the cell density of L. rossiae DPPMA174 (DSM 23214) and P. pentosaceus 2XA3 decreased significantly (P <0.05) (ca. 8.08 and 8.39 log cfu / ml) compared to the conditions of monoculture. In general, µmax values were decreased for all lactic acid bacteria when co-cultured with L. plantarum DC400 (DSM 23213). With the exception of L. plantarum DPPMA20, the latency phase Î »is also increased for all lactic bacteria. Strains of lactic acid bacteria that showed inhibition (L. rossiae DPPMA174 (DSM 23214) and P. pentosaceus 2XA) following co-cultivation with L. plantarum DC400 (DSM 23213) and some of the strains that were not affected by the conditions of co-culture (L. plantarum DPPMA20 and L. pentosus 12H5) were used in subsequent experiments.

In agreement with the previous results, the number of live cells of L. rossiae DPPMA174 (DSM 23214) decreased from 9.0 ± 0.28 to 8.32 ± 0.25 log cells / ml passing from mono conditions -culture under the conditions of co-culture with L. plantarum DC400 (DSM 23213). No significant differences (P> 0.05) were observed between the number of live cells and that of culturable cells. Again compared to the mono-culture conditions, the number of dead or damaged cells of L. rossiae DPPMA174 (DSM 23214) is significantly (P <0.05) increased under co-culture conditions with L. plantarum DC400 (DSM 23213) ). The number of cultivable cells of P. pentosaceus was also significantly (P <0.05) decreased when the lactic bacterium was co-cultured with L. plantarum DC400 (DSM 23213).

After cell removal, the supernatants of the mono- and co-cultures were used for the determination of the plantaricin-type pheromone peptides by MDLC analysis coupled with a nano-ESI / MS-MS mass spectrometer. Figure 1a shows the full-scan chromatogram of the sample derived from the co-culture between L. plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214). Due to the complexity of the matrix, it was possible to identify some species, while it was difficult to completely separate the adjacent peaks. However, it was possible to separate the coeluent species by filtering the signal at particular values of m / z. An example of the identified species is shown in Figure 1b. An MS / MS spectrum was obtained for each species. For example, Figure 1c shows the spectrum at the m / z value of 1493.7, which was selected for the analysis of the sample coming from the co-culture between L. plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214). For the search of the sequences on the NCBInr database the following parameters were specified: gender (Lactobacillus), tolerance ratio M / z for the recognition of ions (0.2 Da) and the instrumentation used for the analysis. The presence of plantaricin A (SEQ ID NO: 1 Lys-Ser-Ser-Ala-Tyr-Ser-Leu-Gln-Met-Gly-Ala-Thr-Ala-Ile-Lysâ € "Gln-Val-Lys-Lys- Leu-Phe-Lys-Lys-Trp-Gly-Trp) was observed both on mono-cultures of L. plantarum DC400 (DSM 23213) and DPPMA20 and in all co-cultures in which the strain was grown DC400 with other lactic bacteria.

On the basis of the previous results, the samples deriving from the mono- and co-cultures were purified by 4 chromatographic steps and further analyzed by nano-ESI-MS to exclude contamination by other peptides. The concentration of plantaricin A synthesized by L. plantarum DC400 (DSM 23213) was determined by chromatographic analysis, using a reverse phase column of type C18 XTerra and the OPA method. Figure 2 shows the concentration of plantaricin A under different conditions. It is possible to observe how the synthesis of the pheromone peptide is increased by passing from the mono-culture conditions (ca. 0.06 µg / ml) to the co-culture in the presence of L. rossiae DPPMA174 (DSM 23214) (ca. 2, 5 µg / ml). In some experimental conditions the concentration of plantaricin A was equal to approx. 4.0 µg / ml. Although plantaricin A production has been observed under co-culture conditions with other lactic acid bacteria species, the synthesized quantity is decidedly lower than that found in the co-culture between L. plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214).

This result indicates that the synthesis of the pheromone peptide is specific in the presence of specific microbial interactions that are able to induce the release of signal molecules. The synthesis of plantaricin A begins during the intermediate exponential growth phase (approx. 7 h) and increases until the end of the exponential phase (approx. 12 h). Although at lower concentrations and in the order of 0.02-0.06 µg / ml, plantaricins of type K and N were also observed only in the co-culture between L. plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214). The same results were obtained using CDM, grape must, whey and aqueous extracts of fruit and vegetable products as substrate for the cultivation of co-culture.

(2) Growth kinetics of Lactobacillus rossiae DPPMA174 (DSM 23214)

L. rossiae DPPMA174 (DSM 23214) was grown on WFH culture medium with the addition of 2.5 µg / ml of purified or chemically synthesized plantaricin A. From Figure 3 it can be seen how the presence of purified plantaricin A caused a significant decrease in the number of cells which ranged from 9.18 ± 0.26 (mono-culture condition) to 8.4 ± 0.14 log cfu / ml. Similar results were obtained using chemically synthesized plantaricin A. In both of these cases similar results were observed to those found during the co-cultivation of L. plantarum DC400 (DSM 23213) with L. rossiae DPPMA174 (DSM 23214). Damaged or dead cells of L. rossiae DPPMA174 (DSM 23214) cultured in the presence of purified or chemically synthesized plantaricin A were significantly (P <0.05) higher than those found in mono-culture (approx. 8.80 ± 0 , 14 vs.

6.08 ± 0.22 log cells / ml).

The results obtained show how the inhibitory effect of L. plantarum DC400 (DSM 23213) against L. rossiae DPPMA174 (DSM 23214) is to be attributed to the synthesis of plantaricin A and, probably, to the synthesis of other pheromone peptides belonging to the same chemical class.

(3) Change in protein expression levels in L. rossiae DPPMA174 (DSM 23214)

In comparison with the mono-culture, the two-dimensional electrophoretic analysis of cytosolic extracts of L. rossiae DPPMA174 (DSM 23214) cultivated in coculture with L. plantarum DC400 (DSM 23213) or in the presence of purified plantaricin A showed the variation of expression level of 51 and 27 proteins, respectively. All proteins over-expressed in the presence of plantaricin A were also over-expressed under co-culture conditions. By way of example, Figure 4 shows gel portions referring to the conditions of mono-culture, co-culture with L. plantarum DC440 and mono-culture in the presence of purified plantaricin A. Some of the most over-expressed proteins have been identified by spectrometry and have been found to be involved in protein biosynthesis (seryl-tRNA synthetase), energy metabolism (glucose-6-phosphate dehydrogenase, phosphoglycerate mutase, acetaldehyde-CoA dehydrogenase, 6-phospho -gluconate dehydrogenase and β-phospho-gluco-mutase), in the catabolism of proteins and amino acids (ATP-dependent Clp proteinase and aminopeptidase R), in the response to environmental stress (GroEL, GroES, S2 and S5 ribosome proteins) and in ™ homeostats of the redox potential (NADH oxidase). Most of these proteins have also been identified in other lactic bacteria as a response to environmental stress conditions and / or as a response to cellular communication mechanisms (Di Cagno et al., 2007. a protome study in Lactobacillus sanfranciscensis CB1. Proteomics 7: 2430-2446). In particular, the glucose-6-phosphate dehydrogenase enzyme is able to catalyze the release of a fratricidal pentapeptide in the programmed death mechanisms of Escherichia coli (Kolodkin-Gal et al., 2007. A linear pentapeptide is a quorum- sensing factor required for mazef-mediated cell death in Escherichia coli. Science 318: 652-655).

The results obtained show how the inhibitory effect of L. rossiae DPPMA174 (DSM 23214) following the synthesis of plantaricin A has as its basis a cellular communication mechanism and is probably capable of triggering reactions that lead to the death of the microorganism target. These results indicate a bactericidal valence of the signal molecule.

(4) Assays on reconstituted epidermis and measurement of TEER (Transepithelial Electric Resistence)

Both a biomass sample deriving from the co-culture between L. plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214) with a concentration of pantaricin A of 2.5 µg / ml, and plantaricin were tested. A purified from the co-culture, at the same concentration of 2.5 µg / ml, for the treatment of human epidermis reconstituted according to the SkinEthic® model. This model has been widely tested and accepted by the scientific community (Di Cagno et al., 2009. Synthesis of γ-amino butyric acid (GABA) by Lactobacillus plantarum DSMZ19463: functional grape must beverage and dermatological application. Appl Biotechnol Microbiol DOI: 10.1007 / s00253-009-23704). After treatment for 24 h, the TEER measurement was performed. This type of analysis, widely recognized by the international scientific community, evaluates the corrosivity of the tissue taking as a reference the integrity of the stratum corneum and the barrier function. In particular, by means of this survey it is possible to obtain information about the presence of a compact lamellar structure at the level of the stratum corneum, intact tight junctions and the epidermal thickness. All these factors together define an efficient barrier function. Figure 5 shows how both in the presence of biomass containing plantaricin and plantaricin A purified by the co-culture in question there is a significant increase (P <0.05) of the TEER value, demonstrating a protective action of the molecule a skin level. The same result was obtained with the chemically synthesized plantaricin A.

In the current state of knowledge, this is the first example of application of a pheromone peptide, involved in the mechanisms of cellular communication between bacteria, which is able to be perceived by human epidermal cells with consequent stimulation of barrier functions. .

(5) Caco-2 / TC7 cell assays

The viability of Caco-2 / TC7 cells was measured as the absorption capacity of the Neutral Red dye. Compared with DMEM medium (negative control), incubation for 24 - 72 h with purified plantaricin A (2.5 µg / ml) significantly increased the viability of Caco-2 / TC7 cells (Figure 6). The same result was obtained by using chemically synthesized plantaricin A. No induction was observed by treatment with the sample deriving from the purified fraction from the mono-culture of L. rossiae DPPMA174 (DSM 23214) which is eluted under the same chromatographic conditions as plantaricin A. As expected, exposure to interferon γ (IFN-γ) of Caco-2 / TC7 cells caused a significant decrease (P <0.05) in viability (Figure 7). On the contrary, the negative effect of IFN-γ was completely abolished in the presence of a simultaneous treatment with purified or chemically synthesized plantaricin A. The addition of purified plantaricin A was found to significantly increase (P <0.05) the TEER values of the Caco-2 / TC7 cells (Figure 8). The same result was observed with chemically synthesized plantaricin A. In comparison with the DMEM medium, the addition of IFN-γ significantly (P <0.05) reduced the TEER values. However, the addition of purified or chemically synthesized plantaricin A also eliminated the negative effects in this case. Caco-2 cells are one of the most widely used in vitro systems to simulate the intestinal mucosa. Despite their neoplastic origin, they are capable of spontaneously differentiating into mature enterocytes and expressing enzymes of the brush border. Under cultivation conditions, Caco-2 cells are capable of developing morphological and functional characteristics, including intercellular tight junctions, the integrity of which is determined by measuring TEER (Sambuy et al., 2005. The Caco-2 cell linea s a model of the intestinal barrier: influence of cell and culture-related factors on Cao-2 cell functional characteristics. Cell Biol Toxicol 21: 1-26). The results of this study show that plantaricin A purified from the coculture between L. plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214) or chemically synthesized is able to stimulate the barrier functions of the intestinal mucosa and prevent negative effects of treatments with interferon γ.

(6) Development of a biotechnological protocol for the synthesis of plantaricin A and its use in the dermatological field

As previously outlined in another part of the text, a biotechnological process has been developed for the synthesis of plantaricin A and for its use in the dermatological field. It provides:

a) Cultivation of L. plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214) in pure culture on mMRS culture medium;

b) collection, washing, resuspension of cells in WFH culture medium, CDM, grape must, whey or aqueous extracts of fruit and vegetable products, suitably integrating for the availability of nutrients;

c) incubation of the co-culture for 18 - 24 h, preferentially 24 h at 30 - 37 ° C, preferentially 30 ° C;

d) cell separation by centrifugation.

In a variant of the process, the preparation may also contain lactic acid bacteria cells;

e) dehydration of the preparation by drying or lyophilization process;

f) preparation of the dermatological preparation.

Example 2: Study of the effect of Biomass according to the invention and of Plantaricin A in wound healing

Method

Human keratinocytes placed in culture were incubated with Plantaricin A (2.5 µg / ml) or Biomass containing Plantaricin A for 1 hour.

At the end of the incubation, the cells were washed and the culture medium restored.

Wound healing

The assay consists in practicing a mechanical interruption in the continuity of the cellular monolayer in order to evaluate the effect of the treatment in favoring or not the ability of the keratinocytes to migrate beyond the damage margin and therefore to â € œscrew the lesionâ €.

To this end, the monolayer of keratinocytes, treated as described above, was incubated for a further 24 hours after the application of the stimulus and then fixed and stained with hematoxylin / eosin. The images were observed under an optical microscope with a 5X objective.

For each image, the maximum cell migration limits were identified and the distance between them calculated (Fig. 9-10).

Results

In Fig. 9, the results related to the wound healing assay are reported. After an incubation of 24 hours from the interruption of cell continuity, the monolayer of keratinocytes not subjected to any stimulus (control) shows some cells that extend from the wound margins. However, cell migration is particularly evident when cells are stimulated with hyaluronic acid. In this case, in fact, the lesion space is narrower than untreated cells (control), highlighting the ability of the cells to migrate to heal the wound.

In order to analytically evaluate the cell migration capacity, the wound margins were delineated, measured and analyzed as reported in Fig.10.

The keratinocytes incubated with Plantaricin A or Biomass containing Plantaricin A reduce the distance between the edges of the wound compared to the control cells, the percentage increase in healing compared to the control is shown in Fig. 11.

ASSAY SYSTEM: HUMAN reconstructed EPIDERMIS The epidermis model used is produced by the Skinethic <®>, Nice (F) laboratories and is used in the format of 0.5 cm <2> from the 17th day of differentiation with an average thickness of the batch of 120 µ (stratum corneum and vital epidermis).

A completely differentiated epidermis is obtained starting from human keratinocytes cultured in a chemically defined medium (MCDB 153) without the addition of fetal bovine serum, on an inert support of porous polycarbonate at the air-liquid interface for 17 days; at this stage of differentiation the morphological analysis shows a multi-layered vital epidermis and a stratum corneum formed by more than 10 compact cell layers.

TEER MEASUREMENT

Trans-epithelial electrical resistance (TEER) is a direct measure of the functionality of the skin barrier: it reflects all the overall resistance of the tissue due to both its thickness and structure. It reflects the integrity of the intercellular contacts at the level of tight junctions, of the bi-lamellar lipid structure that oppose the penetration of external substances.

TEER is the discriminating parameter of the test - Rat skin electrical resistance (B 40) - EU validated test for the evaluation of corrosivity taking as end-points the integrity of the stratum corneum and the barrier function.

It is inversely proportional to the TEWL measured in vivo which is the measure of transepidermal water loss: the greater the TEWL the greater the damage to the barrier function while the greater the TEER the less damage to the barrier function.

1 ml of PBS is dispensed on top of the insert and the transepithelial electrical resistance is measured with the Millicell-ERS instrument (range 0-20 kâ „¦).

Multiple measurements were made for each fabric.

Figure 5 shows the TEER values that refer to the average of 3 tissues, on which 3 measurements were made.

The following are relevant for the TEER value: the presence of a compact lamellar structure at the level of the stratum corneum, of intact tight junctions and the epidermal thickness that globally define an efficient barrier function. Each fabric is its own reference with measurements at t = 0 and t = 24 hours.

The result obtained is particularly interesting, in fact a significant increase in TEER is observed both in the presence of Plantaricin A and of Biomass containing Plantaricin A.

This increase is a direct consequence both of the increase in epidermal thickness and of a better compactness and integrity of the stratum corneum at the level of the tight junctions.

Example 3: Plantaricin A: study of its role in maintaining and restoring the skin barrier function

The study is divided into the use of Plantaricin A obtained from the co-culture of L. plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214) to achieve the following objectives:

- Evaluation of the effects of biomass on wound repair, studying its effects on the migration and proliferation of a human keratinocyte monolayer (NCTC2544) compared to a positive control (untreated cells) and a commercial / negative control with known action. The treatments will be carried out at different concentrations of the Plantaricin A under examination (after determination by cytotoxicity test) for three successive times (24, 48, 72h).

- Analysis of the cellular response to damage, in the times and concentrations considered, evaluating the modulation of mediators such as IL-8, KGF (keratinocyte growth factor), TGF-β (transforming growth factor-β), compared to a positive control (cells untreated) and a commercial / negative control with known action, using Real-Time PCR.

MATERIALS AND METHODS

Cell cultures

The line used is a NCTC 2544 human keratinocyte cell line (Perry, V.P., Sanford, K.K., Evans, V.J., Hyatt, G.W., Earle, W.R., 1957. Establishment of clones of epithelial cells from human skin. J Natl Cancer Inst . 18 (5): 709â € “717) kept in culture in sterile flasks, incubated at 37 ° C in a humid atmosphere with 5% CO2 in culture medium MEM (Minimum Essential Medium) supplemented with 10% of 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.

STUDY OF THE RESOLUTION OF TISSUE DAMAGE OVER TIME THROUGH WOUND HEALING

Principle of the method:

The experiment initially requires the development of a monolayer of confluent cells. Subsequently we proceed to the creation of the cut and to the observation of the â € œgapâ € created through the use of the microscope as the moving cells repair the damage. This healing process (called â € œhealingâ €) can take from a few hours to over a day depending on the cell type, condition and extent of the wound created.

Experimental procedure

o NCTC 2544 cells are piled and cultured at 37 ° C, 5% CO2 for 24h.

o After 24h the complete growth medium is replaced with serum-free medium in order to avoid the effect of the latter on cell proliferation and the cells are incubated for a further 24h at 37 ° C, 5% CO2.

o After 24h the cellular monolayer is mechanically damaged by lightly sliding a 200µl tip which traces a horizontal line about 1mm wide. The monolayer is then washed and after adding the substances to be tested, the plates are incubated at 37 ° C, 5% CO2 for 72h.

o The effect of the substances on the motility of the cells is evaluated by means of an inverted phase contrast microscope, acquiring images at different analysis times appropriately chosen.

o Damage repair is determined by measuring the free area between the two migration fronts of the cut at 0, 4, 8, 12, 24, 48, 72 h after cutting, using image processing software (Leica application Suite).

o All the data of each experiment are statistically processed in Excel in order to determine at 0, 4, 8, 12, 24, 48 and 72h.

ANALYSIS OF THE CELLULAR RESPONSE TO DAMAGE BY EVALUATING THE GENE EXPRESSION OF IL-8, KGF, TGF-β USING REAL-TIME PCR

The procedure consists of 3 basic phases:

I. Extraction of total RNA from cells

II. Reverse transcription in cDNA

III. Real-time PCR

I. Extraction of total RNA from cells

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 cell line will have been subjected to scraping by means of a 200µl tip and to treatments at different times and concentrations.

II. Reverse transcription of RNA into cDNA

The procedure involves amplification of the extracted RNA samples using the â € œHigh Capacity cDNA Reverse Transcription Kitâ € (Applied Byosistem) kit. III. Real-time PCR

The procedure involves the amplification of the cDNAs through the use of specific Taqman Gene assays and the â € œTaqMan Universal PCR Master Mix with Amperase UNG 2Xâ € (Applied Biosystem) kit.

A relative quantification will be used to determine a possible variation of the gene expression of the considered targets with respect to a positive control (untreated cells), using a housekeeping gene as data normalizer and analyzing the data according to the method of 2 <-∠† ∠† Ct>.

RESULTS

Wound healing

The ability of the coculture plantaricia to modify the migration of human keratinocytes NCTC 2544 by wound healing assay was tested.

The study also concerned the evaluation of the effects of a synthetic Plantaricin with respect to the co-culture Plantaricin obtained from the co-culture of L. plantarum DC400 and L. rossiae DPPMA174.

After making a cut on the cell monolayer with a 200µl tip and having completely removed the cells in the space between the two cutting lines, the cells are treated with co-culture Plantaricin and synthetic Plantaricin at the following concentrations: 0, 1- 1- 10 µg / ml. The positive control and the negative control (100 µg / ml of Connettivina) were also tested in duplicate.

Images on the same cutting area were captured at time 0 and after 4, 8, 12, 24, 48 and 72h, in order to monitor cell migration in the cutting area.

Figures 12-16 report the temporal analysis of the effect of co-culture Plantaricin (0.1, 1 and 10 µg / ml) (a) and synthetic Plantaricin (0.1, 1 and 10 µg / ml) (b) on the cell migration of NCTC 2544.

Figure 12 shows the progression of healing (∠† µm / time) at each considered time (every 4 hours) for both the active ingredients tested and the positive and negative control.

In the early stages of cellular healing it is not possible to detect significant changes in cell migration between the controls and the active ingredients tested with respect to time 0. As shown in figure 12a, Plantaricin from co-culture at a concentration of 0.1 and 1 µg / ml is able to significantly accelerate cell migration compared to the control. The effect is significant already 4h after cutting and remains constant and significant up to 72h. Treatments with plantaricin from co-culture at a concentration of 10 µg / ml instead produce a repair of tissue damage comparable to the positive control, therefore to the untreated cells, at all the time intervals considered.

The same experiment, conducted by treating the cells for the same time span with equivalent concentrations of synthetic Plantaricin, showed that also in this case the treatments with (synthetic) Plantaricin have a greater ability to accelerate cell migration between the two cut lines compared to the negative control.

In particular, even in this case, in the early stages of cellular healing it is not possible to highlight significant changes in cell migration both in the two controls used and in the cells treated with the active ingredients to be tested, with respect to time 0.

Figure 12b shows how at concentrations of 0.1 and 10 µg / ml, respectively, the synthetic Plantaricin produces a greater increase in cell migration than both the positive and negative control, already 4h after the production of the cut and this positive effect is maintained for all the time intervals considered, up to 72h. Treatments with a concentration of synthetic Plantaricin equal to 1 µg / ml instead produce a repair of tissue damage comparable to the repair operated by the positive and negative control. In order to better understand and analyze the effects of treatments with coculture and synthetic Plantaricin with respect to positive and negative controls in all the time intervals considered, we statistically analyzed the measurements deriving from the analysis of the images.

Figures 13 and 14 report the data as a percentage of cells migrated with respect to the positive control through the two cutting lines both in relation to treatments with plantaricin from co-culture (Fig. 13) and with synthetic Plantaricin (Fig. 14), respectively. The results are also reported as a percentage of the area between the two cutting lines with respect to the initial cutting area (Fig. 15-16).

The graph shown in figures 13 and 14 shows the data relating to the percentage of cells migrated during the different time intervals considered for the positive and negative controls, for the co-culture Plantaricin (0.1-1 and 10µg / ml) and the synthetic Plantaricin (0.1-1 and 10 µg / ml), respectively. In accordance with the data deriving from image processing, after the first 8 hours of treatment, the negative control does not persist in its cut-healing action and maintains a percentage of migrated cells close to that of the positive control.

With regard to plantaricin from co-culture (Fig. 13) in general it is possible to state that, at all three treatment concentrations used, it is possible to find a greater increase in cell migration from time 0 up to 72h of treatment, even at the lowest treatment concentration.

In particular, treatments with plantaricin from coculture at a concentration of 10µg / ml have the highest migration percentage values for all the time intervals considered except after 72h of treatment, but these data are not significant, with percentage values of cells migrated by 165.56%, 138.18%, 134.64%, 118.08%, 139.07% and 149.04%, respectively.

Figure 14 shows the percentage of cells migrated after treatment, at the different time intervals considered, with equivalent concentrations of synthetic Plantaricin.

Treatment with synthetic Plantaricin at a concentration of 0.1 µg / ml produces the highest percentage values of migrated cells for all the time intervals considered, with values from the initial time up to 72h of treatment of 158.30%, 115.15%, 124.32%, 141.82%, 141.91% and 128.51%, respectively. Treatment with synthetic Plantaricin at a concentration of 10 µg / ml produces a percentage of migrated cells for all the time intervals considered, compared to treatment with the same active at a concentration of 0.1 µg / ml, but at the same time produces a greater increase in the percentage of migrated cells compared to the negative control after 8h of treatment up to 72h with a percentage increase of 4.62%, 20.28%, 14.84% and 7.47%, respectively. On the other hand, treatment with synthetic Plantaricin at a concentration of 1µg / ml produces a greater increase in the percentage of migrated cells compared to the negative control only after 12 and 24h of treatment with percentage values of 9.86% and 15.48%, respectively.

Even if treating the cells with the same concentrations of synthetic Plantaricin compared to co-culture Plantaricin does not produce an increase in the percentage of migrated cells greater than the negative control at all the time intervals considered, the comparison of the results obtained from the experiment of wound healing on human keratinocytes NCTC 2544 treated with plantaricin from co-culture with the results obtained from the same experiment treating the cells instead with synthetic plantaricin, allowed to demonstrate that plantaricin from co-culture has a greater effect on cell migration through the cut area created with respect to the negative control considered.

The TGFβ1 gene is the gene most involved in the healing process.

Of all the times considered for the monitoring of wound healing, the gene expression evaluation was carried out after 8 hours of treatment as confirmation of the data obtained from the wound healing evaluation.

The results obtained highlight and confirm the significant effect of connectivin on scarring.

As regards the data on synthetic Plantaricin, it is highlighted that gene expression is significantly increased even if it is possible to note that at the same concentrations (1 and 10 µg / ml) the plantaricin from co-culture stimulates a greater increase in Expression of the gene, again confirming the action of biomass.

Claims (12)

CLAIMS 1) Biotechnological process for the synthesis of a biomass comprising or consisting of at least one plantaricin chosen from plantaricin A, K or N or their mixtures and the lactic bacteria Lactobacillus plantarum DSM 23213 and Lactobacillus rossiae DSM 23214 or for the preparation of one or more selected plantaricins between plantaricin A, K or N or their mixtures, said process comprising the or being consisting 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. 2) Process according to claim 1, wherein, the suspension of step 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. 3) Process according to any one of the preceding claims, in which the incubation is carried out at a temperature from 30 to 37 ° C, preferably 30 ° C, for 18 - 24 h, preferably 18 h. 4) Process according to any one of the preceding claims in which the centrifugation is carried out at 10,000 x g for 15 min at 4 ° C. 5) Process according to any one of the preceding claims, which further comprises a step e) of dehydration of the supernatant obtained in step d) by drying or lyophilization. 6) Biomass obtainable or obtained by the process as defined in each of claims 1-5, said biomass comprising or consisting of at least one plantaricin selected from plantaricin A, K or N, preferably A, or their mixtures and the lactic bacteria Lactobacillus plantarum DSM 23213 and Lactobacillus rossiae DSM 23214. 7) Pharmaceutical or cosmetic composition comprising or consisting of biomass as defined in claim 6, as active principle, in association with one or more pharmaceutically acceptable excipients and / or adjuvants. 8) Use of the biomass as defined in claim 6 or of the composition as defined in claim 7 for the preparation of a medicament to reinforce the barrier function at the level of epidermis or intestinal wall. 9) Use of the biomass as defined in claim 6 or of the composition as defined in claim 7 for the preparation of a wound healing medicament. 10) Lactic bacterium Lactobacillus plantarum DSM 23213 or Lactobacillus rossiae DSM 23214 or their mixture. 11) Use of one or more plantaricins chosen from plantaricin A, K or N, preferably A, for the preparation of a drug to reinforce the barrier function at the level of the epidermis. 12) Use of one or more plantaricins chosen from plantaricin A, K or N, preferably A, for the preparation of a medicament for the healing of wounds.