Barbara Marzani

Plantaricin A synthesized by Lactobacillus plantarum induces in vitro proliferation and migration of human keratinocytes and increases the expression of TGF-β1, FGF7, VEGF-A and IL-8 genes.

This work showed the effect of pheromone plantaricin A (PlnA) on the proliferation and migration of the human keratinocytes NCTC 2544. PlnA was chemically synthesized and used as pure peptide or biologically synthesized during co-cultivation of Lactobacillus plantarum DC400 and Lactobacillus sanfranciscensis DPPMA174. The cell-free supernatant (CFS) was used as the crude preparation containing PlnA. The inductive effect of PlnA on the proliferation of NCTC 2544 cells was higher than that found for hyaluronic acid, a well known skin protective compound. As shown by scratch assay and image analyses, PlnA enhanced the migration of NCTC 2544 cells. Compared to the basal serum free medium (control), the highest inductive effect was found using 10μg/ml of chemically synthesized PlnA. Similar results (P>0.05) were found for CFS. In agreement, the percentage of the starting scratch area was decreased after treatment (24h) with PlnA. The expression of transforming growth factor-β1 (TGF-β1), keratinocyte growth factor 7 (FGF7), vascular endothelial growth factor (VEGF-A), and interleukin-8 (IL-8) genes was affected by PlnA. Compared to control, TGF-β1 gene was under expressed in the first 4h of treatments and up-regulated after 8-24h. On the contrary, FGF7 gene was strongly up-regulated in the first 4h of treatments. Compared to control, VEGF-A and IL-8 genes were always up-regulated during the 4-24h from scratching. Since capable of promoting the proliferation and migration of the human keratinocytes and of stimulating IL-8 cytokine, the use of PlnA for dermatological purposes should be considered.

Lactic acid fermentation as a tool to enhance the antioxidant properties of Myrtus communis berries

BackgroundMyrtle (Myrtus communis L.) is a medicinal and aromatic plant belonging to Myrtaceae family, which is largely diffused in the Mediterranean areas and mainly cultivated in Tunisia and Italy. To the best of our knowledge, no studies have already considered the use of the lactic acid fermentation to enhance the functional features of M. communis. This study aimed at using a selected lactic acid bacterium for increasing the antioxidant features of myrtle berries, with the perspective of producing a functional ingredient, dietary supplement or pharmaceutical preparation. The antioxidant activity was preliminarily evaluated through in vitro assays, further confirmed through ex vivo analysis on murine fibroblasts, and the profile of phenol compounds was characterized.ResultsMyrtle berries homogenate, containing yeast extract (0.4%, wt/vol), was fermented with Lactobacillus plantarum C2, previously selected from plant matrix. Chemically acidified homogenate, without bacterial inoculum and incubated under the same conditions, was used as the control. Compared to the control, fermented myrtle homogenate exhibited a marked antioxidant activity in vitro. The radical scavenging activity towards DPPH increased by 30%, and the inhibition of linoleic acid peroxidation was twice. The increased antioxidant activity was confirmed using Balb 3xa0T3 mouse fibroblasts, after inducing oxidative stress, and determining cell viability and radical scavenging activity through MTT and DCFH-DA assays, respectively. The lactic acid fermentation allowed increased concentrations of total phenols, flavonoids and anthocyanins, which were 5–10 times higher than those found for the non-fermented and chemically acidified control. As shown by HPLC analysis, the main increases were found for gallic and ellagic acids, and flavonols (myricetin and quercetin). The release of these antioxidant compounds would be strictly related to the esterase activities of L. plantarum.ConclusionsThe lactic acid fermentation of myrtle berries is a suitable tool for novel applications as functional food dietary supplements or pharmaceutical preparations.

Lactic acid fermentation as a tool to enhance the functional features of Echinacea spp

BackgroundExtracts and products (roots and/or aerial parts) from Echinacea ssp. represent a profitable market sector for herbal medicines thanks to different functional features. Alkamides and polyacetylenes, phenols like caffeic acid and its derivatives, polysaccharides and glycoproteins are the main bioactive compounds of Echinacea spp. This study aimed at investigating the capacity of selected lactic acid bacteria to enhance the antimicrobial, antioxidant and immune-modulatory features of E. purpurea with the prospect of its application as functional food, dietary supplement or pharmaceutical preparation.ResultsEchinacea purpurea suspension (5%, wt/vol) in distilled water, containing 0.4% (wt/vol) yeast extract, was fermented with Lactobacillus plantarum POM1, 1MR20 or C2, previously selected from plant materials. Chemically acidified suspension, without bacterial inoculum, was used as the control to investigate functional features. Echinacea suspension fermented with Lb. plantarum C2 exhibited a marked antimicrobial activity towards Gram-positive and -negative bacteria. Compared to control, the water-soluble extract from Echinacea suspension fermented with Lactobacillus plantarum 1MR20 showed twice time higher radical scavenging activity on DPPH. Almost the same was found for the inhibition of oleic acid peroxidation. The methanol extract from Echinacea suspension had inherent antioxidant features but the activity of extract from the sample fermented with strain 1MR20 was the highest. The antioxidant activities were confirmed on Balb 3T3 mouse fibroblasts. Lactobacillus plantarum C2 and 1MR20 were used in association to ferment Echinacea suspension, and the water-soluble extract was subjected to ultra-filtration and purification through RP-FPLC. The antioxidant activity was distributed in a large number of fractions and proportional to the peptide concentration. The antimicrobial activity was detected only in one fraction, further subjected to nano-LC-ESI-MS/MS. A mixture of eight peptides was identified, corresponding to fragments of plantaricins PlnH or PlnG. Treatments with fermented Echinacea suspension exerted immune-modulatory effects on Caco-2 cells. The fermentation with Lb. plantarum 1MR20 or with the association between strains C2 and 1MR20 had the highest effect on the expression of TNF-α gene.ConclusionsE. purpurea subjected to lactic acid fermentation could be suitable for novel applications as functional food dietary supplements or pharmaceutical preparations.

Italian legumes: effect of sourdough fermentation on lunasin-like polypeptides.

AbstractBackgroundThere is an increasing interest toward the use of legumes in food industry, mainly due to the quality of their protein fraction. Many legumes are cultivated and consumed around the world, but few data is available regarding the chemical or technological characteristics, and especially on their suitability to be fermented. Nevertheless,n sourdough fermentation with selected lactic acid bacteria has been recognized as the most efficient tool to improve some nutritional and functional properties. This study investigated the presence of lunasin-like polypeptides in nineteen traditional Italian legumes, exploiting the potential of the fermentation with selected lactic acid bacteria to increase the native concentration. An integrated approach based on chemical, immunological and ex vivo (human adenocarcinoma Caco-2 cell cultures) analyses was used to show the physiological potential of the lunasin-like polypeptides.ResultsItalian legume varieties, belonging to Phaseulus vulgaris, Cicer arietinum, Lathyrus sativus, Lens culinaris and Pisum sativum species, were milled and flours were chemically characterized and subjected to sourdough fermentation with selected Lactobacillus plantarum C48 and Lactobacillus brevis AM7, expressing different peptidase activities. Extracts from legume doughs (unfermented) and sourdoughs were subjected to western blot analysis, using an anti-lunasin primary antibody. Despite the absence of lunasin, different immunoreactive polypeptide bands were found. The number and the intensity of lunasin-like polypeptides increased during sourdough fermentation, as the consequence of the proteolysis of the native proteins carried out by the selected lactic acid bacteria. A marked inhibitory effect on the proliferation of human adenocarcinoma Caco-2 cells was observed using extracts from legume sourdoughs. In particular, sourdoughs from Fagiolo di Lamon, Cece dell’Alta Valle di Misa, and Pisello riccio di Sannicola flours were the most active, showing a decrease of Caco-2 cells viability up to 70xa0%. The over-expression of Caco-2 filaggrin and involucrin genes was also induced. Nine lunasin-like polypeptides, having similarity to lunasin, were identified.ConclusionsThe features of the sourdough fermented legume flours suggested the use for the manufacture of novel functional foods and/or pharmaceuticals preparations.

Improving the antioxidant properties of quinoa flour through fermentation with selected autochthonous lactic acid bacteria

Lactic acid bacteria strains, previously isolated from the same matrix, were used to ferment quinoa flour aiming at exploiting the antioxidant potential. As in vitro determined on DPPH and ABTS radicals, the scavenging activity of water/salt-soluble extracts (WSE) from fermented doughs was significantly (P<0.05) higher than that of non-inoculated doughs. The highest inhibition of linoleic acid autoxidation was found for the quinoa dough fermented with Lactobacillus plantarum T0A10. The corresponding WSE was subjected to Reverse Phase Fast Protein Liquid Chromatography, and 32 fractions were collected and subjected to in vitro assays. The most active fraction was resistant to further hydrolysis by digestive enzymes. Five peptides, having sizes from 5 to 9 amino acid residues, were identified by nano-Liquid Chromatography-Electrospray Ionisation-Mass Spectra/Mass Spectra. The sequences shared compositional features which are typical of antioxidant peptides. As shown by determining cell viability and radical scavenging activity (MTT and DCFH-DA assays, respectively), the purified fraction showed antioxidant activity on human keratinocytes NCTC 2544 artificially subjected to oxidative stress. This study demonstrated the capacity of autochthonous lactic acid bacteria to release peptides with antioxidant activity through proteolysis of native quinoa proteins. Fermentation of the quinoa flour with a selected starter might be considered suitable for novel applications as functional food ingredient, dietary supplement or pharmaceutical preparations.

The antimicrobial peptide pheromone Plantaricin A increases antioxidant defenses of human keratinocytes and modulates the expression of filaggrin, involucrin, β-defensin 2 and tumor necrosis factor-α genes.

Plantaricin A (PlnA) is a peptide with antimicrobial and pheromone activities. PlnA was synthesized chemically and used as a pure peptide or synthesized biologically using Lactobacillus plantarum DC400 co‐cultured with Lactobacillus sanfranciscensis DPPMA174. Cell‐free supernatant (CFS) was used as a crude PlnA preparation. As estimated using the 3‐(4,5‐dimethyl‐2‐yl)‐2,5‐diphenyltetrazolium bromide and the 2’,7’–dichlorofluorescein diacetate assays, both PlnA preparations increased the antioxidant defenses of human NCTC 2544 keratinocytes. PlnA (10 μg/ml) had a higher activity than hyaluronic acid or 125 μg/ml α‐tocopherol. Effects on the transcriptional regulation of filaggrin (FLG), involucrin (IVL), hyaluronan synthase (HAS2), human β‐defensin‐2 (HBD‐2) and tumor necrosis factor‐alpha (TNF‐α) genes were assayed. Compared with the control, expression of the FLG gene in NCTC 2544 cells increased in cells treated with hyaluronic acid, 1 or 10 μg/ml PlnA. Compared with the control, the level of IVL gene expression increased in NCTC 2544 cells treated with 10 μg/ml PlnA. No significant difference was found between the level of the HAS2 gene expressed by control cells and cells treated with PlnA. Compared with chemically synthesized PlnA, the up‐regulation of the HBD‐2 gene by CFS was higher. Compared with the control, expression of TNF‐α decreased in NCTC 2544 cells after treatment with 1 or 10 μg/ml of chemically synthesized PlnA. In contrast, the level of TNF‐α was highest in the presence of 10 μg/ml CFS‐PlnA. These findings suggest that the PlnA was positively sensed by human keratinocytes, promoting antioxidant defenses, barrier functions and antimicrobial activity of the skin.

Exploitation of grape marc as functional substrate for lactic acid bacteria and bifidobacteria growth and enhanced antioxidant activity

This study aimed at using grape marc for the growth of lactic acid bacteria and bifidobacteria with the perspective of producing a functional ingredient having antioxidant activity. Lactobacillus plantarum 12A and PU1, Lactobacillus paracasei 14A, and Bifidobacterium breve 15A showed the ability to grow on grape marc (GM) based media. The highest bacterial cell density (>9.0xa0CFU/g) was found in GM added of 1% of glucose (GMG). Compared to un-inoculated and incubated control fermented GMG showed a decrease of carbohydrates and citric acid together with an increase of lactic acid. The content of several free amino acids and phenol compounds differed between samples. Based on the survival under simulated gastro-intestinal conditions, GMG was a suitable carrier of lactic acid bacteria and bifidobacteria strains. Compared to the control, cell-free supernatant (CFS) of fermented GMG exhibited a marked antioxidant activity inxa0vitro. The increased antioxidant activity was confirmed using Caco-2xa0cell line after inducing oxidative stress, and determining cell viability and radical scavenging activity through MTT and DCFH-DA assays, respectively. Supporting these founding, the SOD-2 gene expression of Caco-2xa0cells also showed a lowest pro-oxidant effect induced by the four CFS of GMG fermented by lactic acid bacteria and bifidobacteria.

A specific combination of zeaxanthin, spermidine and rutin prevents apoptosis in human dermal papilla cells

Hair follicle (HF) regression is characterized by the activation of apoptosis in HF cells. Dermal papilla cells play a leading role in the regulation of HF development and cycling. Human follicular dermal papilla cells (HFDPC) were used to investigate the protective activities of rutin, sperimidine and zeaxanthine. HFDP cell incubation with staurosporine caused apoptosis, which was completely inhibited by exposure to rutin (2.2 μm), spermidine (1 μm) and zeaxanthin (80 μm). These agents were much less effective when applied as single compounds. Moreover, treatment preserved the expression of anti‐apoptotic molecules such as Bcl‐2, MAP‐kinases and their phosphorylated forms. In conclusion, the investigated agents may represent an effective treatment for the prevention of apoptosis, one of the leading events involved in hair bulb regression.

N(1)-methylspermidine, a stable spermidine analog, prolongs anagen and regulates epithelial stem cell functions in human hair follicles.

Spermidine (Spd), the prototypic polyamine, has been shown to be essential for hair follicle (HF) growth. However, Spd can be readily converted into other polyamines, and is physiologically unstable. Therefore, to assess its individual functions on HFs, we used the metabolically stable Spd analog N1-methylspermidine (N1-MeSpd). N1-MeSpd was confirmed to be a metabolically stable compound, with a half life of 90xa0h. 0.5xa0µM N1-MeSpd strongly prolonged anagen and decreased cell apoptosis in HFs in culture after 6xa0days, accompanied by specific stimulation of the expression of the epithelial stem cell-associated keratin, K15. N1-MeSpd also reduced lactate dehydrogenase activity in the culture supernatant, a parameter of cell death and cell lysis. N1-MeSpd diminished intracellular reactive oxygen species production in cultured keratinocytes, and reduced tumor necrosis factor-α, interleukin (IL)-1β and IL-6 gene and protein expression after lipopolysaccharide stimulation. This suggests that some effects of N1-MeSpd may be mediated by anti-oxidative and anti-inflammatory effects. These additional properties of N1-MeSpd could be clinically important for the treatment of inflammatory alopecias and inflammatory scalp diseases.

IL-17 inhibition: is it the long-awaited savior for alopecia areata?

Interleukin-17 (IL-17) has been implicated in the pathogenesis of a large number of inflammatory and autoimmune conditions, including skin disorders such as psoriasis. Recently, much data have accumulated on the possible role of IL-17 in the pathogenesis of alopecia areata (AA). In this review, the available information on the connection between AA and IL-17 is described. While IL-17 levels are consistently reported to be elevated in the serum and lesional skin of AA patients, there is no clear connection between IL-17 levels and disease severity or duration. Some evidence has suggested an association between IL-17 and an early-onset disease, although this awaits further confirmation. While there is enough information to support clinical trials with IL-17-targeted treatments, it is possible that they will be effective only in a subset of AA patients. Further studies are warranted to better delineate the exact role of IL-17 in AA pathogenesis.