Daniela Pinto

Selected lactic acid bacteria synthesize antioxidant peptides during sourdough fermentation of cereal flours

ABSTRACT A pool of selected lactic acid bacteria was used for the sourdough fermentation of various cereal flours with the aim of synthesizing antioxidant peptides. The radical-scavenging activity of water/salt-soluble extracts (WSE) from sourdoughs was significantly (P < 0.05) higher than that of chemically acidified doughs. The highest activity was found for whole wheat, spelt, rye, and kamut sourdoughs. Almost the same results were found for the inhibition of linoleic acid autoxidation. WSE were subjected to reverse-phase fast protein liquid chromatography. Thirty-seven fractions were collected and assayed in vitro. The most active fractions were resistant to further hydrolysis by digestive enzymes. Twenty-five peptides of 8 to 57 amino acid residues were identified by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry. Almost all of the sequences shared compositional features which are typical of antioxidant peptides. All of the purified fractions showed ex vivo antioxidant activity on mouse fibroblasts artificially subjected to oxidative stress. This study demonstrates the capacity of sourdough lactic acid bacteria to release peptides with antioxidant activity through the proteolysis of native cereal proteins.

Robustness of Lactobacillus plantarum starters during daily propagation of wheat flour sourdough type I.

This study aimed at investigating the robustness of selected sourdough strains of Lactobacillus plantarum. Seven strains were singly used as sourdough type I starters under daily back-slopping propagation (ten days) using wheat flour. Cell numbers of presumptive lactic acid bacteria varied slightly (median values of 9.13-9.46 log cfu g(-1)) between and within started sourdoughs, as well as the acidifying activity (median values of 1.24-1.33). After three days also the control sourdough (unstarted) had the same values. As shown by RAPD-PCR analysis, five (DB200, 3DM, G10C3, 12H1 and LP20) out of seven strains maintained elevated cell numbers (ca. 9 log cfu g(-1)) throughout ten days. The other two strains progressively decreased to less than 5 log cfu g(-1). As identified by partial sequencing of 16S rRNA and recA genes, L. plantarum (11 isolates), pediococci (7), Lactobacillus casei (3) and Lactobacillus rossiae (2) dominated the flour microbiota. Monitoring of lactic acid bacteria during sourdough propagation was carried out by culture dependent approach and using PCR-DGGE (Denaturing Gradient Gel Electrophoresis). Except for the sourdough started with L. plantarum LP20, in all other sourdoughs at least one autochthonous strain of L. plantarum emerged. All emerging strains of L. plantarum showed different RAPD-PCR profiles. L. rossiae and Pediococcus pentosaceus were only found in the control and sourdough started with strain 12H1. The characterization of the catabolic profiles of sourdoughs (Biolog System) showed that sourdoughs containing persistent starters behaved similarly and their profiles were clearly differentiated from the others. One persistent strain (DB200) of L. plantarum and Lactobacillus sanfranciscensis LS44, previously shown to be persistent (Siragusa et al., 2009), were used as the mixed starter to produce a wheat flour sourdough. Both strains cohabited and dominated during ten days of propagation.

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 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.

The crystal structure of vurroite, Pb20Sn2(Bi, As)22S54Cl6: OD-character, polytypism, twinning, and modular description

Abstract The crystal structure of the type specimen of vurroite from Vulcano (Aeolian Islands, Italy) has been solved and refined using single-crystal X-ray diffraction data collected at the Elettra synchrotron facility (Basovizza, Trieste). Vurroite has an OD (order-disorder) structure belonging to the category III of OD structures composed of equivalent layers. The OD-groupoid family (λ and σ partial operations) and MDO structures were derived by means of the application of the OD theory. The two theoretically possible polytypes with maximum degree of order (MDO polytypes) have pseudo-orthorhombic metric, with lattice parameters a ≈ 45.6, b ≈ 8.4, c ≈ 54 Å, and a ≈ 22.8, b ≈ 8.4, c ≈ 54 Å, and space group symmetries F2/d11 and A2/d11, respectively. Only the former polytype (MDO1) could be identified in the analyzed crystals. The MDO1 structure was solved and refined to R = 6.26% for 4968 reflections with Fo > 4σ(Fo). In the standard C2/c setting of the space group the unit-cell parameters of MDO1 are a = 8.371(2), b = 45.502(9), c = 27.273(6) Å, β = 98.83(3)°, V = 10265(4) Å3, Z = 4. Frequent twinning with (001) as the twin plane, together with the occurrence of disordered domains in the structure, was observed. The crystal structure of vurroite contains lozenge-shaped composite rods made of coordination polyhedra of Pb and Sn, interconnected into layers parallel to (010) of the standard monoclinic setting. These layers are separated by ribbons of As and Bi, each in distorted octahedral coordination. The ribbons form wavy, discontinuous double layers of PbS archetype. Lone electron pairs of As and Bi are accommodated in the central portions of the PbS-like layers. The structure of vurroite contains building blocks topologically similar to those found in the zinckenite group and in the structure of kirkiite. It can be considered a box-work structure containing the smallest possible pseudo-hexagonal block in the form of a sole octahedron.

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.

First occurrence of iodine in natural sulfosalts : The case of mutnovskite, Pb2AsS3(I,Cl,Br), a new mineral from the Mutnovsky volcano, Kamchatka Peninsula, Russian Federation

Abstract Mutnovskite, ideally Pb2AsS3(I,Cl,Br), is a new mineral from the high-temperature fumaroles of the Mutnovsky volcano, Kamchatka Peninsula, Russian Federation. It occurs as microscopic rubycolored short-prismatic crystals up to 100 μm across, closely associated with halogen-sulfosalts of Pb, Bi, and As, Cd-Pb-Bi sulfosalts, pyrite, anhydrite, and cristobalite. Mutnovskite is transparent in thin fragments with a dark-red to blue color. The crystals are soft and fragile. Cleavage and fracture were not observed and the Mohs hardness is approximately 2. In reflected light mutnovskite is silvery lead-grey in color with an iridescent tarnish. Pleochroism and anisotropy are not visible because of the strong orange internal reflections, especially in immersion. Reflectance percentages measured in air in the range 400.700 nm were tabulated. Reflectance percentages (Rmin and Rmax) for the four COM wavelengths are 34.2, 34.6 (470 nm), 33.2, 33.5 (546 nm), 32.5, 32.7 (589 nm), and 31.4, 31.7 (650 nm), respectively. A mean of four electron microprobe analyses gave Pb 62.0(3), As 11.0(4), Bi 0.6(1), S 14.4(2), Se 0.2(3), I 8.9(3), Cl 2.44(9), Br 1.1(7), Cu 0.03(2), Fe 0.01(1), total 100.7 wt%, corresponding, on the basis of a total of 7 atoms, to Pb1.99(As0.98Bi0.02)Σ1.00(S2.98Se0.02)Σ3.00(I0.47Cl0.46Br0.09)Σ1.02. The nine strongest powder-diffraction lines [d in Å (I/I0) (hkl)] are: 4.69 (32) (002); 4.37 (67) (210); 3.34 (73) (020); 3.19 (100) (212); 2.715 (61) (022); 2.648 (66) (410); 2.539 (31) (213); 2.455 (29) (402); 1.894 (30) (232). Mutnovskite is orthorhombic, space group Pnma, with a = 11.543(1), b = 6.6764(7), and c = 9.359(1) Å, V = 721.3(1) Å3, Z = 4. The crystal structure was solved and refined to R = 4.14%. It consists of three independent cation positions: Pb1 and Pb2 have tricapped trigonal prismatic coordinations with S and I atoms (completed with one As atom in the case of Pb2), while As has threefold coordination with S atoms, which form the base of a trigonal pyramid with As at the apex. Pairs of Pb1-Pb2 prisms are connected in columns which extend along c. AsS3 coordinations are isolated from each other. S atoms and half of the Pb atoms form wavy close-packed layers. Two kinds of channels parallel to boccur between the layers. The smaller channels host As atoms close to the channel walls, with their lone-electron pairs occupying the median part, while the bigger ones accommodate rows of alternating halogen and Pb atoms. The new mineral is named after the type locality, the Mutnovsky volcano, Kamchatka Peninsula, Russian Federation.

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.

First occurrence of close-to-ideal kirkiite at Vulcano (Aeolian Islands, Italy) : chemical data and single-crystal X-ray study

Samples of kirkiite from the high temperature fumaroles of La Fossa crater of Vulcano (Aeolian islands, Italy) were chemically and structurally investigated in this work. Associated minerals are vurroite, bismuthinite, galenobismutite, cannizzarite, lillianite, heyrovskýite, galena, and other less characterized Pb(Bi)-sulfochlorides. Electron-microprobe analyses gave the average chemical formula Pb 10.00 Bi 3.01 As 3.01 (S 18.47 Se 0.44 C 10.06 ) which is very close to the ideal composition of kirkiite, Pb 10 Bi 3 As 3 S 19 , and indeed significantly closer than the composition of the type specimen, Pb 10.08 Bi 2.55 Sb 0.13 As 2.91 S 19 . Lattice parameters are: a = 8.700(2) A, β = 26.237(6) A, c = 8.774(3) A, β = 119.653(4)°, V = 1740.2(9) A 3 . A twinned structure was refined using single-crystal data (Mo K α X-ray diffraction, CCD detector). The refinement converges to R = 0.074 for 1443 reflections with F 0 > 4σ(F 0 ). The structure of the close-to-ideal kirkiite from Vulcano has been compared with the structure of the type specimen. The comparison reveals a variation in As-Bi substitution, with samples from Vulcano probably being close to the maximum possible Bi and the minimum As content for this structure type. This is reflected in more regular and symmetric coordination polyhedra than in the holotype, as well as in the overall regularity of the structure. The increased Bi:As ratio produces an elongation of the a and b lattice periods, and a shortening of the c period, and increases the frequency of twinning in kirkiite.

Lucabindiite, (K,NH4)As4O6(Cl,Br), a new fumarole mineral from the “La Fossa” crater at Vulcano, Aeolian Islands, Italy

Abstract Lucabindiite, ideally (K,NH4)As4O6(Cl,Br), is a new mineral found as a medium-temperature fumarole encrustation (T = 170 °C) at “La Fossa” crater of Vulcano, Aeolian Islands, Italy. The mineral deposited as aggregates of micrometer-sized hexagonal and platy crystals on the surface of the pyroclastic breccia in association with arsenolite, sal ammoniac, sulfur, and amorphous arsenic-rich sulfurite. The new mineral is colorless to white, transparent, non-fluorescent, has a vitreous luster and a white streak. The calculated density is 3.68 g/cm3. Lucabindiite is hexagonal, space group P6/mmm, with a = 5.2386(7) Å, c = 9.014(2) Å, V = 214.23(7) Å3, and Z = 1. The eight strongest reflections in the X-ray powderdiffraction data [d in Å (I) (hkl)] are: 3.20 (100) (102), 2.62 (67) (110), 4.51 (52) (002), 4.54 (30) (100), 1.97 (28) (113), 1.49 (21) (115), 1.60 (21) (212), 2.26 (19) (112). Lucabindiite’s average chemical composition is (wt%): K2O 5.14, As2O3 84.71, Cl 3.63, Br 6.92, F 0.77, (NH4)2O 2.73, O=F,Cl,Br -1.84, total 102.06. The empirical chemical formula, calculated on the basis of 7 anions pfu, is [K0.51(NH4)0.49]Σ1.00 As4.00O5.93(Cl0.48Br0.40F0.19)Σ1.07. According to chemical analyses and X-ray data, lucabindiite is the natural analog of synthetic phases with general formula MAs4O6X where M = K, NH4 and X = Cl, Br, I. The crystal structure is characterized by neutral As2O3 sheets arranged parallel to (001). The As atoms of two neighboring sheets point at each other and the sheets are separated by interlayer M (=K, NH4) and X (=Cl, Br, F) atoms. The name is in honor of Luca Bindi (b. 1971), Professor of Mineralogy and former Head of the Division of Mineralogy of the Natural History Museum of the University of Florence. Both the mineral and the mineral name have been approved by the IMA-CNMNC Commission (IMA 2011-010).