Tannat grape pomace as an ingredient for potential functional biscuits: bioactive compound identification, in vitro bioactivity, food safety, and sensory evaluation.

Grape pomace, the main by-product of wine process, shows high potential for the development of functional foods, being a natural source of bioactive compounds and dietary fiber. Thus, the present study proposes the development of five potential functional biscuits. The five formulations were achieved by varying the Tannat grape pomace powder (TGP, 10-20% w/w total wet dough) and sweetener sucralose (2-4% w/w total wet dough) content through a factorial design with central points. TGP microbiological and pesticides analysis were performed as a food safety requirement. Identification of bioactive compounds by HPLC-DAD-MS, in vitro bioactivity (total phenol content, antioxidant by ABTS and ORAC-FL, antidiabetic and antiobesity by inhibition of α-glucosidase and pancreatic lipase, respectively) and sensory properties of the biscuits were evaluated. TGP microbiological and pesticides showed values within food safety criteria. Sensory profiles of TGP biscuits were obtained, showing biscuits with 20% TGP good sensory quality (7.3, scale 1-9) in a cluster of 37 out of 101 consumers. TGP addition in biscuits had a significant (p < 0.05) effect on total phenolic content (0.893-1.858 mg GAE/g biscuit) and bioactive properties when compared to controls: 11.467-50.491 and 4.342-50.912 µmol TE/g biscuit for ABTS and ORAC-FL, respectively; inhibition of α-glucosidase and pancreatic lipase, IC50 35.572-64.268 and 7.197-47.135 mg/mL, respectively. HPLC-DAD-MS results showed all the identified phenolic compounds in 20/4% biscuit (TGP/sucralose%) were degraded during baking. Malvidin-3-O-(6'-p-coumaroyl) glucoside, (+)-catechin, malvidin-3-O-glucoside, and (-)-epicatechin were the main phenolic compounds (in descendent order of content) found. The bioactive properties could be attributed to the remaining phenolic compounds in the biscuits. In conclusion, TGP biscuits seemed to be a promising functional food with potential for ameliorating oxidative stress, glucose and fatty acids levels with good sensory quality.

Bioprospecting the Antibiofilm and Antimicrobial Activity of Soil and Insect Gut Bacteria.

Antimicrobial resistance is a growing concern in public health and current research shows an important role for bacterial biofilms in recurrent or chronic infections. New strategies, therefore, are necessary to overcome antimicrobial resistance, through the development of new therapies that could alter or inhibit biofilm formation. In this sense, antibiofilm natural products are very promising. In this work, a bioprospection of antimicrobial and antibiofilm extracts from Uruguayan soil bacteria and insect gut bacteria was carried out. Extracts from extracellular broths were tested for their ability to inhibit planktonic cell growth and biofilm formation. Genomic analysis of Bacillus cereus ILBB55 was carried out. All extracts were able to inhibit the growth of, at least, one microorganism and several extracts showed MICs lower than 500 µg mL-1 against microorganisms of clinical relevance (Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacter cloacae). Among the extracts evaluated for biofilm inhibition only ILBB55, from B. cereus, was able to inhibit, S. aureus (99%) and P. aeruginosa (62%) biofilms. Genomic analysis of this strain showed gene clusters similar to other clusters that code for known antimicrobial compounds. Our study revealed that extracts from soil bacteria and insect gut bacteria, especially from B. cereus ILBB55, could be potential candidates for drug discovery to treat infectious diseases and inhibit S. aureus and P. aeruginosa biofilms.

Biogenic Silver Nanoparticles as a Strategy in the Fight Against Multi-Resistant Salmonella enterica Isolated From Dairy Calves.

Infectious diseases are one of the most important health problems worldwide, one of the main causes being the development of multi-resistant microorganisms. Likewise, the zoonotic potential of some pathogens and their ability to transfer resistance mechanisms, reduce the therapeutic options in both humans and animals. Salmonella enterica is an important pathogen that affects a wide range of animal species and humans, being Salmonella Typhimurium one of the most frequent serotypes affecting cattle, causing enteritis, diarrhea, and septicemia. The search for alternative therapeutic approaches has gained importance since the emergence of multidrug resistance to antibiotics and periodic outbreaks of salmonellosis. In this sense, the discovery of new drugs and the development of new strategies, such as the use of nanoparticles with antimicrobial activity, are very promising. The aim of this work was the extracellular production of biogenic silver nanoparticles using fungal extracts and the evaluation of their antimicrobial activity against resistant and multi-resistant Salmonella Typhimurium strains. We here demonstrated the potential of the biogenic nanoparticles as effective bacteriostatic and bactericidal agents for use in biomedical applications. In addition, Confocal Raman Microscopy and Atomic Force Microscopy were used to advance the understanding of the antimicrobial mechanism of biogenic nanoparticles against these pathogenic strains, the results of which suggested that the nanoparticles produced damage in several bacterial cell structures.

Biofilm Eradication Using Biogenic Silver Nanoparticles.

Microorganisms offer an alternative green and scalable technology for the synthesis of value added products. Fungi secrete high quantities of bioactive substances, which play dual-functional roles as both reducing and stabilizing agents in the synthesis of colloidal metal nanoparticles such as silver nanoparticles, which display potent antimicrobial properties that can be harnessed for a number of industrial applications. The aim of this work was the production of silver nanoparticles using the extracellular cell free extracts of Phanerochaete chrysosporium, and to evaluate their activity as antimicrobial and antibiofilm agents. The 45-nm diameter silver nanoparticles synthesized using this methodology possessed a high negative surface charge close to -30 mV and showed colloidal stability from pH 3-9 and under conditions of high ionic strength ([NaCl] = 10-500 mM). A combination of environmental SEM, TEM, and confocal Raman microscopy was used to study the nanoparticle-E. coli interactions to gain a first insight into their antimicrobial mechanisms. Raman data demonstrate a significant decrease in the fatty acid content of E. coli cells, which suggests a loss of the cell membrane integrity after exposure to the PchNPs, which is also commensurate with ESEM and TEM images. Additionally, these biogenic PchNPs displayed biofilm disruption activity for the eradication of E. coli and C. albicans biofilms.

Biogenic nanoparticles: Synthesis, stability and biocompatibility mediated by proteins of Pseudomonas aeruginosa.

The development of environmental friendly new procedures for the synthesis of metallic nanoparticles is one of the main objectives of nanotechnology. Plants, algae, fungi and bacteria for the production of nanomaterials are viable alternatives due to their low cost, the absence of toxic waste production and their highly energy efficiency. It is also known that biosynthesized silver nanoparticles (AgNPs) show higher biocompatibility compared to the chemically-synthesized ones. In previous results, biosynthesized AgNPs were obtained from the supernatant of Pseudomonas aeruginosa, and they showed a bigger antimicrobial activity against different bacterial species compared to the chemically-synthesized ones. The aim of this work was to analyze the capping of biosynthesized AgNPs using techniques such as transmission electron microscopy (TEM), infrared spectroscopy (IR), and protein identification through mass spectrometry (MS) in order to identify the compounds responsible for their formation, stability and biocompatibility. The TEM images showed that AgNPs were surrounded by an irregular coverage. The IR spectrum showed that this coverage was composed of carbohydrates and/or proteins. Different proteins were identified in the capping associated to biosynthesized AgNPs. Some proteins seem to be important for their formation (Alkyl hydroperoxide reductase and Azurin) and stabilization (Outer membrane protein OprG and Glycine zipper 2 T M domain-containing protein). The proteins identified with the capability to interact with some biomolecules can be responsible for the biocompatibility and may be responsible for the bigger antimicrobial activity than AgNPs have previously shown. These results are pioneers in the identification of proteins in the capping of biosynthesized AgNPs.

Effects of Culture Conditions on Antimicrobial Activity of Ganoderma resinaceum (Agaricomycetes) Extracts.

Among many sources of natural bioactive substances, mushrooms constitute a huge and mostly unexplored group. Biologically active secondary metabolites of Ganoderma, a group of wood-degrading mushrooms, have recently been reviewed. Our previous study revealed the antimicrobial activity of extracts from G. resinaceum grown in submerged culture against phytopathogens. Different factors can influence the production of secondary metabolites, including nutritional factors. In this study we evaluated the influence of different culture conditions on the antimicrobial activity of extracts from liquid cultures of G. resinaceum, through use of a factorial design. Minimum inhibitory concentrations for extracts produced under different culture conditions were determined against Staphylococcus aureus and Xanthomonas vesicatoria. Based on the results of these assays, larger-scale cultures in malt extract broth supplemented with 20 g/L glucose and a 15-day incubation time should be performed in order to isolate from G. resinaceum antibiotic compound(s) that are potentially useful against S. aureus. In addition, pH 5 should be considered for the production of antimicrobial metabolites against X. vesicatoria from supernatant broths or extracts from G. resinaceum.

Anti-MRSA Activity of Fruiting Body Extracts of Spectacular Rustgill Mushroom, Gymnopilus junonius (Agaricomycetes).

Despite the great advances in chemotherapeutics, infectious diseases are still one of the leading causes of death worldwide. Among some of the clinically relevant pathogens, methicillin-resistant Staphylococcus aureus (MRSA) ranks as one of the most difficult bacteria to treat. It is a common cause of skin, soft-tissue, and endovascular infections, as well as pneumonia, septic arthritis, endocarditis, osteomyelitis, and sepsis. The research on Basidiomycota is extensive; many species show a broad spectrum of pharmacological activities, including antimicrobial activity. The vast majority of the literature to date generally focuses on screening the antibacterial properties of mushroom extracts. A gap still exists in the identification of the individual compounds responsible for these properties, and few low molecular weight compounds have been described. Gymnopilus junonius, the big laughter mushroom, grows wild in Uruguay, especially on Eucalyptus spp. plantations; it is known as the "eucalyptus fungus." In this work, we report the bioguided isolation, structural elucidation, and antistaphylococcal activity of the main antimicrobial components of fresh basidiocarps of G. junonius.

Screening for Antimicrobial Activity of Wood Rotting Higher Basidiomycetes Mushrooms from Uruguay against Phytopathogens.

In this work, the antimicrobial activity of extracts of wood rotting higher Basidiomycetes mushrooms isolated from Eucalyptus plantations in Uruguay was studied using bacterial and fungal phytopathogens as targets. Fifty-one extracts from mycelia and growth broth were prepared from higher Basidiomycetes mushrooms, from which eight extracts (from Ganoderma resinaceum, Laetiporus sulphureus, Dictyopanus pusillus, and Bjerkandera adusta) showed antimicrobial activity against Xanthomonas vesicatoria, Aspergillus oryzae, Penicillium expansum, Botrytis cinerea, and Rhizopus stolonifer as assayed in the qualitative test. The minimum inhibitory concentration (MIC) for those fungal extracts was determined and the results showed that L. sulphureus deserved further study, with low MIC values against X. vesicatoria. The antimicrobial activity of L. sulphureus culture broth extracts grown under different culture conditions was evaluated against X. vesicatoria. From the results of these assays, larger-scale cultures for the production of the compound(s) with antimicrobial activity should be performed using malt extract broth, at pH 5, at 20°C and static culture conditions.

A Lectin Purified from Blood Red Bracket Mushroom, Pycnoporus sanguineus (Agaricomycetidae), Mycelium Displayed Affinity Toward Bovine Transferrin.

Fungal lectins constitute excellent ligands for development of affinity adsorbents useful in affinity chromatography. In this work, a lectin was purified from Pycnoporus sanguineus (PSL) mycelium using 3 procedures: by affinity chromatography, using magnetic galactosyl-nanoparticles or galactose coupled to Sepharose, and by ionic exchange chromatography (IEC). The highest lectin yield was achieved by IEC (55%); SDS-PAGE of PSL showed 2 bands with molecular mass of 68.7 and 55.2 kDa and IEC displayed 2 bands at pi 5.5 and 5.2. The lectin agglutinates rat erythrocytes, exhibiting broad specificity toward several monosaccharides, including galactose. The agglutination was also inhibited by the glycoproteins fetal calf fetuin, bovine lactoferrin, bovine transferrin, and horseradish peroxidase. The lectin was then used to synthesize an affinity adsorbent (PSL-Sepharose) and the interaction with glycoproteins was evaluated by analyzing their chromatographic behaviors. The strongest interaction with the PSL-derivative was observed with transferrin, although lower interactions were also displayed toward fetuin and lactoferrin. These results indicate that the purified PSL constitutes an interesting ligand for the design of affinity adsorbents to be used (i.e., in glycoprotein purification).

Purification and applications of a lectin from the mushroom Gymnopilus spectabilis.

A lectin was isolated from fruiting bodies of the mushroom Gymnopilus spectabilis (GSL) by ionic exchange chromatography. The lectin agglutinates mouse red cells exhibiting broad specificity towards several monosaccharides including the N-acetylneuraminic acid. Agglutination was also inhibited by the glycoproteins: fetuin, lactoferrin, and recombinant erythropoietin. GSL is a glycoprotein possessing 16 % of carbohydrates; the SDS-PAGE showed two bands with molecular mass of 52.1 and 64.4 kDa. Isoelectric focusing displayed microheterogeneity, with two bands at pIs 5.1 and 5.3. The lectin was stable between pH 2 and pH 8 while at pH 10, the agglutination decayed to 50 % of initial activity. Incubation at 40 and 80 °C led to 50 and 100 % loss in activity of the lectin, respectively. Synthesized GSL-Sepharose interacts with serum pregnant mare gonadotropin, and at least two subpopulations of this glycoprotein were separated. There was no interaction between transferrin and soluble GSL while a partial recognition was achieved with GSL-Sepharose. The terminal sialic acid seems to play an active role in modifying the interaction with GSL, depending if the lectin is in a soluble or immobilized form. The purified lectin inhibited in vitro the growth of Staphylococcus aureus and Aspergillus niger.

Screening for lectins from basidiomycetes and isolation of Punctularia atropurpurascens lectin.

Aqueous extracts of basidiomycete fungi were screened for the presence of lectins by hemagglutination (HA) assays with mouse, rabbit, and sheep red blood cells. From mycelia and/or fruiting bodies, 23 extracts were prepared; 15 extracts exhibited HA activity towards mouse erythrocytes, with specific activities ranging from 12 to 440 lectin units (LU) mg(-1) protein. In HA inhibition assays, 43 carbohydrates including mono-, di-, tri-, tetrasaccharides, glycoproteins, and polysaccharides were tested as haptens, to determine the saccharide-binding specificities of the lectins. A novel lectin with specificity towards N-acetyl-glucosamine was purified from mycelia of Punctularia atropurpurascens using affinity chromatography on chitosan-Sepharose. The lectin has a subunit molecular mass of 67 kDa determined by SDS-PAGE and a pI of 5.0.

Biodegradation of agroindustrial wastes by Pleurotus spp for its use as ruminant feed

The increasing expansion of agro-industrial activity has led to the accumulation of a large quantity of lignocellulosic residues all over the world. In particular, large quantities of rice straw (300.000 t) and citric bagasse (50.000 t) are annually produced in Uruguay. In this work we present the study of the bioconversion of these substrates with the edible mushroom Pleurotus spp so as to increase nutritional values and digestibility for its use as animal feed. The SSF process was optimized and the products after different periods of mushroom growth were evaluated. The microbial counts (cfu/g) for the inoculated substrates 44 days after incubation were 15 x 10(4), < 10 and < 10 for aerobic microorganisms, coliforms and E. coli, respectively. After 14 days of SSF the percentage of dry matter, ADF and NDF decreased, and the content of protein increased. These results show that vegetal cell-wall components were degraded during the period of mushroom incubation. PCR - RFLP analysis of the ITS region was used to characterize the Pleurotus species produced in Uruguay and discriminate between DNAs of Pleurotus ostreatus 814 and other fungi from the different substrates.