Hydrolytic enzyme production from açai palm (Euterpe precatoria) endophytic fungi and characterization of the amylolytic and cellulolytic extracts.

Enzymes are biocatalysts that are widely used in different industries and generate billions of dollars annually. With the advancement of biotechnology, new enzymatic sources are being evaluated, especially microbial ones, in order to find efficient producers. Endophytic fungi are promising sources of biomolecules; however, Amazonian species are still poorly studied as to their enzymatic production potential. In this sense, the production of hydrolases (amylases, lipases, cellulases and pectinases) was evaluated in endophytic fungi isolated from the leaves, roots and stems of açai palms (Euterpe precatoria). A qualitative test was carried out to detect the enzymatic synthesis in each isolate, and the most promising ones were cultivated using submerged fermentation. The enzyme extracts were quantified to determine those with the greatest activity. Cellulolytic and amylolytic extracts showed the highest enzymatic activities and were partially characterized. Among 50 isolates, 82.9% produced pectinase, 58.5% produced cellulase, 31.7% produced amylase, and 12.2% produced lipase. Penicillium sp. L3 was the best producer of amylase and Colletotrichum sp. S1 was the best producer of cellulase in liquid medium cultivation. The amylolytic extract showed the highest enzymatic activity at pH 8.0 and 45 °C, and the cellulolytic extract at pH 5.0 and 35 °C. The cellulase and amylase produced by the endophytes had their molecular masses estimated between 38 and 76 kDa. These results indicate that endophytic fungi from the açai palm can be used as a new source of hydrolytic enzymes, which can be applied in numerous biotechnological processes.

Solvent Effect on the Structural, Optical, Morphology, and Antimicrobial Activity of Silver Phosphate Microcrystals by Conventional Hydrothermal Method.

The design of particle size and morphology are a promising approach to investigating the properties exhibited by different types of materials. In the present study, the silver phosphate microcrystals (Ag3PO4) were first time synthesized using the hydrothermal and solvothermal method by combination of the solvents water/isopropyl alcohol (SP-IA), water/acetone (SP-AC), water/ammonium hydroxide (AP-AH), all in a ratio of 1:1 (v/v). The synthesized materials were structurally characterized by X-ray diffraction (XRD), Rietveld refinement, and Raman vibrational spectroscopy, where it was confirmed that the pure phase was achieved for all prepared samples. The study of the optical properties by UV-vis diffuse reflectance spectroscopy (UV-vis/DRS) and colorimetry revealed that the obtained materials have an optical bandgap between 2.30 and 2.32 eV. The FE-SEM images collected revealed different morphologies for the synthesized materials, with a predominance of tetraploid-shaped microcrystals for the SP-AC sample, rods for the SP-IA sample, cubes and polyhedral for the SP-WT sample and condensed polyhedral for the SP-AH sample. The photocatalytic performance against the Rhodamine B dye (RhB) was 100%, 98.2%, 94.2%, and 87.8%, using the samples SP-AC, SP-IA, SP-WT, and SP-AH as photocatalyst at time of 12 min. On the other hand, the antimicrobial performance of SP-AC sample showed superior performance, resulting in the minimum inhibitory concentration-MIC of 7.81 µg mL-1 for the strain of E. coli, 7.81 µg mL-1 for the strain of E. aureus, 15.62 µg mL-1 for the strain of P. auruginosa, and 15.62 µg mL-1 for the strains of C. albicans. In this way, was synthesized a promissory antimicrobial and photocatalyst material, through an easy and cost-effective method.

Isolation and Identification of Pigment-Producing Endophytic Fungi from the Amazonian Species Fridericia chica.

Pigments of fungal origin have aroused increasing interest in the food dye and cosmetic industries since the global demand for natural dyes has grown. Endophytic microorganisms are a source of bioactive compounds, and Amazonian plant species can harbor fungi with a wide range of biotechnological applications. Popularly known in Brazil as crajiru, Fridericia chica is a medicinal plant that produces a red pigment. In this study, a total of 121 fungi were isolated in potato dextrose agar from three plants. We identified nine pigment-producing endophytic fungi isolated from branches and leaves of F. chica. The isolates that showed pigment production in solid media were molecularly identified via multilocus analysis as Aspergillus welwitschiae, A. sydowii, Curvularia sp., Diaporthe cerradensis (two strains), Hypoxylon investiens, Neoscytalidium sp. (two strains) and Penicillium rubens. These isolates were subjected to submerged fermentation in two culture media to obtain metabolic extracts. The extracts obtained were analyzed in terms of their absorbance between 400 and 700 nm. The pigmented extract produced by H. investiens in medium containing yeast extract showed maximum absorbance in the red absorption range (UA700 = 0.550) and significant antioxidant and antimicrobial activity. This isolate can thus be considered a new source of extracellular pigment.

Development of cassava starch-based films incorporated with phenolic compounds produced by an Amazonian fungus.

Drug-release systems have attracted attention over the last few years since they can be used as a substitute for traditional methods of drug delivery. These have the advantage of being directly administered at the treatment site and can maintain the drug at adequate levels for a longer period, thus increasing their efficacy. Starch-based films are interesting candidates for use as matrices for drug release, especially due to starch's non-toxic properties and its biocompatibility. Endophytic fungi are an important source of bioactive molecules, including secondary metabolites such as phenolic compounds with antioxidant activity. In the present study, cassava starch-based films were developed to act as release systems of phenolic compounds with antioxidant activity. The Amazonian endophytic fungus Aspergillus niger MgF2 was cultivated in liquid media, and the fungal extract was obtained by liquid-liquid partition with ethyl acetate. The starch-based films incorporated with the fungal extract were characterized in regards to their physicochemical properties. The release kinetics of the extract from the film and its antioxidant and cytotoxic properties were also evaluated. The films incorporated with the extract presented maximum release after 25 min at 37 °C and pH 6.8. In addition, it was observed that the antioxidant compounds of the fungal extract maintain their activity after being released from the film, and were non-toxic. Therefore, considering the promising physicochemical properties of the extract-incorporated films, and their considerable antioxidant capacity, the films demonstrate great biotechnological potential with diverse applications in the pharmacological and cosmetic industries.

Antimicrobial activity and rutin identification of honey produced by the stingless bee Melipona compressipes manaosensis and commercial honey.

BACKGROUND: Honey has been identified as a potential alternative to the widespread use of antibiotics, which are of significant concern considering the emergence of resistant bacteria. In this context, this study aimed to evaluate the antimicrobial activity of honey samples produced by a stingless bee species and by Apis sp. against pathogenic bacteria, as well as to identify the presence of phenolic compounds. METHODS: Honey samples from the stingless bee M. compressipes manaosensis were collected twice, during the dry and rainy seasons. Three commercial honey samples from Apis sp. were also included in this study. Two different assays were performed to evaluate the antibacterial potential of the honey samples: agar-well diffusion and broth macrodilution. Liquid-liquid extraction was used to assess phenolic compounds from honey. HPLC analysis was performed in order to identify rutin and apigenin on honey samples. Chromatograms were recorded at 340 and 290 nm. RESULTS: Two honey samples were identified as having the highest antimicrobial activity using the agar diffusion method. Honey produced by Melipona compressipes manaosensis inhibited the growth of Staphylococcus aureus, Escherichia coli (0157: H7), Proteus vulgaris, Shigella sonnei and Klebsiella sp. A sample of honey produced by Apis sp. also inhibited the growth of Salmonella paratyphi. The macrodilution technique presented greater sensitivity for the antibacterial testing, since all honey samples showed activity. Flavonoid rutin was identified in the honey sample produced by the stingless bee. CONCLUSIONS: Honey samples tested in this work showed antibacterial activity against Gram-positive and Gram-negative bacteria. The results reported herein highlight the potential of using honey to control bacterial growth.

Antimicrobial and Antioxidant Activities of Endophytic Fungi Associated with Arrabidaea chica (Bignoniaceae).

The endophytic fungal community of the Amazonian medicinal plant Arrabidaea chica (Bignoniaceae) was evaluated based on the hypothesis that microbial communities associated with plant species in the Amazon region may produce metabolites with interesting bioactive properties. Therefore, the antimicrobial and antioxidant activities of the fungal extracts were investigated. A total of 107 endophytic fungi were grown in liquid medium and the metabolites were extracted with ethyl acetate. In the screening of fungal extracts for antimicrobial activity, the fungus identified as Botryosphaeria mamane CF2-13 was the most promising, with activity against E. coli, S. epidermidis, P. mirabilis, B. subtilis, S. marcescens, K. pneumoniae, S. enterica, A. brasiliensis, C. albicans, C. tropicalis and, especially, against S. aureus and C. parapsilosis (MIC = 0.312 mg/mL). Screening for antioxidant potential using the DPPH elimination assay showed that the Colletotrichum sp. CG1-7 endophyte extract exhibited potential activity with an EC50 of 11 µg/mL, which is equivalent to quercetin (8 µg/mL). The FRAP method confirmed the antioxidant potential of the fungal extracts. The presence of phenolic compounds and flavonoids in the active extracts was confirmed using TLC. These results indicate that two of the fungi isolated from A. chica exhibit significant antimicrobial and antioxidant potential.

Biotechnological Applications of Nanoencapsulated Essential Oils: A Review.

Essential oils (EOs) are complex mixtures of volatile and semi-volatile organic compounds that originate from different plant tissues, including flowers, buds, leaves and bark. According to their chemical composition, EOs have a characteristic aroma and present a wide spectrum of applications, namely in the food, agricultural, environmental, cosmetic and pharmaceutical sectors. These applications are mainly due to their biological properties. However, EOs are unstable and easily degradable if not protected from external factors such as oxidation, heat and light. Therefore, there is growing interest in the encapsulation of EOs, since polymeric nanocarriers serve as a barrier between the oil and the environment. In this context, nanoencapsulation seems to be an interesting approach as it not only prevents the exposure and degradation of EOs and their bioactive constituents by creating a physical barrier, but it also facilitates their controlled release, thus resulting in greater bioavailability and efficiency. In this review, we focused on selecting recent articles whose objective concerned the nanoencapsulation of essential oils from different plant species and highlighted their chemical constituents and their potential biotechnological applications. We also present the fundamentals of the most commonly used encapsulation methods, and the biopolymer carriers that are suitable for encapsulating EOs.

Starch as a Matrix for Incorporation and Release of Bioactive Compounds: Fundamentals and Applications.

Due to its abundance in nature and low cost, starch is one of the most relevant raw materials for replacing synthetic polymers in a number of applications. It is generally regarded as non-toxic, biocompatible, and biodegradable and, therefore, a safe option for biomedical, food, and packaging applications. In this review, we focused on studies that report the use of starch as a matrix for stabilization, incorporation, or release of bioactive compounds, and explore a wide range of applications of starch-based materials. One of the key application areas for bioactive compounds incorporated in starch matrices is the pharmaceutical industry, especially in orally disintegrating films. The packaging industry has also shown great interest in using starch films, especially those with antioxidant activity. Regarding food technology, starch can be used as a stabilizer in nanoemulsions, thus allowing the incorporation of bioactive compounds in a variety of food types. Starch also presents potential in the cosmetic industry as a delivery system. However, there are still several types of industry that could benefit from the incorporation of starch matrices with bioactive compounds, which are described in this review. In addition, the use of microbial bioactive compounds in starch matrices represents an almost unexplored field still to be investigated.

Degradation of Staphylococcus aureus Biofilm Using Hydrolytic Enzymes Produced by Amazonian Endophytic Fungi.

Microbial biofilms can cause serious health problems, since, due to their persistent character, they often function as spreaders of contaminants. Hydrolytic enzymes have a number of industrial applications and have been indicated as an alternative to the traditional chemical methods that are used to eradicate microbial biofilms. In this study, we evaluated the ability of enzymatic extracts produced by endophytic fungi isolated from the Amazonian species Myrcia guianensis to remove Staphylococcus aureus biofilms. After culture in liquid medium, the fungal hydrolytic extracts showed amylase (3.77 U/mL), lipase (3.84 U/mL), protease (3.63 U/mL), and xylanase (2.91 U/mL) activity. A 24 h mature S. aureus ATCC6538 biofilm was exposed to each enzyme extract with standardized enzyme activities for 10, 30, and 60 min. The optical density at 630 nm was used to calculate the growth rate (GR%) and the residual biofilm rate (RBR%). The most promising solutions were used in combination, based on a 24 factorial design for 0, 10, 20, and 30 min of exposure. Lipase and protease solutions, when applied separately, were the most effective, and promoted the complete removal of S. aureus biofilms in t10 (lipase) and t30 and t60 (lipase and protease). Of the combined treatments using 1.0 U/mL protease and 0.4 U/mL lipase, total biofilm degradation was observed for all exposure times. Thus, the hydrolases produced by the Amazonian endophytic fungi evaluated here are highlighted as an interesting tool in the fight against microbial biofilms.

Production of Biosurfactants by Ascomycetes.

Surfactants are utilized to reduce surface tension in aqueous and nonaqueous systems. Currently, most synthetic surfactants are derived from petroleum. However, these surfactants are usually highly toxic and are poorly degraded by microorganisms. To overcome these problems associated with synthetic surfactants, the production of microbial surfactants (called biosurfactants) has been studied in recent years. Most studies investigating the production of biosurfactants have been associated mainly with bacteria and yeasts; however, there is emerging evidence that those derived from fungi are promising. The filamentous fungi ascomycetes have been studied for the production of biosurfactants from renewable substrates. However, the yield of biosurfactants by ascomycetes depends on several factors, such as the species, nutritional sources, and environmental conditions. In this review, we explored the production, chemical characterization, and application of biosurfactants by ascomycetes.

Endophytic fungi from Myrcia guianensis at the Brazilian Amazon: distribution and bioactivity.

Beneficial interactions between plants and microorganisms have been investigated under different ecological, physiological, biochemical, and genetic aspects. However, the systematic exploration of biomolecules with potential for biotechnological products from this interaction still is relatively scarce. Therefore, this study aimed the evaluation of the diversity and antimicrobial activity of the endophytic fungi obtained from roots, stems and leafs of Myrcia guianensis (Myrtaceae) from the Brazilian Amazon. 156 endophytic fungi were isolated and above 80% were identified by morphological examination as belonging to the genera Pestalotiopsis, Phomopsis, Aspergillus, Xylaria, Nectria, Penicillium and Fusarium. Fermented broth of those fungi were assayed for antimicrobial activity and four inhibited the growth of Staphylococcus aureus, Enterococcus faecalis, Candida albicans and Penicillium avellaneum. As the strain named MgRe2.2.3B (Nectria haematococca) had shown the most promising results against those pathogenic strains, its fermented broth was fractioned and only its two low polar fractions demonstrated to be active. Both fractions exhibited a minimum bactericidal concentration of 50 µg.mL(-1) against S. aureus and a minimum fungicidal concentration of 100 µg.mL(-1) against P. avellaneum. These results demonstrate the diversity of fungal genera in M. guianensis and the potential of these endophytic fungi for the production of new antibiotics.

Endophytic fungi from Myrcia guianensis at the Brazilian Amazon: distribution and bioactivity

Beneficial interactions between plants and microorganisms have been investigated under different ecological, physiological, biochemical, and genetic aspects. However, the systematic exploration of biomolecules with potential for biotechnological products from this interaction still is relatively scarce. Therefore, this study aimed the evaluation of the diversity and antimicrobial activity of the endophytic fungi obtained from roots, stems and leafs of Myrcia guianensis (Myrtaceae) from the Brazilian Amazon. 156 endophytic fungi were isolated and above 80% were identified by morphological examination as belonging to the genera Pestalotiopsis, Phomopsis, Aspergillus, Xylaria, Nectria, Penicillium and Fusarium. Fermented broth of those fungi were assayed for antimicrobial activity and four inhibited the growth of Staphylococcus aureus, Enterococcus faecalis, Candida albicans and Penicillium avellaneum. As the strain named MgRe2.2.3B (Nectria haematococca) had shown the most promising results against those pathogenic strains, its fermented broth was fractioned and only its two low polar fractions demonstrated to be active. Both fractions exhibited a minimum bactericidal concentration of 50 µg.mL-1 against S. aureus and a minimum fungicidal concentration of 100 µg.mL-1 against P. avellaneum. These results demonstrate the diversity of fungal genera in M. guianensis and the potential of these endophytic fungi for the production of new antibiotics.

Physicochemical properties of three food proteins treated with transglutaminase

Três fontes protéicas alimentares foram tratadas com transglutaminase microbiana (EC 2.3.2.13) e as características físico-químicas como reatividade, solubilidade, emulsificação e grupos amino livres dos polímeros formados foram avaliadas. Amostras de caseína láctea (CL), proteína isolada de soja (PIS) e de proteína animal hidrolisada (PAH), foram incubadas com a enzima por uma ou duas horas. CL e PIS mostraram uma redução na solubilidade de 15% e 24% respectivamente, enquanto PAH não sofreu alteração de solubilidade. A quantidade de nitrogênio livre na forma amina apresentou uma redução de 7%, 3% e 2% para PAH, CL e PIS respectivamente. CL e PIS demonstraram baixa atividade emulsificante quando tratadas enzimaticamente, porém as emulsões formadas se mostraram estáveis, em contraste com PAH que não alterou suas propriedades emulsificantes.