Production, Kinetic/Thermodynamic Study, and Evaluation of the Influence of Static Magnetic Field on Kinetic Parameters of ß-Fructofuranosidase from Aspergillus tamarii Kita UCP 1279 Produced by Solid-State Fermentation.

ß-fructofuranosidases (FFases) are enzymes involved in sucrose hydrolysis and can be used in the production of invert sugar and fructo-oligosaccharides (FOS). This last is an important prebiotic extensively used in the food industry. In the present study, the FFase production by Aspergillus tamarii Kita UCP 1279 was assessed by solid-state fermentation using a mixture of wheat and soy brans as substrate. The FFase presents optimum pH and temperature at 5.0-7.0 and 60 °C, respectively. According to the kinetic/thermodynamic study, the FFase was relatively stable at 50 °C, a temperature frequently used in industrial FOS synthesis, using sucrose as substrate, evidenced by the parameters half-life (115.52 min) and D-value (383.76 min) and confirmed by thermodynamic parameters evaluated. The influence of static magnetic field with a 1450 G magnetic flux density presented a positive impact on FFase kinetic parameters evidenced by an increase of affinity of enzyme by substrate after exposition, observed by a decrease of 149.70 to 81.73 mM on Km. The results obtained indicate that FFases present suitable characteristics for further use in food industry applications. Moreover, the positive influence of a magnetic field is an indicator for further developments of bioprocesses with the presence of a magnetic field.

Enhanced production of prodigiosin by Serratia marcescens UCP 1549 using agrosubstrates in solid-state fermentation.

This work aimed to investigate the production of prodigiosin by S. marcescens UCP 1549 in solid-state fermentation (SSF), as a sustainable alternative for reducing the production costs and environmental impact. Thus, different agro-industrial substrates were used in the formulation of the prodigiosin production medium, obtaining the maximum yield of pigment (119.8 g/kg dry substrate) in medium consisting of 5 g wheat bran, 5% waste soybean oil and saline solution. The pigment was confirmed as prodigiosin by the maximum absorbance peak at 535 nm, Rf 0.9 in TLC, and the functional groups by infrared spectrum (FTIR). Prodigiosin demonstrated stability at different values of temperature, pH and NaCl concentrations and antimicrobial properties, as well as not show any toxicity. These results confirm the applicability of SSF as a sustainable and promising technology and wheat bran as potential agrosubstrate to produce prodigiosin, making the bioprocess economic and competitive for industrial purposes.

Biotechnological Strategies for Chitosan Production by Mucoralean Strains and Dimorphism Using Renewable Substrates.

We investigated the influence of corn steep liquor (CSL) and cassava waste water (CWW) as carbon and nitrogen sources on the morphology and production of biomass and chitosan by Mucor subtilissimus UCP 1262 and Lichtheimia hyalospora UCP 1266. The highest biomass yields of 4.832 g/L (M. subtilissimus UCP 1262) and 6.345 g/L (L. hyalospora UCP 1266) were produced in assay 2 (6% CSL and 4% CWW), factorial design 22, and also favored higher chitosan production (32.471 mg/g) for M. subtilissimus. The highest chitosan production (44.91 mg/g) by L. hyalospora (UCP 1266) was obtained at the central point (4% of CWW and 6% of CSL). The statistical analysis, the higher concentration of CSL, and lower concentration of CWW significantly contributed to the growth of the strains. The FTIR bands confirmed the deacetylation degree of 80.29% and 83.61% of the chitosan produced by M. subtilissimus (UCP 1262) and L. hyalospora (UCP 1266), respectively. M. subtilissimus (UCP 1262) showed dimorphism in assay 4-6% CSL and 8% CWW and central point. L. hyalospora (UCP 1266) was optimized using a central composite rotational design, and the highest yield of chitosan (63.18 mg/g) was obtained in medium containing 8.82% CSL and 7% CWW. The experimental data suggest that the use of CSL and CWW is a promising association to chitosan production.

Monohexosylceramides from Rhizopus Species Isolated from Brazilian Caatinga: Chemical Characterization and Evaluation of Their Anti-Biofilm and Antibacterial Activities.

Monohexosylceramides (CMHs) are highly conserved fungal glycosphingolipids playing a role in several cellular processes such as growth, differentiation and morphological transition. In this study, we report the isolation, purification and chemical characterization of CMHs from Rhizopus stolonifer and R. microspores. Using positive ion mode ESI-MS, two major ion species were observed at m/z 750 and m/z 766, respectively. Both ion species consisted of a glucose/galactose residue attached to a ceramide moiety containing 9-methyl-4,8-sphingadienine with an amidic linkage to a hydroxylated C16:0 fatty acid. The antimicrobial activity of CMH was evaluated against Gram positive and Gram negative bacteria using the agar diffusion assay. CMH from both Rhizopus species inhibited the growth of Bacillus terrae, Micrococcus luteus (M. luteus) and Pseudomonas stutzeri (P. stutzeri) with a MIC50 of 6.25, 6.25 and 3.13 mg/mL, respectively. The bactericidal effect was detected only for M. luteus and P. stutzeri, with MBC values of 25 and 6.25 mg/mL, respectively. Furthermore, the action of CMH on the biofilm produced by methicillin-resistant Staphylococcus aureus (MRSA) was analyzed using 12.5 and 25 mg/mL of CMH from R. microsporus. Total biofilm biomass, biofilm matrix and viability of the cells that form the biofilm structure were evaluated. CMH from R. microsporus was able to inhibit the MRSA biofilm formation in all parameters tested.

Physicochemical and Antibacterial Properties of Chitosan Extracted from Waste Shrimp Shells.

This research aims to study the production of chitosan from shrimp shell (Litopenaeus vannamei) of waste origin using two chemical methodologies involving demineralization, deproteinization, and the degree of deacetylation. The evaluation of the quality of chitosan from waste shrimp shells includes parameters for the yield, physical chemistry characteristics by infrared spectroscopy (FT-IR), the degree of deacetylation, and antibacterial activity. The results showed (by Method 1) extraction yields for chitin of 33% and for chitosan of 49% and a 76% degree of deacetylation. Chitosan obtained by Method 2 was more efficient: chitin (36%) and chitosan (63%), with a high degree of deacetylation (81.7%). The antibacterial activity was tested against Gram-negative bacteria (Stenotrophomonas maltophilia and Enterobacter cloacae) and Gram-positive Bacillus subtilis and the Minimum Inhibitory Concentrations (MIC) and the Minimum Bactericidal Concentration (MBC) were determined. Method 2 showed that extracted chitosan has good antimicrobial potential against Gram-positive and Gram-negative bacteria and that the process is viable.

Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate).

A fibrinolytic protease from M. subtilissimus UCP 1262 was recovered and partially purified by polyethylene glycol (PEG)/sodium sulfate aqueous two-phase systems (ATPS). The simultaneous influence of PEG molar mass, PEG concentration and sulfate concentration on the enzyme recovery was first investigated using a 2(3) full factorial design, and the Response Surface Methodology used to identify the optimum conditions for enzyme extraction by ATPS. Once the best PEG molar mass for the process had been selected (6000g/mol), a two-factor central composite rotary design was applied to better evaluate the effects of the other two independent variables. The fibrinolytic enzyme was shown to preferentially partition to the bottom phase with a partition coefficient (K) ranging from 0.2 to 0.7. The best results in terms of enzyme purification were obtained with the system formed by 30.0% (w/w) PEG 6000g/mol and 13.2% (w/w) sodium sulfate, which ensured a purification factor of 10.0, K of 0.2 and activity yield of 102.0%. SDS-PAGE and fibrin zymography showed that the purified protease has a molecular mass of 97kDa and an apparent isoelectric point of 5.4. When submitted to assays with different substrates and inhibitors, it showed selectivity for succinyl-l-ala-ala-pro-l-phenylalanine-p-nitroanilide and was almost completely inhibited by phenylmethylsulfonyl fluoride, behaving as a chymotrypsin-like protease. At the optimum temperature of 37°C, the enzyme residual activity was 94 and 68% of the initial one after 120 and 150min of incubation, respectively. This study demonstrated that M. subtilissimus protease has potent fibrinolytic activity compared with similar enzymes produced by solid-state fermentation, therefore it may be used as an agent for the prevention and therapy of thrombosis. Furthermore, it appears to have the advantages of low cost production and simple purification.

Isolation and bioelectrochemical characterization of novel fungal sources with oxidasic activity applied in situ for the cathodic oxygen reduction in microbial fuel cells.

Brazilian filamentous fungi Rhizopus sp. (SIS-31), Aspergillus sp. (SIS-18) and Penicillium sp. (SIS-21), sources of oxidases were isolated from Caatinga's soils and applied during the in situ cathodic oxygen reduction in fuel cells. All strains were cultivated in submerged cultures using an optimized saline medium enriched with 10 g L(-1) of glucose, 3.0 g L(-1) of peptone and 0.0005 g L(-1) of CuSO4 as enzyme inducer. Parameters of oxidase activity, glucose consumption and microbial growth were evaluated. In-cell experiments evaluated by chronoamperometry were performed and two different electrode compositions were also compared. Maximum current densities of 125.7, 98.7 and 11.5 µA cm(-2) were observed before 24 h and coulombic efficiencies of 56.5, 46.5 and 23.8% were obtained for SIS-31, SIS-21 and SIS-18, respectively. Conversely, maximum power outputs of 328.73, 288.80 and 197.77 mW m(-3) were observed for SIS-18, SIS-21 and SIS-31, respectively. This work provides the primary experimental evidences that fungi isolated from the Caatinga region in Brazil can serve as efficient biocatalysts during the oxygen reduction in air-cathodes to improve electricity generation in MFCs.

Green conversion of agroindustrial wastes into chitin and chitosan by Rhizopus arrhizus and Cunninghamella elegans strains.

This article sets out a method for producing chitin and chitosan by Cunninghamella elegans and Rhizopus arrhizus strains using a green metabolic conversion of agroindustrial wastes (corn steep liquor and molasses). The physicochemical characteristics of the biopolymers and antimicrobial activity are described. Chitin and chitosan were extracted by alkali-acid treatment, and characterized by infrared spectroscopy, viscosity and X-ray diffraction. The effectiveness of chitosan from C. elegans and R. arrhizus in inhibiting the growth of Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica, Escherichia coli and Yersinia enterocolitica were evaluated by determining the minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC). The highest production of biomass (24.60 g/L), chitin (83.20 mg/g) and chitosan (49.31 mg/g) was obtained by R. arrhizus. Chitin and chitosan from both fungi showed a similar degree of deacetylation, respectively of 25% and 82%, crystallinity indices of 33.80% and 32.80% for chitin, and 20.30% and 17.80% for chitosan. Both chitin and chitosan presented similar viscosimetry of 3.79-3.40 cP and low molecular weight of 5.08×10³ and 4.68×10³ g/mol. They both showed identical MIC and MBC for all bacteria assayed. These results suggest that: agricultural wastes can be produced in an environmentally friendly way; chitin and chitosan can be produced economically; and that chitosan has antimicrobial potential against pathogenic bacteria.

Effect of corn steep liquor (CSL) and cassava wastewater (CW) on chitin and chitosan production by Cunninghamella elegans and their physicochemical characteristics and cytotoxicity.

Microbiological processes were used for chitin and chitosan production with Cunninghamella elegans UCP/WFCC 0542 grown in different concentrations of two agro-industrial wastes, corn steep liquor (CSL) and cassava wastewater (CW) established using a 2² full factorial design. The polysaccharides were extracted by alkali-acid treatment and characterized by infrared spectroscopy, viscosity, thermal analysis, elemental analysis, scanning electron microscopy and X-ray diffraction. The cytotoxicity of chitosan was evaluated for signs of vascular change on the chorioallantoic membrane of chicken eggs. The highest biomass (9.93 g/L) was obtained in trial 3 (5% CW, 8% CSL), the greatest chitin and chitosan yields were 89.39 mg/g and 57.82 mg/g, respectively, and both were obtained in trial 2 (10% CW, 4% CSL). Chitin and chitosan showed a degree of deacetylation of 40.98% and 88.24%, and a crystalline index of 35.80% and 23.82%, respectively, and chitosan showed low molecular weight (LMW 5.2 × 10³ Da). Chitin and chitosan can be considered non-irritating, due to the fact they do not promote vascular change. It was demonstrated that CSL and CW are effective renewable agroindustrial alternative substrates for the production of chitin and chitosan.

Characterization and properties of the biosurfactant produced by Candida lipolytica UCP 0988

Background: Biotechnological processes are costly, especially for the production of biosurfactants. The successful production of a biosurfactant is dependent on the development of processes using low cost raw materials. Considering the importance of the characteristics of a biosurfactant to facilitate its industrial application, the properties of the biosurfactant produced by Candida lipolytica through previously optimized medium have been established. Results: The yeast was grown for 72 h to determine the kinetics of growth and production. The surface tension of the cell-free broth was reduced from 55 to 25 mN/m. The yield of biosurfactant was 8.0 g/l with a CMC of 0.03%. The biosurfactant was characterized as an anionic lipopeptide composed of 50% protein, 20% lipids, and 8% of carbohydrates. Conclusions: The isolated biosurfactant showed no toxicity against different vegetable seeds: Brassica oleracea, Solanum gilo and Lactuca sativa L. and the micro-crustacean Artemia salina. The properties of the biosurfactant produced suggest its potential application in industries that require the use of effective compounds at low cost.

Assessment of the effectiveness of the PPD-mallein produced in Brazil for diagnosing glanders in mules.

To assess the potency of the PPD-mallein produced in Brazil, five animals were from a property identified as a focus of glanders. These animals had suggestive clinical signs of the disease and the other five, from a property free from glanders, showed no clinical signs and were serology negative (control group). PPD-mallein from Burkholderia mallei was obtained by precipitation with trichloroacetic acid and ammonium sulfate. The animals were inoculated according to the criteria established by Department of Agriculture, Livestock and Supply (MAPA) for the diagnosis of glanders. After 48 h of application of PPD-mallein, there was swelling in the area of application, presence of ocular secretion and tears in sick animals. The control group showed no inflammatory reaction at the site of inoculation of PPD-mallein. This immunogen produced in Brazil and still being tested was effective for identifying the infection in true positive animals and excluding the truly negative ones, being a new possibility for diagnosis and control of glanders.

Sialoglycoproteins in morphological distinct stages of Mucor polymorphosporus and their influence on phagocytosis by human blood phagocytes.

The possible role of sialic acids in host cells-fungi interaction and their association with glycoproteins were evaluated using a clinical isolate of the dimorphic fungus Mucor polymorphosporus. Lectin-binding assays with spores and yeast cells denoted the presence of surface sialoglycoconjugates containing 2,3- and 2,6-linked sialylglycosyl groups. Western blotting with peroxidase-labeled Limulus polyphemus agglutinin revealed the occurrence of different sialoglycoprotein types in both cell lysates and cell wall protein extracts of mycelia, spores, and yeasts of M. polymorphosporus. Sialic acids contributed to the surface negative charge of spores and yeast forms as evaluated by adherence to a cationic substrate. Sialidase-treated spores were less resistant to phagocytosis by human neutrophils and monocytes from healthy individuals than control (untreated) fungal suspensions. The results suggest that sialic acids are terminal units of various glycoproteins of M. polymorphosporus, contributing to negative charge of yeasts and spore cells and protecting infectious propagules from destruction by host cells.

On-line biomass estimation in biosurfactant production process by Candida lipolytica UCP 988.

Biomass is an important variable in biosurfactant production process. However, such bioprocess variable, usually, is collected by sampling and determined by off-line analysis, with significant time delay. Therefore, simple and reliable on-line biomass estimation procedures are highly desirable. An artificial neural network model (ANN) is presented for the on-line estimation of biomass concentration, in biosurfactant production by Candida lipolytica UCP 988, as a nonlinear function of pH and dissolved oxygen. Several configurations were evaluated while developing the optimal ANN model. The optimal ANN model consists of one hidden layer with four neurons. The performance of the ANN was checked using experimental data. The results obtained indicate a very good predictive capacity for the ANN-based software sensor with values of R2 of 0.969 and RMSE of 0.021 for biomass concentration. Estimated biomass using the ANN was proved to be a simple, robust and accurate method.