Prebiotic effect of sorghum biomass xylooligosaccharides employing immobilized endoxylanase from Thermomyces lanuginosus PC7S1T.

Purified endoxylanase from Thermomyces lanuginosus PC7S1T was immobilized in calcium alginate, resulting in a yield of 78.5% and a reusability for 11 cycles. The stability of the immobilized enzyme was given for a pH range of 4 to 9 for 96 h. Endoxylanase immobilized in calcium alginate at 65 °C exhibited thermal stability equal to the soluble enzyme for 5 h, and at high temperatures of 75 °C and 85 °C showed half-lives of 4 and 3 h, respectively. Both soluble endoxylanase and immobilized forms were able to hydrolyze hemicellulose, obtained from low-lignin sorghum biomass pretreated with 5% H2O2 and 2% NaOH, after 1 h of incubation at 65 °C, releasing a mixture of short-chain xylooligosaccharides (X2-X6). The highest amounts of XOS generated were those for X5 (24 to 40%), X4 (33 to 39%), and X3 (11 to 22%). These XOS acted as prebiotics, promoting the growth of the probiotic L. acidophilus, similar to glucose in the MRS broth. These results show the potential of low-lignin sorghum to generate XOS with prebiotic activity, suggesting the application of these compounds in the food industry.

Production, immobilization and application of invertase from new wild strain Cunninghamella echinulata PA3S12MM.

AIMS: The objective of this study was to determine the best conditions to produce invertase by Cunninghamella echinulata PA3S12MM and to immobilize and apply the enzyme. METHODS AND RESULTS: The maximum production was verified in 8 days of cultivation at 28°C supplemented with 10 g L-1 apple peel, reaching 1054.85 U ml-1 . The invertase was purified from the DEAE-Sephadex column. The derivative immobilized in alginate-gelatin-calcium phosphate showed reusability >50% for 19 cycles. The derivative immobilized in glutaraldehyde-chitosan showed greater thermostability and at a different pH. The hydrolysis of 15 ml of sucrose 500 g L-1 in a fixed bed reactor (total volume of 31 ml) produced 24.44 µmol min-1 of glucose and fructose at a residence time of 30 min and a conversion factor of 0.5. CONCLUSIONS: The new wild strain C. echinulata PA3S12MM presents high invertase production in medium supplemented with an agro-industrial residue and the immobilized enzyme showed high thermal stability and resistance at a different pH. SIGNIFICANCE AND IMPACT OF THE STUDY: The fungus C. echinulata PA3S12MM is an excellent producer of invertases in Vogel medium supplemented with apple peel. The enzyme is promising for industrial application since it has good performance in reusability and inverted sugar production.

Cunninghamella echinulata PA3S12MM invertase: Biochemical characterization of a promiscuous enzyme.

The Cunninghamella echinulata PA3S12MM fungus is a great producer of invertases in a growth medium supplemented by apple peels. The enzyme was purified 4.5 times after two chromatographic processes, and it presented a relative molecular mass of 89.2 kDa. The invertase reached maximum activity at pH of 6 and at 60°C, in addition to presenting stability in alkaline pH and thermal activation at 50°C. The enzymatic activity increased in the presence of Mn2+ and dithiothreitol (DTT), while Cu2+ and Z2+ ions inhibited it. Also, DTT showed to protect enzymatic activity. The apparent values for Km , Vmáx , and Kcat for the sucrose hydrolysis were, respectively, 173.8 mmol/L, 908.7 mmol/L min-1 , and 1,388.79 s-1 . The carbohydrate content was of 83.13%. The invertase presented hydrolytic activity over different types of glycosidic bonds, such as α1 â†” 2ß (sucrose), α1 â†’ 4 (polygalacturonic acid), α1 â†’ 4 and α1 â†’ 2 (pectin), and α1 â†” 1 (trehalose), indicating that the enzyme is multifunctional. Thus, the biochemical properties showed by the C. echinulata PA3S12MM suggest a broad industrial application, such as in the biomass hydrolysis or in the food industry. PRACTICAL APPLICATIONS: Invertases are hydrolytic enzymes employed in several industrial sectors. Given their great importance for the economy and several industrial sectors, there is a growing interest in microorganisms producing this enzyme. The analysis of the biochemical properties of invertase in C. echinulata PA3S12MM suggest applications in the food industry. Due to its increased hydrolytic activity, the hydrolysis process of the sucrose may employ invertase for the production of invert sugar. The stability at alkaline pH suggests an application in the development of enzymatic electrodes for the quantification of sucrose in food and beverage. The multifunctional activity may work in the biomass hydrolysis or saccharification of by-products for the extraction of fermentable sugars. The high level of invertase N-linked glycosylation of invertase grants this enzyme thermal stability at high temperatures, in addition to resistance against the action of proteases, which are desirable characteristics for the application of this enzyme in industrial processes.

Susceptibility of Candida albicans Isolated from Blood to Wickerhamomyces anomalous Mycocins.

The occurrence of infections caused by Candida albicans in developed and developing countries and their resistance to some available antifungal drugs have been viewed as causing a great problem to human health worldwide. In order to find new researched molecules, there are some mycoses secreted by yeasts, especially mycocins produced by Wickerhamomyces anomalus with a broad antimicrobial spectrum of activity. Thus, this trial aimed at evaluating mycocins' activity obtained from environmental W. anomalus cell wall compared to thirty C. albicans strains isolated from blood. Mycocins were extracted from cell walls of three W. anomalus strains (WA40, WA45, and WA92). The 400 µg mL-1 concentration of WA40M1, WA45M2, and WA92M3 mycocin extracts showed the following respective activity results: 96.6, 96.6, and 90.0 % C. albicans strains. WA45M2 and WA92M3 mycocin extracts showed some activity in 3.3 % of C. albicans strains at 50 µg mL-1 concentration. Mycocins extracted from cell walls of three W. anomalus strains named as WA40, WA45, and WA92 showed antifungal activity compared to C. albicans and low degree of hemolysis.

ß-(1→3)-Glucan of the Southern Bracket Mushroom, Ganoderma australe (Agaricomycetes), Stimulates Phagocytosis and Interleukin-6 Production in Mouse Peritoneal Macrophages.

Ganoderma australe was studied to determine the composition of the cell wall, and polysaccharide fraction SK5 was obtained after freeze-thawing an aqueous 5% potassium hydroxide extraction. The monosaccharide composition of the SK5 fraction revealed by gas chromatography-mass spectrometry showed 81.3% glucose, and analyses by 13C nuclear magnetic resonance spectroscopy confirmed a ß-glucan with glycosidic links of the (1→3)-ß type and most likely 4-O substituted. In addition, the biological effect of the ß-glucan from G. australe was evaluated via in vitro cell cultures of peritoneal macrophages isolated from Swiss mice. Biological assays were assessed for toxicity and cell activation, interleukin-6 cytokine concentrations, and the ability to stimulate phagocytic activity. There was an increase in interleukin-6 by approximately 111% with 1.0 µg/mL of polysaccharide, and phagocyte activity was increased in all concentrations examined, obtaining 52.3% with 0.25 µg/mL polysaccharide. The results indicate that a ß-(1→3)-glucan isolated from G. australe can be classified as a biological response modifier.

Proteomic profile of hemolymph and detection of induced antimicrobial peptides in response to microbial challenge in Diatraea saccharalis (Lepidoptera: Crambidae).

Insects are organisms extremely well adapted to diverse habitats, primarily due to their innate immune system, which provides them with a range of cellular and humoral responses against microorganisms. Lepidoptera hemolymph proteins involved in humoral responses are well known; however, there is a lack of knowledge about the sugarcane borer Diatraea saccharalis. In this present work, the hemolymph proteins of this pest insect were studied by applying proteomic methodologies. Two-dimensional electrophoresis (2-DE) gels of proteins extracted from naive larvae and larvae challenged with Escherichia coli (ATCC 11224) and Bacillus subtilis (ATCC 6623) showed an average of 300 spots, and 92 of these spots corresponded in all three 2-DE gels. Forty-one spots were excised and digested with trypsin and analyzed using mass spectrometry. After analysis, 10 proteins were identified, including some proteins of the immune system: ß-defensin-like protein, Turandot A-like protein, attacin-like protein, peptidoglycan recognition protein and cyclophilin-like protein. Nine proteins were present in both experimental conditions; however, ß-defensin-like protein was present only in hemolymph challenged by B. subtilis. Notably, attacin-like protein was strongly induced by challenge with E. coli, suggesting an immune response against the infection. However, antimicrobial activity was observed in the test zone of microbial growth inhibition of B. subtilis solely with the hemolymph extract of the larvae challenged with B. subtilis. We made for the first time a proteomic profile of the hemolymph of D. saccharalis in which it was possible to identify the presence of important proteins involved in the immune response.

Malassezia spp. in acoustic meatus of bats (Molossus molossus) of the Amazon Region, Brazil.

The yeasts of the Malassezia genus are opportunistic microorganisms and can cause human and animal infections. They are commonly isolated from the skin and auricular canal of mammalians, mainly dogs and cats. The present study was aimed to isolate Malassezia spp. from the acoustic meatus of bats (Molossus molossus) in the Montenegro region, "Rondônia", Brazil. From a total of 30 bats studied Malassezia spp. were isolated in 24 (80%) animals, the breakdown by species being as follows (one Malassezia sp. per bat, N = 24): 15 (62.5%) M. pachydermatis, 5 (20.8%) M. furfur, 3 (12.5%) M. globosa and 1 (4.2%) M. sympodialis. This study establishes a new host and anatomic place for Malassezia spp., as it presents the first report ever of the isolation of this genus of yeasts in the acoustic meatus of bats.