Potential Applicability of Cocoa Pulp (Theobroma cacao L) as an Adjunct for Beer Production.

The aim of this study was to evaluate the application of cocoa pulp as an adjunct for malt in beer production. The cocoa pulp was analyzed for humidity, proteins, lipids, sugars, total soluble solids, organic acids, and minerals. A study was carried out to reduce the cocoa pulp viscosity by enzymatic depectinization, making its use viable in beer production. The cocoa pulp showed relevant quantities of compounds important in fermentation, such as sugars, acids, and minerals. In fermentation using the adjunct, the proportions of pulp used were 10, 30, and 49%. A significant difference was found between the adjunct and all-malt worts. The 30% cocoa pulp concentration as an adjunct for malt in the fermentation medium contributed the most to the fermentative performance of the yeasts at both 15 and 22°C based on the consumption of apparent extract (°Plato), ethanol production, and cellular growth.

Genomic analysis and D-xylose fermentation of three novel Spathaspora species: Spathaspora girioi sp. nov., Spathaspora hagerdaliae f. a., sp. nov. and Spathaspora gorwiae f. a., sp. nov.

Three novel D-xylose-fermenting yeast species of Spathaspora clade were recovered from rotting wood in regions of the Atlantic Rainforest ecosystem in Brazil. Differentiation of new species was based on analyses of the gene encoding the D1/D2 sequences of large subunit of rRNA and on 642 conserved, single-copy, orthologous genes from genome sequence assemblies from the newly described species and 15 closely-related Debaryomycetaceae/Metschnikowiaceae species. Spathaspora girioi sp. nov. produced unconjugated asci with a single elongated ascospore with curved ends; ascospore formation was not observed for the other two species. The three novel species ferment D-xylose with different efficiencies. Spathaspora hagerdaliae sp. nov. and Sp. girioi sp. nov. showed xylose reductase (XR) activity strictly dependent on NADPH, whereas Sp. gorwiae sp. nov. had XR activity that used both NADH and NADPH as co-factors. The genes that encode enzymes involved in D-xylose metabolism (XR, xylitol dehydrogenase and xylulokinase) were also identified for these novel species. The type strains are Sp. girioi sp. nov. UFMG-CM-Y302(T) (=CBS 13476), Sp. hagerdaliae f.a., sp. nov. UFMG-CM-Y303(T) (=CBS 13475) and Sp. gorwiae f.a., sp. nov. UFMG-CM-Y312(T) (=CBS 13472).

Three novel ascomycetous yeast species of the Kazachstania clade, Kazachstania saulgeensis sp. nov., Kazachstaniaserrabonitensis sp. nov. and Kazachstania australis sp. nov. Reassignment of Candida humilis to Kazachstania humilis f.a. comb. nov. and Candida pseudohumilis to Kazachstania pseudohumilis f.a. comb. nov.

Five ascosporogenous yeast strains related to the genus Kazachstania were isolated. Two strains (CLIB 1764T and CLIB 1780) were isolated from French sourdoughs; three others (UFMG-CM-Y273T, UFMG-CM-Y451 and UFMG-CM-Y452) were from rotting wood in Brazil. The sequences of the French and Brazilian strains differed by one and three substitutions, respectively, in the D1/D2 large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS). The D1/D2 LSU rRNA sequence of these strains differed by 0.5 and 0.7 % from Kazachstania exigua, but their ITS sequences diverged by 8.1 and 8.3 %, respectively, from that of the closest described species Kazachstania barnettii. Analysis of protein coding sequences of RPB1, RPB2 and EF-1α distinguished the French from the Brazilian strains, with respectively 3.3, 6 and 11.7 % substitutions. Two novel species are described to accommodate these newly isolated strains: Kazachstania saulgeensis sp. nov. (type strain CLIB 1764T=CBS 14374T) and Kazachstania serrabonitensis sp. nov. (type strain UFMG-CM-Y273T=CLIB 1783T=CBS 14236T). Further analysis of culture collections revealed a strain previously assigned to the K. exigua species, but having 3.8 % difference (22 substitutions and 2 indels) in its ITS with respect to K. exigua. Hence, we describe a new taxon, Kazachstania australis sp. nov. (type strain CLIB 162T=CBS 2141T), to accommodate this strain. Finally, Candida humilis and Candida pseudohumilis are reassigned to the genus Kazachstania as new combinations. On the basis of sequence analysis, we also propose that Candida milleri and Kazachstania humilis comb. nov. are conspecific.

D-xylose-fermenting and xylanase-producing yeast species from rotting wood of two Atlantic Rainforest habitats in Brazil.

This study investigated the yeast species associated with rotting wood in Brazilian Atlantic Rainforest ecosystems focusing on the identification of D-xylose-fermenting and/or xylanase-producing species. A total of 321 yeast strains were isolated from rotting wood samples collected in two Atlantic Rainforest areas. These samples were cultured in yeast nitrogen base (YNB)-D-xylose or YNB-xylan media. Schwanniomyces polymorphus, Scheffersomyces queiroziae, Barnettozyma californica, and Candida (Ogataea) boidinii were the most frequently isolated yeasts. The rarefaction curves for the yeast communities isolated in YNB-D-xylose and YNB-xylan from both areas continued to rise and did not reach an asymptote, indicating that not all yeast diversity had been recovered. Additionally, the yeast composition was variable among the samples and areas, which was confirmed by the values of the Sorensen index. Among the 69 species identified, only 12 were found in both areas sampled. Fifteen possible new species were obtained. Among them, two species (Sugiyamaella sp. 1 and Sugiyamaella xylanicola) showed the ability to ferment D-xylose into ethanol, and three species (Spencermartinsiella sp. 1, Su. xylanicola and Tremella sp.) were able to produce extracellular xylanases. Indeed, most of the xylanase-producing isolates belong to the new species Su. xylanicola, which was also positive for D-xylose fermentation. S.queiroziae and S. stipitis were the main D-xylose-fermenting yeasts identified. The results of this work showed that rotting wood collected from the Atlantic Rainforests is a huge source of yeasts, including new species, with promising biotechnological properties.

Volatiles, a glutarimide alkaloid and antimicrobial effects of Croton pullei (Euphorbiaceae).

Chemical investigation of Croton pullei (Euphorbiaceae) collected in the Brazilian Amazon region was revisited. The chemical composition of the essential oils of leaves and stems was analyzed by GC/MS. It was found that both the oils comprise mainly terpenes, among which linalool was the major one (24.90 and 39.72%, respectively). Phytochemical investigation of the stem methanol extract led to the isolation of a new natural product from the glutarimide alkaloid group named N-[2,6-dioxo-1-(2-phenylethyl)-3-piperidinyl]-acetamide, confirming that C. pullei is a rich source of this class of alkaloids. The hexane and methanol extracts of the stems of C. pullei showed moderate antibacterial and antifungal activity and the highest inhibition was observed when the methanol extract was tested against Staphylococcus aureus CCMB 262 and CCMB 263.

Evaluation of a Functional Craft Wheat Beer Fermented with Saccharomyces cerevisiae UFMG A-905 to treat Salmonella Typhimurium infection in mice.

Functional foods containing probiotics are generally administered as dairy products. Non-dairy beverages are another possibility, but probiotic functionality must be confirmed in such vehicles. In the present study, a craft wheat beer brewed with the probiotic yeast Saccharomyces cerevisiae UFMG A-905 (905) was evaluated in a murine model of Salmonella Typhimurium infection. Unfiltered or filtered beer brewed with 905, a commercial wheat beer used as a negative control, or saline were administered orally to mice before and during oral S. Typhimurium challenge. High fecal levels of yeast were only counted in mice treated with the unfiltered 905 beer, which also had reduced mortality and body weight loss due to S. Typhimurium infection. Increased levels of intestinal IgA, translocation to liver and spleen, liver and intestinal lesions, pro-inflammatory cytokines in liver and ileum, and hepatic and intestinal myeloperoxidase and eosinophilic peroxidase activities were observed in animals infected with S. Typhimurium. All these parameters were reduced by the treatment with unfiltered 905 beer. In conclusion, the results show that a craft wheat beer brewed with S. cerevisiae UFMG A-905 maintained the probiotic properties of this yeast when administered orally to mice challenged with S. Typhimurium.

Sex differences in the development of conditioned place preference induced by intragastric alcohol administration in mice.

BACKGROUND: The present study aimed to identify for the first time sex differences in the development of CPP induced by intragastric alcohol administration in mice. METHODS: Male and female adult Swiss mice were submitted to 16 days of conditioning with alcohol (0.5-3.0 g/kg, N = 8/dose/sex), with 2 post-conditioning tests (after 8 and 16 sessions) during the protocol. RESULTS: 8 days of conditioning (4 alcohol sessions, 4 saline sessions) with intragastric alcohol administration were sufficient to induce CPP in male mice at the doses of 1.0, 1.5 and 2.0 g/kg. However, only higher doses (2.0, 2.5 and 3.0 g/kg) induced CPP in female mice using an 8-day conditioning protocol, while a 16-day conditioning protocol was necessary for the development of intragastric alcohol-induced CPP at the doses of 1.0 and 1.5 g/kg. Regardless of the conditioning protocol, higher doses or alcohol that had rewarding effects in females (2.5 and 3.0 g/kg) did not induce CPP in males, with a significant difference between males and females at those doses. Analysis of the potency (EC50) and efficacy (Emax) of alcohol in inducing CPP when administered intragastrically in male and female mice showed significant sex differences with 8 conditioning sessions. CONCLUSIONS: Our data show a clear protocol (8 vs 16 days) and dose difference between male and female Swiss mice regarding the development of CPP induced by intragastric alcohol administration. Intragastric alcohol administration is closer to human drinking, and our protocol provides a more translational approach to studying the rewarding effects of alcohol in mice.

Citral modulates virulence factors in methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for high morbidity and mortality rates. Citral has been studied in the pharmaceutical industry and has shown antimicrobial activity. This study aimed to analyze the antimicrobial activity of citral in inhibiting biofilm formation and modulating virulence genes, with the ultimate goal of finding a strategy for treating infections caused by MRSA strains. Citral showed antimicrobial activity against MRSA isolates with minimum inhibitory concentration (MIC) values between 5 mg/mL (0.5%) and 40 mg/mL (4%), and minimum bactericidal concentration (MBC) values between 10 mg/mL (1%) and 40 mg/mL (4%). The sub-inhibitory dose was 2.5 mg/mL (0.25%). Citral, in an antibiogram, modulated synergistically, antagonistically, or indifferent to the different antibiotics tested. Prior to evaluating the antibiofilm effects of citral, we classified the bacteria according to their biofilm production capacity. Citral showed greater efficacy in the initial stage, and there was a significant reduction in biofilm formation compared to the mature biofilm. qPCR was used to assess the modulation of virulence factor genes, and icaA underexpression was observed in isolates 20 and 48. For icaD, seg, and sei, an increase was observed in the expression of ATCC 33,591. No significant differences were found for eta and etb. Citral could be used as a supplement to conventional antibiotics for MRSA infections.

Production of ethanol and xylanolytic enzymes by yeasts inhabiting rotting wood isolated in sugarcane bagasse hydrolysate.

Yeasts associated with rotting wood from four Atlantic Rain forest sites in Brazil were investigated using a culture medium based on sugarcane bagasse hydrolysate. A total of 330 yeast strains were isolated. Pichia manshurica, Candida pseudolambica, and Wickerhamomyces sp. 3 were the most frequently isolated species. Fourteen novel species were obtained in this study. All isolates were tested for their ability to ferment d-xylose and to produce xylanases. In the fermentation assays using d-xylose (30 g L-1), the main ethanol producers were Scheffersomyces stipitis (14.08 g L-1), Scheffersomyces sp. (7.94 g L-1) and Spathaspora boniae (7.16 g L-1). Sc. stipitis showed the highest ethanol yield (0.42 g g-1) and the highest productivity (0.39 g L-1h-1). The fermentation results using hemicellulosic hydrolysate showed that Sc. stipitis was the best ethanol producer, achieving a yield of 0.32 g g-1, while Sp. boniae and Scheffersomyces sp. were excellent xylitol producers. The best xylanase-producing yeasts at 50 °C belonged to the species Su. xylanicola (0.487 U mg-1) and Saitozyma podzolica (0.384 U mg-1). The results showed that rotting wood collected from the Atlantic Rainforest is a valuable source of yeasts able to grow in sugarcane bagasse hydrolysate, including species with promising biotechnological properties.

Polygalacturonase secreted by yeasts from Brazilian semi-arid environments.

Microbial pectinolytic enzymes are known to play a commercially important role in a number of industrial processes. The objective of this study was to investigate the extracellular polygalacturonases of yeasts isolated from Brazilian semi-arid environments. Among the 250 colonies tested, only 33 produced extracellular polygalacturonases: Aureobasidium pullulans (18 isolates), Candida boidinii (one isolate), Trichosporonoides sp. (three isolates), Kluyveromyces marxianus (one isolate), Cryptococcus liquefaciens (one isolate), Pseudozyma sp. (four isolates), and yeast-like related to fungal endophyte (five isolates). The highest activity of polygalacturonase was observed in Pseudozyma sp. CCMB 300 (14.17+/-0.08 micromol acid galacturonic released/min/mg protein). This study shows the potential of yeasts and yeast-like organisms isolated from Brazilian semi-arid environments to produce pectinolytic enzymes.

Characterisation of the diversity and physiology of cellobiose-fermenting yeasts isolated from rotting wood in Brazilian ecosystems.

We investigated the yeast species associated with rotting wood samples obtained from Brazilian ecosystems, with a special focus on cellobiose-fermenting species. About 647 yeast strains were isolated from rotting wood samples collected from the areas of Atlantic rainforest, Cerrado, and Amazonian forest. Eighty-six known species and 47 novel species of yeasts were isolated. Candida boidinii, Cyberlindnera subsufficiens, Meyerozyma guilliermondii, Schwanniomyces polymorphus, Candida natalensis, and Debaryomyces hansenii were the most frequently isolated species. Among the cellobiose-fermenting yeasts, 14 known and three novel yeast species were identified. Scheffersomyces queiroziae, Sc. amazonensis, Yamadazyma sp.1, Hanseniaspora opuntiae, C. jaroonii, and Candida tammaniensis were the main ethanol-producing yeasts. These species also produced an intracellular ß-glucosidase responsible for cellobiose hydrolysis. In fermentation assays using a culture medium containing 50 g L-1 cellobiose, ethanol production was observed in all cases; Sc. queiroziae and Sc. amazonensis showed the highest yield, efficiency, and productivity. Candida jaroonii and Yamadazyma sp.1 strains also showed high efficiency in cellobiose fermentation, while C. tammaniensis and H. opuntiae strains produced low amounts of ethanol. This study shows the potential of rotting wood samples from Brazilian ecosystems as a source of yeasts, including new species as well as those with promising biotechnological properties.

Thermoresistant xylanases from Trichoderma stromaticum: Application in bread making and manufacturing xylo-oligosaccharides.

The enzymes Xyl1 and Xyl2 from T. stromaticum were purified and identified by mass spectrometry (MALDI-TOF/MS). Xyl1 contained three proteins with similarity to xylanase family 10, 62 and anarabinofuranosidase of the Trichoderma genus and Xyl2 contained a protein with similarity to endo-1,4-ß-xylanase. High xylanase activity was found at 50°C for Xyl1 and 60°C for Xyl2 and pH 5.0 for both, retaining more than 80% of activities for one hour at 60°C and pH 5-8. Ag2+ and ß-mercaptoethanol increased while SDS and EDTA inhibited the xylanase activity of both Xyl1 and Xyl2 extracts. The Km and Vmax values for purified Xyl2 were 9.6mg/mL and 28.57µmol/min/mg, respectively. In application tests, both Xyl1 and Xyl2 were effective in degrading beechwood xylan to produce xylo-oligosaccharides. In baking, adding Xyl1 increased the softness and volume of wheat bread and whole grain bread, qualities increasingly desired by consumers in this segment.

Anti-Inflammatory Activity of the Essential Oil Citral in Experimental Infection with Staphylococcus aureus in a Model Air Pouch.

This study proposes to implement an alternative and effective strategy for local treatment of disease provoked by S. aureus. For the analysis of possible anti-inflammatory activity of essential oil, after establishing an air pouch model, 48 male mice of Balb/c were treated, infected, and euthanized at 4 and 8 h. Thus, the total and differential white blood cells were counted in the animal's blood, and cytokines IL-1ß, IL-6, and TNF-α were titrated using ELISA in the air pouch lavage. Moreover, TNF-α, IL-1ß, and IL-6 gene expression was analyzed through an RT-qPCR array, and S. aureus was quantified using qPCR. Our results, p < 0.05, showed that EOC reduced the quantity of microorganisms. The group of mice treated with essential oil citral showed a significant decrease in TNF-α levels in tests demonstrating anti-inflammatory activity. There is no data about the mutual influence of the air pouch model, essential oil citral, and S. aureus. Thus, considering the interaction of these variables and the anti-inflammatory activity of the essential oil citral, we demonstrated, by alternative local treatment, a new antimicrobial agent that is not an antibiotic.

Chemical composition and antibacterial activity of essential oils from Myrcia alagoensis (Myrtaceae).

The chemical composition and antibacterial activity of essential oils obtained from fresh and dried leaves of Myrcia alagoensis O. Berg, collected in a secondary forest remnant in north-eastern Brazil, was compared. The essential oils were obtained by hydrodistillation from fresh and dried leaves, and analysed by GC/FID and GC/MS. The antimicrobial properties of the oils were investigated against five bacteria by determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). The essential oils were rich in cyclic sesquiterpenes, such as germacrene B, with antibiotic action against Gram-positive and Gram-negative bacteria. The drying process after collection interfered with the chemical composition and antibacterial activity of the assessed samples.

Identification and characterization of a class III chitin synthase gene of Moniliophthora perniciosa, the fungus that causes witches' broom disease of cacao.

Chitin synthase (CHS) is a glucosyltransferase that converts UDP-N-acetylglucosamine into chitin, one of the main components of fungal cell wall. Class III chitin synthases act directly in the formation of the cell wall. They catalyze the conversion of the immediate precursor of chitin and are responsible for the majority of chitin synthesis in fungi. As such, they are highly specific molecular targets for drugs that can inhibit the growth and development of fungal pathogens. In this work, we have identified and characterized a chitin synthase gene of Moniliophthora perniciosa (Mopchs) by primer walking. The complete gene sequence is 3,443 bp, interrupted by 13 small introns, and comprises a cDNA with an ORF with 2,739 bp, whose terminal region was experimentally determined, encoding a protein with 913 aa that harbors all the motifs and domains typically found in class III chitin synthases. This is the first report on the characterization of a chitin synthase gene, its mature transcription product, and its putative protein in basidioma and secondary mycelium stages of M. perniciosa, a basidiomycotan fungus that causes witches' broom disease of cacao.