Unraveling potential enzymes and their functional role in fine cocoa beans fermentation using temporal shotgun metagenomics.

Cocoa beans fermentation is a spontaneous process, essential for the generation of quality starting material for fine chocolate production. The understanding of this process has been studied by the application of high-throughput sequencing technologies, which grants a better assessment of the different microbial taxa and their genes involved in this microbial succession. The present study used shotgun metagenomics to determine the enzyme-coding genes of the microbiota found in two different groups of cocoa beans varieties during the fermentation process. The statistical evaluation of the most abundant genes in each group and time studied allowed us to identify the potential metabolic pathways involved in the success of the different microorganisms. The results showed that, albeit the distinction between the initial (0 h) microbiota of each varietal group was clear, throughout fermentation (24-144 h) this difference disappeared, indicating the existence of selection pressures. Changes in the microbiota enzyme-coding genes over time pointed to the distinct ordering of fermentation at 24-48 h (T1), 72-96 h (T2), and 120-144 h (T3). At T1, the significantly more abundant enzyme-coding genes were related to threonine metabolism and those genes related to the glycolytic pathway, explained by the abundance of sugars in the medium. At T2, the genes linked to the metabolism of ceramides and hopanoids lipids were clearly dominant, which are associated with the resistance of microbial species to extreme temperatures and pH values. In T3, genes linked to trehalose metabolism, related to the response to heat stress, dominated. The results obtained in this study provided insights into the potential functionality of microbial community succession correlated to gene function, which could improve cocoa processing practices to ensure the production of more stable quality end products.

Pectinases Secretion by Saccharomyces cerevisiae: Optimization in Solid-State Fermentation and Identification by a Shotgun Proteomics Approach.

A sequential design strategy was applied to optimize the secretion of pectinases by a Saccharomyces cerevisiae strain, from Brazilian sugarcane liquor vat, on passion fruit residue flour (PFRF), through solid-state fermentation (SSF). A factorial design was performed to determine the influence variables and two rotational central composite designs were executed. The validated experimental result was of 7.1 U mL-1 using 50% PFRF (w/w), pH 5, 30 °C for 24 h, under static SSF. Polygalacturonase, pectin methyl esterase, pectin-lyase and pectate-lyase activities were 3.5; 0.08; 3.1 and 0.8 U mL-1, respectively. Shotgun proteomics analysis of the crude extract enabled the identification of two pectin-lyases, one pectate-lyase and a glucosidase. The crude enzymatic extract maintained at least 80% of its original activity at pH values and temperatures ranging from 2 to 8 and 30 to 80 °C, respectively, over 60 min incubation. Results revealed that PFRF might be a cost-effective and eco-friendly substrate to produce pectinases. Statistical optimization led to fermentation conditions wherein pectin active proteins predominated. To the extent of our knowledge, this is the first study reporting the synthesis of pectate lyase by S. cerevisiae.

Pestalotiopsis mangiferae isolated from cocoa leaves and concomitant tannase and gallic acid production.

The enzyme tannase is of great industrial and biotechnological importance for the hydrolysis of vegetable tannins, reducing their undesirable effects and generating products for a wide range of processes. Thus, the search for new microorganisms that permit more stable tannase production is of considerable importance. A strain of P. mangiferae isolated from cocoa leaves was selected and investigated for its capacity to produce tannase enzymes and gallic acid through submerged fermentation. The assessment of the variables affecting tannase production by P. mangiferae showed that tannic acid, ammonium nitrate and temperature were the most significant (8.4 U/mL). The variables were analyzed using Response Surface Methodology - RSM (Box-Behnken design), with the best conditions for tannase production being: 1.9% carbon source, 1% nitrogen source and temperature of 23 °C. Tannase activity doubled (16.9 U/mL) after the optimization process when compared to the initial fermentation. A pH of 7.0 was optimal for the tannase and it presented stability above 80% with pH between 4.0 and 7.0 after 2h of incubation. The optimal temperature was 30 °C and activity remained at above 80% at 40-60 °C after 1 h. Production of gallic acid was achieved with 1% tannic acid (0.9 mg/mL) and P. mangiferae had not used up the gallic acid produced by tannic acid hydrolysis after 144 h of fermentation. A 5% tannic acid concentration was the best for gallic acid production (1.6 mg/mL). These results demonstrate P. mangiferae's potential for tannase and gallic acid production for biotechnological applications.

High-throughput screening for distinguishing nitrilases from nitrile hydratases in Aspergillus and application of a Box-Behnken design for the optimization of nitrilase.

Nitrilases and nitrile hydratases/amidases hydrolyze nitriles into carboxylic acids and/or amides, which are used in industrial chemical processes. In the present study, 26 microorganisms, including yeasts and filamentous fungi, in a minimum solid mineral medium supplemented with glucose and phenylacetonitrile were screened to evaluate their biocatalytic potential. Of these microorganisms, five fungi of the genus Aspergillus were selected and subjected to colorimetry studies to evaluate the production and distinction of nitrilase and nitrile hydratase/amidase enzymes. Aspergillus parasiticus Speare 7967 and A. niger Tiegh. 8285 produced nitrilases and nitrile hydratase, respectively. Nitrilase optimization was performed using a Box-Behnken design (BBD) and fungus A. parasiticus Speare 7967 with phenylacetonitrile volume (µl), pH, and carbohydrate source (starch:glucose; g/g) as independent variables and nitrilase activity (U ml-1 ) as dependent variable. Maximum activity (2.97 × 10-3  U ml-1 ) was obtained at pH 5.5, 80 µl of phenylacetonitrile, and 15 g of glucose. A. parasiticus Speare 7967 showed promise in the biotransformation of nitriles to carboxylic acids.

Lactiplantibacillus plantarum strains isolated from spontaneously fermented cocoa exhibit potential probiotic properties against Gardnerella vaginalis and Neisseria gonorrhoeae.

BACKGROUND: Probiotics are important tools in therapies against vaginal infections and can assist traditional antibiotic therapies in restoring healthy microbiota. Recent research has shown that microorganisms belonging to the genus Lactobacillus have probiotic potential. Thus, this study evaluated the potential in vitro probiotic properties of three strains of Lactiplantibacillus plantarum, isolated during the fermentation of high-quality cocoa, against Gardnerella vaginalis and Neisseria gonorrhoeae. Strains were evaluated for their physiological, safety, and antimicrobial characteristics. RESULTS: The hydrophobicity of L. plantarum strains varied from 26.67 to 91.67%, and their autoaggregation varied from 18.10 to 30.64%. The co-aggregation of L. plantarum strains with G. vaginalis ranged from 14.73 to 16.31%, and from 29.14 to 45.76% with N. gonorrhoeae. All L. plantarum strains could moderately or strongly produce biofilms. L. plantarum strains did not show haemolytic activity and were generally sensitive to the tested antimicrobials. All lactobacillus strains were tolerant to heat and pH resistance tests. All three strains of L. plantarum showed antimicrobial activity against the tested pathogens. The coincubation of L. plantarum strains with pathogens showed that the culture pH remained below 4.5 after 24 h. All cell-free culture supernatants (CFCS) demonstrated activity against the two pathogens tested, and all L. plantarum strains produced hydrogen peroxide. CFCS characterisation in conjunction with gas chromatography revealed that organic acids, especially lactic acid, were responsible for the antimicrobial activity against the pathogens evaluated. CONCLUSION: The three strains of L. plantarum presented significant probiotic characteristics against the two pathogens of clinical importance. In vitro screening identified strong probiotic candidates for in vivo studies for the treatment of vaginal infections.

Gardnerella vaginalis and Neisseria gonorrhoeae Are Effectively Inhibited by Lactobacilli with Probiotic Properties Isolated from Brazilian Cupuaçu (Theobroma grandiflorum) Fruit.

In recent years, certain Lactobacillus sp. have emerged in health care as an alternative therapy for various diseases. Based on this, this study is aimed at evaluating in vitro the potential probiotics of five lactobacilli strains isolated from pulp of cupuaçu fruit fermentation against Gardnerella vaginalis and Neisseria gonorrhoeae. Our lactobacilli strains were classified as safe for use in humans, and they were tolerant to heat and pH. Our strains were biofilm producers, while hydrophobicity and autoaggregation varied from 13% to 86% and 13% to 25%, respectively. The coaggregation of lactobacilli used in this study with G. vaginalis and N. gonorrhoeae ranged from 15% to 36% and 32% to 52%, respectively. Antimicrobial activity was present in all tested Lactobacillus strains against both pathogens, and the growth of pathogens in coculture was reduced by the presence of our lactobacilli. Also, all tested lactobacilli reduced the pH of the culture, even in incubation with pathogens after 24 hours. The cell-free culture supernatants (CFCS) of all five lactobacilli demonstrated activity against the two pathogens with a halo presence and CFCS characterization assay together with gas chromatography revealed that lactic acid was the most abundant organic acid in the samples (50% to 62%). Our results demonstrated that the organic acid production profile is strain-specific. This study revealed that cupuaçu is a promising source of microorganisms with probiotic properties against genital pathogens. We demonstrated by in vitro tests that our Lactobacillus strains have probiotic properties. However, the absence of in vivo tests is a limitation of our work due to the need to evaluate the interaction of our lactobacilli with pathogens in the vaginal mucosa. We believe that these findings may be useful in developing a product containing our lactobacilli and their supernatants in order to support with vaginal health.

Differential Immune Response of Lactobacillus plantarum 286 Against Salmonella Typhimurium Infection in Conventional and Germ-Free Mice.

We aimed at evaluating in vivo the probiotic potential of Lactobacillus plantarum 286 against Salmonella enterica serov. Typhimurium. Colonization capacity and antagonistic activity were determined in feces of gnotobiotic mice. Survival to infection, translocation, histopathology, IgA and cytokine levels (IL-10, IL-6, IFN-γ, TNF-α, TGF-ß) were determined both in conventional and germ-free mice followed L. plantarum 286 administration and Salmonella infection. L. plantarum 286 colonized the intestine of gnotobiotic mice, where it produced antagonistic substances against S. Typhimurium. In conventional animals, the administration of this strain increased intestinal IgA levels and reduced the inflammatory response and the tissue damage caused by S. Typhimurium. Reduction of tissue damage in the intestine and liver of germ-free animals was also observed, however the immune response elicited was different in either model. L. plantarum 286 showed in vivo probiotic properties in both murine models. Probiotic capacity results may depend on the animal model chosen.

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.

Cuticle-induced endo/exoacting chitinase CHIT30 from Metarhizium anisopliae is encoded by an ortholog of the chi3 gene.

The characterization of chitinase genes and enzymes is an important step toward global understanding of the chitinolytic system in entomopathogenic fungi. Chitinase CHIT30 from Metarhizium anisopliae var. anisopliae (strain E6) has both endo- and exochitinase activities and is a potential determinant of pathogenicity. Serum anti-CHIT30 specifically detected this chitinase amongst five isoenzymes shown in glycol-chitin activity gels. Chitinase CHIT30 secretion is upregulated by chitin, tick cuticle and low concentrations of N-acetylglucosamine (0.25%) and is downregulated by both high N-acetylglucosamine (1%) and glucose (1%) concentrations. Chitinase CHIT30 was produced at tick cuticle during fungal infection. The chi3 gene was assigned to code chitinase CHIT30 in M. anisopliae var. anisopliae.