Production and biochemical and biophysical characterization of fibrinolytic protease of a Mucor subtilissimus strain isolated from the caatinga biome.

Cardiovascular diseases, resulting from the deposition of clots in blood vessels, are the leading cause of death worldwide. Fibrinolytic enzymatic activity can catalyze blood clot degradation. Findings show that 36 fungal isolates recovered from Caatinga soils have the potential to produce fibrinolytic protease under submerged conditions. About 58 % of the isolates displayed fibrinolytic activity above 100 U/mL, with Mucor subtilissimus UCP 1262 being the most active. The protease was biochemically and biophysically characterized, showing that the enzyme had a high affinity for SAApNA substrate and was significantly inhibited by fluoride methyl phenyl sulfonyl-C7H7FO2S, suggesting that it is a chymotrypsin-like serine protease. The highest enzyme activity was detected at pH 5.0 and 28 °C. This fibrinolytic protease's far-UV circular dichroism (CD) showed that its secondary structure was primarily α-helical. The purified fibrinolytic enzyme may represent a novel therapeutic agent for treating thrombosis. At temperatures above 65 °C, the enzyme lost all its secondary structure. Its melting temperature was 58.1 °C, the denaturation enthalpy 85.1 kcal/mol, and the denaturation entropy 0.26 kcal/K∙mol.

Novel Bio-Functional Aloe vera Beverages Fermented by Probiotic Enterococcus faecium and Lactobacillus lactis.

Aloe vera has been medicinally used for centuries. Its bioactive compounds have been shown to be very effective in the treatment of numerous diseases. In this work, a novel functional beverage was developed and characterized to combine the health benefits of probiotic bacteria with the Aloe vera plant itself. Two Aloe vera juices were obtained by fermentation either by a novel isolated Enterococcus faecium or a commercial Lactococcus lactis. The extraction of Aloe vera biocompounds for further fermentation was optimized. Extraction with water plus cellulase enhanced the carbohydrates and phenolic compounds in the obtained extracts. The biotransformation of the bioactive compounds from the extracts during fermentation was assessed. Both probiotic bacteria were able to grow on the Aloe vera extract. Lactic acid and short-chain fatty acids (SCFA) together with fourteen individual phenolic compounds were quantified in the produced Aloe vera juice, mainly epicatechin, aloin, ellagic acid, and hesperidin. The amount of total phenolic compounds was maintained through fermentation. The antioxidant activity was significantly increased in the produced juice by the ABTS method. The novel produced Aloe vera juice showed great potential as a functional beverage containing probiotics, prebiotics, SCFA, and phenolic compounds in its final composition.

Purification of chitosanases produced by Bacillus toyonensis CCT 7899 and functional oligosaccharides production.

Chitooligosaccharides (COS) have a great potential to be used by pharmaceutical industry due to their many biological activities. The use of enzymes to produce them is very advantageous, however it still faces many challenges, such as discovering new strains capable to produce enzymes that are able to generate bioactive oligosaccharides. In the present study a purification protein protocol was performed to purify chitosanases produced by Bacillus toyonensis CCT 7899 for further chitosan hydrolysis. The produced chitooligosaccharides were characterized by mass spectroscopy (MS) and their antiedematogenic effect was investigated through carrageenan-induced paw edema model. The animals were treated previously to inflammation by intragastric route with COS at 30, 300 and 600 mg/kg. The purification protocol showed a good performance for the chitosanases purification using 0.20 M NaCl solution to elute it, with a 9.54-fold purification factor. The treatment with COS promoted a decrease of paw edema at all evaluated times and the AUC0-4h, proving that COS produced showed activity in acute inflammation like commercial anti-inflammatory Dexamethasone (corticosteroid). Therefore, the strategy used to purification was successfully applied and it was possible to generate bioactive oligosaccharides with potential pharmacological use.

Purification and characterization of two new antimicrobial molecules produced by an endophytic strain of Paenibacillus polymyxa.

An endophytic bacterium inhibiting pathogenic bacteria was isolated and the strain was genetically identified as Paenibacillus polymyxa. Biochemical characterization of fermentation broth indicated the presence of peptidic antimicrobial molecules. Liquid-liquid partition resulted in an organic fraction (OF) and an aqueous fraction (AF). OF presented a broad spectrum of activity against a panel of pathogenic bacteria and a fungus whereas the AF was active only against Gram-negative bacteria. AF was sequentially submitted to ion-exchange, desalting and reverse phase (RP) chromatography. A molecule with an RT of 2.45 min exhibited activity against all Gram-negative pathogenic strains tested beside P. mirabilis. The primary structure of the molecule, named AMP-Pp, was determined as Gly-Glu-Hyp-Gly-Ala by N-terminal sequencing. The molecular mass and amino acid sequence were confirmed by MS/MS. With a molecular mass of 463 Da, AMP-Pp is one of the smallest active natural peptides reported, yet. RP chromatography of OF resulted in four peaks. The first three peaks corresponded to known antimicrobials. MS analysis of peak 4 revealed the presence of an ion with m/z 3,376.4 Da, whose proposed molecular formula is C182H321N29O29. The compound, named polycerradin, showed a spectrum of activity against Gram-positive bacteria, Gram-negative bacteria (beside P. mirabilis) and a fungus.

Diretriz Brasileira de Reabilitação Cardiovascular ­ 2020 / Brazilian Cardiovascular Rehabilitation Guideline - 2020

INTRODUÇÃO: Está cientificamente comprovado, sendo algo incorporado ao senso comum, que ser fisicamente ativo contribui para preservar e recuperar a boa saúde do corpo e da mente. Os efeitos favoráveis da reabilitação cardiovascular (RCV) com ênfase nos exercícios físicos têm sido consistentemente documentados, inclusive em meta-análises de estudos clínicos randomizados, que demonstram significativas reduções da morbimortalidade cardiovascular e global,1 bem como da taxa de hospitalização,1,2 com expressivo ganho de qualidade de vida,1,2 justificando a sua consensual e enfática recomendação pelas principais sociedades médicas mundiais.3-6 O sedentarismo, que apresenta elevada prevalência no Brasil e no mundo, está fortemente relacionado às doenças cardiovasculares (DCV) e à mortalidade precoce.7,8 Em contrapartida, maiores volumes de atividade física são positivamente associados à melhor qualidade e à maior expectativa de vida,9-13 existindo uma forte e inversa associação dos diferentes componentes da aptidão física com a mortalidade por todas as causas e com a ocorrência de eventos cardiovasculares desfavoráveis. Ou seja, quanto menor o nível de aptidão física, maior tende ser a taxa de mortalidade.14-21 Portanto, o principal objetivo da RCV com ênfase nos exercícios físicos é propiciar uma melhora dos componentes da aptidão física, tanto aeróbico quanto não aeróbicos (força/ potência muscular, flexibilidade, equilíbrio), algo que exige a combinação de diferentes modalidades de treinamento. Assim, a RCV deve proporcionar os mais elevados níveis de aptidão física passíveis de obtenção, de modo a reduzir o risco de eventos cardiovasculares e promover todos os outros benefícios a serem auferidos pela prática regular de exercícios físicos, culminando com a redução da mortalidade geral.

Purification of a lectin from Cratylia mollis crude extract seed by a single step PEG/phosphate aqueous two-phase system.

The partitioning and purification of lectins from the crude extract of Cratylia mollis seeds (Cramoll 1,4) was investigated in aqueous two-phase systems (ATPS). A factorial design model (24) was used to evaluate the influence of polyethylene glycol (PEG) molar mass (1500-8000 g/mol), PEG concentration (12.5-17.5% w/w), phosphate (10-15% w/w) concentration, and pH (6-8) on the differential partitioning, purification factor, and yield of the lectin. Polymer and salt concentration were the most important variables affecting partition of lectin and used to find optimum purification factor by experimental Box-Behnken design together with the response surface methodology (RSM). ATPS showed best conditions composed by 13.9% PEG1500, 15.3% phosphate buffer at pH 6, which ensured purification factor of 4.70. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of protein with 26.1 kDa. Furthermore, results demonstrated a thermostable lectin presenting activity until 60 °C and lost hemagglutinating activity at 80 °C. According to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of lectins.

Hydrogel as an alternative structure for food packaging systems.

Hydrogels are three-dimensional, hydrophilic networks, comprising polymeric chains linked through physical or chemical bonds. In the area of food, hydrogels have great potential to be used in food packaging systems or as carriers of bioactive components. This paper reviews the nature of hydrogels, their 3D network conformation, their functional properties, and their potential applications in food packaging systems. Regarding their potential food packaging applications, hydrogels can present a conformation which allows their use as part of a packaging system to control the humidity generated by food products with high water content. Moreover, the incorporation of nanoparticles into hydrogels may grant them antimicrobial activity. Finally, although the current research in this field is still limited, the results obtained so far are promising for innovative and potential applications in the food field, which also include their integration into intelligent food packaging systems and their direct incorporation into food matrices as a flavor carrier system.

Estudo por analisador de gases do Teste de Caminhada de seis minutos na insuficiência cardíaca com fração de ejeção normal / Study with a portable gas analyzer of the 6-minutes walk test in heart failure with normal ejection fraction

Background: Few studies have used portable gas analyzers during the 6-minute walk test (6MWT) in patients with heart failure and normal ejection fraction (HFNEF). Objectives: To analyze the kinetics of hemodynamic, ventilatory, and metabolic variables in patients with HFNEF during the T6m using a portable gas analyzer. Methods: Prospective, analytical study with an intentional, non-probabilistic, convenience sample. In total, 24 patients with HFNEF and past hospital admissions due to a clinical diagnosis of heart failure (HF) were included using the 2007 criteria established by the European Society of Cardiology. Three assessments were performed: 6MWT familiarization, 6MWT with the portable gas analyzer, and cardiopulmonary exercise test (CPET). Results: The heart rates (HRs) and the peak VO 2 at the end of the 6MWT corresponded to 85.7% and 86.45% of the values obtained during the CPET. The final HRs after the T6m were equivalent to those obtained at the CPET anaerobic threshold (AT), with relative VO 2 values at the end of the 6MWT above the VO 2 of the CPET AT. There was no difference between the maximum respiratory quotient (RQ) values in these two tests, which were both above 1.0. The VE/VO 2 slope descended initially and then ascended significantly after the fifth minute of the test, estimating the identification of the AT. Conclusions: In patients with HFNEF, the 6MWT represents an almost maximum effort, and is performed above the CPET AT and 85% above the maximum HR and the CPET peak VO 2 , with a maximum RQ similar to that in the CPET

Fundamentos: Poucos estudos utilizaram analisadores de gases portáteis no teste da caminhada de seis minutos (T6m) em portadores de insuficiência cardíaca com fração de ejeção normal (ICFEN). Objetivos: Analisar a cinética das variáveis hemodinâmicas, ventilatórias e metabólicas utilizando analisador de gases portátil em portadores de ICFEN durante o T6m. Métodos: Estudo prospectivo, analítico, com amostra não probabilística, intencional e por conveniência. Foram estudados 24 pacientes portadores de ICFEN com passado de internação por clínica de insuficiência cardíaca (IC), incluídos pelos critérios da European Society of Cardiology 2007. Realizaram-se três avaliações: T6m de aprendizado, T6m com o analisador de gases portátil e teste de esforço cardiopulmonar (TECP). Resultados: As frequências cardíacas (FC) e o consumo de oxigênio (VO 2 ) pico ao final do T6m corresponderam a 85,7% e 86,45% dos valores obtidos no TECP. As FC finais no T6m foram equivalentes às obtidas no limiar anaeróbio (LA) do TECP, com valores de VO 2 relativo ao final do T6m acima do VO 2 no LA do TECP. Não houve diferença entre os valores máximos do quociente respiratório (QR) entre os dois testes, ambos acima de 1,0. A curva de VE/VO 2 demonstrou descenso com posterior ascensão significativa após o quinto minuto de teste, estimando-se a identificação do LA. Conclusões: Para pacientes com ICFEN, o T6m representa um esforço quase máximo, sendo executado acima do LA do TECP e acima dos 85% da FC máxima e do VO 2 pico do TECP, com QR máximo semelhante ao do TECP

Antioxidant Compounds Recovery from Juçara Residue by Thermal Assisted Extraction.

This study aimed to recover bioactive compounds by solid-liquid extraction from the agro-industrial residue obtained during juçara fruits processing into pulp. A preliminary study using different solvents (methanol, ethanol and water) indicated ethanol in aqueous solution as the best solvent for antioxidants recovery. Then, a Box-Behnken design was applied considering as independent variables the solvent composition (30-70% ethanol in water), temperature (30-70 °C) and time (30-60 min), in order to evaluate the effects of these factors on antioxidant activity in juçara extract. Results showed that the extracts with higher antioxidant activity were obtained using 30% ethanol at 70 °C for 60 min; measurements included ABTS and DPPH assays, determination of total phenolic content and total monomeric anthocyanins. Furthermore, the effect of pH in antioxidants recovery was evaluated. For this purpose, the 30% ethanol solution was acidified to pH 1 and 2 with HCl. Principal component analysis showed the formation of three distinct groups: one characterized by high bioactive compounds content (pH 1.0), another with superior antioxidant activity (pH 5.75, non-acidified), and finally the group at pH 2 presenting the worst concentrations in the evaluated responses. HPLC analysis showed the presence of cyanidin-3-O-rutinoside and cyanidin-3-O-glucoside in the extracts. Therefore, the conventional solid-liquid extraction using renewable solvent can be successfully applied to recover bioactive compounds from juçara residue, which can be used by different food industries.

Brazilian Kefir-Fermented Sheep's Milk, a Source of Antimicrobial and Antioxidant Peptides.

Fermented milks are a source of bioactive peptides and may be considered as functional foods. Among these, sheep's milk fermented with kefir has not been widely studied and its most relevant properties need to be more thoroughly characterized. This research study is set out to investigate and evaluate the antioxidant and antimicrobial properties of peptides from fermented sheep's milk in Brazil when produced by using kefir. For this, the chemical and microbiological composition of the sheep's milk before and after the fermentation was evaluated. The changes in the fermented milk and the peptides extracted before the fermentation and in the fermented milk during its shelf life were verified. The antimicrobial and antioxidant activities of the peptides from the fermented milk were evaluated and identified according to the literature. The physicochemical properties and mineral profile of the fermented milk were like those of fresh milk. The peptide extract presented antimicrobial activity and it was detected that 13 of the 46 peptides were able to inhibit the growth of pathogenic microorganisms. A high antioxidant activity was observed in the peptides extracted from fermented milk (3.125 mg/mL) on the 28th day of storage. Two fractions displayed efficient radical scavenging properties by DPPH and ABTS methods. At least 11 peptides distributed in the different fractions were identified by tandem mass spectrometry. This sheep's milk fermented by Brazilian kefir grains, which has antioxidant and antimicrobial activities and probiotic microorganisms, is a good candidate for further investigation as a source for bioactive peptides. The fermentation process was thus a means by which to produce potential bioactive peptides.

Saccharomyces cerevisiae from Brazilian kefir-fermented milk: An in vitro evaluation of probiotic properties.

The therapeutic use of probiotics for supporting the antibiotic action against gastrointestinal disorders is a current trend and emerging applications have gained popularity because of their support for various microbiological activities in digestive processes. Microorganisms isolated from kefir with great probiotic properties, in addition to high resistance to harsh environmental conditions, have been widely researched. Administration of probiotic yeasts offers a number of advantages, when compared to bacteria, because of particular characteristics as their larger cell size. In the present study, 28 strains of Saccharomyces cerevisiae were isolated, after in vitro digestion of kefir-fermented milk, and identified by molecular based approaches. A screening was performed to determine important quality requirements for probiotics including: antagonistic and antioxidant activities, ß-galactosidase synthesis, autoaggregation, surface hydrophobicity and adhesion to epithelial cells. The results showed strains: with antagonistic activity against microbial pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis; able to produce ß-galactosidase; with antioxidant activity levels higher than 90%; with hydrophobicity activity and autoaggregation ability (evaluated by adhesion test, where all the strains presented adhesion to mice ileal epithelial cells). These findings are relevant and the strains are recommended for further in vivo studies as well as for potential therapeutic applications.

Trypsin purification using magnetic particles of azocasein-iron composite.

This work presents an inexpensive, simple and fast procedure to purify trypsin based on affinity binding with ferromagnetic particles of azocasein composite (mAzo). Crude extract was obtained from intestines of fish Nile tilapia (Oreochromis niloticus) homogenized in buffer (01g tissue/ml). This extract was exposed to 100mg of mAzo and washed to remove unbound proteins by magnetic field. Trypsin was leached off under high ionic strength (3M NaCl). Preparation was achieved containing specific activity about 60 times higher than that of the crude extract. SDS-PAGE showed that the purified protein had molecular weight (24kDa) in concordance with the literature for the Nile tilapia trypsin. The mAzo composite can be reused and applied to purify trypsin from other sources.

Purification and characterization of a collagenase from Penicillium sp. UCP 1286 by polyethylene glycol-phosphate aqueous two-phase system.

Collagenases are proteolytic enzymes capable of degrading both native and denatured collagen, reported to be applied in industrial, medical and biotechnological sectors. Liquid-liquid extraction using aqueous two-phase system (ATPS) is one of the most promising bioseparation techniques, which can substitute difficult solid-liquid separation processes, offering many advantages over conventional methods including low-processing time, low-cost material and low-energy consumption. The collagenase produced by Penicillium sp. UCP 1286 showed a stronger affinity for the bottom salt-rich phase, where the highest levels of collagenolytic activity were observed at the center point runs, using 15.0% (w/w) PEG 3350 g/mol and 12.5% (w/w) phosphate salt at pH 7.0 and concentration. The enzyme was characterized by thermal stability, pH tolerance and effect of inhibitors, showing optimal collagenolytic activity at 37 °C and pH 9.0 and proved to be a serine protease. ATPS showed high efficiency in the collagenase purification, confirmed by a single band in SDS/PAGE, and can in fact be applied as a quick and inexpensive alternative method.

Collagenolytic enzymes produced by fungi: a systematic review

Abstract Specific proteases capable of degrading native triple helical or denatured collagen have been required for many years and have a large spectrum of applications. There are few complete reports that fully uncover production, characterization and purification of fungi collagenases. In this review, authors searched through four scientific on line data bases using the following keywords (collagenolytic OR collagenase) AND (fungi OR fungus OR fungal) AND (production OR synthesis OR synthesize) AND (characterization). Scientific criteria were adopted in this review to classify found articles by score (from 0 to 10). After exclusion criteria, 21 articles were selected. None obtained the maximum of 10 points defined by the methodology, which indicates a deficiency in studies dealing simultaneously with production, characterization and purification of collagenase by fungi. Among microorganisms studied the non-pathogenic fungi Penicillium aurantiogriseum and Rhizoctonia solani stood out in volumetric and specific collagenase activity. The only article found that made sequencing of a true collagenase showed 100% homology with several metalloproteinases fungi. A clear gap in literature about collagenase production by fungi was verified, which prevents further development in the area and increases the need for further studies, particularly full characterization of fungal collagenases with high specificity to collagen.

Collagenolytic enzymes produced by fungi: a systematic review

Specific proteases capable of degrading native triple helical or denatured collagen have been required for many years and have a large spectrum of applications. There are few complete reports that fully uncover production, characterization and purification of fungi collagenases. In this review, authors searched through four scientific on line data bases using the following keywords (collagenolytic OR collagenase) AND (fungi OR fungus OR fungal) AND (production OR synthesis OR synthesize) AND (characterization). Scientific criteria were adopted in this review to classify found articles by score (from 0 to 10). After exclusion criteria, 21 articles were selected. None obtained the maximum of 10 points defined by the methodology, which indicates a deficiency in studies dealing simultaneously with production, characterization and purification of collagenase by fungi. Among microorganisms studied the non-pathogenic fungi Penicillium aurantiogriseum and Rhizoctonia solani stood out in volumetric and specific collagenase activity. The only article found that made sequencing of a true collagenase showed 100% homology with several metalloproteinases fungi. A clear gap in literature about collagenase production by fungi was verified, which prevents further development in the area and increases the need for further studies, particularly full characterization of fungal collagenases with high specificity to collagen.(AU)