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.

In vitro models as a tool to study the role of gut microbiota in obesity.

Obesity, a highly prevalent condition worldwide that leads to the development of multiple metabolic diseases, has been related to gut microbial dysbiosis. To understand this correlation, in vivo models have been extremely useful. However, its use is limited by associated ethical concerns, high costs, low representativeness, and low reproducibility. Therefore, new and improved in vitro models have been developed in recent years, representing a promising tool in the study of the role of gut microbiota modulation in weight management and metabolic health. This review aims to provide an update on the main findings obtained in vitro regarding gut microbiota modulation with probiotics, and food compounds, and its interaction with the host metabolism, associated with obesity. Available in vitro colon models currently used to study obesity are discussed, including batch and dynamic fermentation systems, and models that allow the study of microbiota-host interactions using cell cultures. In vitro models have demonstrated that homeostatic microbiota may help overcome obesity by producing satiety-related neurotransmitters and metabolites that protect the gut barrier and improve the metabolic activity of adipose tissue. In vitro models may be the key to finding new treatments for obesity-related disorders.

Revealing the Beauty Potential of Grape Stems: Harnessing Phenolic Compounds for Cosmetics.

Grape stems have emerged as a promising natural ingredient in the cosmetics industry due to their abundance of phenolic compounds, known for their antioxidant and anti-inflammatory properties. These compounds have shown great potential in promoting skin health, fighting signs of aging, and shielding against environmental stressors. With high concentrations of resveratrol, flavonoids, and tannins, grape stems have garnered attention from cosmetic scientists. Research has indicated that phenolic compounds extracted from grape stems possess potent antioxidant abilities, effectively combating free radicals that accelerate aging. Moreover, these compounds have demonstrated the capacity to shield the skin from UV damage, boost collagen production, and enhance skin elasticity. Cosmetic formulations incorporating grape stem extracts have displayed promising results in addressing various skin concerns, including reducing wrinkles, fine lines, and age spots, leading to a more youthful appearance. Additionally, grape stem extracts have exhibited anti-inflammatory properties, soothing irritated skin and diminishing redness. Exploring the potential of grape stem phenolic compounds for cosmetics paves the way for sustainable and natural beauty products. By harnessing the beauty benefits of grape stems, the cosmetics industry can provide effective and eco-friendly solutions for consumers seeking natural alternatives. Ongoing research holds the promise of innovative grape stem-based formulations that could revolutionize the cosmetics market, fully unlocking the potential of these extraordinary botanical treasures.

Microbial Hyaluronic Acid Production: A Review.

Microbial production of hyaluronic acid (HA) is an area of research that has been gaining attention in recent years due to the increasing demand for this biopolymer for several industrial applications. Hyaluronic acid is a linear, non-sulfated glycosaminoglycan that is widely distributed in nature and is mainly composed of repeating units of N-acetylglucosamine and glucuronic acid. It has a wide and unique range of properties such as viscoelasticity, lubrication, and hydration, which makes it an attractive material for several industrial applications such as cosmetics, pharmaceuticals, and medical devices. This review presents and discusses the available fermentation strategies to produce hyaluronic acid.

Development of Meat Substitutes from Filamentous Fungi Cultivated on Residual Water of Tempeh Factories.

In recent years, there has been an increased motivation to reduce meat consumption globally due to environmental and health concerns, which has driven the development of meat substitutes. Filamentous fungal biomass, commonly known as mycoprotein, is a potential meat substitute since it is nutritious and has filaments to mimic meat fibrils. The current study aimed to investigate the potential use of a cheap substrate derived from the food industry, i.e., residual water in a tempeh factory, for mycoprotein production. The type of residual water, nutrient supplementation, optimum conditions for biomass production, and characteristics of the mycoprotein were determined. The results showed that the residual water from the first boiling with yeast extract addition gave the highest mycoprotein content. The optimum growth condition was a pH of 4.5 and agitation of 125 rpm, and it resulted in 7.76 g/L biomass. The mycoprotein contains 19.44% (w/w) protein with a high crude fiber content of 8.51% (w/w) and a low fat content of 1.56% (w/w). In addition, the amino acid and fatty acid contents are dominated by glutamic acid and polyunsaturated fatty acids, which are associated with an umami taste and are considered healthier foods. The current work reveals that the residual boiling water from the tempeh factory can be used to produce high-quality mycoprotein.

Structural and Physicochemical Properties of Starch from Rejected Chestnut: Hydrothermal and High-Pressure Processing Dependence.

The quality standards for the export of chestnuts generate large quantities of rejected fruits, which require novel processing technologies for their safe industrial utilization. This study aimed to investigate the impact of high-pressure processing (HPP) and hydrothermal treatments (HT) on the physicochemical properties of rejected chestnut starch. Chestnuts were treated by HPP at 400, 500, and 600 MPa for 5 min and HT at 50 °C for 45 min. In general, all HPP treatments did not induce starch gelatinization, and their granules preserved the integrity and Maltese-cross. Moreover, starch granules' size and resistant starch content increased with the intensity of pressure. Native and HT chestnut starches were the most susceptible to digestion. HPP treatments did not affect the C-type crystalline pattern of native starch, but the crystalline region was gradually modified to become amorphous. HPP-600 MPa treated starch showed modified pasting properties and exhibited the highest values of peak viscosity. This study demonstrates for the first time that after HPP-600 MPa treatment, a novel chestnut starch gel structure is obtained. Moreover, HPP treatments could increase the slow-digesting starch, which benefits the development of healthier products. HPP can be considered an interesting technology to obtain added-value starch from rejected chestnut fruits.

Exploring the Palynological, Chemical, and Bioactive Properties of Non-Studied Bee Pollen and Honey from Morocco.

Bee products are known for their beneficial properties widely used in complementary medicine. This study aims to unveil the physicochemical, nutritional value, and phenolic profile of bee pollen and honey collected from Boulemane-Morocco, and to evaluate their antioxidant and antihyperglycemic activity. The results indicate that Citrus aurantium pollen grains were the majority pollen in both samples. Bee pollen was richer in proteins than honey while the inverse was observed for carbohydrate content. Potassium and calcium were the predominant minerals in the studied samples. Seven similar phenolic compounds were found in honey and bee pollen. Three phenolic compounds were identified only in honey (catechin, caffeic acid, vanillic acid) and six phenolic compounds were identified only in bee pollen (hesperidin, cinnamic acid, apigenin, rutin, chlorogenic acid, kaempferol). Naringin is the predominant phenolic in honey while hesperidin is predominant in bee pollen. The results of bioactivities revealed that bee pollen exhibited stronger antioxidant activity and effective α-amylase and α-glycosidase inhibitory action. These bee products show interesting nutritional and bioactive capabilities due to their chemical constituents. These features may allow these bee products to be used in food formulation, as functional and bioactive ingredients, as well as the potential for the nutraceutical sector.

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.

Green Extraction Techniques as Advanced Sample Preparation Approaches in Biological, Food, and Environmental Matrices: A Review.

Green extraction techniques (GreETs) emerged in the last decade as greener and sustainable alternatives to classical sample preparation procedures aiming to improve the selectivity and sensitivity of analytical methods, simultaneously reducing the deleterious side effects of classical extraction techniques (CETs) for both the operator and the environment. The implementation of improved processes that overcome the main constraints of classical methods in terms of efficiency and ability to minimize or eliminate the use and generation of harmful substances will promote more efficient use of energy and resources in close association with the principles supporting the concept of green chemistry. The current review aims to update the state of the art of some cutting-edge GreETs developed and implemented in recent years focusing on the improvement of the main analytical features, practical aspects, and relevant applications in the biological, food, and environmental fields. Approaches to improve and accelerate the extraction efficiency and to lower solvent consumption, including sorbent-based techniques, such as solid-phase microextraction (SPME) and fabric-phase sorbent extraction (FPSE), and solvent-based techniques (µQuEChERS; micro quick, easy, cheap, effective, rugged, and safe), ultrasound-assisted extraction (UAE), and microwave-assisted extraction (MAE), in addition to supercritical fluid extraction (SFE) and pressurized solvent extraction (PSE), are highlighted.

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.

Anthocyanin Recovery from Grape by-Products by Combining Ohmic Heating with Food-Grade Solvents: Phenolic Composition, Antioxidant, and Antimicrobial Properties.

Usually, wine-making by-products are discarded, presenting a significant environmental impact. However, they can be used as a source of bioactive compounds. Moreover, consumers' increasing demand for naturally nutritious and healthy products requires new formulations and food product improvement, together with sustainable, environmentally friendly extraction methods. Thus, this work aimed to compare ohmic heating (OH) with conventional methodology (CONV), using food-grade solvents, mainly water, compared to standard methanol extraction of anthocyanins. No significant differences were found between the CONV and OH for total phenolic compounds, which were 2.84 ± 0.037 and 3.28 ± 0.46 mg/g DW gallic acid equivalent, respectively. The same tendency was found for antioxidant capacity, where CONV and OH presented values of 2.02 ± 0.007 g/100 g and 2.34 ± 0.066 g/100 g ascorbic acid equivalent, respectively. The major anthocyanins identified were malvidin-3-O-acetylglucoside, delphinidin-3-O-glucoside, petunidine-3-O-glucoside, cyanidin-3-O-glucoside, and peonidine-3-O-glucoside. These extracts displayed antimicrobial potential against microorganisms such as Yersinia enterocolitica, Pseudomonas aeruginosa, Salmonella enteritidis, methicillin-sensitive Staphylococcus aureus, a methicillin-resistant Staph. aureus (MRSA), and Bacillus cereus. In conclusion, OH provides similar recovery yields with reduced treatment times, less energy consumption, and no need for organic solvents (green extraction routes). Thus, OH combined with water and citric acid allows a safe anthocyanin extraction from grape by-products, thus avoiding the use of toxic solvents such as methanol, and with high biological potential, including antimicrobial and antioxidant activity.

Daily intake of wheat germ-enriched bread may promote a healthy gut bacterial microbiota: a randomised controlled trial.

PURPOSE: Wheat bran fibre has a beneficial effect on gastrointestinal function, but evidence for wheat germ is scarce. Accordingly, we evaluated the effects of daily intake of wheat germ on gastrointestinal discomfort and gut microbiota by adding wheat germ to refined (white) wheat bread, the most consumed bread type. We hypothesised that an improvement in the composition of refined bread could beneficially affect intestinal health without compromising consumers' acceptance. METHODS: Fifty-five healthy adults were recruited for a randomised, double-blind, crossover, controlled trial comprising two 4-week intervention periods separated by a 5-week washout stage. During the first 4-week period, one group consumed wheat bread enriched with 6 g of wheat germ and the control group consumed non-enriched wheat bread. RESULTS: Wheat germ-enriched bread was well-appreciated and the number of participants that demonstrated minimal gastrointestinal improvements after wheat-germ intake was higher than in the control arm. Importantly, intake of wheat germ-enriched bread decreased the perceived gastrointestinal discomfort-related quality of life (subscale worries and concerns) over refined white bread. The improvements in the gastrointestinal function were accompanied by favourable changes in gut microbiota, increasing the number of Bacteroides spp. and Bifidobacterium spp. CONCLUSIONS: Adding wheat germ to industrially made white bread without altering sensory properties may promote a healthy gut bacterial microbiota and the gastrointestinal health.

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.

Oleaginous yeasts for sustainable lipid production-from biodiesel to surf boards, a wide range of "green" applications.

A growing world population and a growing number of applications for vegetable oils are generating an increasing demand for these oils, causing serious environmental problems. A sustainable lipid production is then fundamental to address these problems. Oleaginous yeasts are a promising solution for sustainable lipid production, but, with the current knowledge and technology, they are still not a serious alternative in the market. In this review, the potential of these yeasts is highlighted and a discussion is made mainly focused on the economics of the oleaginous yeast oil production and identification of the key points to be improved to achieve lower production costs and higher income. Three main stages of the production process, where costs are higher, were identified. To render economically feasible the production of oils using oleaginous yeasts, a reduction in production costs must occur in all stages, lipid yields and productivities must be improved, and production must be targeted to high-value product applications.

In vitro gastrointestinal evaluation of a juçara-based smoothie: effect of processing on phenolic compounds bioaccessibility.

In the present work, the bioaccessibility of the main phenolic compounds of a juçara, banana and strawberry homogenized smoothie (control), subjected to pasteurization and sonication, was evaluated. The smoothie was also evaluated in terms of its main chemical and physical characteristics. Pasteurized smoothie showed higher apparent viscosity, as well as higher initial shear stress when compared to the control and sonicated samples. The increase in the apparent viscosity of the pasteurized smoothie was associated with the smaller particle size of this sample (68 µm). These characteristics conferred to the pasteurized smoothie higher physical stability than the control and sonicated smoothies. Phenolic compounds bioaccessibility was higher in the pasteurized and sonicated smoothies than in the control sample, which confirmed the positive effect of the treatments for the preservation of these compounds after gastrointestinal digestion. Compared to the sonication process, the pasteurization provided higher total phenolic compounds bioaccessibility (47%), as well as of ferulic (16%) and ellagic (80%) acids. Antioxidant capacity was higher in gastric digest for all the samples evaluated by ABTS assay. These results confirm the importance of processing on the physical stability and phenolic compounds bioaccessibility of the juçara-based smoothie, standing out the thermally treated product.

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.

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.

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.

Vinegar production from fruit concentrates: effect on volatile composition and antioxidant activity.

Vinegar stands as a highly appreciated fermented food product due to several functional properties and multiple applications. This work focuses on vinegar production from fruit wines derived from fruit concentrates, to attain a food product with nutritional added value. Four fruit vinegars (orange, mango, cherry and banana), were produced and characterized, with total acidities of 5.3 ± 0.3% for orange, 5.6 ± 0.2% for mango, 4.9 ± 0.4% for cherry and 5.4 ± 0.4% for banana. Acetification showed impact on aroma volatiles, mainly related to oxidative reactions. Minor volatiles associated with varietal aroma were identified, monoterpenic alcohols in orange vinegar, esters in banana vinegar, C13-norisoprenoids in cherry vinegar and lactones in mango vinegar, indicating fruit vinegars differentiated sensory quality. Total antioxidant activity analysis by FRAP, revealed fruit vinegars potential to preserve and deliver fruit functional properties. Antioxidant activity of fruit vinegars, expressed as equivalents of Fe2SO4, was of 11.0 ± 1.67 mmol L-1 for orange, 4.8 ± 0.5 mmol L-1 for mango, 18.6 ± 2.33 mmol L-1 for cherry and 3.7 ± 0.3 mmol L-1 for banana. Therefore, fruit vinegars presented antioxidant activity close to the reported for the corresponding fruit, and between 8 and 40 folds higher than the one found in commercial cider vinegar, demonstrating the high functional potential of these novel vinegar products.