Development and characterization of films from Campomanesia xanthocarpa and commercial citrus pectins with different degrees of methyl-esterification.

In this study, pectins from commercial citrus and isolated from gabiroba (Campomanesia xanthocarpa) fruits, were obtained with different degrees of methyl-esterification (DM) and applied in the films. The DM ranged from 0 % to 62.5 % and the gradual de-esterification process was confirmed by mono-dimensional analysis (1H NMR). In order to investigate the influence of DM values in pectin film properties, PCP (DM: 62.5 %); PCP-5 (DM: 37.4 %); PCP-15 (DM: 19.1 %), and a fully de-esterified sample PCP-35 (DM: 0 %) were selected. The functional properties of the films clearly showed that the DM and cross-linking process are necessary to obtain a material with water resistance. Furthermore, pectin isolated from the fruits of gabiroba was purified (GW-Na, DM: 51.9 %) and partially de-esterified (GW-Na-5, DM: 37.1 %). These pectins were used, for the first time, in development of films and the physical and mechanical properties were compared with films made with PCP and PCP-5 samples. GW-Na and GW-Na-5 films presented suitable properties, with reduced solubility reduced (57.1 and 26.2 %), high degree of swelling (2.14 and 2.26), low flexibility (18.05 and 6.11 MPa), respectively. High strength and rigidity (99.36 and 1040.9 MPa), for both films (GW-Na and GW-Na-5) were demonstrated, similar to that obtained by analyzed citrus pectin.

Seminal quality comparison of first and second cryopreserved ejaculates of Alpine kid goats by flow cytometry.

Discarding the first ejaculate is recommended as an alternative for improving seminal quality after long sexual resting, especially when semen should be used for cryopreservation. However, when the males are not in sexual resting the necessity to discarding the first ejaculate is still unknown. Therefore, this study aimed to compare by flow cytometry the quality of the first and second ejaculates. Ten kids and uniform goats between 5 and 6 months of age were used in a completely randomized design. Semen collection was carried out every 4 days, until a total of five ejaculates per animal in each treatment was completed. The fresh and frozen semen collected were processed and analyzed using macroscopic and microscopic parameters, resistance test, hypo-osmotic medium test, and flow cytometry (FC). The FC parameters were production of reactive oxygen species, plasma and acrosomal membrane integrity, and lipid peroxidation of the plasma membrane. The ejaculates did not differ for the resistance test, the reactivity in the hypo-osmotic medium and for the macroscopic and microscopic seminal parameters, except for sperm volume and concentration. The first ejaculate had a higher percentage of minor and total defects. None of the FC parameters analyzed differed between the first and second ejaculates. The first and second ejaculates demonstrated similar seminal qualities, so for Alpine kid goats without a sexual resting period, discarding the first ejaculate it is not recommended.

Glycoside hydrolase subfamily GH5_57 features a highly redesigned catalytic interface to process complex hetero-ß-mannans.

Glycoside hydrolase family 5 (GH5) harbors diverse substrate specificities and modes of action, exhibiting notable molecular adaptations to cope with the stereochemical complexity imposed by glycosides and carbohydrates such as cellulose, xyloglucan, mixed-linkage ß-glucan, laminarin, (hetero)xylan, (hetero)mannan, galactan, chitosan, N-glycan, rutin and hesperidin. GH5 has been divided into subfamilies, many with higher functional specificity, several of which have not been characterized to date and some that have yet to be discovered with the exploration of sequence/taxonomic diversity. In this work, the current GH5 subfamily inventory is expanded with the discovery of the GH5_57 subfamily by describing an endo-ß-mannanase (CapGH5_57) from an uncultured Bacteroidales bacterium recovered from the capybara gut microbiota. Biochemical characterization showed that CapGH5_57 is active on glucomannan, releasing oligosaccharides with a degree of polymerization from 2 to 6, indicating it to be an endo-ß-mannanase. The crystal structure, which was solved using single-wavelength anomalous diffraction, revealed a massively redesigned catalytic interface compared with GH5 mannanases. The typical aromatic platforms and the characteristic α-helix-containing ß6-α6 loop in the positive-subsite region of GH5_7 mannanases are absent in CapGH5_57, generating a large and open catalytic interface that might favor the binding of branched substrates. Supporting this, CapGH5_57 contains a tryptophan residue adjacent and perpendicular to the cleavage site, indicative of an anchoring site for a substrate with a substitution at the -1 glycosyl moiety. Taken together, these results suggest that despite presenting endo activity on glucomannan, CapGH5_57 may have a new type of substituted heteromannan as its natural substrate. This work demonstrates the still great potential for discoveries regarding the mechanistic and functional diversity of this large and polyspecific GH family by unveiling a novel catalytic interface sculpted to recognize complex heteromannans, which led to the establishment of the GH5_57 subfamily.

Rheological Behavior of Pectin Gels Obtained from Araçá (Psidium cattleianum Sabine) Fruits: Influence of DM, Pectin and Calcium Concentrations.

In this work, purified pectins from Araçá fruits (Psidium cattleianum Sabine) were obtained and characterized after partial demethylation. On each prepared sample, the carboxylic yield was obtained by titration, the degree of methylation (DM) by 1H-NMR, and the molecular weight distribution by steric exclusion chromatography (SEC). Then, the gelation ability in the presence of calcium counterions was investigated and related to DM (59-0%); the pectin concentration (2-10 g L-1); and the CaCl2 concentration (0.1-1 mol L-1) used for dialysis. The critical pectin concentration for homogeneous gelation was above 2 g L-1 when formed against 1 mol L-1 CaCl2. The elastic modulus (G') increased with pectin concentration following the relationship G'~C2.8 in agreement with rigid physical gel network predictions. The purified samples APP and APP-A with DM ≥ 40% in the same conditions released heterogeneous systems formed of large aggregates. Gels formed against lower concentrations of CaCl2 down to 0.1 mol L-1 had a higher degree of swelling, indicating electrostatic repulsions between charged chains, thus, counterbalancing the Ca2+ cross-linkage. Compression/traction experiments demonstrated that an irreversible change in the gel structure occurred during small compression with an enhancement of the G' modulus.

Isolation, NMR characterization and bioactivity of a (4-O-methyl-α-D-glucurono)-ß-D-xylan from Campomanesia xanthocarpa Berg fruits.

Hemicellulose-type polysaccharides were isolated from Campomanesia xanthocarpa fruits by alkaline extraction and submitted to fractionation processes giving rise to eluted (GE-300) and retained (GR-300) fractions. GE-300 presented a mixture of galactoglucomannans (GGM) and glucuronoxylans (MGX), while the GR-300 fraction is composed only of MGX. In this way, the chemical structure of MGX, investigated by 1D 1H, 13C and 2D 1H-13C HSQC, 1H-1H COSY and 1H-13C HMBC NMR spectroscopy, revealed that the chemical structure of polysaccharide is a (4-O-methyl-α-D-glucurono)-D-xylan. Deep and precise NMR chemical shift determination of clean and specific 1H NMR glycosyl units were developed by 1D TOCSY and 1D NOESY analysis. This approach demonstrated unequivocally that 4-O-methyl-α-D-glucopyranosyl uronic acid group is linked to O-2 of a (1 â†’ 4)-ß-D-xylan in the main chain. Furthermore, MGX scavenged DPPH radical (0.5 to 1.0 mg mL-1) and was not cytotoxic to human dermal fibroblasts at concentrations up to 1.0 mg mL-1, as demonstrated by neutral red and crystal violet assays, evidencing in vitro biocompatibility. The structure elucidation of GR-300 together with its bioactivity assessment contributed to better understand the chemical characteristics of C. xanthocarpa hemicelluloses and may provide structural basis for future structure-property studies.

Effects of pressurized hot water extraction on the yield and chemical characterization of pectins from Campomanesia xanthocarpa Berg fruits.

Pressurized hot water extraction (PHWE), known as a "green" extraction technique, was used to obtain polysaccharide from the pulp of gabiroba (Campomanesia xanthocarpa Berg) fruits. The effects of pressure, temperature, and flow rate on pectin yields were analyzed through a full factorial design experiment 23. The optimal extraction conditions to achieve maximum pectin yield (5.70 wt%) were pressure of 150 bar, temperature of 120 °C, and flow rate of 1.5 mL min-1. The high pressure (100 bar) promoted an increase in galacturonic acid content (36.0%) compared to conventional hot water extraction (CEGP) with 25.7%. Differences in the proportion of homogalacturonan (HG) and rhamnogalacturonan (RG-I) domains ranging from 16.3 to 35.4% and 61.7 to 80.1%, respectively, were observed for each pectin sample according to the extraction conditions. The mono-dimensional (13C-NMR) and bi-dimensional (1H/13C HSQC-NMR) analyses confirmed the presence of HG and RG-I regions and indicated the presence of arabinogalactans type I (AG-I) and arabinogalactans type II (AG-II) in the PHWE pectin samples, which was not found for pectins from gabiroba pulp obtained by CEGP. The results showed that PHWE proved to be a promising method for extracting pectins from gabiroba fruits.

Cytotoxic effect of crude and purified pectins from Campomanesia xanthocarpa Berg on human glioblastoma cells.

A new source of pectin with a cytotoxic effect on glioblastoma cells is presented. A homogeneous GWP-FP-S fraction (Mw of 29,170 g mol-1) was obtained by fractionating the crude pectin extract (GW) from Campomanesia xanthocarpa pulp. According to the monosaccharide composition, the GWP-FP-S was composed of galacturonic acid (58.8%), arabinose (28.5%), galactose (11.3%) and rhamnose (1.1%), comprising 57.7% of homogalacturonans (HG) and 42.0% of type I rhamnogalacturonans (RG-I). These structures were characterized by chromatographic and spectroscopic methods; GW and GWP-FP-S fractions were evaluated by MTT and crystal violet assays for their cytotoxic effects. Both fractions induced cytotoxicity (15.55-37.65%) with concomitant increase in the cellular ROS levels in human glioblastoma cells at 25-400 µg mL-1, after 48 h of treatment, whereas no cytotoxicity was observed for normal NIH 3T3 cells. This is the first report of in vitro bioactivity and the first investigation of the antitumor potential of gabiroba pectins.

Pectins from the pulp of gabiroba (Campomanesia xanthocarpa Berg): Structural characterization and rheological behavior.

The pulp of gabiroba fruits was submitted to a hot water extraction, giving rise to a crude pectin named GW. GW was shown to be composed mainly of arabinose (54.5%), galacturonic acid (33.5%), galactose (7.6%), and rhamnose (1.6%). GW was characterized by chromatographic and spectroscopic methods indicating the presence of homogalacturonans (HG) with a degree of methyl-esterification (DM) of 60% and rhamnogalacturonans I (RG-I). HG domain represents 31.9% and RG-I domain 65.3%. Furthermore, GW was submitted to sequential fractionation methods, giving rise to GWP-TEP fraction, structurally characterized by the predominance of HG regions, and confirmed by NMR analysis. The rheological behavior of GW was analyzed at 1%, 3%, and 5% (w/v) concentration with 0.1 mol L-1 NaCl. All samples showed shear thinning behavior. In the oscillatory measurements, the 1% GW showed a liquid-like behavior, while the 3% presented a concentrated solution behavior and the 5% GW a gel behavior.

Pulp and Jam of Gabiroba (Campomanesia xanthocarpa Berg): Characterization and Rheological Properties.

This study evaluated the physicochemical characterization and rheological behavior of gabiroba pulp, and a gabiroba jam formulation. Gabiroba pulp presented a heterogeneous ultrastructure with a denser area formed by a compact mesh and a porous interface containing fibers. The fibers' presence promoted a slip effect when the gabiroba pulp was subjected to shear. Gabiroba pulp showed a gel behavior with thermal stability. Gabiroba jam, developed using pulp as the raw material, had shear thinning behavior exhibiting yield stress described by the Herschel-Bulkley model. The dynamic oscillatory analysis showed that gabiroba jam typically behaved like a gel, i.e., G' values higher than the G″ in all frequency ranges evaluated. The results showed that gabiroba pulp is suitable for use as a raw material in the development of food products such as jam, encouraging the preservation of this native Brazilian species.

Extraction, purification and structural characterization of a galactoglucomannan from the gabiroba fruit (Campomanesia xanthocarpa Berg), Myrtaceae family.

In this study, we isolated and structurally characterized, for the first time, a galactoglucomannan (GGM) from the pulp of gabiroba, a Myrtaceae family species. The HPSEC-MALLS-RI analysis showed a homogeneous polysaccharide with molar mass of 25,340gmol-1. The monosaccharide composition showed that the GGM consisted of Man:Glc:Gal in a molar ratio of 1:1:0.6. Methylation and 1D and 2D NMR analyses suggested that the main chain of the GGM consisted of ß-d-Glcp and ß-d-Manp units (1→4)-linked. The α-d-Galp substitutions occur mainly at O-6 position of ß-d-Manp units. The glycosidic linkages of the GGM were evident by the presence of the characteristic signals of 4-O-substituted residues at δ 78.6/3.69 for both ß-d-Glcp and ß-d-Manp. Furthermore, the O-6 substitutions for both ß-d-Glcp and ß-d-Manp units were confirmed by signals at δ 67.1/4.00 and 3.93. The interglycosidic correlations, obtained through the analysis of the HMBC spectrum, further confirm the structure.

Determination of heat-set gelation capacity of a quinoa protein isolate (Chenopodium quinoa) by dynamic oscillatory rheological analysis.

This work aimed to study the influence of pH (3.5 and 7.0) and CaCl2 and MgCl2 addition on heat-set gelation of a quinoa protein isolate at 10% and 15% (w/w). The protein isolate obtained was composed mainly of 11S globulin as was observed by electrophoresis and mass spectrometry analysis. Heat-set gelation occurred at both pH values studied. Nevertheless, the gels formed at pH 3.5 were more viscoelastic and denser than those formed at pH 7.0, that was coarser and presented syneresis. The CaCl2 and MgCl2 addition increased the gel strength during rheological analysis at pH 3.5, possibly due to the formation of fiber-like connections in the gel network. At pH 7.0, the divalent salts resulted in weaker gels formed by agglomerates, suggesting a neutralization of the protein surface charges. The differences in quinoa protein gelation were attributed to solubility, and the flexibility of proteins secondary structure at the pH studied.

Analysis of the Biotechnological Potential of a Lentinus crinitus Isolate in the Light of Its Secretome.

Analysis of fungal secretomes is a prospection tool for the discovery of new catalysts with biotechnological applications. Since enzyme secretion is strongly modulated by environmental factors, evaluation of growth conditions is of utmost importance to achieve optimal enzyme production. In this work, a nonsequenced wood-rotting fungus, Lentinus crinitus, was used for secretome analysis by enzymatic assays and a proteomics approach. Enzyme production was assessed after the fungus was cultured in seven different carbon sources and three nitrogen-containing compounds. The biomass yields and secreted protein arrays differed drastically among growing conditions. A mixture of secreted extracts derived from solid and liquid cultures was inspected by shotgun mass spectrometry and two-dimensional gel electrophoresis (2-DE) prior to analysis via LC-MS/MS. Proteins were identified using mass spectrometry (MS)-driven BLAST. The spectrum of secreted proteins comprised CAZymes, oxidase/reductases, proteases, and lipase/esterases. Although preseparation by 2-DE improved the number of identifications (162) compared with the shotgun approach (98 identifications), the two strategies revealed similar protein patterns. Culture media with reduced water content stimulated the expression of oxidases/reductases, while hydrolases were induced during submerged fermentation. The diversity of proteins observed within both the CAZyme and oxidoreductase groups revealed in this fungus a powerful arsenal of enzymes dedicated to the breakdown and consumption of lignocellulose.

Nanoemulsion as a carrier to improve the topical anti-inflammatory activity of stem bark extract of Rapanea ferruginea.

The aim of this study was to develop nanoemulsion containing soft extract of stem bark of Rapanea ferruginea to improve the topical delivery and anti-inflammatory activity. The extract of R. ferruginea stem bark was incorporated into the oily phase of the nanoemulsion by the method of phase inversion at low energy. The developed nanoemulsion had an average droplet size of 47.88±8.20 nm and a polydispersibility index of 0.228. Uniformity of size, spherical shape of droplet, and absence of clusters were confirmed by transmission electronic microscopy. The zeta potential was -34.7±1.15 mV. The nanoemulsion showed a moderate degree of skin irritation in the agarose overlay assay in vitro. The content of the extract markers, myrsinoic acids A and B, was 54.10±0.08 and 53.03 µg/g in the formulation, respectively. The formulation demonstrated pseudoplastic and thixotropic rheological behavior. In vitro release of chemical markers was controlled by diffusion mechanism. An extract-loaded nanoemulsion showed a topical anti-inflammatory activity in a croton oil-induced edema ear model, with a decrease in tumor necrosis factor release and myeloperoxidase activity. The nanoemulsion was 160% more efficient than the conventional cream containing 0.13% of the extract. The nanoemulsion showed suitable properties as a carrier for topical use of R. ferruginea extract and the approach for improving the topical anti-inflammatory activity.

Time-dependent viscometry study of endoglucanase action on xyloglucan: A real-time approach.

Hydrolysis of xyloglucan from Tamarindus indica and Hymenaea courbaril seeds with endoglucanase (EGII), which randomly breaks the (1→4)-linked ß-glycosidic bonds of the polymer chain, was monitored in real time using time-dependent viscometry analysis (TDV). For both samples there was a decrease in the intrinsic viscosity ([η]), viscosity average molar mass (Mv), radius de gyration (Rg) and persistence length (Lp) immediately after the addition of the enzyme. It was observed the formation of oligosaccharides and oligomers composed of ∼2 units, up to 140min. Galactose-containing side chains two positions away from the non-substituted glucose, modulated the action of EGII, and the complete hydrolysis of the XG oligomers occurred after 24h. The results demonstrate for the first time the real-time degradation of xyloglucan as well the macromolecular and oligosaccharide composition during the EGII hydrolysis process.

Influence of the extraction time on macromolecular parameters of galactomannans.

This study evaluated the aqueous extraction of galactomannans from the seeds of Mimosa scabrella (GM), Stryphnodendron adstringens (GS) and Schizolobium parahybae (GG) for 1, 2, 3, 4, 6, 24 and 48 h. The efficiency of extraction processes was assessed in terms of yield, carbohydrate and protein content. The extraction process, as well as the source of the galactomananns generated molecules with differences in molar mass, viscosity and rigidity analyzed by HPSEC-MALLS/RI/VIS. The extraction time results for each species, based on minimum extraction time and HPSEC-MALLS/RI/VIS results, were 4 h (GM4h), 6 h (GS6h) and 2 h (GG2h) for GM, GS and GG, respectively. In most cases, the apparent persistence length, as determined by viscometry, indicated that aggregates remained in galactomannans after centrifugation and filtration. Results suggest an effective extraction time for each plant source of galactomannan based on its performance and its macromolecular behavior in solution.

Topical curcumin-loaded hydrogels obtained using galactomannan from Schizolobium parahybae and xanthan.

The curcumin (CUR)-loaded binary hydrogel was formulated using xanthan and galactomannan from Schizolobium parahybae (guapuruvu). The binary hydrogels presented gel characteristics, stable pH values and mechanical stress resistance even after 45 days of heat exposure (45 °C). The CUR-loaded hydrogel content was 98.6% for XGMC (xanthan and galactomannan with CUR-microemulsion) after the stability test. The in vitro cytotoxicity analysis suggested non-cutaneous membrane irritation, and the in vitro skin permeation analysis indicated 2.15 to 2.50 µg mL(-1) CUR at the stratum corneum, epidermal and dermal levels. The XGEC (xanthan and galactomannan with CUR solubilized in ethanol) and XGMC hydrogels presented 76.8 and 63.2% inhibition of topical inflammation, respectively. Chemical stability and non-cytotoxicity analysis confirm the safety of prolonged exposure of the skin during the topical treatment, offering long-lasting XGEC and XGMC action.

Rheological characterization of O/W emulsions incorporated with neutral and charged polysaccharides.

The effects of polysaccharides, including xyloglucan from Hymenaea courbaril (XG), galactomannans from Schizolobium parahybae (GMSP) and Mimosa scabrella (GMMS), xanthan gum (XT), sodium hyaluronate (HNa) and Fucogel(®) (FG), on the rheological behavior of cosmetic emulsions were evaluated. These incorporations gave rise to six emulsified systems, denoted XGE, GMSPE, GMMSE, XTE, HNaE and FGE, respectively. The emulsion consistency was found to follow the trend GMSPE>XGE>HNaE>FGE>XTE>GMMSE. In general, the addition of polysaccharides increased the viscoelastic properties of the emulsions and decreased the creep compliance. The neutral polysaccharides (GMSPE, GMMSE) led to better stability of the emulsions after storing for 20 days relative to charged polymers. It was found that polysaccharides XG, GMSP and GMMS, which come from the seeds of native Brazilian plant species, might be used to modify the flow properties and stabilities of oil-water emulsions.

Curcumin/xanthan-galactomannan hydrogels: rheological analysis and biocompatibility.

Curcumin, a lipophilic compound found in the plant Curcuma longa L., exhibits a wide range of pharmacological activity; however, its therapeutic use has been limited because of its low bioavailability following oral administration. The aim of this study was to evaluate the viscoelastic characteristics and biocompatibility of a curcumin/xanthan:galactomannan hydrogel (X:G) system after topical application on chick embryo chorioallantoic membrane (CAM), a system established with a view toward curcumin nasal or topical pharmaceutical applications or possible administration in cosmetics or foods. A rheological analysis indicated that incorporation of curcumin did not alter the viscoelastic characteristics of the X:G hydrogel, suggesting that there was no change in the structure of the gel network. X:G hydrogels did not induce CAM tissue injury and the curcumin/X:G hydrogel system was also highly biocompatible. We conclude that the X:G hydrogel represents a potential matrix for curcumin formulations.

Sulfonation and anticoagulant activity of fungal exocellular ß-(1→6)-D-glucan (lasiodiplodan).

An exocellular ß-(1→6)-D-glucan (lasiodiplodan) produced by a strain of Lasiodiplodia theobromae (MMLR) grown on sucrose was derivatized by sulfonation to promote anticoagulant activity. The structural features of the sulfonated ß-(1→6)-D-glucan were investigated by UV-vis, FT-IR and (13)C NMR spectroscopy, and the anticoagulant activity was investigated by the classical coagulation assays APTT, PT and TT using heparin as standard. The content of sulfur and degree of substitution of the sulfonated glucan was 11.73% and 0.95, respectively. UV spectroscopy showed a band at 261 nm due to the unsaturated bond formed in the sulfonation reaction. Results of FT-IR and (13)C NMR indicated that sulfonyl groups were inserted on the polysaccharide. The sulfonated ß-(1→6)-D-glucan presented anticoagulant activity as demonstrated by the increase in dose dependence of APTT and TT, and these actions most likely occurred because of the inserted sulfonate groups on the polysaccharide. The lasiodiplodan did not inhibit the coagulation tests.

Rheological characterization of a xanthan-galactomannan hydrogel loaded with lipophilic substances.

We compared the structures and rheology of xanthan-galactomannan (X:G) hydrogels with the addition of curcumin in microemulsion (X:GMC) and ethanol (X:GEC). X:GMC hydrogels have gel characteristics and exhibited a significantly higher elastic response than the X:GEC and X:G hydrogels at room temperature, but after heating, an increase in the elastic modulus was observed for the last two systems. The visualization of the hydrogel microstructures by cryo-scanning electronic microscopy revealed pores within the lamellar structure only for X:GMC. In vitro skin permeation tests showed a more pronounced lag time for X:GMC; however, a more efficient permeation from X:GMC than from X:GEC. This study demonstrates that the X:G system is an alternative to traditional gels for the topical applications of hydrophobic drugs.