Innovative Approach to Accelerate Wound Healing: Synthesis and Validation of Enzymatically Cross-Linked COL-rGO Biocomposite Hydrogels.

In this study, an innovative conductive hybrid biomaterial was synthetized using collagen (COL) and reduced graphene oxide (rGO) in order for it to be used as a wound dressing. The hydrogels were plasticized with glycerol and enzymatically cross-linked with horseradish peroxidase (HRP). A successful interaction among the components was demonstrated by FTIR, XRD, and XPS. It was demonstrated that increasing the rGO concentration led to higher conductivity and negative charge density values. Moreover, rGO also improved the stability of hydrogels, which was expressed by a reduction in the biodegradation rate. Furthermore, the hydrogel's stability against the enzymatic action of collagenase type I was also strengthened by both the enzymatic cross-linking and the polymerization of dopamine. However, their absorption capacity, reaching values of 215 g/g, indicates the high potential of the hydrogels to absorb fluids. The rise of these properties positively influenced the wound closure process, achieving an 84.5% in vitro closure rate after 48 h. These findings clearly demonstrate that these original composite biomaterials can be a viable choice for wound healing purposes.

Enhancing Alginate Hydrogels as Possible Wound-Healing Patches: The Synergistic Impact of Reduced Graphene Oxide and Tannins on Mechanical and Adhesive Properties.

Hydrogels are three-dimensional crosslinked materials known for their ability to absorb water, exhibit high flexibility, their biodegradability and biocompatibility, and their ability to mimic properties of different tissues in the body. However, their application is limited by inherent deficiencies in their mechanical properties. To address this issue, reduced graphene oxide (rGO) and tannins (TA) were incorporated into alginate hydrogels (Alg) to evaluate the impact of the concentration of these nanomaterials on mechanical and adhesive, as well as cytotoxicity and wound-healing properties. Tensile mechanical tests demonstrated improvements in tensile strength, elastic modulus, and toughness upon the incorporation of rGO and TA. Additionally, the inclusion of these materials allowed for a greater energy dissipation during continuous charge-discharge cycles. However, the samples did not exhibit self-recovery under environmental conditions. Adhesion was evaluated on pig skin, revealing that higher concentrations of rGO led to enhanced adhesion, while the concentration of TA did not significantly affect this property. Moreover, adhesion remained consistent after 10 adhesion cycles, and the contact time before the separation between the material and the surface did not affect this property. The materials were not cytotoxic and promoted healing in human fibroblast-model cells. Thus, an Alg/rGO/TA hydrogel with enhanced mechanical, adhesive, and wound-healing properties was successfully developed.

Multifunctional Chitosan Scaffold Platforms Loaded with Natural Polyphenolic Extracts for Wound Dressing Applications.

Chitosan (CS)-based scaffolds loaded with Pinus radiata extract bark (PE) and grape seed extract (GSE) were successfully developed for wound dressing applications. The effects of incorporating GSE and PE in CS scaffolds were investigated in relation to their physicochemical and biological properties. All scaffolds exhibited porous structures with the ability to absorb more than 70 times their weight when contacted with blood and phosphate buffer solution. The incorporation of GSE and PE into the CS scaffolds increased their blood absorption ability and degradation rates over time. All scaffolds showed a clotting ability above 95%, with their surfaces being favorable for red blood cell attachment. Both GSE and PE were released from the CS scaffolds in a sustained manner. Scaffolds loaded with GSE and PE inhibited the bacterial activity of S. aureus and E. coli by 40% and 44% after 24 h testing. In vitro cell viability studies demonstrated that all scaffolds were nontoxic to HaCaT cells. Importantly, the addition of GSE and PE further increased cell viability compared to that of the CS scaffold. This study provides a new synthesis method to immobilize GSE and PE on CS scaffolds, enabling the formation of novel material platforms with a high potential for wound dressing applications.

Chitosan Microparticles Enhance the Intestinal Release and Immune Response of an Immune Stimulant Peptide in Oncorhynchus mykiss.

The aquaculture industry is constantly increasing its fish production to provide enough products to maintain fish consumption worldwide. However, the increased production generates susceptibility to infectious diseases that cause losses of millions of dollars to the industry. Conventional treatments are based on antibiotics and antivirals to reduce the incidence of pathogens, but they have disadvantages, such as antibiotic resistance generation, antibiotic residues in fish, and environmental damage. Instead, functional foods with active compounds, especially antimicrobial peptides that allow the generation of prophylaxis against infections, provide an interesting alternative, but protection against gastric degradation is challenging. In this study, we evaluated a new immunomodulatory recombinant peptide, CATH-FLA, which is encapsulated in chitosan microparticles to avoid gastric degradation. The microparticles were prepared using a spray drying method. The peptide release from the microparticles was evaluated at gastric and intestinal pH, both in vitro and in vivo. Finally, the biological activity of the formulation was evaluated by measuring the expression of il-1ß, il-8, ifn-γ, Ifn-α, and mx1 in the head kidney and intestinal tissues of rainbow trout (Oncorhynchus mykiss). The results showed that the chitosan microparticles protect the CATH-FLA recombinant peptide from gastric degradation, allowing its release in the intestinal portion of rainbow trout. The microparticle-protected CATH-FLA recombinant peptide increased the expression of il-1ß, il-8, ifn-γ, ifn-α, and mx1 in the head kidney and intestine and improved the antiprotease activity in rainbow trout. These results suggest that the chitosan microparticle/CATH-FLA recombinant peptide could be a potential prophylactic alternative to conventional antibiotics for the treatment of infectious diseases in aquaculture.

Self-Assembled CNF/rGO/Tannin Composite: Study of the Physicochemical and Wound Healing Properties.

In this study, a conductive composite material, based on graphene oxide (GO), nanocellulose (CNF), and tannins (TA) from pine bark, reduced using polydopamine (PDA), was developed for wound dressing. The amount of CNF and TA was varied in the composite material, and a complete characterization including SEM, FTIR, XRD, XPS, and TGA was performed. Additionally, the conductivity, mechanical properties, cytotoxicity, and in vitro wound healing of the materials were evaluated. A successful physical interaction between CNF, TA, and GO was achieved. Increasing CNF amount in the composite reduced the thermal properties, surface charge, and conductivity, but its strength, cytotoxicity, and wound healing performance were improved. The TA incorporation slightly reduced the cell viability and migration, which may be associated with the doses used and the extract's chemical composition. However, the in-vitro-obtained results demonstrated that these composite materials can be suitable for wound healing.

Long-term release of bioactive interferon-alpha from PLGA-chitosan microparticles: in vitro and in vivo studies.

Effective cytokine treatments often require high- and multiple-dose due to the short half-life of these molecules. Here, porcine interferon-alpha (IFNα) is encapsulated in PLGA-chitosan microparticles (IFNα-MPs) to accomplish both slow drug release and drug protection from degradation. A procedure that combines emulsion and spray-drying techniques yielded almost spherical microspheres with an average diameter of 3.00 ± 1.50 µm. SEM, Microtrac, and Z-potential analyses of three IFNα-MP batches showed similar results, indicating the process is reproducible. These studies supported molecular evidence obtained in FTIR analysis, which indicated a compact structure of IFNα-MPs. Consistently, IFNα release kinetics assessed in vitro followed a zero-order behavior typical of sustained release from a polymeric matrix. This study showed that IFNα-MPs released bioactive molecules for at least 15 days, achieving IFNα protection. In addition, pigs treated with IFNα-MPs exhibited overexpression of IFNα-stimulated genes 16 days after treatment. Instead, the expression levels of these genes decreased after day 4th in pigs treated with non-encapsulated IFNα. In vitro and in vivo experiments demonstrated that the formulation improved the prophylactic and therapeutic potential of IFNα, accomplishing molecule protection and long-term release for at least two weeks. The procedure used to obtain IFNα-MPs is reproducible, scalable, and suitable for encapsulating other drugs.

The Effect on Hemostasis of Gelatin-Graphene Oxide Aerogels Loaded with Grape Skin Proanthocyanidins: In Vitro and In Vivo Evaluation.

Using in vitro and in vivo models, this study investigated the hemostatic potential to control bleeding of both unloaded gelatin-graphene oxide aerogels and the same loaded with proanthocyanidins (PAs) from Vitis vinifera grape skin extract. Our results showed that the physicochemical and mechanical properties of the aerogels were not affected by PA inclusion. In vitro studies showed that PA-loaded aerogels increased the surface charge, blood absorption capacity and cell viability compared to unloaded ones. These results are relevant for hemostasis, since a greater accumulation of blood cells on the aerogel surface favors aerogel-blood cell interactions. Although PAs alone were not able to promote hemostasis through extrinsic and intrinsic pathways, their incorporation into aerogels did not affect the in vitro hemostatic activity of these composites. In vivo studies demonstrated that both aerogels had significantly increased hemostatic performance compared to SpongostanTM and gauze sponge, and no noticeable effects of PA alone on the in vivo hemostatic performance of aerogels were observed; this may have been related to its poor diffusion from the aerogel matrix. Thus, PAs have a positive effect on hemostasis when incorporated into aerogels, although further studies should be conducted to elucidate the role of this extract in the different stages of hemostasis.

Novel and effective hemostats based on graphene oxide-polymer aerogels: In vitro and in vivo evaluation.

In this study, graphene oxide (GO)-based aerogels cross-linked with chitosan (CS), gelatin (GEL), and polyvinyl alcohol (PVA) were characterized and their hemostatic efficiencies both in vitro and in vivo were investigated and compared to commercial materials (ChitoGauze®XR and Spongostan™). All aerogels exhibited highly porous structures and a negative surface charge density favorable to their interaction with blood cells. The in vitro studies showed that all aerogels coagulated >60 % of the blood contained in their structures after 240 s of the whole-blood clotting assay, the GO-CS aerogel being the one with the highest blood clotting. All aerogels showed high hemocompatibility, with hemolytic rates <5 %, indicating their use as biomaterials. Among them, the GO-GEL aerogel exhibited the lowest hemolytic activity, due possibly to its high GEL content compared to the GO amount. According to their blood clotting activity, aerogels did not promote coagulation through extrinsic and intrinsic pathways. However, their surfaces are suitable for accelerating hemostasis by promoting alternative routes. All aerogels adhered platelets and gathered RBCs on their surfaces, and in addition the GO-CS aerogel surface also promoted the formation of filamentous fibrin networks adhered on its structure. Furthermore, in vivo evaluations revealed that all aerogels significantly shortened the hemostatic times and reduced the blood loss amounts compared both to the Spongostan™ and ChitoGauze®XR commercial materials and to the gauze sponge (control group). The hemostatic performance in vitro and in vivo of these aerogels suggests that they could be used as hemostats for controlling profuse bleedings.

Fabrication of Biocompatible Electrospun Poly(ε-caprolactone)/Gelatin Nanofibers Loaded with Pinus radiata Bark Extracts for Wound Healing Applications.

In this study, poly(ε-caprolactone) (PCL)/gelatin (GEL) electrospun nanofibers loaded with two different concentrations of Pinus radiata bark extracts (PEs) were fabricated via electrospinning for wound healing applications. The effects of incorporating PE into PCL/GEL electrospun nanofibers were investigated regarding their physicochemical properties and in vitro biocompatibility. All electrospun nanofibers showed smooth, uniform, and bead-free surfaces. Their functional groups were detected by ATR-FTIR spectroscopy, and their total phenol content was measured by a Folin-Ciocalteu assay. With PE addition, the electrospun nanofibers exhibited an increase in their wettability and degradation rates over time and a decrease in their tensile stress values from 20 ± 4 to 8 ± 2 MPa for PCL/GEL and PCL/GEL/0.36%PE samples, respectively. PE was also released from the fibrous mats in a rather controlled fashion. The PCL/GEL/0.18%PE and PCL/GEL/0.36%PE electrospun nanofibers inhibited bacterial activity at around 6 ± 0.1% and 23 ± 0.3% against E. coli and 14 ± 0.1% and 18 ± 0.2% against S. aureus after 24 h incubation, respectively. In vitro cell studies showed that PE-loaded electrospun nanofibers enhanced HaCaT cell growth, attachment, and proliferation, favoring cell migration towards the scratch area in the wound healing assay and allowing a complete wound closure after 72 h treatment. These findings suggested that PE-loaded electrospun nanofibers are promising materials for antibiotic-free dressings for wound healing applications.

Improved hemocompatibility for gelatin-graphene oxide composite aerogels reinforced with proanthocyanidins for wound dressing applications.

Aerogels based on gelatin and graphene oxide (GO) were synthesized by microwave-assisted reactions, incorporating grape skin extracts -high in proanthocyanidins (PAs)- to develop a hemostatic device with improved properties. The effects of incorporating PAs into the aerogels were investigated in relation to their physicochemical properties, absorption ability, clotting activity and cytotoxicity in human dermal fibroblast (HDF) cells. The aerogels presented highly resistant porous structures, capable of absorbing more than 50 times their weight when in contact with a phosphate saline solution (PBS) and fresh human blood. Interestingly, the addition of PAs increased the negative surface charges and the blood absorption ability of the aerogels, which may make them suitable for hemostasis. The incorporation of 5% and 10% (w/w) of extracts into the aerogels increased the total coagulated blood content by 36.6% and 24.5% compared with gelatin-GO aerogel, respectively. These improvements in the hemostatic properties of the aerogels were greater with the inclusion of 5% (w/w) of grape skin extracts into the aerogels. The aerogels were also able to adhere red blood cells onto their surfaces, which could favor the formation of stable fibrin networks to promote hemostasis. Their clotting activity suggested the activation of alternative routes based on complement coagulation systems. Finally, the aerogels were non-toxic for HDF cells and the PAs were successfully released from their matrices. Thus, gelatin-GO aerogels reinforced with PAs are promising as topical phytodrug delivery systems, with great potential for wound healing processes.

Synthesis and characterization of graphene oxide chitosan aerogels reinforced with flavan-3-ols as hemostatic agents.

The natural mechanisms of the body cannot control massive hemorrhaging, resulting in a requirement for hemostatic intervention. In this study, Graphene oxide and Chitosan aerogels reinforced with grape seed (SD) and skin (SK) extracts were developed for use as hemostatic agents by evaluating the influence of pH on their synthesis, and the amount of grape extract added on the physical and chemical properties of the aerogels. The material was evaluated by FTIR, XRD, Raman spectroscopy, DLS, uniaxial compression tests and SEM. The capacity of the aerogels to absorb water, PBS and blood, as well as their coagulation capacity, were determined. In addition, the release profile for grape extracts in PBS and the material's cytotoxicity were determined. The aerogels that were synthesized under basic conditions and loaded with grape extracts were more rigid and negatively charged, and they presented smaller pores than the un-loaded acidic aerogels. For all aerogels, the hemoglobin absorption was greater than 90 % in the first 30 s. A higher density of adsorbed blood cells was observed on aerogels loaded with a higher amount of grape extract. The maximum release of extract from the aerogels occurred for those loaded with SK extracts at a basic pH; the aerogels that were prepared under acidic conditions dissolved in the media. Aerogels loaded with SK extracts under alkaline conditions were not cytotoxic toward human dermal fibroblasts and exhibited cell viabilities above 90 %. These findings suggest that these aerogels have the potential for use as hemostatic agents in wound management.

Polymeric nanoencapsulation of alpha interferon increases drug bioavailability and induces a sustained antiviral response in vivo.

Polymeric nanoparticulate systems allow the encapsulation of bio-active substances, giving them protection against external agents and increasing the drug's bioavailability. The use of biocompatible and biodegradable polymers usually guarantees the harmless character of the formulation, and a controlled drug release is also assured. A relatively easy procedure to obtain polymeric formulations of bioactive agents is ionotropic gelation, which allows the synthesis of chitosan (CS) - sodium tri-polyphosphate nanoparticles (NPs) loading encapsulated proteins. In this work, Bovine serum albumin (BSA) model protein and a recombinant porcine alpha interferon variant were used to obtain nanoparticulate formulations. The internalization of the encapsulated material by cells was studied using a BSA-fluorescein system; the fluorescent conjugate was observable inside the cells after 20 h of incubation. The therapeutic CS-alpha interferon formulation showed a maximum of protein released in vitro at around 90 h. This system was found to be safe in a cytotoxicity assay, while biological activity experiments in vitro showed antiviral protection of cells in the presence of encapsulated porcine alpha interferon. In vivo experiments in pigs revealed a significant and sustained antiviral response through overexpression of the antiviral markers OAS2 and PKR. This proves the preservation of porcine alpha interferon biological activity, and also that a lasting response was obtained. This procedure is an effective and safe method to formulate drugs in nanoparticulate systems, representing a significant contribution to the search for more effective drug delivery strategies.

Design and Characterization of Chitosan-Graphene Oxide Nanocomposites for the Delivery of Proanthocyanidins.

INTRODUCTION: In the last years, the utilization of phytomedicines has increased given their good therapeutic activity and fewer side effects compared to allopathic medicines. However, concerns associated with the biocompatibility and toxicity of natural compounds, limit the phytochemical therapeutic action, opening the opportunity to develop new systems that will be able to effectively deliver these substances. This study has developed a nanocomposite of chitosan (CS) functionalized with graphene oxide (GO) for the delivery of proanthocyanidins (PAs), obtained from a grape seed extract (Ext.). METHODS: The GO-CS nanocomposite was covalently bonded and was characterized by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), atomic force microscopy (AFM) and by dynamic light scattering (DLS). The loading and release of Ext. from the GO-CS nanocomposite were performed in simulated physiological, and the cytotoxicity of the raw materials (GO and Ext.) and nanocomposites (GO-CS and GO-CS-Ext.) was determined using a human kidney cell line (HEK 293). RESULTS: The chemical characterization indicated that the covalent union was successfully achieved between the GO and CS, with 44 wt. % CS in the nanocomposite. The GO-CS nanocomposite was thermostable and presented an average diameter of 480 nm (by DLS). The Ext. loading capacity was approximately 20 wt. %, and under simulated physiological conditions, 28.4 wt.% Ext. (g) was released per g of the nanocomposite. GO-CS-Ext. was noncytotoxic, presenting a 97% survival rate compared with 11% for the raw extract and 48% for the GO-CS nanocomposite at a concentration of 500 µg mL-1 after 24 hrs. CONCLUSION: Due to π-π stacking and hydrophilic interactions, GO-CS was reasonably efficient in binding Ext., with high loading capacity and Ext. release from the nanocomposite. The GO-CS nanocomposite also increased the biocompatibility of PAs-rich Ext., representing a new platform for the sustained release of phytodrugs.

Efficient poultry manure management: anaerobic digestion with short hydraulic retention time to achieve high methane production.

The efficient treatment or appropriate final disposal of poultry manure (PM) to avoid serious environmental impacts is a great challenge. In this work, the optimization of a 2-stage anaerobic digestion system (ADS) for PM was studied with the aim of reaching a maximal methane yield with a short hydraulic retention time (HRT). Three activities were performed: The first activity, ADS 1, consisted of evaluating the effect of the substrate concentration and the HRT on the process, with a constant organic loading rate (OLR) of 3.66 ± 0.21 gVS L-1 d-1. The second activity, ADS 2, consisted of decreasing the HRT from 9.09 to 2.74 d with a constant substrate concentration. In the third activity, ADS 3, the substrate concentration was increased from 10.09 ± 1.41 to 35.25 ± 6.20 gVS L-1 with an average HRT of 4.66 ± 0.11 d. Maximal methane yields of 0.22, 0.21, and 0.22 LCH4 gVS-1 were reached for ADS 1, ADS 2, and ADS 3, respectively, at a low HRT (3.38 to 4.66 d) and high free ammonia concentration (between 323.05 ± 56.48 and 460.93 ± 135.40 mgN-NH3 L-1). These methane yields correspond to the production of 40.36 and 42.28 cubic meters of methane per ton of PM, respectively, and a laying hen produces between 47.45 and 54.75 kg of PM per year in Chile. Finally, this is the first study of the separate and combined effects of OLR, HRT and substrate concentration on the anaerobic digestion of PM. The results demonstrate the technical feasibility of the two-stage ADS treatment of PM with a short HRT; the system tolerates variations in the total ammonia nitrogen concentration of PM throughout the year and achieves a high methane yield when the correct operational conditions are selected.

Development of Graphene Oxide Composite Aerogel with Proanthocyanidins with Hemostatic Properties As a Delivery System.

The graphene aerogels' potential for use as both a hemostatic agent and dermal delivery system has scarcely been investigated. In this study, we used a sol-gel process for generating dry and stable composite aerogels based on graphene oxide (GO) and poly(vinyl alcohol) (PVA). Furthermore, we incorporated natural extract of País grape seed (SD) and skin (SK), rich in proanthocyanidins (PAs or condensed tannins). The effect of the incorporation of the grape extracts was investigated in relation to the aerogels' structure, coagulation performance and the release of the extracts. The results demonstrated that they have a porous structure and low density, capable of absorbing water and blood. The incorporation of 12% (w/w) of PA extracts into the aerogel increased the negative zeta potential of the material by 33% (-18.3 ± 1.3 mV), and the coagulation time was reduced by 37% and 28% during the first 30 and 60 s of contact between the aerogel and whole blood, respectively. The release of extracts from the GO-PVA-SD and GO-PVA-SK aerogels was prolonged to 3 h with 20%, probably due to the existence of strong binding between PAs andGO-PVA, both characterized by the presence of aromatic and hydroxyl groups that can form noncovalent bonds but are strong and stable enough to avoid a greater release into the medium. This study provides a new GO-based aerogel, which has a great potential use in the field of dermal delivery, wound healing and/or the treatment of trauma bleeding.

Modeling of the Nanoparticles Absorption Under a Gastrointestinal Simulated Ambient Condition.

Proanthocyanidins (PAs) have several bioactivities, but they are unstable in the digestive tract and possess low bioavailability. Nanoencapsulation stabilizes these compounds for oral administration. The intestinal absorption of grape seed and skin extracts, and the poly-lactic acid (PLA) nanoparticles loaded with such extracts was modeled, taking into consideration physicochemical process parameters, evaluating the PAs concentration profile in the human small intestine. Density (ρ), solubility, viscosity (µ), diffusion coefficient (D), and the global mass transfer coefficient (K) for both substrates were estimated, simulating their passing from the intestine into the blood at 37°C. For the seed and skin extracts encapsulated in PLA the physicochemical parameters were: D = 1.81 × 10^-5 and D = 5.72 × 10^-5 cm2/s; K = 3.4 × 10^-3 and K = 2.47 × 10^-4 cm/s, respectively. Lower resistance was offered by the seed extract than by skin extracts (nanoencapsulated), which was explained by differences in structural composition, and average molecular weight of the two kinds of extracts, which should be more favorable to the mass transfer in comparison to the raw extracts. The concentration profile of grape extracts in the small intestine was modeled through a pure convection model, and the encapsulated extract on PLA nanoparticles using a mixed regime model, which described the process of dissolution and absorption of the grape extracts from the intestine to the blood stream. The absorbed fraction predicted by the model was 42.7 and 24.2% for seed and skin extracts, respectively. Those values increased to 100% for both extracts after the simulation with the nanoencapsulated extracts. Consequently, extract encapsulation should produce a significant increase in intestinal absorption.

Factorial design optimization and characterization of poly-lactic acid (PLA) nanoparticle formation for the delivery of grape extracts.

Proanthocyanidins (PAs) extracted from grapes have several bioactive properties, giving them potential medical uses. However, PAs are unstable in the digestive tract and must be stabilized to allow oral administration, which can be accomplished by nanoencapsulation. In this study, PAs extracted from grape seed and skin were stabilized with poly-d,l-lactide (PLA) polymer by the emulsion-evaporation method. An experimental Box-Behnken design was implemented, evaluating the influence of three factors: sonication time (30-360s) for the emulsion formation, loading of grape extracts (5-20%) and concentration of stabilizing agent (polyvinyl alcohol, PVA: 1-3%). The process was optimized to achieve higher encapsulation efficiency (EE=82.7%) and a smaller size (256 nm). The nanoparticles (NPs) were physically analyzed by TEM, FT-IR, TGA and DTG to characterize the nanoencapsulation process. In vitro release studies, through stomach and intestinal simulation, showed a sustained release of PAs from PLA-NPs.

Technical and economical optimization of a full-scale poultry manure treatment process: total ammonia nitrogen balance.

A full-scale process for the treatment of 80 tons per day of poultry manure was designed and optimized. A total ammonia nitrogen (TAN) balance was performed at steady state, considering the stoichiometry and the kinetic data from the anaerobic digestion and the anaerobic ammonia oxidation. The equipment, reactor design, investment costs, and operational costs were considered. The volume and cost objective functions optimized the process in terms of three variables: the water recycle ratio, the protein conversion during AD, and the TAN conversion in the process. The processes were compared with and without water recycle; savings of 70% and 43% in the annual fresh water consumption and the heating costs, respectively, were achieved. The optimal process complies with the Chilean environmental legislation limit of 0.05 g total nitrogen/L.

Startup and oxygen concentration effects in a continuous granular mixed flow autotrophic nitrogen removal reactor.

The startup and performance of the completely autotrophic nitrogen removal over nitrite (CANON) process was tested in a continuously fed granular bubble column reactor (BCR) with two different aeration strategies: controlling the oxygen volumetric flow and oxygen concentration. During the startup with the control of oxygen volumetric flow, the air volume was adjusted to 60mL/h and the CANON reactor had volumetric N loadings ranging from 7.35 to 100.90mgN/Ld with 36-71% total nitrogen removal and high instability. In the second stage, the reactor was operated at oxygen concentrations of 0.6, 0.4 and 0.2mg/L. The best condition was 0.2 mgO2/L with a total nitrogen removal of 75.36% with a CANON reactor activity of 0.1149gN/gVVSd and high stability. The feasibility and effectiveness of CANON processes with oxygen control was demonstrated, showing an alternative design tool for efficiently removing nitrogen species.

An enzymatic extraction of proanthocyanidins from País grape seeds and skins.

Proanthocyanidins (PAs) from the skins and seeds of País grapes were obtained by enzymatic extraction by using three enzymes (pectinase, cellulase and tannase) and an enzymatic blend to increase the phenol concentrations and reduce the PA molecular size. The total phenol concentrations (as indicated by Folin-Ciocalteu reagent), mean degree of polymerisation (mDP), galloylation percentage (%G) and structural proportion (phloroglucinolysis) were analysed, in addition to the extract's capacity to inhibit angiotensin I-converting enzyme (ACE) activity. On grape skins, pectinase had the greatest effective on the release of total phenols, to 0.01g/ml solid/liquid (S/L) and 1% enzyme/substrate (E/S). On grape seed, the three enzymes were effective in increasing the phenolic extraction (p<0.05). The effects of enzymes on the mDP and %G of the extracts were related to their enzymatic activity. All the extracts inhibited ACE, but ACE inhibition was thought to be improved by the increased number of terminal units in the seed samples.