Publications by authors named "Bruna Aparecida Souza Machado"

29 Publications

  • Page 1 of 1

Collaborative and Structured Network for Maintenance of Mechanical Ventilators during the SARS-CoV-2 Pandemic.

Healthcare (Basel) 2021 Jun 18;9(6). Epub 2021 Jun 18.

Postgraduate Program MPDS/GETEC/MCTI, University Center SENAI CIMATEC, SENAI CIMATEC, Av. Orlando Gomes, 1845 Piatã, Salvador 41650-010, BA, Brazil.

The SARS-CoV-2 pandemic in Brazil has grown rapidly since the first case was reported on 26 February 2020. As the pandemic has spread, the low availability of medical equipment has increased, especially mechanical ventilators. The Brazilian Unified Health System (SUS) claimed to have only 40,508 mechanical ventilators, which would be insufficient to support the Brazilian population at the pandemic peak. This lack of ventilators, especially in public hospitals, required quick, assertive, and effective actions to minimize the health crisis. This work provides an overview of the rapid deployment of a network for maintaining disused mechanical ventilators in public and private healthcare units in some regions of Brazil during the SARS-CoV-2 pandemic. Data referring to the processes of maintaining equipment, acquiring parts, and conducting national and international training were collected and analyzed. In total, 4047 ventilators were received by the maintenance sites, and 2516 ventilators were successfully repaired and returned to the healthcare units, which represents a success rate of 62.17%. The results show that the maintenance initiative directly impacted the availability and reliability of the equipment, allowing access to ventilators in the public and private health system and increasing the capacity of beds during the pandemic.
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http://dx.doi.org/10.3390/healthcare9060754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8234581PMC
June 2021

Ozonized Water in Microbial Control: Analysis of the Stability, In Vitro Biocidal Potential, and Cytotoxicity.

Biology (Basel) 2021 Jun 12;10(6). Epub 2021 Jun 12.

University Center SENAI/CIMATEC, SENAI Institute of Innovation in Health Advanced Systems (ISI SAS), Salvador 41650-010, Bahia, Brazil.

O dissolved in water (or ozonized water) has been considered a potent antimicrobial agent, and this study aimed to test this through microbiological and in vitro assays. The stability of O was accessed following modifications of the physicochemical parameters of water, such as the temperature and pH, with or without buffering. Three concentrations of O (0.4, 0.6, and 0.8 ppm) dissolved in water were tested against different microorganisms, and an analysis of the cytotoxic effects was also conducted using the human ear fibroblast cell line (Hfib). Under the physicochemical conditions of 4 °C and pH 5, O remained the most stable and concentrated compared to pH 7 and water at 25 °C. Exposure to ozonized water resulted in high mortality rates for , , , , and . Scanning electron micrograph images indicate that the effects on osmotic stability due to cell wall lysis might be one of the killing mechanisms of ozonized water. The biocidal agent was biocompatible and presented no cytotoxic effect against Hfib cells. Therefore, due to its cytocompatibility and biocidal action, ozonized water can be considered a viable alternative for microbial control, being possible, for example, its use in disinfection processes.
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http://dx.doi.org/10.3390/biology10060525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8231602PMC
June 2021

Potential application of novel technology developed for instant decontamination of personal protective equipment before the doffing step.

PLoS One 2021 4;16(6):e0250854. Epub 2021 Jun 4.

SENAI CIMATEC, SENAI Institute of Innovation (ISI) in Health Advanced Systems (CIMATEC ISI SAS), University Center SENAI/CIMATEC, Salvador, Bahia, Brazil.

The use of personal protective equipment (PPE) has been considered the most effective way to avoid the contamination of healthcare workers by different microorganisms, including SARS-CoV-2. A spray disinfection technology (chamber) was developed, and its efficacy in instant decontamination of previously contaminated surfaces was evaluated in two exposure times. Seven test microorganisms were prepared and inoculated on the surface of seven types of PPE (respirator mask, face shield, shoe, glove, cap, safety glasses and lab coat). The tests were performed on previously contaminated PPE using a manikin with a motion device for exposure to the chamber with biocidal agent (sodium hypochlorite) for 10 and 30s. In 96.93% of the experimental conditions analyzed, the percentage reduction was >99% (the number of viable cells found on the surface ranged from 4.3x106 to <10 CFU/mL). The samples of E. faecalis collected from the glove showed the lowest percentages reduction, with 86.000 and 86.500% for exposure times of 10 and 30 s, respectively. The log10 reduction values varied between 0.85 log10 (E. faecalis at 30 s in glove surface) and 9.69 log10 (E. coli at 10 and 30 s in lab coat surface). In general, E. coli, S. aureus, C. freundii, P. mirabilis, C. albicans and C. parapsilosis showed susceptibility to the biocidal agent under the tested conditions, with >99% reduction after 10 and 30s, while E. faecalis and P. aeruginosa showed a lower susceptibility. The 30s exposure time was more effective for the inactivation of the tested microorganisms. The results show that the spray disinfection technology has the potential for instant decontamination of PPE, which can contribute to an additional barrier for infection control of healthcare workers in the hospital environment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0250854PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8177472PMC
June 2021

Numerical and experimental analyses for the improvement of surface instant decontamination technology through biocidal agent dispersion: Potential of application during pandemic.

PLoS One 2021 19;16(5):e0251817. Epub 2021 May 19.

SENAI CIMATEC, National Service of Industrial Learning-SENAI, Computational Modeling and Industrial Technology, University Center SENAI/CIMATEC, Salvador, Bahia, Brazil.

The transmission of SARS-CoV-2 through contact with contaminated surfaces or objects is an important form of transmissibility. Thus, in this study, we evaluated the performance of a disinfection chamber designed for instantaneous dispersion of the biocidal agent solution, in order to characterize a new device that can be used to protect individuals by reducing the transmissibility of the disease through contaminated surfaces. We proposed the necessary adjustments in the configuration to improve the dispersion on surfaces and the effectiveness of the developed equipment. Computational Fluid Dynamics (CFD) simulations of the present technology with a chamber having six nebulizer nozzles were performed and validated through qualitative and quantitative comparisons, and experimental tests were conducted using the method Water-Sensitive Paper (WSP), with an exposure to the biocidal agent for 10 and 30 s. After evaluation, a new passage procedure for the chamber with six nozzles and a new configuration of the disinfection chamber were proposed. In the chamber with six nozzles, a deficiency was identified in its central region, where the suspended droplet concentration was close to zero. However, with the new passage procedure, there was a significant increase in wettability of the surface. With the proposition of the chamber with 12 nozzles, the suspended droplet concentration in different regions increased, with an average increase of 266%. The experimental results of the new configuration proved that there was an increase in wettability at all times of exposure, and it was more significant for an exposure of 30 s. Additionally, even in different passage procedures, there were no significant differences in the results for an exposure of 10 s, thereby showing the effectiveness of the new configuration or improved spraying and wettability by the biocidal agent, as well as in minimizing the impact caused by human factor in the performance of the disinfection technology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0251817PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8133442PMC
June 2021

Prospective study on microencapsulation of oils and its application in foodstuffs.

Recent Pat Nanotechnol 2021 Apr 22. Epub 2021 Apr 22.

Faculty of Pharmacy, Federal University of Bahia, Salvador, Brazil.

Background: Edible oils have gained the interest of several industrial sectors for the different health benefits they offer, such as the supply of bioactive compounds and essential fatty acids. Microencapsulation is one of the techniques that has been adopted by industries to minimize the degradation of oils, facilitating their processing.

Objective: To evaluate the intellectual property related to patent documents referring to microencapsulated oils used in foods.

Methods And Results: This prospective study investigated the dynamics of patents filed in the Espacenet and National Institute of Industrial Property (INPI) databases, and it mapped technological developments in microencapsulation in comparison with scientific literature. The years 2015 and 2018 showed the greatest growth in the number of patents filed in the Espacenet and INPI databases, respectively with China leading the domains of origin, inventors, and owners of microencapsulation technology. The largest number of applications of microcapsules were observed in the food industry, and the foods containing microencapsulated oils were powdered seasonings, dairy products, rice flour, nutritional formulae, pasta, nutritional supplements, and bread. The increase in oxidative stabilities of oils was the most cited objective to microencapsulate oils. Spray drying was the most widely used microencapsulation technique, and maltodextrin, gum arabic, and modified starch were the most widely used wall materials.

Conclusion: Microencapsulation of oils has been expanding over the years and increasing the possibilities of the use of microcapsules, but further investments and development of policies and incentive programs to boost this technology need to be made in less developed countries. For future perspectives, the microencapsulation technique is already a worldwide trend in the food industry, enabling the development of new products to facilitate their insertion in the consumer market.
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http://dx.doi.org/10.2174/1872210515666210422123001DOI Listing
April 2021

A Patent Data Analysis in Nanotechnology Applied to Essential Oils.

Recent Pat Nanotechnol 2021 Feb 16. Epub 2021 Feb 16.

Faculty of Pharmacy, Federal University of Bahia, Salvador. Brazil.

Background: Essential oils (EOs) are natural substances that serve as sources of bioactive compounds with antioxidant and antimicrobial properties.

Objective: This prospecting aimed to understand intellectual property related to patent documents on EOs and nanotechnology.

Methods And Results: The nanotechnology growth curve applied to EOs demonstrated that the period from 2015 to 2017 was the most prominent, with a peak in 2016. China is the dominant country, mainly through research developed in the academic area. The food industry area had the highest number of patents filed, highlighting the preservation line. Ginger essential oil, chitosan and Tween 80 were preferentially used as a core, wall material and an emulsifier, respectively. In the market, the use of EOs is more associated with the pharmaceutical/cosmetics industry. In addition, the food industry market is betting more on products based on hemp oil nanocapsules.

Conclusion: EO nanotechnology is promising for the development of sustainable food systems. However, this nanotechnology in Brazil has not yet advanced enough in the food industry, although there are government incentives that may change this paradigm in the future. The profile of the patent documents and the products in the market differ between the application and the types of oils. In addition, there is a gap between the volume of patent documents investigated and the transfer of technology to the commercial sector, but this constitution could be better explored, given the properties of EOs.
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http://dx.doi.org/10.2174/1872210515666210217090541DOI Listing
February 2021

Development of Bacterial Cellulose Biocomposites Combined with Starch and Collagen and Evaluation of Their Properties.

Materials (Basel) 2021 Jan 19;14(2). Epub 2021 Jan 19.

PPG GETEC, University Center SENAI CIMATEC, National Service of Industrial Learning, SENAI CIMATEC, Salvador 41650-010, Brazil.

One of the major benefits of biomedicine is the use of biocomposites as wound dressings to help improve the treatment of injuries. Therefore, the main objective of this study was to develop and characterize biocomposites based on bacterial cellulose (BC) with different concentrations of collagen and starch and characterize their thermal, morphological, mechanical, physical, and barrier properties. In total, nine samples were produced with fixed amounts of glycerol and BC and variations in the amount of collagen and starch. The water activity (0.400-0.480), water solubility (12.94-69.7%), moisture (10.75-20.60%), thickness (0.04-0.11 mm), water vapor permeability (5.59-14.06 × 10 g·mm/m·h·Pa), grammage (8.91-39.58 g·cm), opacity (8.37-36.67 Abs 600 nm·mm), elongation (4.81-169.54%), and tensile strength (0.99-16.32 MPa) were evaluated and defined. In addition, scanning electron microscopy showed that adding biopolymers in the cellulose matrix made the surface compact, which also influenced the visual appearance. Thus, the performance of the biocomposites was directly influenced by their composition. The performance of the different samples obtained resulted in them having different potentials for application considering the injury type. This provides a solution for the ineffectiveness of traditional dressings, which is one of the great problems of the biomedical sector.
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http://dx.doi.org/10.3390/ma14020458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7833372PMC
January 2021

The Main Molecular and Serological Methods for Diagnosing COVID-19: An Overview Based on the Literature.

Viruses 2020 12 29;13(1). Epub 2020 Dec 29.

SENAI Institute of Innovation (ISI) in Health Advanced Systems (CIMATEC ISI SAS), University Center SENAI/CIMATEC, Salvador 41650-010, Bahia, Brazil.

Diagnostic tests have been considered as the main alternative for the control of coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as a correct diagnosis allows for decision making when facing the disease, particularly as there is a lack of effective therapeutic protocols and vaccines. Thus, in this review, we summarized the main diagnostic approaches currently available for the diagnosis of SARS-CoV-2 infection in humans based on studies available in article databases. The tests can be organized into two main categories: nucleic acid-based tests, recommended for the initial detection of the virus, and serological tests, recommended for assessing the disease progression. The studies have shown that the performance of diagnostic methods depends on different factors, such as the type of samples and the characteristics of each assay. It was identified that the positivity of the tests is mainly related to the onset of symptoms. We also observed that point-of-care diagnoses are considered as one of the main trends in this area, due to the low-cost and simplicity of the assay; however, the analytical performance must be critically analyzed. Thus, the COVID-19 pandemic has highlighted the critical role of diagnostic technologies in the control of infectious diseases.
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http://dx.doi.org/10.3390/v13010040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823618PMC
December 2020

Combination of carotenoids from Spirulina and PLA/PLGA or PHB: New options to obtain bioactive nanoparticles.

Food Chem 2021 Jun 8;346:128742. Epub 2020 Dec 8.

Nutrition School, Federal University of Bahia, Rua Basilio da Gama-s/n-Campus Canela, Salvador, Bahia 40110-907, Brazil; Department of Food Science and Technology, Federal University of Santa Catarina, Admar Gonzaga Highway, 1346, Itacorubi, Florianópolis, Santa Catarina 88034-000, Brazil. Electronic address:

The use of poly-β-hydroxybutyrate (PHB) is an alternative polymer that can be considered environment friendly and renewable to prepare nanoparticles of carotenoids. This study aimed to develop and characterize aqueous dispersion nanoparticles and lyophilized nanoparticles of carotenoid extract obtained from Spirulina sp. LEB 18 by nanoprecipitation, using poly lactic acid (PLA)/poly lactic-co-glycolic acid (PLGA) (75:25 w/w) or PHB as encapsulants. The samples were characterized for the particle size, polydispersity index, zeta potential, apparent viscosity, pH, color parameters, ultraviolet-visible (UV/Vis) spectrophotometry, carotenoid profile, encapsulation efficiency, morphology, and thermal analysis. Nanoparticles containing microalgae carotenoid extract showed average particle diameter on a nanoscale (<200 nm), high homogeneity and stability, high thermal stability, and encapsulation efficiency carotenoid (>80%) when compared to nanoparticles containing β-carotene synthetic. PHB or PLA/PLGA as encapsulating material in the production of nanoparticles from microalgae carotenoids can be a polymeric alternative capable of promoting greater stability and application of carotenoids.
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http://dx.doi.org/10.1016/j.foodchem.2020.128742DOI Listing
June 2021

Application of Liposomes in Cancer Therapy: An Assessment of the Advancement of Technology Through Patent Documents.

Recent Pat Nanotechnol 2020 Dec 22. Epub 2020 Dec 22.

SENAI Institute of Innovation (ISI) in Advanced Health Systems (CIMATEC ISI SAS), University Center SENAI/CIMATEC, National Service of Industrial Learning - SENAI, Salvador, Bahia. Brazil.

Background: Cancer is caused by mutations in oncogenes and loss of tumor suppressor activity. Despite the efforts of the pharmaceutical industry to fight cancer, it is necessary to investigate new methods to enhance the action against this disease. In this scenario, liposome technology has emerged as a drug delivery system for cancer treatment. The objective of this study was to evaluate the use of liposome technology in the treatment of cancer through analysis of patent documents.

Methods: We analyzed prospective documents in the Derwent Innovation Index database.

Results: A total of 203 patent documents were related to the area of interest, published between 2000 and 2020, and the USA and Japan were shown as countries with the largest number of publications. Moreover, most of these publications came from companies, however, also with representation of important universities, mainly the University of California.. According to the International Patent Classification, the codes most applicable to the documents were the area of human necessities and chemistry.

Conclusion: The results of this study showed that the study and application of liposomes for the development of cancer treatment tended to grow, along with the number of related patents.
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http://dx.doi.org/10.2174/1872210514666201223093321DOI Listing
December 2020

Supercritical Extraction of Red Propolis: Operational Conditions and Chemical Characterization.

Molecules 2020 Oct 20;25(20). Epub 2020 Oct 20.

School of Pharmacy, Federal University of Bahia (UFBA), Barão de Jeremoabo Street, 147, Salvador 40110-100, Bahia, Brazil.

The objective of this study was to determine the best operational conditions for obtaining red propolis extract with high antioxidant potential through supercritical fluid extraction (SFE) technology, using carbon dioxide (CO) as the supercritical fluid and ethanol as the cosolvent. The following parameters were studied: overall extraction curve, S/F (mass of CO/mass of sample), cosolvent percentage (0, 1, 2 and 4%) and global yield isotherms as a function of different pressures (250, 350 and 450 bar) and temperatures (31.7, 40 and 50 °C). Within the investigated parameters, the best conditions found were an S/F of 131 and the use of ethanol at the highest concentration (4% /), which resulted in higher extract yields and higher content of antioxidant compounds. Formononetin, the main biomarker of red propolis, was the compound found at the highest amounts in the extracts. As expected, the temperature and pressure conditions also influenced the process yield, with 350 bar and 40 °C being the best conditions for obtaining bioactive compounds from a sample of red propolis. The novel results for red propolis found in this study show that it is possible to obtain extracts with high antioxidant potential using a clean technology under the defined conditions.
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http://dx.doi.org/10.3390/molecules25204816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587948PMC
October 2020

Effect of Drying Methods on Bioactive Compounds and Antioxidant Capacity in Grape Skin Residues from the New Hybrid Variety "BRS Magna".

Molecules 2020 Aug 14;25(16). Epub 2020 Aug 14.

Department of Biotechnology, Health Science Institute, Federal University of Bahia (UFBA), Salvador 40170-115, Bahia, Brazil.

The effects of the drying process using the conventional oven and freeze-drying on the thermogravimetric profile, proximate composition, color parameters, individual bioactive compounds, and antioxidant activity in the grape residue (skin) were evaluated. Twenty individual phenolic compounds were identified, where a variation in concentration was observed for flavonols, stilbenes, phenolic acids, flavonoids, procyanidins, and particularly anthocyanins (malvidin-3,5-di--glucoside of 253.2-536.9 mg/kg) due to the drying process. Drying in a conventional oven caused a decrease of 23% of the total polyphenols. The skin of the BRS magna grape has a high concentration of total phenolic content of 489.5-148.3 mg.GAE/100 g, total anthocyanin content of 124.9-260.1 mg.CE/100 g, and total flavonoid content of 12.7-26.0 mg.QE/100 g. The results of free radical scavenging activity (1.26-4.91 μg/mL, as EC) and ferric reducing antioxidant power (82.93-184.10 μmol/g of skin as equivalent to FeSO) indicate high antioxidant activity, independently of the drying process applied. It was concluded that, if the application is directed to anthocyanin compounds, the use of lyophilization is recommended. On the other hand, if the interest is in bioactive compounds that exert antioxidant activity, conventional oven-drying can be used.
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http://dx.doi.org/10.3390/molecules25163701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7466153PMC
August 2020

Optimization of convolutional neural network hyperparameters for automatic classification of adult mosquitoes.

PLoS One 2020 14;15(7):e0234959. Epub 2020 Jul 14.

University Center SENAI/CIMATEC, SENAI Institute of Innovation (ISI) in Health Advanced Systems (CIMATEC ISI SAS), Salvador, Bahia, Brazil.

The economic and social impacts due to diseases transmitted by mosquitoes in the latest years have been significant. Currently, no specific treatment or commercial vaccine exists for the control and prevention of arboviruses, thereby making entomological characterization fundamental in combating diseases such as dengue, chikungunya, and Zika. The morphological identification of mosquitos includes a visual exam of the samples. It is time consuming and requires adequately trained professionals. Accordingly, the development of a new automated method for realizing mosquito-perception and -classification is becoming increasingly essential. Therefore, in this study, a computational model based on a convolutional neural network (CNN) was developed to extract features from the images of mosquitoes and then classify the species Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus. In addition, the model was trained to detect the mosquitoes of the genus Aedes. To train CNNs to perform the automatic morphological classification of mosquitoes, a dataset, which included 7,561 images of the target mosquitoes and 1,187 images of other insects, was acquired. Various neural networks, such as Xception and DenseNet, were used for developing the automatic-classification model based on images. A structured optimization process of random search and grid search was developed to select the hyperparameters set and increase the accuracy of the model. In addition, strategies to eliminate overfitting were implemented to increase the generalization of the model. The optimized model, during the test phase, obtained the balanced accuracy (BA) of 93.5% in classifying the target mosquitoes and other insects and the BA of 97.3% in detecting the mosquitoes of the genus Aedes in comparison to Culex. The results provide fundamental information for performing the automatic morphological classification of mosquito species. Using a CNN-embedded entomological tool is a valuable and accessible resource for health workers and non-taxonomists for identifying insects that can transmit infectious diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0234959PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360088PMC
September 2020

Effect of Experimental Parameters on the Extraction of Grape Seed Oil Obtained by Low Pressure and Supercritical Fluid Extraction.

Molecules 2020 Apr 2;25(7). Epub 2020 Apr 2.

Federal University of Bahia, Bahia, Salvador 40170-115, Brazil.

Grape seeds are an important byproduct from the grape process. The objective of this work was to evaluate the effect of experimental parameters (temperature and time of pretreatment with ultrasound) to obtain grape seed oil using low pressure (Soxhlet-Sox and Bligh Dyer-BD) and high pressure (supercritical carbon dioxide-SFE) methods. The best condition for pretreatment of samples was 30 min of sonication at 30 °C before extraction by Sox or BD. Ultrasound pretreatment was efficient to increase oil extraction yield by 32.10 (Sox), 20.31 (BD) and 12.54% (SFE), depending on the extraction method used as well as, and certainly influenced the total phenolic concentration in 311 (Sox), 234 (BD), and 184 (SFE)%. Ten fatty acids were identified in the oils, the major ones being 18:2ω-6cis (linoleic 52.39%-63.12%), 16:0 (palmitic 20.22%-26.80%) and 18:0 (stearic 8.52%-13.68%). The highest epicatechin concentration was identified in the BD sample: 30-30 (150.49 ± 5.98mg/kg), which presented a concentration of ≥3 times compared to the control (56.68 ± 1.81mg/kg). Ultrasound pretreatment also contributed positively (56% and 99% increase) in the α-tocopherol content of the SFE: 30-30 and BD: 30-30 samples, respectively. The results indicate that the ultrasound pretreatment is a suitable technology to improve the quality of the oil from the grape seed.
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http://dx.doi.org/10.3390/molecules25071634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7180707PMC
April 2020

Evaluation of Different Methods for Cultivating for Bacterial Cellulose and Montmorillonite Biocomposite Production: Wound-Dressing Applications.

Polymers (Basel) 2020 Jan 26;12(2). Epub 2020 Jan 26.

University Center SENAI CIMATEC, National Service of Industrial Learning, Laboratory of Pharmaceutical's Formulations, Health Institute of Technologies (ITS CIMATEC), Salvador 41650-010, Brazil.

Bacterial cellulose (BC) has received considerable attention due to its unique properties, including an ultrafine network structure with high purity, mechanical strength, inherent biodegradability, biocompatibility, high water-holding capacity and high crystallinity. These properties allow BC to be used in biomedical and industrial applications, such as medical product. This research investigated the production of BC by ATCC 23769 using different carbon sources (glucose, mannitol, sucrose and xylose) at two different concentrations (25 and 50 g∙L). The BC produced was used to develop a biocomposite with montmorillonite (MMT), a clay mineral that possesses interesting characteristics for enhancing BC physical-chemical properties, at 0.5, 1, 2 and 3% concentrations. The resulting biocomposites were characterized in terms of their physical and barrier properties, morphologies, water-uptake capacities, and thermal stabilities. Our results show that bacteria presented higher BC yields in media with higher glucose concentrations (50 g∙L) after a 14-day incubation period. Additionally, the incorporation of MMT significantly improved the mechanical and thermal properties of the BC membranes. The degradation temperature of the composites was extended, and a decrease in the water holding capacity (WHC) and an improvement in the water release rate (WRR) were noted. Determining a cost-effective medium for the production of BC and the characterization of the produced composites are extremely important for the biomedical applications of BC, such as in wound dressing materials.
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http://dx.doi.org/10.3390/polym12020267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077264PMC
January 2020

Susceptibility of Clinical Isolates to Allopathic Antifungals and Brazilian Red, Green, and Brown Propolis Extracts.

Front Vet Sci 2019 13;6:460. Epub 2019 Dec 13.

Instituto de Ciências da Saúde, Universidade Federal da Bahia, Salvador, Brazil.

Clinical mycoses treatment is associated with issues such as negative side effects, high cost, prolonged treatment, and resistant strain selection. is the most frequently isolated yeast in cases of canine otitis and dermatitis. The number of fungal strains exhibiting primary resistance to several drugs is increasing. Propolis has a diverse chemical composition and well-known therapeutic properties against mycoses. An alternative method for producing propolis extracts using supercritical fluid has higher selectivity, yielding extracts with fewer pollutant residues. This study therefore aimed to evaluate the susceptibility profile of clinical isolates to precharacterized supercritical and ethanolic extracts. Three types of Brazilian propolis extracts (green, red, and brown) and commercial allopathic antifungals were used in this investigation. We used the microdilution broth technique to evaluate the susceptibility profile of the yeasts. The minimum inhibitory concentration (MIC) of the brown propolis ethanolic extract was ≥16 μg/mL for all isolates. The MICs of fluconazole, ketoconazole, itraconazole, and amphotericin B ranged from 8 to >64 μg/mL, 0.032-4 μg/mL, 0.0313-16 μg/mL, and 1-2 μg/mL, respectively. The MICs of ethanolic red propolis extracts were lower than those of supercritical red propolis extracts. However, the green propolis ethanolic extract had more pronounced fungicidal activity. Isolates with lower susceptibility to commercial fungicides were inhibited by red and green propolis extracts. These results indicate that propolis can potentially be used in experiments as a promising therapeutic agent against infections.
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http://dx.doi.org/10.3389/fvets.2019.00460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6923270PMC
December 2019

Physicochemical and sensory profile of Beauregard sweet potato beer.

Food Chem 2020 May 24;312:126087. Epub 2019 Dec 24.

Institute of Technology and Research (ITP), Av. Murilo Dantas, 300, Aracaju, Sergipe CEP 49032-490, Brazil; Tiradentes University (UNIT), Av. Murilo Dantas, 300, Aracaju, Sergipe CEP 49032-490, Brazil.

Beer has been one of the most consumed alcoholic beverages worldwide. However, the incorporation of adjuncts in the beer can add new organoleptic and functional characteristics to the beverage. For this, Beauregard sweet potato shows high potential due to being a rich source of starch and many bioactive compounds. The aim of this study was to develop the best process condition to produce a Sweet potato beer with enhanced nutritional and antioxidant properties and good sensory characteristics. Beer samples showed increased antioxidant activity especially due to β-carotene and their total phenolic content. The phytochemical profile of sweet potato biocompounds demonstrated a direct effect of this adjunct on sensory and functional characteristics of the finished beer. In conclusion, it was found that Beauregard sweet potato is a promising adjunct for beer brewing with nutraceutical properties due to its rich composition of bioactive compounds.
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http://dx.doi.org/10.1016/j.foodchem.2019.126087DOI Listing
May 2020

Antiviral effects of Brazilian green and red propolis extracts on Enterovirus surrogates.

Environ Sci Pollut Res Int 2020 Aug 30;27(23):28510-28517. Epub 2019 Dec 30.

National Service of Industrial Learning, University Center SENAI CIMATEC, Salvador, Bahia, Brazil.

Propolis is a natural product of bees with biological activities that are mainly associated with bee type and geographic origin. Propolis extract has been proposed with several applications in environmental health. The ethanol extracts have shown good antimicrobial activity. The association of this technique with ultrasound-assisted extraction has been studied to improve the characteristics of the obtained extracts. Thus, the objective of this work is to verify the antiviral action against two strains of bacteriophages of two extracts of Brazilian propolis (green and red) obtained by conventional extraction and ultrasonic extraction. The activities of the propolis red and green extracts were confirmed by the significant ~3 and ~4.5 Log 10 PFU/mL reduction in the concentrations of the MS2 and Av-08 bacteriophages, respectively. It was found that ultrasound-assisted extraction is comparable to the maceration process and demonstrated the best antiviral activities. Brazilian red propolis was more effective than green propolis in viral reduction in all treatments.
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http://dx.doi.org/10.1007/s11356-019-07458-zDOI Listing
August 2020

Brazilian Green Propolis as a Therapeutic Agent for the Post-surgical Treatment of Caseous Lymphadenitis in Sheep.

Front Vet Sci 2019 26;6:399. Epub 2019 Nov 26.

Instituto de Ciências da Saúde, Universidade Federal da Bahia, Salvador, Brazil.

As antibiotics are ineffective when used to treat caseous lymphadenitis, the surgical excision of lesions is often required. Iodine solution (10%) is currently the choice for the post-surgical treatment; however, it may cause histotoxicity. Propolis are resinous substances composed by a mixture of different plants parts and molecules secreted by bees. As green propolis has already proven to possess anti-bacterial and wound healing properties, this study aimed to evaluate the use of a green propolis-based ointment as a therapeutic agent for the post-surgical treatment of caseous lymphadenitis. The caseous lesions of 28 sheep were surgically excised before dividing animals into two groups: (1) iodine-treated animals and (2) sheep treated with an ointment made with a previously characterized green propolis extract. Clinical data of animals, size of the scar area, the presence of moisture and secretion in the surgical wound, the humoral immune response against the bacterium and the susceptibility of clinical isolates to the green propolis extract were analyzed. The green propolis-treated group presented complete healing of the surgical wound 1 week before the iodine-treated group. Additionally, animals treated with the green propolis ointment had fewer cases of wound secretion, but it was not statistically different from the iodine-treated group. No clinical signs indicating green propolis toxicity or other side effects were found, associated with a faster and more organized hair recovery by propolis use. The green propolis extract was able to inhibit the growth of 23 from the 27 clinical isolates, with minimum inhibitory and minimum bactericide concentrations ranging from 01 to 08 mg/mL, and did not interfere with the humoral immune response against the bacterium. In addition, green propolis was able to inhibit biofilm formation by four of the clinical isolates. We concluded that green propolis is a promising therapeutic agent to be used in the post-surgical treatment of caseous lymphadenitis in small ruminants due to its effects on surgical wound healing, hair recovery, inhibition of wound contamination and bacterial growth.
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http://dx.doi.org/10.3389/fvets.2019.00399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6887654PMC
November 2019

Evaluation of the antioxidant profile and cytotoxic activity of red propolis extracts from different regions of northeastern Brazil obtained by conventional and ultrasound-assisted extraction.

PLoS One 2019 5;14(7):e0219063. Epub 2019 Jul 5.

University Center SENAI/CIMATEC, National Service of Industrial Learning - SENAI, Heath Institute of Technology (ITS CIMATEC), Salvador, Bahia, Brazil.

Propolis is a complex mixture of resinous and balsamic material collected from the exudates of plants, shoots, and leaves by bees. This study evaluated red propolis extracts obtained by conventional (ethanolic) extraction and ultrasound-assisted extraction of six samples from different regions of northeastern Brazil. The total phenolic compounds and flavonoids, in vitro antioxidant activity, concentration of formononetin and kaempferol and the cytotoxicity against four human tumor cell lines were determined for all twelve obtained extracts. Significant variations in the levels of the investigated compounds were identified in the red propolis extracts, confirming that the chemical composition varied according to the sampling region. The extraction method used also influenced the resulting propolis compounds. The highest concentration of the compounds of interest and the highest in vitro antioxidant activity were exhibited by the extracts obtained from samples from state of Alagoas. Formononetin and kaempferol were identified in all samples. The highest formononetin concentrations were identified in extracts obtained by ultrasound, thus indicating a greater selectivity for the extraction of this compound by this method. Regarding cytotoxic activity, for the HCT-116 line, all of the extracts showed an inhibition of greater than 90%, whereas for the HL-60 and PC3 lines, the minimum identified was 80%. In general, there was no significant difference (p>0.05) in the antiproliferative potential when comparing the extraction methods. The results showed that the composition of Brazilian red propolis varies significantly depending on the geographical origin and that the method used influences the resulting compounds that are present in propolis. However, regardless of the geographical origin and the extraction method used, all the red propolis samples studied presented great biological potential and high antioxidant activity. Furthermore, the ultrasound-assisted method can be efficiently applied to obtain extracts of red propolis more quickly and with high concentration of biomarkers of interest.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0219063PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611595PMC
February 2020

Grape peel (Syrah var.) jam as a polyphenol-enriched functional food ingredient.

Food Sci Nutr 2019 May 12;7(5):1584-1594. Epub 2019 Apr 12.

Federal University of Bahia (UFBA) Salvador Brazil.

In this study, we evaluated the effects of the storage time on the physicochemical properties, bioactive compound content, and antioxidant capacity of jam prepared from grape peel extract to explore its potential as a supplementary food and/or functional ingredient. The ethanolic extract from Syrah var. grape peel exhibited high bioactive compound concentrations and antioxidant activity. The jam stability (prepared with 8.9% of extract) at 14°C was evaluated at 0, 15, 30, 45, and 60 days. The jam was found to contain high concentrations of polyphenolic compounds (137.0 ± 3.2 mg of gallic acid equivalent/100 g), total flavonoids (128.5 ± 23.0 mg of equivalent/100 g), and total anthocyanins (92.5 ± 4.0 mg of cyanidin equivalent/100 g). However, a large reduction in the flavonoid (70%-90%), anthocyanin (29%-35%), and phenolic (23%-30%) content was observed during storage. The free radical-scavenging activity (DPPH), ferric reducing antioxidant power (FRAP), and β-carotene-linoleic acid assays revealed the great antioxidant potential of the jam prepared from grape peel extract, which exhibited significant levels of radical-neutralizing activity, especially as determined by the DPPH method with EC values ranging from 2.3 ± 0.1 to 3.9 ± 0.1 µg/ml. High values (> 0.90) were obtained for the correlation between the DPPH results and the concentrations of the compounds of interest. In summary, the high bioactive compound contents and antioxidant capacity of the jam produced from grape peel suggest that it may provide health benefits as a source of natural antioxidants upon incorporation to several food industry products.
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http://dx.doi.org/10.1002/fsn3.981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6526673PMC
May 2019

Effect of Cellulose Nanocrystals from Different Lignocellulosic Residues to Chitosan/Glycerol Films.

Polymers (Basel) 2019 Apr 10;11(4). Epub 2019 Apr 10.

Department of Food and Beverages, National Service of Industrial Learning, Applied Research Laboratory of Biotechnology and Food, University Center SENAI CIMATEC, Salvador 41650-010, Brazil.

Interest in nanocellulose obtained from natural resources has grown, mainly due to the characteristics that these materials provide when incorporated in biodegradable films as an alternative for the improvement of the properties of nanocomposites. The main purpose of this work was to investigate the effect of the incorporation of nanocellulose obtained from different fibers (corncob, corn husk, coconut shell, and wheat bran) into the chitosan/glycerol films. The nanocellulose were obtained through acid hydrolysis. The properties of the different nanobiocomposites were comparatively evaluated, including their barrier and mechanical properties. The nanocrystals obtained for coconut shell (CS), corn husk (CH), and corncob (CC) presented a length / diameter ratio of 40.18, 40.86, and 32.19, respectively. Wheat bran (WB) was not considered an interesting source of nanocrystals, which may be justified due to the low percentage of cellulose. Significant differences were observed in the properties of the films studied. The water activity varied from 0.601 (WB Film) to 0.658 (CH Film) and the moisture content from 15.13 (CS Film) to 20.86 (WB Film). The highest values for tensile strength were presented for CC (11.43 MPa) and CS (11.38 MPa) films, and this propriety was significantly increased by nanocellulose addition. The results showed that the source of the nanocrystal determined the properties of the chitosan/glycerol films.
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http://dx.doi.org/10.3390/polym11040658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6523815PMC
April 2019

Application of convolutional neural networks for classification of adult mosquitoes in the field.

PLoS One 2019 14;14(1):e0210829. Epub 2019 Jan 14.

University Center SENAI CIMATEC, National Service of Industrial Learning-SENAI, Salvador, Bahia, Brazil.

Dengue, chikungunya and Zika are arboviruses transmitted by mosquitos of the genus Aedes and have caused several outbreaks in world over the past ten years. Morphological identification of mosquitos is currently restricted due to the small number of adequately trained professionals. We implemented a computational model based on a convolutional neural network (CNN) to extract features from mosquito images to identify adult mosquitoes from the species Aedes aegypti, Aedes albopictus and Culex quinquefasciatus. To train the CNN to perform automatic morphological classification of mosquitoes, we used a dataset that included 4,056 mosquito images. Three neural networks, including LeNet, AlexNet and GoogleNet, were used. During the validation phase, the accuracy of the mosquito classification was 57.5% using LeNet, 74.7% using AlexNet and 83.9% using GoogleNet. During the testing phase, the best result (76.2%) was obtained using GoogleNet; results of 52.4% and 51.2% were obtained using LeNet and AlexNet, respectively. Significantly, accuracies of 100% and 90% were achieved for the classification of Aedes and Culex, respectively. A classification accuracy of 82% was achieved for Aedes females. Our results provide information that is fundamental for the automatic morphological classification of adult mosquito species in field. The use of CNN's is an important method for autonomous identification and is a valuable and accessible resource for health workers and taxonomists for the identification of some insects that can transmit infectious agents to humans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0210829PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331110PMC
October 2019

Chemical characterization and biological activity of six different extracts of propolis through conventional methods and supercritical extraction.

PLoS One 2018 4;13(12):e0207676. Epub 2018 Dec 4.

Federal University of Bahia, Salvador, Bahia, Brazil.

Propolis is a natural product with many demonstrated biological activities and propolis extract has been used in the food, pharmaceutical and cosmetics industries. Different works have showed the variations in the chemical composition, and consequently, on the biological activity of the propolis that are associated with its type and geographic origin. Due to this study evaluated propolis extracts obtained through supercritical extraction and ethanolic extraction (conventional) in three samples of different types of propolis (red, green and brown), collected from different regions in Brazil (state of Bahia). Analyses were performed to determine the humidity, water activity, the content of total ash, proteins, lipids and fiber in raw propolis samples. The content of phenolic compounds, flavonoids, in vitro antioxidant activity (DPPH), catechin, ferulic acid and luteolin and antimicrobial activity against two bacteria (Staphylococcus aureus and Escherichia coli) were determined for all extracts. For the green and red ethanolic extracts the anti-leishmanicidal potential was also evaluated. The physicochemical profiles showed agreement in relation to the literature. The results identified significant differences among the extracts (p>0.05), which are in conformity with their extraction method, as well as with type and botanical origin of the samples. The extraction with supercritical fluid was not efficient to obtain extracts with the highest contents of antioxidants compounds, when compared with the ethanolic extracts. The best results were shown for the extracts obtained through the conventional extraction method (ethanolic) indicating a higher selectivity for the extraction of antioxidants compounds. The red variety showed the largest biological potential, which included the content of antioxidants compounds. The results found in this study confirm the influence of the type of the raw material on the composition and characteristics of the extracts. The parameters analysis were important to characterize and evaluate the quality of the different Brazilian propolis extracts based on the increased use of propolis by the natural products industry.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0207676PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279037PMC
May 2019

Cocoa pulp in beer production: Applicability and fermentative process performance.

PLoS One 2017 18;12(4):e0175677. Epub 2017 Apr 18.

Department of Biology and Biotechnology of Microorganisms, State University of Santa Cruz, Ilhéus, Bahia, Brazil.

This work evaluated the effect of cocoa pulp as a malt adjunct on the parameters of fermentation for beer production on a pilot scale. For this purpose, yeast isolated from the spontaneous fermentation of cachaça (SC52), belonging to the strain bank of the State University of Feira de Santana-Ba (Brazil), and a commercial strain of ale yeast (Safale S-04 Belgium) were used. The beer produced was subjected to acceptance and purchase intention tests for sensorial analysis. At the beginning of fermentation, 30% cocoa pulp (adjunct) was added to the wort at 12°P concentration. The production of beer on a pilot scale was carried out in a bioreactor with a 100-liter capacity, a usable volume of 60 liters, a temperature of 22°C and a fermentation time of 96 hours. The fermentation parameters evaluated were consumption of fermentable sugars and production of ethanol, glycerol and esters. The beer produced using the adjunct and yeast SC52 showed better fermentation performance and better acceptance according to sensorial analysis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0175677PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5395165PMC
May 2017

Antioxidant, antimicrobial, antiparasitic, and cytotoxic properties of various Brazilian propolis extracts.

PLoS One 2017 30;12(3):e0172585. Epub 2017 Mar 30.

Department of Biotechnology, Federal University of Bahia, Salvador, Bahia, Brazil.

Propolis is known for its biological properties and its preparations have been continuously investigated in an attempt to solve the problem of their standardization, an issue that limits the use of propolis in food and pharmaceutical industries. The aim of this study was to evaluate in vitro antioxidant, antimicrobial, antiparasitic, and cytotoxic effects of extracts of red, green, and brown propolis from different regions of Brazil, obtained by ethanolic and supercritical extraction methods. We found that propolis extracts obtained by both these methods showed concentration-dependent antioxidant activity. The extracts obtained by ethanolic extraction showed higher antioxidant activity than that shown by the extracts obtained by supercritical extraction. Ethanolic extracts of red propolis exhibited up to 98% of the maximum antioxidant activity at the highest extract concentration. Red propolis extracts obtained by ethanolic and supercritical methods showed the highest levels of antimicrobial activity against several bacteria. Most extracts demonstrated antimicrobial activity against Staphylococcus aureus. None of the extracts analyzed showed activity against Escherichia coli or Candida albicans. An inhibitory effect of all tested ethanolic extracts on the growth of Trypanosoma cruzi Y strain epimastigotes was observed in the first 24 h. However, after 96 h, a persistent inhibitory effect was detected only for red propolis samples. Only ethanolic extracts of red propolis samples R01Et.B2 and R02Et.B2 showed a cytotoxic effect against all four cancer cell lines tested (HL-60, HCT-116, OVCAR-8, and SF-295), indicating that red propolis extracts have great cytotoxic potential. The biological effects of ethanolic extracts of red propolis revealed in the present study suggest that red propolis can be a potential alternative therapeutic treatment against Chagas disease and some types of cancer, although high activity of red propolis in vitro needs to be confirmed by future in vivo investigations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0172585PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373518PMC
August 2017

Chemical Composition and Biological Activity of Extracts Obtained by Supercritical Extraction and Ethanolic Extraction of Brown, Green and Red Propolis Derived from Different Geographic Regions in Brazil.

PLoS One 2016 8;11(1):e0145954. Epub 2016 Jan 8.

Institute of Research and Technology, Tiradentes University, Aracaju, Sergipe, Brazil.

The variations in the chemical composition, and consequently, on the biological activity of the propolis, are associated with its type and geographic origin. Considering this fact, this study evaluated propolis extracts obtained by supercritical extraction (SCO2) and ethanolic extraction (EtOH), in eight samples of different types of propolis (red, green and brown), collected from different regions in Brazil. The content of phenolic compounds, flavonoids, in vitro antioxidant activity (DPPH and ABTS), Artepillin C, p-coumaric acid and antimicrobial activity against two bacteria were determined for all extracts. For the EtOH extracts, the anti-proliferative activity regarding the cell lines of B16F10, were also evaluated. Amongst the samples evaluated, the red propolis from the Brazilian Northeast (states of Sergipe and Alagoas) showed the higher biological potential, as well as the larger content of antioxidant compounds. The best results were shown for the extracts obtained through the conventional extraction method (EtOH). However, the highest concentrations of Artepillin C and p-coumaric acid were identified in the extracts from SCO2, indicating a higher selectivity for the extraction of these compounds. It was verified that the composition and biological activity of the Brazilian propolis vary significantly, depending on the type of sample and geographical area of collection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0145954PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4706314PMC
July 2016

Determination of Parameters for the Supercritical Extraction of Antioxidant Compounds from Green Propolis Using Carbon Dioxide and Ethanol as Co-Solvent.

PLoS One 2015 7;10(8):e0134489. Epub 2015 Aug 7.

Institute of Research and Technology, Tiradentes University, Aracaju, Sergipe, Brazil.

The aim of this study was to determine the best processing conditions to extract Brazilian green propolis using a supercritical extraction technology. For this purpose, the influence of different parameters was evaluated such as S/F (solvent mass in relation to solute mass), percentage of co-solvent (1 and 2% ethanol), temperature (40 and 50°C) and pressure (250, 350 and 400 bar) using supercritical carbon dioxide. The Global Yield Isotherms (GYIs) were obtained through the evaluation of the yield, and the chemical composition of the extracts was also obtained in relation to the total phenolic compounds, flavonoids, antioxidant activity and 3,5-diprenyl-4-hydroxicinnamic acid (Artepillin C) and acid 4-hydroxycinnamic (p-coumaric acid). The best results were identified at 50°C, 350 bar, 1% ethanol (co-solvent) and S/F of 110. These conditions, a content of 8.93±0.01 and 0.40±0.05 g/100 g of Artepillin C and p-coumaric acid, respectively, were identified indicating the efficiency of the extraction process. Despite of low yield of the process, the extracts obtained had high contents of relevant compounds, proving the viability of the process to obtain green propolis extracts with important biological applications due to the extracts composition.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0134489PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4529176PMC
May 2016

Bi-functional biobased packing of the cassava starch, glycerol, licuri nanocellulose and red propolis.

PLoS One 2014 10;9(11):e112554. Epub 2014 Nov 10.

Faculty of Pharmacy, Department of Bromatological Analysis, Federal University of Bahia, Rua Barão de Geremoabo, s/n, Ondina, CEP 40171-970, Salvador, Bahia, Brasil.

The aim of this study was to characterize and determine the bi-functional efficacy of active packaging films produced with starch (4%) and glycerol (1.0%), reinforced with cellulose nanocrystals (0-1%) and activated with alcoholic extracts of red propolis (0.4 to 1.0%). The cellulose nanocrystals used in this study were extracted from licuri leaves. The films were characterized using moisture, water-activity analyses and water vapor-permeability tests and were tested regarding their total phenolic compounds and mechanical properties. The antimicrobial and antioxidant efficacy of the films were evaluated by monitoring the use of the active films for packaging cheese curds and butter, respectively. The cellulose nanocrystals increased the mechanical strength of the films and reduced the water permeability and water activity. The active film had an antimicrobial effect on coagulase-positive staphylococci in cheese curds and reduced the oxidation of butter during storage.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0112554PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226550PMC
December 2015
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