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Cell-based assays utilizing reporter gene technology have been widely exploited for biosensing, as they provide useful information about the bioavailability and cell toxicity of target analytes. The long assay time due to gene transcription and translation is one of the main drawbacks of cell biosensors. We report the development of two yeast biosensors stably expressing human estrogen receptors α and ß and employing NanoLuc as the reporter protein to upgrade the widely used yeast estrogen screening (YES) assays. A viability control strain was also developed based on a chimeric green-emitting luciferase, PLG2, expressed for the first time in Saccharomycescerevisiae. Thanks to their brightness, NanoLuc and PLG2 provided excellent sensitivity, enabling the implementation of these biosensors into low-cost smartphone-based devices. The developed biosensors had a rapid (1 h) response and reported on (anti)estrogenic activity via human estrogen receptors α and ß as well as general sample toxicity. Under optimized conditions, we obtained LODs of 7.1 ± 0.4 nM and 0.38 ± 0.08 nM for E2 with nanoYESα and nanoYESß, respectively. As a proof of concept, we analyzed real samples from plants showing significant estrogenic activity or known to contain significant amounts of phytoestrogens. Graphical abstract.
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Técnicas Biossensoriais , Disruptores Endócrinos/análise , Medições Luminescentes/métodos , Nanotecnologia , Saccharomyces cerevisiae/metabolismo , Smartphone , Genes Reporter , Limite de Detecção , Luciferases/genética , Medicago sativa/química , Extratos Vegetais/química , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Glycine max/química , Poluentes Químicos da Água/análiseRESUMO
The availability of smartphones with high-performance digital image sensors and processing power has completely reshaped the landscape of point-of-need analysis. Thanks to the high maturity level of reporter gene technology and the availability of several bioluminescent proteins with improved features, we were able to develop a bioluminescence smartphone-based biosensing platform exploiting the highly sensitive NanoLuc luciferase as reporter. A 3D-printed smartphone-integrated cell biosensor based on genetically engineered Hek293T cells was developed. Quantitative assessment of (anti)-inflammatory activity and toxicity of liquid samples was performed with a simple and rapid add-and-measure procedure. White grape pomace extracts, known to contain several bioactive compounds, were analyzed, confirming the suitability of the smartphone biosensing platform for analysis of untreated complex biological matrices. Such approach could meet the needs of small medium enterprises lacking fully equipped laboratories for first-level safety tests and rapid screening of new bioactive products. Graphical abstract Smartphone-based bioluminescence cell biosensor.
Assuntos
Anti-Inflamatórios/farmacologia , Técnicas Biossensoriais/instrumentação , Luciferases/genética , Medições Luminescentes/instrumentação , Extratos Vegetais/farmacologia , Smartphone/instrumentação , Anti-Inflamatórios/química , Desenho de Equipamento , Genes Reporter , Engenharia Genética , Células HEK293 , Humanos , Limite de Detecção , Luciferases/metabolismo , Extratos Vegetais/química , Reprodutibilidade dos Testes , Vitis/químicaRESUMO
Grapevine is a large source of healthy polyphenols for human diet, and red table-grapes and wines are the main source of stilbenes. These compounds are important both in the plant defence system and for human health. In the present study, Vitis vinifera cv. Barbera cell cultures were treated with 50 µg/mL chitosan and proteomic analyses on soluble and membrane subcellular fractions were performed against suitable controls. Three soluble stilbene synthase protein spots, four stilbene synthase spots in the microsomal fraction and four spots of membrane ATPase subunits were identified, the accumulation of which was modulated in response to chitosan treatment. Present proteomic and immunolocalisation data seem to provide evidence supporting the hypothesis that a stilbene biosynthetic multi-enzyme complex is associated with the intracellular membrane. In addition, proteomic analyses showed a general decrease in the accumulation of proteins belonging to different primary metabolism pathways, both in the soluble and membrane fractions. In particular, energy, sugar and amino acid metabolisms were down-regulated as a consequence of chitosan and acetic acid treatments. These metabolic modifications could lead to a consistent change in the profile and amount of metabolites stored in grape berries, with consequent effects on taste, flavour, organoleptic and nutraceutical properties of derived food products.
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Quitosana/metabolismo , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo , Proteômica , Vitis/citologia , Vitis/metabolismo , Aciltransferases/análise , Aciltransferases/metabolismo , Técnicas de Cultura de Células/métodos , Estilbenos/metabolismo , Frações Subcelulares/química , Frações Subcelulares/metabolismoRESUMO
Wild food plants are widely consumed all over the world and many have both nutritional and therapeutic value due to the presence of biologically active compounds. The present research, for the first time, aims to compare primary and secondary metabolite levels among different plant organs (flower, leaf, stem, root, bark) of seven species (Borago officinalis L., Cynodon dactylon (L.) Pers., Foeniculum vulgare Mill., Hypericum perforatum L., Malva sylvestris L., Sambucus nigra L., Urtica dioica L.) collected in three different Italian regions (Liguria, Tuscany, Apulia). Plant organ samples were extracted with water or 95% (v/v) methanol and liquid fractions were analyzed using spectrophotometric assays. The best results were obtained for Hypericum perforatum L. samples, followed by Sambucus nigra L. and Borago officinalis L. As also confirmed via PCA analysis on normalized data, flower and leaf extracts of all species exhibited higher levels of polyphenols (up to 105.7 mg GA eq/gDW), reducing sugars (up to 389.2 mg GLUC eq/gDW), proteins (up to 675.7 mg BSA eq/gDW) and of antioxidant capacity (up to 263.5 mg AA eq/gDW). No differences among the regions of gathering were detected after spectrophotometric assays, which was confirmed via PCA analysis. These data contribute to further validate the traditionally reported healing effects of these species on human health.
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The effects of polyphenol-rich extract obtained from non-compliant defatted green coffee beans (dGCBs) on physicochemical and antioxidant properties, as well as on the sensory profile of vacuum-packed pork burgers stored at 4 °C for 14 days and after cooking were assessed. The dGCB extract obtained by means of supercritical water extraction was analyzed for its polyphenol profile, total phenolic content, radical scavenging, and ferric-reducing antioxidant activities (DPPH and FRAP), Fe2+-chelating capacity, and total iron. The most abundant polyphenol component observed in the dGCB extract was chlorogenic acid, and the alkaloid caffeine was also present. This extract showed antioxidant properties. Thereafter, five formulations of pork meat burgers with added NaCl (1%) were prepared; one without the antioxidant (negative control, C) and one with the use of a synthetic antioxidant (0.05% ascorbic acid = positive control, A), while the other three were supplemented with a different amount of dGCB extract (P15 = 0.15%; P30 = 0.30%; P60 = 0.60%). The addition of dGCB extract increased the antioxidant activity of the raw and cooked burgers and reduced the lipid oxidation of the cooked burgers (0.47, 0.21, and 0.20 vs. 1.28 and 0.55 mg MDA eq./Kg, for P15, P30, and P60 vs. C and A, respectively). No negative effects were observed on the meat's color parameters and its stability during refrigerated storage and after cooking, nor on sensory attributes (color and aroma) for the lowest concentration of coffee extract. The results obtained indicate that 0.15% dGCB extract is a promising alternative to commercial synthetic antioxidants to improve the quality of refrigerated pork burgers.
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In the last 20 years, an increased interest has been shown in the application of different types and combinations of enzymes to obtain phenolic extracts from grape pomace in order to maximize its valorization. Within this framework, the present study aims at improving the recovery of phenolic compounds from Merlot and Garganega pomace and at contributing to the scientific background of enzyme-assisted extraction. Five commercial cellulolytic enzymes were tested in different conditions. Phenolic compound extraction yields were analyzed via a Design of Experiments (DoE) methodology and a second extraction step with acetone was sequentially added. According to DoE, 2% w/w enzyme/substrate ratio was more effective than 1%, allowing a higher total phenol recovery, while the effect of incubation time (2 or 4 h) variation was more enzyme-dependent. Extracts were characterized via spectrophotometric and HPLC-DAD analyses. The results proved that enzymatic and acetone Merlot and Garganega pomace extracts were complex mixtures of compounds. The use of different cellulolytic enzymes led to different extract compositions, as demonstrated using PCA models. The enzyme effects were observed both in water enzymatic and in the subsequent acetone extracts, probably due to their specific grape cell wall degradation and leading to the recovery of different molecule arrays.
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Over the centuries, wild plants have constituted the main food ingredients and traditional medicine in rural communities. In the last decades, thousands of ethnobotanical studies have been conducted, with the aim of documenting the traditional knowledge on wild and cultivated plants both for food and therapeutic purposes. In the present work, 75 published papers related to Italian ethnobotanical knowledge on wild and cultivated plants traditionally used for medical purposes were analyzed and data on 1117 different species organized in the first dataset to target medicinal applications only. For each plant species, the Italian region of use, plant organs, mode of preparation, specific pathological group of application, citation index, and use index were listed. The different therapeutic applications were subdivided into nine main pathological groups according to the targeted human apparatus. Overall, the cited species with highest number of uses were related to the treatment of the digestive system and skin-ears-eyes-hair diseases, followed by diseases of the genito-urinary and respiratory systems. The 13 most relevant species were identified on the basis of their citation and use indexes. The present review on Italian medicinal flora aims to provide valuable information on wild and cultivated species, which are potential sources of plant-based therapeutic remedies, to preserve and reevaluate endangered traditional folk knowledge.
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One of the major challenges for the modern society, is the development of a sustainable economy also aiming at the valorization of agro-industrial by-products in conjunction with at a significant reduction of generated residues from farm to retail. In this context, the present study demonstrates a biotechnological approach to yield bioactive peptides from a protein fraction obtained as a by-product of the rice starch production. Enzymatic hydrolysis, with the commercial proteases Alcalase and Protamex, were optimized in bioreactor up to 2 L of volume. The two best digestates, selected with respect to peptide release and extract antioxidant capacity, were further fractionated (cut-offs of 10, 5, and 1 kDa) via cross-flow filtration. Amino acid composition indicated that most of the fractions showed positive nutritional characteristics, but a putative bitter taste. A fraction obtained with Alcalase enzyme (retentate 8 kDa) exerted anti-inflammatory potential, while the smaller molecular weight fractions (retentate 1-5 kDa and permeate < 1 kDa) were more active in tyrosinase inhibition. The latter were further sub-fractionated by size-exclusion chromatography. From the 15 most anti-tyrosinase sub-fractions, 365 peptide sequences were identified via liquid chromatography coupled with high resolution mass spectrometry. The present data support the possible exploitation of bioactive peptide from rice starch by-product as ingredients into food, nutraceutical, pharmaceutical, and cosmetic formulations.
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In the past, wild edible alimurgic plants became an important alternative food source when poverty, wars or drought made it difficult to access crops. These plants were considered rich in highly nutritional compounds and also frequently used as food-medicine given their health-promoting properties. With the aim of improving our knowledge on the content of beneficial or detrimental compounds in relation with past local dietary and curative traditions, 12 wild food plant species were collected from two study areas selected for their very different degree of industrialization, urbanization, and conservation of local past traditions among the population: the Bologna province (Northern Italy) and the Middle Agri Valley (Southern Italy). Protein, polyphenol flavonoid and biogenic amine (both free and conjugated) contents and antioxidant activity of raw and boiled wild food plant extracts, and of cooking water were analyzed by means of spectrophotometric and high-performance liquid chromatography methods. The results demonstrated that most of the phenolic compounds were released in the cooking water which also showed the highest antioxidant activity. Seventeen different phenolic compounds were identified, of which the health-related luteolin, luteolin-7-glucoside and rutin were the most abundant (e.g., S. pratensis L. and C. intybus L.). On the other hand, biogenic amines were absent or present at very low levels in cooking water of those very same species (e.g., S. pratensis L., T. officinalis Weber, C. vesicaria subsp. taraxacifolia and C. intybus L.) of which traditionally a decoction is used for therapeutic purposes. Free and conjugated spermidine and spermine were generally the most abundant biogenic amines, while none of the known detrimental monoamines (e.g., histamine) was detected. In conclusion, the present results seem to support past local popular traditions which indicated beneficial medical properties of some wild edible plant, as well as of their cooking water.
Assuntos
Etnobotânica , Compostos Fitoquímicos/química , Extratos Vegetais/química , Plantas Comestíveis/química , Antioxidantes , Cromatografia Líquida de Alta Pressão , Culinária , Flavonoides/química , Flavonoides/isolamento & purificação , Glucosídeos/química , Glucosídeos/isolamento & purificação , Humanos , Itália , Luteolina/química , Luteolina/isolamento & purificação , Fenóis/química , Fenóis/isolamento & purificação , Polifenóis/química , Polifenóis/isolamento & purificaçãoRESUMO
The food waste reduction through an efficient recovery of its valuable building molecules has become an important topic with a positive effect on the economy and the environment. In this work, the revalorization of slaughterhouse calf fleshing meat through its enzymatic hydrolysis is proposed. The proteolytic activity of 11 enzymes was initially screened and the four most efficient enzymes (papain, trypsin, pancreatin, and bromelain) were selected. The molecular profiling of the different protein/peptide fractions by the Linear Trap Quadrupole-OrbiTrap technique showed compositional differences due to the specificity of the enzymes' cleavage sites. In order to find a potential reuse of these hydrolysates, the analysis of antioxidant and, for the first time on fleshing meat hydrolysates, of anti-tyrosinase activities, was performed. Papain-digested samples were those showing the highest inhibition activity of tyrosinase enzyme (55.6%) as well as the highest antioxidant activity (3.52 g TEAC/L). In addition, the composition analysis of the lipid fraction was performed. The mono-unsaturated fatty acids resulted to be the most abundant lipid in all the samples with the exception of pancreatin-treated hydrolysates in which poly-unsaturated fatty acids were predominant. The present results seemed to support a possible valorization of isolated fractions from calf fleshing by-products, as food or feed ingredients, by the implementation of fraction isolation within the meat-processing pipeline.
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Mercury contamination in the environment has reached alarming levels. Due to its persistence and bioaccumulation, mercury is one of the most widespread toxic heavy metals found in air, water and food. Thus, it is mandatory to monitor mercury and its compounds, and the availability of sensitive and rapid biosensors is highly valuable. We developed a low-cost biosensor for orthogonal detection of mercury(II) integrating three different biorecognition principles on a three-leaf paper: i) a mercury-specific bioluminescent Escherichia coli bioreporter strain expressing NanoLuc luciferase as reporter protein, ii) a purified ß-galactosidase (ß-gal) enzyme which is irreversibly inhibited by mercury and other metal ions, and iii) an Aliivibrio fischeri bioluminescent strain which is used to quantitatively assess sample toxicity and correct the analytical signal accordingly. Both sensory elements and substrates, Furimazine for the bioluminescent reporter strain and chlorophenol red-ß-D-galactopyranoside for colorimetric detection of ß-gal, were integrated in the paper sensor to provide a stable all-in-one disposable cartridge which can be easily snapped into a smartphone with a clover-shaped 3D printed housing. This is the first integration of bioluminescence and colorimetric detection on a smartphone-paper sensor, providing a readout within 15 and 60 min for the colorimetric and bioluminescent detection respectively. The biosensor was applied to water samples spiked with different concentrations of mercury, interferents and toxic chemicals providing a limit of detection for Hg(II) at the ppb levels.
Assuntos
Técnicas Biossensoriais , Mercúrio , Aliivibrio fischeri , Colorimetria , SmartphoneRESUMO
Not all the coffee produced goes to the roasting stage, because non-compliant green coffee beans are usually discarded by roasters and the silverskin of the coffee is usually removed and discarded. In the present work, non-compliant green coffee beans and coffee silverskins were fully characterized from a chemical point of view. In addition, enzyme-assisted extraction was applied to recover a fraction rich in proteins and polyphenols, tested for antimicrobial, antityrosinase, and antioxidant activities. Non-compliant green coffee beans showed higher amounts of polyphenols, flavanols, flavonoids, and caffeine than coffee silverskins (which were richer in tannins). The enzymatic extraction of non-compliant coffee green beans produced extracts with a good protein content and with a consistent quantity of polyphenols. The extract showed antioxidant, antityrosinase, and antimicrobial activity, thus representing a promising strategy to recover defective green coffee beans. The antioxidant and antimicrobial activity of coffee silver skins is lower than that of non-compliant coffee green beans extracts, while the antityrosinase activity is comparable.
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Coffea , Antioxidantes , Fenóis , PolifenóisRESUMO
The paper aims at optimising and validating possible routes toward the full valorisation of grape agrowaste to produce bioactive molecules and new materials. Starting from Merlot red pomace, phenol complex mixtures were successfully extracted by using two different approaches. Extracts obtained by solvent-based (SE) technique contained up to 46.9 gGAeq/kgDW of total phenols. Depending on the used solvent, the prevalence of compounds belonging to different phenol families was achieved. Pressurized liquid extraction (PLE) gave higher total phenol yields (up to 79 gGAeq/kgDW) but a lower range of extracted compounds. All liquid extracts exerted strong antioxidant properties. Moreover, both SE and PLE extraction solid residues were directly exploited (between 5 and 20% w/w) to prepare biocomposite materials by direct mixing via an eco-friendly approach with PHBV polymer. The final composites showed mechanical characteristics similar to PHVB matrix. The use of pomace residues in biocomposites could therefore bring both to the reduction of the cost of the final material, as a lower amount of costly PHBV is used. The present research demonstrated the full valorisation of grape pomace, an agrowaste produced every year in large amounts and having a significant environmental impact.
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Wheat bran could be utilised as feedstock for innovative and sustainable biorefinery processes. Here, an enzymatic hydrolysis process for ferulic acid (FA) extraction was optimised step by step for total wheat bran (Tritello) and then also applied to the outer bran layer (Bran 1). Proteins, reducing sugars, total phenols and FA were quantified. The highest FA yields (0.82-1.05â¯g/kg bran) were obtained either by rehydrating the bran by autoclaving (Tritello) or by steam explosion (Bran 1) using a bran/water ratio of 1:20, followed by enzymatic pre-treatment with Alcalase and Termamyl, to remove protein and sugars, and a final enzymatic hydrolysis with Pentopan and feruloyl esterase to solubilise phenol. FA was recovered from the final digestate via solid phase extraction. A 40-fold scale-up was also performed and the release of compounds along all the process steps and at increasing incubation times was monitored. Results showed that FA was initially present at a minimum level while it was specifically released during the enzymatic treatment. In the final optimized process, the FA extraction yield was higher than that obtained with NaOH control hydrolysis while, in comparison with other FA enzymatic extraction methods, fewer process steps were required and no buffers, strong acid/alkali nor toxic compounds were used. Furthermore, the proposed process may be easily scaled-up, confirming the feasibility of wheat bran valorisation by biorefinery processes to obtain valuable compounds having several areas of potential industrial exploitation.
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Ácidos Cumáricos/isolamento & purificação , Fibras na Dieta/metabolismo , Subtilisinas/metabolismo , alfa-Amilases/metabolismo , Ácidos Cumáricos/química , Ácidos Cumáricos/metabolismo , HidróliseRESUMO
Agro-waste reduction and reuse are among the current main social challenges. In this perspective, the present research was aimed at the complete valorisation of Garganega grape pomace by recovering bioactive phenol extracts and by testing the solid fibre extract residues in composite formulation for packaging applications. The pomace was derived from white wine production, therefore, respect to red pomace, it was promptly removed from must after pressing, and its exploitation can be particularly interesting and valuable as still rich in active compounds. Phenol extracts were obtained both via solvent-based and pressurised liquid extractions and their phytochemical compositions were compared in terms of total amount of phenols, flavonoids, flavanols, anthocyanins, hydroxycinnamic acids, and reducing sugars. Antioxidant activity and detailed phenol profiles were also achieved. The highest phenol yield was obtained via solvent-based extraction with 75% acetone (v/v), solid/liquid ratio 1:5, 2h incubation at 50°C (77.9 gGAeq/kgDW). The fibrous solid residue of the extraction was characterized via thermogravimetric analysis and used for composite preparation by melt mixing with the renewable and biodegradable PHBV polymer through a green approach (solvent-less process). The composites resulted thermally stable at high temperatures, showing initial degradation processes only at temperatures higher than 250°C. Differential scanning calorimetry analyses were carried out to study melting and crystallization phenomena, while mechanical properties were investigated by tensile tests. The materials finally showed properties similar to those of the matrix. The bio-composites can be considered as an alternative to plain PHBV, since they are less expensive and eco-friendlier thanks to a reduced polymeric content, and they could represent a suitable way for full agro-waste exploitation.
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Antioxidantes/química , Tecnologia de Alimentos/métodos , Resíduos Industriais , Extratos Vegetais/química , Vitis/química , Gerenciamento de Resíduos/métodos , Flavonoides/química , Fenóis/química , VinhoRESUMO
The rice-starch processing industry produces large amounts of a protein-rich byproducts during the conversion of broken rice to powder and crystal starch. Given the poor protein solubility, this material is currently discarded or used as animal feed. To fully exploit rice's nutritional properties and reduce this waste, a biotechnological approach was adopted, inducing fermentation with selected microorganisms capable of converting the substrate into peptide fractions with health-related bioactivity. Lactic acid bacteria were preferred to other microorganisms for their safety, efficient proteolytic system, and adaptability to different environments. Peptide fractions with different molecular weight ranges were recovered from the fermented substrate by means of cross-flow membrane filtration. The fractions displayed in vitro antioxidant, antihypertensive, and anti-tyrosinase activities as well as cell-based anti-inflammatory and anti-aging effects. In the future, the peptide fractions isolated from this rice byproduct could be directly exploited as health-promoting functional foods, dietary supplements, and pharmaceutical preparations. The suggested biotechnological process harnessing microbial bioconversion may represent a potential solution for many different protein-containing substrates currently treated as byproducts (or worse, waste) by the food industry.
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Polyphenols, including stilbenes and flavonoids, are an essential part of human diet and constitute one of the most abundant and ubiquitous groups of plant secondary metabolites, and their level is inducible by stress, fungal attack or biotic and abiotic elicitors. Proteomic analysis of Vitis vinifera (L.) cultivar (cv.) Barbera grape cell suspensions, showed that the amount of 73 proteins consistently changed in 50 microg/mL chitosan-treated samples compared with controls, or between the two controls, of which 56 were identified by MS analyses. In particular, de-novo synthesis and/or accumulation of stilbene synthase proteins were promoted by chitosan which also stimulated trans-resveratrol endogenous accumulation and decreased its release into the culture medium. No influence was shown on cis-resveratrol. There was no effect on the accumulation of total resveratrol mono-glucosides (trans- and cis-piceid and trans- and cis-resveratroloside). Throughout the observation period the upregulation of phenylalanine ammonia lyase, chalcone synthase, chalcone-flavanone isomerase (CHI) transcript expression levels well correlated with CHI protein amount and with the accumulation of anthocyanins. Chitosan treatment strongly increased the expression of eleven proteins of the pathogenesis related protein-10 family, as well as their mRNA levels.
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Quitosana/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Vitis/efeitos dos fármacos , Vitis/metabolismo , Aciltransferases/metabolismo , Antocianinas/metabolismo , Northern Blotting , Células Cultivadas , Eletroforese em Gel Bidimensional , Flavonoides/metabolismo , Fenóis/metabolismo , Polifenóis , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estilbenos/metabolismoRESUMO
The present work reports the enzymatic valorisation of the protein fraction of scotta, a dairy by-product representing the exhausted liquid residue of ricotta production. Scotta was subjected to ultra-filtration with membrane cut-offs from 500 to 4 kDa and the obtained protein-enriched fractions were used for the optimization of enzyme-based digestions aimed at producing potentially bioactive peptides. Nine different commercial proteases were tested and the best digestion conditions were selected based on protein yield, fraction bioactivity and foreseen scale up processing costs. Scale up of the 3% Pancreatin or 5% Papain processes was performed up to 2 L (37°C or 60°C respectively, 1 h incubation), and the digestion efficiency increased with the reaction volume as well as antioxidant activity (up to 60 gBSA eq/L and to 1.7 gAA eq/L). Retentate 1 digested fractions also showed, for the first time in dairy-based peptides, anti-tyrosinase activity, up to 0.14 gKA eq/L. Digested proteins were sub-fractionated by means of physical membrane separations and 30-10 kDa fraction from Papain treatment showed the highest antioxidant and anti-tyrosinase activities. The peptide sequence of the most bioactive fractions was achieved.
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Antioxidantes , Queijo , Monofenol Mono-Oxigenase/antagonistas & inibidores , Peptídeo Hidrolases/metabolismo , Peptídeos/análise , Sequência de Aminoácidos , Animais , Pancreatina/metabolismo , Papaína/metabolismo , Peptídeos/farmacologia , Proteínas/metabolismo , UltrafiltraçãoRESUMO
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