Structure of the human NK cell NKR-P1:LLT1 receptor:ligand complex reveals clustering in the immune synapse.

Signaling by the human C-type lectin-like receptor, natural killer (NK) cell inhibitory receptor NKR-P1, has a critical role in many immune-related diseases and cancer. C-type lectin-like receptors have weak affinities to their ligands; therefore, setting up a comprehensive model of NKR-P1-LLT1 interactions that considers the natural state of the receptor on the cell surface is necessary to understand its functions. Here we report the crystal structures of the NKR-P1 and NKR-P1:LLT1 complexes, which provides evidence that NKR-P1 forms homodimers in an unexpected arrangement to enable LLT1 binding in two modes, bridging two LLT1 molecules. These interaction clusters are suggestive of an inhibitory immune synapse. By observing the formation of these clusters in solution using SEC-SAXS analysis, by dSTORM super-resolution microscopy on the cell surface, and by following their role in receptor signaling with freshly isolated NK cells, we show that only the ligation of both LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. In summary, our findings collectively support a model of NKR-P1:LLT1 clustering, which allows the interacting proteins to overcome weak ligand-receptor affinity and to trigger signal transduction upon cellular contact in the immune synapse.

Protein purification strategies must consider downstream applications and individual biological characteristics.

BACKGROUND: Proteins are used as reagents in a broad range of scientific fields. The reliability and reproducibility of experimental data will largely depend on the quality of the (recombinant) proteins and, consequently, these should undergo thorough structural and functional controls. Depending on the downstream application and the biochemical characteristics of the protein, different sets of specific features will need to be checked. RESULTS: A number of examples, representative of recurrent issues and previously published strategies, has been reported that illustrate real cases of recombinant protein production in which careful strategy design at the start of the project combined with quality controls throughout the production process was imperative to obtain high-quality samples compatible with the planned downstream applications. Some proteins possess intrinsic properties (e.g., prone to aggregation, rich in cysteines, or a high affinity for nucleic acids) that require certain precautions during the expression and purification process. For other proteins, the downstream application might demand specific conditions, such as for proteins intended for animal use that need to be endotoxin-free. CONCLUSIONS: This review has been designed to act as a practical reference list for researchers who wish to produce and evaluate recombinant proteins with certain specific requirements or that need particular care for their preparation and storage.

Natural killer cell-based strategies for immunotherapy of cancer.

Natural killer (NK) cells are a family of lymphocytes with a natural ability to kill infected, harmed, or malignantly transformed cells. As these cells are part of the innate immunity, the cytotoxic mechanisms are activated upon recognizing specific patterns without prior antigen sensitization. This recognition is crucial for NK cell function in the maintenance of homeostasis and immunosurveillance. NK cells not only act directly toward malignant cells but also participate in the complex immune response by producing cytokines or cross-talk with other immune cells. Cancer may be seen as a break of all immune defenses when malignant cells escape the immunity and invade surrounding tissues creating a microenvironment supporting tumor progression. This process may be reverted by intervening immune response with immunotherapy, which may restore immune recognition. NK cells are important effector cells for immunotherapy. They may be used for adoptive cell transfer, genetically modified with chimeric antigen receptors, or triggered with appropriate antibodies and other antibody-fragment-based recombinant therapeutic proteins tailored specifically for NK cell engagement. NK cell receptors, responsible for target recognition and activation of cytotoxic response, could also be targeted in immunotherapy, for example, by various bi-, tri-, or multi-specific fusion proteins designed to bridge the gap between tumor markers present on target cells and activation receptors expressed on NK cells. However, this kind of immunoactive therapeutics may be developed only with a deep functional and structural knowledge of NK cell receptor: ligand interactions. This review describes the recent developments in the fascinating protein-engineering field of NK cell immunotherapeutics.

Tumor Marker B7-H6 Bound to the Coiled Coil Peptide-Polymer Conjugate Enables Targeted Therapy by Activating Human Natural Killer Cells.

Targeted cancer immunotherapy is a promising tool for restoring immune surveillance and eradicating cancer cells. Hydrophilic polymers modified with coiled coil peptide tags can be used as universal carriers designed for cell-specific delivery of such biologically active proteins. Here, we describe the preparation of pHPMA-based copolymer conjugated with immunologically active protein B7-H6 via complementary coiled coil VAALEKE (peptide E) and VAALKEK (peptide K) sequences. Receptor B7-H6 was described as a binding partner of NKp30, and its expression has been proven for various tumor cell lines. The binding of B7-H6 to NKp30 activates NK cells and results in Fas ligand or granzyme-mediated apoptosis of target tumor cells. In this work, we optimized the expression of coiled coil tagged B7-H6, its ability to bind activating receptor NKp30 has been confirmed by isothermal titration calorimetry, and the binding stoichiometry of prepared chimeric biopolymer has been characterized by analytical ultracentrifugation. Furthermore, this coiled coil B7-H6-loaded polymer conjugate activates NK cells in vitro and, in combination with coiled coil scFv, enables their targeting towards a model tumor cell line. Prepared chimeric biopolymer represents a promising precursor for targeted cancer immunotherapy by activating the cytotoxic activity of natural killer cells.

Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study.

Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.

Natural Killer Cell Activation Receptor NKp30 Oligomerization Depends on Its N-Glycosylation.

NKp30 is one of the main human natural killer (NK) cell activating receptors used in directed immunotherapy. The oligomerization of the NKp30 ligand binding domain depends on the length of the C-terminal stalk region, but our structural knowledge of NKp30 oligomerization and its role in signal transduction remains limited. Moreover, ligand binding of NKp30 is affected by the presence and type of N-glycosylation. In this study, we assessed whether NKp30 oligomerization depends on its N-glycosylation. Our results show that NKp30 forms oligomers when expressed in HEK293S GnTI- cell lines with simple N-glycans. However, NKp30 was detected only as monomers after enzymatic deglycosylation. Furthermore, we characterized the interaction between NKp30 and its best-studied cognate ligand, B7-H6, with respect to glycosylation and oligomerization, and we solved the crystal structure of this complex with glycosylated NKp30, revealing a new glycosylation-induced mode of NKp30 dimerization. Overall, this study provides new insights into the structural basis of NKp30 oligomerization and explains how the stalk region and glycosylation of NKp30 affect its ligand affinity. This furthers our understanding of the molecular mechanisms involved in NK cell activation, which is crucial for the successful design of novel NK cell-based targeted immunotherapeutics.

In vitro enzyme activity and in vivo healing activity of the protein extract from pineapple peel

The pineapple is a fruit that has wide acceptance worldwide both in natural form, as industrialized. Your peel is a residue generated by food industries and from this residue can obtain a protein extract which is a good source of bromelain. This study aimed to obtain a protein extract from pineapple peel, evaluate its enzyme activity and its healing properties in skin lesions in rats. Seven animal groups were used: control, treated with 5% of protein extract, 10% of protein extract and pure protein extract; 5% of commercial bromelain, 10% of commercial bromelain and pure commercial bromelain. The animals were subjected to a tissue incision and treated for 21 days. Proteolytic and specific activities of the protein extract were 1.30 U mg-1 and 45 x 10-3 U μg-1 and, for commercial bromelain, 1.04 U mg-1 and 6 x 10-3 U μg-1, respectively. In the histology of the lesion, there was no significant difference between the control and treated groups; however, macroscopically, the prepared topical formulations assisted in the recovery of skin lesions, providing a significant reduction in their width, in the groups treated with pure protein extract, 5 and 10% commercial bromelain, and pure bromelain.

Mechanism of firmness loss in guava cv. Pedro Sato during ripening at room temperature

Abstract Guava (Psidium guajava L.) is fruit with very short shelf life due to associated with the loss of firmness in the pulp and information on the activity of enzymes that degrade pectic substances, as well as the amount of pectin, is very contradictory and not clearly defined. This work showed that the firmness of the fruit decreased sharply on the first four day­s of ripening. The identified phenolic compounds had their content increased with fruit maturation. The addition of the enzymes cellulase and hemicellulase in the pectin extraction in fruits of Psidium guajava revealed higher contents of this polyssacaride that the ones reported in literature, highlighting pectin as the responsible for the firmness of these fruits at more adequate contents. β-D-glucosidase was identified as one of the responsible enzymes for the maturation of P. guajava fruits. Thus, studies about possible inhibitory effects of this enzyme in P. guajava fruits may reveal an important tool to reduce pectin release and early maturation of these fruits.

Lychee (Litchi chinensis Sonn) peel flour: effects on hepatoprotection and dyslipidemia induced by a hypercholesterolemic diet

ABSTRACT Dyslipidemias are associated with the incidence of cardiovascular diseases, obesity, diabetes, hypertension and hepatic steatosis, being the cause of morbidity and mortality. This study investigated the effects of lychee peel flour (PF) on serum levels of total cholesterol (TC), low-density lipoprotein (LDL-c), triacylglycerols (TAG) and various parameters related to obesity, in rats fed a hypercholesterolemic diet. Therefore, 20 male rats were used. In the first 21 days, the animals were fed a hypercholesterolemic diet, except for control group. In the following 21 days, their diets were modified, and they received a standard diet (Control); hypercholesterolemic (Hyper); hypercholesterolemic + 5% PF (PF5) and hypercholesterolemic + 10% PF (PF10). The results revealed that PF intake attenuated weight gain, reduced body mass index, glucose and the levels of TAG, TC, LDL-c, hepatic enzymes and leptin, besides the percentage of hepatic lipids, liver lipid peroxidation and frequency of severe steatosis. Histological studies of the aorta did not show the formation of the atheromatous plaque. These results reinforce its potential to reduce the risk of diseases associated with obesity.

Lychee (Litchi chinensis Sonn.) peel flour: effects on hepatoprotection and dyslipidemia induced by a hypercholesterolemic diet.

Dyslipidemias are associated with the incidence of cardiovascular diseases, obesity, diabetes, hypertension and hepatic steatosis, being the cause of morbidity and mortality. This study investigated the effects of lychee peel flour (PF) on serum levels of total cholesterol (TC), low-density lipoprotein (LDL-c), triacylglycerols (TAG) and various parameters related to obesity, in rats fed a hypercholesterolemic diet. Therefore, 20 male rats were used. In the first 21 days, the animals were fed a hypercholesterolemic diet, except for control group. In the following 21 days, their diets were modified, and they received a standard diet (Control); hypercholesterolemic (Hyper); hypercholesterolemic + 5% PF (PF5) and hypercholesterolemic + 10% PF (PF10). The results revealed that PF intake attenuated weight gain, reduced body mass index, glucose and the levels of TAG, TC, LDL-c, hepatic enzymes and leptin, besides the percentage of hepatic lipids, liver lipid peroxidation and frequency of severe steatosis. Histological studies of the aorta did not show the formation of the atheromatous plaque. These results reinforce its potential to reduce the risk of diseases associated with obesity.

Composição química e fitoquímica das farinhas da casca e da semente de lichias (Litchi chinensis Sonn) cultivar 'Bengal' / Chemical and phytochemical composition of flours from skin and seeds of 'Bengal' lychee (Litchi chinensis Sonn)

Este estudo teve como objetivo determinar a composição química e fitoquímica das farinhas da casca e da semente de lichias (Litchi chinensis Sonn), família Sapindaceae, da cultivar 'Bengal'. A composição centesimal, o valor energético total, sólidos solúveis, pH, acidez e os teores de compostos fenólicos, antocianinas, flavonoides, minerais, açúcares, vitamina C e a triagem fitoquímica foram determinados. A farinha da casca de lichia apresentou os maiores teores de flavonoides, vitamina C, compostos fenólicos, antocianinas, lipídios, proteínas, cinzas e fibras, enquanto a farinha da semente se destacou pelos teores de potássio, enxofre, cobre e zinco. Ambas as farinhas apresentaram resultado positivo para açúcares redutores, proteína e aminoácidos, taninos, catequinas, flavonoides, depsídeo e depsidonas. Somente a farinha da casca apresentou resultado positivo para alcaloides. As farinhas dos subprodutos da lichia apresentaram-se como fontes promissoras de nutriente e substâncias bioativas, no entanto suas propriedades biológicas devem ser estudadas.

This study had the objective to determine the chemical and phytochemical composition of flours from skin and seeds of 'Bengal' lychees (Litchi chinensis Sonn), Sapindaceae family. Proximate composition, total energy value, soluble solids, pH, acidity and levels of phenolic compounds, anthocyanins, flavonoids, minerals, sugars, vitamin C and the phytochemical screening were determined. Lychee skin flour presented the highest levels of flavonoids, vitamin C, phenolic compounds, anthocyanins, lipids, proteins, ash and fiber, while the seed flour stood out for the contents of potassium, sulfur, copper, and zinc. Both flours were positive for reducing sugars, protein and amino acids, tannins, catechins, flavonoids, depsides and depsidones. Only the skin flour showed a positive result for alkaloids. The flours made from lychee by-products were promising sources of nutrients and bioactive substances; however, their biological properties need to be further studied.

Embalagem a vácuo: efeito no escurecimento e endurecimento do feijão durante o armazenamento / Vacuum package: its effect on the hardening and darkening of the beans during storage

A influência do uso de embalagem a vácuo no controle do escurecimento e endurecimento do tegumento foi avaliada em feijões da cv. 'Pérola'. Os feijões após secos foram submetidos a diferentes embalagens: polietileno seladas a vácuo (espessura de 80µm), polietileno seladas sem vácuo (espessura de 80µm) e embalagem comercial (espessura de 20µm), armazenados por oito meses em condições ambiente. Foram realizadas análises de umidade, tempo de cozimento, cor, atividade enzimática e compostos fenólicos, logo após a secagem e em intervalos de dois meses até o oitavo mês. O tempo de cozimento foi maior para os grãos acondicionados em embalagem comercial do que nas outras embalagens, ao final do armazenamento. Os grãos embalados em sacos de polietileno selados a vácuo apresentaram menor queda nos valores de L*. As amostras da embalagem selada a vácuo apresentaram atividade da Peroxidase e Polifenoloxidase inferior aos grãos da embalagem comercial. O teor de fenólicos totais não apresentou diferença significativa. O uso da embalagem selada a vácuo foi eficiente em retardar o aumento no tempo de cozimento, o escurecimento do tegumento, a atividade das enzimas Polifenoloxidase e Peroxidase da cultivar 'Pérola', durante o armazenamento por oito meses em condição ambiente.

The influence of using vacuum packaging to control browning and hardening of the seed coat was evaluated in beans cv. 'Pérola'. The beans after dried, were subjected to different packaging: vacuum sealed polyethylene (thickness: 80μm), no vacuum sealed polyethylene (thickness: 80μm) and commercial packaging (thickness: 20μm), stored for eight months under ambient conditions. Analyses of moisture, cooking time, color, enzymatic activity and phenolic compounds, shortly after drying and at intervals of two months until the eighth month. Cooking time was longer for the beans packed in commercial packaging, than in the other containers at the end of storage. The grains packed in polyethylene bags sealed under vacuum showed less decline in the values of L *. The samples will vacuum sealed package showed peroxidase activity and polyphenoloxidase below the grain of commercial packaging. The total phenolic content was not significantly different. The use of vacuum sealed package will was effective in slowing the increase in cooking time, the darkening of the integument, the activity of the enzymes polyphenoloxidase and peroxidase of bean cv. 'Perola' during storage for eight months at ambient condition.

Evaluation of the protective effect of guava fruits and leaves on oxidative stress / Avaliação do efeito protetor de folhas e frutos da goiabeira no estresse oxidativo

Oxidative stress is an imbalance between reactive oxygen species and antioxidant capacity of action. Thus, research, alternative to mitigate the damaging effects of oxidative stress, improving the body´s antioxidant capacity, prevented the disease and its complicatons. The leaves and fruits of guava are rich in the antioxidants. This work aimed to study the effect of flour and ethanolic/acetone extracts leaves and fruits of guava on lipid oxidation in rats with hypercholesterolemic. The flour and extracts decreased leves of triglycerides and non ­ HDL cholesterol and increased in the HDL. Cholesterol levels decreased only leaves and fruits. The activity of GPx decreased in samples serum in all treatments and the SOD only the extracts were effective. There was no difference in levels the MDA in relation hypercholesterolemic control. For the samples of liver, there was no difference in activity SOD. In relation of activity GPx, only the flour was effective. There were differences in levels the MDA of the hypercholesterolemic animals treated with flour and extracts with the animals of the hypercholesterolemic control.

O estresse oxidativo é um desequilíbrio entre as espécies reativas de oxigênio e capacidade antioxidante de ação. Dessa forma, pesquisas buscam alternativas para amenizar os efeitos prejudiciais do estresse oxidativo, melhorando a capacidade antioxidante do organismo, prevenido as enfermidades e suas complicações. As folhas e frutos de goiaba são ricos em antioxidantes. Este trabalho teve como objetivo estudar o efeito da farinha e folhas etanólicos/acetona extratos e frutos de goiabeira na oxidação lipídica em ratos com hipercolesterolemia. A farinha e extratos de folhas diminuiu de triglicérides e colesterol não - HDL e aumento do HDL. Os níveis de colesterol diminuiram apenas folhas e frutas. A actividade da GPx diminuiu em amostras de soro, em todos os tratamentos e a SOD apenas os extratos foram eficazes. Não houve nenhuma diferença nos níveis de MDA no controle da hipercolesterolemia relação. Para as amostras de fígado, não houve diferença na atividade da SOD. Na relação de GPx atividade, apenas a farinha foi eficaz. Houve diferenças nos níveis do MDA dos animais hipercolesterolêmicos tratados com farinha e extratos com os animais do controle hipercolesterolêmica.

Quantificação de compostos fenólicos e ácido ascórbico em frutos e polpas congeladas de acerola, caju, goiaba e morango / Quantification of phenolic compounds and ascorbic acid in fruits and frozen pulp of acerola, cashew, strawberry and guava

O crescente interesse da população pela promoção da saúde vem despertando a atenção dos consumidores por alimentos ricos em antioxidantes, combinado com a praticidade do consumo de sucos de frutos processados. O presente trabalho objetivou quantificar e avaliar a capacidade antioxidante de compostos fenólicos e ácido ascórbico em frutas in natura e polpas congeladas. As frutas in natura e polpas congeladas obtidas na região de Lavras - MG foram submetidas à quantificação de compostos fenólicos e ácido ascórbico e os respectivos extratos acetônico-etanólico e acetônico-metanólico. Foram determinadas suas atividades antioxidantes pelos métodos radicalares DPPH e ABTS. O extrato acetônico-metanólico foi mais efetivo para extrair os compostos antioxidantes das amostras. Apenas as polpas congeladas de acerola apresentaram uma redução do teor de compostos fenólicos em relação ao fruto. Para o teor de ácido ascórbico, houve uma redução nas polpas congeladas de acerola e goiaba. As amostras de acerola apresentaram os maiores teores de compostos fenólicos, ácido ascórbico e atividade antioxidante em relação aos demais frutos analisados.

The growing public interest in health- promotion, has called the attention of consumers for foods rich in antioxidants, combined with the practicality of the consumption of processed fruit juices. Therefore, this study aimed to quantify and evaluate the antioxidant capacity of phenolic compounds and ascorbic acid in fruits "in natura" and frozen pulps. The fruits "in natura" and frozen pulps from region of Lavras-MG, underwent quantification of phenolic compounds, ascorbic acid and the corresponding extracts acetonic-ethanolic and acetonic-methanolic. Their antioxidant activities were evaluated by DPPH and ABTS radical methods. The extract acetonic-methanolic was more effective to extract the antioxidant compounds from samples. Only frozen acerola pulps showed lower content of phenolic compounds in relation to fruit. For ascorbic acid, there was a reduction in the frozen pulp of acerola and guava. Acerola samples showed higher content of phenolic compounds, ascorbic acid and antioxidant activity compared to other fruits analyzed.

Caracterização física e química das frações do fruto atemoia Gefner / Physical and chemical characterization of fractions of fruit atemoya Gefner

A atemoia é um fruto híbrido derivado do cruzamento entre a fruta-do-conde, mais conhecida como ata (Annona squamosa L.), com a cherimoia (Annona cherimola Mill.). Este trabalho foi realizado com o objetivo de caracterizar os constituintes químicos das frações casca, polpa e semente de atemoia Gefner. Os frutos adquiridos foram selecionados, pesados e medidos. Foram separados em casca, polpa, semente e eixo floral, que também foram pesados para determinar suas proporções. Para o restante dos frutos, foram separadas as frações em sete repetições de 13 frutos. Em seguida, as frações casca, polpa e sementes foram liofilizadas e armazenadas em freezer. Foi determinada a proporção das frações do fruto, a composição centesimal, o ácido ascórbico, os açúcares totais, os minerais e alguns compostos bioativos. A polpa representou cerca de 60% do peso do fruto, enquanto a casca 28,13% e as sementes 8,34%. O fruto apresentou, em média, 56 sementes, com diâmetro longitudinal de 10,79cm e diâmetro transversal de 26,64cm. Os maiores teores de proteína bruta, extrato etéreo e fibra alimentar foram encontrados nas sementes e casca. A casca se destacou nos teores de cinzas. A polpa apresentou os maiores teores de ácido ascórbico e açúcares totais em relação às outras frações. A ordem da composição de macronutrientes na casca, na polpa e nas sementes da atemoia foi K>P>Ca>Mg; para os micronutrientes na casca e nas sementes, foi Fe>Zn>Cu>Mn>S e, na polpa, foi Fe>Zn>Mn>Cu>S. A casca apresentou os níveis mais altos de inibidores de tripsina e atividade hemaglutinante. Os teores de compostos fenólicos foram relativamente baixos no fruto.

The atemoya is a hybrid fruit derived from the cross between the sugar apple (fruta-do-conde), better known as ata (Annona squamosa L.) with the cherimoya (Annona cherimola Mill.). The aim of this research was to characterize the chemical constituents of the fractions skin, pulp and seed of the hybrid fruit atemoya, variety Gefner. The fruits purchased and selected were weighed and measured. They were separated into skin, pulp, seed and floral axis which were also weighted to determine their proportions. For the rest of the fruits, the fractions were separated into 7 replicates of 13 fruits. Then, the fractions skin, pulp and seeds were freeze-dried and stored in freezer. The proportion of the fruit fractions, the centesimal composition, ascorbic acid, total sugars, minerals and some bioactive compounds were determined. The pulp represented about 60% of the fruit weight, while the skin 28.13% and the seeds 8.34%. The fruit presented on the average 56 seeds, with longitudinal diameter of 10.79 cm and transversal diameter of 26.64 cm. The largest contents of crude protein, ether extract and dietary fiber were found in both seeds and skin. The skin stood out in the ash contents. The pulp showed the highest contents of ascorbic acid and total sugars in relation to the other fractions. The order of the composition of macronutrients in the skin, pulp and seeds of the atemoya was K>P>Ca>Mg; for the skin and seed micronutrients were Fe>Zn>Cu>Mn>S and in the pulp were Fe>Zn>Mn>Cu>S. The skin presented the highest levels of trypsin inhibitors and hemagglutinating activity. The phenolic compounds were relatively low in the fruit.

Activity of fungal phytases stored in two ways in response to the period of storage at room temperature / Atividade de fitases fúngicas armazenadas de duas maneiras em resposta ao período de armazenamento à temperatura ambiente

Two distinct experiments were conducted simultaneously with phytases of Aspergillus oryzae and A. niger for determining enzyme activity in response to storage period (up to 180 days at room temperature - RT). In the first experiment, enzymes were stored as they were acquired (pure form) and the activity was measured periodically during 180 days of storage at RT. In the second experiment, the phytases were incorporated to a supplement containing vitamins, minerals, and amino acids; and then this supplement was stored at RT up to 180 days, so that every 30 days of storage was collected one aliquot from each replicate for determining enzymatic activity. In conclusion, the phytase activity is affected by storage duration. To ensure 80 percent of the initial activity, the phytases of A. oryzae and A. niger can be stored in the pure forms for up to 53 and 135 days at RT, respectively. However, if the phytases of A. oryzae and A. niger are incorporated to a supplement containing vitamins, minerals, and amino acids then the storage period at RT should not exceed 67 and 77 days, respectively.

Dois experimentos distintos foram realizados simultaneamente com fitases de Aspergillus oryzae e A. niger para a determinação da atividade enzimática em resposta ao período de armazenamento (por até 180 dias em temperatura ambiente - TA). No primeiro experimento, as enzimas foram armazenadas como adquiridas (forma pura) e a atividade foi determinada periodicamente durante 180 dias de armazenamento em TA. No segundo experimento, as fitases foram incorporadas a um suplemento contendo vitaminas, minerais e aminoácidos. Então este suplemento foi armazenado em TA por até 180 dias, de modo que, a cada 30 dias de armazenamento, uma alíquota de cada repetição foi coletada para a determinação da atividade enzimática. Conclui-se que a atividade da fitase é afetada pela duração do armazenamento. Para assegurar 80 por cento da atividade inicial, as fitases de A. oryzae e A. niger podem ser armazenadas nas formas pura por até 53 e 135 dias em TA, respectivamente. Entretanto, se as fitases de A. oryzae e A. niger são incorporadas a um suplemento contendo vitaminas, minerais e aminoácidos, o período de armazenamento em TA não deve exceder 67 e 77 dias, respectivamente.

Estudo dos métodos de extração de carotenóides em cenoura por fluido supercrítico (efs) e convencional / A study of the methods of carotenoid extraction in carrots using supercritical fluid extraction (sfe) and conventional methods

A cenoura (Daucus carota L. ), planta da família das umbelíferas, produz uma raiz aromática e comestível, sendo uma das hortaliças mais cultivadas no Brasil. Representa a principal fonte de origem vegetal em carotenóides pró-vitamínicos A, especialmente o á e o β-caroteno, sendo, também, uma grande fonte de fibra dietética, antioxidantes e minerais. Conduziu-se este trabalho, com o objetivo de avaliar dois processos de extração de β-caroteno, fluído super crítico (EFS) e o convencional e analisar a composição centesimal da cenoura in natura. O teor de β-caroteno obtido pela extração por EFS e por convencional foi de 2.457 e 2.455 µg/100g, respectivamente. Os valores médios encontrados para a matéria-seca da cenoura foram de: matéria-seca = 8,9 por cento; extrato etéreo = 0,29 por cento; cinzas = 8,11 por cento; fibra bruta = 14,57 por cento, proteína bruta = 6,4 por cento, extrato não nitrogenado = 6,3 por cento e valor calórico = 27,7kcal. Conclui-se que a extração de carotenóides em cenoura pelo fluido supercritico é uma técnica de separação viável, pois este fluido é inerte, não deixa resíduo final e não gera resíduo ambiental. Pelos teores encontrados conclui-se também que as cenouras são boas fontes de fibras, apresentam alto teor de umidade e baixo teor de gorduras, cinzas e valor calórico.

The carrot (Daucus carota L.) is a plant of the Umbelliferae family. It produces an aromatic and edible root, and is one of the most widely cultivated vegetable in Brazil. It represents the main source of plant-origin, pro-vitamin A carotenoids, especially α and β-carotene, and is also a great source of dietary fiber, antioxidants and minerals. This study aimed to evaluate two methods of extraction of β-carotene, supercritical fluid (SFE) and conventional, and to examine the proximate composition of the carrot in nature. The contents of β-carotene obtained by SFE and by conventional extraction were 2,457 and 2,455 µg/100g, respectively. The average values found for the dry-matter of the carrot were: dry matter = 8.9 percent; ether extract = 0.29 percent; ashes = 8.11 percent, crude fiber = 14.57 percent, crude protein = 6,4 percent, non- nitrogen extract = 6.3 percent and caloric value = 27.7 kcal. It is concluded that the extraction of carotenoids using the supercritical fluid is a viable separation technique since this fluid is inert and does not generate waste or final environmental waste. It can be concluded from the levels found that carrots are good sources of fiber, present high moisture content and low fat, ash and calorific value.

Embalagens e recobrimento em lichias (Litchi chinensis Sonn.) armazenadas sob condições não controladas / Packing and covering in lychee (Litchi chinensis Sonn.) stored under uncontrolled conditions

A lichia (Litchi chinensis Sonn.) é um fruto subtropical de alto potencial comercial devido ao seu sabor levemente acidificado, excelente aroma, alto valor nutritivo e atrativa cor vermelha da casca. Ainda no campo, a cor da casca altera-se facilmente, tornando-se escurecida em resposta a estresses (alterações da umidade relativa e ataque de patógenos). Após colhida, a casca da lichia perde rapidamente sua cor vermelha. O escurecimento da casca tem sido atribuído a rápida degradação da antocianina, assim como: à rápida perda de umidade pelo fruto e à atividade de enzimas oxidativas como polifenoloxidases e peroxidases. Dada essa alta perecibilidade, o controle do escurecimento é fundamental para o aumento na vida útil pós-colheita, visando ao mercado interno e à exportação de frutas. Avaliou-se, neste trabalho, o comportamento pós-colheita de lichias da cv. Bengal quanto ao escurecimento da casca e às modificações relacionadas com a qualidade da polpa de lichias submetidas a diferentes embalagens: bandeja recoberta com filme de polietileno de baixa densidade com e sem perfurações e frutos recobertos com película de fécula de mandioca (3 por cento). Os frutos foram mantidos em temperatura ambiente (25,1ºC±1,5ºC; 69,3 por cento±8,62 por centoUR) e armazenados por seis dias. Foram realizadas análises físicas, químicas e bioquímicas no dia 0 e a cada 2 dias, até osexto dia de armazenamento. A embalagem recoberta com filme de polietileno perfurado apresentou-se eficiente na redução da perda de massa, escurecimento da casca e na manutenção do teor de antocianina da casca de lichias durante seis dias de armazenamento, em temperatura ambiente.

Lychee is a subtropical fruit of high commercial value, due to its slightly acid taste, excellent aroma, high nutritive value and attractive deep bright red color of its peel. Also in the field, skin color changes easily, becoming dark in response to stress (changes in relative humidity and the incidence of pathogens). Harvested lychee fruit rapidly loses its bright red skin colour. Peel browning of harvested lychee has largely been attributed to the rapid degradation of the anthocyanin pigments as well as the rapid loss of moisture from the fruit and the activity of oxidative enzymes such polyphenoloxidase and peroxidases. Due to it high perishability, browning control is a fundamental process to the increase of post-harvest useful life aiming the home market and fruit exportation. This study evaluated the postharvest behavior of Litchi cv. Bengal fruits regarding pericarp browning and changes on lychee fruits pulp quality when different packing is applied: plastic trays covered with parafilm or solely parafilm, and fruits covered with cassava starch film (3 percent). Lychee fruits were stored for six days at room temperature (25.1ºC±1.5; 69.3 percentUR). Physical, chemical and biochemical analyses were performed on day 0 and on every two days till the end of the storage period. The package covered with perforated polyethylene film was efficient in reducing weight loss, skin browning and maintenance of the anthocyanin content of the bark of leeches for six days of storage at room temperature.