Methoxypyrazine removal in grape juice: Development of a removal system using odorant and pheromone binding proteins coupled with bentonite fining

Multicoloured Asian Lady Beetles (MALB) and 7-spot Lady Beetles that infect vineyards can secrete alkyl-methoxypyrazines when they are processed with the grapes, resulting in wines containing a taint. The main methoxypyrazine associated with this taint is 3-isopropyl-2-methoxypyrazine (IPMP). The wines are described as having aroma and flavours of peanut butter, peanut shells, asparagus and earthy which collectively, have become known as “ladybug taint”. To date, there are no known fining agents used commercially added to juice or wine that are effective in removing this taint. The goal of this project was to use previously identified proteins with an ability to bind to methoxypyrazines at low pH, and subsequently develop a binding assay to test the ability of these proteins to bind to and remove methoxypyrazines from grape juice. The piglet odorant binding protein (plOBP) and mouse major urinary protein (mMUP) were identified, cloned and expressed in the Pichia pastoris expression system. Protein expression was induced using methanol and the proteins were subsequently purified from the induction media using anion exchange chromatography. The purified proteins were freeze-dried and rehydrated prior to use in the methoxypyrazine removal assay. The expression and purification system resulted in yields of approximately 78% of purified plOBP and 62% of purified mMUP from expression to rehydration. Purified protein values were 87 mg of purified plOPB per litre of induction media and 19 mg of purified mMUP per litre of induction medium. In order to test the ability of the protein to bind to the MPs, an MP removal assay was developed. In the assay, the purified protein is incubated with either IPMP or 3-isobutyl-2-methoxypyrazine (IBMP) for two hours in either buffer or grape juice. Bentonite is then used to capture the protein-MP complex and the bentonite-protein-MP complex is then removed from solution by filtration. Residual MP is measured in solution following the MP removal assay and compared to that in the starting solution by Gas Chromatography Mass Spectrometry (GC/MS). GC/MS results indicated that the mMUP was capable of removing IBMP and IPMP from 300 ng/L in buffer pH 4.0, buffer pH 3.5 and Riesling Juice pH 3.5 down to the limit of quantification of the instrument, which is 6ng/L and 2ng/L for IBMP and IPMP, respectively. The results for the plOBP showed that although it could remove some IBMP, it was only approximately 50-70 ng/L more than bentonite treatment followed by filtration, resulting in approximately 100 ng/L of the MPs being left in solution. pIOBP was not able to remove IPMP in buffer pH 3.5 using this system above that removed by bentonite alone. As well, the pIOBP was not able to remove any additional MPs from Chardonnay juice pH 3.5 above that already removed by the bentonite and filtration alone. The mouse MUP was shown to be a better candidate protein for removal of MPs from juice using this system.

L’immunothérapie orale pour le traitement des allergies alimentaires multiples

La prévalence des allergies alimentaires IgE-médiées aurait triplé au cours de la dernière décennie avec des études Nord-Américaines atteignant les 8% chez les enfants. Quoiqu’il n’y ait à ce jour aucun traitement curatif pour les allergies alimentaires, l’immunothérapie oral (OIT) constitue une nouvelle approche expérimentale prometteuse. Cette dernière consiste en l’administration de doses progressive d’allergènes par voie orale sur une période prolongée dans le but d’instaurer un état de désensibilisation et possiblement une tolérance orale soutenue. Cette approche a été démontrée sécuritaire et permettrait la désensibilisation à haute dose de plus de 80% des participants allergiques aux arachides, lait ou œufs. Dans cette thèse, nous présentons 2 études de phase 1 portant sur des protocoles d’OIT, destinés à optimiser l’efficience du traitement chez les sujets avec allergies alimentaires multiples. Près de 30% des enfants avec allergie alimentaire sont allergiques à plus d’un aliment, une proportion qui augmente à 70% lorsqu’on considère les cas les plus sévères. Ces enfants sont à risque augmenté de réactions accidentelles et souffrent d’un impact plus grand sur leur qualité de vie. Dans la première étude, en créant un mélange individualisé avec un ratio stochiométrique 1:1 entre les protéines des aliments allergiques de l’enfant, nous démontrons qu’il est possible de désensibiliser jusqu’à 5 aliments simultanément avec un profil d’innocuité similaire à une monothérapie. Dans la seconde étude, nous utilisons un traitement à l’omalizumab, un anticorps monoclonal anti-IgE, pour permettre une désensibilisation orale multi-allergénique fortement accélérée. Lorsque comparé à l’approche sans omalizumab, ce protocole s’associe à une nette diminution du temps requis pour atteindre les doses d’entretien, passant d’une médiane de 21 à 4 mois, sans affecter le profil d’innocuité. Alors que ces études fournissent des approches cliniques raisonnables pour désensibiliser la population multi-allergique, plusieurs questions persistent, notamment en ce qui a trait à l’induction de tolérance permanente. Une barrière majeure à cet égard réside dans notre piètre compréhension des mécanismes sous-jacents à l’immunothérapie. Prenant avantage d’échantillons cliniques bien caractérisés provenant des essais cliniques ci-haut mentionnés, nous utilisons les nouvelles technologies de séquençage TCR pour suivre la distribution clonale des lymphocytes T spécifiques aux arachides durant une immunothérapie orale. Nous démontrons que l’OIT s’associe à des changements significatifs dans les fréquences des clones spécifiques, suggérant un processus d’épuisement clonal et de remplacement. Nous démontrons par ailleurs que le test de prolifération lymphocytaire, traditionnellement utilisé pour évaluer la réponse cellulaire allergique, est dominé par une distribution polyclonale hautement non-spécifique. Cette observation a des implications majeures considérant que la plupart de la littérature actuelle sur la réponse T se base sur cette technique. En somme, cette thèse jette les bases pour des programmes de recherche translationnelle pour optimiser et personnaliser les protocoles cliniques actuels et développer de nouvelles avenues d’investigation et de traitement pour améliorer la prise en charge des sujets avec allergies alimentaires.

Crop yield reduction in the tropics under climate change: Processes and uncertainties

Many modelling studies examine the impacts of climate change on crop yield, but few explore either the underlying bio-physical processes, or the uncertainty inherent in the parameterisation of crop growth and development. We used a perturbed-parameter crop modelling method together with a regional climate model (PRECIS) driven by the 2071-2100 SRES A2 emissions scenario in order to examine processes and uncertainties in yield simulation. Crop simulations used the groundnut (i.e. peanut; Arachis hypogaea L.) version of the General Large-Area Model for annual crops (GLAM). Two sets of GLAM simulations were carried out: control simulations and fixed-duration simulations, where the impact of mean temperature on crop development rate was removed. Model results were compared to sensitivity tests using two other crop models of differing levels of complexity: CROPGRO, and the groundnut model of Hammer et al. [Hammer, G.L., Sinclair, T.R., Boote, K.J., Wright, G.C., Meinke, H., and Bell, M.J., 1995, A peanut simulation model: I. Model development and testing. Agron. J. 87, 1085-1093]. GLAM simulations were particularly sensitive to two processes. First, elevated vapour pressure deficit (VPD) consistently reduced yield. The same result was seen in some simulations using both other crop models. Second, GLAM crop duration was longer, and yield greater, when the optimal temperature for the rate of development was exceeded. Yield increases were also seen in one other crop model. Overall, the models differed in their response to super-optimal temperatures, and that difference increased with mean temperature; percentage changes in yield between current and future climates were as diverse as -50% and over +30% for the same input data. The first process has been observed in many crop experiments, whilst the second has not. Thus, we conclude that there is a need for: (i) more process-based modelling studies of the impact of VPD on assimilation, and (ii) more experimental studies at super-optimal temperatures. Using the GLAM results, central values and uncertainty ranges were projected for mean 2071-2100 crop yields in India. In the fixed-duration simulations, ensemble mean yields mostly rose by 10-30%. The full ensemble range was greater than this mean change (20-60% over most of India). In the control simulations, yield stimulation by elevated CO2 was more than offset by other processes-principally accelerated crop development rates at elevated, but sub-optimal, mean temperatures. Hence, the quantification of uncertainty can facilitate relatively robust indications of the likely sign of crop yield changes in future climates. (C) 2007 Elsevier B.V. All rights reserved.

Use of a crop model ensemble to quantify CO2 stimulation of water-stressed and well-watered crops

Increased atmospheric concentrations of carbon dioxide (CO2) will benefit the yield of most crops. Two free air CO2 enrichment (FACE) meta-analyses have shown increases in yield of between 0 and 73% for C3 crops. Despite this large range, few crop modelling studies quantify the uncertainty inherent in the parameterisation of crop growth and development. We present a novel perturbed-parameter method of crop model simulation, which uses some constraints from observations, that does this. The model used is the groundnut (i.e. peanut; Arachis hypogaea L.) version of the general large-area model for annual crops (GLAM). The conclusions are of relevance to C3 crops in general. The increases in yield simulated by GLAM for doubled CO2 were between 16 and 62%. The difference in mean percentage increase between well-watered and water-stressed simulations was 6.8. These results were compared to FACE and controlled environment studies, and to sensitivity tests on two other crop models of differing levels of complexity: CROPGRO, and the groundnut model of Hammer et al. [Hammer, G.L., Sinclair, T.R., Boote, K.J., Wright, G.C., Meinke, H., Bell, M.J., 1995. A peanut simulation model. I. Model development and testing. Agron. J. 87, 1085-1093]. The relationship between CO2 and water stress in the experiments and in the models was examined. From a physiological perspective, water-stressed crops are expected to show greater CO2 stimulation than well-watered crops. This expectation has been cited in literature. However, this result is not seen consistently in either the FACE studies or in the crop models. In contrast, leaf-level models of assimilation do consistently show this result. An analysis of the evidence from these models and from the data suggests that scale (canopy versus leaf), model calibration, and model complexity are factors in determining the sign and magnitude of the interaction between CO2 and water stress. We conclude from our study that the statement that 'water-stressed crops show greater CO2 stimulation than well-watered crops' cannot be held to be universally true. We also conclude, preliminarily, that the relationship between water stress and assimilation varies with scale. Accordingly, we provide some suggestions on how studies of a similar nature, using crop models of a range of complexity, could contribute further to understanding the roles of model calibration, model complexity and scale. (C) 2008 Elsevier B.V. All rights reserved.

Simulation of the impact of high temperature stress on annual crop yields

Brief periods of high temperature which occur near flowering can severely reduce the yield of annual crops such as wheat and groundnut. A parameterisation of this well-documented effect is presented for groundnut (i.e. peanut; Arachis hypogaeaL.). This parameterisation was combined with an existing crop model, allowing the impact of season-mean temperature, and of brief high-temperature episodes at various times near flowering, to be both independently and jointly examined. The extended crop model was tested with independent data from controlled environment experiments and field experiments. The impact of total crop duration was captured, with simulated duration being within 5% of observations for the range of season-mean temperatures used (20-28 degrees C). In simulations across nine differently timed high temperature events, eight of the absolute differences between observed and simulated yield were less than 10% of the control (no-stress) yield. The parameterisation of high temperature stress also allows the simulation of heat tolerance across different genotypes. Three parameter sets, representing tolerant, moderately sensitive and sensitive genotypes were developed and assessed. The new parameterisation can be used in climate change studies to estimate the impact of heat stress on yield. It can also be used to assess the potential for adaptation of cropping systems to increased temperature threshold exceedance via the choice of genotype characteristics. (c) 2005 Elsevier B.V. All rights reserved.

Design and optimisation of a large-area process-based model for annual crops

The formulation of a new process-based crop model, the general large-area model (GLAM) for annual crops is presented. The model has been designed to operate on spatial scales commensurate with those of global and regional climate models. It aims to simulate the impact of climate on crop yield. Procedures for model parameter determination and optimisation are described, and demonstrated for the prediction of groundnut (i.e. peanut; Arachis hypogaea L.) yields across India for the period 1966-1989. Optimal parameters (e.g. extinction coefficient, transpiration efficiency, rate of change of harvest index) were stable over space and time, provided the estimate of the yield technology trend was based on the full 24-year period. The model has two location-specific parameters, the planting date, and the yield gap parameter. The latter varies spatially and is determined by calibration. The optimal value varies slightly when different input data are used. The model was tested using a historical data set on a 2.5degrees x 2.5degrees grid to simulate yields. Three sites are examined in detail-grid cells from Gujarat in the west, Andhra Pradesh towards the south, and Uttar Pradesh in the north. Agreement between observed and modelled yield was variable, with correlation coefficients of 0.74, 0.42 and 0, respectively. Skill was highest where the climate signal was greatest, and correlations were comparable to or greater than correlations with seasonal mean rainfall. Yields from all 35 cells were aggregated to simulate all-India yield. The correlation coefficient between observed and simulated yields was 0.76, and the root mean square error was 8.4% of the mean yield. The model can be easily extended to any annual crop for the investigation of the impacts of climate variability (or change) on crop yield over large areas. (C) 2004 Elsevier B.V. All rights reserved.

Diversity of cultivars and other plant resources used at habitation sites in the Llanos de Mojos, Beni, Bolivia: Evidence from macrobotanical remains, starch grains, and phytoliths

Although sparsely populated today, the Llanos de Mojos, Bolivia, sustained large sedentary societies in the Late Holocene (ca. 500 to 1400 AD). In order to gain insight into the subsistence of these people, we undertook macrobotanical and phytolith analyses of sediment samples, and starch grain and phytolith analyses of artifact residues, from four large habitation sites within this region. Macrobotanical remains show the presence of maize (Zea mays), squash (Cucurbita sp.), peanut (Arachis hypogaea), cotton (Gossypium sp.), and palm fruits (Arecaceae). Microbotanical results confirm the widespread use of maize at all sites, along with manioc (Manihot esculenta), squash, and yam (Dioscorea sp.). These integrated results present the first comprehensive archaeobotanical evidence of the diversity of plants cultivated, processed, and consumed, by the pre-Hispanic inhabitants of the Amazonian lowlands of Bolivia.

Amaranth bars enriched with fructans: acceptability and nutritional value

Amaranth bars enriched with fructans: acceptability and nutritional value. There is an increasing appeal for convenience foods with potential health benefits to the consumer. Raw materials with high nutritional value and functional properties must be used on the development of these food products. Amaranth is a gluten-free grain with high nutrition value. Inulin and oligofructose are prebiotic ingredients presenting effects as the enhancement of calcium absorption. Amaranth bars enriched with inulin and oligofructose were developed in the flavors: banana, Brazilian nuts and dried grape, coconut, peach, strawberry and wall nut. The proximate composition were determined and compared to commercial cereal bars, available in traditional (n=59), light (n=60), diet (n=8), with soy (n=10) and quinoa (n=1) categories. Amaranth bars present mean global acceptance values from 6.3 to 7.6 on a 9-point hedonic scale, nutritional advantages as compared to commercial cereal bars (caloric reduction and higher levels of dietary fiber). Although amaranth is an unknown raw material in Brazil, it shows good potential to be used in the manufacturing of ready-to-eat products. As they are gluten free, these amaranth bars are also an alternative product for celiacs, also contributing to the enhancement of calcium absorption, a problem frequently observed in these patients.

Crystal structure and statistical coupling analysis of highly glycosylated peroxidase from royal palm tree (Roystonea regia)

Royal palm tree peroxidase (RPTP) is a very stable enzyme in regards to acidity, temperature, H(2)O(2), and organic solvents. Thus, RPTP is a promising candidate for developing H(2)O(2)-sensitive biosensors for diverse applications in industry and analytical chemistry. RPTP belongs to the family of class III secretory plant peroxidases, which include horseradish peroxidase isozyme C, soybean and peanut peroxidases. Here we report the X-ray structure of native RPTP isolated from royal palm tree (Roystonea regia) refined to a resolution of 1.85 angstrom. RPTP has the same overall folding pattern of the plant peroxidase superfamily, and it contains one heme group and two calcium-binding sites in similar locations. The three-dimensional structure of RPTP was solved for a hydroperoxide complex state, and it revealed a bound 2-(N-morpholino) ethanesulfonic acid molecule (MES) positioned at a putative substrate-binding secondary site. Nine N-glycosylation sites are clearly defined in the RPTP electron-density maps, revealing for the first time conformations of the glycan chains of this highly glycosylated enzyme. Furthermore, statistical coupling analysis (SCA) of the plant peroxidase superfamily was performed. This sequence-based method identified a set of evolutionarily conserved sites that mapped to regions surrounding the heme prosthetic group. The SCA matrix also predicted a set of energetically coupled residues that are involved in the maintenance of the structural folding of plant peroxidases. The combination of crystallographic data and SCA analysis provides information about the key structural elements that could contribute to explaining the unique stability of RPTP. (C) 2009 Elsevier Inc. All rights reserved.

Efeito sacietogênico de um novo inibidor de tripsina da paçoca do amendoim com aumento plasmático de colecistocinina (cck)

Obesity is increasing, reaching epidemic levels in many regions of the world. Studies have shown that consumption of peanuts influences on weight control and this influence may be due to the action of trypsin inhibitors sacietogênica that condition increased plasma colescistocinina (CCK). Moreover, the peanut has other health benefits, and these assignments are guaranteed to increase their production and consumption of several of its products, including the paçoca peanut. The aim of this study was to identify the presence of a trypsin inhibitor in paçoca peanut and evaluate its effect on food intake, weight gain and histomorphological changes in swiss mice (n = 8) and Wistar rats (n = 6). Experimental diets were prepared based on the AIN-93G and supplemented with tack or peanut trypsin inhibitor partially purified paçoca peanut (AHTI). After each treatment, the animals were anesthetized and euthanized, their bloods were collected by cardiac puncture for the determination of CCK and other biochemical parameters (glucose, triglycerides, total cholesterol, high density lipoprotein, low density lipoprotein, glutamic-pyruvic transaminase, glutamic oxaloacetic transaminase and albumin) and their pancreas removed for histologic and morphometric analysis. The supplementation with paçoca peanut and the AHTI showed a decrease of body weight gain and food intake in both mice and rats, due to the satiety, since the animals showed no evidence of impairment of nutritional status conditioned by consumption the AHTI. There were also observed biochemical or morphological important when compared with controls. However, AHTI led to increased secretion of CCK, a peptide sacietogênico. Thus, these results indicate that AHTI present in paçoca peanut, is able to enhance the secretion of plasma CCK and thereby reduce the weight gain associated with lower food intake of experimenta animals

Conversão térmica e termocatalítica à baixa temperatura do óleo de girassol para obtenção de bio-óleo

The use of biofuels remotes to the eighteenth century, when Rudolf Diesel made the first trials using peanut oil as fuel in a compression ignition engine. Based on these trials, there was the need for some chemical change to vegetable oil. Among these chemical transformations, we can mention the cracking and transesterification. This work aims at conducting a study using the thermocatalytic and thermal cracking of sunflower oil, using the Al-MCM-41 catalyst. The material type mesoporous Al-MCM-41 was synthesized and characterized by Hydrothermical methods of X-ray diffraction, scanning electron microscopy, nitrogen adsorption, absorption spectroscopy in the infrared and thermal gravimetric analysis (TG / DTG).The study was conducted on the thermogravimetric behavior of sunflower oil on the mesoporous catalyst cited. Activation energy, conversion, and oil degradation as a function of temperature were estimated based on the integral curves of thermogravimetric analysis and the kinetic method of Vyazovkin. The mesoporous material Al-MCM-41 showed one-dimensional hexagonal formation. The study of the kinetic behavior of sunflower oil with the catalyst showed a lower activation energy against the activation energy of pure sunflower oil. Two liquid fractions of sunflower oil were obtained, both in thermal and thermocatalytic pyrolisis. The first fraction obtained was called bio-oil and the second fraction obtained was called acid fraction. The acid fraction collected, in thermal and thermocatalytic pyrolisis, showed very high level of acidity, which is why it was called acid fraction. The first fraction was collected bio-called because it presented results in the range similar to petroleum diesel

Seletividade do imazapic para dois cultivares de amendoim (Arachis hypogaea) cultivados na ausência e na presença de palha de cana-de-açúcar

Neste trabalho, objetivou-se estudar a seletividade de imazapic a dois cultivares de amendoim (Tatu Vermelho e IAC-5), em condições de ausência e de presença de palha de cana-de-açúcar. O experimento foi instalado em vasos, em delineamento inteiramente casualizado, com os tratamentos dispostos em esquema fatorial 3x2, com quatro repetições. Constituíram os fatores principais três doses de imazapic (0, 98 e 140 g ha-1), aplicadas em pré-emergência sobre duas condições de palha de cana-de-açúcar (0 e 15 t ha-1). Constatou-se que a camada de palha possui potencial em reter o herbicida, especialmente sob períodos de pouca precipitação ou irrigação; as plantas de amendoim sob esta condição apresentaram menos sintomas de intoxicação. Entretanto, na ausência da palha, as plantas, independentemente do cultivar, apresentaram maiores injúrias visuais e redução na altura, massa seca da parte aérea e raiz.

Adubação e o período anterior à interferência das plantas daninhas na cultura do amendoim

O presente trabalho teve como objetivo determinar o efeito da adubação sobre a comunidade infestante e sobre o período anterior à interferência (PAI) das plantas daninhas na cultura do amendoim, cv. IAC Runner 886, em sistema convencional. Os períodos de convivência estudados foram: 0, 7, 15, 20, 30, 45, 55 e 126 dias após a emergência (DAE), totalizando oito tratamentos, dispostos em blocos casualizados, com quatro repetições, em dois níveis de adubação: 0 e 150 kg ha-1 da formulação 00-20-20 (N-P-K). A adubação não alterou a composição da comunidade infestante, destacando-se Cyperus rotundus, Eleusine indica, Indigofera hirsuta, Portulaca oleraceae e Sida rhombifolia como as espécies de maior importância relativa. Contudo, a densidade de plantas daninhas foi maior na área não adubada, mas sem afetar de modo diferenciado a massa seca acumulada por elas. O cultivar de amendoim IAC Runner 886 pode conviver com essa comunidade com e sem adubação por até 15 e 17 DAE (PAI), respectivamente, sem sofrer perdas significativas na produção. A interferência das plantas daninhas reduziu entre 31 e 34% a produtividade do amendoim para as áreas sem adubação e com adubação, respectivamente.

Análise de crescimento em amendoinzeiro (Arachis hypogaea L.) submetido à matocompetição

Foram verificados os efeitos de diferentes períodos de controle e convivência de plantas daninhas (0; 14; 28; 42; 56; 70; 84 e 98 dias) na cultura do amendoinzeiro (Arachis hypogaea L.) em Moji-Guaçu (SP). As principais espécies invasoras da área experimental foram: Acanthospermum australe (Loef.) O. Kuntz, Waltheria indica L., Borreria alata DC., Richardia scabra L., Rhynchelytrum repens (Nees) Stapf. et Hubb. e Diodia teres Walt. de maneira geral, durante quase todo o ciclo, a espécie de maior importância relativa foi A. australe, sendo suplantada por outras espécies apenas na colheita, quando já estava encerrando seu ciclo de desenvolvimento. O período inicial de controle das plantas daninhas, além de reduzir a densidade da comunidade infestante, diminuiu sua riqueza em espécies e alterou a importância relativa das populações. A matocompetição resultou na redução do crescimento do amendoinzeiro, principalmente a partir de 42 dias de convivência, afetando a altura das plantas, a área foliar, o índice de área foliar e o acúmulo epígeo de matéria seca. Não foram afetados os números de ramos e de folíolos por planta, nem a qualidade dos grãos e o vigor das sementes do amendoim. A produção de frutos foi reduzida ao redor de 31,5% em decorrência da interferência das plantas daninhas. O período total de prevenção da interferência na produção foi de 14 dias e o período anterior à interferência foi de 70 dias.

Efeito do espaçamento entre fileiras de amendoim rasteiro na interferência de plantas daninhas na cultura

Objetivou-se com este trabalho avaliar o efeito da redução do espaçamento entre fileiras nos períodos de interferência e na produtividade do amendoim rasteiro (Arachis hypogaea). O experimento foi instalado no município de Jaboticabal-SP. Os tratamentos constaram de dois espaçamentos entre fileiras (80 e 90 cm), divididos em dois grupos. No primeiro, as plantas daninhas foram controladas desde a emergência até 0 (interferência constante), 30, 45, 60, 82, 97 e 112 dias depois. No segundo, as plantas daninhas conviveram com a cultura pelos mesmos períodos do grupo anterior. O delineamento experimental adotado foi o de blocos casualizados, em arranjo de parcelas subdivididas, com quatro repetições. As principais plantas daninhas presentes na área foram Digitaria sp., Xanthium strumarium, Acanthospermum hispidum e Cenchrus echinatus. Para uma perda tolerável de 5% de produtividade, o período crítico de prevenção à interferência foi dos 27 aos 76 e dos 35 aos 96 dias após a emergência para os espaçamentos de 80 e 90 cm, respectivamente; a queda de produtividade das parcelas mantidas com interferência de plantas daninhas em relação àquelas no limpo foi superior a 80%, independentemente do espaçamento.