Coffee Species Research Articles

Antiradical Activity, Phenolics Profile, and Hydroxymethylfurfural in Espresso Coffee: Influence of Technological Factors

The influence of technological factors (decaffeination, brew volume, coffee species, and roast degree) on antiradical activity and phenolics content of espresso coffee is described. The screenings of phenolics profile and other compounds (caffeine and trigonelline), as well as the quantification of hydroxymethylfurfural, were performed by LC-DAD-ESI-MS. Significantly lower (p < 0.05) scavenging activities and phenolics contents were found in decaffeinated espressos when compared with regular ones (32 vs 38% and 324 vs 410 mg/30 mL cup, respectively). A long espresso (70 mL) offers more than twice the phenolics amount of a short one (20 mL). Robusta brews showed higher (p < 0.05) antiradical activity and phenolic contents than arabica ones, for all roast degrees (light, medium, and dark). No significant differences (p > 0.05) were observed for scavenging activities of differently roasted robusta brews, whereas an increase in medium-dark brews was observed for arabica samples. Total phenolics in robusta espressos decreased (p < 0.05) with the increase of roast degree, but no significant differences (p > 0.05) were found between arabica espressos from different roasts. By LC-DAD-ESI-MS, 23 hydroxycinnamic derivatives were found, including chlorogenic acids, lactones, and cinnamoyl-amino acid conjugates. The amount of each compound was differently affected by species and roast. Robusta brews presented superior levels of caffeine and chlorogenic acids, whereas arabica ones contained more trigonelline. Hydroxymethylfurfural contents in the brew (30 mL) varied from 2.60 to 0.84 mg for light- and dark-roasted arabicas and from 1.29 to 0.68 mg for light- and dark-roasted robustas, respectively.

Use of high-resolution satellite imagery in an integrated model to predict the distribution of shade coffee tree hybrid zones

In New Caledonia (21°S, 165°E), shade-grown coffee plantations were abandoned for economic reasons in the middle of the 20th century. Coffee species ( Coffea arabica, C. canephora and C. liberica) were introduced from Africa in the late 19th century, they survived in the wild and spontaneously cross-hybridized. Coffee species were originally planted in native forest in association with leguminous trees (mostly introduced species) to improve their growth. Thus the canopy cover over rustic shade coffee plantations is heterogeneous with a majority of large crowns, attributed to leguminous trees. The aim of this study was to identify suitable areas for coffee inter-specific hybridization in New Caledonia using field based environmental parameters and remotely sensed predictors. Due to the complex structure of tropical vegetation, remote sensing imagery needs to be spatially accurate and to have the appropriate bands for monitoring vegetation cover. Quickbird panchromatic (black and white) imagery at 0.6 to 0.7 m spatial resolutions and multispectral imagery at 2.4 m spatial resolution were pansharpened and used for this study. The two most suitable remotely sensed indicators, canopy heterogeneity and tree crown size, were acquired by the sequential use of tree crown detection (neural network), image processing (such as textural analysis) and classification. All models were supervised and trained on learning data determined by human expertise. The final model has two remotely sensed indicators and three physical parameters based on the Digital Elevation Model: elevation, slope and water flow accumulation. Using these five predictive variables as inputs, two modelling methods, a decision tree and a neural network, were implemented. The decision tree, which showed 96.9% accuracy on the test set, revealed the involvement of ecological parameters in the hybridization of Coffea species. We showed that hybrid zones could be characterized by combinations of modalities, underlining the complexity of the environment concerned. For instance, forest heterogeneity and large crown size, steep slopes (> 53.5%) and elevation between 194 and 429 m asl, are favourable factors for Coffea inter-specific hybridization. The application of the neural network on the whole area gave a predictive map that distinguished the most suitable areas by means of a nonlinear continuous indicator. The map provides a confidence level for each area. The most favourable areas were geographically localized, providing a clue for the detection and conservation of favourable areas for Coffea species neo-diversity.

Tocopherols in coffee brews: Influence of coffee species, roast degree and brewing procedure

Tocopherols in roasted coffee and coffee brews were analysed by HPLC/diode-array/fluorescence detection. Only α and β vitamers were identified in the samples. The influence of several factors on espresso coffee content, as well as on tocopherol extractability, was ascertained, namely, coffee species, roast degree, brew volume and preparation method. Pure arabica espressos contained 2.2 and 3.9 μg of α- and β-tocopherol, respectively, while robusta espressos showed significantly lower amounts ( p < 0.05): 1.3 and 1.1 μg, correspondingly. Total contents (α + β) in espressos prepared from commercial blends ranged from 2.2 to 4.8 μg. Overall, tocopherol amounts in the brews corresponded to only 1% of those found in the coffee cake. However, espressos prepared from servings contained ∼5-fold higher amounts of tocopherol than those prepared by the classic method. The medium-roast degree seems to be more favourable for the extraction of compounds than light or dark roasts. Compared with other coffee beverages, an arabica espresso cup contained an intermediate level of total tocopherols: 7.0 μg/30 mL, against 4.4 μg for filter (150 mL), 13.6 μg for press-pot (60 mL), 19.7 μg for moka (60 mL), 25.4 μg for Turkish, and 40.1 μg for boiled (150 mL) coffees.

Genetic and physical mapping of the SH3 region that confers resistance to leaf rust in coffee tree (Coffea arabica L.)

Resistance to coffee leaf rust is conferred by SH3, a major dominant gene that has been introgressed from a wild coffee species Coffea liberica (genome L) into the allotetraploid cultivated species, Coffea arabica (genome CaEa). As the first step toward the map-based cloning of the SH3 gene, using a bacterial artificial chromosome (BAC) library, we describe the construction of a physical map in C. arabica spanning the resistance locus. This physical map consists in two homeologous BAC-contigs of 1,170 and 1,208 kb corresponding to the subgenomes Ca and Ea, respectively. Genetic analysis was performed using a single nucleotide polymorphism detection assay based on Sanger sequencing of amplicons. The C. liberica-derived chromosome segment that carries the SH3 resistance gene appeared to be introgressed on the sub-genome Ca. The position of the SH3 locus was delimited within an interval of 550 kb on the physical map. In addition, our results indicated a sixfold reduction in recombination frequency in the introgressed SH3 region compared to the orthologous region in Coffea canephora.

Adaptive radiation in Coffea subgenus Coffea L. (Rubiaceae) in Africa and Madagascar

Phylogeographic analysis of the Coffea subgenus Coffea was performed using data on plastid DNA sequences and interpreted in relation to biogeographic data on African rain forest flora. Parsimony and Bayesian analyses of trnL-F, trnT-L and atpB-rbcL intergenic spacers from 24 African species revealed two main clades in the Coffea subgenus Coffea whose distribution overlaps in west equatorial Africa. Comparison of trnL-F sequences obtained from GenBank for 45 Coffea species from Cameroon, Madagascar, Grande Comore and the Mascarenes revealed low divergence between African and Madagascan species, suggesting a rapid and radial mode of speciation. A chronological history of the dispersal of the Coffea subgenus Coffea from its centre of origin in Lower Guinea is proposed. No relation was found between phylogenetic topology and the age of emergence of the volcanic islands that Coffea species have colonised in the Indian Ocean, suggesting dispersal from mainland Africa after the emergence of the youngest island, Grande Comore, 500,000 years ago. Additional sequences were obtained from GenBank for 24 species of other Rubiaceae genera, including the Rubia genus whose origin has been dated from the Upper Miocene. Estimates of substitution rates suggested that diversification in Coffea subgenus Coffea occurred about 460,000 years ago or as recently as the last 100,000 years, depending on the cpDNA region considered and calibration. The phylogenetic relationships based on plastid sequences confirmed biogeographic differentiation of coffee species, but they were not congruent with morphological and biochemical classifications, or with the capacity to grow in specific environments. Examples of convergent evolution in the main clades are given using characters of leaf size, caffeine content and reproductive mode.

Isoflavones in Coffee: Influence of Species, Roast Degree, and Brewing Method

This paper reports the isoflavone contents of roasted coffee beans and brews, as influenced by coffee species, roast degree, and brewing procedure. Total isoflavone level is 6-fold higher in robusta coffees than in arabica ones, mainly due to formononetin. During roasting, the content of isoflavones decreases, whereas their extractability increases (especially for formononetin). Total isoflavones in espresso coffee (30 mL) varied from approximately 40 microg (100% arabica) to approximately 285 microg (100% robusta), with long espressos (70 mL) attaining more than double isoflavones of short ones (20 mL). Espressos (30 mL) prepared from commercial blends contained average amounts of 6, 17, and 78 microg of genistein, daidzein, and formononetin, respectively. Comparison of different brewing methods revealed that espresso contained more isoflavones ( approximately 170 microg/30 mL) than a cup of press-pot coffee ( approximately 130 microg/60 mL), less than a mocha coffee ( approximately 360 microg/60 mL), and amounts similar to those of a filtered coffee cup ( approximately 180 microg/120 mL).

Discrimination of coffee species using kahweol and cafestol: effects of roasting and of defects

The two most commercialized coffee species worldwide are: Coffea arabica L. (arabica) and Coffea canephora Pierre ex A. Froehner (robusta). Since these coffees differ in their commercial value and acceptability, adulteration and mislabeling are major concerns. The diterpenes kahweol and cafestol are considered potential indicators of conilon coffee addition, as they are present in different contents in the species. The degree of roasting and the presence of defective beans may affect the theor of several coffee constituents. The aim of this work was to evaluate the possibility of discriminating the coffee species arabica and robusta through their kahweol and cafestol contents. Samples of arabica, robusta, and of their blends, with different amounts of defects and degrees of roasting (light, medium and dark) were studied. After direct saponification and extraction with terc-butyl methyl ether, the samples were analyzed by reverse-phase HPLC with UV detection. The kahweol content varied between 661 and 923 mg/100 g in the arabica coffee, and its presence was not observed in the conilon. Cafestol ranged from 360 to 478 mg in arabica, and from 163 to 275 mg/100 g in conilon coffee. The addition of conilon coffee reduced diterpene contents, but this effect varied according to the amount of defects and roasting degrees. A higher intensity roast did not affect diterpene degradation. No differences in the kahweol and cafestol levels, comparing defective or regular beans, were observed. In the analysis of coffee samples with different degrees of roasting and defects, the parameters kahweol and cafestol showed potential for discriminating between the species.

Acrylamide in espresso coffee: Influence of species, roast degree and brew length

Espresso coffees were analysed for acrylamide contents by matrix solid-phase dispersion and GC–MS. The influence of coffee species, roast degree, and brew length were ascertained. Mean acrylamide contents of medium roasted espressos (30 mL) were 1.16 ± 0.25 and 2.31 ± 0.43 μg for pure arabica and robusta samples, respectively. Espressos prepared from commercial blends contained an average acrylamide level of 1.26 ± 0.28 μg. A 25% decrease was observed when comparing espressos prepared with medium and dark roasted coffee. The extraction efficacy of acrylamide for standard espressos of 30 mL was near 80%, being only affected by brew volume, with long espressos (70 mL) containing practically all acrylamide of the coffee cake (99%), almost double that of short ones (20 mL). When compared with other common coffee beverages, espresso acrylamide concentration (μg/L) was higher. However, due to the small volume per cup, it may contribute less to acrylamide ingestion.

Orchid fleck symptoms may be caused naturally by two different viruses transmitted by Brevipalpus

Brevipalpus-transmitted viruses (BTV) cause chlorotic, necrotic and/or ringspot lesions in leaves and stems of orchids, citrus, coffee and several other plant species. There are two different types of BTVs, the nuclear and the cytoplasmic, based on maturation locale in the cell and particle morphology. The orchid fleck virus (OFV) is a BTV that infects orchids. Its short rodlike particles are 32–40 nm in diameter, 100–150 nm in length. OFV is found in the nucleus and is associated with intranuclear electronlucent viroplasms. In 1999, transmission electron microscopy analysis revealed a distinct type of virus causing orchid fleck symptoms. The bacilliform particles, 70–80 nm in diameter and 110–120 nm in length, induced electron-dense viroplasm inclusions in infected cells and resembled the cytoplasmic type associated with BTV, such as the citrus leprosis virus C. Our objective in the present study was to verify whether the cytoplasmic type virus found in orchids could be amplified using primers for other cytoplasmic BTVs, such as CiLV-C and Solanumviolaefolium ringspot virus (SvRSV). Additionally, we aimed to differentiate the two BTVs found in orchids: the nuclear and the cytoplasmic types of OFV using microscopy and molecular and serological tools. This virus was not amplified by the CiLV-C and SvRSV primers, and neither the molecular nor the serological tools available to the OFV diagnosis reacted with it, demonstrating that they are definitely different viruses.

Brewing trouble: coffee invasion in relation to edges and forest structure in tropical rainforest fragments of the Western Ghats, India

While the conservation impacts of invasive plant species on tropical biodiversity is widely recognised, little is known of the potential for cultivated crops turning invasive in tropical forest regions. In the Western Ghats biodiversity hotspot, India, fragmented rainforests often adjoin coffee plantations. This study in the Anamalai hills assessed the effects of distance from edges and forest structure on the occurrence and abundance of shade-tolerant coffee (Arabica Coffea arabica and Robusta C. canephora) in four fragments (32–200 ha) using replicate line transects laid from the edges into the interiors. The coffee species cultivated in adjoining plantations was more abundant than the other coffee species inside study fragments, showing a clear decline in stem density from edge (0 m) to interior (250 m), suggesting the influence of propagule pressure of adjoining plantations, coupled with edge effects and seed dispersal by animals. Significant positive correlations of coffee density with canopy cover indicate the potential threat of coffee invasion even in closed canopy rainforests. Stem density of Coffea arabica (150–1,825 stems/ha) was higher in more disturbed fragments, whereas Coffea canephora had spread in disturbed and undisturbed sites achieving much higher densities (6.3–11,486 stems/ha). In addition, a negative relationship between C. canephora and native shrub density indicates its potential detrimental effects on native plants.

Physical characterization of non-defective and defective Arabica and Robusta coffees before and after roasting

The presence of defective coffee beans is known to depreciate the quality of coffee beverage consumed worldwide. These beans represent about 15–20% of the total coffee produced in Brazil and, although they are separated from the non-defective beans, they are still commercialized in the coffee trading market. Thus, it was the aim of this work to verify if physical characteristics provide an effective discrimination of defective and non-defective coffees of both Arabica and Robusta species, before and after roasting. The evaluated parameters were size (volume), density and color. The results obtained in the present study showed that, within a given coffee species, defective beans can be separated from non-defective ones by sieving, before roasting. In the case of Arabica coffees, size separation between non-defective and defective can also be accomplished after roasting. Prior to roasting, color separation will be effective for Robusta coffees, but ineffective for separation of non-defective, immature and light sour Arabica beans.

Spent Coffee Grounds as a Versatile Source of Green Energy

The production of energy from renewable and waste materials is an attractive alternative to the conventional agricultural feed stocks such as corn and soybean. This paper describes an approach to extract oil from spent coffee grounds and to further transesterify the processed oil to convert it into biodiesel. This process yields 10-15% oil depending on the coffee species (Arabica or Robusta). The biodiesel derived from the coffee grounds (100% conversion of oil to biodiesel) was found to be stable for more than 1 month under ambient conditions. It is projected that 340 million gallons of biodiesel can be produced from the waste coffee grounds around the world. The coffee grounds after oil extraction are ideal materials for garden fertilizer, feedstock for ethanol, and as fuel pellets.

Discrimination between arabica and robusta coffee species on the basis of their tocopherol profiles

The tocopherol profiles of arabica and robusta coffee beans, both green and roasted, were ascertained. A solid-liquid micro-extraction method was used, and the quantification performed by normal-phase HPLC/diode-array/fluorescence detection. Regarding green arabicas, the mean contents were 2.7 ± 0.4 mg/100 g, for α-tocopherol, and 8.0 ± 0.9 mg/100 g, for β-tocopherol. For green robustas, mean values of 1.7 ± 0.3 and 2.1 ± 0.3 mg/100 g were found for α- and β-tocopherol, respectively. Generally, more than 90% of tocopherols remained after the roasting procedure, except for β-tocopherol in robustas, whose mean degradation was approximately 25% when expressed as dry weight. No γ-tocopherol was detected in any sample. The results show that tocopherol profile could be useful in the discrimination of arabica and robusta coffees (either green or roasted).

Determination of Acrylamide during Roasting of Coffee

In this study different Arabica and Robusta coffee beans from different regions of the world were analyzed for acrylamide after roasting in a laboratory roaster. Due to the complex matrix and the comparably low selectivity of the LC-MS at m/ z 72, acrylamide was analyzed after derivatization with 2-mercaptobenzoic acid at m/ z 226. Additionally, the potential precursors of acrylamide (3-aminopropionamide, carbohydrates, and amino acids) were studied. The highest amounts of acrylamide formed in coffee were found during the first minutes of the roasting process [3800 ng/g in Robusta ( Coffea canephora robusta) and 500 ng/g in Arabica ( Coffea arabica)]. When the roasting time was increased, the concentration of acrylamide decreased. It was shown that especially the roasting time and temperature, species of coffee, and amount of precursors in raw material had an influence on acrylamide formation. Robusta coffee contained significantly larger amounts of acrylamide (mean = 708 ng/g) than Arabica coffee (mean = 374 ng/g). Asparagine is the limiting factor for acrylamide formation in coffee. 3-Aminopropionamide formation was observed in a dry model system with mixtures of asparagine with sugars (sucrose, glucose). Thermal decarboxylation and elimination of the alpha-amino group of asparagine at high temperatures (>220 degrees C) led to a measurable but low formation of acrylamide.

Spatial and temporal analysis of coffee wilt disease caused by Fusarium xylarioides in Coffea canephora

Coffee wilt disease (CWD) caused by Fusarium xylarioides, considered to be a soil-inhabiting fungus, is endemic in several African countries, affecting commercially important coffee species and causing serious economic losses. Coffee wilt disease development in naturally infected Coffea canephora fields at the Coffee Research Institute in Uganda was assessed from April 2001 to March 2006 to generate information about temporal and spatial spread of the disease. Maps of diseased trees were also generated from the data. Semi-variance analysis was performed on the data to show the spatio-temporal structure of disease. Host influence on the spatio-temporal structure was deduced from the distribution pattern of diseased and healthy trees and analysis of variance. Results show that the temporal disease epidemic progress was slow. The disease was found to spread from initial infections to healthy neighbouring trees, resulting in an aggregated pattern. An infected tree could infect up to three healthy trees away, in any direction. Disease foci formed and expanded with time, coalescing but punctuated in spots planted with resistant hosts. There were varying levels of susceptibility among host genotypes, affecting the rates and levels of epidemic development. The implications of the findings to the control of CWD are discussed.

Diversity in coffee assessed with SSR markers: structure of the genus Coffea and perspectives for breeding

The present study shows transferability of microsatellite markers developed in the two cultivated coffee species (Coffea arabica L. and C. canephora Pierre ex Froehn.) to 15 species representing the previously identified main groups of the genus Coffea. Evaluation of the genetic diversity and available resources within Coffea and development of molecular markers transferable across species are important steps for breeding of the two cultivated species. We worked on 15 species with 60 microsatellite markers developed using different strategies (SSR-enriched libraries, BAC libraries, gene sequences). We focused our analysis on 4 species used for commercial or breeding purposes. Our results establish the high transferability of microsatellite markers within Coffea. We show the large amount of diversity available within wild species for breeding applications. Finally we discuss the consequences for future comparative mapping studies and breeding of the two cultivated species.

Development of genomic microsatellite markers in Coffea canephora and their transferability to other coffee species

Of the 103 accepted Coffea species, 70% are threatened with extinction but only a few of them have been studied. A set of 40 polymorphic microsatellite markers was developed using a GA/GT-enriched Coffea canephora genomic library. Amplification of these markers was tested in accessions of C. heterocalyx (a Critically Endangered species) and C. pseudozanguebariae (a Vulnerable species) belonging to different African geographical clades. All microsatellites were polymorphic in C. canephora, with a mean allele number per polymorphic locus of more than 3 (at least 9 genotypes were tested). Observed and expected heterozygosities calculated for C. canephora and C. pseudozanguebariae varied from 0.10 to 0.91 and from 0.20 to 0.77, respectively. In total, 38 primer pairs (95%) were amplified in C. heterocalyx and C. pseudozanguebariae, indicating their high level of transferability across the genus Coffea. This large marker set will be useful for more extensive genetic studies of threatened Coffea species.

Sucrose metabolism during fruit development in Coffea racemosa

AbstractSucrose is one of the compounds in the raw coffee bean that has been identified as an important precursor of coffee flavour and aroma. In order to increase our knowledge of sucrose metabolism in coffee species, sucrose and reducing sugar content were investigated in the fast fruit‐ripening coffee species Coffea racemosa. Fruits were harvested regularly from anthesis, until the point of complete fruit maturation and tissue development, followed by measurement of fruit tissue weight. Pericarp was the most abundant tissue, and always represented around 70–80% of fruit fresh weight. The perisperm present in young fruits was rapidly replaced by the endosperm at around 40 days after flowering (DAF). In the latter tissue, total and reducing sugars decreased during development. However, sucrose content was maintained at a relatively high level throughout fruit development, with a peak at 40 DAF that coincided with the highest level of sucrose synthase (SUS) activity detected in this tissue. For all endosperm developmental stages analysed, very low invertase activity was observed, suggesting a limited role for this enzyme in sucrose metabolism. Northern blot experiments using SUS1 and SUS2 cDNA sequences from Coffea arabica as probes were carried out to study the expression of SUS‐encoding genes. The SUS1 transcripts of C. racemosa overlapped with the peak of SUS activity in the endosperm, whereas SUS2 transcripts accumulated mainly during the latest stages of endosperm development. Altogether, these results suggest that the SUS1 isoform of SUS is essential for sucrose accumulation in the fruits of C. racemosa.

Sub-genomic origin and regulation patterns of a duplicated WRKY gene in the allotetraploid species Coffea arabica

The extensively cultivated coffee species Coffea arabica is an allotetraploid resulting from a recent hybridization between two wild diploid Coffea species. We describe in this paper the first identification and functional assessment of homoeologous gene copies in C. arabica. When cloning the CaWRKY1 gene encoding a transcription factor of the WRKY superfamily associated with plant defense responses to pathogens (Ganesh et al. in Plant Sci 170:1045–1051, 2006), two distinct gene copies (CaWRKY1a and CaWRKY1b) were obtained from C. arabica. Southern blots experiments and phylogenetic analysis of the WRKY1 gene in related diploid Coffea species showed that CaWRKY1a and CaWRKY1b are homoeologous sequences in the allopolyploid coffee genome and are probably close descents of the extant Coffea canephora and C. eugenioides WRKY1 genes. To verify if CaWRKY1a and CaWRKY1b were both functional, gene expressions were monitored in C. arabica plants challenged by the rust fungus Hemileia vastatrix, the root-knot nematode Meloidogyne exigua, and after several abiotic treatments. Real-time quantitative polymerase chain reaction (PCR) assays showed that CaWRKY1 homoeologs were concomitantly expressed and displayed the same altered patterns of expression in leaves and roots during biotic and abiotic treatments. These results suggest that CaWRKY1a and CaWRKY1b were functionally retained in the coffee genome after allopolyploidization and that no functional divergence occurred between the duplicated genes in the C. arabica genome. This study provides the first molecular data on sub-genome-specific expression in allopolyploid coffee. The origin of the C. arabica sub-genomes is discussed with regards on the probable progenitors of this important crop species.

Teor de zinco no café como variável da disponibilidade no solo e extrator

No Brasil são cultivadas duas espécies de café: Coffea arabica L. e Coffea canephora Pierre. As pesquisas com adubação com Zn têm sido feitas, principalmente, com cultivares arábica. Esta pesquisa foi realizada com objetivo de obter a relação entre os teores de Zn disponível pelos extratores DTPA e Mehlich-1 em plantas de C. arabica cv. Mundo Novo IAC 379-19 (MN) e C. canephora cv. Apoatã IAC 2258 (AP), bem como em plantas enxertadas (MN/AP). Os tratamentos foram obtidos pelo fatorial 3 x 3, entre materiais genéticos (MN, AP e MN/AP) e doses de Zn (0, 10 e 20 mg dm-3). O teor de Zn na planta, em relação à disponibilidade do nutriente no solo, independentemente dos extratores, varia com a espécie e a combinação entre espécies e a enxertia. Os extratores DTPA e Mehlich-1 apresentaram eficiência semelhante para determinar o teor de Zn disponível no solo, independentemente da espécie (C. arabica e C. canephora). A absorção máxima de Zn ocorreu com teor disponível de 5,6 mg dm-3 (DTPA) e 6,6 mg dm-3 (Mehlich-1) para a cultivar Mundo Novo (C. arabica). Para a espécie C. canephora cv. Apoatã e nas plantas enxertadas (MN/AP), não foi estabelecida a máxima absorção de Zn, independentemente dos extratores, nas doses utilizadas no experimento.