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Research Paper: PH—Postharvest Technology
Ultrastructural analysis of drying damage in parchment Arabica coffee endosperm cells F.M. Bore´ma,, E.R. Marquesb, E. Alvesc a
Department of Engineering, University Federal of Lavras, P.O. Box 37, 37200-000 Lavras, Minas Gerias, Brazil Department of Food Science, University Federal of Lavras, P.O. Box 37, 37200-000 Lavras, Minas Gerais, Brazil c Department of Plant Pathology, University Federal of Lavras, P.O. Box 37, 37200-000 Lavras, Minas Gerias, Brazil b
ar t ic l e i n f o
The objective of this work was to evaluate and compare the alterations in the structure of coffee seed endosperm subjected to different temperatures and drying conditions. The
Article history:
seeds were dried at 40, 50 and 60, with an airflow of 0.33 m3 s
1
m 2. After drying, 10 seeds
Received 14 September 2006
were randomly selected and prepared for the histochemical tests with Sudan IV and
Received in revised form
scanning and transmission electron microscopy, according to the laboratory’s routine
4 August 2007
techniques. The histochemical results showed that, for the coffee parchment beans dried
Accepted 28 September 2007
at 40 1C, there was no change in the cellular integrity of the plasma membrane and vesicles.
Available online 19 November 2007
In contrast, in the endosperm of parchment coffee beans dried at 60 1C, fused oil bodies that gave rise to large droplets in the intercellular space were observed, indicating a rupture of the vesicles and plasma membrane. Scanning electron microscopy showed that, for the parchment Arabica coffee beans dried at 40 1C, the internal cellular content remained intact and full of cellular material and the space between the plasma membrane and the cell wall was empty. However, in seeds dried at 60 1C, a rupture of the cells was observed, represented by occluded intercellular spaces, indicating a leaking of part of the protoplasm. The results from the transmission electron microscopy corroborated the undamaged and the damaged structure of the coffee parchment beans dried at 40 and 60 1C, respectively. & 2007 IAgrE. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
The drying of coffee is one of the most important processing stages, and involves the removal of water from the coffee parchment beans into the surrounding environment until the beans reach an equilibrium moisture content. Several researches (Berbert et al., 1994; De Grandi et al., 2000; Freire, 1998; Guimara˜es et al., 1998) have evaluated the drying system, the reduction of the energy consumption and the dryer’s efficiency. Recently, the importance of post-harvest treatments for coffee bean quality has received increasing
attention, and several studies describe the impact of the wet and dry processing on the physiology and quality of coffee (Bytof et al., 2000). Nevertheless, ultrastructural analyses which occur upon drying are not well understood. Inappropriate handling of coffee cherry before and after picking, such as the use of high temperatures and drying rates, can lead to a degeneration of the plasma membrane. The alterations in the structure of the plasma membrane and its deteriorating capacity to act as a semipermeable barrier are the main factors responsible for the decrease of quality in coffee (Amorim, 1978; Salazar et al., 1994). Studies have shown
Corresponding author. Department of Engineering, University Federal of Lavras, P.O. Box 37, 37200-000 Lavras, Minas Gerias, Brazil. Tel.:+ 55 35 38291488. E-mail address:
[email protected] (F.M. Bore´m). 1537-5110/$ - see front matter & 2007 IAgrE. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biosystemseng.2007.09.027
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Table 1 – Summary description of the coffee cup classification system Flavour Strictly soft Soft
Softish Hard Rioysh
Rio
Classification characteristics Low acidity, mellow sweetness, pleasant mouth-feel
Same characteristics as strictly soft, only less accentuated Same characteristics as soft, only less accentuated Lacks sweetness and softness Iodine, medicine-like inky flavour from microbetainted beans Same characteristics as rioysh, only more accentuated
that, after desiccation, the plasma membrane is one of the first points of damage. Ultrastructural analysis of the endosperm tissues is essential to verify these works. In a study of the sensitivity of coffee embryo to desiccation, Branda˜o Ju´nior (2000) found that greater ultrastructural damage, such as coalescence of lipids and probable rupture of the membrane, occurred in unripe than semi-ripe coffee berries. The degree of the damage was also influenced by the coffee species. The study found that the species Coffea canephora was more sensitive to desiccation than the Coffea arabica and its cells presented advanced deterioration of the membrane structures, even after reaching maturity. On the other hand, the C. arabica seeds became more tolerant to desiccation during the maturation process. The beverage and presence of defects are the most important criteria to evaluate the quality of coffee. In Brazil, coffees are officially categorised by reference to a flavour scale (Brasil, 2003) as presented in Table 1. The concentration of lipids in the endosperm tissue is related to the quality of the beverage. Studies have shown a greater concentration of lipids in the coffee soft beverage with well-defined droplets inside the protoplasts in the seeds’ outer rims. Nevertheless, the lipids were observed to be homogeneously distributed throughout the whole tissue surface of the hard and rioysh coffee beverage (Goulart, 2002). In these types of coffee, the lipids fill the intercellular spaces without forming well-defined droplets. Despite these works, ultrastructural analyses of the endosperm membrane submitted to different drying temperatures are still lacking. The objective of this study was to evaluate the effect of different drying air temperatures on the resultant structure of the endosperm cells of parchment Arabica coffee.
2.
Material and methods
2.1.
Experimental design
The Arabica coffee, variety Catucai, was harvested manually using the stripping system. Four replications were done in 45
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days with two pickings of approximately 0.9 m3 of coffee fruits for each repetition. The coffee was then separated according to the density of the fruits using a mechanical washer. The cherry fruits were peeled to obtain the parchment coffee which was carried to a concrete ground on the evening it was picked and spread in layers of approximately 1 cm depth. The coffee was separated into 12 equal portions, six of which remained on the ground for 1 day and the remaining six for 3 days to reduce the moisture content. The samples were stirred during the day every 30 min. Thus, the parchment coffee remained on the ground for 1 and 3 days before artificial drying to obtain two levels of moisture content. After solar drying, the moisture content was determined at the start of the drying process in the dryers. This was carried out by the oven method at 10573 1C for 24 h (Brasil, 1992). The coffee was then dried in three dryers of fixed layers 0.13 m thick, using an airflow of 0.33 m3 s 1 m 2 (Agullo & Marenya, 2005) and three average temperatures (40, 50 and 60 1C). The temperature and relative humidity of the ambient air were monitored using a thermohygrograph.
2.2.
Experimental dryer
The apparatus used in this study is shown in Fig. 1. It consisted of a fan (A), an air duct (B), a plenum chamber (C) and a drying chamber (D) measuring 0.61 m by 0.61 m by 0.61 m. The plenum chamber contained a group of 3400 kW electrical circuits to heat the air. The drying chamber, which received four of the 12 samples dried on the ground, was composed of four removable sections (Fig. 1E). Each section received an average of 0.01 m3 of coffee. When the final moisture content of 11% (w.b.) was achieved, the coffee was cooled with ambient air to interrupt the waterremoving process. After cooling, samples were taken to determine the coffee’s final moisture content (Brasil, 1992). The dried coffee was then stored in polythene bags until cleaning and the histochemical and ultrastructural analyses. In this study, the defective grains were removed before the analyses were carried out so that they would not interfere with the results.
2.3.
Histochemical and ultrastructural analyses
For each drying temperature and solar drying period, samples composed of 10 parchment coffee beans were randomly removed from the bulk coffee.
Fig. 1 – Schematic of the experimental apparatus used to dry the parchment coffee: (A) fan; (B) air duct; (C) plenum chamber; (D) drying chamber and (E) removable sections of drying chamber.
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The samples were prepared and observed in the Cytology Laboratory in the Biology Department/UFLA (Federal University of Lavras). For histochemical analysis, the coffee beans were submerged in distilled water for 24 h at room temperature. Cuttings of fresh tissue, obtained with a hand microtome and kept in the open air, were treated for three minutes with the Sudan IV reagent in an ethanol solution at 80% to visualise the lipids (Jensen, 1962).
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set up permanently in a Permalt environment. The ultra-thin sections were taken in golden slot grids and dried on aluminium racks covered with formvar. The sections were post contrasted in uranyl acetate, followed by lead citrate for three minutes, and then examined with transmission electron microscopes (TEM) Zeiss Mod. EM-109.
3. 2.4. Preparation of the samples for scanning electron microscopy (SEM) Samples of parchment coffee were cut lengthways and immersed in a modified Karnovisky solution (glutaraldehyde 2.5%, paraformaldehyde 2.5% in sodium cacodylate buffer 0.05 M, pH 7.2, CaCl2 0.001 M) and stored in a cold chamber until analysis. They were then infiltrated with a cryoprotector, an aqueous solution consisting of 30% glycerol, for 30 min and transversally sectioned in liquid nitrogen using a scalpel blade. The cuts obtained were transferred to a 1% aqueous solution of osmium tetroxide for 1 h and were subsequently dehydrated for 10 min in a series of acetone solutions (25%, 50%, 75%, 90% and 100% three times) before being taken to the critical point apparatus (Baltec CPD 030). The specimens were placed on aluminium support stubs placed over a film of aluminium foil using a carbon tape, sputter covered with gold (Baltec SCD 050) and observed in a LEO EVO 40 XVP scanning electron microscope (Leo Electron Microscopy). Images were digitally generated and registered at varying magnifications in a set up to 20 kV and a working distance of 9 mm. The images were processed using the Corel Draw 9 Photopaint Software, where they were selected and arranged.
2.5. Preparation of the samples for transmission electron microscopy (TEM) Samples of parchment coffee were cut lengthways and immersed in a fixative solution (Karnovisky’s modified), pH 7.2, and stored in a cold chamber until analysis. They were then washed in cacodylate buffer 0.05 M, pH 7.2 (three times for 10 min), post-fixed in 1% aqueous osmium tetroxide solution for 1 h, washed twice for 15 min in distilled water, transferred to a 0.5% uranyl acetate solution for 12 h at 4 1C and then washed once more in distilled water and dehydrated in a series of acetone solutions (25%, 50%, 75%, 90% and 100% three times). The dehydrated tissue was gradually infiltrated with spur/acetone, 30% for 8 h, 70% for 12 h and 100% twice for 24 h each. The specimens obtained were set in moulds and polymerised at 70 1C for 48 h forming blocks that were used in ultramicrotomy.
2.6.
Ultramicrotomy
The blocks were trimmed. Thin (4100 nm) and ultra thin sections (o100 nm) were obtained using a diamond knife in a Reichrt-jung ultramicrotome. The thin sections were selected using a gold ring, placed in glass microscope slides, coloured with toluidine blue (1 g of toluidine blue, 1 g of sodium borate and 100 ml of water), filtered in a Millipore filter (0.2 mm) and
Results and discussion
The ambient temperature varied from 17.2 to 21.5 1C and the relative humidity from 48.5% to 81.5%, because drying was carried out over 15 h beginning at 7:00 am. As a result, the relative humidity of the heated air for the temperature of 40 1C varied from 21.8% to 28.2%; for the temperature of 50 1C, it varied from 14.1% to 26.2%; and for the temperature of 60 1C, it varied from 14.5% to 22.4%. The relative humidity for each level of heated air varied over a fairly narrow range and this is not considered to have had a substantial effect on the results of this work (Bore´m et al., 1992; Afonso Ju´nior et al., 2004). The results of the histochemical test with Sudan IV are presented in Fig. 2. The scanning electron micrographs of parchment coffee are presented in Fig. 3 and the transmission electron micrographs are presented in Fig. 4. These latter results not only confirm the light micrograph observations but add further relevant information. As the SEM results were carried out on dry seeds, any doubts as to the methodology used in the preparation of the fresh cuts, such as the possibility of the membrane ruptures occurring during the steeping process, are eliminated. There was a greater concentration of oils in a globular shape inside the membrane of Arabica coffee parchment beans dried at 40 1C (Fig. 2a). At this temperature, the oils were preserved in this shape due to the integrity of the membrane (Fig. 3a) and the vesicles remained undamaged (Fig. 4a). It is also possible to observe that the content of the cells remained intact and the intercellular spaces remained empty. These results show that there was no ultrastrucutral damage in the endosperm cells of the parchment Arabica coffee dried with air at 40 1C. Fig. 2(b) shows some cells with oils in the extremities of the endosperm tissue while, in others, endosperm cells were full of cellular material, indicating a rupture of the vesicles and of the plasma membrane when the parchment coffee was dried at 50 1C. At this temperature (Fig. 3b), in some cells the cellular content remained intact, while in others there was a rupture of the membrane, partial occlusion of the intercellular spaces and some vesicles were ruptured (Fig. 4b). This indicates that, at 50 1C, an initial disorganisation of the membrane system occurred, leading to a loss of quality in Arabica parchment coffee. The drying of parchment coffee at 60 1C (Fig. 2c) results in major ultrastructural damage. The vesicles and the membrane were ruptured and the cellular material was spread throughout the whole endosperm tissue surface. The oils were not as well defined as they were at 40 1C, forming large droplets in the intercellular spaces. At 60 1C (Fig. 3c), there was a complete occlusion of the intercellular spaces following the rupture of the cellular membranes. Inside the cells, a coalescence and rupture of the vesicles was observed (Fig. 4c).
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Fig. 2 – Light micrographs of coffee seeds dried at 40 1C, (arrow) oils in globular shape inside the cells (a); coffee seeds dried at 50 1C (arrow) oils in the extremity of the endosperm tissue, occlusion of the intercellular spaces (b); coffee seeds dried at 60 1C (arrow) oils spread throughout the cellular surface forming large droplets in the intercellular spaces (c).
Fig. 3 – Scanning electron micrographs of coffee seeds dried at 40 1C (arrow) empty intercellular spaces (a); coffee seeds dried at 50 1C (arrow) partially occluded intercellular spaces (b); coffee seeds dried at 60 1C (arrow ) intercellular spaces completely occluded (c).
Fig. 4 – Transmission electron micrographs of coffee seeds dried at 40 1C (a), at 50 1C (b) and at 60 1C (c).
These observations are essential to the preservation of the quality of coffee, as the rupture of the membranes can expose the oils to oxidation and rankness, which in turn leads to the formation of undesirable compounds that alter the coffee’s aroma and flavour. These results confirm the results of fatty acid analysis obtained by Marques (2006), who observed higher fatty acid values in coffee seeds submitted to higher drying temperatures. In this work, although different moisture contents were used in the beginning of the artificial drying, no ultrastructural differences were observed in the coffee endosperm.
the putative oil bodies uniformly distributed in the cell. (2) At 50 1C, an initial disorganisation of the membrane system occurred, leading to a loss of quality in Arabica parchment coffee. (3) The drying of parchment coffee at 60 1C leads to a coalescence and a rupture of the vesicles and of the membranes, causing occlusion of the intercellular spaces. Independent of the temperature, the pre-drying period does not interfere with the integrity of the cellular membrane of coffee seeds.
4.
Acknowledgements
Conclusions
(1) The drying parchment coffee at 40 1C preserves the integrity of the plasma membrane, keeping
This work was partially supported by grants from FAPEMIG, CNPQ and Federal University of Lavras, Brazil.
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