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Anatomical study of the effect of cooking on differently pigmented rice varieties
Food Structure 7 (2016) 6–12
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Anatomical study of the effect of cooking on differently pigmented rice varieties Maria Zaupa a, Tommaso Ganino a,*, Lucia Dramis b, Nicoletta Pellegrini a a b
Department of Food Science, University of Parma, Parco Area delle Scienze 59/A, 43124 Parma, Italy Department of Bioscience, University of Parma, Parco Area delle Scienze 11/A, 43124 Parma, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 February 2015 Received in revised form 4 December 2015 Accepted 15 December 2015
In recent years, the consumption of wholemeal rice, and in particular pigmented varieties, has received increased interest because of its content in nutritionally relevant compounds. However, thermal treatment can modify the structure of the matrix, influencing the accessibility and possibly the localization of antioxidants and other compounds. Therefore, in this study the effect of two different cooking methods (i.e. ‘‘risotto’’ and boiling) on the anatomical structure of three differently pigmented wholemeal rice varieties was evaluated. The presence and the localization of tannin inclusions were also analyzed. Cooking caused the formation of voids in the grains and, in particular, black rice presented the highest proportion of voids among the varieties analyzed. After both thermal treatments, a significant increase in the tannin inclusions in endosperm was observed, suggesting a partial resorption of the leached compounds. These observations suggest that an evaluation of the anatomical structure may help to better understand the behavior of cereals during domestic cooking, which in turn, affects their nutritional quality also in terms of compound accessibility. ß 2015 Elsevier Ltd. All rights reserved.
Keywords: Oryza sativa L. Ribe rice Ermes rice Venere rice Cooking Tannins
1. Introduction Rice (Oryza sativa L.) is one of the most important cereal crops for human consumption in the world. Usually rice is consumed in the polished form, in which the outer layers of the caryopsis are removed. However, from the nutritional point of view, the consumption of wholemeal rice is preferable, because a large amount of nutritionally relevant compounds, such as fiber, proteins, vitamins, and minerals, are located in the bran (Fardet, Rock, & Re´me´sy, 2008). In particular, although not widespread, in recent years pigmented rice varieties have received increased attention because of their antioxidant properties, related to the presence of phenolic compounds located mainly in the outer layer of the caryopsis (Fardet et al., 2008; Finocchiaro, Ferrari, & Gianinetti, 2010). The thermal treatment can modify the structure of the matrix, influencing the accessibility and probably the localization of antioxidants and other compounds (Parada & Aguilera, 2007). However, analysing the behavior and the localization of individual compounds during cooking is not simple. In fact, the most common
* Corresponding author at: Department of Food Science, University of Parma, Parco Area delle Scienze 11/A, 43124 Parma, Italy. E-mail address: [email protected] (T. Ganino). http://dx.doi.org/10.1016/j.foostr.2015.12.001 2213-3291/ß 2015 Elsevier Ltd. All rights reserved.
techniques for a qualitative and quantitative analysis of phenolic and antioxidant compounds are unable to localize these molecules. Therefore, evaluating the anatomical structure and changes occurring in matrix components during thermal treatment may help to understand the effect of cooking on these compounds. Moreover, observing total tannins may indicate the localization of antioxidant compounds in the caryopsis. The term tannins refers to inclusions that react with the tannin solution (Ruzin, 1999) and the molecules visualized with this technique present red-ox properties and may be comparable to those considered in the antioxidant capacity analysis. The analysis of food structure is a topical issue and several recent papers have been published on the subject. In previous studies, different techniques, such as microscopy, NMR microimaging and spectroscopic methods, have been employed to observe the morphology of cooked rice grains and the effect of thermal treatments on cereal matrix (Briffaz, Mestres, Escoute, Lartaud, & Dornier, 2012; Horigane et al., 1999; Ogawa, Glenn, Orts, & Wood, 2003; Tamura & Ogawa, 2012; Witek et al., 2010). These studies mainly focus on starch gelatinization of milled rice and analyzed the structure of rice in order to evaluate a relationship with the texture and other quality parameters, such as sensorial properties (Leelayuthsoontorn & Thipayarat, 2006; Mestres, Ribeyre, Pons, Fallet, & Matencio, 2011; Rewthong, Soponronnart, Taechapairoj, Tungtrakul, & Prachayawarakorn,
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2011). In particular, the authors observed the formation of cracks and hollows inside the caryopsis (Horigane et al., 1999; Ogawa et al., 2003; Tamura et al., 2014) and the presence of a coated layer on the surface of the rice grain (Tamura & Ogawa, 2012). However, all these works analyzed milled rice, whereas, to our knowledge, no study has evaluated the anatomical structure of cooked wholemeal rice, in which the presence of pericarp might influence the behavior of rice during cooking. Moreover, in the literature there are no studies observing the presence and localization of tannin inclusions in cereal caryopses. Therefore, the aim of this study was to evaluate the effect of cooking on the caryopsis rice structure of three differently pigmented wholemeal rice varieties, using two cooking treatments (i.e. boiling and ‘‘risotto’’). The anatomical changes that occurred during cooking and the formation of internal hollows were observed. The presence and localization of total tannins were also analyzed histochemically. 2. Materials and methods 2.1. Materials Three commercial varieties of wholemeal rice were analyzed: a white variety (Ribe), a red variety (Ermes), and a black variety (Venere). The grains were purchased at a local market in Parma (Italy).
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optical microscope (Leica DM 4000B, Wetzlar, Germany) equipped with a Leica DC 100 digital camera, which was also used for image capturing. The quantity of tannin inclusions (area) was measured using an image analysis system (QWIN3 Leica Imaging Systems Ltd., Wetzlar, Germany). Features were measured on binary images; feature data included linear dimension, areas and shapes. Data used for the present analysis included only the feature areas in square micrometers. The feature data file was opened in a statistical program for further analysis. Images (1024 1536 pixel grayscale) were saved as 300 dpi TIFF files. 2.5. Statistical analysis The statistical analysis was carried out using SPSS Statistics 21.0 software (SPSS Inc., Chicago, IL, USA). Descriptive statistics were applied to the dataset. All dependent variables were analyzed using two-way ANOVA, with two factors: ‘‘Cultivar’’ and ‘‘Cooking treatment’’. If a statistically significant interaction effect was found, the two factors were evaluated simultaneously by plotting the estimated marginal means for all levels of each factor. For each statistical factor, comparisons of the means were performed using Tukey’s post hoc tests. The statistical tests were performed at a 5% significance level. 3. Results and discussion
2.2. Chemicals
3.1. Raw samples
All chemicals and solvents were analytical grade. Tolouidine Blue O (TBO) solution was purchased from Carlo Erba (Milan, Italy – C.I. 52040), Periodic Acid Schiff (PAS) staining kit was from BioOptica (Milan, Italy). Paraffin for processing and embedding was purchased from Sherwood Medical Co. (St. Luis, MO, USA). Ultrapure water from MilliQ system (Millipore, Bedford, MA, USA) was used throughout the experiments.
Rice samples showed the typical structure of cereal caryopsis; from the outside inwards pericarp, perisperm, aleurone and endosperm were visible (Bechtel & Pomeranz, 1977). The outer layers, or pericarp, consisted of compressed cells and appeared fused together, with a pericarp thickness of 20.6 3.9, 21.8 3.5, 19.1 5.3 mm in the black (Fig. 1A), red (Fig. 1B) and white (Fig. 1C) varieties, respectively. The aleurone consisted of one or more cell layers. In rice this characteristic occurs within a single grain (Evers & Millar, 2002), with single- or multi-cellular layers observed near the embryo. In white rice, aleurone cells were mostly distributed in a single layer, whereas in black rice the aleurone was distribuited in two cells layer; the red variety commonly showed a mono or bicellular layer, within a single grain. Aleurone cells, in transversal section, appeared roughly square, with a more elongated form along the tangential diameter in the black variety, whereas in the white variety the radial and tangential diameters were similar. The red rice presented both squared and elongated aleurone cells. The area of the aleurone cells ranged between 370 and 4300 mm2 in the black rice, between 333 and 3300 mm2 in the red rice and between 390 and 4150 mm2 in the white variety. Moreover, the aleurone layer thickness was higher in the white (51.0 11.6 mm) and black (48.8 10.2 mm) varieties with respect to the red one (35.9 5.7 mm). In aleurone cells the cytoplasm was visible, because in raw rice the cells are partially dehydrated due to the maturation process of the caryopsis. Aleurone cells contained some inclusions (Fig. 1A–C): small starch granules (confirmed by PAS staining) and others, likely proteins and other molecules, such as phytates (Krishnan, Ebenezer, & Dayanandan, 2001). In endosperm, the size of the cells gradually increases from small cells in the subaleurone layer to large cells in the inner zone of the endosperm tissue, in accordance with the observations of Briffaz et al. (2012). These cells contain mainly starch inclusions, which were present in the form of granules and clusters of granules (Yu, Zhou, Xiong, & Wang, 2014). In the subaleurone region, starch was in the form of small spherical starch granules, as observed by Bechtel and Pomeranz (1978). Moreover, higher protein and vitamin content was reported in this region compared to the central part of the endosperm (Bechtel & Pomeranz,
2.3. Cooking procedure Two different domestic cooking techniques were employed: boiling and ‘‘risotto’’. Both cooking methods were designed to achieve the complete starch gelatinization. Briefly, 110–120 mg of rice was weighed into a 10 mL glass tube. The tube was placed in a water bath at 100 8C, after adding a specific amount of boiling water, and covered. The water/rice ratio was 3.6:1 (w/w) for ‘‘risotto’’ and 20:1 (w/w) for boiling. The cooking time was 40 min for all studied varieties and for both cooking procedure. The cooked rice was then cooled in an ice bath for 5 min. Cooking tests were performed in triplicate for each variety. 2.4. Histological and histochemical analyses Rice samples (uncooked and cooked) were fixed in a formalin: acetic acid: 60%-ethanol solution (2:1:17, v/v; FAA solution) and after at least 2 weeks they were dehydrated with gradual alcohol concentrations. Dehydrated samples were immersed in liquid paraffin for at least 7 days. Samples were embedded in paraffin blocks and the blocks cut into 5 mm thick cross sections with a microtome Leitz 1512 (Wetzlar, Germany). The sections were stained with: (i) TBO (0.1% w/v) and PAS solutions (Ruzin, 1999) to detect changes in anatomical structure during the cooking process for all varieties; (ii) tannin solution (a mixture of 89 mL of water, 0.25 mL acetic acid, 10 mL formalin and 2 g ferrous sulphate) to detect total tannins (Ruzin, 1999). For each staining technique, at least three replicate sections for each variety and treatment were stained. The sections were examined using an
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cause expansion and swelling followed by fracturing visible particularly in the outer layers (Fig. 2). This fracturing was probably caused by the pericarp resisting expansion, which in boiled rice appeared especially along the dorsal/ventral area, whereas in risotto rice it appeared at the extremities. This effect has not previously been reported, probably because previous studies analyzed the effect of cooking on milled rice, in which the pericarp was removed. In the areas near the fractures, where the cooking water penetrated, cooking produced another considerable effect in all varieties, i.e. disruption of the cells around of the fractures. Ogawa et al. (2003) previously observed in milled rice that thermal treatment caused more damage to cells in the grain regions where the water penetrated, especially the outermost layers. In our samples, with both cooking methods, the external layers were only slightly damaged by thermal treatment, and cell walls remained almost intact, with no noticeable differences between the two treatments. Conversely, the endosperm was the most damaged tissue, especially near the fractures (Fig. 3A), whereas the subaleurone layers were less affected. Conversely, Ogawa et al. (2003) observed in milled rice that the central endosperm was only minimally affected. The different behavior observed in our samples compared to the milled rice can likely be explained by the thick and robust cell walls of tegument and pericarp, which may have protected the outer layers (including the aleurone and subaleurone cells) from heat damage. 3.2.2. Cellular structure With respect to cellular dimensions, cells expanded due to water absorption and starch gelatinization with a consequent cell lysis. In the endosperm in particular, cell walls were ruptured, as observed by Tamura et al. (2014), and cell contents released. Starch gelatinization after both cooking methods was different in the three varieties. In the white and red rice, it occurred throughout the caryopsis, causing a melted appearance of the material present in the endosperm (Fig. 3B). In the black variety, starch gelatinization was more evident near the fractures (Fig. 3C and D); in the subaleurone layer, where water interacted less with the cells, the starch was partially still present in the form of granules. In fact, water absorption was observed to occur gradually from outside inwards (Briffaz et al., 2012; Stapley, Hyde, Gladden, & Fryer, 1997). Moreover, in the black rice and inside the endosperm, in particular at the margins of the fractures and the void areas, starch granules were observed in the form of chains, in which the profile of the starch granules was slightly visible. It has been reported that different rice cultivars can present different starch gelatinization (Varavinit, Shobsngob, Varanyanond, Chinachoti, & Naivikul, 2003). This is due to the different granule architecture (crystalline to amorphous ratio) (Singh, Singh, Kaur, Singh Sodhi, & Singh Gill, 2003) and the different characteristics of starch, such as the composition in amylose and amylopectin, that can vary greatly among rice varieties (Sompong, Siebenhandl-Ehn, LinsbergerMartin, & Berghofer, 2011). Fig. 1. Cross-sections of rice caryopside samples stained with TBO: (A) Venere rice raw/uncooked; (B) Ermes rice raw/uncooked; (C) Ribe rice raw/uncooked. Legend: p = pericarp; a = aleuronic layer; e = endosperm.
1978). In the central endosperm region, starch was present as large granules and clusters of granules. 3.2. Effect of cooking on anatomical structure 3.2.1. Macroscopic structure With regards to the effect of cooking on the macroscopic dimension of rice caryopsis, thermal treatments were observed to
3.2.3. Fractures and voids Voids were observed inside the caryopsis, in both cooking methods. In general, in risotto rice there was a large number of smaller voids (Fig. 3E and F), along with a few large voids (cracks ranged in size from 2476.46 mm2 to 579,845.44 mm2 in Venere, 176.89 mm2 to 18,750.34 mm2 in Ermes and 1415.12 mm2 to 109,141.13 mm2 in Ribe). In boiled rice, the proportion of voids with a middle size was higher than in risotto rice. The minimum and maximum areas were, respectively, 1768.90 mm2 and 1,397,961.75 mm2 in Venere, 1238.23 mm2 and 28,302.40 mm2 in Ermes, 884.45 mm2 and 69,163.99 mm2 in Ribe. Of the three varieties, black rice presented bigger voids than the others (mean area 59,410.64 mm2 and 137,089.76 mm2, in risotto and boiled
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Fig. 2. Venere caryopsides after risotto cooking; pericarp fractures visible in the extremities of the rice samples.
Fig. 3. Cross-sections of rice caryopside samples: (A) Endosperm damage in Ribe rice after boiling. Sample stained using TBO solution; (B) High magnification view of gelification in endosperm of Ribe rice after boiling. Sample stained using TBO solution; (C) Gelification in endosperm of Venere rice after boiling. Cross-section stained using TBO solution; (D) High magnification view of starch grain gelification (PAS stain) in Venere boiled rice. PAS discoloration indicates gelification; (E) Appearance of voids in boiled Ribe rice. Cross-section stained using TBO solution; (F) Voids in risotto Ribe rice. Cross-section stained using TBO solution. Legend: e = endosperm; v = voids, g = starch gelification.
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Table 1 Mean void size (expressed in percentage) and average tannin area, cultivar effect, cooking effect and interaction of ‘‘cultivar’’ ‘‘cooking treatment’’ factors. Data analyzed using two-way ANOVA statistical analysis, and means for each statistical using Tukey’s post hoc test. Significance is expressed in the column with different letters and significance expressed by Pr > Diff. Bold characters indicate significant variablesa.
Cultivar
Cooking Treatment
Cultivar Cooking Treatment
Venere Ermes Ribe P-value Boiling Risotto Uncooked P-value P-value
% voids vs. total area
Tannins
10.47a 1.19b 3.77ab 0.01 8.51a 6.91a – 0.643 0.161
96,194.24a 58,625.58c 85,369.83b 0.0003 106,230.55a 112,407.37a 21,551.73b <0.0001 0.0001
a The measures of tannins are expressed in mm2 and referred to a total area of 300,000 mm2.
samples, respectively), whereas red rice had the smallest voids (mean area 5565.23 mm2 and 8628.30 mm2, in risotto and boiled samples, respectively). In the white variety, the voids had a mean area of 23,897.00 mm2 and 20,840.86 mm2, in risotto and boiled specimens, respectively. The same is behavior was also reflected in the percentage of voids in the caryopsis, as reported in Tables 1 and 2. Table 1 shows the mean void size (expressed in percentage) for each cultivar in the two cooking treatments, as well as all the cultivar average values per cooking treatment. The factor interaction (cultivar cooking treatment) was not significant for this variable (P = 0.161). Moreover, there were no significant differences between the two cooking treatments (risotto and boiling). With reference to varietal differences, it was observed that the cultivar Venere presented higher values than the others. In our samples, the variety was seen to be an important factor that determines resistance to heat treatment and the formation of voids. Table 2 shows % void variability in all cultivar samples for all cooking treatments. Black rice had the highest percentage of voids, despite considerable variability among the specimens analyzed, with a void proportion of 12.8 and 18.6% in risotto and boiled samples, respectively. Red rice maintained its structure better, with a total void area of less than 3% for both thermal treatments. Cracks were probably formed by the combined effect of pressure build-up, subsequent expansion and partial solubilization of cell wall components, determining consequent cell lysis and the release of its content (Briffaz et al., 2012). In particular, in a recent work it was observed that cracks were generated in the initial stages of cooking and became voids as temperatures inside the caryopsis increased (Tamura et al., 2014). Table 2 Percentage of voids and tannins calculated in all cultivar samples for all cooking treatmentsa. CV
Treatment
% voids vs. total area
Tannins
Venere
Boiling Risotto Uncooked Boiling Risotto Uncooked Boiling Risotto Uncooked
18.61a 12.81ab – 2.26ab 1.30b – 4.67ab 6.63ab –
125,533.45a 137,434.40a 25,614.87cd 69,569.63b 76,569.18b 29,737.92c 123,588.57a 123,218.55a 9302.38d
Ermes
Ribe
a Data are presented as means. The measures of tannins are expressed in mm2 and referred to a total area of 300,000 mm2. Different letters in the same column correspond to significantly (p < 0.05) different samples.
Several works observed that, during cooking, cell components such as amylose and amylopectin, damaged cell walls, and other molecules may leach into the cooking medium (Ogawa et al., 2003; Rewthong et al., 2011; Tamura & Ogawa, 2012). Similarly, some voids near the exterior surface, caused by starch leaching into cooking water, have been observed in milled rice cooked by boiling (Rewthong et al., 2011). It has been argued that cracks act as channels for water to flow into the grain during cooking (Ogawa et al., 2003). In addition, cracks probably also allow material to leach into the cooking water because cell wall disruptions release more compounds from the cell matrix (Leelayuthsoontorn & Thipayarat, 2006; Tamura et al., 2014). In both thermal processes, a part of the cellular content of endosperm was eluted in the cooking water, forming voids. In the second stage of cooking, a part of the leached compounds may be resorbed by the grain. However, in boiling only a certain percentage is probably resorbed, whereas most remains in the cooking medium. In risotto cooking, in the second stage of cooking, water is completely absorbed by the grain, with probable resorption of leached compounds. The interior cellular organization and integrity of rice grain cells were better preserved in boiled rice (Fig. 3E) than in rice cooked with the risotto method (Fig. 3F). This occurs probably because boiled rice, after initial absorption, reaches an equilibrium between the inside of the grain and the outside. Conversely, complete water absorption during the final stages of risotto cooking may affect the organization of the matrix cellular compounds. 3.3. Tannins In rice caryopses, the presence and localization of tannins were observed with the tannin solution (Ruzin, 1999). Because this solution is non-specific for a single class of compounds, but can reveal different compounds with red-ox properties, histochemical analysis using the tannin solution may provide clues as to the localization of compounds with red-ox properties before and after cooking. In the analyzed rice caryopsis, tannin inclusions (dark) were measured in raw and cooked samples and the results are reported in Tables 1 and 2. Tannin was measured in the outermost layers of the caryopsis, including the pericarp and teguments, and the outermost part of the endosperm. This decision was based on the fact that in raw rice tannins are concentrated in the outer layers, whereas in the endosperm they are scarce. In fact, it was observed that in all raw varieties, tannins were mainly concentrated in the outermost layers, especially in Venere and Ermes rice pericarp, as was expected (Fig. 4A and B). Table 1 shows the mean tannin area for each rice cultivar, as well as for both cooking treatments. Notably, cultivar x cooking treatment interaction was positive (P 0.0001) for this variable, therefore the cultivar factor appears to play an important role in cooking treatment. Considering only the cultivar factor, Venere and Ribe varieties presented a higher concentration of tannins (Table 1) than Ermes. Moreover, the cooking treatment seems to be responsible for the increase in tannins. Table 2 shows the variability in tannins in all cultivar samples for both cooking treatments. In all varieties, thermal treatments induced an increase in tannins and the area of the inclusions was significantly higher in Venere and Ribe varieties after both cooking methods (137,434 mm2 and 125,533 mm2 in Venere, risotto and boiled, respectively; 123,218 mm2 and 123,588 mm2 in Ribe, risotto and boiled, respectively) than in Ermes variety. However, it should be noted that the lowest tannin level was observed in uncooked Ribe (9302 mm2). Considering the form of tannins found in raw samples, in the white variety, tannin in the pericarp appeared as crystalline dust
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Fig. 4. Cross-sections of rice caryopside samples stained with tannin solution. (A) Venere rice; (B) Ribe rice; (C) Particular of endosperm of Venere rice after risotto treatment; (D) Particular of endosperm of Ribe rice after risotto treatment; (E) Particular of antioxidant compound absorption (stained with tannin solution) in Venere rice sample. Legend: dt = dark granular form of tannins; cdt = crystalline dust form of tannins.
(Fig. 4B), whereas in the black (Fig. 4A) and red varieties the tannin appeared in dark granular form. Some tannins were also noted in the cytoplasm of aleurone cells and in the endosperm of all varieties. After both cooking treatments, tannins were also observed in a remarkable concentration in endosperm (Fig. 4C and D). In particular, a significant increase in the size of the cross-section that reacted with tannin solution was observed in the black and white varieties, in which tannins were present throughout the
endosperm cross-section. A lower but still significant increase in tannins was also measured in the red variety, where tannins were not observed throughout the endosperm but only in the outermost layer of the cross-section, explaining the lower amounts measured in this cooked rice. In particular, in the white rice, the tannin solution reacted in proximity with the starch granules (Fig. 4D), whereas in the black (Fig. 4C) and red rice varieties tannins were more scattered in the caryopsis. In the samples analyzed, on average, risotto samples presented a higher tannin content
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(Tables 1 and 2) than boiled samples, although the difference between the two treatments was not statistically significant. In causing partial solubilization of cell wall components, thermal treatment probably releases some compounds and components previously bound to the cell wall, thus increasing detectable tannins. A release of bound phenolics after cooking was observed in millet grains by Chandrasekara, Naczk, and Shahidi (2012). Moreover, some authors suggest the possible migration of phenolic compounds (present to a larger extent in the external layers of the caryopsis of cereals) into the endosperm during cooking and the formation of complexes with the macromolecules present in the internal part of the grain (Chandrasekara et al., 2012; Towo, Svanberg, & Ndossi, 2003). The presence of tannins in endosperm after cooking could be explained by the above-mentioned hypothesis of compounds leaching into cooking water and their subsequent partial resorption during the second stage of cooking. Molecules are probably reabsorbed through the fractures because the outermost layers were not severely damaged by cooking and did not permit the migration of compounds from water to endosperm. Indeed, the absorption of compounds that react with tannin solution is visible near a fracture (Fig. 4E). In polished rice it has been observed that during boiling, starch, cell wall fragments and other materials are eluted into cooking water (Tamura & Ogawa, 2012), and, when water is absorbed, eluted materials condense on the surface of grains without fractures, forming a coated layer. In our samples we observed that the two thermal treatments had different effects on tannin inclusions in the three rice varieties. In particular, the white variety showed a significant increase in tannin content after cooking, suggesting that during cooking certain compounds with red-ox properties were released from the fiber to which they were bound and could then react with the tannin solution. Conversely, the red rice variety had the lowest tannin content after cooking, despite having a higher content in raw samples. The different behavior among the varieties could be due to the different compounds with red-ox properties present in the three varieties. In the literature there are reports indicating that rice varieties with different pigmentation contain different phenolic compounds (Finocchiaro et al., 2010). In fact, the different forms of tannin inclusions (crystalline dust, dark granular) observed in our samples were probably formed by different compounds (that react differently with the tannin solution). These compounds are probably affected differently by cooking, causing different behavior of the tannin inclusions in the three varieties. 4. Conclusions In evaluating the effects of cooking on rice, we observed that in all the varieties analyzed, risotto cooking damaged the grain structure more than boiling, and the three varieties presented different proportions and areas of voids, with the black rice being the most affected by cooking. Black rice also presented differences in starch gelatinization. With regards to tannins, the black and red rice varieties displayed a higher content of these compounds in raw samples than the white variety. After both thermal treatments, tannins were measured in very high concentrations also in the endosperm, indicating the possible a resorption of some previously leached compounds. Moreover, a significant increase was observed in the white variety. These observations suggest that an evaluation of the anatomical structure might help to better understand the behavior of cereals during domestic cooking, which, in turn, affects their nutritional quality also in terms of compound accessibility. In fact the release
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International Journal of Food Science and Technology, 32, 355–375. Tamura, M., & Ogawa, Y. (2012). Visualization of the coated layer at the surface of rice grain cooked with varying amounts of cooking water. Journal of Cereal Science, 56, 404–409. Tamura, M., Nagai, T., Hidaka, Y., Noda, T., Yokoe, M., & Ogawa, Y. (2014). Changes in histological tissue structure and textural characteristics of rice grain during cooking process. Food Structure, 1, 164–170. Towo, E. E., Svanberg, U., & Ndossi, G. D. (2003). Effect of grain pre-treatment on different extractable phenolic groups in cereals and legumes in Tanzania. Journal of the Science of Food and Agriculture, 83, 980–986. Varavinit, S., Shobsngob, S., Varanyanond, W., Chinachoti, P., & Naivikul, O. (2003). Effect of amylose content on gelatinization, retrogradation and pasting properties of flours from different cultivars of Thai rice. Starch/Sta¨rke, 55, 410–415. Witek, M., We˛glarz, W. P., de Jong, L., van Dalen, G., Blonk, J. C. G., Heussen, P., et al. (2010). The structural and hydration properties of heat-treated rice studied at multiple length scales. Food Chemistry, 120, 1031–1040. Yu, X. R., Zhou, L., Xiong, F., & Wang, Z. (2014). Structural and histochemical characterization of developing rice caryopsis. Rice Science, 21, 142–149.
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Food Structure journal homepage: www.elsevier.com/locate/foostr
Anatomical study of the effect of cooking on differently pigmented rice varieties Maria Zaupa a, Tommaso Ganino a,*, Lucia Dramis b, Nicoletta Pellegrini a a b
Department of Food Science, University of Parma, Parco Area delle Scienze 59/A, 43124 Parma, Italy Department of Bioscience, University of Parma, Parco Area delle Scienze 11/A, 43124 Parma, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 February 2015 Received in revised form 4 December 2015 Accepted 15 December 2015
In recent years, the consumption of wholemeal rice, and in particular pigmented varieties, has received increased interest because of its content in nutritionally relevant compounds. However, thermal treatment can modify the structure of the matrix, influencing the accessibility and possibly the localization of antioxidants and other compounds. Therefore, in this study the effect of two different cooking methods (i.e. ‘‘risotto’’ and boiling) on the anatomical structure of three differently pigmented wholemeal rice varieties was evaluated. The presence and the localization of tannin inclusions were also analyzed. Cooking caused the formation of voids in the grains and, in particular, black rice presented the highest proportion of voids among the varieties analyzed. After both thermal treatments, a significant increase in the tannin inclusions in endosperm was observed, suggesting a partial resorption of the leached compounds. These observations suggest that an evaluation of the anatomical structure may help to better understand the behavior of cereals during domestic cooking, which in turn, affects their nutritional quality also in terms of compound accessibility. ß 2015 Elsevier Ltd. All rights reserved.
Keywords: Oryza sativa L. Ribe rice Ermes rice Venere rice Cooking Tannins
1. Introduction Rice (Oryza sativa L.) is one of the most important cereal crops for human consumption in the world. Usually rice is consumed in the polished form, in which the outer layers of the caryopsis are removed. However, from the nutritional point of view, the consumption of wholemeal rice is preferable, because a large amount of nutritionally relevant compounds, such as fiber, proteins, vitamins, and minerals, are located in the bran (Fardet, Rock, & Re´me´sy, 2008). In particular, although not widespread, in recent years pigmented rice varieties have received increased attention because of their antioxidant properties, related to the presence of phenolic compounds located mainly in the outer layer of the caryopsis (Fardet et al., 2008; Finocchiaro, Ferrari, & Gianinetti, 2010). The thermal treatment can modify the structure of the matrix, influencing the accessibility and probably the localization of antioxidants and other compounds (Parada & Aguilera, 2007). However, analysing the behavior and the localization of individual compounds during cooking is not simple. In fact, the most common
* Corresponding author at: Department of Food Science, University of Parma, Parco Area delle Scienze 11/A, 43124 Parma, Italy. E-mail address: [email protected] (T. Ganino). http://dx.doi.org/10.1016/j.foostr.2015.12.001 2213-3291/ß 2015 Elsevier Ltd. All rights reserved.
techniques for a qualitative and quantitative analysis of phenolic and antioxidant compounds are unable to localize these molecules. Therefore, evaluating the anatomical structure and changes occurring in matrix components during thermal treatment may help to understand the effect of cooking on these compounds. Moreover, observing total tannins may indicate the localization of antioxidant compounds in the caryopsis. The term tannins refers to inclusions that react with the tannin solution (Ruzin, 1999) and the molecules visualized with this technique present red-ox properties and may be comparable to those considered in the antioxidant capacity analysis. The analysis of food structure is a topical issue and several recent papers have been published on the subject. In previous studies, different techniques, such as microscopy, NMR microimaging and spectroscopic methods, have been employed to observe the morphology of cooked rice grains and the effect of thermal treatments on cereal matrix (Briffaz, Mestres, Escoute, Lartaud, & Dornier, 2012; Horigane et al., 1999; Ogawa, Glenn, Orts, & Wood, 2003; Tamura & Ogawa, 2012; Witek et al., 2010). These studies mainly focus on starch gelatinization of milled rice and analyzed the structure of rice in order to evaluate a relationship with the texture and other quality parameters, such as sensorial properties (Leelayuthsoontorn & Thipayarat, 2006; Mestres, Ribeyre, Pons, Fallet, & Matencio, 2011; Rewthong, Soponronnart, Taechapairoj, Tungtrakul, & Prachayawarakorn,
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2011). In particular, the authors observed the formation of cracks and hollows inside the caryopsis (Horigane et al., 1999; Ogawa et al., 2003; Tamura et al., 2014) and the presence of a coated layer on the surface of the rice grain (Tamura & Ogawa, 2012). However, all these works analyzed milled rice, whereas, to our knowledge, no study has evaluated the anatomical structure of cooked wholemeal rice, in which the presence of pericarp might influence the behavior of rice during cooking. Moreover, in the literature there are no studies observing the presence and localization of tannin inclusions in cereal caryopses. Therefore, the aim of this study was to evaluate the effect of cooking on the caryopsis rice structure of three differently pigmented wholemeal rice varieties, using two cooking treatments (i.e. boiling and ‘‘risotto’’). The anatomical changes that occurred during cooking and the formation of internal hollows were observed. The presence and localization of total tannins were also analyzed histochemically. 2. Materials and methods 2.1. Materials Three commercial varieties of wholemeal rice were analyzed: a white variety (Ribe), a red variety (Ermes), and a black variety (Venere). The grains were purchased at a local market in Parma (Italy).
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optical microscope (Leica DM 4000B, Wetzlar, Germany) equipped with a Leica DC 100 digital camera, which was also used for image capturing. The quantity of tannin inclusions (area) was measured using an image analysis system (QWIN3 Leica Imaging Systems Ltd., Wetzlar, Germany). Features were measured on binary images; feature data included linear dimension, areas and shapes. Data used for the present analysis included only the feature areas in square micrometers. The feature data file was opened in a statistical program for further analysis. Images (1024 1536 pixel grayscale) were saved as 300 dpi TIFF files. 2.5. Statistical analysis The statistical analysis was carried out using SPSS Statistics 21.0 software (SPSS Inc., Chicago, IL, USA). Descriptive statistics were applied to the dataset. All dependent variables were analyzed using two-way ANOVA, with two factors: ‘‘Cultivar’’ and ‘‘Cooking treatment’’. If a statistically significant interaction effect was found, the two factors were evaluated simultaneously by plotting the estimated marginal means for all levels of each factor. For each statistical factor, comparisons of the means were performed using Tukey’s post hoc tests. The statistical tests were performed at a 5% significance level. 3. Results and discussion
2.2. Chemicals
3.1. Raw samples
All chemicals and solvents were analytical grade. Tolouidine Blue O (TBO) solution was purchased from Carlo Erba (Milan, Italy – C.I. 52040), Periodic Acid Schiff (PAS) staining kit was from BioOptica (Milan, Italy). Paraffin for processing and embedding was purchased from Sherwood Medical Co. (St. Luis, MO, USA). Ultrapure water from MilliQ system (Millipore, Bedford, MA, USA) was used throughout the experiments.
Rice samples showed the typical structure of cereal caryopsis; from the outside inwards pericarp, perisperm, aleurone and endosperm were visible (Bechtel & Pomeranz, 1977). The outer layers, or pericarp, consisted of compressed cells and appeared fused together, with a pericarp thickness of 20.6 3.9, 21.8 3.5, 19.1 5.3 mm in the black (Fig. 1A), red (Fig. 1B) and white (Fig. 1C) varieties, respectively. The aleurone consisted of one or more cell layers. In rice this characteristic occurs within a single grain (Evers & Millar, 2002), with single- or multi-cellular layers observed near the embryo. In white rice, aleurone cells were mostly distributed in a single layer, whereas in black rice the aleurone was distribuited in two cells layer; the red variety commonly showed a mono or bicellular layer, within a single grain. Aleurone cells, in transversal section, appeared roughly square, with a more elongated form along the tangential diameter in the black variety, whereas in the white variety the radial and tangential diameters were similar. The red rice presented both squared and elongated aleurone cells. The area of the aleurone cells ranged between 370 and 4300 mm2 in the black rice, between 333 and 3300 mm2 in the red rice and between 390 and 4150 mm2 in the white variety. Moreover, the aleurone layer thickness was higher in the white (51.0 11.6 mm) and black (48.8 10.2 mm) varieties with respect to the red one (35.9 5.7 mm). In aleurone cells the cytoplasm was visible, because in raw rice the cells are partially dehydrated due to the maturation process of the caryopsis. Aleurone cells contained some inclusions (Fig. 1A–C): small starch granules (confirmed by PAS staining) and others, likely proteins and other molecules, such as phytates (Krishnan, Ebenezer, & Dayanandan, 2001). In endosperm, the size of the cells gradually increases from small cells in the subaleurone layer to large cells in the inner zone of the endosperm tissue, in accordance with the observations of Briffaz et al. (2012). These cells contain mainly starch inclusions, which were present in the form of granules and clusters of granules (Yu, Zhou, Xiong, & Wang, 2014). In the subaleurone region, starch was in the form of small spherical starch granules, as observed by Bechtel and Pomeranz (1978). Moreover, higher protein and vitamin content was reported in this region compared to the central part of the endosperm (Bechtel & Pomeranz,
2.3. Cooking procedure Two different domestic cooking techniques were employed: boiling and ‘‘risotto’’. Both cooking methods were designed to achieve the complete starch gelatinization. Briefly, 110–120 mg of rice was weighed into a 10 mL glass tube. The tube was placed in a water bath at 100 8C, after adding a specific amount of boiling water, and covered. The water/rice ratio was 3.6:1 (w/w) for ‘‘risotto’’ and 20:1 (w/w) for boiling. The cooking time was 40 min for all studied varieties and for both cooking procedure. The cooked rice was then cooled in an ice bath for 5 min. Cooking tests were performed in triplicate for each variety. 2.4. Histological and histochemical analyses Rice samples (uncooked and cooked) were fixed in a formalin: acetic acid: 60%-ethanol solution (2:1:17, v/v; FAA solution) and after at least 2 weeks they were dehydrated with gradual alcohol concentrations. Dehydrated samples were immersed in liquid paraffin for at least 7 days. Samples were embedded in paraffin blocks and the blocks cut into 5 mm thick cross sections with a microtome Leitz 1512 (Wetzlar, Germany). The sections were stained with: (i) TBO (0.1% w/v) and PAS solutions (Ruzin, 1999) to detect changes in anatomical structure during the cooking process for all varieties; (ii) tannin solution (a mixture of 89 mL of water, 0.25 mL acetic acid, 10 mL formalin and 2 g ferrous sulphate) to detect total tannins (Ruzin, 1999). For each staining technique, at least three replicate sections for each variety and treatment were stained. The sections were examined using an
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cause expansion and swelling followed by fracturing visible particularly in the outer layers (Fig. 2). This fracturing was probably caused by the pericarp resisting expansion, which in boiled rice appeared especially along the dorsal/ventral area, whereas in risotto rice it appeared at the extremities. This effect has not previously been reported, probably because previous studies analyzed the effect of cooking on milled rice, in which the pericarp was removed. In the areas near the fractures, where the cooking water penetrated, cooking produced another considerable effect in all varieties, i.e. disruption of the cells around of the fractures. Ogawa et al. (2003) previously observed in milled rice that thermal treatment caused more damage to cells in the grain regions where the water penetrated, especially the outermost layers. In our samples, with both cooking methods, the external layers were only slightly damaged by thermal treatment, and cell walls remained almost intact, with no noticeable differences between the two treatments. Conversely, the endosperm was the most damaged tissue, especially near the fractures (Fig. 3A), whereas the subaleurone layers were less affected. Conversely, Ogawa et al. (2003) observed in milled rice that the central endosperm was only minimally affected. The different behavior observed in our samples compared to the milled rice can likely be explained by the thick and robust cell walls of tegument and pericarp, which may have protected the outer layers (including the aleurone and subaleurone cells) from heat damage. 3.2.2. Cellular structure With respect to cellular dimensions, cells expanded due to water absorption and starch gelatinization with a consequent cell lysis. In the endosperm in particular, cell walls were ruptured, as observed by Tamura et al. (2014), and cell contents released. Starch gelatinization after both cooking methods was different in the three varieties. In the white and red rice, it occurred throughout the caryopsis, causing a melted appearance of the material present in the endosperm (Fig. 3B). In the black variety, starch gelatinization was more evident near the fractures (Fig. 3C and D); in the subaleurone layer, where water interacted less with the cells, the starch was partially still present in the form of granules. In fact, water absorption was observed to occur gradually from outside inwards (Briffaz et al., 2012; Stapley, Hyde, Gladden, & Fryer, 1997). Moreover, in the black rice and inside the endosperm, in particular at the margins of the fractures and the void areas, starch granules were observed in the form of chains, in which the profile of the starch granules was slightly visible. It has been reported that different rice cultivars can present different starch gelatinization (Varavinit, Shobsngob, Varanyanond, Chinachoti, & Naivikul, 2003). This is due to the different granule architecture (crystalline to amorphous ratio) (Singh, Singh, Kaur, Singh Sodhi, & Singh Gill, 2003) and the different characteristics of starch, such as the composition in amylose and amylopectin, that can vary greatly among rice varieties (Sompong, Siebenhandl-Ehn, LinsbergerMartin, & Berghofer, 2011). Fig. 1. Cross-sections of rice caryopside samples stained with TBO: (A) Venere rice raw/uncooked; (B) Ermes rice raw/uncooked; (C) Ribe rice raw/uncooked. Legend: p = pericarp; a = aleuronic layer; e = endosperm.
1978). In the central endosperm region, starch was present as large granules and clusters of granules. 3.2. Effect of cooking on anatomical structure 3.2.1. Macroscopic structure With regards to the effect of cooking on the macroscopic dimension of rice caryopsis, thermal treatments were observed to
3.2.3. Fractures and voids Voids were observed inside the caryopsis, in both cooking methods. In general, in risotto rice there was a large number of smaller voids (Fig. 3E and F), along with a few large voids (cracks ranged in size from 2476.46 mm2 to 579,845.44 mm2 in Venere, 176.89 mm2 to 18,750.34 mm2 in Ermes and 1415.12 mm2 to 109,141.13 mm2 in Ribe). In boiled rice, the proportion of voids with a middle size was higher than in risotto rice. The minimum and maximum areas were, respectively, 1768.90 mm2 and 1,397,961.75 mm2 in Venere, 1238.23 mm2 and 28,302.40 mm2 in Ermes, 884.45 mm2 and 69,163.99 mm2 in Ribe. Of the three varieties, black rice presented bigger voids than the others (mean area 59,410.64 mm2 and 137,089.76 mm2, in risotto and boiled
M. Zaupa et al. / Food Structure 7 (2016) 6–12
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Fig. 2. Venere caryopsides after risotto cooking; pericarp fractures visible in the extremities of the rice samples.
Fig. 3. Cross-sections of rice caryopside samples: (A) Endosperm damage in Ribe rice after boiling. Sample stained using TBO solution; (B) High magnification view of gelification in endosperm of Ribe rice after boiling. Sample stained using TBO solution; (C) Gelification in endosperm of Venere rice after boiling. Cross-section stained using TBO solution; (D) High magnification view of starch grain gelification (PAS stain) in Venere boiled rice. PAS discoloration indicates gelification; (E) Appearance of voids in boiled Ribe rice. Cross-section stained using TBO solution; (F) Voids in risotto Ribe rice. Cross-section stained using TBO solution. Legend: e = endosperm; v = voids, g = starch gelification.
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Table 1 Mean void size (expressed in percentage) and average tannin area, cultivar effect, cooking effect and interaction of ‘‘cultivar’’ ‘‘cooking treatment’’ factors. Data analyzed using two-way ANOVA statistical analysis, and means for each statistical using Tukey’s post hoc test. Significance is expressed in the column with different letters and significance expressed by Pr > Diff. Bold characters indicate significant variablesa.
Cultivar
Cooking Treatment
Cultivar Cooking Treatment
Venere Ermes Ribe P-value Boiling Risotto Uncooked P-value P-value
% voids vs. total area
Tannins
10.47a 1.19b 3.77ab 0.01 8.51a 6.91a – 0.643 0.161
96,194.24a 58,625.58c 85,369.83b 0.0003 106,230.55a 112,407.37a 21,551.73b <0.0001 0.0001
a The measures of tannins are expressed in mm2 and referred to a total area of 300,000 mm2.
samples, respectively), whereas red rice had the smallest voids (mean area 5565.23 mm2 and 8628.30 mm2, in risotto and boiled samples, respectively). In the white variety, the voids had a mean area of 23,897.00 mm2 and 20,840.86 mm2, in risotto and boiled specimens, respectively. The same is behavior was also reflected in the percentage of voids in the caryopsis, as reported in Tables 1 and 2. Table 1 shows the mean void size (expressed in percentage) for each cultivar in the two cooking treatments, as well as all the cultivar average values per cooking treatment. The factor interaction (cultivar cooking treatment) was not significant for this variable (P = 0.161). Moreover, there were no significant differences between the two cooking treatments (risotto and boiling). With reference to varietal differences, it was observed that the cultivar Venere presented higher values than the others. In our samples, the variety was seen to be an important factor that determines resistance to heat treatment and the formation of voids. Table 2 shows % void variability in all cultivar samples for all cooking treatments. Black rice had the highest percentage of voids, despite considerable variability among the specimens analyzed, with a void proportion of 12.8 and 18.6% in risotto and boiled samples, respectively. Red rice maintained its structure better, with a total void area of less than 3% for both thermal treatments. Cracks were probably formed by the combined effect of pressure build-up, subsequent expansion and partial solubilization of cell wall components, determining consequent cell lysis and the release of its content (Briffaz et al., 2012). In particular, in a recent work it was observed that cracks were generated in the initial stages of cooking and became voids as temperatures inside the caryopsis increased (Tamura et al., 2014). Table 2 Percentage of voids and tannins calculated in all cultivar samples for all cooking treatmentsa. CV
Treatment
% voids vs. total area
Tannins
Venere
Boiling Risotto Uncooked Boiling Risotto Uncooked Boiling Risotto Uncooked
18.61a 12.81ab – 2.26ab 1.30b – 4.67ab 6.63ab –
125,533.45a 137,434.40a 25,614.87cd 69,569.63b 76,569.18b 29,737.92c 123,588.57a 123,218.55a 9302.38d
Ermes
Ribe
a Data are presented as means. The measures of tannins are expressed in mm2 and referred to a total area of 300,000 mm2. Different letters in the same column correspond to significantly (p < 0.05) different samples.
Several works observed that, during cooking, cell components such as amylose and amylopectin, damaged cell walls, and other molecules may leach into the cooking medium (Ogawa et al., 2003; Rewthong et al., 2011; Tamura & Ogawa, 2012). Similarly, some voids near the exterior surface, caused by starch leaching into cooking water, have been observed in milled rice cooked by boiling (Rewthong et al., 2011). It has been argued that cracks act as channels for water to flow into the grain during cooking (Ogawa et al., 2003). In addition, cracks probably also allow material to leach into the cooking water because cell wall disruptions release more compounds from the cell matrix (Leelayuthsoontorn & Thipayarat, 2006; Tamura et al., 2014). In both thermal processes, a part of the cellular content of endosperm was eluted in the cooking water, forming voids. In the second stage of cooking, a part of the leached compounds may be resorbed by the grain. However, in boiling only a certain percentage is probably resorbed, whereas most remains in the cooking medium. In risotto cooking, in the second stage of cooking, water is completely absorbed by the grain, with probable resorption of leached compounds. The interior cellular organization and integrity of rice grain cells were better preserved in boiled rice (Fig. 3E) than in rice cooked with the risotto method (Fig. 3F). This occurs probably because boiled rice, after initial absorption, reaches an equilibrium between the inside of the grain and the outside. Conversely, complete water absorption during the final stages of risotto cooking may affect the organization of the matrix cellular compounds. 3.3. Tannins In rice caryopses, the presence and localization of tannins were observed with the tannin solution (Ruzin, 1999). Because this solution is non-specific for a single class of compounds, but can reveal different compounds with red-ox properties, histochemical analysis using the tannin solution may provide clues as to the localization of compounds with red-ox properties before and after cooking. In the analyzed rice caryopsis, tannin inclusions (dark) were measured in raw and cooked samples and the results are reported in Tables 1 and 2. Tannin was measured in the outermost layers of the caryopsis, including the pericarp and teguments, and the outermost part of the endosperm. This decision was based on the fact that in raw rice tannins are concentrated in the outer layers, whereas in the endosperm they are scarce. In fact, it was observed that in all raw varieties, tannins were mainly concentrated in the outermost layers, especially in Venere and Ermes rice pericarp, as was expected (Fig. 4A and B). Table 1 shows the mean tannin area for each rice cultivar, as well as for both cooking treatments. Notably, cultivar x cooking treatment interaction was positive (P 0.0001) for this variable, therefore the cultivar factor appears to play an important role in cooking treatment. Considering only the cultivar factor, Venere and Ribe varieties presented a higher concentration of tannins (Table 1) than Ermes. Moreover, the cooking treatment seems to be responsible for the increase in tannins. Table 2 shows the variability in tannins in all cultivar samples for both cooking treatments. In all varieties, thermal treatments induced an increase in tannins and the area of the inclusions was significantly higher in Venere and Ribe varieties after both cooking methods (137,434 mm2 and 125,533 mm2 in Venere, risotto and boiled, respectively; 123,218 mm2 and 123,588 mm2 in Ribe, risotto and boiled, respectively) than in Ermes variety. However, it should be noted that the lowest tannin level was observed in uncooked Ribe (9302 mm2). Considering the form of tannins found in raw samples, in the white variety, tannin in the pericarp appeared as crystalline dust
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Fig. 4. Cross-sections of rice caryopside samples stained with tannin solution. (A) Venere rice; (B) Ribe rice; (C) Particular of endosperm of Venere rice after risotto treatment; (D) Particular of endosperm of Ribe rice after risotto treatment; (E) Particular of antioxidant compound absorption (stained with tannin solution) in Venere rice sample. Legend: dt = dark granular form of tannins; cdt = crystalline dust form of tannins.
(Fig. 4B), whereas in the black (Fig. 4A) and red varieties the tannin appeared in dark granular form. Some tannins were also noted in the cytoplasm of aleurone cells and in the endosperm of all varieties. After both cooking treatments, tannins were also observed in a remarkable concentration in endosperm (Fig. 4C and D). In particular, a significant increase in the size of the cross-section that reacted with tannin solution was observed in the black and white varieties, in which tannins were present throughout the
endosperm cross-section. A lower but still significant increase in tannins was also measured in the red variety, where tannins were not observed throughout the endosperm but only in the outermost layer of the cross-section, explaining the lower amounts measured in this cooked rice. In particular, in the white rice, the tannin solution reacted in proximity with the starch granules (Fig. 4D), whereas in the black (Fig. 4C) and red rice varieties tannins were more scattered in the caryopsis. In the samples analyzed, on average, risotto samples presented a higher tannin content
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(Tables 1 and 2) than boiled samples, although the difference between the two treatments was not statistically significant. In causing partial solubilization of cell wall components, thermal treatment probably releases some compounds and components previously bound to the cell wall, thus increasing detectable tannins. A release of bound phenolics after cooking was observed in millet grains by Chandrasekara, Naczk, and Shahidi (2012). Moreover, some authors suggest the possible migration of phenolic compounds (present to a larger extent in the external layers of the caryopsis of cereals) into the endosperm during cooking and the formation of complexes with the macromolecules present in the internal part of the grain (Chandrasekara et al., 2012; Towo, Svanberg, & Ndossi, 2003). The presence of tannins in endosperm after cooking could be explained by the above-mentioned hypothesis of compounds leaching into cooking water and their subsequent partial resorption during the second stage of cooking. Molecules are probably reabsorbed through the fractures because the outermost layers were not severely damaged by cooking and did not permit the migration of compounds from water to endosperm. Indeed, the absorption of compounds that react with tannin solution is visible near a fracture (Fig. 4E). In polished rice it has been observed that during boiling, starch, cell wall fragments and other materials are eluted into cooking water (Tamura & Ogawa, 2012), and, when water is absorbed, eluted materials condense on the surface of grains without fractures, forming a coated layer. In our samples we observed that the two thermal treatments had different effects on tannin inclusions in the three rice varieties. In particular, the white variety showed a significant increase in tannin content after cooking, suggesting that during cooking certain compounds with red-ox properties were released from the fiber to which they were bound and could then react with the tannin solution. Conversely, the red rice variety had the lowest tannin content after cooking, despite having a higher content in raw samples. The different behavior among the varieties could be due to the different compounds with red-ox properties present in the three varieties. In the literature there are reports indicating that rice varieties with different pigmentation contain different phenolic compounds (Finocchiaro et al., 2010). In fact, the different forms of tannin inclusions (crystalline dust, dark granular) observed in our samples were probably formed by different compounds (that react differently with the tannin solution). These compounds are probably affected differently by cooking, causing different behavior of the tannin inclusions in the three varieties. 4. Conclusions In evaluating the effects of cooking on rice, we observed that in all the varieties analyzed, risotto cooking damaged the grain structure more than boiling, and the three varieties presented different proportions and areas of voids, with the black rice being the most affected by cooking. Black rice also presented differences in starch gelatinization. With regards to tannins, the black and red rice varieties displayed a higher content of these compounds in raw samples than the white variety. After both thermal treatments, tannins were measured in very high concentrations also in the endosperm, indicating the possible a resorption of some previously leached compounds. Moreover, a significant increase was observed in the white variety. These observations suggest that an evaluation of the anatomical structure might help to better understand the behavior of cereals during domestic cooking, which, in turn, affects their nutritional quality also in terms of compound accessibility. In fact the release
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